Achaete regulation in imaginal discs: Role of genes other than wingless
Fas II is required for the control of proneural gene expression. Clusters of cells in the eye-antennal imaginal disc express the achaete proneural gene and give rise to mechanosensory neurons; other clusters of cells express the atonal gene and give rise to ocellar photoreceptor neurons. In FasII loss-of-function mutants, the expression of both proneural genes is absent in certain locations, and, as a result, the corresponding sensory precursors fail to develop. In FasII gain-of-function mutations, extra sensory structures arise from this same region of the imaginal disc. Mutations in the Abelson tyrosine kinase gene show dominant interactions with FasII mutations, suggesting that Abl and Fas II function in a signaling pathway that controls proneural gene expression (Garcia-Alonso, 1995).
Expression of the neurogenic gene Delta has been studied during microchaeta development in Drosophila as well as in the regulatory relationships between the Delta-Notch signaling pathway and the proneural gene, achaete. The adult notum is derived from the fusion of two heminota found at the anterior ends of the two wing/notal imaginal discs. Fusion takes place between 6 and 8 hours after puparium formation (APF). Within each notum, microchaeta sensory organ precursors (SOPs) arise within stripes of proneural cells arrayed from anterior to posterior. The stripe that develops in the center of the adult notum is designated stripe1, and the stripe that contains the dorsocentral macrochaetae is designated stripe 5. Microchaeta stripes 1, 3, and 5 develop first, followed by stripes 2 and 4. The majority of microchaeta SOPs arise within proneural stripes between 10 and 12 hours APF. Delta is expressed in all microchaeta proneural cells and microchaeta SOPs, and is expressed dynamically in SOP progeny. Delta expression in microchaeta proneural cells is detected prior to the onset of achaete expression and arises normally in the absence of achaete/scute function, indicating that the initial Delta expression in the notum is not dependent on proneural gene function (Parks, 1997).
The Delta-Notch signaling pathway is required at two steps during proneural cluster formation and SOP specification. (1) Dl prevents specification of supernumerary SOPS. In this function Delta represses achaete expression between microchaeta proneural stripes. Thus Delta function is required to help define microchaeta proneural stripe boundaries. (2) Dl is required later within proneural stripes to repress achaete expression. This second function involves the choice between neural (neuronal and thecogen/glial) and non-neural (tormogen and trichogen support cells) cell fate in the two cells decendent from a single SOP. In the first stage of Delta function, the expression data indicate that Delta is transcribed and Delta protein is localized throughout the entire microchaeta proneural stripe. There are no asymmetries in Delta accumulation within proneural stripes at the transcriptional level; nor is there a decrease in Notch protein levels in nascent SOPs. This is in contrast to the predictions of lateral inhibition models that suggest a Delta-Notch feedback loop might result in higher Delta expression in the cell adopting the SOP fate and higher Notch expression in immediately surrounding cells. Within proneural stripes, therefore the expression data is most consistent with the idea of mutual inhibition, i. e., that microchaeta proneural cells within the entire equivalence group interact via Delta and Notch to inhibit adoption of the SOP fate (Parks, 1997).
After Achaete protein expression has, for the most part, resolved to cells adopting the SOP fate, there do appear to be asymmetries in subcellular Delta protein localization in the vicinity of SOPs. Activation of the Delta-Notch pathway results in loss of Delta protein accumulation, suggesting that Delta expression is post-transcriptionally regulated, in part, by Delta-Notch signaling activity. Thus, Delta signaling is required for correct delineation of early proneural gene expression in developing nota. Later, within microchaeta proneural stripes, Delta-Notch signaling prohibits adoption of the SOP fate by repressing expression of the proneural gene achaete (Parks, 1997).
Several genes that regulate ac-sc gene expression have been characterized. For example, hairy (h) and extramacrochaete (emc) act as negative regulators of ac-sc since mutations in these genes result in the generation of ectopic SOPs. Proteins encoded by these genes, as well as AC-SC, contain basic helix-loop-helix domains that have been found in a number of proteins involved in transcriptional regulation. Hairy has been shown to be a direct transcriptional regulator of ac-sc, while Emc appears to down-regulate ac-sc indirectly by interacting with other factors. Pannier (Pnr), a zinc finger protein with homology to the vertebrate transcription factor GATA-1, also acts as a negative regulator of ac-sc. The u-shaped (ush) gene is involved in transregulation of ac-sc in the dorsal region of the notum. Ush, a zinc finger protein, heterodimerizes with Pnr as a cofactor and negatively regulates the transcriptional activity of Pnr. Two clustered genes isolated from the iroquois region, araucan (ara) and caupolican (caup), show similar spatial expression and function in the wing. iroquois (iro) has been recently identified as a candidate for prepattern genes, that is, these genes are expressed in a pattern which preceeds neurogeneis in the wing imaginal disc. Since the Ara protein has been shown to directly bind to an ac-sc enhancer element, it is suggested that the pattern of expression of iro genes determines the pattern of proneural gene expression and thus the pattern of neural development in the wing disc. Therefore, the iro genes fulfill the characteristics of prepattern genes that direct sensory organ formation in the notum. In addition to the eye, mirr is expressed in the wing disc in a similar, but not identical, pattern as seen with iro genes. The role of the mirr gene has been investigated in the formation of alula and sensory organs in the wing. This study suggests that mirr acts together with other iroquois genes in the prepatterning of sensory organs and alula development (Kehl, 1998).
mirr expression was examined in the imaginal discs by in situ mRNA hybridization and immunohistological detection of the lacZ reporter expression. In the wing disc, Mirror mRNA is expressed in several regions, including the notum and pleura. mirr expression is also detected in the alula region, an accessory basal structure of the wing. Since the alula is lost in mirr mutants, it is suggested that mirr is required for alula formation. The expression pattern of mirr is very similar to that of ara and caup (Gomez-Skarmeta, 1996). However, mirr is not expressed in the precursors for L3/L5 wing veins, tegula and dorsal radius while ara and caup are expressed in these regions. The tegula is the most proximal part of the anterior wing margin. lacZ expression in the wing disc is similar to the mRNA expression pattern, suggesting that the lacZ reporter reflects the pattern of mirr expression (Kehl, 1998).
An individual bristle on the notum can be easily identified by its specific position. mirror mutations caused specific loss of macrobristles only in the lateral domain of the notum. mirr Sai1 /TM3 Sb or mirr Sai1 /TM6 heterozygotes show a dominant bristle phenotype: deletion of presutural (PS) and/or posterior supraalar (pSA) bristles. In many cases (31%), both PS and pSA bristles are absent. Approximately 90% of mirr B1-12 /mirr B1-12 flies examined were missing one bristle, although occasionally two bristles were deleted. The deletion of bristles is specifically restricted to two of the seven macrobristles in the region; the pSA and anterior postalar bristles (aPA). The strongest phenotype observed is found in mirr Sai1 /mirr B1-12 , in which up to four lateral bristles were missing. This is consistent with the observation that Mirror mRNA level is greatly reduced in the notum of this mutant wing disc. These results indicate that the elimination of lateral macrobristles by mirr mutations are allele-specific and are mainly restricted to four bristles: the PS, pSA, aPA and pPA. This is consistent with the expression of mirr in this subset of sensory organ precursors (SOPs). It is concluded that like ara and caup, mirror expression establishes the prepattern of several macrobristles in the notum (Kehl, 1998).
Ara and Caup are expressed in SOPs, acting as positive transcriptional regulators of achaete in the wing disc epithelium (Gomez-Skarmeta, 1996). Mirr is also expressed in the SOPs in the notum. Mirr and Ac expression overlap only in a subset of bristle SOPs in the lateral heminotum, including the PS and PA bristles, which are affected by mirr mutations. In contrast, SOPs for the notopleural and anterior supraalar bristles are stained with anti-Achaete, while Mirr is either not expressed or is expressed at low levels in the notopleural (NP) and aSA bristles. This is consistent with the finding that these bristles form normally in mirr mutations. These results suggest that Mirr as well as Ara and Caup might control ac-sc expression, and the loss of a subset of bristles in different mirr alleles might result from the loss of the corresponding SOPs rather than the degeneration of bristles (Kehl, 1998).
The Bar homeobox genes function as latitudinal prepattern genes in the developing Drosophila notum. In Drosophila notum, the expression of achaete-scute proneural genes and bristle formation have been shown to be regulated by putative prepattern genes expressed longitudinally. The two Bar locus genes may belong to a different class of prepattern genes expressed latitudinally: it is suggested that the developing notum consists of checker-square- like subdomains, each governed by a different combination of prepattern genes. BarH1 and BarH2 are coexpressed in the anterior-most notal region and regulate the formation of microchaetae within the region of BarH1/BarH2 expression through activating achaete-scute. Presutural macrochaetae formation also requires Bar gene activity. Bar gene expression is restricted in dorsal and posterior regions by Decapentaplegic signaling, while the ventral limit of the expression domain of Bar genes is determined by wingless, whose expression is under the control of Decapentaplegic signaling (Sato, 1999).
Seven Enhancer of split complex genes in Drosophila melanogaster encode basic-helix-loop-helix transcription factors that are components of the Notch signaling pathway. They are expressed in response to Notch activation and mediate some effects of the pathway by regulating the expression of target genes. Using random oligonucleotide selection, the optimal DNA binding site for the Enhancer of split proteins has been determined to be a palindromic 12-bp sequence, 5'-TGGCACGTG(C/T)(C/T)A-3', which contains an E-box core (CACGTG). This site is recognized by all of the individual Enhancer of split basic helix-loop-helix proteins, consistent with their ability to regulate similar target genes in vivo. The 3 base pairs flanking the E-box core are intrinsic to DNA recognition by these proteins and the Enhancer of split and proneural proteins can compete for binding on specific DNA sequences. Furthermore, the regulation conferred on a reporter gene in Drosophila by three closely related sequences demonstrates that even subtle sequence changes within an E box or flanking bases have dramatic consequences on the overall repertoire of proteins that can bind in vivo (Jennings, 1999).
The overall similarity in the binding of different E(spl) proteins in vitro suggests that they are capable of recognizing the same targets in vivo and is consistent with the phenotypes observed when the individual proteins are expressed ectopically. Ectopic expression of M8, M5, Mbeta, Mdelta, and M7 all produce phenotypes of vein and bristle loss. Both Mbeta and M7 are able to interact with DNA sequences regulating achaete. The ability to recognize the same DNA target sequences could explain the apparent redundancy between the E(spl) genes, as they would all have the potential to act in the same processes. The observation that specific E(spl)bHLH proteins are more or less efficient in regulating different processes (e.g., Mbeta more effective at suppressing veins and M8 more effective at suppressing bristles) is thus more likely to be consequence of differences in protein:protein interactions than of differences in target recognition (Jennings, 1999 and references).
In the absence of E(spl)bHLH proteins, proneural protein expression persists at high levels in all cells of a proneural cluster. Thus, one action of E(spl)bHLH proteins is to antagonize the proneural proteins, with the ultimate consequence that proneural gene expression is repressed. It has been proposed that E(spl)bHLH proteins exert their influence by binding to regulatory regions within the AS-C and repressing transcription of the proneural genes. This hypothesis is supported by the observations that expression of Achaete is induced by M7ACT and MbetaACT and that induction of ectopic bristles in the Drosophila wing and notum by M7ACT is abolished in the absence of proneural proteins. One putative binding site for the E(spl)bHLH proteins, that upstream of the achaete gene, has the sequence 5'-CGGCACGCGACA-3' (Hairy site). Mgamma will bind this site in vitro, and M7 can bind this sequence and repress transcription in a cotransfection assay in Drosophila S2 cultured cells. However, mutation of this site in vivo results in a phenotype resembling that caused by mutations in hairy rather than in the E(spl)-C. This fits with the observation that this sequence conforms to an optimal Hairy DNA binding site but is a suboptimal site for the E(spl) proteins and indicates that the E(spl) proteins do not recognize this sequence in vivo. Thus, if E(spl) proteins are directly repressing achaete expression, there should be more optimal target sites elsewhere within the AS-C. Indeed, a search of recently available AS-C genomic sequence identifies >10 sequences with good matches to ESE boxes, in addition to the sites that have been identified by in vitro binding assays (Jennings, 1999 and references).
An alternative hypothesis is that the primary function of the E(spl)bHLH proteins is to antagonize the actions of proneural proteins posttranscriptionally. Evidence in support of this comes from experiments in which L'sc is ectopically expressed using a heterologous promoter that is not subject to direct regulation by E(spl)bHLH proteins. Under these conditions L'sc expression results in isolated ectopic bristles, rather than clusters of bristles, demonstrating that lateral inhibition is still able to restrict neural fate to a single cell even though l'sc transcription is insensitive to Notch signaling. This implies that E(spl)bHLH proteins are able to antagonize proneural genes in ways other than by repressing their transcription. One possibility is that the E(spl) proteins can interact with the same targets as proneural proteins, but that they repress rather than activate transcription. The ability of E(spl) proteins to bind to the B1 and A1 sequences and repress transcription from a heterologous promoter is consistent with this model, as is the observation that M7ACT can induce certain ectopic leg bristles in the absence of the achaete and scute genes. In the latter context, M7ACT is likely to be acting on genes with functions downstream of the proneural proteins to cause neural differentiation. In addition, the E(spl)bHLH proteins are involved with developmental processes that do not involve the proneural proteins, e.g. wing vein development; thus, they cannot act solely to repress proneural gene transcription during development (Jennings, 1999 and references).
In vertebrates and invertebrates, spatially defined proneural gene expression is an early and essential event in neuronal patterning. This study investigates the mechanisms involved in establishing proneural gene expression in the primordia of a group of small mechanosensory bristles (microchaetae), which on the legs of the Drosophila adult are arranged in a series of longitudinal rows along the leg circumference. In prepupal legs, the proneural gene achaete (ac) is expressed in longitudinal stripes, which comprise the leg microchaete primordia. It has been shown that periodic ac expression is partially established by the prepattern gene, hairy, which represses ac expression in four of eight interstripe domains. This study identifies Delta (Dl), which encodes a Notch (N) ligand, as a second leg prepattern gene. Hairy and Dl function concertedly and nonredundantly to define periodic ac expression. The regulation of periodic hairy expression was explored. In prior studies, it was found that expression of two hairy stripes along the D/V axis is induced in response to the Hedgehog (Hh), Decapentaplegic (Dpp) and Wingless (Wg) morphogens. This study shows that expression of two other hairy stripes along the orthogonal A/P axis is established through a distinct mechanism which involves uniform activation combined with repressive influences from Dpp and Wg. These findings allow for the formulation of a general model for generation of periodic pattern in the adult leg. This process involves broad and late activation of ac expression combined with refinement in response to a prepattern of repression, established by Hairy and Dl, which unfolds progressively during larval and early prepupal stages (Joshi, 2006).
Patterning of the leg imaginal disc along its circumference axis is controlled by the Hh, Dpp and Wg morphogens. In this and previous studies, attempts have been made to elucidate the molecular mechanisms through which these signals give rise to specific morphological features of the leg, the mechanosensory microchaetae. Patterning of leg mechanosensory microchaetae requires spatially defined expression of the proneural gene ac and its repressor Hairy. Expression of hairy in two pairs of longitudinal stripes, the D/V-hairy and A/P-hairy stripes, is directed by separate enhancers that are Hh-, Dpp- and Wg-responsive. In this study, it is reported that the D/V-hairy and A/P-hairy stripes are differentially regulated by Dpp and Wg and that distinct mechanisms are utilized to control hairy expression along the A/P and D/V axes. D/V-hairy expression is locally induced near the A/P compartment boundary by Hh signaling. In addition, Dpp and Wg positively influence expression of the dorsal and ventral components of the D/V-hairy stripes, respectively, by acting together with Hh to define the register of these stripes relative to the compartment boundary. In contrast, the A/P-hairy stripes, which are expressed orthogonal to the D/V-hairy stripes and A/P compartment boundary, are not activated via local induction. Rather, it appears that they are broadly activated along the leg circumference and repressed by Dpp dorsally and Wg ventrally to define their dorsal and ventral boundaries. This model for A/P-hairy regulation is supported by the observations that hairy is ectopically expressed in dorsal, but not ventral, clones lacking tkv or Mad function and that A/P-hairy expression is compromised by elevation of Dpp signaling. Furthermore, ventral, but not dorsal, clones lacking dsh function also ectopically express hairy and high-level Wg signaling results in loss of A/P-hairy expression (Joshi, 2006).
A potential caveat to this model for regulation of A/P-hairy expression is that conclusions were drawn from analysis of endogenous hairy expression rather than by examining expression directed by isolated A/P-hairy enhancer(s). Hence, it is possible that the ectopic hairy expression seen in tkv, Mad and dsh mutant clones is a result of expansion of D/V-hairy rather than A/P-hairy expression. However, several lines of evidence argue against this interpretation. First, through genetic and molecular analyses of D/V-hairy enhancer function, it has been demonstrated that Dpp and Wg positively regulate D/V-hairy expression, an observation that is inconsistent with the suggestion that D/V-hairy is ectopically expressed in clones unable to respond to Dpp or Wg signaling. Furthermore, in 3rd instar and early prepupal leg discs, stages at which the A/P-hairy stripes are not expressed, ectopic hairy expression is not observed in tkv mutant clones. Second, it has been found that the D/V-hairy stripes can only be expressed in anterior compartment cells near the A/P boundary, which are the cells that receive and respond to Hh signal. Thus, it is unlikely that ectopic hairy expression observed in clones at distance from the compartment boundary, which receive little or no Hh signal, and in the posterior compartment, in which cells do not respond to Hh signal, corresponds to D/V-hairy expression. Finally, it has been found that elevation of Dpp or Wg signaling specifically disrupts A/P-hairy but not D/V-hairy expression. Taken together, these findings are consistent with the conclusion that A/P-hairy rather than D/V-hairy is expressed in clones compromised in their response to Dpp and Wg signaling (Joshi, 2006).
The expression of the A/P-hairy stripes at a distance from the dorsal and ventral organizers implies that A/P-hairy expression is repressed even at low threshold levels of Dpp and Wg signaling. This raises questions regarding the mechanisms through which Dpp and Wg define the sharp boundaries of the A/P-hairy stripes. A mechanism for Dpp-mediated repression in imaginal discs has been described, in which a complex of activated Mad with the Schnurri transcription factor acts directly through a repression element in the brinker (brk) gene. However, Dpp does not establish sharp boundaries of brk expression. Rather, brk expression drops off in a graded fashion toward the source of Dpp. Since the dorsal boundary of A/P-hairy expression is sharp and at distance from the Dpp source, this would imply that A/P-hairy expression is very sensitive to Dpp-mediated repression. Hence, it will be of interest to further investigate this process. Also of interest are the mechanisms of Wg-mediated repression, which are poorly understood (Joshi, 2006).
In this study, Dl was identified as a second prepattern gene that functions together with hairy to establish ac expression in the leg microchaete proneural fields. Several lines of evidence that support this conclusion. (1) It was found that, beginning at 4 h APF, Dl expression is up-regulated in domains overlapping the microchaete proneural fields. This distribution of Dl is similar to that, in the notum, where Dl has been shown to regulate proneural ac expression. (2) It was shown that ac expression is expanded in legs with reduced Dl function. (3) It was found that elevated N signaling throughout the tarsus results in severely reduced ac expression. (4) Activation of N signaling was observed within the hairy-OFF interstripes, in agreement with the genetic requirement for Dl/N signaling in these domains. Based on these results, it is proposed that ac expression is activated broadly during mid-prepupal leg development but is confined to the microchaete proneural fields by a previously generated prepattern of repression, established by Hairy and Dl/N signaling. This hypothesis is supported by analysis of cis-regulatory elements that direct ac expression in the leg microchaete proneural fields. By generating rescue and reporter constructs, an enhancer was identified that specifically controls expression of ac in the microchaete proneural fields. Unlike the hairy leg enhancers, no modular organization is observed of the cis-regulatory elements that control expression of ac stripes in different regions of the leg. Rather, preliminary analyses suggest that there is one enhancer consisting of an activation element that directs broad expression of ac along the leg circumference and two repression elements, which are N- or Hairy-responsive. This finding is consistent with genetic studies and the model for regulation of ac expression in the leg microchaete proneural fields (Joshi, 2006).
hairy and Dl function to repress ac expression in complementary domains. hairy encodes a transcriptional repressor which has been shown to directly repress ac expression in the wing by binding a specific site in the ac promoter. It is likely that Hairy acts through a similar site to repress ac expression in the leg. Dl represses ac expression via a different mechanism: presumably, cells of the microchaete proneural fields, which express high levels of Dl, signal to adjacent cells to activate N. This suggestion is supported by the observation that expression of two N-responsive reporters is specifically activated in cells corresponding to the hairy-OFF interstripes. One of the reporters used in this study, E(spl)mβ-CD2, and other similar reporters recapitulate endogenous E(spl)mβ-CD2 expression in wing and leg imaginal discs. E(spl)mβ is one of seven genes in the E(spl)-C that encode bHLH repressors related to Hairy. Hence, it appears that ac expression in the leg microchaete proneural fields may be established by a prepattern of periodically expressed bHLH repressors (Joshi, 2006).
N signaling is not activated within ac-expressing cells, even though these cells express high levels of Dl. This could be explained by a dominant-negative effect of Notch ligands on N signaling, which has been observed in the wing. In the wing, it has been shown that N signaling is not activated within cells expressing high levels of Dl and Ser but, rather, that these cells signal to adjacent cells to activate N signaling within the wing margin. Consistent with the hypothesis of a potential dominant-negative function for Dl in the leg microchaete proneural fields is the observation that over-expression of Dl along the leg circumference results in expansion of ac expression into the hairy-OFF interstripes, which would be expected if N signaling was disabled. Over-expression of N ligand expression has been shown to exert a similar effect in other tissues (Joshi, 2006).
A curious observation of this study is that, as suggested by genetic evidence and the expression of two N-responsive reporters, N signaling, with one exception, is not activated within the hairy-ON interstripes, even though each Hairy stripe is straddled on either side by a Dl stripe. This suggests either that Dl signals asymmetrically or that there is an asymmetric response to N signaling and raises questions regarding the underlying mechanism of asymmetric activation of N-target gene expression. A potential mechanism for asymmetric signaling by Dl is suggested by studies in the notum, in which it has been shown that the N receptor is distributed in a pattern complementary to Dl. If N levels were higher within the hairy-OFF vs. the hairy-ON interstripes in the leg, this could allow for preferential signaling within these domains. However, N expression was assayed in prepupal legs and it was found that N appears to be uniformly distributed along the leg circumference. Hence, either there is an asymmetric response to N or alternative mechanisms are responsible for establishing the directionality of Dl signaling in the leg, such as post-translational modification N signaling pathway components. For example, glycosylation of N by the Fringe glycosyltransferase influences its interactions with its ligands (Joshi, 2006).
Another intriguing finding is the overlap of N signaling with the V-Hairy stripe. This result was surprising because it would suggest redundancy between hairy and Dl/N signaling in this region. However, an absolute requirement for hairy function was observed in the ventral leg. An explanation for this puzzling finding is suggested by the specific loss of the V-Gbe+Su(H)m8-lacZ stripe in hairy mutant legs, which indicates that Dl/N signaling or responsiveness in the ventral leg is dependent on hairy function. The specific loss of N signaling in the ventral leg could be a result of the expansion of Dl expression in hairy mutant legs, which as explained earlier might have a dominant-negative effect on N signaling. This proposal is corroborated by the expansion of ac expression along the circumference of legs ectopically expressing Dl throughout the tarsus. The overlap of hairy and Dl/N signaling in the ventral leg raises questions regarding the function of Dl/N signaling in this domain. It was observed that V-hairy and Gbe+Su(H)m8-lacZ expression overlap only partially, suggesting that combined function of Dl and Hairy in the ventral leg could serve to establish a broader domain of repression in this region in comparison to other interstripe domains. This idea is supported by the morphology of the adult leg tarsus in which the spacing of bristles is most pronounced along the ventral midline. However, the function of N in the ventral leg is not as yet clear. It is plausible that there is a role for Dl/N signaling in the ventral leg that is unrelated to regulation of ac expression (Joshi, 2006).
The potential function of Dl as a regulator of proneural ac expression in the leg was suggested by studies in the notum, on which mechanosensory microchaetae are also organized in longitudinal rows. In the notum, Dl/Notch signaling, rather than Hairy, regulates periodic ac expression. The current studies suggest a distinct mechanism for leg microchaete patterning in which Hairy and Dl act together and nonredundantly to define periodic ac expression. In both the leg and notum, Dl signals to adjacent cells to repress ac expression. However, whereas in the notum Dl activates N signaling in cells on either side of each Dl/Ac stripe, in the leg, N signaling is activated (with one exception) only within the hairy-OFF interstripes. Although the pattern of mechanosensory bristles on the leg and notum is overtly similar, the bristle rows are more precisely aligned in the leg. The more organized pattern on the leg may be a consequence of the combined function of Hairy and Dl which might more precisely define the domains of proneural gene expression (Joshi, 2006).
Dl function is essential for proper patterning of ac expression, and it is suggested that accurate positioning of the Dl stripes is necessary for activation of Notch signaling within appropriate domains. Hence, regulation of Dl expression is an important aspect of leg microchaete patterning. In legs lacking hairy function, Dl expression expands into four broad domains and ectopic hairy expression greatly reduces Dl expression, indicating that periodic expression of Dl is regulated in part by hairy. Concomitant with the expansion of Dl expression, there is loss of N signaling in the ventral leg, suggesting that hairy functions to create an apposition of cells expressing high levels of Dl to cells expressing low levels of Dl, which allows for activation of N signaling in the ventral leg. Regulation of Dl expression in proneural fields is not understood. A plausible hypothesis is that, like hairy, Dl expression is established in response to the morphogens that control pattern formation during leg development (Joshi, 2006).
This and previous studies suggest an outline of a general genetic pathway for the regulation of ac expression in the leg microchaete proneural fields. This process involves broad and late activation, by an unknown factor, of ac expression along the leg circumference combined with refinement in response to a prepattern of repressors, which is established during larval and early prepupal stages. Hairy and Dl have been identified as the primary prepattern factors that regulate ac expression along the leg circumference. Position-specific expression of both hairy and Dl in longitudinal stripes is essential for proper ac expression. It has been determined that the longitudinal stripes of hairy are established in direct response to the Hh, Dpp and Wg signals, which globally pattern the leg, indicating that hairy acts as an interface between ac and these morphogens. Dl expression is regulated by Hairy, but its regulation is otherwise poorly understood. In addition to elucidating a pathway for establishment of periodic ac expression during leg development, these studies also provide insight into the mechanisms through which morphogens function to generate leg morphology (Joshi, 2006).
Periodic ac expression is established progressively. The first evidence of periodicity is expression of the longitudinal stripes of hairy expression. The D/V-hairy stripes are expressed first in the early 3rd instar leg disc followed by the A/P-stripes between 3 and 4 h APF. Between 4 and 6 h APF, Dl expression within the mechanosensory microchaete primordia is established. Then, ac expression is activated uniformly along the leg circumference. By the time that ac expression is activated, the interstripe domains have been defined by the four Hairy stripes and Dl/N signaling (Joshi, 2006).
The delay of ac expression in the microchaete proneural fields until mid-prepupal stages is likely due to the requirement of ac function for formation of all leg sensory organs. Leg sensory bristles can be grouped into two broad categories based on their time of specification: one group includes the early-specified mechanosensory macrochaetae (large bristles) and chemosensory microchaetae, and the second group includes the more numerous late-specified mechanosensory microchaetae. During the 3rd instar and early prepupal stages, ac is expressed in small clusters of cells that define the primordia of early-specified bristles, while expression of ac in the mechanosensory microchaete primordia is activated later in the mid-prepupal stage. This late expression of ac is activated broadly along the leg circumference and is presumably delayed to allow for expression of the hairy and Dl stripes during earlier stages. Premature expression of this normally late ac expression would likely lead to disturbances in sensory organ patterning, suggesting that temporal control of ac expression is an important aspect of its regulation (Joshi, 2006).
Mi-2, the central component of the nucleosome remodeling and histone deacetylation (NuRD) complex, is known as an SNF2-type ATP-dependent nucleosome remodeling factor. No morphological mutant phenotype of Drosophila Mi-2 (dMi-2) has been reported previously; however, it was found that rare escapers develop into adult flies showing an extra bristle phenotype. dMi-2 enhances the phenotype of acHw49c, which is a dominant gain-of-function allele of achaete (ac) and produces extra bristles. Consistent with these observations, the ac-expressing proneural clusters are expanded, and extra sensory organ precursors (SOP) are formed in the dMi-2 mutant wing discs. Immunostaining of polytene chromosomes showed that dMi-2 binds to the ac locus, and dMi-2 and acetylated histones distribute on polytene chromosomes in a mutually exclusive manner. Chromatin immunoprecipitation assay of the wing imaginal disc also demonstrated a binding of dMi-2 on the ac locus. These results suggest that the Drosophila Mi-2/NuRD complex functions in neuronal differentiation through the repression of proneural gene expression by chromatin remodeling and histone deacetylation (Yamasaki, 2006).
Many studies have shown that morphological diversity among homologous animal structures is generated by the homeotic (Hox) genes. However, the mechanisms through which Hox genes specify particular morphological features are not fully understood. This issue was addressed by investigating how diverse sensory organ patterns are formed among the legs of the Drosophila adult. The Drosophila adult has one pair of legs on each of its three thoracic segments (the T1-T3 segments). Although homologous, legs from different segments have distinct morphological features. Focus was placed is on the formation of diverse patterns of small mechanosensory bristles or microchaetae (mCs) among the legs. On T2 legs, the mCs are organized into a series of longitudinal rows (L-rows) precisely positioned along the leg circumference. The L-rows are observed on all three pairs of legs, but additional and novel pattern elements are found on T1 and T3 legs. For example, at specific positions on T1 and T3 legs, some mCs are organized into transverse rows (T-rows). The T-rows on T1 and T3 legs are established as a result of Hox gene modulation of the pathway for patterning the L-row mC bristles. The findings suggest that the Hox genes, Sex combs reduced (Scr) and Ultrabithorax (Ubx), establish differential expression of the proneural gene achaete (ac) by modifying expression of the ac prepattern regulator, Delta (Dl), in T1 and T3 legs, respectively. This study identifies Dl as a potential link between Hox genes and the sensory organ patterning hierarchy, providing insight into the connection between Hox gene function and the formation of specific morphological features (Shroff, 2007).
It is proposed that T-rows are formed on T1 and T3 legs in response to Scr or Ubx alteration of the L-row prepattern via repression of Dl expression. Dl is expressed in narrow longitudinal stripes that correspond to the L-row primordia. Dl-expressing cells in the L-row primordia signal to adjacent cells to activate N signaling and repress ac expression in the hairy-OFF interstripes and in one hairy-ON interstripe, between the L-row 1 and 8 proneural fields. It is suggested that in T1 and T3 legs, reduction of Dl expression in cells with high-levels of Scr or Ubx establishes a zone where there is no repressive influence on ac expression, resulting in expression of Ac in broad domains from which the T-row precursors will be selected. Cells in the center of T-row primordia are presumably out of range of the Dl signaling that takes place at the interface of Dl-expressing and Dl-non-expressing cells. The anterior and posterior boundaries of Ac expression in the T-row primordia of T1 prepupal legs are likely established by Dl/N signaling. In T3 legs, in contrast, it appears that Hairy rather than Dl/N signaling establishes the boundaries of ac on either side of the T-row primordia. Reduced Dl expression in the T-row primordia of T3 legs, however, is likely required to establish a broader domain of Ac expression than would be observed in the corresponding domain of T2 legs (Shroff, 2007).
A key feature of the model for mC patterning is that position-specific expression of ac expression in the mC proneural fields is established mainly by repression and that differential mC patterns are generated by altering expression or function of the repressive factors, Hairy and/or Dl. It is suggested that altered Dl expression is required in order to reduce N signaling, which allows proneural gene expression within the T-row primordia. An alternative hypothesis is that Dl function in during leg mC development is limited to selection of SOPs via lateral inhibition and that regulation of Dl by Scr/Ubx alters lateral inhibition within the T-row proneural fields. However, the hypothesis that Scr/Ubx regulation of Dl alters the proneural prepattern is supported by several observations. A prepattern function for Dl in mC patterning has been previously demonstrated in the notum. Similarly, it has been observed that in prepupal legs with reduced Dl function, proneural Ac expression expands along the leg circumference and is excluded only from Hairy-expressing cells. Furthermore, in prepupal legs, proneural Ac expression fills the center of large clones lacking Dl function. It is also observed that N signaling is activated only in narrow stripes on either side, but not within the T-row proneural fields. The genetic observations are substantiated by analysis of an enhancer that directs ac expression in both the L-row and T-row proneural fields. This enhancer consists of an activation element that directs uniform expression of ac along the leg circumference and two associated repression elements, one that is N-responsive and another that is Hairy-responsive. This is consistent with genetic studies suggesting that in the absence of repressive influences from Hairy and Dl, proneural ac expression would be uniformly along the leg circumference. Combined, these observations suggest that the mC patterning pathway is modified upstream of proneural gene expression by establishment of differential Dl expression in legs from different thoracic segments (Shroff, 2007).
The finding that Dl expression is down-regulated in the T-row primordia, does not necessarily imply that Dl expression is incompatible with mC formation. That this is not the case is suggested by the observation that Dl is expressed in the L-row primordia. Previous studies in a number of tissues have shown that high-level N-ligand expression renders cells non-responsive to N signaling. Hence, it appears that Dl/N signaling at the boundary of Dl-expressing and Dl-non-expressing cells, not Dl expression per se, is incompatible with mC development (Shroff, 2007).
Many studies have made clear the importance of establishing spatially defined proneural gene expression, largely via transcriptional regulation, for patterning of both the vertebrate and invertebrate nervous system. For example, it has been shown that ectopic proneural gene expression causes disruption of the sense organ pattern in adults. In the leg, compromised hairy function results in ectopic proneural ac expression and disorganization of the adult mC pattern, including formation of extranumerary mCs. However, other studies have implicated post-transcriptional regulation of proneural gene function in neural patterning. This was suggested by a study that showed that generalized and transient sc expression in a background devoid of ac and sc function results in an almost normal sense organ pattern in adult flies. Studies in the notum have provided an explanation for this observation by identification of the Extra macrochaetae (Emc) protein as a post-transcriptional regulator of proneural gene function. Emc, an HLH protein that lacks a basic DNA binding domain, binds proneural bHLH proteins, such as Ac, and inhibits their activity. In the notum, emc is expressed in a complex pattern that partially overlaps proneural gene expression, and it appears that SOPs are selected from cells with the lowest levels of Emc. This would suggest that on the notum, competence to acquire a neural fate depends on the balance of proneural protein to Emc levels. It is probable that similar mechanisms function in leg mC patterning as well, since largely normal sense organ patterns are found in legs ubiquitously expressing Sc. These observations indicate that sense organ patterning is a complex process that involves regulation of both proneural gene expression and function. Hence, it would be of interest to assess the relative contribution of post-transcriptional regulation of proneural gene function on leg mC patterning (Shroff, 2007).
It is proposed that T-row mCs are selected from domains of up-regulated Scr or Ubx expression and that one essential function for Scr and Ubx in T-row development is repression of Dl expression. This proposal is supported by several lines of evidence. The requirement of Scr and Ubx in T-row formation was suggested by prior reports that loss of Scr or Ubx function results in transformation of T1 or T3 legs, respectively, toward a T2 fate. This study shows that adult legs heterozygous for reduced function alleles of Scr (ScrEdK6/ScrEfW22) exhibit almost complete loss of T-rows in the adult. Moreover, ectopic expression of Scr or Ubx induces T-row formation on T2 legs, on which T-rows are never normally observed. The domains of elevated Scr and Ubx expression in T1 and T3 prepupal legs correspond to the respective positions of T-rows in adult T1 and T3 legs. Furthermore, comparison of Scr expression to that of an SOP marker, sca-Gal4, shows that T-row mCs are selected from groups of cells that express high-level Scr on T1 legs (Shroff, 2007).
Strong evidence is provided that Scr and Ubx repress Dl expression in the T-row primordia. First, a correlation is observed between up-regulated Scr and Ubx expression and domains of reduced Dl expression. Second, loss and gain of function studies indicate that Scr and Ubx negatively regulate Dl expression. In ScrEdK6/ScrEfW22, prepupal legs, Dl is expressed in two longitudinal stripes overlapping the region of high-level Scr expression, whereas in wild type legs Dl stripes flank but do not overlap this domain. In addition, Dl is ectopically expressed in either Scr or Ubx loss of function clones within the T-row primordia. Consistent with loss of function results, it was found that ectopic high-level expression of Scr or Ubx results in repression of Dl expression (Shroff, 2007).
Taken together, these observations suggest a function for Scr and Ubx in specification of a T-row fate. However, the finding that the formation of T-rows in response to ectopic Scr or Ubx is confined to ventro-lateral regions along the circumference implies that there may be other positional cues, in addition to elevated Scr or Ubx expression, that are required for T-row specification. Hence, it is plausible that these genes function combinatorially with other factors to induce T-row formation. Wg, for example, is a good candidate since it is expressed in ventro-lateral regions of the leg. In addition, ectopic Wg expression results in expansion of T-row bristles in T1 legs. It is also plausible that, in addition to or instead of T-row promoting factors in ventro-lateral leg regions, there are factors outside these domains that inhibit T-row formation (Shroff, 2007).
These studies have elucidated a general pathway for leg mC patterning in which an early event is establishment of position-specific expression of the prepattern genes hairy and Delta, presumably in response to the global regulators of limb patterning. The spatial regulation of hairy expression during leg development has been investigated ant it has been determined that hairy expression is controlled by modular enhancer elements that integrate patterning information provided by the signaling molecules known to pattern the leg along its circumference, Hedgehog (Hh), Decapentaplegic (Dpp) and Wingless (Wg). Periodic Dl expression is partially established by Hairy. However, it is likely that Dl expression in the mC proneural fields is also regulated, like hairy, by genes that pattern the leg along the circumference (circumferential patterning genes), such as hh, dpp and wg (Shroff, 2007).
Up-regulated expression of Scr and Ubx at specific positions along the circumference and P/D axis of T1 and T3 prepupal legs is key to generating the T-row pattern, raising the question of how Scr and Ubx expression in the T-row primordia is regulated. It is proposed that Scr and Ubx expression is controlled by the circumferential patterning genes and genes that pattern the leg along the P/D axis (P/D patterning genes). For example, Scr and/or Ubx expression might be regulated by Wg, which is known to specify ventral leg identity and patterns the ventral leg along the A/P axis in a concentration dependent manner. Along the P/D axis, a number of genes, such as Distal-less and dachshund, are expressed in defined and partially overlapping domains and might function to define the extent of up-regulated Scr and/or Ubx expression. This model would suggest that circumferential and P/D patterning information is integrated by Scr and Ubx, implying that these Hox genes link the global regulators of leg development to local acting genes, such as ac, that specify a neural fate (Shroff, 2007).
These studies indicate that Scr and Ubx function early in T-row development to repress Dl expression, which allows formation of the T-row proneural fields. It will be of interest to determine whether Dl is a direct target of these Hox genes, especially since few direct Hox-gene targets have been identified to date. However, it is probable that Scr and Ubx have other functions in T-row development. Establishment of ac expression in the T-row primordia is an early and essential step of T-row development, but, while ac specifies a neural fate, it does not specify sensory organ type. Hence, it is likely that Scr and Ubx function, in conjunction with other factors, to specify 'T-row-type' mCs. For example, since the T-row mCs are less pigmented than L-row mCs, a potential role of Scr and Ubx in T-row development is to regulate genes involved bristle pigmentation. A second potential function for these Hox genes is in controlling growth in the regions of the legs where T-rows are formed. In T1 legs, for example, the region between L-rows 7 and 8, in which the T-rows are found, is larger than the corresponding region on T2 legs, implying that there is additional growth in this domain. Inconsistent with this hypothesis, however, is the observation that posterior compartment clones lacking Ubx function in the T3 basitarsus did not have a significant effect on basitarsal width (Shroff, 2007).
Another potential role for Scr and Ubx in patterning T-row bristles is to implement a mechanism for selection and organization of T-row mCs into transverse rows. The mechanisms through which the L-row and T-row bristles are selected and organized within their respective proneural fields are likely to differ substantially. The regular spacing of L-row mCs suggests that the L-row SOPs send inhibitory signals in all directions to establish their proper spacing. This is also suggested by the observation that in hairy mutant legs, Ac is expressed in four broad domains, similar to the broad T-row proneural fields, and the supernumerary mCs that are formed on hairy mutant legs are well spaced along the leg circumference. This would suggest that the lateral inhibitory signals are sent along both the leg circumference and P/D axis. Unlike the L-row bristles, the T-rows mCs are positioned directly adjacent to one another in straight regularly spaced transverse rows. How the T-row precursors are selected from a broad field of ac-expressing cells and are arranged in tandem in straight rows is not understood. Previous studies have implicated N and EGFR signaling in formation of organized T-rows. Although the current studies indicate that N-signaling is down-regulated in the T-row primordia, it is conceivable that N functions at later stages of T-row development to pattern the T-row bristles. For example, N might function to set the register and spacing of the T-rows. Also of interest is how the T-rows are aligned in tandem within the rows. It has been suggested that homophilic adhesion between mC SOPs might be involved in organizing T-row bristles. Hence, it is plausible that Hox genes regulate expression of genes involved in adhesion, N signaling and/or EGFR signaling. Investigation of the mechanisms of T-row SOP selection and organization will provide an opportunity to uncover a potential connection between Hox gene function and morphogenesis (Shroff, 2007).
The proposed function for Scr and Ubx in T-row patterning bears some similarity to that described for Ubx in generation of diverse trichome patterns among the T2 legs of various Drosophila species. It has been shown that late pupal expression of Ubx in the T2 femur primordia correlates with lack of trichome formation in different Drosophila species, implying that Ubx inhibits formation of these structures. This role for Ubx, which has been termed a 'micromanaging role' is analogous to the function described here for Scr and Ubx in directing formation of T-rows in specific domains of T1 and T3 legs. Hence, micromanaging functions for Hox genes in generating complex and detailed morphologies may be a general phenomenon (Shroff, 2007).
Another common theme that has emerged from studies of the mechanisms through which Hox genes generate morphological diversity is that, in many cases, Hox genes function to suppress specific developmental pathways. For example, in legs, Antennapedia functions to repress expression of genes that promote antennal development, and Ubx functions to prevent development of specific macrochaete bristles on T3 legs. Furthermore, Ubx is known to act at several levels of the wing patterning hierarchy to suppress wing development in the haltere disc and as mentioned, Ubx functions late in leg development to suppress trichome formation. Less well understood, in contrast, is how and whether Hox genes act positively to direct the formation of morphological novelties among homologous structures, e.g., the T-row bristles on T1 and T3 legs. Further analysis of the mechanisms involved in T-row specification and morphogenesis is likely to provide insight into this question (Shroff, 2007).
The GATA factor Pannier (Pnr) activates proneural expression through binding to a remote enhancer of the achaete-scute (ac-sc) complex. Chip associates both with Pnr and with the (Ac-Sc)-Daughterless heterodimer bound to the ac-sc promoters to give a proneural complex that facilitates enhancer-promoter communication during development. Using a yeast two-hybrid screening, Toutatis (Tou; see Teutates the supposed deified spirit of male tribal unity in ancient Celtic polytheism, best known under the name Toutatis, through the Gaulish catchphrase "By Toutatis!", invented for the Asterix comics by Goscinni and Uderzo), which physically interacts with both Pnr and Chip, was identified. Loss-of-function and gain-of-function experiments indicate that Tou cooperates with Pnr and Chip during neural development. Tou shares functional domains with chromatin remodelling proteins, including TIP5 (termination factor TTFI-interacting protein 5) of NoRC (nucleolar remodelling complex), which mediates repression of RNA polymerase 1 transcription. In contrast, Tou acts positively to activate proneural gene expression. Moreover, Iswi associates with Tou, Pnr and Chip, and is also required during Pnr-driven neural development. The results suggest that Tou and Iswi may belong to a complex that directly regulates the activity of Pnr and Chip during enhancer-promoter communication, possibly through chromatin remodelling (Vanolst, 2005).
Transcriptional activation of many developmentally regulated genes is mediated by proteins binding to enhancers scattered over the genome, raising the question on how long-range activation is restricted to the relevant target promoter. Numerous studies have highlighted the essential role of boundaries, which maintain domains independent of their surrounding (Vanolst, 2005).
The patterning of the large sensory bristles (macrochaetae) on the thorax of Drosophila melanogaster is a powerful model to study how enhancers communicate with promoters during regulation of gene expression. Each macrochaeta derives from a precursor cell selected from a group of equivalent ac-sc-expressing cells, the proneural cluster. ac and sc encode basic helix-loop-helix proteins (bHLH) that heterodimerize with Daughterless (Da) to activate expression of downstream genes required for neural fate. Transcription of ac and sc in the different sites of the imaginal disc is initiated by enhancers of the ac-sc complex and the expression is maintained throughout development by autoregulation mediated by the (Ac-Sc)-Da heterodimers binding to E boxes within the ac-sc promoters. Each enhancer interacts with specific transcription factors that are expressed in broader domains than the proneural clusters and define the bristle prepattern. Thus, the GATA factor Pannier (Pnr) binds to the dorsocentral (DC) enhancer and activates proneural expression to promote development of DC sensory organs. The Drosophila LIM-domain-binding protein 1 (Ldb1), Chip physically interacts both with Pnr and the (Ac-Sc)-Da heterodimer to give a multiprotein proneural complex which facilitates the enhancer-promoter communication (Vanolst, 2005 and references therein).
Chromatin plays a crucial role in control of eukaryotic gene expression and is a highly dynamic structure at promoters. In Drosophila, the polycomb (Pc) group and the trithorax (Trx) group proteins are chromatin components that maintain stable states of gene expression and are involved in various complexes. The Pc group proteins are required to maintain repression of homeotic genes such as Ultrabithorax, presumably by inducing a repressive chromatin structure. Members of the Trx group were identified by their ability to suppress dominant Polycomb phenotypes. Evidence has been provided that enhancer-promoter communication during Pnr-driven proneural development is negatively regulated by the Brahma (Brm) chromatin remodelling complex, homologous to the yeast SWI/SNF complex (Vanolst, 2005).
Evidence is presented that Toutatis (Tou), a protein that associates both with Pnr and Chip and that positively regulates activity of the proneural complex encompassing Pnr and Chip during enhancer-promoter communication. Tou has been previously identified in a genetic screen for dominant modifiers of the extra-sex-combs phenotype displayed by mutant of polyhomeotic (ph), a member of the Pc group in Drosophila. Tou shares functional domains with Acf1, a subunit of both the human and Drosophila ACF (ATP-utilizing chromatin assembly and remodelling factor) and CHRAC (chromatin accessibility complex), and with TIP5 of NoRC (nucleolar remodelling complex). Hence, Tou regulates activity of the proneural complex during enhancer-promoter communication, possibly through chromatin remodelling. Moreover, Iswi, a highly conserved member of the SWI2/SNF2 family of ATPases, is also necessary for activation of ac-sc and neural development. Since Iswi is shown to physically interact with Tou, Pnr and Chip, it is suggested that a complex encompassing Tou and Iswi directly regulates activity of the proneural complex during enhancer-promoter communication, possibly through chromatin remodelling (Vanolst, 2005).
In Drosophila, Chip has been postulated to be a facilitator required both for activity of the DC enhancer of the ac-sc complex. Enhancer-promoter communication at the ac-sc complex is negatively regulated by the Brm complex whose activity is targeted to the ac-sc promoter sequences through dimerization of the Osa subunit with both Pnr and Chip. The Brm complex is thought to remodel chromatin in a way that represses transcription (Vanolst, 2005).
Tou and Iswi appear to act together as subunits of a multiprotein complex to positively regulate activity of Pnr and Chip during enhancer-promoter communication. Tou and Iswi therefore display opposite activity to that of the Brm complex, raising questions about their molecular function during neural development. Tou shares essential functional domains with members of the WAL family of chromatin remodelling proteins, including Acf1 of ACF and CHRAC. Importantly, Acf1 and TIP5 associate in vivo with Iswi, showing that Iswi can mediate both activation and repression of gene expression. Tou positively regulates Pnr/Chip function during the period of ac-sc expression in neural development, and it associates with Iswi. Since Iswi also positively regulates Pnr/Chip function, it is hypothesized that a complex encompassing Tou and Iswi acts during long-range activation of proneural expression, possibly through chromatin remodelling. Further studies will help to resolve this issue (Vanolst, 2005).
Interestingly, Chip and Pnr seem to play similar roles both during recruitment of the Brm complex and recruitment of Tou and Iswi, since they dimerize with Osa, Tou and Iswi. In addition, Pnr and Chip apparently cooperate to strengthen the physical association with Osa and Tou. However, Osa, on the one hand, and Tou and Iswi, on the other, display antagonistic activities during neural development. Since they are ubiquitously expressed, accurate regulation of ac-sc expression would require a strict control of the stoichiometry between Osa, Tou and Iswi. It remains to be investigated whether the functional antagonism between Osa and Tou/Iswi relies on a molecular competition for association with Pnr and Chip. Determination of this would require a complete molecular definition of the putative complex encompassing Tou and Iswi, together with a full understanding of how this complex and the Brm complex molecularly interact with the proneural complex to regulate enhancer-promoter communication during development (Vanolst, 2005).
Biochemical analysis of Iswi and Iswi-containing complexes, together with genetic studies of Iswi and associated proteins in flies and in budding yeast, has revealed roles for Iswi in a wide variety of nuclear processes, including transcriptional regulation, chromosome organization and DNA replication. Accordingly, Iswi was found to be a subunit of various complexes, including NURF (nucleosome remodelling factor), ACF and CHRAC. Iswi-containing complexes were primarily recognized as factors that facilitate in vitro transcription from chromatin templates. However, genetic analysis in Drosophila and in Saccharomyces cerevisiae have provided evidence that Iswi-containing complexes are involved in both transcriptional activation and repression in vivo. For example, immunostaining of Drosophila polytene chromosomes of salivary glands showed that Iswi is associated with hundreds of euchromatic sites in a pattern that is non-overlapping with RNA polymerase II. It suggests that Iswi may play a general role in transcriptional repression. In contrast, it was also demonstrated that expression of engrailed and Ultrabithorax are severely compromised in Iswi-mutant Drosophila larvae. Recent studies have also shown that a mouse Iswi-containing complex, NoRC, plays an essential role during repression of transcription of the rDNA locus by RNA polymerase I. Tou, a protein that is structurally related to the TIP5 subunit of NoRC. Tou positively regulates enhancer-promoter communication during Pnr-driven proneural development and its activity is targeted to the ac-sc promoter sequences through dimerization with Pnr and Chip. Evidence is provided that Iswi is required during neural development. Overexpression of IswiK159R in the precursor cells of the sensory organs using the scaGal4 driver leads to flies lacking multiple bristles, suggesting that Iswi functions late during neural development, essential for either cell viability or division of the precursor cell. Using the Iswi1/Iswi2 transheterozygous combination and individuals overexpressing IswiK159R in earlier stages of development and in less restricted patterns, it has been shown that Iswi also regulates ac-sc expression. Interestingly, the regulation is probably direct since Iswi associates with the transcription factors Pnr and Chip, known to promote ac-sc expression at the DC site. Since Iswi interacts with Tou, it is proposed that Tou and Iswi may positively regulate activity of Pnr and Chip during enhancer-promoter communication, possibly as subunits of a multiprotein complex involved in chromatin remodelling (Vanolst, 2005).
pannier encodes a GATA transcription factor that is involved in various biological processes, including heart development, dorsal closure during embryogenesis as well as neurogenesis and regulation of wingless (wg) expression during imaginal development. This study demonstrates that pnr encodes two highly related isoforms that share functional domains but are differentially expressed during development. Moreover, two genomic regions of the pnr locus are described that drive expression of a reporter in transgenic flies in patterns that recapitulate essential features of the expression of the isoforms, suggesting that these regions encompass crucial regulatory elements. These elements contain, in particular, sequences mediating regulation of expression by Decapentaplegic (Dpp) signaling, during both embryogenesis and imaginal development. Analysis of pnr alleles reveals that the isoforms differentially regulate expression of both wg and proneural achaete/scute (as/sc) targets during imaginal development. Pnr function has been demonstrated to be necessary both for activation of wg and, together with U-shaped (Ush), for its repression in the dorsal-most region of the presumptive notum. Expression of the isoforms define distinct longitudinal domains and, in this regard, it is shown that the dual function of pnr during regulation of wg is achieved by one isoform repressing expression of the morphogen in the dorsal-most region of the disc while the other laterally promotes activation of the notal wg expression. This study provides novel insights into pnr function during Drosophila development and extends the knowledge of the roles of prepattern factors during thorax patterning (Fromental-Ramain, 2008).
Focus was placed on reporter expression in the wing disc where pnr is necessary for the development of thoracic macrochaetae. A DNA fragment, 15.7 kilobases (kb) in length and including the 5′ untranslated sequences of exon 1 (construct A15.7), directs expression of lacZ in the dorsal-most domain of the disc. The 15.7 kb DNA fragment was dissected by 5′-end deletion, it was observed that the genomic sequences contain two distinct regions responsible for reporter expression. The 3.2 kb DNA fragment adjacent to pnr (construct E3.2) drives expression of the reporter along the A/P border of the notal region of the disc where Dpp is expressed, and also in a central cluster of cells. Expression remains similar in lines carrying the reporter under the control of the 9.3 kb fragment (construct C9.3), suggesting that the supplementary 6.1 kb DNA fragment does not contain essential regulatory sequences. When the DNA fragment inserted upstream of the reporter is the 12 kb fragment (construct B12), lacZ expression is reinforced in comparison of expression seen with lines carrying construct C9.3. Expression of the reporter fully covers dorsal domain of the disc when the promoter sequences include the distal DNA fragment (construct A15.7). Thus, a second domain responsible for expression in the disc appears to be located in the distal region of construct A15.7 (Fromental-Ramain, 2008).
It is concluded that reporter expression depends on activity of two domains, a proximal one located in the 3.2 kb fragment adjacent to pnr (construct E3.2) and a distal one corresponding to the 5′-end of construct A15.7. These observations are reinforced by the fact that both the distal fragment (construct H6.4) and the proximal fragment (construct J1.8) inserted in front of an heterologous hsp43 (heat shock protein43) minimal promoter direct reporter expression in the wing disc. In contrast, the intervening fragment (construct I6.1) does not promote expression when placed in front of this heterologous promoter (Fromental-Ramain, 2008).
Interestingly, the location of the two domains suggests that they may correspond to alternate promoters of the pnr isoforms. Indeed, sequence analysis of the pnr locus and characterization of the mRNAs expressed during development led to the prediction that pnr may encode two isoforms. Isoform-α (pnr-α) encodes the Pnr protein as it has been identified, whereas the putative isoform-β (pnr-β) encodes a truncated version of the Pnr-α protein, lacking the 52 N-terminal amino acids. However, Pnr-α and Pnr-β share functional domains and the N-terminus of Pnr-α does not contain any obvious functional signature. In vitro experiments revealed that both Pnr-α and -β associate with Ush and equivalently activate a reporter driven by promoter sequences including GATA sites in a cultured cell line (Fromental-Ramain, 2008).
Several reports have implicated Pnr as a key transcriptional regulator during expression of both ac/sc and wg in the presumptive notum. The current study extends previous work and importantly demonstrates that Pnr function is achieved by two structurally related isoforms with distinct expression domains. Moreover, the isoforms display distinct transcriptional activities, including antagonism during regulation of wg expression (Fromental-Ramain, 2008).
Identification of the isoforms led to revisiting the role of pnr during regulation of ac/sc and ush targets in the wing disc. Both overexpressed pnr-α and overexpressed pnr-β lead to activation of proneural expression and development of ectopic sensory bristles suggesting that the isoforms may act as subunits of the multiprotein proneural complex as it has been previously identified. However, the current analysis of the pnrV1 and pnrGal4 alleles do not argue in favor of such a model during regulation of ac/sc expression. Both the reduced pnr-β expression associated with homozygous pnrGal4 animals and the increased pnr-α expression observed in homozygous pnrV1 animals exhibit a loss of DC bristles and impaired proneural expression at the DC site of the wing/thorax discs. As the domains of isoform expression stay the same in mutants animals, this suggest that the mutant phenotypes result from antagonistic activities of the Pnr proteins. This hypothesis is reinforced by the observation that proneural expression is reduced in both (pnrGal4/+) and (pnrV1/+) animals and is totally abolished in homozygous mutant animals. Thus, proneural expression at the DC site of the imaginal disc relies on the stoichiometry between Pnr-α/Pnr-β. Additional evidence is provided by molecular analysis of the vertebrate complex, homologous to the proneural complex encompassing Pnr, Chip and the heterodimer (Ac/Sc)-Da. Indeed, the vertebrate hematopoietic-specific complex contains only one GATA molecule, that does not support the notion that both the Pnr-α and Pnr-β isoforms simultaneously belong to the Drosophila complex necessary for ac/sc activation during Pnr-driven proneural development (Fromental-Ramain, 2008).
Previous analysis have shown that ush expression is also regulated by Pnr. ush expression is abolished in the dorsal-most domain of (pnrVX6/pnrV1) disc. Since the (pnrVX6/pnrV1) combination was predicted to correspond to a loss of pnr function, it was postulated that Pnr mediates activation of notal ush expression. It has also been reported that ush expression is lost in (pnrD1/pnrV1) disc, except at the A/P border of the notal region. Since pnrD1 encodes a mutant protein carrying a single amino acid exchange in the DNA binding domain that disrupts interaction with the negative regulator Ush, it was hypothesized that the (Pnr-Ush) complex serves as a transcriptional activator of ush expression. However, the current analysis revealed a strong induction of pnr-α expression at the A/P border of the disc while pnr-β expression is not modified. Hence, expression of the (PnrD1-α) protein is induced at the A/P border in (pnrD1/pnrV1) discs and it is suggested that Pnr-α-Ush is involved in the repression of ush expression. Moreover, it is also suggested that the ush expression depends on the stoichiometry between Pnr-α and Pnr-β since ush expression is abolished in the dorsal-most domain of the pnrD1/pnrV1 discs outside the A/P border. The pnrV1/pnrD1 combination is consequently characterized by ectopic sensory bristles and increased proneural expression in the DC area (Fromental-Ramain, 2008).
Pnr is involved in regulation of both the ac/sc and ush targets during neural development and the stoichiometry of the isoforms is a crucial determinant during regulation of gene expression. These characteristics may explain the paradoxical observations that increased pnr-α expression in homozygous pnrV1 displays reduced ac/sc expression and loss of DC bristles whereas overexpressed pnr-α in (pnrGal4/UAS pnr α) leads to activated ac/sc expression and additional macrochaetae. The DC enhancer would require lower Pnr-α concentration for repression than the notal ush enhancer, probably reflecting different affinities of the binding sites for the Pnr-α-Ush effector. At low concentration, the Pnr-α-Ush heterodimer antagonizes Pnr-β activity, leading to reduced ac/sc expression at the DC site and loss of DC bristles. Overexpressed pnr-α mediates repression of ush, leading to reduced concentration of the Pnr-α-Ush heterodimer and consequently, ac/sc expression at the DC site results from activating Pnr-β. Hence, overexpressed pnr-α displays ectopic sensory organs. In contrast, overexpressed pnr-β would repress pnr-α involved together with Ush in repression of ac/sc and would also directly activate proneural expression to promote development of ectopic sensory organs. Both overexpressed pnr-α or pnr-β activates proneural expression, leading to ectopic sensory organs but they act by distinct mechanisms. During activation of proneural expression, overexpressed pnr-β appears to directly stimulate ac/sc through binding to their regulatory sequences whereas overexpressed pnr-α indirectly acts in repressing ush expression (Fromental-Ramain, 2008).
The present data highlight the merit of revisiting pnr function during development since pnr isoforms are expressed in domains that define a novel subdivision of the wing disc. The biological significance of the subdivision is of critical importance since the isoforms exhibit antagonistic activities during regulation of targets genes. A challenging issue will be to understand how the Pnr isoforms molecularly interact with the regulatory sequences of the target genes ac/sc, ush and wg. Sequence analysis revealed that the DC enhancer contains several Pnr binding sites and some of them are involved in regulation of ac/sc expression during neural development (Garcia-Garcia, 1999). These binding sites may correspond to targets for Pnr-β and (Pnr-α)-Ush complexes. Mutagenesis of the Pnr binding sites would be required to understand how the isoforms interact with the regulatory element to antagonistically regulate proneural expression, to clarify the role of Ush during regulation of Pnr target genes, and to resolve the question on how upon dimerization Ush can convert Pnr from an activator to a repressor (Fromental-Ramain, 2008).
The Drosophila bHLH proneural factors Achaete (Ac) and Scute (Sc) are expressed in clusters of cells (proneural clusters), providing the cells with the potential to develop a neural fate. Mediodorsal proneural patterning is mediated through the GATA transcription factor Pannier (Pnr) that activates ac/sc directly through binding to the dorsocentral (DC) enhancer of ac/sc. Besides, the Gfi transcription factor Senseless (Sens), a target of Ac/Sc, synergizes with ac/sc in the presumptive sensory organ precursors (SOPs). This study investigated, through new genetic tools, the function of dLMO, the Drosophila LIM only transcription factor that was already known to control wing development. dLMO gene encodes two isoforms, dLMO-RA and dLMO-RB. dLMO null and dLMO-RA− deletions have similar phenotypes, lacking thoracic and wing margin sensory organs (SO), while dLMO-RB− deletion has normal SOs. At early stages, dLMO-RA is expressed in proneural clusters, however later it is excluded from the SOPs. dLMO functions as a Pnr coactivator to promote ac/sc expression. In the late SOPs, where dLMO-PA is not expressed, Pnr participates to the Sens-dependent regulation of ac/sc. Taken together these results suggest that dLMO-PA is the major isoform that is required for early activation of ac/sc expression (Asmar, 2008).
The lack of dLMO protein leads to very distinctive phenotypes. The mutant animals are not able to fly, they have a short life span and show an abnormal gait behaviour. In addition, they show a discreet bristle phenotype. In Drosophila, there are two paralogous LMO factors, dLMO and CG5708. These genes are expressed in the CNS where redundancy is not excluded. However CG5708 is not expressed in the wing discs and presumptive SOPs. Therefore it is concluded that the mild phenotype observed for the adult PNS in dLMO mutants, is not attributed to gene redundancy. dLMO encodes two distinct isoforms, dLMO-PA and -PB, which only differ from their N-terminus. Only dLMO-RA is broadly expressed in the notum, and contributes to the PNS phenotype. dLMO function is also critical in the developing central nervous system for the activity of the ventral lateral neurons, LNvs. It is highly probable that dLMO-RB has some subtle biological activities in the brain, where it has a specific pattern (Asmar, 2008).
In vertebrate, multiproteic complexes composed by GATA-1, LMO2, Ldb-1 and the bHLHs E47 and SCL, are required for normal differentiation of haematopoietic cells. The current results highlight several evidences in favour of dLMO as a GATA coactivator in Drosophila . (1) A genetic synergism exists between pnr− and dLMO− null alleles. (2) dLMO modulates the activity of a DC:ac-lacZ reporter, the model target of Pnr, in vivo. Loss of function dLMO mutants show reduced level of the DC:ac-lacZ expression, whereas in gain-of-function dLMO mutants the DC:ac-lacZ expression is increased. (3) dLMO-PA isoform directly interact with Pnr in GST pull down assay. Therefore it is concluded that dLMO might enhance the proneural activity of Pnr through direct interaction with the GATA factor. Consistently, dLMO expression overlaps with the dorsal-most domain of Pnr during third instar larval stages. Though Pnr controls the development of both DC and SC bristles, dLMO null alleles affect only DC bristles. dLMO expression, that overlaps both SC and DC proneural clusters in the notum, is significantly weaker in the SC region, suggesting that regulation of proneural ac/sc expression is differentially sensitive and responds to local combinations of transcription factors. These data support previously published studies demonstrating that the proneural activity of Pnr is prominently repressed in the SC region by the LIM-HD transcription factor Isl (Asmar, 2008).
At later stages, dLMO expression is excluded from the corresponding SOP and its derivative cells. In contrast, the proneural factor Sens, that plays an important role for sensory organ specification, is first broadly expressed in proneural clusters at low levels where it functions as a repressor of ac/sc, and then later, is expressed at high levels in the presumptive SOPs, where it acts as a transcriptional activator that directly interacts and synergizes with the proneural proteins, Ac and Sc. It has been shown that both Gfi-1 and GATA-1, the mammalian ortholog of Sens and Pnr respectively, are essential for development of the closed related erythroid and megakaryocytic lineages. The Sens/Pnr interaction is evolutionary conserved in Drosophila neurogenesis. It is suggestd that Pnr could participate to the Sens-dependent positive autoregulation of Ac/Sc in late SOPs where dLMO is not expressed. The synergism between Pnr and Sens would need more detailed investigations. Taken together, these studies have shown dLMO-PA as a co-activator for Pnr during the establishment of proneural fields and revealed another level of proneural ac/sc regulation during late neurogenesis in the Drosophila PNS (Asmar, 2008).
Hox genes control regional identity along the anterior-posterior axis in various animals. Each region contains morphological characteristics specific to that region as well as some that are shared by several different regions. The mechanism by which one Hox gene regulates region-specific characteristics has been extensively analyzed. However, little attention has been paid to the mechanism by which different Hox genes regulate the same characteristics in different regions. This study shows that two Hox genes in Drosophila, Sex combs reduced and Ultrabithorax, employ different mechanisms to achieve the same out-put, the absence of sternopleural bristles, in the prothorax and metathorax, respectively. Sternopleural bristles are characteristics of the mesothorax, and it was found that spineless is involved in their development. Analysis of the regulatory relationship between Hox genes and spineless indicated that ss expression is repressed by Sex combs reduced in the prothorax. Since sole misexpression of ss could induce ectopic sternopleural bristle formation in the prothorax irrespective of the expression of Sex combs reduced, spineless repression appears to be critical for inhibition of sternopleural bristles by Sex combs reduced. In contrast, spineless is expressed in the metathorax independently of Ultrabithorax activity, indicating that Ultrabithorax blocks sternopleural bristle formation through mechanisms other than spineless repression. This finding indicates that the same characteristics can be achieved in different segments by different Hox genes acting in different ways (Tsubota, 2008).
This study found that three genes, Antp, ss and al, are involved in sternopleural bristle formation. In the al mutant, no appreciable Ac expression in the T2 leg disc is detected and sternopleural bristles are not formed, indicating that the requirement of al is absolute. In contrast, Ac expression is detectable in the ss mutant T2 leg disc and in the Antp mutant clones, indicating that the requirement of both ss and Antp for ac expression is not absolute. However, sternopleural bristles were never found in the ss mutant, despite the fact that Antp expression was unaffected in the ss mutant clone in the T2 leg disc. In contrast, Antp mutant cells, in which ss is expressed normally, formed sternopleural bristles. In addition, sole misexpression of ss in the T1 segment produces sternopleural bristles ectopically, while that of Antp did not. Therefore, ss appears to be necessary and sufficient for sternopleural bristle formation, while Antp appears to be insufficient and not necessarily required. Moreover, Ac expression is ectopically induced in the T1 leg disc by misexpression of ss but not of Antp and in the ss mutant T2 leg disc is very weak, highly restricted, and only transient. This indicates that ss but not Antp appears to be one of the major activators of ac expression. Taken together, ss appears to be much more fundamental for sternopleural bristle formation than Antp (Tsubota, 2008).
The initiation of ac expression coincides with the initiation of ss expression. Since al and Antp are already expressed before ac induction in the early third instar stage, the timing of ac induction may be determined by the regulation of ss expression. Interestingly, the residual Ac expression seen in the ss mutant leg disc is first observed in the mid third instar as in the wild-type leg disc. This implies that at least one additional gene (referred to as X hereafter), whose expression or function is activated at the same stage as the initiation of ss expression, may be involved in ac induction. One possibility may be a gene functioning in hormonal regulation. Nonetheless, the ability of the sole misexpression of ss to induce ectopic ac expression and sternopleural bristle formation strongly indicates that ss is much more fundamental than X (Tsubota, 2008).
The restriction of ac expression to the overlap between the ss and al expression domains indicates the importance of determining the distal limit of ss expression and the proximal limit of al expression. Analysis of clones lacking ss activity or misexpressing ss indicates that ss has a repressive activity on al expression. How can al be expressed in the overlap domain? In the overlap domain, ss represses al expression when misexpressed at high levels but does not when misexpressed at approximately endogenous levels. The level of ectopic Al expression in the ss mutant clone located in a region proximal to the normal al expression domain is lower than that of endogenous Al expression. Moreover, Al expression in the wild-type leg disc gradually decays at its proximal edges. Considering all of these observations, the following hypothesis is suggested: al expression is activated according to the proximodistal information and the proximal limit of the al expression domain may be determined by a balance between activation according to the proximodistal information and repression by ss. The activation force may dominate the repressive activity of ss in the overlapping region but may gradually decay towards the proximal edges of the al expression domain. In contrast, ss expression does not appear to be regulated by al. As with the case of al activation, it may be possible that ss is repressed according to the proximodistal information (Tsubota, 2008).
The morphological identities of the T1 and T3 segments, including the absence of sternopleural bristles, are determined by Scr and Ubx, respectively. Analyses of the T1 leg disc with Scr mutant clones and the T2 leg disc with ectopic Scr activity indicate that both ss and Antp are repressed by Scr in the T1 leg disc. In addition, there is a possibility that the expression or function of gene X is repressed by Scr. Weak Ac expression is transiently observed in the ss mutant T2 leg disc, indicating that ac expression can be weakly activated without ss activity in the presence of gene X and Antp activity. In addition, Scr does not appear to repress ac expression directly, since ectopic induction of ac by ss misexpression in the T1 leg disc was not associated with an alteration in Scr expression. If gene X is active in the T1 leg disc, sole misexpression of Antp is expected to activate ac expression at least weakly and transiently. However, no ectopic Ac expression was found upon sole misexpression of Antp. Therefore, the activity of gene X is likely to be repressed in the T1 leg disc. For evaluating the significance of these three genes on Scr-dependent inhibition of sternopleural bristle formation, the ability of ss misexpression to induce ectopic ac expression and sternopleural bristle formation without affecting Scr expression is of crucial importance. At present, whether ac expression and sternopleural bristle formation can be induced solely by ss or only in a combination of ss and Antp and/or gene X is unclear. However, ss misexpression induced Antp expression and, thus, at least ss and Antp were coexpressed upon sole misexpression of ss. As for gene X, if it is not activated by ss misexpression, the results indicate that ac expression and sternopleural bristle formation can be induced without gene X activity at least in the presence of both ss and Antp expression. In contrast, if ac expression and sternopleural bristle formation require gene X activity, ss misexpression must activate gene X. After all, the results indicate that sole misexpression of ss can fulfill at least a minimum requirement for ac expression and sternopleural bristle formation. In other words, if Scr could not repress ss expression, ac expression would be activated and sternopleural bristles would be formed irrespective of the expression and function of Antp and gene X. Therefore, Scr must repress ss expression and this appears to be a key step to block sternopleural bristle formation in the T1 segment (Tsubota, 2008).
In contrast to the T1 leg disc, strong Ss expression was observed in the wild-type T3 leg disc and it is unaltered in Ubx mutant clones. Therefore, Ubx appears to act through a mechanism unrelated to ss expression. How does Ubx function? Simultaneous expression of both ss and Antp seemed insufficient for ac expression and sternopleural bristle formation in the T3 segment, since Antp misexpression failed to induce Ac expression in the T3 leg disc, in which ss is prominently expressed. It may be possible that Ubx represses ac expression directly. Alternatively, Ubx may compromise the function of the Ss protein directly or indirectly through regulation of its downstream gene products. Another possibility is that Ubx acts through repression of gene X activity. These possibilities are not mutually exclusive with each other (Tsubota, 2008).
The occurrence of ac expression and sternopleural bristle formation in the absence of Antp activity indicates that the absence of sternopleural bristles is not the ground state. However, the number of sternopleural bristles is variable in that condition, indicating that the complete formation of sternopleural bristles is not also the ground state. Since ss misexpression experiment suggests that sternopleural bristles can be formed as long as ss is expressed, one possible aspect of the ground state may be the expression of ss and the production of at least some kind of bristles. Antp may have acquired the ability to modify this state to produce the current-type of sternopleural bristles. On the other hand, Scr may have evolved the ability to block sternopleural bristle formation by acquiring the activity to repress ss expression and Ubx by acquiring another, yet unknown function. Taken together, the current state of sternopleural bristles in all three thoracic segments appears to be the derived state (Tsubota, 2008).
The Insulin Receptor (InR) in Drosophila presents features conserved in its mammalian counterparts. InR is required for growth; it is expressed in the central and embryonic nervous system and modulates the time of differentiation of the eye photoreceptor without altering cell fate. This study shows that the InR is required for the formation of the peripheral nervous system during larval development and more particularly for the formation of sensory organ precursors (SOPs) on the fly notum and scutellum. SOPs arise in the proneural cluster that expresses high levels of the proneural proteins Achaete (Ac) and Scute (Sc). The other cells will become epidermis due to lateral inhibition induced by the Notch (N) receptor signal that prevents its neighbors from adopting a neural fate. In addition, misexpression of the InR or of other components of the pathway (PTEN, Akt, FOXO) induces the development of an abnormal number of macrochaetes, which are Drosophila mechanoreceptors. These data suggest that InR regulates the neural genes ac, sc and sens. The FOXO transcription factor, which becomes localized in the cytoplasm upon insulin uptake, displays strong genetic interaction with the InR and is involved in Ac regulation. The genetic interactions between the epidermal growth factor receptor (EGFR), Ras and InR/FOXO suggest that these proteins cooperate to induce neural gene expression. Moreover, InR/FOXO is probably involved in the lateral inhibition process, since genetic interactions with N are highly significant. These results show that the InR can alter cell fate, independently of its function in cell growth and proliferation (Dutrieux, 2013).
A model is proposed in which the InR receptor plays a role in the development of the peripheral nervous system mainly through FOXO cell localization independently of its role in proliferation and apoptosis. The role of the InR/FOXO pathway appears early in PNS development before SOP formation. The use of different mutants involved in growth indicates that the TOR pathway does not play a major role in the phenotypes observed. The results using genetic and molecular methods strongly suggest that InR/FOXO controls the level of proneuronal genes such as ac, sc and Sens early in PNS development. This explains the interaction observed with N55e11 (Dutrieux, 2013).
Several arguments indicate that the phenotypes observed when InR is overexpressed are not due, at least for the most part, to proliferation, growth or lack of apoptosis. First using anti-PH3 staining that allows to visualize mitotic cells, no extra mitoses are observed in the clusterOverexpression of genes such as dE2F1, or dacapo did not lead to a significant increase or decrease in the number of macrochaetes. In addition co-expression of these genes with InR indicates no interaction. Moreover, the effects of InR and FOXO when overexpressed on respectively the increase and the decrease in cell number, could be estimated by the number of Ac-positive cells in the DC and SC clusters. No significant differences were observed between the control and the overexpressed strain (either InR or FOXO) in the number of cells positive for Ac. If the possibility that proliferation is somehow involved in cluster size cannot be discarded, it does not account for the effects observed since the ratio of Sens-positive cells when InR is overexpressed over the control strain is much higher than the ratio of Ac-positive cells. A similar role for FOXO in apoptosis could also be discarded on the same basis. No clear interactions were observed between FOXO and genes involved in inhibition of apoptosis like diap1 (Dutrieux, 2013).
Along the same line it has been shown that the InR/TOR pathway plays a role in controlling the time of neural differentiation. This has been observed in photoreceptor formation but also in the chordotonal organs of the leg that develop on the same basis as thoracic bristles. The dynamic formation of the SOPs, particularly after a block of InR signaling was undertaken. No differences were observed before the end third larval instar in the test and in the overexpressed strain. Only an increase in the number of positive Sens stained cells are observed in the sca>InR strain (Dutrieux, 2013).
Using Pros staining that marks pIIb cells, this study shows that staining appears in the late third instar larvae at the level of DC SOPs in sca>InR; this is not observed in the control strain. In addition in sca>FOXO RNAi wing discs it also leads to Pros staining. This indicates that the time of differentiation is advanced in the InR strain through the absence of nuclear FOXO. However it was verified that in very early third instar larvae the first scutellar SOP appears at the same time in the control and in the overexpressed strains and that no differences were observed in mid third instar (Dutrieux, 2013).
In addition the observations show that the increase in the number of macrochaetes in sca>InR is independent of the TOR pathway since none of the members induces a similar phenotype as does InR or interacts either with InR or FOXO in this process. However, some interactions were observed with raptor and Rheb that could be the consequence for the latter of its role in PIIa and PIIb formation regulating N (Dutrieux, 2013).
Are InR and FOXO acting on the same target in SOP formation? Several arguments are in favor of this possibility. First underexpression experiments (InR clones, InR RNAi or FOXO RNAi overexpression and FOXO homozygotes and even heterozygotes,) induce exactly opposite phenotypes. This is also true for overexpression experiments with InR and hFOXO3a-TM. Moreover overexpression of both transgenes leads to an intermediate phenotype, very different from the control phenotype. Finally, overexpression of InR in a heterozygote FOXO mutant background leads to an increase in the number of macrochaetes compared to InR alone. FOXO null flies are fully viable and do not usually display any phenotype. However an increase in the number of pDC and aSC macrochaetes is observed in some FOXO homozygotes and even heterozygotes that are nor observed in the control strain. This could indicate that FOXO function is in part dispensable. Even if the InR/FOXO double heterozygote is completely normal, the double null mutant InR/FOXO shows either an excess or a lack of macrochaetes, that is in favor of the hypothesis that InR acts through FOXO. FOXO null clones do not display any phenotype comparable to FOXO RNAi overexpression. However overexpression of InR in a FOXO null clone leads to stronger phenotypes than overexpression of InR alone in a clone. Yet, it cannot be excluded that part of the InR overexpression phenotype is not due to the absence of FOXO or its cytoplasmic retention (Dutrieux, 2013).
The absence of FOXO, using FOXO RNAi, or its retention in the cytoplasm by InR or Akt overexpression produces the same neurogenic phenotypes that are exactly the opposite when nuclear hFOXO3a-TM is overexpressed. In addition overexpression of both hFOXO3a-TM and InR leads to a decrease in the number of highly positive Ac and Sens expressing cells compared to overexpression of InR alone. Finally, overexpression of FOXO RNAi in dpp regulatory sequences, induces Ac expression. All these results should be explained by the same molecular process. One possibility would be that InR/FOXO regulates one or several neural genes involved in cluster formation and maintenance. The results are in favor of the hypothesis that genes of the Ac/Sc complex could be regulated by InR. Either InR via nuclear FOXO represses the Ac/Sc pathway, or FOXO activates a repressor of the pathway (Dutrieux, 2013).
Since it has been well established that InR induces cell proliferation, it remains possible that these functions could affect the size of the proneural clusters when the genes are overexpressed. However, when the number of the Ac-positive cells in the DC and SC clusters in the different genotypes was estimated, it was not significantly different (Dutrieux, 2013).
Several relevant arguments exist suggesting that InR is necessary for SOP formation and regulation of neural gene expression. (1) The phenotype of the overexpression experiments either with InR or with InR RNAi suggests that InR perturbs the normal pattern of singling out a cell in the proneural cluster that will become an SOP. The fact that the sensitive period occurs in the late second/beginning third instar is in accordance with this hypothesis. The phenotype of the InR null clones comfort this hypothesis. (2) When InR is overexpressed the level of Ac is significantly higher. This is confirmed by the IMARIS technique that estimated that in this genotype, the number of cells with the highest scores (106 and 107 units) is larger than in the control strain. These 'highly Ac-positive cells' seem to also be Sens positive cells indicating a correlation between the two events. (3) In sca>InR the level of Sens, measured by the IMARIS technique is higher than in the test raising the possibility that InR regulates several neural genes independently. However another possibility would be that this high Sens expression level would be indirectly due to the induction by InR of a Sens-positive regulator such as sc. (4) Several sc enhancers are regulated by InR, the sc promoter, and the SRV and DC enhancers. As sc is auto-regulated through its different enhancers, it is difficult to evaluate if a specific enhancer is involved although the effect on the 3.8 kb sc promoter is the most striking. For FOXO the absence of FOXO using the FOXO RNAi strain shows that Ac is induced. The double expression of InR and hFOXO3a-TM produces an intermediate phenotype and decreases the effects of InR, on Ac and Sens expression. The results using the sc enhancers when hFOXO3a-TM is overexpressed showed that only a decrease in the expression of the SRV enhancer is observed. However, the phenotypes observed in sca>hFOXO3a-TM agree with the hypothesis of repression of ac and sc by hFOXO3a-TM. As expected, overexpression of FOXO RNAi induces sc-lacZ enhancer. (5) Overexpression of both InR and sc leads to a significant increase in the effect of a single transgene. This indicates that both transgenes have a common target; one of them could be sc itself. An opposite effect is observed with constitutively active hFOXO3a-TM. This favors the model whereby InR and FOXO act in opposite ways on the sc target in SOP formation. (6) Highly significant genetic interactions are observed between sc and InR, and sc and FOXO. (7) Another gene charlatan (chn) which is both upstream and downstream of sc, strongly interacts genetically with InR (Dutrieux, 2013).
Lateral inhibition is determined by the activity of the N receptor. When N is mutated, cell fate changes and extra macrochaete singling appear. Using the N deletion (N55e11) to test possible genetic interaction with InR and with FOXO in heterozygote females, interaction was observed with the InR RNAi strain. Moreover strong interaction is observed with InR overexpression. This indicates that InR impairs lateral inhibition and cooperates with N in this process. In parallel, as for Inr overexpression, the absence of nuclear FOXO either using FOXO25 homozygotes (or even heterozygotes) or FOXO RNAi overexpression induces an increase in the neurogenic phenotype. With this latter strain, tufted microchaetes were observed, indicating that FOXO could also act later in development. Overexpression of hFOXO3a-TM displays highly significant interaction with N55e11 as the neurogenic phenotype is increased compared to overexpression in a wild type background. However, overexpression of InR RNAi in a N55e11 heterozygote background leads to a significant increase but only at the level of aSC, raising the possibility of a local interaction or appearing at a specific time for the different clusters (Dutrieux, 2013).
Moreover the fact that there is no differences when Suppressor of Hairless (Su(H)) which transduces the N pathway, is expressed with or without the InR, indicates that lateral inhibition is not affected. In addition in the InR strain, Sens stained cells were clearly individualized and separated from one another. These results clearly indicate that InR and FOXO act with N on the choice of the cell that will become an SOP (Dutrieux, 2013).
EGFR has also been implicated in macrochaete development. Indeed EGFR mutants and EGFR null clones display macrochaete phenotypes. This could be explained since in EGFR hypomorphic mutants the level of Sc is reduced in some clusters and increased in others suggesting a different requirement of EGFR for the different SOPs. If RasV12 was overexpressed with an ubiquitous driver, sc was ectopically expressed. Thus, Ac/Sc induction by Ras overrules lateral inhibition due to N. Moreover N downregulation enhances EGFR signaling. A model has been established of antagonist interaction between EGFR and N in which Ac/Sc activates both pathways that in turn act on the same SOP specific enhancers (Dutrieux, 2013).
Moreover, the InR/TOR pathway regulates the expression of some of the components of the EGFR signaling pathway such as argos, rhomboid and pointed. The results suggest that both the InR and the EGFR/Ras pathways induce sc in a synergic manner and this further overrules the lateral inhibition mechanism induced by N. The fact that overexpression of RasV12 in an InR null heterozygote background significantly lowers the phenotype observed with RasV12 only, is in agreement with this hypothesis. The interactions observed with the EGFR RNAi strain seem to be FOXO independent (Dutrieux, 2013).
Taken together these results show that InR and several components of the pathway such as PTEN, Akt and FOXO are involved in PNS development independently of their role in growth, proliferation and delay in the time of neural differentiation. The function of InR in PNS development seems to be independent of TOR/4E-BP. FOXO cytoplasmic retention either by InR activation or by the use of FOXO RNAi produces opposite phenotypes suggesting that nuclear FOXO could be a repressor of PNS development. These results using antibody staining and reporters of sc enhancers indicate that InR targets are the neural genes ac, sc and sens. However, as most of these neural genes display a complex co-regulation, it is difficult to demonstrate whether or not sc is the primary target of the pathway. A strong interaction is observed between the EGFR/Ras pathways and InR suggesting that both could act together to induce neural gene expression and this would explain the strong interaction observed between InR/FOXO and N (Dutrieux, 2013).
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