hedgehog
Hedgehog (Hh) is a secreted morphogen, involved in both short and long range signaling necessary for tissue patterning during development. It is unclear how this dually lipidated protein is transported over a long range in the aqueous milieu of interstitial spaces. Previous work has shown that the long range signaling of Hh requires its oligomerization. This study shows that Hh is secreted in the form of exovesicles. These are derived by the endocytic delivery of cell surface Hh to multi vesicular bodies (MVBs) via an endosomal sorting complex required for transport (ECSRT: see Hrs)-dependent process. Perturbations of ESCRT proteins have a selective effect on long-range Hh signaling in Drosophila wing imaginal discs. Importantly oligomerization-defective Hh is inefficiently incorporated into exovesicles due to its poor endocytic delivery to MVBs. These results provide evidence that nanoscale organization of Hh regulates the secretion of Hh on ESCRT-derived exovesicles, which in turn act as a vehicle for long range signaling (Parchure, 2015).
The patterning activity of a morphogen depends on secretion and dispersal mechanisms that shape its distribution to the cells of a receptive field. In the case of the protein Hedgehog (Hh), these mechanisms of secretion and transmission remain unclear. In the developing Drosophila visual system, Hedgehog is partitioned for release at opposite poles of photoreceptor neurons. Release into the retina regulates the progression of eye development; axon transport and release at axon termini trigger the development of postsynaptic neurons in the brain. This study shows that this binary targeting decision is controlled by a C-terminal proteolysis. Hh with an intact C-terminus undergoes axonal transport, whereas a C-terminal proteolysis enables Hedgehog to remain in the retina, creating a balance between eye and brain development. Thus, a novel mechanism is defined for the apical/basal targeting of this developmentally important protein ,and it is posited that similar post-translational regulation could underlie the polarity of related ligands (Daniele, 2017a).
The Drosophila eye is an ideal model to study development, intracellular signaling, behavior, and neurodegenerative disease.Using axonal transport of the morphogen Hedgehog (Hh), which is integral to eye-brain development and implicated in stem cell maintenance and neoplastic disease, this study demonstrated the ability to quantify and characterize its trafficking in various neuron types and a neurodegeneration model in live early 3rd instar larval Drosophila. Neuronal Hh was found to favor fast anterograde transport and varies in speed and flux with respect to axonal position. This suggests distinct trafficking pathways along the axon. Lastly, abnormal transport is rorported of Hh in an accepted model of photoreceptor neurodegeneration. As a technical complement to existing eye-specific disease models, the ability to directly visualize transport in real time was demonstrated in intact and live animals, and secreted cargoes were tracked from the axon to their release points. Particle dynamics can now be precisely calculated and it is posited that this method could be conveniently applied to characterizing disease pathogenesis and genetic screening in other established models of neurodegeneration (Daniele, 2017b).
In Drosophila, the sine oculis (so) gene is
important for the development of the entire visual system, including Bolwig's
organ, compound eyes and ocelli. Together with twin of eyeless,
eyeless, eyes absent and dachshund, so
belongs to a network of genes that by complex interactions initiate eye
development. Although much is known about the genetic interactions of the
genes belonging to this retinal determination network, only a few such
regulatory interactions have been analysed down to the level of DNA-protein
interactions. An eye/ocellus
specific enhancer of the sine oculis gene has been identified that is directly regulated
by eyeless and twin of eyeless. This regulatory element has been further characterized
and a minimal enhancer fragment of
so has been identified that sets up an autoregulatory feedback loop crucial for proper
ocelli development. By systematic analysis of the DNA-binding specificity of
so the most important nucleotides for this interaction have been identified.
Using the emerging consensus sequence for SO-DNA binding a
genome-wide search was performed and eyeless has been identified as
well as the signalling gene hedgehog as putative targets of
so. These results strengthen the general assumption that feedback loops
among the genes of the retinal determination network are crucial for proper
development of eyes and ocelli (Pauli, 2005).
In-vitro data on the autoregulatory element with the known so
target sequence of lz and the AREC3/Six4-binding site, the consensus
sequence GTAANYNGANAYC/G was identified as necessary for SO binding to DNA. This consensus
sequence was taken as a basis for scanning the Drosophila genome for similar
sites. In total, 1632 putative so targets emerged
from this survey. Out of the affected genes several candidates are already known
to be involved in eye development (Pauli, 2005).
decapentaplegic (dpp) signalling plays an important role
in the complex regulatory network of eye development. In dpp mutant
eye discs, so, eya and dac are not expressed, whereas
dpp is able to initiate ectopic expression of so and
dac when expressed at the anterior margin of the eye disc.
Conversely, dpp expression is patchy in eye discs of eya and
so loss-of-function mutants, suggesting that eya and
so are required for either initiation or maintenance of dpp
at the posterior disc margin before MF initiation (Pauli, 2005).
hh is required for dpp expression at the posterior margin
before MF initiation, and dpp expression is induced by hh in
the MF, supporting the assumption that dpp is downstream
of hh signalling. Since dpp alone is not able to rescue
posterior margin clones of hh, there have to be more eye-relevant
target genes of hh signalling during third instar larval development.
dpp in combination with eya can restore photoreceptor
differentiation in posterior margin clones lacking smoothened
(smo) expression (smo is a cell-autonomous receptor of
hh signalling). This shows that dpp, in combination with
eya, is able to bypass the requirement of hh during eye
development. Taken together, it is evident that hh is necessary
for proper eya and dpp expression, both of which can induce
so, and it contains two so target sites. It is therefore
hypothesized that the transcriptional complex consisting of Eya and So, as
with ey, might also feed back on hh in order to drive the
furrow during late eye development. In this model the genetic cascade starts
with hh, which induces dpp and eya, moves on to
so and through the So/Eya complex feeds back to hh in order
to maintain hh expression as a driving force of the MF (Pauli, 2005).
The impact of these so-binding sites in the hh enhancer
on eye development becomes evident from the fact that hh1
(bar-3) mutant flies have smaller eyes. The severity of the
hh1 mutant phenotype is probably diminished by an
additional putative So-binding site that resides outside the area covered by
the hh1 deletion. If functional, this region (5' to
the hh1 deletion) might mediate a residual
hh-expression that overcomes the loss of the other sites to some
extent. Another possible explanation for the rather weak
hh1 phenotype might be that the feedback of so on
hh is not crucial for MF initiation but still might be of importance
for the well-balanced expression of hh during MF propagation (Pauli, 2005).
Drosophila development depends on stable boundaries between cellular territories, such as the embryonic parasegment boundaries and the compartment boundaries in the imaginal discs. Patterning in the compound eye is fundamentally different: the boundary is not stable, but moves (the morphogenetic furrow). Paradoxically, Hedgehog signaling is essential to both: Hedgehog is expressed in the posterior compartments in the embryo and in imaginal discs, and posterior to the morphogenetic furrow in the eye. Therefore, uniquely in the eye, cells receiving a Hedgehog signal will eventually produce the same protein. The mechanism that underlies this difference is the special regulation of hedgehog (hh) transcription through the dual regulation of an eye specific enhancer. This enhancer requires the Egfr/Ras pathway transcription factor Pointed. Recently, others have shown that this same enhancer also requires the eye determining transcription factor Sine oculis (So). These data are discussed in terms of a model for a combinatorial code of furrow movement (Rogers, 2005).
There are two known eye-specific hedgehog (hh) mutations:
hhbar3 (also known as hh1) and
hhfurrow stops early (or hhfse). Both
are associated with deletions in the first intron.
hhbar3 is a homozygous viable allele with a strong
recessive eye phenotype resulting from arrest of the morphogenetic furrow.
hhfse is a gamma-induced viable allele with a weaker eye
phenotype. PCR and direct sequencing were used to determine the precise end-points of the deletions. The hhbar3 deletion
is 1885 bp and the hhfse deletion lies within the span of
hhbar3, but is shorter. Both
hhbar3 and hhfse are viable and can be
maintained as homozygous stocks, although they are not as vigorous as wild
type. This is probably not due to second-site recessive lethal mutations, since
lines were derived that are isogenic for the X and major autosomes and they are
no more vigorous. The cuticles and nervous system (by anti-Elav
and anti-Futsch stains) of the isogenic hhbar3 and
hhfse embryos were examined, and no detectable phenotypes were found (Rogers, 2005).
To determine if either of these two eye-specific alleles are null for
hedgehog function in the eye, all viable pair-wise
combinations of these alleles, wild-type and two zygotic lethal alleles
(hhAC and hh8), were derived.
hhAC is a single gene deletion that removes both the start
sites for transcription and translation.
hh8 (also known as hh13C) is a
chain-terminating mutation in the coding sequence. Both
alleles are zygotic lethal with strong cuticle phenotypes.
hhAC is thought to be a null because of the strength of
its phenotype and the nature of its lesion. On
phenotypic grounds and comparison with other alleles, other groups have also
reported hh8 to be functionally amorphic (Rogers, 2005).
These alleles form a series for adult eye phenotype. This
was quantified by counting eye facets in adult females; hhfse, hhbar3 and hh8
heterozygotes are not significantly different from wild type. However,
hhAC is slightly dominant, with an eye that is about 10%
smaller than wild type (although this difference is not statistically
significant) (Rogers, 2005).
By facet number, hhbar3 is a strong, eye-specific
hypomorph. It is fully recessive in trans to wild type, has a severely reduced
eye when homozygous (68% smaller than
hhbar3/hh+) and in trans to the null
hhAC it is smaller still (82% smaller than
hhbar3/hh+). This suggests that
hhbar3 is not an amorph for eye size by Muller's test: the
phenotype becomes stronger in trans to the null.
hhfse is similar to but weaker than
hhbar3: the hhfse homozygous eye is
only 32% smaller than hhfse/hh+ and in
trans the null (hhAC), it is further reduced to 78%. Thus,
by both measures (phenotype as a homozygote and in trans to a null),
hhbar3 is a strong hypomorphic allele and
hhfse is a weaker hypomorph. From the 95% confidence
limits, all these results are statistically significant (Rogers, 2005).
Probably hhbar3 and hhfse affect a
transcriptional enhancer and not the protein itself or the gene promoter,
because neither lesion directly affects the coding sequence. In sequencing 23
cDNAs from eye-imaginal discs, no alternative first exon or start
site was found in the region of the two mutations (Rogers, 2005).
These two eye-specific alleles of hedgehog were characterized and they were found to delete elements that are specifically necessary for expression
in the developing eye, posterior to the morphogenetic furrow. This
hedgehog eye enhancer drives expression in all of the developing
ommatidial cells except the R8. This element was reduced to a 203 bp minimal
fragment that is sufficient for reporter expression. The
hedgehog eye enhancer is regulated by pointed in vivo and
bound by Pointed in vitro. Since Egfr/Ras-driven Pointed activates reporters in
all the cells except the R8, it is suggested that the hedgehog expression
in the developing eye is driven by this enhancer and that Hedgehog is
expressed in the developing ommatidial cells excepting the R8 (Rogers, 2005).
It is proposed that hhbar3 is indeed null for
hedgehog expression in the developing eye, consistent with the loss
of detectable antigen. This appears to contradict facet count data, which
show that hhbar3 is not null for eye size. It is suggested that
hedgehog functions elsewhere (probably in the eye disc margin),
expressed at some lower level, and acts redundantly with Decapentaplegic to
drive the early phases of furrow progression. This is consistent with data
from others for an early role for hedgehog in the eye margin for
furrow initiation, and with a proposed redundancy between
hedgehog and dpp in the furrow.
The enhancement of the hhbar3 phenotype when it is placed
in trans to a null (hhAC) suggests that hhbar3 may reduce, but not eliminate this early function (Rogers, 2005).
Several examples of eye-specific transcriptional enhancers have been
characterized. A number of these are in genes that act early in retinal
determination (eyes absent, dachshund and sine oculis), and
are not directly involved in the morphogenetic furrow. Some
enhancers that function in and posterior to the morphogenetic furrow have also
been studied. One example is the atonal gene, which has been shown to
have two regulatory enhancers with specific and different activities in the
furrow.
Interestingly the atonal enhancers produce almost the reciprocal
expression pattern of the hedgehog eye enhancer described here:
hedgehog is expressed in all cells except the R8 and atonal
expression is in only the R8, posterior to the furrow. Furthermore,
atonal mutations can affect hedgehog signaling, although
this may be indirect, and indeed, hedgehog is also known to regulate
atonal. Other enhancers that act posterior to the furrow have been
characterized in the rough, sevenless and prospero genes,
but none of these appears to show the particular type of regulation
described in this study (Rogers, 2005).
A similar DNA fragment from the hhbar3 region confers post-furrow, eye-specific expression on a lacZ reporter (Pauli, 2005). The consensus binding site for another transcription factor was characterized: the retinal determination protein Sine oculis (So). Two So-binding sites were found in the hhbar3 region, and it was shown that these are necessary for the normal function of the hedgehog eye enhancer. A So site tetramer is
sufficient to drive reporter expression in the entire presumptive eye field in
the third instar disc. One of the two So sites lies
within the 203 bp minimal element (Rogers, 2005).
Taken together, both sets of data suggest that Pointed and So activation
at the minimal element are each necessary, but that neither is sufficient for
the specific activation of the hedgehog eye enhancer posterior to the
furrow. It is proposed that they act together to confer this dual regulation. This
is consistent with the following model: that special dual regulation of hedgehog is the mechanism which makes the morphogenetic furrow move, unlike the stable compartment boundaries. It is suggested that this dual regulation depends on one 'selector' signal that is eye specific (So), to differentiate the furrow from boundaries in other organs. The second component must act to close a loop such that cells which receive the furrow inducing signal will later send it, after a delay, to make the boundary move forward. This 'signal' component is Pointed, acting downstream of Egfr/Ras signaling in the assembling ommatidia. This may be a case of 'selector' and 'signal' transcriptional integration. Indeed, pointed itself has been shown to integrate 'selector' factors in muscle development. It is proposed that by this dual regulatory mechanism, a system that first evolved to divide the bauplan into metameric parasegments has been co-opted to drive a moving wave of differentiation in the developing eye (Rogers, 2005).
In Drosophila, trunk metamerization is established by a cascade of segmentation gene activities: the gap genes, the pair rule genes, and the segment polarity genes. In the anterior head, metamerization requires also gap-like genes and segment polarity genes. However, because the pair rule genes are not active in this part of the embryo, the question of which gene activities fulfill the role of the second order regulators still remains to be solved. This study provides first molecular evidence that the Helix-Loop-Helix-COE transcription factor Collier fulfills this role by directly activating the expression of the segment polarity gene hedgehog in the posterior part of the intercalary segment. Collier thereby occupies a newly identified binding site within an intercalary-specific cis-regulatory element. Moreover, a direct physical association has been identified between Collier and the basic-leucine-zipper transcription factor Cap'n'collar B, which seems to restrict the activating input of Collier to the posterior part of the intercalary segment and to lead to the attenuation of hedgehog expression in the intercalary lobes at later stages (Ntini, 2011b).
In the context of an analysis to identify cis-regulatory elements controlling expression of segment polarity genes in the embryonic head, an intercalary-specific cis-regulatory element of hh—ic-CRE—was isolated within the upstream 6.43 kb region (Ntini, 2011a). The ~ 1 kb enhancer fragment (− 4085 to − 3077 bp) mediates reporter expression in the hh expressing cells of the posterior part of the intercalary segment, when combined with the endogenous hh promoter (− 120 to + 99 bp;). Further functional dissection of this element showed that the 450 bp ?1mF5 subfragment (− 3914 to − 3465 bp) mediates the intercalary-specific expression with slightly delayed onset, while the 335 bp F5_R4 subfragment (− 3799 to − 3465 bp) constitutes the minimum sequence required for the intercalary expression, but mediates an additional spotty metameric pattern in the trunk (Ntini, 2011). Because a high degree of phylogenetic conservation in non-coding DNA sequence implicates a functional role in vivo, such as recognition and DNA-binding by sequence-specific transcription factors, the sequence of the ic-CRE was subjected to phylogenetic conservation analysis within the genome of twelve Drosophila species, and different in silico analyses were performed to detect putative transcription factor binding sites. The minimum 335 bp ic-CRE consists of six highly conserved sequence blocks. A series of complete block deletions designed in the context of the minimum ic-CRE in combination with the endogenous hh promoter resulted in non-functional elements. This could be either because individual binding motifs were disrupted or inter-motif distances crucial for transcription factor binding and operation were disturbed. A point mutagenesis screen was conducted in the context of the 450 bp ic-CRE to extract crucial cis-regulatory information in respect to the conserved in silico identified transcription factor binding sites (Ntini, 2011b).
The ic-CRE responds to the homeotic transformation of the mandibular into an intercalary segment resulting from ectopic ems expression by a duplication of its expression pattern. However, despite this and the fact that the Hox gene labial is active in the intercalary segment, disrupting the homeodomain binding sites in conserved sequence blocks III or IV by point mutations did not abolish the ic-CRE mediated reporter expression. In contrast, disrupting a putative binding site for the fork head transcription factor Sloppy paired 1 (Slp1) in block IV eliminated the ic-CRE-mediated reporter expression. This is consistent with the reduced reporter expression in an RNAi-mediated knock-down of slp1, which is a proposed head gap-like and pair rule segmentation gene (Ntini, 2011b).
Another in silico prediction was found in conserved block II at position − 3771 to − 3755 bp that scores the binding matrix of the mammalian COE factor Olf1. Disrupting this site by point-mutation resulted in the complete abolishment of the ic-CRE mediated reporter expression, indicating that the site is absolutely required for the function of the 450 bp ic-CRE. Olf1 is the mammalian COE homolog of Collier and the endogenous hh expression in the intercalary segment is abolished in a col loss-of-function mutant (col1. Likewise, the ic-CRE-mediated expression pattern is abolished in col1 or col knock-down. In addition, the DNA-binding domain of Collier displays a high degree of primary sequence identity (86%) to the mammalian homolog. High degree of primary sequence identity in the DNA-binding domain, shared among the members of the COE family allows for a similar DNA-binding specificity: both Collier and the Xenopus homologs recognize the mammalian DNA target sequences in vitro. Therefore, the Olf1 prediction identified in silico within the ic-CRE is regarded as a putative Collier binding site and referred to as a Collier recognition site (Ntini, 2011b).
Apart from this functionally required Collier recognition site at − 3773 to − 3751 bp, scanning in silico the 6.43 kb upstream hh enhancer using MatInspector with a similarity cut-off of 1, 0.8 (core, matrix) identifies one more Olf1 prediction within the ic-CRE at position − 3967 to − 3945 bp. The 6.43 kb upstream enhancer of hh was also submitted to rVISTA using the nucleotide positions 3–19 of the binding matrix of Olf1. When setting the highest possible similarity cut-off 0.95, 0.85 (core, matrix), so that at least one prediction is generated, then only the functionally required Collier recognition site CAATTCCCCAATGGCAT (at − 3771 to − 3755) within the ic-CRE is detected. Lowering the matrix similarity threshold by 0.05, using cut-off 0.95, 0.8, generates three additional predictions. These are two distant sites, GAGACACTTGGGATGAG at − 3963 to − 3947 and CACACCACGGGGAAGCG at − 2872 to − 2856, and one promoter-proximal site CACTTCCCTTGCGCATA at − 212 to − 196. The first distant site is within the ic-CRE, 190 bp upstream of the functionally required Collier recognition site, and is also predicted by the MatInspector. Interestingly, in contrast to the functionally required Collier recognition site within the ic-CRE, none of the other predicted sites are phylogenetically conserved among the twelve Drosophila species. Considering the displayed short-range homotypic clustering (within 200 bp), it is, however, possible that the weaker predictions may contribute to the transcriptional outcome of the ic-CRE, even though they might be recognized with minor affinity by Collier in vivo (Ntini, 2011b).
In order to verify that the in silico identified and functionally required Collier recognition site within the ic-CRE is indeed occupied by Collier in vivo, chromatin immunoprecipitations (ChIP) from Drosophila embryonic nuclear extracts were performed with an antibody against Collier. In the anti-Col ChIPs, the functionally required Collier binding site within the ic-CRE was specifically enriched in comparison to mock ChIPs, which indicates that the site is indeed occupied by Collier in vivo (Ntini, 2011b).
In the case of the mammalian COE homolog of Collier, it was previously deciphered that the mouse transcription factor EBF contains two distinct and functionally independent transcription activation domains, the second one within the C-terminal region. Although Drosophila Collier has been genetically implicated as an activator of downstream segment polarity gene expression, its transcriptional activation potential had not yet been analyzed. In Drosophila two Collier isoforms are expressed from the col gene locus. The cDNAs encoding Collier A (also termed Col2) and Collier B (Col1) differ from each other by 465 bp due to alternative splicing. The two protein isoforms share the same first 528 N-terminal amino acids and differ in the C-terminal 29 amino acids for Collier A and 47 amino acids for Collier B. No specific expression pattern of collier A could be detected by double in situ hybridization using an RNA probe specific for collier B and a probe that hybridizes with both transcripts (Ntini, 2011b).
Therefore the transcriptional activation potential of each of the two Collier isoforms was examined by reporter assays in Drosophila S2 R+ cell transfections. In the reporter construct the functionally required and in vivo occupied Collier site was cloned in a single copy upstream of the endogenous hh promoter (− 120 to + 99 bp) driving luciferase gene expression. Both Collier isoforms activate luciferase expression when independently co-transfected with the reporter construct, indicating that both isoforms possess transcriptional activation potential. A truncated form of ColA lacking the last 23 C-terminal amino acids (ColA 1–534) displays a significantly reduced activation potential (~ 84% decrease), which indicates that a transcriptional activation domain must reside within either C-terminal region of both isoforms. Disrupting the Collier recognition site by point mutations decreased the mediated reporter activation by ~ 48% in the case of Collier A and ~ 44% in the case of Collier B. Taking into consideration that disrupting the Collier binding site in the context of the ic-CRE resulted in a complete abolishment of the mediated reporter expression in vivo, and that the same mutation does not support Collier DNA-binding in vitro, it is assumed that part of the reporter activation assessed in cell transfection may be achieved by Collier transactivating via unknown system-provided DNA-binding activities on the regulatory sequences of the reporter plasmid. Moreover, Collier carries a perfect SUMOylation motif within the N-terminus, predicted with the highest threshold value. The protein sequence TSLKEEP at amino acid position 44-50 matches the SUMOylation motif. Additional members of the COE transcription factor family contain also a SUMOylation motif at this conserved position. Apart from antagonizing ubiquitin-mediated degradation, sumoylation has been implicated in modifying transcriptional activation/repression potential of transcription factors. Mutant versions of Collier A and Collier B where the K within the SUMOylation motif is mutated towards R (ColA RK and ColB RK) display reduced activation potential, implying a possible role for sumoylation in regulation of Collier transcriptional activity (Ntini, 2011b).
Data is presented consistent with the cap-n-collar isoform CncB performing as a sequestering factor or inhibitor of Collier DNA-binding to its cognate site found within the ic-CRE. Furthermore, fluorescent immunostaining revealed that only a small fraction of the expressed Collier protein is nuclear localized in vivo. Conversely, CncB protein greatly accumulates in the nuclei. Prediction of nuclear localization signals (NLS) in silico generates no results for Collier, while CncB contains an NLS within the bZIP domain (aa 617–680). Interestingly, Collier carries a perfect SUMOylation motif in the very N-terminus, predicted with the highest threshold value. Apart from antagonizing ubiquitin-mediated degradation and modifying transcriptional activation/repression potential of transcription factors, sumoylation has also been implicated in protein nucleo-cytoplasmic translocation. Alternatively, in the absence of a nuclear localization signal, Collier import in the nucleus may be realized by heterodimerization with a protein that carries an NLS. This would increase the probability that Collier is recruited into combinatorial control mechanisms, which has already been implicated in muscle specification. Furthermore, nuclear accumulation of CncB, in converse to a relatively low concentration of nuclear Collier protein, indicated by the fluorescent immunostainings, may facilitate the sequestering function of CncB to antagonize and overcome the DNA-binding activity of Collier on the ic-CRE in the cells of the anterior most part of the mandibular segment during the establishment of procephalic hh expression, and at later stages in the hh expressing cells of the intercalary lobes (Ntini, 2011b).
In this respect it is interesting to note that despite the intrinsic transcriptional activation properties of the Cnc homologs, CncB acts to suppress both the expression and the homeotic selector (maxillary structures promoting) function of Deformed (Dfd) in the mandibular segment. In particular, CncB represses the maintenance phase of Dfd transcription in the mandibular cells, most probably by interfering with the positive regulatory function of Deformed within the Dfd autoactivation circuit. Overexpression of CncB partially represses Dfd-responsive transcriptional target elements in vivo. Interestingly, interaction between CncB and Dfd proteins has been reported. Perhaps the negative regulation of Dfd expression and function caused by CncB results from CncB interfering with Dfd binding to its cognate target cis-regulatory elements in vivo, as a consequence of a direct physical interaction at protein level with a sequestering effect similar to the interaction with Collier reported in this study (Ntini, 2011b).
The isolation of an intercalary-specific cis-regulatory element from the hh upstream region supports a unique mode for anterior head segment-specific transcriptional control of segment polarity gene expression. Thus, not only cross-regulatory interactions among segment polarity genes during the maintenance phase, but also the initial establishment of procephalic segment polarity gene expression seems to be unique for each of the anterior head segments. The previously proposed mode of second order regulation in anterior head patterning, resulting in activation of hh in the posterior part of the intercalary segment, is mediated by the HLH-COE factor Collier evidently via direct DNA binding. The reported physical interaction between Collier and CncB is likely to attenuate the activating function of Collier in the hh expressing cells of the posterior part of the intercalary segment at a later developmental stage, and it might also be involved in eliminating the potential of target activation by Collier in the anterior most part of the mandibular segment where the two factors are co-expressed (Ntini, 2011b).
The Drosophila wing is formed by two cell populations, the anterior and posterior compartments,
distinguished by the activity of the selector gene engrailed (en) in posterior cells. EN governs growth and patterning in both compartments by controlling the expression
of the secreted proteins HH and Decapentaplegic (DPP) as well as the response of cells
to these signaling molecules. EN activity programs wing cells to express
hh, whereas in the absence of EN activity they are able to respond to HH by expressing dpp. As a
consequence, posterior cells secrete HH and induce a stripe of neighboring anterior cells across the
compartment boundary to secrete DPP (Zecca, 1995).
The normal growth of the wing disc requires
that posterior-specific genes, such as hedgehog and engrailed are not expressed in cells of the
anterior compartment. Hedgehog has the capacity to activate engrailed in the anterior
compartment but both hedgehog and engrailed are specifically repressed in anterior cells by
the activity of the neurogenic gene groucho. In groucho mutant discs, hedgehog and engrailed are
expressed at the dorsoventral boundary of the anterior compartment, leading to the ectopic
activation of decapentaplegic and patched and to a localised increase in cell growth associated with
pattern duplications. The presence of Engrailed in the anterior compartment causes the
transformation of anterior into posterior structures (de Celis, 1995).
The identity of anterior cells in the wing imaginal disc requires cubitus interruptus function. Anterior cells lacking ci express hedgehog and adopt posterior properties without expressing engrailed. Most clones cause an up-regulation of CI protein levels in surrounding cells, in a manner that is similar to that of CI along the A/P compartment boundary. Increased levels of CI can induce the expression of the HH target gene decapentaplegic in a HH-independent manner, suggesting that dpp is a target gene of CI. Thus, expression of CI in anterior cells controls limb development by restriction HH transcription to posterior cells and by conferring competence to respond to HH by mediating the transduction of this signal. The multiple role of CI in the anterior compartment suggests that anterior cell identity is not a default fate that imaginal cells adopt in the absence of engrailed (Domínguez, 1996).
wingless and hedgehog are not regulated by pair rule genes in the head as they are in the trunk. Instead they are regulated by head gap genes. Both wg and hh are normally expressed at blastoderm stage
in two broad domains anterior to the segmental stripes of the trunk region. At the blastoderm stage,
each gap gene acts specifically to regulate the expression of either wg or hh in the anterior cephalic
region: huckebein, orthodenticle and buttonhead regulate the anterior blastoderm expression of wg, while tailless and empty spiracles
regulate hh blastoderm expression. Additionally, btd is required for the first segmental stripe
(mandibular segment) of both hh and wg at blastoderm stages. The subsequent segmentation of the
cephalic segments (preantennal, antennal and intercalary) appears to be dependent on the overlap
of the wg and hh cephalic domains as defined by these gap genes at the blastoderm stage (Mohler, 1995a).
Segment polarity genes are not activated in the anterior by pair-rule genes, as they are in the trunk but instead they are activated by gap genes. The segment polarity genes hedgehog and wingless are two important targets of cap'n'collar and forkhead, expressed in the anterior and posterior gut anlagen. cnc is expressed in the labral region of the foregut, fated to give rise to the dorsal pharynx and fkh is expressed in the adjacent esophagus. fkh is responsible for the maintenance but not the initiation of wg synthesis in the invaginating esophageal primordium. cnc is responsbile for the maintenance of wg in the dorsal pharyngeal domain of wg expression. Expression of hedgehog is similarly affected in cnc and fkh mutants. It is not known whether the actions of cnc and fkh on hh and wg are direct or indirect (Mohler, 1995 b).
Whereas the segmental nature of the insect head is well
established, relatively little is known about the genetic and
molecular mechanisms governing this process. The phenotypic analysis is reported of mutations in
collier (col), which encodes the Drosophila member of the
COE family of HLH transcription factors and is activated
at the blastoderm stage in a region overlapping a
parasegment (PS0: posterior intercalary and anterior
mandibular segments) and a mitotic domain, MD2. col
mutant embryos specifically lack intercalary ectodermal
structures. col activity is required for intercalary-segment
expression both of the segment polarity genes hedgehog,
engrailed, and wingless, and of the segment identity gene
cap and collar. The embryonic head phenotype of col1 hemizygous mutant
embryos indicates a loss of skeletal structures derived from the
intercalary, and possibly mandibular, segments without transformation toward another
segment identity. To investigate this segmentation phenotype
in more detail, col expression was compared with that of
the segment polarity genes hh and wg. At the blastoderm stage,
the posterior limit of col expression is parasegmental
(PS0/PS1), since it precisely abuts the mandibular stripe of hh-expressing
cells. Whether its anterior limit is also
parasegmental cannot be answered at this stage because the
expression of segment polarity genes in pre-gnathal segments is not yet
established at this stage. Examination of early stage 11 embryos
shows that col expression overlaps the intercalary hh stripe and
abuts the intercalary Wg spot, indicating a parasegmental
anterior border for col expression. At this stage however, col
expression has been lost from the posterior part of PS0, since it
does not overlap mandibular Wg expression. The
cnc gene, which codes for a b-ZIP transcription factor, has been
postulated to act as a segment identity gene in the mandibular
segment. Consistent
with col being expressed in PS0, col and cnc expression only
partly overlap, in the region corresponding to the anterior
mandibular segment. Together, these data indicate a
parasegmental register of col expression at the blastoderm
stage, which is subsequently restricted to anterior PS0 (Crozatier, 1999b).
A determination was made of whether col mutations affect the
expression of wg and En, which mark the anterior and posterior
compartments of each segment, respectively. In col1 hemizygous embryos, both the
intercalary stripe of En and the spot of wg expression are
missing. Since col expression does not overlap the
intercalary Wg spot, the loss of this spot in col mutant
embryos suggested that col does not regulate wg expression
directly but possibly by an hh-dependent mechanism. It has indeed been found
that in col mutant embryos, the intercalary stripe of hh is also
absent, or much reduced. Together, these results show
that col controls hh, en and wg expression in the intercalary
segment and is required for establishing the PS(-1)/PS0
parasegmental border. The head skeleton structures ventral arm (VA) and lateral-gräten (LG), which are, respectively, either missing or
reduced in col mutant embryos, are also affected
in two other head mutants: crocodile (croc), which codes for a
forkhead-domain protein, and cnc. These
structures are also affected in embryos mutant for the homeotic
genes Dfd and lab, which are expressed, respectively, in the mandibular and
maxillary segments, and in the intercalary segment. col expression was examined in embryos
mutant for croc, cnc, Dfd or lab. In none of these embryos was there a change in col transcription. Conversely, no
changes could be detected for croc, Dfd or Lb expression in
col1 hemizygous embryos, indicating that expression of each
of these three genes is independent of col. In
contrast, col is required for cnc transcription in the posterior
intercalary segment at stage 9-10. Because this region
is anterior to the region of overlap between col and cnc
expression at the blastoderm stage, it is concluded that
this region corresponds to a secondary site of cnc expression initiated at
stage 9, under control of col activity. In cnc mutant embryos,
intercalary hh expression is normal,
indicating that hh and cnc are regulated by col, independent
of one another (Crozatier, 1999b).
forkhead is required for the activation of wingless, hedgehog and decapentaplegic in both the foregut and hindgut, considered to be ectodermal tissues. wingless is expressed initially in the whole hindgut primordium, but becomes restricted to a ring in the small intestine anterior to the outgrowing Malpighian tubules, and to a ring in the posterior region of the rectum. hedgehog is also expressed in the hindgut primordium but becomes restricted to a ring of cells posterior to the outgrowing Malpighian tubules in the future small intestine of the hindgut. A second hh expression domain is located in the anterior portion of the rectum. These two expression domains are adjacent to the wg expression domains. dpp is expressed in the hindgut primordium and later on one side in the large intestine of the hindgut tube, in between the small intestine and the rectum. Thus the expression domains of wg, hh and dpp subdivide the hindgut tube into a central portion (the large intestine) where dpp is expressed, and two flanking regions (the small intestine and the rectum) where wg and hh are expressed. In fkh mutant embryos, the foregut, the midgut and the hindgut epithelia are disrupted, and fkh is required for the activation of each of these genes in the fore- and hindgut primordia. fkh is expressed in the entire foregut and hindgut, whereas wg, hh and dpp are expressed only in restricted domains. Since the expression of these genes appear not to be established through cross-regulatory interactions, there must be other factors which act to spatially regulate wg, hh and dpp expression along the hindgut (Hoch, 1996).
Single-minded represses wingless, hedgehog and vnd gene expression in developing midline cells. By doing this sim plays a key role in proper patterning of the neuroetoderm by helping to generate the boundary between mesectoderm and ventral ectoderm. This process likely requires simultaneous function of
SIM as both a transcriptional activator (of slit and Toll) and transcriptional repressor within the developing midline cells (Xiao, 1996).
The regulation and function of the Hedgehog pathway activity has been compared in eye and wing discs, and there are significant differences. Whereas in the wing disc, engrailed function is required for hedgehog expression, in the eye disc activation and maintenance of hedgehog expression is achieved independently of engrailed. Nevertheless, engrailed functions in the eye disc, as elevated engrailed expression represses dpp, patched and cubitus interruptus in the eye disc, but does not disrupt morphogenesis. Regulation of decapentaplegic expression also differs: in the wing disc it is repressed in the anterior compartment by patched and in the posterior compartment by engrailed. In the eye disc, however, it is repressed posterior to the morphogenetic furrow in the absence of either patched or engrailed activity (Strutt, 1996).
Anterior terminal development is controlled by several
zygotic genes that are positively regulated at the anterior
pole of Drosophila blastoderm embryos by the anterior
(bicoid) and the terminal (torso) maternal determinants.
Most Bicoid target genes, however, are first expressed at
syncitial blastoderm as anterior caps, which retract from
the anterior pole upon activation of Torso. To better
understand the interaction between Bicoid and Torso, a
derivative of the Gal4/UAS system was used to selectively
express the best characterized Bicoid target gene,
hunchback, at the anterior pole when its expression should
be repressed by Torso. Persistence of hunchback at the pole
mimics most of the torso phenotype and leads to repression
at early stages of a labral (cap'n'collar) and two foregut
(wingless and hedgehog) determinants that are positively
controlled by bicoid and torso. These results uncovered an
antagonism between hunchback and bicoid at the anterior
pole, whereas the two genes are known to act in concert for
most anterior segmented development. They suggest that
the repression of hunchback by torso is required to prevent
this antagonism and to promote anterior terminal
development, depending mostly on bicoid activity (Janody, 2000).
The results indicate that early anterior expression of a labral
determinant, cnc, and of two foregut determinants, wg and hh,
is repressed when zygotic expression of hb is allowed to persist
at the anterior pole of the Drosophila blastoderm embryo.
Expression of cnc, wg and hh is under the positive regulation
of bcd and torso but no zygotic gene has yet been implicated
in this control. This suggests that the Hb protein is able to repress the three genes cnc, wg and hh, and
that torso-induced anterior repression of hb is necessary for
their positive control by torso. To determine whether the
positive control of cnc, wg and hh by torso could be the result
of a double negative control involving hb, expression of these
genes was analysed in hb zygotic mutant embryos derived from
torso females. If the lack of early anterior expression of cnc, wg and hh was solely due to the absence of repression of hb
at the pole, expression of these genes should be recovered in
hb minus embryos derived from torso females. Early anterior expression of cnc, wg and hh is
not recovered in hb minus embryos derived from torso females
whereas it is normal in hb minus embryos. This indicates
that, although necessary, the anterior repression of hb is not
sufficient to mediate Torso positive control on cnc, wg and hh
early anterior expression (Janody, 2000).
A function of Gro in imaginal development has been investigated, namely the repression of hedgehog in anterior wing pouch cells. hh is repressed in anterior compartments at least partly via Ci[rep], a form of the multifunctional transcription factor Cubitus interruptus (Ci). Cells in the
wing primordium close to the AP boundary need gro activity to maintain repression of hh transcription, whereas in more anterior cells gro is dispensable. This repressive function of Gro does not appear to be mediated by Ci[rep]. Analysis of mutant gro transgenes has revealed that the Q
and WD40 domains are both necessary for hh repression. Yet, deletion of the WD40 repeats does not always abolish Gro activity. These findings
provide new insights both into the mechanisms of AP patterning of the wing and into the function of Gro (Apidianakis, 2001).
Although Ci[rep]-mediated repression can account for the lack of hh expression away from the AP boundary, it has not been firmly established that Ci[rep] is operational close to the AP boundary. These cells receive high Hh signal and as a result not only do they not process Ci to Ci[rep], but also they activate full-length Ci into a strong activator, Ci[act], by post-translational modification. There is indirect evidence that Hh-receiving cells do not contain sufficient Ci[rep] levels to repress hh: in posterior cells, ci is repressed by En; other than this, the cellular mechanism for Hh signal transduction is present. When full-length ci is provided by ectopic expression in the posterior compartment, hh-lacZ is not repressed. This suggests that these cells cannot produce appreciable amounts of Ci[rep], consistent with their responding to Hh signaling. That this is indeed the case was shown by the fact that ectopic expression of ci does repress posterior hh-lacZ in smo loss-of-function clones, where the Hh signal transduction has been disrupted. If anterior cells that are exposed to Hh behave similarly, then the lack of hh expression there cannot be attributed to Ci[rep]. It is proposed that a Gro-dependent repression complex supplies this function, since gro- clones exhibit strong derepression of hh-lacZ near the AP boundary. The Gro complex is not required in anterior cells far from the boundary, because those receive no Hh signal and thus contain sufficient Ci[rep] to repress hh. Accordingly, by supplying increased levels of Ci[rep] near the AP boundary via the ciCe2 allele, the need for Gro-mediated hh repression is able to be largely abolished, with the exception of the DV boundary. Since Gro is a ubiquitous co-repressor, one has to postulate the existence of a DNA-tethering factor, which will be referred to as 'X' for the purpose of this discussion, and some process of spatial regulation of the X-Gro complex activity. The possibility that X is a form of Ci itself was tested and the answer was negative: using three different assays -- GST pulldowns, yeast two-hybrid and transfection colocalization -- no interaction between Gro and either form of Ci could be shown. Most importantly, the fact that Ci[rep] does not require Gro to repress hh in anterior cells away from the boundary supports a model where Ci and Gro repress hh independently of each other (Apidianakis, 2001).
The quantitative aspect of hh derepression in gro- clones is intriguing: clones abutting the AP boundary (type I) express the highest hh-lacZ levels, which drop gradually as clones arise further from the P compartment. This might reflect the fact that Ci[rep]-dependent repression gradually increases away from the boundary, and this is independent of gro. This interpretation assumes that basal (unrepressed) hh transcription in the A compartment would be high and subject to the dual repressors (Ci and X-Gro). Alternatively, basal hh transcription could be low, but, in addition to the repression control, hh could display a positive response to Hh signaling at the AP boundary. The latter model is consistent with the fact that in ci- cells, basal hh expression appears to be low. It also agrees with the behavior of large type I gro- clones in the present study. In these clones, high levels of hh-lacZ could be observed throughout the clone, even at a distance from the AP boundary. This could be accounted for by Hh signaling, which, having risen over some threshold owing to hh derepression, further stimulates hh transcription to a high level. This effect would spread to the edge of the clone, beyond which activation of the X-Gro repressor would silence hh transcription. The putative inducer of hh by Hh signaling may be Ci[act], as with all other direct Hh target genes; alternatively, it may be another factor induced by Ci[act]. The hypothesis that Ci[act] itself can activate hh transcription is not unreasonable, since hh should contain a regulatory region(s) that bind(s) Ci[rep]. Ci[act] and Ci[rep] contain the same DNA-binding domain and recent work has shown that the two forms of Ci bind the same target sites, although some enhancers may be configured in such a way as to respond preferentially to either the activator or repressor form (Apidianakis, 2001).
For the sake of simplicity, the existence of a low level ubiquitous activator of hh (basal levels) with a stronger activator located in P cells is postulated to account for the high levels of hh expression in P cells. In A cells that do not receive the Hh signal, the basal activity of hh is repressed by Ci[rep] and gro is not required. In A cells close to the Hh source, the basal transcription of hh would be enhanced by positive autoregulation; however, the presence of the repressive X-Gro complex does not allow this activation to take place. Implicit in this model is that X is itself activated by Hh (e.g. transcriptionally induced via Ci[act]), so that it only functions in Hh-receiving cells. In addition X production/activity should be spatially limited to the A compartment (e.g. repressed by En), since ectopic expression of full-length ci in the posterior cannot induce X-Gro activity to repress endogenous hh. According to this model, ci- clones close to the AP boundary express basal hh levels, since they lack both the X-Gro repressor (no activation of X in the absence of Ci[act]) and the activator of hh transcription (Ci[act] itself or a downstream target). By contrast, gro- clones in the same region only lack the repressive X-Gro complex and thus actively transcribe hh in response to Ci[act]; the high levels of hh produced are sufficient to initiate Hh signaling, which can propagate this effect of hh derepression throughout the clone (Apidianakis, 2001).
gro- clones near the DV boundary behave somewhat aberrantly. hh-lacZ derepression there is more efficient, observable in further anteriorly arising clones, compared with equivalent clones away from the DV boundary -- it even occurs in the presence of increased Ci[rep]. Although the mechanism remains to be discovered, one way to account for this special behavior, without invoking additional regulators, is that Ci[rep] is less active near the DV boundary and/or Ci[act] is more active, and this modulation of Ci activity in favor of the activator form allows high level hh expression at a greater distance from the Hh source and even in the ciCe2/+ background. Interestingly, ci- clones show little or no hh-lacZ derepression at the DV boundary, consistent with Gro, rather than Ci[rep], being the major hh repressor there (Apidianakis, 2001).
The model put forward here is perhaps the simplest, but by no means the only one that fits the existing data. For example, Gro might interact with Ci[act] itself, switching it from an activator into a repressor, given the right enhancer context, much like the effect Gro has on other activators, such as Dorsal. This interaction may be weak and/or require additional factors, accounting for the inability to detect it. To resolve the mechanism of hh repression at the AP boundary will necessitate detailed molecular dissection of the hh regulatory regions and characterization of relevant trans acting factors. Whatever the mechanism, it appears that a Gro-containing complex is deployed in the wing to block the spread of hh expression anteriorly from the AP compartment boundary. This should ensure a spatially fixed organizer (dpp expression stripe), in contrast to a moving one, as found in the fly retina (Apidianakis, 2001).
Gro is the founder of a family of transcriptional co-repressors encountered in invertebrates and vertebrates. Gro proteins are multipurpose co-repressors, since they can interact with a good number of DNA-binding repressors. A number of Gro mutants were tested both for subcellular localization. Grocdc2- and GroDeltaQ show the same nuclear accumulation as wild-type Gro. GroDeltaWD40 is also nuclear, but it shows a striking departure from the rather uniform wild-type pattern, since it localizes predominantly to a small number of subnuclear particles. GroNLS- is both nuclear and cytoplasmic, whereas GroDeltaGCS is exclusively cytoplasmic. This suggests that the GP, CcN and SP domains contain at least two different regions needed for efficient nuclear accumulation, one of which is the canonical NLS. It can be speculated that other such regions might be those necessary for association with histones or with DNA-bound repressors, which might promote nuclear accumulation of Gro even in the absence of the NLS (Apidianakis, 2001).
In vivo activity was tested by assaying the ability of mutant Gro proteins to repress anterior hh-lacZ expression. GroDeltaQ and GroDeltaWD40 proteins were inactive in this assay. In contrast, Grocdc2- was as active as wild-type Gro. The inability of GroDeltaQ to function as a co-repressor is expected, since the Q domain is the strongest repression domain and is needed both for tetramerization as well as for histone interaction. The inactivity of the GroDeltaWD40 mutant might be accounted by its inability to interact with the X-factor tether. Or one could suggest an alternative explanation based on the localization data: that GroDeltaWD40 is retained in subnuclear particles and as a result cannot gain access to target genetic loci. Whether the aberrant subnuclear localization of GroDeltaWD40 is a cause or a consequence of its inactivity is a matter for further study. Despite its aberrant localization, GroDeltaWD40 is as active as wild-type Gro and Grocdc2- when overexpressed by omb-Gal4: all three transgenes results in abnormal leg development. Gro-DeltaQ, -NLS- and -DeltaGCS did not have such an effect. This shows that GroDeltaWD40 retains some activity, although in the absence of data regarding the cause of defects in leg patterning, the function of the mutant protein is as yet unknown. 'Short' Gro family proteins that lack WD40 repeats exist in vertebrates. These, human AES and mouse Grg5, contain only Q and GP domains, thus they are not entirely equivalent to the DeltaWD40 mutant. It has been shown that these proteins are cytoplasmic, although they are readily transported to the nucleus upon interaction with a Tcf partner. Their role in transcription seems to be context dependent, since they can act as co-repressors in some cases, whereas in others they might counter repression by 'long' Gro proteins. One study suggests that this anti-repression effect is not necessarily due to the absence of the CcN/SP/WD40 domains, but rather due to the inability of the GP domain of the 'short' proteins to interact with HDAC1. In this study, GroDeltaWD40 was active in one assay and inactive in another. It will be interesting to determine its activity in additional biological contexts where Gro is required (Apidianakis, 2001).
Photoreceptor differentiation in the Drosophila eye disc progresses from posterior to anterior in a wave driven by
the Hedgehog and Decapentaplegic signals. Cells mutant for the hyperplastic discs (hyd) gene misexpress both of these
signaling molecules in anterior regions of the disc, leading to premature photoreceptor differentiation and
overgrowth of surrounding tissue. hyperplastic discs encodes a HECT domain E3 ubiquitin ligase that is likely to act by targeting Cubitus interruptus and an unknown activator of
hedgehog expression for proteolysis (Lee, 2002).
Since hyd is expressed in the wing disc and is required for its
normal growth, whether its effects on wing development might
be mediated by alterations in hh expression and Ci levels was examined. In wild-type wing discs, hh is expressed uniformly throughout the
posterior compartment of the wing pouch, while dpp is expressed in
the anterior compartment in a stripe along the AP border.
Ci155 is present at high levels in a similar stripe at the AP
border and at lower levels elsewhere in the anterior compartment.
Expression of hh, dpp and Ci155 in hyd clones
remains restricted to the correct compartment. However, some hyd
mutant clones in the posterior compartment express elevated levels of
hh-lacZ. This misexpression of hh is correlated with a rounded shape and
apparent overgrowth of the clones. The only known regulator of hh
expression in the wing disc is Ci, which is restricted to the anterior
compartment by En-mediated repression; Ci76 represses hh
there. These results suggest that a Ci-independent activator of hh expression must be present in the posterior compartment and kept in check by Hyd activity (Lee, 2002).
In Drosophila, the Trithorax-group (trxG) and Polycomb-group (PcG) proteins interact with chromosomal elements, termed Cellular
Memory Modules (CMMs). By modifying chromatin, this ensures a stable heritable maintenance of the transcriptional state of
developmental regulators, like the homeotic genes, that is defined embryonically. It was asked whether such CMMs could also control
expression of genes involved in patterning imaginal discs during larval development. The results demonstrate that expression of the
hedgehog gene, once activated, is maintained by a CMM. In addition, the experiments indicate that the switching of such CMMs to an
active state during larval stages, in contrast to embryonic stages, may require specific trans-activators. Thes results suggest that the patterning of cells in particular
developmental fields in the imaginal discs does not only rely on external cues from morphogens, but also depends on the previous history of the cells, since the control
by CMMs ensures a preformatted gene expression pattern (Maurange, 2002).
Immunoprecipitation using cross-linked chromatin (XChIP) allows the mapping of in vivo DNA target sites of chromatin proteins. Because one Polycomb (PC, a member of the PcG) binding site on polytene chromosomes coincides with the
cytological position of hh at 94E, this method was applied to ask whether there are PC and GAGA factor (GAF/Trl, a member of the trxG) binding sites in the hh genomic region. These two factors had previously been found to be hallmarks of CMMs, and the GAF has been shown to be associated with some PcG complexes and necessary for the silencing function of PREs. Initially the immunoprecipitated material was hybridized to a genomic stretch of 45 kb encompassing the hh gene. This led to the identification of PC/GAF-binding sites in regions close to the transcription unit. To
further fine-map the location of the PC/GAF-binding sites,
the region around the hh gene was subdivided into 1-kb-sized PCR fragments (from 4 kb upstream of the hh transcription start site according to the transcript CG4637 from Flybase, to
13.4 kb downstream to the end of the gene). Slot-blot hybridizations of
immunoprecipitated material revealed two main sites where PC
and GAF are strongly enriched. The first site (A) is located
in a region between 0.07 and 1.06 kb upstream of the transcription
start site, whereas the second binding site (B) is found in a region
spanning the second exon of the hh gene and spreading about
0.4 kb on both sides of the exon. On both sites a
substantial overlap was observed between PC- and GAF-binding sites. The presence of this particular arrangement of PC- and GAF-binding sites in the
hh genomic region suggests that these PcG and trxG proteins
directly control hh expression (Maurange, 2002).
To investigate this at the functional level, the
accessibility of the hh promoter region to a
trans-activating factor was assessed. It is known that a PRE placed in the vicinity of an Upstream Activating Sequence (UAS) is able to counteract
GAL4 binding, preventing expression of the reporter gene (Zink,
1995; Fitzgerald, 2001). Advantage was taken of the
availability of an EP line possessing a UAS site close to the
endogenous hh transcription start site to
test whether the hh-PREs could inhibit the activation of
transcription induced by GAL4. The EP3521 line (termed here
EP-hh) possesses an EP transposon containing several UAS
sites, and is inserted in the hh promoter region (-0.36 kb). The endogenous hh gene is not transcribed in
salivary glands. By using an hs-GAL4 line, which is known to
be leaky at 25°C, weak expression of GAL4 in larval salivary glands
is observed. When hs-GAL4 is crossed to a line containing
UAS-hh integrated randomly in the genome, in situ stainings reveal that at 25°C, by the action of GAL4, the hh mRNA is
present in high amounts in all the salivary gland cells. However, when hs-GAL4 is crossed to the EP-hh line, in which the UAS sites are
juxtaposed to the presumptive PRE, hh transcription was
observed in only a very few cells situated mainly at the base of the
glands. It was reasoned, because in most cells transcription is inhibited, that the PcG proteins binding the PREs in the vicinity of
the hh promoter block the accessibility of GAL4 to the UAS sites. Accordingly, reducing the amount of some of the PcG proteins in
the cells by repeating the experiment with flies heterozygous for the
Pc3 allele or with males hemizygous for
the ph409 allele induces partial
derepression of transcription of the endogenous hh gene in a
substantial number of gland cells. These results indicate that the
repression observed in most of the salivary gland cells in the EP line
is caused by the action of the PcG proteins through their binding to the identified PREs. As such, these experiments demonstrate that the transcription of hh is directly repressed by the PcG proteins (Maurange, 2002).
Having shown that the hh gene is controlled by the PcG
proteins, it was of interest to see whether the mapped PC/GAF-binding sites could function as CMMs. Transgenic flies were produced using the
vector that allows for a test of the maintenance of the reporter gene
expression through cell divisions. A 3.4-kb
fragment, starting from position -3760 to -402 bp upstream of the
hh transcription start site (according to transcript
CG4637 from Flybase), and containing the PRE identified in the
hh promoter region, was linked to a
GAL4/UAS-inducible lacZ gene (UAS-lacZ) and
miniwhite as a reporter and transformation marker. Most of the lines obtained (15/22)
exhibit pairing-sensitive silencing, a phenomenon often associated with
PREs, when homozygous for the construct, indicated by the variegated
expression of miniwhite in the eyes. A short GAL4 pulse
produced in these flies during embryogenesis by activation of the
hs-GAL4 driver leads to homogeneous expression of the
lacZ gene in the entire embryo. When these
embryos are transferred back to 21°C and are allowed to develop to
adulthood, >90% of the offspring of the two lines tested displayed
partial or homogeneous miniwhite derepression in the eyes. These results show that the upstream 3.4-kb fragment is able to maintain the initial state of transcription of the reporter gene throughout development and therefore exhibits CMM properties (Maurange, 2002).
Having shown that the hh gene is controlled by PcG proteins
and that a DNA fragment upstream of the hh transcription start site can function as a CMM in a transgenic assay, tests were performed to see
whether the hh gene itself, in its original chromatin
environment, is regulated by CMM activity during imaginal disc
development, when cells undergo a high number of divisions. It is known
that all wing pouch cells are progenies of the cells determined at the
dorso-ventral (D-V) boundary at early larval stages. It was
hypothesized that if the transcription of a gene possessing a CMM is
activated in cells during early larval development at the D-V boundary, then transcription should be inherited to daughter cells after mitosis, resulting in expression of the gene in all wing pouch cells (Maurange, 2002).
During embryonic and larval development, En induces transcription of
hh in the posterior compartment of leg and wing imaginal discs, where the two factors substantially colocalize. Even though it is not presently clear whether En directly activates hh expression, this regulatory feature provides a tool to test for CMM activity at the hh gene. UAS-en was expressed at the D-V boundary using a
vestigial-GAL4 driver (vg-GAL4). This transgene combination allows expression of GAL4 in a thin
stripe (1 or 2 cells thick) along the D-V boundary during wing disc
development. Double stainings of such late third-instar wing
discs reveal that, surprisingly, En not only induces a thin stripe
of hh-lacZ expression (reflecting the hh expression
pattern in the P30 enhancer trap line) in cells along the D-V boundary
as expected, but also in all the posterior and anterior wing pouch
cells (except in a stripe along the A-P boundary). Strong
UAS-en expression is detected in cells at the D-V boundary
and lower levels of En in some regions of the anterior wing pouch. The repression of the endogenous en observed in some
parts of the posterior compartment is explained by the fact that high
levels of En could cause repression of the endogenous en in
the P compartment. Strikingly, the overlay of Hh-LacZ and En stainings clearly reveals large domains, in both anterior and posterior wing pouch, with strong hh expression in the absence of En, suggesting that the transcription of hh in these cells becomes independent of En. Furthermore, it is
known that En represses cubitus interruptus (ci) expression, and it has been shown that clones of A cells lacking Ci express low levels of Hh protein. These observations suggest that hh expression is activated by En at the D-V boundary in early larval development, and is inherited, even in the absence of the initial trans-activator (En), through mitosis in the cells forming, in later stages, the wing pouch (Maurange, 2002).
Alternatively, hh inheritance of transcription to daughter cells could be
explained by the existence of a positive feedback loop
allowing continuous maintenance of hh expression. This
positive feedback loop would be activated once hh is
expressed, either by autoactivation or cross-activation with another
factor, like En, for instance. To investigate this possibility, hh was
misexpressed along the D-V boundary, using the
vg-GAL4 driver and a UAS-hh transgene. Although
UAS-hh is continuously strongly expressed at the D-V boundary
from the second instar larval stage, in situ stainings do not reveal
any inheritance of hh transcription to daughter cells, because the presence of hh mRNA is always restricted to a thin row of cells at the D-V boundary, even in late third-instar wing discs. This result demonstrates that the previously observed inheritance of hh expression in wing pouch cells of vg-GAL4; UAS-en flies is not caused by autoactivation by Hh itself nor by any positive feedback loop (Maurange, 2002).
Furthermore, antibody stainings in such discs display a progressive
activation of en expression along the D-V boundary during development. In late third-instar larvae, a strong En signal is observed, testifying to the functional activity of the protein produced
by UAS-hh. Higher magnification shows that in these discs, Hh
is able to induce en expression non-cell-autonomously in a stripe of ~7 rows of cells. However, the fact that at this
stage, hh expression is only limited to a stripe of 2 rows of
cells indicates that En is no longer able to induce transcription of
the endogenous hh gene, in contrast with early larval stages. It implies that the low levels of En protein observed in some of the
anterior wing pouch cells of vg-GAL4; UAS-en
third-instar larvae is most probably caused by a late
activation of en transcription by Hh. In addition, hh
expression in these cells cannot be due to activation by low or
undetectable levels of En protein, because even
strong doses of En do not activate hh transcription in this
region at this stage of development (Maurange, 2002).
When UAS-en is misexpressed at the D-V boundary in a wild-type genetic background using vg-GAL4, it induces hh expression in most of the cells of the wing pouch except in a stripe along the A-P boundary where hh seems to be repressed. Whereas UAS-en is strongly misexpressed at the D-V boundary, the endogenous en gene is weakly misactivated in some cells of the anterior wing pouch (Maurange, 2002).
Repeating the same experiment in a genetic background hemizygous mutant
for an hypomorphic allele of polyhomeotic (ph409) leads to a broader domain of expression of hh. Remarkably, the region along the A-P boundary seems to be less refractory to activation of hh transcription, given that the territory of the repressed domain is reduced. Endogenous en is itself overexpressed in the anterior compartment. This is consistent with the findings demonstrating that en expression can be derepressed in a PcG gene mutant background. In this case in the anterior wing pouch cells, the activation of en transcription by Hh is probably more efficient than in a wild-type background because en cannot be correctly silenced by PH (Maurange, 2002).
The same experiment repeated in a genetic background now doubly
heterozygote for the trxG genes trithorax (trxE2) and brahma (brm2) consistently shows that hh
expression is activated at the D-V boundary, but can hardly be
maintained through cell divisions in the anterior compartment, because
with in situ staining, the Hh signal progressively fades away from the
D-V boundary. As expected, in such a case, en
expression in the anterior compartment is restricted to the D-V
boundary, because Hh might not be present in a sufficient amount to
activate transcription of the endogenous en gene in the
subsequent wing pouch cells (Maurange, 2002).
Furthermore, it is known that PcG-mediated silencing is enhanced at
higher temperature, and this hyperrepressed
state can be inherited through cell divisions.
Based on these observations, it was reasoned that raising embryos at 28°C
instead of 18°C would make the Pc-mediated silencing more difficult
to derepress, and influence the activation of hh transcription
by En. vg-GAL4; UAS-en embryos were allowed to
develop at 28°C until the beginning of second instar larvae, when the
D-V boundary is established in wing discs and UAS-en is
expressed there. As expected, stainings on third instar imaginal discs
reveal ectopic clones of wing pouch cells expressing hh. However, the frequency of cells expressing hh is lower than in discs of larvae grown at 18°C, indicating that the Pc-mediated silencing was harder to erase at 28°C. Nevertheless, in contrast with trxG mutant flies, once the transcription has initially been activated in this case, it is maintained in the subsequent daughter cells as suggested by the presence of clones spreading from the D-V midline to the limits of the wing pouch (Maurange, 2002).
These experiments demonstrate that once initiated by En, the
maintenance of the transcriptional state of hh to the daughter cells can be attributed to the action of the PcG and trxG proteins. It is
concluded that the CMM activity of the hh upstream region described in the transgenic assay is also efficient when considered in its natural chromatin environment and is responsible for
the inheritance of the initial transcriptional state of hh from the initiation to the completion of the wing pouch development (Maurange, 2002).
In the GAL4/UAS system, a GAL4 pulse,
when provided in larval stages, is only able to transiently activate
transcription of the reporter gene, but no heritable switching of the
Fab7-CMM is observed because transcription is lost as soon
as the trans-activator (GAL4) is down-regulated. These observations led to the hypothesis that Pc-mediated
silencing might be more stable in larval stages than in embryonic
stages, and CMMs cannot be switched to mitotically heritable activity
at these later stages. Consistent with these data,
the upstream 3.4-kb fragment showing a CMM activity cannot not be
switched to an active state through a GAL4 pulse produced during larval
stages as demonstrated by the lack of miniwhite derepression
in the eyes of the adult flies (Maurange, 2002).
However, in contrast to these experiments, the
endogenous hh CMM can be switched to an active state in larval
wing pouch cells upon an En pulse. The switch occurs in second instar
larval stages, when the D-V boundary is established through the action
of the Notch pathway and GAL4 expressed
by the vg driver. At this moment, en misexpression induces a
switch of the endogenous hh CMM at the D-V boundary to an
active state, leading to maintenance of hh transcription in
all wing pouch cells. It was of interest to test whether GAL4 is also able to
directly switch the endogenous hh CMM, in its natural
chromatin environment, in larval stages or whether this feature is
restricted to specific trans-activators like En. To perform
this experiment, the previously described line containing an
EP-element inserted into the hh promoter region (EP-hh) was used. By inducing GAL4 in the cells it is possible to
activate expression of the endogenous hh gene. It was postulated
that, by promoting transcription of the endogenous hh gene,
the hh CMM may be switched to an active state in wing pouch
cells. As observed on in situ preparations of late third-instar discs,
endogenous hh transcription is activated by GAL4 at the D-V
boundary, but is not maintained through cell division in wing pouch
cells. In comparison, also the
well-characterized Fab7-CMM is itself not switched to the
active state after GAL4 induction at the D-V boundary because expression of the reporter gene is not maintained in daughter wing
pouch cells. It is concluded that the GAL4 trans-activator is not able to switch a CMM in larval stages, although this can be carried out by the action of a gene-specific trans-activator, alone or more likely in association with other factors (Maurange, 2002).
Initially, CMMs were found to maintain the embryonically defined
expression of selector genes encoding the HOX/HOM factors, used to
established long-term cellular identities. However, CMMs appear to be
also used to freeze developmental decisions taken at later stages.
Indeed, the expression pattern of hh is subject to substantial
changes over time, depending on the morphogenetic field needed to be
patterned. Yet, the finding that hh expression, once activated, is also maintained by CMM mechanisms suggests that this type of control through chromatin-based epigenetic features is much more widespread and influenced by external signals. The results indicate that CMMs, if controlled by the correct trans-activator, can be switched and maintained in the active state at any time during development (Maurange, 2002).
Very little is known about how the gene expression pattern of cells
building compartments in imaginal discs is inherited through cell
divisions. Except for some homeotic genes, it is generally assumed that
auto- and cross-regulations allow selector and segmentation gene
expression to be maintained until the adult stage. However, this study
shows that at least in the case of hh, a cellular memory system
can take over to carry out the maintenance. It had already been
proposed that trxG proteins might be needed to allow a proper inheritance of En expression in the cells of the posterior compartment. It was also suggested that with a positive feedback loop between en and hh, these genes could achieve their own maintenance. The results presented here indicate that
this does not seem to be the case because the windows of time in which
En can activate hh and Hh can activate en seem not to
overlap over the entire wing development. During embryogenesis and
early larval development (at least until the D-V boundary is
established in wing disc), En is able to activate hh. This competence disappears later, in particular in third instar larvae, when even high amounts of En cannot activate hh transcription in at least the anterior compartment of the disc. However, Hh seems to acquire the competence to activate en transcription in late larval stages. These results are consistent with the fact that in late larval stages, the Hh gradient is able to induce a stripe of en expression at the A-P boundary, whereas En
does not in turn induce hh expression in this domain. Thus, because no feedback loop seems to exist, the data suggest that the hh CMM has a role in maintaining hh expression in the posterior domain during late stages of development (Maurange, 2002).
A domain along the A-P boundary exists that
seems to be refractory to a switch of the hh CMM to an active
state. Interestingly, it appears that in this region
Groucho and PH contribute to a strong repression system preventing
hh expression from being activated in the anterior compartment
in wild-type flies. Thus, these proteins may counteract a stable switch of the CMM to an active state. Consistent with this result is the reduction of the thickness of this refractory domain in flies mutant for ph (Maurange, 2002).
Large clones lacking en/inv expression in the posterior compartment of wing discs show reduced or no Hh protein, although this was not a universal feature of small clones. Apparently, in this
situation the loss of en/inv in the cells, especially when induced early in development, might cause a substantial
reprogramming of the gene expression pattern leading to repression of
hh, perhaps owing to the appearance of new repressors. In this
case, the initially activated CMM would not be able to overcome the repression (Maurange, 2002).
From these results, it is likely that CMMs have major direct roles in the
inheritance of the expression of hh in the development of
wing imaginal discs (it could also be imagined that the well-defined en-PRE could also act as a CMM). Furthermore, hh
and its vertebrate homologs are expressed in many other tissues during
development, in which its activation and/or maintenance are
independent of En and not yet elucidated (i.e., eye, gut, lung). Further studies will help develop an understanding of how the hh CMM may be involved in regulating the gene in different tissues (Maurange, 2002).
The finding that genes necessary to pattern imaginal discs can be
regulated by CMMs is in disagreement with models in which the
elaboration of pattern in multicellular fields is solely based on
information conferred by the local concentration of secreted signaling
molecules (morphogen model). In addition to this, it is proposed that the
establishment of a specific gene expression program in cells at various
developmental stages depends on both the information conferred by the
morphogens surrounding the cell and its history. Thus, a cell fate will
be specified by the transcriptional activation or repression of new
genes, as a result of surrounding information, as well as by the
maintenance of old transcriptional states established earlier and
inherited by CMMs through the action of the PcG and trxG proteins. It
has already been suggested that the gene optomotor-blind could
be regulated by a cellular memory mechanism in imaginal discs, although it was not directly demonstrated which mechanism
could allow inheritance of transcription (Maurange, 2002 and references therein).
It is important to note that the state of activation of a CMM does not
have to be established, once and for all, during embryogenesis, but can
be modified or stably switched later in development. This may be
especially true for genes patterning imaginal discs for which the
expression pattern is established during larval development in contrast
to homeotic genes defining the A-P axis during embryogenesis. However,
it seems that general trans-activating factors like GAL4, which are able to establish the active state of a CMM during
embryogenesis, are not able to modify or switch the CMM state later in
development, suggesting that the chromatin state of a CMM is more
difficult to reprogram at late developmental stages. During larval
stages, many cell divisions have been accomplished and cells are
getting more and more restricted in their determination state. The
chromatin could then be in a 'mature' conformation stable enough to
transmit a previously established transcriptional state despite the
potentially contradictory actions of other transcription factors found
simultaneously in the nucleus. Nevertheless, other transcription
factors such as En (in the case where En directly activates
hh) seem to be able, alone or by recruiting cofactors, to
stably switch a CMM from a repressed to an active state during larval
stages. At these stages, the switching of CMMs could require specific
factors to set epigenetic marks. It could be envisaged that the En
complex is able to attract some kind of chromatin-remodeling machinery that would have the potency to erase the memory and leave the chromatin
competent to be reprogrammed (Maurange, 2002).
In this way, it seems that the cell memory system is a complex and
dynamic process during development, in which the role of CMMs is to
heritably maintain a previously established transcriptional state until
new specific patterning cues are able to redirect the epigenetic marks
of the CMMs. However, this also makes it quite clear that during the
establishment of a morphogenetic field, besides the local specifying
signaling events, the previous history of a determining gene should be
taken into account (Maurange, 2002).
Steroid hormones fulfil important functions in animal development. In Drosophila, ecdysone triggers molting and metamorphosis through its effects on gene expression. Ecdysone works by binding to a nuclear receptor, EcR, which heterodimerizes with the retinoid X receptor homolog Ultraspiracle. Both partners are required for binding to ligand or DNA. Like most DNA-binding transcription factors, nuclear receptors activate or repress gene expression by recruiting co-regulators, some of which function as chromatin-modifying complexes. For example, p160 class coactivators associate with histone acetyltransferases and arginine histone methyltransferases. The Trithorax-related gene of Drosophila encodes the SET domain protein TRR. TRR is a histone methyltransferase capable of trimethylating lysine 4 of histone H3 (H3-K4). trr acts upstream of hedgehog (hh) in progression of the morphogenetic furrow, and is required for retinal differentiation. Mutations in trr interact in eye development with EcR, and EcR and TRR can be co-immunoprecipitated on ecdysone treatment. TRR, EcR and trimethylated H3-K4 are detected at the ecdysone-inducible promoters of hh and BR-C in cultured cells, and H3-K4 trimethylation at these promoters is decreased in embryos lacking a functional copy of trr. It is proposed that TRR functions as a coactivator of EcR by altering the chromatin structure at ecdysone-responsive promoters (Sedkov, 2003).
The cohesin complex is a key player in regulating cell division. Cohesin proteins SMC1, SMC3, Rad21, and stromalin (SA), along with associated proteins Nipped-B, Pds5, and EcoI, maintain sister chromatid cohesion before segregation to daughter cells during anaphase. Recent chromatin immunoprecipitation (ChIP) data reveal extensive overlap of Nipped-B and cohesin components with RNA polymerase II binding at active genes in Drosophila. These and other data strongly suggest a role for cohesion in transcription; however, there is no clear evidence for any specific mechanisms by which cohesin and associated proteins regulate transcription. This study reports a link between cohesin components and trithorax group (trxG) function, thus implicating these proteins in transcription activation and/or elongation. The Drosophila Rad21 protein is encoded by verthandi (vtd), a member of the trxG gene family that is also involved in regulating the hedgehog (hh) gene. In addition, mutations in the associated protein Nipped-B show similar trxG activity i.e., like vtd, they act as dominant suppressors of Pc and hhMrt without impairing cell division. These results provide a framework to further investigate how cohesin and associated components might regulate transcription (Hallson, 2008).
In eukaryotic mitosis, accurate chromosome segregation requires paired sister chromatids to attach to opposite spindle poles. Sister chromatids are held together by the cohesin protein complex, which consists of four core subunits, Rad21/SCC1, stromalin (SA) and structural maintenance of chromosome (SMC) proteins SMC1 and SMC3. A widely accepted model postulates that cohesin forms a ring-like structure via interaction of the N- and C-termini of Rad21 with a SMC1/SMC3 heterodimer. With the participation of SCC2/Nipped-B, SCC4, EcoI/Ctf7, and Pds5 proteins, sister-chromatid cohesion is maintained until the onset of mitosis. Cleavage of Rad21 and the resulting removal of cohesin then allow separation of sister chromatids in anaphase. Mutation of genes encoding these subunits leads to errors in chromosome segregation and aneuploidy, which are hallmarks of cancer and a leading cause of birth defects in humans (Hallson, 2008).
Given the highly conserved role for cohesin in sister chromatid cohesion, it was unexpected to discover that cohesin and associated proteins might also play a distinct, independent role in regulating gene expression. Reduction in Nipped-B expression in Drosophila affects expression of the cut and Ultrabithorax genes, and mutations in the human orthologue, NIPBL, result in Cornelia de Lange Syndrome. In zebrafish, mutations in rad21 or Smc3 affect embryonic runx gene transcription in heterozygous mutant animals without compromising cell division, suggesting that these proteins may have functions in transcription that are distinct from a mitotic role. Recently, extensive overlap has been found of Nipped-B and cohesin components with RNA polymerase II binding at active genes and apparent exclusion from genes silenced by Polycomb group (PcG) genes. This intriguing chromatin immunoprecipitation (ChIP) result strongly suggests a role in transcription for cohesin and Nipped-B, although the mechanisms are unknown (Hallson, 2008).
Trithorax group (trxG) genes encode proteins implicated in transcriptional regulation. These genes were initially characterized as regulators of homeotic genes in Drosophila. The trxG genes are required to maintain activation of homeotic and other genes; many that have been molecularly characterized encode members of multimeric complexes with roles in transcriptional initiation and/or elongation. Typically, mutations in trxG genes suppress the phenotypes of mutations in PcG genes, whose function is to maintain the repressed state of homeotic genes and other developmentally important genes like hedgehog (hh), a gene required for cell signaling (Hallson, 2008).
As part of work toward a functional annotation of heterochromatin of Drosophila, the verthandi (vtd) locus, a member of the trxG gene family with Suppressor of Polycomb [Su(Pc)] activity, was characterized (Kennison, 1988; Schulze, 2001). The vtd locus also affects hh expression; vtd mutations are dominant suppressors of Moonrat (Mrt), a dominant gain of function allele of hh (Schulze, 2001; Felsenfeld, 1995). However, because of its location deep within the centric heterochromatin of the left arm of chromosome, vtd has resisted characterization at the molecular level (Hallson, 2008).
This study reports that vtd mutations, isolated on the basis of their trxG phenotypes, map to the gene encoding the cohesin subunit Rad21 and exhibit corresponding defects in mitosis and sister chromatid cohesion. Mutations in Nipped-B also show trxG phenotypes, and as is the case for vtd, heterozygous mutant flies show trxG phenotypes without significantly affecting cell division. These results provide a link between sister chromatid cohesion proteins and trxG functions, thus suggesting that cohesion factors may act by facilitating transcription activation and/or elongation (Hallson, 2008).
Alleles of vtd have lesions in rad21, mutations or knockdowns of rad21 have vtd phenotypes, and vice versa, and a transgene containing rad21 rescues the lethality of vtd. It is also noteworthy that reductions in Rad21 or Nipped-B dosage alter gene expression without seriously affecting chromatid cohesion, suggesting that these may be separable functions for cohesin and associated proteins. Evidence has accumulated that cohesin and associated proteins have important roles in gene regulation, but the functional basis for this has been unclear. The simplest model that explains the existing data is that Rad21, like most other trxG proteins, facilitates transcription (Hallson, 2008).
In Drosophila, many trxG proteins are subunits of complexes with diverse roles in transcriptional activation. Trx and Ash1 encode SET domain proteins that methylate lysine 4 of histone H3 (H3K4), and Ash2 is a member of a complex that also methylates H3K4. Other trxG proteins (e.g., Brahma, Osa, Moira, Kismet) are members of ATP-dependent nucleosome remodeling complexes. However, despite concerted efforts from many laboratories, the precise mechanisms by which trxG proteins regulate transcription remain unclear. In addition to chromatin modification, trxG proteins appear to be directly involved in recruiting factors required for transcription elongation, and noncoding RNAs may also play a role in regulating some of the affected genes (Hallson, 2008).
The hypothesis that cohesin facilitates transcription is supported by the results of a recent genome-wide ChIP study, which shows preferential binding of Nipped-B and the cohesin subunits SMC1 and SA to transcribed regions, overlapping with RNA polymerase II (Pol II) binding sites (Misulovin, 2008). The colocalization of Nipped-B with cohesin on chromosomes, and physical association with SA and Rad21 in extracts further suggests that Nipped-B and cohesin act together (Hallson, 2008).
There are strong correlations between binding of cohesin components and active gene expression. The dosage sensitive suppression of the hhMrt gain of function allele by both vtd and Nipped-B mutations suggests that Nipped-B and cohesin both promote expression of hh. It is unknown, however, if this effect is direct. Cohesin or Nipped-B do not bind to the hh gene in any of the three cell lines examined, however, in at least two of these, PcG proteins actively silence hh. Genome-wide, PcG silencing and the resulting histone H3 lysine 27 trimethylation strongly anti-correlates with Nipped-B and cohesin binding. Thus, it would not be expected that cohesin binds hh in these cell lines even if it directly regulates hh. For example, although Nipped-B regulates Ubx expression in vivo, Nipped-B and cohesin are excluded from the silenced Ubx and Abd-A genes in Sg4 cells, but bind to the transcribed Abd-B gene. In cells in which Abd-B is silenced, cohesin does not bind to the Abd-B promoter region. Thus, it remains possible that Nipped-B and cohesin directly stimulate hh transcription in vivo (Hallson, 2008).
Identification of loss of function zebrafish rad21 alleles in a genetic screen for mutations that reduce expression of runx genes also suggests that cohesin promotes gene expression, but again, it is unknown if this effect is direct (Horsfield, 2007). Stronger evidence supporting the idea that cohesin directly stimulates transcription arises from a recent study on axon pruning in the Drosophila mushroom body (Schuldiner, 2008). In this study, loss of function alleles of the Smc1 and SA genes were isolated in a screen for mutations that block pruning. The lack of pruning correlated with reduced expression of the ecdysone receptor (EcR) gene, and could be partially rescued by ectopic EcR expression. Nipped-B and cohesin bind to the transcribed portion of the EcR gene in all three cell lines examined, including the ML-DmBG3 line derived from third instar central nervous system, suggesting that they directly facilitate EcR expression (Hallson, 2008).
The question remains as to whether the same cohesin complexes required for cohesion of sister chromatids also function in transcription regulation, or whether, analogous to trxG proteins, different cohesin subunits have different functions in transcription, presumably because they are members of different complexes. One might conclude the latter based upon the observation that reductions in Rad21, SA, or SMC1 all increase cut expression, whereas decreases in Nipped-B reduce cut expression. These effects are likely direct because cohesin and Nipped-B bind to a 180 kb region that encompasses the entire upstream regulatory and transcribed regions of cut in ML-DmBG3 cells. The expression of RNAi transgenes encoding for SA and Rad21 decreases the severity of the cutK allele, whereas Nipped-B mutations enhance the cutK phenotype, also suggesting that they have opposite effects at cut. Finally, in contrast to results with Nipped-B mutations, no consistent effects on cut expression were observed for vtd mutant heterozygotes; moreover, mutations in vtd and Nipped-B both suppress the phenotypes of Pc4 and Mrt, but mutations in Smc1 or pds5 did not. Similarly, Dorsett (2005) has reported that null alleles of the cohesion factors sans and deco have no effect on the expression of cut when a functional chromosomal copy is present. Based on all of the above evidence, one might therefore conclude that different cohesin components may act differentially, possibly because, like trxG proteins, they are members of different regulatory complexes (Hallson, 2008).
However, it is also possible that the same cohesin complex involved in chromatid cohesion also regulates transcription, if binding at different loci results in different, gene specific consequences. Thus, in cut, which is activated by a remote wing margin enhancer located >80 kb upstream of the promoter, it has been proposed that cohesin could inhibit long range activation, and that Nipped-B facilitates activation by maintaining a dynamic cohesin binding equilibrium (Dorsett, 2005). In other genes, such as EcR or hh, cohesin might help maintain open chromatin to facilitate transcription by encircling a 10-nm fiber and preventing refolding to a higher order structure. As for the differences observed in the genetics of cohesin components, there are likewise other plausible explanations: differences in genetic background of mutant lines tested, differences in maternal expression/loading of required gene products in different heterozygous flies, or the possibility that the cutK or Pc alleles are less sensitive to changes in rad21/vtd, SMC1, or pds5 gene dosage than they are to the gene dosage of Nipped-B. Consistent with this idea, effects of rad21 dosage on cut expression were observed when RNAi was used to deplete rad21 mRNA, presumably to levels lower than those available in vtd(+) heterozygotes. It was also reported that Nipped-B expression is not directly proportional to gene dosage. The data in this study also show that reductions in Nipped-B and rad21 dosage act in the same direction i.e., suppress Mrt and Pc, suggesting that both genes may contribute to gene activation. The fact that both the rad21 and Nipped-B genes are resident within a late-replicating, heterochromatic environment may also explain some differences in outcomes of genetics tests of cohesin subunit function (Hallson, 2008).
These results provide a link between cohesin binding and trxG gene function. It will be an interesting challenge for the future to determine how components involved in chromatid cohesion act at the molecular level to regulate transcription, particularly given other very recent evidence implicating cohesin in gene regulation. The discovery that vtd encodes the Rad21 cohesin subunit expands the known roles of cohesin and Nipped-B in Drosophila development to include regulation of hh, which like cut, Ubx, and EcR, has many developmental roles. Similar modulation of key developmental regulators in humans, each with multiple roles, could explain why Cornelia de Lange syndrome patients have multiple diverse developmental deficits (Hallson, 2008).
The lymph gland is a specialized organ for hematopoiesis, utilized during larval development in Drosophila. This tissue is composed of distinct cellular domains populated by blood cell progenitors (the medullary zone), niche cells that regulate the choice between progenitor quiescence and hemocyte differentiation [the posterior signaling center (PSC)], and mature blood cells of distinct lineages (the cortical zone). Cells of the PSC express the Hedgehog (Hh) signaling molecule, which instructs cells within the neighboring medullary zone to maintain a hematopoietic precursor state while preventing hemocyte differentiation. As a means to understand the regulatory mechanisms controlling Hh production, a PSC-active transcriptional enhancer was characterized that drives hh expression in supportive niche cells. The findings indicate that a combination of positive and negative transcriptional inputs program the precise PSC expression of the instructive Hh signal. The GATA factor Serpent (Srp) is essential for hh activation in niche cells, whereas the Suppressor of Hairless [Su(H)] and U-shaped (Ush) transcriptional regulators prevent hh expression in blood cell progenitors and differentiated hemocytes. Furthermore, Srp function is required for the proper differentiation of niche cells. Phenotypic analyses also indicated that the normal activity of all three transcriptional regulators is essential for maintaining the progenitor population and preventing premature hemocyte differentiation. Together, these studies provide mechanistic insights into hh transcriptional regulation in hematopoietic progenitor niche cells, and demonstrate the requirement of the Srp, Su(H) and Ush proteins in the control of niche cell differentiation and blood cell precursor maintenance (Tokusumi, 2010).
The lymph gland hematopoietic organ is formed near the end of embryogenesis from two clusters of cells derived from anterior cardiogenic mesoderm (Crozatier, 2004; Mandal, 2004). About 20 pairs of hemangioblast-like cells give rise to three distinct lineages that will form the lymph glands and anterior part of the dorsal vessel. Notch (N) pathway signaling serves as the genetic switch that differentially programs these progenitors towards cell fates that generate the lymph glands (blood lineage), heart tube (vascular lineage), or heart tube-associated pericardial cells (nephrocytic lineage). An essential requirement has also been proven for Tailup (Islet1) in lymph gland formation, in which it functions as an early-acting regulator of serpent, odd-skipped and Hand hematopoietic transcription factor gene expression (Tokusumi, 2010).
By the end of the third larval instar, each anterior lymph gland is composed of three morphologically and molecularly distinct regions (Jung, 2005). The posterior signaling center (PSC) is a cellular domain formed during late embryogenesis due to the specification function of the homeotic gene Antennapedia (Antp) (Mandal, 2007) and the maintenance function of Collier, the Drosophila ortholog of the vertebrate transcription factor early B-cell factor. PSC cells selectively express the Hedgehog (Hh) and Serrate (Ser) signaling molecules and extend numerous thin filopodia into the neighboring medullary zone. This latter lymph gland domain is populated by undifferentiated and slowly proliferating blood cell progenitors (Mandal, 2007). Prohemocytes within the medullary zone express the Hh receptor Patched (Ptc) and the Hh pathway transcriptional effector Cubitus interruptus (Ci). Medullary zone cells also express components of the Jak/Stat signaling pathway. By contrast, the third lymph gland domain -- the cortical zone -- solely contains differentiating and mature hemocytes, such as plasmatocytes and crystal cells. Upon wasp parasitization, or in certain altered genetic backgrounds, lamellocytes will also appear in the cortical zone as a third type of differentiated hemocyte (Tokusumi, 2010).
Two independent studies have provided compelling data to support the contention that the PSC functions as a hematopoietic progenitor niche within the lymph gland, with this cellular domain being essential for maintaining normal hemocyte homeostasis (Krzemien, 2007; Mandal, 2007). These investigations showed that communication between the PSC and prohemocytes present in the medullary zone is crucial for the preservation of the progenitor population and to prevent these cells from becoming abnormally programmed to differentiate into mature hemocytes. Seminal findings from these studies can be summarized as follows: Col expression must be restricted to the PSC by the localized expression of Ser; Hh must be expressed selectively in the PSC, coupled with the non-autonomous activation of the Hh signaling pathway in prohemocytes of the medullary zone; and the PSC triggers activation of the Jak/Stat pathway within cells of the medullary zone. With the perturbation of any of these molecular events, the precursor population of the medullary zone is lost owing to the premature differentiation of hemocytes, which swell the cortical zone. Although the exact interrelationship of Ser, Hh and Jak/Stat signaling within the lymph gland is currently unknown, the cytoplasmic extensions emanating from PSC cells might facilitate instructive signaling between these niche cells and hematopoietic progenitors present in the medullary zone (Krzemien, 2007; Mandal, 2007). A more recent study showed that components of the Wingless (Wg) signaling pathway are expressed in the stem-like prohemocytes to reciprocally regulate the proliferation and maintenance of cells within the supportive PSC niche (Sinenko, 2009). The cellular organization and molecular signaling of the Drosophila lymph gland are remarkably similar to those of the hematopoietic stem cell niches of vertebrate animals, including several mammals (Tokusumi, 2010 and references therein).
Through detailed molecular and gene expression analyses this study has identified the PSC-active transcriptional enhancer within hh intron 1 and delimited its location to a minimal 190 bp region. The hh enhancer-GFP transgene faithfully recapitulates the niche cell expression of Hh derived from the endogenous gene, as double-labeling experiments with the GFP marker and Antp or Hh show a clear co-expression in PSC domain cells. Appropriately, GFP expression is not detected in Antp loss-of-function or TCFDN genetic backgrounds, which culminate in an absence of niche cells from the lymph gland. The hematopoietic GATA factor Srp serves as a positive activator of hh PSC expression, as mutation of two evolutionarily conserved GATA elements in the enhancer abrogates its function and Srp functional knockdown via srp RNAi results in hh enhancer-GFP transgene inactivity and the absence of Hh protein expression. An additional intriguing phenotype was observed in lymph glands expressing the srp RNAi transgene, that being a strong reduction in the number of filopodial extensions emerging from cells of the PSC. This phenotype suggests a functional role for Srp in the correct differentiation of niche cells, via a requirement for normal Hh presentation from these cells and/or the transcriptional regulation by Srp of additional genes needed for the formation of filopodia (Tokusumi, 2010).
As Srp accumulates in all cells of the lymph gland, a question arose as to how hh expression is restricted to cells of the PSC. This paradox could be explained by a mechanism in which hh expression is also under some means of negative transcriptional control in non-PSC cells of the lymph gland. This possibility proved to be correct, with the analyses identifying two negative regulators of hh lymph gland expression. The first is Su(H). Mutation of the evolutionarily conserved GTGGGAA element, a predicted recognition sequence for this transcriptional repressor, resulted in an expanded activity of the hh PSC enhancer-GFP transgene; that is, the de novo appearance of GFP was observed in prohemocytes of the medullary zone. Likewise, ectopic medullary zone expression of the wild-type PSC enhancer-GFP transgene and of Hh protein was seen in lymph glands mutant for Su(H). These findings, coupled with the detection of Su(H) in blood cell progenitors, strongly implicate this factor as a transcriptional repressor of the hh PSC enhancer, restricting its expression to niche cells (Tokusumi, 2010).
Additional studies identified Ush as a second negative regulator of hh expression. Ush is expressed in most cells of the lymph gland, with the exception of those cells resident within the PSC domain. Previous research demonstrated that ush expression in the lymph gland is under the positive control of both Srp. Why Ush protein fails to be expressed in the PSC remains to be determined. Forced expression of ush in niche cells resulted in inactivation of the hh PSC enhancer and reduced the formation of filopodia. It was hypothesized that Ush might be forming an inhibitory complex with the SrpNC protein, changing Srp from a positive transcriptional activator to a negative regulator of hh lymph gland expression. Such a mechanism has been demonstrated previously in the negative regulation by Ush of crystal cell lineage commitment. The expansion of wild-type hh enhancer-GFP transgene and Hh protein expression to prohemocytes within the medullary zone and to differentiated hemocytes within the cortical zone in lymph glands mutant for ush is also supportive of Ush functioning as a negative regulator of hh expression (Tokusumi, 2010).
Bringing these results together, a model can be proposed for the regulatory events that culminate in the precise expression of the vital Hh signaling molecule in niche cells. Srp is a direct transcriptional activator of hh in the lymph gland and Hh protein is detected in niche cells due to this activity. hh expression is inhibited in prohemocytes of the medullary zone by Su(H) action, while a repressive SrpNC-Ush transcriptional complex prevents Srp from activating hh expression in prohemocytes and in differentiated hemocytes of the medullary zone and cortical zone. Together, these positive and negative modes of regulation would allow for the niche cell-specific expression of Hh and facilitate the localized presentation of this crucial signaling molecule to neighboring hematopoietic progenitors (Tokusumi, 2010).
The identification of Srp and Su(H) as key regulators of Hh expression in the larval hematopoietic organ prompted an investigation into the functional requirement of these proteins in the control of blood cell homeostasis. Since Srp knockdown by RNAi leads to an absence of the crucial Hh signal, it was not surprising to find that normal Srp function is required for prohemocyte maintenance and the control of hemocyte differentiation within the lymph gland; that is, a severe reduction of Ptc-positive hematopoietic progenitors and a strong increase in differentiated plasmatocytes and crystal cells was observed in srp mutant tissue (Tokusumi, 2010).
Likewise, Ptc-positive prohemocytes were lost and large numbers of plasmatocytes were prematurely formed in Su(H) mutant lymph glands. This disruption of prohemocyte maintenance occurred even though Hh protein expression was expanded throughout the medullary zone. This raised the question as to why expanded Hh protein and possible Hh pathway activation did not increase the progenitor population in Su(H) mutant lymph glands, instead of the observed loss of prohemocytes and appearance of differentiated plasmatocytes. One explanation might be that the PSC niche is not expanded in Su(H) mutant lymph glands and Hh might only function in promoting blood cell precursor maintenance within the context of the highly ordered progenitor-niche microenvironment. It has been hypothesized that the filopodial extensions that emanate from differentiated niche cells are crucial for Hh signal transduction from the PSC to progenitor cells of the medullary zone. The possibility exists that ectopic Hh protein, which is not produced or presented by niche cells, is unable to positively regulate prohemocyte homeostasis. An experimental result consistent with this hypothesis is that expression of UAS-hh under the control of the medullary zone-specific tepIV-Gal4 driver failed to expand the blood cell progenitor population. A second possibility is that the Hh pathway transcriptional effector Ci might require the co-function of Su(H) in its control of prohemocyte maintenance. This model would predict that, in the absence of Su(H) function, Hh signaling would be less (or non) effective in controlling the genetic and cellular events needed for the maintenance of the prohemocyte state. Third, Su(H) might regulate additional target genes, the expression (or repression) of which is crucial for normal blood cell precursor maintenance and the prevention of premature hemocyte differentiation. Finally, it cannot be ruled out that the expression of ectopic Hh in medullary zone cells, in the context of the adverse effects of Su(H) loss of function in these cells, culminates in the disruption of normal Hh pathway signaling due to an unforeseen dominant-negative effect (Tokusumi, 2010).
In summary, these findings add significantly to knowledge of hematopoietic transcription factors that function to control stem-like progenitor maintenance and blood cell differentiation in the lymph gland. An additional conclusion from these studies is that the hh enhancer-GFP transgene can serve as a beneficial reagent to identify and characterize genes and physiological conditions that control the cellular organization of the hematopoietic progenitor-niche cell microenvironment. RNAi-based genetic screens could be undertaken using this high-precision marker to determine signaling pathways and/or environmental stress conditions that might alter niche cell number and function, leading to an alteration in hematopoietic progenitor maintenance coupled with the robust production of differentiated blood cells. Much remains to be determined about the regulated control of these critical hematopoietic changes and their likely relevance to hematopoietic stem cell-niche interactions in mammals (Tokusumi, 2010).
Hematopoiesis occurs in two phases in Drosophila, with the first completed during embryogenesis and the second accomplished during larval development. The lymph gland serves as the venue for the final hematopoietic program, with this larval tissue well-studied as to its cellular organization and genetic regulation. While the medullary zone contains stem-like hematopoietic progenitors, the posterior signaling center (PSC) functions as a niche microenvironment essential for controlling the decision between progenitor maintenance versus cellular differentiation. This study used PSC-specific GAL4 driver and UAS-gene RNAi strains, to selectively knockdown individual gene functions in PSC cells. The effect of abrogating the function of 820 genes was assessed as to their requirement for niche cell production and differentiation. 100 genes were shown to be essential for normal niche development, with various loci placed into sub-groups based on the functions of their encoded protein products and known genetic interactions. For members of three of these groups, loss- and gain-of-function phenotypes were characterized. Gene function knockdown of members of the BAP chromatin-remodeling complex resulted in niche cells that do not express the hedgehog (hh) gene and fail to differentiate filopodia believed important for Hh signaling from the niche to progenitors. Abrogating gene function of various members of the insulin-like growth factor and TOR signaling pathways resulted in anomalous PSC cell production, leading to a defective niche organization. Further analysis of the Pten, TSC1, and TSC2 tumor suppressor genes demonstrated their loss-of-function condition resulted in severely altered blood cell homeostasis, including the abundant production of lamellocytes, specialized hemocytes involved in innate immune responses. Together, this cell-specific RNAi knockdown survey and mutant phenotype analyses identified multiple genes and their regulatory networks required for the normal organization and function of the hematopoietic progenitor niche within the lymph gland (Tokusumi, 2012).
The discovery of a stem cell-like hematopoietic progenitor niche in Drosophila represents a significant contribution of this model organism to the study of stem cell biology and blood cell development. Extensive findings support the belief that the PSC functions as the niche within the larval lymph gland, with this cellular domain essential to the control of blood cell homeostasis within this hematopoietic organ. Molecular communication between the PSC and prohemocytes present in the lymph gland medullary zone is crucial for controlling the decision as to maintaining a pluri-potent progenitor state versus initiating a hemocyte differentiation program. This lymph gland cellular organization and the signaling pathways controlling hematopoieis therein have prompted several researchers in the field to point out its functional similarity to the HSC niche present in mammalian (Tokusumi, 2012).
As a means to discover new information on genetic and molecular mechanisms at work within a hematopoietic progenitor niche microenvironment, an RNAi-based loss-of-function analysis was carried out to selectively eliminate individual gene functions in PSC cells. The effect of knocking-down the function of 820 lymph gland-expressed genes was assessed as to their requirement for niche cell production and differentiation, and 100 of these genes were shown to be required for one or more aspects of niche development. The distinguishable phenotypes observed in these analyses included change in number of Hh-expressing cells, change in number of Antp-expressing cells, scattered and disorganized niche cells, rounded cells lacking extended filopodia, and lamellocyte induction in the absence of a normal PSC. The genes were placed into sub-groups based on their coding capacity and known genetic interactions, and the phenotypes associated with the functional knockdown of members of three of these gene regulatory networks were characterized (Tokusumi, 2012).
Previous studies have demonstrated that the PSC-specific ablation of srp function resulted in a lack of expression of the crucial Hh signaling molecule in these cells, the inactivity of the hh-GFP transgene in the niche, failure of niche cells to properly differentiate filopodial extensions, and the loss of hematopoietic progenitor maintenance coupled with the abundant production of differentiated hemocytes. Thus it was intriguing when it was observed that RNAi function knockdown of several members of the BAP chromatin-remodeling complex resulted in the identical phenotypes of lack of hh-GFP transgene expression and absence of filopodia formation in PSC cells. A convincing functional interaction was observed between srp encoding the hematopoietic GATA factor and osa encoding the DNA-binding Trithorax group protein in the inability of niche cells to express hh-GFP in double-heterozygous mutant lymph glands. Thus one working model is that the BAP chromatin-remodeling complex establishes a chromatin environment around and within the hh gene that allows access of the Srp transcriptional activator to the PSC-specific enhancer, facilitating Hh expression in these cells. It will be of interest to determine if there exists a direct physical interaction between Osa and Srp in this positive regulation of hh niche transcription and if so, what are the functional domains of the proteins essential for this critical regulatory event in progenitor cell maintenance. It is also likely that these functional interactions are important for Srp's transcriptional regulation of additional genes needed for the formation of niche cell filapodia (Tokusumi, 2012).
In this study, a total of 33 gain- or loss-of-function genetic conditions were analyzed that enhanced or eliminated the function of various positive or negatively-acting components of the insulin-like growth factor and TOR signaling pathways. A conclusion to be drawn from these analyses is that genetic conditions that have an end effect of enhancing translation activity and protein synthesis result in supernumerary PSC cell numbers in disorganized niche domains, while conditions that promote growth suppression lead to substantially reduced populations of niche cells. The same conclusion was obtained from recent studies performed by Benmimoun (2012). The Wg and Dpp signaling pathways have also been shown to be important for the formation of a PSC niche of normal size and function, and it is possible that the insulin-like growth factor and TOR signaling networks regulate the translation of one or more members of the Wg and/or Dpp pathways. These analyses have also shown that mutation of the Pten, TSC1, and TSC2 tumor suppressor genes results in severely altered blood cell homeostasis in lymph glands and in circulation, including the prolific induction of lamellocytes. A recent report demonstrated that in response to larval wasp infestation, the PSC secretes the Spitz cytokine signal, which triggers an EGFR-mediated signal transduction cascade in the generation of dpERK-positive lamellocytes in circulation. As dpERK activity is known to inhibit TSC2 function, inactivation of the TSC complex may be a downstream regulatory event leading to robust lamellocyte production in larvae in response to wasp immune challenge (Tokusumi, 2012).
To summarize, an RNAi-based loss-of-function analysis has been undertaken to identify new genes and their signaling networks vital for normal PSC niche formation and function. While information has been gained on the requirements of three such networks for PSC development and blood cell homeostasis within the lymph gland, numerous other genes have been discovered that likewise play key roles in these hematopoietic events. Their characterization is warranted as well to further enhance knowledge of genetic and molecular mechanisms at work within an accessible and easily manipulated hematopoietic progenitor niche microenvironment (Tokusumi, 2012).
The development of the Drosophila wing depends on its subdivision into anterior and posterior compartments, which constitute two independent cell lineages since their origin in the embryonic ectoderm. The anterior-posterior compartment boundary is the place where signaling by the Hedgehog pathway takes place, and this requires pathway activation in anterior cells by ligand expressed exclusively in posterior cells. Several mechanisms ensure the confinement of hedgehog expression to posterior cells, including repression by Cubitus interruptus, the co-repressor Groucho and Master of thick veins. This work identifies Kismet, a chromodomain-containing protein of the SNF2-like family of ATPases, as a novel component of the hedgehog transcriptional repression mechanism in anterior compartment cells. In kismet mutants, hedgehog is ectopically expressed in a domain of anterior cells close to the anterior-posterior compartment boundary, causing inappropriate activation of the pathway and changes in the development of the central region of the wing. The contribution of Kismet to the silencing of hedgehog expression is limited to anterior cells with low levels of the repressor form of Cubitus interruptus. Knockdown of CHD8, the kismet homolog in Xenopus tropicalis, is also associated with ectopic sonic hedgehog expression and up-regulation of one of its target genes in the eye, Pax2, indicating the evolutionary conservation of Kismet/CHD8 function in negatively controlling hedgehog expression (Terriente-Félix, 2011).
This work has used a genetic approach to analyse the role of Kis in the patterning of the Drosophila wing. The main finding is that Kis is required, among other processes, for the repression of hh in anterior cells close to the A/P boundary. This conclusion is based in the phenotype of kis clones in the wing, the changes in the expression of Hh-target genes in these mutant cells, and more directly, in the observation of hh ectopic expression in wing discs of kis loss-of-function alleles. A similar requirement was identified for CHD8, a Kis homolog in X. tropicalis, suggesting conservation in the mechanisms of hh/Shh regulation during evolution. Finally, it was determined that the repression of hh mediated by Kis is not needed when the repressor form of Ci, Ci75, is present in the cell (Terriente-Félix, 2011).
As a way to identify the functional requirements of Kis, the phenotype caused by several kis loss-of-function alleles was studied. For six of these alleles similar results were found , and consequently this study will refer to all of them together. Two main alterations were identified in wings mutant for kis: the formation of ectopic veins and defects in the patterning of the central region of the wing. These phenotypes are diagnostic of failures in the regulation of the level or domain of activity of two signaling pathways, EGFR and Hh, and it is likely that they identify independent requirements for Kis in the modulation of these pathways during wing development. The formation of ectopic veins is observed in all situations when the activity of the EGFR pathway is not correctly restricted to the positions occupied by the normal veins. Thus, over-activation of EGFR and loss-of-function alleles in a variety of the known antagonists of the pathway, such as MKP3 and sprouty, result in the formation of ectopic veins in similar positions to those observed in kis alleles. The implication of Kis in regulating EGFR signaling is also supported by the identification of kis alleles in several genetic screens searching for modifiers of EGFR phenotypes in different developmental stages and tissues. For example, kis was found in a screen of kinase-suppressor of Ras modifiers in the eye and in a screen of EGFR modifiers affecting border cells migration during oogenesis. kis alleles were also identified as modifiers of the Notch phenotype caused by dominant-negative mastermind over-expression in the wing disc. However, in this study the vein phenotype of loss of kis does not appear related to Notch signalling, because Notch-related defects such as thickened veins or loss of wing margin were never pbserved in kis mutant wings (Terriente-Félix, 2011).
The implication of Kis in the modulation of EGFR signalling has never been directly analyzed. However, it is interesting to notice in this context that the two SNF2-familiy chromatin-remodeling complexes containing Brm as the catalytic subunit are also involved in this pathway. In this manner, the BAP (Brahma associated proteins) and PBAP (Polybromo-Brahma associated proteins) complexes are required to modulate positively or negatively, respectively, EGFR signaling in the wing. Furthermore, Brm and Kis share some domain architecture, as they both have an ATPase domain N-terminal to a BRK (Brahma related to Kismet) domain. They also bind to identical sites in polytenic chromosomes, and both are part of the Trithorax group of genes (TrxG). Therefore, it is possible that BAP, PBAP and Kis participate in the transcriptional regulation of EGFR targets using a conserved mechanism involving chromatin modifications in collaboration with other PcG and TrxG proteins. Similarly, the function of CHD7 in the regulation of neural crest cell identities requires the function of PBAF (Polybromo, Brg1-Associated Factors), the homolog of PBAP, suggesting that Kis and its vertebrate homologs can collaborate with other SNF2-helicases (Terriente-Félix, 2011).
This work focussed in the second phenotype observed in kis mutant cells, which consists in duplications of the L3 vein and increase in the distance between the L3 and L4 veins. These two defects are limited to the pattern elements regulated by the Hh pathway, and correspond to an enlargement of the domain of Hh signalling. Hh activity and diffusion in anterior cells are linked to each other by the function of the receptor Ptc, because ptc expression is activated by Ci155 and Hh diffusion is prevented by Ptc. In this manner the phenotype of kis clones might be caused by a reduction in Hh signaling leading to loss of Ptc and consequently to an increase in the range of Hh diffusion. However, it was found that kis clones do not affect the normal domain of ptc expression, but cause ectopic ptc expression in cells localised anterior to this domain. This observation indicates that Hh signaling itself is not affected in kis mutant clones, and points towards changes in hh expression as a likely cause for the kis phenotype. Indeed, ectopic expression of hh is found in kis mutant cells localised in the anterior compartment close to the A/P boundary. Furthermore, ectopic and cell autonomous expression of hh-lacZ reporters is observed in small kis clones (less than 5 cells) localised in this region. These results suggest that Kis is needed during wing imaginal development to maintain hh expression turned-off in anterior cells that, due to Hh signalling, don't have enough levels of the repressor Ci75. This requirement for Kis readily explains both the phenotype of kis alleles in the central region of the wing and the changes in the expression of hh and its target genes observed in kis mutant cells. Interestingly, this activity of Kis in the repression of hh expression appears conserved in its Xenopus homolog CHD8 (Terriente-Félix, 2011).
The regulation of hh expression relies on a combination of several mechanisms acting in different domains of the wing disc. First, Ci75, a form of Ci that is produced by proteolysis from Ci155 when Hh signaling is not active, represses hh in anterior cells. In those anterior cells exposed to Hh the levels of Ci75 are low, and in this domain a second mechanism involving Mtv and Gro represses hh expression. Finally, several genes of the Polycomb group (PcG), such as Polycomb (Pc) and Polyhomeotic (Ph) are also involved in the repression of hh transcription in a tissue-specific manner. In this way, the PcG is involved in maintaining the repressive transcriptional state of hh in anterior cells, while the TrxG maintains the active transcriptional state of hh in posterior cells. This regulation seems direct, because the Pc protein and the TrxG member GAF/Trl bind two regions of the hh gene, and one of them, situated upstream of the hh transcription start site, exhibits cellular memory module (CMM) characteristics. The activity of this CMM is also regulated by PcG and TrxG proteins in experimental situations in which hh is activated by ectopic En in anterior cells, or turndown by loss of En in posterior cells. Interestingly, ectopic expression of En in anterior cells located along the dorso-ventral boundary induces hh in most of the cells of the wing blade except those nearest to the A/P boundary, implying that the activity of this hh CMM in the anterior compartment is excluded or less efficient in the territory where Kis represses hh (Terriente-Félix, 2011).
How Kis regulates hh in anterior cells is not known, but several arguments suggest that Kis is related to the repression mediated by Mtv/Gro. Thus, mtv/gro and kis mutations cause ectopic expression of hh in a similar domain of the wing disc, and in both cases they are not required when the repressor Ci75 is present. In this scenario, it is proposed that the chromatin remodeling activity of Kis could make the hh regulatory region accessible to the Mtv/Gro repressor complex. The putative function of Kis as part of the Mtv/Gro repressor complex would be independent of other functions assigned for the protein in, for example, the control of transcriptional elongation and Histone methylation. Similarly, this function of Kis on hh regulation would be independent of its role as a TrxG protein, because it is only effective in a spatial domain complementary to that in which PcG and TrxG regulate hh expression (Terriente-Félix, 2011).
Interestingly, heterozygous mutations in human CHD7 result in congenital anomalies called CHARGE syndrome, which is caused by the abnormal development of the neural crest. The function of CHD7 in the regulation of neural crest cell identities implies the regulation of several transcription factors expressed in these cells, and requires the function of the PBAF chromatin remodeler. In this manner, Kis and other CHD proteins might form part of different multiprotein complexes regulating different promoters using independent molecular mechanisms. It is remarkable that CHD8 is required for the regulation of Shh, as this implies a strong conservation in the mechanism of hh and Shh transcriptional regulation during evolution. Future experiments should address the mechanisms by which Kis/CHD8 are recruited to the hh/Shh regulatory regions (Terriente-Félix, 2011).
Nociceptive sensitization is a tissue damage response whereby sensory neurons near damaged tissue enhance their responsiveness to external stimuli. This sensitization manifests as allodynia (aversive withdrawal to previously nonnoxious stimuli) and/or hyperalgesia (exaggerated responsiveness to noxious stimuli). Although some factors mediating nociceptive sensitization are known, inadequacies of current analgesic drugs have prompted a search for additional targets. This study used a Drosophila model of thermal nociceptive sensitization to show that Hedgehog (Hh) signaling is required for both thermal allodynia and hyperalgesia following ultraviolet irradiation (UV)-induced tissue damage. Sensitization does not appear to result from developmental changes in the differentiation or arborization of nociceptive sensory neurons. Genetic analysis shows that Hh signaling acts in parallel to tumor necrosis factor (TNF) signaling to mediate allodynia and that distinct transient receptor potential (TRP) channels mediate allodynia and hyperalgesia downstream of these pathways. Hh has a demonstrated role in analgesic signaling in mammals. Intrathecal or peripheral administration of cyclopamine (CP), a specific inhibitor of Sonic Hedgehog signaling, blocked the development of analgesic tolerance to morphine (MS) or morphine antinociception in standard assays of inflammatory pain in rats and synergistically augmented and sustained morphine analgesia in assays of neuropathic pain. This study demonstrates a novel physiological role for Hh signaling, which has not previously been implicated in nociception. The results also identify new potential therapeutic targets for pain treatment (Babcock, 2011).
These results identify canonical Hh signaling as a major new pathway in modulation of nociception. Hh has long been known as a developmental signaling molecule responsible for patterning many developing tissues in both Drosophila and vertebrates. Many of these effects are exerted through Hh's abilities to induce cell proliferation or the expression of specific target genes in signal-receiving cells. Hh can also signal through a noncanonical pathway to mediate cell migration and axon guidance as in the developing spinal cord, where it helps guide spinal commissural neurons to the ventral midline. In the Drosophila system, it is highly unlikely that Hh is exerting its effects through control of cell proliferation because both the UV-injured epidermis and nociceptive sensory neurons are postmitotic. It is also unlikely that Hh is exerting its effects through control of neuronal remodeling, because the nociceptive sensory neurons do not appreciably change their arborization or branching pattern following epidermal damage and both baseline nociception and neuronal arbors are not affected when Hh signaling components are knocked down in nociceptive sensory neurons. These results thus represent a novel physiological function of Hh that is independent of its established roles in control of proliferation and neuronal morphology (Babcock, 2011).
In both vertebrates and Drosophila, distinct TRP channels mediate the detection of both ambient and noxious temperatures across the full range that animals experience. For instance, in Drosophila, Painless in nociceptive sensory neurons detects stimuli at or near the nociceptive threshold, dTRPA1 mediates ambient thermal preference in the ambient range, and Brivido channels mediate cool avoidance in antennal sensory neurons. Likewise, in vertebrates, TRPV1 mediates noxious detection of heat, whereas TRPM8 mediates detection of noxious cold. The current results provide evidence for a similar division of labor during sensitization. Painless appears to be the target of both Eiger/TNF signaling and Hh signaling at the lower end of the nociceptive threshold because it is required for the allodynia induced by ectopic activation of either pathway. Unlike Eiger/TNF, Hh can also mediate thermal hyperalgesia, a response that appears to function through the TRP channel dTRPA1. The more natural case of UV-induced allodynia appears to be somewhat more complicated because it seems to require both Painless and dTRPA1. An interesting question for future studies is how dTRPA1 can mediate temperature preference in an ambient range, UV-induced allodynia at slightly subthreshold temperatures, and both UV- and genetically-induced hyperalgesia at much higher noxious temperatures (Babcock, 2011).
Hh (from an as yet undetermined source tissue) acts through Patched (whose overexpression blocks sensitization), which subsequently inhibits Smoothened (required for sensitization) to activate signal transduction. The subsequent signaling steps likely involve the transcriptional activity of Cubitus interruptus (required for sensitization) and other components of the canonical Hh signaling pathway. For thermal allodynia, this pathway acts through the transient receptor potential (TRP) channel Painless, and for thermal hyperalgesia it requires the TRP channel dTRPA1 (Babcock, 2011).
It will also be important to determine how Hh signaling modifies Painless and dTRPA1 in the development of allodynia and hyperalgesia, respectively. In mammals, TRPV1 is phosphorylated by multiple kinases, which can increase the opening probability of the channel or reverse desensitization. Activation of Hh signaling could cause similar effects. Other possible mechanisms for this effect could include an increase of transcription or translation of TRP channels, or perhaps increased translocation of channels to the plasma membrane. The requirement of two Hh-pathway transcription factors, Cubitus interruptus and Engrailed, for development of both thermal allodynia and hyperalgesia suggests that nociceptor-specific gene transcription is a component of the sensitization response, although the precise gene targets remain to be determined. It is not yet known whether there is a transcriptional component to Eiger/TNF-induced allodynia, but the shared requirement of Painless in thermal allodynia suggests that Hh and TNF must both somehow converge on this channel, possibly in mechanistically distinct manners, to alter its activity (Babcock, 2011).
These results establish the power of using model genetic organisms to identify important new pathways in nociceptive biology. Once a pharmacologically targetable pathway is genetically identified in Drosophila, the translation to vertebrate systems using established nociceptive assays, as performed in this study, is both rapid and straightforward. In rats, central or peripheral administration of cyclopamine prevents the development of morphine analgesic tolerance or enhances morphine antinociception in models of both inflammatory and neuropathic pain. A particularly striking finding was that cyclopamine appeared to markedly augment the analgesic effect of morphine in the neuropathic pain model. This could have profound clinical implications, because neuropathic pain is often resistant to treatment with opioids in humans. These findings also suggest the intriguing possibility that central modulation of nociceptive signaling by Hh may be a conserved feature of sensitization in Drosophila and neuropathic pain in mammals. Behavioral modulation of nociception by opiates has not been observed in Drosophila (Babcock, 2011).
In conclusion, these surprising findings establish a completely unexpected role for Hedgehog signaling in the modulation of nociception and analgesia. The results suggest that blocking Hh signaling could eliminate the major shortcomings of opioids in chronic pain treatment, namely, poor efficacy against neuropathic pain, respiratory depression with increasing dose, and the development of analgesic tolerance. Thus, Hh signaling components could represent a major new set of potential therapeutic targets for clinical pain treatment (Babcock, 2011).
Studying the dynamic of gene regulatory networks is essential in order to understand the specific signals and factors that govern cell proliferation and differentiation during development. This also has direct implication in human health and cancer biology. The general transcriptional elongation regulator P-TEFb regulates the transcriptional status of many developmental genes. Its biological activity is controlled by an inhibitory complex composed of HEXIM and the 7SK snRNA. This study examines the function of HEXIM during Drosophila development. It was found that HEXIM affects the Hedgehog signaling pathway. HEXIM knockdown flies display strong phenotypes and organ failures. In the wing imaginal disc, HEXIM knockdown initially induces ectopic expression of Hedgehog (Hh) and its transcriptional effector Cubitus interuptus (Ci). In turn, deregulated Hedgehog signaling provokes apoptosis, which is continuously compensated by apoptosis-induced cell proliferation. Thus, the HEXIM knockdown mutant phenotype does not result from the apoptotic ablation of imaginal disc but rather from the failure of dividing cells to commit to a proper developmental program due to Hedgehog signaling defects. Furthermore, ci was shown to be a genetic suppressor of hexim. Thus, HEXIM ensures the integrity of Hedgehog signaling in wing imaginal disc, by a yet unknown mechanism (Nguyen, 2016).
Transcription of protein-coding genes is mediated by RNA polymerase II (RNA Pol II) whose processivity is tightly controlled by the positive transcription elongation factor b (P-TEFb) after transcriptional initiation. This kinase promotes productive transcription elongation by catalyzing the phosphorylation of a number of regulatory factors, namely the Negative elongation factor (NELF), the DRB-sensitivity inducing factor (DSIF), as well as the C-terminal domain (CTD) of RNA Pol II.
In human cells, P-TEFb forms two alternative complexes, which differ in size, components, and enzymatic activity. A 'small complex' (SC), composed of CyclinT and CDK9, corresponds to the catalytically active P-TEFb. In contrast, P-TEFb is kept in a catalytically inactive state and forms a 'large complex' (LC) when bound by a macromolecular complex containing the 7SK snRNA, Bicoid-interacting protein 3 (BCDIN3), La-related protein 7 (LARP7), and Hexamethylene bis-acetamide inducible protein 1 (HEXIM1). The formation of the LC is reversible and P-TEFb can switch back and forth between LC and SC in a very dynamic manner. Thus, HEXIM, together with other factors, acts as a sink of active P-TEFb which regulates its biological availability at target genes in response to the transcriptional demand of the cell. Although HEXIM target genes are not known, many lines of evidence strongly support a connection between developmental pathways or diseases and the control of transcription by HEXIM (Nguyen, 2016).
Transcriptional pause was initially described in the late 80s for the Drosophila HSP90 gene, where transcription stalls shortly after the elongation start and RNA Pol II accumulates at the 5' end of the gene, which is thus poised for transcription. It has been proposed that this phenomenon may be more general, as virtually all developmental genes in Drosophila and approximately 20 to 30 percent of genes in human and mouse show similar properties. The release from pause and the transition to productive elongation is under the control of the NELF factor, and so to P-TEFb, which is in turn controlled by HEXIM. Given that these genes already completed transcriptional initiation and that mRNA synthesis started, release from pause allows for a very fast and synchronized transcriptional response with low transcriptional noise (Nguyen, 2016).
It has been proposed that sustained pause may be a potent mechanism to actually repress gene transcription. This leads to the apparent paradox where transcriptional repression requires transcriptional initiation. Therefore, knockdown of the transcriptional pausing factor HEXIM would release transcription and reveal the regulation of poised genes (Nguyen, 2016).
HEXIM1 has been initially identified as a 359 aa protein whose expression is induced in human vascular smooth muscle cells (VSMCs) following treatment with hexamethylene bis-acetamide (HMBA) which is a differentiating agent. It is also called estrogen down-regulated gene 1 (EDG1) due to its decreased expression by estrogen in breast cancer cells. Ortholog of HEXIM1 in mice and chickens is activated in heart tissue during early embryogenesis, and was so named cardiac lineage protein 1 (CLP-1). HEXIM1 is involved in many kinds of cancer, viral transcription of HIV-1, cardiac hypertrophy, and inflammation. Overall, HEXIM defects are strongly associated with imbalance in the control of proliferation and differentiation. The CLP-1/HEXIM1 null mutation is embryonic lethal in mice, and results in early cardiac hypertrophy. Heterozygous littermates are still affected but with a less severe phenotype and survived up to adulthood. Moreover, Mutation in the carboxy-terminal domain of HEXIM1 causes severe defects during heart and vascular development by reducing the expression of vascular endothelial growth factor (VEGF), which is essential for myocardial proliferation and survival. Overexpression of HEXIM1 in breast epithelial cells and mammary gland decreases estrogen-driven VEGF expression, whereas it is strongly increased in loss of function mutant. As reported recently, HEXIM1 expression is required for enhancing the response to tamoxifen treatment in breast cancer patients. In addition, increased HEXIM1 expression correlates with a better prognosis and decreases probability of breast cancer recurrence. Additionally, terminal differentiation of murine erythroleukemia cells induced by HMBA or DMSO correlates with elevated levels of both HEXIM1 mRNA and protein. Furthermore, in neuroblastoma cells, HEXIM1 overexpression inhibits cell proliferation and promotes differentiation. Moreover, HEXIM1 modulates the transcription rate of NF-κB, an important regulator of apoptosis, cell proliferation, differentiation, and inflammation. However, despite theses advances, the dissection of HEXIM functions was mostly approached on a biochemical basis, and to date, very little is known about its physiological and developmental relevance in an integrated model. In order to address this important point, an in vivo model was developed (Nguyen, 2012) and it shown that a similar P-TEFb regulation pathway also exists in Drosophila, and that HEXIM is essential for proper development (Nguyen, 2016 and references therein).
In Drosophila, the Hedgehog (Hh) signaling pathway controls cell proliferation, differentiation and embryo patterning. The Hh activity is transduced to a single transcription factor, Cubitus interruptus (Ci), the Drosophila homolog of Gli. Wing imaginal discs can be subdivided into two compartments based on the presence of Hh protein. The posterior compartment (P) expresses Engrailed (En), which activates Hh and represses ci expression. The anterior compartment (A) expresses ci. The full length Ci protein (called Ci155) is constitutively cleaved into a truncated protein acting as a transcriptional repressor (Ci75) of hh and Decapentaplegic (dpp) genes. Hh inhibits the proteolytic cleavage of Ci, which then acts as a transcriptional activator of a number of target genes [Patched (ptc) and dpp, to name a few]. Thus, Ci155 is accumulated at the boundary between the A and P compartments where there are high levels of Hh, and it is absent in P compartment. Ci regulates the expression of Hh target genes in a manner dependent on Hh levels. In addition to proteolytic cleavage, the biological activity of Ci is also modulated by phosphorylation and nucleo-cytoplasmic partitioning. The mis-regulation of any components of the Hh pathway usually modifies the Ci155 levels, and results in developmental defects (Nguyen, 2016).
This paper examines the function of HEXIM during Drosophila development. HEXIM knockdown is shown to disrupt organ formation. In the wing disc, this latter effect is mediated by a strong ectopic induction of Hh signaling followed by apoptosis. The death of proliferative cells is subsequently compensated by proliferation of the neighboring cells: this is the mechanism of apoptosis-induced cell proliferation. Ci, the transcriptional effector of Hh pathway, is highly accumulated at both mRNA and protein levels in cells where HEXIM is knocked-down. Thus, the severe phenotype of HEXIM mutants resulted from Hh-related wing patterning defects. Furthermore, it was also shown that ci acts as a genetic suppressor of hexim, suggesting that HEXIM is an interacting factor of the Hh signaling pathway. This is the first time that the physiological function of HEXIM has been addressed in a whole organism (Nguyen, 2016).
Given that HEXIM is a general regulator of transcription elongation, the transcription machinery of mutant cells is eventually expected to be strongly affected that leads to cell death. One would argue that the rn>RNAi Hex mutant phenotype (undeveloped wing) is likely to be a simple consequence of a severe demolition of the wing pouch. However, this study clearly shows that the whole tissue is not ablated, although HEXIM mutant displays significant levels of apoptosis. Indeed, dying cells are efficiently replaced by new ones through apoptosis-induced proliferation (AIP) to such extent that the wing disc, including the wing pouch, increases strongly in size but still fails to promote the proper development of the wing. Thus, the phenotype is not a consequence of reduced size of the wing pouch, but rather cells fail to commit to a proper developmental program. The ectopic induction of Hh is one (among other) clear signature of abnormal development (Nguyen, 2016).
Two lines of evidence support a functional connection between HEXIM and Hedgehog signaling: (1) Ci expression is induced early, and (2) ci is a genetic suppressor of hexim.
Although Hh is supposed to be mainly anti-apoptotic, there are a few reports indicating that it can promote apoptosis during development. For example when Ptc is deleted, there is increasing apoptosis in hematopoietic cells or Shh increases cell death in posterior limb cells. In this study, the induction of Hedgehog signaling is a primary event that precedes the wave of apoptosis, in HEXIM knockdown mutants. Given that cells subject to patterning defects often undergo apoptosis, the ectopic expression of Hh is probably the molecular event that triggers apoptosis in the wing disc. Then, the subsequent AIP will produce new cells and fuel a self-reinforcing loop of Hh activation and apoptosis (since HEXIM expression is continuously repressed). Accordingly, in rn>RNAi Hex mutant, cells undergoing AIP survive but fail to differentiate. This is supported by previous reports where deregulation of Hedgehog signaling, through modifications of Ci expression levels, leads to developmental defects. The phenotype of double knockdown mutants of Ci and HEXIM can be simply explained as following: cells lack the ability to respond to Hedgehog signaling and become blind to Hh patterning defect, thus leading to a Ci-like phenotype (Nguyen, 2016).
Although it cannot be excluded that Ci155 expression is directly affected by HEXIM, the extended expression domain of 155 in rn>RNAi Hex mutants may also indirectly result from increased levels of Hh. Indeed, the breadth of the AP stripe is defined in part by a morphogenetic gradient of Hh, with a decreasing concentration towards the anterior part of the wing disc. Thus, the augmented levels of Hh induced in rn>RNAi mutants could in principle explain the broader Ci expression at the AP stripe. To summarize, HEXIM knockdown increases Hh expression, potentially through regulation of P-TEFb complex, leading to patterning defects and a wave of apoptosis followed by compensatory proliferation (Nguyen, 2016).
A genetic screen in Drosophila showed that the two components of the small P-TEFb complex, Cdk9 and Cyclin T, are strong activators of the Hh pathway, but so far, no evidence directly connects HEXIM to Hh pathway. To this regard, the current work clearly establishes this connection. It is then tempting to speculate that by knocking-down HEXIM, the levels of active P-TEFb will be eventually increased that leads to an ectopic activation of the Hh pathway. More work is needed to specifically address this mechanistic point (Nguyen, 2016).
Interestingly, when carried out in the eye discs, GMR>RNAi Hex mutants display an extreme but rare phenotype with protuberances of proliferating cells piercing through the eyes. Although these few events could not be characterized any further, the parallel with the proliferating cells, which fail to differentiate in the wing disc, is striking. Of note, the role of HEXIM in the balance between proliferation and differentiation is not quite novel. Indeed, HEXIM was previously reported to be up-regulated upon treatment of HMBA, a well known inducer of differentiation. This paper shows that the regulatory role of HEXIM during development is mediated via controlling the Hedgehog signaling pathway. This is the first study that has addressed this phenomenon in vivo and in a non-pathological context (Nguyen, 2016).
Among other functions, HEXIM acts as a regulator of the P-TEFb activity which is in turn a general regulator of elongation (Nguyen, 2012). The availability of the P-TEFb activity mediates transcriptional pausing, a mechanism by which RNA pol II pauses shortly after transcription initiation and accumulates at the 5' end of genes. Transcription may then or may not resume, depending on a number of inputs. In these cases, RNA pol II appears 'stalled' at the 5' end of genes. Release from transcriptional pausing is fast and allows a more homogeneous and synchronized transcription at the scale of an imaginal disc or organ. In the other hand, a lack of release from transcriptional pausing is also a potent way to silence transcription. Interestingly, genome wide profiling of RNA pol II revealed a strong accumulation at the 5' end of 20% to 30% of the genes, most of which involved in development, cell proliferation and differentiation. In this context, HEXIM knockdown would be expected to have strong developmental defects. Such effects have been seen in all tissues tested so far (Nguyen, 2016).
The patterning of WT wing disc is set by a morphogenetic gradient of Hh, with high levels in the P compartments and no expression in the A compartment. It is therefore tempting to speculate that the Hh coding gene would be in a transcriptionally paused state in the anterior part of the wing pouch, that would be released upon HEXIM knockdown. This simple molecular mechanism, although speculative, would account for the induction of the ectopic expression of Hh in the anterior part of the wing pouch and the subsequent loops of apoptosis and AIP, ultimately leading to the wing developmental defects. Attempts were made to see whether the distribution of RNA Pol II along hh and ci is compatible with a transcriptional pause by using a number of RNA Pol II ChIP-Seq datasets that have been generated, together with RNA-Seq data, over the past few years. This study has processed these datasets and computed the stalling index (SI) for all genes. The SI is computed after mapping ChIP-Seq reads on the reference genome and corresponds to the log ratio of the reads density at the 5' end of the gene over the reads density along the gene body. Although these datasets clearly reveal a number of 'stalled' genes (>>100), no evidence of paused RNA Pol II was found for hh and ci (SI value of order 0), which were instead being transcribed. It is noted, however, that these datasets have been generated from whole embryos and S2 cell line. Given that Hh and Ci define morphogenetic gradients, their expression (and their transcriptional status) is likely highly variable between cells located in the different sub-regions of a disc, which may therefore not be reflected in these datasets (Nguyen, 2016).
Apart from the developmental function of HEXIM that is addressed in this work and the connection between HEXIM and Hedgehog signaling, the current results may also be of interest for human health studies. First, Hedgehog is a major signaling pathway that mediates liver organogenesis and adult liver regeneration after injury. In a murine model of liver regeneration, the Hedgehog pathway promotes replication of fully differentiated (mature) hepatocytes. Thus, addressing whether a connection between HEXIM and Hh exists would provide a mechanistic link between the control of gene expression and adult liver regeneration. Second, deregulated Hedgehog signaling is a common feature of many human tumors, and is found in at least 25% of cancers. In addition, recent data showed that aberrant Hedgehog signaling activates proliferation and increases resistance to apoptosis of neighboring cells and thus helps create a micro-environment favorable for tumorigenesis. Since its discovery, deregulated HEXIM expression is often associated to cancers and other diseases. Adding a new connection between HEXIM and Hedgehog signaling will shed more light into the role of HEXIM in abnormal development and cancer (Nguyen, 2016).
Surprisingly, although the biochemical interactions between HEXIM and its partners have been thoroughly described, very little is known about its biological function. Thus, this is the first time that the functional impact of HEXIM has been addressed in an integrated system (Nguyen, 2016).
Hedgehog (Hh) signaling pathway and Cyclin E are key players in cell proliferation and organ development. Hyperactivation of hh and cyclin E has been linked to several types of cancer. However, coordination of the expression of hh and cyclin E was not well understood. This study shows that an evolutionarily conserved transcription factor Apontic (Apt) directly activates hh and cyclin E through its binding site in the promoter regions of hh and cyclin E. This Apt-dependent proper expression of hh and cyclin E is required for cell proliferation and development of the Drosophila wing. Furthermore, Fibrinogen silencer-binding protein (FSBP), a mammalian homolog of Apt, also positively regulates Sonic hh (Shh), Desert hh (Dhh), Cyclin E1 (CCNE1) and Cyclin E2 (CCNE2) in cultured human cells, suggesting evolutionary conservation of the mechanism. Apt-mediated expression of hh and cyclin E can direct proliferation of Hh-expressing cells and simultaneous growth, patterning and differentiation of Hh-recipient cells. The discovery of the simultaneous expression of Hh and principal cell-cycle regulator Cyclin E by Apt implicates insight into the mechanism by which deregulated hh and cyclin E promotes tumor formation (Wang, 2017).
Continued, see Hedgehog Regulation: part 2/2
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