The Interactive Fly
Genes involved in tissue and organ development
The wing is derived from the wing imaginal disc, formed from the embryonic ectoderm by an invagination at the intersection of a dorsal/ventral stripe of Wingless with an anterior-posterior stripe of Decapentaplegic. These cells come from the posterior compartment of the second thoracic parasegment and the anterior compartment of the third (Cohen, 1993). One or two cells expressing aristaless, a homeobox protein, invaginate along with the presumptive imaginal disc. The aristaless expressing cells are fated to become the distal most cells of the wing (Campbell, 1993).
The nuclear proteins, Distal-less and Vestigial are the earliest known markers for the leg and wing imaginal discs, and are required for pattern formation along the proximal-distal axis in the adult. However, their involvement in imaginal disc formation is not clear since imaginal discs are formed in the absence of Dll and vg. Ventral leg and dorsal wing primordia appear to originate from a common imaginal primordium. Cell lineage tracing study has shown that in stage 12, the wing disc cells expressing vg segregate and move dorsally away from Dll expressing cells. Of major importance is the role of escargot and snail in the initial specification of the wing disc. Although Snail is known best for its role in mesoderm formation, it is expressed later in the ectodermal wing primordium. In an esg sna double mutant, the apical constriction of the wing primordial cells is not observed. This supports the idea that in the absence of esg and sna the wing primordium is transformed into epidermis. A two step model for wing disc formation is proposed. In the first step, an extrinsic signal, such as the combined activity of Dpp and Wg, induces vg, esg and sna expression. In a second step esg and sna initiate a program of auto- and crossactivation to stabilize their own expression. This second step is likely to be responsible for irreversible and autonomous fate commitment of the wing primordium (Fuse, 1996).
The disc is structured into three axes. The anterior/posterior axis is structured by the segment polarity genes engrailed, hedgehog and dishevelled on either side of a stripe expressing decapentaplegic. The proximal/distal axis is strucured by the genes distal-less and aristaless. The dorso-ventral axis is structured by vestigial. wingless serves to structure the sensory hairs at the edges of the wing.
For a discussion of the hierarchy of genes involved in wing vein formation (Sturtevant, 1995), see the biological overview of rhomboid. Essential information is also found at the vein site.
The origin of insect wings has long been debated. Central to this debate is whether wings are a novel structure on the body wall resulting from gene co-option, or evolved from an exite (outgrowth; for example, a gill) on the leg of an ancestral crustacean. This study reports the phenotypes for the knockout of five leg patterning genes in the crustacean Parhyale hawaiensis and compares these with their previously published phenotypes in Drosophila and other insects. This leads to an alignment of insect and crustacean legs that suggests that two leg segments that were present in the common ancestor of insects and crustaceans were incorporated into the insect body wall, moving the proximal exite of the leg dorsally, up onto the back, to later form insect wings. These results suggest that insect wings are not novel structures, but instead evolved from existing, ancestral structures (Bruce, 2020).
The Drosophila wing disc is divided along the proximaldistal
axis into regions giving rise to the body wall (proximal), wing hinge
(central) and wing blade (distal). DNA microarray analysis has been applied to
discover genes with potential roles in the development of these regions. A set of 94 transcripts, enriched two fold or greater, were identified in the body
wall and 56 enriched transcripts in the wing/hinge region. Transcripts
that are known to have highly restricted expression patterns, such as
pannier, twist and Bar-H1 (body wall) and knot,
nubbin and Distal-less (wing/hinge), show strong differential
expression on the arrays. In situ hybridization for 50 previously
uncharacterized genes similarly revealed that transcript enrichment identified
by the array analysis is consistent with the observed spatial expression.
There was a broad spectrum of patterns, in some cases suggesting that the
genes could be targets of known signaling pathways. Three of
these genes respond to wingless signaling. Genes
likely to play specific roles in tracheal and myoblast cell types were also discovered, since these cells are part of the body wall fragment. In summary, the identification of
genes with restricted expression patterns using whole genome profiling
suggests that many genes with potential roles in wing disc development remain
to be characterized (Butler, 2003).
To identify genes with expression patterns enriched in the presumptive
wing/hinge or body wall regions, wing imaginal discs were cut into two
fragments at the boundary between the body wall and the wing hinge. Folds associated with the hinge provide morphological features to allow precise cutting. RNA expression
profiles of these samples were determined using oligonucleotide microarrays
representing approximately 13,500 known and predicted genes in the
Drosophila genome (Genechip Drosophila Genome Array 1,
Affymetrix). Information for all genes is available at http://dev.biologists.org/supplemental/. Ninety-four transcripts show two-fold or greater enrichment in the body wall and 56 transcripts show two-fold or greater enrichment in the wing/hinge. Several of these
genes were also found to be more highly expressed in
wing discs than leg discs or eye-antennal discs, suggesting they may also have
appendage-specific roles (Butler, 2003).
The rank order of transcripts correlates well with the spatial expression
patterns of characterized genes. In the body wall, pannier
(pan), twist (twi) and BarH1, which are
enriched in the body-wall sample, are all known to be highly expressed in the presumptive body wall. In the wing, knot (kn), nubbin (nub) and
Distal-less (Dll) are expressed at levels greater than
10-fold above those in the body wall. kn is expressed in the wing 3/4 intervein and hinge regions; nub is strongly expressed in the entire wing pouch and
Dll is expressed along the dorsal-ventral (DV) margin exclusively in
the wing pouch (Butler, 2003).
Other genes, known to have important roles in disc development, appear
lower down the rank order. vestigial (vg), a key gene for development
of the wing and hinge regions, shows only two-fold enrichment but this is consistent
with the expression pattern of vg in the wing disc that extends into
the body wall region. Transcripts with expression patterns restricted to the
posterior compartment, [engrailed (en), invected
(inv) and hedgehog (hh)], show
approximately two-fold enrichment in the wing/hinge sample. The
anterior-posterior compartment boundary splits the wing/hinge region into two
equally sized compartments, but the position of the boundary in the body wall
region produces a small posterior compartment representing approximately
one-quarter of the total tissue. This is consistent with the approximately two-fold enrichment of posterior-specific transcripts found in the wing/hinge tissue sample. The
E(spl)-Complex genes are expressed in developing sensory organs found
in both the body wall and wing margin regions. Hence, these genes are not
enriched in any one sample. The m6 gene is an exception (enriched in the body wall sample) and is known to be
expressed only in the body wall region. In
contrast, genes that show ubiquitous expression such as Ras or tubulin show no
enrichment on the arrays (Butler, 2003).
Microarray analysis can therefore identify transcripts known to be
differentially expressed in the wing/hinge and body wall regions of the disc. Few expression patterns of the identified genes have been
described, so to verify the validity of the approach, and to discover more
genes with potential roles in the development of these specific regions, in situ hybridizations were made for some of these uncharacterized genes (Butler, 2003).
Fifty transcripts that had strong enrichment
(mostly three-fold or greater) were examined. For the body wall-enriched transcripts, the larger set (only transcripts for which clones are available in the
Drosophila gene collections -- DGC1 and DGC2, Berkeley Drosophila
Genome Project) were examined. For the wing/hinge region, transcripts were examined with
three-fold or greater enrichment, systematically in rank order from the top,
and PCR probes were generated when clones were not available. All
transcripts tested showed expression patterns that were consistent with the
microarray data, providing confirmation that the microarray analysis mirrors
the spatial distribution of transcripts in vivo (Butler, 2003).
The wing disc comprises three cell layers: the squamous epithelium of the
peripodial membrane; the columnar epithelium that becomes the adult epidermis,
and the adepithelial layer that includes myoblast cells that give rise to
adult thoracic muscles and tracheal cells that form air passages. The adepithelial
layer extends from the proximal disc dorsally into the hinge region. The body wall
fragment includes cells of all three layers, so the arrays also identified
transcripts specific to muscle and tracheal cells (Butler, 2003).
pan and BarH1, which encode transcription factors, are
expressed in the body wall epidermis and are involved in bristle patterning. Both
transcripts were highly enriched on the arrays. Also highly enriched
was tailup (tup), which encodes a LIM domain homeobox protein,
and is expressed in the epithelium in a large region of the posterior body
wall encompassing the presumptive postnotum, scutellum and scutum. No role for
tup in patterning the mesothorax has been described. Another
transcript with broad expression was thrombospondin/CG11326 (tsp),
which is expressed in a similar region of the body wall to tup. tsp is also expressed in the ventral hinge and hence shows lower enrichment on the arrays.
The other genes found to be specific to the epithelium showed highly localized
expression: Obp56a/CG11797, CG10126, CG3244 and Glucose dehydrogenase. Obp56a/CG11797 encodes an odorant-binding protein and interestingly three other odorant-binding proteins showed enrichment on the arrays: Obp99a, CG9358 and Obp56d/CG1128. Idgf4, encoding an imaginal disc growth factor, is
expressed in the peripodial membrane, primarily in dorsal cells. Presumably secretion
of Idgf4 could influence development of the columnar epithelium (Butler, 2003).
Myoblast cells of the adepithelial layer develop into the direct and
indirect flight muscles of the thorax, and genes involved in the development
of these muscles have been shown to be expressed in the myoblasts during wing
disc development. Several of these transcripts are enriched on the arrays: Mef2,
twist (twi) and heartless (htl).
Act57B is known to be regulated by Mef2 in the embryo, and Act57B is expressed in the myoblasts, suggesting this
relationship also exists in these adult muscle precursors. Mef2
expression is activated by twi and may
be inhibited by the transcriptional repressor, zinc finger homology 1
(zfh1). zfh1 is expressed in the myoblasts. stumps is
also enriched on the arrays and expressed in the myoblasts.
Together with htl, stumps has a role in the development of the
tracheal cells. Viking (Vkg) encodes a
component of collagen type IV and is known to be coexpressed with
Cg25C, another collagen IV subunit in the embryo and in blood cells. Both transcripts are enriched on the arrays and show similar
expression patterns in the adepithelial myoblasts and blood cells. Other genes
showing specific expression in the myoblasts are BM-40/SPARC, a
calcium-binding glycoprotein, which is expressed in the embryonic mesoderm, Elongation factor 1 alpha 100E (Ef1 alpha), CG8689, an
alpha-amylase, and two transcripts encoding predicted proteins with unknown function
CG11100 and CG15064 (Butler, 2003).
In the wing disc, cells of the larval and developing adult tracheal systems
require activity of genes in the FGF pathway.
Some of the key genes are expressed in the myoblasts (for example,
htl and stumps), others in the epithelium (for example,
branchless, bnl), and others in the tracheal cells themselves (for
example, breathless, btl). htl and stumps showed
enrichment on the arrays but bnl and btl were not
detectable. For bnl this may be because expression is highly
localized and apparently at very low levels. However, it is not clear why the arrays failed to detect btl expression because six genes were identified that are also expressed specifically in tracheal cells -- these are CG5397, an
O-acyltransferase, CG4386, a serine-type endopeptidase, CG2663, an
alpha-tocopherol transfer-like protein, and CG15353, CG6921 and CG9338 that have no known homologies. In particular, CG4386 is interesting since it is only expressed in the dorsal branch, and CG6921 is distinguished because it is very strongly expressed in the most proximal cells (Butler, 2003).
The wing/hinge fragment of the wing disc primarily contains cells of the
peripodial membrane and the columnar epithelium, with only a few
myoblasts that extend into the hinge region. Thus the genes detected by the
arrays as enriched in this disc fragment are expressed in cells of one of the
two epithelial layers (Butler, 2003).
Transcription factors comprise the largest category of genes (18/56) with
elevated expression in the wing/hinge region. These are expected to have
regulatory roles in patterning the region. Transcription factors with known
expression domains and roles in wing development are present: kn, pox-n,
nub, Dll, bifid/optomotor blind, rotund, ventral veins lacking, en, vg
and in. pdm2, which is highly related to nub,
also shows wing-enriched expression on the arrays and is expressed in a
similar domain to nub. pdm2 apparently has no significant function in the
wing. The roles of the remaining seven predicted transcription factors are unknown,
although the expression pattern of zinc finger homology 2
(zfh2) and Sox 15 have been described and both are expressed
specifically in the hinge region. defective proventriculous (dve), which
encodes a homeodomain protein, and CG15000, which is similar to
NGFI-A-binding protein 2, are broadly expressed in the wing pouch, although
dve is downregulated at the DV compartment boundary. odd
paired (opa), known for a role in embryonic segmentation, is
discretely expressed in cells of the presumptive mesopleura and dorsal hinge. No role for opa in wing disc development has been reported. Dorsocross1
(Doc1) and Doc2/CG5187 are T-Box related factors that are
expressed in what appears to be an identical domain in the wing disc. Both transcripts
also accumulate in body wall cells and this probably lowers their position in
the overall ranked list (Butler, 2003).
Eight transcripts encoding enzymes are enriched two-fold or greater in the
wing/hinge region. This group includes the most highly enriched transcript detected in the analysis, a kazal-type serpin gene CG17278 (68-fold). CG17278
shows a strong and specific expression pattern in the wing encompassing most
of the wing pouch. One of the potentially most interesting wing-enriched
enzymes is a cytochrome P450 gene, Cyp310al. This gene is strongly
expressed in the dorsal and ventral parts of the wing pouch but excluded from
the DV and AP boundaries. Variable expression in anterior body wall cells is also
observed that is consistent with the array data that indicate
Cyp310al transcripts are also present in body wall RNA. Surprisingly, the
ß-galactosidase gene (CG3132) was found to be enriched in the
wing/hinge region. Weak expression was found in a cluster of cells in the hinge but the
majority of expression is in blood cells, which adhere preferentially to the
distal disc margin. Thus the ß-gal transcript probably appears as wing/hinge enriched primarily because it is expressed in blood cells. The
expression patterns of two other enzymes were also determined: the metalloendopeptidase
Nep1/CG5894 and UDP-glucosyl transferase (Ugt86Di) (Butler, 2003).
The alpha-integrin, inflated, which has a role in cell adhesion,
is expressed in the ventral compartment and is
thus enriched on the wing/hinge arrays. A novel gene,
CG5758, is potentially involved in cell adhesion since it encodes a
predicted protein with ß-Ig-H3/Fas domains and its expression is
restricted to the dorsal hinge. CG8381 encodes a proline-rich protein with repeated 'PEVK' motifs also found in titin. This gene is strongly expressed in the wing
pouch but repressed in cells of the future veins and cells at the DV margin. Despite intense expression in the wing pouch, CG8381 shows only modest enrichment on
the arrays, probably reflecting the fact that the gene is also expressed in several groups of cells in the body wall region (Butler, 2003).
The expression of two receptors was determined. CG4861 encodes an
ldl-receptor-like protein and is expressed at very low levels throughout the
wing pouch. wengen
/CG6531, which is a receptor of the TNFR family, is
expressed strongly in the wing pouch and weakly in the body wall. On the arrays, its
ligand, eiger, was undetectable in the wing/hinge region sample but
enriched in the body wall sample (Butler, 2003).
Two structural proteins, CG6469, a larval cuticle protein, and
CG14301, a chitin-binding protein, are the only genes identified
as being expressed in the ventral peripodial membrane. CG6469 is
expressed broadly in the peripodial membrane but at a higher level in the
ventral region. CG14301 is expressed in cells of both epithelial layers, in the
columnar epithelium at the anterior disc margin and in four patches of cells
in the wing pouch and the overlying peripodial membrane (Butler, 2003).
In a group of genes with miscellaneous functions the expression of three genes was determined. anachronism (ana), a secreted glycoprotein, is expressed in five clusters of cells including one in the body wall region and in some individual neuroblasts. ana null mutants are viable and have no observable defects suggesting it is not required, or functions redundantly, in the wing.
CG14534, which has a domain that has been recognized in several
proteins but has an unknown function (DUF243), is expressed only in cells that
will give rise to the posterior wing margin. CG8483,
which has homology to a venom allergen, is expressed in a complex pattern
suggestive of expression in peripheral sense organ precursors (Butler, 2003).
The expression pattern is described for five of eight genes for which the
sequence reveals no homology to known protein domains. CG15489 and
CG15488
are in a cluster of genes also including nub and pdm-2 that
are expressed in similar domains and are adjacent in the genome.
CG15001, consisting of only a single exon, is adjacent to another
gene (CG15000), also discovered on the arrays, with a similar
expression domain.
BG:DS00797.2/CG9008 is expressed strongly in the wing pouch and also
in the adepithelial cell layer. CG8780 is highly enriched on the arrays (31-fold), and expressed specifically in the hinge and ventral pleura (Butler, 2003).
The genes CG17278, Cyp310a1 and CG8381 all show very
intense expression in the wing pouch but reduced expression at the DV margin. Wg is expressed at the DV margin forming a gradient that regulates the expression of target
genes in a concentration-dependent manner. To
determine whether Wg signaling represses the expression of CG17278,
Cyp310a1 and CG8381, wg was ectopically expressed in the
dorsal and ventral wing-pouch regions (71B-gal4; UAS-wg), or Wg function
was inhibited at the DV margin by expressing a dominant-negative form
of TCF (Pangolin), a transcription factor required for Wg-signal transduction
(C96-GAL4; UAS-DN-dTCF). With higher levels of Wg activity
in the wing pouch, expression of all three genes was inhibited. In contrast,
inhibition of Wg signaling at the DV margin allowed ectopic expression of
Cyp310a1 in all margin cells and increased the number of cells
expressing CG17278 and CG8381. In the
presumptive margin, cells continue to express wg in the absence of Wg
activity; cell replication increases,
and ectopic expression of dmyc appears in margin cells.
Therefore, ectopic expression of the genes studied here is caused by loss of
Wg-dependent repression rather than loss of the non-expressing cells from the
presumptive margin. This does not imply that Wg-dependent repression must be
direct. Without functional data on these potential target genes, their
relationship to wg and their role in wing patterning remain
unknown (Butler, 2003).
The Drosophila wing disc has been a fundamental model system for the discovery of key signaling pathways and for understanding of developmental processes. However, a complete map of gene expression in this tissue is lacking. To obtain a gene expression atlas in the wing disc, single cell RNA sequencing (scRNA-seq) was used, and a method was developed for analyzing scRNA-seq data based on gene expression correlations rather than cell mapping. This enables computation of expression maps for all detected genes in the wing disc and to discover 824 genes with spatially restricted expression patterns. This approach identifies clusters of genes with similar expression patterns and functional relevance. As proof of concept, the previously unstudied gene CG5151 was characterized and was show to regulate Wnt signaling. This method will enable the leveraging of scRNA-seq data for generating expression atlases of undifferentiated tissues during development (Bageritz, 2019).
To construct a gene expression map of the Drosophila wing disc, cells from wing discs of 3rd instar female larvae and their mRNA using DropSeq. This yielded RNA sequences from 1,468 cells with a median depth of 3,774 transcripts and 1,134 genes per cell, in line with or better than what others have reported for Drosophila cells using this method. True cell barcodes could be unambiguously identify indicating a low level of ambient mRNA, and hence cell breakage, during sample preparation. Gene expression values of two biological replicate DropSeq libraries correlated very highly to each other indicating reproducibility of the data. The average gene expression values obtained by combining together all the single-cell reads correlated well with RNA-seq data of whole, non-dissociated wing discs, suggesting that the DropSeq data captured most of the gene expression in the wing disc and that the DropSeq procedure, including the cell dissociation, did not strongly alter gene expression in the disc (Bageritz, 2019).
To identify sub-populations of cells in the wing disc, cells using a graphical approach implemented in the Seurat R package for single-cell sequencing data. Visualization by t-Distributed Stochastic Neighbor Embedding (t-SNE) identified two distinct cell populations. Inspection of the main marker genes distinguishing these populations revealed that the two clusters correspond to cells of the wing disc proper (and adult muscle precursor cells which are attached to the basal surface of the dorsal wing disc. Since this study focused on the wing disc proper, AMP cells were excluded from all subsequent analyses (Bageritz, 2019).
To identify genes with a spatially restricted expression patterns (which were termed Spatially Restricted Genes, SRGs), plots were carried out for every gene the number of cells in which it was detected versus the average expression level
of the gene in those expressing cells. The rationale is that for ubiquitously expressed genes, the stronger the gene is expressed, the higher the chance the mRNA will be captured by the DropSeq beads, and hence the higher the number of cells in which it will be detected. Indeed, it was found that most genes lie on a curve that progressively increases and asymptotes near the total number of cells sequenced. The SRGs are the genes that are observed in fewer cells than expected, given the level of expression of the gene. These were identified as genes with residuals smaller than 1 standard deviation below the mean on the inverse graph, yielding a set of 824 SRGs. As a benchmark, a list was compiled of 28 genes well-known from the literature to be expressed in specific domains of the wing disc, such as engrailed, dpp, apterous, or wingless. The 824 SRGs included all 28 of these known patterning genes and almost all genes known to have a spatially restricted expression pattern in the wing disc. In comparison, a similarly sized set of 829 'Highly Variable Genes' (HVGs) identified using the Seurat R package only contained 6 of the benchmark genes, suggesting the analysis presented in this study is well suited for the specific goal of identifying genes with spatially restricted expression domains (Bageritz, 2019).
By using this set of SRGs for dimensional reduction and clustering, wing disc cells clustered into five clusters along the proximal-distal axis of the wing, corresponding to the wing margin, the wing blade, the proximal wing, the hinge, and the notum, as could be seen by the expression levels of
wing margin (Wnt4), pouch (nub), or hinge/notum genes (tsh, zfh2, pnr, hth) in the five clusters. It was confirmed that there were no biases in these clusters in terms of the number of Unique Molecular Identifiers (UMIs)/cell, read alignment rate, fraction of mitochondrial RNA or representation of the two biological replicates. The major wing disc regions were retrieved by the clustering approach, indicating a successful cell isolation from the entire tissue (Bageritz, 2019).
Whether it was possible to determine the location in the wing disc of the sequenced cells, based on the presence or absence of expression of genes with known expression patterns, such as engrailed (for the posterior of the wing), ci (for the anterior), apterous (for dorsal) and so on. Since the expression pattern of many genes is known in the wing disc, the intersection of these gene expression domains could allow precise placement of sequenced cells. However, although the DropSeq data are of high quality, it was not possible to confidently map the location of the sequenced cells because the transcriptome coverage of current single-cell approaches does not allow distinguishing whether a gene is not expressed or not detected in any given cell: Although roughly 35% of wing disc cells should express engrailed (estimated by measuring the area of the engrailed expression domain of a wing disc), and 65% of disc cells should express the gene ci with complementary expression pattern, in the entire library only 14% of cells were en+ (>0 reads for en) and 28% were ci+. Attempts were made to tested whether this could be solved by setting a minimum UMI per cell threshold. Setting a minimum requirement of 12,000 UMIs/cell, however, still
resulted in only 84% of cells being en+ or ci+, and only 45 of the 948 sequenced wing disc cells passed this threshold. Therefore an alternate method was sought to leverage these data and build a wing disc expression map (Bageritz, 2019).
It was noticed that correlations in gene expression between genes, based on their expression across the hundreds of sequenced cells, are quite good. For instance, correlation coefficients were calculated between en and all other genes in the genome across the sequenced cells and it was found that, as expected, the top genes genome-wide correlating to en are inv and hh, and the top anti-correlating gene to en is ci. Likewise, the top genes either correlating or anti-correlating to wg or dpp are also known to be expressed in either overlapping or complementary expression patterns, respectively, in the wing disc. The underlying data can be visualized using 2-dimensional histograms. For instance, in the case of wingless (wg) and frizzled 2 (fz2) which are expressed in largely complementary domains, many cells have detectable transcripts for fz2 or for wg, but few cells express both. In contrast, a good number of cells have detectable transcripts for both wg and Wnt6, as expected given that they are expressed in overlapping domains. Likewise, few cells are en+/ci+, whereas many cells are en+/inv+ or en+/hh+, as expected from their relative expression domains. Interestingly, this correlation analysis also identifies novel genes which correlate strongly with en and therefore likely have a similar expression pattern, such as the non-coding RNA CR44334 (Bageritz, 2019).
A method for generating gene expression maps based on gene correlations, which does not necessitate mapping the location of the sequenced cells in the tissue. This method uses the concept that the correlation coefficient between two genes indicates whether the expression domain of the two genes is overlapping (positive correlation), complementary (negative correlation), or orthogonal (no correlation). Therefore, for a given cell within the expression domain of Gene 1 with known expression pattern, uncharacterized Gene 2 is likely also expressed if the two genes correlate, and not expressed if they anti-correlate. If the correlation coefficient is close to zero, then the expression domain of Gene 1 is not informative with regards to Gene 2. A virtual map was compiled of the wing disc containing the expression domains of 58 genes known from the literature to have distinct expression patterns which were term 'mapping genes (Fig. 2e), and a cross-correlation matrix was calculated between these 58 mapping genes and all genes in the genome. To compute an expression map of a gene, for each position in the wing disc the correlation coefficients between this gene and the mapping genes were mapped with a weighting factor of either +1 or -1 depending on whether the mapping gene is expressed in that position or not. These maps are called 'computed expression maps'. This approach was tested by performing fluorescent in situ hybridizations (FISH) to assay whether the computed maps and the in situs agree with each other (Bageritz, 2019).
The method described above generates computed expression maps for all genes in the genome. Based on these, there are multiple different ways to sort out genes of interest based on similarity of their expression patterns to known genes of interest. Three different ways are presented: (1) clustering genes using a two-dimensional dendrogram, (2) searching for genes that correlate or anti-correlate with one specific gene of interest, and (3) generating an interaction network based on gene expression similarities. To cluster genes by expression pattern, a cross-correlation matrix of gene expression was calculated for all 824 SRGs against each other, and then this was used to hierarchically cluster the genes according to their expression patterns. Visual inspection of this dendrogram confirmed that genes that cluster together have similar expression patterns in the wing. For instance, the 'dark blue' cluster consists of en, inv and hh, which are co-expressed throughout the posterior compartment of the wing disc. The 'medium blue' cluster consists of genes expressed in the proximal region of the wing disc such as hth and zfh2, together with other genes of unknown expression pattern or function. The 'yellow' cluster consists of wg, Wnt4, Wnt6 and cut (ct), which are all expressed on or near the dorsal/ventral boundary of the wing disc. Three clusters were selected that contain both characterized and uncharacterized genes and in situs were performed on all genes in the cluster. The 'red' cluster consists of genes enriched in the wing pouch with a pattern along the anterior-posterior axis. The gene 'kn' is one of the 58 'mapping genes' hence the computed map matches the in situ because it is one of the inputs into the mapping algorithm. CG9850, a gene of unknown function and expression pattern, is predicted by the computed map to also have a mild 'kn-like' stripe that is less accentuated than kn, and indeed this matched the fluorescent in
situ. The uncharacterized gene CG3168 was predicted according to the computed map to have a broader expression pattern in the wing pouch that is repressed at the dorsal/ventral boundary. Indeed, this pattern was confirmed by in situ hybridization. The gene Trim9, involved in neurogenesis in the central nervous system, but of unknown expression pattern or function in the wing, was predicted to be expressed predominantly in the wing pouch with an inverse venation pattern and inhibition at the D/V boundary. This complex expression pattern was also confirmed by in situ hybridization. The in situ for the last gene in the cluster, CG7201, had some elements of the predicted map, such as higher expression medially and broad repression at the D/V boundary, but it also differed somewhat from the map. Thus, overall, the computed maps are able to predict the main features of the gene expression patterns. Along the same lines, in situs were performed for genes in the orange and light-blue clusters, and the in situs confirmed the broad characteristics of the computed maps. Interestingly, due to their expression patterns, this implicates a number of genes with previously uncharacterized functions and/or expression patterns in anterior-posterior patterning and ptc or dpp signaling. For instance, the expression pattern of the functionally uncharacterized gene nord is largely overlapping with the expression patterns of dpp or ptc (Bageritz, 2019).
A second way to identify genes of interest is to select genes that have expression patterns that either correlate or anti-correlate with a specific gene of interest, such as senseless, wg or dpp. Amongst these are many genes
that have previously been implicated in the respective signaling pathways. Hence, in situs were performed only for the top correlating/anti-correlating genes with distinct expression patterns that had not previously been characterized in the wing disc. In all cases, the in situ confirmed the pattern predicted by the computed maps, thereby implicating novel genes in wing neurogenesis. For instance, Fhos is involved in actin stress fiber formation 17 and hence may play a role in neurogenesis, and ImpL3 is the metabolic enzyme lactate dehydrogenase. Rau and cpo have previously been implicated in neurogenesis in other organs. Amongst the genes correlating with wingless, CG10249 (Kank) has been linked to attachment sites between muscle and epidermal cells in the embryo. One additional uncharacterized gene correlating with Dpp is CG9689 (Bageritz, 2019).
CG5151 was selected as a gene to study in more detail, as it is functionally uncharacterized and has a human ortholog LDLRAD4 (also known as C18ORF1). The computed map predicts CG5151 to be expressed weakly along the dorsal/ventral boundary and in a more proximal ring, coinciding with wingless expression. In situ hybridizations confirmed this expression pattern, and also detected expression of CG5151 in the Adult Muscle Precursor cells, AMPs, which are not part of the disc proper and are not included in the computed maps. This expression pattern was also observed with a GFP transcript trap in the endogenous CG5151 locus. Next tests were performed to see if CG5151 might be involved in wingless or notch signaling. Knockdown of CG5151 in the posterior half of the wing caused wing notching (a phenotype typical for Notch or wingless loss-of-function, and strongly reduced wingless expression. In sum, as proof of principle, the mapping strategy allowed identification of a novel uncharacterized gene, CG5151, which has an expression pattern that overlaps with that of wingless, and is functionally involved in wingless/notch signaling. Interestingly, the human ortholog LDLRAD4 is functionally not well characterized but its expression is elevated in hepatic cancers and it promotes tumorigenesis. It will be interesting to test whether Wnt or Notch signaling are involved in its tumorigenic activity (Bageritz, 2019).
The simple cellular composition and array of distally pointing hairs has made
the Drosophila wing a favored system for studying planar polarity and the
coordination of cellular and tissue level morphogenesis. A gene expression screen was carried out to identify candidate genes that functioned in wing and wing hair morphogenesis. Pupal wing RNA was isolated from tissue prior to, during and after hair growth and used to probe Affymetrix Drosophila gene chips. 435 genes were identified whose expression changed at least 5 fold during this period and 1335 whose expression changed at least 2 fold. As a functional validation, 10 genes were chosen where genetic reagents existed but where there was little or no evidence for a wing phenotype. New phenotypes were found for 9 of these genes providing functional validation for the collection of identified genes. Among the phenotypes seen were a delay in hair initiation, defects in hair maturation, defects in cuticle formation and pigmentation and abnormal wing hair polarity. The collection of identified genes should be a valuable data set for future studies on hair and bristle morphogenesis, cuticle synthesis and planar polarity (Ren, 2005).
The primary goal in characterizing pupal wing gene expression was to identify genes that play an important role in pupal wing morphogenesis. ken and barbie (ken) encodes a DNA binding transcription factor that contains an N terminal BTB/POZ domain and 3 C2H2 zinc fingers. Its expression increased 6.8 fold from 32 to 40 hrs. Loss of function mutations in ken are semilethal. Escaper adults have been described as having unpigmented aristae and often lack external genitalia (hence the gene name). Wings were examined from ken mutant escapers and also in genetic mosaics. The triple row bristles on the wing margin were lightly pigmented reminiscent of the arista phenotype. This is most obvious in mosaics where the lightly pigmented bristles stand out from their wild type neighbors. No hair phenotype was seen, but a subtle hair pigmentation phenotype would be difficult to see (Ren, 2005).
The HMGS gene encodes the Drosophila HMG Coenzyme A synthase, a key enzyme in steroid and isoprenoid metabolism. Its expression increased 8.4 fold from 32 to 40 hrs. Individuals homozygous for a P insertion allele die as pharate adults or pupae. The pharate adults are notable for a melanotic liquid that accumulates principally near the ventral head. Mutations that result in weak cuticle often show such melanotic leakage, suggesting that HMGS may be required for normal cuticle elaboration. The reason for the phenotype being seen primarily in the ventral head is unclear. No evidence was seen for a specific wing phenotype (Ren, 2005).
The expression karst gene, which encodes betaHeavy-spectrin, increased 5.5 fold from 32 to 40 hrs. Spectrin typically contains 4 chains, 2 alpha and 2 beta; these chains are known to link the actin cytoskeleton to the plasma membrane. Somewhat surprisingly, kst mutants are viable (at reduced levels) and female sterile due to defects in the follicular epithelium. Adult kst mutants have rough eyes and their wings often are cupped downward. kst wings were examined and an additional mutant phenotype was found that is nicely correlated with its expression profile. kst wing cells produce normal looking hairs but the hairs are often found on a small pedestal. The wing cell surface (that is not hair) is rough and at times remnants of cell outlines are visible. This phenotype can also be seen in mosaic clones. The clones can be recognized under the stereo microscope because they are often associated with a dimpling of the wing surface (Ren, 2005).
The krotzkopf verkehrt (kkv) and knickkopf (knk) genes were both identified in a screen for having an unusual defect in embryonic cuticle, known as the blimp phenotype. Mutant embryo cuticles were seen to expand in cuticle preparations. The kkv gene encodes a chitin synthase implicating it in cuticle synthesis and its expression increased 4.9 fold from 32 to 40 hrs. The knk gene encodes a novel gene that is only well conserved in the ecdysozoa, suggesting a role in cuticle metabolism. The amino acid sequence shows homology to what is thought to be a dopamine binding domain suggesting Knk might be involved in cross linking of cuticle. The expression of knk increased 7 fold between 32 and 40 hrs. Mutations in both of these genes are embryonic lethals so mosaic clones of cells carrying mutations in either of these genes were examined. The phenotypes seen in the adult cuticle were quite similar to one another. Most notably wing mutant wing hairs displayed a lack of pigmentation and were thinner and flimsier than normal. This phenotype is dramatic and at low magnification it often appears as if hairs were not formed by mutant cells. The hairs appeared normal in size and shape when clones were examined in pupal wings arguing that the mutations affect a process after hair outgrowth (e.g., cuticle synthesis or maturation). Clones in other body regions such as the abdomen and thorax also showed a dramatic loss of pigmentation. In all of these cases the borders between pigmented and unpigmented were relatively sharp. Consistent with these mutations resulting in weak cuticle phenotypes, areas were often seen where internal tissues and hemolymph appeared to be erupting from the animal. This was usually seen on the dorsal abdomen, particularly in the region of the intersegmental membrane. The eruptions could be related to the blimp phenotype seen in embryos (Ren, 2005).
The expression of the brain tumor (brat) decreased 5.5 fold from 24 to 40 hrs. This gene has been studied primarily due to the neural tumor phenotype seen in loss of function mutants. The wings of bratts/Df brat flies raised at semi-permissive conditions were examined. No hair phenotype was seen but the occasional loss of sensory bristle shaft cells (principally distally along the anterior margin) was seen and occasional duplicated bristle cells (principally in the costa). These phenotypes are suggestive of a role for brat in specifying cell fate or in Notch mediated lateral inhibition (Ren, 2005).
The expression of dopa decarboxylase (Ddc) increased 6 fold from 24 to 32 hrs and then decreased 1.9 fold from 32 to 40 hrs. This well characterized gene is known to function in the epidermis for the cross linking of cuticle and in the formation of melanin. Loss of Ddc function results in fragile and pale cuticle with thin bristles. No detailed description of the wing phenotype had been reported previously. Ddc null alleles are recessive embryonic lethals adults that contained clones mutant for Ddc were examined. On the abdomen (and some other parts of the body) clones could be seen where there was lightly pigmented cuticle and bristles. No wing phenotype was seen other than apparent clones resulting in lighter triple row bristles. The abdominal clone boundaries were not sharp as seen for grh, knk or kkv, which also give rise to lightly pigmented cuticle suggesting that the Ddc cells might be rescued by the diffusion of dopamine from neighboring cells. Therefore adults homozygous for a temperature sensitive Ddc allele were examined. Animals raised at 25°C showed a much stronger phenotype in general than was in clones suggesting that Ddc acts nonautonously in the wing. The phenotype was even stronger in animals raised at 29°C. The wings of Ddc mutants were characterized by very thin wispy hairs, occasional multiple hair cells and an overall faint appearance. When Ddcts pupal wings were examined, the early hairs appeared normal in morphology. Thus, the wispy appearance of the adult wing hairs is presumably due to a late defect. It is suggested that Ddc dependent cross linking of the cuticle is essential for maintaining the structure of the hair and in the absence of this cross linking the hair collapses after the actin cytoskeleton is disassembled. Occasional multiple hair cells were seen in the Ddcts pupal wings; thus that defect is likely due to a different process also being affected in the mutant. The formation of multiple hair cells has previously been associated with planar polarity defects or due to disruptions of the cytoskeleton (Ren, 2005).
The HR46 gene (also known as DHR3) encodes a nuclear receptor and is an essential gene known to be important for the ecdysone cascade. Large clones of loss of function alleles result in wing (folded and curved) and notum defects (rough short bristles and pale pigmentation). The expression of this gene increased 250 fold from 24 to 32 hr and then decreased 4.3 fold from 32 to 40 hr. Moderate sized wing clones of cells lacking HR46 were examined, but no clear cut phenotype was seen. In pupal wing clones examined a couple of hours after hair formation mutant hairs appeared somewhat thicker but this alteration was transient (Ren, 2005).
The Eip78CD gene encodes a related nuclear receptor. The expression of this non-essential gene increased 3 fold from 24 to 32 hr followed by a three fold drop from 32 to 40 hr (but the differences were not significant) suggesting it might be functionally redundant with HR46. To test this hypothesis Eip78CD mutants, which also contained HR46 mutant clones, were examined. No mutant phenotypes were seen in the clones, suggesting either that there is an alternative redundant gene or that HR46 is not essential for hair morphogenesis. Since the level of HR46 expression fell dramatically between 32 and 40 hrs it seemed possible that declining HR46 expression could be important for hair development. To test this the overexpression of HR46 from a transgene containing a hs promoter was induced. This resulted in a dramatic loss of hair formation leading to wings with extensive bald regions. The strongest phenotype was seen when the transgene was induced by heat shocking 6-8 hrs prior to the time of hair initiation. The phenotype was dose sensitive and directly related to the number of transgenes and length and temperature of transgene induction (Ren, 2005).
The expression of the non-stop (not) gene decreased 3.9 fold from 24 to 40 hrs. Mutations in not result in photoreceptor neurons projecting through the lamina instead of terminating there. The mutations also result in approximately 20% of ommatidia being misoriented -- a planar polarity phenotype. Strong alleles of not die as prepupae so not clones were examined in both adult and pupal wings. Large numbers of clones were induced. Perhaps 25% of wing cells are found in clones. All adult wings of this genotype had regions where there were cells that failed to form hairs or that had very small hairs. These were found only in proximal medial regions on the ventral wing surface. All such wings also had subtle polarity abnormalities; small groups of hairs with slightly abnormal polarity in all regions of the wing. Consistently finding such defects leads to the conclusion that these were due to not clones. Of 47 such wings examined 27 also contained multiple hair cells and a further 10 contained regions with planar polarity defects reminiscent of genes such as fz and dsh. When marked not clones were examined in pupal wings most, but not all, showed cells where hair differentiation was delayed or absent. Such clones were seen in all wing regions. It is suggested that all not clones have delayed hair formation. When the clones are located in wing regions where hairs normally form first (distal or peripheral regions) the hairs form later than normal but still have enough time to reach a relatively normal length. In contrast, when clones are located in regions where hair formation is normally late (proximal and medial regions on the ventral wing surface) not enough time remains prior to cuticle deposition to produce a normal hair. The not gene encodes a ubiquitin carboxyterminal hydrolase likely to function in the removal of ubiquitin from proteins during protein degradation (Ren, 2005).
The Uch-L3 gene also encodes a ubiquitin carboxy hydrolase and its expression decreased 2.9 fold between 24 and 40 hrs. A P insertion mutation in this gene is semi-lethal and escapers have an abnormal eye. No homozygous Uch- L3J2b8 flies were found that eclosed but it was possible to examine animals that died as pharate adults. These animals displayed several morphological defects such as loss of tarsal leg joints, shorter and fatter leg segments, the loss of a discrete antennal segment 4 and a fatter arista that could be due to defects in cell shape or movement. Pupal wings from such animals were examined and wings were found that were wider and shorter than normal and regions were found with a loss of hairs. All of the phenotypes seen in Uch-L3 pupae and pharate adults showed variable expressivity (Ren, 2005).
The manner by which genetic diversity within a population generates individual phenotypes is a fundamental question of biology. To advance the understanding of the genotype-phenotype relationships towards the level of biochemical processes, a proteome-wide association study (PWAS) was performed of a complex quantitative phenotype. The variation of wing imaginal disc proteomes was quantified in Drosophila genetic reference panel (DGRP) lines using SWATH mass spectrometry. In spite of the very large genetic variation (1/36 bp) between the lines, proteome variability is surprisingly small, indicating strong molecular resilience of protein expression patterns. Proteins associated with adult wing size form tight co-variation clusters that are enriched in fundamental biochemical processes. Wing size correlates with some basic metabolic functions, positively with glucose metabolism but negatively with mitochondrial respiration and not with ribosome biogenesis. This study highlights the power of PWAS to filter functional variants from the large genetic variability in natural populations (Okada, 2016).
Post-eclosion elimination of the Drosophila wing epithelium was studied in vivo where collective 'suicide waves' promote sudden, coordinated death of epithelial sheets without a final engulfment step (see Collective Cell Death and Canonical Pathways). Like apoptosis in earlier developmental stages, this unique communal form of cell death is controlled through the apoptosome proteins, Dronc and Dark, together with the IAP antagonists, Reaper, Grim, and Hid. Genetic lesions in these pathways caused intervein epithelial cells to persist, prompting a characteristic late-onset blemishing phenotype throughout the wing blade. This phenotype was leveraged in mosaic animals to discover relevant genes. This study establish that homeodomain interacting protein kinase (HIPK) is required for collective death of the wing epithelium. Extra cells also persisted in other tissues, establishing a more generalized requirement for HIPK in the regulation of cell death and cell numbers (Link, 2007).
Elimination of the wing epithelium in newly eclosed adults is predictable, easily visualized, and experimentally tractable. The major histomorphologic events involve cell death, delamination, and clearance of corpses and cell remnants. Recent studies established that post-eclosion PCD is under hormonal control and involves the cAMP/PKA pathway (Kimura, 2004). While dying cells in the adult wing present apoptotic features (e.g., sensitivity to p35 and TUNEL positive), elimination of the epithelium is distinct from classical apoptosis in several important respects. First, unlike most in vivo models, overt engulfment of cell corpses does not occur at the site of death. Instead, dead or dying cells and their remnants are washed into the thoracic cavity via streaming of material along and through wing veins. Second, extensive vacuolization is seen in ultrastructural analyses, which could indicate elevated autophagic activity. Third, widespread and near synchronous death that occurs in this context defines an abrupt group behavior. The process affects dramatic change at the tissue level, causing wholesale loss of intervein cells and coordinated elimination of the entire layer of epithelium. Rather than die independently, these cells die communally, as if responding to coordinated signals propagated throughout the entire epithelium, perhaps involving intercellular gap junctions. This group behavior contrasts with canonical in vivo models where a single cell, surrounded by viable neighbors, sporadically initiates apoptosis (Link, 2007).
One study proposed that an epithelial-to-mesenchymal transition (EMT) accounts for the removal of epithelial cells after eclosion (Kiger, 2007). Although the results do not exclude EMT associated changes in the newly eclosed wing epithelium, compelling lines of evidence establish that post-eclosion loss of the wing epithelium occurs by PCD in situ—before cells are removed from the wing (Kimura, 2004 and this study). First, before elimination, wing epithelial cells label prominently with TUNEL. Second, every mutation in canonical PCD genes so far tested failed to effectively eliminate the wing epithelium, and at least two of these were recovered in the current screen. Third, elimination of the wing epithelium was reversed by induction of p35, a broad-spectrum caspase inhibitor (Kimura, 2004). Fourth, using time-lapse microscopy, condensing or pycnotic nuclei were clearly detected, followed by the rapid removal of all cell debris in time frames (minutes) not consistent with active migration. Instead, removal of cell remnants occurred by a passive streaming process, involving perhaps hydrostatic flow of the hemolymph (Link, 2007).
This study sampled over one fifth of all lethal genes and nearly 10% of all genes in the fly genome for the progressive blemish phenotype, a reliable indicator of PCD failure in the wing epithelium. Nearly half of the mutants that produced melanized wing blemishing also displayed a cell death–defective phenotype when examined with the vg:DsRed reporter. The precise link between these defects is unclear, but a likely explanation suggests that as the surrounding cuticle fuses, persisting cells, now deprived for nutrients and oxygen, become necrotic and may initiate melanization. Mutants could arrest at upstream steps, involving the specification or execution of PCD, or they might affect proper clearance of cell corpses from the epithelium. New alleles were recovered of dark (l(2)SH0173) and a likely hypermorph of thread (l(3)S048915), which provides reassuring validation of this prediction (Link, 2007).
By leveraging this distinct phenotype, novel cell death genes, were captured including the Drosophila orthologue of HIPK. Though first identified as an NK homeodomain binding partner, this gene was found to be an essential regulator of PCD and cell numbers in diverse tissue contexts. Of the four mammalian HIPK genes, HIPK2, the predicted orthologue of Drosophila HIPK, has been placed in the p53 stress-response apoptotic pathway, but whether the Drosophila counterpart similarly impacts this network is not yet known (Link, 2007).
For animal development it is necessary that organs stop growing after they reach a certain size. However, it is still largely unknown how this termination of growth is regulated. The wing imaginal disc of Drosophila serves as a commonly used model system to study the regulation of growth. Paradoxically, it has been observed that growth occurs uniformly throughout the disc, even though Decapentaplegic (Dpp), a key inducer of growth, forms a gradient. This paper presents a model for the control of growth in the wing imaginal disc, which can account for the uniform occurrence and termination of growth. A central feature of the model is that net growth is not only regulated by growth factors, but by mechanical forces as well. According to the model, growth factors like Dpp induce growth in the center of the disc, which subsequently causes a tangential stretching of surrounding peripheral regions. Above a certain threshold, this stretching stimulates growth in these peripheral regions. Since the stretching is not completely compensated for by the induced growth, the peripheral regions will compress the center of the disc, leading to an inhibition of growth in the center. The larger the disc, the stronger this compression becomes and hence the stronger the inhibiting effect. Growth ceases when the growth factors can no longer overcome this inhibition. With numerical simulations it was shown that the model indeed yields uniform growth. Furthermore, the model can also account for other experimental data on growth in the wing disc (Aegerter-Wilmsen, 2007).
Since the wing imaginal disc serves as a model system to study the regulation of growth, a large amount of experimental data is already available. The model has been evaluated with experimental results from the literature. When clones with increased Dpp signaling are generated, they grow larger in the lateral regions than in the medial part. Furthermore, clones with decreased Dpp signaling survive better laterally than medially. A common explanation for these findings is that the medial cells are more competitive than the lateral cells because they receive higher levels of Dpp. Therefore, a clone with a fixed level of Dpp signaling is hindered more when growing in the medial part than when growing more laterally. The model may offer an additional, alternative explanation. A clone is stretched more and compressed less when growing laterally than when growing medially. Therefore, it grows faster laterally as long as its level of Dpp signaling is fixed. It is expected that both competition and differences in compression contribute to the difference of size among different clones (Aegerter-Wilmsen, 2007 and references therein).
Discs with homogeneous Dpp signaling are expanded along the dorsoventral boundary. According to the model, the total growth factor activity in these discs is highest along the dorsoventral boundary, thus accounting for the expansion along this boundary. Furthermore, it has been found that discs with homogeneous Dpp signaling do not show uniform growth. Instead the growth rate of cells in the lateral regions, close to the dorsoventral boundary, is higher than the growth rate of cells in the medial part of the disc. According to the model, the high growth factor activity along the dorsoventral boundary will promote additional growth along the whole boundary. This stretches the regions further away from the dorsoventral boundary. This stretching pulls the cells along the dorsoventral boundary toward the center of the disc. The cells in the center are thus being compressed. The closer the cells are located to the center, the more they are compressed and the more growth is inhibited, thus leading to the observed differences in growth rate (Aegerter-Wilmsen, 2007).
The Dpp pathway can be activated locally by expressing a constitutively active form of one of its receptors (tkvQ-D). Recently, it has been shown that activating the Dpp pathway in clones in this way can stimulate transient non-autonomous cell proliferation. When inhibiting the pathway, similar effects were seen. Clones with increased Dpp activity were modeled as a region with increased Dpp activity compared to its surrounding tissue with lower homogeneous Dpp activity. In that case, the cells with high Dpp signaling initially grow faster than the surrounding cells, thus stretching them. As in the wild-type situation at the start of growth, the stretching is highest in the cells closest to the region with high Dpp signaling and therefore growth is induced in these cells. This non-autonomous growth increases the stretching in the cells further away from the clone, which will increase their growth. Therefore, after some time, growth in the cells surrounding the clone will be homogeneous again, comparable with the situation in the wild type disc. Thus, the model accounts for the non-autonomous effect as well as for the observation that it only occurs transiently (Aegerter-Wilmsen, 2007).
Clones with decreased Dpp activity were modeled in a similar way. The cells surrounding the clone get stretched between the slow growing cells in the clone and the faster growing cells further away from the clone. Therefore growth is also induced non-autonomously in cells surrounding clones with decreased Dpp signaling, which is again in agreement with the data (Aegerter-Wilmsen, 2007).
Non-autonomous effects on cell proliferation were also assessed for clones in which growth is increased by overexpressing CyclinD and Cdk4 instead of by increased Dpp signaling. The non-autonomous proliferation was not observed in that case, even though this would in principle be expected based on the model. However, cell divisions are only slightly increased in these clones and apoptosis is increased, which is generally accompanied by basal extrusion. Therefore, it seems as if co-expression of CyclinD and Cdk4 causes only very little net overgrowth at the stage measured. For such clones the non-autonomous stimulation of proliferation is expected to be less pronounced and to occur at a relatively late point in time, which may explain why it has not been observed (Aegerter-Wilmsen, 2007).
Experimentally induced alterations in cell proliferation are often compensated for by changes in cell size, such that the final wing disc size is not changed. This suggests that wing disc size is not a function of cell numbers. In the model, the wing disc is considered as an elastic sheet with certain mechanical properties. As long as the mechanical properties of the tissue as a whole are not influenced by cell size, the final disc size is indeed not a function of cell numbers according to the model. Furthermore, according to the model, it would be expected that a reduction of growth in the center of the disc automatically leads to a reduction of growth in the peripheral regions. Indeed, when the size of the wing blade was decreased by down-regulating vestigial (vg) expression, non-autonomous reductions in surrounding WT territories were observed along all axes of growth. Lastly, the model predicts that stretching occurs in the peripheral regions. Therefore, it also predicts that, upon cutting the disc from the end toward the middle, tissue at both sides of the cut moves apart. In wound healing experiments, this was indeed observed. In contrast, the model predicts that the central region of the disc becomes compressed. The increased thickness of the (columnar layer of the) wing disc could be seen as an indication that compression indeed occurs (Aegerter-Wilmsen, 2007).
This paper has presented a model for the determination of final size in the wing imaginal disc. In the model, growth is negatively regulated by mechanical stresses, which are automatically generated as a result of growth rate differences in an elastic tissue. With the use of numerical simulations, it was demonstrated that the model naturally leads to uniform growth as was shown experimentally and that it leads to the observed final size of the wing disc. Furthermore, it was argued that the model can also account for other experimental data in literature (Aegerter-Wilmsen, 2007).
The stereotyped dimensions of animal bodies and their component parts result from tight constraints on growth. Yet, the mechanisms that stop growth when organs reach the right size are unknown. Growth of the Drosophila wing-a classic paradigm-is governed by two morphogens, Decapentaplegic (Dpp, a BMP) and Wingless (Wg, a Wnt). Wing growth during larval life ceases when the primordium attains full size, concomitant with the larval-to-pupal molt orchestrated by the steroid hormone ecdysone. This study blocked the molt by genetically dampening ecdysone production, creating an experimental paradigm in which the wing stops growing at the correct size while the larva continues to feed and gain body mass. Under these conditions, wing growth is limited by the ranges of Dpp and Wg, and by ecdysone, which regulates the cellular response to their signaling activities. Further, evidence is presented that growth terminates because of the loss of two distinct modes of morphogen action: 1) maintenance of growth within the wing proper and 2) induced growth of surrounding "pre-wing" cells and their recruitment into the wing. These results provide a precedent for the control of organ size by morphogen range and the hormonal gating of morphogen action (Parker, 2020).
The evagination of Drosophila imaginal discs is a classic system for studying tissue level morphogenesis. Evagination involves a dramatic change in morphology and published data argue that this is mediated by cell shape changes. The evagination of both the leg and wing discs has been reexamined and it has been found that the process involves cell rearrangement and that cell divisions take place during the process. The number of cells across the width of the ptc domain in the wing and the omb domain in the leg decreases as the tissue extends during evagination and cell rearrangement was observed to be common during this period. In addition, almost half of the cells in the region of the leg examined divided between 4 and 8 h after white prepupae formation. Interestingly, these divisions were not typically oriented parallel to the axis of elongation. These observations show that disc evagination involves multiple cellular behaviors, as is the case for many other morphogenetic processes (Taylor, 2008).
This study established that cell rearrangement takes place during leg and wing evagination and contributes to the thinning and extension of the appendages. These observations are consistent with the pioneering results of Fristrom (1976) on evagination. The current data also established that cell rearrangement takes place throughout the appendage and is not restricted to a particular region along the proximal/distal axis. However, the observations are also consistent with cell rearrangement being non-uniform as some regions appeared to 'thin' more than others. For example, in the wing the width of the ptc domain at position M5 thinned more than at position M4 (refering to neuronal landmarks). The evaginating leg and wing cells retain their epithelial morphology with extensive apical junctional complexes. Rearrangement requires that cells change neighbors and hence must remove old junctions and generate new ones while maintaining tissue integrity. This problem is not restricted to evaginating discs but is a general one for epithelial tissues and is an issue that has concerned developmental/cell biologists for many years. Important insights into how this could be accomplished come from recent observations on germ band elongation in the Drosophila embryo. Several groups have provided evidence that junctional remodeling plays a key role in cell rearrangement in this epithelial tissue. This mechanism also appears to function in the repacking of pupal wing cells. It is suggested that it also plays a role in leg and wing evagination. No clear evidence is seen for the multicellular rosettes that have been implicated in germ band extension. Perhaps this is due to disc evagination being substantially slower than germ band extension (Taylor, 2008).
No evidence was seen of dramatic coordinated changes in cell shape. There was a small but significant increase in the length along the proximal/distal axis of evaginating omb domain tibia cells that should contribute to elongation. However, the change was not large enough to account for leg morphogenesis. No significant change was seen in cell shape in evaginating ptc domain wing cells although there was a hint of a possible small effect. It is worth noting that in these measurements cells from all positions along the relevant part of the proximal/distal axis were included. Casual observation suggested that there might be small regions with consistent changes but these would likely be counterbalanced by changes in shape elsewhere in the domain (Taylor, 2008).
It was not possible to image the earliest stages of leg disc evagination or the disc cells that form ventral thorax. Thus, these observations were not able to distinguish between the two proposed mechanisms of eversion (i.e., spreading vs. invasion hypotheses). Patterned cell death could in principle play an important role in disc evagination. Previous studies have not seen evidence for patterned cell death during wing blade evagination and the current observations support this conclusion. Cell death has been detected in evaginating legs but this is restricted to the regions of the tarsal segments where the leg joints form and hence is unlikely to contribute to the overall thinning of the omb domain of leg segments (Taylor, 2008).
Based on the literature, it was not expected that cell division takes place during evagination, but the current observations showed that it occurred. The most definitive experiments involved generating clones of cells marked by GFP expression and following these in vivo. These experiments provided compelling evidence for cell division. This was only done for the leg but other experiments provided strong evidence for cell division in evaginating wings. The size of wing clones was larger when they were induced at white prepupae than at the formation of the definitive pupae. Cell division was not rare in evaginating legs, and on average about 40% of the cells divided. Indeed, a majority of the cells divided in about 1/3 of clones examined. This amount of cell division is sufficient to account for the thickening of the omb domain that was observed from 6 to 8 h in developing legs. Observations on the size of wing clones suggested a similar fraction of wing cells divided during evagination. A limitation is that the in vivo imaging technique only allowed effective imaging of clones on the leg surface juxtaposed to the pupal case in the basitarsus and tibia (and occasionally tarsal) segments. Thus, data could not be obtained for much of the leg disc derivatives, and hence the overall proportion of evaginating leg cells that divide cannot be confidently estimated. The spindle in these dividing cells was not imaged but it was inferred that the spindle was not oriented parallel to the elongating axis, based on the position of the resulting daughter cells shortly after division. The two daughter cells usually filled up the area taken up by the parental cell prior to division, which helped in assigning a lineage. The leg epidermis is continuous without free 'space'. Hence, that daughter cells would occupy the space of the parental cell is not surprising. A parallel orientation for the spindle might be expected if the cell division plane was tightly linked to the mechanism of elongation. The inferred orientation of the cell divisions was most often between 46o and 60o. Thus, they would increase the number of cells both along the proximal/distal and anterior/posterior (and dorsal/ventral) axes. In the second day pupal leg, the width of the omb domain was narrower than it was in the evaginating leg. This could be a reflection of a later stage of convergent extension. However, legs were not followed throughout this period, other possibilities cannot be ruled out. It is interesting to note that cells in the pupal tibia and basitarsus have a spiral arrangement, and this appears to arise from 6 to 8 h after white prepupae. Thus, this arrangement could be at least in part a consequence of the orientation of the cell divisions (Taylor, 2008).
The fraction of dividing cells varied widely from one clone to another. This was not correlated with particular pupae or legs as both clones where a majority of the cells divided and clones where no cells divided were found in the same pupae and on the same leg. One possibility is that the variation is due to region specific differences. For example, cells in one region of the leg might never divide during evagination while a majority of cells in another region might always divide. No evidence is seen for this but the experiments were not compelling on this point. The observations on the omb domain did not examine a majority of leg cells and in the experiments where MARCM clones were followed, it could not be routinely said exactly where on the leg a clone was located. A second possibility is that the variation is due to the clustered distribution of S phase and mitotic cells in wing and leg discs. Any small clone could comprise a cluster (or not contain a cluster) and this could lead to a great deal of variation in observed cell division. The basis for the clustering is uncertain but could simply represent a pseudo-synchronization due to neighboring sister cells having been born at the same time (Taylor, 2008).
The observations suggest that several different factors play a role in evagination. At the start of evagination, the leg and wing discs are folded and some of the initial elongation is due to an unfolding of the tissue that presumably results from changes in the shape of cells along the apical/basal axis. During the period when leg discs evert and present the apical surface of their epithelial cells to the outside, elongation is also taking place and there is active pulsatile movement. This appears to be related to the movement of hemolymph in the prepupae and blood cells can often be seen to move in step with the pulses. This suggests that hydraulic pressure could be playing a role in eversion and elongation. The leg resembles a cylinder closed on one side (distal tip) and open to the body on the other (proximal). Thus, it is expected that hemolymph is pumped by the heart to produce a mechanical force that could help evert and/or elongate the leg. The pulsatile movement starts to decrease at about 4-4.5 h after white prepupae and largely ends by about 5 h. This is around the time of eversion, but the slowing clearly precedes eversion. It is suggested that the hydraulic pressure of the hemolymph helps drive the early stages of evagination, when the leg is short and unfolding of the tissue plays a major role. It is possible that after this time the increased leg length or increased leg stiffness limits the effectiveness of hemolymph hydraulic pressure. Alternatively, it is possible that there is a decline in the hydraulic pressure due to changes in heart pumping or other prepupal events. The lack of hydraulic pressure may be one reason for the less than optimal evagination of discs seen during in vitro culture (Taylor, 2008).
Mutations in many Drosophila genes result in changes in appendage morphology. It is expected that some of these produce their phenotype by interfering with the observed cell rearrangement. A particularly interesting candidate for such a gene is dachsous (ds), which encodes a large protein with many cadherin domains. Mutations in this gene result in shorter fatter wings and legs with an altered distribution of cells (e.g. an increase in the number of cells along the anterior posterior axis of the wing and a decrease in the number of cells along the proximal/distal axis). However, mutations in this gene are known to alter disc patterning and growth and this may be the cause of the altered shape (Taylor, 2008).
Another group of interesting candidate genes for altering cell rearrangement in evaginating legs is the cellular myosin encoded by zipper and the interacting Sqh (myosin regulatory light chain) and RhoA proteins. Mutations in these genes give rise to a crooked leg phenotype that has been interpreted as being due to the mutations altering cell shape. However, myosin has been implicated in the junctional remodeling associated with cell rearrangements in the extending germ band and it is possible that the leg phenotype is also due to an effect on junctional remodeling required for cell rearrangement. One of the interesting properties of extending germ band cells is the planar polarization of membranes so that the anterior/posterior edges of cells are distinct from the dorsal/ventral edges of cells in their content of proteins such as myosin. No evidence was seen for this in prepupal legs and wings but this point deserves further study as it is possible the experimental conditions were not favorable for seeing this (Taylor, 2008).
This paper reports experiments aimed at understanding the connections between cell competition and growth in the Drosophila wing disc. The principal assay has been to generate discs containing marked cells that proliferate at different rates and to study their interactions and their contribution to the final structure. It is known that single clones of fast-dividing cells within a compartment may occupy the larger part of the compartment without affecting its size. This has suggested the existence of interactions involving cell competition between fast- and slow-dividing cells directed to accommodate the contribution of each cell to the final compartment. This study shows that indeed fast-dividing cells can outcompete slow-dividing ones in their proximity. However, it is argued that this elimination is of little consequence because preventing apoptosis, and therefore cell competition, in those compartments does not affect the size of the clones or the size of the compartments. These experiments indicate that cells within a compartment proliferate autonomously at their own rate. The contribution of each cell to the compartment is exclusively determined by its division rate within the frame of a size control mechanism that stops growth once the compartment has reached the final arresting size. This is supported by a computer simulation of the contribution of individual fast clones growing within a population of slower dividing cells and without interacting with them. The values predicted by the simulation are very close to those obtained experimentally (Martín, 2009).
The main objective of this work was to study the role of cell competition in regulating growth and size in the wing disc of Drosophila. As the disc is composed of two (A and P) compartments, which behave as independent units of size control, these experiments refer to mechanisms operating within compartments (Martín, 2009).
To make some precise statements about the growth dynamics of the disc several developmental parameters were calculated, some of which had not been described in detail in previous publications. According to the data, the wing disc starts growth with about 55 cells, of which 34 would belong to the A and 21 to the P compartment. The final cell number is around 30,000 (about 19,000 A and 11,000 P). The total number of cell divisions is about 9.1. These estimates coincide well with previous ones, although the final number of 30,000 cells is somewhat lower than previous measurements (Martín, 2009).
This study has shown that the growth rate of the wild-type wing disc changes markedly through development: during the second larval period wild type (M+) cells divide at about 5.5-5.7 hours per cycle, then the length of the cell division cycle increases as development progresses, and in the second half of the third larval period it is as high as 30 hours. Thus most of the growth occurs during the second and early third larval period. A similar growth pattern is found in developmentally delayed M/+ discs, the cell doubling time (CDT) of which increases from 10-12 hours in the second larval period to 34-40 at the prepupal stage. It is not surprising that the major difference between wild-type and M/+
discs occurs during the early periods. Possibly the metabolic demand is greater in fast-proliferating cells and therefore the limitation in protein synthesis of M/+ cells is more noticeable (Martín, 2009).
Normally there is little apoptosis in the wing disc; therefore, cell competition cannot have a major role in normal circumstances. Nevertheless, it may be a safeguard mechanism to eliminate abnormal cells or to deal with unusual situations such as cells with different division rates, which may interfere with the growth of the disc. The significance was examined of these events of cell competition in the overgrowth of M+ clones, and in the control of compartment size (Martín, 2009).
The fact that M+ clones growing in M/+ discs can reach an average size more than ten times their normal size (when they grow in a M+ disc) while not altering the final size of the compartment, suggested that both clone overgrowth and size control may depend on cell competition. In this view the M+ clones would overgrow at the expense of the elimination of neighbouring M/+
cells. Moreover, the removal of the latter would ensure that they do not produce progeny that would give rise to an excess of cells in the compartment (Martín, 2009).
However, the experiments indicate that cell competition does not play a significant role in these processes; in the absence of cell competition, the M+ clones are able to overgrow as much as in the normal situation. Besides, the size of compartments is not affected by the presence of these clones, even though they can occupy the larger part of the compartment (Martín, 2009).
The authors believe that the key element to explain these results is the mechanism that controls the overall size of the compartment and that arrests growth once it has reached the final dimension. This mechanism would function without regard to the lineage of the cells or of their relative contribution to the final structure. It would also function autonomously in each compartment (Martín, 2009).
The authors ask us to consider a compartment containing M+ and M/+ cells from early in development. The cells proliferate at the rate dictated by their metabolic activity, according to their Minute genotype, and their division rate is not affected by interactions with neighbour cells. Because of their proliferation advantage the M+ cells occupy a large part of the compartment. In principle, if the M/+ cells were to proliferate at their normal rhythm for the whole duration of the M/+ larval period, the sum of the contribution of the M+ and M/+ cells would produce an excess of tissue. The reason why this is not the case is that the size control mechanism arrests growth as soon as the final size has been reached. In the presence of a large M+ clone the final arresting size of the compartment is reached earlier than in a one entirely made of M/+ cells. For this reason the M/+ cells contribute less than they would have in the absence of a M+ clone. That is, the developmental delay associated with the M/+ condition is partially abolished by the presence of the M+ clone. This is predicted by the computer simulation and is supported by the results. Using the expression of the vg and wg genes to monitor the developmental stage of the compartment, it was found that compartments with M+ clones are ahead and are therefore expected to reach the final arresting size earlier than those that are entirely M/+ (Martín, 2009).
The existence of a non-cell-autonomous mechanism governing the growth of the compartment is also suggested by observations about vg expression in compartments containing M+ clones. M+ clones can sometimes split the vg domain into M+ and M/+ territories. The significant finding is that both territories show the same pattern of expression. This very strongly suggests that the control of vg expression is determined by an overall mechanism probably measuring the size of the compartment in each moment and regardless of the individual lineages (Martín, 2009).
What, then, is the role of cell competition in regulating growth and size in the wing disc? Cell competition results from interactions between two types of viable cells and causes the elimination of one of them. That is, it is a mechanism to remove viable cells that are not developmentally adapted to the growing tissue. Unlike other situations that cause apoptosis, the 'loser' cells in the competition process are not necessarily damaged; they are poor competitors. In the cases reported here, it is the relatively slow proliferating cells that are eliminated, which may have a beneficial effect on the general fitness of the disc. Nevertheless, there may be other safeguard functions of greater biological significance. Cell competition may be instrumental in removing viable but developmentally abnormal cells, which, for instance, do not interpret developmental cues correctly. This would include tumour or transformed cells that may arise in development. The process would ensure that tumour cells would normally be outcompeted by non-tumour cells. In certain circumstances, however, the acquisition by the latter of some additional property may turn tumour cells into super-competitors, thus reversing the situation. It has been argued that cell competition may be a major factor in tumour progression in circumstances in which tumour cells are able to outcompete normal cells (Martín, 2009).
In spite of conceptual views of how differential gene expression is used to define different cell identities, it is still not understood how different cell identities are translated into actual cell properties. The fly wing is composed of two main cell types, vein and intervein. These two types differ in many features, including their adhesive properties. One of the major differences is that intervein cells express integrins, which are required for the attachment of the two wing layers to each other, whereas vein cells are devoid of integrin expression. The major signaling pathways that divide the wing to vein and intervein domains have been characterized. However, the genetic programs that execute these alternative differentiation programs are still very roughly drawn. This study identifies the bHLH protein Delilah (Dei) as a mediator between signaling pathways that specify intervein cell-fate and one of the most significant realizators of this fate, βPS integrin. Dei's expression is restricted to intervein territories where it acts as a potent activator of βPS integrin expression. In the absence of normal Dei activity the level of βPS integrin is reduced, leading to a failure of adhesion between the dorsal and ventral wing layers and a consequent formation of wing blisters. The effect of Dei on βPS expression is not restricted to the wing, suggesting that Dei functions as a general genetic switch, which is turned on wherever a sticky cell-identity is determined and integrin-based adhesion is required (Egoz-Matia, 2011).
This study has identified the bHLH transcription factor Dei as an important positive regulator of the expression of βPS, the major β subunit in Drosophila. During embryonic development Dei's expression is confined mainly to cells that adhere strongly to other cells and are able to withstand mechanical strain, for instance, tendon cells that attach body wall muscle to the cuticle. Moreover, when different types of cells arise from within a uniform cell population, or through asymmetric cell division, Dei's expression is restricted to the ‘stickier’ types of cells. For example, in the chordotonal organ lineage, Dei is expressed in the four types of support cells (cap, ligament, cap-attachment and ligament-attachment), but is excluded from the neuron and glia. Similar phenomenon is seen in the developing wing where Dei is expressed only in intervein territories, where the ventral and dorsal layers adhere to each other, and is not expressed in vein cells that do not adhere to cells of the opposite layer. In all these systems, Dei does not function as a selector of cell identity, but it is required to realize the selected fate by activating a developmental program that specifies adhesive properties of cells (Egoz-Matia, 2011).
Although dei's expression has not been characterized in all developmental stages and tissues, published data of various microarray analyses suggest that dei is expressed in other developmental and physiological contexts where up-regulation of βPS integrin is required. For example, dei was up-regulated when larvae were exposed to immune challenge, or when mutant larvae exhibited an increase in lamellocyte cell population. Lamellocytes represent a subset of hemocytes in Drosophila, which differentiate in response to specific immune challenge. The lamellocytes aggregate around large pathogens to form a rigid laminated capsule that confines the pathogen and enables its elimination. This encapsulation process requires members of the integrin family that presumably mediate the lamellocyte's attachment (Egoz-Matia, 2011).
This work focused mainly on the role of dei in intervein cells and showed that dei provides a missing link between the genetic specification of these epithelial cells and their differentiation. The data place dei downstream to the major signaling pathways that divide the wing to regions of veins and interveins and downstream to Bs, which works as a selector of intervein identity. It remains to be determined whether dei is a direct target of Bs, and whether it is a direct regulator of βPS, however the results of the rescue experiment suggest that the effect of Bs on integrin expression is mediated, at least in part, by the activity of Dei (Egoz-Matia, 2011).
The venation phenotypes caused by weak dei alleles could be also attributed to the effects of Dei on βPS expression. Even though vein and intervein territories are established during early stages of wing development, the decision remains plastic for at least 24-h APF. Maintenance of the right fates depends on both vein-specific and intervein-specific genes. Appropriate levels of integrin expression are required for the maintenance of intervein fate, as suggested by the ectopic vein phenotype of certain mys alleles, which is very similar to the venation phenotype of weak dei alleles (Egoz-Matia, 2011).
It is reasonable to assume that Dei regulates multiple target genes in different cells and tissues. However, as for integrins, Dei regulates specifically βPS integrin. No evidence was found for regulation of αPS1 or αPS2, which are expressed differentially in the two wing layers, by Dei. Since βPS is the dimerization partner of both αPS1 and αPS2, by regulating its expression Dei practically affects all integrin-based adhesion processes at both the dorsal and ventral wing layers. The data also suggest that the effects of Dei on integrin-dependent adhesion are not restricted to the wing. Ectopic expression of Dei led to up-regulation of βPS expression in embryonic tissues, whereas loss of Dei's activity caused a reduction in the level of βPS expression in the cone cells of the eye (Egoz-Matia, 2011).
In summary, Dei is thought of as a general switch that turns on βPS integrin expression wherever a sticky cell has to develop. Since such a switch needs to be turned on in different tissues and different developmental and physiological contexts, it is predicted that the dei gene can respond to various signaling pathways and transcription factors. Indeed, analysis of the regulatory region of the dei locus demonstrated that it harbors multiple regulatory modules that respond to different transcription factors working in different developmental contexts (Egoz-Matia, 2011).
Cell competition is an emerging principle that eliminates suboptimal or potentially dangerous cells. For 'unfit' cells to be detected, their competitive status needs to be compared to the collective fitness of cells within a tissue. This study reports that the NMDA receptor controls cell competition of epithelial cells and Myc supercompetitors in the Drosophila wing disc. While clonal depletion of the NMDA receptor subunit NR2 results in their rapid elimination via the TNF/Eiger>JNK signalling pathway, local over-expression of NR2 causes NR2 cells to acquire supercompetitor-like behaviour that enables them to overtake the tissue through clonal expansion that causes, but also relies on, the killing of surrounding cells. Consistently, NR2 is utilised by Myc clones to provide them with supercompetitor status. Mechanistically, this study found that the JNK>PDK signalling axis in 'loser' cells reprograms their metabolism, driving them to produce and transfer lactate to winners. Preventing lactate transfer from losers to winners abrogates NMDAR-mediated cell competition. These findings demonstrate a functional repurposing of NMDAR in the surveillance of tissue fitness (Banreti, 2020).
Cell competition is an evolutionary conserved quality control process, which ensures that suboptimal, but otherwise viable, cells do not accumulate during development and aging. How relative fitness disparities are measured across groups of cells, and how the decision is taken whether a particular cell will persist in the tissue ('winner cell') or is killed ('loser cell') is not completely understood. This is an important issue as competitive behaviour can be exploited by cancer cells (Banreti, 2020).
Various types of cell competition exist. While structural cell competition is triggered upon loss of cellular adhesion or changes in epithelial apico-basal polarity, metabolic cell competition occurs in response to alterations in cellular metabolic states. Growth signalling pathways involved in metabolic cell competition seem to funnel through Myc, which functions as an essential signalling hub in many types of cancers. Myc regulates expression of components that control proliferation, cell death, differentiation, and central metabolic pathways. Particularly, acute changes in cellular metabolism appear to be critical for the winner phenotype during Myc supercompetition in Drosophila, where robustly growing Myc-expressing cells are able to not only outgrow but also actively trigger the elimination of nearby wild-type cells from the tissue (Banreti, 2020).
Recent in vivo data demonstrate that some tumours can uptake lactate and preferentially utilize it over glucose to fuel tricarboxylic acid (TCA) cycle and sustain tumour metabolism. Moreover, the growth-promoting effect of stromal cells is impaired by glycolytic inhibition, suggesting that the stroma provides nutritional support to malignant cells by transferring lactate from cancer-associated fibroblasts (CAFs) to cancer cells. Such energy transfer from glycolytic stromal cells to epithelial cancer cells closely resembles the physiological processes of metabolic cooperativity, such as in 'neuron-astrocyte metabolic coupling' in the brain, and the 'lactate shuttle' in the skeletal muscle. Activation of glycolysis in astrocytes and MCT-mediated transfer of lactate to neurons supports neuron mitochondrial oxidative phosphorylation and energy demand. These observations raise the intriguing possibility that lactate serves as fuel to complement glucose metabolism during cell competition (Banreti, 2020).
This study reports that the NMDA receptor controls the competitiveness of epithelial cells in the Drosophila wing discs. While tissue-wide depletion of NR2 has no effect on cell viability and growth, clonal depletion of NR2 results in their rapid elimination via the TNF>JNK signalling pathway. Conversely, local over-expression of NR2 causes NR2-overexpressing cells to acquire supercompetitor-like behaviour that enables them to overtake the tissue. These data indicate that relative levels of NR2 underpins cell competitive behaviour in the wing epithelia. Moreover, this study finds that Myc-induced supercompetition also depends on upregulation of NMDAR. Genetic depletion of NR2 abrogates Myc-induced supercompetition. Mechanistically, this study finds that the JNK>PDK signalling axis in 'loser' cells (lower NMDAR) results in phosphorylation and inactivation of PDH, the enzyme that converts pyruvate to Acetyl-CoA to fuel the TCA in the mitochondria. In such loser cells, phospho-dependent inactivation of PDH causes mitochondrial shutdown and metabolic reprogramming, thus loser cells produce and secrete lactate to winners. Preventing lactate transfer from losers to winners removes fitness disparities and abrogates NMDAR-mediated cell competition. Together these data are consistent with the notion that NMDAR underpins cell competition and that targeting NMDAR converts Myc supercompetitor clones into superlosers (Banreti, 2020).
The elimination of unfit cells via competitive interactions plays an important role for the maintenance of tissue health during development and adulthood. The data indicate that the NMDA receptor NR2 influences the competitive behaviour of epithelia cells and Myc supercompetitors in the Drosophila wing disc. Genetic depletion of NR2 reprograms metabolism via TNF-dependent and JNK-mediated activation of PDK, which in turn phosphorylates and inactivates PDH. This causes a shutdown of pyruvate catalysis and results in a switch to aerobic glycolysis. Upon phospho-dependent inhibition of PDH, pyruvate is reduced to lactate via LDH, and secreted. While lactate exits cells to avoid acidification, it can be recaptured and used as carbon source by other cells, leading to metabolic compartmentalisation between adjacent cells. In normal physiology as well as in murine and human tumours, lactate is an important energy source that fuels mitochondrial metabolism. For example, lactate produced and secreted by astrocytes is transported to neighbouring neurons where it is used as source of energy to support neuronal function. This is akin of the 'reverse Warburg effect', also named 'two-compartment metabolic coupling' model, where cancer-associated fibroblasts (CAFs) undergo aerobic glycolysis and production of high energy metabolites, especially lactate, which is then transported to adjacent cancer cells to sustain their anabolic need (Banreti, 2020).
These data suggest that the epithelial NMDA receptor is responsible for fitness surveillance and to provide Myc clones with supercompetitor status. Cells with decreased epithelial NMDA receptor are metabolically reprogrammed to transfer their carbon fuel to their neighbours. According to the current model, differential NMDAR signalling in adjacent cells triggers lactate-mediated metabolic coupling, and underpins cell competition in epithelia. Consistently, preventing loser cells from 'transferring' lactate to their neighbours, via inhibition of MCT1, Impl3 or PDK, removes the fitness disparity and nullifies cell competition. Likewise, exposure to elevated levels of systemic lactate, blocks elimination of NR2 loser clones. This suggests that cell competition may be based on NMDAR-mediated metabolic coupling between winners and losers. Importantly, this metabolic coupling only occurs under competitive conditions. Consistently, NR2 losers are only eliminated if they are surrounded by cells with functional NMDAR. This is evident as tissue-wide inhibition of NMDAR by AP5, a selective inhibitor of NR2, blocks elimination of NR2 loser clones in a heterotypic genetic setting (Banreti, 2020).
NR2 is upregulated in Myc expressing clones and Myc cells co-opt epithelial NR2 to promote cell competition, subduing their neighbouring wild-type cells that become re-classified as 'unfit'. Interestingly, Myc clones lose their supercompetitor status upon tissue-wide depletion of NR2. Under this condition, WT cells are no longer eliminated and survive among Myc supercompetitors. This indicates that NR2 underpins Myc-induced supercompetition. Given that Myc is a major driver of cancer cell growth, and is a hallmark of the disease in nearly seven out of ten cases, blocking Myc's function would be a powerful approach to treat many types of cancer. However, the properties of the Myc protein itself make it difficult to design a drug against it. Since the NMDAR signalling circuit is hijacked in many types of human cancers, and its expression level is associated with poor patient survival, it is attractive to speculate that targeting NMDAR may be a promising strategy to improve patient care (Banreti, 2020).
The control of organ size presents a fundamental open problem in biology. A declining growth rate is observed in all studied higher animals, and the growth limiting mechanism may therefore be evolutionary conserved. Most studies of organ growth control have been carried out in Drosophila imaginal discs. Previous studies have shown that the area growth rate in the Drosophila eye primordium declines inversely proportional to the increase in its area, which is consistent with a dilution mechanism for growth control. This study shows that a dilution mechanism cannot explain growth control in the Drosophila wing disc. A range of alternative candidate mechanisms were computationally evaluate, and the experimental data can be best explained by a biphasic growth law. However, also logistic growth and an exponentially declining growth rate fit the data very well. The three growth laws correspond to fundamentally different growth mechanisms that are discussed. Since a fit to the available experimental growth kinetics is insufficient to define the underlying mechanism of growth control, future experimental studies must focus on the molecular mechanisms to define the mechanism of growth control (Vollmer, 2016).
A fundamental question of biology is what determines organ size. Despite demonstrations that factors within organs determine their sizes, intrinsic size control mechanisms remain elusive. This study shows that Drosophila wing size is regulated by JNK signaling during development. JNK is active in a stripe along the center of developing wings, and modulating JNK signaling within this stripe changes organ size. This JNK stripe influences proliferation in a non-canonical, Jun-independent manner by inhibiting the Hippo pathway. Localized JNK activity is established by Hedgehog signaling, where Ci elevates dTRAF1 expression. As the dTRAF1 homolog, TRAF4, is amplified in numerous cancers, these findings provide a new mechanism for how the Hedgehog pathway could contribute to tumorigenesis, and, more importantly, provides a new strategy for cancer therapies. Finally, modulation of JNK signaling centers in developing antennae and legs changes their sizes, suggesting a more generalizable role for JNK signaling in developmental organ size control (Willsey, 2016).
Two independently generated antibodies that recognize the phosphorylated, active form of JNK (pJNK) specifically label a stripe in the pouch of developing wildtype third instar wing discs. Importantly, localized pJNK staining is not detected in hemizygous JNKK mutant discs, in clones of JNKK mutant cells within the stripe, following over-expression of the JNK phosphatase puckered (puc), or following RNAi-mediated knockdown of bsk using two independent, functionally validated RNAi lines (Willsey, 2016).
The stripe of localized pJNK staining appeared to be adjacent to the anterior-posterior (A/P) compartment boundary, a location known to play a key role in organizing wing growth, and a site of active Hedgehog (Hh) signaling. Indeed, pJNK co-localizes with the Hh target gene patched (ptc). Expression of the JNK phosphatase puc in these cells specifically abrogated pJNK staining, as did RNAi-mediated knockdown of bsk. Together, these data indicate that the detected pJNK signal reflects endogenous JNK signaling activity in the ptc domain, a region of great importance to growth control. Indeed, while at earlier developmental stages pJNK staining is detected in all wing pouch cells, the presence of a localized stripe of pJNK correlates with the time when the majority of wing disc growth occurs (1000 cells/disc at mid-L3 stage to 50,000 cells/disc at 20 hr after pupation, so it is hypothesized that localized pJNK plays a role in regulating growth (Willsey, 2016).
Inhibition of JNK signaling in the posterior compartment previously led to the conclusion that JNK does not play a role in wing development. The discovery of an anterior stripe of JNK activity spurred a reexamination of the issue. Since bsk null mutant animals are embryonic lethal, JNK signaling was conditionally inhibited in three independent ways in the developing wing disc. JNK inhibition was achieved by RNAi-mediated knockdown of bsk (bskRNAi#1or2), by expression of JNK phosphatase (puc), or by expression of a dominant negative bsk (bskDN). These lines have been independently validated as JNK inhibitors. Inhibition of JNK in all wing blade cells (rotund-Gal4, rn-Gal4) or specifically in ptc-expressing cells (ptc-Gal4) resulted in smaller adult wings in all cases, up to 40% reduced in the strongest cases. Importantly, expression of a control transgene (UAS-GFP) did not affect wing size. This contribution of JNK signaling to size control is likely an underestimate, as the embryonic lethality of bsk mutations necessitates conditional, hypomorphic analysis. Nevertheless, hypomorphic hepr75/Y animals, while pupal lethal, also have smaller wing discs, as do animals with reduced JNK signaling due to bskDN expression. Importantly, total body size is not affected by inhibiting JNK in the wing. Even for the smallest wings generated (rn-Gal4, UAS-bskDN), total animal body size is not altered (Willsey, 2016).
To test whether elevation of this signal can increase organ size, eiger (egr), a potent JNK activator, was expressed within the ptc domain (ptc-Gal4, UAS-egr). Despite induction of cell death as previously reporte and late larval lethality, a dramatic increase was observed in wing disc size without apparent duplications or changes in the shape of the disc. While changes in organ size could be due to changing developmental time, wing discs with elevated JNK signaling were already larger than controls assayed at the same time point. Similarly, inhibition of JNK did not shorten developmental time. Thus, changes in organ size by modulating JNK activity do not directly result from altering developmental time. Finally, the observed increase in organ size is not due to induction of apoptosis, as expression of the pro-apoptotic gene hid does not increase organ size. In contrast, it causes a decrease in wing size. Furthermore, co-expression of diap1 or p35 did not significantly affect the growth effect of egr expression, while the effect was dependent on Bsk activity (Willsey, 2016).
In stark contrast to known developmental morphogens, no obvious defects were observed in wing venation pattern following JNK inhibition, suggesting that localized pJNK may control growth in a pattern formation-independent manner. Indeed, even a slight reduction in Dpp signaling results in dramatic wing vein patterning defects. Second, inhibiting Dpp signaling causes a reduction in wing size along the A-P axis, while JNK inhibition causes a global reduction. Furthermore, ectopic Dpp expression increases growth in the form of duplicated structures, while increased JNK signaling results in a global increase in size. Molecularly, it was confirmed that reducing Dpp signaling abolishes pSMAD staining, while quantitative data shows that inhibiting JNK signaling does not. Furthermore, it was also found that Dpp is not upstream of pJNK, as reduction in Dpp signaling does not affect pJNK. Together, the molecular data are consistent with the phenotypic results indicating that pJNK and Dpp are separate programs in regulating growth. Consistent with these findings it has been suggested that Dpp does not play a primary role in later larval wing growth control (Akiyama, 2015). Finally, it was found that inhibition of JNK does not affect EGFR signaling (pERK) and that inhibition of EGFR does not affect the establishment of pJNK (Willsey, 2016).
A difference in size could be due to changes in cell size and/or number. Wings with reduced size due to JNK inhibition were examined and no changes in cell size or apoptosis were found, suggesting that pJNK controls organ size by regulating cell number. Consistently, the cell death inhibitor p35 was unable to rescue the decreased size following JNK inhibition. Indeed, inhibition of JNK signaling resulted in a decrease in proliferation, while elevation of JNK signaling in the ptc domain resulted in an increase in cell proliferation in the enlarged wing disc. Importantly, this increased proliferation is not restricted to the ptc domain, consistent with previous reports that JNK can promote proliferation non-autonomously (Willsey, 2016).
To determine the mechanism by which pJNK controls organ size, canonical JNK signaling through its target Jun was considered. Interestingly, RNAi-mediated knockdown of jun in ptc cells does not change wing size, consistent with previous analysis of jun mutant clones in the wing disc. Furthermore, in agreement with this, a reporter of canonical JNK signaling downstream of jun (puc-lacZ) is not expressed in the pJNK stripe. Finally, knockdown of fos (kayak, kay) alone or with junRNAi did not affect wing size. Together, these data indicate that canonical JNK signaling through jun does not function in the pJNK stripe to regulate wing size (Willsey, 2016).
In search of such a non-canonical mechanism of JNK-mediated size control, the Hippo pathway was considered. JNK signaling regulates the Hippo pathway to induce autonomous and non-autonomous proliferation during tumorigenesis and regeneration via activation of the transcriptional regulator Yorkie (Yki). Recently it has been shown that JNK activates Yki via direct phosphorylation of Jub. To test whether this link between JNK and Jub could account for the role of localized pJNK in organ size control during development, RNAi-mediated knockdown of jub was performed in the ptc stripe, and adults with smaller wings were observed. Indeed, the effect of JNK loss on wing size can be partially suppressed in a heterozygous lats mutant background and increasing downstream yki expression in all wing cells or just within the ptc domain can rescue wing size following JNK inhibition. These results suggest that pJNK controls Yki activity autonomously within the ptc stripe, leading to a global change in cell proliferation. This hypothesis predicts that the Yki activity level within the ptc stripe influences overall wing size. Consistently, inhibition of JNK in the ptc stripe translates to homogeneous changes in anterior and posterior wing growth. Similarly, overexpression or inhibition of Yki signaling in the ptc stripe also results in a global change in wing size (Willsey, 2016).
It is important to note that the yki expression line used is wild-type Yki, which is still affected by JNK signaling. For this reason, the epistasis experiment was also performed with activated Yki, which is independent of JNK signaling. Expression of this activated Yki in the ptc stripe caused very large tumors and lethality. Importantly, inhibiting JNK in this context did not affect the formation of these tumors or the lethality. Furthermore, inhibiting both JNK and Yki together does not enhance the phenotype of Yki inhibition alone, further supporting the idea that Yki is epistatic to JNK, instead of acting in parallel processes (Willsey, 2016).
Mutants of the Yki downstream target four-jointed (fj) have small wings with normal patterning, and fj is known to propagate Hippo signaling and affect proliferation non-autonomously. Although RNAi-mediated knockdown of fj in ptc cells does not cause an obvious change in wing size, it is sufficient to block the Yki-induced effect on increasing wing size . However, overexpression of fj also reduces wing size, which makes it not possible to test for a simple epistatic relationship. Overall, these data are consistent with the notion that localized pJNK regulates wing size not by Jun-dependent canonical JNK signaling, but rather by Jun-independent non-canonical JNK signaling involving the Hippo pathway (Willsey, 2016).
While morphogens direct both patterning and growth of developing organs, a link between patterning molecules and growth control pathways has not been established. pJNK staining is coincident with ptc expression, suggesting it could be established by Hh signaling. During development, posterior Hh protein travels across the A/P boundary, leading to activation of the transcription factor Cubitus interruptus (Ci) in the stripe of anterior cells. To test whether localized activation of JNK is a consequence of Hh signaling through Ci, RNAi-mediated knockdown of ci was performed, and it was found that the pJNK stripe is eliminated. Consistently, adult wing size is globally reduced. In contrast, no change was observed in pJNK stripe staining following RNAi-mediated knockdown of dpp or EGFR. Expression of non-processable Ci leads to increased Hh signaling. Expression of this active Ci in ptc cells leads to an increase in pJNK signal and larger, well-patterned adult wings. The modest size increase shown for ptc>CiACT is likely due to the fact that higher expression of this transgene (at 25 ° C) leads to such large wings that pupae cannot emerge from their cases. For measuring wing size, this experiment was performed at a lower temperature so that the animals were still viable. Furthermore, inhibition of JNK in wings expressing active Ci blocks Ci's effects, and resulting wings are similar in size to JNK inhibition alone . Together, these data indicate that Hh signaling through Ci is responsible for establishing the pJNK stripe (Willsey, 2016).
To determine the mechanism by which Ci activates the JNK pathway, transcriptional profiles of posterior and ptc domain cells isolated by FACS from third instar wing discs were compared. Of the total 12,676 unique genes represented on the microarray, 50.4% (6,397) are expressed in ptc domain cells, posterior cells, or both. Hh pathway genes known to be differentially expressed were identified. It was next asked whether any JNK pathway genes are differentially expressed, and and it was found that dTRAF1 expression is more than five-fold increased in ptc cells, while other JNK pathway members are not differentially expressed (Willsey, 2016).
dTRAF1 is expressed along the A/P boundary and ectopic expression of dTRAF1 activates JNK signaling. Thus, positive regulation of dTRAF1 expression by Ci could establish a stripe of pJNK that regulates wing size. Indeed, Ci binding motifs were identified in the dTRAF1 gene, and a previous large-scale ChIP study confirms a Ci binding site within the dTRAF1 gene. Consistently, a reduction in Ci led to a 29% reduction in dTRAF1 expression in wing discs. Given that the reduction of dTRAF1 expression in the ptc stripe is buffered by Hh-independent dTRAF1 expression elsewhere in the disc, this 29% reduction is significant. Furthermore, inhibition of dTRAF1 by RNAi knockdown abolished pJNK staining. Finally, these animals have smaller wings without obvious pattern defects. Conversely, overexpression of dTRAF1 causes embryonic lethality, making it not possible to attempt to rescue a dTRAF1 overexpression wing phenotype by knockdown of bsk. Nevertheless, it has been shown that dTRAF1 function in the eye is Bsk-dependent. Finally, inhibition of dTRAF1 modulates the phenotype of activated Ci signaling. Together, these data reveal that the pJNK stripe in the developing wing is established by Hh signaling through Ci-mediated induction of dTRAF1 expression (Willsey, 2016).
Finally, localized centers of pJNK activity were detected during the development of other imaginal discs including the eye/antenna and leg. Inhibition of localized JNK signaling during development caused a decrease in adult antenna size and leg size. Conversely, increasing JNK signaling during development resulted in pupal lethality; nevertheless, overall sizes of antenna and leg discs were increased. Together, these data indicate that localized JNK signaling regulates size in other organs in addition to the wing, suggesting a more universal effect of JNK on size control (Willsey, 2016).
Intrinsic mechanisms of organ size control have long been proposed and sought after. This study reveals that in developing Drosophila tissues, localized, organ-specific centers of JNK signaling contribute to organ size in an activity level-dependent manner. Such a size control mechanism is qualitatively distinct from developmental morphogen mechanisms, which affect both patterning and growth. Aptly, this mechanism is still integrated in the overall framework of developmental regulation, as it is established in the wing by the Hh pathway. These data indicate that localized JNK signaling is activated by Ci-mediated induction of dTRAF1 expression. Furthermore,it is not canonical Jun-dependent JNK signaling, but rather non-canonical JNK signaling that regulates size, possibly through Jub-dependent regulation of Yki signaling, as described for regeneration. As the human dTRAF1 homolog, TRAF4, and Hippo components are amplified in numerous cancers, these findings provide a new mechanism for how the Hh pathway could contribute to tumorigenesis. More importantly, these findings offer a new strategy for potential cancer therapies, as reactivating Jun in Hh-driven tumors could lead tumor cells towards an apoptotic fate (Willsey, 2016).
This study has shown that the exoribonuclease Dis3L2 is required for regulation of proliferation in the wing imaginal discs in Drosophila. Dis3L2 is a member of a highly conserved family of exoribonucleases that degrade RNA in a 3'-5' direction. Knockdown of Dis3L2 results in substantial wing overgrowth due to increased cellular proliferation rather than an increase in cell size. Imaginal discs are specified in the embryo before proliferating and differentiating to form the adult structures of the fly. Using RNA-seq, a small set of mRNAs was identified that are sensitive to Dis3L2 activity. Of the mRNAs which increase in levels and are therefore potential targets of Dis3L2, two were identified that change at the post-transcriptional level but not at the transcriptional level, namely CG2678 (a transcription factor) and pyrexia (a TRP cation channel). A compensatory effect between Dis3L2 and the 5'-3' exoribonuclease Pacman was identified, demonstrating that these two exoribonucleases function to regulate opposing pathways within the developing tissue. This work provides the first description of the molecular and developmental consequences of Dis3L2 inactivation in a non-human animal model. The work is directly relevant to the understanding of human overgrowth syndromes such as Perlman syndrome (Towler, 2016).
The development of the Drosophila wing depends on the correct regulation of cell survival, growth, proliferation, differentiation, and pattern formation. These processes, and the genes controlling then, are common to the development of epithelia in many different organisms. To identify additional genes contributing to wing development a genetic screen was performed in mosaic wings carrying clones of homozygous mutant cells. Twelve complementation groups were obtained corresponding to genes with a proven role in wing formation such as smoothened, thick veins, mothers against dpp, expanded, and fat and 71 new complementation groups were obtained affecting the pattern of veins and the size of wing. One of these groups mapped to the mediator15 gene (med15), a component of the Mediator complex. Med15 and other members of the Mediator complex were shown to be required, among other processes, for the transcription of decapentaplegic target genes (Terriente-Félix, 2010).
The complementation group formed by the 77A and 133A1 mutants was analyzed in some detail. These mutations are alleles of med15, a gene encoding one component of the Mediator complex. Thus, they fail to complement other med15 alleles, and med15133A1 is associated with a stop codon that could truncate the protein in the N-terminal region after the KIX domain. The Mediator multiprotein complex promotes the transcription of inducible genes, acting as a link between the RNApolII holoenzyme and several sequence-specific transcription factors. The human homolog of Med15, MED105, is included in all Mediator complexes identified so far and forms part of a module named the tail that is the main target for the transcriptional activators. Thus, Med15 homologs can bid to different transcription factors such as Gcn4 and Gal4 in Saccharomyces cerevisiae, and, more interesting from the perspective of these data, to Smad2/3 and Smad4 in Xenopus. Other members of the Mediator complex that were previously analyzed are kohtalo and skuld (Med12 and Med13, respectively), which form part of the conserved Cdk8 module. Interestingly, mouse Cdk8 and Cdk9 phosphorylate Smad proteins, regulating their transcriptional activity and turnover (Alarcón). However, kohtalo and skuld are required for sensory organ development, for some aspects of Notch and Hedgehog signaling, and for the transcription of Wingless downstream genes (Terriente-Félix, 2010).
Med15 mutations result in smaller than normal wings and loss of mainly the L2 vein. They also affect the fusion between the left and the right hemithorax and leg morphogenesis. The reduction in the level of expression of other components of the Mediator complex, most notably med20, med27, and med30, also results in smaller than normal wings and failures in vein differentiation, in addition to causing some levels of cell death. Although these phenotypes were similar, they are not identical, which might indicate specific requirements of these subunits or, alternatively, a different degree in the effectiveness of each interference RNA used. Mutant med15 cells display specific defects in gene expression, suggesting a requirement limited to particular enhancer-promotor interactions. In particular, the expression of spalt, a direct target of Dpp signaling, is compromised in med15 mutant cells. There are no known transcriptional targets of TGFβ signaling in the wing, and consequently it could not be determined directly whether the activity of this pathway is diminished in med15 mutants. A direct requirement of Med15 for the transcription of TGFβ target genes is nonetheless suggested by the similar phenotypes of wing size reduction observed in med15 mutants and in baboon mutations (Terriente-Félix, 2010).
Wing disc compartments were generated that contain marked fast growing M+ clones surrounded by slow dividing M/+ cells. Under these conditions the interactions between fast and slow dividing cells at the clone borders frequently lead to cell competition. However, an assay suppressing apoptosis indicates that cell competition plays no major role in size control. It is argued that cells within a compartment proliferate according to their genotype independently of each other and that their contribution to the final structure will depend solely on their proliferation rate. This model is supported by a computer simulation that predicts values similar to those found experimentally. The results on the growth of M+ clones within compartments and on the expression of developmental genes like vestigial and wingless suggest the existence of a non-cell autonomous mechanism that functions at the level of the entire cell population. It measures the population size in each moment, determines the corresponding expression levels of developmental genes and establishes the time to arrest growth (Morata, 2010).
The regulation of organ size constitutes a major unsolved question in developmental biology. The wing imaginal disc of Drosophila serves as a widely used model system to study this question. Several mechanisms have been proposed to have an impact on final size, but they are either contradicted by experimental data or they cannot explain a number of key experimental observations and may thus be missing crucial elements. This study has modeled a regulatory network that integrates the experimentally confirmed molecular interactions underlying other available models. Furthermore, the network includes hypothetical interactions between mechanical forces and specific growth regulators, leading to a size regulation mechanism that conceptually combines elements of existing models, and can be understood in terms of a compression gradient model. According to this model, compression increases in the center of the disc during growth. Growth stops once compression levels in the disc center reach a certain threshold and the compression gradient drops below a certain level in the rest of the disc. This model can account for growth termination as well as for the paradoxical observation that growth occurs uniformly in the presence of a growth factor gradient and non-uniformly in the presence of a uniform growth factor distribution. Furthermore, it can account for other experimental observations that argue either in favor or against other models. The model also makes specific predictions about the distribution of cell shape and size in the developing disc, which were confirmed experimentally (Aegerter-Wilmsen, 2012).
This paper presents a new model for the regulation of wing disc size. The
model contains a rather complex regulatory network, which
consists of a considerable number of interactions, receives nonuniform
input of protein activities, and interacts with a mechanical
stress pattern that emerges over time and space. It is assumed that the regulatory network represents protein activities and interactions that regulate these
activities. The model does not distinguish between interactions at the transcriptional and protein activity level, but considers effects on net activities.
All protein activities emerge from the network, except for those of Dpp, Wg and N, which are implemented in the model. In the regulatory network, differences in Ds and Fj concentrations between neighboring cells lead to activation of Dichate (D) by changing its intracellular
localization. In addition, it is assumed that a weighted average of the area of a cell and its neighbors is a good readout for mechanical stress,
that cells do not rearrange when exposed to mechanical tension, and that the planar polarization of D imposes a bias on the direction of the division plane. The interactions are hypothetical and form the main untested assumptions underlying the model. The regulation of ds by mechanical compression is not essential for the principle behind size regulation in the model, but improves the fit of simulation results with experimental data (Aegerter-Wilmsen, 2012).
A qualitative understanding can be gained by considering it in terms
of a compression gradient model. During growth, compression
increases in the center of the disc. Growth ceases when
compression in the center reaches a certain threshold and the
gradient of the compression gradient drops below a certain
threshold in the rest of the disc. Read-out of the compression
gradient is accomplished by a mechanism that involves Vg and the
Hippo pathway. Numerical simulations were used to show that
the model can account for growth termination and that it
reproduces a large range of additional data on growth regulation,
including some emergent properties of the system.
Based upon the principle underlying the model, predictions
can be made with respect to cell shape patterns. In order to take
into account the curved surface of the wing pouch, an open source image analysis program was developed. The results showed that the general dynamics of the formation of cell shape
patterns is indeed similar to the one predicted by the model. This
analysis is, however, based on images from different discs and,
especially during the early stages, there is variation among discs.
It would therefore be interesting to assess whether the predicted
dynamics is also present in the temporal evolution of single
discs. However, this first requires the development of experimental methods with which single discs can be followed over time (Aegerter-Wilmsen, 2012).
Even though the development of cell shape patterns
constitutes a fundamental prediction of the model, it would be
an interesting future experimental challenge to test the model's
basic assumptions directly, i.e., the regulation of Yki, Arm and
ds by mechanical forces. The regulation of Yki by mechanical
compression is most relevant for the model's behavior and
appears necessary to obtain growth termination in combination
with roughly uniform growth. The regulation of Arm by
compression seems to be involved in stabilizing the Vg gradient,
which could be relatively unstable if it would be regulated by Vg
autoregulation alone. In addition, this interaction smoothens the
compression gradient, which might have implications for the 3D
structure of the wing disc. Last, the regulation of ds by
mechanical forces is not essential for the principle behind size
regulation, but improves the modeling results and also
contributes to smoothening of the compression gradient.
While developing the model, focus was placed on its ability to
reproduce specific features of growth dynamics, as well as a
number of key experiments that are used to argue in favor and
against current models. One of the latter results, the decrease of
medial growth upon induction of uniform Dpp signaling, could not
be reproduced. In
the simulations, these discs grow very fast. It is conceivable that
such growth rates cannot be sustained in vivo because of a limited
availability of nutrients and oxygen. When imposing a maximum
total growth rate on disc growth, it is indeed possible to obtain
growth rates in the medial part that are lower than those in wildtype
discs, whereas lateral growth rates are higher, in agreement
with experiments. Thus, with this additional assumption, the model can reproduce the results it was aimed to reproduce (Aegerter-Wilmsen, 2012).
There are currently no experimental data available on the
parameters underlying the model and therefore they were fitted
manually. As has become clear from the parameter analysis, there
are only a few parameter combinations that can reproduce all
results. However, it is not known whether this set is reproduced
robustly in vivo and there is no natural selection on reproducing
experimental manipulations robustly. Nevertheless, it is entirely
possible that a larger set of parameter values should reproduce the
results. In addition, even though the model can reproduce the
selected set of experimentally observed features, there are related
observations it cannot reproduce. For example, the final size
reached in the model is too small, the experimentally observed nonautonomous
growth induction by clones overexpressing brk
is nearly absent in the model, and
growth induction along the boundary of ds overexpressing clones
extends further inside the clone than measured experimentally. It
would be interesting to study whether there are factors missing in
the model, which would make the parameter space less strict. For
example, the parameter space was strongly restricted by the
stipulation to reproduce the absence of Vg-BE activity in ap0
mutants upon ectopic wg expression. If it could be assumed that
smaller discs have a different geometry in vivo than larger ones,
the number of possible parameter combinations would increase. It
will be interesting to assess the geometrical properties of discs in
young larvae and evaluate whether the model should be adjusted
in this respect (Aegerter-Wilmsen, 2012).
Very recently, another model has been formulated for growth
regulation that assumes that growth is regulated by increases of
Dpp signaling levels over time. However,
growth is increased in wing discs in which Brk and Dpp signaling
are removed. This either contradicts this
model or the current understanding of Dpp signaling needs to be
revised. The current model reproduces increased growth in such mutants,
including its non-uniformity (Aegerter-Wilmsen, 2012).
The adult wing is covered by bristles, which point towards the
distal part of the wing. This orientation is regulated by planar
polarity genes. Regulation of planar polarity seems
to be related to growth regulation. For example, Ds and Fj are not
only important for growth regulation, but are also required for the
development of a proximodistal polarity pattern. It is currently not clear whether Ds and Fj are
directly involved in regulating planar polarity. If this were the case, then the
model would suggest that planar polarity may, at least in part, arise
from an interplay between morphogens and mechanical forces.
The model presented in this study was developed for the wing imaginal
disc of Drosophila. It would be interesting to see whether a similar
model could also reproduce size regulation and additional
experimental results in other systems. For other imaginal discs, it
has been shown that their centers are also compressed at the end of
growth. The precise regulatory networks
involved in growth and size regulation are different for the different
discs, but it would be interesting to see whether certain principles
are conserved. In mammals, mechanical forces regulate growth in
many tissues. However, the situation
is often very different from that in the wing disc in that most
mammalian tissues reach their final size while they perform a
biological function. Thus, it would be interesting to study whether
principles similar to those described here apply for mammalian
organs early during development (Aegerter-Wilmsen, 2012).
Planar cell polarity (PCP) is cellular polarity within the plane of an epithelial tissue or organ. PCP is established through interactions of the core Frizzled (Fz)/PCP factors and, although their molecular interactions are beginning to be understood, the upstream input providing the directional bias and polarity axis remains unknown. Among core PCP genes, Fz is unique as it regulates PCP both cell-autonomously and non-autonomously, with its extracellular domain acting as a ligand for Van Gogh (Vang). This study demonstrates in Drosophila wings that Wg (Wingless) and dWnt4 (Drosophila Wnt homologue) provide instructive regulatory input for PCP axis determination, establishing polarity axes along their graded distribution and perpendicular to their expression domain borders. Loss-of-function studies reveal that Wg and dWnt4 act redundantly in PCP determination. They affect PCP by modulating the intercellular interaction between Fz and Vang, which is thought to be a key step in setting up initial polarity, thus providing directionality to the PCP process (Wu, 2013).
The data indicate that Wg/dWnt4 regulate the establishment of Fz–PCP axes by modulating the Fz–Vang intercellular interactions in a graded, dosage dependent manner. Consequently they might generate different levels of Fz–Vang interactions across a Wg/dWnt4 gradient experienced by cells. This process is reiterated across the tissue, and the directionality of Fz–Vang binding is subsequently reinforced by intracellular core PCP factor interactions. The data are consistent with a model in which Wg/dWnt4 generate a Fz ‘activity'), suggesting that both of these light sensors are necessary for light avoidance behavior.' gradient models. Accordingly, PCP axes are orientated towards the Wg/dWnt4 source, which is evident in (at least) the wing and eye. The early wing PCP axis (late larval to early pupal stages) correlates well with Wg/dWnt4 margin expression and, similarly, in the eye polarity is oriented in the dorsoventral axis towards the poles where Wg/Wnt4 are expressed. This model, relying on a Fz–Vang interaction, is also compatible with the addition of Fmi to this scenario, with intercellular (homophilic) Fmi–Fmi interactions also being required for PCP specification. As Fmi forms complexes with both Fz and Vang, the full complement of intercellular interactions includes Fz/Fmi–Fmi/Vang complexes, and these interactions would also be modulated by Wnt binding to Fz, either directly as proposed in this model or possibly by modulating the Fmi–Fmi interactions by Fmi being associated with Fz that is bound to different levels of Wg/Wnt4. In vivo, Fmi helps to enrich both Fz and Vang to the subapical junctional region, and Fmi–Fmi interactions bring Fz and Vang to close molecular proximity (Wu, 2013).
Intercellular Fmi–Fmi interactions are strong, as Fmi-expressing S2 cells form cell aggregates through homophilic Fmi interactions. The interaction between Fz and Vang is weaker, and cell–cell contacts between the two cell groups are infrequent. It was suggested that PCP signal sensing complexes include both Fmi and Fz on one cell interacting with Fmi/Vang at the surface of a neighbouring cell. Within these complexes, Fz is required for sending a polarity signal, whereas Fmi and Vang are involved in its reception, consistent with the data and model. Although it has been suggested that Fmi is capable of sensing Fz/Fmi signals in the absence of Vang, the 'Fz-sensing' capability of cells with Fmi alone (lacking Vang) is much weaker than that of cells with Vang. It will be interesting to determine if there are other PCP regulators directly involved in modifying Fmi–Fmi interactions (Wu, 2013).
How do these data relate to previous models and why was the Wg/Wnt4 requirement not observed before? Previous work attempted to address the role for the wing margin in PCP by examining either mutants affecting wing margin cells without eliminating wg/Wnt expression or in clones. Although cellular hairs near the site of wing margin loss point towards remaining wing margin areas, the effect Is considered weak. Potential effects were examined of Wnt LOF clones of Df(2L)NL, lacking wnt4, wg, wnt6 and wnt10. In contrast to the global reduction of Wg/Wnt4 through the temperature sensitive wg allele, such clones cause only mild PCP perturbations. There are several reasons why clonal loss of Wnt expression in the margin only mildly affects PCP orientation: cells can respond to Wnts from several sources/cells from remaining Wnt-expressing wing margin regions; polarization strengths (measured by nematic order) in the first few rows of cells near the margin are much weaker than those in cells further away (at 14-17 h APF) and weak PCP reorientation in cells neighbouring wing margin clones could thus reflect the initial weak polarization in these cells; and PCP orientation changes from its initial radial polarity towards the proximodistal polarity during hinge contraction morphogenesis and associated cell flow, probably leading to significant corrections of subtle defects near the margin. Similarly, PCP orientation in cells near the margin is only very weak early (at 14-16 h APF), probably because cells close to the Wnt-producing cells are exposed to saturated Wnt levels (and not a Wnt gradient), or because the presence of other organizers (directing polarity parallel to the margin) weakens the effect of Wnts. PCP in these cells is established/corrected through more local interactions during the feedback loops among neighbouring cells (Wu, 2013).
To determine the direct role for Wg/Wnts on Fz–PCP signalling, it was examined at pupal stages, as the patterning role for canonical Wg signalling is much reduced then and PCP still correlates well with Wg/Wnt4 expression. Importantly, Wnt4 does not affect expression of patterning genes through canonical signalling at larval or pupal stages, yet Wnt4 alters PCP orientation, consistent with the model that Wnt4/Wg act directly on Fz-PCP interactions. The observation that Wnt4 requires Fz to affect neighbouring cells further supports this model. It is likely that, as well as the Wg/Wnt4 input and mechanism identified in this study, both early and late PCP axes depend on further cues, provided for instance by the parallel Ft/Ds-PCP system or other morphogenetic organizers. Strikingly, such a scenario would suggest that Wg regulates PCP directionality through both PCP systems, affecting Fz-PCP interactions directly and through canonical Wg signalling transcriptionally regulating graded fj and ds expression in eyes and wings. In summary, these data provide insight into Wnt-mediated mechanisms to directly regulate long-range Fz–PCP orientation by modulating Fz–Vang/PCP interactions during tissue morphogenesis (Wu, 2013).
Drosophila imaginal disc cells exhibit preferred cell division orientations according to location within the disc. These orientations are altered if cell death occurs within the epithelium, such as is caused by cell competition or by genotypes affecting cell survival. Both normal cell division orientations, and their orientations after cell death, depend on the Fat-Dachsous pathway of planar cell polarity (PCP). The hypothesis that cell death initiates a planar polarity signal was investigated. When clones homozygous for the pineapple eye (pie) mutation were made to initiate cell death, neither Dachsous nor Fat was required in pie cells for the re-orientation of nearby cells, indicating a distinct signal for this PCP pathway. Dpp and Wg were also not needed for pie clones to re-orient cell division. Cell shapes were evaluated in wild type and mosaic wing discs to assess mechanical consequences of cell loss. Although proximal wing disc cells and cells close to the dorso-ventral boundary were elongated in their preferred cell division axes in wild type discs, cell shapes in much of the wing pouch were symmetrical on average and did not predict their preferred division axis. Cells in pie mutant clones were slightly larger than their normal counterparts, consistent with mechanical stretching following cell loss, but no bias in cell shape was detected in the surrounding cells. These findings indicate that an unidentified signal influences PCP-dependent cell division orientation in imaginal discs (Kale, 2016).
This paper made use of the observation that clones of imaginal disc cells mutant for pie, which exhibit an elevated rate of apoptosis, bias the cell division orientation of other cells nearby in a search for a signal responsible for cell division orientation. It is hypothesized that dying pie cells may be the source of a polarizing signal that is detected by other cells, and the roles of candidate signals were evaluated by removing them genetically from pie mutant cells. It is further hypothesized that the result may also be relevant to the orientation of cell divisions in normal development (Kale, 2016).
Since cell division orientation requires the PCP receptor Fat, this study tested whether its PCP ligand Dachsous was required, but this model was excluded. Since cell division orientation also requires Dachsous in the dividing cells, tests were performed to see whether Fat itself was a signal required in the apoptotic clones, but this was also excluded. In fact both Fat and Dacshous could be eliminated together from the dying cell population without preventing the orientation of nearby cells. The possibility was considered that rather than expressing Fat or Dachsous, apoptotic cells might down-regulate one or both proteins and that this might affect nearby cells, but it was found that eliminating one or both genes was not sufficient to orient nearby cell divisions. The possible contribution of Four-jointed, a kinase that phosphorylates Fat and Dachsous proteins in the Golgi, was not tested because Four-jointed should be unable to signal in cells already mutated for both ft and ds. Altogether, the experiments eliminated known ligands for the Fat/Dachsous PCP pathway, suggesting that the pathway must be required to orient cell division in response to some other signal (Kale, 2016).
It has been suggested that apoptotic imaginal disc cells secrete the morphogens Dpp and Wg in the process of stimulating compensatory proliferation. Since Dpp and Wg pattern many aspects of imaginal disc development, including the expression of some PCP genes, they were candidates to orient the division of imaginal disc cells. Contrary to this prediction, clones of apoptotic cells lacking Dpp and Wg continued to orient nearby cell divisions. It cannot be excluded that there may be other biochemical signals from dying cells that orient cell division. For example, there are other Wnt proteins in Drosophila that might affect cell polarity (Kale, 2016).
One other model consistent with these results is that cell division is oriented by physical constraints rather than biochemical signals. It is thought that in the wild type wing disc, the characteristic circumferential division pattern of the peripheral cells is a result of their being stretched around the growing wing pouch. Consistent with this conclusion, it has been reported that when a clone of cells grows more rapidly than the surrounding epithelium, cells around the clone are stretched circumferentially to accommodate the hyperplastic region, and this change in shape tends to orient cell divisions in a circumferential pattern around the hyperplastic clones. By analogy to these findings concerning enhanced growth, it might be expected that clones of cells experiencing high rates of cell death would expand more slowly than surrounding cells, and that this would stretch the cells around the clone inwards towards the slow growing region, leading to a reorientation of cell divisions towards the slow growing clone, opposite to the case of more rapidly growing clones. As expected given their persistent cell death, clones of pie homozygous cells grow more slowly than control clones, and exhibit a small increase in apical cell size, consistent with local tension in the epithelium. The changed orientation of cell division near to pie clones has been reported previously. This study was unable, however, to measure a consistent change in shape of the wing cells adjacent to pie homozygous clones, the population of cells where the altered division orientation is measured. This lack of correlation between cell shape and cell division orientation is also seen for wing pouch cells in the wild type wing disc, which show a proximo-distal division preference but no obvious proximo-distal polarization. The shapes of mitotic cells were not measured separately, and so the possibility cannot be excluded that only the mitotic cells exhibit altered shapes in the wing pouch. Recently, it has been reported that the orientation of epithelial cell division is determined by microtubule interactions with cell junction vertices, and that cell shape is a poor predictor of cell division in rounded cells, where the disposition of cell junction vertices varies. This may explain why both the normal cell division orientation and the response to cell death do not correlate with cell shape within the wing pouch region, where cells are more rounded than in peripheral regions of the wing disc (Kale, 2016).
Oriented cell divisions are suggested to contribute to organogenesis. It was suggested that oriented cell divisions are responsible for the shape of cell clones in the wing disc, which ultimately determines the shape of the whole tissue (which is a collection of clones). Oriented cell divisions may have other functions, for example they may represent a homeostatic mechanism that ameliorates growth-induced mechanical stress (Kale, 2016).
The shape of cell clones becomes less regular during cell competition, and the interfaces between wild type and Minute cell populations become more convoluted and interdigitated. Previously, it was suggested that oriented cell division could be responsible for the intermingling of wild type and Minute cells. Recently, Levayer described very similar intermingling between cells in the pupal notum that are induced to compete by expression of different levels of Myc protein (Levayer, 2015). Very little cell division occurs in pupal notum, and Levayer describe cell neighbor exchanges that are responsible for intermingling the cell populations. They propose these exchanges are promoted by mechanical effects of differential growth rates. Wild type and Minute cells also grow at different rates, but the apoptotic protein baculovirus p35 reduces the degree of intermixing between wild type and Minute cells. There is now evidence that p35 also stimulates Minute growth rate, while having less effect on wild type cells. Although the precise mechanism is unclear, Minute cell growth is possibly stimulated by signals from the undead Rp/Rp cells that are preserved when p35 is expressed. Together these data raise the possibility that p35 may affect both cell division orientation and intermingling of wild type and Minute cells by equalizing their relative growth rates. In the case of pie clones that expand slowly, differential growth might result in local mechanical stretching which influences nearby cell divisions, although it cannot be excluded that the pie mutant clones have other differences from wild type (Kale, 2016).
Fat has a role as an upstream regulator of the Salvador-Warts-Hippo (SWH) pathway of tumor suppressors. There is substantial evidence that the SWH pathway responds to mechanical cues. Inputs are reported from actin polymerization status and from adhesion junctions via α-catenin and Juba proteins. Recent studies indicate that the SWH pathway itself promotes epithelial junctional tension, which is reduced in clones of ft or wts mutants. Cell division orientation also depends on atro, however, which has been thought not to affect SWH activity, since it does not affect growth. Recent studies suggest that mutations in the Fat-Dachsous pathway may affect PCP through a disruption of the Spiny Leg protein by de-repressed Dachs that is not a reflection of normal Dachs function. This does not explain how cell division orientation is affected by Fat or Dachsous but it does raise the possibility that Fat and Dachsous mutations might affect processes that depend little on their normal alleles. What this study reports is that the model developed for planar cell polarity, in which ligand-receptor interactions between Fat and gradients of Dachsous control cell polarity, do not seem applicable to the orientation of cell division in the wing disc, where mechanical factors may be important (Kale, 2016).
The Crumbs (Crb) complex is a key epithelial determinant. To understand its role in morphogenesis, this study examined its function in the Drosophila pupal wing, an epithelium undergoing hexagonal packing and formation of planar-oriented hairs. Crb distribution is dynamic, being stabilized to the subapical region just before hair formation. Lack of crb or stardust, but not DPatj, affects hexagonal packing and delays hair formation, without impairing epithelial polarities but with increased fluctuations in cell junctions and perimeter length, fragmentation of adherens junctions and the actomyosin cytoskeleton. Crb interacts with Moesin and Yurt, FERM proteins regulating the actomyosin network. Moesin and Yurt distribution at the subapical region depends on Crb. In contrast to previous reports, yurt, but not moesin, mutants phenocopy crb junctional defects. Moreover, while unaffected in crb mutants, cell perimeter increases in yurt mutant cells and decreases in the absence of moesin function. These data suggest that Crb coordinates proper hexagonal packing and hair formation, by modulating junction integrity via Yurt and stabilizing cell perimeter via both Yurt and Moesin. The Drosophila pupal wing thus appears as a useful system to investigate the functional diversification of the Crb complex during morphogenesis, independently of its role in polarity (Salis, 2017).
This study aimed at unveiling the function of the Crumbs complex in epithelial morphogenesis. Although Crb was discovered several decades ago in Drosophila, the severe apico-basal polarity defects associated to crb inactivation in embryos have hampered the full exploration of its function during epithelia development. The results indicate that Crb also acts during pupal wing morphogenesis, where the absence of crb function does not impair AP/BL polarity and does not lead to the dramatic tissue alterations often seen in other tissues. The pupal wing thus represents an attractive model system, well suited to dissect additional functions of the Crb complex during epithelial morphogenesis, independently of its role in polarity (Salis, 2017).
The redistribution of Crb at the subapical region (SAR) at the end of hexagonal packing, as well as the defects in cells orientation observed in crb mutants suggest that Crb is required to stabilize the actin cytoskeleton and E-cadherin at the adherens junctions at the end of tissue rearrangement. Alterations in F-actin and Myosin II (Myo) distribution in crb mutant cells strikingly mimic those observed in embryos mutant for the actin-binding protein Canoe/Afadin, which links the actomyosin network to AJs. Canoe loss diminishes this coupling leading to reduced cell shape anisometry and defects in germ band elongation. As for crb, canoe mutant cells still retain some ability to change their shape and germ band elongation is delayed and not completely impaired. The defects observed in crb mutant cells support the hypothesis that Crb is a crucial regulator of the interconnection between the actomyosin cytoskeleton and AJs (Salis, 2017).
The fragmentation of AJs upon Crb depletion has been already described, for example in embryo or during follicular morphogenesis. However, in these two systems the function of Crb has been related to the role of Moe in the regulation of the actomyosin cytoskeleton, while the role of Yurt has never been addressed or has been excluded. The current data support that in pupal wing cells the role of Crb in the stability of the AJs is likely established via Yurt. Crb is shown to modulate Yurt localization at the SAR at the end of hexagonal packing and yurt mutant cells phenocopy crb mutant cortical defects. Nonetheless, previous studies in cultured cells have established that Yurt participates in epithelial polarity and organization of apical membranes by negatively regulating the activity of the Crb complex. On the contrary, this study shows that, whereas Crb modulates Yurt distribution at the SAR at the end of hexagonal packing of wing cells, Yurt depletion does not impact Crb association to the SAR, with the exception of the E-cad- and F-actin-devoid gaps. Yurt and Crb similarly act on actomyosin and E-cad organization at the cell-cell junctions suggesting that the coordinated function of these two proteins is regulated by different mechanisms in different tissues. On the other hand, moe depletion does not specifically modify Crb distribution at the SAR, a finding coherent with the evidence that Moe is not implicated in stability of AJs in this tissue, as opposed to other models (Salis, 2017).
Studies based on in vivo mechanical measurements or mathematical/physical modeling have proposed that epithelial cell packing results from a balance between intrinsic cell tension and extrinsic tissue-wide forces to establish a correct and robust order in the tissue. Hence, the tension generated by the actomyosin cortex and the pressure transmitted through adherens junctions are the two main self-organizing forces driving tissue morphogenesis. Tension shortens cell-cell contacts and pressure of individual cells counteracts tension to maintain cell size. The current data indicate that Crb recruits at SAR Moe and Yurt, which show opposite effects on pupal wing morphogenesis. While Moe promotes cell expansion, Yurt controls cell constriction and the stability of the AJs and of the actomyosin network. In crb mutant cells, the absence of variation in the cell perimeter might be explained by the simultaneous loss of positive and negative regulators. Therefore, Crb acts as a coordinator of the two self-organizing mechanisms implicated in morphogenesis. Additionally, the dynamic redistribution of Crb at the SAR at the end of hexagonal packing, together with the disruption of cell orientation in crb mutants, is consistent with the hypothesis that Crb is required to stabilize cell shape and pattern in order to properly progress throughout tissue development (Salis, 2017).
In conclusion, these functional analyses during pupal wing morphogenesis allowed the unraveling Crb-dependent mechanisms that are integrated to produce shape changes during development independently of epithelial polarity. Furthermore, the results show that the interplay between Crb and FERM proteins is tissue-regulated and that their epistatic interactions differ in a spatio-temporal manner (Salis, 2017).
Coordinated rearrangements of cytoskeletal structures are the principal source of forces that govern cell and tissue morphogenesis. However, unlike for actin-based mechanical forces, knowledge about the contribution of forces originating from other cytoskeletal components remains scarce. This study has establish microtubules as central components of cell mechanics during tissue morphogenesis. Individual cells were found to be mechanically autonomous during early Drosophila wing epithelium development. Each cell contains a polarized apical non-centrosomal microtubule cytoskeleton that bears compressive forces, whereby acute elimination of microtubule-based forces leads to cell shortening. It was further established that the Fat planar cell polarity (Ft-PCP) signalling pathway couples microtubules at adherens junctions (AJs) and patterns microtubule-based forces across a tissue via polarized transcellular stability, thus revealing a molecular mechanism bridging single cell and tissue mechanics. Together, these results provide a physical basis to explain how global patterning of microtubules controls cell mechanics to coordinate collective cell behaviour during tissue remodelling. These results also offer alternative paradigms towards the interplay of contractile and protrusive cytoskeletal forces at the single cell and tissue levels (Singh, 2018).
During development individual cells assemble into complex tissues and organs with specialized forms and functions. Tissue morphogenesis is driven by mechanical forces that are generated by the cytoskeleton within cells and transmitted in a coordinated manner through adhesion molecules across neighbouring cells. The best-studied cytoskeletal component is actin, which, together with other proteins, forms protrusive and contractile arrays, a fundamental constituent of rearrangements on the single cell and tissue levels. Recent work has suggested that microtubules, similar to actin, can also generate forces in cells. However, understanding of the contribution of microtubules to cell mechanics, cell shape changes and force coordination during morphogenesis remains poor. This is mainly due to the fact that many current models describing the mechanical state of tissues during shape changes focus on actomyosin dynamics and/or rely on continuum mechanics. These studies, which are based on coarse-grain observations of cell movements or cell shape changes, reveal only part of the physical mechanisms that drive morphogenesis and do not directly investigate the physicomechanical context of tissue remodelling. To understand the relationship between cell mechanics, force patterning and molecular structure, this study investigated the mechanical properties of microtubules at high spatiotemporal resolution using wing development in Drosophila melanogaster as a paradigm (Singh, 2018).
During pupal wing development, non-centrosomal microtubules form an apical array of parallel microtubule bundles that are globally aligned along the proximal-distal (P-D) axis. Patterning of the microtubule cytoskeleton depends on the Ft-PCP signalling pathway and occurs during the early phase of wing reshaping (that is, between 14 and 18 h after puparium formation, or APF). This patterning is associated with extensive changes in cell shape, cell divisions and cell-cell rearrangements. In the Drosophila wing, the Ft-PCP pathway further orients cell elongation and cell divisions along the P-D axis to induce wing tissue elongation. Intriguingly, rescue of the Hippo pathway in Ft-PCP mutant animals, in which microtubule alignment is impaired, aberrant development results in perturbed cell elongation and an abnormal rounded wing shape, suggesting that there is an interdependence between these events. Therefore this study explored the possibility that microtubule-based cell mechanics control cell and tissue shape during early wing development between 16 and 18 h APF (Singh, 2018).
Tissue remodelling is driven by intrinsic and extrinsic mechanisms, and it has previously been shown that extrinsic mechanical forces act during the late phase of wing reshaping (starting 18 h APF). These forces are generated by hinge contraction of the wing that is attached to the cuticle on the distal side. This study evaluated the mechanical autonomy of individual cells before hinge contraction at an earlier developmental stage (that is, between 16 and 18 h APF). This was done by isolating a single cell (or a small patch of cells) using a single-pulse multipoint procedure to cut AJs, thus mechanically uncoupling individual cells from their surrounding. Strikingly, the shape of individual isolated cells did not change significantly upon laser ablation at 17-18 h APF, when cells in the wing are already elongated. The same result was obtained when patches of cells were isolated. Additional analyses of the Feret's diameter before and after ablation showed a small isotropic decrease in cell size, providing evidence that at this early stage, individual cells are not influenced by the neighbouring cells or by tissue-scale forces in a polarized fashion. Consistently, analysis of animals expressing a mutant form of dumpy protein, an extracellular matrix protein associated with tissue remodelling at later developmental stages, showed no substantial differences in wing shape compared to wild-type wings at 18 h APF. Together, these experiments argue that unlike later stages, cell autonomous forces are the major drivers of initial cell shape changes between 16 and 18 h APF (Singh, 2018).
To identify the molecular mechanism underlying cell autonomous shape formation, the distribution and dynamics of two cytoskeletal force-generators were investigated: microtubules and non-muscle myosin II (MyoII) as a component of the actomyosin cytoskeleton. MyoII was detected at the apical cell cortex at the level of AJs. A subsequent analysis of the signal distribution within single cells revealed a planar polarized distribution of MyoII along the P-D axis, which correlated with increased tension along the same junctions. As MyoII provides contractile forces, this should result in P-D junctional shortening upon laser ablation. However, this is inconsistent with the current ablation experiments, suggesting that there is an opposing force present. Interestingly, staining of microtubules showed planar polarized apical microtubules along the P-D axis at the level of AJs. Microtubules are the stiffest cytoskeletal filaments, with a persistence length on the order of millimetres. Microtubules are therefore well suited to balance the tension generated by actomyosin contraction. Consistently, the distribution of microtubules and MyoII in mechanically isolated cells remain polarized. In addition, microtubule and MyoII polarity was preserved in dumpy mutant wings at 18 h APF, indicating that they are polarized in a cell autonomous fashion. The possible role of the atypical myosin Dachs, a downstream component of the Fat signalling pathway, was also analyzed. Dachs mutant wings showed no change in cell elongation or microtubule polarity, which is consistent with recent work reporting that recombinant Dachs does not have ATPase activity and can therefore not function as a molecular motor. Together, these observations argue that planar polarized microtubules may balance actomyosin tensional forces that pull on P-D junctions and stabilize cell shape (Singh, 2018).
To validate this hypothesis, and to elucidate the dynamic and functional role of microtubules in cell mechanics, their properties were investigated during wing development. Live cell imaging of EOS-α-tubulin (EOS-Tub) showed that microtubules were not static but engaged in complex and dynamic rearrangements. An analysis of microtubule straightness showed that in wing cells, virtually all microtubules along the P-D axis were bent, consistently undergoing short wavelength buckling (~3 μm) near the cell cortex. It was further observed that growing microtubules remain straight and only start to buckle after they reach the cell cortex, exhibiting local short wavelength buckling near these sites. This result indicates that microtubule polymerization can generate considerable compressive forces to induce microtubule buckling (Singh, 2018).
Next, whether buckling of microtubules in Drosophila wing epithelium is indeed a result of forces acting on microtubules was also investigated, as suggested by the current experiments and in vitro studies, or whether the cellular environment yields more flexible microtubules. This is important, as buckling of flexible microtubules would rule out a role in balancing actomyosin contractility. To probe the forces of single microtubule filaments in vivo, individual microtubules were cut by laser ablation and the subsequent relaxation was monitored using live imaging. Previously curved microtubules rapidly straighten out, thus verifying that microtubules are indeed loaded with compressive forces. Finally, it was also observed that local ablation of microtubules triggers a rapid translocation of the adjacent junction. This finding supports the idea that non-centrosomal microtubules continuously generate pushing forces via polymerization that may then be stored as compressive forces in a polarized fashion to balance contractile forces generated by junctional actomyosin (Singh, 2018).
How are microtubules polarized along the P-D axis? While the molecular mechanism has remained elusive, previous work has established that the Ft-PCP signalling pathway aligns the apical microtubule network along the P-D axis by regulating association sites of microtubules with AJs. Considering the observed stability of aligned microtubules, whether directional differences in microtubule dynamics could serve as a mechanism for the planar polarization of microtubules was tested. Monitoring of EB1 tagged with green fluorescent protein (EB1-GFP) revealed two populations of microtubule-plus ends: fast growing microtubules with a growth velocity of 24.43 ± 0.43 7mi;m min-1 (mean ± s.e.m.), and slow growing microtubules with a velocity of 17.06 ± 0.26 7mi;m min-1. A further analysis showed that the microtubule growth rates depended on relative localization within cells as well as the growth angle relative to the P-D axis. Microtubule growth rates in the cell interior were higher compared to the cell cortex. Similarly, microtubules along the P-D axis grew faster than microtubules growing perpendicular to the P-D axis along the A-P axis, establishing a spatial gradient in microtubule growth velocity. The lower growth rate along the A-P axis close to the cell periphery suggests that there is more frequent pausing and switching between polymerization and depolymerization of microtubules, thus indicating a decreased stability of A-P oriented microtubules (Singh, 2018).
It was reasoned that over time, such differences in dynamics and stability may result in predominantly P-D aligned microtubules. To test this hypothesis, the cortical residence time was analyzed of microtubules as a function of their angle with respect to the P-D axis. Intriguingly, it found that microtubules that interact with the P-D cell cortex have a longer lifetime than microtubules interacting with the A-P cortex. Upon closer inspection, dynamic cycles of short-lived interactions of microtubules with A-P junctions were noted followed by depolymerization. Importantly, A-P oriented microtubules do not show buckling behaviour, which is in contrast to P-D oriented microtubules, but rather undergo catastrophe soon after interaction with A-P oriented cell junctions. This result suggests that microtubule-plus ends are less stable at these sites and thus cannot sustain long-lasting interactions with the cell cortex, which are required to generate compressive forces. Building on these observations, in silico probing was performed to see whether the angular difference in lifetime may indeed be sufficient for microtubule polarization. Assuming a random orientation for de novo formed microtubules, the lifetime of each microtubule was defined as a function of the angle with a maximal lifetime along the P-D axis. Upon expiration, individual microtubules were re-introduced into the system at random angles, therefore keeping the total number of microtubules constant. Consistent with the in vivo observations, the simulation reached a steady-state at which a constant fraction of microtubules polarized along the P-D axis. Taken together, these observations point to a mechanism whereby microtubule stability regulates the planar alignment of the microtubule cytoskeleton along the P-D axis, which in turn directs cell mechanics along this axis. These data place directional microtubule stability upstream of proposed mechanisms of how cell shape influences microtubule alignment. Furthermore, these results are consistent with previous findings that microtubule association with P-D oriented AJs during the initial stage of wing development depends on Ft-PCP signalling (Singh, 2018).
Having established that planar polarized microtubule-based forces shape single cells, their mechanical coupling and integration into tissue-level mechanics were investigated. In a first round of experiments, transcellular coupling of microtubules were investigated on the ultrastructural level using transmission electron microscopy (TEM). In agreement with previous work, AJs were juxtaposed in neighbouring cells associated with microtubule filaments that span across cells in wild-type wings, forming supracellular cables analogous to myosin cables. Notably, no such association was observed in ftl(2)fdd1 / ftl(2)fd dGC13 (ft d) and ftl2 fd/ftGRV;ActP-Gal4/UAS-FtΔECDΔN-1 (N1) mutant wings, in which microtubules are randomly oriented in wing cells, therefore providing structural support for the Ft-PCP-dependent stabilization of microtubule-based forces at P-D oriented AJs. Consistently, ft mutant clones showed a fragmented microtubule cytoskeleton, arguing that there is Ft-PCP-dependent stabilization of microtubules via coupling at AJs (Singh, 2018).
To further validate the role of polarized transcellular microtubule stability in tissue mechanics and organization, tissue shape changes were observed upon acute perturbation of microtubule-based forces. To control microtubule dynamics in a precise spatial and temporal manner, he recently developed photostatin (PST1)35, a photo-switchable analogue of combretastatin A-4 (CA4)36 was used. The drug was applied to directly test the requirement of microtubules for cell shape maintenance. Notably, it was found that the exposed wing area contracted along the P-D axis upon microtubule inhibition. Quantitative cell shape analysis showed a small but significant reduction in the elongation index (EI) in selective regions where the drug was activated, arguing that polarized tissue stabilization is via microtubule-based forces. Finally, overexpression of the microtubule-severing protein Spastin increased cell shape heterogeneity. These results are consistent with the hypothesis that an intact polarized microtubule cytoskeleton is not only required for the maintenance of anisotropic cell shape but also critically involved in shaping the whole tissue during morphogenesis via polarized transcellular force stability (Singh, 2018).
Understanding the role of microtubules during animal development has so far been limited, especially because of a shortage of methods suitable to demonstrate causality in vivo. Taking advantage of complementary genetic, chemical, numerical and microscopy approaches, these experiments unveil polarized microtubule-based compressive forces as an alternative principle for stabilizing and maintaining cell and tissue shape during morphogenesis. Alignment of microtubules along the P-D axis was found to be based on increased longevity and polymerization of microtubules interacting with P-D oriented AJs compared to non-polarized microtubules. The result of this microtubule patterning along the P-D axis is an asymmetric distribution of protruding forces, which are stored in a polarized fashion via compressive loads on microtubules. Considering that actomyosin and microtubules are both planar polarized, it is plausible to envision that the observed compressive load on microtubules plays an active role in balancing actin-based contractile forces, resulting in the cell mechanical autonomy observed in the laser ablation experiments. Intriguingly, it was recently shown that acetylation of microtubules increases their mechanical resistance and that microtubules undergo self-repair upon damage. These important features support the role of the microtubule cytoskeleton as a site of long-term compressive force storage. Finally, evidence is provided that planar polarized microtubules are coupled at AJs across individual cells, bridging forces on the tissue level via polarized transcellular stability. Although the molecular identity remains elusive, the data suggests an involvement of AJ-associated proteins organized by the Ft-PCP pathway in this process (Singh, 2018).
Collectively, this work provides evidence that PCP-based planar patterning of the microtubule cytoskeleton not only results in polarized cell-autonomous forces but also coordinates global force patterning during tissue morphogenesis. The proposed mechanism establishes the Ft-PCP pathway at the onset of cell and wing elongation, before shape changes, due to extrinsic mechanical forces. Consistently, in a Ft-PCP mutant, in which initial elongation fails, consecutive remodelling by extrinsic tensile forces cannot rescue these length defects, therefore leading to shorter and rounder adult wings. Considering that the Ft-PCP signalling pathway controls a variety of dynamic cell population in vertebrates, the microtubule-based mechanism described in this study is likely to be physiologically relevant beyond wing development (Singh, 2018).
How tissues acquire their characteristic shape is a fundamental unresolved question in biology. While genes have been characterized that control local mechanical forces to elongate epithelial tissues, genes controlling global forces in epithelia have yet to be identified. This study describes a genetic pathway that shapes appendages in Drosophila by defining the pattern of global tensile forces in the tissue. In the appendages, shape arises from tension generated by cell constriction and localized anchorage of the epithelium to the cuticle via the apical extracellular-matrix protein Dumpy (Dp). Altering Dp expression in the developing wing results in predictable changes in wing shape that can be simulated by a computational model that incorporates only tissue contraction and localized anchorage. Three other wing shape genes, narrow, tapered, and lanceolate, encode components of a pathway that modulates Dp distribution in the wing to refine the global force pattern and thus wing shape (Ray, 2015).
This study has identified a group of genes that define the global force patterns that shape the appendages in Drosophila. During pupal development, shape is determined by a general contraction of the tissue in combination with localized anchorage to the pupal cuticle, which is mediated by the apical extracellular matrix (aECM) protein Dp. In the developing wing, Dp is localized to the wing margin such that, as tissue contraction proceeds, tension along the P-D axis elongates the wing and also draws the two wing surfaces together. Indeed, manipulating the pattern of Dp localization at this stage leads to dramatic changes in wing shape that reflect the underlying change in tissue anchorage. In the legs and antennae, Dp is found in a dense plaque at the distal tip of the appendage, and, as in the wing, tissue contraction results in tapering and elongation of the structure. Thus, this study has identified a genetic mechanism that determines shape by regulating the pattern of global tensile forces that the epithelium experiences during tissue contraction (Ray, 2015).
While the mechanism that this study has uncovered is clearly important for proper anchorage of the wing epithelium to the cuticle, it is only one part of the regulatory mechanism that leads to the localized attachment. Indeed, in a nw mutant, while the distribution of Dp is altered, it is still localized to the margin, thus other inputs must be involved in defining where Dp is localized during pupal development. In the wing, the localization of Dp to the margin is reminiscent of the expression of genes controlled by the Notch and Wingless pathways that define the dorsal-ventral compartment boundary. Indeed, the gene Dll is a downstream target of Wg, and knocking down dp in the cells that express Dll phenocopies the dp loss-of-function phenotype. Moreover, the notching associated with mutations in the Notch and Wg pathways, as well as their targets such as cut, are, in essence, defects in the anchorage of the margin during pupal development: the failure to specify the margin results in a gap in the expression of Dp which produces a phenotype not unlike that which is observed in dpp-Gal4>dpRNAi. Consistent with this, it has previously been shown that the notching associated with cut arises during pupal development during the period of hinge contraction. Thus, it may be that these phenotypes arise from a failure to localize Dp to specific regions of the margin rather than to cell death, as has been suggested previously (Ray, 2015).
In the leg and antenna, the localization of Dp to the extreme distal tip of the appendage is presumably under control of the P-D patterning system that operates in these tissues. Indeed, as in the wing, this study hasa shown that the retraction of the leg and antenna is produced by knocking down dp with Dll-Gal4, which is expressed in the most distal segments of both appendages. Moreover, the observed phenotypes are also associated with mutations in genes that affect specification of the distal most tarsal segments. Classical loss-of-function mutations of the Paired-type homeodomain protein Aristaless result in the same shortening of the arista as is observed with dp-RNAi. Similarly, loss-of-function alleles of the transcription factor Lim1 result in a shortening of the arista and the distal leg segments, which presumably reflects the failure to anchor the distal tip of the appendage to the pupal cuticle. Thus, it is speculated that for wings, legs, and antennae, the localized function of Dp appears to depend on cues from the developmental programs that pattern the appendages (Ray, 2015).
While Dp localization clearly depends on the positional cues set down in the imaginal discs, how these signaling molecules and transcription factors result in a localized pattern of Dp protein in the pupa remains unclear. Previous studies have shown that the dp mRNA is expressed throughout the developing wing in early pupal development (Wilkin, 2000), suggesting that the localized pattern of Dp expression that is evident at 18 hr APF results by a post-transcriptional mechanism. Throughout development, Dp functions as a link between the ectodermal epithelium and the cuticle, and, in order for the animal to molt, this connection must be periodically broken. During larval development, molting (or ecdysis) is initiated by apolysis, a process that separates epidermal cells from the old cuticle by secretion of a complex mixture of chitinases and proteases that degrade the carbohydrate and protein components of the exoskeleton, respectively. Given that Dp is essential for the link between epidermis and cuticle, it is a key target of the apolytic machinery. In the pupa, apolysis of the pupal cuticle occurs just prior to the onset of tissue contraction and is a prerequisite for generating global forces by differential anchorage. Given this, the localization of Dp and the localized anchorage of the appendage tissues to the cuticle presumably arise by protection of Dp at the margin and its degradation throughout the rest of the wing blade. These results suggest that in the wing, Nw acts to extend this protection more proximally to give rise to the wild-type shape of the wing (Ray, 2015).
The results show that coordination of the behavior of thousands of individual cells in the pupal wing is achieved by the precise regulation of global extrinsic forces that determine appendage shape. In this system, the wing hinge undergoes apical constriction to generate a pulling force that is transmitted through the tissue. As shown in this study, resistance to this force is provided by anchorage of the wing margin to the overlying pupal cuticle, resulting in anisotropic tension oriented predominantly along the P-D axis. As has been shown previously, cells respond to this tension via cell shape changes, oriented cell divisions, and cell rearrangements to drive tissue elongation and determine the final shape the wing. Thus, as a mechanical process, tissue shaping during pupal development depends on the magnitude of the force generated by the constricting cells, the strength of the anchorage resisting this force, and the fluidity of the tissue to relieve the tension (Ray, 2015).
Several lines of evidence suggest that these different components of the system operate toward an equilibrium state where opposing forces come to balance. For instance, if the force produced by the cell constriction exceeds the resistance, the anchorage ruptures, releasing the tissue. When this occurs, as in wings mutant for the hypomorphic allele dpov1, the margin collapses and the cells in the middle of the wing blade constrict further than they would otherwise. Similarly, the range of phenotypes observed in the interactions between nwD and dpov1 suggest that if the level of Dp falls below a certain threshold, the anchorage ruptures after hinge contraction has started, resulting in intermediate phenotypes that are flattened and retain some taper, but have nevertheless retracted. These observations suggest that cells in the wing blade actively respond to the pattern of tensile force with significant consequences on wing shape (Ray, 2015).
The data also indicate that hinge cells respond to the tension in the blade and constrict accordingly. Morphometric analysis of the nw mutant shows that the narrowing and extension of the wing blade is accompanied by a isometric contraction of the hinge, such that in the adult wing, the hinge is significantly smaller than in wild-type. Similarly, in the various dp mutants this study had examined, any change in the wing shape is accompanied by a corresponding change in hinge size and shape. The implication from these observations is that in the absence of sufficient resistance, hinge cells continue to undergo constriction, resulting in a corresponding change in hinge shape. Indeed, in a computational model, whenever the anchorage is released by any significant amount, the hinge is also found to contract further than in the wild-type simulation (Ray, 2015).
Taken together, these observations suggest that the system operates toward an equilibrium point where the force generated by hinge contraction is balanced with the resistance coming from the distal anchorage and the deformation properties of the cell matrix. Initially, the force generated by hinge contraction is offset by cell division and cell rearrangement, but when the cell division ceases, resistance in the epithelium feeds back on the hinge and eventually blocks further constriction. At this point, the system comes to equilibrium and the tension along the cell junctions equalizes. Notably, this model is consistent with previous reports, which have shown that after the initial phase of hinge contraction is completed, equalization of junction tension initiates the shift toward the hexagonal packing geometry that is characteristic of late stage pupal wings. The attractiveness of this kind of model is that it ensures robustness of the shaping mechanism and avoids the complications of tears or buckles that might arise from stochastic perturbations occurring during development. The nature of these feedbacks and how the equilibrium is achieved remain a question for further study (Ray, 2015).
As orthologs of Dp and Nw have been found, and in all sequenced insect genomes and in the crustacean Daphnia, yhr results suggest that the Nw-Dp system may be a key target for evolution of appendage shape in Arthropods. Indeed, given the tremendous variety of wing shapes that are found in insects, it is tempting to speculate that some of this variation is achieved by modulation of the Nw-Dp system, resulting in changes in the force patterns in the developing wing. For instance, in a recent report, wing shape differences in males of Nasonia vitripennis and Nasonia giraulti were attributed to differences in transcriptional regulation of an unpaired-like gene that was proposed to regulate proliferation in the developing wing. Significantly, the expression of this gene in both species was confined to the margin at the distal tip of the wing and strikingly prefigures the radius of curvature of the adult wing. Given these results, it is plausible that the difference in wing shape in these two species may arise from differences in anchorage of the wing tip, with the difference in cell number arising as a secondary consequence as in the case of nw. Similarly, the evolution of the diverse, specialized wing shapes in butterflies and moths has been attributed to defined expression of margin specific genes that prefigure the adult wing shape. While little is known of the elaboration of this prepattern during pupal development, the scalloping of the adult wing margin observed in many species—reminiscent of notching in the Drosophila wing—may well arise from precise deployment of the Nw-Dp system. Exploration of how Dp is regulated in different species may shed light on how the variety of insect wing shapes has evolved (Ray, 2015).
In all metazoans, assembly of the aECM is dependent on proteins that contain a common protein motif, the Zona Pellucida (ZP) domain, which is thought to act as a polymerization module promoting the formation of homo and heterotypic filaments. Genetic studies have implicated ZP-domain proteins in a variety of morphogenetic processes that typically involve shaping or remodeling of the apical domain of the cells. In Drosophila, ZP-domain proteins shape the embryonic denticles and hairs and the wing trichomes that form on the apical surface of the cells, and in C. elegans, the Cuticlulins are ZP-domain proteins involved in the formation of the alae. Similarly, in flies and nematodes, aECM proteins have been implicated as anchors in the cellular morphogenesis of sensory neurons, with Drosophila NompA functioning to anchor neural dendrites to the cuticular structures in sensory organs while in the nematode, the aECM proteins DEX-1 and DYF-7 anchor the dendritic tips during cell body migration to shape the amphid sense organs. Among these examples, these findings represent a different paradigm for how aECM proteins can influence morphogenesis: rather than affecting the behavior of individual cells, and consequently the shape of the tissue, the Nw-Dp mechanism defines global force patterns across the tissue to which the individual cells respond to give rise to appendage shape (Ray, 2015).
In vertebrates, the most well studied ZP-domain proteins are the eponymous zona pellucida proteins that form the extracellular coat of mammalian ova and the α- and β-tectorins that are required for the formation of the tectorial membrane in the ear. However, despite their importance for fertility and hearing, respectively, these proteins do not affect morphogenesis of the tissue per se. On the other hand, in the human kidney, the ZP-domain protein hensin/DMPT1 regulates morphogenesis of α- and β-Intercalated cells in the collecting tubules, and results from at least one study have found that global deletion of hensin results in embryonic lethality, suggesting a more general role in epithelial differentiation. Thus, there is mounting evidence from both invertebrates and vertebrates that the aECM plays an important role in morphogenesis and further studies on aECM proteins will undoubtedly reveal other roles they play in development and disease (Ray, 2015).
Hedgehog (Hh) signalling is important in development, stem cell biology and disease. In a variety of tissues, Hh acts as a morphogen to regulate growth and cell fate specification. Several hypotheses have been proposed to explain morphogen movement, one of which is transport along filopodia-like protrusions called cytonemes. This study analysed the mechanism underlying Hh movement in the wing disc and the abdominal epidermis of Drosophila melanogaster. In both epithelia, cells were shown to generate cytonemes in regions of Hh signalling. These protrusions are actin-based and span several cell diameters. Various Hh signalling components localize to cytonemes, as well as to punctate structures that move along cytonemes and are probably exovesicles. In vivo imaging was used show that cytonemes are dynamic structures and that Hh gradient establishment correlates with cytoneme formation in space and time. Indeed, mutant conditions that affect cytoneme formation reduce both cytoneme length and Hh gradient length. The results suggest that cytoneme-mediated Hh transport is the mechanistic basis for Hh gradient formation (Bischoff, 2013).
The localization of several Hh signalling components at cytonemes has suggested a role for these structures in Hh signalling. This study characterize cytonemes in two Drosophila paradigms, the wing disc and the abdominal epidermis, and investigate their role in Hh gradient formation. Evidence is presented that cytonemes play an active role in gradient formation: the establishment of the Hh signalling gradient correlates dynamically in space and time with cytoneme formation in vivo; experimental shortening and lengthening of cytonemes affects the gradient accordingly; the analysis of ttv−/−,botv−/− mutant clones implicates cytonemes in Hh transport. Overall, the results support a model in which cytonemes of signal-producing cells are involved in long-range Hh transport (Bischoff, 2013).
In wing discs, however, both sending and receiving cells generate cytonemes raising the question of which role the cytonemes of receiving cells play. Expression of Ihog in A compartment cells leads to a depletion of Hh from the P compartment cells close to the A/P border, which suggests that A compartment cytonemes might actively engage in Hh reception. Hence, cytonemes of both sending and receiving cells might contribute to Hh transport. Interestingly, A compartment cytonemes are rare in histoblasts, suggesting that cytonemes of receiving cells play a minor role in the abdomen (Bischoff, 2013).
Ihog–RFP puncta were observed that associated with and moved along cytonemes. Frequently, such puncta were released from cytonemes. Puncta were also observed when labelling cytonemes with CD4–Tomato. This suggests that cytonemes might transport exovesicles that act as a vehicle for Hh or are the structure where exovesicles are being released. Accordingly, the knockdown of genes involved in exovesicle production/release has a significant effect on Hh gradient length, and Ihog can be detected in baso-lateral exovesicles at the ultrastructural level. However, the characterization of these exovesicles as well as their implication in Hh gradient formation requires further analysis. A role of exosomes in morphogen gradient formation has recently been suggested. Active Wnt proteins are secreted in exosomes in cultured cells and in the wing disc. In addition, vesicular release of SonicHh has been implicated in the determination of left–right asymmetry in vertebrates. Very recently, particles containing SonicHh and CDO (the vertebrate homologue of Ihog) that travel along filopodia-like extensions have been described in the chicken limb bud (Bischoff, 2013).
The mechanisms by which cytonemes could transport morphogens to their targets must ensure specificity and accuracy. One possibility is that cytonemes established contact between sending and receiving cells. Alternatively, cytonemes could act as a structure of morphogen release and uptake without cell–cell contacts involved. In vivo imaging showed that cytonemes are dynamic structures. Cytonemes might grow towards a receiving cell and then retract after a signalling event has taken place, or their dynamics could be determined intrinsically by the stability of their cytoskeleton. Moreover, not just cytoneme length but also their number could shape the gradient, as its brightest section coincides with the dense array of shorter cytonemes. This cytoneme-based model challenges the previous diffusion-based models (Bischoff, 2013).
Cytonemes have been described in a variety of signalling pathways. The Dpp receptor Thickveins is present in punctate structures moving along cytonemes. Air sac precursors extend cytonemes towards FGF-expressing cells. Tracheal cells were reported to have at least two types of cytoneme; one type that carries an FGF receptor, and another type that carries the Dpp receptor. This suggests that cytonemes are ligand specific. In the context of Notch signalling, filopodia mediate lateral inhibition between non-neighbouring cells of the pupal notum. Interestingly, the dynamic behaviour of these processes is crucial for signalling. Spitz/EGF is delivered through polarized actin protrusions to spatially bias the specification of a particular cell of the Drosophila leg. In another example, short cytonemes mediate the delivery of a juxtacrine Hh signal to maintain germline stem cells in the Drosophila ovary. This study has shown that cytonemes also play a pivotal role in long-range Hh signalling in wing disc cells, histoblasts and LECs. Therefore, it is believed that cytonemes are a general feature of signalling events of all epithelial cells (Bischoff, 2013).
While the membrane potential of cells has been shown to be patterned in some tissues, specific roles for membrane potential in regulating signalling pathways that function during development are still being established. In the Drosophila wing imaginal disc, Hedgehog (Hh) from posterior cells activates a signalling pathway in anterior cells near the boundary which is necessary for boundary maintenance. This study shows that membrane potential is patterned in the wing disc. Anterior cells near the boundary, where Hh signalling is most active, are more depolarized than posterior cells across the boundary. Elevated expression of the ENaC channel Ripped Pocket (Rpk), observed in these anterior cells, requires Hh. Antagonizing Rpk reduces depolarization and Hh signal transduction. Using genetic and optogenetic manipulations, in both the wing disc and the salivary gland, it was shown that membrane depolarization promotes membrane localization of Smoothened and augments Hh signalling, independently of Patched. Thus, membrane depolarization and Hh-dependent signalling mutually reinforce each other in cells immediately anterior to the compartment boundary (Emmons-Bell, 2021).
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Initially discovered for its role in regulating segment polarity in Drosophila, Hh signalling has since been implicated in a multitude of developmental processes. Among the best characterized is the signalling between two populations of cells that make up the Drosophila wing imaginal disc, the larval primordium of the adult wing and thorax. The wing disc consists of two compartments of lineage-restricted cells separated by a smooth boundary. Posterior (P) cells make the morphogen Hedgehog, which binds to its receptor Patched (Ptc), which is expressed exclusively in anterior (A) cells. Hh has a relatively short range either because of its limited diffusion, or because it is taken up by nearby target cells via filopodia-like protrusions known as cytonemes. Hh alleviates the repressive effect of Ptc on the seven-transmembrane protein Smoothened (Smo) in A cells near the boundary, initiating a signalling cascade that culminates in the stabilization of the activator form of the transcription factor Cubitus interruptus (Ci), and expression of target genes such as the long-range morphogen Dpp. In turn, Dpp regulates imaginal disc patterning and growth in both compartments (Emmons-Bell, 2021).
While the role of cell-cell interactions, diffusible morphogens and even mechanical forces have been studied in regulating the growth and patterning of the wing disc, relatively little attention has been paid to another cellular parameter, membrane potential or Vmem. Vmem is determined by the relative concentrations of different species of ions across the cell membrane, as well as the permeability of the membrane to each of these ions. These parameters are influenced by the abundance and permeability of ion channels, the activity of pumps, and gap junctions. While changes in Vmem have been studied most extensively in excitable cells, there is increasing evidence that the Vmem of all cells, including epithelial cells, can vary depending on cell-cycle status and differentiation status. Mutations in genes encoding ion channels in humans ('channelopathies') can result in congenital malformations. Similarly, experimental manipulation of ion channel permeability can cause developmental abnormalities in mice as well as in Drosophila. Only more recently has evidence emerged that Vmem can be patterned during normal development. Using fluorescent reporters of membrane potential, it has been shown that specific cells during Xenopus gastrulation and Drosophila oogenesis appear more depolarized than neighbouring cells. A recent study established that cells in the vertebrate limb mesenchyme become more depolarized as they differentiate into chondrocytes, and that this depolarization is essential for the expression of genes necessary for chondrocyte fate. However, in many of these cases, the relationship between changes in Vmem and specific pathways that regulate developmental patterning have not been established (Emmons-Bell, 2021).
This study investigated the patterning of Vmem during wing disc development and showed that the regulation of Vmem has an important role in regulating Hh signalling. The cells immediately anterior to the compartment boundary, a zone of active Hh signalling, are more depolarized than surrounding cells, and Hh signalling and depolarized Vmem mutually reinforce each other. This results in an abrupt change in Vmem at the compartment boundary (Emmons-Bell, 2021).
This study shows that Vmem is patterned in a spatiotemporal manner during development of the wing disc of Drosophila and that it regulates Hedgehog signalling at the compartment boundary. First, it was shown that cells immediately anterior to the compartment boundary are relatively more depolarized than cells elsewhere in the wing pouch. This region coincides with the A cells where Hh signalling is most active, as evidenced by upregulation of Ptc. Second, the expression of at least two regulators of Vmem, the ENaC channel Rpk and the alpha subunit of the Na+/K+ ATPase were shown to be expressed at higher levels in this same portion of the disc. Third, by altering Hh signalling, this study demonstrated that the expression of both Rpk and ATPα is increased in cells with increased Hh signalling. Fourth, by manipulating Hh signalling in the disc and using optogenetic methods, both in the salivary gland and wing disc, it was shown that membrane depolarization promotes Hh signalling as assessed by increased membrane localization of Smo, and expression of the target gene ptc. Thus, Hh-induced signalling and membrane depolarization appear to mutually reinforce each other and thus contribute to the mechanisms that maintain the segregation of A and P cells at the compartment boundary (Emmons-Bell, 2021).
Two regions of increased DiBAC fluorescence were observed in the wing imaginal disc. No obvious upregulation of Rpk and ATPα was observed in other discs, and therefore, these studies have focused on the region immediately anterior to the A-P compartment boundary in the wing disc. In the late L3 wing disc, a region of increased DiBAC fluorescence was observed in the A compartment in the vicinity of the D-V boundary. This corresponds to a 'zone of non-proliferating cells' (ZNC). Interestingly, the ZNC is different in the two compartments. In the A compartment, two rows of cells are arrested in G2 while in the P compartment, a single row of cells is arrested in G1. The observation of increased DiBAC fluorescence in the DV boundary of only the A compartment is consistent with previous reports that cells become increasingly depolarized as they traverse S-phase and enter G2. In contrast, cells in G1 are thought to be more hyperpolarized. Additionally, increased expression of the ENaC channel Rpk was observed in two rows of cells at the D-V boundary in the anterior compartment, indicating that increased expression of Rpk could contribute to the depolarization observed in those cells. It is noted, however, that the increased DiBAC fluorescence in these cells was not entirely eliminated by exposing discs to amiloride, indicating that other factors are also likely to contribute (Emmons-Bell, 2021).
These data are consistent with a model where membrane depolarization and Hh-induced signalling mutually reinforce each other in the cells immediately anterior to the compartment boundary. Both membrane depolarization and the presence of Hh seem necessary for normal levels of activation of the Hh signalling pathway in this region; neither alone is sufficient. First, it was shown that Hh signalling promotes membrane depolarization. It was also shown that the expression of Rpk just anterior to the A-P compartment boundary is dependent upon Hh signalling. Elevated Rpk expression is not observed when a hhts allele is shifted to the restrictive temperature, and cells become more depolarized when Hh signalling is constitutively activated through expression of the ci3m allele. Previously published microarray data suggest that Rpk as well as another ENaC family channel Ppk29 are both enriched in cells that also express ptc. However, there is no antibody to assess Ppk29 expression currently. The sensitivity of the depolarization to amiloride indicates that these and other ENaC channels make an important contribution to the membrane depolarization (Emmons-Bell, 2021).
Second, this study has shown that the depolarization increases Hh signalling. The early stages of Hh signalling are still incompletely understood. Hh is thought to bind to a complex of proteins that includes Ptc together with either Ihog or Boi. This alleviates an inhibitory effect on Smo, possibly by enabling its access to specific membrane sterols. Interestingly, it has recently been proposed that Ptc might function in its inhibitory capacity by a chemiosmotic mechanism where it functions as a Na+ channel. An early outcome of Smo activation is its localization to the membrane where its C-terminal tail becomes phosphorylated and its ubiquitylation and internalization are prevented. By manipulating channel expression in the wing disc, and by optogenetic experiments in both the salivary gland and wing disc, this study has shown that membrane depolarization can promote Hh signalling as assessed by increased Smo membrane localization and increased expression of the target gene ptc. The time course of Smo activation is relatively rapid (over minutes) and is therefore unlikely to require new transcription and translation. In the P compartment, membrane Smo levels are elevated likely because of the complete absence of Ptc, and some downstream components of the Hh signalling pathway are known to be activated. However, since Ci is not expressed in P cells, target gene expression is not induced. In the cells just anterior to the boundary, the partial inhibition of Ptc by Hh together with membrane depolarization seem to combine to achieve similar levels of Smo membrane localization. More anteriorly, the absence of this mutually reinforcing mechanism appears to result in Smo internalization (Emmons-Bell, 2021).
The experiments do not point to a single mechanism by which depolarization promotes Hh signalling. It is possible that depolarization results in increased Ca2+ levels by opening Ca2+channels at the plasma membrane or by promoting release from intracellular sources (e.g. the ER or mitochondria). Indeed, there is evidence that Ca2+ entry into the primary cilium promotes Hh signalling, and recent work shows that targets of Sonic Hedgehog (Shh) signalling during mammalian development is augmented by Ca2+ influx. A second possibility is that membrane depolarization could, by a variety of mechanisms, activate the kinases that phosphorylate the C-terminal tail of Smo and maintain it at the plasma membrane in an activated state. Depolarization could also impact electrostatic interactions at the membrane that make the localization of Smo at the membrane more favourable. Since Rpk and ATPα are expressed at higher levels in the cells that receive Hh, which have been postulated to make synapse-like projections with cells that produce Hh, it is conceivable that these channels could modulate synapse function. Additionally, while this work was under review, it has been reported that reducing glycolysis depletes ATP levels and results in depolarization in the wing imaginal disc, reducing the uptake of Hh pathway inhibitors and stabilizing Smo at the cell membrane (Spannl, 2020). Importantly, all these mechanisms are not mutually exclusive and their roles in Hh signalling are avenues for future research (Emmons-Bell, 2021).
It is now generally accepted that both cell-cell signalling and mechanical forces have important roles in cell fate specification and morphogenesis. This work adds to a growing body of literature suggesting that changes in Vmem, a relatively understudied parameter, may also have important roles in development. Integrating such biophysical inputs with information about gene expression and gene regulation will lead to a more holistic understanding of development and morphogenesis (Emmons-Bell, 2021).
This study investigated the roles of components of neuronal synapses for development of the Drosophila air sac primordium (ASP). The ASP, an epithelial tube, extends specialized signaling filopodia called cytonemes that take up signals such as Dpp (Decapentaplegic, a homolog of the vertebrate bone morphogenetic protein) from the wing imaginal disc. Dpp signaling in the ASP was compromised if disc cells lacked Synaptobrevin and Synaptotagmin-1 (which function in vesicle transport at neuronal synapses), the glutamate transporter, and a voltage-gated calcium channel, or if ASP cells lacked Synaptotagmin-4 or the glutamate receptor GluRII. Transient elevations of intracellular calcium in ASP cytonemes correlate with signaling activity. Calcium transients in ASP cells depend on GluRII, are activated by l-glutamate and by stimulation of an optogenetic ion channel expressed in the wing disc, and are inhibited by EGTA and by the GluR inhibitor NASPM (1-naphthylacetyl spermine trihydrochloride). Activation of GluRII is essential but not sufficient for signaling. Cytoneme-mediated signaling is glutamatergic (Huang, 2019).
Drosophila wings mainly consist of two cell types, vein and intervein cells. Acquisition of either fate depends on specific expression of genes that are controlled by several signaling pathways. The nuclear mechanisms that translate signaling into regulation of gene expression are not completely understood, but they involve chromatin factors from the Trithorax (TrxG) and Enhancers of Trithorax and Polycomb (ETP) families. One of these is the ETP Corto that participates in intervein fate through interaction with the Drosophila EGF Receptor -- MAP kinase ERK pathway. Precise mechanisms and molecular targets of Corto in this process are not known. This study shows that Corto interacts with the Elongin transcription elongation complex. This complex, that consists of three subunits (Elongin A, B, C), increases RNA polymerase II elongation rate in vitro by suppressing transient pausing. Analysis of phenotypes induced by EloA, B, or C deregulation as well as genetic interactions suggest that the Elongin complex might participate in vein vs intervein specification, and antagonizes corto as well as several TrxG genes in this process. Chromatin immunoprecipitation experiments indicate that Elongin C and Corto bind the vein-promoting gene rhomboid in wing imaginal discs. It is proposed that Corto and the Elongin complex participate together in vein vs intervein fate, possibly through tissue-specific transcriptional regulation of rhomboid (Rougeot, 2013).
In Drosophila as in mammals, the three Elongin proteins Elo A, B, and C are mainly nuclear and interact two by two. EloC/B and EloC/A interactions may be direct, as they were observed without cross-linking treatment. By contrast, EloA/B interaction is more labile and may thus be indirect. It is possible that Drosophila EloC mediates the interaction between EloA and EloB, as previously shown in mammals. This study also showed that the ETP Corto interacts with all three Elo proteins, suggesting that Corto interacts with the Elongin complex. Hence, Corto and the Elongin Complex could share transcriptional targets. Several studies have shown that EloC binds its partners through a degenerate BC box motif, defined as (L,M)XXX(C,S)XXX(Í). Two putative BC boxes (aa 357-365 and aa 542-550) are present in the C-terminal part of Corto. However, deletion of these sequences did not impair co-immunoprecipitation between Corto and EloC, suggesting that these two proteins interact through another unidentified sequence (Rougeot, 2013).
This study presents the first characterization of lines allowing deregulation of EloB or EloC expression. EloB or EloC loss-of-function mutations induce early lethality (before the third larval instar), demonstrating that EloB and EloC, like EloA (Gerber, 2004), are essential proteins. Clonal and tissue-specific analyses of EloC mutant cells reveal that EloC is critically required all through wing development. By contrast, RNAi-mediated EloA down-regulation only induced lethality during the pupal stage (Gerber, 2004), indicating either a less efficient reduction of EloA mRNA or a longer perdurance of maternal EloA. Alternatively, requirement of EloB and EloC in other complexes, such as an E3 ubiquitin ligase complex, might explain this difference (Rougeot, 2013).
EloB/C loss-of-function as well as EloA over-expression induced wing phenotypes, mostly vein phenotypes. Interestingly, these loss-of-function and over-expression phenotypes are opposite (i.e truncated L5 vein for loss-of-function, ectopic veins for over-expression). Furthermore, whereas EloA over-expression induced ectopic veins, no phenotype was observed when over-expressing EloB and EloC. This result suggests that the amount of catalytic subunit EloA might be critical for Elongin complex function. In mammals, EloA is indeed the limiting component of the Elongin complex, EloB and EloC being in large excess (100 to 1000-fold more abundant than EloA). Curiously, a previous study reported that mitotic clones for a deficiency that uncovers EloA, produced ectopic wing veins. As this deletion uncovers more than 10 genes that may influence vein formation, the hypothesis is favored, in agreement with all data presented above, that EloA loss- of-function leads to loss of vein tissue. Alternatively, EloB and EloC, which also belong to ubiquitin ligase complexes, might modulate vein vs intervein cell fate in this context (Rougeot, 2013).
Altogether, the observations suggest that the Elongin A, B, C subunits promote vein cell identity. On the opposite, Corto maintains intervein cell identity, possibly via interaction with TrxG complexes. As Corto and EloC co-localize at a few sites on polytene chromosomes, they might have common transcriptional targets. A balance between Corto and the Elongin complex might fine-tune transcription of such genes (Rougeot, 2013).
In corto mutants, previous study has shown that ectopic veins perfectly match with ectopic expression of rho, the first vein-promoting gene to be expressed (Mouchel-Vielh, 2011). As Elo gene mutations counteract corto mutations during formation of ectopic veins, it is proposed that rho could be a common target of Corto and the Elongin complex in intervein cells. In agreement with this hypothesis, immunoprecipitation using chromatin from late third instar wing imaginal discs, that can be assimilated to chromatin of intervein cells, revealed the presence of both Corto and EloC on rho. Two independent genome-wide studies on whole embryos and embryonic S2 cells have shown that poised RNA-PolII binds the rho promoter, suggesting that rho expression is controlled by 'pause and release' of the transcriptional machinery. Interestingly, this studu found that Corto is slightly enriched just after the rho TSS, a position usually occupied by paused RNA-PolII. Corto shares many sites on polytene chromosomes with paused RNA-PolII-S5p, suggesting that it is involved in transcriptional pausing. On the other hand, this study found that EloC co-localizes with H3K36me3, that characterizes transcriptional elongation, and the Elongin complex was shown to suppress transient RNA-PolII pausing. Hence, in future intervein cells, Corto and the Elongin complex could apply opposite forces on the transcriptional machinery at the rho promoter. Corto would block rho transcription whereas the Elongin complex would be ready to accompany rho elongation if release should occur. In future vein cells on the other hand, the Elongin complex could actively participate in rho transcriptional elongation, since loss of function mutants for EloB and EloC exhibit loss of vein tissue. In these cells, rho expression would be independent of Corto, since corto mutants never present truncated veins (Rougeot, 2013).
The results suggest that the Elongin complex might participate in determination of vein and intervein cell identity during wing development. It is proposed that this complex might interact with the ETP Corto at certain target genes and fine-tune their transcription in a cell-type specific manner. One of these targets could be the vein-promoting gene rho. In intervein cells, binding of Corto to the Elongin complex could prevent transcription of rho. Corto could also recruit other chromatin factors, such as the BAP chromatin-remodeling complex that was previously shown to inhibit rho expression in intervein cells. By contrast, in vein cells, the Elongin complex could participate in rho transcriptional elongation independently of Corto (Rougeot, 2013).
Vein patterning in the Drosophila wing provides a powerful tool to study regulation of various signaling pathways. This study shows that the ADAMTS extracellular protease AdamTS-B (CG4096) is expressed in the embryonic wing imaginal disc precursor cells and the wing imaginal disc, and functions to inhibit wing vein formation. Knock-down of AdamTS-B displayed posterior crossveins (PCVs) with either extra branches or deltas, or wider PCVs, and a wandering distal tip of the L5 longitudinal vein. Conversely, over-expression of AdamTS-B resulted in a complete absence of the PCV, an incomplete anterior crossvein (ACV), and missing distal end of the L5 longitudinal vein. It is concluded that AdamTS-B inhibits wing vein formation through negative regulation of signaling pathways, possibly BMP as well as Egfr, displaying the complexity of roles for this family of extracellular proteases (Pham, 2018).
Drosophila Phosphatase of Regenerating Liver-1 (PRL-1) is the only homolog of the mammalian PRLs with which it shares high sequence and structural similarities. Whilst PRLs are most notable for their high expression in malignant cancers and related promotion of cancer progression, the specific biological functions of the PRLs remain largely elusive. Using a gain-of-function approach, it was found that PRL-1 functions during wing vein development in Drosophila melanogaster. Overexpression of Drosophila PRL-1 caused dose-dependent wing vein proliferation. Genetic screening of the main TGF-;beta; signaling factors, Mad and Smox, showed that the RNAi-mediated knockdown of Mad could alleviate the extra vein phenotype caused by overexpressed PRL-1 and lead to loss of the posterior section of longitudinal veins. However, knockdown of Smox resulted in an identical phenotype with or without the overexpression of Drosophila PRL-1. Clonal analyses revealed that overexpression of PRL-1 led to decreased expressions of activated phospho-Mad protein, as measured by immunostaining. Real-time PCR showed that the transcriptional levels of Smox were significantly increased upon overexpression of the Drosophila PRL-1 in wing discs, with a dose dependent effect. This study proposed that the main function of Drosophila PRL-1 in wing development is to affect the phospho-Mad levels and Smox transcriptional levels, therefore influencing the competitive balance for Medea between Mad and Smox. This study demonstrates the novel role for Drosophila PRL-1 in regulating TGF-β signaling to influence wing vein formation which may also provide insight into the understanding of the relationship between PRLs and TGF-β signaling in mammals (Zheng, 2022).
How mechanisms of pattern formation evolve has remained a central research theme in the field of evolutionary and developmental biology. The mechanism of wing vein differentiation in Drosophila is a classic text-book example of pattern formation using a system of positional-information, yet very little is known about how species with a different number of veins pattern their wings, and how insect venation patterns evolved. This study examine the expression pattern of genes previously implicated in vein differentiation in Drosophila in two butterfly species with more complex venation Bicyclus anynana and Pieris canidia. The function of some of these genes was tested in B. anynana. Both conserved as well as new domains of decapentaplegic, engrailed, invected, spalt, optix, wingless, armadillo, blistered, and rhomboid gene expression in butterflies were identified, and a proposal is made about how the simplified venation in Drosophila might have evolved via loss of decapentaplegic, spalt and optix gene expression domains, along with silencing of vein inducing programs at Spalt-expression boundaries, and changes in gene expression of vein maintenance genes (Banerjee, 2020).
In vertebrates, TFEB (transcription factor EB) and MITF (microphthalmia-associated transcription factor) family of basic Helix-Loop-Helix (bHLH) transcription factors regulate both lysosomal function and organ development. However, it is not clear whether these 2 processes are interconnected. This study shows that Mitf, the single TFEB and MITF ortholog in Drosophila, controls expression of vacuolar-type H+-ATPase pump (V-ATPase) subunits. Remarkably, it was also found that expression of Vha16-1 and Vha13, encoding 2 key components of V-ATPase, is patterned in the wing imaginal disc. In particular, Vha16-1 expression follows differentiation of proneural regions of the disc. These regions, that will form sensory organs in the adult, appear to possess a distinctive endo-lysosomal compartment and Notch (N) localization. Modulation of Mitf activity in the disc in vivo alters endo-lysosomal function and disrupts proneural patterning. Similar to these findings in Drosophila, in human breast epithelial cells, it was observed that the impairment of the Vha16-1 human ortholog ATP6V0C changes the size and function of the endo-lysosomal compartment and depletion of TFEB reduces ligand-independent N signaling activity. These data suggest that lysosomal-associated functions regulated by the TFEB-V-ATPase axis might play a conserved role in shaping cell fate (Tognon, 2016).
Regulation of cell growth and cell division plays fundamental roles in tissue morphogenesis. However, the mechanisms of regulating tissue elongation through cell growth and cell division are still not well understood. The wing imaginal disc of Drosophila provides a model system that has been widely used to study tissue morphogenesis. This study used a recently developed two-dimensional cellular model to study the mechanisms of regulating tissue elongation in Drosophila wing. The effects of directional cues on tissue elongation were simulated. Also the role of reduced cell size was computationally analyzed. The simulation results indicate that oriented cell divisions, oriented mechanical forces, and reduced cell size can all mediate tissue elongation, but they function differently. Oriented cell divisions and oriented mechanical forces were shown to act as directional cues during tissue elongation. Between these two directional cues, oriented mechanical forces have a stronger influence than oriented cell divisions. In addition, the novel hypothesis is raised that reduced cell size may significantly promote tissue elongation. It was found that reduced cell size alone cannot drive tissue elongation. However, when combined with directional cues, such as oriented cell divisions or oriented mechanical forces, reduced cell size can significantly enhance tissue elongation in Drosophila wing. Furthermore, the simulation results suggest that reduced cell size has a short-term effect on cell topology by decreasing the frequency of hexagonal cells, which is consistent with experimental observations. Thse simulation results suggest that cell divisions without cell growth play essential roles in tissue elongation (Li, 2014).
Animals have body parts made of similar cell types located at different axial positions, such as limbs. The identity and distinct morphology of each structure is often specified by the activity of different 'master regulator' transcription factors. Although similarities in gene expression have been observed between body parts made of similar cell types, how regulatory information in the genome is differentially utilized to create morphologically diverse structures in development is not known. This study used genome-wide open chromatin profiling to show that among the Drosophila appendages, the same DNA regulatory modules are accessible throughout the genome at a given stage of development, except at the loci encoding the master regulators themselves. In addition, open chromatin profiles change over developmental time, and these changes are coordinated between different appendages. It is proposed that master regulators create morphologically distinct structures by differentially influencing the function of the same set of DNA regulatory modules (McKay, 2013).
This paper addresses a long-standing question in developmental biology:
how does a single genome give rise to a diversity of structures?
The results indicate that the combination of transcription factors
expressed in each thoracic appendage acts upon a shared set of
enhancers to create different morphological outputs, rather than
operating on a set of enhancers that is specific to each tissue. This conclusion is based upon the surprising observation
that the open chromatin profiles of the developing appendages
are nearly identical at a given developmental stage.
Therefore, rather than each master regulator operating on a set
of enhancers that is specific to each tissue, the master regulators
instead have access to the same set of enhancers in different tissues,
which they differentially regulate. It was also found that tissues
composed of similar combinations of cell types have very similar
open chromatin profiles, suggesting that a limited number of
distinct open chromatin profiles may exist at a given stage of
development, dependent on cell-type identity (McKay, 2013).
Different tissues were dissected from developing flies to compare
their open chromatin profiles. These tissues are composed of
different cell types, each with its own gene expression profile.
Formaldehyde-assisted isolation of regulatory elements (FAIRE) data thus represent the average signal across all cells
present in a sample. However, data from embryos and imaginal
discs indicate that FAIRE is a very sensitive detector of functional
DNA regulatory elements. For example, the Dll01 enhancer is
active in 2–4 neurons of the leg imaginal disc; yet, the FAIRE
signal at Dll01 is as strong as the Dll04 enhancer, which is active
in hundreds of cells of the wing pouch. Thus, FAIRE may detect nearly all of the DNA regulatory elements that are in use among the
cells of an imaginal disc. This study does not rule out the existence of DNA regulatory elements that are not marked by open chromatin or are otherwise not
detected by FAIRE (McKay, 2013).
Despite this sensitivity, the approach of this study
does not identify which cells within the
tissue have a particular open chromatin
profile. For a given locus, it is possible that all cells in the tissue
share a single open chromatin profile or that the FAIRE signal
originates from only a subset of cells in which a given enhancer
is active. Comparisons between eye-antennal discs, larval
CNS, and thoracic discs suggest that the latter
scenario is most likely, with open chromatin profiles among cells within a tissue shared by cells with similar identities at a given developmental stage (McKay, 2013).
The observation that halteres and wings share open chromatin
profiles demonstrates that Hox proteins like Ubx can differentially
interpret the DNA sequence within the same subset of enhancers
to modify one structure into another. This is consistent
with the idea that morphological differences are largely dependent
on the precise location, duration, and magnitude of expression
of similar genes, and it is further supported by the similarity in gene
expression profiles observed between Drosophila appendages and observed between vertebrate
limbs. However, that such dramatic differences
in morphology could be achieved by using the same
subset of DNA regulatory modules in different tissues genome-wide
was not known. The current findings provide a molecular framework
to support the hypothesis that Hox factors function as
'versatile generalists,' rather than stable binary switches. The similarity in open chromatin profiles between wings and legs suggests that this framework also extends to
other classes of master regulators beyond the Hox genes. It is also noted that, like the Drosophila appendages, vertebrate
limbs are composed of similar combinations of cell types that
differ in their pattern of organization. Moreover, the Drosophila
appendage master regulators share a common evolutionary
origin with the master regulators of vertebrate limb development, suggesting that the concept of shared open chromatin profiles may also apply to human development (McKay, 2013).
The data suggest that open chromatin profiles vary both over
time for a given lineage and between cell types at a given stage
of development. Given the dramatic differences in the FAIRE landscape
observed during embryogenesis and between the CNS and
the appendage imaginal discs during larval stages, it appears as
though the alteration of the chromatin landscape is especially
important for specifying different cell types from a single genome.
After cell-type specification, open chromatin profiles in the
appendages continued to change as they proceeded toward terminal
differentiation, suggesting that stage-specific functions
require significant opening of new sites or the closing of existing
sites. These findings contrast with those investigating hormone-induced
changes in chromatin accessibility,
in which the majority of open chromatin sites did not change after
hormone treatment, including sites of de novo hormone-receptor
binding. Thus, it may be that genome-wide remodeling of chromatin
accessibility is reserved for the longer timescales and eventual
permanence of developmental processes rather than the
shorter timescales and transience of environmental responses (McKay, 2013).
Different combinations of 'master regulator' transcription factors,
often termed selector genes, are expressed in the developing
appendages. Selectors are thought to specify the identity
of distinct regions of developing animals by regulating the
expression of transcription factors, signaling pathway components,
and other genes that act as effectors of identity.
One property attributed to selectors to
explain their unique power to specify identity during development
is the ability to act as pioneer transcription factors. In such models, selectors
are the first factors to bind target genes; once bound, selectors
then create a permissive chromatin environment for other transcription
factors to bind. The finding that the same set of
enhancers are accessible for use in all three appendages, with
the exception of the enhancers that control expression of the
selector genes themselves and other primary determinants of
appendage identity, suggests that the selectors expressed in
each appendage do not absolutely control the chromatin
accessibility profile; otherwise, the haltere chromatin profile (for example) would differ from that of the wing because of the expression of Ubx (McKay, 2013).
What then determines the appendage open chromatin profiles?
Because open chromatin is likely a consequence of transcription
factor binding, two nonexclusive models are possible.
First, different combinations of transcription factors could
specify the same open chromatin profiles. In this scenario,
each appendage's selectors would bind to the same enhancers
across the genome. For example, the wing selector Vg, with its
DNA binding partner Sd, would bind the same enhancers in
the wing as Dll and Sp1 bind in the leg. In the second model, transcription
factors other than the selectors could specify the
appendage open chromatin profiles. Selector genes are a small
fraction of the total number of transcription factors expressed in
the appendages. Many of the non-selector transcription
factors are expressed at similar levels in each appendage,
and thermodynamic models would predict them to bind the
same enhancers. This model could also help to
explain how the appendage open chromatin profiles coordinately
change over developmental time despite the steady
expression of the appendage selector genes during this same
period. It is possible that stage-specific transcription factors
determine which enhancers are accessible at a given stage of
development. This would help to explain the temporal specificity
of target genes observed for selectors such as Ubx. Recent work supports the role of hormone-dependent
transcription factors in specifying the temporal identity
of target genes in the developing appendages (Mou,
2012). Further experiments, including ChIP of the selectors
from each of the appendages, will be required to determine the
extent to which either of these models is correct (McKay, 2013).
Binding of Ubx results in differential activity of enhancers
in the haltere imaginal disc relative to the wing, despite
equivalent accessibility of the enhancers in both discs, indicating
that master regulators control morphogenesis by differentially
regulating the activity of the same set of enhancers. It is likely
that functional specificity of enhancers is achieved through
multiple mechanisms. These include differential recruitment of
coactivators and corepressors, modulation of binding specificity
through interactions with cofactors, differential
utilization of binding sites within a single enhancer, or regulation of binding dynamics through an altered
chromatin context. This last mechanism
would allow for epigenetic modifications early in development
to affect subsequent gene regulatory events. For example, the
activity of Ubx enhancers in the early embryo may
control recruitment of Trithorax or Polycomb complexes to the
PREs within the Ubx locus, which then maintain Ubx in the ON
or OFF state at subsequent stages of development. Consistent with this model,
Ubx enhancers active in the early embryo are only accessible
in the 2-4 hr time point, whereas the accessibility of Ubx PREs
varies little across developmental time or between tissues at a
given developmental stage (McKay, 2013).
The current results also have implications for the evolution of morphological
diversity. Halteres and wings are considered to have a
common evolutionary origin, but the relationship between insect
wings and legs is unresolved. The observation that wings and legs share open
chromatin profiles supports the hypothesis that wings and legs
also share a common evolutionary origin in flies. Because legs
appear in the fossil record before wings, the similarity in their
open chromatin profiles suggests that the existing leg cis-regulatory
network was co-opted for use in creation of dorsal
appendages during insect evolution (McKay, 2013).
Changes in gene expression during animal development are largely responsible for the evolution of morphological diversity. However, the genetic and molecular mechanisms responsible for the origins of new gene-expression domains have been difficult to elucidate. This study sought to identify molecular events underlying the origins of three novel features of wingless (wg) gene expression that are associated with distinct pigmentation patterns in Drosophila guttifera. The activity of cis-regulatory sequences (enhancers) across the wg locus in D. guttifera and Drosophila melanogaster were compared, and strong functional conservation was found among the enhancers that control similar patterns of wg expression in larval imaginal discs that are essential for appendage development. For pupal tissues, however, three novel wg enhancer activities were found in D. guttifera associated with novel domains of wg expression, including two enhancers located surprisingly far away in an intron of the distant Wnt10 gene. Detailed analysis of one enhancer (the vein-tip enhancer) revealed that it overlapped with a region controlling wg expression in wing crossveins (crossvein enhancer) in D. guttifera and other species. These results indicate that one novel domain of wg expression in D. guttifera wings evolved by co-opting pre-existing regulatory sequences governing gene activity in the developing wing. It is suggested that the modification of existing enhancers is a common path to the evolution of new gene-expression domains and enhancers (Koshikawa, 2015).
A large body of comparative studies has shown that changes in the spatiotemporal expression of toolkit genes and the target genes they regulate correlate with the evolution of morphological traits. In a considerable number of instances, these spatiotemporal changes in gene expression have been demonstrated to involve the modification of enhancers. However, there are relatively few cases in which the origins of new enhancers have been elucidated, and none involving regulatory genes themselves (Koshikawa, 2015).
This study has shown that three novel domains of wg expression in D. guttifera are governed by three novel enhancers, respectively. The evolution of wg cis-regulatory sequences within the D. guttifera lineage played a role in the gain of each enhancer activity, and the evolution of trans-acting regulatory factors was also necessary for the activity of two elements (gutCS and gutTS). Detailed analysis of the D. guttifera vein-tip enhancer revealed that it evolved within another conserved enhancer, whereas two other enhancers (the campaniform sensilla and thoracic stripe enhancers) arose within in an intron of the distant Wnt10 locus. These results bear on the understanding of the mechanisms underlying the evolution of new enhancers and domains of gene expression (Koshikawa, 2015).
The D. guttifera vein-tip enhancer activity was localized within a 756-bp DNA segment that was also active in the developing pupal crossveins. This DNA segment is orthologous to segments of DNA in D. melanogaster and D. deflecta that were only active in the crossveins. The segments are all collinear, and contain numerous blocks of identical sequence, which suggests that the vein-tip enhancer activity evolved within the pre-existing crossvein enhancer (Koshikawa, 2015).
One explanation for the presence of two activities in this one fragment is that they share functional sites: that is, binding sites for common transcription factors. Because both activities appear in the pupal wing, it is likely that they use common tissue-specific (wing) and temporal (pupal) inputs. The evolution of a new activity in the vein tips could have arisen through the addition of DNA-binding sites for transcription factors that were already present active in cells at vein tips. In this scenario, the novel enhancer activity would have resulted from the evolutionary co-option of an existing enhancer (Koshikawa, 2015).
There is precedent for multifunctional enhancers and for this mechanism of co-option. For example, one enhancer of the D. melanogaster even-skipped gene governs two domains of gene expression that are controlled by shared inputs. In addition, it has been demonstrated that a novel optic lobe enhancer of the Drosophila santomea Neprilysin-1 gene arose via co-option of an existing enhancer. Moreover, it was shown that co-option had occurred in just a few mutational steps. The co-option of existing elements is an attractive explanation for the evolution of novel enhancers because it requires a relatively short mutational path (Koshikawa, 2015).
One surprising property of enhancers is their ability to control gene transcription at promoters located at considerable linear distances away in the genome. For example, the enhancer that drives Sonic hedgehog (Shh) expression in the developing amniote limb bud is located in the intron of another gene ~1 Mb from the Shh locus. A growing body of evidence indicates that long segments of DNA are looped out in accommodating long-range enhancer-promoter interactions. The ability of enhancers to act over such long ranges suggests that new enhancers could evolve at considerable distances from the promoters that they regulate (Koshikawa, 2015).
This study identified two enhancers in an intron of the D. guttifera Wnt10 gene that control transcription of the wg gene from a distance of ~70 kb, and separated by the Wnt6 locus. The data suggest that the gutTS enhancer preferentially regulates wg transcription and not Wnt10 or Wnt6 transcription, although the authors cannot explain this preference. The origins of the gutCS and gutTS enhancers are not as clear as the vein-tip enhancer. No pupal enhancer activity was detected in the orthologous DNA segments of D. melanogaster, there was no evidence of enhancer co-option. Nor were any obvious insertions found in these DNA segments such as a transposon. Nevertheless, the discovery of these novel, distant elements reflects the functional flexibility of cis-regulatory elements and their contribution to the evolution of gene regulation and morphological diversity (Koshikawa, 2015).
Proteasome-dependent and autophagy-mediated degradation of eukaryotic cellular proteins represent the two major proteostatic mechanisms that are critically implicated in a number of signaling pathways and cellular processes. Deregulation of functions engaged in protein elimination frequently leads to development of morbid states and diseases. In this context, and through the utilization of GAL4/UAS genetic tool, this study examined the in vivo contribution of proteasome and autophagy systems in Drosophila eye and wing morphogenesis. By exploiting the ability of GAL4-ninaE. GMR and P{GawB}Bx(MS1096) genetic drivers to be strongly and preferentially expressed in the eye and wing discs, respectively, this study proved that proteasomal integrity and ubiquitination proficiency essentially control fly's eye and wing development. Indeed, subunit- and regulator-specific patterns of severe organ dysmorphia were obtained after the RNAi-induced downregulation of critical proteasome components (Rpn1, Rpn2, alpha5, beta5 and beta6) or distinct protein-ubiquitin conjugators (UbcD6, but not UbcD1 and UbcD4). Proteasome deficient eyes presented with either rough phenotypes or strongly dysmorphic shapes, while transgenic mutant wings were severely folded and carried blistered structures together with loss of vein differentiation. Moreover, transgenic fly eyes overexpressing the UBP2-yeast deubiquitinase enzyme were characterized by an eyeless-like phenotype. Therefore, the proteasome/ubiquitin proteolytic activities are undoubtedly required for the normal course of eye and wing development. In contrast, the RNAi-mediated downregulation of critical Atg (1, 4, 7, 9 and 18) autophagic proteins revealed their non-essential, or redundant, functional roles in Drosophila eye and wing formation under physiological growth conditions, since their reduced expression levels could only marginally disturb wing's, but not eye's, morphogenetic organization and architecture. However, Atg9 proved indispensable for the maintenance of structural integrity of adult wings in aged flies. In all, these findings clearly demonstrate the gene-specific fundamental contribution of proteasome, but not autophagy, in invertebrate eye and wing organ development (Velentzas, 2013).
Viable yet damaged cells can accumulate during development and
aging. Although eliminating those cells may benefit organ
function, identification of this less fit cell population remains
challenging. Previously, a molecular mechanism, based on 'fitness
fingerprints' displayed on cell membranes, was identifed that allows direct fitness comparison among cells in Drosophila. This study reports the physiological consequences of efficient cell selection for the whole organism. The study found that fitness-based cell culling is naturally used to maintain tissue
health, delay aging, and extend lifespan in Drosophila.
A gene, ahuizotl (azot), was identified that ensures the elimination of less fit cells. Lack of azot increases morphological malformations and susceptibility to random
mutations and accelerates tissue degeneration. On the contrary,
improving the efficiency of cell selection is beneficial for
tissue health and extends lifespan (Merino, 2015).
Individual cells can suffer insults that affect their normal functioning, a situation often aggravated by exposure to external damaging agents. A fraction of damaged cells will critically lose their ability to live, but a different subset of cells may be more difficult to identify and eliminate: viable but suboptimal cells that, if unnoticed, may adversely affect the whole organism (Merino, 2015).
What is the evidence that viable but damaged cells accumulate within tissues? The somatic mutation theory of aging proposes that over time cells suffer insults that affect their fitness, for example, diminishing their proliferation and growth rates, or forming deficient structures and connections. This creates increasingly heterogeneous and dysfunctional cell populations disturbing tissue and organ function. Once organ function falls below a critical threshold, the individual dies. The theory is supported by the experimental finding that clonal mosaicism occurs at unexpectedly high frequency in human tissues as a function of time, not only in adults an embryos (Merino, 2015).
Does the high prevalence of mosaicism in our tissues mean that it is impossible to recognize and eliminate cells with subtle mutations and that suboptimal cells are bound to accumulate within organs? Or, on the contrary, can animal bodies identify and get rid of unfit viable cells (Merino, 2015)?
One indirect mode through which suboptimal cells could be eliminated is proposed by the 'trophic theory,' which suggested that Darwinian-like competition among cells for limiting amounts of surv ead to removal of less fit cells. However, it is apparent from recent work that trophic theories are not sufficient to explain fitness-based cell selection, because there are direct mechanisms that allow cells to exchange 'cell-fitness' information at the local multicellular level (Merino, 2015).
In Drosophila, cells can compare their fitness using different isoforms of the transmembrane protein Flower. The 'fitness fingerprints' are therefore defined as combinations of Flower isoforms present at the cell membrane that reveal optimal or reduced fitness. The isoforms that indicate reduced fitness have been called FlowerLose isoforms, because they are expressed in cells marked to be eliminated by apoptosis called 'Loser cells.' However, the presence of FlowerLose isoforms at the cell membrane of a particular cell does not imply that the cell will be culled, because at least two other parameters are taken into account: (1) the levels of FlowerLose isoforms in neighboring cells: if neighboring cells have similar levels of Lose isoforms, no cell will be killed; (2) the levels of a secreted protein called Sparc, the homolog of the Sparc/Osteonectin protein family, which counteracts the action of the Lose isoforms (Merino, 2015 and references therein).
Remarkably, the levels of Flower isoforms and Sparc can be altered by various insults in several cell types, including: (1) the appearance of slowly proliferating cells due to partial loss of ribosomal proteins, a phenomenon known as cell competition; (2) the interaction between cells with slightly higher levels of d-Myc and normal cells, a process termed supercompetition; (3) mutations in signal transduction pathways like Dpp signaling; or (4) viable neurons forming part of incomplete ommatidia. Intriguingly, the role of Flower isoforms is cell type specific, because certain isoforms acting as Lose marks in epithelial cells are part of the fitness fingerprint of healthy neurons. Therefore, an exciting picture starts to appear, in which varying levels of Sparc and different isoforms of Flower are produced by many cell types, acting as direct molecular determinants of cell fitness.
This study aimed to clarify how cells integrate fitness information in order to identify and eliminate suboptimal cells. Subsequently, the physiological consequences were analyzed of efficient cell selection for the whole organism (Merino, 2015).
In order to discover the molecular mechanisms underlying cell selection in Drosophila, this study analyzed genes transcriptionally induced using an assay where WT cells (tub>Gal4) are outcompeted by dMyc-overexpressing supercompetitors (tub>dmyc) due to the increased fitness of these dMyc-overexpressing cells. The expression of CG11165 was strongly induced 24 hr after the peak of flower and sparc expression. In situ hybridization revealed that CG11165 mRNA was specifically detected in Loser cells that were going to be eliminated from wing imaginal discs due to cell competition. The gene, which was named ahuizotl (azot) after a multihanded Aztec creature selectively targeting fishing boats to protect lakes, consists of one exon. azot's single exon encodes for a four EF-hand-containing cytoplasmic protein of the canonical family that is conserved, but uncharacterized, in multicellular animals (Merino, 2015).
To monitor Azot expression, a translational reporter was designed resulting in the expression of Azot::dsRed under the control of the endogenous azot promoter in transgenic flies. Azot expression was not detectable in most wing imaginal discs under physiological conditions in the absence of competition. Mosaic tissue was generated of two clonal populations, which are known to trigger competitive interactions resulting in elimination of otherwise viable cells. Cells with lower fitness were created by confronting WT cells with dMyc-overexpressing cells, by downregulating Dpp signaling, by overexpressing FlowerLose isoforms, in cells with reduced Wg signaling, by suppressing Jak-Stat signaling or by generating Minute clones. Azot expression was not detectable in nonmosaic tissue of identical genotype, nor in control clones overexpressing UASlacZ. On the contrary, Azot was specifically activated in all tested scenarios of cell competition, specifically in the cells undergoing negative selection. Azot expression was not repressed by the caspase inhibitor protein P35 (Merino, 2015).
Because Flower proteins are conserved in mammals, tests were made to see if they are also able to regulate azot. Mouse Flower isoform 3 (mFlower3) has been shown to act as a 'classical' Lose isoform, driving cell elimination when expressed in scattered groups of cells, a situation where azot was induced in Loser cells but is not inducing cell selection when expressed ubiquitously a scenario where azot was not expressed. This shows that the mouse FlowerLose isoforms function in Drosophila similarly to their fly homologs (Merino, 2015).
Interestingly, azot is not a general apoptosis-activated gene because its expression is not induced upon eiger, hid, or bax activation, which trigger cell death. Azot was also not expressed during elimination of cells with defects in apicobasal polarity or undergoing epithelial exclusion-mediated apoptosis (dCsk) (Merino, 2015).
azot expression was analyzed during the elimination of peripheral photoreceptors in the pupal retina, a process mediated by Flower-encoded fitness fingerprints. Thirty-six to 38hr after pupal formation (APF), when FlowerLose-B expression begins in peripheral neurons, no Azot expression was detected in the peripheral edge. At later time points (40 and 44hr APF), Azot expression is visible and restricted to the peripheral edge where photoreceptor neurons are eliminated. This expression was confirmed with another reporter line, azot{KO; gfp}, where gfp was directly inserted at the azot locus using genomic engineering techniques (Merino, 2015).
From these results, it is concluded that Azot expression is activated in several contexts where suboptimal and viable cells are normally recognized and eliminated (Merino, 2015).
To understand Azot function in cell elimination, azot knockout (KO) flies were generated by deleting the entire azot gene. Next, Azot function was analyzed using dmyc-induced competition. In the absence of Azot function, loser cells were no longer eliminated, showing a dramatic 100-fold increase in the number of surviving clones. Loser cells occupied more than 20% of the tissue 72hr after clone induction (ACI). Moreover, using azot{KO; gfp} homozygous flies (that express GFP under the azot promoter but lack Azot protein), it was found that loser cells survived and showed accumulation of GFP. From these results, it is concluded that azot is expressed by loser cells and is essential for their elimination.
In addition, clone removal was delayed in an azot heterozygous background (50-fold increase, 15%), compared to control flies with normal levels of Azot. Cell elimination capacity was fully restored by crossing two copies of Azot::dsRed into the azot-/- background demonstrating the functionality of the fusion protein. Silencing azot with two different RNAis was similarly able to halt selection during dmyc-induced competition. Next, in order to determine the role of Azot's EF hands, a mutated isoform of Azot (Pm4Q12) was generated and overexpressed, that carryed, in each EF hand, a point mutation known to abolish Ca2+ binding. Although overexpression of wild-type azot in negatively selected cells did not rescue the elimination, overexpression of the mutant AzotPm4Q12 reduced cell selection, functioning as a dominant-negative mutant. This shows that Ca2+ binding is important for Azot function. Finally, staining for apoptotic cells corroborated that the lack of Azot prevents cell elimination, because cell death was reduced 8-fold in mosaic epithelia containing loser cells (Merino, 2015).
The role of azot in elimination of peripheral photoreceptor neurons in the pupal retina was examined using homozygous azot KO flies. Pupal retinas undergoing photoreceptor culling (44hr APF) of azot+/+ and azot-/- flies were stained for the cell death marker and the proapoptotic factor. Consistent with the expression pattern of Azot, the number of Hid and TUNEL-positive cells was dramatically decreased in azot-/- retinas compared to azot+/+ retinas (Merino, 2015).
Those results show that Azot is required to induce cell death and Hid expression during neuronal culling. Therefore, tests were performed to see that was also the case in the wing epithelia during dmyc-induced competition. Hid was found to be expressed in loser cells and the expression was found to be strongly reduced in the absence of Azot function (Merino, 2015).
Finally, forced overexpression of FlowerLose isoforms from Drosophila were unable to mediate WT cell elimination when Azot function was impaired by mutation or silenced by RNAi (Merino, 2015).
These results suggested that azot function is dose sensitive, because heterozygous azot mutant flies display delayed elimination of loser cells when compared with azot WT flies. Therefore advantage was taken of the functional reporter Azot::dsRed to test whether cell elimination could be enhanced by increasing the number of genomic copies of azot. Tissues with three functional copies of azot were more efficient eliminating loser cells during dmyc-induced competition and most of the clones were culled 48hr ACI. From these results, it is concluded that azot expression is required for the elimination of Loser cells and unwanted neurons (Merino, 2015).
Next, it was asked what could be the consequences of decreased cell selection at the tissue and organismal level. To this end, advantage was taken of the viability of homozygous azot KO flies. An increase of several developmental aberrations was observed. Focus was placed on the wings, where cell competition is best studied and, because aberrations, including melanotic areas, blisters, and wing margin nicks, were quantified. Wing defects of azot mutant flies could be rescued by introducing two copies of azot::dsRed, showing that the phenotypes are specifically caused by loss of Azot function (Merino, 2015).
Next, it was reasoned that mild tissue stress should increase the need for fitness-based cell selection after damage. First, in order to generate multicellular tissues scattered with suboptimal cells, larvae were exposed to UV light and Azot expression was monitored in wing discs of UV-irradiated WT larvae that were stained for cleaved caspase-3, 24hr after treatment. Under such conditions, Azot was found to be expressed in cleaved caspase-3-positive cells. All Azot-positive cells showed caspase activation and 17% of cleaved caspase-positive cells expressed Azot. This suggested that Azot-expressing cells are culled from the tissue. To confirm this, later time points (3 days after irradiation) were examined; the increase in Azot-positive cells was no longer detectable. The elimination of azot-expressing cells after UV irradiation required azot function, because cells revealed by reporter azot{KO; gfp}, that express GFP instead of Azot, persisted in wing imaginal discs from azot-null larvae. Tests were performeed to see if lack of azot leads to a faster accumulation of tissue defects during organ development upon external damage. azot-/- pupae 0 stage were irradiated, and the number of morphological defects in adult wings was compared to those in nonirradiated azot KO flies. It was found that aberrations increased more than 2-fold when compared to nonirradiated azot-/- flies (Merino, 2015).
In order to functionally discriminate whether azot belongs to genes regulating apoptosis in general or is dedicated to fitness-based cell selection, whether azot silencing prevents Eiger/TNF-induced cell death was exanubed. Inhibiting apoptosis (UASp35) or eiger (UASRNAieiger) rescued eye ablation, whereas azot silencing and overexpression of AzotPm4Q12 did not. Furthermore, azot silencing did not impair apoptosis during genitalia rotation or cell death of epithelial precursors in the retina. These results highlight the consequences of nonfunctional cell-quality control within developing tissues (Merino, 2015).
The next part of the analysis demonstrated that the azot promoter computes relative FlowerLose and Sparc Levels. Epistasis analyses were performed to understand at which level azot is transcriptionally regulated. For this purpose, the assay where WT cells are outcompeted by dMyc-overexpressing supercompetitors was used. It was previously observed that azot induction is triggered upstream of caspase-3 activation and accumulates in outcompeted cells unable to die. Then, upstream events of cell selection were genetically modified. Silencing fweLose transcripts by RNAi or overexpressing Sparc both blocked the induction of Azot::dsRed in WT loser cells. In contrast, when outcompeted WT cells were additionally 'weakened' by Sparc downregulation using RNAi, Azot is detected in almost all loser cells compared to its more limited induction in the presence of endogenous Sparc. Inhibiting JNK signaling with UASpuc did not suppress Azot expression (Merino, 2015).
The activation of Azot upon irradiation was examined. Strikingly, it was found that all Azot expression after irradiation was eliminated when Flower Lose was silenced and also when relative differences of Flower Lose where diminished by overexpressing high levels of Lose isoforms ubiquitously. On the contrary, Azot was not suppressed after irradiation by expressing the prosurvival factor Bcl-2 or a p53 dominant negative. These results show that Azot expression during competition and upon irradiation requires differences in Flower Lose relative levels (Merino, 2015).
Finally, the regulation of Azot expression in neurons was analyzed. Silencing fwe transcripts by RNAi blocked the induction of Azot::dsRed in peripheral photoreceptors. Because Wingless signaling induces FlowerLose-B expression in peripheral photoreceptors, tests were performed to see if overexpression of Daxin, a negative regulator of the pathway, affected Azot levels. Axin overespression completely inhibited Azot expression. Similarly, overexpression of the cell competition inhibitor Sparc also fully blocked Azot endogenous expression in the retina. Finally, ectopic overexpression of FlowerLose-B in scattered cells of the retina was sufficient to trigger ectopic Azot activation. These results show that photoreceptor cells also can monitor the levels of Sparc and the relative levels of FlowerLose-B before triggering Azot expression (Merino, 2015).
These results suggest that the azot promoter integrates fitness information from neighboring cells, acting as a relative 'cell-fitness checkpoint.'
To test if fitness-based cell selection is a mechanism active not only during development, but also during adult stages, WT adult flies were exposed to UV light and monitor Azot and Flower expression were monitored in adult tissues. UV irradiation of adult flies triggered cytoplasmic Azot expression in several adult tissues including the gut and the adult brain. Likewise, UV irradiation of adult flies triggered Flower Lose expression in the gut and in the brain. Irradiation-induced Azot expression was unaffected by Bcl-2 but was eliminated when Flower Lose was silenced or when relative differences of Flower Lose where diminished in the gut. This suggests that the process of cell selection is active throughout the life history of the animal. Further confirming this conclusion, Azot function was essential for survival after irradiation, because more than 99% of azot mutant adults died 6 days after irradiation, whereas only 62.4% of WT flies died after the same treatment. The percentage of survival correlated with the dose of azot because adults with three functional copies of azot had higher median survival and maximum lifespan than WT flies, or null mutant flies rescued with two functional azot transgenes (Merino, 2015).
The next part of the study addressed the role of cell selection during aging. Lack of cell selection could affect the whole organism by two nonexclusive mechanisms. First, the failure to detect precancerous cells, which could lead to cancer formation and death of the individual. Second, the time-dependent accumulation of unfit but viable cells could lead to accelerated tissue and organ decay. We therefore tested both hypotheses (Merino, 2015).
It has been previously shown that cells with reduced levels for cell polarity genes like scrib or dlg are eliminated but can give rise to tumors when surviving. Therefore this study checked if azot functions as a tumor suppressing mechanism in those cells. Elimination of dlg and scrib mutant cells was not affected by RNAi against azot or when Azot function was impaired by mutation, in agreement with the absence of azot induction in these mutant cells. However, azot RNAi or the same azot mutant background efficiently rescued the elimination of clones with reduced Wg signaling (Merino, 2015).
Moreover, the high number of suboptimal cells produced by UV treatment did not lead to tumoral growth in azot-null background. Thus, tumor suppression mechanisms are not impaired in azot mutant backgrounds, and tumors are not more likely to arise in azot-null mutants (Merino, 2015).
Also tests were performed to see whether the absence of azot accelerates tissue fitness decay in adult tissues. Focused was placed on the adult brain, where neurodegenerative vacuoles develop over time and can be used as a marker of aging. The number was compared of vacuoles appearing in the brain of flies lacking azot (azot-/-), WT flies (azot+/+), flies with one extra genomic copy of the gene (azot+/+; azot+), and mutant flies rescued with two genomic copies of azot (azot-/-;azot+/+). For all the genotypes analyzed, a progressive increase was observed in the number and size of vacuoles in the brain over time. Interestingly, azot-/- brains showed higher number of vacuoles compared to control flies (azot+/+ and azot-/-;azot+/+) and a higher rate of vacuole accumulation developing over time. In the case of flies with three genomic copies of the gene (azot+/+; azot+), vacuole number tended to be the lowest (Merino, 2015).
The cumulative expression of azot was analyzed during aging of the adult brain. Positive cells were detected as revealed by reporter azot{KO; gfp}, in homozygosis, that express GFP instead of Azot. A time-dependent accumulation of azot-positive cells was observed (Merino, 2015).
From this, it is concluded that azot is required to prevent tissue degeneration in the adult brain and lack of azot showed signs of accelerated aging. This suggested that azot could affect the longevity of adult flies. Flies lacking azot (azot-/-) had a shortened lifespan with a median survival of 7.8 days, which represented a 52% decrease when compared to WT flies (azot+/+), and a maximum lifespan of 18 days, 25% less than WT flies (azot+/+). This effect on lifespan was azot dependent because it was completely rescued by introducing two functional copies of azot. On the contrary, flies with three functional copies of the gene (azot+/+; azot+) showed an increase in median survival and maximum lifespan of 54% and 17%, respectively (Merino, 2015).
In conclusion, azot is necessary and sufficient to slow down aging, and active selection of viable cells is critical for a long lifespan in multicellular animals (Merino, 2015).
The next part of the study demonstrates that death of unfit cells is sufficient and required for multicellular fitness maintenance.
The results cited above show the genetic mechanism through which cell selection mediates elimination of suboptimal but viable cells. However, using flip-out clones and MARCM, this study found that Azot overexpression was not sufficient to induce cell death in wing imaginal discs. Because Hid is downstream of Azot, it was wondered whether expressing Hid under the control of the azot regulatory regions could substitute for Azot function (Merino, 2015).
In order to test this hypothesis, the whole endogenous azot protein-coding sequence was replaced by the cDNA of the proapoptotic gene hid (azot{KO; hid}) flies. In a second strategy, the whole endogenous azot protein-coding sequence was replaced by the cDNA of transcription factor Gal4, so that the azot promoter can activate any UAS driven transgene (azot{KO; Gal4} flies. The number of morphological aberrations was compared in the adult wings of six genotypes: first, homozygous azot{KO; Gal4} flies that lacked Azot; second, azot{KO; hid} homozygous flies that express Hid with the azot pattern in complete absence of Azot; third, azot+/+ WT flies as a control; and finally three genotypes where the azot{KO; Gal4} flies were crossed with UAShid, UASsickle, another proapoptotic gene, or UASp35, an apoptosis inhibitor. In the case of UASsickle flies, a second azot mutation was introduced to eliminate azot function. Interestingly, the number of morphological aberrations was brought back to WT levels in all the situations where the azot promoter was driving proapoptotic genes (azot{KO; hid}, azot{KO; Gal4} × UAShid, azot{KO; Gal4} × UASsickle with or without irradiation. On the contrary, expressing p35 with the azot promoter was sufficient to produce morphological aberrations despite the presence of one functional copy of azot. Likewise, p35-expressing flies (azot{KO; Gal4}/azot+; UASp35) did not survive UV treatments, whereas a percentage of the flies expressing hid (26%) or sickle (28%) in azot-positive cells were able to survive (Merino, 2015).
From this, it is concluded that specifically killing those cells selected by the azot promoter is sufficient and required to prevent morphological malformations and provide resistance to UV irradiation (Merino, 2015).
The next part of the study demonstrated that death of unfit cells extends lifespan
It was asked whether the shortened longevity observed in azot-/- flies could be also rescued by killing azot-expressing cells with hid in the absence of Azot protein. It was found that azot{KO; hid} homozygous flies had dramatically improved lifespan with a median survival of 27 days at 29°C, which represented a 125% increase when compared to azot-/- flies, and a maximum lifespan of 34 days, 41% more than mutant flies (Merino, 2015).
Similar results were obtained at 25°C. It was found that flies lacking azot (azot-/-) had a shortened lifespan with a median survival of 25days, which represented a 24% decrease when compared to WT flies (azot+/+), and a maximum lifespan of 40 days, 31% less than WT flies (azot+/+). On the contrary, flies with three functional copies of the gene (azot+/+; azot+) or flies where azot is replaced by hid (azot{KO; hid} homozygous flies) showed an increase in median survival of 54% and 63% and maximum lifespan of 12% and 24%, respectively (Merino, 2015).
Finally, the effects of dietary restriction on longevity of those flies was tested. It was found that dietary restriction could extend both the median survival and the maximum lifespan of all genotypes. Interestingly, dietary restricted flies with three copies of the gene azot showed a further increase in maximum lifespan of 35%. This shows that dietary restriction and elimination of unfit cells can be combined to maximize lifespan (Merino, 2015).
In conclusion, eliminating unfit cells is sufficient to increase longevity, showing that cell selection is critical for a long lifespan in Drosophila (Merino, 2015).
This study has shown that active elimination of unfit cells is required to maintain tissue health during development and adulthood. The gene (azot), whose expression is confined to suboptimal or misspecified but morphologically normal and viable cells. When tissues become scattered with suboptimal cells, lack of azot increases morphological malformations and susceptibility to random mutations and accelerates age-dependent tissue degeneration. On the contrary, experimental stimulation of azot function is beneficial for tissue health and extends lifespan. Therefore, elimination of less fit cells fulfils the criteria for a hallmark of aging (Merino, 2015).
Although cancer and aging can both be considered consequences of cellular damage, no evidence was found for fitness-based cell selection having a role as a tumor suppressor in Drosophila. The results rather support that accumulation of unfit cells affect organ integrity and that, once organ function falls below a critical threshold, the individual dies (Merino, 2015).
Azot expression in a wide range of 'less fit' cells, such as WT cells challenged by the presence of 'supercompetitors,' slow proliferating cells confronted with normal proliferating cells, cells with mutations in several signaling pathways (i.e., Wingless, JAK/STAT, Dpp), or photoreceptor neurons forming incomplete ommatidia. In order to be expressed specifically in 'less fit' cells, the transcriptional regulation of azot integrates fitness information from at least three levels: (1) the cell's own levels of FlowerLose isoforms, (2) the levels of Sparc, and (3) the levels of Lose isoforms in neighboring cells. Therefore, Azot ON/OFF regulation acts as a cell-fitness checkpoint deciding which viable cells are eliminated. It is proposed that by implementing a cell-fitness checkpoint, multicellular communities became more robust and less sensitive to several mutations that create viable but potentially harmful cells. Moreover, azot is not involved in other types of apoptosis, suggesting a dedicated function, and - given the evolutionary conservation of Azot - pointing to the existence of central cell selection pathways in multicellular animals (Merino, 2015).
Cell-cell intercalation is used in several developmental processes to shape the normal body plan. There is no clear evidence that intercalation is involved in pathologies. This study used the proto-oncogene myc to study a process analogous to early phase of tumour expansion: myc-induced cell competition. Cell competition is a conserved mechanism driving the elimination of slow-proliferating cells (so-called 'losers') by faster-proliferating neighbours (so-called 'winners') through apoptosis and is important in preventing developmental malformations and maintain tissue fitness. Using long-term live imaging of myc-driven competition in the Drosophila pupal notum and in the wing imaginal disc, this study showed that the probability of elimination of loser cells correlates with the surface of contact shared with winners. As such, modifying loser-winner interface morphology can modulate the strength of competition. Elimination of loser clones requires winner-loser cell mixing through cell-cell intercalation. Cell mixing is driven by differential growth and the high tension at winner-winner interfaces relative to winner-loser and loser-loser interfaces, which leads to a preferential stabilization of winner-loser contacts and reduction of clone compactness over time. Differences in tension are generated by a relative difference in F-actin levels between loser and winner junctions, induced by differential levels of the membrane lipid phosphatidylinositol (3,4,5)-trisphosphate. These results establish the first link between cell-cell intercalation induced by a proto-oncogene and how it promotes invasiveness and destruction of healthy tissues (Levayer, 2015).
To analyse quantitatively loser cell elimination, long-term live imaging was performed of clones showing a relative decrease of the proto-oncogene myc in the Drosophila pupal notum, a condition known to induce cell competition in the wing disc. Every loser cell delamination was counted over 10 h, and the probability of cell elimination was calculated for a given surface of contact shared with winner cells. A significant increase was observed of the proportion of delamination with winner-loser shared contact, whereas this proportion remained constant for control clones. The same correlation was observed in ex vivo culture of larval wing disc. Cell delamination in the notum was apoptosis dependent and expression of flowerlose (fwelose) This suggests that winner-loser interface morphology could modulate the probability of eliminating loser clones. Using the wing imaginal disc, winner-loser contact was reduced by inducing adhesion- or tension-dependent cell sorting and observed a significant reduction of loser clone elimination. This rescue was not driven by a cell-autonomous effect of E-cadherin (E-cad) or active myosin II regulatory light chain (MRLC) on growth, death or cell fitness but rather by a general diminution of winner-loser contact. Competition is ineffective across the antero-posterior compartment boundary, a frontier that prevents cell mixing through high line tension. Accordingly, there was no increase in death at the antero-posterior boundary in wing discs overexpressing fweloseA in the anterior compartment. However, reducing tension by reducing levels of myosin II heavy chains was sufficient to increase the shared surface of contact between cells of the anterior and posterior compartments, and induced fwelose death at the boundary. Altogether, it is concluded that the reduction in surface contact between winners and losers is sufficient to block competition, which explains how compartment boundaries prevent competition (Levayer, 2015).
Loser clones have been reported to fragment more often than controls, whereas winner clones show convoluted morphology, suggesting that winner-y actin levels, and F-actin levels were higher in WT clones than M-/+ cells. This prompted a test of the role of actin organization in winner-loser mixing. Downregulating the formin Diaphanous (Dia, a filamentous actin polymerization factor) by RNA interference (RNAi) or by using a hypomorphic mutant was sufficient to obtain a high proportion of fragmen-cell intercalation independent of death. To assess the contribution of each phenomenon, the proportion of clones fragmented 48 h after clone induction (ACI) was systematically counted. A twofold increase was observed in the frequency of split clones in losers (wild type (WT) in tub-dmyc) versus WT in WT controls. Overexpressing E-cad or active myosin II was sufficient to prevent loser clone splitting, whereas blocking apoptosis or blocking loser fate by silencing fwelose did not reduce splitting. Finally, the proportion of split clones was also increased for winner clones either during myc-driven competition (UAS-myc, UAS-p35) or during Minute-dependent competition (WT clones in M-/+ background). Altogether, this suggested that winner-loser mixing is increased independently of loser cell death or clone size by a factor upstream of fwe, and could be driven by cell-cell intercalation. Accordingly, junction remodelling events leading to disappearance of a loser-loser junction were three times more frequent at loser clone boundaries than control clone boundaries in the pupal notum. The rate of junction remodelling was higher in loser-loser junctions and in winner-winner junctions than in winner-loser junctions. The preferential stabilization of winner-loser interfaces should increase the surface of contact between winner and loser cells over time. Accordingly, loser clone compactness in the notum decreased over time whereas it remains constant on average for WT clones in WT background. Similarly, the compactness of clones in the notum also decreased over time for conditions showing high frequency of clone splitting in the wing disc, whereas clone compactness remained constant for conditions rescuing clone splitting. Altogether, it is concluded that both Minute- and myc-dependent competition increase loser-winner mixing through cell-cell intercalation (Levayer, 2015).
It was then asked what could modulate the rate of junction remodelling during competition. The rate of junction remodelling can be cell-autonomously increased by myc. Interestingly, downregulation of the tumour suppressor PTEN is also sufficient to increase the rate of junction remodelling through the upregulation of phosphatidylinositol (3,4,5)-trisphosphate (PIP3). It was reasoned that differences in PIP3 levels could also modulate junction remodelling during competition. Using a live reporter of PIP3 that could detect modulations of PIP3 in the notum, a significant increase of PIP3 was observed in the apico-lateral membrane of tub-dmyc-tub-dmyc interfaces compared with WT-WT and WT-tub-dmyc interfaces. Moreover, increasing/reducing Myc levels in a full compartment of the wing disc was sufficient to increase/decrease the levels of phospho-Akt (a downstream target of PIP3, whereas fweloseA overexpression had no effect. Similarly, levels of phospho-Akt were relatively higher in WT clones than in the surrounding M-/+ cells. Thus differences in PIP3 levels might be responsible for winner-loser mixing (Levayer, 2015).
Accordingly, reducing PIP3 levels by overexpressing a PI3 kinase dominant negative (PI3K-DN) or increasing PIP3 levels by knocking down PTEN (UAS-pten RNAi) were both sufficient to induce a high proportion of fragmented clones and to reduce clone compactness over time in the notum, whereas increasing PIP3 in loser clones was sufficient to prevent cell mixing. Moreover, abolishing winner-loser PIP3 differences through larval starvation prevented loser clone fragmentation, the reduction of clone compactness over time in the notum and could rescue WT clone elimination in tub-dmyc background. It is therefore concluded that differences in PIP3 levels are necessary and sufficient for loser-winner mixing and required for loser cell elimination (Levayer, 2015).
It was then asked which downstream effectors of PIP3 could affect junction stability. A relative growth decrease can generate mechanical stress that can be released by cell-cell intercalation. Accordingly, growth reduction through Akt downregulation is sufficient to increase clone splitting and could contribute to loser clone splitting. However, Akt is not sufficient to explain winner-loser mixing because, unlike PIP3, increasing Akt had no effect on clone splitting. PIP3 could also modulate junction remodelling through its effect on cytoskeleton and the modulation of intercellular adhesion or tension. No obvious modifications of E-cad, MRLC or Dachs (another regulator of tension) was detected in loser cells. However, a significant reduction of F-actin levels and a reduction of actin turnover/polymerization rate were observed in loser-loser and loser-winner junctions in the notum. Similarly, modifying Myc levels in a full wing disc compartment was sufficient to modify actin levels, and F-actin levels were higher in WT clones than M-/+ cells. This prompted a test of the role of actin organization in winner-loser mixing. Downregulating the formin Diaphanous (Dia, a filamentous actin polymerization factor) by RNA interference (RNAi) or by using a hypomorphic mutant was sufficient to obtain a high proportion of fragmented clones and to reduce clone compactness over time, whereas overexpressing Dia in loser clones prevented clone splitting (UAS-dia::GFP) and compactness reduction. This effect was specific to Dia as modulating Arp2/3 complex (a regulator of dendritic actin network) had no effect on clone splitting. Thus, impaired filamentous actin organization was necessary and sufficient to drive loser-winner mixing. These actin defects were driven by the differences in PIP3 levels between losers and winners. Thus Dia could be an important regulator of competition through its effect on cell mixing. Overexpression of Dia was indeed sufficient to reduce loser clone elimination significantly (Levayer, 2015).
Filamentous actin has been associated with tension regulation. It was therefore asked whether junction tension was modified in winner and loser junctions. The maximum speed of relaxation of junction after laser nanoablation (which is proportional to tension) was significantly reduced in loser-loser and winner-loser junctions compared with winner-winner junctions. This distribution of tension has been proposed to promote cell mixing. Accordingly, decreasing PIP3 in clones reduced tension both in low-PIP3-low-PIP3 and low-PIP3-normal-PIP3 junctions, whereas overexpressing Dia in loser clones or starvation were both sufficient to abolish differences in tension, in agreement with their effect on winner-loser mixing and the distribution of F-actin. Thus the lower tension at winner-loser and loser-loser junctions is responsible for winner-loser mixing. Altogether, it is concluded that the relative PIP3 decrease in losers increases winner-loser mixing through Akt-dependent differential growth and the modulation of tension through F-actin downregulation in winner-loser and loser-loser junctions (Levayer, 2015).
Several modes of tissue invasion by cancer cells have been described, most of them relying on the departure of the tumour cells from the epithelial layer. This study suggests that some oncogenes may also drive tissue destruction and invasion by inducing ectopic cell intercalation between cancerous and healthy cells, and subsequent healthy cell elimination. myc-dependent invasion could be enhanced by other mutations further promoting intercalation (such as PTEN). Stiffness is increased in many tumours, suggesting that healthy cell-cancer cell mixing by intercalation might be a general process (Levayer, 2015).
Cell competition is a cell selection process that arises in growing tissues as a result of interactions between cells of different fitness. This behavior is also observed in Myc super-competition, where healthy wild type cells in growing wing discs of Drosophila are outcompeted by nearby cells that express higher levels of the Myc oncogene. Most work on Myc super-competition has examined it in mixed populations of male and female larvae. However, as physiological and genetic differences between Drosophila males and females could affect the competitive behavior of cells, this study investigated whether sex differences affect the process. Both male and female wing disc cells were shown to be subject to Myc super-competition. Female disc cells appear to be more sensitive to competitive elimination than male cells, potentially due to differences in baseline cellular Myc levels between the sexes. This paper also reports sexual dimorphism of cell size and number between male and female growing wing discs that is independent of competition; wing discs and wing pouches from females are larger than males' due to larger cell size and cell number. It is suggested that separately examining male and female tissues in cell competition assays could enhance understanding of the effects of sex-specific pathways on cell and super-competition (Svoysky, 2021).
In Drosophila, differential development of wing and haltere, which differ in cell size, number and morphology, is dependent on the function of Hox gene Ultrabithorax (Ubx). This paper reports studies on Ubx-mediated regulation of the Fat/Hippo and IIS/dAkt pathways, which control cell number and cell size during development. Over-expression of Yki or down regulation of negative components of the Fat/Hippo pathway, such as expanded, caused considerable increase in haltere size, mainly due to increase in cell number. These phenotypes were also associated with the activation of Akt pathways in developing haltere. Although activation of Akt alone did not affect the cell size or the organ size, dramatic increase was observed in haltere size when Akt was activated in the background where expanded is down regulated. This was associated with the increase in both cell size and cell number. The organ appeared flatter than wildtype haltere and the trichome morphology and spacing resembled that of wing suggesting homeotic transformations. Thus, these results suggest a link between cellular growth and pattern formation and the final differentiated state of the organ (Singh, 2015).
Wing and haltere are the dorsal appendages of second and third thoracic segments, respectively, of adult Drosophila. They are homologous structures, although differ greatly in their morphology. The homeotic gene Ultrabithorax (Ubx), which is required and sufficient to confer haltere fate to epithelial cells, is known to regulate many wing patterning genes to specify haltere, but the mechanism is still poorly understood (Singh, 2015).
There are a number of differences between wing and haltere at the cellular and organ levels. Wing is a large, flat and thin structure, while haltere is a small globular structure, although both are made up of 2-layered sheet of epithelial cells. Space between the two layers of cells in haltere is filled with haemocytes. Cuticle area of each wing cell is 8 fold more than a haltere cell. Haltere has smaller and fewer cells than the wing. Trichomes of wing cells are long and thin, while haltere trichomes are short and stout in morphology. The ratio of anterior to posterior compartment size in the haltere (~2.5:1) is much different from that in the wing (~1.2:1). Haltere also lacks wing-type vein and sensory bristles. Haltere cells are more cuboidal compared to flatter wing cells (Roch, 2000). Thus, cell number, size and shape all add to the differences in the size and shape of the two organs (Singh, 2015).
However, cells of the third instar larval wing and haltere discs are similar in size and shape (Makhijani, 2007). The difference between cell size and shape becomes evident at late pupal stages (Roch, 2000). Wing cells become much larger, compared to haltere cells. At pupal stages, they also exhibit differences in the organization of actin cytoskeleton elements viz. F-actin levels are much higher in haltere cells compared to wing cells (Roch, 2000) (Singh, 2015).
In the context of final shape of wings and halteres, one needs to understand the mechanism by which Ubx influences cell size, shape and arrangement. It is possible that Ubx regulates overall shape of the haltere by regulating either cell size and/or shape. The current understanding of mechanisms by which wing and haltere differ at cellular, tissue and organ level is ambiguous (Sanchez-Herrero, 2013). For example, while removal of Ubx from the entire haltere, or at least from one entire compartment, leads to haltere to wing transformation with increased growth of Ubx minus tissues, mitotic clones of Ubx (using the null allele Ubx6.28) show similar sized twin spot in small clones (Crickmore, 2006, De Navas, 2006; Makhijani, 2007). Only when very large clones of Ubx6.28Ubx6.28 are generated, one can see increased growth compared to their twin spots (Crickmore, 2006). This suggests that unless a certain threshold level of growth factors is de-repressed, the haltere does not show any overgrowth phenotype (Singh, 2015).
There have been several efforts to identify functional and molecular mechanisms by which Ubx regulates genes/pathways to provide haltere its distinct morphology. Various approaches have been used to identify targets of Ubx that are expected to differentially express between wing and haltere, e.g., loss-of-function genetics, deficiency screens, enhancer-trap screening and genome wide approaches such as microarray analysis and chromatin immunoprecipitation (ChIP). Targets include genes involved in diverse cellular functions like components of the cuticle and extracellular matrix, genes involved in cell specification, cell proliferation, cell survival, cell adhesion, or cell differentiation, structural components of actin and microtubule filaments, and accessory proteins controlling filament dynamics (reviewed in Sanchez-Herrero, 2013; Singh, 2015).
Decapentaplegic (Dpp), Wingless (Wg), and Epidermal growth factor receptor (EGFR) are some of the major growth and pattern regulating pathways that are repressed by Ubx in the haltere (Weatherbee, 1998, Shashidhara, 1999; Prasad, 2003; Mohit, 2006; Crickmore, 2006, Pallavi, 2006; De Navas, 2006; Makhijani, 2007). However, over-expression of Dpp, Wg, Vestigial (Vg) or Vein (Vn) provides only marginal growth advantage to haltere compared to the wildtype. In this context, additional growth regulating pathways amongst the targets of Ubx were examined. Genome wide studies have identified many components of Fat/Hippo and Insulin-insulin like/dAkt signalling (IIS/dAkt) pathways as potential targets of Ubx. The Fat/Hippo pathway is a crucial determinant of organ size in both Drosophila and mammals. It regulates cell proliferation, cell death, and cell fate decisions and coordinates these events to specify organ size. In contrast, the IIS/dAkt pathway is known to regulate cell size (Singh, 2015).
Recent studies have revealed that the Fat/Hippo pathway networks with other signalling pathways. For example, during wing development, Fat/Hippo pathway activities are dependent on Four-jointed (Fj) and Dachous (Ds) gradients, which are influenced by Dpp, Notch, Wg and Vg. Glypicans, which play a prominent role in morphogen signalling, are regulated by Fat/Hippo signalling (Baena-Lopez, 2008). EGFR activates Yorkie (Yki; effector of Fat/Hippo pathway) through its EGFR-RAS-MAPK signalling by promoting the phosphorylation of Ajuba family protein WTIP (Reddy, 2013). However, EGFR negatively regulates events downstream of Yki (Herranz, 2012). The Fat/Hippo pathway is also known to inhibit EGFR signalling, which makes the interaction between the two pathways very complex and context-dependent. IIS/dAkt pathway is also known to activate Yki signalling and vice-versa. Thus, Fat/Hippo pathway may specify organ size by regulating both cell number (directly) and cell size (via regulating IIS/dAkt pathway) (Singh, 2015).
This study reports studies on the functional implication of regulation of Fat/Hippo and IIS/dAkt pathways by Ubx in specifying haltere development. Over-expression of Yki or down regulation of negative components of the Fat/Hippo pathway, such as expanded (ex), induced considerable increase in haltere size, mainly due to increase in cell number. Although activation of dAkt alone did not affect the organ size or the cell size, activation of Yki or down regulation of ex in the background of over-expressed dAkt caused dramatic increase in haltere size, much severe than Yki or ex alone. In this background, increase was observed in both cell size and cell number. The resulted haltere appeared flatter than wildtype haltere and the morphology of trichomes and their spacing resembled that of wing suggesting homeotic transformations. Thus, these results suggest a link between cellular growth and pattern formation and the final differentiated state of the organ (Singh, 2015).
The findings suggest that, downstream of Ubx, the Fat/Hippo pathway is critical for haltere specification. It is required for Ubx-mediated specification of organ size, sensory bristle repression, trichome morphology and arrangement. The Fat/Hippo pathway cooperates with the IIS/dAkt pathway, which is also a target of Ubx, in specifying cell size and compartment size in developing haltere. The fact that over-expression of Yki or downregulation of ex show haltere-to-wing transformations at the levels of organ size and shape, and trichome morphology and arrangement, suggest that regulation of the Fat/Hippo pathway by Ubx is central to the modification of wing identity to that of the haltere (Singh, 2015).
The observations made in this study pose new questions and suggest various interesting possibilities to study the Fat/Hippo pathway with a new perspective.
(1) It was observed that while Yki is nuclear in haltere discs, it appears to be non-functional. Yki is a transcriptional co-activator protein, which requires other DNA-binding partners for its activity. In this context, understanding the precise relationship between Yki and Ubx may provide an insight into mechanism of haltere specification (Singh, 2015).
(2) The Fat/Hippo pathway (along with the IIS/dAkt pathway) may be involved in the specification of cell size, trichome morphology and their arrangement, all of which are important parameters in determining organ morphology. Recent studies indicate that the Fat/Hippo pathway regulates cellular architecture and the mechanical properties of cells in response to the environment. It would be interesting to study the role of the Fat/Hippo pathway in regulating the cytoskeleton of epithelial cells during development. Haltere cells at pupal stages exhibit higher levels of F-actin than wing cells. One possible mechanism that is currently being investigated is lowering of F-actin levels in transformed haltere cells due to over-expression of Yki or down regulation of ex. This may cause flattening of cells during morphogenesis leading to larger organ size (Singh, 2015).
(3) Reversing cell size and number was sufficient to induce homeotic transformations at the level of haltere morphology. This suggests the importance of negative regulation of genetic mechanisms that determine cell size and number, in specifying an organ size and shape. As a corollary, Ubx-mediated regulation of Fat/Hippo and IIS/dAkt pathways provides an opportunity to study cooperative repression of cell number and cell size during organ specification (Singh, 2015).
(4) Certain genetic backgrounds investigated in this study showed severe effect on cell proliferation in haltere discs than in wing discs. This could be due to the fact that, the wing disc has already attained a specific size by the third instar larval stage (the developmental stage examined in this study), which is controlled by several pathways. Any change to this size may need more drastic alteration to the controlling mechanisms. As Ubx specifies haltere by modulating various wing-patterning events, there may still exist a certain degree of plasticity in mechanisms that determine the size of the haltere. However, in absolute terms, the haltere is also resistant to changes in growth control due to regulation by Ubx at multiple levels. Thus, differential development of wing and haltere provides a very good assay system to study not only growth control, but also to dissect out function of important growth regulators (tumour suppressor pathways) such as the Fat/Hippo pathway using various genome-wide approaches (Singh, 2015).
The developing crossveins of the wing of Drosophila melanogaster are specified by long-range BMP signaling and are especially sensitive to loss of extracellular modulators of BMP signaling such as the Chordin homolog Short gastrulation (Sog). However, the role of the extracellular matrix in BMP signaling and Sog activity in the crossveins has been poorly explored. Using a genetic mosaic screen for mutations that disrupt BMP signaling and posterior crossvein development, this study has identified Gyc76C, a member of the receptor guanylyl cyclase family that includes mammalian natriuretic peptide receptors. Gyc76C and the soluble cGMP-dependent kinase Foraging, likely linked by cGMP, are necessary for normal refinement and maintenance of long-range BMP signaling in the posterior crossvein. This does not occur through cell-autonomous crosstalk between cGMP and BMP signal transduction, but likely through altered extracellular activity of Sog. This study identified a novel pathway leading from Gyc76C to the organization of the wing extracellular matrix by matrix metalloproteinases and shows that both the extracellular matrix and BMP signaling effects are largely mediated by changes in the activity of matrix metalloproteinases. Parallels and differences between this pathway and other examples of cGMP activity in both Drosophila melanogaster and mammalian cells and tissues are discussed (Schleede, 2015).
The vein cells that develop from the ectodermal epithelia of the Drosophila melanogaster wing are positioned, elaborated and maintained by a series of well-characterized intercellular signaling pathways. The wing is easily visualized, and specific mutant vein phenotypes have been linked to changes in specific signals, making the wing an ideal tissue for examining signaling mechanisms, for identifying intracellular and extracellular crosstalk between different pathways, and for isolating new pathway components (Schleede, 2015).
The venation defect, the loss of the posterior crossvein (PCV), is used to identify and characterize participants in Bone Morphogenetic Protein (BMP) signaling. The PCV is formed during the end of the first day of pupal wing development, well after the formation of the longitudinal veins (LVs, numbered L1-L6), and requires localized BMP signaling in the PCV region between L4 and L5. Many of the homozygous viable crossveinless mutants identified in early genetic screens have now been shown to disrupt direct regulators of BMP signaling, especially those that bind BMPs and regulate BMP movement and activity in the extracellular space. The PCV is especially sensitive to loss of these regulators because of the long range over which signaling must take place, and the role many of these BMP regulators play in the assembly or disassembly of a BMP-carrying 'shuttle' (Schleede, 2015).
The BMP Decapentaplegic (Dpp) is secreted by the pupal LVs, possibly as a heterodimer with the BMP Glass bottom boat (Gbb). This stimulates autocrine and short-range BMP signaling in the LVs that is relatively insensitive to extracellular BMP regulators. However, Dpp and Gbb also signal over a long range by moving into the intervein tissues where the PCV forms. In order for this to occur, the secreted BMPs must bind the D. melanogaster Chordin homolog Short gastrulation (Sog) and the Twisted gastrulation family member Crossveinless (Cv, termed here Cv-Tsg2 to avoid confusion with other 'Cv' gene names). The Sog/Cv-Tsg2 complex facilitates the movement of BMPs from the LVs through the extracellular space, likely by protecting BMPs from binding to cell bound molecules such as their receptors. In order to stimulate signaling in the PCV, BMPs must also be freed from the complex. The Tolloid-related protease (Tlr, also known as Tolkin) cleaves Sog, lowering its affinity for BMPs, and Tsg family proteins help stimulate this cleavage. Signaling is further aided in the PCV region by a positive feedback loop, as BMP signaling increases localized expression of the BMP-binding protein Crossveinless 2 (Cv-2, recently renamed BMPER in vertebrates). Cv-2 also binds Sog, cell surface glypicans and the BMP receptor complex, and likely acts as a co-receptor and a transfer protein that frees BMPs from Sog. The lipoprotein Crossveinless-d (Cv-d) also binds BMPs and glypicans and helps signaling by an unknown mechanism (Schleede, 2015).
PCV development takes place in a complex and changing extracellular environment, but while there is some evidence that PCV-specific BMP signaling can be influenced by changes in tissue morphology or loss of the cell-bound glypican heparan sulfate proteoglycans, other aspects of the environment have not been greatly investigated. During the initial stages of BMP signaling in the PCV, at 15-18 hours after pupariation (AP), the dorsal and ventral wing epithelia form a sack that retains only a few dorsal to ventral connections from earlier stages; the inner, basal side of the sack is filled with extracellular matrix (ECM) proteins, both diffusely and in laminar aggregates. As BMP signaling in the PCV is maintained and refined, from 18-30 hours AP, increasing numbers of dorsal and ventral epithelial cells adhere, basal to basal, flattening the sack. The veins form as ECM-filled channels between the two epithelia, while in intervein regions scattered pockets of ECM are retained basolaterally between the cells within each epithelium; a small amount of ECM is also retained at the sites of basal-to-basal contact. This changing ECM environment could potentially alter BMP movement, assembly of BMP-containing complexes, and signal reception, as has been demonstrated in other developmental contexts in Drosophila (Schleede, 2015).
This study demonstrates the strong influence of the pupal ECM on PCV-specific long-range BMP signaling, through the identification of a previously unknown ECM-regulating pathway in the wing. In a screen conducted for novel crossveinless mutations on the third chromosome, a mutation was found in the guanylyl cyclase at 76C (gyc76C) locus, which encodes one of five transmembrane, receptor class guanylyl cyclases in D. melanogaster. Gyc76C has been previously characterized for its role in Semaphorin-mediated axon guidance; Malpighian tubule physiology, and the development of embryonic muscles and salivary glands. Like the similar mammalian natriuretic peptide receptors NPR1 and NPR2, the guanylyl cyclase activity of Gyc76C is likely regulated by secreted peptides, and can act via a variety of downstream cGMP sensors (Schleede, 2015).
The evidence suggests that Gyc76C influences BMP signaling in the pupal wing by changing the activity of the cGMP-dependent kinase Foraging (For; also known as Dg2 or Pkg24A), also a novel role for this kinase. But rather than controlling BMP signal transduction in a cell-autonomous manner, evidence is provided that Gyc76C and Foraging regulate BMP signaling non-autonomously by dramatically altering the wing ECM during the period of BMP signaling in the PCV. This effect is largely mediated by changing the levels and activity of matrix metalloproteinases (Mmps), especially Drosophila Mmp2. Genetic interactions suggest that the ECM alterations affect the extracellular mobility and activity of the BMP-binding protein Sog (Schleede, 2015).
This provides the first demonstration of Gyc76C and For activity in the developing wing, and the first evidence these proteins can act by affecting Mmp activity. Moreover, the demonstration of in vivo link from a guanylyl cyclase to Mmps and the ECM, and from there to long-range BMP signaling, may have parallels with findings in mammalian cells and tissues. NPR and NO-mediated changes in cGMP activity can on the one hand change matrix metalloproteinase expression secretion and activity, and on the other change in BMP and TGFβ signaling (Schleede, 2015).
Mutation 3L043, uncovered by a genetic screen to identify homozygous lethal mutations required for PCV development, is a novel allele of gyc76C, a transmembrane peptide receptor that, like vertebrate NPRs, acts as a guanylyl cyclase. gyc76C is likely linked by cGMP production to the activity of the cGMP-dependent kinase For, and that Gyc76C and For define a new pathway for the regulation of wing ECM. This pathway appears to act largely through changes in the activity of ECM-remodeling Mmp enzymes. Loss of gyc76C or For alter both the organization of the wing ECM and the levels of the two D. melanogaster Mmps, and the gyc76C knockdown phenotype can be largely reversed by knockdown of Mmp2. This is the first indication of a role for cGMP, Gyc76C and For function in the developing wing, and their effects on the ECM provides a novel molecular output for each (Schleede, 2015).
Gyc76C and For are necessary for the normal refinement and maintenance of long-range BMP signaling in the posterior crossvein region of the pupal wing; in fact, crossvein loss is the most prominent aspect of the adult gyc76C knockdown phenotype. The evidence suggests that this effect is also mediated by changes in Mmp activity, and most likely the Mmp-dependent reorganization of the ECM. In fact, analysis using genetic mosaics finds no evidence for a reliable, cell autonomous effect of cGMP activity on BMP signal transduction in the wing. Thus, this apparent crosstalk between receptor guanylyl cyclase activity and BMP signaling in the wing is mediated by extracellular effects (Schleede, 2015).
It is noteworthy that the cGMP activity mediated by NPR or nitric oxide signaling can change also Mmp gene expression, secretion or activation in many different mammalian cells and tissues. Both positive and negative effects have been noted, depending on the cells, the context, and the specific Mmp. Given the strong role of the ECM in cell-cell signaling, the contribution of cGMP-mediated changes in Mmp activity to extracellular signaling may be significant.
There is also precedent for cGMP activity specifically affecting BMP and TGFβ signaling in mammals. cGMP-dependent kinase activity increases BMP signaling in C2C12 cells, and this effect has been suggested to underlie some of the effects of nitric oxide-induced cGMP on BMP-dependent pulmonary arterial hypertension. Conversely, atrial natriuretic peptide stimulates the guanylyl cyclase activities of NPR1 and NPR2 and can inhibit TGFβ activity in myofibroblasts; this inhibition has been suggested to underlie the opposing roles of atrial natriuretic peptide and TGFβ during hypoxia-induced remodeling of the pulmonary vasculature. However, unlike the pathway observed in the fly wing, these mammalian effects are thought to be mediated by the intracellular modulation of signal transduction, with cGMP-dependent kinases altering BMP receptor activity or the phosphorylation and nuclear accumulation of receptor-activated Smads. Nonetheless, it remains possible that there are additional layers of regulation mediated through extracellular effects, underscoring the importance of testing cell autonomy (Schleede, 2015).
Aside from its role in adult Malpighian tubule physiology, Gyc76C was previously shown to have three developmental effects: in the embryo it regulates the repulsive axon guidance mediated by Semaphorin 1A and Plexin A, the proper formation and arrangement of somatic muscles, and lumen formation in the salivary gland. All these may have links to the ECM. Loss of gyc76C from embryonic muscles affects the distribution and vesicular accumulation of the βintegrin Mys, and reduces laminins and the integrin regulator Talin in the salivary gland. The axon defects likely involve a physical interaction between Gyc76C and semaphorin receptors that affects cGMP levels; nonetheless, gyc76C mutant axon defects are very similar to those caused by loss of the perlecan Trol (Schleede, 2015).
The parallels between the different contexts of Gyc76C action are not exact, however. First, only the wing phenotype has been linked to a change in Mmp activity. Second, unlike the muscle phenotype, the wing phenotype is not accompanied by any obvious changes in integrin levels or distribution, beyond those caused by altered venation. Finally, most gyc76C mutant phenotypes are reproduced by loss of the Pkg21D (Dg1) cytoplasmic cGMP-dependent kinase, instead of For (Dg2, Pkg24A) as found in the wing, and thus may be mediated by different kinase targets (Schleede, 2015).
For has been largely analyzed for behavioral mutant phenotypes, and the overlap between Pkg21D and For targets is unknown. While many targets have been identified for the two mammalian cGMP-dependent kinases, PRKG1 (which exists in alpha and beta isoforms) and PRKG2, it is not clear if either of these is functionally equivalent to For. One of the protein isoforms generated by the for locus has a putative protein interaction/dimerization motif with slight similarity to the N-terminal binding/dimerization domains of alpha and beta PRKG1, but all three For isoforms have long N-terminal regions that are lacking from PRKG1 and PRKG2. In fact, a recent study suggested that For is instead functionally equivalent to PRKG2: Like PRKG2, For can stimulate phosphorylation of FOXO, and is localized to cell membranes in vitro. But For apparently lacks the canonical myristoylation site that is thought to account for the membrane localization and thus much of the target specificity of PRKG2. FOXO remains the only identified For target, and foxo null mutants are viable with normal wings (Schleede, 2015).
The loss of long range BMP signaling in the PCV region caused by knockdown of gyc76C can, like the ECM, be largely rescued by knockdown of Mmp2. Two results suggest that it is the alteration to the ECM that affects long-range BMP signaling, rather than some independent effect of Mmp2. First, the BMP signaling defects caused by gyc76C knockdown were rescued by directly manipulating the ECM through the overexpression of the perlecan Trol. Second, when Mmp activity is inhibited by overexpression of the diffusible Mmp inhibitor TIMP, this not only rescued the PCV BMP signaling defects caused by gyc76C knockdown, but also led to ectopic BMP signaling, not throughout the region of TIMP expression, but only in those regions with abnormal accumulation of ECM (Schleede, 2015).
The Mmp2-mediated changes in the ECM likely affect long-range BMP signaling by altering the activity of extracellular BMP-binding proteins, particularly Sog. The BMPs Dpp and Gbb produced in the LVs bind Sog and Cv-Tsg2, shuttle into the PCV region, and are released there by Tlr-mediated cleavage of Sog and transfer to Cv-2 and the receptors. Genetic interaction experiments suggest that knockdown of gyc76C both increases Sog's affinity for BMPs and reduces the movement of the Sog/Cv-Tsg2/BMP complex into the crossvein region (Schleede, 2015).
Collagen IV provides the best-studied example for how the ECM might affect Sog activity. The two D. melanogaster collagen IV chains regulate BMP signaling in other contexts, and they bind both Sog and the BMP Dpp. Results suggest that collagen IV helps assemble and release a Dpp/Sog/Tsg shuttling complex, and also recruits the Tld protease that cleaves Sog cleavage and releases Dpp for signaling. D. melanogaster Mmp1 can cleave vertebrate Collagen IV. Since reduced Gyc76C and For activity increases abnormal Collagen IV aggregates throughout the wing and diffuse Collagen IV in the veins, it ishypothesized that these Collagen IV changes both foster the assembly or stability of Sog/Cv-Tsg2/BMP complexes and tether them to the ECM, favoring the sequestration of BMPs in the complex and reducing thelong-range movement of the complex into the region of the PCV (Schleede, 2015).
While few other D. melanogaster Mmp targets have been identified, it is likely that Mmp1 and Mmp2 share the broad specificity of their mammalian counterparts, so other ECM components, known or unknown, might be involved. For instance, vertebrate Perlecan and can be cleaved by Mmps. Trol regulates BMP signaling in other D. melanogaster contexts, and Trol overexpression rescue gyc76C knockdown's effects on BMP signaling. But while null trol alleles are lethal before pupal stages, normal PCVs were formed in viable and even adult lethal alleles like trolG0023, and actin-Gal 4-driven expression of trol-RNAi using any of four different trol-RNAi lines did not alter adult wing venation. Loss of the D. melanogaster laminin B chain shared by all laminin trimers strongly disrupts wing venation, and a zebrafish laminin mutation can reduce BMP signaling (Schleede, 2015).
Finally, it was recently shown that Dlp, one of the two D. melanogaster glypicans, can be removed from the cell surface by Mmp2. While gyc76C knockdown did not detectably alter anti-Dlp staining in the pupal wing, it is noteworthy that Dlp and the second glypican Dally are required non-autonomously for BMP signaling in the PCV and that they bind BMPs and other BMP-binding proteins.
In polarized epithelial cells, receptor-ligand interactions can be restricted by different spatial distributions of the 2 interacting components, giving rise to an underappreciated layer of regulatory complexity. This study explored whether such regulation occurs in the Drosophila wing disc, an epithelial tissue featuring the TGF-β family member Decapentaplegic (Dpp) as a morphogen controlling growth and patterning. Dpp protein has been observed in an extracellular gradient within the columnar cell layer of the disc, but also uniformly in the disc lumen, leading to the question of how graded signaling is achieved in the face of 2 distinctly localized ligand pools. The Dpp Type II receptor Punt was found to be enriched at the basolateral membrane and depleted at the junctions and apical surface. Wit, a second Type II receptor, shows a markedly different behavior, with the protein detected on all membrane regions but enriched at the apical side. Mutational studies identified a short juxtamembrane sequence required for basolateral restriction of Punt in both wing discs and mammalian Madin-Darby canine kidney (MDCK) cells. This basolateral targeting (BLT) determinant can dominantly confer basolateral localization on an otherwise apical receptor. Rescue of punt mutants with transgenes altered in the targeting motif showed that flies expressing apicalized Punt due to the lack of a functional BLT displayed developmental defects, female sterility, and significant lethality. This study found that basolateral presentation of Punt is required for optimal signaling. Finally, evidence is presented that the BLT acts through polarized sorting machinery that differs between types of epithelia. This suggests a code whereby each epithelial cell type may differentially traffic common receptors to enable distinctive responses to spatially localized pools of extracellular ligands (Peterson, 2022).
Tissue organization requires the interplay between biochemical signaling and cellular force generation. The formation of straight boundaries separating cells with different fates into compartments is important for growth and patterning during tissue development. In the developing Drosophila wing disc, maintenance of the straight anteroposterior (AP) compartment boundary involves a local increase in mechanical tension at cell bonds along the boundary. The biochemical signals that regulate mechanical tension along the AP boundary, however, remain unknown. This study shows that a local difference in Hedgehog signal transduction activity between anterior and posterior cells is necessary and sufficient to increase mechanical tension along the AP boundary. This difference in Hedgehog signal transduction is also required to bias cell rearrangements during cell intercalations to keep the characteristic straight shape of the AP boundary. Moreover, severing cell bonds along the AP boundary does not reduce tension at neighboring bonds, implying that active mechanical tension is upregulated, cell bond by cell bond. Finally, differences in the expression of the homeodomain-containing protein Engrailed also contribute to the straight shape of the AP boundary, independently of Hedgehog signal transduction and without modulating cell bond tension. The data reveal a novel link between local differences in Hedgehog signal transduction and a local increase in active mechanical tension of cell bonds that biases junctional rearrangements. The large-scale shape of the AP boundary thus emerges from biochemical signals inducing patterns of active tension on cell bonds (Rudolf, 2015).
This study has analyzed the links between the determination of cell fate and the physical and mechanical mechanisms shaping the AP boundary of larval Drosophila wing discs. Previous work has shown a role for the transcription factors Engrailed and Invected and the Hedgehog signal transduction pathway in organizing the segregation of anterior and posterior cells of the wing disc. This study now shows that a difference in Hedgehog signal transduction between anterior and posterior cells significantly contributes to the straight shape of the AP boundary by autonomously and locally increasing mechanical cell bond tension that in turn biases the asymmetry of cell rearrangements during cell intercalations. Furthermore, Engrailed and Invected also contribute to maintaining the characteristic straight shape of the AP boundary by mechanisms that are independent of Hedgehog signal transduction and do not appear to modulate cell bond tension (Rudolf, 2015).
In the wild-type wing disc, anterior cells transducing the Hedgehog signal are juxtaposed to posterior cells that do not transduce the Hedgehog signal. Three cases were genereated to test whether this difference in Hedgehog signal transduction is important for the straight shape of the AP boundary, the morphological and molecular signature of cells along the AP boundary, and the local increase in cell bond tension. In case I, Hedgehog signal transduction was low (or absent) in both A and P cells. In case II, Hedgehog signal transduction was high in both A and P cells. And in case III, Hedgehog signal transduction was high in P cells, but low in A cells, reversing the normal situation. In cases I and II the AP boundary was no longer as straight as in the wild-type situation. Moreover, the increased apical cross-section area of cells along the AP boundary that is characteristic for the wild type was no longer seen. Finally, the levels of F-actin and cell bond tension were no longer increased along the AP boundary. In case III, it was found that the difference in Hedgehog signal transduction is sufficient to maintain the characteristic straight shape of the AP boundary, to induce the morphological signatures of cells along the AP boundary and to increase F-actin and mechanical tension. Taken together, these experiments establish that the difference in Hedgehog signal transduction between anterior and posterior cells plays a key role in increasing cell bond tension along the AP boundary, in maintaining the characteristic shape of the AP boundary, and in defining the molecular and morphological signatures of cells along the AP boundary. These findings account for the observation that while Hedgehog signal transduction is active within the strip of anterior cells, the increase in mechanical tension is confined to cell bonds along the AP boundary, where cells with highly different Hedgehog signal transduction activities are apposed. The small differences in Hedgehog signal transduction activity that might exist between neighboring rows of anterior cells in the vicinity of the AP boundary appear to be insufficient to increase cell bond tension. Importantly, Hedgehog signal transduction per se does not increase cell bond tension along the AP boundary. The role of Hedgehog signal transduction along the AP boundary thus differs from its roles during other morphogenetic processes in which all cells that transduce the Hedgehog signal, for example, respond by accumulation of F-actin and a change in shape. It will be interesting to elucidate the molecular mechanisms by which cells perceive a difference in Hedgehog signal transduction, and how such a difference in Hedgehog signal transduction results in increased cell bond tension (Rudolf, 2015).
F-actin and Myosin II are enriched along the AP boundary. Based on similar observations, the existence of actomyosin cables has been proposed for several compartment boundaries, including the AP boundary in the Drosophila embryonic epidermis, the DV boundary of Drosophila wing discs and the rhombomeric boundaries in zebrafish embryos. Actomyosin cables have been proposed to maintain the straight shape of compartment boundaries by acting as barriers of cell mixing between cells of the adjacent compartments. Actomyosin cables are also characteristic of additional processes, e.g. dorsal closure and germband extension in the Drosophila embryo, tracheal tube invagination and neural plate bending and elongation. During Drosophila germ band extension, it has been shown that mechanical tension is higher at cell bonds that are part of an actomyosin cable compared with isolated cell bonds, indicating that cell bond tension is influenced by higher-order cellular organization during this process. The results, based on laser ablation experiments, show that the increased cell bond tension along the AP boundary can be induced by single cells and does not depend on the integrity of the actomyosin cable. Thus, these data instead indicate that increased cell bond tension is autonomously generated cell bond by cell bond along the AP boundary. This suggests that differences in Hedgehog signal transduction activity regulate the structure and mechanical properties of cell junctions between adjacent cells and in particular upregulate an active mechanical tension, mediated by actomyosin contractility (Rudolf, 2015).
The cell cortex is a thin layer of active material that is under mechanical tension. In addition to viscous and elastic stresses, active stresses generated by actomyosin contractility are an important contribution. Adherens junctions are adhesive structures that include elements of the cell cortices of the adhering cells. Locally generated active tension, therefore, can largely determine the cell bond tension as long as cell bonds do not change length or rearrange. As a consequence, locally generated active tension also sets the cell bond tension at the actomyosin cable along the AP boundary. This view is consistent with experiments in which cell bond tension remains high even if the integrity of the actomyosin cable is lost. These mechanical properties of cell junctions along the AP boundary are thus different from those of a conventional string or cable in which elastic stresses are associated with stretching deformations. Such elastic stresses relax and largely disappear when the cable is severed. Thus, this work suggests that the mechanical properties of the actomyosin cable along the AP boundary are very different from those of a conventional cable, but fit well in the concepts of active tension studied in the cell cortex, e.g., in Caenorhabditis elegans. This active tension is a local property that can be set by local signals irrespective of the local force balances. Force balances rather determine movements and rearrangements, e.g. upon laser ablation (Rudolf, 2015).
How does a local increase in actively generated cell bond tension contribute to the straight shape of the AP boundary? Previous work showed that cell intercalations promote irregularities in the shape of compartment boundaries. The local increase in active cell bond tension enters the force balances during cell rearrangements. During cell intercalation, differences in active cell bond tension between junctions along the AP boundary and neighboring junctions are balanced by frictional forces associated with vertex movements. As a result, vertex movements are biased such that the AP boundary remains straight and cell mixing between neighboring compartments is suppressed. The observation that a local difference in Hedgehog signal transduction upregulates active cell bond tension leads to the prediction that cell rearrangements along the AP boundary should not be biased if there is no difference in Hedgehog signal transduction. This is indeed what was found in case II (Rudolf, 2015).
It has been previously suggested that the engrailed and invected selector genes play a role in maintaining the separation of anterior and posterior cells that is independent of Hedgehog signal transduction. Quantitative analysis of clone shapes in this study supports this notion. It is speculated that this Hedgehog-independent pathway contributes to the remarkably straight shape of the AP boundary in cases I and II, in which Hedgehog signal transduction activities between anterior and posterior cells have been nearly equalized. Two lines of evidence indicate that the Hedgehog-independent pathway shapes the AP boundary without modulating cell bond tension. First,several cases have been generated in which neighboring cell populations differed in the expression of Engrailed and Invected, but not in Hedgehog signal transduction activity. In none of these cases was an increase in cell bond tension detected along the interface of these two cell populations. Second, in cases in which a difference was created in Hedgehog signal transduction between two cell populations in the absence of differences in Engrailed and Invected expression, the same increase was detected in cell bond tension between these cell populations compared with the wild-type compartment boundary (Rudolf, 2015).
Previously studies have described several physical mechanisms that shape the DV boundary of wing discs. In addition to a local increase in mechanical tension along the DV boundary, evidence was provided that oriented cell division and cell elongation created by anisotropic stress contribute to the characteristic shape of the DV boundary. It is therefore conceivable that the Hedgehog-independent pathway influences the shape of the AP boundary by one or more of these mechanisms (Rudolf, 2015).
It is proposed that the AP boundary is shaped by mechano-biochemical processes that integrate signaling pathways with patterns of cell mechanical properties. In tjos model, Engrailed and Invected shape the AP boundary with the help of two different mechanisms. (1) Engrailed and Invected result in a difference in Hedgehog signal transduction between anterior and posterior cells. This difference leads to a cell-autonomous increase in F-actin and active cell bond tension along the AP boundary. The local increase in active cell bond tension then biases the asymmetry of cell rearrangements during cell intercalations and thereby contributes to maintaining the straight shape of the AP boundary. (2) Engrailed and Invected contribute independently of Hedgehog signal transduction to the straight shape of the AP boundary by an as yet unknown mechanism not involving the modulation of cell bond tension. The first mechanism uses biochemical signals to create mechanical patterns that subsequently guide junctional dynamics to organize a straight compartment boundary. It is speculated that the second mechanism also involves a mechano-chemical process, even though the nature of this process is currently unknown. The current work suggests that the large-scale shape of the AP boundary thus emerges from the collective behavior of many cells that locally exchange biochemical signals and regulate active mechanical tension (Rudolf, 2015).
Decades of research on the highly modified wings of Drosophila melanogaster has suggested that insect wings are divided into two Anterior-Posterior (A-P) compartments separated by an axis of symmetry. This axis of symmetry is created by a developmental organizer that establishes symmetrical patterns of gene expression that in turn pattern the A-P axis of the wing. Butterflies possess more typical insect wings and butterfly wing colour patterns provide many landmarks for studies of wing structure and development. Using eyespot colour pattern variation in Vanessa butterflies, this study shows an additional A-P axis of symmetry running between wing sectors 3 and 4. Boundaries of Drosophila mitotic clones suggest the existence of a previously undetected Far-Posterior (F-P) compartment boundary that coincides with this additional A-P axis. A similar compartment boundary is evident in butterfly mosaic gynandromorphs. It is suggested that this additional compartment boundary and its associated developmental organizer create an axis of wing colour pattern symmetry and a gene expression-based combinatorial code, permitting each insect wing compartment to acquire a unique identity and allowing for the individuation of butterfly eyespots (Abbasi, 2017).
This is a response to a recent report by Abbasi and Marcus who present two main findings: first that study argued that there is an organiser and a compartment boundary within the posterior compartment of the butterfly wing. Second, that study presented evidence for a previously undiscovered lineage boundary near wing vein 5 in Drosophila, a boundary that delineates a "far posterior" compartment. Clones of cells were marked with the yellow mutation and that study reported that these clones always fail to cross a line close to vein 5 on the Drosophila wing. In the current study yellow proved an unusable marker for clones in the wing blade and therefore the matter was reexamined. Clones of cells were marked with multiple wing hairs or forked, and a substantial proportion of these clones were found to cross the proposed lineage boundary near vein 5. As internal controls, these same clones were shown to respect the other two well established compartment boundaries: the anteroposterior compartment boundary is always respected. The dorsoventral boundary is mostly respected, and is crossed only by clones that are induced early in development, consistent with many reports (Lawrence, 2019).
The subdivision of cell populations in compartments is a key event during animal development. In Drosophila, the gene apterous (ap) divides the wing imaginal disc in dorsal vs ventral cell lineages and is required for wing formation. ap function as a dorsal selector gene has been extensively studied. However, the regulation of its expression during wing development is poorly understood. This study analyzed ap transcriptional regulation at the endogenous locus and identified three cis-regulatory modules (CRMs) essential for wing development. Only when the three CRMs are combined, robust ap expression is obtained. In addition, the trans-factors that regulate these CRMs were genetically and molecularly analyzed. The results propose a three-step mechanism for the cell lineage compartment expression of ap that includes initial activation, positive autoregulation and Trithorax-mediated maintenance through separable CRMs (Bieli, 2015).
Genetic and cis-regulatory analysis has provided information about the logic of ap expression during wing development. It is proposed that ap expression is controlled by at least three CRMs that act in combination. The first element, apE is the earliest to be activated in proximal wing disc cells via the EGFR pathway; its expression subsequently weakens in the wing pouch. Deletion of this early enhancer (e.g., apDG12 or apC1345) completely abolishes wing formation. The asymmetry of ap expression to the proximal domain of the wing disc is probably due to the localized activation of the EGFR pathway by its ligand Vn and a distal repression by Wg signaling. The initial activation of the apE by the EGFR pathway was genetically and molecularly confirmed; however, other inputs are required for the continuous activation of this CRM in later wing discs (Bieli, 2015).
A few hours after apE activation, a second CRM, apDV, is activated in a subset of apE positive cells. In contrast to apE, apDV is restricted to the dorsal-distal domain of the wing pouch by direct positive inputs from Ap and Vg/Sd. The direct Ap autoregulatory input defines the time window when the apDV element is activated; apDV can only be active after the induction of Ap by the early enhancer (apE). It has been shown that Ap induces vg expression by triggering Notch signaling at the D/V boundary. Thus, the (direct) input of Vg/Sd on apDV can be regarded as an indirect positive autoregulation, which delimits the spatial domain where apDV can be actived. Consequently, the interface of Ap and Vg expression defines the region of apDV activity via positive autoregulation (Bieli, 2015).
The third ap CRM is the ap PRE/TRE region (apP), that, when deleted, leads to a strong hypomorphic wing phenotype (apc1.2b). The apP requires Trx input and maintains ap expression when placed in cis with the apDV and apE CRMs. Only the combination of the three CRMs faithfully reproduces ap expression in the wing disc. Moreover, the regulatory in locus deletion and in situ rescue analysis provide strong functional relevance for these CRMs (Bieli, 2015).
Ultimately, this cascade of ap CRMs provides a mechanism to initiate, refine and maintain ap expression during wing imaginal disc development, in which the later CRMs depend on the activity of the early ones. A similar mechanism has been described for Distal-less (Dll) regulation in the leg primordia where separate CRMs trigger and maintain Dll expression in part by an autoregulatory mechanism (Bieli, 2015).
It has been proposed that positive autoregulation may help to maintain the epigenetic memory of differentiation. In the case of ap, this study demonstrates that autoregulation works in conjunction with a PRE/TRE system; this might make the system very robust and refractory to perturbations (Bieli, 2015).
ChIP experiments have shown that many developmentally important genes are associated with a promoter proximal PRE as found at ap. The role of such a PRE has been studied at the engrailed (en) locus. It has been demonstrated that in imaginal discs, the promoter as well as the promoter proximal PRE are important for the long-range action of en enhancers. It has been proposed that this PRE brings chromatin together, allowing both positive and negative regulatory interactions between distantly located DNA fragments (Bieli, 2015).
The current results indicate that sequences around the transcription start of ap (apP) may serve a similar function. First, this element, when placed in cis with the ap CRMs (apE and apDV), maintains the ap expression pattern and keeps reporter gene expression off in cells where low or no activity of apDV and apE has been observed. Second, in the absence of trx, the expression of ap and apDV+E+P-lacZ is strongly reduced. All these data suggest that sequences within the apP integrate Trx input, thereby maintaining ap expression in a highly proliferative tissue such as the wing disc. Interestingly, trx mutant clones were not round and did not show ectopic wg activation, which is a hallmark of ap loss-of-function clones. This suggests that in trx mutant clones enough Ap protein is still present to maintain wg expression off. However, derepression of the ventral-specific integrin αPS2 was found in trx mutant clones in the wing pouch as previously described for ap mutant clones (Bieli, 2015).
It has been suggested that TrxG proteins could act passively antagonizing PcG silencing, rather than playing an active role as co-activators of gene transcription. For example, Ubx expression in the leg and haltere does not require Trx in the absence of Polycomb repression. These possibilities were tested and trx mutant clones were generated that were also mutant for the PcG member Sex combs on midlegs (Scm). Dorsally-located Scm- trx- double mutant clones still downregulate ap-lacZ expression while ventral-induced ones are unable to derepress ap-lacZ as was observed for Scm- single mutant clones. Therefore, the results suggest that TrxG maintains ap expression in dorsal cells, while ap expression is repressed in the ventral compartment by PcG proteins. Moreover, it has been shown that the sequences around the ap transcription start, including the PRE, are occupied by PcG complexes PRC1 and PRC2, as well as Trx (Bieli, 2015).
Enhancers-promoter interactions initiate transcription but their dynamics during development have remained poorly understood. A Chromosome conformation capture (3C) experiment provides evidence for the direct interaction between the ap CRMs apE and apDV with the maintenance element encoded by the apP. Beyond this, it was also found that these elements cooperate continuously during wing development. Flip-out experiments, in which the apDV and apE CRMs were removed at different time points, suggest that these elements need to be present continuously to ensure correct ap expression. Additionally, flies carrying apE only on one chromosome and apDV only on the homologue were unable to fully rescue wing development suggesting that these CRMs need to be in cis. It is conceivable that in cis configuration of the three ap CRMs facilitates and stabilizes enhancer-promoter looping. It could also help to rapidly establish relevant chromatin contacts after each cell division. These results are in accordance with previous observations, in which constant interactions between ap enhancers and promoter during embryogenesis have been described. The current results extend these observations to the wing disc, a highly proliferative tissue, where the expression of the trans-factors that regulate the activity of the apE and apDV is very dynamic. This raises the question on how this contact is re-assembled over many cell generations. It is possible that some epigenetic modifications are laid down in the activated apE and apDV CRMs, which are then inherited during cell divisions to ensure contact with apP. Studies of the chromatin status of these elements will be required to fully understand this process (Bieli, 2015).
A key question in developmental biology is how transcriptional regulation is coupled to tissue growth to precisely regulate gene expression in a spatio-temporal manner. For example, during Drosophila leg development, initial activation of the ventral appendage gene Dll by high levels of Wg and Dpp initiates a cascade of cross-regulation between Dll and Dachshund (Dac) and positive feedback loops that patterns the proximo-distal axis. Other mechanisms to expand gene expression patterns depend on memory modules such as PREs, as it is the case for the Hox genes or other developmental genes like hh. To direct wing formation, expression of ap in the highly proliferative tissue of the wing disc must be precisely induced to generate and maintain the D/V border. These in-depth analyses at the ap locus provide a functional and molecular explanation of how expression of this dorsal selector gene is initiated, refined at the D/V border, and maintained during wing disc development. It is proposed that this three-step mechanism may be common for developmental patterning genes to make the developmental program robust to perturbations (Bieli, 2015).
In higher organisms, the phenotypic impacts of potentially harmful or beneficial mutations are often modulated by complex developmental networks. Stabilizing selection may favor the evolution of developmental canalization-that is, robustness despite perturbation-to insulate development against environmental and genetic variability. In contrast, directional selection acts to alter the developmental process, possibly undermining the molecular mechanisms that buffer a trait's development, but this scenario has not been shown in nature. This study examined the developmental consequences of size increase in highland Ethiopian Drosophila melanogaster. Ethiopian inbred strains exhibited much higher frequencies of wing abnormalities than lowland populations, consistent with an elevated susceptibility to the genetic perturbation of inbreeding. Mutagenesis was then used to test whether Ethiopian wing development is, indeed, decanalized. Ethiopian strains were far more susceptible to this genetic disruption of development, yielding 26 times more novel wing abnormalities than lowland strains in F2 males. Wing size and developmental perturbability cosegregated in the offspring of between-population crosses, suggesting that genes conferring size differences had undermined developmental buffering mechanisms. These findings represent the first observation of morphological evolution associated with decanalization in the same tissue, underscoring the sensitivity of development to adaptive change (Lack, 2016).
The cuticular exoskeleton of insects and other arthropods is a remarkably
versatile material with a complex multilayer structure. This study
isolated cuticle synthesizing cells in relatively pure form by dissecting
pupal wings and used RNAseq to identify
genes expressed during the formation of the adult wing cuticle. Dramatic
changes in gene expression during cuticle deposition were observed, and
combined with transmission electron microscopy, candidate genes for the
deposition of the different cuticular layers were identified. Among genes
of interest that dramatically change their expression during the cuticle
deposition program are ones that encode cuticle proteins, ZP domain
proteins, cuticle modifying proteins and transcription factors, as well as
genes of unknown function. A striking finding is that mutations in a
number of genes that are expressed almost exclusively during the
deposition of the envelope (the thin outermost layer that is deposited
first) result in gross defects in the procuticle (the thick chitinous
layer that is deposited last). An attractive hypothesis to explain this is
that the deposition of the different cuticle layers is not independent
with the envelope instructing the formation of later layers.
Alternatively, some of the genes expressed during the deposition of the
envelope could form a platform that is essential for the deposition of all
cuticle layers (Sobala, 2016).
Wings are essential for insect fitness. A number of proteins and enzymes have been identified to be involved in wing terminal differentiation, which is characterized by the formation of the wing cuticle. This study addressed the question whether Chitinase 10 (Cht10) may play an important role in chitin organization in the wings of the fruit fly Drosophila melanogaster. Cht10 expression was found to coincide with the expression of the chitin synthase coding gene kkv. This suggests that the respective proteins may cooperate during wing differentiation. In tissue-specific RNA interference experiments, it was demonstrated that suppression of Cht10 causes an excess in chitin amounts in the wing cuticle. Chitin organization is severely disrupted in these wings. Based on these data, it is hypothesized that Cht10 restricts chitin amounts produced by Kkv in order to ensure normal chitin organization and wing cuticle formation. In addition, it was found by scanning electron microscopy that Cht10 suppression also affects the cuticle surface. In turn, cuticle inward permeability is enhanced in Cht10-less wings. Moreover, flies with reduced Cht10 function are unable to fly. In conclusion, Cht10 is essential for wing terminal differentiation and function (Dong, 2020).
The polysaccharide chitin is a major scaffolding molecule in the insect cuticle. In order to be functional, both chitin amounts and chitin organization have been shown to be important parameters. Despite great advances in the past decade, the molecular mechanisms of chitin synthesis and organization are not fully understood. This study has characterized the function of the Chitinase 6 (Cht6) in the formation of the wing, which is a simple flat cuticle organ, in the fruit fly Drosophila melanogaster. Reduction of Cht6 function by RNA interference during wing development does not affect chitin organization, but entails a thinner cuticle suggesting reduced chitin amounts. This phenotype is opposed to the one reported recently to be caused by reduction of Cht10 expression. Probably as a consequence, cuticle permeability to xenobiotics is enhanced in Cht6-less wings. Massive deformation of these wings was also observed. In addition, the shape of the abdomen is markedly changed upon abdominal suppression of Cht6. Finally, it was found that suppression of Cht6 transcript levels influences the expression of genes coding for enzymes of the chitin biosynthesis pathway. This finding indicates that wing epidermal cells respond to activity changes of Cht6 probably trying to adjust chitin amounts. Together, in a working model, it is proposed that Cht6-introduced modifications of chitin are needed for chitin synthesis to proceed correctly. Cuticle thickness, according to this hypothesis, is in turn required for correct organ or body part shape. The molecular mechanisms of this processes shall be characterized in the future (Dong, 2022).
Chitin is an aminopolysaccharide present in insects as a major structural component of the cuticle. However, current knowledge on the chitin biosynthetic machinery, especially its constituents and mechanism, is limited. Using three independent binding assays, including co-immunoprecipitation, split-ubiquitin membrane yeast two-hybrid assay, and pull-down assay, this study demonstrated that choline transporter-like protein 2 (Ctl2) interacts with krotzkopf verkehrt (kkv) in Drosophila melanogaster. The global knockdown of Ctl2 by RNA interference (RNAi) induced lethality at the larval stage. Tissue-specific RNAi to silence Ctl2 in the tracheal system and in the epidermis of the flies resulted in lethality at the first larval instar. The knockdown of Ctl2 in wings led to shrunken wings containing accumulated fluid. Calcofluor White staining demonstrated reduced chitin content in the first longitudinal vein of Ctl2 knockdown wings. The pro-cuticle, which was thinner compared to wildtype, exhibited a reduced number of chitin laminar layers. Phylogenetic analyses revealed orthologues of Ctl2 in different insect orders with highly conserved domains. These findings provide new insights into cuticle formation, wherein Ctl2 plays an important role as a chitin-synthase interacting protein (Duan, 2022).
Chitin, the major structural polysaccharide in arthropods such as insects and mites, is a linear polymer of N-acetylglucosamine units. The growth and development of insects are intimately coupled with chitin biosynthesis. The membrane-bound β-glycosyltransferase chitin synthase is known to catalyze the key polymerization step of N-acetylglucosamine. However, the additional proteins that might assist chitin synthase during chitin biosynthesis are not well understood. Recently, fatty acid binding protein (Fabp) has been suggested as a candidate that interacts with the chitin synthase Krotzkopf verkehrt (Kkv) in Drosophila melanogaster. Using split-ubiquitin membrane yeast two-hybrid and pull-down assays, this study has demonstrated that the Fabp-B splice variant physically interacts with Kkv in vitro. The global knockdown of Fabp in D. melanogaster using RNA interference (RNAi) induced lethality at the larval stage. Moreover, in tissue-specific RNAi experiments, silenced Fabp expression in the epidermis and tracheal system caused a lethal larval phenotype. Fabp knockdown in the wings resulted in an abnormal wing development and uneven cuticular surface. In addition to reducing the chitin content in the first longitudinal vein of wings, Fabp silencing also caused the loss of procuticle laminate structures. This study revealed that Fabp plays an important role in chitin synthesis and contributes to a comprehensive understanding of the complex insect chitin biosynthesis (Chen, 2023).
Resilin is a protein matrix in movable regions of the cuticle conferring resistance to fatigue. The main component of Resilin is Pro-Resilin that polymerises via covalent di- and tri-tyrosine bounds (DT). Loss of Pro-Resilin is nonlethal and causes a held-down wing phenotype (hdw) in the fruit fly Drosophila melanogaster. To test whether this mild phenotype is recurrent in other insect species, resilin was analyzed in the spotted-wing fruit fly Drosophila suzukii. As quantified by DT autofluorescence by microscopy, DT intensities in the trochanter and the wing hinge are higher in D. suzukii than in D. melanogaster, while in the proboscis the DT signal is stronger in D. melanogaster compared to D. suzukii. To study the function of Pro-Resilin in D. suzukii, a mutation was generated in the proresilin gene applying the Crispr/Cas9 technique. D. suzukii pro-resilin mutant flies are flight-less and show a hdw phenotype resembling respective D. melanogaster mutants. DT signal intensity at the wing hinge is reduced but not eliminated in D. suzukii hdw flies. Either residual Pro-Resilin accounts for the remaining DT signal or, as proposed for the hdw phenotype in D. melanogaster, other DT forming proteins might be present in Resilin matrices. Interestingly, DT signal intensity reduction rates in D. suzukii and D. melanogaster are somehow different. Taken together, in general, the function of Pro-Resilin seems to be conserved in the Drosophila genus; small differences in DT quantity, however, lead to the hypothesis that Resilin matrices might be modulated during evolution probably to accommodate the species-specific lifestyle (Lerch, 2022).
Regulatory mechanisms for tissue repair and regeneration within damaged tissue have been extensively studied. However, the systemic regulation of tissue repair remains poorly understood. To elucidate tissue nonautonomous control of repair process, it is essential to induce local damage, independent of genetic manipulations in uninjured parts of the body. This study developed a system in Drosophila for spatiotemporal tissue injury using a temperature-sensitive form of diphtheria toxin A domain driven by the Q system to study factors contributing to imaginal disc repair. Using this technique, it was demonstrated that methionine metabolism in the fat body, a counterpart of mammalian liver and adipose tissue, supports the repair processes of wing discs. Local injury to wing discs decreases methionine and S-adenosylmethionine, whereas it increases S-adenosylhomocysteine in the fat body. Fat body-specific genetic manipulation of methionine metabolism results in defective disc repair but does not affect normal wing development. The data indicate the contribution of tissue interactions to tissue repair in Drosophila, as local damage to wing discs influences fat body metabolism, and proper control of methionine metabolism in the fat body, in turn, affects wing regeneration (Kashio, 2016).
Morphogenesis of the adult structures of holometabolous insects is regulated by ecdysteroids and juvenile hormones and involves cell-cell interactions mediated in part by the cell surface integrin receptors and their extracellular matrix (ECM) ligands. These adhesion molecules and their regulation by hormones are not well characterized. This study describes the gene structure of a newly described ECM molecule, tenectin, and demonstrate that it is a hormonally regulated ECM protein required for proper morphogenesis of the adult wing and male genitalia. Tenectin's function as a new ligand of the PS2 integrins is demonstrated by both genetic interactions in the fly and by cell spreading and cell adhesion assays in cultured cells. Its interaction with the PS2 integrins is dependent on RGD and RGD-like motifs. Tenectin's function in looping morphogenesis in the development of the male genitalia led to experiments that demonstrate a role for PS integrins in the execution of left-right asymmetry (Fraichard, 2010).
Tenectin is a protein localized to the ECM during Drosophila embryonic development. The presence of an integrin-binding RGD motif led to a speculation that tenectin could be a new integrin ligand. To study the function of tenectin during Drosophila development, tenectin knockdowns were generated by RNA interference. Two strains of tenectin knockdown flies were selected that gave visible hypomorphic phenotypes. Flies were also characterized that give phenotypes due to overexpression of the endogenous tenectin gene. Lowering mRNA level by RNAi partially rescued the effects of tenectin overexpression and overexpression of tenectin partially rescues tenectin knockdown phenotypes. Thus, the authors are confident that the tenectin knockdown phenotypes result specifically from reduced tenectin expression (Fraichard, 2010).
Lethality is the most prevalent phenotype displayed by ubiquitous reduction in tenectin expression but this study focused on adult phenotypes to ascertain tenectin's function in morphogenetic processes of metamorphosis. The most striking adult phenotype observed in adult flies with reduced tenectin expression is deformed wings including blisters, nicks, lack of expansion and malformation. These phenotypes resemble those associated with mutations in integrin subunits, their extracellular ligands, and genes encoding intracellular proteins that interact with integrins. Three lines of evidence support tenectin functioning as a PS integrin ligand to facilitate wing morphogenesis. First, tenectin protein was found to localize between the dorsal and ventral epithelial cell layers in prepupal wings. Integrins function at this location to promote adhesion of these cell layers. Second, a mutation of mys, encoding the βPS subunit, interacts genetically to increase the frequency of blisters in flies with reduced tenectin expression. Finally, in vitro experiments demonstrate that tenectin, through multiple RGD motifs, can function to promote αPS2βPS-mediated cell spreading and adhesion. Taken together, these genetic and biochemical data provide strong evidence that tenectin is a new ligand of αPS2βPS integrin in the wing (Fraichard, 2010).
Perhaps relevant to tenectin's function in the wing, Syed (2008), using a bioinformatics approach, identified tenectin as being a mucin-related-protein. In an analysis of the tenectin protein this study also notice mucin like repeats. Mucins are highly hydrated O-glycosylated macromolecules that are important to the mucosal lining of mammalian organs. In addition to serving a protective function, various mucins interact with growth factors and cell surface receptors to modulate signaling. It has been shown in vertebrates that mucins also modulate cell adhesion. For example, MUC4 was found to sterically reduce the accessibility of integrins to extracellular matrix ligands and thereby interfere with adhesion. Interestingly, a mucin-type glycosyltransferase, PGANT3, glycosylates another PS2 integrin ligand, tiggrin. Moreover, mutation of the pgant3 gene results in a wing-blistering phenotype. In the developing wing disc PGANT3 glycosylates tiggrin and other matrix molecules, thus potentially modulating cell adhesion through integrin-ECM interactions. Future biochemical experiments will be needed to determine if tenectin is a bona fide mucin, glycosylated by PGANT3, and whether glycosylation down- or up-regulates its adhesive function (Fraichard, 2010).
The formation of the flat bi-layered wing from a folded imaginal disc involves several steps of apposition and separation of the ventral and dorsal epidermal sheets followed ultimately by an epithelial to mesenchymal transition and migration of the cells out of the wing. The resulting wing is predominantly two layers of cuticle cemented together by ECM. These studies point out the importance of regulating the adhesive properties of the wing epidermal cells by modulating the activity of integrins and their intracellular and extracellular binding partners. One mode of regulation is at the transcriptional level and several studies have demonstrated that the hormone 20E plays an important role in regulating at least some of these morphogenetic events including integrin expression levels. Consistent with tenectin's role in wing morphogenesis this study found that during metamorphosis tenectin mRNA expression correlates with the ecdysone titer profile. In vitro, imaginal disc cultures demonstrate that tenectin is a 20E target gene. The comparison of the developmental tenectin expression profile with those of early (E74A, E74B) and prepupal (β-Ftz-F1) genes defined more precisely the temporal expression pattern of tenectin. E74B is a class I transcript, induced in mid-third instar larvae in response to a low concentration of 20E and repressed at higher ecdysone concentrations. In contrast, the class II transcripts, including E74A, are induced by high 20E concentration and their expressions are unaffected by higher 20E concentrations. The temporal profile of tenectin is similar to those of E74A, with a slight delay in the peak levels of tenectin mRNA accumulation. This temporal delay in tenectin is similar to the delay observed in the early-late gene profiles. The early-late genes appear to share properties with both the early genes and late genes. Early-late genes respond directly to ecdysone even in the presence of protein synthesis inhibitors like cycloheximide but unlike early genes their full induction requires protein synthesis due to a requirement for other ecdysone induced gene products. It is proposed that tenectin is an early-late gene as its expression in cultured larval organs was induced by 20E in the presence of cycloheximide but maximal induction required protein synthesis. In the wing, it is proposed that 20E also regulates morphogenesis by regulation of tenectin mRNA levels, suggesting that ecdysone controls wing morphogenesis and cell adhesion not only by regulating integrin expression but also their ECM ligand expression. Just as E74A and E75B do not display identical expression profiles, the tenectin expression pattern is complicated and likely involves additional modes of regulation that will need to be elucidated (Fraichard, 2010).
Tenectin knockdown resulted in reduced rotation of male genitalia. Looping morphogenesis of the male genitalia occurs during the pupal stage as the genital disc undergoes a 360° dextral (clockwise) looping around the hindgut. A variety of genes expressed in larval posterior abdominal segments A8, A9 and A10 have been identified that affect male genital rotation. These include genes encoding a signaling protein (Pvf1), a transcription factor (Taf1, formerly TAF250), and a pro-apoptosis gene (hid). One adhesion molecule, fasciclin-2, was genetically demonstrated to be involved in genital rotation. However, the effect was indirect as Fas2spin mutant alters the synapses connecting neurosecretory cells to the organ that produces juvenile hormone (the corpora allata), and genitalia under-rotation is due to an excess of juvenile hormone. The effects on genitalia rotation have been shown to be mediated by an excess of juvenile hormone, a retinoic-like molecule, establishing a parallel between vertebrate and invertebrate left right asymmetry, since the retinoic acid is involved in the control of asymmetry in vertebrates. In Drosophila, excessive juvenile hormone may result in the attenuation of ecdysone regulated processes required for male genital rotation as mutations in Broad-Complex, an ecdysone early-response gene, also result in malrotation of male genitalia. Mutations of the unconventional myosin 31DF gene (Myo31DF) have been shown to uniquely reverse the looping direction of genitalia. Knockdown of tenectin in imaginal discs, but not in neuronal cells, resulted in incomplete rotation of the genitalia but not in direction of looping. Thus, this study has for the first time identified a Drosophila ECM component required for genital looping morphogenesis (Fraichard, 2010).
The tenectin mutant phenotype in male genitalia prompted a re-examination ofe integrin hypomorphic mutations for a similar phenotype. Males bearing 3 different hypomorphic mutations in the gene encoding the βPS integrin subunit, mysb13, mysb47, and mysb69 displayed under-rotated male genitalia when raised at elevated temperatures. A mutation has been described that was likely in myospheroid that produced under-rotated male genitalia when larvae and pupae were raised at elevated temperatures. Combining mysb13 with the if3 mutation in the gene encoding the αPS2 integrin subunit caused a dramatic increase in the expressivity of the rotated genitalia phenotype. Therefore, tenectin's proposed cell surface adhesion receptor is also required for the execution of looping morphogenesis. In addition to adhesion, the PS integrins function in the regulation of intracellular signaling pathways and specifically the JNK pathway. JNK signaling pathway has also been suggested to function in apoptosis required for rotation of male genitalia. Thus, tenectin and PS integrin function in looping morphogenesis could be at the level of adhesion and/or signaling. Additional experiments are required to distinguish between these two models (Fraichard, 2010).
Tenectin's RGD sequence in the 3rd von Willebrand factor type-C (VWC) domain is conserved in the beetle homolog, tenebrin, and supported PS2 integrin-mediated cell spreading. This result is expected given that RGD is a well known integrin-binding motif of the PS2 integrins. More novel is the presence in the identical location in the 5th VWC of the sequence RSD and elsewhere in this 5th repeat the occurrence of RDD and RYE sequences. The biological importance of the 5th VWC domain is supported by the extraordinary high degree of conservation in this domain between Drosophila tenectin and Tenebrio tenebrin. The two proteins share 92% (62/67) sequence identity in the 5th VWC repeat and this includes the RDD, RSD, and RYE sequences. To date, this domain is found conserved, with greater than 84% sequence identity, in mosquitoes, honey bees, crickets, wasps, the beetle, and aphids (not shown). While RGD is the best studied integrin-binding motif, experimental evidence is accumulating that variants of this sequence are also important. These variants include KQAGD, KGD, RSD, WGD, MVD and RYD found in fibrinogen, thrombospondin, tenascin-W, CD40, snake venom disintegrins, viral coat proteins, and ligand mimetic monoclonal antibodies. Cell adhesion assays demonstrate that VWC#5 as well as VWC#3 promotes cell adhesion mediated by PS2 integrins. Mutations of the individual RGD-variant motifs in VWF#5 suggest that they have differing effects on different integrins. The RDD is required for strong adhesion by both the PS2m8 and PS2c integrin isoforms as mutation of this sequence reduced adhesion of cells expressing either integrin. This is the first time the RDD tripeptide in an ECM protein has been found to function in integrin-mediated adhesion. It also appears that the RSD and RYE motifs may be inhibitory for adhesion mediated by the PS2c isoform as their mutations increased cell adhesion. With multiple integrin-binding domains, both positive and inhibitory, tenectin potentially functions in multiple processes in development and specifically in metamorphosis (Fraichard, 2010).
Future experiments will be required to address the many unanswered issues regarding tenectin–PS integrin interactions including: which PS integrin(s) interact with tenectin in vivo; how the function of the motifs may be affected by the context of other ECM proteins; and how other regions of tenectin and modifications, such as glycosylation or cleavage, influence the functionality of the putative integrin-binding motifs. The presence of multiple motifs also raises the possibility that tenectin can bridge integrins on neighboring cells, or on the surface of the same cell. Finally, the different motifs may be needed to bind different integrins at different times in development and this binding of different motifs may have different adhesive and/or signaling consequences (Fraichard, 2010).
Neuroanatomical evidence argues for the presence of taste sensilla in Drosophila wings; however, the taste physiology of insect wings remains hypothetical, and a comprehensive link to mechanical functions, such as flight, wing flapping, and grooming, is lacking. This study shows that the sensilla of the Drosophila anterior wing margin respond to both sweet and bitter molecules through an increase in cytosolic Ca2+ levels. Conversely, genetically modified flies presenting a wing-specific reduction in chemosensory cells show severe defects in both wing taste signaling and the exploratory guidance associated with chemodetection. In Drosophila, the chemodetection machinery includes mechanical grooming, which facilitates the contact between tastants and wing chemoreceptors, and the vibrations of flapping wings that nebulize volatile molecules as carboxylic acids. Together, these data demonstrate that the Drosophila wing chemosensory sensilla are a functional taste organ and that they may have a role in the exploration of ecological niches (Raad, 2016). Tissue folding is a fundamental process that shapes epithelia into complex 3D organs. The initial positioning of folds is the foundation for the emergence of correct tissue morphology. Mechanisms forming individual folds have been studied, but the precise positioning of folds in complex, multi-folded epithelia is less well-understood. This paper present a computational model of morphogenesis, encompassing local differential growth and tissue mechanics, to investigate tissue fold positioning. The Drosophila wing disc was used as a model system; there was shown to be spatial-temporal heterogeneity in its planar growth rates. This differential growth, especially at the early stages of development, is the main driver for fold positioning. Increased apical layer stiffness and confinement by the basement membrane drive fold formation but influence positioning to a lesser degree. The model successfully predicts the in vivo morphology of overgrowth clones and wingless mutants via perturbations solely on planar differential growth in silico (Tozluoglu, 2019).
Morphogen signaling proteins disperse across tissues to activate signal transduction in target cells. This study investigated dispersion of Hedgehog (Hh), Wnt homolog Wingless (Wg), and Bone morphogenic protein homolog Decapentaplegic (Dpp) in the Drosophila wing imaginal disc. Delivery of Hh, Wg, and Dpp to their respective targets was found to be regulated. <5% of Hh and <25% of Wg are taken up by disc cells and activate signaling. The amount of morphogen that is taken up and initiates signaling did not change when the level of morphogen expression was varied between 50-200% (Hh) or 50-350% (Wg). Similar properties were observed for Dpp. An area of 150 mm x 150 mm was analyzed that includes Hh-responding cells of the disc as well as overlying tracheal cells and myoblasts that are also activated by disc-produced Hh. The extent of signaling in the disc was unaffected by the presence or absence of the tracheal and myoblast cells, suggesting that the mechanism that disperses Hh specifies its destinations to particular cells, and that target cells do not take up Hh from a common pool (Hatori, 2021).
Adenosine triphosphate (ATP) production and utilization is critically important for animal development. How these processes are regulated in space and time during tissue growth remains largely unclear. This study used a FRET-based sensor to dynamically monitor ATP levels across a growing tissue, using the Drosophila larval wing disc. Although steady-state levels of ATP are spatially uniform across the wing pouch, inhibiting oxidative phosphorylation reveals spatial differences in metabolic behavior, whereby signaling centers at compartment boundaries produce more ATP from glycolysis than the rest of the tissue. Genetic perturbations indicate that the conserved Hedgehog signaling pathway can enhance ATP production by glycolysis. Collectively, this work suggests the existence of a homeostatic feedback loop between Hh signaling and glycolysis, advancing understanding of the connection between conserved developmental patterning genes and ATP production during animal tissue development (Nellas, 2022).
The evolutionarily conserved Hedgehog (Hh) signaling plays a critical role in embryogenesis and adult tissue homeostasis. Aberrant Hh signaling often leads to various forms of developmental anomalies and cancer. Since altered microRNA (miRNA) expression is associated with developmental defects and tumorigenesis, it is not surprising that several miRNAs have been found to regulate Hh signaling. However, these miRNAs are mainly identified through small-scale in vivo screening or in vitro assays. As miRNAs preferentially reduce target gene expression via the 3' untranslated region, this study analyzed the effect of reduced expression of core components of the Hh signaling cascade on downstream signaling activity, and generated a transgenic Drosophila toolbox of in vivo miRNA sensors for core components of Hh signaling, including hh, patched (ptc), smoothened (smo), costal 2 (cos2), fused (fu), Suppressor of fused (Su(fu)), and cubitus interruptus (ci). With these tools in hand, a genome-wide in vivo miRNA overexpression screen was performed in the developing Drosophila wing imaginal disc. Of the twelve miRNAs identified, seven were not previously reported in the in vivo Hh regulatory network. Moreover, these miRNAs may act as general regulators of Hh signaling, as their overexpression disrupts Hh signaling-mediated cyst stem cell maintenance during spermatogenesis. To identify direct targets of these newly discovered miRNAs, the miRNA sensor toolbox was used to show that The oncogenic G-protein-coupled receptor (GPCR) Smoothened (SMO) is a key transducer of the Hedgehog (HH) morphogen, which plays an essential role in the patterning of epithelial structures. This study examined how HH controls SMO subcellular localization and activity in a polarized epithelium using the Drosophila wing imaginal disc as a model. Evidence is provided that HH promotes the stabilization of SMO by switching its fate after endocytosis toward recycling. This effect involves the sequential and additive action of protein kinase A, casein kinase I, and the Fused (FU) kinase. Moreover, in the presence of very high levels of HH, the second effect of FU leads to the local enrichment of SMO in the most basal domain of the cell membrane. Together, these results link the morphogenetic effects of HH to the apico-basal distribution of SMO and provide a novel mechanism for the regulation of a GPCR (Antunes, 2022).
This study has screened a collection of UAS-RNAi lines targeting 10920 Drosophila protein-coding genes for phenotypes in the adult wing. 3653 genes (33%) were identified whose knock-down causes either larval/pupal lethality or a mutant phenotype affecting the formation of a normal wing. The most frequent phenotypes consist in changes in wing size, vein differentiation and patterning, defects in the wing margin and in the apposition of the dorsal and ventral wing surfaces. This study also defined 16 functional categories encompassing the most relevant aspect of each protein function, and assigned each Drosophila gene to one of these functional groups. This allowed identification of which mutant phenotypes are enriched within each functional group. Finally, this study used previously published gene expression datasets to determine which genes are or are not expressed in the wing disc. Integrating expression, phenotypic and molecular information offers considerable precision to identify the relevant genes affecting wing formation and the biological processes regulated by them (Lapez-Varea, 2021).
Identifying the genetic architecture of complex traits is important to many geneticists, including those interested in human disease, plant and animal breeding, and evolutionary genetics. Advances in sequencing technology and statistical methods for genome-wide association studies (GWAS) have allowed for the identification of more variants with smaller effect sizes, however, many of these identified polymorphisms fail to be replicated in subsequent studies. In addition to sampling variation, this failure to replicate reflects the complexities introduced by factors including environmental variation, genetic background, and differences in allele frequencies among populations. Using Drosophila melanogaster wing shape, it was asked if it were possible to replicate allelic effects of polymorphisms first identified in a GWAS in three genes: dachsous (ds),extra-macrochaete (emc) and neuralized (neur), using artificial selection in the lab, and bulk segregant mapping in natural populations. It was demonstrated that multivariate wing shape changes associated with these genes are aligned with major axes of phenotypic and genetic variation in natural populations. Following seven generations of artificial selection along the ds shape change vector, genetic differentiation of variants was observed in ds and genomic regions containing other genes in the hippo signaling pathway. This suggests a shared direction of effects within a developmental network. Artificial selection was also performed with the emc shape change vector, which is not a part of the hippo signaling network, but which exhibited a largely shared direction of effects. The response to selection along the emc vector was similar to that of ds, suggesting that the available genetic diversity of a population, summarized by the genetic (co)variance matrix (G), influenced alleles captured by selection. Despite the success with artificial selection, bulk segregant analysis using natural populations did not detect these same variants, likely due to the contribution of environmental variation and low minor allele frequencies, coupled with small effect sizes of the contributing variants (Pelletier, 2023).
Scaling between specific organs and overall body size has long fascinated biologists, being a primary mechanism by which organ shapes evolve. Yet, the genetic mechanisms that underlie the evolution of scaling relationships remain elusive. This study compared wing and fore tibia lengths (the latter as a proxy of body size) in Drosophila melanogaster, Drosophila simulans, Drosophila ananassae, and Drosophila virilis, and showed that the first three of these species have roughly a similar wing-to-tibia scaling behavior. In contrast, D. virilis exhibits much smaller wings relative their body size compared to the other species and this is reflected in the intercept of the wing-to-tibia allometry. It was then asked whether the evolution of this relationship could be explained by changes in a specific cis-regulatory region or enhancer that drives expression of the wing selector gene, vestigial (vg), whose function is broadly conserved in insects and contributes to wing size. To test this hypothesis directly, CRISPR/Cas9 was used to replace the DNA sequence of the predicted Quadrant Enhancer (vgQE) from D. virilis for the corresponding vgQE sequence in the genome of D. melanogaster. Strikingly, it was discovered that D. melanogaster flies carrying the D. virilis vgQE sequence have wings that are significantly smaller with respect to controls, partially shifting the intercept of the wing-to-tibia scaling relationship towards that observed in D. virilis. It is concluded that a single cis-regulatory element in D. virilis contributes to constraining wing size in this species, supporting the hypothesis that scaling could evolve through genetic variations in cis-regulatory elements (Farfan-Pira, 2023).
Secretory Wnt trafficking can be studied in the polarized epithelial monolayer of Drosophila wing imaginal discs (WID). In this tissue, Wg (Drosophila Wnt-I) is presented on the apical surface of its source cells before being internalized into the endosomal pathway. Long-range Wg secretion and spread depend on secondary secretion from endosomal compartments, but the exact post-endocytic fate of Wg is poorly understood. This paper summarizes and presents three protocols for the immunofluorescence-based visualization and quantitation of different pools of intracellular and extracellular Wg in WID: (1) steady-state extracellular Wg; (2) dynamic Wg trafficking inside endosomal compartments; and (3) dynamic Wg release to the cell surface. Using a genetic driver system for gene manipulation specifically at the posterior part of the WID (EnGal4) provides a robust internal control that allows for direct comparison of signal intensities of control and manipulated compartments of the same WID. Therefore, it also circumvents the high degree of staining variability usually associated with whole-tissue samples. In combination with the genetic manipulation of Wg pathway components that is easily feasible in Drosophila, these methods provide a tool-set for the dissection of secretory Wg trafficking and can help understanding of how Wnt proteins travel along endosomal compartments for short- and long-range signal secretion (Witte, 2021).
Organismal development is a complex process, involving a vast number of molecular constituents interacting on multiple spatio-temporal scales in the formation of intricate body structures. Despite this complexity, development is remarkably reproducible and displays tolerance to both genetic and environmental perturbations. This robustness implies the existence of hidden simplicities in developmental programs. Using the Drosophila wing as a model system, a new quantitative strategy was developed that enables a robust description of biologically salient phenotypic variation. Analyzing natural phenotypic variation across a highly outbred population and variation generated by weak perturbations in genetic and environmental conditions, a highly constrained set of wing phenotypes was observed. Remarkably, the phenotypic variants can be described by a single integrated mode that corresponds to a non-intuitive combination of structural variations across the wing. This work demonstrates the presence of constraints that funnel environmental inputs and genetic variation into phenotypes stretched along a single axis in morphological space. These results provide quantitative insights into the nature of robustness in complex forms while yet accommodating the potential for evolutionary variations. Methodologically, this study introduced a general strategy for finding such invariances in other developmental contexts (Alba, 2021).
Cholinergic signaling dominates the insect central nervous system, contributing to numerous fundamental pathways and behavioral circuits. However, the diverse roles different cholinergic receptors may play are only beginning to be understood. Historically, insect nicotinic acetylcholine receptors have received attention due to several subunits being key insecticide targets. More recently, there has been a focus on teasing apart the roles of these receptors, and their constituent subunits, in native signaling pathways. In this study, CRISPR-Cas9 genome editing was used to generate germline and somatic deletions of the Dβ1 nicotinic acetylcholine receptor subunit and investigate the consequences of loss of function in Drosophila melanogaster. Severe impacts on movement, male courtship, longevity, and wing expansion were found. Loss of Dβ1 was also associated with a reduction in transcript levels for the wing expansion hormone bursicon. Neuron-specific somatic deletion of Dβ1 in bursicon-producing neurons (CCAP-GAL4) was sufficient to disrupt wing expansion. Furthermore, CCAP-GAL4-specific expression of Dβ1 in a germline deletion background was sufficient to rescue the wing phenotype, pinpointing CCAP neurons as the neuronal subset requiring Dβ1 for the wing expansion pathway. Dβ1 is a known target of multiple commercially important insecticides, and the fitness costs exposed in this study explain why field-isolated target-site resistance has only been reported for amino acid replacements and not loss of function. This work reveals the importance of Dβ1-containing nicotinic acetylcholine receptors in CCAP neurons for robust bursicon-driven wing expansion (Christesen, 2021).
Phosphatidylinositol 4 phosphate (PI4P) and phosphatidylinositol 4,5 bisphosphate [PI(4,5)P2] are enriched on the inner leaflet of the plasma membrane and proposed to be key determinants of its function. PI4P is also the biochemical precursor for the synthesis of PI(4,5)P2 but can itself also bind to and regulate protein function. However, the independent function of PI4P at the plasma membrane in supporting cell function in metazoans during development in vivo remains unclear. Conserved components of a multi-protein complex composed of phosphatidylinositol 4-kinase IIIalpha (PI4KIIIalpha), TTC7, and Efr3 were found to required for normal vein patterning and wing development. Depletion of each of these three components of the PI4KIIIalpha, complex in developing wing cells results in altered wing morphology. These effects are associated with an increase in apoptosis and can be rescued by expression of an inhibitor of Drosophila caspase. In contrast to previous reports, PI4KIIIalphaa depletion does not alter key outputs of hedgehog signalling in developing wing discs. Depletion of PI4KIIIalphae results in reduced PI4P levels at the plasma membrane of developing wing disc cells while levels of PI(4,5)P2, the downstream metabolite of PI4P are not altered. Thus, PI4P itself generated by the activity of the PI4KIIIalpha complex plays an essential role in supporting cell viability in the developing Drosophila wing disc (Basu, 2020).
The vacuolar ATPases (V-ATPases) are ATP-dependent proton pumps that play vital roles in eukaryotic cells. Insect V-ATPases are required in nearly all epithelial tissues to regulate a multiplicity of processes including receptor-mediated endocytosis, protein degradation, fluid secretion, and neurotransmission. Composed of fourteen different subunits, several V-ATPase subunits exist in distinct isoforms to perform cell type specific functions. The 100 kD a subunit (see Vha100) of V-ATPases are encoded by a family of five genes in Drosophila, but their assignments are not fully understood. This study reports an experimental survey of the Vha100 gene family during Drosophila wing development. A combination of CRISPR-Cas9 mutagenesis, somatic clonal analysis and in vivo RNAi assays is used to characterize the requirement of Vha100 isoforms, and mutants of Vha100-2, Vha100-3, Vha100-4, and Vha100-5 genes were generated. Vha100-3 and Vha100-5 were shown to be dispensable for fly development, while Vha100-1 is not critically required in the wing. As for the other two isoforms, Vha100-2 was found to regulate wing cuticle maturation, while Vha100-4 is the single isoform involved in developmental patterning. More specifically, Vha100-4 is required for proper activation of the Wingless signaling pathway during fly wing development. Interestingly, a specific genetic interaction was found between Vha100-1 and Vha100-4 during wing development. These results revealed the distinct roles of Vha100 isoforms during insect wing development, providing a rationale for understanding the diverse roles of V-ATPases (Mo, 2020).
Highly reproducible tissue development is achieved by robust, time-dependent coordination of cell proliferation and cell death. To study the mechanisms underlying robust tissue growth, this study analyzed the developmental process of wing imaginal discs in Drosophila Minute mutants, a series of heterozygous mutants for a ribosomal protein gene. Minute animals show significant developmental delay during the larval period but develop into essentially normal flies, suggesting there exists a mechanism ensuring robust tissue growth during abnormally prolonged developmental time. Surprisingly, this study found that both cell death and compensatory cell proliferation were dramatically increased in developing wing pouches of Minute animals. Blocking the cell-turnover by inhibiting cell death resulted in morphological defects, indicating the essential role of cell-turnover in Minute wing morphogenesis. These analyses showed that Minute wing discs elevate Wg expression and JNK-mediated Dilp8 expression that causes developmental delay, both of which are necessary for the induction of cell-turnover. Furthermore, forced increase in Wg expression together with developmental delay caused by ecdysone depletion induced cell-turnover in the wing pouches of non-Minute animals. These findings suggest a novel paradigm for robust coordination of tissue growth by cell-turnover, which is induced when developmental time axis is distorted (Akai, 2021).
The folding of epithelial sheets is important for tissues, organs and embryos to attain their proper shapes. Epithelial folding requires subcellular modulations of mechanical forces in cells. Fold formation has mainly been attributed to mechanical force generation at apical cell sides, but several studies indicate a role of mechanical tension at lateral cell sides in this process. However, whether lateral tension increase is sufficient to drive epithelial folding remains unclear. This study used optogenetics to locally increase mechanical force generation at apical, lateral or basal sides of epithelial Drosophila wing disc cells, an important model for studying morphogenesis. Optogenetic recruitment of RhoGEF2 to apical, lateral or basal cell sides leads to local accumulation of F-actin and increase in mechanical tension. Increased lateral tension, but not increased apical or basal tension, results in sizeable fold formation. These results stress the diversification of folding mechanisms between different tissues and highlight the importance of lateral tension increase for epithelial folding (Sui, 2020).
Cell extrusion is a crucial regulator of epithelial tissue development and homeostasis. Epithelial cells undergoing apoptosis, bearing pathological mutations or possessing developmental defects are actively extruded toward elimination. However, the molecular mechanisms of Drosophila epithelial cell extrusion are not fully understood. This study reports that activation of the conserved Hippo (Hpo) signaling pathway induces both apical and basal cell extrusion in the Drosophila wing disc epithelia. Canonical Yorkie targets Diap1, Myc and Cyclin E are not required for either apical or basal cell extrusion (ACE and BCE) induced by activation of this pathway. Another target gene, bantam, is only involved in basal cell extrusion, suggesting novel Hpo-regulated apical cell extrusion mechanisms. Using RNA-seq analysis, it was found that JNK signaling is activated in the extruding cells. Genetic evidence is provided that JNK signaling activation is both sufficient and necessary for Hpo-regulated cell extrusion. Furthermore, it was demonstrate that the ETS-domain transcription factor Ets21c, an ortholog of proto-oncogenes FLI1 and ERG, acts downstream of JNK signaling to mediate apical cell extrusion. These findings reveal a novel molecular link between Hpo signaling and cell extrusion (Ai, 2020).
Cell extrusion plays an important role in epithelial homeostasis and development as well as in cancer cell metastasis. In Drosophila epithelia, BCE occurs during dorsal closure and epithelial-mesenchymal transition (EMT) as well as in apoptosis, whereas ACE occurs in tumor invasion and extrusion of apoptotic enterocytes in the Drosophila adult midgut. However, the molecular mechanisms underlying BCE and ACE in Drosophila epithelia are not well understood. The current results demonstrate that inappropriate Hpo-Yki-JNK signaling induces ACE and BCE in Drosophila wing disc epithelia. This study also shows that in the wing disc epithelia, ban acts downstream of Yki to regulate BCE and Ets21c acts downstream of JNK to regulate ACE (Ai, 2020).
The Hpo pathway regulates tissue growth in Drosophila. It has been reported that ykiB5 mutant clones grow poorly in the wing and eye discs. Consistent with these reports, the current results showed small ykiRNAi and ykiB5 mutant clones. Cells with depleted yki expression are extruded either apically or basally from the epithelia independently of apoptosis, indicating that cell extrusion is one explanation for the low recovery rate of Yki-depleted clones. In the Drosophila wing disc, overexpression of hpo by MS1096-Gal4 and nub-Gal4 dramatically decreases adult wing size. Meanwhile, overexpression of wts by nub-Gal4 also reduces the wing size. When hpo and wts expression, using C765-Gal4, cells were intensively extruded to the lumen and the basal side of the epithelia. Therefore, in addition to the proliferation defect, cell extrusion is one reason for the reduced tissue size induced by Hpo pathway activation. Diap1 levels are decreased in the small yki mutant clones, and co-expression of Diap1 and ykiRNAi could not block ACE or BCE. These results indicate that Diap1 does not regulate cell extrusion downstream of Yki. Hpo, wts mutant and yki overexpression in clones confers on cells supercompetitive properties that can lead to elimination of surrounding wild-type cells. This suggests that cell competition could promote elimination of Yki-depleted clones. In the current results, however, elimination of Yki-depleted cells could be triggered autonomously, even when Yki was depleted in the whole wing pouch. Cells expressing low levels of Myc are extruded basally through cell competition. Expressing Myc alone is not sufficient to prevent the elimination of yki mutant cells. Consistently, overexpression of Myc could not block BCE induced by silenced yki, indicating that other factors regulate BCE downstream of Yki. ban could inhibit ykiRNAi-mediated BCE but not ACE. It is known that activated Hpo plays a role in cell migration. Cells with depleted yki expression migrated across the AP boundary and were extruded basally, and this cell migration was suppressed by ban. These results show that ban can suppress ykiRNAi-induced BCE in the Drosophila wing disc but does not regulate ykiRNAi-induced ACE (Ai, 2020).
In vertebrate epithelia, cells dying through apoptosis or crowding stress are extruded apically into the lumen. The S1P-S1P2 pathway regulates both apoptosis-induced and apoptosis-independent ACE. The oncogenic KRASV12G mutation in MDCK (Madin-Darby canine kidney) epithelial cell monolayers can downregulate both S1P (sphingosine 1-phosphate) and its receptor S1P2 (also known as S1PR2) to promote basal extrusion. In Drosophila epithelia, the direction of apoptotic cell extrusion is reversed with most apoptotic cells undergoing BCE. Apoptosis-induced BCE is regulated by JNK signaling. One exception is in Drosophila adult midgut, where enterocytes are lost through apical extrusion. However, little is known about the mechanism of ACE in Drosophila epithelia (Ai, 2020).
In Drosophila epithelia, apical extrusion of scrib mutant cells is mediated by the Slit-Robo2-Ena complex, reduced E-cadherin and elevated Sqh levels. In normal cells, slit, robo2 and ena overexpression only results in BCE when cell death is blocked. More importantly, in the RNA-seq results, expression of slit, robo2 and ena were not changed in the Yki-depleted Drosophila wing disc, which means Slit-Robo2-Ena does not associate with the Hpo pathway to regulate ACE. scrib mutant cells activate Jak-Stat signaling and undergo ACE in the 'tumor hotspot' located in the dorsal hinge region of the Drosophila wing disc. Moreover, ACE can precede M6-deficient RasV12 tumor invasion following elevation of Cno-RhoA-MyoII. RNA-seq results revealed that the expression of Jak-Stat pathway genes and RhoA (Rho1) were not altered, indicating that ACE can be regulated by novel signaling pathways (Ai, 2020).
In Drosophila, the JNK signaling pathway is essential for regulating cell extrusion in phenomena including wound healing, cell competition, apoptosis and dorsal closure. JNK signaling mediates the role of Dpp and its downstream targets in cell survival regulation in the Drosophila wing. Cell extrusion and retraction toward the basal side of the wing epithelia induced by the lack of Dpp activity is independent of JNK. In one case of ectopic fold formation at the AP boundary of the Drosophila wing, loss of Omb activates both Yki and JNK signaling. In this case, JNK signaling induces the AP fold by cell shortening, and Yki signaling suppresses JNK-dependent apoptosis in the folded cells. During cell competition induced by Myc manipulation, JNK-dependent apoptosis mediates the death of 'loser' cells and their extrusion to the basal side of the epithelia. Apoptosis-induced BCE can be blocked by Diap1, which suppresses JNK-dependent apoptosis. Taken together, these results show that JNK signaling mediates or interacts with Yki signaling in a cellular context-dependent manner during the regulation of wing epithelial morphogenesis and apoptosis (Ai, 2020).
JNK is required for the migration of Csk mutant cells across the AP boundary and for their extrusion to the basal side of the epithelia. puc encodes a JNK-specific phosphatase that provides feedback inhibition to specifically repress JNK activity. Expression of puc can prevent ptc>CskRNAi cells from spreading at the AP boundary. JNK activity is also needed for ykiRNAi cells to invade across the wing disc AP boundary, and co-expression of bskDN and ykiRNAi blocks this invasion. Consistent with the role of JNK in BCE regulation, blocking JNK signaling by bskDN expression prevented ykiRNAi cells from being extruded to the basal side of the wing epithelia. More importantly, this study found that JNK activation by hepCA was sufficient to induce BCE, independently of apoptosis. Furthermore, few JNK targets have been shown to regulate cell migration and BCE. An exception to this are caspases that function downstream of JNK, which can promote cell migration when activated at a mild level (Ai, 2020).
In Drosophila eye imaginal discs, elevated JNK signaling in scrib mutant cells regulates both ACE and BCE. JNK and Robo2-Ena constitute a positive-feedback loop that promotes the apical and basal extrusion of scrib mutant cells through E-cadherin reduction. Meanwhile, in normal cells, p35 upregulation when Robo2 and Ena are overexpressed only induces BCE. The current results showed that blocking JNK signaling could suppress ACE induced by silenced yki. Meanwhile, activation of JNK by hepCA was sufficient to induce the extrusion of cells into the lumen. Cell debris may be trapped in the disc lumen when overexpressing hepCA. Apoptosis was suppressed by co-expressing p35, to confirm that the ACE observed was independent of cell death. Taken together, these results indicate that there are additional regulators downstream of JNK to mediate ACE in normal cells (Ai, 2020).
E-twenty-six (ETS) family transcription factors have conserved functions in metazoans. These include apoptosis regulation, cell differentiation promotion, cell fate regulation and cellular senescence. Ets21c encodes a member of the ETS-domain transcription factor family and is the ortholog of the human proto-oncogenes FLI1 and ERG. In Drosophila eye imaginal discs, 30-fold increased Ets21c expression is induced by RasV12 and eiger, an activator of JNK. In the Drosophila adult midgut, Ets21c expression is increased when JNK is activated by the JNK kinase hep. Ets21c can also promote tumor growth downstream of the JNK pathway. These results have confirmed that Ets21c functions downstream of JNK. Indeed, this study showed that Ets21c-GFP level was elevated following JNK activation. Expression of Ets21cHA was sufficient to induce ACE and silencing of Ets21c was sufficient to rescue ykiRNAi-induced ACE in the wing discs. However, the mechanism through which yki regulates JNK-Ets21c remains to be determined (Ai, 2020).
In Drosophila imaginal discs, ACE promotes polarity-impaired cells to grow into tumors. Therefore, it is possible that Ets21c can promote Hpo-Yki-JNK-related tumorigenesis by facilitating ACE in Drosophila. It is difficult to infer a putative pro-tumoral function of Et21c in mammals through its effect on ACE. ACE is rather associated with the elimination of tumor cells in mammals, whereas BCE is traditionally associated with higher invasive capacity. Yki/YAP gain-of-function promotes cancer cell invasion in non-small-cell lung cancer, neoplastic transformation, uveal melanoma and pancreatic cancer. Additionally, Yki/YAP loss of function helps tumor cells to escape from apoptosis in hematologic malignancies, including multiple myeloma, lymphoma and leukemia. Consistent with the latter role, Yki suppressed cell extrusion from the Drosophila wing epithelia by suppressing Ets21c. Therefore, the role of Ets21c in Hpo-Yki-related tumor models should be further examined (Ai, 2020).
Spatial boundaries formed during animal development originate from the pre-patterning of tissues by signaling molecules, called morphogens. The accuracy of boundary location is limited by the fluctuations of morphogen concentration that thresholds the expression level of target gene. Producing more morphogen molecules, which gives rise to smaller relative fluctuations, would better serve to shape more precise target boundaries; however, it incurs more thermodynamic cost. In the classical diffusion-depletion model of morphogen profile formation, the morphogen molecules synthesized from a local source display an exponentially decaying concentration profile with a characteristic length λ. It is hypothesized that in order to attain a precise profile with the minimal cost, λ should be roughly half the distance to the target boundary position from the source. Remarkably, it was found that the profiles of Bcd, Wg, Hh, and Dpp, morphogens that pattern the Drosophila embryo and wing imaginal disk, are formed with nearly optimal λ. This finding underscores the thermodynamic cost as a key physical constraint in the morphogen profile formation in Drosophila development (Song, 2021).
Protein kinases and phosphatases constitute a large family of conserved enzymes that control a variety of biological processes by regulating the phosphorylation state of target proteins. They play fundamental regulatory roles during cell cycle progression and signaling, among other key aspects of multicellular development. The complement of protein kinases and phosphatases includes approximately 326 members in Drosophila, and they have been the subject of several functional screens searching for novel components of signaling pathways and regulators of cell division and survival. These approaches have been carried out mostly in cell cultures using RNA interference to evaluate the contribution of each protein in different functional assays, and have contributed significantly to assign specific roles to the corresponding genes. The results are described of an evaluation of the Drosophila complement of kinases and phosphatases using the wing as a system to identify their functional requirements in vivo. This study also describes the results of several modifying screens aiming to identify among the set of protein kinases and phosphatases additional components or regulators of the activities of the Epidermal Growth Factor and Insulin receptors signaling pathways (Ostala, 2021).
Cell-cell junctions are dynamic structures that maintain cell cohesion and shape in epithelial tissues. During development, junctions undergo extensive rearrangements to drive the epithelial remodelling required for morphogenesis. This is particularly evident during axis elongation, where neighbour exchanges, cell-cell rearrangements and oriented cell divisions lead to large-scale alterations in tissue shape. Polarised vesicle trafficking of junctional components by the exocyst complex has been proposed to promote junctional rearrangements during epithelial remodelling, but the receptors that allow exocyst docking to the target membranes remain poorly understood. Here, this study shows that the adherens junction component Ras Association domain family 8 (RASSF8) is required for the epithelial re-ordering that occurs during Drosophila pupal wing proximo-distal elongation. The exocyst component Sec15 was identfied as a RASSF8 interactor. Loss of RASSF8 elicits cytoplasmic accumulation of Sec15 and Rab11-containing vesicles. These vesicles also contain the nectin-like homophilic adhesion molecule Echinoid, the depletion of which phenocopies the wing elongation and epithelial packing defects observed in RASSF8 mutants. Thus, these results suggest that RASSF8 promotes exocyst-dependent docking of Echinoid-containing vesicles during morphogenesis (Chan, 2021).
Wnt signalling is a core pathway involved in a wide range of developmental processes throughout the metazoa. In vitro studies have suggested that the small GTP binding protein Arf6 regulates upstream steps of Wnt transduction, by promoting the phosphorylation of the Wnt co-receptor, LRP6, and the release of β-catenin from the adherens junctions. To assess the relevance of these previous findings in vivo, this study analysed the consequence of the absence of Arf6 activity on Drosophila wing patterning, a developmental model of Wnt/Wingless signalling. A dominant loss of wing margin bristles and Senseless expression was observed in Arf6 mutant flies, phenotypes characteristic of a defect in high level Wingless signalling. In contrast to previous findings, this study showa that Arf6 is required downstream of Armadillo/β-catenin stabilisation in Wingless signal transduction. These data suggest that Arf6 modulates the activity of a downstream nuclear regulator of Pangolin activity in order to control the induction of high level Wingless signalling. These findings represent a novel regulatory role for Arf6 in Wingless signalling (Marcetteau, 2021).
Information flow within and between cells depends significantly on calcium (Ca2+) signaling dynamics. However, the biophysical mechanisms that govern emergent patterns of Ca2+ signaling dynamics at the organ level remain elusive. Recent experimental studies in developing Drosophila wing imaginal discs demonstrate the emergence of four distinct patterns of Ca2+ activity: Ca2+ spikes, intercellular Ca2+ transients, tissue-level Ca2+ waves, and a global "fluttering" state. This study used a combination of computational modeling and experimental approaches to identify two different populations of cells within tissues that are connected by gap junction proteins. These two subpopulations were termed "initiator cells," defined by elevated levels of Phospholipase C (PLC) activity, and "standby cells," which exhibit baseline activity. The type and strength of hormonal stimulation and extent of gap junctional communication were found to jointly determine the predominate class of Ca2+ signaling activity. Further, single-cell Ca2+ spikes are stimulated by insulin, while intercellular Ca2+ waves depend on Gαq activity. A computational model successfully reproduces how the dynamics of Ca2+ transients varies during organ growth. Phenotypic analysis of perturbations to Gαq and insulin signaling support an integrated model of cytoplasmic Ca2+ as a dynamic reporter of overall tissue growth. Further, perturbations to Ca2+ signaling tuned the final size of organs. This work provides a platform to further study how organ size regulation emerges from the crosstalk between biochemical growth signals and heterogeneous cell signaling states (Soundarrajan, 2021).
Hedgehog (Hh) and bone morphogenetic proteins (BMPs) pattern the developing Drosophila wing by functioning as short- and long-range morphogens, respectively. This study shows that a previously unknown Hh-dependent mechanism fine-tunes the activity of BMPs. Through genome-wide expression profiling of the Drosophila wing imaginal discs, this study identified nord as a novel target gene of the Hh signaling pathway. Nord is related to the vertebrate Neuron Derived Neurotrophic Factor (NDNF) involved in Congenital Hypogonadotropic Hypogonadism and several types of cancer. Loss- and gain-of-function analyses implicate Nord in the regulation of wing growth and proper crossvein patterning. At the molecular level, biochemical evidence ia presented that Nord is a secreted BMP-binding protein and localizes to the extracellular matrix. Nord binds to Decapentaplegic (Dpp) or the heterodimer Dpp-Glass bottom boat (Gbb) to modulate their release and activity. Furthermore, this study demonstrates that Nord is a dosage-depend BMP modulator, where low levels of Nord promote and high levels inhibit BMP signaling. Taken together, it is proposed that Hh-induced Nord expression fine tunes both the range and strength of BMP signaling in the developing Drosophila wing (Yang, 2022).
In Drosophila, the short-range morphogen Hh and the long-range morphogen BMP function together to organize wing patterning. It has been previously shown that the Hh signal shapes the activity gradient of BMP by both inducing the expression of Dpp and simultaneously downregulating the Dpp receptor Tkv, resulting in lower responsiveness to Dpp in cells at the A/P compartment border. This study showed that the activity of BMP is further fine-tuned by another previously unknown Hh-dependent mechanism. Using a genome-wide expression profiling of the Drosophila wing imaginal discs, this study identfied nord as a novel target gene of the Hh signaling pathway. Nord and its homolog NDNF belong to a family of secreted proteins that can exist in two distinct pools: diffusible Nord/NDNF proteins that can reach a longer distance and membrane/matrix-associated Nord/NDNF proteins spreading within a short distance from the source cells. During larval and early pupal wing development, Nord is expressed together or in close proximity with the BMP ligand Dpp along the A/P compartment boundary. Elimination of nord caused a reduction of overall wing size and resulted in ectopic posterior crossvein (PCV) formation. Both of these phenotypes are attributable to alterations of BMP signaling activity as monitored by the level of Mad phosphorylation, yet in opposite directions: loss of nord led to decreased pMad in larval wing discs, whereas ectopic pMad surrounded the primordial PCV in nord mutant pupal wings. Moreover, expressing exogenous Nord at different levels and during different developmental stages and contexts showed that Nord is a dosage-dependent modulator of BMP signaling both in wing growth and crossvein patterning. At the molecular level, it was further demonstrated that Nord is a BMP-binding protein that directly enhances or inhibits BMP signaling in cultured S2 cells (Yang, 2022).
Combining the genetic and biochemical evidence, it is proposes that Nord mediates BMP signaling activity through binding of the BMP ligands Dpp and Dpp-Gbb. Depending on the levels of Nord proteins and the source/types of BMP ligands, Nord-mediated binding of Dpp and Dpp-Gbb may promote or repress BMP signaling activity. Additionally, the existence of two spatially distinct pools of diffusible and membrane/matrix-associated Nord proteins may introduce further complications in Nord-mediated BMP signaling regulation. In the wild-type wing discs, expressed in a subset of Dpp-secreting cells along the A/P boundary, Nord binds and enhances the local BMP signaling activity by augmenting ligand concentration near the Nord/Dpp-secreting cells. Meanwhile, Nord also impedes the mobilization of Dpp, especially the long-range BMP signaling mediator Dpp-Gbb heterodimer. Loss of nord simultaneously led to reduced local BMP and increased long-range BMP activities, and therefore gave rise to the seemingly opposite phenotypes of reduced wing size and ectopic PCV. In contrast, low levels of ectopic Nord in the P compartment autonomously increased BMP signaling activity, whereas high levels of Nord, either in the P compartment or throughout the wing pouch, inhibited BMP signaling activity likely through interfering with the normal BMP reception. Taken together, it is proposed that Hh-induced Nord expression provides an exquisite regulation of the strength and range of BMP signaling in the developing Drosophila wing (Yang, 2022).
The activity of TGF-β type factors, including the BMP subfamily, is modulated by a large variety of binding proteins that can either enhance or inhibit their signaling in a context-dependent manner. These modulator proteins vary broadly in structure, location, and mechanism of action. Well-known extracellular and freely diffusible proteins include Noggin, Tsg, Follistatin, the CR (cysteine-rich) domain containing proteins such as Chordin/Sog, and the Can family named after two founding members, Dan and Cerberus. With the exception of Tsg and Tsg/Sog or Tsg/Chordin complexes that in some cases can promote BMP signaling, all of these factors behave as antagonists, where BMP binding prevents association of the ligand with the receptor complex (Yang, 2022).
The other broad category of BMP-binding proteins includes membrane-bound or matrix-associated proteins and, in contrast to the highly diffusible class of BMP-binding factors, these proteins often act as either agonists or antagonists depending on context. These proteins are also structurally diverse, but to date, none contain FN3 or DUF2369 domains that are characteristic of Nord and NDNF, its vertebrate counterpart. From a mechanistic point of view, perhaps the two most instructive Drosophila members of this class of modulators are the heparan sulfate proteoglycan (HSPG) Dally and the CR-containing protein Cv-2. HSPGs are well characterized as modulators of growth factor signaling. In the case of FGFs, HSPGs act as true co-receptors in which they form a tripartite complex with ligand and FGFR, the signaling receptor. However, they can also mediate signaling in other ways. Analysis of dally loss-of-function clones in imaginal discs demonstrates that it has both cell-autonomous and non-autonomous effects with respect to BMP signaling. In general, low levels tend to promote signaling while high doses attenuate signaling. Many models have been put forth to explain these opposing effects and often come down to balancing ligand sequestration and diffusion properties. For instance, in the absence of HSPGs, Dpp may more freely diffuse away from the disc epithelial cell surface. In this case, HSPG acts to enhance signaling by keeping Dpp tethered to the cell surface where it can engage its signaling receptors. On the other hand, a high level of HSPG may compete with signaling receptors for BMP binding and thereby reduce signal (Yang, 2022).
The situation with respect to signal modulation becomes even more complex for factors such as Nord that bind both HSPGs and BMPs. An instructive example to consider is Cv-2, a secreted factor that, like Nord, binds both to HSPGs and BMPs and is also induced by BMP signaling. Like Dally, Cv-2 also has dose-dependent effects on signaling in wing imaginal discs, where low levels enhance while high levels inhibit BMP signaling. By virtue of being bound to HSPGs, it may simply function as an additional tethering molecule that keeps BMPs localized near the cell surface. However, Cv-2 has the unique property that it is also able to bind Tkv, a Drosophila BMPR type I receptor. This has led to speculation that it could act as an exchange factor that aids in handing off a BMP ligand from the HSPG pool to the type I receptor. Mathematical modeling showed that this mechanism can produce a biphasic signal depending on affinities of the various BMP-binding proteins involved and their concentrations (Yang, 2022).
In the case of Nord, its mechanism of action is likely compatible with a variety of these and/or alternative models. While this study has shown that Nord is a BMP-binding protein and Akiyama (2021) have shown that it also binds HSPGs, it is not clear whether the BMP and HSPG-binding sites overlap or are distinct and where they are positioned relative to the FN3 and DUF2369 domains. This is an important issue to consider with respect to the two CRISPR mutants that were generated that truncate Nord within the DUF2369 domain. Interestingly, the nord3D allele appears to retain some function since it does not generate ectopic crossveins as do the nordMI06414 or nord22A alleles, yet nord3D still produces small wings in transheterozygous combination with a deficiency or nord22A, consistent with having lost the BMP growth-promoting ability. The discrepancy in crossvein patterning between the different nord alleles may be explained by a difference in residual function of the various truncated Nord protein products. Because the nordMI06414 allele yields a much shorter predicted Nord peptide compared to the two CRISPR alleles, it is likely to behave as a protein null with a stronger phenotype. The two nord CRISPR alleles, although similar in the sequence deleted from the C-terminus, differ in how many non-nord encoded amino acids occur between the frameshift and the stop codon. The nord22A allele has additional 14 amino acids relative to nord3D. Perhaps this extension of the truncated fragment destabilizes or interferes with residual function found in the nord3D allele. Additional biochemical studies defining the BMP and HSPG-binding sites, the stability of truncated Nord fragments, and whether Nord can also associate with either the type I or II receptors will aid in formulating a more precise mechanistic model (Yang, 2022).
Nord shows some sequence similarity to the NDNF family of proteins. Based on a very recent study, like many other neurotrophic factors, NDNF arose in the ancestor of bilaterians or even later. In agreement, by analyzing the genome and EST sequences from various organisms, this study found that nearly all bilaterian animals have either single or multiple orthologous genes for Nord/Ndnf. Of note, no Ndnf homologs were identiied in the flatworm Planarian, but these factors are highly conserved across vertebrates. All vertebrate family members contain a signal peptide, two FN3-like repeats, and a domain of unknown function (DUF2369) that is now referred to as the NDNF domain. The NDNF domain partially overlaps with the first FN3 but shows some additional conservation that extends between the two FN3 domains. The FN3 module is quite diverse in sequence but is thought to exhibit a common fold that is used as an interaction surface or spacer. The function of the NDNF domain is not clear, but it may also provide a protein interaction surface (Yang, 2022).
Although the vertebrate NDNFs are highly conserved throughout the entire protein length, the Caenorhabditis elegans and Drosophila relatives are quite divergent in primary sequence and show little conservation beyond a few key residues that define the second FN3 and NDNF domains. Notably, the Drosophila protein is missing the first FN3 domain, and therefore it is not clear the extent to which Nord and the vertebrate NDNFs may exhibit functional conservation. Ironically, the original human NDNF clone was identified on the basis of domain structure conservation with Drosophila Nord, which was identified via enhancer trapping to be a gene expressed in mushroom bodies and whose loss leads to defects in olfactory learning and memory (Dubnau et al., 2003). Unfortunately, that particular LacZ enhancer trap line that disrupted the nord locus is no longer available. The use of these new alleles should prove helpful for either confirming or eliminating the involvement of Nord as a modulator of learning and memory and/or other neuronal functions in larva and adult Drosophila (Yang, 2022).
In the mouse, NDNF is highly expressed in many neurons of the brain and spinal cord. Studies using cultured mouse hippocampal neurons revealed that it promotes neuron migration and neurite outgrowth, hence its name. In later studies, NDNF was also found to be upregulated in mouse endothelial cells in response to hindlimb ischemia, where it promotes endothelial cell and cardiomyocyte survival through integrin-mediated activation of AKT/endothelial NOS signaling. Additionally, recent studies have shown that NDNF expression is significantly downregulated in human lung adenocarcinoma (LUAD) and renal cell carcinoma (RCC), indicating that NDNF may also provide a beneficial function as a tumor suppressor (Yang, 2022).
Taken together, these studies have suggested some possible functions for vertebrate NDNF. However, they have primarily relied on in vitro cell culture models, and only recently have in vivo loss-of-function studies been reported. Remarkably, NDNF mutants were discovered in the genomes of several probands with congenital hypogonadotropic hypogonadism (CHH), a rare genetic disorder that is characterized by absence of puberty, infertility, and anosmia (loss of smell). This phenotype is very similar to that produced by loss of the anos1, which also encodes an FN3 superfamily member and is responsible for Kallmann syndrome, a condition that similarly presents with CHH and anosmia due to lack of proper GnRH and olfactory neuron migration. Although in vitro studies indicated that NDNF modulates FGFR1 signaling after FGF8 stimulation, the in vivo molecular mechanism responsible for the neuronal migration defects is not clear. The results of the current study on the function of Drosophila Nord raise the issue of whether any of the ascribed vertebrate NDNF functions could involve alterations in BMP signaling. In the case of angiogenesis and EMT, BMPs, as well as other TGF-β family members, participate at many levels. At present, however, no involvement of BMP or TGF-β signaling has been implicated in migration of the GnRH neurons, although BMP signaling does define neurogenic permissive areas in which the olfactory placode forms. A clear objective for the future is to determine if the vertebrate NDNF factors bind BMPs and/or HSPG proteins such as Dally-like glypicans to modulate BMP signaling activity. On the Drosophila side, additional non-BMP-modulating roles for Nord should also be examined (Yang, 2022).
Adhesion to the extracellular matrix (ECM) is required for normal epithelial cell survival. Disruption of this interaction leads to a specific type of apoptosis known as anoikis. Yet, there are physiological and pathological situations in which cells not connected to the ECM are protected from anoikis, such as during cell migration or metastasis. The main receptors transmitting signals from the ECM are members of the integrin family. However, although integrin-mediated cell-ECM anchorage has been long recognized as crucial for epithelial cell survival, the in vivo significance of this interaction remains to be weighed. This study used the Drosophila wing imaginal disc epithelium to analyze the importance of integrins as survival factors during epithelia morphogenesis. Reducing integrin expression in the wing disc induces caspase-dependent cell death and basal extrusion of the dead cells. In this case, anoikis is mediated by the activation of the JNK pathway, which in turn triggers expression of the proapoptotic protein Hid. In addition, the results strongly suggest that, during wing disc morphogenesis, the EGFR pathway protects cells undergoing cell shape changes upon ECM detachment from anoikis. Furthermore, it was shown that oncogenic activation of the EGFR/Ras pathway in integrin mutant cells rescues them from apoptosis while promoting their extrusion from the epithelium. Altogether, these results support the idea that integrins promote cell survival during normal tissue morphogenesis and prevent the extrusion of transformed cells (Valencia-Exposito, 2022).
Morphogen gradients need to be robust, but may also need to be tailored for specific tissues. Often this type of regulation is carried out by negative regulators and negative feedback loops. In the Hedgehog (Hh) pathway, activation of patched (ptc) in response to Hh is part of a negative feedback loop limiting the range of the Hh morphogen. This study shows that in the Drosophila wing imaginal disc two other known Hh targets genes feed back to modulate Hh signaling. First, anterior expression of the transcriptional repressor Engrailed modifies the Hh gradient by attenuating the expression of the Hh pathway transcription factor cubitus interruptus (ci), leading to lower levels of ptc expression. Second, the E-3 ligase Roadkill shifts the competition between the full-length activator and truncated repressor forms of Ci by preferentially targeting full-length Ci for degradation. Finally, evidence is provided that Suppressor of fused, a negative regulator of Hh signaling, has an unexpected positive role, specifically protecting full-length Ci but not the Ci repressor from Roadkill (Roberto, 2022).
This study examined the roles of three potential negative regulators of Hh signal transduction, two of which are themselves encoded by Hh target genes. In each case interesting new aspects about the pathway's regulation.
Anterior expression of en likely extends the range of the Hh gradient were discovered (Roberto, 2022).
Anterior expression of en in the wing imaginal disc was first observed 30 years ago and en is the Hh target gene requiring the highest level of Hh signaling. Its domain of expression exactly correlates with a region of lower full-length Ci protein levels. It had been proposed that the lower Ci protein levels are a consequence of Ci being particularly active and labile in this region. This study shows that the lower levels of Ci are not primarily due to it being particularly labile, but rather are a consequence of negative transcriptional regulation by En. The role of this negative feedback loop appears to be to modulate the Hh gradient by downregulating the expression of ptc in addition to its effects on dpp. This leads to Hh signaling extending further into the anterior compartment, with a corresponding anterior shift in the location of LV3 and the expression of dpp. A model is prefered in which the attenuation of ptc expression by anterior en is indirect via Ci, but in principle en could also directly negatively regulate ptc. This is thought less likely as, 'flip-out' clones expressing Ci activate high levels of ptc in the posterior compartment in the presence of en. The anterior expression of en occurs late in third instar larvae, which correlates with the downregulation of ci expression as visualized using the UAS-TT transcriptional timer and the refinement of wing vein specification (Roberto, 2022).
Ci function is modulated by two feedback loops acting at different levels. Anterior expression of the En protein attenuates Ci activity directly adjacent to the compartment boundary of the wing disc by downregulating the expression of the ci gene. Rdx and Su(fu) act at the protein level modulating the competition between the full-length (Ci FL) and repressor forms (Ci R) of Ci. Rdx specifically targets full-length Ci, whereas Su(fu) partially protects full-length Ci from Rdx-mediated degradation. Rdx degradation of full-length Ci appears to help downregulate Hh target genes in cells no longer receiving the Hh signal (Roberto, 2022).
Why did this mechanism evolve to modulate the Hh gradient? Morphogen gradients, by virtue of their central roles in the development of multiple tissues, must be robust and resistant to perturbation. Therefore, to specifically expand the range of the Hh gradient in the wing disc a new component was added, anterior expression of the ci repressor en (Roberto, 2022).
The lack of the C-terminal domain in the Ci repressor has multiple consequences. It loses the binding site for the co-activator CBP, and it loses C-terminal binding sites for Su(fu), Cos2 and Rdx. As a consequence, the Ci repressor is not sequestered in the cytoplasm by Cos2 in the absence of Hh signaling and enters the nucleus without Su(fu), whereas full-length Ci enters the nucleus only in the presence of Hh signaling and as a complex with Su(fu) (Roberto, 2022).
In order to better understand the roles of Su(fu) and Rdx, animals heterozygous for the ciCe2 mutation were examined. In this context, overexpression of rdx or loss of Su(fu) function leads to a complete fusion between LV3 and LV4. In addition, clones mutant for Su(fu) show dramatic reduction in the expression of the Hh target genes ptc and dpp. These results show that Su(fu) has a potential novel positive role in Hh signal transduction, improving the ability of full-length Ci to compete with the repressor form. A positive role for Su(fu) has also been found in mammals where Su(fu) appears to function as a chaperone for the full-length Gli proteins, but not the repressor forms, and is required for full activation of Gli target genes. The requirement for Drosophila Su(fu) is obviated in the absence of Rdx, suggesting that Rdx primarily targets full-length Ci and not Ci repressor, even though the repressor is not protected by Su(fu). These results are analogous to what is seen with the mammalian homologue of Rdx, SPOP, indicating that this mechanism has been conserved during evolution. SPOP is opposed by Su(fu) and degrades the full-length forms of the mammalian GLI2 and GLI3 but not the GLI3 repressor form. The competition between Rdx and Su(fu) appears to be rather finely balanced as either increasing the expression of rdx or reducing the expression of Su(fu) enhances the ability of CiCe2 to compete with full-length Ci. This function of protecting full-length Ci from Rdx presumably takes place in the nucleus, as this is where the Rdx protein primarily localizes (Roberto, 2022).
However, the functional relevance of rdx being an Hh target gene has been unclear. Zygotic loss of rdx in the embryo has no visible effect on segmental patterning of the cuticle and, unlike en, knockdown of rdx along the compartment boundary in the wing disc has little effect on wing patterning. Perhaps its role is to clear full-length Ci from cells that were once within the domain of Hh signaling and have moved outside the domain of Hh signaling. Perdurance of Rdx could target full-length Ci in the nucleus allowing the Ci repressor to shut off Hh target genes. This is the situation in the eye disc with the progression of the morphogenetic furrow. Cells that recently received high level Hh signaling and activated Ci must now downregulate Ci to allow proper differentiation of the ommatidia. Rdx appears to be important for this process, as loss of rdx leads to defects in the eye. A similar situation may exist in other tissues. Looking at the temporal regulation of ptc expression with UAS-TT, cells removed from the compartment boundary in the wing disc have lower levels of destabilized GFP relative to RFP and appear to be in the process of shutting off ptc. This distinction is lost following downregulation of rdx by RNAi (Roberto, 2022).
In the domain of modest level Hh signaling (in which dpp is expressed), both full-length Ci and Ci repressor must be present in some form of reciprocal gradients. In this domain, enhancers with perfect Ci consensus binding sites are silent due to binding of Ci repressor. The dpp enhancer with imperfect Ci binding sites is expressed, and for it to be completely active, full-length Ci must be bound. Why is full-length Ci able to better compete with Ci repressor for the imperfect binding sites? Full-length Ci and the Ci repressor share the same DNA binding domain, and it would be expected that the repressor would outcompete full-length Ci for binding to target sites because the repressor is primarily nuclear, whereas full-length Ci is primarily cytoplasmic, even in the presence of Hh signaling, due to a strong nuclear export signal (NES). I suggest that cooperativity between Ci repressor proteins at perfect Ci binding sites can account for this distinction. Another potential mechanism for preferentially recruiting full-length Ci to imperfect binding sites might be suggested by the different protein interactions observed with full-length Ci and CiCe2. Full-length Ci enters the nucleus with Su(fu) while the Ci repressor is not bound to Su(fu). In addition, the Ci repressor is missing the CBP binding site. As a consequence, full-length Ci could engage in protein-protein interactions with other transcription factors that are not available to the Ci repressor. This added affinity to other proteins within the enhanceosome could allow the preferential recruitment of full-length Ci to enhancers with imperfect Ci binding sites. Differential protein-protein interactions may also explain why full-length Ci is still able to activate ptc-lacZ expression along the compartment boundary in ciCe2/+ heterozygotes (Fig. S4) but not the artificial enhancer 4bs-lacZ. The ptc-lacZ enhancer is a bona fide Drosophila enhancer and is likely to recruit a constellation of proteins that could interact with full-length Ci, whereas protein-protein interactions are likely to be much less robust at 4bs (Roberto, 2022).
In conclusion, these results highlight the complexity of Hh signal transduction and its modulation. Expressing en in the anterior compartment of the wing pouch modulates the Hh gradient, whereas Su(fu) has a surprising positive role in the pathway, acting to partially protect full-length Ci from the E-3 ligase Rdx that Ci activates (Roberto, 2022).
Mitochondrial ribosomal proteins (MRPs) assemble as specialized ribosome to synthesize mtDNA-encoded proteins, which are essential for mitochondrial bioenergetic and metabolic processes. MRPs are required for fundamental cellular activities during animal development, but their roles beyond mitochondrial protein translation are poorly understood. This study reports a conserved role of the mitochondrial ribosomal protein L4 (mRpL4) in Notch signaling. Genetic analyses demonstrate that mRpL4 is required in the Notch signal-receiving cells to permit target gene transcription during Drosophila wing development. mRpL4 physically and genetically interacts with the WD40 repeat protein Wings apart (Wap) and activates the transcription of Notch signaling targets. This study shows that human mRpL4 is capable of replacing fly mRpL4 during wing development. Furthermore, knockout of mRpL4 in zebrafish leads to downregulated expression of Notch signaling components. Thus, this study has discovered a previously unknown function of mRpL4 during animal development (Mo, 2023).
Mutations in the Ultrabithorax (Ubx) gene cause homeotic transformation of the normally two-winged Drosophila into a four-winged mutant fly. Ubx encodes a HOX family transcription factor that specifies segment identity, including transformation of the second set of wings into rudimentary halteres. Ubx is known to control the expression of many genes that regulate tissue growth and patterning, but how it regulates tissue morphogenesis to reshape the wing into a haltere is still unclear. This study shows that Ubx acts by repressing the expression of two genes in the haltere, Stubble and Notopleural, both of which encode transmembrane proteases that remodel the apical extracellular matrix to promote wing morphogenesis. In addition, Ubx induces expression of the Tissue inhibitor of metalloproteases in the haltere, which prevents the basal extracellular matrix remodelling necessary for wing morphogenesis. These results provide a long-awaited explanation for how Ubx controls morphogenetic transformation (Diaz-de-la-Loza, 2020).
The results reveal how Ubx – a homeotic gene that encodes the founding member of the HOX-family of transcription factors – regulates apical and basal matrix remodelling to control epithelial morphogenesis (see Ubx controls apical and basal ECM degradation to regulate morphogenesis). Ubx strongly represses two genes encoding apical matrix proteases (Np and Sb), as well as partially repressing two genes encoding basal matrix metalloproteases (Mmp1 and Mmp2), while inducing an inhibitor of Mmp1/2 (Timp) in the haltere. In this way, Ubx prevents both apical and basal matrix remodelling in the haltere, a key event in the homeotic wing-to-haltere transformation. In addition to regulating morphogenesis, Ubx controls many other genes affecting wing growth and pattern. Together, the combined repression of morphogenesis, growth and patterning by Ubx is responsible for the full transformation of wing to haltere (Diaz-de-la-Loza, 2020).
Ubx controls apical and basal ECM degradation to regulate morphogenesis. Schematic of Ubx expression and function in Drosophila and a hypothetical four-winged ancestor. Ubx controls organ shape via regulation of aECM and bECM proteases, in addition to its known functions in regulating organ growth and patterning. These target genes have presumably evolved to be specifically regulated in the Drosophila wing and/or haltere, and must be insensitive to Ubx in four-winged ancestors (Diaz-de-la-Loza, 2020).
These findings also support the general view that transcriptional control of matrix synthesis and degradation is a conserved mechanism by which information encoded in the genome is deployed to govern the shape of tissues and organs in animals. Although this concept is broadly appreciated for the regulation of the bECM, the notion that the aECM is also developmentally regulated during tissue morphogenesis needs further investigation, particularly in mammals. Beyond animals, morphogenesis of plants, fungi and bacteria is also known to be fundamentally dependent on patterned synthesis and degradation of the cell wall, a type of ECM. Thus, genetic control of the matrix appears to be a general principle that shapes all life forms (Diaz-de-la-Loza, 2020).
Epithelial cells remodel cell adhesion and change their neighbors to shape a tissue. This cellular rearrangement proceeds in three steps: the shrinkage of a junction, exchange of junctions, and elongation of the newly generated junction. By combining live imaging and physical modeling, this study showed that the formation of myosin-II (myo-II) cables around the cell vertices underlies the exchange of junctions in the Drosophila wing epithelium. The local and transient detachment of myo-II from the cell cortex is regulated by the LIM domain-containing protein Jub and the tricellular septate junction protein M6. Moreover, M6 shifts to the adherens junction plane on jub RNAi and that Jub is persistently retained at reconnecting junctions in m6 RNAi cells. This interplay between Jub and M6 can depend on the junction length and thereby couples the detachment of cortical myo-II cables and the shrinkage/elongation of the junction during cell rearrangement. Furthermore, this study developed a mechanical model based on the wetting theory and clarified how the physical properties of myo-II cables are integrated with the junction geometry to induce the transition between the attached and detached states and support the unidirectionality of cell rearrangement. Collectively, this study elucidates the orchestration of geometry, mechanics, and signaling for exchanging junctions (Ikawa, 2023).
Cell rearrangement plays a fundamental role in shaping a tissue and developing multicellular patterns.Cell rearrangement proceeds in three steps: the shrinkage of a junction, exchange of junctions around the cell vertex (four-way or higher-folded), and elongation of the newly generated junction. The molecular mechanisms underlying junction shrinkage and elongation have been well characterized. At the apical level, medial actomyosin flow and junctional recruitment of myosin-II (myo-II) lead to the generation of forces that shrink and elongate a junction. At the basolateral level, actin-rich protrusions deform the cell membrane. In sharp contrast, little is known regarding how epithelial cells exchange junctions during cell rearrangement (Ikawa, 2023).
Molecules comprising the junctional structure play essential roles in the development of epithelial tissue, where the bicellular junction (bCJ) including the adherence junction (AJ) adheres cells, and the tricellular junction (TCJ) seals the tissue at the cell vertex. During cell rearrangement, as the bCJ dynamically changes its length, the position of the TCJ changes. The junction deformation and vertex displacement are determined by the balance between the constriction force generated by myo-II and the adhesive force generated by cell adhesion molecules such as E-cadherin. The mechanical force balance is tuned by actin-AJ linkers, which are responsible for the linkage between actomyosin and E-cadherin, and TCJ proteins, which are a class of proteins comprising the cell vertex structures, to maintain cell adhesion during the junction shrinkage and control the speed of junction elongation (Ikawa, 2023).
The localization and activity of these molecules need to be coordinated such that cell adhesion is weakened specifically at short junctions around the cell vertex and such that de novo formation of cell adhesion is initiated between the correct pair of cells. The mechanism underlying the coordination of cell adhesion and the regulatory molecules during the junction exchange is unclear (Ikawa, 2023).
Recent studies identified a cytoskeleton structure that could be critical in the junction exchange. It has been shown that myo-II transiently forms rectangle-shaped cables around the cell vertices at the AJ plane in Drosophila wing epithelium (hereafter called rsMCs [rectangle-shaped myo-II cables]). Such local detachment of myo-II from short junctions has been reported in other epithelial tissues.The rsMC may represent a temporally loosened junctional structure, which facilitates the reconnection of junctions. However, the molecular and physical basis of rsMC dynamics and function have not been studied extensively (Ikawa, 2023).
This study sought to identify the mechanism by which apical junctions are exchanged during cell rearrangement in the Drosophila wing epithelium. The findings demonstrate that the precise control of rsMC formation supports the exchange of junctions and the stabilization of newly generated junctions. The rsMC formation is coupled with the junction shrinkage and elongation via an interplay between the LIM domain-containing protein Jub and the TCJ protein M6. A mechanical model based on the wetting theory can explain the junction-length-dependent transition between the detached and attached states of cortical myo-II and the unidirectionality of cell rearrangement. The coupling between junction geometry, mechanics, and signaling identified in the present study may function in other aspects of morphogenesis, as all morphogenetic events are associated with spatio-temporal changes in these three aspects (Ikawa, 2023).
This study delineates the mechanism by which junctions are exchanged in epithelial cells (see Summary and working hypothesis of the mechanism of junction exchange). The size and duration time of the rectangle-shaped myo-II cables (rsMC) are autonomously coordinated through the junction geometry and mechanics to loosen the junctional structure locally and transiently. The transient formation of the rsMC may prevent dilution of DE-cad at loosened junctions as suggested by the decrease of DE-cad signal at long-lasting rsMCs and the observation that jub RNAi resulted in the increase in the gap in junctional DE-cad at 27 h APF. In addition, the directional information of the shrinking junction is inherited by the differential myo-II accumulation along the vertical and horizontal edges of the rsMC, which ensures unidirectional cell rearrangement by inducing the reattachment to junctions along edges with lower myo-II levels. This represents an advantage of the rsMC-mediated mechanism over in situ regulation at cell-cell contact surfaces in supporting the fidelity of junction exchange (Ikawa, 2023).
M6 is not detected in immature epithelial cells, including germband cells, and cells start to express M6 as the epithelium matures. Thus, immature epithelial cells such as germband cells may deform cell membranes via actin-rich basolateral protrusions to form a multicellular rosette.
In contrast, mature epithelial cells such as pupal wing cells may reconnect junctions at more apical sides via M6. The mechanism whereby M6 regulates AJ components such as Jub in wing cells and Cno in RasV12-overexpressing eye disc cells remains unclear. Since the mammalian homolog of M6 is involved in the actin regulation, M6 may control actin remodeling, which is known to affect the structure and dynamics of AJ. Alternatively, M6 may directly interact with the AJ components through a rapid, short-term translocation to the AJ, as the position of junction compartments can dynamically change during the development (Ikawa, 2023).
More experiments such as simultaneous tracking of the AJ and SJ components will be necessary to elucidate how AJ and SJ interact and coordinate during junction exchange.
A simple theoretical model was introduced, and the mechanical and geometrical conditions were uncovered under which cortical myo-II cables attach to and detach from the junction. These conditions are compatible with the temporal changes in myo-II and jub levels in WT and RNAi cells. However, the model considers only the static force balance and thus cannot describe the entire process of junction exchange. To understand the dynamics of junction exchange, a more comprehensive model, including the turnover of actomyosin and junction components, needs to be developed. Recently, it has been proposed the apposed-cortex adhesion model, which investigates how the duration of cell-cell membrane linkage at the molecular scale affects cell rearrangement (Ikawa, 2023).
Extending the model to incorporate the concept of apposed-cortex adhesion model is an interesting direction for future research (Ikawa, 2023).
In conclusion, the present study shed light on the orchestration of geometry, mechanics, and signaling around the cell vertex for reconnection of junctions. The proposed mechanism can potentially contribute to other aspects of morphogenesis. For instance, the rsMC-like structure at the vertex of a non-remodeling junction implies its relevance in a ratchet-like movement of the vertex along the junction. In addition, because loss of function of M6 causes excess extrusion of cancer cells, it is possible that M6 and actin-AJ linkers function to sense the risk of fracture at cell vertices, thereby preventing the formation of wounds in the epithelium. Given recent findings on the reorganization of the cytoskeleton and information sensing at the cell vertex, further dissecting the complex interplay between actin-AJ regulators, TCJ proteins, and the geometry and mechanics at the vertices will clarify the origin of collective cell behaviors in epithelial development and plasticity (Ikawa, 2023).
In the early 20th century, Calvin Bridges and Thomas Morgan identified a number of spontaneous mutations that displayed visible phenotypes in adult flies and subsequent analysis of these mutations over the past century have provided fundamental insights into subdisciplines of biology such as genetics, developmental, and cell biology. One of the mutations they identified in 1915 was named tilt (tt) and was described by Bridges and Morgan as having two visible phenotype characteristics in the wing. The wings were "held out at a wider angle from the body" and had a break in wing vein L3. Subsequent analysis of the tilt phenotype identified another phenotype: the wings were missing a varying number of campaniform sensilla on L3. Though Bridges and Morgan provided an ink drawing of the wing posture phenotype, only the vein and campaniform sensilla loss images have been published. This study confirmed and documented the tilt phenotypes that have been previously described. Yhe penetrance of these phenotypes was also shown: the vein break and the distinct outward wing posture have decreased since its discovery (Houtman, 2023).
The steroid hormone 20-hydroxy-ecdysone (20E) promotes proliferation in Drosophila wing precursors at low titer but triggers proliferation arrest at high doses. Remarkably, wing precursors proliferate normally in the complete absence of the 20E receptor, suggesting that low-level 20E promotes proliferation by overriding the default anti-proliferative activity of the receptor. By contrast, 20E needs its receptor to arrest proliferation. Dose-response RNA sequencing (RNA-seq) analysis of ex vivo cultured wing precursors identifies genes that are quantitatively activated by 20E across the physiological range, likely comprising positive modulators of proliferation and other genes that are only activated at high doses. It is suggested that some of these 'high-threshold' genes dominantly suppress the activity of the pro-proliferation genes. It was then shown mathematically and with synthetic reporters that combinations of basic regulatory elements can recapitulate the behavior of both types of target genes. Thus, a relatively simple genetic circuit can account for the bimodal activity of this hormone (Perez-Mockus, 2023).
WNK (With no Lysine [K]) kinases have critical roles in the maintenance of ion homeostasis and the regulation of cell volume. Their overactivation leads to pseudohypoaldosteronism type II (Gordon syndrome) characterized by hyperkalemia and high blood pressure. More recently, WNK family members have been shown to be required for the development of the nervous system in mice, zebrafish, and flies, and the cardiovascular system of mice and fish. Furthermore, human WNK2 and Drosophila Wnk modulate canonical Wnt signaling. In addition to a well-conserved kinase domain, animal WNKs have a large, poorly conserved C-terminal domain whose function has been largely mysterious. In most but not all cases, WNKs bind and activate downstream kinases OSR1/SPAK, which in turn regulate the activity of various ion transporters and channels. This study shows that Drosophila Wnk regulates Wnt signaling and cell size during the development of the wing in a manner dependent on Fray, the fly homolog of OSR1/SPAK. The only canonical RF(X)V/I motif of Wnk, thought to be essential for WNK interactions with OSR1/SPAK, is required to interact with Fray in vitro. However, this motif is unexpectedly dispensable for Fray-dependent Wnk functions in vivo during fly development and fluid secretion in the Malpighian (renal) tubules. In contrast, a structure function analysis of Wnk revealed that the less-conserved C-terminus of Wnk, that recently has been shown to promote phase transitions in cell culture, is required for viability in vivo. These data thus provide novel insights into unexpected in vivo roles of specific WNK domains (Yarikipati, 2023).
The morphogen Sonic Hedgehog (SHH) plays an important role in coordinating embryonic development. Short- and long-range SHH signalling occurs through a variety of membrane-associated and membrane-free forms. However, the molecular mechanisms that govern the early events of the trafficking of neosynthesised SHH in mammalian cells are still poorly understood. This study employed the retention using selective hooks (RUSH) system to show that newly-synthesized SHH is trafficked through the classical biosynthetic secretory pathway, using TMED10 as an endoplasmic reticulum (ER) cargo receptor for efficient ER-to-Golgi transport and Rab6 vesicles for Golgi-to-cell surface trafficking. TMED10 and SHH colocalized at ER exit sites (ERES), and TMED10 depletion significantly delays SHH loading onto ERES and subsequent exit leading to significant SHH release defects. Finally, the Drosophila wing imaginal disc model to demonstrate that the homologue of TMED10, Baiser (Bai), participates in Hedgehog (Hh) secretion and signalling in vivo. In conclusion, this work highlights the role of TMED10 in cargo-specific egress from the ER and sheds light on novel important partners of neosynthesized SHH secretion with potential impact on embryonic development (Bare, 2023).
The exact mechanism controlling cell growth remains a grand challenge in developmental biology and regenerative medicine. The Drosophila wing disc tissue serves as an ideal biological model to study mechanisms involved in growth regulation. Most existing computational models for studying tissue growth focus specifically on either chemical signals or mechanical forces. This study developed a multiscale chemical-mechanical model to investigate the growth regulation mechanism based on the dynamics of a morphogen gradient. By comparing the spatial distribution of dividing cells and the overall tissue shape obtained in model simulations with experimental data of the wing disc, it is shown that the size of the domain of the Dpp morphogen is critical in determining tissue size and shape. A larger tissue size with a faster growth rate and more symmetric shape can be achieved if the Dpp gradient spreads in a larger domain. Together with Dpp absorbance at the peripheral zone, the feedback regulation that downregulates Dpp receptors on the cell membrane allows for further spreading of the morphogen away from its source region, resulting in prolonged tissue growth at a more spatially homogeneous growth rate (Ramezani, 2023).
While the membrane potential of cells has been shown to be patterned in some tissues, specific roles for membrane potential in regulating signalling pathways that function during development are still being established. In the Drosophila wing imaginal disc, Hedgehog (Hh) from posterior cells activates a signalling pathway in anterior cells near the boundary which is necessary for boundary maintenance. This study shows that membrane potential is patterned in the wing disc. Anterior cells near the boundary, where Hh signalling is most active, are more depolarized than posterior cells across the boundary. Elevated expression of the ENaC channel Ripped Pocket (Rpk), observed in these anterior cells, requires Hh. Antagonizing Rpk reduces depolarization and Hh signal transduction. Using genetic and optogenetic manipulations, in both the wing disc and the salivary gland, it was shown that membrane depolarization promotes membrane localization of Smoothened and augments Hh signalling, independently of Patched. Thus, membrane depolarization and Hh-dependent signalling mutually reinforce each other in cells immediately anterior to the compartment boundary (Emmons-Bell, 2021).
Initially discovered for its role in regulating segment polarity in Drosophila, Hh signalling has since been implicated in a multitude of developmental processes. Among the best characterized is the signalling between two populations of cells that make up the Drosophila wing imaginal disc, the larval primordium of the adult wing and thorax. The wing disc consists of two compartments of lineage-restricted cells separated by a smooth boundary. Posterior (P) cells make the morphogen Hedgehog, which binds to its receptor Patched (Ptc), which is expressed exclusively in anterior (A) cells. Hh has a relatively short range either because of its limited diffusion, or because it is taken up by nearby target cells via filopodia-like protrusions known as cytonemes. Hh alleviates the repressive effect of Ptc on the seven-transmembrane protein Smoothened (Smo) in A cells near the boundary, initiating a signalling cascade that culminates in the stabilization of the activator form of the transcription factor Cubitus interruptus (Ci), and expression of target genes such as the long-range morphogen Dpp. In turn, Dpp regulates imaginal disc patterning and growth in both compartments (Emmons-Bell, 2021).
While the role of cell-cell interactions, diffusible morphogens and even mechanical forces have been studied in regulating the growth and patterning of the wing disc, relatively little attention has been paid to another cellular parameter, membrane potential or Vmem. Vmem is determined by the relative concentrations of different species of ions across the cell membrane, as well as the permeability of the membrane to each of these ions. These parameters are influenced by the abundance and permeability of ion channels, the activity of pumps, and gap junctions. While changes in Vmem have been studied most extensively in excitable cells, there is increasing evidence that the Vmem of all cells, including epithelial cells, can vary depending on cell-cycle status and differentiation status. Mutations in genes encoding ion channels in humans ('channelopathies') can result in congenital malformations. Similarly, experimental manipulation of ion channel permeability can cause developmental abnormalities in mice as well as in Drosophila. Only more recently has evidence emerged that Vmem can be patterned during normal development. Using fluorescent reporters of membrane potential, it has been shown that specific cells during Xenopus gastrulation and Drosophila oogenesis appear more depolarized than neighbouring cells. A recent study established that cells in the vertebrate limb mesenchyme become more depolarized as they differentiate into chondrocytes, and that this depolarization is essential for the expression of genes necessary for chondrocyte fate. However, in many of these cases, the relationship between changes in Vmem and specific pathways that regulate developmental patterning have not been established (Emmons-Bell, 2021).
This study investigated the patterning of Vmem during wing disc development and showed that the regulation of Vmem has an important role in regulating Hh signalling. The cells immediately anterior to the compartment boundary, a zone of active Hh signalling, are more depolarized than surrounding cells, and Hh signalling and depolarized Vmem mutually reinforce each other. This results in an abrupt change in Vmem at the compartment boundary (Emmons-Bell, 2021).
This study shows that Vmem is patterned in a spatiotemporal manner during development of the wing disc of Drosophila and that it regulates Hedgehog signalling at the compartment boundary. First, it was shown that cells immediately anterior to the compartment boundary are relatively more depolarized than cells elsewhere in the wing pouch. This region coincides with the A cells where Hh signalling is most active, as evidenced by upregulation of Ptc. Second, the expression of at least two regulators of Vmem, the ENaC channel Rpk and the alpha subunit of the Na+/K+ ATPase were shown to be expressed at higher levels in this same portion of the disc. Third, by altering Hh signalling, this study demonstrated that the expression of both Rpk and ATPα is increased in cells with increased Hh signalling. Fourth, by manipulating Hh signalling in the disc and using optogenetic methods, both in the salivary gland and wing disc, it was shown that membrane depolarization promotes Hh signalling as assessed by increased membrane localization of Smo, and expression of the target gene ptc. Thus, Hh-induced signalling and membrane depolarization appear to mutually reinforce each other and thus contribute to the mechanisms that maintain the segregation of A and P cells at the compartment boundary (Emmons-Bell, 2021).
Two regions of increased DiBAC fluorescence were observed in the wing imaginal disc. No obvious upregulation of Rpk and ATPα was observed in other discs, and therefore, these studies have focused on the region immediately anterior to the A-P compartment boundary in the wing disc. In the late L3 wing disc, a region of increased DiBAC fluorescence was observed in the A compartment in the vicinity of the D-V boundary. This corresponds to a 'zone of non-proliferating cells' (ZNC). Interestingly, the ZNC is different in the two compartments. In the A compartment, two rows of cells are arrested in G2 while in the P compartment, a single row of cells is arrested in G1. The observation of increased DiBAC fluorescence in the DV boundary of only the A compartment is consistent with previous reports that cells become increasingly depolarized as they traverse S-phase and enter G2. In contrast, cells in G1 are thought to be more hyperpolarized. Additionally, increased expression of the ENaC channel Rpk was observed in two rows of cells at the D-V boundary in the anterior compartment, indicating that increased expression of Rpk could contribute to the depolarization observed in those cells. It is noted, however, that the increased DiBAC fluorescence in these cells was not entirely eliminated by exposing discs to amiloride, indicating that other factors are also likely to contribute (Emmons-Bell, 2021).
These data are consistent with a model where membrane depolarization and Hh-induced signalling mutually reinforce each other in the cells immediately anterior to the compartment boundary. Both membrane depolarization and the presence of Hh seem necessary for normal levels of activation of the Hh signalling pathway in this region; neither alone is sufficient. First, it was shown that Hh signalling promotes membrane depolarization. It was also shown that the expression of Rpk just anterior to the A-P compartment boundary is dependent upon Hh signalling. Elevated Rpk expression is not observed when a hhts allele is shifted to the restrictive temperature, and cells become more depolarized when Hh signalling is constitutively activated through expression of the ci3m allele. Previously published microarray data suggest that Rpk as well as another ENaC family channel Ppk29 are both enriched in cells that also express ptc. However, there is no antibody to assess Ppk29 expression currently. The sensitivity of the depolarization to amiloride indicates that these and other ENaC channels make an important contribution to the membrane depolarization (Emmons-Bell, 2021).
Second, this study has shown that the depolarization increases Hh signalling. The early stages of Hh signalling are still incompletely understood. Hh is thought to bind to a complex of proteins that includes Ptc together with either Ihog or Boi. This alleviates an inhibitory effect on Smo, possibly by enabling its access to specific membrane sterols. Interestingly, it has recently been proposed that Ptc might function in its inhibitory capacity by a chemiosmotic mechanism where it functions as a Na+ channel. An early outcome of Smo activation is its localization to the membrane where its C-terminal tail becomes phosphorylated and its ubiquitylation and internalization are prevented. By manipulating channel expression in the wing disc, and by optogenetic experiments in both the salivary gland and wing disc, this study has shown that membrane depolarization can promote Hh signalling as assessed by increased Smo membrane localization and increased expression of the target gene ptc. The time course of Smo activation is relatively rapid (over minutes) and is therefore unlikely to require new transcription and translation. In the P compartment, membrane Smo levels are elevated likely because of the complete absence of Ptc, and some downstream components of the Hh signalling pathway are known to be activated. However, since Ci is not expressed in P cells, target gene expression is not induced. In the cells just anterior to the boundary, the partial inhibition of Ptc by Hh together with membrane depolarization seem to combine to achieve similar levels of Smo membrane localization. More anteriorly, the absence of this mutually reinforcing mechanism appears to result in Smo internalization (Emmons-Bell, 2021).
The experiments do not point to a single mechanism by which depolarization promotes Hh signalling. It is possible that depolarization results in increased Ca2+ levels by opening Ca2+channels at the plasma membrane or by promoting release from intracellular sources (e.g. the ER or mitochondria). Indeed, there is evidence that Ca2+ entry into the primary cilium promotes Hh signalling, and recent work shows that targets of Sonic Hedgehog (Shh) signalling during mammalian development is augmented by Ca2+ influx. A second possibility is that membrane depolarization could, by a variety of mechanisms, activate the kinases that phosphorylate the C-terminal tail of Smo and maintain it at the plasma membrane in an activated state. Depolarization could also impact electrostatic interactions at the membrane that make the localization of Smo at the membrane more favourable. Since Rpk and ATPα are expressed at higher levels in the cells that receive Hh, which have been postulated to make synapse-like projections with cells that produce Hh, it is conceivable that these channels could modulate synapse function. Additionally, while this work was under review, it has been reported that reducing glycolysis depletes ATP levels and results in depolarization in the wing imaginal disc, reducing the uptake of Hh pathway inhibitors and stabilizing Smo at the cell membrane (Spannl, 2020). Importantly, all these mechanisms are not mutually exclusive and their roles in Hh signalling are avenues for future research (Emmons-Bell, 2021).
It is now generally accepted that both cell-cell signalling and mechanical forces have important roles in cell fate specification and morphogenesis. This work adds to a growing body of literature suggesting that changes in Vmem, a relatively understudied parameter, may also have important roles in development. Integrating such biophysical inputs with information about gene expression and gene regulation will lead to a more holistic understanding of development and morphogenesis (Emmons-Bell, 2021).
Organ formation relies on the orchestration of pattern formation, proliferation and growth during development. How these processes are integrated at individual cell level remains unclear. Studies using Drosophila wing imaginal discs as a model system have provided valuable insights into pattern formation, growth control and regeneration in the past decades. This study provides single cell transcriptomic landscapes of pattern formation, proliferation and growth of wing imaginal discs. Patterning information is robustly maintained in the single cell transcriptomic data and can provide reference matrices to computationally map single cells into discrete spatial domains. Assignment of wing disc single cells to spatial sub-regions facilitates examination of patterning refinement processes. Single cells were clustered into different proliferation and growth states, and the correlation was evaluated between cell proliferation/growth states and spatial patterning. Furthermore, the single cell transcriptomic analysis allowed quantitative examination of the disturbance of differentiation, proliferation and growth in a well-established tumor model. A database explores these datasets at: http://drosophilayanlab-virtual-wingdisc.ust.hk:3838/v2/ (Deng, 2019).
'Developmental robustness' is the ability of biological systems to maintain a stable phenotype despite genetic, environmental or physiological perturbations. In holometabolous insects, accurate patterning and development is guaranteed by alignment of final gene expression patterns in tissues at specific developmental stage such as molting and pupariation, irrespective of individual rate of development. In the present study, faster developing Drosophila melanogaster populations were used that show reduction of ~22% in egg to adult development time. Flies from the faster developing population exhibit phenotype constancy, although significantly small in size. The reduction in development time in faster developing flies is possibly due to coordination between higher ecdysteroid release and higher expression of developmental genes. The two together might be ensuring appropriate pattern formation and early exit at each development stage in the populations selected for faster pre-adult development compared to their ancestral controls. This study reports that apart from plasticity in the rate of pattern progression, alteration in the level of gene expression may be responsible for pattern integrity even under reduced development time (Chauhan, 2020).
The Drosophila wing has served as a paradigm to mechanistically characterize the role of morphogens in patterning and growth. Wingless (Wg) and Decapentaplegic (Dpp) are expressed in two orthogonal signaling centers, and their gradients organize patterning by regulating the expression of well-defined target genes. By contrast, graded activity of these morphogens is not an absolute requirement for wing growth. Despite their permissive role in regulating growth, this study shows that Wg and Dpp are utilized in a non-interchangeable manner by the two existing orthogonal signaling centers to promote preferential growth along the two different axes of the developing wing. The data indicate that these morphogens promote anisotropic growth by making use of distinct and non-interchangeable molecular mechanisms. Whereas Dpp drives growth along the anterior-posterior axis by maintaining Brinker levels below a growth-repressing threshold, Wg exerts its action along the proximal-distal axis through a double repression mechanism involving the T cell factor (TCF) Pangolin (Barrio, 2020).
Two orthogonal signaling centers, corresponding to the AP and DV compartment boundaries and expressing the Dpp and Wg morphogens, regulate growth and patterning of the developing wing along the AP and PD axes, respectively. Whereas graded activity of these morphogens defines the spatial location of longitudinal veins and sensory organs that decorate the adult wing along these two axes, their graded activity is not an absolute requirement for its growth-promoting role. Despite the non-instrumental role of Wg and Dpp gradients in regulating tissue size, this study presents evidence that these two morphogens control the size of the adult wing along two orthogonal axes by mediating the growth-promoting activities of compartment boundaries in a non-interchangeable manner through the use of morphogen-specific molecular mechanisms. While Dpp regulates growth along the AP axis by maintaining the levels of the transcriptional repressor Brinker below a growth-repressing threshold, Wg regulates growth along the PD axis by counteracting the activity of TCF as a transcriptional repressor. At the time TCF was molecularly identified in flies, it was shown that clones of cells mutant for TCF are poorly recovered in the primordium of the wing pouch and proposed to be a consequence of TCF promoting proliferative growth. However, later studies identified cell competition as the mechanism to eliminate cells with steep differences in Wg signaling in the wing primordium. The Warts-Hippo signaling pathway governs organ size in animals, and the upstream regulators include the atypical cadherins Fat and Dachsous. Surprisingly, inactivation of the Warts-Hippo signaling pathway was unable to rescue the tissue size defects caused by morphogen depletion. These data indicate that for wing blade cells to grow along the PD and AP axes, cells need first to lose TCF and Brinker, and it is proposed that Hippo signaling can then modulate the amount of growth of those cells in which these two repressors are not active or expressed. The experimental data are consistent with a model whereby a minimal amount of signaling from the two morphogens, sufficient to maintain the activity levels of the two transcriptional repressors below a growth-repressing threshold, regulate the physical size of the adult wing primordium along the AP and PD axes. The mechanistic similarities of how Dpp and Wg morphogens, their gradients, and their range of activity regulate the patterning and growth of the fly wing are remarkable and might shed light on the role of morphogens in regulating proliferative growth and patterning in vertebrates (Barrio, 2020).
Experimental conditions in developing wings in which proliferation rates are either increased or reduced have shown that a perfectly normal-sized wing can be obtained with fewer or more cells. Similarly, experimental randomization of the orientation of cell divisions in the growing wing primordium can give rise to well-shaped adult wings. These results suggest that the ability of compartment boundaries, and their dedicated morphogens, to drive anisotropic growth and regulate the width and length of the adult wing blade does not rely only on the control of cell division or oriented cell divisions. Several experimental data indicate that it is the range of the morphogen and not the total amount of it that regulates the physical size, and not the number of cells, of each axis. How do Wg and Dpp regulate growth preferentially along a certain axis and not the other? Restricted expression of these two morphogens along the two existing orthogonal boundaries does not appear to be essential as their ability to drive anisotropic growth is still observed when they are ubiquitously overexpressed in all wing cells. The experimental data indicate that the capacity of Wg and Dpp to drive anisotropic growth relies on the existence of morphogen-specific and non-interchangeable molecular mechanisms mediating their growth-promoting activities and the requirement of the presence of the two of them to drive growth. In this regard, each morphogen promotes growth only along a particular axis, as the distance to the source of the other morphogen has to be maintained to get sufficient levels of the two of them to promote wing growth. The data also indicate that the Wg gradient contributes to orient growth along the PD axis. However, this contribution does not appear to play an essential role since well-shaped elongated wings can be obtained upon uniform expression of Wg (Barrio, 2020).
While the growth-promoting role of Dpp emanating from the AP compartment boundary has been experimentally validated and recently clarified, previous experimental characterization of the growth-promoting role of Wg emanating from the DV compartment boundary reached opposing conclusions. This study presents experimental evidence that Wg mediates the organizing activity of the DV boundary in terms of growth, as uniform expression of this morphogen rescues the extreme growth defects caused by the absence of a DV signaling center. Moreover, the data indicate that Wg is the main growth-promoting Wnt in the developing wing, the DV boundary is the main source of Wg driving proliferative growth of the primordium of the wing appendage, and boundary Wg regulates tissue growth and proliferation rates equally in distal and proximal regions of the developing wing appendage, throughout development and independently of its potential role as survival factor. This latter observation questions the proposal that Wg drives wing growth, at least in part, by promoting cell survival. This proposal was based on the ability of apoptotic inhibitors to rescue the poor recovery and growth of clones of cells unable to transduce the Wg signal, but cell competition was subsequently shown to be the mechanism used to eliminate cells with steep differences in Wg signaling. The experimental observation that even late depletion of Wg expression has an effect on wing size questions the proposal that continuous exposure to Wg is not an absolute requirement for wing cells to grow. Recently, a membrane-tethered form of the Wg protein was shown to be able to substitute for the endogenous Wg protein in producing normally patterned wings of nearly the right size. Either the activity of cellular extensions at a distance, higher stability of the membrane-tethered form of Wg, or emerging compensatory mechanisms should be able to facilitate or extend in time the exposure of all wing cells to the morphogen in the absence of secretion, thus fulfilling its continuous growth-promoting role (Barrio, 2020).
Archipelago (Ago) is a Drosophila homolog of mammalian F-box and WD repeat domain-containing 7 (FBW7, also known as FBXW7). In previous studies, FBW7 has been addressed as a tumor suppressor mediating ubiquitin-dependent proteolysis of several oncogenic proteins. Ubiquitination is a type of protein modification that directs protein for degradation as well as sorting. The level of beta-catenin (β-cat), an intracellular signal transducer in Wnt signaling pathway, is reduced upon overexpression of FBW7 in human cancer cell lines. Loss of function mutations in FBW7 and overactive Wnt signaling have been reported to be responsible for human cancers. This study found that Ago is important for the formation of shafts in chemosensory bristles at wing margin. This loss of shaft phenotype by knockdown of ago was rescued by knockdown of wingless (wg) whereas wing notching phenotype by knockdown of wg was rescued by knockdown of ago, establishing an antagonistic relationship between ago and wg. In line with this finding, knockdown of ago increased the level of Armadillo (Arm), a homolog of β-cat, in Drosophila tissue. Furthermore, knockdown of ago increased the level of Distal-less (Dll) and extracellular Wg in wing discs. In S2 cells, the amount of secreted Wg was increased by knockdown of Ago but decreased by Ago overexpression. Therefore, Ago plays a previously unidentified role in the inhibition of Wg secretion. Ago-overexpressing clones in wing discs exhibited accumulation of Wg in endoplasmic reticulum (ER), suggesting that Ago prevents Wg protein from moving to Golgi from ER. It is concluded that Ago plays dual roles in inhibiting Wg signaling. First, Ago decreases the level of Arm, by which Wg signaling is downregulated in Wg-responding cells. Second, Ago decreases the level of extracellular Wg by inhibiting movement of Wg from ER to Golgi in Wg-producing cells (Nam, 2020).
Developmental patterning is thought to be regulated by conserved signalling pathways. Initial patterns are often broad before refining to only those cells that commit to a particular fate. However, the mechanisms by which pattern refinement takes place remain to be addressed. Using the posterior crossvein (PCV) of the Drosophila pupal wing as a model, into which bone morphogenetic protein (BMP) ligand is extracellularly transported to instruct vein patterning, this study investigate how pattern refinement is regulated. It was found that BMP signalling induces apical enrichment of Myosin II in developing crossvein cells to regulate apical constriction. Live imaging of cellular behaviour indicates that changes in cell shape are dynamic and transient, only being maintained in those cells that retain vein fate competence after refinement. Disrupting cell shape changes throughout the PCV inhibits pattern refinement. In contrast, disrupting cell shape in only a subset of vein cells can result in a loss of BMP signalling. It is proposed that mechano-chemical feedback leads to competition for the developmental signal which plays a critical role in pattern refinement (Toddie-Moore, 2021).
How morphogen gradients control patterning and growth in developing tissues remains largely unknown due to lack of tools manipulating morphogen gradients. This study generated two membrane-tethered protein binders that manipulate different aspects of Decapentaplegic (Dpp), a morphogen required for overall patterning and growth of the Drosophila wing. One is "HA trap" based on a single-chain variable fragment (scFv) against the HA tag that traps HA-Dpp to mainly block its dispersal, the other is "Dpp trap" based on a Designed Ankyrin Repeat Protein (DARPin) against Dpp that traps Dpp to block both its dispersal and signaling. Using these tools, it was found that, while posterior patterning and growth require Dpp dispersal, anterior patterning and growth largely proceed without Dpp dispersal. dpp transcriptional refinement is shown from an initially uniform to a localized expression and persistent signaling in transient dpp source cells render the anterior compartment robust against the absence of Dpp dispersal. Furthermore, despite a critical requirement of dpp for the overall wing growth, neither Dpp dispersal nor direct signaling is critical for lateral wing growth after wing pouch specification. These results challenge the long-standing dogma that Dpp dispersal is strictly required to control and coordinate overall wing patterning and growth (Matsuda, 2021).
Within developing tissues, cell proliferation, cell motility and other cell behaviors vary spatially, and this variability gives a complexity to the morphogenesis. Recently, novel formalisms have been developed to quantify tissue deformation and underlying cellular processes. A major challenge for the study of morphogenesis now is to objectively define tissue sub-regions exhibiting different dynamics. This paper proposes a method to automatically divide a tissue into regions where the local deformation rate is homogeneous. This was achieved by several steps including image segmentation, clustering and region boundary smoothing. The use of the pipeline is demonstrated using a large dataset obtained during the metamorphosis of the Drosophila pupal notum. It was also adapted to determine regions in which the time evolution of the local deformation rate is homogeneous. Finally, its use generalized to find homogeneous regions for cellular processes such as cell division, cell rearrangement, or cell size and shape changes. Its use is also demonstrated on wing blade morphogenesis. This pipeline will contribute substantially to the analysis of complex tissue shaping, and the biochemical and biomechanical regulations driving tissue morphogenesis (Yamashita, 2021).
How signaling proteins generate a multitude of information to organize tissue patterns is critical to understanding morphogenesis. In Drosophila, FGF produced in wing-disc cells regulates the development of the disc-associated air-sac-primordium (ASP). This study shows that FGF is Glycosylphosphatidylinositol-anchored to the producing cell surface and that this modification both inhibits free FGF secretion and promotes target-specific cytoneme contacts and contact-dependent FGF release. FGF-source and ASP cells extend cytonemes that present FGF and FGFR on their surfaces and reciprocally recognize each other over distance by contacting through cell-adhesion-molecule (CAM)-like FGF-FGFR binding. Contact-mediated FGF-FGFR interactions induce bidirectional responses in ASP and source cells that, in turn, polarize FGF-sending and FGF-receiving cytonemes toward each other to reinforce signaling contacts. Subsequent un-anchoring of FGFR-bound-FGF from the source membrane dissociates cytoneme contacts and delivers FGF target-specifically to ASP cytonemes for paracrine functions. Thus, GPI-anchored FGF organizes both source and recipient cells and self-regulates its cytoneme-mediated tissue-specific dispersion (Du, 2022).
The establishment of body pattern is a fundamental process in developmental biology. In Drosophila, the wing disc is subdivided into dorsal (D) and ventral (V) compartments by the D/V boundary. The dorsal fate is adopted by expressing the selector gene apterous (ap). ap expression is regulated by three combinational cis-regulatory modules which are activated by EGFR pathway, Ap-Vg auto-regulatory and epigenetic mechanisms. This study found that the Tbx family transcription factor Optomotor-blind (Omb) restricted ap expression in the ventral compartment. Loss of omb induced autonomous initiation of ap expression in the middle third instar larvae in the ventral compartment. Oppositely, over-activation of omb inhibited ap in the medial pouch. All three enhancers apE, apDV and apP were upregulated in omb null mutants, indicating a combinational regulation of ap modulators. However, Omb affected ap expression neither by directly regulating EGFR signaling, nor via Vg regulation. Therefore, a genetic screen of epigenetic regulators, including the Trithorax group (TrxG) and Polycomb group (PcG) genes was performed. Knocking down the TrxG gene kohtalo (kto), domino (dom) or expressing the PcG gene grainy head (grh), the ectopic ap in omb mutants was repressed. The inhibition of apDV by kto knockdown and grh activation could contribute to ap repression. Moreover, Omb and the EGFR pathway are genetically parallel in ap regulation in the ventral compartment. Collectively, Omb is a repressive signal for ap expression in the ventral compartment, which requires TrxG and PcG genes (Chen, 2023).
Klipa, O., El Gammal, M. and Hamaratoglu, F. (2023). Elimination of aberrantly specified cell clones is independent of interfacial Myosin II accumulation. J Cell Sci 136(13). PubMed ID: 37309190
Spatial organization within an organ is essential and needs to be maintained during development. This is largely implemented via compartment boundaries that serve as barriers between distinct cell types. Biased accumulation of junctional non-muscle Myosin II along the interface between differently fated groups of cells contributes to boundary integrity and maintains its shape via increased tension. Using the Drosophila wing imaginal disc, whether interfacial tension driven by accumulation of Myosin is responsible for the elimination of aberrantly specified cells that would otherwise compromise compartment organization was tested. To this end, Myosin II levels were genetically reduced in three different patterns: in both wild-type and misspecified cells, only in misspecified cells, and specifically at the interface between wild-type and aberrantly specified cells. The recognition and elimination of aberrantly specified cells do not strictly rely on tensile forces driven by interfacial Myosin cables. Moreover, apical constriction of misspecified cells and their separation from wild-type neighbours occurred even when Myosin levels were greatly reduced. Thus, it is concluded that the forces that drive elimination of aberrantly specified cells are largely independent of Myosin II accumulation (Klipa, 2023).
Programmed cell death (apoptosis) is a homeostasis program of animal tissues designed to remove cells that are unwanted or are damaged by physiological insults. To assess the functional role of apoptosis, the consequences were studied of subjecting Drosophila epithelial cells defective in apoptosis to stress or genetic perturbations that normally cause massive cell death. Many of those cells acquire persistent activity of the JNK pathway, which drives them into senescent status, characterized by arrest of cell division, cell hypertrophy, Senescent Associated β-gal activity (SA-β-gal), reactive oxygen species (ROS) production, Senescent Associated Secretory Phenotype (SASP) and migratory behaviour. Two classes of senescent cells were identified in the wing disc: 1) those that localize to the appendage part of the disc, express the upd, wg and dpp signalling genes and generate tumour overgrowths, and 2) those located in the thoracic region do not express wg and dpp nor they induce tumour overgrowths. Whether to become tumorigenic or non-tumorigenic depends on the original identity of the cell prior to the transformation. The p53 gene was also found to contribute to senescence by enhancing the activity of JNK (Garcia-Arias, 2023).
In Drosophila, wing epidermal cells undergo programmed cell death as the last step of metamorphosis. The aim of this study was to evaluate the role of hid, particularly the Wrinkled mutation (hidW), an allele of hid, in the cell death. The wing epithelial cell death is suppressed by loss-of-function mutation of hid, indicating that the death is governed by a cascade involving hid. Examination of the cell death in hidW showed that precocious death started at G stage, 3 h before eclosion. Thus, mutated-HID in the hidW mutant was activated at G stage, supporting the gain-of-function effect of hidW mutation (Ohta, 2023).
engrailed (en) encodes a homeodomain transcription factor crucial for the proper development of Drosophila embryos and adults. Like many developmental transcription factors, en expression is regulated by many enhancers, some of overlapping function, that drive expression in spatially and temporally restricted patterns. The en embryonic enhancers are located in discrete DNA fragments that can function correctly in small reporter transgenes. In contrast, the en imaginal disc enhancers (IDEs) do not function correctly in small reporter transgenes. En is expressed in the posterior compartment of wing imaginal disks; small IDE-reporter transgenes are expressed in the anterior compartment, the opposite of what is expected. The data show that the En protein binds to en IDEs, and it is suggested that En directly represses IDE function. Two en IDEs, 'O' and 'S' were identified. Deletion of either of these IDEs from a 79kb HA-en rescue transgene (HAen79) caused a loss-of-function en phenotype when the HAen79 transgene was the sole source of En. In contrast, flies with a deletion of the same IDEs from the endogenous en gene had no phenotype, suggesting a resiliency not seen in the HAen79 rescue transgene. Inserting a gypsy insulator in HAen79 between en regulatory DNA and flanking sequences strengthened the activity of HAen79, giving better function in both the ON and OFF transcriptional states. Altogether these data show that the en IDEs stimulate expression in the entire imaginal disc, and that the ON/OFF state is set by epigenetic regulators. Further, the endogenous locus imparts a stability to en function not seen even in a large transgene, reflecting the importance of both positive and negative epigenetic influences that act over relatively large distances in chromatin (Cheng, 2023).
In developing Drosophila, perturbing the growth of one imaginal disc - the parts of a holometabolous larva that become the external adult organs - has been shown to retard growth of other discs and delays development, resulting in tight inter-organ growth coordination and the generation of a correctly proportioned adult. This study used the wing imaginal disc in Drosophila to study and identify mechanisms of intra-organ growth coordination. Larvae were generated in which the two compartments of the wing imaginal disc have ostensibly different growth rates (wild-type or growth-perturbed). It was found that there is tightly coordinated growth between the wild-type and growth-perturbed compartments, where growth of the wild-type compartment is retarded to match that of the growth-perturbed compartment. Crucially, this coordination is disrupted by application of exogenous 20-hydroxyecdysone (20E), which accelerates growth of the wild-type compartment. The role of 20E signaling in growth coordination was further elucidate by showing that in wild-type discs, compartment-autonomous up-regulation of 20E signaling accelerates compartment growth and disrupts coordination. Interestingly, growth acceleration through exogenous application of 20E is inhibited with suppression of the Insulin/Insulin-like Growth Factor Signaling (IIS) pathway. This suggests that an active IIS pathway is necessary for ecdysone to accelerate compartment growth. Collectively, these data indicate that discs utilize systemic mechanisms, specifically ecdysone signaling, to coordinate intra-organ growth (Gokhale, 2016).
The results reveal that growth among developmental compartments in an organ is tightly coordinated, such that even if the growth of one compartment is perturbed, both compartments grow at more-or-less the same relative rate as observed in wild-type flies. This growth coordination between compartments is disrupted by exogenously feeding 20E to growth-perturbed larvae, resulting in acceleration in the growth rate of the unperturbed compartment. This growth acceleration upon feeding 20E is dependent on IIS in the unperturbed compartment. Collectively these data support a model of imaginal disc growth regulation whereby growth perturbation in one compartment causes a systemic reduction in circulating ecdysteroids, which results in reduction in growth rate of the adjacent compartment (Gokhale, 2016).
These data are surprising in light of previous studies that suggest that imaginal discs and individual compartments within imaginal discs can autonomously grow to their target size. A previous study cultured WT imaginal discs in the abdomen of adults hosts and found that these discs grow autonomously to their normal size. Another study generated 'fast' discs and compartments in M-/+ larvae and demonstrated that these compartments have higher growth rates relative to the body as a whole and to adjacent compartments. It was further demonstrated that the 'fast' compartments and discs are developmentally advanced as compared to M-/+controls. Collectively, these data support the hypothesis that imaginal disc possesses an autonomous mechanism for arresting growth once they reach a target size, and that this mechanism operates at the level of developmental compartments. Whilst compartments may possess a target size, the current data suggest that they do not grow independently to this size, at least in vivo. Rather growth between developmental compartments is coordinated even when one compartment is growth perturbed, and this growth coordination appears to be regulated by systemic rather than disc-autonomous mechanisms, at least in part (Gokhale, 2016).
The conclusions are supported by data from Mesquita (2010), who also looked at inter-compartmental growth in the Drosophila wing imaginal disc. They observed that slowing the growth of one compartment non-autonomously slowed the growth of the adjacent compartment. They further demonstrate that the signal from the growth-perturbed compartment is dependent on Drosophila p53. However, they do not elucidate what the signal is. The current results suggest that the signal involves ecdysone. This is surprising given the current understanding of wing imaginal disc growth. Recent models of disc growth suggest that growth of the wing imaginal disc is driven mainly by morphogen gradients formed by the patterning genes Wg, Dpp, and Vg, which drive cellular proliferation within the disc. Recent studies further implicate disc-autonomous mechanisms in regulating the relative size of different compartments within the wing (Ferreira, 2015). The current data show that systemic signaling, mediated by ecdysone, is also critical for regulating growth rates among different parts of the disc (Gokhale, 2016).
The involvement of ecdysone in intra-organ growth coordination echoes its known role in inter-organ growth coordination. As noted above, growth among organs is tightly coordinated when one organ is growth perturbed-a consequence of the growth-perturbed organ suppressing ecdysone synthesis. Addition of ecdysone to these growth-perturbed larvae is able to rescue the growth rate of undamaged imaginal discs. Ecydsone is however not able to rescue the growth rate of the growth perturbed tissues, most likely because the inherent growth perturbation of these tissues prevents them from responding to ecdysone. Similar to these studies on inter-organ growth coordination, the current data suggest ecdysone is able to rescue the growth rate of wild-type compartments in M-/+larvae, and this is mediated by compartment-autonomous ecdysone signaling (Gokhale, 2016).
While the current data indicate that ecdysone is an important growth-coordinating signal among developmental compartments, it is unclear precisely which tissue is influencing ecdysone synthesis. It is possible that in larvae with antfast:postslow discs the limitation on ecdysone synthesis might be an autonomous effect of the Minute mutation on the prothoracic gland, since the whole of the rest of the larvae is Minute. However, the data demonstrate that knock-down of RpS3 using engrailed-GAL4, which is not expressed in the prothoracic gland, still retards disc growth. This suggests that the growth coordination mechanism is regulated by a signal from the compartments themselves. As discussed above, in studies where systemic growth is retarded through localized tissue damage, including knock-down of ribosomal proteins, it is the damaged/growth-perturbed tissue itself that inhibits ecdysone synthesis by signaling via dILP8. Therefore, in larvae with antfast:postslow discs, ecdysteroidgenesis could be limited via a dILP8-dependent mechanism. Which compartment is generating a putative dILP8 signal is, however, unclear. dILP8 levels are highest at the L2-L3 transition and decline during L3, before increasing somewhat before pupariation. It is possible, therefore, that in larvae with antfast:postslow discs, it is the immature slow-growing posterior compartment that is secreting dILP8. Conversely, the residual generation and death of M-/- cells in the anterior compartment through mitotic recombination early in L3 may also drive dILP8 synthesis. Further experiments exploring the role of dILP8 in intra-organ growth coordination are clearly necessary (Gokhale, 2016).
A key feature of growth coordination is that ecdysone acts as a promoter of growth for imaginal discs. This appears contrary to previous findings that show that ecdysone inhibits larval body growth by inhibiting IIS or Myc in the fat body. However, evidence from other insect species suggests that ecdysone can function as either a growth promoter or inhibitor, depending on its concentration. Specifically, in vitro evidence from Manduca shows that low concentrations of ecdysone can promote growth of imaginal tissues, while higher concentrations stimulate differentiation, and stop cell proliferation. Further evidence from Manduca suggests that ecdysone promotes mitosis by regulating the cell cycle, and thus acts as a mitogen. These data echo data from Drosophila that suggests that ecdysone regulates cell cycle progression and promotes imaginal disc growth via the ecdysone inducible gene crooked legs. Collectively, it is apparent, therefore, that ecdysone is a central regulator of larval and imaginal tissue growth, although the tissue-specific effects and molecular mechanisms involved have not yet been completely elucidated. Research from this and other labs supports the hypothesis that imaginal discs reduce their growth rates in response to low levels of ecdysone. At the same time, low levels of ecdysone increase body growth rate and final adult body size. Together these data suggest that ecdysone suppresses the growth of larval tissue (which comprises the majority of the larva) but promotes growth of imaginal tissues. This hypothesis has intuitive appeal in that a key function of ecdysone is to 'prepare' the larva for pupariation and metamorphosis, a process that involves breakdown and autophagy of the larval tissues to provide nutrients for final growth and differentiation of the imaginal discs (Gokhale, 2016).
Research over the past decade has elucidated mechanisms by which ecdysone functions as a suppressor of larval growth. These studies demonstrate a role for IIS in ecdysone-mediated suppression of larval growth. Specifically, ecdysone signaling in the fat body suppresses IIS, which in turn inhibits systemic IIS and larval growth through repression of dILP2 release from the brain and promotes fat body autophagy. How ecdysone promotes imaginal disc growth is less clear, however. A recent paper by Herboso (2015), indicated that ecdysone promotes growth by suppressing Thor signaling in the imaginal discs. Discs from larvae with reduced ecdysone synthesis have elevated levels of Thor, a repressor of growth that is a target of the IIS pathway. The hypothesis that ecdysone regulates and coordinates growth via IIS/TOR signaling is further supported by the observation that down-regulation of Inr activity prevents the wild-type compartment of antfast:postslow discs from increasing its relative growth rate in response to ecdysone (Gokhale, 2016).
However, additional data suggest a more nuanced role for IIS in coordinating growth among developmental compartments. In particular, changes in Inr activity in the anterior compartment do not affect relative compartment growth rate in larvae that are otherwise wholly wild-type. Rather, changes in Inr activity increase or decrease relative compartment size, presumably due to changes in compartment growth earlier in development. This is surprising, given that mutations of Inr reduce the growth and proliferation of clones in the wing imaginal disc during L3. In antfast:postslow discs, however, changes in Inr activity does alter growth coordination during L3, but in a counterintuitive way: reduced Inr activity increases relative growth rate, whilst increased Inr activity decreases relative growth rate. This is the opposite of what would be predicted if ecdysone promotes growth by directly upregulating IIS. One interpretation of these data is that the anterior compartments of the antfast:postslow disc adjust their relative growth rate to rescue the final anterior:posterior size ratio, presumably using a mechanism independent of ecdysone. Why this rescue is not evident in wild-type larvae is unclear, but suggest that the rescue mechanism is able to override the ecdysone-regulated mechanism that coordinates growth rates between compartments with different potential growth rates (Gokhale, 2016).
From the current study and those of others, it seems unlikely that ecdysone promotes imaginal disc growth only through its effects on IIS. In particular, the role of ecdysone in the regulation of differentiation and patterning genes such as broad, senseless, and cut has been well elucidated. Patterning genes are known to regulate cell proliferation. It is therefore possible that ecdysone also regulates imaginal disc growth by regulating the expression of patterning genes in the imaginal disc. One of the challenges in elucidating the role of ecdysone signaling in imaginal disc development is that manipulating ecdysone-signaling organ-autonomously in imaginal discs is technically difficult. This study likely only subtly up-regulated ecdysone signaling by knocking down EcR compartment-autonomously and found that this mild knockdown accelerated compartment growth. It is seems likely that this effect is related to the degree of the knockdown, however, for two reasons. First, complete knockdown of EcR will ultimately block ecdysone signaling, even if it de-represses the expression of certain genes. Second, ecdysone levels can both promote and inhibit insect growth and development depending on its level. As discussed above, moderate level of ecdysone are sufficient to stimulate imaginal disc growth in vitro, while high levels suppress cell proliferation. More precise methods of manipulating ecdysone signaling at a cellular and tissue level are therefore needed (Gokhale, 2016).
In summary, this study provides evidence for an ecdysone-dependent mechanism that coordinates growth between compartments in the wing imaginal disc of Drosophila. The data suggest that the control of cell proliferation across the imaginal disc is not an entirely autonomous process, but is coordinated through humoral signaling. This research also highlights the crosstalk between different systemic signaling mechanisms - insulin/IGF- and ecdysone-signaling - in the generation of correctly proportioned organs. The developmental mechanisms regulating organ size, while best studied in Drosophila, are conserved across all animals. There is considerable evidence that localized growth perturbation causes systemic growth retardation in humans. For example, children suffering from chronic inflammatory diseases such as Crohn's disease have systemic growth hormone insensitivity and experience severe growth retardation as a complication of the disease. The utilization of systemic signaling mechanisms to coordinate growth within and between organs may thus be a conserved mechanism across all animals (Gokhale, 2016).
The Drosophila wing imaginal disc is a sac-like structure that is composed of two opposing cell layers: peripodial epithelium (PE, also known as squamous epithelia) and disc proper (DP, also known as pseudostratified columnar epithelia). The molecular mechanism of cell morphogenesis has been well studied in the DP but not in the PE. Although proper Dpp signalling activity is required for proper PE formation, the detailed regulation mechanism is poorly understood. This study found that the Dpp target gene spalt (sal) is only expressed in DP cells, not in PE cells, although pMad is present in the PE. Increasing Dpp signalling activity cannot activate Sal in PE cells. The absence of Sal in the PE is essential for PE formation. The ectopic expression of sal in PE cells is sufficient to increase the PE cell height. Down-regulation of sal in the DP reduced DP cell height. It was further demonstrated that the known PE cell height regulator Lines, which can convert PE into a DP cell fate, is mediated by sal mis-activation in PE because sal-RNAi and lines co-expression largely restores PE cell morphology. By revealing the microtubule distribution, it was demonstrated that Lines- and Sal-heightened PE cells are morphologically similar to the intermediate cell with cuboidal morphology (Tang, 2016).
The wing disc is a sac-like structure composed of PE, DP, and intermediate cells linking the PE and DP. To investigate the potential role of Dpp signalling in PE morphogenesis, the distribution of Dpp signalling activity in late 3rd instar (L3) wing imaginal discs was revealed in both the x-y and x-z views. Using an antibody of phospho-Mothers against dpp (pMad) to reveal Dpp signal transduction activity, it was found that Dpp signal transduction was ubiquitously present in both the PE and DP. The pMad level was relatively reduced in the central PE compared with the central DP. Dpp target gene expression patterns were detected in the PE. The main Dpp target genes are brinker (brk), omb, and sal in the L3 wing discs. brk was transcribed in both the PE and DP, with a relatively weaker level in the PE, as indicated by a brk-lacZ reporter. However, both omb and sal were transcribed only in the DP, not in the PE. These data indicate that the Dpp target genes omb and sal are asymmetrically expressed in the PE and DP. Brk is also a repressor of other Dpp target genes, including sal and omb, and thereby restricts their expression domains to the medial DP region. The presence of Brk in the PE might be a direct cause of the absence of Omb and Sal in the PE. To assess this possibility, brk-RNAi was expressed in the PE. Sal expression was not detectable in the central PE. The efficiency of brk-RNAi was demonstrated by the elevation of Brk targets Omb and Sal in lateral wing discs of C765>brk-RNAi. To further confirm that Dpp signalling cannot induce sal expression in the PE, a constitutive active form of the Dpp receptor tkvQD was expressed in the PE. Sal was not induced in central PE. When tkvQD clones were generated, Sal was induced only in clones within the DP and not in clones located in the PE). Similarly, Omb was not induced in clones located in the PE. Ubiquitous expression of tkvQD failed to induce Omb in the PE. These data demonstrate that Dpp signalling cannot activate omb and sal in PE (Tang, 2016).
The expression patterns of Dpp target genes are well studied in wing DP. Dpp controls target genes (sal and omb) expression indirectly through repression of the transcriptional repressor Brk. Dpp target gene expression patterns have not been studied in wing PE to date. The results revealed that pMad is ubiquitously present in both DP and PE. However, brk-lacZ was still present in PE. Either suppressing brk or elevating Dpp signalling by expressing tkvQD cannot induce Sal and Omb in the PE. Except for Lin, other factors, such as Bowl, Wg, and EGFR, cannot induce Sal in the PE. The factors that suppress sal in the PE require further investigation (Tang, 2016).
As omb and sal are expressed only in the DP, not the PE, it was therefore asked whether this asymmetric transcription of omb and sal is essential for correct PE formation. To test this possibility, sal was mis-expressed in the PE using the Gal4-UAS system. dpp-Gal4 line is expressed in narrow stripes in the lateral PE and in the middle DP. When sal was ectopically expressed in the dpp-Gal4 domain, the height of lateral PE was notably elongated to a height similar to that of intermediate cells. Then, sal was expressed driven by C765-Gal4, which is ubiquitously expressed in both DP and PE. A similar elongation phenotype was observed in the PE. The cell height of the central PE was elongated to a height similar to that of cuboidal cells. The extent of elongation as a result of dpp-Gal4 was stronger than that of C765-Gal4. This difference might be due to the differences in Gal4 activity because dpp-Gal4 is stronger than C765-Gal4. The quantification of cell height, using the ratio between PE and DP cells within one wing disc, revealed a significant increase in sal mis-expression discs. Consistently, the PE height ratio between sal mis-expression and control also revealed a significant increase. To confirm this result, sal over-expression clones were generated in PE. From the x-z cross view, the clonal height was apparently elongated to a height similar to that for cuboidal cells. Therefore, it is concluded that sal is sufficient to elongate PE height (Tang, 2016).
Unlike the effect of sal mis-expression, the elongation of PE height in case of omb ectopic expression was not apparent, however, the differences in the PE/DP ratio and normalized PE height for C765>omb wing discs are statistically significant. Strong overexpression of omb induces severe extrusion and basal delamination, and cell motility can thicken the wing disc. Although a relatively weaker UAS-omb line was used, the side effect from cell movement may remain, thus leading to the statistic difference in the PE measurement. A previous report demonstrates that if Dpp signalling is suppressed in the PE by Ubx-Gal4 driven dad, a portion of the PE cells are elongated to a cuboidal shape. Therefore, suppressing Dpp signalling in the PE and expressing sal in the PE exhibit similar effects. When carefully assessing the Ubx-Gal4 expression domain, a portion of the expressing cells were, surprisingly, located in the DP. Thus, a non-autonomous effect from a loss of Dpp signalling in the DP in the Ubx>dad wing disc is reasonable. Because when dad-expressing clones were generated within PE, the height of PE did not increase. When Omb-RNAi was driven by hh-Gal4 which is expressed in PE and the posterior compartment of DP, the posterior DP height was reduced. Interestingly, the height of opposite PE was increased. Thus, it is possible that there is a connection between DP and PE during cell morphogenesis. To directly confirm the non-autonomous effect on PE elongation, the DP height was shortened by expression of either dad or brk within the DP specific nub-Gal4 domain. Consistently, the PE height was apparently increased (Tang, 2016).
Previous studies have revealed that mis-expressing lin induces ectopic sal expression in the PE. Thus, sal may mediate lin’s role in PE elongation. First, the experiment of lin mis-expression in the PE was repeated and the elongation phenotype was consistently observed in the PE. Then, the transcription state of sal was revealed using a sal-lacZ reporter. sal was apparently transcribed in the PE. The sal gene complex is composed of two functionally redundant genes: spalt major (salm) and spalt-related (salr). A rescue experiment was performed by co-expressing lin and salm-RNAi. The morphology of the wing imaginal discs was rescued to an approximate normal state, and PE height was no longer elongated. The cell heights of the corresponding genotypes were also measured. sal down-regulation largely rescued the abnormal cell height induced by lin mis-expression. These results indicate that sal mediates the role of lin in promoting PE elongation. However, Lin elongated PE to a greater extent than Sal did, according to the statistic measurements. Other mediators may be involved downstream of Lin. Since that Ubx-Gal4 line is also expressed in part of the DP cells, potential non-autonomous effects between DP and PE can not be ruled out (Tang, 2016).
Given that the mis-expression of sal in the PE elongates cell height, whether down-regulating Dpp-Sal signalling in DP is sufficient to shorten the DP was assessed. nub-Gal4 is only expressed in the wing pouch region in the DP. When Dpp signalling was mildly inhibited by expressing a dominant negative form of the Dpp receptor, tkvDN, in the nub-Gal4 domain, DP cell height was reduced. Unlike the strong inhibition of Dpp signalling by expressing dad, the non-autonomous effect on PE height was not apparent in nub>tkvDN. The Dpp signalling activities in discs of nub>tkvDNand nub>dad were revealed by anti-Sal staining. The DP height was slightly reduced when sal was down-regulated either by salm-RNAi or salr-RNAi; however, the extent of this reduction was weaker than that of tkvDN. Then, sal mutant clones were generated, marked by the loss of GFP in the DP. The intensity of F-actin labelled by Phalloidin was much stronger in the clone regions. The x-z cross-section showed that the apical side of sal mutant clones in the DP was retracted toward the basal side. These data suggested that Dpp-Sal signalling is required to maintain DP elongation. Interestingly, a similar retraction phenotype was also observed in sal-overexpressing clones. Therefore, both sal loss- and gain-of-function clones induce an apical retraction phenotype in the DP. This phenotype is observed in both omb loss- and gain-of-function clones in the DP. Omb exhibits a graded distribution in the DP along the A/P axis and specifies unknown apically distributed adhesion molecules. A continuous Omb level is essential for maintaining the epithelial integrity of the wing disc. Therefore, sharp discontinuity in either Omb or Sal levels in the DP induces apical retraction of cells. To confirm this conclusion, a sharp discontinuity of Sal was generated in the DP using dpp-Gal4 driven UAS-sal. Sal continuity was disrupted at the A/P boundary and where deep apical folds were formed. The expression domain of sal in the DP is narrower than that of omb and vg. Beyond the sal domain, omb and vg can ensure the correct cell morphogenesis in the DP. Clones lacking Vg function are also extruded from the DP layer (Tang, 2016).
Microtubule cytoskeleton is polarized during cell morphogenesis in the wing imaginal disc. To reveal microtubule-based cytoskeleton changes induced by either lin or sal mis-expression and the microtubule dynamics during normal development, the microtubule level was monitored via antibody staining. In the 2nd instar, all cells were cuboidal shape, and microtubules were uniformly distributed. During the early 3rd instar, the cell shape begins to differentiate. PE cells were largely shortened, whereas DP cells were remarkably elongated. Correlating with DP elongation, the microtubule network was asymmetrically enriched to the apical side of the DP. When lin was mis-expressed in the PE by Ubx-Gal4, sal was activated in the PE. Both direct and indirect sal expression induced PE height elongation with an even microtubule distribution. The microtubule levels of PE were increased compared with the wild type PE. The microtubule distribution in Sal-elongated PE was similar to that in very lateral PE and intermediate cells in the L3 stage or undifferentiated cells in earlier larval stages. In the rescue experiment, lin and salm-RNAi were co-expressed. PE height was restored were similar to that in wild type PE. Therefore, based on the cell height and microtubule distribution, Sal mis-expression converts the PE into a cuboidal cell shape (Tang, 2016).
During tissue morphogenesis, cell-shape changes always accompany microtubule cytoskeleton rearrangement. Dpp signalling activity has been proposed to play a basic function in microtubule organization. Dpp signalling is graded in the DP along the A/P axis, with higher levels in the medial DP region, which is enriched in apical microtubules. Thus, a correlation is noted between Dpp signalling activity and microtubule levels in the DP. Clones with loss-of-function of Dpp receptors in the DP appear extruded (cell height is severely shortened) and exhibit reduced apical microtubule levels. Consistently, clones with both loss- and gain-of-function sal and omb in the DP consistently exhibit severe apical retraction with shortened cell height and loss of apical microtubule enrichment. Therefore, the data support the hypothesis that Dpp-Omb/Sal signalling activity plays a more general function in microtubule-based cell morphogenesis. Other transcription factors that induce reductions in DP cell height also correlate with the loss of apical microtubule enrichment. The Tbx6 subfamily gene cluster Dorsocross (Doc) initiates wing hinge/blade fold formation. In the Doc expression domain in the DP, cells are shortened from the apical side with severe loss of apical microtubules (Tang, 2016).
During development cell proliferation and differentiation must be tightly
coordinated to ensure proper tissue morphogenesis. Because steroid
hormones are central regulators of developmental timing, understanding the
links between steroid hormone signaling and cell proliferation is crucial
to understanding the molecular basis of morphogenesis. This study examined
the mechanism by which the steroid hormone ecdysone regulates the cell cycle in Drosophila. A cell cycle arrest induced by ecdysone in Drosophila cell culture is analogous to a G2 cell cycle arrest observed in the early pupa.
In the wing, ecdysone signaling at the
larva to puparium transition induces Broad
which in turn represses the cdc25c phosphatase String. The repression of String generates a temporary G2 arrest that synchronizes the cell cycle in the wing epithelium during early pupa wing elongation
and flattening. As ecdysone levels decline after the larva to puparium
pulse during early metamorphosis, Broad expression plummets allowing
String to become re-activated, which promotes rapid G2/M progression and a
subsequent synchronized final cell cycle in the wing. In this manner,
pulses of ecdysone can both synchronize the final cell cycle and promote the coordinated acquisition of terminal differentiation characteristics in the wing (Guo, 2016).
This study presents a model for how the pulse of ecdysone at the larval to pupal transition
impacts the cell cycle dynamics in the wing during metamorphosis.
Ecdysone signaling at the larva to puparium transition induces Broad, which in turn
represses Stg to generate a temporary G2 arrest, which synchronizes the cell cycle
in the wing epithelium. As ecdysone levels decline, Broad expression plummets,
allowing Stg to be re-activated resulting in a pulse of cdc2 activity that promotes a
rapid G2/M progression during the final cell cycle in the wing. This ultimately
culminates in the relatively synchronized cell cycle exit at 24h APF, coinciding with the second large pulse of
ecdysone. This second pulse in the pupa activates a different set of transcription
factors (not Broad), promoting the acquisition of terminal differentiation
characteristics in the wing. In this way, two pulses of ecdysone signaling can both
synchronize the final cell cycle by a temporary G2 arrest and coordinate permanent
cell cycle exit with the acquisition of terminal differentiation characteristics in the
wing (Guo, 2016).
Over 30 years ago it was shown that 20-HE exposure in
Drosophila tissue culture cells induces a cell cycle arrest in G2-phase. This response appears to be shared among 3 different cell lines, Cl-8, Kc and S2. This study shows that in Kc cells, pulsed 20-HE
exposure also leads to a G2 arrest followed by rapid cell cycle re-entry after 20-HE
removal and a subsequent prolonged G1. This cell cycle response to a pulse
of 20-HE is reminiscent of the cell cycle changes that occur during early
metamorphosis in the pupal wings and legs (Guo, 2016).
It is worth considering why Kc and S2 cells, which are thought to be derived
from embryonic hemocytes would exhibit a similar cell cycle response to 20-HE to
the imaginal discs. Relatively little is known about how ecdysone signaling impacts
embryonic hemocytes, although recent work suggests that ecdysone signaling
induces embryonic hemocyte cell death under sensitized conditions. More is known about larval hemocytes, which differentiate into phagocytic macrophages and disperse into the hemolymph during the first 8h of
metamorphosis. Ecdysone is involved in this maturation process, as lymph glands of
ecdysoneless (ecd) mutants fail to disperse mature
hemocytes and become hypertrophic in the developmentally arrested mutants. This suggests that the high levels of systemic ecdysone signaling at the larval-puparium transition mediates a switch from proliferation to
cell cycle arrest and terminal differentiation for lymph gland hemocytes during
metamorphosis. Without ecdysone signaling, hemocytes may continue to proliferate
and fail to undergo terminal differentiation leading to the hypertrophic lymph gland
phenotype observed. Interestingly, while the loss of
broad also prevents proper differentiation of hemocytes similar to loss of
ecd, loss of broad does not lead to the
hypertrophy observed in ecd mutants. Further studies will
be needed to examine whether the ecdysone induced cell cycle arrest in larval
hemocytes occurs in the G2 phase, or whether their cell cycle arrest proceeds via a
similar pathway to that shown in this study for the wing (Guo, 2016).
Multiple lines of evidence suggest that the ecdysone receptor complex in the larval
wing acts as a repressor for certain early pupa targets and that the binding of
ecdysone to the receptor relieves this repression. For example loss of EcR by
RNAi or loss of the EcR dimerization partner USP, de-represses ecdysone target
genes that are high in the early pupal wing such as Broad-Z1 and βFtz-F1. The EcR/USP heterodimer also cooperates with the SMRTR co-repressor in the wing to prevent precocious
expression of ecdysone target genes such as Broad-Z1. Consistent
with the hypothesis that a repressive EcR/USP complex prevents precocious
expression of Broad-Z1 and thereby a precocious G2 arrest, inhibition of SMRTR can
also cause a G2 arrest. Thus, in the context of the early pupal wing,
it is proposed that the significant pulse of ecdysone at the larval to puparium
transition relieves the inhibition of a repressive receptor complex, leading to Broad-Z1 activation. Consistent with this model, high levels of Broad-Z1 in the larval wing lead to precocious neural differentiation at the margin and precocious inhibition of stg
expression in the wing pouch. Interestingly, a switch in Broad isoform expression also occurs during the
final cell cycle in the larval eye, such that Broad-Z1 becomes high in cells undergoing
their final cell cycle and entering into terminal differentiation.
However in this case, Broad-Z1 expression is not associated with a G2 arrest and
occurs in an area of high Stg expression, suggesting the downstream Broad-Z1
targets in the eye may be distinct or regulated differently from those in the wing (Guo, 2016).
The ecdysone receptor has also been shown to down regulate Wingless
expression via the transcription factor Crol at the wing margin, to indirectly
promote CycB expression. While a loss of EcR at the margin
decreased CycB protein levels, the effects of EcR loss on CycB levels in the wing
blade outside of the margin area were not obvious. It is suggested
that in the wing, the role for EcR outside of the margin acts on the cell cycle via a
different mechanism through stg. Consistent with a distinct mechanism acting in the
wing blade, over-expression of Cyclin B in the early prepupal wing could not
promote increased G2 progression or bypass the prepupal G2 arrest. Instead
the results on the prepupal G2 arrest are consistent with previous findings that Stg
is the rate-limiting component for G2-M cell cycle progression in the fly wing pouch
and blade (Guo, 2016).
In order to identify the gene expression changes in the wing that occur in response
to the major peaks of ecdysone during metamorphosis, RNAseq was performed on a
timecourse of pupal wings. Major changes were observed in gene expression in this
tissue during metamorphosis. In addition, known ecdysone targets were identified that are affected differently in the wing during the first larval-to-pupal ecdysone pulse and the second, larger pulse at 24h APF.
Ecdysone signaling induces different direct targets with distinct kinetics. Furthermore specific targets, for example Ftz-F1 can modulate the expression of other ecdysone targets, to shape the response to the
hormone. Thus, it is expected that a pulse of ecdysone
signaling leads to sustained effects on gene expression and the cell cycle, even after
the ecdysone titer returns to its initial state. These factors together with the
differences in the magnitude of the ecdysone pulse may contribute to the differences
in the response to the early vs. later pulses in the wing (Guo, 2016).
Ecdysone signaling can also affect the cell cycle and cell cycle exit via indirect
mechanisms such as altering cellular metabolism. This is used to promote cell cycle
exit and terminal differentiation in neuroblasts, where a switch toward oxidative
phosphorylation leads to progressive reductive divisions, (divisions in the absence
of growth) leading to reduced neuroblast cell size and eventually terminal
differentiation. Although reductive divisions do occur in the final cell cycle of the pupa wing, this
type of mechanism does not provide a temporary arrest to synchronize the final cell
cycle in neuroblasts as is see in wings. Importantly, a striking reduction is seen
in the expression of genes involved in protein synthesis and ribosome biogenesis in
the wing during metamorphosis, consistent with the lack of cellular growth. Instead the increased surface area of the pupal wing comes from a flattening, elongation and apical expansion of the cells due to interactions
with the extracellular matrix creating tension and influencing cell shape changes. This is also consistent with the findings that a significant number of genes associated with protein targeting to the membrane are increased as the wing begins elongation in the early pupa. Further studies will be
needed to determine whether the changes in expression of genes involved in
ribosome biogenesis and protein targeting to the membrane are controlled by
ecdysone signaling, or some other downstream event during early wing metamorphosis (Guo, 2016).
Perhaps the most interesting and least understood aspect of steroid hormone
signaling is how a diversity of cell-type and tissue-specific responses are generated
to an individual hormone. Cell cycle responses to ecdysone signaling are highly cell
type specific. For example abdominal histoblasts, the progenitors of the adult
abdominal epidermis, become specified during embryogenesis and remain
quiescent in G2 phase during larval stages. During pupal development, the
abdominal histoblasts must be triggered to proliferate rapidly by a pulse of
ecdysone to quickly replace the dying larval abdominal epidermis. This is in contrast
to the behavior of the wing imaginal disc, where epithelial cells undergo
asynchronous rapid proliferation during larval stages, but during metamorphosis
the cell cycle dynamics become restructured to include a G2 arrest followed by a
final cell cycle and entry into a permanently postmitotic state, in a manner
coordinated with tissue morphogenesis and terminal differentiation (Guo, 2016).
How does the same system-wide pulse of ecdysone at the larval to puparium
transition lead to such divergent effects on the cell cycle in adult progenitors?
Surprisingly it seems to be through divergent effects on tissue specific pathways
that act on the same cell cycle targets. In the abdominal histoblasts the larval to
puparium pulse of ecdysone triggers cell cycle re-entry and proliferation via indirect
activation of Stg, by modulating the expression of a microRNA
miR-965 that targets Stg. This addition of the microRNA essentially allows ecdysone signaling to act oppositely on the same cell cycle regulatory target as Broad-Z1 does in the wing. Thus, tissue specific programs of gene regulatory networks can create divergent outcomes from the same system-
wide hormonal signal, even when they ultimately act on the same target (Guo, 2016).
Animals develop in unpredictable, variable environments. In response to environmental change, some aspects of development adjust to generate plastic phenotypes. Other aspects of development, however, are buffered against environmental change to produce robust phenotypes. How organ development is coordinated to accommodate both plastic and robust developmental responses is poorly understood. This study demonstrates that the steroid hormone ecdysone coordinates both plasticity of organ size and robustness of organ pattern in the developing wings of the fruit fly Drosophila melanogaster. Using fed and starved larvae that lack prothoracic glands, which synthesize ecdysone, this study showed that nutrition regulates growth both via ecdysone and via an ecdysone-independent mechanism, while nutrition regulates patterning only via ecdysone. It was then demonstrated that growth shows a graded response to ecdysone concentration, while patterning shows a threshold response. Collectively, these data support a model where nutritionally regulated ecdysone fluctuations confer plasticity by regulating disc growth in response to basal ecdysone levels and confer robustness by initiating patterning only once ecdysone peaks exceed a threshold concentration. This could represent a generalizable mechanism through which hormones coordinate plastic growth with robust patterning in the face of environmental change (Alves, 2022).
Biological systems are inherently noisy; however, they produce highly stereotyped tissue morphology. Drosophila pupal wings show a highly stereotypic folding through uniform expansion and subsequent buckling of wing epithelium within a surrounding cuticle sac (see Emergence of stereotypic folding within a cuticle sac). The folding pattern produced by buckling is generally stochastic; it is thus unclear how buckling leads to stereotypic tissue folding of the wings. This study found that the extracellular matrix (ECM) protein, Dumpy, guides the position and direction of buckling-induced folds. Dumpy anchors the wing epithelium to the overlying cuticle at specific tissue positions. Tissue-wide alterations of Dumpy deposition and degradation yielded different buckling patterns. In summary, it is proposed that spatiotemporal ECM remodeling shapes stereotyped tissue folding through dynamic interactions between the epithelium and its external structures (Tsuboi, 2023).
This study has revealed that the spatiotemporally coordinated deposition and destruction of the ECM protein (Dpy) guide the position and direction of buckling. The results demonstrate that although cell populations show spatially homogeneous cellular behaviors (i.e., cell flattening), they can yield stereotypic tissue buckling morphology through the positional information encoded by ECM remodeling (Tsuboi, 2023).
Dpy, which anchors the wing tissue and cuticle, is constructed at specific positions (see Dumpy regulates the stereotypic buckling direction by connecting the wing epithelium and the pupal cuticle along veins). The position of Dpy anchorage is likely controlled posttranslationally, because Dpy overexpression did not result in the ectopic formation of Dpy anchorage. The construction of the Dpy matrix involves multiple processes, including synthesis, secretion, assembly into polymers, and association of the cuticle with the apical cell surface. Although the signaling pathway regulating Dpy anchoring remains unknown, the loss of vein Dpy in the mutant wing lacking veins suggests that positional information and signaling pathways related to wing vein patterning may contribute to the spatial differences in Dpy structure formation (Tsuboi, 2023).
Dpy degradation was found to initiate in the distal-posterior region, and the spatial pattern of degradation controls the position of the marginal fold (see Dumpy degradation is indispensable for the marginal fold formation). The degradation of Dpy is regulated by the action of Stubble (Sb) and Np apical transmembrane proteases, suggesting that the spatial pattern of Np/Sb expression or localization to the apical surface may control the degradation pattern of Dpy. Alternatively, another possible mechanism for the propagation pattern of Dpy degradation could be attributed to the spatial pattern of proteolytic activation of the proteases, because Np and Sb are expressed as catalytically inactive zymogens and require proteolytic cleavage to become active. In addition, regional differences in the physical properties of Dpy and its sensitivity to the proteases could contribute to the propagation pattern of Dpy degradation. It has been reported that Dpy undergoes filamentous conversion in response to increasing tension during indirect flight muscle development. Considering that Dpy filaments in wing tissue are formed under anisotropic proximal-distal–oriented tension caused by hinge contraction, the extent of Dpy filamentous conversion may vary regionally within the tissue depending on the applied stress. It would be interesting to investigate the regional difference in the physical properties of Dpy filaments within a tissue and their relevance to the propagation pattern of Dpy degradation (Tsuboi, 2023).
These findings are unique as they reveal the potential of external cues in generating stereotypic 3D tissue shapes within a spatially confined environment. This ECM-based mechanism should confer a potential means to generate diverse and controllable 3D tissue shaping in parallel with cell-intrinsic genetic programming. Future application of this ECM modification to tissue engineering would pave the way for manufacturing precisely folded tissues in any desired manner (Tsuboi, 2023).
In Drosophila, loss of regenerative capacity in wing imaginal discs coincides with an increase in systemic levels of the steroid hormone ecdysone. Regenerating discs release the relaxin hormone Dilp8 ) to limit ecdysone synthesis and extend the regenerative period. This study describes how regenerating tissues produce a biphasic response to ecdysone levels: lower concentrations of ecdysone promote local and systemic regenerative signaling, whereas higher concentrations suppress regeneration through the expression of broad splice isoforms. Ecdysone also promotes the expression of wingless during both regeneration and normal development through a distinct regulatory pathway. This dual role for ecdysone explains how regeneration can still be completed successfully in dilp8(-) mutant larvae: higher ecdysone levels increase the regenerative activity of tissues, allowing regeneration to reach completion in a shorter time (Karanja. 2022).
Specification of tissue identity during development requires precise coordination of gene expression in both space and time. Spatially, master regulatory transcription factors are required to control tissue-specific gene expression programs. However, the mechanisms controlling how tissue-specific gene expression changes over time are less well understood. This study shows that hormone-induced transcription factors control temporal gene expression by regulating the accessibility of DNA regulatory elements. Using the Drosophila wing, it was demonstrated that temporal changes in gene expression are accompanied by genome-wide changes in chromatin accessibility at temporal-specific enhancers. A temporal cascade of transcription factors was uncovered following a pulse of the steroid hormone ecdysone such that different times in wing development can be defined by distinct combinations of hormone-induced transcription factors. Finally, the ecdysone-induced transcription factor E93 was shown to control temporal identity by directly regulating chromatin accessibility across the genome. Notably, it was found that E93 controls enhancer activity through three different modalities, including promoting accessibility of late-acting enhancers and decreasing accessibility of early-acting enhancers. Together, this work supports a model in which an extrinsic signal triggers an intrinsic transcription factor cascade that drives development forward in time through regulation of chromatin accessibility (Uyehara, 2017).
The importance of master transcription factors in specifying spatial identity during development suggests that they may control where other transcription factors bind in the genome. One prediction of this model is that tissues whose identities are determined by different master transcription factors would exhibit different genome-wide DNA-binding profiles. However, it was recently found that the Drosophila appendages (wings, legs, and halteres), which use different transcription factors to determine their identities, share nearly identical open chromatin profiles. Moreover, these shared open chromatin profiles change coordinately over developmental time. There are two possible explanations for these findings. Either (1) different transcription factors produce the same open chromatin profiles in different appendages or (2) transcription factors shared by each appendage control open chromatin profiles instead of the master transcription factors of appendage identity. The second model is favored for several reasons. Since the appendage master transcription factors possess different DNA-binding domains with distinct DNA-binding specificities, it is unlikely for them to bind the same sites in the genome. Supporting this expectation, ChIP for Scalloped and Homothorax, two transcription factors important for appendage identity, shows clear tissue-specific binding in both the wing and eye–antennal imaginal discs. The second model is also preferred because it provides a relatively straightforward mechanism for the observed temporal changes in open chromatin: By changing the expression of the shared temporal transcription factor over time, the open chromatin profiles that it controls would change as well. In contrast, expression of appendage master transcription factors is relatively stable over time, making it unlikely for them to be sufficient for temporal changes in open chromatin (Uyehara, 2017).
It is proposed that control of chromatin accessibility in the appendages is mediated at least in part by transcription factors downstream from ecdysone signaling. According to this model, a systemic pulse of ecdysone initiates a temporal cascade of hormone-induced transcription factor expression in each of the appendages. These are referred to as 'temporal' transcription factors. Temporal transcription factors can directly regulate the accessibility of transcriptional enhancers by opening or closing them, thereby conferring temporal specificity to their activity and driving development forward in time. Master transcription factors then bind accessible enhancers depending on their DNA-binding preferences (or other means of binding DNA) and differentially regulate the activity of these enhancers to control spatial patterns of gene expression, thus shaping the unique identities of individual appendages (Uyehara, 2017).
The experiments with E93 provide direct support for this model. In wild-type wings, thousands of changes in open chromatin occur after the large pulse of ecdysone that triggers the end of larval development. In E93 mutants, ~40% of these open chromatin changes fail to occur. Importantly, nearly three-quarters of sites that depend on E93 for accessibility correspond to temporally dynamic sites in wild-type wings. Thus, chromatin accessibility is not grossly defective across the genome; instead, defects occur specifically in sites that change in accessibility over time. This finding, combined with the large fraction of temporally dynamic sites that depend on E93 for accessibility, indicates that E93 controls a genome-wide shift in the availability of temporal-specific transcriptional enhancers. Supporting this hypothesis, temporal-specific enhancers depend on E93 for both accessibility and activity. Since it is proposed that the response to ecdysone is shared across the appendages, it is predicted that similar defects occur in appendages besides the wing. It remains to be seen whether other ecdysone-induced transcription factors besides E93 control accessibility of enhancers at different developmental times. It also remains to be seen how the temporal transcription factors work with the appendage master transcription factors to control appendage-specific enhancer activity (Uyehara, 2017).
The findings suggest that E93 controls temporal-specific gene expression through three different modalities that potentially rely on three distinct biochemical activities. The enrichment of E93 motifs and binding of E93 to temporally dynamic sites indicate that it contributes to this regulation directly. It is proposed that these combined activities drive development forward in time by turning off early-acting enhancers and simultaneously turning on late-acting enhancers (Uyehara, 2017).
First, as in the case of the tenectin tncblade enhancer, active most strongly in the interveins between the first and second and between the fourth and fifth longitudinal veins and in cells near the proximal posterior margin, E93 appears to function as a conventional activator. In the absence of E93, tncblade fails to express at high levels, but the accessibility of the enhancer does not measurably change. This suggests that binding of E93 to tncblade is required to recruit an essential coactivator. Importantly, this finding demonstrates that E93 is not solely a regulator of chromatin accessibility. E93 binds many open chromatin sites in the genome without regulating their accessibility and thus may regulate the temporal-specific activity of many other enhancers. In addition, since the tncblade enhancer opens between L3 and 24 h even in the absence of E93, there must be other factors that control its accessibility, perhaps, for example, transcription factors induced by ecdysone earlier in the temporal cascade (Uyehara, 2017).
Second, as in the case of the nubvein enhancer, E93 is required to promote chromatin accessibility. In this capacity, E93 may function as a pioneer transcription factor to open previously inaccessible chromatin. Alternatively, E93 may combine with other transcription factors, such as the wing master transcription factors, to compete nucleosomes off DNA. Testing the ability of E93 to bind nucleosomal DNA will help to discriminate between these two alternatives. In either case, it is proposed that this function of E93 is necessary to activate late-acting enhancers across the genome. Since only half of E93-dependent enhancers are directly bound by E93 at 24 h, it is also possible that E93 regulates the expression of other transcription factors that control chromatin accessibility. Alternatively, if E93 uses a “hit and run” mechanism to open these enhancers, the ChIP time point may have been too late to capture E93 binding at these sites (Uyehara, 2017).
Finally, as in the case of the broad brdisc enhancer, E93 is required to decrease chromatin accessibility. It is proposed that this function of E93 is necessary to inactivate early-acting enhancers across the genome. Current models of gene regulation do not adequately explain how sites of open chromatin are rendered inaccessible, but the ability to turn off early-acting enhancers is clearly an important requirement in developmental gene regulation. It may also be an important contributor to diseases such as cancer, which exhibits widespread changes in chromatin accessibility relative to matched normal cells. Thus, this role of E93 may represent a new functional class of transcription factor (“reverse pioneer”) or conventional transcriptional repressor activity. Additional work is required to decipher the underlying mechanisms. Notably, recent work on the temporal dynamics of iPS cell reprogramming suggest a similar role for Oct4, Sox2, and Klf4 in closing open chromatin to inactivate somatic enhancers (Chronis, 2017; Uyehara, 2017 and references therein).
Steroid hormones perform diverse biological functions in developing and adult animals. However, the mechanistic basis for their tissue specificity remains unclear. In Drosophila, the ecdysone steroid hormone is essential for coordinating developmental timing across physically separated tissues. Ecdysone directly impacts genome function through its nuclear receptor, a heterodimer of the EcR and ultraspiracle proteins. Ligand binding to EcR triggers a transcriptional cascade, including activation of a set of primary response transcription factors. The hierarchical organization of this pathway has left the direct role of EcR in mediating ecdysone responses obscured. This study investigates the role of EcR in controlling tissue-specific ecdysone responses, focusing on two tissues that diverge in their response to rising ecdysone titers: the larval salivary gland, which undergoes programmed destruction, and the wing imaginal disc, which initiates morphogenesis. EcR was found to function bimodally, with both gene repressive and activating functions, even at the same developmental stage. EcR DNA binding profiles are highly tissue-specific, and transgenic reporter analyses demonstrate that EcR plays a direct role in controlling enhancer activity. Finally, despite a strong correlation between tissue-specific EcR binding and tissue-specific open chromatin, it was found that EcR does not control chromatin accessibility at genomic targets. It is concluded that EcR contributes extensively to tissue-specific ecdysone responses. However, control over access to its binding sites is subordinated to other transcription factors (Uyehara, 2022).
Organ size and pattern results from the integration of two positional information systems. One global, encoded by the Hox genes, links organ type with position along the main body axis. Within specific organs, local information is conveyed by signaling molecules that regulate organ growth and pattern. The mesothoracic (T2) wing and the metathoracic (T3) haltere of Drosophila represent a paradigmatic example of this coordination. The Hox gene Ultrabithorax (Ubx), expressed in the developing T3, selects haltere identity by, among other processes, modulating the production and signaling efficiency of Dpp, a BMP2-like molecule that acts as a major regulator of size and pattern. Still, the mechanisms of the Hox-signal integration even in this well-studied system are incomplete. This study has investigated this issue by studying the expression and function of the Six3 transcription factor optix during the development of the Drosophila wing and haltere development. In both organs Dpp defines the expression domain of optix through repression, and the specific position of this domain in wing and haltere seems to reflect the differential signaling profile among these organs. optix expression in wing and haltere primordia is conserved beyond Drosophila in other higher diptera. In Drosophila, optix is necessary for the growth of wing and haltere: In the wing, optix is required for the growth of the most anterior/proximal region (the 'marginal cell') and for the correct formation of sensory structures along the proximal anterior wing margin, and the halteres of optix mutants are also significantly reduced. In addition, in the haltere optix is necessary for the suppression of sensory bristles (Al Khatib, 2017).
In the haltere, Ubx modifies the wing developmental program in two ways. First, as a transcription factor, Ubx regulates the expression of some targets. For example, Ubx represses sal expression (Weatherbee, 1998). Second, Ubx modifies the shape of the Dpp-generated signaling gradient indirectly, by controlling the expression of proteoglycans required for Dpp dispersion (Crickmore, 2006; de Navas, 2006). Globally,
these modifications of Dpp signaling and target gene activation by Ubx have been related to the size and patterning differences between halteres and wings (Al Khatib, 2017).
Since Dpp signaling generates a signaling gradient that spans the whole wing pouch and its activity is required throughout the wing, it is expected to control the expression of target genes not only in central region of the pouch, but also in more lateral ones. The Six3-type transcription factor optix has been reported to be expressed in the lateral region of the wing pouch, as well as in the haltere (Seimiya, 2000). Functional studies show that optix is required for the normal patterning of the anterior portion of the wing and that its expression is negatively regulated by sal genes (Organista, 2015). The fact that sal genes are Dpp signaling targets in the wing, places optix downstream of Dpp regulation. However, since sal genes are not expressed in haltere discs (Weatherbee, 1998), the mechanism of optix regulation in this organ is still unknown. This study analyzed comparatively the expression, function and regulation of optix in wing and haltere discs. In both discs, optix expression is anteriorly restricted by Dpp signaling, although in the wing the precise expression boundary may be set with the collaboration of wing specific Dpp targets, such as sal. optix shows organ-specific functions: in the wing, previous results were confirmed showing it is necessary for the growth of the anterior/proximal wing ('marginal cell') and the development of wing margin sensory bristles. However, in the haltere optix is required for the suppression of sensory bristle formation. Overexpression of optix in the entire wing pouch affects only anterior wing development, suggesting that other parts of the wing cannot integrate ectopic Optix input. This observation may provide a mechanistic explanation for a widespread re-deployment of optix expression in wing spot formation in various butterfly species (Al Khatib, 2017).
The Dpp signaling gradient is required for the patterning of the whole wing, from the center to its margin. This gradient is translated into a series of contiguous domains expressing distinct transcription factors, each required for the specification of specific features in the adult organ. However, while the transcription factors acting in the central wing were known, the most anterior region of the wing -- the region comprised between the longitudinal vein 2 (L2) and the anterior margin (L1) -- lacked a specific transcription factor. This paper shows that this transcription factor, or at least one of them, is Optix (Al Khatib, 2017).
The results confirm previous findings (Organista, 2015) that optix is expressed in, and required for the growth of this most anterior sector of the wing, the so-called margin cell. This study now shows that optix is also required for the growth of the wing's serially homologous organ: the haltere. This role is in agreement with previous results showing that Six3 regulates cell proliferation in vertebrate systems. This study further shows that Dpp signaling plays a major role in setting the optix expression domain. Although it has been reported before that sal genes are required to set the central limit of this domain, in discs lacking sal function optix does not extend all the way to the AP border (Organista, 2015), suggesting additional mechanisms involved in optix repression. The fact that sal is not expressed in the haltere pouch and still optix does not extend all the way to the AP border, the exclusion of optix expression from intermediate/high Dpp signaling in both wing and haltere, and the requirement of Dpp signaling to repress optix in any position of the anterior wing compartment globally suggested that either Dpp activates a different repressor closer to the AP border, or that Dpp signaling represses directly optix transcription. The current work cannot distinguish between these possibilities. Regarding another well characterized Dpp target, omb, the extensive coexpression of omb and optix in the haltere also seems to exclude omb as a repressor. Therefore, either another unknown repressor exists, or Dpp signaling acts as a direct optix repressor. While in the haltere, the domain of optix would be set directly by Dpp, in the wing sal would be an additional repressor. By intercalating sal, the Dpp positioning system may be able to push the limit of optix expression farther away from the AP border of the wing. The Sal proteins have been previously shown to act as transcriptional repressors of knirps (kni) to position vein L2. Thus, adding sal repression may help to align the optix domain with L2. This additional repression would not be operating in the haltere, which lacks venation (Al Khatib, 2017).
Interestingly, the logic of optix regulation by Dpp is different from that of other Dpp targets. The activation of the sal paralogs (sal-m and sal-r) and aristaless (al), another target required for vein L2 formation, proceeds through a double repression mechanism: In the absence of signal, the Brinker repressor keeps sal and al off. Activation of the pathway leads to the phosphorylation of the nuclear transducer Mad (pMad) which, in turn, represses brk, thus relieving the repression on sal and al. Therefore, optix regulation by Dpp signaling could be more direct similarly to that of brk (Al Khatib, 2017).
One interesting aspect of optix function is that it plays an additional specific role in the haltere. While in the wing optix is required for the development of the anterior-most portion of the wing (including the margin bristles), in the haltere optix serves to suppress the development of sensory bristles, a task known to be carried out by the Hox gene Ubx. A role for optix in regulating Ubx expression has been ruled out, at least when judged from Ubx protein levels. Therefore, optix is required for a subset of Ubx's normal functions. Since optix encodes a Six3-type transcription factor, this interaction could be happening at the level of target enhancers, where the combination of Ubx and Optix would allow the activation or repression of specific sets of genes (Al Khatib, 2017).
Finally, it was observed that the expression of optix in wing and haltere primordia is conserved across higher Diptera. Interestingly, optix is expressed in the developing wings of passion vine butterflies (genus Heliconius). In Heliconius species, optix has been co-opted for red color patterning in wings. However, the ancestral pattern found in basal Heliconiini is in the proximal complex, a region that runs along the base of the forewing costa, the most anterior region of the forewing. This similarity between optix expression patterns in forewings of Diptera and Lepidoptera leads to the hypothesis that an ancestral role of optix might have been 'structural', being required for the development of the anterior wing. Once expressed in the wings, recruitment of red pigmentation genes allowed optix co-option for color pattern diversification through regulatory evolution. It is noted that a pre-requisite for this co-option in wing pigmentation patterning must have been that optix would not interfere with the developmental pathway leading to the formation of a normal wing in the first place. The fact that the effects of optix overexpression throughout the wing primordium in Drosophila are restricted to the anterior/proximal wing -its normal expression domain- indicates that optix cannot engage in promiscuous gene regulation, and that its function depends on other competence factors, which would limit its gene expression regulatory potential (Al Khatib, 2017).
The Drosophila body comprises a central part, the trunk, and outgrowths of the trunk, the appendages. Much is known about appendage regeneration, but little about the trunk. As the wing imaginal disc contains a trunk component, the notum, and a wing appendage, this study has investigated the response to ablation of these two components. In contrast with the strong regenerative response of the wing, the notum does not regenerate. Nevertheless, the elimination of the wing primordium elicits a proliferative response of notum cells, but they do not regenerate wing; they form a notum duplicate. Conversely, the wing cells cannot regenerate an ablated notum; they over-proliferate and generate a hinge overgrowth. These results suggest that trunk and appendages cannot be reprogrammed to generate each other. These experiments demonstrate that the proliferative response is mediated by JNK signalling from dying cells, but JNK functions differently in the trunk and the appendages, explaining their distinct regenerative potential (Martin, 2017).
Basement membranes (BMs) are extracellular matrix polymers basally underlying epithelia, where they regulate cell signaling and tissue mechanics. Constriction by the BM shapes Drosophila wing discs, a well-characterized model of tissue growth. Recently, the hypothesis that mechanical factors govern wing growth has received much attention, but it has not been definitively tested. This study manipulated BM composition to cause dramatic changes in tissue tension. Increased tissue compression was found when perlecan was knocked down did not affect adult wing size. BM elimination, decreasing compression, reduced wing size but did not visibly affect Hippo signaling, widely postulated to mediate growth mechanoregulation. BM elimination, in contrast, attenuated signaling by bone morphogenetic protein/transforming growth factor beta ligand Dpp, which was not efficiently retained within the tissue and escaped to the body cavity. These results challenge mechanoregulation of wing growth, while uncovering a function of BMs in preserving a growth-promoting tissue environment (Ma, 2017).
Basement membranes (BMs) are laminar polymers of extracellular matrix proteins which underlie epithelia and surround organs in all animals. The main components of BMs are collagen IV, nidogen, laminin, and perlecan, all conserved from insects to humans. Despite long-known conservation, ubiquity in animal tissues, and extensive biochemical knowledge, understanding of the developmental roles of BMs is comparatively poor. Nonetheless, significant progress has been made in recent years with the help of model organisms, such as Drosophila melanogaster and Caenorhabditis elegans, thanks to limited genetic redundancy of BM components in these systems. In this way, it has been shown in the fruit fly Drosophila that collagen IV is required for full Dpp activity in dorsal cells of the embryo and for the response to Dpp of renal tubules. In addition, BMs are now known to play an essential role in mechanically shaping tissues: in the absence of a BM, tissues such as the egg follicleand the larval imaginal discs uffer profound deformations (Ma, 2017).
Drosophila adult wings develop from the pouch region of the wing imaginal disc, a widely studied model for tissue growth regulation. The wing pouch of the third instar larva (L3 stage) is a highly columnar monolayered epithelium where each cell attaches to the BM. Recently, the hypothesis that mechanical factors contribute to the regulation of wing growth has gathered considerable momentum. The observations that cell compression is higher at the center of the pouch and that compression increases during larval development have led to several models postulating a negative effect of compression on growth. This negative effect of compression on growth is invoked to solve the apparent paradox that combined concentration of growth promoters Dpp and Wingless (Wg) is higher at the center of the pouch, yet the distribution of cell proliferation is roughly homogeneous throughout the disc. In this context, the Hippo signaling pathway, known to respond to cell contact, cell crowding, and cytoskeletal tension has been postulated as a mediator of mechanical inputs into wing growth. However, the difficulty of experimentally changing tissue constriction in an internally developing organ has precluded definitive testing of this hypothesis (Ma, 2017).
To investigate the developmental role of the BM and explore the influence of mechanical factors on wing growth, this study subjected wing discs to different BM manipulations changing tissue constriction in order to assess their effect on disc development and adult wing size. The results show a lack of effect of mechanical constriction on Hippo signaling and wing growth. In contrast, BM was foudn to contribute to tissue growth by enhancing tissular retention of Dpp (Ma, 2017).
The results of the experiments changing tissue constriction through BM manipulation are difficult to reconcile with a physiological role of cell compression in regulation of normal wing growth, a central tenet of wing growth mechanoregulation models. Increase in compression when perlecan was knocked down, and decreased compression when the BM was degraded, both failed to produce the predicted effects: smaller and larger wings, respectively. In contrast to the results in the larval wing, tissue size regulation by cell crowding and apoptosis has been shown to occur in the notum during metamorphosis. Since both the wing and the notum derive from the same imaginal disc, it follows that mechanical effects on size must be highly dependent on the specific developmental context (Ma, 2017).
The failure to observe changes in Hippo activity after dramatic changes in tissue shape also challenges the role of Hippo signaling in regulating wing growth in response to compression. Nonetheless, several manipulations of cytoskeletal components clearly influence Hippo signaling in the wing, affecting growth. Because the actin-rich zonula adherens is the physical locus where Hippo signaling complexes assemble, Hippo signaling may act as a critical sensor of cell polarity or cell contact. According to the current results, however, it does not act in the wing as a tension-growth feedback regulator slowing growth in response to cell crowding (Ma, 2017).
Discs made of larger, fewer cells have long been known to give rise to normally sized adult wings, indicating that some parameter different from cell numbers contributes to defining final wing size, for instance some physical dimension of the tissue such as planar area or tissue volume. BM manipulations dramatically changed apical area and height of individual cells and of the tissue as a whole, but they may not have changed cell size, as suggested by the fact that cell density in the adult wing did not change. These findings, therefore, would be consistent with a model in which tissue mass or volume contributes to determination of final wing size. Normally sized discs and adult wings made of larger, fewer cells, in addition, offer a further argument against mechanical regulation of wing growth, as these larger cells would display very different physical properties in terms of their apical areas and the tensions supported by their membranes and cytoskeletons (Ma, 2017).
Even though no mechanical effects on Hippo signaling or wing growth were detected following profound tissue deformations, it cannot be completely rule out that BM manipulations caused secondary effects that negated putative effects of mechanical signals. Such is the case, it is arguee, of the discs flattened by BM elimination. These discs gave rise to smaller adult wings, an effect that further experiments indicate is a result of the specific requirement of the BM in Dpp signaling. Nonetheless, this study also failed to detect changes in cell proliferation or adult wing size when discs were flattened in vivo through direct application of force. Importantly, a contribution of the directionality of compression is also a possibility that cannot be rule out, as cells in the periphery of both act > troli and rn > Mmp2 discs change their apical discs change their apical area, but maintain the tendency of the wild-type to align their major axis tangentially to the center of the disc. Therefore, if the vector of the compression rather than its magnitude is readable by a cell or its neighbors, the results cannot rule out a role for this in regulating wing growth. This pattern of cell orientation has been attributed to a slightly higher proliferation rate in the center of the wing pouch, a fact overlooked in the past and possibly responsible in the first place for the higher cell compression in the center of the wing. BM modifications, therefore, would not affect this intrinsically different proliferation rate in the central and peripheral wing regions. The results, finally, do not rule out the possibility that more extreme mechanical inputs could impact wing growth, for instance in wound healing or damage-stimulated growth (Ma, 2017).
Despite the lack of influence on Hippo signaling in the BM manipulations, the data show that the BM itself is required to preserve a growth-promoting environment by hindering diffusion of Dpp out of the disc. Collagen IV, the main component of BMs, physically interacts with Dpp through the C-terminal NC1 domains of both collagen IV chains. The effects of collagen IV loss on Dpp signaling in the wing, the dorsal blastoderm and germarium, and renal tubules are all consistent with a role of collagen IV in Dpp concentration. Elimination of the BM, however, did not seem to affect signaling by the other diffusible ligands Wg and Hh, which are, unlike Dpp, quite hydrophobic and may not require a mechanism preventing their escape from the tissue. The role of the BM in maintaining the concentration of extracellular ligands, therefore, may not be general, but ligand specific or specific to Dpp (Ma, 2017).
A role has been attributed to Dpp signaling in modulating cell height in the wing epithelium. Even though the current experiments eliminating the BM caused both a Dpp deficit and decreased cell height, it is unlikely that the effects on cell height in this experiment are caused by the Dpp deficit. First, the effects of collagenase treatment on disc morphology are immediate, which is difficult to explain as a deficit in Dpp signaling, specially a transcriptionally mediated effect. Second, discs in which the BM was simultaneously degraded and Dpp signaling was activated were still flattened, supporting the idea that effects on tissue shape elicited by BM degradation are not due to a Dpp deficit (Ma, 2017).
Since Dpp does not seem to accumulate basally in the wing disc, it is hypothesized that transient binding of Dpp allows the wing BM to act as a semipermeable barrier hindering Dpp diffusion, although not completely preventing it. This is a function that other BMs are long known to serve in the vertebrate kidney or the blood-brain barrier. Indeed, the results showing homogeneously high levels of Dpp signaling in the disc when Dpp was expressed in the fat body demonstrate an ability of Dpp to cross the BM. This result has also implications for understanding of Dpp signaling in the wing, as it shows that Dpp presentation by apical cytonemes is not absolutely required for signaling. A function of the BM in limiting basal escape of Dpp is, in addition, highly consistent with recent findings showing that a Dpp.GFP fusion could be immobilized at the BM, with effects on patterning and growth similar to the ones observed when the BM was eliminated. The findings support a critical role for basolaterally diffusing Dpp against a competing hypothesis stating that the functional Dpp gradient forms apically. It must be noted, however, that the role of the medial Dpp stripe in regulating growth has been called into question during the third larval instar, when a non-stripe source in the anterior compartment would serve this growth-promoting function instead. Because BM elimination reduces not just medial spalt and pMad, but also growth, it follows that the BM is required to maintain the concentration of Dpp from both sources: the medial stripe and the unknown anterior non-stripe source (Ma, 2017).
Given the conservation of BM components and Dpp, BM degradation and epithelial-to-mesenchymal transitions may enhance BMP/TGF-β signaling across tissue layers in development. The results also suggest a way in which tumoral BM degradation could contribute to tissue signaling misregulation in cancer by allowing escape of these diffusible signals. Finally, the visualization of an apico-basal gradient of Dpp in this highly columnar epithelium calls for the inclusion of the apico-basal dimension in future quantitative studies of Dpp gradient formation (Ma, 2017).
The molecular mechanisms concerning the ubiquitin-related dynamic regulation of TGF-β signaling are not thoroughly understood. Using a combination of proteomics and an siRNA screen, this study identified pVHL as an E3 ligase for SMAD3 ubiquitination. pVHL directly interacts with conserved lysine and proline residues in the MH2 domain of SMAD3, triggering degradation. As a result, the level of pVHL expression negatively correlates with the expression and activity of SMAD3 in cells, Drosophila wing, and patient tissues. In Drosophila, loss of pVHL leads to the up-regulation of TGF-β targets visible in a downward wing blade phenotype, which is rescued by inhibition of SMAD activity. Drosophila pVHL expression exhibited ectopic veinlets and reduced wing growth in a similar manner as upon loss of TGF-β/SMAD signaling. Thus, this study demonstrates a conserved role of pVHL in the regulation of TGF-β/SMAD3 signaling in human cells and Drosophila wing development (Zhou, 2022).
The control of organ growth is critical for correct animal development. From flies to mammals, the mechanisms regulating growth are conserved and the role of microRNAs in this process is emerging. The conserved miR-7 has been described to control several aspects of development. This study has analyzed the function of miR-7 during Drosophila wing development. Loss of miR-7 function results in a reduction of wing size and produces wing cells that are smaller than wild type cells. It was also found that loss of miR-7 function interferes with the cell cycle by affecting the G1 to S phase transition. Further, evidence is presented that miR-7 is expressed in the wing imaginal discs and that the inactivation of miR-7 increases the expression of Cut and Senseless proteins in wing discs. Finally, these results show that the simultaneous inactivation of miR-7 and either cut, Notch, or dacapo rescues miR-7 loss of function wing size reduction phenotype. The results from this work reveal that miR-7 functions to regulate Drosophila wing growth by controlling cell cycle phasing and cell mass through its regulation of the expression of dacapo and the Notch signaling pathway (Aparicio, 2015).
The function of miR-7 in eye morphogenesis and ovariole development has been analyzed using the miR-7Δ1 and Df(2R)exu1 deletions and the genetic allelic combination Df(2R)exu1/miR-7Δ1. In contrast, the function of miR-7 in wing development has only been studied analyzing the phenotypes of miR-7 over-expression in the wing disc. These wing studies indicated that miR-7 regulates Notch signaling pathway targets like Enhancer of split to control sensory organ precursor determination. The analysis of the loss of miR-7 function using the Df(2R)exu1/miR-7Δ1 allelic combination has not been performed. Furthermore, the use of miR-7 sponges has been uninformative. As the Df(2R)exu1/miR-7Δ1 allelic combination, used by this study, might also partially inactivate the function of the bancal gene where miR-7 is located, attempts were made to analyze the contribution of miR-7 to the Df(2R)exu1/miR-7Δ1 wing phenotype. This study shows that the over-expression of miR-7 and the constitutive and ubiquitous expression of miR-7 rescue, at least partially, the wing phenotype of Df(2R)exu1/miR-7Δ1, thereby demonstrating that lack of miR-7 function contributes to the wing phenotype observed in Df(2R)exu1/miR-7Δ1 flies and indicating that miR-7 is involved in the control of wing growth. This is consistent with the findings that miR-7 is expressed in the wing imaginal discs and that the G1 to S cell cycle transition is arrested in Df(2R)exu1/miR-7Δ1 wing imaginal discs. Expression of mature miR-7 in the wing imaginal discs has been previously reported. The current results show that in situ miR-7 expression in the wing disc is weak but still significant compared to Df (2R) exu1/miR-7Δ1 wing discs. Moreover, miR-7 expression in the 'non-boundary cells' along with the absence of its expression in the 'boundary cells' suggests that miR-7 is involved in the regulation of the factors that control the cell interactions regulating growth at the D/V boundary (Herranz, 2008). Although these results strongly suggest that miR-7 has a function in wing growth control, definitive proof will require either a precise miR-7 deletion or an effective miR-7 sponge (Aparicio, 2015).
Bioinformatic analyses have predicted at least 150 miR-7 putative target genes in Drosophila genome. Among these, only a few have been validated using in vivo experiments. To date, miR-7 has been shown to promote photoreceptor differentiation by regulating the expression of yan as well as to control chordotonal organ differentiation through the control of the Enhancer of split-complex genes (Stark, 2003; Li, 2009). Moreover, miR-7 has been shown to regulate dacapo expression to maintain the GSCs proliferation as well as to regulate bag of marbles expression to control the GSCs testis differentiation. The current results show that the Df (2R) exu1/miR-7Δ1 wing phenotype is rescued by reducing the levels of expression of the genes Notch, cut, and dacapo. These results indicate that miR-7 functions together with the Notch pathway and with dacapo to control wing growth. Curiously, the shape of the Df (2R) exu1/miR-7Δ1 wings appears slightly different from wild type wings perhaps due the Insulin-like receptor signaling control of dacapo expression. Neither the Notch nor cut genes have been predicted to be targets of miR-7. However, effectors of the Notch pathway such as components of the Enhancer of split complex genes have been shown to be regulated by high levels of miR-7 to control chordotonal organ differentiation (Aparicio, 2015).
How could miR-7 be involved in the control of wing growth and the regulation of the Notch pathway? This study has shown that the G1-S cell cycle transition is delayed in miR-7 loss of function mutants perhaps as a consequence of the reduced cell size to compensate for the loss of cell mass. Also, this study showed that miR-7 regulates Notch activity. Considering that Notch has been shown to negatively regulate the G1-S transition, a mechanism consistent with all these results is that miR-7 regulates Notch activity, which in turn regulates cell cycle progression and ultimately wing growth (Aparicio, 2015).
dacapo is an inhibitor of the cell cycle progression that is expressed ubiquitously in the wing imaginal cells and is required for normal wing growth. The dacapo 3'UTR contains functional miR-7 binding sites that are required for the maintenance of GSC division. Interestingly, reducing the activity in the wing imaginal discs of dicer, the central element in miRNA biogenesis, produces small wings whose size is rescued when dacapo levels of expression are decreased. Thus, wing growth control mediated by dacapo depends on microRNAs activity. The current results show that reducing the levels of dacapo expression rescues the small wing phenotype produced by miR-7 loss of function. Therefore, the results strongly suggest that miR-7 regulates dacapo expression to control wing growth and, furthermore, that this control is direct as it has been shown using luciferase assays that miR-7 directly regulates the expression of dacapo (Aparicio, 2015).
In the current model, miR-7 controls the expression of Notch pathway components as well as dacapo to allow cell cycle progression and regulate wing growth. A role of miR-7 in the direct regulation of dacapo as well as the direct or indirect regulation of Notch activity is supported by these results. Additionally, Notch has also been proposed to regulate dacapo expression in the follicle cells and it is not unreasonable to think that this regulation might also take place in the wing to control organ growth. These investigations add a new component, the microRNA miR-7, to the group of factors modulating proliferation/growth. Thus, the study of their interactions may help to resolve the intricate relationship between cell cycle and cell growth. Further analysis will be necessary to study the mechanisms involved in this regulation and the factors that together with miR-7, dacapo, and Notch control both the initiation and the termination of cell growth of the wing imaginal disc (Aparicio, 2015).
Mechanical forces are critical but poorly understood inputs for organogenesis and wound healing. Calcium ions (Ca2+) are critical second messengers in cells for integrating environmental and mechanical cues, but the regulation of Ca2+ signaling is poorly understood in developing epithelial tissues. This study reports a chip-based regulated environment for microorgans that enables systematic investigations of the crosstalk between an organ's mechanical stress environment and biochemical signaling under genetic and chemical perturbations. This method enabled definition of the essential conditions for generating organ-scale intercellular Ca2+ waves in Drosophila wing discs that are also observed in vivo during organ development. Mechanically induced intercellular Ca2+ waves are shown to require fly extract growth serum as a chemical stimulus. Using the chip-based regulated environment for microorgans, it was demonstrated that not the initial application but instead the release of mechanical loading is sufficient, but not necessary, to initiate intercellular Ca2+ waves. The Ca2+ response depends on the prestress intercellular Ca2+ activity and not on the magnitude or duration of the mechanical stimulation applied. Mechanically induced intercellular Ca2+ waves rely on IP3R-mediated Ca2+-induced Ca2+ release and propagation through gap junctions. Thus, intercellular Ca2+ waves in developing epithelia may be a consequence of stress dissipation during organ growth (Narciso, 2017).
Quantitative analysis of the dynamic cellular mechanisms shaping the Drosophila wing during its larval growth phase has been limited, impeding the ability to understand how morphogen patterns regulate tissue shape. Such analysis requires explants to be imaged under conditions that maintain both growth and patterning, as well as methods to quantify how much cellular behaviors change tissue shape. This study demonstrates a key requirement for the steroid hormone 20-hydroxyecdysone (20E) in the maintenance of numerous patterning systems in vivo and in explant culture. Low concentrations of 20E support prolonged proliferation in explanted wing discs in the absence of insulin, incidentally providing novel insight into the hormonal regulation of imaginal growth. 20E-containing media was used to observe growth directly and to apply recently developed methods for quantitatively decomposing tissue shape changes into cellular contributions. Whereas cell divisions drive tissue expansion along one axis, their contribution to expansion along the orthogonal axis is cancelled by cell rearrangements and cell shape changes. This finding raises the possibility that anisotropic mechanical constraints contribute to growth orientation in the wing disc (Dye, 2017).
The Drosophila wing exhibits a well-ordered cell pattern, especially along the posterior margin, where hair cells are arranged in a zigzag pattern in the lateral view. Based on an experimental result observed during metamorphosis of Drosophila, it was considered that a pattern of initial cells autonomously develops to the zigzag pattern through cell differentiation, intercellular communication, and cell death (apoptosis), and computer simulations were performed of a cell-based model of vertex dynamics for tissues. The model describes the epithelial tissue as a monolayer cell sheet of polyhedral cells. Their vertices move according to equations of motion, minimizing the sum total of the interfacial and elastic energies of cells. The interfacial energy densities between cells are introduced consistently with an ideal zigzag cell pattern, extracted from the experimental result. The apoptosis of cells is modeled by gradually reducing their equilibrium volume to zero and by assuming that the hair cells prohibit neighboring cells from undergoing apoptosis. Based on experimental observations, wing elongation along the proximal-distal axis was also assumed. Starting with an initial cell pattern similar to the micrograph experimentally obtained just before apoptosis, the simulations were carried out according to the model mentioned above, and the ideal zigzag cell pattern was successfully reproduced. This elucidates a physical mechanism of patterning triggered by cell apoptosis theoretically and exemplifies a new framework to study apoptosis-induced patterning. It is concluded that the zigzag cell pattern is formed by an autonomous communicative process among the participant cells (Nagai, 2018).
Epithelial folding transforms simple sheets of cells into complex three-dimensional tissues and organs during animal development. Epithelial folding has mainly been attributed to mechanical forces generated by an apically localized actomyosin network, however, contributions of forces generated at basal and lateral cell surfaces remain largely unknown. This study shows that a local decrease of basal tension and an increased lateral tension, but not apical constriction, drive the formation of two neighboring folds in developing Drosophila wing imaginal discs. Spatially defined reduction of extracellular matrix density results in local decrease of basal tension in the first fold; fluctuations in F-actin lead to increased lateral tension in the second fold. Simulations using a 3D vertex model show that the two distinct mechanisms can drive epithelial folding. This combination of lateral and basal tension measurements with a mechanical tissue model reveals how simple modulations of surface and edge tension drive complex three-dimensional morphological changes (Sui, 2018).
This work has uncovered two new mechanisms of epithelial fold formation. First, a locally defined basal decrease of surface and edge tension, associated with local reduction of ECM density, leads to basal cell expansion and folding. Second, a lateral increase of surface tension at the future fold location, associated with F-actin flows and pulsatile contractions, leads to a local reduction of tissue height and fold formation. It is conceivable that both mechanisms may also operate in combination during epithelial folding (Sui, 2018).
A simplified picture resulting from mechanical analysis of how basal tension reduction can induce fold formation is as follows. Higher basal tension in the cells outside the fold compared to cells inside the fold stretches the basal surface areas of fold cells. Consequently, fold cells widen basally and reduce cell height to maintain cell volume. The new force balance state is characterized by apical indentation and wedge-shaped, shortened cells. How is ECM depletion linked to a decrease in basal cell edge and surface tension? In one scenario, following ECM depletion, the actomyosin network lacks stabilization via binding to integrins, reducing the active tension it can generate with myosin molecular motors. Alternatively, the ECM and cortical actomyosin network, linked together via integrins and other molecules, can be seen as a single composite material under tension. Elastic straining of the ECM, e.g. during tissue growth, could give rise to a passive mechanical tension within the ECM. As the ECM is depleted, the composite material is reorganized and passive ECM stress due to ECM straining could be lost, also contributing to the overall decrease in basal tension in the fold (Sui, 2018).
Lateral tension increase can also induce fold formation. This can be outlined in a simplified picture (see Differential lateral and basal tension drive folding of Drosophila wing discs through two distinct mechanisms). Increased lateral tension leads to a reduction in cell height. Since basal tension is high, the shortened cells deform the apical surface inwards, while the basal surface resists deformation. As the cells resist volume changes, they widen. Conceivably, increased apical tension in the fold cells favors further basal expansion of the fold cells (Sui, 2018).
Folding requires the transition of cells from a columnar to a wedge-shape where the apical surface is smaller than the basal surface. Previous work has stressed the role of mechanical stresses generated by apical actomyosin networks driving apical constriction during folding. This work shows that for the epithelial folds, in the case of the wing, apical constriction is not important. Instead, they rely either on the basal widening of cells due to the decrease of basal tension or alternatively on increased lateral tension. Interestingly, two fundamentally different mechanisms generate similar morphologies of neighboring folds. This implies that the mechanical processes shaping a tissue cannot be deduced from the tissue morphology alone. Cell shortening and an active role for the ECM is also required for the folding of the zebrafish embryonic brain. Basal decrease of tension and lateral increase of tension may therefore represent two important mechanisms driving the folding of epithelia in different organisms (Sui, 2018).
The robust specification of organ development depends on coordinated cell-cell communication. This process requires signal integration among multiple pathways, relying on second messengers such as calcium ions. Calcium signaling encodes a significant portion of the cellular state by regulating transcription factors, enzymes, and cytoskeletal proteins. However, the relationships between the inputs specifying cell and organ development, calcium signaling dynamics, and final organ morphology are poorly understood. In this study a quantitative image-analysis pipeline was designed for decoding organ-level calcium signaling. With this pipeline, spatiotemporal features were extracted of calcium signaling dynamics during the development of the Drosophila larval wing disc, a genetic model for organogenesis. Specific classes of wing phenotypes were identified that resulted from calcium signaling pathway perturbations, including defects in gross morphology, vein differentiation, and overall size. Four qualitative classes of calcium signaling activity were. These classes can be ordered based on agonist stimulation strength Galphaq-mediated signaling. In vivo calcium signaling dynamics depend on both receptor tyrosine kinase/phospholipase C gamma and G protein-coupled receptor/phospholipase C beta activities. Spatially patterned calcium dynamics were found to correlate with known differential growth rates between anterior and posterior compartments. Integrated calcium signaling activity decreases with increasing tissue size, and it responds to morphogenetic perturbations that impact organ growth. Together, these findings define how calcium signaling dynamics integrate upstream inputs to mediate multiple response outputs in developing epithelial organs (Brodskiy, 2019).
The molecular mechanisms regulating animal tissue size during development are unclear. This question has been extensively studied in the Drosophila wing disc. Although cell growth is regulated by the kinase TORC1, no readout exists to visualize TORC1 activity in situ in Drosophila. Both the cell cycle and the morphogen Dpp are linked to tissue growth, but whether they regulate TORC1 activity is not known. This study developed an anti-phospho-dRpS6 antibody that detects TORC1 activity in situ. Unexpectedly, it was found that TORC1 activity in the wing disc is patchy. This is caused by elevated TORC1 activity at the cell cycle G1/S transition due to CycD/Cdk4 phosphorylating TSC1/2.TORC1 is also activated independently of CycD/Cdk4 when cells with different levels of Dpp signaling or Brinker protein are juxtaposed. This study has thereby characterize the spatial distribution of TORC1 activity in a developing organ (Romero-Pozuelo, 2017).
During animal development, tissues increase tremendously in mass, yet stop growing at very stereotyped sizes in a robust manner. For instance, the Drosophila wing is specified as a cluster of circa 50 cells, which increases in mass ~500-fold before terminating growth. Once growth has ceased, the left and right wings of an individual fly are virtually identical in size, to within 1%, illustrating the robustness of this process. How animal tissue size is regulated is a fundamental open question in developmental biology (Romero-Pozuelo, 2017).
As mitotically growing tissues develop, two independent cellular processes occur in a coordinated manner: proliferation and cell growth. By itself, proliferation -- the division of cells -- does not lead to mass accumulation. This was nicely shown in the Drosophila wing where overexpression of E2F speeds up the cell cycle, but leads to a normally sized tissue containing more, smaller cells. For a tissue to grow, cells need to accumulate biomass. The mechanisms interconnecting cell proliferation and cell growth are not completely understood. In organisms from yeast to humans, growth is in large part regulated by the target of rapamycin complex 1 (TORC1) kinase. TORC1 promotes biomass accumulation by promoting anabolic metabolic pathways such as protein, lipid, and nucleotide biosynthesis, while repressing catabolic processes such as autophagy. Hence, to understand tissue growth it would be of interest to study the spatial distribution of TORC1 activity in a developing tissue. This line of investigation has been hampered, however, by the lack of readouts for TORC1 activity that can be used in situ (Romero-Pozuelo, 2017).
One signaling pathway that strongly affects tissue size is the Dpp pathway. Dpp is expressed and secreted by a stripe of cells in the medial region of the wing imaginal disc, and forms an extracellular morphogen gradient that both helps to pattern the wing and affects its size. In the absence of Dpp signaling during development, only small rudimentary wings are formed. In contrast, overexpression of Dpp leads to strong tissue overgrowth, in particular along the axis of the morphogen gradient. Several models have been proposed for how Dpp signaling regulates wing size. The exact mechanism by which Dpp regulates tissue size, however, is an unresolved issue. Dpp signaling acts to repress expression of a transcription factor called Brinker. Brinker appears to mediate most of the size effects of Dpp signaling. When Brinker is genetically removed, Dpp signaling becomes dispensable for wing growth. Given that Dpp signaling promotes tissue growth, an open question is whether Dpp signaling promotes TORC1 activity (Romero-Pozuelo, 2017).
Thia study examined whether Dpp signaling promotes TORC1 activity in the Drosophila wing disc. To this end, a phospho-RpS6 (pS6) antibody was developed that allows TORC1 activity to be assayed in situ in tissue. This reagent reveals unexpectedly that TORC1 activity in the growing wing disc is neither uniform nor graded, but is instead patchy. This patchiness is mediated via CycD/Cdk4 and the tuberous sclerosis 1 (TSC1)-TSC2 complex in response to cell cycle stage. Using this pS6 antibody, this study found that TORC1 activity is also induced by discontinuities in Dpp signaling or discontinuities in Brinker levels. It is proposed that these discontinuous conditions may be analogous to regenerative conditions that happen in the wing disc in response to tissue damage. In sum, this work reveals the pattern of TORC1 activity in the context of a developing organ (Romero-Pozuelo, 2017).
TORC1 activity in the wing disc is modulated by the cell cycle, with cells in early S phase showing the highest TORC1 activity. Interestingly, an accompanying paper finds similar results in the Drosophila eye disc (Kim, 2017). This might reflect a metabolic requirement by early S-phase cells for large amounts of nucleotide biosynthesis, an anabolic process promoted by TORC1. Indeed, in various contexts S6K and TORC1 activity were found to be required for the transition from G1 to S. Connections between mechanistic TOR (mTOR) and the cell cycle have previously been found in cultured cells. In human fibroblasts, mTOR shuttles in and out of the nucleus in a cell cycle-dependent manner, peaking in the nucleus shortly before S phase. The relevance of this subcellular relocalization to what is observe in this study, however, is unclear. In fibroblasts, S6K1 activity was found to be highest during early G1, whereas in HeLa cells it was found to be highest during M phase. In sum, it is unclear to what extent cells in culture recapitulate endogenous development, or whether the influence of the cell cycle on TORC1 activity is very context dependent. The TSC1/2 complex has been reported to be phosphorylated by cell cycle-dependent kinases.TSC1 is phosphorylated on Thr417 by Cdk1 during the G2/M transition. This inhibitory phosphorylation would lead to elevated TORC1 activity during G2/M, which does not fit with what was observe here, and thus might be relevant in a different developmental context. Instead, this study found that TSC2 can be phosphorylated by the CycD/Cdk4 complex on Ser1046, and possibly other sites as well, and that this leads to activation of TORC1. This fits with several observations in the literature. Firstly, in U2OS cells the TSC complex was also found to bind cyclin D, leading to its phosphorylation at unknown sites. In U2OS cells, this causes destabilization of the Tsc1 and Tsc2 proteins, which was not observed in this study. Secondly, Tsc1/2 and CycD/Cdk4 were previously found to interact genetically in Drosophila: The reduced tissue growth caused by Tsc1 + Tsc2 overexpression was found to be fully suppressed by expression of CycD + Cdk4. This fits well with the current data suggesting that CycD/Cdk4 directly inhibits the TSC complex via phosphorylation. Thirdly, Cyclin D and Cdk4 were previously reported in Drosophila to promote cell and tissue growth, fitting with activation of the TORC1 complex by CycD/Cdk4. It is worth noting that some patchy TORC1 activity is still seen in CycD- or Cdk4-null discs and in discs with the single phospho-site mutations in TSC2. Hence it is possible that Cdk4 may not be the only factor regulating TORC1 activity in response to the cell cycle, and that Cdk4 might phosphorylate TSC2 on additional sites (Romero-Pozuelo, 2017).
What are the roles of CycD/Cdk4 in cell cycle progression and cell growth? Whereas mammals have three cyclin D genes, CycD1-3, and two CycD binding kinases, Cdk4 and Cdk6, Drosophila has a single CycD, a single Cdk4, and no Cdk6. Hence Drosophila provides an opportunity to elucidate the function of the CycD/Cdk4 complex without difficulties arising from redundancy. Indeed, results in Drosophila clearly show that CycD/Cdk4 promotes cell growth and not cell cycle progression. Both CycD- and Cdk4-null animals are viable, and fluorescence-activated cell sorting (FACS) analysis of null cells revealed that they have a normal cell cycle profile, indicating that they are dispensable for normal cell cycle progression. Instead, Cdk4- and CycD-null animals are 10%-20% smaller than controls, indicating that they promote cell growth. The finding that CycD/Cdk4 activates TORC1 during the G1/S transition can provide one mechanism by which the CycD/Cdk4 complex promotes growth. Hence, from these data it is proposed that in Drosophila the CycD/Cdk4 complex is not part of the core machinery required for cell cycling, but is rather an effector 'side branch' activated at G1/S to promote cell growth. Data from the mouse suggest something similar. CycD1, CycD2, and CycD3 knockout mice are all viable. One could imagine this to be due to redundancy between these three genes, but actually CycD1, CycD2, CycD3 triple-knockout mice survive to mid-gestation, and the triple-knockout mouse embryonic fibroblasts proliferate relatively normally. The mid-gestation lethality of the triple knockouts appears to be due to specific effects in hematopoietic and myocardial cells. Hence, cyclins D1-D3 are also dispensable for cell cycle progression in mice. Interestingly, CycD1 knockout mice and CycD1, CycD2 double-knockout mice are viable but have reduced body size, reminiscent of the size phenotype observed in CycD knockout flies. In sum, despite CycD/Cdk4 being claimed in most reviews on the cell cycle as playing an important role in G1/S progression, it appears that this complex may function rather to promote cell growth in a cell cycle-dependent manner (Romero-Pozuelo, 2017).
Does Dpp control growth in the wing? When discontinuities in Dpp activity or in Brinker levels were genetically induce, activation was observed of TORC1 at the site of discontinuity. Hence, Dpp signaling per se does not appear to activate TORC1; rather, the comparison between high Dpp signaling and low Dpp signaling cells does. In an unperturbed disc, no pattern of pS6 staining was observed that correlates with the Dpp activity gradient, which is highest medially and drops toward the anterior and posterior extremities. This might be due to the fact that in an unperturbed disc the Dpp and Brinker gradients are smooth and do not have such discontinuities. A similar effect of Dpp was previously observed on cell prolife ration, except that in this case the effect of the Dpp discontinuity was very transient, lasting only a few hours after clone induction, whereas the effect seen on growth is sustained. Dpp signaling is, nonetheless, required for growth, because in the absence of Dpp, small vestigial wings are formed. Hence one interpretation might be that low levels of Dpp signaling are continuously required for growth, but that Dpp signaling becomes instructive for tissue growth only when discontinuities in the gradient arise, perhaps as a result of tissue damage or cell delamination, to initiate a regenerative response (Romero-Pozuelo, 2017).
One additional interesting non-autonomous phenomenon observed is that sometimes when a region of the wing disc has high pS6 levels, the rest of the disc loses its typically patchy pS6 pattern and becomes pS6 negative. This phenomenon is not understood, and future work will be necessary to understand it molecularly (Romero-Pozuelo, 2017).
During morphogenesis, cells communicate with each other to shape tissues and organs. Several lines of recent evidence indicate that ion channels play a key role in cellular signaling and tissue morphogenesis. However, little is known about the scope of specific ion-channel types that impinge upon developmental pathways. The Drosophila melanogaster wing is an excellent model in which to address this problem as wing vein patterning is acutely sensitive to changes in developmental pathways. A screen was carried out of 180 ion channels expressed in the wing using loss-of-function mutant and RNAi lines. This study identified 44 candidates that significantly impacted development of the Drosophila melanogaster wing. Calcium, sodium, potassium, chloride, and ligand-gated cation channels were all identified in the screen, suggesting that a wide variety of ion channel types are important for development. Ion channels belonging to the pickpocket family, the ionotropic receptor family, and the bestrophin family were highly represented among the candidates of the screen. Seven new ion channels with human orthologs that have been implicated in human channelopathies were also identified. Many of the human orthologs of the channels identified in this screen are targets of common general anesthetics, anti-seizure and anti-hypertension drugs, as well as alcohol and nicotine. These results confirm the importance of ion channels in morphogenesis and identify a number of ion channels that will provide the basis for future studies to understand the role of ion channels in development (George, 2019).
Due to the complexity of genotype-phenotype relationships, simultaneous analyses of genomic associations with multiple traits will be more powerful and informative than a series of univariate analyses. In most cases, however, studies of genotype-phenotype relationships have been analyzed only one trait at a time. This paper reports the results of a fully integrated multivariate genome-wide association analysis of the shape of the Drosophila melanogaster wing in the Drosophila Genetic Reference Panel. Genotypic effects on wing shape were highly correlated between two different labs. 2,396 significant SNPs were found using a 5% FDR cutoff in the multivariate analyses, but just 4 significant SNPs were found in univariate analyses of scores on the first 20 principal component axes. One quarter of these initially significant SNPs retain their effects in regularized models that take into account population structure and linkage disequilibrium. A key advantage of multivariate analysis is that the direction of the estimated phenotypic effect is much more informative than in a univariate one. This fact was exploited to show that the effects of knockdowns of genes implicated in the initial screen were on average more similar than expected under a null model. A subset of SNP effects were replicable in an unrelated panel of inbred lines. Association studies that take a phenomic approach in considering many traits simultaneously are an important complement to the power of genomics (Pitchers, 2019).
The final step in morphogenesis of the adult fly is wing maturation, a process not well understood at the cellular level due to the impermeable and refractive nature of cuticle synthesized some 30 h prior to eclosion from the pupal case. Advances in GFP technology now make it possible to visualize cells using fluorescence after cuticle synthesis is complete. Between eclosion and wing expansion, the epithelia within the folded wing begin to delaminate from the cuticle and that delamination is complete when the wing has fully expanded. After expansion, epithelial cells lose contact with each other, adherens junctions are disrupted, and nuclei become pycnotic. The cells then change shape, elongate, and migrate from the wing into the thorax. During wing maturation, the Timp gene product, tissue inhibitor of metalloproteinases, and probably other components of an extracellular matrix are expressed that bond the dorsal and ventral cuticular surfaces of the wing following migration of the cells. These steps are dissected using the batone and Timp genes and ectopic expression of αPS integrin, inhibitors of Armadillo/β-catenin nuclear activity and baculovirus caspase inhibitor p35. It is concluded that an epithelial-mesenchymal transition is responsible for epithelial delamination and dissolution (Kiger, 2007; full text of article).
The following outline is proposed of that program based upon cell behavior: delamination and severing contacts; changing cell shape; and migration and ECM synthesis.
Stage 1, delamination and severing contacts
A signaling role for integrins during the prepupal apposition has been proposed that prepares cells for integrin-based adhesion of the epithelia at the pupal apposition. The observation that wing epithelial cells persist in the blistered regions produced by ectopic αPS integrin expression suggests that the integrin interaction also prepares cells to respond to the later signal that induces epithelial delamination and dissolution. This signal is also blocked in the mutant batone, which prevents wing expansion. Some cells begin to delaminate from the cuticle before wing expansion has begun, and all have delaminated by the time expansion is complete. Delamination must involve severing of ECM contacts. The precision of the cellular array in a newly open wing must derive from cell–cell contacts between stretched cells that are maintained following delamination. Each cell then compacts and becomes round (as judged by the increase in fluorescence intensity). The round cells have evidently severed their junctions with adjacent cells because the precise array of cells begins to break up and Arm-GFP moves from the cell membrane to the cytoplasm (Kiger, 2007).
It would appear that disturbing the normal state of Arm/β-catenin signaling activity in epithelial cells blocks delamination. Delamination is blocked by ectopic expression of Pygo in the epithelial cells, which blocks expression of Arm target genes in a variety of tissues, and by ectopic expression of Shaggy, which blocks expression of Arm target genes by phosphorylating cytoplasmic Arm, promoting its degradation and depleting nuclear Arm. Ectopic expression of stabilized forms of Arm not subject to Shaggy phosphorylation evidently has a dominant-negative effect on Arm signaling activity in the maturing wing, blocking delamination of epithelial cells. This interpretation is supported by the following observations. First, no effect is produced by ectopic expression of wild-type Arm using the same Gal4-30A driver, consistent with other reports, very likely indicating the efficiency with which wild-type Arm is eliminated by phosphorylation and degradation through the proteasome. Second, a very low level of nuclear Arm is sufficient for target gene expression. The Arm-GFP fusion protein used here is fully active and completely covers homozygosity for a null arm allele, yet nuclear Arm-GFP cannot be detected in cells receiving a Wingless signal. Thus, it is reasonable that non-physiologically high levels of stable forms of Arm could have a dominant-negative effect, not unlike the inhibitory effect of over-expression of Pygo on Arm-directed transcription. (Kiger, 2007).
Arguing against an interpretation that the effects of ectopic gene expression might be non-specific, note that Gal4-30A-driven expression of p35 does not block delamination. Nor does Gal4-30A-driven expression of either αPS integrin or wild-type transcription factor Pangolin/dTCF/LEF-1, or a dominant-negative form of CREB have any effect on wing maturation (Kiger, 2007).
Stage 2, changing cell shape
The round cells then begin to change shape, extending thin cytoplasmic filaments, and elongate into spindles that associate with similarly shaped cells forming streams. The fact that p35 expression interrupts developmental progression at the round cell stage clearly separates Stage 1 from the changes in cell shape, cell migration, and ECM synthesis events that follow. In some cellular contexts caspase inhibition prevents cell migration independently of blocking apoptosis. It has been shown that the nuclei of wing cells cease to retain nuclear-targeted GFP and begin to fragment their DNA at what appears to be the round cell stage, consistent with the observation of pycnotic nuclei at this stage (Kiger, 2007).
Stage 3, migration and ECM synthesis
The cells migrate toward the hinge and into the body of the fly, leaving behind components, perhaps including tissue inhibitor of metalloproteinases, of an ECM that will bond dorsal and ventral cuticular surfaces. It is noteworthy that Timp deficiency does not interfere with cell migration. ECM assembly must be the final step in the developmental program. The nonautonomous action of Timp in bonding cuticle secreted by mutant Timp clones suggests that Timp is present in abundance and diffuses over large distances in the wing to participate in ECM formation (Kiger, 2007).
Precisely how ectopic expression of the various UAS transgenes studied in this paper produces wing blisters or collapsed wings is not wholly clear. It seems doubtful that cells that fail to delaminate during early phases of tissue remodeling would secrete ECM components normally. Yet a variable number of cells in these wings do delaminate and leave the wing, presumably because of variation in the level of Gal4-30A expression. These cells might be expected to secrete the necessary ECM components, although the level of critical component(s) may be insufficient for normal bonding to occur in some cases. Blister formation might also be caused by the presence of numbers of undelaminated cells physically preventing ECM from bonding the underlying cuticle. Note that when ectopic p35 expression is limited, a moderate number of round, delaminated cells can become bound in the wing without producing blisters (Kiger, 2007).
The presence of true hemocytes in the wing raises the question of whether these cells play a role in wing maturation. If Gal4-30A was to be expressed in these cells, as well as in epithelial cells, interpretation of ectopic expression studies would be complicated. No cells were detected expressing DsRedGFP fluorescence that did not express ywing-GFP fluorescence, suggesting that Gal4-30A is not expressed in true hemocytes. The observations that Hemese-Gal4-driven expression of Shaggy or of Pygo has no effect on wing maturation strongly suggest that the effects of Shaggy and Pygo on wing maturation are not mediated by true hemocytes exclusively, if at all. While the possibility that Timp and/or other ECM components are supplied by true hemocytes cannot be ruled out, the bulk of the evidence supports an active role for epithelial cells in bonding the wing surfaces. Precocious death of epithelial cells induced by Gal4-30A-driven expression of Ricin A in late pupal epithelial cells prevents bonding of dorsal and ventral cuticle after eclosion. Because the wing cuticle is fully formed, the induced cell death must have occurred after cuticle deposition but before eclosion. UV irradiation after eclosion blocks both epithelial cell delamination and bonding of the wing surfaces. In addition, it is clear that mitotic clones of defective epithelial cells affect bonding of the wing surfaces. Mitotic clones mutant for an integrin gene produce blisters in the wing cuticle as do mitotic clones ectopically expressing PKAc (Kiger, 2007).
These studies describe for the first time the developmental program that completes morphogenesis of the adult fly. The requirement for a normal state of Arm/β-catenin signaling activity suggests that an epithelial–mesenchymal transition (EMT) transforms epithelial cells into mobile fibroblasts in the wing (Kiger, 2007).
The best known example of an EMT in Drosophila is neuroblast delamination. In embryonic central nervous system formation, Wingless signaling has been shown to induce nonautonomously the delamination of specific neuroectoderm cells to form S2 neuroblasts. In peripheral nervous system formation, Wingless signaling is required for bristle formation at the wing margin, and ectopic expression of Wingless induces ectopic bristles in the wing blade. The ability of Wingless to induce neuroectoderm cells to form neuroblasts is tightly regulated by Notch in both the central and peripheral nervous systems. Evidence supports the idea that Notch modulates Wingless signaling by associating directly with Arm/β-catenin to regulate its transcriptional activity (Kiger, 2007).
Arm/β-catenin signaling appears to be characteristic of EMTs. Translocation of Arm/β-catenin into the nucleus precedes gastrulation in Drosophila, the sea urchin, and zebrafish. EMTs occur in the vertebrate neural crest when cells delaminate from the neural epithelium and migrate throughout the embryo. In the avian neural crest, dominant-negative forms of β-catenin and LEF/TCF inhibit delamination of cells from the epithelium, G1/S transition, and transcription of target genes. β-Catenin and LEF/TCF proteins are observed to translocate to the nuclei of avian neural crest cells only during delamination and to be absent during advanced stages of migration. EMTs are also a characteristic of cancer formation and can be initiated in some cancers by aberrant β-catenin activity (Kiger, 2007).
Multiple ways of activating Arm/β-catenin signaling exist. There are two independently regulated pathways that can target Arm/β-catenin to the proteasome, the Shaggy/Glycogen synthase kinase 3 degradation complex and the Seven in Absentia Homologue/ubiquitin ligase degradation complex. Multiple G-protein-coupled receptors target the Shaggy/Glycogen synthase kinase 3 degradation complex for inhibition. Further studies are necessary to identify the hormone(s), receptor(s) and signal transduction mechanisms acting in the wing maturation program and to relate this work to the extensive studies of the hormonal signals controlling wing expansion and cuticle tanning (Kiger, 2007).
In addition to the heart proper, insects possess wing hearts in the thorax to ensure regular hemolymph flow through the narrow wings. In Drosophila, the wing hearts consist of two bilateral muscular pumps of unknown origin. This paper presents the first developmental study on these organs and reports that the wing hearts originate from eight embryonic progenitor cells arising in two pairs in parasegments 4 and 5. These progenitors represent a so far undescribed subset of the Even-skipped positive pericardial cells (EPC) and are characterized by the early loss of tinman expression in contrast to the continuously Tinman positive classical EPCs. Ectopic expression of Tinman in the wing heart progenitors omits organ formation, indicating a crucial role for Tinman during progenitor specification. The subsequent postembryonic development is a highly dynamic process, which includes proliferation and two relocation events. Adults lacking wing hearts display a severe wing phenotype and are unable to fly. The phenotype is caused by omitted clearance of the epidermal cells from the wings during maturation, which inhibits the formation of a flexible wing blade. This indicates that wing hearts are required for proper wing morphogenesis and functionality (Tögel, 2008).
Unlike in vertebrates, where an elaborate closed blood vessel system extends throughout the whole body, insects possess only one vessel, the tubular heart, in their otherwise open circulatory system. Once the hemolymph has left the heart, it moves freely between the internal organs and can not be directed into narrow body appendages such as antennae, legs or wings. To ensure sufficient hemolymph supply of these appendages additional circulatory organs evolved (Pass, 2000; Pass, 2006). In Drosophila, circulation in the wings is maintained by the so-called wing hearts (Krenn, 1995), a pair of autonomous muscular pumps located bilaterally in the scutellum, the dorsal elevation of the second thoracic segment. Due to this location, they are also referred to as scutellar pulsatile organs. Although known for many years, no developmental studies on the origin or morphogenesis of these organs have been performed. Probably, this was due to the lack of available methods to track their differentiation. However, studies on the origin of the thoracic somatic muscles in Drosophila and comparative anatomical investigations in insects suggested that the wing hearts originate from the cardiac mesoderm or from the heart itself (Tögel, 2008).
A previous study identified an enhancer region of the Drosophila hand gene that is able to drive reporter gene activity in the wing hearts (Sellin, 2006). In the present work, this reporter was used to identify the embryonic anlagen of the wing hearts and to elucidate the dynamics of their postembryonic development with in vivo time lapse imaging. It was found that the anlagen of the Drosophila wing hearts indeed derive from the cardiac mesoderm but, astonishingly, not from the muscular cardioblast lineage. Instead, they represent a so far undescribed subpopulation of the well-known Even-skipped (Eve) positive pericardial cells (EPCs) (Tögel, 2008).
In addition to their unknown origin, little is known about the contribution of wing hearts to wing morphogenesis and functionality. After eclosion, wings are unfolded by a sudden influx of hemolymph and subsequently undergo maturation. During this process, the epidermal cells that until then bonded the dorsal and ventral wing surfaces enter programmed cell death, delaminate from the cuticle, and disappear into the thorax (Kimura, 2004). Subsequently, the cuticles of the intervein regions become tightly bonded to form a flexible wing blade, while the cuticles of the vein regions form tubes, lined by living cells, through which hemolymph circulates in mature adult insects. Measurements of hemolymph flow in adult butterflies showed that wing hearts function as suction pumps that draw hemolymph out of the wings starting shortly after wing unfolding. Whether wing hearts might play a role in wing maturation was tested by generating flies lacking wing hearts. The findings demonstrate that the delaminated epidermal cells are removed from the wings by the hemolymph flow generated by the wing hearts. Loss of wing heart function leads to remains of epidermal cells resting between the unbonded dorsal and ventral wing surfaces which results in malformation of the wing blade and flightlessness. It is concluded that wing hearts are essential for wing maturation and, thus, for acquiring flight ability in Drosophila (Tögel, 2008).
A hand-C-GFP reporter was generated (Sellin, 2006) that reflects the described hand expression pattern and was found to be active in wing hearts. To confirm that the hand-C-GFP reporter is expressed in all cells of mature wing hearts, their morphology was examined based on the signal from the reporter in conjunction with histological sections. In the adult fly, wing hearts are located at the lateral angles of the scutellum, which are joined to the posterior wing veins by cuticular tubes. Each organ is curved in anterior–posterior direction as well as dorso-ventrally. It consists of about 7-8 horizontally arranged rows of prominent muscle cells, which are attached at their proximal side to a thin layer of cells that has a greater dorsal extension than the muscle cells. Both cell types are labeled by the reporter. The fine acellular strands that hold the wing hearts to the adjacent epidermal cells were not observed to be marked by the reporter. PubMed ID: Movies are provided to demonstrate the location and the beating of wing hearts (Tögel, 2008).
The hand-C-GFP reporter was tested for expression in earlier stages of wing heart development and it was found to be active throughout the entire organogenesis. This enabled identification of the embryonic anlagen of the wing hearts, which consist of eight progenitor cells located dorsally and anterior to the heart, in two pairs in the second and third thoracic segment from stage 16/17 onward. The progenitors exhibit a flattened triangular shape and are interconnected by thin cytoplasmic extensions. In addition, the second and the fourth pair of the progenitors are closely associated with the dorsal tracheal branches at their interconnection in the second and third thoracic segments. The characteristic pairwise arrangement and the connection to the tracheae are retained during the subsequent three larval stages. Proliferation starts at about the transition from the second to the third larval instar, leading to eight clusters of cells that remain arranged in four pairs in the anterior region until 1h after puparium formation (APF). Between 1 and 10h APF, the cell number increases significantly and the anterior three pairs of cell clusters are retracted to join the last pair of clusters, eventually forming one large median cluster. Between 13 and 50h APF, the single large cluster splits along its anterior-posterior axis into two groups of cells that migrate laterally in the forming scutellum, thereby adopting the characteristic arched appearance of the adult wing hearts. During this process some of the cells on either side form the underlying thin layer while the remaining cells arrange in horizontal rows along that layer. First contractions of the mature organs were observed at about 45-50h APF (Tögel, 2008).
The expression of the bHLH transcription factor Hand in the wing heart progenitors, which serves as a general marker for all classes of heart cells in Drosophila, prompted a to screen for the expression of genes known to be active in cardiac lineages. Analysis of Even-skipped (Eve) expression revealed that the embryonic wing heart progenitors arise through the same lineage as the well described Eve expressing pericardial cells (EPCs). At stage 10 in embryogenesis, 12 Eve clusters are present on either side of the embryo, located in parasegments (PS) 2 to 12. Each cluster gives rise to a pair of EPCs, except for the most posterior cluster in PS 14, which generates only one EPC. During subsequent development, the first and the second pair of EPCs, located in parasegment 2 and 3, turn toward the midline of the embryo to accompany the tip of the heart, which later bends ventrally into the embryo. The third and the fourth pair of EPCs in PS 4 and 5 are shifted anteriorly in relation to the heart. This step is not based on migration but on the remodeling of the embryo during head involution, since the cells remain in their PS close to the likewise Eve positive anlagen of the DA1 muscle. The EPCs in PS 4 and 5 subsequently differentiate into the later wing heart progenitors, while all others become the classical EPCs and accompany the heart in a loosely associated fashion. At least from PS 4 to 12, all pairs of Eve positive cells (wing heart progenitors and classical EPCs) are interconnected by cytoplasmic extensions forming a rope ladder-like strand above the heart after dorsal closure at stage 16/17. This mode of contact between the cells persists in the wing heart progenitors in postembryonic stages and might be essential for proper relocation in the prepupae (Tögel, 2008).
Although the Drosophila wing hearts have been known for many years, their origin and development have remained unknown. This study provides the first developmental approach on these organs using in vivo time lapse imaging as well as genetic and immunohistochemical methods. It was found that the wing hearts develop from embryonic anlagen that consist of eight progenitor cells located anterior to the heart. Analysis of gene expression in these progenitors confirmed the hypothesis that the wing hearts originate from the cardiac mesoderm, but not from the contractile cardioblast lineage, as has been suggested based on anatomical data. Surprisingly, the embryonic anlagen derive from a particular subset of the well-known EPCs. EPCs arise in pairs in PS 2 to 12 from the dorsal progenitor P2, which divides asymmetrically into the founder of the dorsal oblique muscle 2 and the founder of the EPCs in a numb-dependent lineage decision. Additionally, a single EPC arises in PS 14. The subsequent differentiation of the founders into EPCs requires the activity of the transcription factors Zfh1 and Eve. This study shows that the EPCs located in PS 4 and 5 are relocated in relation to the heart during head involution at stage 14/15 of embryogenesis and subsequently differentiate into the wing heart progenitors. Until this step, no difference to the EPCs in the anterior and posterior PS could be detected. Like the classical EPCs, which remain close to the heart, the EPCs that give rise to the wing heart progenitors depend on factors involved in asymmetric cell division, e.g. Insc or Numb, and fail to differentiate in embryos mutant for zfh1 as well as in animals lacking mesodermal Eve. Loss of tinman expression is the only event that could be identified that discriminates between a classical EPC fate and the specification of wing heart progenitors. Consistently, ectopic expression of Tinman in the wing heart progenitors effectively represses their specification, probably by committing them to a classical EPC fate, indicating that Tinman plays a crucial role in the involved regulatory pathway (Tögel, 2008).
So far, the biological role of pericardial cells (PCs), and EPCs in particular, is not well understood. In the embryo, three populations of PCs arise in each segment, which are characterized by the expression of different combinations of genes (Odd positive PCs, Eve positive PCs, and Tinman positive PCs). During postembryonic stages, the number of PCs decreases, raising the question which population contributes to the final set of PCs in the adult and whether all PCs have the same function throughout development. Recent studies have shown that postembryonic PCs express Odd and Eve, a combination which is not observed in the embryo, and are dispensable for cardiac function. Genetic ablation of all larval PCs had no effect on heart rate, but increased sensitivity to toxic stress. In contrast, the specification of the correct number of embryonic PCs is crucial for normal heart function. Loss of mesodermal Eve during embryogenesis results in fewer larval pericardial cells, which causes a reduction in heart rate and lifespan. Conversely, hyperplasia of embryonic PCs has no effect on heart rate but causes decreased cardiac output. This was explained by an excess of Pericardin secreted by the PCs into the extracellular matrix enveloping the heart (Johnson, 2007). Taken together, embryonic PCs seem to influence cardiac development by e.g., secreting substances whereas postembryonic PCs function as nephrocytes. However, in this study, functional data is provided on a subset of embryonic EPCs, which differentiate into adult progenitors giving rise to a myogenic lineage. This represents a completely new function of PCs, raising the question whether EPCs might in general have myogenic potential and rather represent a population of adult progenitors, than PCs in a functional sense (Tögel, 2008).
The organogenesis of the wing hearts is a highly dynamic process, which includes distinct cellular interactions. At first, adjacent EPCs (including the wing heart progenitors) on either side of the embryo establish contact via cytoplasmic extensions. After dorsal closure of the embryo, interconnections are also formed between opposing EPCs resulting in a rope ladder-like strand above the heart. These interconnections are assumed to be needed to retain contact between the wing heart progenitors during the subsequent development. During larval stages, some of the wing heart progenitors establish a second contact to specific tracheal branches and proliferation starts. In the prepupa, a relocation event joins all wing heart progenitors in one large cluster. During this step, the progenitors are probably passively relocated in conjunction with the tracheal branches to which they are connected. Finally, the wing heart progenitors initiate active migration and form the mature wing hearts in the pupa. Considering the complexity of their development, it is proposed that wing hearts provide an ideal model for studying organogenesis on several different levels such as signaling, cell polarity, or path finding (Tögel, 2008).
Elimination of the embryonic progenitors by ectopic expression of tinman or by laser ablation causes the loss of wing hearts, which results in a specific wing phenotype in conjunction with flightlessness. In the identified phenotype, the delaminated epidermal cells are not cleared from the wings during wing maturation and bonding of the dorsal and ventral wing surfaces is omitted. Recently, it was reported that the epidermal cells transform into mobile fibroblasts and actively migrate out of the wings. However, in in vivo time-lapse studies migration of epidermal cells could not be observed during wing clearance. Conversely, their movements correlated with the periods of wing heart beating, indicating that they are passively transported by the hemolymph flow. One-sided ablation of mature wing hearts in pupae, confirms that wing hearts play a crucial physiological role in wing maturation, since the wing phenotype occurs only on the treated side, but in the same genetic background. In contrast, mutations in genes coding for proteins involved in cell adhesion, e.g. integrins, or in adhesion to the extra cellular matrix, cause a blistered wing phenotype. In the latter phenotype, the epidermal cells of the immature wings are not attached to their opposing cells or to the cuticle and the wing surfaces are separated during unfolding by the sudden influx of hemolymph. In contrast, in animals lacking wing hearts the wings resemble those of the wild-type shortly after unfolding. The epidermal cells also delaminate later from the cuticle, as indicated by their disarrayed pattern, but are not removed from the wings due to the missing hemolymph circulation and probably impede spatially the bonding of the dorsal and the ventral cuticle. Thus, the wings remain in their immature state and do not acquire aerodynamic properties, which accounts for the flightlessness. It is concluded that wing hearts are crucial for establishing proper wing morphology and functionality in Drosophila (Tögel, 2008).
Wing hearts occur in all winged insects, but differ considerably in their morphology. However, their function is highly conserved, since they all function as suction pumps that draw hemolymph from the wings. In the basal condition, the heart itself is directly connected to the scutellum and constitutes the pump. This connection was lost several times during evolution and other muscles, e.g. the separate wing hearts in Drosophila, were recruited to retain the function indicating a high selection pressure on wing circulation. It is suggested that this is due to the crucial role of wing hearts during wing maturation. Since proper wing morphogenesis is essential for flight ability, insect flight might not have been possible before the evolution of wing hearts (Tögel, 2008).
Patterning
Flight Behavior