During development, the imaginal wing disc of Drosophila is subdivided into territories separated by developmental boundaries. The best characterized boundaries delimit compartments defined by cell-lineage restrictions. This study analyzes the formation of a boundary that does not rely on such restrictions, namely, that which separates the notum (body wall) and the wing hinge (appendage). It is known that the homeobox genes of the Iroquois complex (Iro-C) define the notum territory and that the distal limit of the Iro-C expression domain demarks the boundary between the notum and the wing hinge. However, it is unclear how this boundary is established and maintained. msh, a homeobox gene of the Msx family, is strongly expressed in the territory of the hinge contiguous to the Iro-C domain. Loss- and gain-of-function analyses show that msh maintains Iro-C repressed in the hinge, while Iro-C prevents high level expression of msh in the notum. Thus, a mutual repression between msh and Iro-C is essential to set the limit between the contiguous domains of expression of these genes and therefore to establish and/or maintain the boundary between body wall and wing. In addition, msh is found to be necessary for proper growth of the hinge territory and the differentiation of hinge structures. msh also participates in the patterning of the notum, where it is expressed at low levels (Villa-Cuesta, 2005).

msh is known to be involved in different processes. Thus, it participates in regional specification of muscle progenitors/founders; together with vnd and ind, it helps subdivide the embryonic neuroectoderm along the dorsoventral axis, and it confers dorsal identity to the dorsal bristles of the anterior margin of the wing. This study reports additional functions of msh, namely, the formation/maintenance of the subdivision between the territories of the wing disc that will give rise to the notum (dorsal mesothoracic trunk) and the dorsal hinge (appendix), the proper growth of the dorsal hinge, and the patterning of this region and of the notum (Villa-Cuesta, 2005).

In the developing wing disc, msh is expressed most strongly in the territory of the dorsal hinge, the region between the notum and the dorsal wing blade territories. Removal of msh in clones results in malformations that range from small defects, such as an outheld wing, to partial or even complete loss of most hinge structures. In the latter cases, the hinge may be posteriorly misplaced and ectopically attached to the scutellum. In addition, in a fraction of flies ectopic notum tissue appears contiguous to the extant hinge. Because at least a large part of the hinge tissue is still present, it is surmised that the absence of recognizable hinge structures is due to the failure of their proper differentiation. This phenotype correlates well with that observed in third instar wing discs displaying msh^- clones. Indeed, even large clones that remove msh from most of the dorsal hinge territory allow the specification of this territory, as demonstrated by the relatively unmodified characteristic patterns of expression of genes such as wg, zfh-2, hth and tsh, and the presence of recognizable proneural clusters of sc expression. Moreover, the presence in mutant hinges of relatively well resolved clusters of sc expression indicate that the prepatterning of the hinge can proceed to a large extent in the absence of msh. It is concluded that msh is largely dispensable for specification of the dorsal hinge territory, but it is required for the final stages of its patterning and differentiation (Villa-Cuesta, 2005).

Mosaic analyses aimed at studying the patterns of cell proliferation in the wing disc have disclosed the presence of the anterior, posterior, dorsal and ventral compartments of the wing with borders that imposed absolute restrictions to cell proliferation. A border of this type has been suggested to exist between the notum and dorsal hinge, as well as between the pleura and the ventral hinge, but the complex morphology of these regions and the unavailability of appropriate cuticular markers made the proposal uncertain. In fact, analyses performed in the wing disc, has shown that clones can straddle the notum/dorsal hinge boundary, this being defined by the distal border of the Iro-C domain. Hence, at this boundary, the descendants of a cell adopt their developmental fate not according to lineage, but depending on the side of the boundary they were located. The issue thus arises of how the boundary between the notum and the dorsal hinge territories is established and maintained. Considering that the extent of the notum territory is defined by the expression of the Iro-C, this issue can be largely resolved by explaining how the distal border of the Iro-C domain of expression is defined (Villa-Cuesta, 2005).

So far, several genetic interactions have been identified that together permit to suggest a mechanism that partially answers this question. In the second instar disc, the EGFR pathway activates ap and Iro-C. The distinct but overlapping domains of expression of these genes, the dorsal compartment (ap) and the notum territory (Iro-C), may be defined by differential sensitivity to EGFR signaling or, alternatively, in the case of Iro-C, by Dpp signaling. In these early stages, Dpp signaling is active only in the distal part of the disc, where it represses the Iro-C and sets its distal limit of expression. Hence the antagonistic actions of the EGFR and the Dpp pathways define the position of the distal limit of the Iro-C domain, and therefore the position of the notum/hinge subdivision (Villa-Cuesta, 2005).

At approximately the time Iro-C starts to be expressed in the more proximal part of the disc, i.e., that which will become the notum, expression of msh (by means of ap) is turned on in the adjacent dorsal hinge territory. These essentially complementary patterns of expression are maintained, with some qualifications, in the third instar disc. Loss- and gain-of-function experiments show that msh prevents ara/caup from being expressed in the hinge, and ara/caup restrain msh from being expressed in the notum at the high levels typical of the hinge (although it is expressed at a low level in part of the notum). This mutual repression also occurs late during development (Villa-Cuesta, 2005).

How relevant is this mutual repression for the establishment of the notum/dorsal hinge territorial subdivision? As indicated above, in ~19% of flies with msh clones, the removal of Msh from the hinge induces extra notum tissue. In the remaining cases, this removal does not substantially affect the identity of the hinge territory. Thus, the mutual repression between msh and Iro-C is crucial for the notum/hinge territorial subdivision in only a small but substantial fraction of the discs. This indicates that additional agents, probably expressed in the hinge, participate in effecting the subdivision. By contrast, notum cells that lose Iro-C always change their fate to hinge cells and, consequently, depending on position, they modify the notum/hinge subdivision or create an ectopic notum/hinge boundary. Hence, the relevance of the msh/Iro-C mutual repression to define/maintain that subdivision relies mainly on its defining/maintaining the border of the Iro-C domain, and thereby preventing the expression of hinge genes within the notum territory. Thus, a 'pronotum' gene (Iro-C) and a 'hinge differentiation' gene (msh), despite their different positions within the genetic hierarchies that govern the development of their respective domains, cross-regulate each other and participate in the early definition of their respective territories. The current data do not permit distinguishing between the possibilities that the mutual repression between msh and Iro-C is instrumental in establishing this territorial border, or, alternatively, that it stabilizes a previous border defined by the antagonistic actions of EGFR and Dpp on the Iro-C (Villa-Cuesta, 2005).

The relevance of the mutual repression between Iro-C and msh is also manifested by their respective overexpression. Ectopic Iro-C products in the hinge impair the proper differentiation of hinge structures (R. Diez del Corral, PhD thesis, Universidad Autónoma de Madrid, 1998). High levels of Msh in the notum turn on a hinge-specific marker like zfh-2 and are detrimental for notum development (Villa-Cuesta, 2005).

In the third instar disc, the distal border of the Iro-C domain is no longer straight and displays a pronounced 'bay' where ara/caup are downregulated. This roughly coincides with the area of highest expression of msh in the lateral notum. msh is probably responsible for this downregulation of ara/caup, as the 'bay' disappears in msh clones. Moreover, the abutting domains of msh and Iro-C in the ventral hinge and pleura, respectively, suggest that a similar mutual repression may occur there to establish the subdivision between these neighboring regions. Finally, the removal of msh does not activate Iro-C in the anterior part of the hinge territory, suggesting again that agents other than msh and Dpp help maintain Iro-C expression confined to the notum territory (Villa-Cuesta, 2005).

Iro-C^– clones located within the medial notum not only undergo an autonomous transformation to dorsal hinge. They also become surrounded by a fold similar to that which separates the notum and hinge territories, and they modify the expression of several markers in the surrounding wild-type tissue in a way consistent with a transformation of this tissue towards lateral notum. These nonautonomous effects suggest that signals emerge from the Iro-C^– clones, and that these signals alter the fate of the aposed notum tissue. Hence, it was inferred that, in the wild-type disc, signaling would take place across the hinge/notum boundary and this would help pattern at least the lateral notum. This is reminiscent of the DV and AP compartment boundaries, where signaling mediated by the diffusible molecules Wg, and Hh and Dpp, respectively, are key to stimulating the growth and pattern of the wing disc. However, in the hinge/notum boundary, the signaling agents have not been identified. They could be either diffusible molecules or cell-bound molecules that mediate this cell to cell communication (Villa-Cuesta, 2005).

The imaginal disc territories flanking the notum/hinge border are reduced in size when they are mutant for msh. It is not known whether this effect is due to decreased cell proliferation, increased cell death or both, and whether it mostly affects the hinge or the lateral notum. However, it is clear that by removing msh and allowing Iro-C to be expressed in the hinge, the msh clones suppress the confrontation of proper hinge cells with notum cells. It is tempting to speculate that this could affect the net growth of the territory by removing positional values and/or by suppressing or making ineffective the postulated signaling associated with the hinge/notum border. Consistently, a reduced size of the notum plus hinge region (and a simplification of the patterning) is also observed in discs overexpressing UAS-ara in the dorsal compartment, a condition that removes most msh expression from the hinge. The failure of Iro-C^– clones within the notum territory to grow and survive when they are also depleted of Msh might result from the absence of proper signaling across a boundary where wild-type notum cells confront Iro-C^– msh^– cells. Considering that the activity of the EGFR signaling pathway is necessary for notum cell proliferation, it would be of interest to examine whether this pathway is involved in, or is modulated by, the presence of the notum/hinge boundary (Villa-Cuesta, 2005).

In Drosophila, the Iro-C genes and msh respectively participate in the DV subdivision of the eye and of the neuroectoderm. In vertebrates, although no instance of mutual repression between homologs of Iro-C and msh has been described, members of each family participate in establishing borders by repression with other genes in the spinal cord, the brain and between rhombomeres. Clearly, both genes are used frequently to subdivide territories and establish alternative differentiation pathways at each side of the border that separates them (Villa-Cuesta, 2005).

Throughout the third instar, msh is expressed at relatively low levels in the posterior notum territory. Here, removal of msh most often results in impaired growth of the scutellum, absence of the scutellum/scutum suture and alterations of the bristle pattern. Interestingly, the lateral/anterior notum macrochaetae are often missing, even though they arise in a region apparently devoid of msh expression. This suggests that either msh is expressed there at very low but functional levels, or that the suppression of macrochaetae results from non-autonomous effects of the absence of Msh from neighboring territories. It should be noted that non-autonomous macrochaetae suppression is also associated with Iro-C^– clones that cause notum to hinge transformations. This has suggested that modification of the putative signaling across the notum/hinge boundary interferes with macrochaetae patterning at the notum. It is possible that the msh clones might also interfere, as indicated above, with signaling from this border. If so, the presence of clusters of sc expression at the anterior lateral notum within large msh clones suggest that this interference might occur at a stage later than the emergence of the proneural clusters (Villa-Cuesta, 2005).

The absence of msh function does not modify the expression of Iro-C in the lateral notum or the characteristic patterns of expression of eyg and hth, genes that are high in the hierarchy that control notum development. But it removes the scutum/scutellar suture and promotes development of extra bristles in the dorsocentral and scutellar regions. Again, these are phenotypes suggestive of an interference with the late patterning and differentiation of these structures (Villa-Cuesta, 2005).

The Drosophila embryonic CNS develops from the ventrolateral region of the embryo, the neuroectoderm. Neuroblasts arise from the neuroectoderm and acquire unique fates based on the positions in which they are formed. Previous work has identified six genes that pattern the dorsoventral axis of the neuroectoderm: Drosophila epidermal growth factor receptor (Egfr), ventral nerve cord defective (vnd), intermediate neuroblast defective (ind), muscle segment homeobox (msh), Dichaete and Sox-Neuro (SoxN). The activities of these genes partition the early neuroectoderm into three parallel longitudinal columns (medial, intermediate, lateral) from which three distinct columns of neural stem cells arise. Most of the knowledge of the regulatory relationships among these genes derives from classical loss of function analyses. To gain a more in depth understanding of Egfr-mediated regulation of vnd, ind and msh and investigate potential cross-regulatory interactions among these genes, loss of function was combined with ectopic activation of Egfr activity. Ubiquitous activation of Egfr expands the expression of vnd and ind into the lateral column and reduces that of msh in the lateral column. This work has identified the genetic criteria required for the development of the medial and intermediate column cell fates. ind appears to repress vnd, adding an additional layer of complexity to the genetic regulatory hierarchy that patterns the dorsoventral axis of the CNS. This study also demonstrates that Egfr and the genes of the achaete-scute complex act in parallel to regulate the individual fate of neural stem cells (Zhao, 2007).

In central nervous system development, the identity of neuroblasts critically depends on the precise spatial patterning of the neuroectoderm in the dorsoventral (DV) axis. This study has uncovered novel gene regulatory network underlying DV patterning in the Drosophila brain; the cephalic gap gene empty spiracles (ems) and the Nk6 homeobox gene (Nkx6) encode key regulators. The regulatory network implicates novel interactions between these and the evolutionarily conserved homeobox genes ventral nervous system defective (vnd), intermediate neuroblasts defective (ind) and muscle segment homeobox (msh). Msh cross-repressively interacts with Nkx6 to sustain the boundary between dorsal and intermediate neuroectoderm in the tritocerebrum (TC) and deutocerebrum (DC), and Vnd positively regulates Nkx6 by suppressing Msh. Remarkably, Ems is required to activate Nkx6, ind and msh in the TC and DC, whereas later Nkx6 and Ind act together to repress ems in the intermediate DC. Furthermore, the initially overlapping expression of Ems and Vnd in the ventral/intermediate TC and DC resolves into complementary expression patterns due to cross-repressive interaction. These results indicate that the anteroposterior patterning gene ems controls the expression of DV genes, and vice versa. In addition, in contrast to regulation in the ventral nerve cord, cross-inhibition between homeodomain factors (between Ems and Vnd, and between Nkx6 and Msh) is essential for the establishment and maintenance of discrete DV gene expression domains in the Drosophila brain. This resembles the mutually repressive relationship between pairs of homeodomain proteins that pattern the vertebrate neural tube in the DV axis (Seibert, 2009).

This study shows that the evolutionarily conserved homeodomain protein Ems is an integral component of the gene regulatory network that governs DV patterning in the posterior brain neuromeres, the TC and DC. This novel function is surprising because ems has hitherto been exclusively connected with patterning functions along the AP axis. It has been proposed that the combined activities of the gap genes ems, buttonhead and orthodenticle (ocelliless - FlyBase) generate head segments and that ems mutants exhibit defects in the formation of the intercalary and antennal segment as well as in the corresponding TC and DC in accordance with the early pattern of ems expression. ems probably also has a homeotic function in specifying aspects of intercalary segment identity. This study provides evidence that another crucial function of Ems is its cross-repressive interaction with Vnd. Previously, it was shown that vnd expression is dynamic and exhibits specific differences in the TC and DC. This study demonstrates that Ems is involved in the regulation of brain-specific differences in vnd expression, and that Vnd acts to repress ems in complementary parts of the TC and DC. These interactions help to refine the pattern into mutually exclusive domains at the onset of neurogenesis, which is important as both genes provide positional information that subsequently specifies the identity of individual brain NBs. Depending on the context, Vnd/Nkx2 can act as a transcriptional activator or repressor, as determined by physical interaction with the co-repressor Groucho, which enhances repression. Interestingly, it was observed that Ems also regulates the expression of two Nkx genes in an opposing manner: it represses vnd/Nkx2 but is necessary to activate Nkx6. The repressor function of Ems most likely also depends on Groucho; Ems has been reported to bind Groucho in vitro (Seibert, 2009).

In ems mutants, defects in proneural gene expression (lethal of scute and achaete) are restricted to NE regions where ems is normally expressed during early neurogenesis, leading to the loss of a subset of NBs in the TC and DC. This contrasts with the phenotype of the late embryonic ems mutant brain, which exhibits a severe reduction, or entire elimination, of the TC and DC, suggesting that the proper development of a larger NE domain and/or fraction of NBs in the TC and DC must be affected. However, in ems mutants the organization of the early procephalic NE appears normal until stages 9/10 and apoptosis is not detected. A possible explanation for the subsequent complete loss of TC and DC is that in ems mutants, vnd becomes derepressed in the ventral/intermediate NE of both neuromeres, and expression of msh, ind and Nkx6 is not activated. It has been shown that ectopic vnd prevents the expression of many NB identity genes. Indeed, the expression of a number of molecular markers has been reported to be absent in the ems mutant brain. It is therefore conceivable that in the TC and DC of ems mutants, as a consequence of lacking ems and ectopic vnd (and the absence of proneural gene activation), some NBs do not form. Additionally, owing to mis-specification of the NE (where neural identity gene expression is absent or altered), the other NBs and their progeny might still form but degenerate at later stages (Seibert, 2009).

It has been largely unclear how expression of Nkx6 is regulated in the brain NE, although Vnd has been suggested to act as a positive regulator. At the blastodermal stage, coexpression of ems and vnd is only observed in the intermediate and ventral NE of the TC and DC, which might account for early Nkx6 expression being limited to the respective NE in the brain and absent from the trunk. The data indicate that Ems and Vnd together facilitate the activation of Nkx6. Ems expression closely prefigures the domain of Nkx6 expression in the TC and DC, and together with the fact that Nkx6 is completely abolished in ems mutants, this suggests that Ems might act as a direct activator to regulate the extension of the Nkx6 domain along the AP axis. Vnd indirectly regulates the enlargement of the Nkx6 domain along the DV axis by repressing the Nkx6-repressor Msh. That DV patterning in the brain NE integrates AP signals is additionally supported by the fact that Ems is also necessary for activation of ind and msh, indicating that ems is a key regulator in DV patterning of the TC and DC. Evidence is also provided for a negative-feedback control in the DV regulatory network, in which Ems is needed to activate its own later-stage repressors, Nkx6 and Ind. Together, these data suggest not only that Ems regulates the expression of all DV genes (activating Nkx6, ind, msh and repressing vnd), but also that DV factors (Nkx6, Ind and Vnd) control expression of ems, indicating that integration of DV and AP patterning signals takes place at different levels in the DV genetic network (Seibert, 2009).

Nkx6 has been identified as specifically involved in DV patterning of the TC and DC. In addition to later suppression of ems (in concert with Ind), a further pivotal function of Nkx6 is to maintain the suppression of msh in the intermediate/ventral TC and DC that was initiated by Vnd. Since in both neuromeres the expression of Nkx6 starts before and persists longer than that of ind, and because msh is ventrally derepressed in Nkx6 but not in ind mutants, this implies that Nkx6 (but not Ind) is the major msh suppressor necessary to prevent intermediate/ventral NE and the descending NBs from adopting dorsal fates. Consequently, Nkx6 indirectly regulates the proper specification of brain NB identity by suppressing msh (and ems). Further experiments are required to show whether Nkx6 is also more directly involved in the fate specification of NBs and progeny cells in the brain, as has been shown in the VNC, where Nkx6 promotes the fate of ventrally projecting, and represses the fate of dorsally projecting, motoneurons (Seibert, 2009).

Additionally, cross-inhibitory interactions were observed between Nkx6 and Msh. It is assumed that this mutually repressive regulation in the TC and DC is necessary to stabilize the boundary between dorsal and intermediate NE, and to ensure the regionalized expression of msh and Nkx6 over time. It is likely that Nkx6 and Msh/Msx interact with the co-repressor Groucho to repress each other at the transcriptional level. Interestingly, aspects of the genetic interactions between Nkx6 and Msh/Msx seem to be evolutionarily conserved, since Msx1, which is expressed in the vertebrate midbrain and functions as a crucial determinant in the specification of dopamine neurons, represses Nkx6.1 in ventral midbrain dopaminergic progenitors of mice (Seibert, 2009).

It had not been shown until now that domains of DV gene expression in the Drosophila brain become established through cross-repressive regulation, and it is possible that such genetic interactions are more common than previously thought (e.g. Ind and Msh act as mutual inhibitors). This suggests that in the fly brain, cross-inhibition between pairs of homeodomain transcription factors is fundamental for establishing and maintaining DV neuroectodermal and corresponding stem cell domains. By contrast, in the NE of the VNC, where DV patterning is much better understood, cross-repressive interactions of homeobox genes are largely omitted. There, DV patterning is proposed to be conducted by a strict ventral-dominant hierarchy according to which ventral genes repress more-dorsal genes. However, one exception to the rule seems to be the cross-inhibitory interaction between Vnd and Ind. Interestingly, in the developing vertebrate neural tube, cross-repressive interactions of homeodomain proteins are common and indeed crucial for the establishment of discrete DV progenitor domains. This bears a marked resemblance to the mutually antagonistic relationship between pairs of homeodomain proteins that dorsoventrally pattern the fly brain (Seibert, 2009).

A predominant feature of the brain-specific DV genetic network described in this study, and a general design feature of gene regulatory networks, is the extensive use of transcriptional repression to regulate target gene expression in spatial and temporal dimensions. All factors involved in the network operate as repressors (except Ems, which may also serve as an activator), via mutual repression (between Ems and Vmd, and between Nkx6 and Msh), a double-negative mechanism (Vnd represses Msh, which represses Nkx6), and a negative-feedback loop (Ems is needed to activate Nkx6 and Ind, which in turn repress Ems). The spatial and temporal complexity of the regulatory interactions that have been deciphered implies similar complexity in the underlying cis-regulatory control of these factors. For example, the domain of msh expression is regulated by the input of at least two transcriptional repressors acting in subsequent time windows (Vnd early and Nkx6 late), and the input of at least three repressors regulates the dynamics of ems expression (Vnd early, Ind and Nkx6 late). The brain-specific DV patterning network probably comprises further genes in addition to those that have been identified, and it is likely that interactions with other putative regulators (e.g. Dorsal, Egfr, Dpp) will complement the present model. Altogether, these data provide the basis for a systematic comparison of the genetic processes underlying DV patterning of the brain between different animal taxa at the level of gene regulatory networks (Seibert, 2009).

The genetic factors considered in this study in the developing fly brain are expressed in similar NE domains from early embryonic stages onwards in the anterior neural plate in vertebrates. Emx2, for example, is expressed in the laterodorsal region, and Nkx2 genes in the ventral region, of the early vertebrate forebrain. At the four-somite stage (~E8), these two domains exhibit a common border, similar to that observed in Drosophila after Ems and Vnd have, through cross-repression, regulated their mutually exclusive expression domains. Moreover, whereas Msx genes are mainly expressed in dorsal regions of the posterior forebrain, midbrain and hindbrain, expression of Nkx6 genes is reported in more lateroventral regions, overlapping ventrally with the expression of Nkx2 genes. However, even though these patterns of gene expression exhibit certain similarities between insects and vertebrates, it remains to be shown whether their genetic interactions are also conserved (Seibert, 2009).

Specification of cell fates across the dorsoventral axis of the central nervous system in Drosophila involves the subdivision of the neuroectoderm into three domains that give rise to three columns of neural precursor cells called neuroblasts. Ventral nervous system defective (Vnd), intermediate neuroblasts defective (Ind) and muscle segment homeobox (Msh) are expressed in the three columns from ventral to dorsal, respectively. The products of these genes play multiple important roles in formation and specification of the embryonic nervous system. Ind, for example, is known to play roles in two important processes. First, Ind is essential for formation of neuroblasts in conjunction with SoxB class transcription factors. Sox class transcription factors are known to specify neural stem cells in vertebrates. Second, Ind plays an important role in patterning the CNS in conjunction with, vnd and msh, which is also similar to how vertebrates pattern their neural tube. This work focuses two important aspects of Ind function. First, multiple approaches were used to identify and characterize specific domains within the protein that confer repressor or activator ability. Currently, little is known about the presence of activation or repression domains within Ind. This study shows that transcriptional repression by Ind requires multiple conserved domains within the protein, and that Ind has a transcriptional activation domain. Specifically, a novel domain, the Pst domain, was identified that has transcriptional repression ability and appears to act independent of interaction with the co-repressor Groucho. This domain is highly conserved among insect species, but is not found in vertebrate Gsh class homeodomain proteins. Second, it was shown that Ind can and does repress vnd expression, but does so in a stage specific manner. It is concluded from this that the function of Ind in regulating vnd expression is one of refinement and maintenance of the dorsal border (Von Ohlen, 2009).

The function of Ind in development of the embryonic nervous system is multifold. Initially, Ind serves to define the intermediate column of the neuroectoderm, this subsequently leads to formation of the corresponding neuroblasts. This study shows that transcriptional repression activity by Ind involves at least two transcriptional repression domains, suggesting that Ind represses transcription via Groucho-dependent and Groucho-independent mechanisms. There are two highly conserved domains in the N-terminal region of the Ind protein. Both appear to be essential for maximal repression activity of Ind. In addition, a third domain was identified that is capable of conferring transcriptional activation ability on a heterologous DNA-binding domain. Also, data is presented demonstrating that Ind functions to define and maintain this domain via transcriptional repression of other columnar genes vnd and msh. Suggesting that, depending on which enhancer it is bound to and possibly association with co-factors, Ind can act as either a transcriptional repressor on as activator. Finally, an Ind protein lacking the Eh1 domain but retaining the Pst domain fails to physically interact with purified Groucho protein. Furthermore, the Gal4-IndδEh1 protein was still a strong repressor of transcription in cultured cells. These results strongly support the hypothesis that the Pst domain confers repressor activity independent of Groucho interaction. However, the possibility that the Pst domain also plays a role in stabilizing the interaction with Groucho or association with other co-factors in vivo cannot be ruled out (Von Ohlen, 2009).

It is not surprising that Ind has incorporated additional repressor activities that are independent of Groucho activity. Formation of the intermediate column of neuroblasts is also dependent on the activity of the Egfr signaling pathway. Specifically, in egfr mutant embryos the intermediate column of neuroblasts fails to form because Ind is not expressed. The readout for activation of the Egfr pathway is the presence of the activated form of Map kinase (dpErk). DpErk is detected in the ventral and intermediate columns of the neuroectoderm at the early stages of development. Interestingly, the activation of DpErk appears to correlate with down-regulation of Groucho activity. Specifically, Map kinase directly phosphorylates Groucho and this phosphorylation of Groucho results in reduced co-repressor activity (Cinnamon, 2008). Since Groucho activity is down-regulated in the region where Ind is expressed and Ind is a Groucho-dependent transcriptional repressor, additional repression activity may be necessary to overcome the effects of Egfr signaling on Groucho activity (Von Ohlen, 2009).

Formation of the proper complement of neuroblasts in the embryonic nervous system of Drosophila and other insects is essential for the proper development of the organism. Initially the neuroblasts form in three columns that correspond to the domains of vnd, ind and msh expression. Therefore, formation of the stripes of homeobox gene expression is essential for the ultimate formation of the CNS. While there is an apparent ventral dominance mechanism in place to initiate the expression of these genes, there is also a cross repressive relationship that is essential for maintaining the boundaries between the domains of gene expression. Ind represses vnd only at stages 9 and 10 of embryonic development and not earlier. This coincides with differences in the ability of Ind to repress vnd reporter gene expression. Thus, Ind can repress transcription from enhancer elements located upstream that regulate expression in neuroblasts. However, Ind is unable to repress transcription of lacZ message from reporter constructs that include the vnd NEE, which is essential for initiation of vnd expression. In conclusion, the temporal differences in the ability of Ind to repress transcription of vnd reflect a role for Ind maintaining the boundary between Vnd and Ind domains. However, Ind was not required for establishing the dorsal border of vnd expression at the earliest stages of embryogenesis (Von Ohlen, 2009).

Subdivision of the neuroectoderm into discrete gene expression domains is essential for the correct specification of neural stem cells (neuroblasts) during central nervous system development. This study extends knowledge on dorsoventral (DV) patterning of the Drosophila embryonic brain and uncovers novel genetic interactions that control expression of the evolutionary conserved homeobox genes ventral nervous system defective (vnd), intermediate neuroblasts defective (ind), and muscle segment homeobox (msh). Cross-repression between Ind and Msh was shown to stabilize the border between intermediate and dorsal tritocerebrum and deutocerebrum, and both transcription factors are competent to inhibit vnd expression. Conversely, Vnd segment-specifically affects ind expression; it represses ind in the tritocerebrum but positively regulates ind in the deutocerebrum by suppressing Msh. These data provide further evidence that in the brain, in contrast to the trunk, the precise boundaries between DV gene expression domains are largely established through mutual inhibition. Moreover, it was found that the segment-polarity gene engrailed (en) regulates the expression of vnd, ind, and msh in a segment-specific manner. En represses msh and ind but maintains vnd expression in the deutocerebrum, is required for down-regulation of Msh in the tritocerebrum to allow activation of ind, and is necessary for maintenance of Ind in truncal segments. These results indicate that input from the anteroposterior patterning system is needed for the spatially restricted expression of DV genes in the brain and ventral nerve cord (Seibert, 2010)

The spatial and temporal order in which the DV genes (vnd, ind, and msh) are activated in neuromeres of the brain differs from their appearance in the trunk neuroectoderm, and those differences seem to be basic for the segment-specific regulation of vnd, ind, and msh expression. In the early trito- and deutocerebrum, Vnd is expressed not only in the ventral but also in the intermediate neuroectoderm, where cross-repression between Vnd and dorsally expressed Msh establishes the border between intermediate and dorsal neuroectoderm. Since Msh was found to be an ind repressor, the repression of msh via Vnd is a prerequisite for ind to become activated in the intermediate tritocerebrum (anterior) and deutocerebrum. In the trunk, ind expression in the intermediate neuroectoderm starts before that of msh in the dorsal neuroectoderm, and msh and vnd domains do not abut; accordingly, repressive interaction between Msh and Vnd is not required (Seibert, 2010)

In the tritocerebrum, Vnd not only acts as repressor of msh but also of ind, in contrast to the deutocerebrum. When the level of Vnd protein in the intermediate tritocerebrum declines with time (down-regulated through the activity of Ems), ind becomes subsequently activated. In the trito- and deutocerebrum, instead of Vnd, increasing levels of Ind, together with the recently uncovered msh-repressor Nkx6, still keep msh expression limited to the dorsal neuroectoderm. Since it was found that Nkx6 expression starts earlier and persists longer than that of ind in both brain neuromeres, and additionally, that msh is expanded into the intermediate neuroectoderm in Nkx6 but not in ind mutants, it is proposes that Nkx6 represses msh more efficiently (Seibert, 2010)

The most striking difference in DV gene regulation leads to the question how vnd and ind can be co-expressed in the anterior deutocerebrum (during stages 6–9), if Vnd is a repressor of ind and, vice versa, Ind is also capable of preventing vnd expression in the neuroectoderm. It has been reported recently that the repressor activity of Ind on vnd seems to be stage-specific, not taking place before stage 9. By contrast, Vnd repression of ind seems independent of the developmental period. In this context, it is interesting that activity of Vnd can be modified by EGFR signalling, which is supposed to affect the selective interaction of Vnd with co-factors necessary to mediate repression or activation of target genes. Availability of co-factors might also account for the specific situation of vnd and ind co-expression in the anterior deutocerebrum that was observed specifically during early stages of development (Seibert, 2010)

Involvement of en, which can act as transcriptional repressor as well as activator, has been implicated in diverse developmental processes in Drosophila such as compartmentalization in the early embryo, modulation of Hox gene expression, or regulation of molecules that directly govern axon growth (e.g. frazzled). This study demonstrates a novel function for En in the early embryo, that is to control the spatially restricted expression of the DV genes in the neuromeres of the posterior brain (trito- and deutocerebrum) and ventral nerve cord. In the posterior compartment of the deutocerebrum, En represses expression of msh and ind, but maintains expression of vnd. Since it was found that Ind (later) becomes a vnd repressor, this indicates that En maintains expression of vnd by repressing ind. In the posterior compartment of the tritocerebrum, En is also required for down-regulation of Msh, but opposite to the deutocerebrum, En is necessary for activation of ind. This study shows that Msh is an ind repressor, its repression by En seems to allow for activation of ind; yet, it cannot be excluded that En in addition directly activates ind expression. Similar to the situation in the tritocerebrum, En seems to negatively regulate expression of msh and to positively regulate expression of ind (as a maintenance factor) in the neuroectoderm of the ventral nerve cord. Together, these data suggest that the AP patterning gene engrailed is crucially involved in fine-tuning the regionalized expression of distinct DV genes in the posterior compartment of neuromeres in the brain and ventral nerve cord. En may act as a positive or negative transcriptional regulator depending on the gene that is regulated and on the segmental context. For DV genes it is known that they control formation and specification of brain neuroblasts. Since all the genetic interactions between En and DV genes take place during the period when neuroblasts develop, it is likely that En, via regulation of DV genes, controls formation and fate specification of neuroblasts in the brain (Seibert, 2010)

It was observed that cross-repressive interaction between pairs of DV gene factors in the brain (i.e. in trito- and deutocerebrum) is essential for the establishment and maintenance of discrete DV gene expression domains. Early, cross-repression between Ems/Vnd pre-patterns the ventral and intermediate neuroectoderm in both neuromeres. Mutual repression between Msh/Nkx6 and Msh/Ind maintains the dorsal/intermediate neuroectodermal border in trito- and deutocerebrum, and between Ind/Vnd the intermediate/ventral border in the tritocerebrum. All these genetic interactions, and the observation that Msh and Vnd act as mutual repressors, are not in compliance with the concept of ventral dominance (as proposed in the neuroectoderm of the ventral nerve cord where the more ventral gene represses the gene expressed more dorsally) but rather support the model that in the brain cross-repression between DV factors is crucial for stabilizing these borders (Seibert, 2010)

However, despite the ability of Msh and Ind to repress vnd, neither factor seems to be sufficient to define the dorsal border of vnd expression in trito- and deutocerebrum, as has been shown for Ind in the ventral nerve cord (from stage 9 onwards). Instead of reinforcing this border through repressive interaction, vnd expression in the brain could also be limited by a (too) low concentration or absence of an activator, like Dorsal (as has been speculated for the trunk), or be regulated by BMP signalling in a dosage-dependent fashion. Neuromere-specific differences are also observed regarding limitation of ind and msh expression domains along the DV axis. Vnd establishes the ventral border of ind expression in the trunk and tritocerebrum, but not in the deutocerebrum or protocerebrum (where the expression domains of ind and vnd do not abut). ind expression was found to be limited dorsally by repression through Msh in the trito- and deutocerebrum, but not in the protocerebrum (where msh is not expressed before stage 11) or trunk, although evidence is available that Msh might act in rendering the dorsal border of ind expression more precisely in the ventral nerve cord. Taking into account that ind expression does not expand into the complete dorsal neuroectoderm of trito- and deutocerebrum in msh mutants, this may also indicate an involvement of the nuclear Dorsal gradient, possibly in concert with graded activity of EGFR (as was shown for the trunk neuroectoderm), or BMP (which can repress ind in the trunk neuroectoderm), in establishing a rough dorsal border of ind expression that is further defined and stabilized via repression by Msh. Whereas Vnd is initially responsible for keeping msh expression confined to the dorsal neuroectoderm in trito- and deutocerebrum, it is only indirectly involved in defining the ventral border of msh expression in the trunk neuroectoderm. Later in development Ind helps to maintain repression of msh in trito- and deutocerebrum (together with Nkx6), which is in contrast to the trunk where Ind directly establishes the ventral limit of msh expression from the beginning (Seibert, 2010)

DV neuroectodermal and corresponding stem cell domains in the Drosophila brain become established and maintained through cross-repressive regulation, and it has been speculated that such genetic interactions are more common in the fly brain. This study has presented further examples supporting this hypothesis. Notably, this is a feature that bears similarity to DV patterning in the neural tube of vertebrates where cross-repressive interactions of homeodomain proteins are common and indeed crucial for the establishment of discrete DV progenitor domains (Seibert, 2010)

All interactions between DV genes in the brain identified so far are based on the interplay of transcriptional repressors. Likewise, this study shows that Vnd does not act as a direct activator to positively regulate ind, but according to a double-negative mechanism, it suppresses the ind-repressor Msh. It has been shown previously, that interactions of the AP patterning gene ems with the DV genes (vnd, ind, msh, and Nkx6) are indispensable for proper development of the trito- and deutocerebrum. This study demonstrates that the segmentation gene en is significantly involved in regionalization of DV gene expression domains, thus representing a further example of an AP patterning gene integrating into the DV gene regulatory network that patterns the brain. This study has shown that En acts differently on the respective DV genes, but no evidence is available that, vice versa, DV genes control en, as has been observed for expression of ems. DV genes, as well as En and Ems, all contain an Eh1 repressor domain and are able to interact with the co-repressor Groucho (Gro), and thus are capable of mediating repression on target genes (including each other). But how could it be possible that all DV genes interact with the same co-factor to stabilize expression domains by conferring repression onto genes expressed in neighboring domains? In the first place, the DV genes display spatio-temporal differences in their respective expression. In addition, conformational changes of the protein seem to be necessary to enable binding of Gro which has been at least shown for Nkx6. It has been observed that Vnd can be phosphorylated by activated MAPK and is present in different isoforms in the developing embryo, which most likely leads to a change in its binding partners. Another critical point could be inactivation of the co-repressor, in case of Gro also through phosphorylation by activated MAPK, or modification of target genes so that binding of the repressor complex is impaired. Still, that the DV genes are able to interact with Groucho, does not exclude that their repressor activity is Gro-independent, since also other repressor domains have been reported for these genes, as well as activator domains, at least for Vnd, Ind, and Nkx6. Whether the DV gene products function as repressors or activators seems to depend on co-factor availability as well as on the respective target gene, since not only the presence of a transcriptional binding site, but also its accessibility is limiting in this context (Seibert, 2010)

Regulatory networks driving morphogenesis of animal genitalia must integrate sexual identity and positional information. Although the genetic hierarchy that controls somatic sexual identity in Drosophila is well understood, there are very few cases in which the mechanism by which it controls tissue-specific gene activity is known. In flies, the sex-determination hierarchy terminates in the doublesex (dsx) gene, which produces sex-specific transcription factors via alternative splicing of its transcripts. To identify sex-specifically expressed genes downstream of dsx that drive the sexually dimorphic development of the genitalia, genome-wide transcriptional profiling was performed of dissected genital imaginal discs of each sex at three time points during early morphogenesis. Using a stringent statistical threshold, 23 genes that have sex-differential transcript levels at all three time points were identified, of which 13 encode transcription factors, a significant enrichment. This study focused on three sex-specifically expressed transcription factors encoded by lozenge (lz), Drop (Dr) and AP-2. In female genital discs, Dsx activates lz and represses Dr and AP-2. It was further shown that the regulation of Dr by Dsx mediates the previously identified expression of the fibroblast growth factor Branchless in male genital discs. The phenotypes observed upon loss of lz or Dr function in genital discs explain the presence or absence of particular structures in dsx mutant flies and thereby clarify previously puzzling observations. This time course of expression data also lays the foundation for elucidating the regulatory networks downstream of the sex-specifically deployed transcription factors (Chatterjee, 2011).

A common theme in the evolution of development is that a limited 'toolkit' of regulatory factors is deployed for different purposes during morphogenesis. It is therefore not surprising that the key regulators of genital morphogenesis that this study identified are pleiotropic factors with roles in other developmental processes (Chatterjee, 2011).

Two genes that are expressed sex-differentially in the genital disc, branchless (bnl) and dachshund (dac), provide the best picture of how dsx controls genital morphogenesis. Bnl, which is the fly fibroblast growth factor (FGF), is expressed in two bowl-like sets of cells in the A9 primordium in male discs; there is no expression in female discs because DsxF cell-autonomously represses bnl. Bnl recruits mesodermal cells expressing the FGF receptor Breathless (Btl) to fill the bowls; these Btl-expressing cells develop into the vas deferens and accessory glands (Chatterjee, 2011 and references therein).

Dac, a transcription factor, is expressed in male discs in lateral domains of the A9 primordium and in female discs in a medial domain of the A8 primordium. These lateral and medial domains correspond to regions exposed to high levels of the morphogens Decapentaplegic (Dpp) and Wingless (Wg), respectively. Dsx determines whether these signals activate or repress dac. Male dac mutants have small claspers with fewer bristles and lack the single, long mechanosensory bristle. Female dac mutants have fused spermathecal ducts (Chatterjee, 2011 and references therein).

As with bnl and dac, it remains to be determined whether these downstream genes are direct Dsx targets. Each contains at least one match within an intron to the consensus Dsx binding sequence ACAATGT. Future work will determine whether these matches are indeed contained within Dsx-regulated genital disc enhancers. Moreover, efforts are underway to define Dsx binding locations genome-wide through experiments rather than bioinformatics (B. Baker and D. Luo, personal communication to Chatterjee, 2011); combined with the current expression data, these binding data could speed the discovery of a large number of sex-regulated genital disc enhancers (Chatterjee, 2011).

An important future direction will be to determine how spatial and temporal cues are integrated with dsx to regulate downstream genes. Because lz is expressed in the anterior medial region of the female disc, it is hypothesized that, like dac, it is activated by Wg and repressed by Dpp. Such combinatorial regulation could explain the spatially restricted competence of cells in the male disc to activate lz in response to DsxF. Although Dr, AP-2 and lz are expressed at L3, P6 and P20, many other genes are differentially expressed at only one or two of these time points. How these timing differences are regulated is an important unanswered question, especially for genes such as ac, which shifts from highly female biased at P6 to highly male biased at P20. The finding that Dsx binding sites are most enriched in genes with sex-biased expression at L3 suggests that indirect regulation through a cascade of interactions might contribute to expression timing differences (Chatterjee, 2011).

It has already been shown that DsxF indirectly represses bnl by repressing Dr. To date, Dr has been shown to repress, but not activate, transcription. Therefore, activation of bnl by Dr might itself be indirect, via repression of a repressor. The regulation of bnl by Dr is sufficient to explain the sex-specific expression of bnl. However, upstream of bnl are two sequence clusters that match the consensus binding motif of Dsx. Thus, bnl might be repressed both directly and indirectly by Dsx, in a coherent feed-forward loop (FFL). FFLs attenuate noisy input signals. An FFL emanating from Dsx could provide a mechanism of robustly preventing bnl activation in female discs, despite potential fluctuations in DsxF levels (Chatterjee, 2011).

Understanding how Dr controls the morphogenesis of external structures is also important. The posterior lobe will be of particular interest because it is the most rapidly evolving morphological feature between D. melanogaster and its sibling species. Mutations in Poxn and sal also impair posterior lobe development. Understanding how these two regulators work with Dr to specify and pattern the developing posterior lobe could substantially advance efforts to understand its morphological divergence. Likewise, understanding how lz governs spermathecal development could advance evolutionary studies, as this organ also shows rapid evolution (Chatterjee, 2011).

The extent to which the regulators that were identified play deeply conserved roles in genital development remains to be determined. Although sex-determination mechanisms evolve rapidly, some features are shared by divergent animal lineages. The observation that FGF signaling is crucial to male differentiation in mammals, or that mutations in a human sal homolog cause anogenital defects, could reflect ancient roles in genital development or convergent draws from the toolkit (Chatterjee, 2011).

Whether AP-2, Dr and lz play conserved roles in vertebrate sexual development is similarly uncertain. In mice, an AP-2 homolog is expressed in the urogenital epithelium (albeit in both sexes) and at least one AP-2 homolog shows sexually dimorphic expression (albeit in the brain). The mouse Dr homolog Msx1 is expressed in the genital ridge and Msx2 functions in female reproductive tract development. In chick embryos, Msx1 and Msx2 are expressed male specifically in the Müllerian ducts. The mouse lz homolog Aml1 (Runx1) is expressed in the Müllerian ducts and genital tubercle. As more data accumulate on the genetic mechanisms controlling genital development in other taxa, the question of how deeply these mechanisms are conserved might be resolved (Chatterjee, 2011).