pebbled
pebbled expression in the midgut is controlled by the maternal and zygotic members of the torso mediated terminal pathway. Embryos produced by homozygous torso loss-of-function mutant females lack Hnt protein in the posterior midgut, which lies within the domain of torso function. Instead of extending their germ bands dorsoanteriorly, most such embryos form spiralled germ bands. Reciprocally, embryos carrying torso gain of function mutations lack dorsal expression (that is, in the presumptive amnioserosa), consistent with conversion of central cell fates to more terminal ones. These embryos also show expanded expression of Hnt protein in the enlarged posterior midgut primordium and a twisted gastrulation phenotype (Yip, 1997).
tailless mutations have little effect on pebbled expression; from analysis of huckebein tailless double mutants, it is clear that the only loss of Hnt protein expression in tailless mutants occurs in the region from which the Malpighian tubule primordia originate, consistent with the reported role for tll and peb in the development of these structures. hkb mutant embryos lack Hnt protein expression in the regions from which the anterior and posterior midgut normally arise; expression remains only in the presumptive ureter of the Malpighian tubules. In hkb tll double mutant embryos, Hnt protein is not present at all in the domains that would form anterior and posterior midgut and Malpighian tubule primordia; however expression does occur in the amnioserosa. Germ-band retraction occurs in tll or hkb single mutants as well as in hkb tll double mutants, suggesting that midgut expression of Hnt is not necessary for germ-band retraction (Yip, 1997).
Hnt protein is present in u-shaped, tailup and Egfr mutants. These results suggest that peb either resides upstream of these three genes in the same hierarchy or one or more of these genes functions in a parallel pathway. In contrast, endodermal expression of Hnt is missing in serpent mutant embryos. This last result is consistent with the fact that serpent is required to establish the identity of the endodermal midgut; loss-of-function mutations in serpent result in transformation of endoderm into ectoderm (Yip, 1997 and references).
pebbled expression in the amnioserosa is regulated by the dorsoventral pathway. Dorsal Hnt protein expression is reduced in genetically ventralized mutant embryos such as those produced by saxophone or cactus null females. Reciprocally, dorsal Hnt expression expands ventrally in dorsalized embryos. Anterior midgut expression of Hnt is also affected by the dorsoventral pathway (Yip, 1997).
The Drosophila tracheal system arises from clusters of ectodermal cells that invaginate and migrate to originate a network of epithelial
tubes. Genetic analyses have identified several genes that are specifically expressed in the tracheal cells and are required for tracheal
development. Among them, trachealess (trh) is able to induce ectopic tracheal pits and therefore it has been suggested that it would act
as an inducer of tracheal cell fates; however, this capacity appears to be spatially restricted. The expression of the tracheal
specific genes in the early steps of tracheal development and their crossinteractions have been examined. There is a set of primary genes including trh
and ventral veinless (vvl) whose expression does not depend on any other tracheal gene and a set of downstream genes whose expression
requires different combinations of the primary genes. The combined expression of primary genes is sufficient to induce some
downstream genes but not others. While tracheal expression of tkv depends on
vvl, it appears to be independent of trh. The opposite
appears to be the case for two other tracheal genes, tracheal defective (tdf) and pebbled (peb) [also known as hindsight
(hnt)], which code for two putative transcription factors. Both genes
appear to be targets of trh but they are present in the tracheal cells of vvl mutant embryos. Thus, some tracheal genes
seem to be common targets of vvl and trh but others seem
to depend only on one of them (Boube, 2000).
The dorsal ectoderm of the Drosophila embryo is
subdivided into different cell types by an activity gradient
of two TGFbeta signaling molecules, Decapentaplegic
and Screw. Patterning responses to this gradient
depend on a secreted inhibitor, Short gastrulation
and a newly identified transcriptional repressor, Brinker, which are expressed in neurogenic regions that abut the dorsal ectoderm. The expression of a
number of Dpp target genes has been examined in transgenic embryos that
contain ectopic stripes of Dpp, Sog and Brk expression.
These studies suggest that the Dpp/Scw activity gradient
directly specifies at least three distinct thresholds of gene
expression in the dorsal ectoderm of gastrulating embryos.
Brk was found to repress two target genes, tailup/islet and
pannier, that exhibit different limits of expression within
the dorsal ectoderm. These results suggest that the Sog
inhibitor and Brk repressor work in concert to establish
sharp dorsolateral limits of gene expression. Evidence is provided that the activation of Dpp/Scw target
genes depends on the Drosophila homolog of the CBP
histone acetyltransferase (Ashe, 2000).
Different dorsal ectoderm genes were examined in a variety of
mutant and transgenic embryos using digoxigenin-labeled
RNA probes and in situ hybridization. The
normal expression patterns suggest the occurrence of at least
three thresholds of gene activity in response to the Dpp/Scw
activity gradient. The Race and hindsight/pebbled (hnt) genes are expressed
in narrow strips in the dorsalmost regions of the embryo, although the anteroposterior limits of the
two patterns are distinct. It is conceivable that early-acting
segmentation genes are responsible for repressing Race in
posterior regions and hnt in anterior regions. Somewhat
broader expression patterns are observed for tup and ush. These patterns encompass the presumptive amnioserosa
and dorsal regions of the dorsal epidermis.
Broad staining patterns are observed for two genes encoding
GATA transcription factors, dGATAc and pnr. pnr is expressed throughout the dorsal
ectoderm in the presumptive thorax and abdomen.
dGATAc exhibits a nearly reciprocal pattern in anterior and
posterior regions; staining is mainly excluded from regions
expressing pnr, although a weak patch of staining is detected
in a portion of the presumptive amnioserosa. Most of the
subsequent analyses on gradient thresholds have focussed on the
regulation of hnt, tup and pnr (Ashe, 2000).
All of the aforementioned genes are virtually silent in the
dorsal ectoderm of dpp-/dpp- embryos, while
changes in dpp+ gene dose cause altered patterns of expression. For example, increasing the number of dpp+ copies
from two to three to four results
in a sequential expansion of the hnt expression pattern, whereas
expression is lost in dpp/+ heterozygotes. In
contrast, ush is expressed in dpp/+ heterozygotes, although
there is a marked narrowing in the expression pattern as
compared with wild-type embryos.
Three copies of dpp+ cause an expansion of the ush pattern. Similarly, the tup expression pattern is narrower in
dpp/+ heterozygotes and expanded in embryos with three
copies of dpp.
Further evidence that hnt and ush are early targets of the Dpp
signaling pathway was obtained by analyzing transgenic
embryos that contain the dpp-coding sequence attached to the
eve stripe 2 enhancer. These embryos exhibit both
the endogenous dpp pattern in the dorsal ectoderm as well as an ectopic stripe of expression (Ashe, 2000).
Additional Dpp/Scw target genes were examined for
repression by the stripe2-brk transgene. Those showing altered
patterns of expression include tup, rho, hnt and Race. The normal tup expression pattern
encompasses both the presumptive amnioserosa and dorsal
regions of the dorsal epidermis. In transgenic
embryos, there is a gap in the pattern in regions where the
stripe2-brk fusion gene is expressed. These results
suggest that Brk represses tup, even though it appears to
respond to a different threshold of Dpp/Scw signaling than pnr.
Additional experiments were done to determine whether Brk
directly represses tup expression, or works indirectly by
inhibiting Dpp signaling (Ashe, 2000).
The limits of the tup expression pattern seem to depend on
both Sog and Brk. The introduction of the stripe2-brk transgene leads to a clear gap in the tup expression pattern, although there is a narrow stripe of repression in gd- mutants lacking the transgene. The stripe2-sog
transgene causes a more extensive gap in the tup pattern. The stripe2-brk transgene was also found to repress Race,
hnt and rho in this assay (Ashe, 2000).
A summary is presented of Dpp signaling thresholds in the embryo. The Dpp/Scw activity
gradient presumably leads to a broad nuclear gradient of Mad and
Medea across the dorsal ectoderm of early embryos. It is conceivable
that the early lateral stripes of brk expression lead to the formation of
an opposing Brk repressor gradient through the limited diffusion of
the protein in the precellular embryo. Peak
levels of Dpp and Scw activity lead to the activation of Race and hnt
at the dorsal midline. The tup and ush patterns represent another
threshold of gene activity. The similar patterns might involve
different mechanisms of Dpp signaling since tup is repressed by Brk,
whereas ush is not. Finally, the broad pnr pattern
represents another threshold of gene activity. It is not inhibited by
Sog but is repressed by Brk. It is possible that tup and pnr are
differentially repressed by a Brk gradient. Low levels of Brk might
be sufficient to direct the lateral limits of the tup pattern, whereas
high levels may be required to repress pnr (Ashe, 2000).
Su(H)/CBF1 is a key component of the evolutionary conserved Notch
signalling pathway. It is a transcription factor that acts as a repressor in
the absence of the Notch signal. If Notch signalling is activated, it
associates with the released intracellular domain of the Notch
receptor and acts as an activator of transcription. During the development of
the mechanosensory bristles of Drosophila, a selection process called
lateral inhibition assures that only a few cells are selected out of a group
to become sensory organ precursors (SOP). During this process, the SOP cell is
thought to suppress the same fate in its surrounding neighbours via the
activation of the Notch/Su(H) pathway in these cells. Although
Su(H) is required to prevent the SOP fate during lateral inhibition, it is
also required to promote the further development of the SOP once it is
selected. Importantly, in this situation Su(H) appears to act independently of
the Notch signalling pathway. Loss of Su(H)
function leads to an arrest of SOP development because of the loss of
sens expression in the SOP. These results suggest that Su(H) acts as a repressor that suppresses the activity of one or more negative regulator(s) of sens expression. This repressor activity is encoded by one or several genes of the E(spl)-complex. These results further suggest that the position of the SOP in a proneural cluster is determined by very precise positional cues, which render the SOP insensitive to Dl (Koelzer, 2003).
Thus Su(H) is required to promote
SOP development. This is based on the fact that most cells of proneural
clusters in the notum that lack Su(H) function do not express SOP
markers such as Sens, Hindsight (Hnt) and partially neurA101-lacZ.
Loss of neurA101-lacZ expression has been attributed to a 'general sickness' of
the mutant discs, since the lack of neurA101-lacZ expression has only been observed in the late developing proneural clusters. The data argue against such an explanation: Presenilin (Psn)
mutant wing imaginal discs exhibit a stronger neurogenic phenotype than do
Su(H) mutants. Similar to Su(H) mutants, homozygous
Psn mutant animals also die during the early pupal phase.
Nevertheless, the cells of the proneural clusters of these mutants express all
tested markers, indicating that SOP development is not affected. The same is
true for kuzbanian (kuz) mutants, whose mutant phenotype is comparable with that
of Su(H) mutants. Hence, general sickness of the wing imaginal disc
cells is not likely to explain the arrest of SOP development in Su(H) mutants (Koelzer, 2003).
A role of Su(H) in development of the SOP is surprising, because it is a
core element of the Notch signalling pathway and the activity of this
pathway is required to prevent SOP development in cells of the proneural
clusters. Importantly, in this new role, Su(H) seems to function
independently of the Notch signalling pathway. This is indicated by the finding that the Su(H) mutant phenotype is epistatic over that of Psn mutants (Koelzer, 2003).
The data presented here indicate that Su(H) appears to be required to
suppress the activity of one or more members of the E(spl)-C, that in turn suppress the expression of genes such as hnt and sens. This conclusion is based on: (1) the failure of Su(H)VP16 to activate
sens; (2) the fact that Psn H double mutants display a
similar loss or reduction of sens expression as Su(H) and
Su(H); Psn double mutants, and (3) the fact that expression of a
Su(H) construct that is unable to bind H (UAS Su(H)DeltaH)
leads to an arrest of SOP development in Psn mutant wing imaginal
discs. Several reports show that H is involved in Su(H)-related
suppression of gene expression in the absence of Notch signalling. Recently, it has been shown that H acts as a bridge between
Su(H) and the general co-repressors CtBP and Gro. It is therefore likely, that this Su(H)/H/Gro/dCtBP complex mediates the repressor function required during SOP development (Koelzer, 2003).
Repression by Su(H) is not strictly required in all proneural clusters to
allow expression of sens and other late SOP markers. Examples are the
clusters in the wing region, such as the clusters of the dorsal radius.
However, even in these clusters, sens and hnt are not
expressed in all cells that express early markers, such as
neurA101. Therefore, it appears that the activity of Su(H) promotes SOP development also in these clusters. The clusters of the dorsal radius give
rise to other types of sense organs, such as companiforme sensilla, and it is
possible that there are different requirements for the activity of
Su(H) for the development of the different types of sense organs (Koelzer, 2003).
In Su(H) mutant cell clones induced during the first
larval instar stage, hnt is expressed in a fraction of cells of
specific proneural clusters, such as the scutellar cluster, but absent or
strongly reduced in other clusters. It was further found that in Su(H)
mutant wing imaginal discs, expression of sens and hnt is either
lost or strongly reduced when compared to mutant cell clones induced during the first larval instar (Koelzer, 2003).
Altogether, these observations suggest that the Notch pathway
might have two separable functions during SOP development. During early phases of a proneural cluster, the activity of the pathway keeps the cells of the cluster undecided, perhaps by mutual repression. Owing to positional cues, one cell becomes insensitive to the inhibitory signal and adopts the SOP fate. Subsequently the SOP inhibits its immediate neighbours by sending an inhibitory signal through Dl (Koelzer, 2003).
The Notch signaling pathway plays important roles in a variety of developmental events. The context-dependent activities of positive and negative modulators dramatically increase the diversity of cellular responses to Notch signaling. In a screen for mutations affecting the Drosophila follicular epithelium, a mutation was isolated in CoREST that disrupts the Notch-dependent mitotic-to-endocycle switch of follicle cells at stage 6 of oogenesis. Drosophila CoREST positively regulates Notch signaling, acting downstream of the proteolytic cleavage of Notch but upstream of Hindsight activity; the Hindsight gene is a Notch target that coordinates responses in the follicle cells. CoREST genetically interacts with components of the Notch repressor complex, Hairless, C-terminal Binding Protein and Groucho. In addition, it was demonstrated that levels of H3K27me3 and H4K16 acetylation are dramatically increased in CoREST mutant follicle cells. The data indicate that CoREST acts as a positive modulator of the Notch pathway in the follicular epithelium as well as in wing tissue, and suggests a previously unidentified role for CoREST in the regulation of Notch signaling. Given its high degree of conservation among species, CoREST probably also functions as a regulator of Notch-dependent cellular events in other organisms (Domanitskaya, 2012).
The highly conserved Notch signaling pathway plays a crucial role in a broad array of developmental events, including the maintenance of stem cells, cell fate specification, control of proliferation and apoptosis. Misregulation of the Notch pathway is associated with a number of diseases, including different types of cancer. The binding of the transmembrane ligands DSL (Delta, Serrate, LAG-2) to the extracellular domain of Notch, exposed on a neighboring cell, activates the signaling cascade by triggering a sequence of proteolytic cleavages of Notch protein. Extracellular cleavage (S2) leads to the formation of an intermediate membrane-bound C-terminal fragment of Notch, called NEXT. This event is followed by an intramembranous cleavage (S3) by the γ-secretase complex. The intracellular domain of Notch (NICD) then translocates to the nucleus and binds to a transcription factor of the CSL family [CBF-1, Su(H), LAG-1], converting it from a transcriptional repressor to an activator. In the canonical Notch pathway, Su(H) directly activates Notch target genes in response to signaling. Despite the relative simplicity of the Notch transduction pathway, the presence of a large number of proteins that positively or negatively influence Notch signaling dramatically increases the complexity of the Notch pathway and its cellular responses. For instance, extracellular modulators, such as Fringe, alter ligand-specific Notch activation, whereas cytoplasmic modulators, such as Numb, restrict signal transduction. Nuclear modulators, for instance Mastermind, influence the transcriptional activity of the NICD-containing complex. In addition, there is increasing evidence of the importance of the epigenetic regulation of Notch targets, which can cause differential cellular responses upon Notch activation (Domanitskaya, 2012).
Drosophila serves as an excellent model system to dissect the regulation of the Notch pathway. The Drosophila genome contains only a single Notch protein and two ligands [Delta (Dl) and Serrate (Ser)]. The Notch pathway is involved in several aspects of Drosophila development. The role of Notch in lateral inhibition during neurogenesis has been extensively studied; it restricts neural cell fates in the embryo, and leads to restriction of sensory-organ formation and induction of boundary formation in the wing discs. Notch activity is also required for many aspects of oogenesis, such as the establishment of egg chamber polarity, polar cell formation, control of follicle cell (FC) proliferation, differentiation, cell fate specification and morphogenesis. The Drosophila FCs are somatically derived epithelial cells that form a monolayer covering the germline cells during oogenesis. FCs divide mitotically from stage 2 to stage 6 of oogenesis, followed by the switch from the mitotic cycle to the endocycle (the M/E transition). Endocycles take place from stage 7 to stage 10A of oogenesis and include three rounds of DNA duplication without subsequent cell division. The M/E switch is triggered upon Notch pathway activation. Dl produced in the germline binds to its receptor Notch, expressed in the FCs, and induces activation of the canonical Notch signaling pathway. Removal of Dl from germline cells, or of Notch from FCs, maintains follicle cells in the mitotic cycle throughout oogenesis. NICD complexed with Su(H) activates transcription of downstream target genes required for the M/E switch, such as Hindsight (Hnt). Hnt then mediates the Notch-dependent downregulation of Cut, String (Stg) and Hedgehog (Hh) signaling in the FCs, thus promoting the M/E switch (Domanitskaya, 2012).
This study describes the identification of the transcriptional cofactor Corepressor for element-1-silencing transcription factor (CoREST) as a positive modulator of Notch signaling in the FCs and during wing development. CoREST is required for the promotion of the M/E switch during oogenesis. CoREST acts downstream of NICD release but upstream of Hnt activity, and it is a previously unidentified modulator of the Notch pathway. The genetic interactions between CoREST and Hairless (H), CtBP and Groucho (Gro), members of the Notch repressor complex, suggest that CoREST might influence the activity of either Notch transcriptional repressor or activator complexes. In addition, CoREST specifically affects tri-methylation of lysine 27 of histone 3 (H3K27) and acetylation of H4K16 in FCs, because these chromatin modifications show elevated levels in the CoREST mutant cells. These findings point to a possible role of CoREST in regulation of the activity of the Notch repressor-activator complexes and/or epigenetic regulation of the components of the repressor-activator complexes or of factors involved in the transduction of the signaling or directly of target genes of the Notch signaling pathway (Domanitskaya, 2012).
Initially, CoREST was identified in humans as a corepressor with REST (RE1 silencing transcription factor) in mediating repression of the proneuronal genes, and thus as an important factor in the establishment of non-neural cell specificity. Subsequently, CoREST was identified in a variety of vertebrate and invertebrate species, and was shown to play a functionally conserved role in neurogenesis. Recent studies show that CoREST regulates a very broad range of genes by both REST-dependent and REST-independent means, including genes encoding members of key neural developmental signaling pathways, such as BMP, SHH, Notch, RA, FGF, EGF and WNT. Analysis of CoREST downstream target genes and their developmental expression profiles suggested that the liberation of CoREST from gene promoters is associated with both gene repression and activation depending on the cell context. In the work reported in this study, a lethal allele of Drosophila CoREST was isolated, and the contribution of CoREST to the development of FCs, a process that involves cell proliferation and differentiation, was analyzed. This study has implicated CoRESTin the regulation of Notch signaling, and acts as a positive modulator of the Notch pathway in Drosophila FCs (Domanitskaya, 2012).
This study has identified a role for CoREST in the Notch-mediated regulation of the M/E switch during stage 6 of oogenesis. Loss of CoREST activity in FCs primarily disrupts the Notch signaling pathway. We further demonstrated that CoREST regulates the Notch pathway downstream of NICD release and upstream of Hnt. The misexpression of Hnt in the CoREST mutant clones rescues the failure in the M/E switch. Furthermore, the role of CoREST in Notch pathway regulation is not restricted to FCs: CoREST also interacts with Notch during wing development. Interestingly, CoREST was identified as a negative modulator of Notch signaling in Caenorhabditis elegans in a genetic screen for suppressors of the developmental defects in sel-12 presenilin mutants. Presenilin is a component of the γ-secretase complex that performs the S3 cleavage of Notch. Mutations in spr-1, the C. elegans homolog of CoREST, suppress the developmental defects observed in sel-12 animals by derepressing the transcription of the other functionally redundant presenilin gene, hop-1. Therefore, CoREST acts as a negative regulator of the γ-secretase complex in C. elegans, and hence proteolytic cleavage of Notch and release of NICD. By contrast, Drosophila CoREST does not affect the processing of the Notch receptor in the follicle cells, and instead acts as a positive modulator of the Notch pathway functioning downstream of NICD release (Domanitskaya, 2012).
CoREST plays transcriptional and epigenetic regulatory roles: it can promote gene activation in addition to repression, as well as being able to modify the epigenetic status of target gene loci distinct from its effects on transcription. Several possible scenarios of how CoREST could be involved in the regulation of Notch signaling are discussed, based on the previous knowledge about CoREST and considering the current data (Domanitskaya, 2012).
hnt, the downstream target gene of Notch signaling in FCs, fails to be properly upregulated upon Notch activation in the CoREST mutant cells. CoREST might therefore act as a transcriptional repressor for an unknown factor, which is in turn involved in the transcriptional repression of hnt. Alternatively, CoREST could be directly involved in the transcriptional regulation of hnt and act as an activator. hnt was shown to be a putative direct target of Notch signaling in DmD8 cells from the analysis of genes for which mRNA levels increase within 30 minutes of Notch activation, and which contain regions occupied by Su(H). If hnt is a direct target of Notch in FCs, its transcription would be regulated by the balance between Notch repressor and activator complexes, and CoREST might be involved in the regulation of stability or activity of either of these. Interestingly, CoREST was shown to interact with CtBP1 in mammals (Kuppuswamy, 2008), and to bind to the SIRT1-LSD1-CtBP1 complex, which is required for the repression of certain Notch target genes (Mulligan, 2011). Thus, Drosophila CoREST might similarly directly bind to the repressor complex containing CtBP and modify its activity or destabilize it. However, CoREST could be involved in the transcriptional regulation of the components of Notch repressor or activator complexes. In this scenario, in CoREST mutant FCs, upregulation of negative regulator(s) would lead to greater activity of negative than positive regulators, resulting in disruption of Notch signaling. Both suggested models of the direct and indirect transcriptional role of CoREST are consistent with the current results, given that the CoREST mutant phenotype could be suppressed by removal of one copy of H, CtBP or Gro, components of the Notch repressor complex (Domanitskaya, 2012).
More recently, epigenetic mechanisms have emerged as an important interface regulating context-dependent and stage-specific gene regulation. Mammalian CoREST acts as a scaffold for recruitment of transcriptional regulators such as REST, and epigenetic factors such as the enzymes HDAC1, HDAC2 and LSD1. In Drosophila, using two-hybrid interaction, CoREST was also shown to interact with Su(VAR)3-3 (Drosophila homolog of LSD1) and Rpd3 (HDAC1). This study has shown that the levels of H3K27me3 and H4K16 acetylation are significantly and specifically increased in the CoREST mutant FCs. Recently, the H3K27me3 demethylase UTX was shown to act as a suppressor of Notch- and Rb-dependent tumors in Drosophila eyes, and in addition to increased level of H3K27me3 staining, an excessive activation of Notch was detected in Utx mutant eye discs. The observation of increased levels of H3K27me3 coupled to cell overproliferation and modified Notch signaling in both of these cases suggests that the increased H3K27me3 results in epigenetic regulation of genes involved in Notch signaling and/or of Notch target genes. However, in the eye tumor system, this increase in H3K27me3 promotes Notch signaling, whereas in the follicle cells, it reduces Notch signaling. This indicates a strong context-dependent effect on Notch signaling by certain chromatin modifications. Thus, these chromatin modifications might be involved in cell-context-dependent Notch target gene silencing and/or activation. Interestingly, many Notch-regulated genes are highly enriched in a characteristic chromatin modification pattern, termed a bivalent domain, consisting of regions of H3K4me3, a marker for actively expressed genes, and H3K27me3, a marker for stably repressed genes; and Notch signaling could be involved in resolving these domains, leading to gene expression (Schwanbeck, 2011). Therefore, the increased level of H3K27me3 in CoREST mutant FCs might lead to a repression of certain Notch target genes, for instance hnt (Domanitskaya, 2012).
To further understand the function of the Drosophila CoREST in Notch pathway regulation, identification of other CoREST essential and specific binding partners would be useful. One previously identified partner for CoREST is Chn (Tsuda, 2006). Given that wild-type expression of Hnt and Cut was observed in chn mutant cells, this factor does not appear to partner CoREST in regulation of Notch signaling in FCs. Using yeast two-hybrid analyses and an embryonic cDNA fusion protein library, it was shown that all three splice variants of Drosophila CoREST interact with the unique C-terminus of Tramtrack88 (Ttk88), a known repressor without homology to REST. In addition, a Ttk69 splice variant can form a complex with CoREST and Ttk88. However, Ttk88 was not detected in the ovary by immunofluorescence or western blot analysis, and disruption of Ttk88 does not have any impact on oogenesis. Conversely, Ttk69 is steadily expressed in FCs before stage 10 and it is required for the M/E transition. However, in contrast to CoREST, which acts upstream of Hnt, Hnt expression is not affected in ttk1e11 mutant FCs, indicating a role of Ttk69 downstream of Hnt in the control of the M/E switch. Additionally, Ttk69 is not required for cell differentiation, as expression of FasIII, a cell fate marker for immature follicle cells, is normal in ttk1e11 mutant FCs. From these important phenotypic differences between Ttk69, Ttk88 and CoREST, it appears that CoREST plays a Ttk-independent role in Notch pathway regulation in the FCs. Future work to identify transcription regulators that act as binding partners of CoREST will help in determining the precise biochemical role of CoREST in modulating Notch signaling (Domanitskaya, 2012).
These results demonstrate an unexpected role for CoREST in positively regulating Notch signaling. The effect of the loss of CoREST is particularly strong in the PFCs and relatively mild in the lateral and anterior follicle cells. This implies that CoREST is crucially required in cells that are more sensitive to loss of Notch signaling. The difference between the PFCs and the other follicle cells is established at approximately stages 6-7 of oogenesis by EGF receptor activation in response to Gurken produced by the oocyte. EGF signaling, therefore, is active around the same time as the Notch pathway and hence it is probable that downstream effector(s) of EGFR signaling result in the increased sensitivity of PFCs to the loss of CoREST. In the model of CoREST negatively affecting a repressor of Notch signaling, EGFR signaling would be expected to act positively to enhance expression and/or activity of a Notch repressor. Thus, loss of CoREST from the PFCs would occur in a cell type where repressor activity is already augmented, which would explain the observation of differential loss of Notch signaling in the PFCs (Domanitskaya, 2012).
In summary this study has shown that CoREST, a component of transcriptional repressor complexes, acts positively in Notch signaling in the ovarian follicle cells of Drosophila. The results also show that different cell types are differentially sensitive to loss of this repressor. Future identification of partners and targets of CoREST in the follicle cells should further elucidate how activity of EGFR and other signaling pathways are integrated in this process (Domanitskaya, 2012).
Absence of Krüppel at stage 11 correlates with the premature apoptosis of the differentiated amnioserosa in pebbled mutants (Frank, 1997).
pebbled may regulate Krüppel. Krüppel, which accumulates in wild-type embryos in the nuclei of amnioserosal cells, is absent from most but not all of these cells in stage 11 peb mutants (Yip, 1997).
As the germ band shortens in Drosophila melanogaster embryos, cell shape changes cause segments to narrow anteroposteriorly and to lengthen dorsoventrally. One
of the genes required for this retraction process is the hindsight (hnt) gene. hnt encodes a nuclear Zinc-finger protein that is expressed in the extraembryonic
amnioserosa and the endodermal midgut prior to and during germ band retraction. Through analysis of hnt genetic mosaic embryos, it has been shown that hnt activity in the amnioserosa, particularly in those cells that are adjacent to the
epidermis, is necessary for germ band retraction. In hnt mutant embryos the amnioserosa undergoes premature cell death. Prevention of premature apoptosis in hnt mutants does not rescue retraction. Thus, failure of this process is not
an indirect consequence of premature amnioserosal apoptosis; instead, hnt must function in a pathway that controls germ band retraction (Lamka, 1999).
The Kruppel
gene is activated by hnt in the amnioserosa while the Drosophila insulin receptor (INR) functions downstream of hnt in the germ band. Kr protein is first detected in the amnioserosa of wild-type embryos during germ band extension; however, in hnt mutants, Kr is clearly absent from most amnioserosal cells by stage 11. Loss of Kr in hnt mutants is not a result of premature apoptosis of the amnioserosa. Rather the Kr gene resides downstram of Hnt in the hnt genetic hierarchy in the amniserosa. A specific role for Kr in germ band retraction remains to be defined. Using a heat shock insulin receptor transgene to overexpress the wild-type insulin receptor, nearly complete rescue of germ band retraction is observed in hnt mutants. The extent of rescue depends on the strength of the hnt allele used. The fact that high levels of the Inr can rescue germ band retraction in hnt mutants is consistent with the possiblility that the Inr functions downstream of hnt in a germ band retraction pathway. Evidence against a
physical model in which the amnioserosa 'pushes' the germ band during retraction is presented. Rather, it is likely that the amnioserosa functions in production, activation, or
presentation of a diffusible signal required for retraction (Lamka, 1999).
The Drosophila Hindsight (hnt) gene encodes a C2H2-type Zinc-finger protein, HNT, that plays multiple developmental roles including control of embryonic germ band retraction and regulation of retinal cell fate and morphogenesis. While the developmental functions of the human HNT homolog, RREB-1, are unknown, it has been shown to function as a transcriptional modulator of several tumor suppressor genes. This study investigated HNT's functional motifs, target genes and its regulatory abilities. The C-terminal region of HNT, containing the last five of its 14 Zinc fingers, binds in vitro to DNA elements very similar to those identified for RREB-1. HNT's in vivo binding sites were mapped on salivary gland polytene chromosomes, and where HNT is bound to two target genes, hnt itself and nervy (nvy), was defined at high resolution. Data from both loss-of-function and over-expression experiments show that HNT attenuates the transcription of these two targets in a tissue-specific manner. RREB-1, when expressed in Drosophila, binds to the same polytene chromosome sites as HNT, attenuates expression of the hnt and nvy genes, and rescues the germ band retraction phenotype. HNT's ninth Zinc finger has degenerated or been lost in the vertebrate lineage. A HNT protein mutant for this finger can also attenuate target gene expression and rescue germ band retraction. Thus HNT and RREB-1 are functional homologs at the level of DNA binding, transcriptional regulation and developmental control (Ming, 2013).
Home page: The Interactive Fly © 1997 Thomas B. Brody, Ph.D.
The Interactive Fly resides on the
pebbled:
Biological Overview
| Developmental Biology
| Effects of Mutation
| References
Society for Developmental Biology's Web server.