pebbled/hindsight
Initial embryonic expression of Pebbled mRNA and protein occurs in the endoderm (midgut) and extraembryonic membrane (amnioserosa) prior to germ-band extension and continues in these tissues beyond the completion of germ-band retraction. HNT mRNA accumulates beginning at stage 5 in the cellular blastoderm, in the posterior-terminal midgut primordium and dorsally in the presumptive amnioserosa. Hnt protein appears in these cells slightly later (stage 6). During stage 7, dorsal expression expands to cover the entire presumptive amnioserosa from the cephalic furrow to the posterior midgut primordium. Anteroventral staining, corresponding to the anterior midgut primordium, is first detected at stage 8. Accumulation in these tissues continues as gastrulation continues. Commencing at stage 11, expression also occurs in the developing tracheal system, glial cells of the central and peripheral nervous systems, and the ureter of the Malpighian tubules. Strikingly, pebbled is not expressed in the epidermal ectoderm, which is the tissue that undergoes the cell shape changes and movements during germ-band retraction (Yip, 1997).
The pebbled gene is X-linked. Hemizygous (peb/Y) embryos fail to retract their germ bands. All such embryos have the correct number of thoracic and abdominal segments that are patterned normally. As a consequence of failed-germ-band retraction, the embryos are U-shaped with their posterior region folded into the dorsal side. Additionally, mutant embryos show defects in head involution and often have a severely disrupted cephalopharyngeal skeleton. A viable peb allele is named pebbled because of its rough eye phenotype. peb alleles can be ordered into a phenytypic series (Yip, 1997).
A group of genes, referred to as the U-shaped-group (ush-group), is required for maintenance of the amnioserosa tissue once it has differentiated. Using several molecular markers, amnioserosa development was examined in the ush-group mutants: u-shaped (ush), pebbled (peb), serpent (srp) and tail-up (tup). The amnioserosa in these mutants is specified correctly and begins to differentiate as in wild type. However, following germ-band extension, there is a premature loss of the amnioserosa. This cell loss is a consequence of programmed cell death (apoptosis) in ush, peb and srp, but not in tup. The ush-group genes are implicated in the maintainance of the amnioserosa's viability. In light of these mutants' unretracted phenotype, the amnioserosa could be involved in signal reception or the initiation of signal transduction with respect to the adjacent ectoderm (Frank, 1996).
Several approaches have been taken to study the relationships between previously identified mutations (u-shaped, serpent, pebbled and tailup) that selectively cause germband retraction defects in homozygous embryos, and a more pleiotropically acting locus, Egfr. The former four loci are elements of at least two parallel and partially redundant cellular pathways that affect germ band retraction by acting in amnioserosal development or maintenance. An additional discrete and unique pathway, represented by Egfr, is likely to function in the germband itself. While the role of the amnioserosa during germband retraction appears to be permissive, the action of Egfr in the germband may be mediated by the cytoskeleton (Goldman-Levi, 1996).
The longitudinal glia (LG), progeny of a single glioblast, form a scaffold that presages the formation of longitudinal tracts in the ventral nerve cord (VNC) of the Drosophila embryo. The LG are used as a substrate during the extension of the first axons of the longitudinal tract. The differentiation of the LG has been examined in six mutations in which the longitudinal tracts are absent, displaced, or interrupted to determine whether the axon tract malformations may be attributable to disruptions in the LG scaffold. Embryos mutant for the gene prospero have no longitudinal tracts, and glial differentiation remains arrested at a preaxonogenic state. Two mutants of the Polycomb group also lacked longitudinal tracts; here the glia fail to form an oriented scaffold, but cytological differentiation of the LG is unperturbed. The longitudinal tracts in embryos mutant for slit fuse at the VNC midline and scaffold formation is normal, except that it is medially displaced. Longitudinal tracts have intersegmental interruptions in embryos mutant for pebbled and midline. In pebbled, there are intersegmental gaps in the glial scaffold. In midline, the glial scaffold retracts after initial extension. LG morphogenesis during axonogenesis is abnormal in midline. Commitment to glial identity and glial differentiation also occurs before scaffold formation. In all mutants examined, the early distribution of the glycoprotein Neuroglian is perturbed. This is indicative of early alterations in VNC pattern present before LG scaffold formation begins. Therefore, some changes in scaffold formation may reflect changes in the placement and differentiation of other cells of the VNC. In all mutants, alterations in scaffold formation precedes longitudinal axon tract formation (Jacobs, 1993).
During animal development, morphogenesis of tissues and organs requires dynamic cell shape changes and movements that
are accomplished without loss of epithelial integrity. Data from vertebrate and invertebrate systems have implicated several
cell surface and cytoskeleton-associated molecules in the establishment and maintenance of epithelial architecture, but
there has been little analysis of the genetic regulatory hierarchies that control epithelial morphogenesis in specific tissues.
The Drosophila Hindsight nuclear zinc-finger protein is required during tracheal morphogenesis for the
maintenance of epithelial integrity and assembly of apical extracellular structures known as taenidia. In hindsight (hnt)
mutants, tracheal placodes form, invaginate, and undergo primary branching as well as early fusion events. However, starting at
midembryogenesis, the tracheal epithelium either collapses or expands to give rise to sacs of tissue. While a subset of hnt
mutant tracheal cells enters the apoptotic pathway, genetic suppression of apoptosis indicates that this is not the cause of
the epithelial defects. Surviving hnt mutant tracheal cells retain cell-cell junctions and a normal subcellular distribution
of apical markers such as Crumbs and DE-Cadherin. However, taenidia do not form on the lumenal surface of tracheal cells.
While loss of epithelial integrity is a common feature of crumbs, stardust, and hnt mutants, defective assembly of taenidia
is unique to hnt mutants. These data suggest that Hnt is a tissue-specific factor that regulates maintenance of the tracheal
epithelium as well as differentiation of taenidia (Wilk, 2000).
During embryogenesis hnt expression commences
in the amnioserosa and midgut at stage 5 and,
subsequently, initiates in a variety of additional cell types,
including the tracheal system. There is no
maternal contribution of HNT RNA or protein to the early
embryo. There is
no detectable accumulation of Hnt protein in the tracheal
placodes at stage 10. Hnt accumulates in the
nuclei of tracheal cells beginning at early stage 11 and
expression continues in all tracheal cells from stage 12
through the remainder of embryogenesis (Wilk, 2000).
The epithelial defects in hnt mutants seen in the amnioserosa and in the tracheal
system show striking similarities to other
Drosophila mutants that disrupt epithelial tissues, suggesting
that Hnt has a direct function in regulating the
maintenance of epithelial structure. Drosophila genes involved
in epithelial differentiation include shotgun (shg)
and crumbs (crb). shg encodes
DE-Cadherin, the major epithelial cadherin in Drosophila,
which is required for the formation and maintenance of
most epithelia in the embryo. crb encodes a
transmembrane protein that localizes to, and that is required
for, maintaining the apical cell surface in ectodermal
epithelia. Mutations in crb, shg, and other genes that affect epithelial
integrity (e.g., stardust) cause very similar terminal phenotypes.
When an epithelial tissue breaks down, a subset of
the epithelial cells loses polarity/structural integrity, enters
the programmed cell death pathway, and degenerates. However,
the majority of cells in such tissues survive until late
embryogenesis, show normal polarity and junctional differentiation,
and form small epithelial (often vesicular) units. In particular, analyses of crb and the phenotypically similar gene stardust (sdt) have revealed that the mechanisms involved in epithelial maintenance change during
development. crb and sdt are not needed for the formation of the blastoderm epithelium. During gastrulation both genes are required for
the formation of the zonula adherens. In crb and sdt
mutants the zonula adherens fails to assemble throughout
the ectoderm and amnioserosa, a defect that is soon followed
by the appearance of gross morphological abnormalities
in all ectodermal and amnioserosal cells. Surprisingly,
recovery of epithelial morphology, including delayed
differentiation of a junctional complex, is observed in crb
and sdt mutants during organogenesis. The extent of recovery
of epithelial morphology differs from tissue to tissue
and ranges from very little (e.g., epidermis) to almost
complete (e.g., hindgut). These findings
suggest that tissue-specific factors contribute to maintenance
of epithelial structure and can partially compensate
for loss of crb and sdt function. Hnt is likely to represent
such a tissue-specific factor required for epithelial differentiation
as suggested by its phenotype and its expression
pattern, which is limited to only a subset of embryonic
epithelia. Since Hnt is a zinc-finger nuclear protein that may
control gene expression, Hnt could modulate the expression
levels, or could alter the repertoire, of structural
components that are needed for epithelial differentiation.
Such components remain to be identified; Hnt does not appear to be essential for the expression of Crb and Shotgun (Wilk, 2000).
The basic structural organization of the tracheal tubes is
similar among insects, consisting of a simple monolayer
epithelium with a lamina on the basal (outer) surface
and cuticle apically (facing the lumen). The cuticle usually
contains regular folds known as taenidia. These are arranged
in a helical pattern and function to keep the tracheal
tube open without compromising flexibility. Tracheal cells of hnt embryos
secrete an epicuticle as well as other components of the
lumen. However, the size of the lumen is variable and
it is discontinuous. Moreover, the taenidial folds are absent
or are highly disorganized in hnt embryos. Since the tracheae
lose their integrity in hnt mutants prior to the
presence of morphologically identifiable taenidia, it is possible
that the absence of regular taenidial folds is a secondary
effect. Molecular components of the taenidia have not
yet been identified; thus it is not known exactly when the
taenidia begin to form during tracheal morphogenesis. Circumstantial
evidence suggests that the taenidia begin to form prior to completion of secretion of the outer epicuticle. It is thus possible that taenidial components are missing from an early stage in hnt mutants and that Hnt plays a direct role in taenidium formation independent of its role in maintenance of tracheal integrity. Consistent with this possibility, regular taenidial folds are present in other mutants, such as sdt and crb, in which the tracheal system disintegrates. Thus abnormal taenidial organization is not an obligatory consequence of loss of integrity of the tracheal epithelium (Wilk, 2000).
Together these results suggest that there are three roles for
Hnt during tracheal morphogenesis: (1) to preserve epithelial integrity; (2) to direct assembly of the extracellular
structures known as taenidia, and (3) to prevent apoptosis.
Since Hnt is likely to be a transcription factor, it is presumed
that Hnt resides in a genetic hierarchy that regulates or
coordinates these processes. In one model, Hnt might have
a tripartite role, independently regulating epithelial integrity,
directing taenidial assembly, and preventing programmed
cell death. In a second model, both
apoptosis and defective taenidial assembly may be indirect
consequences of defects in epithelial architecture.
A third model suggests that Hnt may independently regulate epithelial architecture
and assembly of taenidia, while apoptosis in hnt-mutant
tracheae might be an indirect consequence of defects
in the first of these processes (Wilk, 2000).
The leading edge (LE) is a single row of cells in the Drosophila embryonic epidermis that marks the boundary
between two fields of cells: the amnioserosa and the dorsal ectoderm. LE cells play a crucial role in the morphogenetic process of dorsal closure and eventually form the dorsal midline of the embryo. Mutations that block
LE differentiation result in a failure of dorsal closure and embryonic lethality. How LE cells are specified remains
unclear. To explore whether LE cells are specified in response to early dorsoventral patterning information or whether they arise secondarily, the extent of amnioserosa and dorsal ectoderm was altered genetically, and LE cell fate was assayed. No expansion of LE fate is observed in dorsalized or ventralized mutants. Furthermore, the LE fate arises as a single row of cells, wherever amnioserosa tissue and dorsal epidermis are physically juxtaposed. Taken together these data indicate that LE formation is a secondary consequence of early zygotic dorsal patterning signals. In particular, proper LE specification requires the function of genes such as u-shaped and hindsight, which are direct transcriptional targets of the early Decapentaplegic/Screw patterning gradient, to establish a competency zone from which LE arises. It is proposed that subsequent inductive signaling between amnioserosa and dorsal ectoderm restricts the formation of LE to a single row of cells (Stronach, 2001).
Using mutations that influence DV patterning, it is possible to alter the size and distribution of BMP target gene expression patterns, which indicate the extent of amnioserosa and dorsal ectodermal cell fates. If LE fate was specified directly by a particular threshold level of BMP signal, then one would expect LE fate to be perturbed in concert with amnioserosa and dorsal ectoderm fates in DV mutants. Mutations in genes such as dorsal, Toll, brinker and short gastrulation alter the size of BMP target gene expression domains; however, these mutants failed to alter specification of LE fate. Among these genotypes, brk and sog specifically modulate the shape of the BMP signaling gradient in a region where LE fate might arise, yet LE formation in these mutants is fundamentally normal. Furthermore, in dorsalized embryos, LE cells were observed regularly at the boundary between amnioserosa and dorsal ectoderm even when the morphology of these tissues was severely disrupted. Islands of amnioserosa cells within a field of ectoderm were consistently surrounded with a single row of LE cells, independent of the number of amnioserosa cells constituting the island. The converse situation also occurred; again, a single row of LE cells formed at the boundary between the ectoderm and amnioserosa (Stronach, 2001).
DV mutants were also analyzed to determine whether a decrease in BMP signaling activity converts amnioserosa to LE as predicted by a gradient patterning model. A range of ventralizing mutations (cactus, sog, screw, dpp) displaying progressive loss of amnioserosa tissue did not give rise to embryos with an expanded domain of LE cells. In fact, LE cells were not detected in the absence of amnioserosa. No situation was found in which an altered BMP gradient was associated with expanded LE fate, thus the prediction of a direct gradient response model does not explain LE fate specification (Stronach, 2001).
Notably, DV mutant embryos that perturb the BMP gradient, also perturb the expression domains of target genes, including u-shaped and hindsight, without altering LE specification. However, loss of ush and hnt function results in specific and distinct perturbations in LE formation. Thus, the interpretation that LE fate specification is not a direct early response to the BMP gradient is favored, but rather is a secondary consequence of the specification of dorsal fates through the action of BMP target genes like ush and hnt (Stronach, 2001).
Taken together, these results raise the possibility that amnioserosa may be required for LE formation. To address the function of amnioserosa for LE specification, puc enhancer expression was examined in several mutants of the U-shaped class, including u-shaped (ush) and hindsight (hnt). Incidentally, the dorsal expression domains of these genes are directly regulated by DV patterning signals. In these mutant embryos, the amnioserosa tissue is fated normally and begins to differentiate up to stage 11, but then degenerates prematurely. In both ush and hnt mutants, programmed cell death takes place over the course of a few hours, with elimination of amnioserosa cells by stage 13 -- the time when dorsal closure would normally commence. Unexpectedly, different patterns of expression were observed with the puc enhancer in the two mutants. In ush embryos, ß-gal-positive cells were not detected. In contrast, hnt mutant embryos displayed Puc-positive LE cells at the edge of the dorsal ectoderm, albeit with less uniform expression than normally observed. To confirm these observations, the accumulation of dpp mRNA in the LE was examined. Similar to puc enhancer expression, differential expression of dpp was observed in ush versus hnt mutant embryos. ush mutant embryos show a consistent and significant reduction in LE dpp expression, although residual dpp transcripts are seen. dpp expression appears relatively normal in hnt mutant embryos (Stronach, 2001).
In addition to the differential expression of two LE markers in the U-shaped mutants, ectopic expression of LE markers is observed only in hnt mutant embryos. ß-gal-positive cells were observed in the region of the amnioserosa in hnt mutants as early as stage 11, raising the possibility that this could be an example of expanded LE cell fates. These cells adopt only partial LE cell fate, for the following reasons. These cells do not express the LE marker Fasciclin III, but do express two other LE molecules, albeit aberrantly. puc, for example, is expressed precociously in these cells, preceding Fasciclin III expression in the ectoderm, and dpp is rarely but reproducibly expressed. Additionally, cells in this region express amnioserosa fate markers such as race, through stage 11. Thus, based on the possibility that these cells may co-express LE and amnioserosa markers during stage 11, their identity cannot be unequivocally determined. These results may indicate that these cells are of mixed fate. The presence of ectopic LE-like cells in hnt mutant embryos, coupled with the severe reduction of LE fate markers in ush mutants, suggest that the distinction between amnioserosa and LE is a secondary consequence of Hnt and Ush functions, not a direct result of specific BMP signaling thresholds (Stronach, 2001).
If LE cells are specified as a secondary consequence of DV patterning gradients, then what additional mechanisms are at work to define LE as a single row of cells? The data are consistent with several mechanisms. One possibility is that specification of the LE involves the combinatorial action of nested sets of transcriptional regulators, including Hnt dorsally and Ush in a broader domain. Accordingly, loss of Hnt function is predicted to result in a failure to differentiate amnioserosa, coupled with dorsal expansion of more lateral fates, such as the LE. Consistent with this model, hnt mutant embryos display Puc-positive cells with partial LE character in the region of the dying amnioserosa during stage 11. These results suggest that Hnt may be necessary to distinguish amnioserosa from LE fate at the time of extended germ band stage. This timing is late, relative to the timing of the early BMP threshold response, further supporting the notion that LE specification is a secondary consequence of initial BMP signaling (Stronach, 2001).
Ush may play a role in differentiation of more lateral fates adjacent to the amnioserosa and the Hnt expression domain. Indeed, Ush function is essential for LE development because LE does not form in ush mutant embryos. Based on these results, it is imagined that Ush could define a competency zone from which LE cells arise, or Ush could participate in generating or modulating a signal(s) for communication between the differentiating amnioserosa and dorsal ectoderm. Ush is related to mammalian zinc-finger protein family, Friend of GATA (FOG), which has been shown to participate as a cofactor with GATA transcription factors. Together, these protein complexes regulate cell fate determination multiple times during both mammalian and Drosophila development. Interestingly, FOG2, a mammalian homolog of Ush, appears to be required during an inductive signaling event between two distinct tissues in the mouse heart, suggesting that inductive processes in development may commonly use the function of Ush family members. It has not been determined whether the function of Ush in LE cell specification is localized to the amnioserosa, the dorsal ectoderm, or both. Experiments to replace Ush function in a tissue-specific manner should address this issue (Stronach, 2001).
Although transcriptional targets of BMP signaling, such as ush and hnt, among others, define at least three specific threshold responses, the size difference between the nested expression domains of these markers still fails to account for a cell fate defined by a single row of cells. An additional mechanism to explain the spatially restricted stripe of LE cells is through an inductive signaling event. From the analysis of dorsalized mutants, it is observed that LE forms as a result of the juxtaposition of amnioserosa tissue with dorsal ectoderm, which may provide spatially limited activation of the JNK pathway. Thus, restricted expression of JNK target genes, such as puc and dpp may be a direct result of a signal that specifies LE (Stronach, 2001).
Communication between the amnioserosa and the dorsal ectoderm during embryogenesis has been suggested in two cases recently: (1) Hnt expression in the amnioserosa is required nonautonomously for proper cell rearrangements in the dorsal ectoderm, associated with retraction of the embryonic germband; (2) the raw gene product appears to be expressed in the amnioserosa, though it influences the activity of the JNK pathway in the ectoderm during dorsal closure. As amnioserosa and ectoderm develop, they may acquire different cell affinities, which cause them to sort into separate domains or islands (in the case of dorsalized embryos), displaying smooth borders at their interface. A difference in cell adhesion at the boundary may be sufficient to generate signaling for LE specification similar to inductive mechanisms at work at the compartmental boundaries of larval imaginal discs. The challenge now will be to identify molecules that may participate in an inductive signal (Stronach, 2001).
These results suggest that a multistep process determines the LE as a single row of cells. LE does not form directly in response to discrete intermediate levels of BMP signaling activity, but forms secondarily by the action of transcriptional regulators that are themselves BMP target genes. Among these targets, Hnt and Ush define a LE competency zone that is expanded in hnt mutants and eliminated in ush mutants. It is proposed that from within the competency zone, LE fate is further refined to a single row by an unknown inductive signal generated by the physical juxtaposition of amnioserosa with dorsal ectoderm. This signal activates the JNK pathway that regulates localized expression of dpp and puc (Stronach, 2001).
The hindsight gene regulates cell morphology, cell fate specification, planar cell polarity and epithelial integrity during Drosophila retinal development. In the third instar larval eye imaginal disc, Hnt protein expression begins in the morphogenetic furrow and is refined to cells in the developing photoreceptor cell clusters just before their determination as neurons. In hnt mutant larval eye tissue, furrow markers persist abnormally, posterior to the furrow; there is a delay in specification of preclusters as cells exit the furrow; there are morphological defects in the preclusters, and recruitment of cells into specific R cell fates often does not occur. Additionally, genetically mosaic ommatidia with one or more hnt mutant outer photoreceptor cell, have planar polarity defects that include achirality, reversed chirality and misrotation. Mutants in the JNK pathway act as dominant suppressors of the hnt planar polarity phenotype, suggesting that Hnt functions to downregulate JUN kinase (JNK) signaling during the establishment of ommatidial planar polarity. Hnt expression continues in the photoreceptor cells of the pupal retina. When an ommatidium contains four or more hnt mutant photoreceptor cells, both genetically mutant and genetically wild-type photoreceptor cells fall out of the retinal epithelium, indicating a role for Hnt in maintenance of epithelial integrity. In the late pupal stages, Hnt regulates the morphogenesis of rhabdomeres within individual photoreceptor cells and the separation of the rhabdomeres of adjacent photoreceptor cells. Apical F-actin is depleted in hnt mutant photoreceptor cells before the observed defects in cellular morphogenesis and epithelial integrity. The analyses presented here, together with previous studies in the embryonic amnioserosa and tracheal system, show that during development Hnt has a general role in regulation of the F-actin-based cytoskeleton, JNK signaling, cell morphology and epithelial integrity (Pickup, 2002).
The earliest defects observed in hnt mutant eye tissue occur during the formation of the definitive five-cell ommatidial preclusters. Specifically, there is a 4 hour delay in specification of the ommatidial preclusters relative to wild type, the shape of individual R cells within these preclusters is often abnormal, and the relative orientation of the presumptive R cells within a precluster is disrupted. There are two plausible explanations for the observed defects: a failure in cell cycle synchronization or abnormal cellular morphology. (1) It has been shown that lack of cell cycle synchronization in the furrow can lead to a one to two column delay in R cell precursor determination (e.g. in roughex mutants). Furthermore, a precluster phenotype in which nuclei are inappropriately positioned in the apicobasal plane relative to one another, is seen in mutants disrupted for cell cycle synchronization in the furrow (e.g. thick vein, saxophone, schnurri). Thus, it is possible that failure of cell cycle synchronization underlies the hnt mutant phenotypes described here. (2) Alternatively, the primary defect in hnt mutants might be the abnormal morphology of the R cells. For example, the act up mutant, which alters the apical morphology of the precluster cells, shows premature dpp expression, premature R cell differentiation and uncoordinated neuronal determination. dpp expression persists posterior to the furrow in eye discs of hypomorphic hnt mutants. This phenotype is distinct from that described for act up or any other mutant. It is consistent, however, with an analyses demonstrating a role for Hnt in downregulation of dpp expression in the amnioserosa (Pickup, 2002).
Results obtained from mosaic analysis of hnt alleles clearly demonstrate that Hnt function is necessary in all of the outer R cells in the developing eye disc, particularly in the R3, R4 and R6 precursor cells, for correct planar polarity. A role for the R3 and R4 precursor cells in establishing planar polarity in the eye has been proposed on the basis of analyses of the JNK pathway in this process. Those experiments could not rule out a role for other outer R precursor cells, which also express the JUN transcription factor. hindsight must now be added to the list of genes, such as strabismus, that regulate planar polarity and have a clearly described role in all of the outer R cells (Pickup, 2002).
The fact that all of the symmetrical ommatidia along the borders of hnt clones are of the R3/R3 conformation suggests that Hnt function is necessary for correct R4 fate and orientation. It has been suggested that, owing to its closer proximity to the polarizing signal from the equator, a stronger activation of the JNK pathway occurs in the R3 precursor cell. Activated JUN would then be responsible for the upregulation of the target gene, Delta, in the R3 precursor cell relative to the R4 precursor cell. Since results in the eye and results in the embryo imply that Hnt is necessary for downregulating JNK function, it is proposed that the wild-type function of Hnt is to downregulate JNK activity in the R4 precursor cell. Such downregulation would enhance JNK signaling differences between the R3 and R4 cells. In the absence of the Hnt gene product, JNK signaling would be inappropriately elevated in the R4 precursor cell, thereby upregulating the transcription of JNK targets such as Delta, leading that cell to behave more like an R3 precursor cell. Consistent with this model, it has been found that Delta hypomorphs act as enhancers of the hntpeb rough eye phenotype. R3/R3 symmetric clusters are observed both when the R4 cell is mutant for hnt and the R3 precursor is hnt+, and when the R3 cell is mutant for hnt and the R4 precursor is Hnt+. In the latter case, the above model would lead one to expect normal R3/R4 clusters. Since only R3/R3 clusters are observed, it is speculated that Hnt can affect the R4 precursor cell when expressed only in the neighboring R3 precursor cell (i.e. that there may be some communication feedback between these cells leading to local non-autonomy of the hnt phenotype) (Pickup, 2002).
The early morphological and fate determination defects and the later planar polarity defects seen in hnt eyes may be causally connected. Specification of the early outer R cell precursors (R2, R5, R3 and R4) is often disrupted in hnt tissue. It has been established that early disruption of R3 and R4 fate can perturb planar polarity (e.g. in the seven-up mutant. This result suggests that accurate interpretation of extrinsic polarity signals may require each R cell to already be properly determined. It has also been shown that the relative positioning of outer R cells within a precluster is frequently deviant in hnt mutants before precluster rotation. Therefore the relative distance to the equator, or to a neighboring cluster, may also be crucial for differential reading of the polarity signal by pairs of outer cells within the cluster (R3 versus R4, R2 versus R5) (Pickup, 2002).
Hnt function in the amnioserosa is required for the assembly and/or maintenance of focal complexes in adjacent epithelial cells along the leading edge of the dorsal ectoderm. These F-actin rich structures, which also accumulate high levels of anti-phosphotyrosine
monoclonal antibody reactive proteins, are required for morphogenetic events during normal dorsal closure. In wild-type eye discs, F-actin is enriched at the apical tips of presumptive R cells in ommatidial preclusters. This apical F-actin forms part of a tightly localized signaling complex enriched for receptor and ligand molecules such as Sevenless, Boss, Notch and Delta. In late larval eye discs, hnt mutant R cells have reduced F-actin at their apical tips. Furthermore, although the R8 precursor cell is correctly determined in hnt mutant patches, the Boss ligand is less concentrated at the apical tips of some of the hnt R8 cells. The F-actin phenotype seen in hnt patches in larval eye discs occurs late and is therefore probably not a direct cause of disruption of the apical signaling complex in the eye disc. However, the defects in F-actin accumulation may be a marker indicative of perturbed cytoarchitecture in the apical region of the mutant R cell. Such a perturbation would have serious consequences for correct R cell specification, which requires intimate contacts between adjacent R cells for proper intercellular signaling (Pickup, 2002).
In the pupal retina, the concentration of F-actin in the apical tips of the R cell clusters is depleted in hnt mutant eye tissue. Still later, less F-actin accumulates in the extended rhabdomeres. F-actin and associated proteins in the apical surfaces of the photoreceptors play a key role in the initiation of rhabdomere morphogenesis. In addition, F actin is a component of the rhabdomere terminal web, a structure that anchors the rhabdomere membranes along the length of the differentiating R cell and prevents them from collapsing into the R-cell cytoplasm. When the function of Drac1, which regulates F-actin arrangement, is disrupted, the rhabdomere membranes 'spool out' into the cytoplasm. Interestingly, in addition to its role in rhabdomere morphogenesis, Drac1 is thought to signal through the JNK cascade and, like Hnt, is necessary earlier on for the establishment of planar polarity in the eye and for dorsal closure of the embryo. The pupal photoreceptor phenotype observed in hnt R cells, where rhabdomeres do not extend fully in the apicobasal axis, is qualitatively different from that seen for a dominant negative Drac1 allele (Pickup, 2002).
It is speculated that the depletion of F-actin in hnt mutant R cells may affect morphogenetic events that precede rhabdomere terminal web maturation. For example, there may be defects in extension of the specialized membrane down the length of the R cell or in closure of cone cells over the highly constricted R cell apices. The morphogenetic parallels between closure of the leading edge epidermal cells over the constricted amnioserosa of the embryo and closure of the cone cells over the constricted R cell apices in the pupal eye are striking. Furthermore, both require assembly and function of F-actin-rich complexes at the boundary between the two cell types involved (Pickup, 2002).
hnt mutant R cells are unable to maintain their integrity within the retinal epithelium during retinal differentiation and morphogenesis in pupal discs. By analogy, the hnt phenotype in the tracheal system is first seen at stage 14, when the overtly normal tracheal epithelium begins to disintegrate, and in so doing forms sacs and vesicles from the collapsed dorsal trunk and branches. In hnt hypomorphs, a specific proportion of the cells in the amnioserosa fall out of the epithelium during dorsal closure. Thus, Hnt function is required for maintenance of the integrity of the epithelia in which it is expressed (Pickup, 2002).
In the eye, loss of integrity occurs in clusters containing fewer than five out of eight Hnt-expressing R cells. It is speculated that delamination of clusters may occur because they lack a threshold level of apical F-actin required for inter-photoreceptor communication and/or adhesion. For example, if hnt mutant R cells fail to form focal contacts with the overlying cone cells, a genetically mosaic R cell cluster could slip basally, eventually falling out of the epithelium before consolidation of the fenestrated membrane during the pupal period (Pickup, 2002).
In the eye, patches of tissue mutant for certain of the integrins (myospheroid and inflated) have missing R cells or R cells with shortened rhabdomeres. Detailed studies have shown that integrins are expressed in the cone and pigment cells and that the mutant phenotypes may trace their origin to a structural defect in the cone cell plate at the retinal floor. Hnt is not expressed in cone or pigment cells and there is no gross defect in the retinal floor beneath hnt mutant eye tissue. However, an F-actin defect is observed and extracellular engagement of adhesion molecules is known to physically links F-actin bundles with the cell surface to provide structural integrity; further studies to examine possible requirements of Hnt for extracellular matrix production or function may be revealing (Pickup, 2002).
These results clearly implicate Hnt in regulation of several types of cellular events that are common to the different contexts in which Hnt functions. These include establishment or maintenance of the morphology of individual cells within an epithelium, as well as maintenance of the integrity of the epithelium per se. There are also shared molecular correlates of these Hnt functions. In particular, Hnt is required for establishment of localized F-actin- and phosphotyrosine-rich complexes in the leading edge epidermal cells, as well as in the photoreceptor cells. Hnt functions to regulate two JNK signaling dependent processes (planar polarity in the eye and dorsal closure of the embryo), possibly by downregulating JNK signaling in time and space. A fuller understanding of whether the functions of Hnt in different tissues and at different times during development derive from control of the same molecular pathway will await genetic and molecular analyses of the genes regulated by Hnt (Pickup, 2002).
The nuclear zinc-finger protein encoded by the hindsight (hnt)
locus regulates several cellular processes in Drosophila epithelia, including
the Jun N-terminal kinase (JNK) signaling pathway and actin polymerization.
Defects in these molecular pathways may underlie the abnormal cellular
interactions, loss of epithelial integrity, and apoptosis that occurs in
hnt mutants, in turn causing failure of morphogenetic processes such as
germ band retraction and dorsal closure in the embryo. To define the genetic
pathways regulated by hnt, 124 deficiencies on the second and third
chromosomes and 14 duplications on the second chromosome were assayed for
dose-sensitive modification of a temperature-sensitive rough eye phenotype
caused by the viable allele, hntpeb; 29 interacting regions
were identified. Subsequently, 438 P-element-induced lethal mutations
mapping to these regions and 12 candidate genes were tested for genetic
interaction, leading to identification of 63 dominant modifier loci. A subset of
the identified mutants also dominantly modify hnt308-induced
embryonic lethality and thus represent general rather than tissue-specific
interactors. General interactors include loci encoding transcription factors,
actin-binding proteins, signal transduction proteins, and components of the
extracellular matrix. Expression of several interactors was assessed in
hnt mutant tissue. Five genes -- apontic (apt),
Delta (Dl), decapentaplegic (dpp), karst
(kst), and puckered (puc) -- regulate tissue
autonomously and, thus, may be direct transcriptional targets of Hnt. Three of
these genes -- apt, Dl, and dpp -- are also regulated
nonautonomously in adjacent non-Hnt-expressing tissues. The expression of
several additional interactors -- viking (vkg), Cg25, and
laminin-alpha (LanA) -- is affected only in a nonautonomous manner (Wilk, 2004).
Cell movements represent a major driving force in embryonic development, tissue repair, and tumor metastasis. The migration of single cells has been well studied, predominantly in cell culture; however, in vivo, a greater variety of modes of cell movement occur, including the movements of cells in clusters, strands, sheets, and tubes, also known as collective cell migrations. In spite of the relevance of these types of movements in both normal and pathological conditions, the molecular mechanisms that control them remain predominantly unknown. Epithelial follicle cells of the Drosophila ovary undergo several dynamic morphological changes, providing a genetically tractable model. This study found that anterior follicle cells, including border cells, mutant for the gene hindsight (hnt) accumulated excess cell-cell adhesion molecules and failed to undergo their normal collective movements. In addition, HNT affected border cell cluster cohesion and motility via effects on the JNK and STAT pathways, respectively. Interestingly, reduction of expression of the mammalian homolog of HNT, RREB1, by siRNA inhibited collective cell migration in a scratch-wound healing assay of MCF10A mammary epithelial cells, suppressed surface activity, retarded cell spreading after plating, and led to the formation of immobile, tightly adherent cell colonies. It is proposed that HNT and RREB1 are essential to reduce cell-cell adhesion when epithelial cells within an interconnected group undergo dynamic changes in cell shape (Melani, 2008).
To explore the mechanisms by which HNT affects cluster cohesion and motility, its effects on known signaling pathways were investigated. In the extraembryonic tissue known as the amnioserosa, hnt is a negative regulator of the JNK signaling cascade. Recently, the JNK pathway was shown to be active in the border cells and to affect border cell migration in clusters with reduced PVR activity. In addition, inhibition of the JNK cascade causes a phenotype that strikingly resembles the cluster dissociation phenotype caused by HNT overexpression, suggesting that HNT could be a negative regulator of the JNK pathway or vice versa. By using phospho-Jun antibody staining as a readout of the JNK signaling cascade, the activity of this pathway was seen to be reduced in border cells overexpressing hnt. In clusters in which JNK was reduced by overexpression of either Puckered (the JNK phosphatase) or a dominant-negative form of Basket (Drosophila JNK), cluster disassembly reminiscent of the hnt gain-of-function phenotype was observed. In addition, HNT was upregulated 1.7- and 1.4-fold, respectively. Together, these results indicate that hnt and JNK repress each other. In the embryo, in which HNT also antagonizes JNK, this pathway is required for the turnover of focal complexes and proper dorsal closure. Therefore, HNT appears to play a general role in remodeling of adhesion complexes to facilitate morphogenesis (Melani, 2008).
Although the cluster-disassembly phenotype of HNT could be attributed to effects on JNK signaling, JNK pathway mutations caused milder border cell motility defects than hnt. To determine whether HNT affected, in addition, one of the known border-cell-motility pathways, the effect of hnt on the activity of STAT and its key target SLBO was examined. STAT activation and nuclear translocation is the most upstream event in the differentiation of the border cells and is also required throughout border cell migration. It was found that, in border cells overexpressing HNT, nuclear accumulation of STAT was reduced though not eliminated. In addition, the levels of slbo were dramatically reduced in border cells overexpressing HNT. Because loss of function of either STAT or SLBO causes a dramatic migration defect, the effects of HNT overexpression on STAT and SLBO can account for the severe effect on motility. However, neither stat nor slbo mutant border cells exhibit a cluster-disassembly phenotype. Therefore, it is concluded that HNT mediates its effect on cluster cohesion via JNK and its effect on border cell motility primarily through STAT and SLBO (Melani, 2008).
Although HNT overexpression affects border cell motility via effects on STAT and SLBO, HNT has general effects on cell adhesion and morphogenesis, whereas SLBO appears to be more specific. For example, the effects of hnt on stretched follicle cells and in embryonic tissues are independent of SLBO because this protein is neither expressed nor required in these other cell types. Therefore, it is proposed that HNT plays a general role in regulating cell adhesion and morphogenesis via JNK signaling and a tissue-specific role in motility through STAT and SLBO. In this way, HNT can cooperate with tissue-specific factors to orchestrate a diverse array of collective cell movements (Melani, 2008).
The induction of cone cells in the Drosophila larval eye disc by the determined R1/R6 photoreceptor precursor cells requires integration of the Delta-Notch and EGF receptor signaling pathways with the activity of the Lozenge transcription factor. This study demonstrates that the zinc-finger transcription factor Hindsight (Hnt) is required for normal cone-cell induction. R-cells in which hindsight levels are knocked down using RNAi show normal subtype specification, but these cells have lower levels of the Notch ligand Delta. HNT functions in the determined R1/R6 precursor cells to allow Delta transcription to reach high enough levels at the right time to induce the cone-cell determinants Prospero and D-Pax2 in neighboring cells. The Delta signal emanating from the R1/R6 precursor cells is also required to specify the R7 precursor cell by repressing seven-up. As hindsight mutants have normal R7 cell-fate determination, it is inferred that there is a lower threshold of Delta required for R7 specification than for cone-cell induction (Pickup, 2009).
This study shows that Hnt function is necessary to elevate the Dl ligand
in the R1/R6 precursor cells to a level high enough to achieve cone-cell
induction. Notably, Hnt is not an on/off switch for Dl expression;
rather it potentiates the level of Dl transcription in the R1/R6
precursor cells. The data suggest that this modulation is likely to be
independent of Chn, which is itself a transcriptional repressor of Dl. Although this paper does not show that this Hnt effect is due to direct action, the exact sequence for two Hnt binding sites was found in the upstream and
intronic sequences of the Delta transcription unit (Pickup, 2009).
Earlier reports describing Hnt function in the ovary show that Hnt
expression is regulated by the Notch signaling pathway and controls follicle
cell proliferation and differentiation. This
paper reports that Hnt acts upstream of Notch activation by regulating Dl
ligand expression levels. These two modes of regulation are not necessarily
mutually exclusive, but it is not thought that Notch activates the hnt
gene in the eye. (1) Hnt is expressed in all the R-cell precursors in the
eye, whereas the Notch pathway is activated at high levels only in a subset of
these precursors, as well as in the accessory cone and pigment cell
precursors, where Hnt is not expressed at all. (2) When Notch activity is attenuated by using the Nts mutant, Hnt expression in the furrow expands to all cells that now acquire a neuronal fate. This result cannot be interpreted as a simple repression of
Hnt expression by Notch activation in non-neuronal cells, as Hnt expression is
not complementary to Notch activation in the eye disc. (3) Notch activation cannot be sufficient to induce Hnt expression in the eye disc, since no expansion of Hnt expression into adjacent, non-determined cells is seen when Dl is ectopically expressed early in the cone-cell precursors (with the lz-Gal4 driver). (4) It was shown that the expression of Dl in the R-cell precursors is partly dependent on Hnt function. Others have clearly demonstrated that this late Dl expression does not require Notch activity, since it is unaffected in a Nts1 mutant (Pickup, 2009).
The two-signal model of R7 fate hypothesizes that R7 determination requires
a strong RTK signal (achieved by the additive effects of Sevenless and EGFR
activation) together with Notch activation. These signals are necessary to activate pros and repress svp expression, respectively. Since the cone-cell precursor cells do not contact the determined R8 cell at the appropriate time, they will not 'see' the SEV ligand BOSS. Cone cell precursors, then, will not ordinarily activate their Sev receptors. In this model, different fates have been reinforced in the R7/cone equivalence group by adding a second, activating ligand for EGFR (Pickup, 2009).
This paper suggests a further level of complexity. It was shown, by
manipulating the level of Dl in the R1/R6 signaling cells, that
activation of the key players in cone-cell determination requires high levels
of the Notch activation in the cone-cell precursor cell. Several lines of
evidence support the idea that the level of the Dl ligand is translated into
cell-fate differences in a responding R precursor cell. Since there is low Dl
expression in the R7 precursor cell and only late expression of Dl in the
cone-cell precursor cell, the adjacent R1/R6 precursor cells never activate their
Notch receptors. Both the R7 precursor and the cone-cell precursor cells
receive their ligand signal from the R1/R6 precursor cells. In this hypothesis, the R7 precursor cell requires only a low level of ligand signal to activate the R7-like program: turning on pros and off svp (Pickup, 2009).
It is suggested that the cone-cell precursor requires a high level of ligand
signal to activate the cone-cell program. Expressing a dominant-negative form
of Dl in the R1/R6 signaling cells prevents cone-cell, but not R7-cell,
determination. Since both the cone and R7 precursor cells receive their Dl input from the same R1/R6 cells, it is possible that an intrinsic feature of the R7 precursor cell - possibly the high RTK activation - antagonizes N signaling, so that D-Pax2 transcription does not occur in that cell. The transcriptional repressor, Lola, may also be involved in this distinction, since it is known to bias precursor cells towards R7-over cone-cell fate (Pickup, 2009).
Although a role for Notch signaling in cone-cell induction has been shown
to be necessary for D-Pax2 expression, it has
not been directly demonstrated as necessary for pros regulation in
cone cells. The experiments presented in this study suggest that high levels of Notch signaling may indirectly or directly be required for Pros expression in the cone-precursor cells. This requirement is independent of the role of SU(H) in inducing D-Pax2, since there are normal levels of Pros in the cone-cell
precursors of a D-Pax2 null mutant.
Ectopically activating the Notch pathway in the R1/R6 precursor cells
occasionally induces ectopic Pros (but eliminates ELAV) in these cells.
Although this effect on Pros expression may be a secondary result of a
cell-fate transformation, it could also be interpreted as a more direct effect
of Notch signaling on pros transcription. In a different context,
Pros expression has been shown to be affected by Dl-activated Notch signaling
in a subset of glial cells in the embryonic CNS (Pickup, 2009).
Why would there be two Dl thresholds for different cell fates? There is
some preliminary work that suggests different mechanisms for Notch-activated
transcriptional readout in the responding cell, depending on the level of
signal received. In the cone-cell equivalence group, the cone-cell
determination pathway requires that D-PAX2 and Pros be expressed. It is
hypothesized that D-Pax2 may require a higher level of Notch
activation than Pros, which is also required for R7 determination.
These experiments indicate that there may be coordinated regulation of both
D-Pax2 and Pros expression in the cone cells. It is
postulated that the mechanism of Pros-gene induction in the cone cells
is different from pros regulation in R7. By potentiating the level of
Dl gene expression in the R1/R6 signaling cells, it is possible to
overlay the cone-cell fate over the transcriptional module necessary for
R7-cell fate. This simple change has, thus, allowed for the elaboration of
very different cell fates from the same equivalence group (Pickup, 2009).
Ashe, H. L., Mannervik, M. and Levine, M. (2000). Dpp signaling thresholds in the dorsal ectoderm of the Drosophila embryo. Development 127: 3305-3312. PubMed Citation: 10887086
Boube, M., Llimargas, M. and Casanova, J. (2000). Cross-regulatory interactions among tracheal genes support a co-operative model for the induction of tracheal fates in the Drosophila embryo. Mech. Dev. 91: 271-278. PubMed Citation: 10704851
Domanitskaya, E. and Schupbach, T. (2012). CoREST acts as a positive regulator of Notch signaling in the follicle cells of Drosophila melanogaster. J Cell Sci 125: 399-410. Pubmed: 22331351
Farley, J. E., Burdett, T. C., Barria, R., Neukomm, L. J., Kenna, K. P., Landers, J. E. and Freeman, M. R. (2018). Transcription factor Pebbled/RREB1 regulates injury-induced axon degeneration. Proc Natl Acad Sci U S A. PubMed ID: 29295933
Frank, L. H. and Rushlow, C. (1996). A group of genes required for maintenance of the amnioserosa tissue in Drosophila. Development 122 (5): 1343-1352. PubMed Citation: 8625823
Goldman-Levi, R., et al. (1996). Cellular pathways acting along the germband and in the amnioserosa may participate in germband retraction of the Drosophila melanogaster embryo. Int J Dev Biol 40 (5): 1043-1051. PubMed Citation: 8946251
Jacobs, J. R. (1993). Perturbed glial scaffold formation precedes axon tract malformation in Drosophila mutants. J Neurobiol 24 (5): 611-626. PubMed Citation: 8326301
Koelzer, S. and Klein, T. (2003). A Notch-independent function of Suppressor of Hairless during the development of the bristle sensory organ precursor cell of Drosophila. Development 130: 1973-1988. 12642500
Lamka, M. L. and Lipshitz, H. D. (1999). Role of the amnioserosa in germ band retraction of the Drosophila melanogaster embryo. Dev. Biol. 214(1): 102-12. PubMed Citation:
Martinez Arias, A. (1993). Development and patterning of the larval epidermis of Drosophila. In The Development of Drosophila melanogaster (ed. M. Bate and M. Martinez Arias). pp. 510-608. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
Melani, M., Simpson, K. J., Brugge, J. S. and Montell, D. (2008). Regulation of cell adhesion and collective cell migration by hindsight and its human homolog RREB1. Curr. Biol. 18(7): 532-7. PubMed Citation: 18394891
Ming, L., Wilk, R., Reed, B. H. and Lipshitz, H. D. (2013). Drosophila Hindsight and mammalian RREB-1 are evolutionarily conserved DNA-binding transcriptional attenuators. Differentiation 86(4-5): 159-70. PubMed ID: 24418439
Pickup, A. T., et al. (2002). Control of photoreceptor cell morphology, planar polarity and epithelial integrity during Drosophila eye development. Development 129: 2247-2258. 11959832
Pickup, A. T., Ming, L. and Lipshitz, H. D. (2009). Hindsight modulates Delta expression during Drosophila cone cell induction. Development 136(6): 975-82. PubMed Citation: 19234063
Reed, B. H., Wilk, R. and Lipshitz, H. B. (2001). Downregulation of Jun kinase signaling in the amnioserosa is essential for dorsal closure of the Drosophila embryo. Cur. Bio. 11: 1098-1108. 11269501
Stronach, B. E. and Perrimon, N. (2001). Investigation of leading edge formation at the interface of amnioserosa and dorsal ectoderm in the Drosophila embryo. Development 128: 2905-2913. 11532914
Sun, J. and Deng, W.-M. (2007). Hindsight mediates the role of Notch in suppressing Hedgehog signaling and cell proliferation. Dev. Cell 12: 431-442. Medline abstract: 17336908
Wilk, R., et al. (2000). The hindsight gene is required for epithelial
maintenance and differentiation of the
tracheal system in Drosophila. Dev. Biol. 219: 183-196. Medline abstract: 10694415
Wilk, R., Pickup, A. T., Hamilton, J. K., Reed, B. H. and Lipshitz, H. D. (2004).
Dose-sensitive autosomal modifiers identify candidate genes for tissue autonomous and
tissue nonautonomous regulation by the Drosophila nuclear zinc-finger
protein, Hindsight. Genetics 168(1): 281-300. 15454543
Yip, M.L, Lamka, M. L. and Lipshitz, H. D. (1997). Control of germ-band retraction in Drosophila by the zinc-finger protein HINDSIGHT. Development 124 (11): 2129-2141. PubMed Citation: 9187140
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Biological Overview
| Regulation
| Developmental Biology
| Effects of Mutation
date revised: 20 April 2021
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