decapentaplegic

Table of contents

Regulation in dorsal closure

Dorsal closure requires two signaling pathways: the Drosophila Jun-amino-terminal kinase (DJNK) pathway and the Decapentaplegic pathway. The changes in cell shape in the lateral epidermis occur in two phases. In the first phase, the cells of the leading edge begin to stretch dorsally. In a second phase, the remaining cells ventral to the first row change shape. DJNK, known as Basket, controls dorsal closure by activating DJun and inactivating the ETS repressor Aop/Yan by phosphorylation. The role of Aop/Yan is to hold decapentaplegic transcriptionally silent until Aop/Yan is inactivated by phosphorylation. These phosphorylation events regulate dpp expression in the most dorsal row of cells. Interestingly, mutants in components of the DJNK and Dpp pathways affect the two phases of dorsal closure differently. Whereas loss-of-function mutations in either DJNK or DJun block the cell shape changes of all cells, mutations in thick veins and punt block only the second phase. Thus it is concluded that Dpp functions to instruct more ventrally located cells to stretch. These results provide a causal link between the DJNK and Dpp pathways during dorsal closure (Riesgo-Escovar, 1997).

Dorsal closure depends on the activities of a Jun amino (N)-terminal kinase kinase (JNKK) encoded by the hemipterous gene, and of a JNK encoded by basket. Hep is required for cell determination in the leading edge of migrating epithelia, by controlling specific expression of the puckered gene in these cells. puc encodes a protein related to vertebrate dual-specificity MAPK phosphatases of the CL100 family (E. Martin-Blanco, A. Gampel, and A. Martinez Arias, pers. comm. to Glise, 1997). During dorsal closure, decapentaplegic is expressed in the row of cells making up the leading edge of the epithelia. The small GTPases Dcdc42, Drac1, and the Hep JNKK control dpp expression in this migratory process. Activated Drac1 and Dcdc42 induce distinct, although partly overlapping, responses. Dcdc42 appears to be a good inducer of ectopic puc and dpp expression in ectodermal cells located more ventrally, whereas Drac1 seems more active in cells nearest to the leading edge. Appropriate dpp and puc expression in the leading edge also depends on the inhibitory function of the puc gene. puc acts as a repressor of dpp expression in the ectoderm, likely acting to inhibit Basket, the Jun N-terminal kinase. In puc mutants ectopic expression of puc and dpp is induced in the ectoderm, that is, outside the normal domain of puc expression in the leading edge. In addition, puc (but not dpp) is expressed ectopically in amnioserosa cells. These observations indicate a cell non-autonomous effect of puc mutations. The data suggest that the leading edge is the source of a JNK autocrine signal, and exclude a role of Dpp as such a ligand. Dorsal closure couples JNK and Dpp signaling pathways, a situation that may be conserved in vertebrate development (Glise, 1997).

During the second half of embryogenesis, decapentaplegic is expressed in specific regions in the embryo, particularly in the dorsal-most epidermal cells that express puc. It has been shown recently that the maintenance of decapentaplegic expression along the leading edge of the epidermis depends on the activity of the DJNK pathway. In pucE69 mutant embryos, the expression of dpp in the dorsal-most epidermal cells is enhanced from stage 11. Furthermore, after germ-band shortening there are more epidermal cells expressing dpp than in wild type, but this expression is still mainly limited to dorsal cells. In contrast, ubiquitous expression of Puc leads to a decrease in the expression of dpp in those cells during stage 11 and the complete absence at stages 12 and 13. Puc overexpression does not affect the levels of dpp on the visceral mesoderm or the ventral epidermis. These results suggest a role for puc in the control of dpp expression, which could be mediated by Puc activity on JNK signaling (Martin-Blanco, 1998).

The null phenotype for dpp, a completely ventralized embryo, reflects its initial role over the dorsal epidermis and might obscure the function of later patterns of expression. Mutants for thick veins, however, which encodes a Dpp receptor, display dorsal holes similar to those of hep or bsk mutants. To test if the higher levels of dpp present in puc mutants could be involved in puckering phenotypes, dpp was overexpressed. Such dpp overexpressing embryos undergo an extreme dorsalization of the epidermis, but they still have a dorsal midline; in many cases, phenotypes were observed that were very similar to those observed in pucE69 mutants. This indicates that besides an early function for dpp in epidermal cell stretching, the downregulation of dpp in the dorsal-most epidermal cells is necessary for completion of dorsal closure (Martin-Blanco, 1998).

Drosophila kayak mutant embryos exhibit defects in dorsal closure, a morphogenetic cell sheet movement during embryogenesis. It is shown that kayak encodes D-Fos, the Drosophila homologue of the mammalian proto-oncogene product, c-Fos. D-Fos is shown to act in a similar manner to Drosophila Jun: in the cells of the leading edge it is required for the expression of the TGFbeta-like Decapentaplegic (Dpp) protein, which is believed to control the cell shape changes that take place during dorsal closure. The kayak expression domain include the cells of the amnioserosa and the lateral epidermis during the process of dorsal closure. At the onset of dorsal closure, elevated levels of D-Fos can be detected in the nuclei of leading-edge cells as they initiate elongation. Subsequently, elevated expression of Fos can also be observed in more ventrally located epidermal cells. Concurrently, cell elongation spreads laterally, until the two edges meet at the dorsal midline. At this stage, Fos is strongly expressed throughout the embryonic epidermis, with the highest levels remaining in the cells of the leading edge. This expression pattern is very similar to that of D-Jun and also correlates with the JNK-pathway-dependent stripe of dpp expression in the leading edge, which becomes apparent during the initiation phase of dorsal closure. Therefore, it is conceivable that Drosophila Fos acts in conjunction with Jun to regulate dorsal closure and dpp expression (Zeitlinger, 1998).

Defects observed in mutant embryos, and adults with reduced Fos expression, are reminiscent of phenotypes caused by 'loss of function' mutations in the Drosophila JNKK homologue, hemipterous. Mutant alleles of D-fos have not previously been described. Based on the potential involvement of D-Fos in the process of dorsal closure, known dorsal open mutants were examined for defects in D-fos. kay1 mutant embryos all die during embryogenesis with large dorsal and anterior holes that indicate failed dorsal closure and head involution. In kay2 embryos, dorsal holes are also observed, but at lower penetrance. Depending on the temperature and genetic background, up to ~1% of kay2 homozygotes even develop to adulthood, as seen after recessive markers had been removed from the kay2 mutant chromosome by recombination. The transheterozygous kay1/kay2 allelic combination displays an intermediate phenotype and embryonic, or early larval, lethality. These observations indicate that kay2 is a weaker allele than kay1, and thus is a hypomorph. To examine the cause of the kay defect at the cellular level, mutant and wild-type embryos were stained with an anti-Coracle serum, which outlines the epidermal cells. It was found that the kay mutant phenotype is caused by a failure of the lateral epidermal cells to elongate. As previously observed in the case of D-jun, leading-edge cells of zygotic kay1 mutants initiate elongation transiently, but fail to maintain it and subsequently resume the unelongated shape. The more lateral epidermal cells elongate to a very minor extent and resume the typical polygonal shape after the process has been terminated prematurely. Thus, the kay mutant phenotype closely resembles those described for hep, bsk and D-jun mutant embryos, also at the cellular level (Zeitlinger, 1998)

The phenotypic similarity between D-jun and kay mutants suggests that D-Fos and D-Jun act in concert to mediate dorsal closure. Thus, one may predict that D-fos/kay, like D-jun and the upstream signaling components bsk/JNK and hep/JNKK, is required for the expression of dpp in the leading edge cells. To test this idea, the expression of DPP mRNA was monitored in wild-type and kay-mutant backgrounds. Expression of dpp in the cells of the future leading edge is normally initiated when the germ band is fully extended and is maintained throughout dorsal closure. In contrast, in kay1 homozygous embryos, dpp expression is absent (or reduced in kay2 mutants) in cells of the leading edge. Significantly, other pattern elements of dpp expression are still present in the kay/D-fos mutants, including a more ventral stripe and midgut-specific expression, which have previously been shown to be independent of JNK signaling. Another downstream effect of Bsk signaling, the transcriptional activation of the puckered gene (puc) in the cells of the leading edge is also abrogated in kay1 mutant embryos. Taken together, the requirement of both D-Fos and D-Jun for dpp and puc expression in leading-edge cells suggests that the JNK signal is relayed by a heterodimeric transcription factor composed of D-Jun and D-Fos. These results indicate that D-Fos is required downstream of the Drosophila JNK signal transduction pathway, consistent with a role in heterodimerization with D-Jun, to activate downstream targets such as dpp (Zeitlinger, 1998).

The TAK kinases belong to the MAPKKK group and have been implicated in a variety of signaling events. Originally described as a TGFß activated kinase (TAK), the mammalian protein has, however, been demonstrated to signal through p38, Jun N-terminal kinase (JNK) and Nemo types of MAP kinases, and the NFkappaB inducing kinase. Despite these multiple proposed functions, the in vivo role of TAK family kinases remains unclear. The isolation and genetic characterization of the Drosophila TAK homolog (TGF-ß activated kinase 1: Tak1) is reported in this study. Sequence analysis reveals a 678 amino acid long open reading frame (ORF), which shows the highest similarity to vertebrate TAK proteins. Subsequent conceptual translation displays an N-terminal kinase domain of about 280 amino acids, showing 54% identity and 69% similarity to mTAK1, and a long C-terminal domain. The C-terminal domain is less well conserved. However, a 60 amino acid stretch shows a significant level of conservation as compared to the vertebrate and C. elegans orthologs (36% identity and 60% similarity to mTAK1), constituting a conserved protein-protein interaction interface for putative modulators of TAK activity, such as TAB-2 (Mihaly, 2001).

The use of overexpression and double-stranded RNA interference (RNAi) techniques has allowed analysis of Tak1 function during embryogenesis and larval development. Overexpression of Tak1 in the embryonic epidermis is sufficient to induce the transcription of the JNK target genes decapentaplegic and puckered. Furthermore, overexpression of dominant negative (DN) or wild-type forms of Tak1 in wing and eye imaginal discs, respectively, results in defects in thorax closure and ommatidial planar polarity, two well described phenotypes associated with JNK signaling activity. Surprisingly, RNAi and DN-Tak1 expression studies in the embryo argue for a differential requirement of Tak1 during developmental processes controlled by JNK signaling, and a redundant or minor role of Tak1 in dorsal closure. In addition, Tak1-mediated activation of JNK in the Drosophila eye imaginal disc leads to an eye ablation phenotype due to ectopically induced apoptotic cell death. Genetic analyses in the eye indicate that Tak1 can also act through the p38 and Nemo kinases in imaginal discs. These results suggest that dTAK can act as a JNKKK upstream of JNK in multiple contexts and also other MAPKs in the eye. However, the loss-of-function RNAi studies indicate that it is not strictly required and thus either redundant or playing only a minor role in the context of embryonic dorsal closure (Mihaly, 2001).

Dorsal closure, taking place in mid-embryogenesis, describes the morphogenetic movements of the epidermis in order to replace the amnioserosa on the dorsal side of the embryo. This event is driven by the concerted spreading of epidermal cells towards the dorsal midline, where the two contralateral epidermal cell layers meet and remain connected. The JNK signaling module and nuclear targets of JNK, the AP-1 transcription factors dJun and dFos, control the process of dorsal closure. Uncompleted or failed dorsal closure is indicative of disrupted JNK signaling. Mutations in all known components of the JNK signaling pathway result in dorsal open embryos. During dorsal closure, the expression of the dpp and puc genes in cells of the leading edge is controlled by the JNK kinase module and the AP-1 transcription factors Jun and Fos. Leading edge cells show loss of puc and dpp expression when deficient for JNK signaling. Conversely, constitutive activation of JNK signaling in the embryonic epidermis by overexpressing activated Rac or Cdc42 induces the upregulation of dpp and puc. To address the question of whether dTAK can activate and act through the JNK MAPK module, Tak1 was expressed under the control of the en-GAL4 and pnr-GAL4 drivers and the induction of dpp and puc was monitered in the epidermis of stage 12-15 embryos (pnr is strongly expressed in leading edge cells and cells neighboring the leading edge). In wild-type, dpp is expressed in two lateral stripes along the Drosophila embryo, and the dorsal most stripe corresponds to the leading edge of the epidermis. Overexpression of Tak1 with either GAL4 driver causes ectopic upregulation of dpp, as monitored by RNA in situ hybridization. Similarly, the analysis of embryos carrying one copy of a puc lacZ enhancer trap by ß-galactosidase activity staining shows a clear and robust ectopic puc expression when Tak1 is overexpressed. These patterns of dpp and puc activation by Tak1 are identical to those observed with activated Jun, suggesting that the effect is direct and mediated by the JNK signaling pathway (Mihaly, 2001). In summary, these data indicate that overexpression of Tak1 in the embryonic ectoderm is sufficient to induce high-level expression of both known JNK target genes. Since the same upregulation of puc and dpp is observed with activated JNKK/Hep and Jun (a JNK activated transcription factor), they strongly suggest that Tak1 acts through the JNK/ Jun(AP-1) module in the context of dorsal closure (Mihaly, 2001).

A genetic system has been developed based upon the hobo transposable element in Drosophila melanogaster. hobo, like the better-known P element, is capable of local transposition. A hobo enhancer trap vector has been mobilized and two unique alleles of decapentaplegic (dpp) have been generated . A detailed study of one of those alleles (dpp^F11) is reported. This is the first application of the hobo genetic system to understanding developmental processes. LacZ expression from the dpp^F11 enhancer trap accurately reflects dpp mRNA accumulation in leading edge cells of the dorsal ectoderm. Combinatorial signaling by the Wingless (Wg) pathway, the Dpp pathway, and the transcriptional coactivator Nejire (CBP/p300) regulates dpp^F11 expression in these cells. This analysis of dpp^F11 suggests a model for the integration of Wg and Dpp signals that may be applicable to other developmental systems. This analysis also illustrates several new features of the hobo genetic system and highlights the value of hobo, as an alternative to P, in addressing developmental questions (Newfeld, 2002).

During early stages of embryogenesis, wg and dpp are expressed in undifferentiated dorsal ectoderm. wg mRNA expression, in 15 stripes along the entire dorsal-ventral axis of the embryo (including the dorsal ectoderm), begins at stage 8. wg expression persists in this striped pattern through stage 17. dpp mRNA is expressed on the dorsal side of the embryo along the entire anterior-posterior axis, beginning at stage 4. dpp mRNA expression persists in a large portion of the dorsal ectoderm through stage 8 and resolves into leading edge cell-specific expression in stage 12 embryos. The embryonic expression pattern of nej has not been reported. However, some information can be inferred from nej mutant phenotypes. nej zygotic mutant embryos show visible defects in the tracheal system at stage 12. The tracheal system is derived from the dorsal ectoderm, suggesting that nej is expressed in this tissue prior to stage 12 (Newfeld, 2002).

Analysis of dpp^F11 suggests that dpp expression in leading edge cells is initiated by prior episodes of wg and dpp expression in the undifferentiated dorsal ectoderm. The maintenance of dpp expression in leading edge cells appears to require continuous input from wg and from a dpp feedback loop. The initiation and maintenance of dpp expression in leading edge cells also require continuous nej activity. Overall, these data are consistent with the following combinatorial signaling model: Med (signaling for the Dpp pathway) interacts with Arm (signaling for the Wg pathway) via the transcriptional coactivator Nej. This multimeric complex initiates and, with continuous signaling, maintains dpp expression in leading edge cells (Newfeld, 2002).

These data extend previous studies of dpp expression in leading edge cells and Dpp signaling in several ways. A role for Wg signaling in the regulation of dpp expression in the leading edge has been suggested. dpp leading edge expression is not maintained in arm² zygotic mutants and does not initiate in arm² germline clones. nej and Med are involved in the regulation of dpp expression in leading edge cells. While nej³ enhances dpp wing phenotypes, Med¹ enhances nej³ embryonic phenotypes. This study suggests a role for nej in mediating combinatorial signaling by the Wg and Dpp pathways (Newfeld, 2002).

Several questions remain about the combinatorial regulation of dpp expression by Wg, Dpp, and Nej. One question is, how is Nej recruited to bridge the two pathways? Numerous studies have shown that p300/CBP transcriptional coactivation functions are stimulated by its phosphorylation but the site of phosphorylation has never been mapped. Perhaps Zeste white3 (a serine-threonine kinase in the Wg pathway) or Thickveins (a serine-threonine kinase in the Dpp pathway) are involved in recruiting Nej to participate in combinatorial signaling (Newfeld, 2002).

A second question concerns the nature of the enhancer element that directs dpp expression in leading edge cells. Using reporter genes, a 54-nucleotide candidate enhancer has been identified near the dpp^F11 transgene insertion that drives lacZ expression in a subset of leading edge cells. The region contains two sets of conserved, overlapping consensus-binding sites for dTCF (a transcriptional partner for Arm in the Wg pathway) and Mad (a transcriptional partner for Med in the Dpp pathway). No JNK-pathway-binding sites are in the region, suggesting that JNK regulation of dpp expression in leading edge cells is independent of Wg and Dpp signaling (Newfeld, 2002).

Interestingly, there is also a consensus Brinker (Brk) binding site in the candidate enhancer. Brk is a transcriptional repressor of Dpp target genes and one mechanism by which Dpp signaling activates its target genes is to relieve Brk repression. The genetic data cannot discriminate between the possibility that combinatorial signaling by the Wg and Dpp pathways regulates dpp expression in leading edge cells by direct activation or by relief of Brk repression (Newfeld, 2002).

Various lines of evidence from mammalian tissue culture suggest that Cdc42 functions in regulating the JNK signaling cascade. In Drosophila, the JNK pathway plays an integral role in dorsal closure, a morphogenetic process involving cell shape changes and local signaling events that occurs late in embryogenesis. One demonstrated function of the JNK pathway is to promote expression of the morphogen Decapentaplegic in cells at the leading edge of the lateral epidermis during dorsal closure. Consistent with this notion, previous studies have shown that dpp expression in the leading-edge epidermal cells is disrupted in embryos carrying mutations in members of the JNK signaling pathway. Reduction but not complete loss of maternally contributed hemipterous results in complete loss of dpp expression while loss of a negative regulator, puckered, results in increased dpp expression (Genova, 2000 and references therein).

Thus dpp expression at the leading edge is sensitive to the level of JNK pathway function. Previous studies using ectopic expression of dominant Cdc42 alleles have suggested that Cdc42 is necessary for dorsal closure and functions upstream of the JNK pathway at the leading edge. If this is so, then loss of Cdc42 function should disrupt JNK signaling and therefore dpp expression in these cells. To test this hypothesis, in situ hybridization to dpp mRNA was performed on embryos derived from Cdc42⁴/Cdc42⁶ mothers. Although ~70% of embryos produced by these females displayed epithelial defects and lethality, normal levels of dpp expression were observed in all embryos that developed to the onset of dorsal closure, including those that had arrested development due to insufficient levels of Cdc42 function. Thus, unlike known upstream components of the JNK pathway, reduction in Cdc42 function has no apparent effect on dpp expression by leading-edge cells at the time of dorsal closure (Genova, 2000).

Regulation in the eye disc

Signaling by the secreted Hedgehog, Decapentaplegic and Wingless proteins organizes the pattern of photoreceptor differentiation within the Drosophila eye imaginal disc; hedgehog and decapentaplegic are required for differentiation to initiate at the posterior margin and progress across the disc, while wingless prevents it from initiating at the lateral margins. Wingless and Decapentapegic often have opposing functions in differentiation. This is true in the eye imaginal disc, where Dpp effects are positive, promoting initiation of the morphogenetic furrow and also promoting photoreceptor differentiation. Wg effects are negative, opposing the effects of Dpp, inhibiting furrow initiation and likewise inhibiting differentiation. An analysis of these interactions has shown that initiation requires both the presence of decapentaplegic and the absence of wingless, which inhibits photoreceptor differentiation downstream of the reception of the decapentaplegic signal. The eyes absent and eyegone genes encode members of a group of nuclear proteins required to specify the fate of the eye imaginal disc. Both eyes absent and eyegone are required for normal activation of decapentaplegic expression at the posterior and lateral margins of the disc and also repression of wingless expression in presumptive retinal tissue. The requirement for eyegone can be alleviated by inhibition of the wingless signaling pathway, suggesting that eyegone promotes eye development primarily by repressing wingless. These results provide a link between the early specification and later differentiation of the eye disc (Hazelett, 1998).

As a direct test of the requirement for dpp in furrow initiation and of the inhibitory role of wg, clones of cells were generated of doubly mutant for Mothers against dpp (required to transduce the dpp signal) and wg were examined. Cells in these clones are unable to respond to dpp, but are also unable to produce wg. When such clones of cells occur at the posterior margin of the eye disc, they autonomously fail to initiate photoreceptor development. Clones of cells singly mutant for Mad also fail to differentiate as photoreceptors, but often have an additional non-autonomous inhibitory effect on photoreceptor differentiation by surrounding cells. This inhibitory effect is likely to be mediated by wg. Thus dpp signaling is required not only to repress wg expression, but also independently for morphogenetic furrow initiation (Hazelett, 1998).

wg is shown to inhibit photoreceptor formation downstream of the dpp receptors. wg is known to be required to prevent ectopic morphogenetic furrow initiation from the lateral margins of the eye disc. However, the mechanism by which wg inhibits photoreceptor differentiation is not well understood. It has been suggested that wg acts by preventing dpp expression, since dpp expression is lost in clones of cells lacking the kinase encoded by shaggy/zeste-white 3 (sgg), which normally functions to inhibit the wg pathway. However, a low level of ectopic wg can inhibit photoreceptor differentiation without reducing dpp expression. Since dpp positively autoregulates its own expression, inhibition of dpp function may result in a loss of dpp expression. If wg acted by inhibiting dpp expression, it should be possible to overcome its effects by expressing dpp from a heterologous promoter. However, co-expression of dpp and wg does not allow initiation of photoreceptor development at the posterior margin. Ectopic expression of dpp in the eye disc has been shown to specifically induce initiation of photoreceptor differentiation from the anterior margin of the disc in a non-autonomous fashion. Surprisingly, this ectopic differentiation is not inhibited by wg signaling. Co-expression of dpp and wg throughout the disc results in initiation from the anterior margin at a much higher frequency than from the posterior margin. Thus initiation from the anterior margin must be able to overcome the inhibition normally caused by wg. wg inhibitory function is shown to act downstream of or in parallel to the action of Thickveins, a receptor for Dpp (Hazelett, 1998).

Rather than affecting the Dpp pathway directly, Wg might block photoreceptor differentiation at a stage subsequent to Dpp signaling. Formation of all photoreceptors is known to depend on the EGF receptor and its downstream component Ras. Wg has recently been shown to antagonize EGF receptor signaling during the specification of the cuticle pattern in the embryo. To determine whether wg also acts on this pathway in the eye, a test was performed to determine if a secreted and active form of the ligand Spitz (s-Spi) or a constitutively active form of Ras could bypass the block caused by wg. In discs expressing both Wg and activated Ras ubiquitously, extensive photoreceptor differentiation and growth are observed, as in discs expressing activated ras alone. Thus Wg must act upstream, prior to Ras activation, to block differentiation. Expression of s-Spi also rescues photoreceptor differentiation in discs expressing Wg ectopically. The inhibition of photoreceptor differentiation is mediated by the conventional Wg signal transduction pathway, because a constitutively active form of Armadillo, a mediator of the Wg signal, is shown to block photoreceptor differentiation (Hazelett, 1998).

Since the phenotype caused by ectopic wg is rescued by expressing activated forms of Spi or Ras it is possible that Wg interferes with Egf receptor signaling upstream of (prior to the activation of) Ras. Recently, it has been shown that in the embryonic segments Wg and secreted Spi emanate from distinct sources and promote opposing cell fates. This led to the proposal that Wg antagonizes signaling by Spi through the EGF receptor and the Ras/MAPK cascade. Since EGF receptor signaling is required for the formation of all photoreceptors, it is a possible target for Wg inhibition in the eye disc. However, it does not appear that the effects of ectopic Wg can be completely explained by the antagonism of Spi signaling, since mutations in spi allow the specification of R8 and the progression of the furrow, while the presence of ectopic Wg does not. It is possible that another ligand, such as Vein, normally activates the EGF receptor in R8 and that this ligand is also antagonized by Wg. Another possibility is that Ras activation in R8 is mediated by another tyrosine kinase receptor; one of the identified FGF receptors is expressed in the morphogenetic furrow. The lower effectiveness of rescue by s-Spi than by activated Ras also suggests that Wg has effects both upstream of Spi expression or processing, and downstream of these events. Some factors known to be required between Spi and Ras that could be targets of Wg inhibition are Daughter of sevenless, Downstream of receptor kinases and Son of sevenless. Alternatively, Wg could act by stimulating the expression or function of Argos, a secreted antagonist of Spi (Hazelett, 1998).

Genes implicated in early events of eye development, eyes absent and eyegone, are involved in the regulation of dpp and wg expression. The expression of dpp and wg were examined in eya mutant eye discs. Expression is greatly reduced in early third instar eya mutant discs, prior to the initiation of the morphogenetic furrow, and was completely lost in eya mutant clones, suggesting that eya is required for dpp transcription. Although the initiation of wg expression in early eya mutant eye discs appeared to be normal, ectopic Wg protein was observed in eya mutant clones in late third instar discs. Another gene required for eye formation that has not been placed within the hierarchy of eye development is eyegone (eyg); in its absence, no photoreceptors differentiate and the eye disc does not reach its normal size and shape. Eyegone is a Pax-like protein (C. Desplan and H. Sun, personal communication to Hazelett, 1998). The expression patterns of dpp and wg were examined in early third instar eyg mutant discs. dpp expression is restricted to the posterior margin of eyg mutant discs, in contrast to its expression around the posterior and lateral margins of wild-type discs. On the contrary, wg expression was expanded, especially on the dorsal side of the disc, where it extends to the posterior margin. eyg thus acts to delimit the domains of dpp and wg expression; since it encodes a Pax-like transcription factor, it is possible that this regulation is direct. Since inhibition of the wg pathway at the posterior margin of eyg mutant discs is sufficient to allow photoreceptor formation, it is concluded that the misexpression of wg observed at the posterior of the eyg mutant discs is a major cause of the absence of photoreceptor development. As expected since it can overcome the effect of ectopic wg, activated Ras is also able to rescue photoreceptor differentiation in eyg mutant discs. In summary, these results show that wg inhibits normal photoreceptor differentiation in a manner independent of dpp expression or activation. The expression patterns of both dpp and wg, and perhaps their cross-regulatory interactions, are determined during early eye development by genes including eya and eyg (Hazelett, 1998).

In the anterior compartment of appendage discs and anterior to the morphogenetic furrow in the eye disc, cells that lack cAMP-dependent Protein kinase A (PKA) activity ectopically express decapentaplegic. Pka- cells can influence the fate of neighboring cells to reorganize anterior patterns in appendages and trigger ectopic morphogenetic furrows in the developing retina. This organizing activity of Pka mutant cells depends on dpp activity. These findings suggest that PKA is a component of a signaling pathway that represses dpp expression and that HH antagonizes this pathway to maintain dpp expression at the anterior-posterior compartment border and in the morphogenetic furrow (Pan, 1995).

The dorsal open cuticle phenotype of slipper mutant embryos resembles that caused by mutations in components of the JNK pathway known to regulate the progression of dorsal closure and expression of genes in the leading edge of the epidermis. To determine whether loss of slpr function affects JNK transcriptional targets, the expression of decapentaplegic (dpp) was characterized in slpr mutant embryos. It was found that dpp expression is absent from leading edge cells of approximately one-quarter of the observed embryos, consistent with the expected frequency of zygotically mutant slpr⁹²¹/Y embryos. In the mutant embryos, the other tissue-specific patterns of dpp expression are unaffected. Furthermore, nearly half of all embryos derived from females with germ-line clones of the slpr^3P5 allele show loss of leading edge dpp expression. Thus, the specific loss of dpp from leading edge cells of slpr mutant embryos indicates a significant reduction of JNK signaling and AP-1 activity (Stronach, 2002).

Although Hedgehog (Hh) signaling is essential for morphogenesis of the Drosophila eye, its exact link to the network of tissue-specific genes that regulate retinal determination has remained elusive. In this report, it is demonstrated that the retinal determination gene eyes absent (eya) is the crucial link between the Hedgehog signaling pathway and photoreceptor differentiation. Specifically, it is shown that the mechanism by which Hh signaling controls initiation of photoreceptor differentiation is to alleviate repression of eya and decapentaplegic (dpp) expression by the zinc-finger transcription factor Cubitus interruptus (Ci^rep). Furthermore, the results suggest that stabilized, full length Ci (Ci^act) plays little or no role in Drosophila eye development. Moreover, while the effects of Hh are primarily concentration dependent in other tissues, hh signaling in the eye acts as a binary switch to initiate retinal morphogenesis by inducing expression of the tissue-specific factor Eya (Pappu, 2003).

Misexpression of eyeless (ey) in the wing disc causes ectopic photoreceptor differentiation only in regions where both dpp and hh signaling are normally active. The simplest explanation for this effect invokes a linear regulatory hierarchy where hh induces dpp, which in turn cooperates with ey to initiate retinal morphogenesis. While misexpression of ey and dpp together does indeed lead to synergistic photoreceptor differentiation, this occurs only in the posterior compartment of the wing disc. Notably, Hh signaling is not transduced in the posterior compartment of the wing disc due to the repression of ci by En. Furthermore, dpp and ey expression does not induce Ci expression in the posterior compartment of the wing disc. Thus, it is concluded that dpp and ey can induce Eya expression and photoreceptor differentiation in the posterior compartment of the wing disc in the absence of Hh signaling and Ci^rep. Misexpression of hh and ey induces robust eya expression and photoreceptor differentiation in the wing disc, but only in the anterior compartment. This result is consistent with a model in which Hh signaling normally blocks the production of Ci^rep and converts it into an activated form, Ci^act, in the anterior compartment of the wing disc. Ci^act can induce dpp expression in the anterior compartment and dpp can in turn cooperate with ey to induce robust Eya expression and photoreceptor differentiation. Consistent with this model, co-expression of hh, dpp and ey leads to Eya expression and photoreceptor differentiation in both compartments of the wing disc. Taken together, these results suggest that, in the wing disc, ey and dpp can activate eya expression only in the absence of Ci^rep (Pappu, 2003).

Co-expression of dpp, ey and eya using the 30A-Gal4 driver induces photoreceptor differentiation in both wing compartments, albeit with low penetrance. This effect becomes stronger and more penetrant when dpp, ey, eya and so are misexpressed in a ring around the wing pouch. These results demonstrate that providing ey, dpp and eya from an exogenous source is sufficient to bypass the requirement for Hh signaling during initiation of ectopic photoreceptor differentiation. In addition, these results implicate eya as a key target for Hh signaling during the initiation of normal retinal morphogenesis, most likely by blocking Ci^rep (Pappu, 2003).

It is proposed that Hh signaling acts as a binary switch during Drosophila eye development to control the timing of initiation of photoreceptor differentiation. Specifically, the data suggest that during early larval development Ci^rep normally inhibits retinal morphogenesis by blocking eya and dpp expression. Hh signaling in late second instar larvae blocks production of Ci^rep, which in turn allows dpp and eya expression, MF initiation, progression and photoreceptor differentiation. Rather than regulating the differentiation of multiple cell types in a concentration-dependent manner, the data suggest that Hh signaling acts as a molecular switch that is sufficient to initiate dpp and eya expression and retinal morphogenesis. This model also explains the seemingly contradictory phenotypes of loss of smo (blocks MF initiation) and loss of ci (no effect) during Drosophila eye development. Loss of ci creates a permissive environment for eya and dpp expression and photoreceptor differentiation, rendering eye development Hh independent. By contrast, Ci^rep persists in the absence of smo function and thus photoreceptor morphogenesis does not occur in smo clones. Since ci null mutant clones in the eye develop normally, other Hh independent mechanisms must also act to control the initiation of retinal morphogenesis in Drosophila (Pappu, 2003).

Posterior margin smo mutant clones lack Eya expression and photoreceptor differentiation. The lack of eya expression in these cells is attributed to their inability to block the production of Ci^rep. Furthermore, the data demonstrate that co-expression of dpp and eya in these posterior smo mutant clones rescues photoreceptor differentiation. In addition, dpp and eya co-expression is sufficient to rescue delayed furrow progression in smo clones. However, the precise temporal and spatial order of photoreceptor recruitment may not be rescued in these clones. Thus, the requirement for Hh signaling in the eye can be circumvented by the expression of the downstream targets dpp and eya. These results demonstrate that eya is a crucial eye-specific target of Hh signaling during the initiation of retinal differentiation and has led to a new model for the initiation of retinal morphogenesis. In this model, Hh signaling blocks the proteolytic degradation of Ci^act into Ci^rep, thus allowing initiation of dpp expression. Once dpp expression is established, the absence of Ci^rep allows dpp to act in parallel with ey to initiate eya expression, which in turn leads to so expression. Furthermore, dpp cooperates with eya and so to initiate the expression of dac and extensive feedback regulation among these genes leads to consolidation of retinal cell fates (Pappu, 2003).

Characterization of different alleles of the Hedgehog receptor patched (ptc) indicates that they can be grouped into several classes. Most mutations result in complete loss of Ptc function. However, missense mutations located within the putative sterol-sensing domain (SSD) or C terminus of ptc encode antimorphic proteins that are unable to repress Smo activity and inhibit wild-type Ptc from doing so, but retain the ability to bind and sequester Hh. Analysis of the eye and head phenotypes of Drosophila in various ptc/ptc^tuf1 heteroallelic combinations shows that these two classes of ptc alleles can be easily distinguished by their eye phenotype, but not by their head phenotype. Adult eye size is inversely correlated with head vertex size, suggesting an alteration of cell fate within the eye-antennal disc. A balance between excess cell division and cell death in the mutant eye discs may also contribute to final eye size. In addition, contrary to results reported recently, the role of Hh signaling in the Drosophila head vertex appears to be primarily in patterning rather than in proliferation, with Ptc and Smo having opposing effects on formation of medial structures (Thomas, 2003).

The two major targets of Hh signaling during MF progression are dpp and ato. The data indicate that although dpp is ectopically activated in ptc trans-heterozygotes, ato is not. This is unexpected, since Hh signaling activates the initial expression of ato, so an increase might be anticipated to expand the ato expression domain into more anterior regions, while maintaining or even increasing the level of expression. Conversely, reducing the activity of ptc in the hh¹ mutant rescues the expression of ato, but not that of dpp. There are several possible explanations for these findings. (1) ato is an indirect target of Hh signaling and the mediators of Hh activity in this context are unclear. It is likely that other factors, in addition to those directly induced by Hh, are necessary for ato expression, any one of which may be limiting. (2) dpp may respond to lower levels of Hh pathway activation more than genes upstream of ato. In the wing disc, dpp is activated by relatively low levels of Hh anterior to the AP border, whereas other Hh target genes such as collier require a higher level of pathway activation. In ptc trans-heterozygotes some Ptc activity is retained and hence the very highest level of Hh signaling cannot be reached. (3) Dpp in its role as an inducer of the preproneural state can actually inhibit the expression of ato through activation of the proneural repressors h and emc. In ptc trans-heterozygous discs, the domain of h expression does appear to be expanded, suggesting a possible explanation for the observed downregulation of ato expression. A fourth possibility that has not been tested is that the increased level of Hh signaling in ptc trans-heterozygote discs results in an expansion of the domain of rough (ro) expression. Ro is induced by high-level Hh signaling at the posterior margin of the MF, but, if expressed at excessive levels (as in the ro^Dom mutant), causes a downregulation of ato expression. Although a severe reduction in ato expression such as that caused by ro^Dom can result in furrow arrest, the significance of a mild downregulation of expression is unknown (Thomas, 2003).

The two classes of mutants both show an increase in dpplacZ expression relative to wild type. However, the domain of ectopic expression differs significantly between allele types, suggesting a difference in the way in which the pathway is activated in the two classes of mutant. Because LF ptc alleles cannot sequester Hh efficiently, the broad band of ectopic dpplacZ seen ahead of the MF may be caused by excessive diffusion of Hh anteriorly. In contrast, the AN/ptc^tuf1 trans-heterozygotes can sequester Hh efficiently and consequently demonstrate only phenotypes caused by autonomous ectopic pathway activation (Thomas, 2003).

Despite the rescue of adult-eye phenotype observed in ptc/ptc^tuf1;hh¹ double mutants, the expression of dpplacZ was not restored in the center of the disc. Since Dpp does not play a major role in MF progression, the lack of expression in this situation may not have a significant effect. Alternatively, excessive dpp expression at the margins may allow the protein to diffuse medially into the disc, thus aiding MF progression in an unconventional way (Thomas, 2003).

Dpp is known to have several functions in the eye disc, all of which, when modified, can influence the final size and shape of the adult eye. However, despite the disparity between the patterns of dpplacZ expression in the two types of trans-heterozygote, surprisingly little difference is detected downstream of Dpp. Although ectopic dpp expression has been shown to induce ectopic MFs, this does not occur in the ptc trans-heterozygotes, presumably because the ectopic Dpp is either not high enough or not expressed in the right place (Thomas, 2003).

In addition to its effect on furrow initiation, Dpp is also responsible for defining the eye field via inhibition of Wg and for inducing cell cycle arrest ahead of the furrow. Small-eye mutants do show an increased head vertex size, suggesting that an eye-to-vertex fate change has occurred. However, ptc trans-heterozygotes do not display critical differences either from wild type or between allele classes in the distribution of Wg in second instar eye discs. In addition, dpp expression is actually expanded in eye discs of small-eye mutants, indicating that processes other than Wg/Dpp antagonism must be involved in specification of eye vs. head domains (Thomas, 2003).

Ectopic Dpp ahead of the furrow does not appear to induce premature cell cycle arrest and therefore cannot explain the reduced-eye phenotypes observed in LF/ptc^tuf trans-heterozygotes. However, when compared to wild type, ptc mutants do show an increase in cell divisions ahead of the furrow. It is suggested that in addition to an eye/head vertex specification defect, LF/ptc^tuf1 trans-heterozygotes may exhibit a small-eye phenotype due to excessive cell death, despite some increase in cell divisions ahead of the furrow. Conversely, in DN/ptc^tuf1 trans-heterozygotes, increased proliferation could overcompensate for increased cell death, leading to larger eyes. This suggests that the adult-eye phenotype is at least partially dependent upon a balance between cell division and cell death in the disc, in addition to an eye-to-head fate change (Thomas, 2003).

The nuclear zinc-finger protein encoded by the hindsight (hnt) locus regulates several cellular processes in Drosophila epithelia, including the Jun N-terminal kinase (JNK) signaling pathway and actin polymerization. Defects in these molecular pathways may underlie the abnormal cellular interactions, loss of epithelial integrity, and apoptosis that occurs in hnt mutants, in turn causing failure of morphogenetic processes such as germ band retraction and dorsal closure in the embryo. To define the genetic pathways regulated by hnt, 124 deficiencies on the second and third chromosomes and 14 duplications on the second chromosome were assayed for dose-sensitive modification of a temperature-sensitive rough eye phenotype caused by the viable allele, hnt^peb; 29 interacting regions were identified. Subsequently, 438 P-element-induced lethal mutations mapping to these regions and 12 candidate genes were tested for genetic interaction, leading to identification of 63 dominant modifier loci. A subset of the identified mutants also dominantly modify hnt³⁰⁸-induced embryonic lethality and thus represent general rather than tissue-specific interactors. General interactors include loci encoding transcription factors, actin-binding proteins, signal transduction proteins, and components of the extracellular matrix. Expression of several interactors was assessed in hnt mutant tissue. Five genes -- apontic (apt), Delta (Dl), decapentaplegic (dpp), karst (kst), and puckered (puc) -- regulate tissue autonomously and, thus, may be direct transcriptional targets of Hnt. Three of these genes -- apt, Dl, and dpp -- are also regulated nonautonomously in adjacent non-Hnt-expressing tissues. The expression of several additional interactors -- viking (vkg), Cg25, and laminin-alpha (LanA) -- is affected only in a nonautonomous manner (Wilk, 2004).

Regulation in the leg disc

Slimb (Slmb) is an F-box/WD40 protein that potentially participates in the ubiquitin proteolysis machinery. During development, Slmb is required in limb discs to repress Hedgehog (Hh) target genes, i.e. wingless and decapentaplegic, as well as the Wg signal transduction pathway. These repression functions have been proposed from studies using weak slmb alleles. Interestingly, experiments with strong slmb alleles have revealed additional functions in which slmb is required, such as leg dorsal-ventral restriction. New alleles of the slmb gene have been isolated in a screen for new negative regulators of dpp: several amorphs (characterized by genetic and molecular criteria) and a cold-sensitive hypomorph. By performing somatic clone experiments with these new amorphic slmb alleles, it has been determined that regulation of Dpp and Wg by Slmb could be different from what has already been published. In leg discs, lack of slmb function derepresses the transcription of wg, independent of Hh signaling. Ectopic legs resulting from slmb^- clone induction come only from wg misexpression in the normal dpp domain, since ectopic proximo-distal axes are induced dorsally, and adult ectopic legs are often perfect with respect to antero-posterior polarity. In wing discs, transcription of wg, which is normally independent of Hh signaling, is also derepressed in the absence of slmb function. In discs bearing amorphic slmb clones and in discs of two other slmb^- contexts, a novel pattern of dpp expression is described consisting of an enlargement of the normal dpp domain. Strong evidence indicates that this dpp modification can be linked to imaginal disc regeneration following slmb^- cell elimination. The fate of slmb^- clones, which disappear before adulthood, has been investigated. It was found that two mechanisms of cell elimination can account for imaginal cell regeneration: an early apoptosis and a mechanism of sorting-out that excludes all slmb^- clones from all imaginal discs. This result suggests that Slmb is likely to be involved, in addition to its repression role on Dpp and Wg, in some other essential cellular mechanism, since, in the absence of Slmb, cell affinities are dramatically modified regardless of the deregulated morphogen and of the type of imaginal disc (Miletich, 2000).

The Wingless signaling pathway directs many developmental processes in Drosophila by regulating the expression of specific downstream target genes. The product of the trithorax group gene osa is required to repress such genes in the absence of the Wingless signal. The Wingless-regulated genes nubbin, Distal-less, and decapentaplegic and a minimal enhancer from the Ultrabithorax gene are misexpressed in osa mutants and repressed by ectopic Osa. Osa-mediated repression occurs downstream of the up-regulation of Armadillo but is sensitive both to the relative levels of activating Armadillo/Pangolin and repressing Groucho/Pangolin complexes that are present, and to the responsiveness of the promoter to Wingless. Osa functions as a component of the Brahma chromatin-remodeling complex; other components of this complex are likewise required to repress Wingless target genes. These results suggest that altering the conformation of chromatin is an important mechanism by which Wingless signaling activates gene expression (Collins, 2000).

Whereas many of the genes regulated by the wg pathway require wg for their expression, several genes appear to be repressed by high levels of wg signaling. To determine the effect of Osa on the expression of genes that are normally repressed by wg, the expression of dpp in leg discs was examined with altered dosage of osa. In third-instar leg discs, wg and dpp are expressed along the A/P boundary in the ventral and dorsal compartment, respectively, and mutually antagonize each other's expression. dpp expression is repressed when UAS-Osa is expressed in a broad central domain of the leg disc with a Dll-Gal4 driver and dpp is ectopically expressed in the ventral compartment in osa^eld308/osa^4H leg discs. Clones of cells mutant for osa can also induce leg duplications in the ventral compartment of the leg, consistent with the ectopic expression of dpp. Thus, in addition to repressing the expression of genes that are normally activated by the wg signal, Osa is also required for the repression of at least one of the genes that are repressed by wg (Collins, 2000).

The secreted glycoprotein Wingless (Wg) acts through a conserved signaling pathway to regulate expression of Wingless pathway nuclear targets. Wg signaling causes nuclear translocation of Armadillo, the fly ß-catenin, which then complexes with the DNA-binding protein TCF (Pangolin), enabling it to activate transcription. Though many nuclear factors have been implicated in modulating TCF/Armadillo activity, their importance remains poorly understood. A ubiquitously expressed protein, Pygopus, is required for Wg signaling throughout Drosophila development. Pygopus contains a PHD finger at its C terminus, a motif often found in chromatin remodeling factors. Overexpression of pygopus also blocks the pathway, consistent with the protein acting in a complex. The pygopus mutant phenotype is highly, though not exclusively, specific for Wg signaling. Epistasis experiments indicate that Pygopus acts downstream of Armadillo nuclear import, consistent with the nuclear location of heterologously expressed protein. These data argue strongly that Pygopus is a new core component of the Wg signaling pathway that acts downstream or at the level of TCF (Parker, 2002).

pygo overexpression causes derepression of decapentaplegic (dpp) in leg imaginal discs. In the developing leg, wg and dpp are expressed in wedge-like domains just anterior to the posterior compartment, with wg highly enriched in the ventral half and dpp in the dorsal part. If Wg signaling is blocked, dpp expression becomes derepressed. If pygo is misexpressed using the patched-Gal4 driver, which is active in both the dpp and wg expression domains, then dpp expression (as judged by dpp-lacZ) is extended into the ventral compartment. This derepression of dpp expression is seen in the vast majority of leg discs examined and is again consistent with pygo overexpression antagonizing Wg signaling (Parker, 2002).

Regulation in the wing disc

Localized activation of the Ras/Raf pathway by epidermal growth factor receptor (Egfr) signalling specifies the formation of veins in the Drosophila wing. However, little is known about how the Egfr signal regulates transcriptional responses during the vein/intervein cell fate decision. Evidence is provided that Egfr signaling induces expression of vein-specific genes by inhibiting the Capicua (Cic) HMG-box repressor, a known regulator of embryonic body patterning. Lack of Cic function causes ectopic expression of Egfr targets such as argos, ventral veinless and decapentaplegic and leads to formation of extra vein tissue. In vein cells, Egfr signaling downregulates Cic protein levels in the nucleus and relieves repression of vein-specific genes, whereas intervein cells maintain high levels of Cic throughout larval and pupal development, repressing the expression of vein-specific genes and allowing intervein differentiation. However, regulation of some Egfr targets such as rhomboid appears not to be under direct control of Cic, suggesting that Egfr signaling branches out in the nucleus and controls different targets via distinct mediator factors. These results support the idea that localized inactivation of transcriptional repressors such as Cic is a rather general mechanism for regulation of target gene expression by the Ras/Raf pathway (Roch, 2002).

During Drosophila wing development, Hedgehog (Hh) signaling is required to pattern the imaginal disc epithelium along the anterior-posterior (AP) axis. The Notch (N) and Wingless (Wg) signaling pathways organise the dorsal-ventral (DV) axis, including patterning along the presumptive wing margin. A functional hierarchy of these signaling pathways is described that highlights the importance of the competing influences of Hh, N, and Wg in establishing gene expression domains. Investigation of the modulation of Hh target gene expression along the DV axis of the wing disc has revealed that collier/knot (col/kn), patched, and decapentaplegic are repressed at the DV boundary by N signaling. Attenuation of Hh signaling activity caused by loss of fused function results in a striking down-regulation of col, ptc, and engrailed (en) symmetrically about the DV boundary. This down-regulation depends on activity of the canonical Wg signaling pathway. It is proposed that modulation of the response of cells to Hh along the future proximodistal (PD) axis is necessary for generation of the correctly patterned three-dimensional adult wing. These findings suggest a paradigm of repression of the Hh response by N and/or Wnt signaling that may be applicable to signal integration in vertebrate appendages (Glise, 2002).

Short-range Hh signaling, partly through activation of Col function, is essential for correct AP patterning and differentiation of L3-L4 intervein tissue. N and Wg first define the DV boundary and later subdivide the region near this boundary into a number of distinct subregions that will eventually differentiate into wing margin bristles and vein tissue. These signals overlap spatially and temporally and lead to opposite fates. It is proposed that in and close to the DV boundary, N, Wg, and Hh signaling exist in a delicate balance to allow vein tissue, bristle, and sensory organ differentiation along the adult wing margin (Glise, 2002).

The Hh target genes col/kn and ptc, in contrast to en, are repressed in a wild type wing in cells corresponding to the presumptive wing margin. It has been demonstrated, using both gain- and loss-of-function experiments, that this repression is mediated by N signaling and that its inhibition results in aberrant morphogenesis of the wing. Hh signaling, achieved either by overexpression of Hh or loss of Ptc activity, is not sufficient to give maximum activation of Hh targets in cells of the prospective wing margin, suggesting that a finely tuned balance of activation and repression is required to achieve the appropriate biological output. However, overexpression of a stabilized form of Ci under the ptc-Gal4 driver results in the activation of Col in the prospective wing margin and defects in wing margin differentiation, indicating that N repression can be overcome by hyperactivity of the Hh signaling pathway. N signaling may lead to the repression of col, ptc, and dpp directly or it may act indirectly by affecting the ability of Ci to act as a transcriptional activator. Since expression of en, which requires the highest level of Hh signaling and Ci activity, appears immune to N repression, the former possibility is favored (Glise, 2002).

Sex-lethal (Sxl), the Drosophila sex-determination master switch, is on in females and controls sexual development as a splicing and translational regulator. Hedgehog (Hh) is a secreted protein that specifies cell fate during development. Sxl protein has been shown to be part of the Hh cytoplasmic signaling complex and Hh promotes Sxl nuclear entry (Vied, 2001; Horabin, 2003). In the wing disc anterior compartment, Patched (Ptc), the Hh receptor, acts positively in this process. This study shows that the levels and rate of nuclear entry of full-length Cubitus interruptus (Ci), the Hh signaling target, are enhanced by Sxl. This effect requires the cholesterol but not palmitoyl modification on Hh, and expands the zone of full-length Ci expression. Expansion of Ci activation and its downstream targets, particularly decapentaplegic the Drosophila TGFß homolog, suggests a mechanism for generating different body sizes in the sexes; in Drosophila, females are larger and this difference is controlled by Sxl. Consistent with this proposal, discs expressing ectopic Sxl show an increase in growth. In keeping with the idea of the involvement of a signaling system, this growth effect by Sxl is not cell autonomous. These results have implications for all organisms that are sexually dimorphic and use Hh for patterning (Horabin, 2005) (Horabin, 2005).

The data presented here show that when Sxl is present, the Hh signal is augmented. This is seen as an increase in full-length Ci in whole-mount tissue, and in Western blots which give a more quantitative sense of protein levels. In addition to elevating the levels of full-length Ci, several of the Hh downstream targets, including ptc, dpp and some of the downstream targets of Dpp, show an increase in expression. Conversely, removal of Sxl in female cells shows a reduction in the strength of the Hh signal (Horabin, 2005).

Simply reducing the levels of the repressor form of Ci (which is accomplished by increasing the levels of full-length Ci) should increase the expression of the growth factor dpp. This is because dpp is affected by Ci at two levels: absence of the Ci repressor ameliorates repression to give low levels of dpp expression, while activated full-length Ci further elevates dpp transcription. Indeed, while the wing patterning defect caused by the ectopic expression of C84S-Hh narrows the region between wing veins L3 and L4 equally in the two sexes (due to its dominant-negative effect on endogenous Hh), the overall sexual dimorphic size difference is maintained. Consistent with this idea, co-expressing Sxl and Hh with only the cholesterol modification produces an overgrowth phenotype in discs, indicating Sxl can promote disc growth through this form of Hh (Horabin, 2005).

The growth induced by Dpp has been described as 'balanced', involving both mass accumulation as well as cell cycle progression. The net effect is that cell size does not change, nor does the ploidy. This is in contrast to growth induced by hyperactivation of Ras, Myc or Phosphoinositide 3 kinase, which increase growth but do not induce a progression through the G2/M phase of the cell cycle and, as a result, increase cell size (Horabin, 2005).

Sxl not only elevates expression of dpp and its downstream targets to induce growth, but is able to elevate ptc expression. Enhancing ptc suggests that the Hh signal is 'corrected' for the enlarged patterning field, since short-range patterning has to be controlled by Hh. By enhancing dpp, Sxl indirectly also enhances the long-range patterning system of the disc. Augmenting the Hh signal would thus appear an elegant solution for increasing overall size without changing the basic body plan or pattern. Since Sxl is expressed in all female tissues from very early in development and this expression is maintained for the rest of the life cycle, Sxl is constantly available to upregulate the Hh signal. This augmentation must be kept within check, however, because, as argued above, too high an increase can change the overall slope of the Hh gradient, effectively changing the final patterning of the tissue (Horabin, 2005).

Differential regulation of Hedgehog target gene transcription by Costal2 and Suppressor of Fused

The mechanism by which the secreted signaling molecule Hedgehog (Hh) elicits concentration-dependent transcriptional responses from cells is not well understood. In the Drosophila wing imaginal disc, Hh signaling differentially regulates the transcription of target genes decapentaplegic (dpp), patched (ptc) and engrailed (en) in a dose-responsive manner. Two key components of the Hh signal transduction machinery are the kinesin-related protein Costal2 (Cos2) and the nuclear protein trafficking regulator Suppressor of Fused [Su(fu)]. Both proteins regulate the activity of the transcription factor Cubitus interruptus (Ci) in response to the Hh signal. This study analyzed the activities of mutant forms of Cos2 in vivo and found effects on differential target gene transcription. A point mutation in the motor domain of Cos2 results in a dominant-negative form of the protein that derepresses dpp but not ptc. Repression of ptc in the presence of the dominant-negative form of Cos2 requires Su(fu), which is phosphorylated in response to Hh in vivo. Overexpression of wild-type or dominant-negative cos2 represses en. These results indicate that differential Hh target gene regulation can be accomplished by differential sensitivity of Cos2 and Su(Fu) to Hh (Ho, 2005).

The data suggest that the activities of Cos2 and Su(fu) are independently regulated by different concentrations of Hh along the gradient that forms from posterior to anterior. In the anterior cells distant from the AP boundary, little or no Hh is received and target genes are silent. In these cells, Cos2 is required for proteolytic processing of Ci into its repressor form and possibly for the delivery of Ci^FL for lysosomal degradation. The data suggest that Cos2 requires an intact P-loop for its role in these events. Cos2 ATPase activity may be inhibited in cells receiving very low levels of Hh, preventing Ci proteolysis and stabilizing Ci^FL. The stabilization of Ci^FL results in the activation of dpp. Nearer the AP border, where higher levels of Hh are received, Su(fu) becomes phosphorylated, inactivating its negative regulatory hold on Ci, while inhibition of the ATPase activity of Cos2 continues to allow stabilization of Ci. In this situation, ptc and dpp are transcribed. Finally, at the highest levels of Hh signaling adjacent to the AP border, Cos2 is required for activation of the pathway and the expression of en. S182N expression, or cos2 over-expression, inhibits the induction of en by endogenous Hh in these cells. The elements of this model are addressed below (Ho, 2005).

Ci plays a central role in determining which genes are repressed or activated in response to different concentrations of Hh. In order to activate target genes such as dpp or ptc, Ci must be stabilized in its full-length form. In wild-type discs, Hh stabilizes Ci by antagonizing molecular events that reduce the concentration of nuclear Ci^FL. In addition to the constitutive nuclear export of Ci, there are two ways Ci^FL concentration is reduced: full-length Ci is proteolytically processed into a repressor form; and Ci^FL is degraded by a lysosome-mediated process involving a novel protein called Debra. In these experiments, the stabilization of Ci^FL was accomplished by expressing S182N in responsive cells, which antagonizes Cos2 repressor activity and results in the accumulation of high levels of Ci^FL, with minimal effects on the levels of Ci^R. This same type of differential effect on Ci^R and Ci^FL is accomplished by Debra, which causes the lysosomal degradation of Ci^FL without affecting the production of Ci^R. Cos2 and Debra may act in concert to destabilize Ci^FL, while Cos2 may also aid in the production of Ci^R via a Debra-independent mechanism. This would involve presenting Ci to the kinases, PKA, CKI and GSKß (Shaggy) for phosphorylation and processing by the proteasome. Since Debra regulates Ci stability in limited areas of the wing disc but S182N can stabilize Ci throughout the anterior compartment, it is likely that S182N interferes with both Debra-dependent and Debra-independent mechanisms of Ci stability to achieve the observed effect: cell-autonomous stabilization of Ci^FL leading to derepression of dpp (Ho, 2005).

These results suggest that Cos2 may use its ATPase activity to transport Ci to a location where it becomes phosphorylated in preparation for processing, or to the site of processing itself. Alternatively, the ATPase activity may be important for regulating the conformation of Cos2 and its binding to partners such as Smo, Su(fu), Fu and Ci, which would be a novel role for the P-loop in a kinesin-related protein. The S182N mutation may lock Cos2 in a conformation that changes association with binding partners. For example, S182N may decrease the ability of Cos2 to bind Ci, releasing Ci from the cytoplasm, resulting in an increased level of Ci^FL in the nucleus and the activation of dpp (Ho, 2005).

The human ortholog of Suppressor of fused is a tumor suppressor gene. Su(fu) can associate with Ci, and with the mammalian homologs of Ci, the Gli proteins, through specific protein-protein interactions. Through these interactions, Su(fu) controls the nuclear shuttling of Ci and Gli, as well as the protein stability of Ci^FL and Ci^R. Flies homozygous for Su(fu) loss-of-function mutations are normal, so the importance of Su(fu) becomes evident only when other gene functions are thrown out of balance, as in a fu mutant background, with extra or diminished Hh signaling caused by ptc, slimb and protein kinase A mutations or when altered Cos2 is produced (Ho, 2005).

To activate ptc transcription in the wing disc, two conditions have to be met simultaneously: Ci^FL must be stabilized, and the activity of Su(fu) must be reduced. Removal of Su(fu) changes S182N from a ptc repressor into a ptc activator. Removal of Su(fu) may result in the modification, activation or relocalization of Ci^FL, or in further sensitizing the system to stabilized Ci^FL. In Su(fu) homozygous animals, the quantity of Ci^FL and Ci^R proteins is greatly diminished, and Su(fu) mutant cells are more sensitized to the Hh signal. The lower levels of both Ci^FL and Ci^R in mutant Su(fu) cells may contribute to the sensitivity of these cells to Hh, since a small Hh-driven change in the absolute concentration of either form of Ci would result in a significant change in the ratio between the two proteins. Both Ci^FL and Ci^R bind the same enhancer sites, so their relative ratio is likely to be important in determining target gene expression. S182N expression tips the ratio of Ci^FL to Ci^R toward Ci^FL, and reducing the absolute quantities of both Ci isoforms by removing Su(fu) will enhance this effect. Furthermore, Su(fu) binds Ci and sequesters it in the cytoplasm in a stoichiometric manner Reducing the amount of Su(fu) should release more Ci^FL to the nucleus to activate ptc (Ho, 2005).

The activity of Su(fu) must be regulated or overcome so that target genes can be activated at the right times and places in response to Hh. The regulation of Su(fu) activity may occur by Hh-dependent phosphorylation. A phosphoisoform of Su(fu), Su(fu)-P, was detected in discs where GAL4 was used to drive extra Hh expression. At high concentrations of Hh, the phosphorylation of Su(fu) is not antagonized by overexpression of cos2 or either of the cos2 mutants, suggesting that phosphorylation of Su(fu) occurs independently of Cos2 function. One kinase involved in the phosphorylation of Su(fu) is the Ser/Thr kinase Fused, a well-established component of Hh signal transduction. It is not known whether the phosphorylation of Su(fu) by Fu is direct or indirect (Ho, 2005).

The phosphorylation state of Su(fu) may be an important factor in determining Hh target gene activity. Phosphorylation of an increasing number of Su(fu) molecules with increasing Hh signal may gradually release Ci from all of the known modes of Su(fu)-dependent inhibition, such as nuclear export and recruitment of repressors to nuclear Ci, leading to higher levels of Ci^FL in the nucleus and the activation of Hh target genes such as ptc (Ho, 2005).

Anterior en expression was used as an in vivo reporter of high levels of Hh signaling. cos2 mutant cells at the AP boundary fail to activate en, suggesting that Cos2 plays a positive regulatory role in en regulation. S182N, S182T and Cos2 overexpression mimics the cos2 loss-of-function condition with respect to en: en remains off in these cells. One interpretation of these data is that all the Cos2 proteins are able to associate with another pathway component, such as Smo, and overproduction of any of them inactivates some of the Smo in non-productive complexes not capable of activating en (Ho, 2005).

In contrast to the activity of all the other mutations generated, deletion of the C terminal domain creates a protein (Cos2DeltaC) that represses normal dpp, ptc and en expression in the wing disc. In this in vivo assay, Cos2DeltaC acts just like wild-type Cos2. A similar deletion has been shown to retain function in cell culture assays. This mutant, expressed under the control of its endogenous promoter, rescues the lethality and wing duplication phenotypes of a cos2 loss-of-function allele over a cos2 deficiency. The results of the rescue experiment bring up a new possibility: that the C-terminal domain of Cos2, and the Cos2-Smo interaction via the C terminus of Cos2, is not necessary for repressor activities of Cos2. Alternatively, Cos2DeltaC could complement or boost the activity of the hypomorphic allele cos2¹¹, which was used for the rescue experiment (Ho, 2005).

Modulation of the Suppressor of fused protein regulates the Hedgehog signaling pathway in Drosophila embryo and imaginal discs: Regulation of dpp expression

The Suppressor of fused (Su(fu)) protein is known to be a negative regulator of Hedgehog (Hh) signal transduction in Drosophila imaginal discs and embryonic development. It is antagonized by the kinase Fused (Fu) since Su(fu) null mutations fully suppress the lack of Fu kinase activity. In this study, the Su(fu) gene was overexpressed in imaginal discs and opposing effects were observed depending on the position of the cells, namely a repression of Hh target genes in cells receiving Hh and their ectopic expression in cells not receiving Hh. These effects were all enhanced in a fu mutant context and were suppressed by cubitus interruptus (Ci) overexpression. The Su(fu) protein is poly-phosphorylated during embryonic development and these phosphorylation events are altered in fu mutants. This study thus reveals an unexpected role for Su(fu) as an activator of Hh target gene expression in absence of Hh signal. Both negative and positive roles of Su(fu) are antagonized by Fused. Based on these results, a model is proposed in which Su(fu) protein levels and isoforms are crucial for the modulation of the different Ci states that control Hh target gene expression (Dussillol-Godar, 2006).

Su(fu) plays a negative role in Hh signalization since it participates both in the cytoplasmic retention of Ci and in the inhibition of the activation of Ci¹⁵⁵. This study analyzed the effects of Su(fu) overexpression on appendage development and on the expression of several Hh target genes in the corresponding discs. In parallel, its accumulation and post-translational modifications were examined during embryonic development in fu⁺ and fu mutant backgrounds (Dussillol-Godar, 2006).

The effects of Su(fu) overexpression on the Hh pathway were assessed by examining both the adult appendage development and the transcription of well characterized Hh targets (such as dpp and ptc) and accumulation of full-length Ci (Ci¹⁵⁵) in the corresponding discs. No effect was detected in the posterior compartment, but two apparently opposite effects were observed in the anterior compartment depending on the distance from the source of Hh.

(1) At the A/P border, there was a decrease in the response to low and high levels of Hh signaling. Indeed, dpp and, to a lesser extent, ptc gene expression was reduced. This result is in agreement with the known inhibitory role of the Su(fu) protein in cells transducing the Hh signal (Dussillol-Godar, 2006).

(2) More anteriorly, in cells which do not receive the Hh signal, overexpression of Su(fu) led to anterior duplications in adult appendages. This was correlated with an ectopic expression of dpp in the wing disc or dpp and wg in the leg disc, associated with an accumulation of Ci¹⁵⁵. Ectopic ptc expression was also seen but at a much lower level. These effects phenocopy those of cos2 loss of function mutants or of ectopic hh expression. They can be interpreted as a constitutive activation of the pathway. However, the fact that only low levels of ectopic ptc expression are induced shows that the highest levels of Ci activation are not attained (Dussillol-Godar, 2006).

High Ptc protein levels at the boundary are known to sequester the Hh. Thus, the anterior ectopic dpp expression observed in this study in discs overexpressing Su(fu) could be secondary to the deregulation of the Hh pathway at the A/P border: the initial decrease of Ptc at the A/P boundary would result in a further diffusion of Hh to the neighboring cells in which Ci cleavage would be inhibited, allowing hh and dpp expression. So, step by step, a partial activation of the pathway could be propagated up to the anterior region of the wing pouch. Alternatively, the anterior effects of Su(fu) overexpression could occur independently of events at the A/P border. This latter hypothesis is favored for two reasons: (1) induction of Su(fu) overexpression in the A region, outside the A/P border (using either the vgBE-GAL4 driver or clonal analysis), showed that the ectopic activation of dpp can occur independently of Su(fu) overexpression at the A/P border, (2) no significant ectopic hh expression could be detected (Dussillol-Godar, 2006).

At least three Ci states have been postulated to exist, depending on the Hh signal gradient: (1) a fully active Ci (Ci^act) responsible for high ptc expression in a stripe 4–5 cells wide close to the A/P border, (2) a full-length Ci (Ci¹⁵⁵) sufficient for dpp expression 10–15 cell diameters away from the A/P border, (3) a cleaved Ci form (Ci⁷⁵) in anterior cells not receiving Hh which represses hh and dpp expression. The balance between these forms of Ci depends on the regulation of non-exclusive processes such as cytoplasmic tethering, protein stability, nuclear shuttling and cleavage. At least two complexes that contain Ci have been identified: a tetrameric Su(fu)–Ci–Fu–Cos2 complex (complex A) probably present in cells receiving a high level of Hh and a trimeric Ci–Fu–Cos2 complex (complex B) which is devoid of Su(fu) and bound to microtubules in the absence of Hh. At the molecular level, Su(fu) binds to N-terminal Ci and thus has the capacity to bind both Ci¹⁵⁵ and Ci⁷⁵. Su(fu) was shown to sequester Ci in the cytoplasm thus controlling the nuclear shuttling of Ci. It was also shown to be involved in the stability of Ci¹⁵⁵ and Ci⁷⁵ (Dussillol-Godar, 2006).

This study shows that overexpression of Su(fu) differentially affects the expression of Hh target genes in Hh-receiving and non-receiving cells and that these effects are all reversed by overexpression of Ci. Moreover, the resulting anterior ectopic activation of dpp is associated with an important accumulation of Ci¹⁵⁵. To account for these data, it is hypothesized that Su(fu) overexpression disturbs the balance between the different Ci complexes and thus between the different Ci states. A model is proposed for Hh signaling in imaginal discs in which the effects of Su(fu) over-expression result mainly from the cytoplasmic retention of Ci¹⁵⁵. At the A/P boundary in Hh-receiving cells, Ci¹⁵⁵ is normally present in a tetrameric complex with Su(fu), Fu and Cos2 (complex A). In these cells, Hh signaling via the activation of Fu blocks Cos2 and Su(fu) negative effects in the tetrameric complex, thus preventing Ci cleavage and cytoplasmic retention and favoring the release of Ci, its activation and nuclear access. Su(fu) overexpression could lead to the recruitment of a significant fraction of endogenous Ci¹⁵⁵ into complexes in which Su(fu) is no longer inhibited by Fu. A fraction of Ci is thus sequestered in the cytoplasm as an inactive full-length form. Co-overexpression of Ci along with Su(fu) would provide enough Ci to buffer the excess of Su(fu), leading to the formation of active Ci¹⁵⁵. In the anterior region where Hh is absent, Ci is present in a microtubule-bound trimeric complex (complex B) containing Fu and Cos2 but not Su(fu), leading to Ci cytoplasmic tethering and favoring its cleavage in the Ci⁷⁵ repressive form. This complex would be in equilibrium with a Fu–Su(fu)–Ci complex. In this complex, Su(fu) would act as a safety lock for the cytoplasmic retention of an uncleaved fraction of Ci¹⁵⁵ potentially able to yield some active forms of Ci. When Su(fu) is overexpressed, extra Su(fu) would bind Ci¹⁵⁵, preventing it from joining the microtubule-bound complex. Ci would not be effectively processed, leading to the accumulation of uncleaved Ci¹⁵⁵. The reduction in the amount of Ci⁷⁵ would be sufficient to allow the expression of dpp but not that of hh, which has been reported to be more sensitive to Ci⁷⁵ repression than dpp. There would be an enrichment in the other complex but only a few active Ci forms would be produced in agreement with the almost total absence of ectopic ptc expression (Dussillol-Godar, 2006).

The present data show that all the effects induced by overexpression of Su(fu) were enhanced in fu mutants, namely pupal lethality, ectopic anterior expression of dpp and ptc genes and their decrease at the antero-posterior border (Dussillol-Godar, 2006).

At the A/P border, Fu is normally required to antagonize the negative effect of Su(fu) in Hh receiving cells. In fu mutant discs overexpressing Su(fu), the negative effects that Su(fu) exerts on Ci¹⁵⁵ cytoplasmic retention in the tetrameric complex would no longer be counteracted by Fu. The shifting of the equilibrium towards the inactive Su(fu)–Ci complex is increased. Less active Ci is available and the reduction in dpp and ptc expression is aggravated (Dussillol-Godar, 2006).

The anterior ectopic activation of the pathway seen in discs overexpressing Su(fu) was greatly enhanced in fu mutants. These unexpected results provide evidence for an inhibitory role of Fu on Ci¹⁵⁵ in the absence of the Hh signal. In the absence of Hh, Fu activity could favor the normal restrictive effect of Su(fu) on Ci¹⁵⁵ in the Fu–Su(fu)–Ci complex. In fu⁻ mutants, the negative effect of Su(fu) on the trapped fraction of Ci¹⁵⁵ would be weakened and enough Ci¹⁵⁵ would be active to induce transcription of dpp and of ptc (Dussillol-Godar, 2006).

Strikingly, unlike Su(fu) loss of function mutations, Su(fu) overexpression failed to distinguish between the two classes of fu alleles. Since the regulatory domain is probably necessary for Fu kinase activity, the effects seen are probably all mostly due to a loss of Fu kinase activity which would reduce the level of phosphorylation of Su(fu). As shown here and in several recent reports, the Su(fu) protein is phosphorylated in the embryo. Multiple levels of phosphorylation were detected, with hyperphosphorylated forms that accumulate at a period in embryonic development when Fu is activated by the Hh signal and that are significantly reduced in fu mutants. Thus, Fu could modulate Su(fu) activity by controlling, directly or indirectly, its phosphorylation. In the absence of Hh signaling, a low level of Su(fu) phosphorylation by Fu would reinforce the negative effect of Su(fu), whereas a higher phosphorylation level would inactivate Su(fu) in Hh responding cells at the A/P border (Dussillol-Godar, 2006).

Nevertheless, phosphorylated isoforms were not totally abolished in fu mutants, suggesting that other kinase(s) can phosphorylate Su(fu). In agreement with this point, numerous putative phosphorylation sites for kinases such as Caseine kinase II or PKC, but not PKA, are present in the Su(fu) protein. However, the biological implications of the Su(fu) isoforms and their modulation by the Hh transduction signal remain to be demonstrated (Dussillol-Godar, 2006).

JAK/STAT controls organ size and fate specification by regulating morphogen production and signalling

A stable pool of morphogen-producing cells is critical for the development of any organ or tissue. This study presents evidence that JAK/STAT signalling in the Drosophila wing promotes the cycling and survival of Hedgehog-producing cells, thereby allowing the stable localization of the nearby BMP/Dpp-organizing centre in the developing wing appendage. The inhibitor of apoptosis dIAP1 and Cyclin A were identified as two critical genes regulated by JAK/STAT and contributing to the growth of the Hedgehog-expressing cell population. JAK/STAT was found to have an early role in guaranteeing Wingless-mediated appendage specification, and a later one in restricting the Dpp-organizing activity to the appendage itself. These results unveil a fundamental role of the conserved JAK/STAT pathway in limb specification and growth by regulating morphogen production and signalling, and a function of pro-survival cues and mitogenic signals in the regulation of the pool of morphogen-producing cells in a developing organ (Recasens-Alvarez, 2017).

Morphogens of the Wnt/Wg, Shh/Hh and BMP/Dpp families regulate tissue growth and pattern formation in vertebrate and invertebrate limbs. This study has unraveled a fundamental role of the secreted Upd ligand and the JAK/STAT pathway in facilitating the activities of these three morphogens in exerting their fate- and growth-promoting activities in the Drosophila wing primordium. Early in wing development, two distinct mechanisms ensure the spatial segregation of two alternative cell fates. First, the proximal-distal subdivision of the wing primordium into the wing and the body wall relies on the antagonistic activities of the Wg and Vn signalling molecules. While Wg inhibits the expression of Vn and induces the expression of the wing-determining genes, Vn, through the EGFR pathway, inhibits the cellular response to Wg and instructs cells to acquire body wall fate. Second, growth promoted by Notch pulls the sources of expression of these two morphogens apart, alleviates the repression of wing fate by Vn/EGFR, and contributes to Wg-mediated appendage specification. Expression of Vn is reinforced by a positive amplification feedback loop through the activation of the EGFR pathway. This existing loop predicts that, in the absence of additional repressors, the distal expansion of Vn/EGFR and its targets would potentially impair wing development. The current results indicate that Upd and JAK/STAT restrict the expression of EGFR target genes and Vn to the most proximal part of the wing primordium, thereby interfering with the loop and allowing Wg to correctly trigger wing development. Evidence is presented that JAK/STAT restricts the expression pattern and levels of its own ligand Upd and that ectopic expression of Upd is able to bypass EGFR-mediated repression and trigger wing development de novo. This negative feedback loop between JAK/STAT and its ligand is of biological relevance, since it prevents high levels of JAK/STAT signalling in proximal territories that would otherwise impair the development of the notum or cause the induction of supernumerary wings, as shown by the effects of ectopic activation of the JAK/STAT pathway in the proximal territories. Thus, while Wg plays an instructive role in wing fate specification, the Notch and JAK/STAT pathways play a permissive role in this process by restricting the activity range of the antagonizing signalling molecule Vn to the body wall region (Recasens-Alvarez, 2017).

Later in development, once the wing field is specified, restricted expression of Dpp at the AP compartment boundary organizes the growth and patterning of the whole developing appendage. Dpp expression is induced in A cells by the activity of Hh coming from P cells, which express the En transcriptional repressor. This study shows that JAK/STAT controls overall organ size by maintaining the pool of Hh-producing cells to ensure the stable and localized expression of the Dpp organizer. JAK/STAT does so by promoting the cycling and survival of P cells through the regulation of dIAP1 and CycA, counteracting the negative effects of En on these two genes. Since the initial demonstration of the role of the AP compartment boundary in organizing, through Hh and Dpp, tissue growth and patterning, it was noted that high levels of En interfered with wing development by inducing the loss of the P compartment. The capacity of En to negatively regulate its own expression was subsequently shown to be mediated by the Polycomb-group genes and proposed to be used to finely modulate physiological En expression levels. Consistent with this proposal, an increase was observed in the expression levels of the en-gal4 driver, which is inserted in the en locus and behaves as a transcriptional reporter, in enRNAi-expressing wing discs. The negative effects of En on cell cycling and survival reported in this work might also contribute to the observed loss of the P compartment caused by high levels of En. As is it often the case in development, a discrete number of genes is recurrently used to specify cell fate and regulate gene expression in a context-dependent manner. It is proposed that the capacity of En to block cell cycle and promote cell death might be required in another developmental context and that this capacity is specifically suppressed in the developing Drosophila limbs by JAK/STAT, and is modulated by the negative autoregulation of En, thus allowing En-dependent induction of Hh expression and promoting Dpp-mediated appendage growth. It is interesting to note in this context that En-expressing territories in the embryonic ectoderm are highly enriched in apoptotic cells. Whether this apoptosis plays a biological role and relies on En activity requires further study (Recasens-Alvarez, 2017).

Specific cell cycle checkpoints appear to be recurrently regulated by morphogens and signalling pathways, and this regulation has been unveiled to play a major role in development. Whereas Notch-mediated regulation of CycE in the Drosophila eye and wing primordia is critical to coordinate tissue growth and fate specification by pulling the sources of two antagonistic morphogens apart, the current results indicate that JAK/STAT-mediated regulation of CycA is critical to maintain the pool of Hh-producing cells in the developing wing and to induce stable Dpp expression. The development of the wing hinge region, which connects the developing appendage to the surrounding body wall and depends on JAK/STAT activity, has been previously shown to restrict the Wg organizer and thus delimit the size and position of the developing appendage. The current results support the notion that JAK/STAT and the hinge region are also essential to restrict the organizing activity of the Dpp morphogen to the developing appendage. Taken together, these results reveal a fundamental role of JAK/STAT in promoting appendage specification and growth through the regulation of morphogen production and activity, and a role of pro-survival cues and mitotic cyclins in regulating the pool of morphogen-producing cells in a developing organ. The striking parallelisms in the molecules and mechanisms underlying limb development in vertebrates and invertebrates have contributed to the proposal that an ancient patterning system is being recurrently used to generate body wall outgrowths. Whether the conserved JAK/STAT pathway plays a developmental role also in the specification or growth of vertebrate limbs by regulating morphogen production or activity is a tempting question that remains to be elucidated (Recasens-Alvarez, 2017).

Regulation in the hindgut

The Dichaete gene of Drosophila encodes a group B Sox protein related to mammalian Sox1, -2, and -3. Like these proteins, Dichate is widely and dynamically expressed throughout embryogenesis. In order to unravel new Dichaete functions, the organization of the Dichaete gene was characterized using a combination of regulatory mutant alleles and reporter gene constructs. Dichaete expression is tightly controlled during embryonic development by a complex of regulatory elements distributed over 25 kb downstream and 3 kb upstream of the transcription unit. A series of regulatory alleles which affect tissue-specific domains of Dichaete were used to demonstrate that Dichaete has functions in addition to those during segmentation and midline development that have been previously described. (1) Dichaete has functions in the developing brain. A specific group of neural cells in the tritocerebrum fails to develop correctly in the absence of Dichaete, as revealed by reduced expression of labial, Zn finger homeodomain 2 (zfh-2), wingless, and engrailed. (2) Dichaete is required for the correct differentiation of the hindgut. The Dichaete requirement in hindgut morphogenesis is, in part, via regulation of dpp, since ectopically supplied dpp can rescue Dichaete phenotypes in the hindgut. Taken together, there are now four distinct in vivo functions described for Dichaete that can be used as models for context-dependent comparative studies of Sox function (Sanchez-Soriano, 2000).

The Drosophila embryonic hindgut is a robust system for the study of patterning and morphogenesis of epithelial organs. In a period of about 10 h, and in the absence of significant cell division or apoptosis, the hindgut epithelium undergoes morphogenesis by changes in cell shape and size and by cell rearrangement. The epithelium concomitantly becomes surrounded by visceral mesoderm and is characterized by distinct gene expression patterns that forecast the development of three morphological subdomains: small intestine, large intestine, and rectum. At least three genes encoding putative transcriptional regulators, drumstick (drm), bowl, and lines (lin), are required to establish normal hindgut morphology. The defect in hindgut elongation in drm, bowl, and lin mutants is due, in large part, to the requirement of these genes in the process of cell rearrangement. Further, drm, bowl, and lin are required for patterning of the hindgut, i.e., for correct expression in the prospective small intestine, large intestine, and rectum of genes encoding cell signals (wingless, hedgehog, unpaired, Serrate, dpp) and transcription factors (engrailed, dead ringer). The close association of both cell rearrangement and patterning defects in all three mutants suggest that proper patterning of the hindgut into small intestine and large intestine is likely required for its correct morphogenesis (Iwaki, 2001).

This study shows that drm, bowl, and lin are required for the gene expression patterns that distinguish these three domains: lin is required for expression characteristic of large intestine (dpp, dri, and en) and rectum (Ser, hh, and wg); drm and bowl are required for expression characteristic of small intestine (hh and upd). By both morphological criteria (cell shape, presence or absence of boundary cells) and gene expression patterns (expanded expression of genes expressed in the small intestine), lin hindguts appear to consist of a greatly expanded small intestine and to lack the large intestine and rectum. In contrast, both morphological and gene expression characteristics of drm and bowl hindguts indicate that they lack most or all of the small intestine, and consist only of large intestine (which remains unelongated) and rectum. A model consistent with these data is that lin functions in the hindgut to repress small intestine fate and to promote large intestine and rectum fate, while establishment of the small intestine requires the activity of drm and bowl. The requirement for drm (but not bowl) for wg expression at the most anterior of the hindgut could be explained if the domain of bowl function in the small intestine does not extend to the most anterior of the hindgut (consistent with the expression of bowl). Since they have opposite effects on Ser expression, bowl and drm may function in different ways, possibly in different pathways, to promote small intestine fate (Iwaki, 2001).

The function of lin as both an activator and repressor of gene activity in the developing hindgut is consistent with molecular and genetic characterization of its function in other embryonic tissues. In the developing dorsal epidermis, lin is required for transcriptional regulation (both activation and repression) of targets downstream of wg signaling. In the developing posterior spiracles, lin is required for the activation by Abd-B of its transcriptional targets. lin encodes a novel protein that is expressed globally throughout the embryo, including the developing hindgut. When ectopically expressed, Lin protein is detected in nuclei of cells signaled by Wg. The early expression of wg throughout the hindgut primordium, starting at the blastoderm stage and continuing to stage 10, might, analogous to its effect in the dorsal epidermis, activate Lin. This might be required for Lin to carry out its function, demonstrated here, of promoting expression of genes characteristic of large intestine identity (otp, dpp, en, and dri), and repressing expression of genes characteristic of small intestine identity (hh, upd, and Ser).

The phenotypes described for wg, hh, dpp, dri, Ser, and en do not suggest a role for these genes in hindgut elongation by cell rearrangement. Mutations in Ser, dri, and en do not appear to affect overall hindgut morphology. The hindgut in wg mutants is extremely small, suggesting that the critical function of wg in hindgut development is in establishing and maintaining the primordium, but not in elongation. dpp mutant hindguts are shorter, consistent with the role of dpp in endoreplication in the large intestine; nevertheless, dpp hindguts have a roughly normal diameter. In hh mutant embryos, the rectum degenerates and hindgut length is reduced, but the overall morphology, particularly the narrowing of the large intestine, appears normal (Iwaki, 2001).

Only in upd embryos is a defect in both elongation and narrowing of the hindgut observed; significantly, upd is expressed only in the small intestine, the same domain that is largely missing from drm and bowl mutants. Shorter and wider hindguts are seen in younger upd embryos, but the majority of hindguts in mature upd embryos appear normal. Thus, while upd may at least partially mediate drm and bowl function in the hindgut, there must be other targets of these genes that are required for cell rearrangement in the hindgut (Iwaki, 2001).

Regulation in the brain

The morphogenesis of specialized structures within the CNS relies on the nonautonomous activity of cell populations that play the role of organizers. In the Drosophila visual system, cells on the dorsal and ventral margins of the developing visual cortex express the Wnt family member Wingless (Wg) and the TGF-beta Decapentaplegic (Dpp). The activity of these morphogens in establishing cortical cell fates sets the stage for the guidance of photoreceptor axons to their retinotopic destinations in the Drosophila brain. One role for Wg in cortical development is to induce and maintain the expression of Dpp, a key step in the assignment of dorsoventral cell identities. Dpp is induced early in cortical development, shortly after the onset of Wg expression in a few dorsal and ventral margin cells, and is maintained by Wg activity until at least the time of retinal axon pathfinding. Wg is a developmental signal in many different tissues, and acts by regulating different target gene sets to elicit a constellation of different cell fates. Wingless-controlled targets include distal-less and vestigial in the wing, engrailed in the embryonic ectoderm, labial in the gut, and sloppy-paired in the embryonic CNS. Conversely, Dpp belongs to a Hedgehog-controlled circuit in the wing (Song, 2000 and references therein).

A regulatory mechanism is described that relays Wg signal reception to the tissue-specific expression of target genes in the visual cortex. In a screen for mutants in which photoreceptor axons project aberrantly to their destinations in the brain, a mutation in combgap was discovered. Retinal axon navigation defects in combgap animals are due to the role of cg in the establishment of cortical cell identity. cg represses the expression of Wg target genes in a positionally restricted manner in the visual cortex. wg+ induction of its cortical cell targets occurs via the downregulation of cg. Combgap is thus a tissue-specific relay between Wingless and its target genes for the determination of cell fate in the visual cortex (Song, 2000).

A combgap mutation was recovered in a screen for mutants with aberrations in retinal axon projections. On the basis of its effects on target region gene expression and the outcome of mosaic analysis, it is evident that a role for combgap in the specification of cortical cell identity underlies its requirement for the establishment of retinotopic connectivity in the visual system. In cg loss of function animals, three markers under wg⁺ control are expressed in expanded dorsal and ventral portions of the retinal axon target field. The requirement for cg to repress the markers within these domains is autonomous. The lamina midline region, however, appears phenotypically normal in homozygous or mosaic cg animals. This positionally restricted requirement for cg⁺ activity is correlated with the pattern of cg expression, since cg is not expressed in the midline region where it is not required. Since wg⁺ misexpression is sufficient to induce wg⁺-dependent markers in the midline region, another regulatory system must control these markers there. Hence, the consequences of wg signal reception at different dorsoventral positions within the cortical precursor field would appear to involve a set of regulatory molecules that divide the cortex into specific domains for pattern formation (Song, 2000).

At hatching, approximately 40 cortical cell precursors form a disc-shaped epithelium on the ventrolateral surface of each brain hemisphere. The epithelium is divided into lamina and medulla precursor zones, which can be distinguished by the expression of Cubitus interruptus (Ci) in the prospective lamina cortex. Cells in two domains at the prospective dorsal and ventral margins of the adult optic lobe begin to express wingless in the mid-first instar stage. dpp expression begins after the onset of wg expression and continues in domains immediately adjacent to the Wg-positive cells. Two additional dorsoventral-specific markers are optomotor blind (omb) and aristaless. Omb is expressed in dorsal and ventral domains that include both the Wg- and Dpp-positive cell populations. Omb-positive glia migrate from these domains toward the lamina midline. aristaless, as assayed by the al⁰⁴³⁵² enhancer trap insertion (al-lacZ), is expressed in a graded pattern with respect to distance from the Wg-positive cells. The expression of omb, dpp, and al-lacZ is induced by ectopic wg⁺ expression and absent under conditions of wg loss of function. These observations indicate that Wg is responsible for the expression of these three markers (Song, 2000).

In the wild-type brain, retinal axons project in a crescent-shaped array into the lamina target field. Domains of dpp-expressing cortical cell precursors lie at the ends of the crescent-shaped retinal axon array. In cg^k11504 animals, ingrowing retinal axons form an irregular pattern of projections, with axons often straying outside the normal target field. When assayed by either introduction of the dpp-lacZ reporter construct BS3.0 or by staining with anti-Dpp antibody, dpp expression was found to extend, in the cg mutant, toward the midline beyond the normal positions of its dorsal and ventral domains. The domains of omb expression also extend beyond their usual boundaries toward the midline. Expression of the al-lacZ reporter does not diminish in a graded fashion with distance from the Wg domains in cg brains. With respect to all three markers, and on the basis of morphology, a region centered about the dorsoventral midline is relatively unaffected by cg loss of function. Similar results have been obtained with the stronger cg^Delta10 deletion allele and with cg¹/cg^k11504 heterozygotes. Thus cg loss of function results in an extension of dorsal and ventral cell identities toward the midline, while a region centered about the midline remains relatively unaffected (Song, 2000).

The cell autonomy of combgap function was determined by generating somatic cg^k11504 clones using the FLP, FRT method. Within cg clones outside of the midline region, Omb, Dpp, and al-lacZ are all expressed ectopically. Clones or portions of clones that fall within the midline region (30% of those examined) appeared phenotypically normal, consistent with the lack of a cg requirement for the midline region in homozygous animals. There are also position-specific effects observed within cg clones. For example, not all cells within a cg clone expressed the marker Dpp. The position-specific ectopic gene activation in cg clones might reflect the activity of other signals involved in cortical cell fate determination. cg thus behaves as an autonomous repressor of omb, dpp, and al-lacZ expression, except in the midline region where it is not required (Song, 2000).

The loss of cg function is epistatic to Axn misexpression. The developing visual ganglia of animals homozygous for cg^k11504, harboring the omb-GAL4 and UAS-Axn transgenes, displays ectopic expression of the wg targets Dpp and Omb like that found in cg^k11504 animals. Ectopic Axn does suppress the high level of Omb expression in the normal Omb domains in the cg^k11504 background, consistent with the notion that high-level Omb expression remains wg⁺ dependent in cg^k11504. A second set of experiments were performed utilizing the heat shock-inducible P{hsGAL4} driver to express the P{UAS-Axn} transgene. Multiple heat shocks during the larval period were applied to animals harboring the transgenes in either a wild-type or cg background. Similar results were obtained to those with the omb-GAL4 driver. These observations argue that cg functions downstream of Axn (Song, 2000).

The constellation of genes under Wingless control displays considerable tissue specificity. Wingless-controlled targets include Distal-less and vestigial in the wing, engrailed in the embryonic ectoderm, and sloppy-paired in the embryonic CNS. Though Dpp and Omb belong to a Hedgehog-controlled circuit in the wing, they are under Wg control in the visual cortices of the brain. With respect to the control of cell fate, Wg signal transduction apparently follows a canonical pathway from a pair of redundant receptors at the cell surface to the cytoplasmic control of Armadillo stability and nuclear translocation. This raises the question of how the tissue specificity of wg target gene expression is achieved (Song, 2000).

The observations that cg regulates dpp, optimotor blind and aristaless in the visual cortex place cg in a second tier of regulation, as a component of a tissue-specific relay mechanism between the Wg signal transduction pathway and the target genes that are wg dependent in visual system cortical cells. The evidence in support of this hypothesis is as follows: (1) epistasis analysis with the wg pathway negative regulator Axn places the requirement for cg downstream of the cytoplasmic complex that includes APC, GSK-beta, and Armadillo; (2) the induction of at least three downstream effectors of wg⁺ activity is mediated by negative regulation of cg expression -- cg expression is reduced in the dorsal and ventral domains of the cortical lamina where these wg target genes are expressed and ectopic cg expression blocks wg target gene expression within these domains; (3) ectopic wg⁺ clones repress cg expression, yielding Cg-negative domains in which wg target genes are ectopically expressed. The presence of consensus Pangolin binding sites in the first intron of cg suggests cg may be a direct target of Wg signal transduction. How the Armadillo/Pangolin complex might participate in the negative regulation of cg is unclear. Cg might act by binding directly to wg target gene regulatory elements as a transcriptional repressor (Song, 2000).

Regulation in adult head development

The eye/antennal discs of Drosophila form most of the adult head capsule. The role of the BMP family member decapentaplegic (dpp) in the process of head formation is being analyzed, since a class of cis-regulatory dpp mutations (dpp^s-hc) have been identified that specifically disrupts expression in the lateral peripodial epithelium of eye/antennal discs and is required for ventral head formation. This study describes the recovery of mutations in odd-paired (opa), a zinc finger transcription factor related to the vertebrate Zic family, as dominant enhancers of this dpp head mutation. A single loss-of-function opa allele in combination with a single copy of a dpp^s-hc produces defects in the ventral adult head. Furthermore, postembryonic loss of opa expression alone causes head defects identical to loss of dpp^s-hc/dpp^s-hc, and dpp^hc/+;opa/+ mutant combinations. opa is required for dpp expression in the lateral peripodial epithelium, but not other areas of the eye/antennal disc. Thus a pathway that includes opa and dpp expression in the peripodial epithelium is crucial to the formation of the ventral adult head. Zic proteins and members of the BMP pathway are crucial for vertebrate head development, since mutations in them are associated with midline defects of the head. The interaction of these genes in the morphogenesis of the fruitfly head suggests that the regulation of head formation may be conserved across metazoans (Lee, 2007).

This work demonstrates that opa is an upstream activator of dpp in the peripodial epithelium, and acts in a cell-autonomous fashion. It is not known whether this role is direct, with Opa acting as a transcription factor for dpp, or through other proteins. This ability to activate dpp appears limited to the peripodial epithelium of the eye/antennal disc, since misexpression of Opa in the disc proper does not induce expression. Furthermore, Opa acts only on a dpp reporter that has expression restricted to the peripodial epithelium of the eye/antennal disc. With the exception of antennal defects, loss-of-function clones of opa produce identical head defects to homozygous dpp^s-hc mutants, and ectopic expression of either Dpp or Opa in the peripodial epithelium produces a similar spectrum of misplaced sensory structures. These data suggest that dpp is the major target of opa in the peripodial epithelium (Lee, 2007).

Both opa and dpp are involved in embryonic midgut development, where dpp is a negative regulator of opa in the visceral mesoderm. In addition, BMP2 and BMP4 are negative regulators of Zic proteins in zebrafish, but the exact mechanism of this regulation is unclear. Thus, Zic family proteins are often seen in regulatory networks with BMP proteins, but there does not seem to be a canonical regulatory relationship. These data indicates that during eye/antennal disc development opa exerts a positive effect on peripodial dpp (Lee, 2007).

Both opa and dpp exert their role on ventral head development through expression limited to the peripodial epithelium of the eye/antennal disc. The structures affected in ventral head capsule mutations, such as palps and vibrissae, are reported to arise from the disc proper in the fate map of the eye/antennal disc; thus the effect of Opa-Dpp signal transduction could be to cross epithelial layers, from the peripodial epithelium to the disc proper. Loss of lateral peripodial Dpp expression results in apoptosis in the underlying disc proper, which further suggests a role for peripodial signaling to support disc proper cell viability and morphogenesis. However, when the descendants of peripodial cells are followed by the perdurance of ß-galactosidase expression through metamorphosis, significant contributions of lateral peripodial cells are found in areas of the ventral head where defects are observed in dpp^s-hc or opa mutations, suggesting that the ventral adult head is formed from descendants of both disc proper and peripodial cells. Adult head expression has also been seen with the MS1096-Gal4 driver, of which expression in the eye disc is limited to the lateral and medial peripodial epithelium and margin cells. These data provide further support to the idea that the peripodial epithelium provides more than passive or purely mechanical functions during disc development. The role of the peripodial epithelium in imaginal disc development has begun to receive more attention, and there is evidence that peripodial-specific signaling affects the patterning of the eye, growth control and the fusion of discs at metamorphosis. It now seems likely that in addition to providing such support to cells of the disc proper, peripodial cells contribute directly to the cuticle of the adult head (Lee, 2007).

In mice and humans, Zic genes are associated with holoprosencephaly, a congenital head defect the extreme manifestation of which is cyclopia. In holoprosencephaly there is variable loss or disruption in the development of the ventral forebrain, and midline facial structures. Holoprosencephaly is a common defect in humans, and genes in the TGF-ß and hedgehog pathways are also associated with both the human and mouse condition. Relevant to this work, a significant number of holoprosencephaly cases result from autosomal dominant inheritance, and often, obligate carriers of these autosomal dominant pedigrees are clinically normal. This incomplete penetrance suggests extreme dose sensitivity and the presence of multiple modifying loci. The ability of a genetic screen to recover multiple dominant enhancers of the dpp ventral head defect, including opa, suggests that this may be a model for the kind of digenic inheritance seen with holoprosencephaly. The hedgehog pathway is known to be crucial to adult head development in Drosophila, and this work adds TGF-ß and opa to this process in the fruitfly. It will be of interest to see how many other connections exist between vertebrate and fly head malformations (Lee, 2007).

Hox proteins coordinate peripodial decapentaplegic expression to direct adult head morphogenesis in Drosophila

The Drosophila BMP, decapentaplegic (dpp), controls morphogenesis of the ventral adult head through expression limited to the lateral peripodial epithelium (P e) of the eye-antennal disc by a 3.5 kb enhancer in the 5' end of the gene. A 15 bp deletion mutation within this enhancer was recovered that identified a homeotic (Hox) response element that is a direct target of labial and the homeotic cofactors homothorax and extradenticle. Expression of labial and homothorax are required for dpp expression in the peripodial epithelium, while the Hox gene Deformed represses labial in this location, thus limiting its expression and indirectly that of dpp to the lateral side of the disc. The expression of these homeodomain genes is in turn regulated by the dpp pathway, as dpp signalling is required for labial expression but represses homothorax. This Hox-BMP regulatory network is limited to the peripodial epithelium of the eye-antennal disc, yet is crucial to the morphogenesis of the head, which fate maps suggest arises primarily from the disc proper, not the peripodial epithelium. Thus Hox/BMP interactions in the peripodial epithelium of the eye-antennal disc contribute inductively to the shape of the external form of the adult Drosophila head (Stultz, 2012).

dpp expression in the lateral PE of the eye-antennal disc is necessary for correct morphogenesis of the adult Drosophila head. This study shows that dpp expression related to ventral head formation is part of a Hox/BMP genetic network restricted to the PE of the eye-antennal disc. The homeotic gene lab, and its cofactors hth and exd positively regulate PE dpp expression. This is supported by the observation that Lab, Exd and Hth bind in vitro to the dpp^hc enhancer and the consensus sites for these factors are required in vivo for expression. In addition, individually, lab and hth are both genetically necessary and together demonstrate sufficiency for expression from dpp^hc enhancer, as shown from both LOF and GOF clonal analyses. lab exerts positive control over Dfd expression, as indicated by loss of Dfd expression in lab LOF clones. In contrast, Lab is ectopically expressed in Dfd LOF clones, demonstrating that Dfd represses lab in domains of its expression. Dpp signalling is genetically required for the transcription of lab, as expression from a lab reporter construct is reduced in tkv LOF clones. Expression of a hth enhancer trap increases in tkv LOF clones, and is reduced when activated Tkv is ectopically expressed, indicating that hth transcription is negatively regulated by Dpp signalling. Finally, dpp directly autoregulates its own expression (Stultz, 2006a), and may be spatially limited to domains of signalling by repression by brk, as demonstrated by the ability of ectopically expressed Brk to repress expression from the dpp^hc enhancer. Lab and Hth (acting with Exd) activate the expression of dpp. Lab also contributes to the activation of Dfd, which when expressed, represses lab, acting as a switch to limit the extent of lab expression. It is envisioned that during disc development, lab initiates both dpp and Dfd, and when Dfd reaches a certain threshold level, it turns off lab, establishing the boundary between the two Hox proteins. However, while loss of Dfd is capable of derepressing lab throughout the disc, it does not do so to dpp, so further negative regulation must exist. brk may provide this repression to further ensure the lateral boundary of PE dpp through a potential AE element in the enhancer. These inputs collaborate to define the sharp boundary of PE dpp expression. The level of dpp transcription is positively modulated by feedback between lab and dpp and autoregulation of dpp, presumably through Mad/Med binding to the AE element. Negative feedback between dpp and hth provides a brake on expression; others may exist. For example, the inhibitory Smad protein, daughters against dpp is a target of peripodial Dpp expression (Stultz, 2006a). It is presumed these interactions activate dpp expression rapidly but shut it down when a certain expression level is reached (Stultz, 2012).

The Hox response region represents one of what will likely be many inputs into the expression of this 3.5 kb enhancer. Another input, opa, is homologous to the Zinc Finger Protein of the Cerebellum or Zic family of transcription factors, and was identified due to its genetic interaction with dpp^s-hc mutations. Other transcription factors and signalling pathways display genetic interactions, and their contribution to PE dpp expression is being actively investigated, although it is noteworthy that lab, Dfd, hth, and exd are not among them. It is expected that many transcription factors and signalling pathways impinge on the dpp^hc enhancer. In this regard, the dpp^hc enhancer may resemble the dpp visceral mesoderm enhancer, another identified Hox target, where direct Ubx, Abdominal A, Exd, and Hth homeodomain inputs collaborate with the Fox-F-related factor binou, as well as Dpp and Wingless signalling to control gene expression. Enhancers that respond to signalling pathways often demonstrate characteristic behaviours: 'activator insufficiency', 'cooperative activation', and 'default repression', and the dpp^hc enhancer conforms to this model. No single activator is able to induce expression over the entire disc, as shown by GOF experiments. Ectopically expressed Lab produced activation only in close proximity to the domain of endogenous dpp, while Hth activated only in the PE of the posterior eye disc. Addition of two inputs together (Lab and Hth or Lab and Dpp signalling, activated over a much broader area. Only Opa has broad ability to activate on its own over the PE but only in concert with Lab was it able to activate outside the PE. Thus each activator is insufficient individually; the enhancer requires simultaneous cooperative inputs of multiple factors to produce correct spatial expression. Brk would provide the default repression, preventing Lab and Hth individually from successfully activating in the middle of the disc, away from domains of dpp activity (Stultz, 2012).

Based on the transcriptional inputs so far identified, it is proposed that activation is controlled on the lateral side at a minimum by Lab, Hth, Exd, Mad, Med, and Opa. In the middle of the disc, the presence of only Hth and Exd is insufficient to activate the enhancer, particularly over resident default repression provided by Brk. On the medial (future dorsal) side of the disc, Dpp and phosphorylated Mad expression are observed, controlled by an unknown area of the dpp gene. Lab, Hth, Exd, and Opa are expressed there as well, so an additional repressor was hypothesised to be needed that limits expression driven by the dpp^hc enhancer to the lateral side. In this model, Lab is the activity required for peripodial specificity, with its cofactors Hth and Exd, while Mad/Med and Opa act as necessary collaborative activators of the enhancer (Stultz, 2012).

At the nucleotide level, the Hox response element in and adjacent to the dpp^s-hc1 deficiency bears sequence homology to previously identified Lab response elements: the mouse Hoxb1 autoregulatory enhancer (b1-ARE), which also generates a lab-like pattern, dependent on lab and exd activity, in Drosophila, and the lab autoregulatory enhancer. Both these enhancers have binding sites for Hox (Hoxb1, Lab), PBC (Pbx, Exd) and MEIS (Prep, Hth) proteins. The orientation of the bipartite Exd/Lab site relative to the MEIS site is the same in these elements as seen in the dpp^hc Hox response element, and the relative spacing between the PBC/Hox and MEIS components is very similar. However, the dpp^hc Hox response element has a cluster of three overlapping Hth sites, two residing on the opposite strand, and an additional functional Exd site downstream of the Hth sites, as determined by its requirement for expression in vivo). The expression of mutated reporter constructs in vivo, as well as LOF analyses of lab and hth, indicate that Hth/Exd plays a more critical role in enhancer activity than does Lab, as neither mutations in the Lab binding site nor Lab loss-of-function within somatic clones completely extinguished expression. This suggests that there may be multiple ways that homeodomain transcription factors activate the enhancer, depending on the cellular context. It is noted that the expression driven by the dpp^hc enhancer actually manifests as two separate lines {see also Stultz, 2006b). The level of Lab associated with each of these lines is not equivalent, therefore the control of expression may be specific to each line. This would be reminiscent of a situation seen within dpp itself, where the Ubx responsive visceral mesoderm enhancer is activated by Ubx/Exd/Hth in parasegment seven, but only requires Hth/Exd for activation in parasegment three. The in vitro EMSA data further support this, as Hth and Exd bind synergistically to more locations within the enhancer than Lab. The TALE family homeodomain proteins function independently of Hox proteins in many contexts. An additional explanation for the apparent primacy of hth may be because it plays both direct and indirect roles on enhancer expression. Hth acts with the transcription factor Yorkie (Yki) as part of the Hippo signalling pathway, and the nuclear activity of Yki and Hth are required to specify the PE of the eye-antennal disc. In the absence of hth, the PE is incorrectly fated. This may effect early gene expression upstream of the Hox/BMP interactions described in this study, magnifying the genetic affect of hth (Stultz, 2012).

The Hox/BMP network described in this study plays a prominent role in the external appearance of the adult head, yet is restricted completely to the PE of the eye-antennal disc. The terminal mutant phenotypes of dpp^s-hc, Dfd, and lab have similarities, but are sufficiently distinct that additional targets for each must exist, and for the cell autonomous Dfd and lab, these targets must reside in the PE. Other signalling proteins such as Wingless and Hedgehog, and the Notch pathway ligands Serrate and Delta, are expressed in the PE of the eye-antennal disc. While some adult structures derive from the PE, and PE cells likely contribute to other adult structures, it is likely that much of the effect of the PE on head morphogenesis is via inductive interactions with the DP, either through secreted signalling molecules, or targeted cell protrusions. Based on the cuticular alterations seen in dpp^s-hc, Dfd, and lab mutations, such interactions are capable of exerting structural modifications on the final head shape. Dipterans demonstrate great variety in the external morphology of their heads often with sexually dimorphic alterations within a species. Much of this variety involves changes in the relative proportions of eye and head capsule tissue. BMP expression has been implicated in shaping the jaws of cichlid fish and the beak shape of finches, while dpp expression itself is correlated with the growth of beetle horns, a specialized cuticular structure of the head. It is speculated that the PE specific Hox/BMP network described in this study could be a motor for such types of shape change in the Drosophila species (Stultz, 2012).

Table of contents

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decapentaplegic continued: Biological Overview | Evolutionary Homologs | Targets of activity | Protein Interactions | Post-transcriptional Regulation | Developmental Biology | Effect of mutation | References

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