vestigial


DEVELOPMENTAL BIOLOGY

Embryonic

vestigial expression is first detected in germ band extended embryos in lateral stripes from the first thoracic to the seventh abdominal segments. Three additional ventrolateral clusters of cells in the three thoracic segments also express vestigial. Most of the vg expressing cells in thoracic segments 2 and 3 correspond to the embryonic primordia of the wing and haltere discs. During germ band retraction (stage 12), segmentally repeated expression is detected in discrete cells in the ventral nerve cord; beginning in stage 13, additional head region and ventral nerve cord cells express vg. Clusters of cells in the head and abdominal regions may represent progenitors of sense organs of the peripheral nervous system (Williams, 1991).

Vestigial expression in the Drosophila embryonic central nervous system

The Drosophila central nervous system is an excellent model system in which to resolve the genetic and molecular control of neuronal differentiation. This study shows that the wing selector vestigial is expressed in discrete sets of neurons. The axonal trajectories of Vestigial-expressing cells in the ventral nerve cord were tracked, and these cells were shown to descend from neuroblasts 1-2, 5-1, and 5-6. In addition, along the midline, Vestigial is expressed in ventral unpaired median motorneurons and cells that may descend from the median neuroblast. These studies form the requisite descriptive foundation for functional studies addressing the role of vestigial during interneuron differentiation (Guss, 2008; full text of article).

Larval

VG protein marks most of the cells destined to form wing and hinge regions in the wing disc and haltere structures in the haltere disc, but no labeling is seen in the subregions of these discs that form notum or other thoracic cuticular structures (Williams, 1991).

vestigial expression is dynamic in the wing disc. Expression in early-mid second instar discs is ubiquitous, but later in the second instar, expression is repressed distally. Finally expression resolves to form a well-defined strip by the early third instar. Expression drops off in intensity both proximally and distally. This band of expression corresponds to the early wing region of the disc, as distinguished from regions that give rise to non-wing structures such as the notum (Williams, 1993).

defective proventriculus is required for pattern formation along the proximodistal axis, cell proliferation and formation of veins in the Drosophila wing

Little is known about the genes and mechanisms that pattern the proximodistal (PD) axis of the Drosophila wing. Vestigial (Vg) is instrumental in patterning this axis, but the genes that mediate its effects and the mechanisms that operate during PD patterning are not known. The gene defective proventriculus (dve) is required for a region of the PD axis encompassing the distal region of the proximal wing (PW) and a small part of the adjacent wing pouch. Loss-of-function of dve results in the deletion of this region and, consequently, shortening of the PD axis. dve expression is activated by Vg in a non-autonomous manner, and is repressed at the DV boundary through the combined activity of Nubbin and Wg. Besides its role in the establishment of the distal part of the PW, dve is also required for the formation of the wing veins 2 and 5, and the proliferation of wing pouch cells, especially in regions anterior to wing vein 3 and posterior to wing vein 4. The study of the regulation of dve expression provides information about the strategies employed to subdivide and pattern the PD axis, and reveals the importance of vg during this process (Kölzer, 2003).

The wing imaginal disc is a single-cell layered epithelium and, thus, is a two-dimensional structure. Therefore, establishment and patterning of the PD axis must occur with the help of the existing AP and DV axes. The vg gene is an important translator of the positional values of these axes in corresponding PD values. vg is required for the establishment of distal wing fates. This work gives insight into how Vg organizes the PD axis (Kölzer, 2003).

Vg is required for the establishment of the medial part of the PW. During this process Vg induces the expression of rotund (rn). Expression of rn is in turn required to set up the inner ring-like expression domain of Wg, which subsequently organizes the formation of the medial part of the PW. This work shows that Vg is further required for the establishment of the distal part of the PW. It shows that one crucial event during this process is the establishment of the expression of dve by Vg. Importantly, Vg induces both parts of the PW in a non-autonomous manner. This indicates that Vg controls the expression of a diffusible factor that induces the expression of genes, such as dve and rn, in cells inside and outside of its expression domain, in order to establish the corresponding regions of the PW. Furthermore, the induction of expression of rn and dve occurs independently of each other. The expression domain of rn is larger than that of dve. Taking for granted that expression of both genes is induced by the same diffusible factor, this observation suggests that the factor might act in a concentration dependent manner. In this scenario the induction of rn expression would require less activity than the induction of dve. Expression of nub has been shown to be lost in vg-mutant wing imaginal discs, suggesting that Vg is also required non-autonomously for the activation of nub, in a yet larger domain than dve and rn. However, these results are in conflict with earlier work that reports that nub expression is not dependent on Vg function. Wg, but not Vg, has been shown to be able to induce ectopic expression of nub in the notum of the wing imaginal disc. Furthermore, expression of nub RNA was observed in vg-null mutant wing imaginal discs. These data strongly suggest that Wg is required to activate expression of nub. Hence, further work is necessary to resolve the contradictions, and to determine whether Vg also plays a role during activation of the expression of nub. Despite this uncertainty, all of the mentioned genes are expressed in disc-like domains of different sizes. Their expression leads to concentric areas with different combinations of gene activities. It seems likely that a particular combination of these genes establishes a specific part of the PW (Kölzer, 2003).

These data provide evidence that Vg controls the expression of fj, within an expression domain that corresponds to the wing pouch. Fj is required for the establishment of a proximal region of the wing pouch and also for planar polarity of the wing. Furthermore, Vg regulates the expression of Distal-less (Dll), which is required to pattern the wing margin. Thus, Vg is involved in the patterning of the PD axis inside as well as outside its expression domain (Kölzer, 2003).

It is widely accepted that pattern formation and cell proliferation are closely connected during wing development. However, it has not been clear how these processes are connected. The fact that expression of dve is initiated by one of the central patterning factors, Vg, provides a possible link (Kölzer, 2003).

The data presented in this study reveal how patterning along the PD axis might occur with help of the two other existing axes. Wing development starts at the cross-point of the expression domains of Dpp and Wg in the ventral part of the wing disc. It appears that the combined activity of the two signals define the wing field. Although the activity of Wg is sufficient to establish the proximal-most pattern elements, the hinge and the proximal region, of the PW, the establishment of all distal regions requires the additional activity of vg. In the wing field, the Notch signalling pathway activates the expression of vg in cells at the future compartment boundary. In addition, Wg, perhaps in collaboration with Vg/Sd, activates the expression of nub (Kölzer, 2003).

In the next step Vg induces the expression of wg in cells at the DV boundary, in collaboration with the Notch pathway. In addition, Vg activates an unknown diffusible factor that induces the expression of dve and rn in disc-like domains of different sizes. All these domains are larger than that of Vg, and expression of the three genes is established independently of one another. This fact suggests that the diffusible factor might act in a concentration-dependent manner, as is typical for morphogens. Dve and Rn act in collaboration with Nub to establish the medial and distal parts of the PW (Kölzer, 2003).

When the expression of nub, rn and dve is initiated, Vg is expressed in cells at the DV boundary. These cells will later form the distal-most structure, the wing margin. The wing pouch is formed by the progenies of cells at the DV boundary, and is therefore intercalated between the margin and the anlagen of the PW. During its formation, the pouch will be further subdivided through the combined activity of Vg and Wg. Both proteins generate gradients that further subdivide the pouch along the DV axis (Kölzer, 2003).

In summary, the data suggest that pattern formation along the PD axis occurs in several steps and uses a strategy similar to that observed during leg development. It is initiated by the definition of the proximal (hinge and the distal part of the PW) and the distal-most point (wing margin), with help of the existing AP and DV axes. During development, the intermediate pattern elements (first the anlagen of the medial and distal part of the PW, then the wing blade) are intercalated stepwise with respect to these reference points (Kölzer, 2003).

Regulation of cellular plasticity in Drosophila imaginal disc cells by the Polycomb group, trithorax group and lama genes: Activation of vg gene expression marks leg-to-wing transdetermination

Drosophila imaginal disc cells can switch fates by transdetermining from one determined state to another. The expression profiles of cells induced by ectopic Wingless expression to transdetermine from leg to wing were examined by dissecting transdetermined cells and hybridizing probes generated by linear RNA amplification to DNA microarrays. Changes in expression levels implicated a number of genes: lamina ancestor, CG12534 (a gene orthologous to mouse augmenter of liver regeneration), Notch pathway members, and the Polycomb and trithorax groups of chromatin regulators. Functional tests revealed that transdetermination was significantly affected in mutants for lama and seven different PcG and trxG genes. These results validate the described methods for expression profiling as a way to analyze developmental programs, and they show that modifications to chromatin structure are key to changes in cell fate. These findings are likely to be relevant to the mechanisms that lead to disease when homologs of Wingless are expressed at abnormal levels and to the manifestation of pluripotency of stem cells (Klebes, 2005).

When prothoracic (1st) leg discs are fragmented and cultivated in vivo, cells in a proximodorsal region known as the 'weak point' can switch fate and transdetermine. These 'weak point' cells give rise to cuticular wing structures. The leg-to-wing switch is regulated, in part, by the expression of the vestigial (vg) gene, which encodes a transcriptional activator that is a key regulator of wing development. vg is not expressed during normal leg development, but it is expressed during normal wing development and in 'weak point' cells that transdetermine from leg to wing. Activation of vg gene expression marks leg-to-wing transdetermination (Klebes, 2005).

Sustained proliferation appears to be a prerequisite for fate change, and conditions that stimulate growth increase the frequency and enlarge the area of transdetermined tissue. Transdetermination was discovered when fragments of discs were allowed to grow for an extensive period of in vivo culture. More recently, ways to express Wg ectopically have been used to stimulate cell division and cell cycle changes in 'weak point' cells (Sustar, 2005), and have been shown to induce transdetermination very efficiently. Experiments were performed to characterize the genes involved in or responsible for transdetermination that is induced by ectopic Wg. Focus was placed on leg-to-wing transdetermination because it is well characterized, it can be efficiently induced and it can be monitored by the expression of a real-time GFP reporter. These attributes make it possible to isolate transdetermining cells as a group distinct from dorsal leg cells, which regenerate, and ventral leg cells in the same disc, which do not regenerate; and, in this work, to directly define their expression profiles. This analysis identified unique expression properties for each of these cell populations. It also identified a number of genes whose change in expression levels may be significant to understanding transdetermination and the factors that influence developmental plasticity. One is lamina ancestor (lama), whose expression correlates with undifferentiated cells and is shown to control the area of transdetermination. Another has sequence similarity to the mammalian augmenter of liver regeneration (Alr; Gfer -- Mouse Genome Informatics), which controls regenerative capacity in the liver and is upregulated in mammalian stem cells. Fifteen regulators of chromatin structure [e.g. members of the Polycomb group (PcG) and trithorax group (trxG)] are differentially regulated in transdetermining cells, and mutants in seven of these genes have significant effects on transdetermination. These studies identify two types of functions that transdetermination requires -- functions that promote an undifferentiated cell state and functions that re-set chromatin structure (Klebes, 2005).

The importance of chromatin structure to the transcriptional state of determined cells makes it reasonable to assume that re-programming cells to different fates entails reorganization of the Polycomb group (PcG) and trithorax group (trxG) protein complexes that bind to regulatory elements. Although altering the distribution of proteins that mediate chromatin states for transcriptional repression and activation need not involve changes in the levels of expression of the PcG and trxG proteins, the array hybridization data was examined to determine if they do. The PcG Suppressor of zeste 2 [Su(z)2] gene had a median fold repression of 2.1 in eight TD to DWg/VWg comparisons, but the cut-off settings did not detect significant enrichment or repression of most of the other PcG or trxG protein genes with either clustering analysis or the method of ranking median ratios. Since criteria for assigning biological significance to levels of change are purely subjective, the transdetermination expression data was re-analyzed to identify genes whose median ratio changes within a 95% confidence level. Fourteen percent of the genes satisfied these conditions. Among these genes, 15/32 PcG and trxG genes (47%) had such statistically significant changes. Identification of these 15 genes with differential expression suggests that transdetermination may be correlated with large-scale remodeling of chromatin structure (Klebes, 2005).

To test if the small but statistically significant changes in the expression of PcG and trxG genes are indicative of a functional role in determination, discs from wild-type, Polycomb (Pc), Enhancer of Polycomb [E(Pc)], Sex comb on midleg (Scm), Enhancer of zeste [E(z)], Su(z)2, brahma (brm) and osa (osa) larvae were examined. The level of Wg induction was adjested to reduce the frequency of transdetermination and both frequency of transdetermination and area of transdetermined cells was determined. The frequency of leg discs expressing vg increased significantly in E(z), Pc, E(Pc), brm and osa mutants, and the frequency of leg to wing transdetermination in adult cuticle increased in Scm, E(z), Pc, E(Pc) and osa mutants. Remarkably, Su(z)2 heterozygous discs had no vg expression, suggesting that the loss of Su(z)2 function limits vg expression (Klebes, 2005).

Members of the PcG and trxG are known to act as heteromeric complexes by binding to cellular memory modules (CMMs). The functional tests demonstrate that mutant alleles for members of both groups have the same functional consequence (they increase transdetermination frequency). The findings are consistent with recent observations that the traditional view of PcG members as repressors and trxG factors as activators might be an oversimplification, and that a more complex interplay of a varying composition of PcG and trxG proteins takes place at individual CMMs. Furthermore the opposing effects of Pc and Su(z)2 functions are consistent with the proposal that Su(z)2 is one of a subset of PcG genes that is required to activate as well as to suppress gene expression. In addition to measuring the frequency of transdetermination, the relative area of vg expression was examined in the various PcG and trxG heterozyogous mutant discs. The relative area decreased in E(Pc), brm and osa mutant discs, despite the increased frequency of transdetermination in these mutants. There is no evidence to explain these contrasting effects, but the roles in transdetermination of seven PcG and trxG genes that were identified by these results support the proposition that the transcriptional state of determined cells is implemented through the controls imposed by the regulators of chromatin structure (Klebes, 2005).

The determined states that direct cells to particular fates or lineages can be remarkably stable and can persist after many cell divisions in alien environments, but they are not immune to change. In Drosophila, three experimental systems have provided opportunities to investigate the mechanisms that lead to switches of determined states. These are: (1) the classic homeotic mutants; (2) the PcG and trxG mutants that affect the capacity of cells to maintain homeotic gene expression, and (3) transdetermination. During normal development, the homeotic genes are expressed in spatially restricted regions, and cells that lose (or gain) homeotic gene function presumably change the transcriptional profiles characteristic of the particular body part. In the work reported here, techniques of micro-dissection, RNA amplification and array hybridization were used to monitor the transcription profiles of cells in normal leg and wing imaginal discs, in leg disc cells that regenerate and in cells that transdetermine from leg to wing. The results validate the idea that changing determined states involves global changes in gene expression. They also identify genes whose function may be unrelated to the specific fates of the cells characterized, but instead may correlate with developmental plasticity (Klebes, 2005).

Overlap between the transcriptional profiles in the wing and transdetermination lists (15 genes) and with genes in subcluster IV (high expression in wing discs) is extensive. The overlap is sufficient to indicate that the TD leg disc cells have changed to a wing-like program of development, but interestingly, not all wing-specific genes are activated in the TD cells. The reasons could be related to the incomplete inventory of wing structures produced (only ventral wing) or to the altered state of the TD cells. During normal development, vg expression is activated in the embryo and continues through the 3rd instar. Although the regulatory sequences responsible for activation in the embryo have not been identified, in 2nd instar wing discs, vg expression is dependent upon the vgBE enhancer, and in 3rd instar wing discs expression is dependent upon the vgQE enhancer. Expression of vg in TD cells depends on activation by the vgBE enhancer, indicating that cells that respond to Wg-induction do not revert to an embryonic state. Recent studies of the cell cycle characteristics of TD cells support this conclusion (Sustar, 2005), but the role of the vgBE enhancer in TD cells and the incomplete inventory of 'wing-specific genes' in their expression profile probably indicates as well the stage at which the TD cells were analyzed: they were not equivalent to the cells of late 3rd instar wing discs (Klebes, 2005).

Investigations into the molecular basis of transdetermination have led to a model in which inputs from the Wg, Dpp and Hh signaling pathways alter the chromatin state of key selector genes to activate the transdetermination pathway. The analyses were limited to a period 2-3 days after the cells switched fate, because several cell doublings were necessary to produce sufficient numbers of marked TD cells. As a consequence, these studies did not analyze the initial stages. Despite this technical limitation, this study identified several genes that are interesting novel markers of transdetermination (e.g., ap, CG12534, CG14059 and CG4914), as well as several genes that function in the transdetermination process (e.g., lama and the PcG genes). The results from transcriptional profiling add significant detail to a general model proposed for transdetermination (Klebes, 2005).

(1) It is reported that ectopic wg expression results in statistically significant changes in the expression of 15 PcG and trxG genes. Moreover, although the magnitudes of these changes were very small for most of these genes, functional assays with seven of these genes revealed remarkably large effects on the metrics used to monitor transdetermination -- the fraction of discs with TD cells, the proportion of disc epithelium that TD cells represent, and the fraction of adult legs with wing cuticle. These effects strongly implicate PcG and trxG genes in the process of transdetermination and suggest that the changes in determined states manifested by transdetermination are either driven by or are enabled by changes in chromatin structure. This conclusion is consistent with the demonstrated roles of PcG and trxG genes in the self-renewing capacity of mouse hematopoietic stem cells, in Wg signaling and in the maintenance of determined states. The results now show that the PcG and trxG functions are also crucial to pluripotency in imaginal disc cells, namely that pluripotency by 'weak point' cells is dependent upon precisely regulated levels of PcG and trxG proteins, and is exquisitely sensitive to reductions in gene dose (Klebes, 2005).

The data do not suggest how the PcG and trxG genes affect transdetermination, but several possible mechanisms deserve consideration. A recent study (Sustar, 2005) reported that transdetermination correlates with an extension of the S phase of the cell cycle. Several proteins involved in cell cycle regulation physically associate with PcG and trxG proteins, and Brahma, one of the proteins that affects the metrics of transdetermination, has been shown to dissociate from chromatin in late S-phase and to reassociate in G1. It is possible that changes in the S-phase of TD cells are a consequence of changes in PcG/trxG protein composition (Klebes, 2005).

Another generic explanation is that transdetermination is dependent or sensitive to expression of specific targets of PcG and trxG genes. Among the 167 Pc/Trx response elements (PRE/TREs) predicted to exist in the Drosophila genome, one is in direct proximity to the vg gene. It is possible that upregulation of vg in TD cells is mediated through this element. Another factor may be the contribution of targets of Wg signaling, since targets of Wg signaling have been shown to be upregulated in osa and brm mutants. These are among a number of likely possible targets, and identifying the sites at which the PcG and trxG proteins function will be necessary if an understand is to be gained of how transdetermination is regulated. Importantly, understanding the roles of such targets and establishing whether these roles are direct will be essential to rationalize how expression levels of individual PcG and trxG genes correlate with the effects of PcG and trxG mutants on transdetermination (Klebes, 2005).

(2) The requirement for lama suggests that proliferation of TD cells involves functions that suppress differentiation. lama expression has been correlated with neural and glial progenitors prior to, but not after, differentiation, and it is observed that lama is expressed in imaginal progenitor cells and in early but not late 3rd instar discs. lama expression is re-activated in leg cells that transdetermine. The upregulation of unpaired in TD cells may be relevant in this context, since the JAK/STAT pathway functions to suppress differentiation and to promote self-renewal of stem cells in the Drosophila testis. It is suggested that it has a similar role in TD cells (Klebes, 2005).

(3) A role for Notch is implied by the expression profiles of several Notch pathway genes. Notch may contribute directly to transdetermination through the activation of the vgBE enhancer [which has a binding site for Su(H)] and of similarly configured sequences that were found to be present in the regulatory regions of 45 other TD genes. It will be important to test whether Notch signaling is required to activate these co-expressed genes, and if it is, to learn what cell-cell interactions and 'community effects' regulate activation of the Notch pathway in TD cells (Klebes, 2005).

(4) The upregulation in TD cells of many genes involved in growth and division, and the identification of DNA replication element (DRE) sites in the regulatory region of many of these genes supports the observation that TD cells become re-programmed after passing through a novel proliferative state (Sustar, 2005), and suggests that this change is in part implemented through DRE-dependent regulation (Klebes, 2005).

There was an interesting correlation between transdetermination induced by Wg mis-expression and the role of Wg/Wnt signaling for stem cells. Wg/Wnt signaling functions as a mitogen and maintains both somatic and germline stem cells in the Drosophila ovary, and mammalian hematopoetic stem cells. Although the 'weak point' cells in the Drosophila leg disc might lack the self-renewing capacity that characterizes stem cells, they respond to Wg mis-expression by manifesting a latent potential for growth and transdetermination. It seems likely that many of the genes are conserved that are involved in regulating stem cells and that lead to disease states when relevant regulatory networks lose their effectiveness (Klebes, 2005).

The prevalence of transcription factors among the genes whose relative expression levels differed most in the tissue comparisons was intriguing. It is commonly assumed that transcription factors function catalytically and that they greatly amplify the production of their targets, so the expectation was that the targets of tissue-specific transcription factors would have the highest degree of tissue-specific expression. In these studies, tissue-specific expression of 15 transcription factors among the 40 top-ranking genes in the wing and leg data sets (38%) is consistent with the large number of differentially expressed genes in these tissues, but these rankings suggest that the targets of these transcription factors are expressed at lower relative levels than the transcription factors that regulate their expression. One possible explanation is that the targets are expressed in both wing and leg disc cells, but the transcription factors that regulate them are not. This would imply that the importance of position-specific regulation lies with the regulator, not the level of expression of the target. Another possibility is that these transcription factors do not act catalytically to amplify the levels of their targets, or do so very inefficiently and require a high concentration of transcription factor to regulate the production of a small number of transcripts. Further analysis will be required to distinguish between these or other explanations, but it is noted that the prevalence of transcription factors in such data sets is neither unique to wing-leg comparisons nor universal (Klebes, 2005).

Modulation of AP and DV signaling pathways by the homeotic gene Ultrabithorax during haltere development

Suppression of wing fate and specification of haltere fate in Drosophila by the homeotic gene Ultrabithorax is a classical example of Hox regulation of serial homology (Lewis, E. B. 1978. Nature 276: 565–570) and has served as a paradigm for understanding homeotic gene function. DNA microarray analyses was used to identify potential targets of Ultrabithorax function during haltere specification. Expression patterns of 18 validated target genes and functional analyses of a subset of these genes suggest that down-regulation of both anterior–posterior and dorso-ventral signaling is critical for haltere fate specification. This is further confirmed by the observation that combined over-expression of Decapentaplegic and Vestigial is sufficient to override the effect of Ubx and cause dramatic haltere-to-wing transformations. These results also demonstrate that analysis of the differential development of wing and haltere is a good assay system to identify novel regulators of key signaling pathways (Mohit, 2005).

Suppression of wing fate and specification of haltere fate by Ubx is a classical example of Hox regulation, which has served as a paradigm for understanding the nature of homeotic gene function. Using microarray analyses and subsequent downstream validation by methods other than microarray, 18 potential targets have been identified of Ubx function during haltere specification. In addition, differential expression of Dpp at the transcriptional level has been observed between wing and haltere imaginal discs. Including previously known 13 targets, there are now as many as 32 well-established direct or indirect targets of Ubx function during haltere specification. Although Ubx may regulate additional downstream targets, the expression patterns of the genes identified suggest that negative regulation of D/V and A/P signaling is one of the important mechanisms by which Ubx specifies haltere development (Mohit, 2005).

The functional significance of down-regulation of these signaling pathways is confirmed by the dramatic homeotic transformations caused by ectopic activation of Dpp and/or Vg in developing haltere discs. These transformed halteres still lacked veins and wing margin bristles, indicating that Ubx specifies haltere development by additional mechanisms. Indeed, the EGFR pathway, which plays a significant role in specifying wing veins, is directly repressed by Ubx in haltere discs (S. K. Pallavi, unpublished observations reported in Mohit, 2005). Furthermore, over-expression of Dad in wing discs does not cause any obvious wing-to-haltere transformation nor do dppd6/dppd12 wings show such phenotypes. Thus, while over-expression of Dpp causes partial haltere-to-wing transformations, down-regulation of Dpp in wing discs has no such effect. Further investigation is needed to identify all the critical steps downstream of Ubx required to completely transform haltere to a wing or vice versa. Nevertheless, the dramatic homeotic transformations induced by the co-expression of just two genes (Dpp and Vg) suggest that down-regulation of these two steps by Ubx is critical to specify haltere fate (Mohit, 2005).

Although both Vg and Dpp are known to induce growth, it is believed that the observed homeotic transformation is due to re-patterning and trans-differentiation and not due to simple over-growth. Induction of over-growth in haltere leads to larger appendages, but not homeotic transformations. Furthermore, a recent report suggests that changes in cell division patterns alone do not lead to cell fate changes. Thus, Dpp/Vg-induced homeosis is a specific mechanism that overrides the effect of Ubx and suggests an important mechanism for Ubx function during haltere specification. Interestingly, in the mouse, signaling molecules such as Bmp2, Bmp7 and Fgf8 are downstream targets of Hoxa13 during the development of limbs and genitalia. Thus, down-regulation of Dpp and Wnt/Wg signaling pathways in Drosophila and Bmp and Fgf in mouse suggest a common theme underlying Hox gene function during appendage specification and development (Mohit, 2005).

The results presented in this report are significant in two ways. First, they suggest a mechanism by which halteres may have evolved from hind wings of lepidopteran insects. Ubx protein itself has not evolved among the diverse insect groups, although there are significant differences in Ubx sequences between Drosophila and crustacean Arthropods. Nevertheless, over-expression of Ubx derived from either a non-winged arthropod such as Onychophora or a four-winged insect such as Tribolium is sufficient to induce wing-to-haltere transformations in Drosophila. This suggests that, in the dipteran lineage, certain wing patterning genes have come under the regulation of Ubx. In such a scenario, it is likely that only a small number of genes will have their cis-regulatory sequences modified (converging mutations) to enable their regulation by Ubx. Considering the gross morphological differences between lepidopteran hind wings and halteres, any new target of Ubx will have greater influence on the entire hind wing morphology. Indeed, over-expression of Dpp and/or Vg caused dramatic haltere-to-wing homeotic transformations. Since such transformations were not observed by over-expressing their upstream regulators such as Hh, Ci, N or Wg, it is likely that direct targets of Ubx would be closer to Dpp and Vg in the hierarchy of gene regulation. Currently, chromatin immunoprecipitation experiments using haltere extracts are underway to identify those target genes (Mohit, 2005).

The second significant conclusion from the results described here is on the utility of differential development of wing and haltere as a good model system to identify additional components of both A/P and D/V signaling. Nine such genes have been identified, 8 of which show modulation of their expression patterns along the D/V axis. Based on restricted expression patterns and biochemical features of the encoded proteins, their possible involvement in maintaining the integrity of the D/V boundary as well as differences between dorsal and ventral compartments is predicted. Indeed, preliminary characterization of two genes suggests their probable roles to restrict Wg expression to the D/V boundary (Mohit, 2005).

A recent report has identified 16 potential genes downstream of mouse Hoxd cluster during the development of the most distal parts such as digits and genitals. Most of them have not been previously implicated in the early stages of either limb or genital bud development or as components of the known signal transduction pathways. Considering tissue- and developmental stage-specific expression of those genes, it is possible that those targets too could be novel modulators of known signal transduction pathways. Taken together, these results provide a framework for understanding the mechanisms by which Hox genes specify segment-specific developmental pathways (Mohit, 2005).

Control of growth and positional information by the graded vestigial expression pattern in the wing

The size and shape of organs depend on cellular processes such as cell proliferation, cell survival, and spatial arrangement of cells. In turn, all of these processes are a consequence of positional identity of individual cells in whole organs. Links of positional information with organ growth and pattern expression of genes is a little-addressed question. This study shows that differences in vestigial expression between neighboring cells of the wing blade autonomously and nonautonomously affect cell proliferation along the proximo-distal axis. In contrast, uniform expression of vestigial inhibits cell proliferation and also perturbs the shape of wing blade altering the preferential orientation of cell divisions. These observations provide evidence that local cell interactions, triggered by differences in vestigial expression between neighboring cells, confer positional values operating in the control of growth and shape of the wing (Baena-Lopez, 2006; full text of article).

The complementary activation of two independent enhancers (vg D/V boundary enhancer vgBE and vg quadrant enhancer vgQE) defines the heterogeneous pattern of vg expression along the A/P and P/D axes of growth. Vg is expressed 60 h after egg laying (AEL), forming a symmetrical gradient at both sides of the D/V boundary. Vg expression is also detected in provein territories at higher levels than in interveins, from the middle of the third instar on (96 h AEL). These heterogeneities in Vg distribution remain later during pupal development. This graded expression of vg suggests that it might be related to the positional information in the wing blade cells (Baena-Lopez, 2006).

Previous work has reported that morphogenetic mosaics defective in vg or sd expression grow poorly (3–5 cells) and disappear from the wing blade 24 h after clone initiation. This finding indicates that vg expression is formally required for the survival of wing blade cells. The cell survival and growth of mutant cells for vg were not favored in a Minute (M) genetic background either, dying quickly as it happens in a WT background. Alternatively, mutant clones were generated for vg (vgnull has deleted all exons) that simultaneously express the inhibitor of cell death puckered (UAS-puc2A). The size of these clones depends on their position along the P/D axis of growth, being recovered with higher frequency and larger size in the proximal territories of the vg expression domain. However, these clones express high levels of cell death markers (activated caspase-3) and tend to be extruded from the wing blade in larval stages, thus failing to appear in the adult wing. To better modulate the lack of function of vg, an RNAi construct of vg (vg-RNAi) was generated. The overexpression of vg-RNAi in either several G4 territories [distalless-G4 (dllG4md23), engrailed-G4 (enG4), and apterous-G4 (apG4md544)] or in clones diminishes the expression of vg in a dose (temperature)-dependent manner. The down-regulation of vg expression was correlated with autonomous size reductions of the wing blade. These reductions were not associated with changes in cell death or cell size during imaginal development. Interestingly, nonautonomous reductions (–13.5%) were also observed in surrounding WT territories along all axes of growth. Furthermore, clones of vg-RNAi frequently show an atypical morphology, becoming narrower where the amount of vg is higher. Thus, cell survival and proliferation of vg-expressing cells is closely correlated with the quantity of Vg and its pattern of expression in the wing blade (Baena-Lopez, 2006).

The capacity of vg to promote cell proliferation was investigated by driving the transgene UAS-vestigial in either different Gal4 territories of the wing or in genetic mosaics. Surprisingly, the regional overexpression of vg (under the control of apG4md544, nubbin-G4 (nubG4K), spalt-G4EPv (salG4EPv), and dllG4md23) reduces the size of the wing blade in an autonomous and nonautonomous manner. In these experiments, FACS and adult measurements of cell size were not significantly different from wild type. Cell death does not explain reductions of the wing size either (e.g., 44% in salG4EPv/UAS-vg) because the expression of activated Caspase-3 does not differ from wild type, and the inhibition of cell death (overexpressing UAS-puc2A, UAS-P35, or in a mutant background DfH99) only slightly rescues wing growth (7.4%) . Thus, reductions in wing size are mainly due to a lower cell proliferation such as it happens in the loss of function of vg (Baena-Lopez, 2006).

The effects of vg on cell proliferation in the wing blade was evaluated bt comparing the area and the expression of cell proliferation reporters [BrdU, proliferating cell nuclear antigen (PCNA), and PH3] between vg-overexpressing cells and WT cells. The overexpression of vg in wing blade territories autonomously reduces the area and the expression of cell proliferation reporters. Cell proliferation also diminishes in a dose-dependent manner because the lack of growth is strongly restored at 17°C. The most prominent reduction in size is visible along the P/D axis of growth (Baena-Lopez, 2006).

In contrast to the wing blade, vg-expressing cells in proximal wing territories (wing hinge) causes tubular outgrowths that are composed by mutant and wild type surrounding cells. The overgrowths in the wing hinge are correlated with an autonomous and nonautonomous increment of mitosis, Proliferating cell nuclear antigen (PCNA) expression and BrdU incorporation. These findings indicate that the autonomous and nonautonomous effects of vg on cell proliferation depend on the positional location along the P/D axis (Baena-Lopez, 2006).

To quantitatively evaluate the autonomous and nonautonomous effects of cells overexpressing vg along the axes of growth, an analysis of twin clones (48–72 h AEL) was carried out by using the system FRT/Gal80 (MACRM system). The area of vg-expressing clones increases proportionally with the distance to the wing margin in a continuous manner. Interestingly, the same variation in size is detected in their associated twin clones. These data suggest that differences in vg expression between neighboring cells drive cell proliferation, autonomously and nonautonomously, in the wing blade along the P/D axis. This conclusion is reinforced by data obtained expressing vg under the control of dllG4md23. dllG4md23 expression does not reach more proximal cells of vg expression domain, thus allowing the confrontation of juxtaposed cells with different levels of Vg. In mutant territories, mitoses and PCNA expression are preferably located in mutant cells in contact with or next to WT cells (Baena-Lopez, 2006).

Contrary to what happens in the P/D axis, the growth of vg-expressing clones and associated twins is not affected by their position along the A/P axis. In this axis, the heterogeneous pattern of vg possibly appears later in development or is related to vein differentiation. In fact, vg-expressing clones in adult wings tend to appear on vein territories (Baena-Lopez, 2006).

How are the different phenotypes of vg on growth correlated with the cell cycle? Overexpression of vg in the apG4md544 or enG4 domains accelerates the G1/S transition. However, G2/M transition also is delayed in these experiments, and vg has differential effects on growth in different territories of the wing discs. Thus, vg was overexpressed only in the wing blade (under the control of dllG4md23 or salG4EPv). Paradoxically, dllG4md23 and salG4EPv FACS experiments revealed the same profile of cell cycle than apG4md544 and enG4 experiments. Two conclusions can be drawn from these results. 1) The excess or lack of growth is not explained by the effect of vg on a particular regulatory point of cell cycle (G1/S transition) because compensatory mechanisms retard the G2/M phasing and 2) The control of growth is not cell autonomously determined but rather related to cell interactions between mutant and WT surrounding cells as in normal development (Baena-Lopez, 2006).

How is the length of the whole cell cycle modified in conditions of the lack or excess of function of vg? The cell doubling time of vg-expressing clones and twins (60 AEL) located in the wing hinge or the wing blade territories was calculated. Interestingly, outgrowths of wing hinge were correlated with a higher rate of cell proliferation and a shorter cell doubling time. On the contrary, the lack of function of vg in the wing blade increases the length of cell cycle. These findings suggest that the lack or excess of growth is a consequence of changes in the whole length of cell cycle, not in any particular cell cycle transition (Baena-Lopez, 2006).

The shape of the wing has been correlated with the preferential orientation of cell divisions along the P/D axis of growth. To analyze the possible links between positional identity and the orientation of cell division, a cell lineage study was performed in a genetic background overexpressing vg under the control of several G4 lines (salG4EPv, apG4md544, and nubG4K). In these mutant backgrounds, cell markers reveal that clones grow abnormally with respect to the wild type, and the orientation of cell divisions is altered, leading to rounded wings. These findings explain why the homogenous expression of vg perturbs the shape of the wing (Baena-Lopez, 2006).

In conclusion, this study has focused on the role of patterning genes, such as vg, in the control of cell proliferation of the wing to reach a normal size. The graded expression pattern of vg in the wing blade prompted an investigation pf its relationship with cell proliferation and positional identity in the wing disk (Baena-Lopez, 2006).

The homogenous expression of vg (by lack or excess of function) notably reduces the growth of the wing blade without changes in the cell size or rate of cell death during larval development. However, cell proliferation is locally maintained when different levels of vg between neighboring cells are confronted. These findings suggest that the homogenous rate of clone size throughout the wing blade requires local cell interactions between neighboring cells, and it is not cell autonomously determined by the amount of vg expression in cells. These observations clearly indicate that the autonomous expression of Vg–Sd complex does not stimulate cell proliferation per se (Baena-Lopez, 2006).

Discrepancies in vg expression experimentally induced between neighboring cells can autonomously and nonautonomously (accommodation) affect the wing blade growth in a position-dependent manner. Thus, vg-expressing clones autonomously show extra cell proliferation and outgrowths when they appear in the wing hinge; i.e., territories of the wing farther away from cells expressing high levels of vg. In contrast to examples of extra cell proliferation, reductions in size of the wing blade are observed in loss of function mosaics of vg. All of these effects on growth are continuously graded along the P/D axis in correlation with Vg distribution. These observations support the notion that vg expression is involved with a system of positional values, which is required to drive the growth in the wing blade. The effects on growth of Vg–Sd complex along the A/P axis are much less manifest, because the heterogeneities in its expression pattern possibly appear late during proliferation stages of wing discs (Baena-Lopez, 2006).

Gain or loss of function mosaics of vg have also nonautonomous effects. Interestingly, the whole wing also can be reduced even though the rate of cell proliferation is locally maintained in confronted cells expressing different levels of vg. This result is interpreted as a consequence of the following parameters being affected: (1) significant discrepancies of positional values (or vg expression) between neighboring cells fail to exist; (2) confronted populations of cells in mosaics are unable to reach their maximal positional values; (3) confronted cells exchange their positional information in a continuous and graded manner. In the loss-of-function mosaics of vg, mutant cells never achieve the maximal positional value (maximal expression of vg), nonautonomously diminishing also the reference of positional values to neighboring cells and, hence, to the whole wing. In these mosaics, the exchange of positional information apparently is perturbed, and mutant and WT territories are unable to intercalate the full scale of positional values corresponding to the WT situation, thus reducing the size of the whole wing. In contrast, vg-overexpressing clones in distal territories of the wing blade show nonautonomously reduced cell proliferation because mutant and surrounding cells do not have significant discrepancies in positional values. Conversely, outgrowths of the wing hinge appear in response to vg-expressing cells because the cell proliferation in the mutant cell is locally maintained and surrounding cells intercalate higher values up to a continuous landscape of positional values. According to these interpretations, the proliferation is stimulated by cell interactions between neighboring cells, but the growth of organs is controlled globally by the full scale of positional values locally exchanged between neighboring cells (Baena-Lopez, 2006).

Wg signaling is required to define the expression of vg and, therefore, the contribution of Wg signaling was analyzed in phenotypes of vg-expressing clones. The conclusion of these experiments is that Wg signaling does not contribute to cell proliferation but acts as a cell survival factor in vg-expressing cells (wing blade cells). In light of all these results, the notion and role of Wg as a morphogen perhaps should be revised. In contrast, the internalistic view (Entelechia model) was favored to explain the local control of growth in the wing blade. In this model, the heterogeneous expression of vg might be instrumental in specifying positional values, at least along the P/D axis, leading to the intercalar growth. The molecules involved in the control of short-range signals to exchange positional information between neighboring cells remain unknown; however, they should be able to transform the positional value associated with the amount of Vg in the nucleus to neighboring cells, eliciting cell division and providing intermedial positional values (Baena-Lopez, 2006).

The differential effects of Vg–Sd complex on growth are not correlated with changes in a single regulatory point of cell cycle but on changes in the whole length of the cell cycle. These findings indicate that the length of cell cycle is not cell autonomously determined by the amount of Vg–Sd but, rather, is likely related to local signaling between neighboring cells. This hypothesis agrees with the fact that cells nonclonally related form clusters of cells in the same phase of the cell cycle throughout the wing blade (Baena-Lopez, 2006).

The shape of the wing blade is strongly determined by the orientation of cell divisions. Somehow, the heterogeneous expression of vg contributes to polarize these orientations along the P/D axis because the homogenous expression of vg can randomize the characteristic WT orientations. It is difficult to propose molecular mechanisms to control the orientation of cell divisions; however, adhesion molecules might be implicated, affecting cell affinity and cell recognition properties. Supporting this hypothesis, it is known that vg modulates the level of dachsous (ds) in the membrane of wing blade cells, and ds expression participates in the preferential orientation of cell divisions. Taken as a whole, these results indicate that the orientation of cell division is intimately related to the mechanisms at work in positional identity specification (Baena-Lopez, 2006).

Effects of Mutation and Mis-expression

vestigial mutants undergo extensive cell death in the presumptive wing region of the third-larval instar imaginal discs, resulting in the observed complete elimination of wing structures in adults. Cell death is a consequence of a cell-autonomous inability of mutant discs to differentiate the wing region of the disc (Williams, 1991).

Timing of cell death differs among wingless, apterous, scalloped and vestigial mutants. Cell death in vg and sd discs occurs throughout third instar development, at a time when no death is observed in wg mutant discs. In wg mutant discs, no cell death is detected throughout the second instar; however, elevated cell death is detected at the edges of the presumptive wing region at the second to third instar molt. apterous mutants undergo increasing levels of cell death in the second instar larval period (Williams, 1993).

Mutations and duplications of vestigial and scabrous alter the severity of phenotypes associated with Notch mutations and duplications in a manner that is essentially tissue- and allele-specific. These interactions indicate that the products of vestigial and scabrous act in conjunction with Notch to stimulate the differentiation of specific cell types (Rabinow, 1990).

Lethal mutations in giant discs (lgd) and fat (ft) tumor suppressor genes cause epithelial hypertrophy in all imaginal discs. By contrast, mutations in the vestigial gene adversely affect cell viability in the wing disc. Combining lgd or ft mutations with vg increases the size of the wing disc and partially restores the bristle pattern (Agrawal, 1995). These results, along with the above described effects of antifolates (aminopterin) suggest that an important function of vg is to enhance cell survival and cell division.

Flies bearing a temperature sensitive allele of strawberry notch show a modest loss of wing margin tissue when raised at 23 degrees C. When such flies are also made heterozygous for a single copy loss of wingless, extensive loss of wing margin tissue is observed, suggesting a dominant synergistic interaction between wg and sno. In similar experiments, temperature sno combined with a single copy loss of vestigial also results in a dominant enhancement of wing margin defects; mutants exhibit extensive loss of wing margin tissue. Genetic combination of a weak allele of cut with a sno mutation shows extensive loss of wing margin tissue, suggesting a synergistic interaction between sno and cut. These results are reminiscent of the interaction of Notch with wg, vg and ct and further extablish that sno, like Notch, has a crucial role in the establishment of D/V boundary fate by participating in a common genetic pathway that regulates wing margin-specific genes. In addition to the wing margin defects, sno mutants also exhibit thickening of wing veins. This is likely to be a secondary consequence of defective wing pouch development caused by improper D/V boundary specification. This same phenotype can also be seen in some of the other D/V boundary genes, such as vestigial and Serrate (Majumdar, 1997).

The vestigial gene product is required for the completion of wing development in Drosophila. In the absence of vestigial gene expression, cells within the larval wing and haltere imaginal discs fail to proliferate normally thus producing adults with severely reduced wings. Of a large number of vestigial mutations that have been characterized, only two are currently known to exist: vestigial(U) and vestigial(W), both of which manifest a significant dominant phenotype. Both are associated with chromosomal inversions that fuse the majority of the vestigial coding regions to other genes; mastermind in vestigial(U) and invected in vestigial(W). Examination of vestigial expression in the presence of these dominant alleles shows alterations in the disc-specific expression of vestigial during later stages of larval development. These patterning disruptions are specific to cells of the wing imaginal disc, since significant suppression of total levels of vestigial expression within entire larvae cannot be detected. This dominant interference of vestigial patterning appears to be mediated in part by the vestigial coding sequences that are within the gene fusions. Further evidence that the dominant phenotype is the result of disrupted vestigial patterning comes from observations that the dominant alleles can be partially suppressed by mutations within the Drosophila epidermal growth factor receptor gene. Mutagenesis of vestigial(U) and vestigial(W) produce a series of alleles with partially dominant phenotypes that restored various amounts of the adult wing. These phenotypes can be correlated with alterations in specific portions of the vestigial sequences associated with the dominant alleles. In the presence of these partially dominant alleles, wing imaginal discs have significantly more cells that express vestigial as compared with the number associated with the original dominant phenotype. Eliminating some of the dominant effect causes alterations in the patterns of early stag apoptotic cell death associated with dominant vestigial alleles. It is suggested that the dominant vg alleles interfer with the establishemt of endogeneous vg mediated patterning along the D/V margin. Utilizing these new vestigial alleles, it is possible to correlate the consequence of altered vestigial expression to subsequent changes in patterning of the wing disc (Simmonds, 1997).

The invected gene of Drosophila melanogaster is a homeobox-containing gene that is closely related to engrailed. A dominant gain of function allele, invectedDominant, was derived from mutagenesis of a dominant allele of vestigial, In(2R)vgW. A careful analysis of the phenotype of invectedDominant shows that it is associated with the transformation of the anterior compartment of the wing into a posterior fate. This transformation is normally limited to the wing blade itself and does not involve the remaining tissues derived from the wing imaginal disc, including the wing hinge and dorsal thorax of the fly. The ectopic expression of Invected protein associated with invectedDominant correlates spatially with the normal expression pattern of vestigial in the wing imaginal disc, suggesting that control elements of vestigial are driving ectopic invected expression. This was confirmed by sequence analysis, which has shown that the dominant vestigial activity is eliminated by a deletion that removes the 3' portion of the vestigial coding region. This leaves a gene fusion wherein the vestigial enhancer elements are still juxtaposed immediately 5' to the invected transcriptional start site, but with the vg sequences harboring an additional lesion. Unlike recessive invected alleles, the invectedDominant allele produces an observable phenotype, and as such should prove useful in determining the role of invected in patterning the wing imaginal disc. Genetic analysis has shown that mutations of polyhomeotic, a gene involved in regulating engrailed expression, cause a reproducible alteration in the invectedDominant phenotype. Finally, the invectedDominant allele should prove valuable for identifying and characterizing genes that are activated within the posterior compartment. A screen using various lacZ lines that are asymmetrically expressed in an anterior-posterior manner in the wing imaginal disc has isolated one line that shows posterior-specific expression within the transformed anterior compartment (Simmonds, 1998b).

Dual role for Drosophila epidermal growth factor receptor signaling in early wing disc development

Cell fate decisions in the early Drosophila wing disc assign cells to compartments (anterior or posterior and dorsal or ventral) and distinguish the future wing from the body wall (notum). Egf receptor signaling stimulated by its ligand, Vein, has a fundamental role in regulating two of these cell fate choices: (1) Vn/EGFR signaling directs cells to become notum by antagonizing wing development and by activating notum-specifying genes; (2) Vn/EGFR signaling directs cells to become part of the dorsal compartment by induction of apterous, the dorsal selector gene, and consequently also controls wing development, which depends on an interaction between dorsal and ventral cells (Wang, 2000).

To determine when Vn/EGFR signaling is required for notum development, the temperature-sensitive alleles, Egfrtsla and vntsWB240 were used. Inactivating Vn/Egfr activity during the second instar (a 24 hr period) causes loss of the notum. The wing develops but shows pattern abnormalities characteristic of vn hypomorphs. Later shifts during the third instar does not cause loss of the notum. This demonstrates that Vn/Egfr activity is required for notum development in the second instar when wg is required to specify the wing. Thus, Vn and Wg appear to have complementary roles and this relationship has been examined by following their expression in mutants (Wang, 2000).

In second instar wild-type wing discs, wg is expressed distally in a wedge of anterior ventral cells and vn is expressed proximally. In vn null mutants, the initiation of wg expression is normal as is expression of its target gene optomotor-blind (omb). In wg mutants, however, there is a dramatic and early expansion of vn expression to include distal cells, presaging the development of these cells as an extra notum. Together these results suggest that Vn has an early role in establishing the notum and that Wg signaling is required to define a distal domain that is reduced in Egfr activity to allow wing development (Wang, 2000).

It is suggested that the mechanisms by which wg and vn specify alternate cell fates in the early wing disc, wing, or notum are antagonistic. This is based on the observation that loss of Wg results in the spread of vn expression and the supposition that the resulting ectopic Egfr activity causes loss of the wing and a double notum phenotype. Further evidence that Vn/Egfr signaling represses wing development comes from the results of misexpressing a constitutive receptor, Egfrlambdatop4.2, in the presumptive wing. In these flies, the wing is reduced to a stump covered with sensilla characteristic of the proximal wing (hinge) region and expression of the wing specific gene vestigial (vg) is repressed. Ectopic notal structures also form from the ventral pleura. The ability of ectopic Egfr signaling to suppress wing development is cell autonomous because clones of cells expressing Egfrlambdatop4.2 lack vg expression. In adult wings these clones produced outgrowths lacking wing characteristics but are otherwise difficult to characterize (Wang, 2000).

Although vn expression expands in wg mutants, no reciprocal spread of wg expression was observed in vn mutants that would have been indicative of a double wing phenotype. However, when Vn/Egfr signaling is inhibited in the notum by expressing a ligand antagonist (Vn::Aos-EGF) under the control of ptc-Gal4, ectopic wings are induced in ~10% of the flies. This result demonstrates that presumptive notal tissue can be transformed to wing by reducing Egfr signaling. However, the transformation occurs only when Egfr signaling is reduced in a subset of cells, rather than all cells in the notum (as in a vn mutant). This may reflect the indirect requirement for Egfr activity to also promote wing development (Wang, 2000).

The loss of notum phenotype is characteristic of vn hypomorphs but in null vn alleles and some Egfr alleles both the wing and notum primordia fail to develop and the wing discs remain tiny. Thus, although ectopic activity of Egfr in the distal disc represses wing development, the pathway is nevertheless normally required for wing development. Using the temperature-sensitive Egfrtsla allele it was found that this requirement is restricted to the period from mid-first to mid-second instar. Key genes involved in wing development that are active at this time include wg and apterous (ap). ap is expressed in dorsal cells and acts as a selector gene to divide the disc into dorsal and ventral compartments. Regulation of Notch ligands by Ap leads to Notch signaling at the DV boundary and the formation of an organizer for wing outgrowth and expression of the wing-specific transcription factor vg (Wang, 2000 and references therein).

Of these two candidates, wg and ap, it seemed unlikely that wg was the key gene affected by Egfr signaling from mid-first to mid-second instar because wg expression is normal in vn mutants at mid-second instar. However, later in the second instar, wg expression normally expands to fill the growing wing pouch and it was noted that in vn mutants, wg expression fails to undergo this expansion. A similar defect in wg expression is seen in ap mutants consistent with Ap function being impaired in vn mutants. Remarkably, ap expression is completely absent in second instar vn mutant discs. Thus, loss of Ap can explain why there is no wing in vn mutants. This is supported by the demonstration that ectopic ap is capable of rescuing wing development in vn mutants (Wang, 2000).

Several additional lines of evidence demonstrate that ap is a cell autonomous target of Vn/Egfr signaling and that this relationship exists only transiently in early wing development: (1) ap expression partially overlaps that of vn in the second instar; (2) ap can be induced ectopically in ventral clones misexpressing an activated form of the receptor, Egfrlambdatop4.2; (3) Egfrtsla mutant clones generated in the first instar show autonomous loss of ap expression, whereas clones generated in the second instar express ap normally. Finally, loss of Egfr activity in whole discs from mid-first to mid-second instar results in complete loss of ap expression, whereas ap is still expressed in discs from larvae given a temperature shift slightly later during the second instar (Wang, 2000).

The results described here suggest that division of the early wing disc into presumptive wing and body wall regions is defined by the action of two secreted signaling molecules, Wg and Vn. wg, a pro-wing gene, is required to repress vn expression, which at high levels antagonizes wing development. Antagonism between Wg and Egfr signaling has also been demonstrated in segmental patterning of the embryo and in development of the head and third instar wing pouch, suggesting such a relationship between these pathways may be a common theme in a number of cell fate choices. Finding that one of the main functions of Wg in early wing specification is to repress Vn/Egfr signaling in the distal region of the early disc raises the question as to whether this is the only role of Wg in wing specification and hence if wing-cell fate can be specified in the absence of both signals. This seems unlikely, because nubbin, an early wing cell marker, is not misexpressed proximally in a vn mutant, where cells would lack both signals (Wang, 2000).

Vn/Egfr signaling promotes development of the notum by maintaining its own activity through transcriptional activation of vn itself, and also promotes expression of ap. Thus, both vn and ap appear to be targets of Egfr signaling, but the domain of ap is clearly wider than that of vn, indicating that ap can be activated at a lower signaling threshold than vn. Vn is a secreted molecule and thus could generate a gradient of Egfr activity. This provides an explanation for how Egfr signaling can regulate both wing and notum development: vn autoregulation and notum development requires high Egfr signaling activity while ap expression and subsequent wing development requires lower signaling activity (Wang, 2000).

Interestingly, vertebrate Egfr and its ligands are expressed in the chick limb bud in a pattern that appears to overlap with the vertebrate ap homolog Lhx2, and these factors are required for limb outgrowth in the chick. In light of the present results it will be important to determine whether Egfr signaling controls Lhx2 expression and thus plays a role in regulating outgrowth of the vertebrate limb. These results may also have implications for the evolution of insect wings. If the control of body wall development by Egfr signaling is ancestral, and comparative analysis of other arthropods will be required to assert this, then one of the first steps towards evolution of wings could have occurred when Egfr signaling assumed control of ap (Wang, 2000).

Roles for scalloped and vestigial in regulating cell affinity and interactions between the wing blade and the wing hinge

The scalloped and vestigial genes are both required for the formation of the Drosophila wing, and recent studies have indicated that they can function as a heterodimeric complex to regulate the expression of downstream target genes. The consequences of complete loss of scalloped function, ectopic expression of scalloped, and ectopic expression of vestigial for the development of the Drosophila wing imaginal disc have been analyzed. Clones of cells mutant for a strong allele of scalloped fail to proliferate within the wing pouch, but grow normally in the wing hinge and notum. Cells overexpressing scalloped fail to proliferate in both notal and wing-blade regions of the disc, and this overexpression induces apoptotic cell death. Clones of cells overexpressing vestigial grow smaller or larger than control clones, depending upon their distance from the dorsal-ventral compartment boundary. These studies highlight the importance of correct scalloped and vestigial expression levels to normal wing development. Studies of vestigial-overexpressing clones also reveal two further aspects of wing development. (1) In the hinge region vestigial exerts both a local inhibition and a long-range induction of wingless expression. These and other observations imply that vestigial-expressing cells in the wing blade organize the development of surrounding wing-hinge cells. (2) Clones of cells overexpressing vestigial exhibit altered cell affinities. The analysis of these clones, together with studies of scalloped mutant clones, implies that scalloped- and vestigial-dependent cell adhesion contributes to separation of the wing blade from the wing hinge and to a gradient of cell affinities along the dorsal-ventral axis of the wing (Liu, 2000).

Clones of cells in imaginal tissues generally adopt very irregular shapes. Strikingly, however, clones of cells that are ectopically expressing Vg are more rounded and have smoother borders than control clones. Similar differences in clone behavior have been observed upon misexpression or mutation of a number of different genes in Drosophila and have been attributed to differences in the affinity of cells for their neighbors. This effect of Vg is evident in the notum, hinge, and proximal regions of the wing, as well as in other imaginal discs. Within the wing region of the disc, the influence of Vg overexpression on clone shape is graded: clones that are near the D-V wing border generally continue to have irregular shapes, while clones that are far from the D-V border are more circular. This effect was quantified by calculating the circularity of 117 Vg-expressing clones and then plotting the circularity of each clone against its relative distance from the D-V wing border. Circularity is a ratio of clone area to the square of the clone perimeter. The average circularity of VG-expressing clones increases with distance from the D-V border. The observation that the same, constitutive level of Vg expression induces graded changes in clone shape that depend upon clone location suggests that there are normally graded differences in Sd:Vg-dependent cell affinities. Three aspects of the behavior of sd clones in the wing blade are consistent with the hypothesis that reduction in normal Sd:Vg function also influences cell affinity. (1) In the few instances in which relatively large sd clones were recovered, they tended to be more rounded than their wild-type twins. (2) Over a quarter of the sd clones recovered in the wing pouch were associated with multiple wild-type 'twin' clones. Although in some cases this may occur fortuitously, it is also suggested that affinity differences with surrounding wild-type cells could force independent mutant clones into a coherent patch. (3) sd clones tend to be located farther from the D-V boundary than their wild-type twins. Differential location between mutant and wild-type twins has also been observed in the wing for shaggy mutant clones and in the abdomen for patched and smoothened mutant clones, and in both cases it has been hypothesized to derive from differences in cell affinity (Liu, 2000).

While previous studies have emphasized the autonomous requirement for vg in wing development, these results make clear that this autonomous requirement is restricted to the wing blade and that Sd:Vg has an additional, nonautonomous role in promoting the development of the wing hinge. Null alleles of vg delete the wing blade and most, or sometimes all, of the wing hinge. Even when vg mutant animals retain some hinge tissue, a significant amount of tissue is deleted proximal to the inner Wg expression ring. However, by making clones of cells mutant for sd, it was found that Sd:Vg is autonomously required only distal to the inner Wg expression ring. Similarly, clones of cells that are mutant for a null allele of vg grow normally in the notum, but fail to grow in the wing. The precise border where vg is autonomously required maps to the edge of detectable Vg expression. This places the border distal to the inner Wg expression ring. Altogether, these results suggest that Sd:Vg is required nonautonomously for normal development of the wing hinge. Indeed, clones of cells ectopically expressing Vg frequently reorganize the patterning of surrounding tissue in the wing hinge. This reorganization is visible through changes in the expression of Wg and Nubbin, as well as changes in the folding of the disc epithelia. These studies, along with reports on the function and regulation of hth in the hinge, lead to a model for the regulatory interactions between wing hinge and wing blade (Liu, 2000).

The observation that Sd:Vg is both required nonautonomously for normal hinge development and sufficient to reorganize the normal patterning of surrounding hinge tissue leads to the hypothesis that Sd:Vg-expressing wing blade cells produce a signal (X) that influences gene expression in surrounding wing-hinge cells. Ultimately, one key target of this signal is the inner ring of Wg hinge expression. Wg is essential for wing hinge development; Wg expression is induced non-autonomously by Sd:Vg, and normal Wg hinge expression is reduced or absent in vg mutants. The detection of a spot of Wg expression in some vg mutant discs that appears to correspond to a portion of the inner hinge ring implies that the hypothesized signal X may not be absolutely required for Wg expression. Instead, it may function to maintain and promote Wg hinge expression as the wing pouch grows. Alternatively, it may be, as suggested by the failure of Vg-expressing clones to effectively induce Wg hinge expression near the D-V boundary, that Wg hinge expression near the D-V boundary is regulated by a Vg-independent mechanism, which continues to promote a spot of Wg expression even in vg mutants (Liu, 2000). Although the identity of the signal X is not yet known, nor how direct its regulatory influence on Wg may be, it can be inferred that its action ultimately impinges on enhancers within a 1.2-kb fragment of the wg gene identified as being responsible for the distal ring of Wg hinge expression. Recent studies of Drosophila leg development have implied the existence of signaling from proximal cells to distal cells. Thus, in both legs and wings, normal appendage development appears to rely not just on the direct interpretation of primary signals produced along compartment boundaries, but also on secondary signaling between cells in different domains along the proximal-distal axis (Liu, 2000).

While these studies imply that a Sd:Vg-dependent signal is essential for normal hinge development, hinge cells are uniquely competent to express Wg in response to this signal. This implies that a distinct hinge fate precedes receipt of the signal. In addition, a small amount of wing-hinge tissue, and in some cases Wg expression, remains in vg null mutants. Signaling from the wing blade does not therefore act as an inducer of wing-hinge fate per se, but rather acts to elaborate the patterning and growth of the hinge. hth plays a key role in hinge development, and recent studies have demonstrated that hth is essential for Wg expression in the hinge. Thus Hth, together with its partner protein Extradenticle (Exd), may be at least partially responsible for the distinct responsiveness of hinge cells to Sd:Vg-dependent signaling. Hth expression is itself positively regulated by Wg, and thus the distinct fates of both the wing blade and the wing hinge are maintained in part by positive regulatory loops with Wg. Separate blade and hinge territories are also maintained in part by repressive interactions between Sd:Vg and Exd:Hth. However, while the repression of Hth by Sd:Vg is autonomous, and thus may be direct, Hth does not repress Sd:Vg directly, but instead represses Wg expression along the D-V border, which then indirectly limits Sd:Vg expression (Liu, 2000).

Myoblast diversification and ectodermal signaling in Drosophila

The flight muscles of Drosophila derive from myoblasts found within the third instar disc. These myoblasts already show distinctive properties: how is this diversity generated was examined? In the late larva, Vestigial and low levels of Cut are expressed in myoblasts that will contribute to the indirect flight muscles. Other myoblasts, which express high levels of Cut but no Vestigial, are required for the formation of the direct flight muscles. Vestigial and Cut expression are stabilized by a mutually repressive feedback loop. Vestigial expression begins in the embryo in a subset of adult myoblasts, and Wingless signaling is required later to maintain this expression. Thus, myoblasts are divided into identifiable populations, consistent with their allocation to different muscles, and ectodermal signals act to maintain these differences (Sudarsan, 2001).

The indirect flight muscles (IFMs) are divided into two classes on the basis of their location; the dorsal longitudinal muscles (DLMs) and the dorsoventral muscles (DVMs). These, together with the direct flight muscles (DFMs), constitute the dorsal muscles of the adult thorax and derive from myoblasts that lie over the notal region of the developing wing disc epithelium. They have been considered to be a uniform population of cells, capable of contributing to a variety of muscles. Consistent with this view, they uniformly express the transcription factors encoded by twist (twi) and Dmef2 (Sudarsan, 2001).

These myoblasts are segregated into two distinct groups well before the onset of myoblast fusion. In late third instar discs, a large group of proximally situated myoblasts expresses Vestigial (Vg), while a more distal group located around the hinge region does not express this gene. These two groups of myoblasts also differ in their levels of Cut expression; Vg-expressing cells show low levels of Cut while cells not expressing Vg are marked by high levels of Cut (Sudarsan, 2001).

It was asked whether the segregation of these two groups of myoblasts, expressing different combinations of transcription factors, predicts a distinctive contribution to the development of specific adult flight muscles. The development of the IFMs was examined in flies bearing adult viable alleles of vg. In two strong alleles, vg1 and vg83b27-R, the DLMs are severely reduced and the DVMs are minimal or completely missing. To assess the gain-of-function phenotype, a UAS-vg transgene was expressed in all wing disc-associated myoblasts using the Gal4 driver 1151. This leads to alterations in the development of the DFMs, so that muscles 51 and 52 are always missing. In contrast, the IFMs develop normally. Viable hypomorphic allelic combinations of cut show no muscle phenotype and therefore do not allow the examination of effects of loss of function on adult muscle development. To assess the effects of cut gain of function, uniformly high levels were expressed in all the myoblasts, which results in the virtual complete loss of IFMs, whereas the DFMs are unaffected (Sudarsan, 2001).

Thus, while the overexpression of Vg or Cut leads to the reduction or loss of one class of muscle, it is striking that the alternative class is not expanded, as would be expected if ectopically expressing cells switched their fate and executed the newly specified myogenic program. The alternative possibility, that cells forced into a new developmental program die was investigated. Pupal wing discs in which Cut had been overexpressed in all the myoblasts were stained for the general myoblast marker, Twi, and for acridine orange. Twi staining reveals an approximately 10-fold reduction in the number of myoblasts overlying the IFM larval templates, and acridine orange shows a greatly increased incidence of cell death. Driving maximal levels of Cut (at 29°C) results in a dramatic reduction in disc-associated myoblasts even earlier, by the end of the larval period. Together these results indicate that alterations in the pattern of gene expression in myoblasts leads to cell death (Sudarsan, 2001).

The location of the two populations of myoblasts on the notum, together with phenotypes for the gain and loss of function for vg and for cut gain of function, suggests that the IFMs derive from proximal, Vg-expressing, low Cut myoblasts and the DFMs from more distal, Vg-negative, high Cut myoblasts. Since these distinctive patterns of gene expression are observed in the third instar, before myoblast fusion, this hypothesis can be tested by following the fate of Vg-expressing cells through pupal development. Indeed, myoblasts expressing Vg contribute to the developing IFMs. Vg-expressing myoblasts overlie the larval templates on which the DLMs develop and in the muscles below after myoblast fusion. After the larval templates have split, the DFMs have many Vg-expressing nuclei. In contrast, the vast majority of myoblasts that contribute to DFMs do not express Vg, either during fusion or later (Sudarsan, 2001).

Together these results show that the third instar myoblasts are partitioned into two populations from which cells contribute to the IFMs or DFMs and that the larval patterns of vg and cut expression play an important role in specifying these populations (Sudarsan, 2001).

The notum epithelium is patterned during the second and third instars and could therefore act to pattern the myoblasts associated with it. Since the myoblasts and wing disc arise as separate structures in the embryo, Vg expression was examined in early myoblasts to see whether they adopt different patterns of gene expression only after they associate with the disc epithelium. The progenitors of adult myoblasts are specified in the embryo and are characterized by the persistent expression of twist. Embryos stained for twi-lacZ expression, to identify adult progenitors, and with an antibody against Vg reveal a few Vg-expressing adult myoblasts lying between the leg and wing disc primordia from late stage 12. Vg expression is maintained in a subset of myoblasts during the first and second instars as they become associated with the wing disc. By 74 hr AEL (early third instar), approximately six Vg-expressing adult myoblasts are seen associated with the stalk of the wing disc. Thus, the initial patterning of Vg expression in the adult myoblasts is independent of patterning of the wing disc epithelium (Sudarsan, 2001).

Although embryonic myoblasts are allotted to sets that express Vg and those that do not, the other transcription factor, Cut, whose level of expression also distinguishes myoblast groups, is initiated only later, in mid third instar larvae. During the third instar, all adult myoblasts express Cut, but they are in two distinct groups: a distal group that expresses high levels of Cut and a proximal group, also distinguished by the expression of Vg, that expresses Cut weakly (Sudarsan, 2001).

Together these data show that the adult myoblast progenitors diversify during embryogenesis before they are associated with the wing disc and suggest that these differences are maintained and elaborated after the myoblasts migrate onto the disc. Might the disc epithelium regulate these later stages of myoblast patterning (Sudarsan, 2001)?

If the specification of the adult myoblasts is influenced by the disc epithelium, signals must be able to diffuse between germ layers to stimulate the myoblasts. A striking feature of the presumptive notum of the wing disc is an ectodermal stripe of wingless (wg) expression, which is initiated in the third instar. When conventional fixation procedures are used, Wg is detectable at the apical surface of the disc epithelium, whereas the protein gradient forms on the basolateral domain, adjacent to the myoblast layer. To assess whether Wg secreted from epidermal cells could signal to the associated myoblasts, a truncated, nonfunctional, GPI-linked form of the Wingless receptor, DFrizzled2, was expressed in all the adult myoblasts. Whether Wg protein was detectable in the mesodermal layer was then examined. It was found that Wg can cross between the germ layers and bind to the myoblasts in a graded fashion, covering a domain that is wider than the Wg stripe (Sudarsan, 2001).

It was asked whether myoblasts were receptive to inductive signaling from the epidermis and whether wg is required for the divergence of the two myoblast populations in wild-type third instar discs. Using a temperature-sensitive allele, wgIL114, Wingless function was removed from the second instar onward. This results in a strong reduction of Vg expression in the myoblasts. The importance of signaling through the canonical Wg pathway was tested by expressing a dominant-negative form of the transcriptional effector, TCF, in all the myoblasts of the notum. A complete loss of Vg expression was found, and, in the adult, the IFMs were much reduced in size. In contrast, when the Wg pathway was activated by expressing armS10, a constitutively active form of Armadillo, throughout the notum myoblasts, a small expansion of Vg expression into the most distal population of myoblasts was found. In the adult, some DFMs are lost, while those that remain are very reduced in size. These results show that spatially organized Wg signaling from the ectoderm is required to maintain Vg expression in the adjoining population of myoblasts (Sudarsan, 2001).

In order to compare the levels of Cut in wild-type and genetically manipulated cells within single populations of myoblasts, mitotic clones were generated of myoblasts carrying a mutation in dishevelled (dsh) and were therefore unable to transduce Wg signaling. As expected, dsh clones lose Vg expression, but they also show an increase in the level of Cut expression relative to neighboring wild-type myoblasts. This indicates that, in addition to its role in maintaining Vg expression, Wg signaling is also required, directly or indirectly, to modulate myoblast expression of Cut. Interestingly, changes in gene expression are confined to the mutant cells, indicating a cell-autonomous response; the induction of secondary signaling by Wg-activated cells is not involved (Sudarsan, 2001).

Since high levels of Cut are found only in the absence of Vg and Vg-expressing cells that show depressed levels of Cut, the relationship between Vg and Cut expression was examined by inducing overexpression of each gene in all the notum myoblasts. A general expression of Vg results in uniformly low levels of Cut expression, while the induction of uniformly high Cut virtually abolishes Vg expression. These results reveal a negative regulatory loop between these two gene products, which from the third instar will act to maintain the distinction between the two groups of notum myoblasts, namely, those expressing Vg, that will contribute to the IFMs, and those expressing Cut at high levels that contribute to the DFMs (Sudarsan, 2001).

A characterization of the effects of Dpp signaling on cell growth and proliferation in the Drosophila wing

Cell proliferation and patterning must be coordinated for the development of properly proportioned organs. If the same molecules were to control both processes, such coordination would be ensured. This possibility has been investigated in the Drosophila wing using the Dpp signaling pathway. Previous studies have shown that Dpp forms a gradient along the AP axis that patterns the wing, that Dpp receptors are autonomously required for wing cell proliferation, and that ectopic expression of either Dpp or an activated Dpp receptor, TkvQ253D, causes overgrowth. These findings are extended with a detailed analysis of the effects of Dpp signaling on wing cell growth and proliferation. Increasing Dpp signaling by expressing TkvQ253D accelerates wing cell growth and cell cycle progression in a coordinate and cell-autonomous manner. Conversely, autonomously inhibiting Dpp signaling using a pathway specific inhibitor, Dad, or a mutation in tkv, slows wing cell growth and division, also in a coordinate fashion. Stimulation of cell cycle progression by TkvQ253D is blocked by the cell cycle inhibitor RBF, and requires normal activity of the growth effector, PI3K. Among the known Dpp targets, vestigial was the only one tested that was required for TkvQ253D-induced growth. The growth response to altering Dpp signaling varies regionally and temporally in the wing disc, indicating that other patterned factors modify the response (Martín-Castellanos, 2002).

In the wing imaginal disc, omb, spalt and vestigial (vg) have been reported to respond to Dpp signaling. It was of interest to know which if any of these genes was involved in controlling tissue growth effected by TkvQ253D. spalt is probably not required, since Spalt protein is not induced by TkvQ253D expression in the lateral areas of the wing disc, where the strongest overgrowth effects are observed. In the case of omb and vg, null alleles were used as a genetic background in which the expression of the activated Dpp receptor was induced. TkvQ253D can promote growth in the absence of Omb (Martín-Castellanos, 2002).

By contrast, TkvQ253D is not able to promote tissue growth in a null vg83b27R background. This result points to Vg as a possible effector of growth induced by Dpp signaling. Consistently, ectopic Vg expression induces wing-like outgrowths in imaginal discs. However, it was surprising to find that clones expressing TkvQ253D do not show increased levels of Vg protein, regardless of their position in the disc. Some lateral clones express Vg, but these most probably originate in the Vg expression domain. In fact, clones in lateral positions where Vg is expressed over-grow better than in other regions. These results suggest that activation of Dpp signaling is not sufficient to induce Vg expression, but that TkvQ253D and Vg might synergize to effect tissue growth (Martín-Castellanos, 2002).

The growth response of a cell to altered Dpp signaling varies according to its location in the disc. Ectopic TkvQ253D causes the strongest over-growth phenotypes in lateral regions, far from the source of endogenous Dpp, whereas inhibition of Dpp signaling has the strongest phenotypes in medial areas of the disc, where Dpp levels are normally high. Similar region-specific responses have been observed in experiments in which Notch or Wingless signaling is activated ectopically using cell autonomous effectors, or ligands. What is the significance of these region-specific responses? Without knowing the pertinent growth regulatory targets of these signaling systems, it is only possible to speculate. Perhaps the differential responses reflect cooperation between several regionally expressed signals that affect tissue growth, both positively and negatively, in a combinatorial fashion. Observations relating to vg seem consistent with this possibility. vg is required by TkvQ253D to promote tissue growth, yet Vg protein is not up-regulated by ectopic TkvQ253D, and TkvQ253D is capable of promoting overgrowth in wing regions where Vg is not detectable. The complex growth responses of cells to Dpp signaling illustrate how much is unknown about mechanisms of growth control. New, more global, approaches to studies of growth modulation will be required before its regulation by patterning signals can be understood. Important tasks for future studies include identifying the Dpp targets that stimulate cellular metabolism to effect growth, and determining how these targets integrate input from other patterning signals such as Wingless, Notch, Hedgehog and the Egfr ligands (Martín-Castellanos, 2002).

A characterization of the effects of Dpp signaling on cell growth and proliferation in the Drosophila wing

Compartment formation is a developmental process that requires the existence of barriers against intermixing between cell groups. In the Drosophila wing disc, the dorso-ventral (D/V) compartment boundary is defined by the expression of the apterous selector gene in the dorsal compartment. Ap activity is under control of dLMO (Beadex) which destabilizes the formation of the Ap-Chip complex. D/V boundary formation in the wing disc also depends on early expression of vestigial. These data suggest that vg is already required for wing cell proliferation before D/V compartmentalization. In addition, over-expression of vg can, to some extent, rescue the effect of the absence of ap on D/V boundary formation. Early Vg product regulates Ap activity by inducing dLMO and thus indirectly regulating ap target genes such as fringe and the PSalpha1 and PSalpha2 integrins. It is concluded that normal cell proliferation is necessary for ap expression at the level of the D/V boundary. This would be mediated by vg, which interacts in a dose-dependent way with ap (Delanoue, 2002).

An in vivo analysis of the vestigial gene in Drosophila melanogaster defines the domains required for vg function

Considerable evidence indicates an obligate partnership of Vestigial (Vg) and Scalloped (Sd) proteins within the context of wing development. It is evident that Sd and Vg act together as a transcriptional complex during wing formation, wherein Sd provides the DNA-binding activity and nuclear localization signal, while Vg provides the activation function. A 56-amino-acid motif within Vg is necessary and sufficient for binding of Vg with Sd. While the importance of this Sd-binding domain has been clearly demonstrated both in vitro and in vivo, the remaining portions of Vg have not been examined for their in vivo function(s). Herein, additional regions within Vg were tested for possible in vivo functions. The results identify two additional domains that must be present for optimal Vg function as measured 1) by the loss of ability to rescue vg mutants, 2) by the ability to induce ectopic sd expression, and 3) by the ability to perform other normal Vg functions when these domains are deleted. An in vivo study such as this one is fundamentally important because it identifies domains of Vg that are necessary in the cellular context in which wing development actually occurs. The results also indicate that an additional large portion of Vg, outside of these two domains and the Sd-binding domain, is dispensable in the execution of these normal Vg functions (MacKay, 2003).

From results using ectopic sd-lacZ induction (which measures the ability of ectopic vg to induce ectopic sd expression), the ability to rescue vg mutations, and the ability to carry out other functions associated with normal vg, it can be discerned that certain portions of the vg ORF, in addition to the Sd-binding domain, are necessary to accomplish normal Vg function. These appear to be the critical regions, since other portions can be deleted without effect. More specifically, the N-terminal amino acids (approximately the first 65) and C-terminal residues from 335 to 453 seem to play an important role in the induction of sd-lacZ. When the N-terminal deletion Delta5'-5 (deleting amino acids 2-65) is assayed, the ectopic expression ability is reduced markedly compared to that seen with the full-length vg construct, although it is not eliminated completely. Moreover, the larger N-terminal deletions (amino acids 2-170 and 2-278, respectively) do not further lower the ability to express sd. Thus, it seems that the fundamentally important region is already removed with the Delta5'-5 construct. For C-terminal deletions Delta1-4 and Delta1-2 (amino acids 356-453 and 335-426, respectively), the ability to ectopically express sd is much less than that produced by full-length vg but somewhat stronger than that produced when the N-terminal deletion constructs are assayed. Deletions Delta5'-5, Delta5'-6, and Delta5'-7 retain the encoded amino acids missing from Delta1-4 and Delta1-2 and vice versa. Taken together, these data suggest the presence of two important functional domains for Vg: one within amino acids 1-65 (domain 1) and the other within amino acids 336-453 (domain 2). Although the precise boundaries of these domains have not yet been determined, domain 1 is very likely within the first 65 amino acids (deleted in vgDelta5'-5) since this is the region most highly conserved between D. melanogaster and the mosquito Aedes egyptii. There is 82% identity over the first 66 amino acids, but over the next 20 amino acids the identity drops to 35% and drops even further beyond that. In agreement with this notion, the extent of 'functional' loss in UASDeltavg 5'-6 and 5'-7 is no stronger than that exhibited by UASDeltavg 5'-5, which deletes the first 65 amino acids only. The activity of domain 2 appears to be weaker, since domain 1 deletions produce a slightly more drastic impairment of Vg function than do domain 2 deletions (amino acids 356-453 or 335-426). However, homology between Drosophila and mosquito Vg is also high within the Sd-binding domain of Vg and, in fact, remains strong to the carboxyl terminus of Vg (82% identity from residue 335 to 453. The data define the presence of two necessary functional domains for the Vg protein in vivo. These domains correlate well with data that predict two activation regions using in vitro experiments, including yeast one-hybrid assays. The regions identified in this study also complement more recent in vitro data, implicating these regions of Vg as necessary for binding of the Vg/Sd complex to target genes (MacKay, 2003).

Control of apterous by vestigial drives indirect flight muscle development in Drosophila

Drosophila thoracic muscles are comprised of both direct flight muscles (DFMs) and indirect flight muscles (IFMs). The IFMs can be further subdivided into dorsolongitudinal muscles (DLMs) and dorsoventral muscles (DVMs). The correct patterning of each category of muscles requires the coordination of specific executive regulatory programs. DFM development requires key regulatory genes such as cut (ct) and apterous (ap), whereas IFM development requires vestigial (vg). Using a new vgnull mutant, a total absence of vg is shown to lead to DLM degeneration through an apoptotic process and to a total absence of DVMs in the adult. vg and scalloped (sd), the only known Vg transcriptional coactivator, are coexpressed during IFM development. Moreover, an ectopic expression of ct and ap, two markers of DFM development, is observed in developing IFMs of vgnull pupae. In addition, in vgnull adult flies, degenerating DLMs express twist (twi) ectopically. Evidence is provided that ap ectopic expression can induce per se ectopic twi expression and muscle degeneration. All these data seem to indicate that, in the absence of vg, the IFM developmental program switches into the DFM developmental program. Moreover, the muscle phenotype of vgnull flies can be rescued by using the activity of ap promoter to drive Vg expression. Thus, vg appears to be a key regulatory gene of IFM development (Bernard, 2003).

Thus the absence of Vg leads to IFM degeneration. Some IFM phenotypes have been reported for the vg83b27R allele, a strong allele of vg. In these flies, the DVMs are absent and some DLMs are missing. It has been shown that this phenotype is fully penetrant in vgnull flies and that apoptosis is involved in loss of IFMs. Since muscle attachment sites are normal in vgnull flies, the process of degeneration is different from that described in ap mutants. Phenotypic analysis shows that degeneration occurs during late metamorphosis (after 48 h APF) (Bernard, 2003).

Vg interacts with Sd to form a transcription factor that binds DNA through the Sd TEA/ATTS domain and activates transcription through the Vg activation domain. Since vgnull mutants show drastic muscle degeneration phenotypes, Vg and sd expressions were examined. Vg is expressed in adepithelial cells. Vg is expressed in myoblasts around the forming DLMs and in some of the DLM nuclei. Moreover, sd expression is expressed in adepithelial cells and developing IFMs. Vg is present in all DLM nuclei and sd is coexpressed with Vg. It is therefore likely that in muscle, as in the wing disc, Sd and Vg are obligate partners. This result is supported by indirect arguments: (1) Vg dimerization with Sd is necessary for Vg activity. Protein interaction has been shown between Vg and Strawberry Notch (SNO), but the function of this new partner remains unknown; (2) Vg is localized to the nucleus in muscles, and nuclear relocalization of Vg in S2 cells requires the presence of Sd. However, no muscle phenotypes were found in sd strong hypomorphic viable mutants (sd58 and sd3L). It is concluded that if Sd is required for muscle development, a very low level of sd product is sufficient to fulfill its function. There is some precedent for this type of situation: for example, whereas Ct is necessary for DFMs development, viable ct mutant alleles do not exhibit any muscle phenotypes (Bernard, 2003).

One of the aims of developmental biology is to determine how a given cell population undergoes specific developmental program. This means trying to determine when cell commitment is specified and what are the factors involved. Adepithelial cells were at first considered as a homogenous population that expressed Twi. Adepithelial cells can, however, be considered as two distinct populations. The population, that forms DFMs, expresses a high level of Ct and does not express Vg. The second population forms IFMs and expresses Vg and a low level of Ct. In addition, Ct and Vg levels are stabilized by a repressive feedback loop: overexpression of Ct in all myoblasts using the 1151-GAL4 driver leads to Vg repression and to IFMs-specific apoptotic degeneration; overexpression of Vg in all adepithelial cells using the same driver leads to Ct repression and to DFM degeneration. The data in vgnull flies are consistent with these observations: absence of Vg leads to Ct derepression in adepithelial cells, in myoblasts surrounding DLMs and in developing DLMs. However, Ct overexpression phenotypes in DLMs are slightly different from those observed in vgnull flies. Splitting of the three larval templates (LOMs) into six DLMs occurs normally and at 48 h APF DLMs are morphologically normal in vgnull flies. It is concluded that DLM degeneration is a late event in vgnull mutants. In contrast, Ct overexpression leads to early degeneration of myoblasts and muscle fibers. This suggests that Ct overexpression induces apoptosis independent of Vg repression. Therefore, it would seem that the effect of Ct overexpression using the 1151-GAL4 driver is not equivalent to that observed in the absence of Vg (Bernard, 2003).

In vgnull mutants all adepithelial cells express high levels of Ct, while this is normally only the case of DFM-forming myoblasts. Is DLM degeneration in vgnull mutants the result of engagement of DLMs toward a DFM-like differentiation process? To answer this question, ap expression was examined in vgnull developing and adult DLMs. In wild-type flight muscles, ap expression is specific to DFMs and begins at 17-19 h APF. In vgnull flies, ap expression is found in developing DLMs at 21 h APF, in myoblasts surrounding DLMs and in adult muscles. Moreover, an absence of actin 88F expression was found in vgnull developing IFMs, suggesting that IFM differentiation is disrupted. Interestingly, as in wild-type flies, no expression was found in adepithelial cells. These data show that ap starts to be expressed at the same stage in DLMs of vgnull flies and in DFMs of the wild type strain (Bernard, 2003).

In summary, the following has been demonstrated in vgnull flies: (1) DLM-forming myoblasts express high levels of Ct, an early marker for DFM-forming myoblasts and (2) myoblasts and developing and adult degenerating DLMs express ap, a specific late DFM marker, whereas actin 88F expression, an IFM-specific differentiation marker, is lost. According to these data, it is supposed that in the vgnull mutants, adepithelial cells and developing DLMs enter into a DFM-like development. The suggestion that ap ectopic expression may impose a DFM identity on the IFMs has already been proposed. However, an IFM-to-DFM transformation was not observed; rather, IFMs degenerated through an apoptotic process. Similarly, DFMs were not transformed into IFMs upon overexpression of Vg in DFM-forming myoblasts. Instead, DFM degeneration was obtained. This suggests that Vg and AP are major actors but are not sufficient for IFM and DFM development, respectively. Other signals and factors must be required to specify these muscles. Nerve-muscle interaction is associated with IFM development. Wnt oncogene analog 2 (Dwnt-2) expression is required in the vicinity of the developing DFMs for patterning of DFMs. Thus, it appears that adult muscle development requires complex interactions between several kinds of signals delivered in specific localizations. In vgnull homozygous flies, adepithelial cells and swarming myoblasts express DFM markers, but their position on the wing imaginal disc and in the pupa remains unchanged with respect to wild type. Thus, developing IFMs receive IFM signaling (at least nerve-muscle interactions), but myoblasts express apterous, a DFM maker. Moreover, they lack information necessary for formation of either DFMs or IFMs (absence of vg expression). It is suggested that IFM degeneration in vgnull homozygous flies is the result of this complex interaction between two contradictory signals (IFM and DFM) associated with incomplete signaling for formation of either type of muscle (Bernard, 2003).

Attempts were made to rescue the vgnull muscle phenotype by targeted Vg overexpression using the UAS-GAL4 system. Significant rescue was obtained with the ap-GAL4 driver. It is therefore likely that ectopic activation of the ap-GAL4 transgene in vgnull DLMs and myoblasts occurs when Vg is required for DLM formation. Since ap activation in vgnull myoblasts and developing DLMs occurs after puparium formation, it is concluded that a late Vg expression is sufficient to restore the DLM developmental process. This implies that adepithelial cell determination by the level of Ct at the wing disc is reversible. Thus, even though earlier Ct levels distinguish two adepithelial cell populations that will differentiate into DFMs or IFMs, definitive DFM versus IFM determination is a later event that takes place during metamorphosis. vg and ap could be key genes during specification of IFMs and DFMs, respectively. To support this hypothesis, ubiquitous overexpression of ap was shown to be sufficient to induce specific DLM degeneration. The way in which AP and Vg direct muscle development toward a DFM or IFM fate remains unclear. However, it is well known that muscle fibers express specific structural genes or isoforms. Since ap and vg encode transcription factors, they are probably involved in specific genes activation. For example, misexpression of ap in developing IFMs represses the expression of actin 88F, an IFM-specific actin gene. Moreover, no actin 88F expression is found in a vgnull context. However, it is not currently known whether AP or Vg can directly activate or repress structural genes. Interestingly, the Sd mammalian homolog (Transcription Enhancer Factor-1, TEF-1) has been shown to bind muscle-specific promoters, like the cardiac alpha-Myosin Heavy Chain and the cardiac Troponin T promoters. It is therefore possible that the Sd-Vg dimer plays a similar role in Drosophila, directly activating structural genes. Further studies are necessary to address this question (Bernard, 2003).

Thus DLMs degenerate by apoptosis in homozygous vgnull flies. This degeneration could be due to a misprogramming of myoblasts surrounding DLMs during development. The process that leads to apoptosis in these muscles remains to be determined. DLM degeneration is associated with an ectopic expression of Twi transcription factor. During flight muscle development, Twi expression is restricted to myoblasts and that persistent expression in developing muscles leads to muscle degeneration. Thus, Twi expression in vgnull mutants could be responsible for DLM degeneration. Finally, it has been shown that ectopic ap expression induces Twi expression in DLMs. Since AP and twi are known to be, respectively, activator and target of the N pathway, it can be hypothesized that AP activates Twi ectopically in vgnull DLMs through the N pathway. If this hypothesis is confirmed, it can be asked why AP does not activate Twi during normal DFM development. It is likely that numerous genes, other than vg and ap, are differentially activated during DFM and IFM development. Twi activation by AP could be repressed by one of these genes during DFM development (Bernard, 2003).

In this study, evidence is provided that vg is required to change DFM-forming myoblasts into IFM-forming myoblasts. As in wing development where Vg is considered as a selector gene, Vg could be a key gene in IFM specification. Its function would be equivalent to that of Ap for DFM development. DFM fate inhibition through repression of ct and ap by Vg seems therefore to be a key regulation feature of IFM development. Thus, correct programming and regulation of these three genes are necessary for correct patterning of Drosophila flight muscles (Bernard, 2003). A NAME="Mis">

Mis-expression studies define the genetic requirements of vestigial in the regulation of wing development

The gene vestigial has been proposed to act as a master gene because of its supposed capacity to initiate and drive wing development. The ectopic expression of vestigial only induces ectopic outgrowths with wing cuticular differentiation and wing blade gene expression patterns in specific developmental and genetic contexts. In the process of transformation, wingless seems to be an essential but insufficient co-factor of vestigial. vestigial ectopic expression alone or vestigial plus wingless co-expression in clones differentiate 'mixed' cuticular patterns (they contain wing blade trichomes and chaetae characteristic of the endogenous surrounding tissue) and express wing blade genes only in patches of cells within the clones. In addition, these clones, in the wing imaginal disc, may cause autonomous as well as non-autonomous cuticular transformations and wing blade gene expression patterns. These non-autonomous effects in surrounding cells result from recruitment or 'inductive assimilation' of vestigial or wingless-vestigial overexpressing cells (Baena-López, 2003).

The notion of 'master gene', as applied to the gene eyeless, corresponds to a gene that by itself would trigger a developmental program that is independent of the tissue where it is expressed. Although this definition has been applied to vg, the present results indicate otherwise. The ectopic expression of vg elicits certain characteristics of 'wing blade' development but is not sufficient for a complete transformation. The effect of vg depends on the time and genetic context of the tissue where it is overexpressed. These results reveal a strong dependence of vg on wg to initiate a wing blade developmental pathway. Wg by itself does not lead to tissue transformations. This cooperative effect between wg and vg remains insufficient in all tissues analyzed, suggesting the existence of additional genes necessary to initiate and drive wing development. The molecular mechanisms that underlie the interaction between wg pathway and vg are not known. However, the co-expression of vg with a construct of armadillo (arm) (transcriptional effector of wg pathway) using vg-G4 fails to promote the transformation of eye tissue. This result suggests that the interaction of wg and vg takes place upstream of arm and, therefore, outside of the cell nucleus. Whereas vg requires high levels of wg expression to initiate wing development, the clones of vg overexpression contain in later stages, low or null levels of wg expression. Moreover, wg-vg co-overexpression clones can also show low levels of wg, even when wg is also mobilized in G4 territories or in Flip-out clones. These results suggest that vg may indirectly reduce wg expression once wing development is already initiated, and may explain why the transformed tissue in vg clones does not contain wing margin cuticular elements. The late repression of wg seems to be important to specify territories of the wing blade depending on vg expression outside of the wing margin; if high levels of Wg are maintained, all cells differentiate into wing margin chaetae. It is concluded that wg and vg activities together specify wing margin territories, but vg alone specifies the remaining part of the wing blade (Baena-López, 2003).

The ectopic expression of vg or wg-vg in clones may cause outgrowths with wing histotypic characteristics or patterning perturbations in the notum, leg or eyes. The transformed tissues show 'mixed' phenotypes or 'mosaic' territories where, in a 'salt and pepper' distribution, wing blade trichomes co-exist with notum or leg chaetae. Adult cuticular 'mixed' phenotypes are correlated with the ectopic expression of wing blade genes in particular combinations. However, expression of wing blade genes is detected only in some compact groups of cells within the clones. These results indicate that either vg or wg-vg are insufficient by themselves to displace all endogenous signals of identity, or that reciprocal non-autonomous influences between clonal cells and surrounding cells exist, reducing the expression of wing blade genes to groups of cells within clones. The change of wing blade genes expression in compact groups of cells in the disc and 'mixed' (salt and pepper) cuticular phenotypes in the adult could result from cell interactions during patterning and cell rearrangements in pupal stages (Baena-López, 2003).

Transformations induced by overexpression of vg or wg-vg in clones and G4 territories are, as a rule, cell autonomous, except in the wing hinge, notum and corresponding tissues in the haltere. In the wing hinge the cells of the outgrowths outside the vg clones differentiate into wing blade territories and show gene expression patterns characteristic of the wing blade cells located between the proximal vg expression and the internal ring of wg in the wild-type disc. This suggests that the non-autonomous effects in vg clones could reproduce the wild-type intercalary growth induced by the confrontation of cells expressing proximal genes with distal genes. In the notum, vg clones located simultaneously in territories expressing and not expressing ap, and initiated in the wg expression domain, may non-autonomously recruit surrounding cells to express characteristic wing blade genes at long cell distances, as wg-vg clones do. Thus, vg (together with wg expression) is necessary to induce and extend the transformation over long distances outside the clones. In contrast to vg or wg-vg clones, wg clones do not show non-autonomous transformation phenotypes and expression of wing blade genes at long distances. The issue of whether the recruitment process is caused by Wg diffusion, or whether it results from intercalary growth induced by the confrontation between cells expressing proximal genes (genes of the notum) and cells expressing distal genes (wing blade genes), remains unresolved (Baena-López, 2003).

The expression of selector genes like Ubx and en is not modified by overexpression of vg or wg-vg, but is inherited and maintained. However, the expression of the selector gene ap can be modified or inherited in some tissues, such as the legs, to give DV identity (Baena-López, 2003).

The comparative analysis of vg with other morphogenetic genes suggests that vg acts as Dll, pnr or iro, rather than as a 'master' or 'selector of tissue' gene: vg is simply a component of the genetic combination that is necessary to initiate and drive wing blade development where vg is normally expressed. Interestingly, the function of vg, in addition to conferring territorial identity, may also non-autonomously recruit surrounding cells ('inductive assimilation'), changing their specific cuticular and gene expression patterns. This is related to its function as a local organizer of growth when it is expressed among cells with different positional or regional fates. Later in development, vg, in combination with other genes, activates an inventory of downstream wing genes that specify more discrete territories within the wing blade such as veins, interveins and sensory elements (Baena-López, 2003).


REFERENCES

Agrawal, N., et al. (1995). Epithelial hyperplasia of imaginal discs induced by mutations in Drosophila tumor suppressor genes: growth and pattern formation in genetic mosaics. Dev. Biol 169: 387-398. PubMed Citation: 7781886

Azpiazu, N. and Morata, G. (2000). Function and regulation of homothorax in the wing imaginal disc of Drosophila. Development 127: 2685-2693. Medline abstract: 10821766

Baena-López, L. A. and García-Bellido. A. (2003). Genetic requirements of vestigial in the regulation of Drosophila wing development. Development 130: 197-208. 12441303

Baena-Lopez, L. A. and García-Bellido, A. (2006). Control of growth and positional information by the graded vestigial expression pattern in the wing of Drosophila melanogaster. Proc. Natl. Acad. Sci. 103: 13734-13739. Medline abstract: 16950871

Bate, M. and Rushton, E. (1993). Myogenesis and muscle patterning in Drosophila. C. R. Acad. Sci. III 316: 1047-1061. PubMed Citation: 8076205

Bernard, F., et al. (2003). Control of apterous by vestigial drives indirect flight muscle development in Drosophila. Dev. Biol. 260: 391-403. 12921740

Blair, S. S. (1994). A role for the segment polarity gene shaggy-zeste white 3 in the specification of regional identity in the developing wing of Drosophila. Dev Biol 162: 229-44. PubMed Citation: 8125190

Bonnet, A., Dai, F., Brand-Saberi, B. and Duprez, D. (2010). Vestigial-like 2 acts downstream of MyoD activation and is associated with skeletal muscle differentiation in chick myogenesis. Mech. Dev. 127(1-2): 120-36. PubMed Citation: 19833199

Campbell, G. and Tomlinson, A. (1999). Transducing the Dpp morphogen gradient in the wing of Drosophila: regulation of Dpp targets by brinker. Cell 96(4): 553-62. Medline abstract: 10052457

Carrasco-Rando, M., et al. (2011). Drosophila araucan and caupolican integrate intrinsic and signalling inputs for the acquisition by muscle progenitors of the lateral transverse fate. PLoS Genet. 7(7): e1002186. PubMed Citation: 21811416

Casares, F. and Mann, R. S. (2000). A dual role for homothorax in inhibiting wing blade development and specifying proximal wing identities in Drosophila. Development 127: 1499-1508. Medline abstract: 10704395

Certel, K., et al. (2000). Restricted patterning of vestigial expression in Drosophila wing imaginal discs requires synergistic activation by both Mad and the Drifter POU domain transcription factor. Development 127: 3173-3183. Medline abstract: 10862753

del Alamo Rodriguez, D., Terriente Felix, J., Diaz-Benjumea, F. J. (2004). The role of the T-box gene optomotor-blind in patterning the Drosophila wing. Dev. Biol. 268(2): 481-92. 15063183

Delanoue, R., et al. (2002). Interaction between apterous and early expression of vestigial in formation of the dorso-ventral compartments in the Drosophila wing disc. Genes Cells 7(12): 1255-66. 12485165

Deng, H., Bell, J. B. and Simmonds, A. J. (2010). Vestigial is required during late-stage muscle differentiation in Drosophila melanogaster embryos. Mol. Biol. Cell 21: 3304-3316. PubMed Citation: 20685961

Djiane, A., Zaessinger, S., Babaoglan, A. B., Bray, S. J. (2014). Notch inhibits yorkie activity in Drosophila wing discs. PLoS One 9: e106211. PubMed ID: 25157415

Doherty, D., et al. (1996). Delta is a ventral to dorsal signal complementary to Serrate, another Notch ligand, in Drosophila wing formation. Genes Dev. 10: 421-434. PubMed Citation: 8600026

Felix, J. T., Magarinos, M. and Diaz-Benjumea, F. J. (2007). Nab controls the activity of the zinc-finger transcription factors Squeeze and Rotund in Drosophila development. Development 134(10): 1845-52. Medline abstract: 17428824

Fryer, C. J., White, J. B. and Jones, K. A. (2004). Mastermind recruits CycC:CDK8 to phosphorylate the Notch ICD and coordinate activation with turnover. Mol. Cell 16: 509-520. PubMed Citation: 15546612

Furriols M. and Bray, S. (2000). Dissecting the mechanisms of Suppressor of Hairless function. Dev. Bio. 227: 520-532. PubMed Citation: 11071771

Fuse, N., Hirose, S. and Hayashi, S. (1996). Determination of wing cell fate by the escargot and snail genes in Drosophila. Development 122: 1059-1067. PubMed Citation: 8620833

Go, M. J., Eastman, D. S. and Artavanis-Tsakonas, S. (1998). Cell proliferation control by Notch signaling in Drosophila development. Development 125: 2031-2040. PubMed Citation: 9570768

Goulev, Y., et al. (2008). SCALLOPED interacts with YORKIE, the nuclear effector of the hippo tumor-suppressor pathway in Drosophila. Curr. Biol. 18(6): 435-41. PubMed Citation: 18313299

Guss, K. A. et al. (2001). Control of a genetic regulatory network by a selector gene. Science 292: 1164-1167. 11303087

Guss, K. A., Mistry, H. and Skeath, J. B. (2008). Vestigial expression in the Drosophila embryonic central nervous system. Dev. Dyn. 237(9): 2483-9. PubMed Citation: 18697219

Halder, G., et al. (1998). The Vestigial and Scalloped proteins act together to directly regulate wing-specific gene expression in Drosophila. Genes Dev. 12(24): 3900-9. PubMed Citation: 9869643

Halder, G. and Carroll, S. B. (2001). Binding of the Vestigial co-factor switches the DNA-target selectivity of the Scalloped selector protein. Development 128: 3295-3305. 11546746

Hasson, P., et al. (2001). Brinker requires two corepressors for maximal and versatile repression in Dpp signalling. EMBO J. 20: 5725-5736. 11598015

Hepker, J., Blackman, R. K. and Holmgren, R. (1999). Cubitus interruptus is necessary but not sufficient for direct activation of a wing-specific decapentaplegic enhancer. Development 126: 3669-3677. PubMed Citation: 10409512

Herzog, V. A., Lempradl, A., Trupke, J., Okulski, H., Altmutter, C., Ruge, F., Boidol, B., Kubicek, S., Schmauss, G., Aumayr, K., Ruf, M., Pospisilik, A., Dimond, A., Senergin, H. B., Vargas, M. L., et al. (2014). A strand-specific switch in noncoding transcription switches the function of a Polycomb/Trithorax response element. Nat. Genet. 46(9):973-981. PubMed ID: 25108384

Jaiswal, M., Agrawal, N. and Sinha, P. (2006). Fat and Wingless signaling oppositely regulate epithelial cell-cell adhesion and distal wing development in Drosophila. Development 133(5): 925-35. 16452097

Janody, F. and Treisman, J. E. (2006). Actin capping protein α maintains vestigial-expressing cells within the Drosophila wing disc epithelium. Development 133(17): 3349-57. Medline abstract: 16887822

Janody, F. and Treisman, J. E. (2011). Requirements for mediator complex subunits distinguish three classes of notch target genes at the Drosophila wing margin. Dev. Dyn. 240(9): 2051-9. PubMed Citation: 21793099

Katsuyama, T., Sugawara, T., Tatsumi, M., Oshima, Y., Gehring, W. J., Aigaki, T. and Kurata, S. (2005). Involvement of winged eye encoding a chromatin-associated bromo-adjacent homology domain protein in disc specification. Proc. Natl. Acad. Sci. 102(44): 15918-23. 16247005

Kim, J., et al. (1996). Integration of positional signals and regulation of wing formation and identity by Drosophila vestigial gene. Nature 382: 133-138. PubMed Citation: 8700202

Kim, J., et al. (1997). Drosophila Mad binds to DNA and directly mediates activation of vestigial by Decapentaplegic. Nature 388: 304-8. PubMed Citation: 9230443

Kirkpatrick, H., Johnson, K. and Laughon, A. (2001). Repression of Dpp targets by binding of Brinker to Mad sites. J. Biol. Chem. 276: 18216-18222. 11262410

Klebes, A., et al. (2005). Regulation of cellular plasticity in Drosophila imaginal disc cells by the Polycomb group, trithorax group and lama genes. Development 132: 3753-3765. 16077094

Klein, T. and Martinez Arias, A. (1999). The Vestigial gene product provides a molecular context for the interpretation of signals during the development of the wing in Drosophila. Development 126: 913-925. PubMed Citation: 9927593

Klein, T., et al. (2000). Two different activities of Suppressor of Hairless during wing development in Drosophila. Development 127: 3553-3566. PubMed Citation: 10903180

Kölzer, S., Fuss, B., Hoch, M. and Klein, T. (2003). defective proventriculus is required for pattern formation along the proximodistal axis, cell proliferation and formation of veins in the Drosophila wing. Development 130: 4135-4147. 12874133

Kugler, S. J. and Nagel, A. C. (2010). A novel Pzg-NURF complex regulates Notch target gene activity. Mol. Biol. Cell 21(19): 3443-8. PubMed Citation: 20685964

Kurata, S., et al. (2000). Notch signaling and the determination of appendage identity. Proc. Natl. Acad. Sci. 97: 2117-2122. PubMed Citation: 10681430

Lee, N., Maurange, C., Ringrose, L. and Paro, R. (2005). Suppression of Polycomb group proteins by JNK signalling induces transdetermination in Drosophila imaginal discs. Nature 438(7065): 234-7. 16281037

Legent, K., Dutriaux, A., Delanoue, R. and Silber, J. (2006). Cell cycle genes regulate vestigial and scalloped to ensure normal proliferation in the wing disc of Drosophila melanogaster. Genes Cells 11(8): 907-18. 16866874

Ligoxygakis, P., et al. (1999). Ectopic expression of individual E(spl) genes has differential effects on different cell fate decisions and underscores the biphasic requirement for Notch activity in wing margin establishment in Drosophila. Development 126: 2205-2214. PubMed Citation: 10207145

Liu, X., Grammont, M. and Irvine, K. D. (2000). Roles for scalloped and vestigial in regulating cell affinity and interactions between the wing blade and the wing hinge. Dev. Biol. 228: 287-303. PubMed Citation: 11112330

MacKay, J. O., et al. (2003). An in vivo analysis of the vestigial gene in Drosophila melanogaster defines the domains required for vg function. Genetics 163: 1365-1373. 12702681

McKay, D. J. and Lieb, J. D. (2013). A common set of DNA regulatory elements shapes Drosophila appendages. Dev Cell 27: 306-318. PubMed ID: 24229644

Maeda, T., Chapman, D. L. and Stewart, A. F. (2002). Mammalian Vestigial-like 2, a cofactor of TEF-1 and MEF2 transcription factors that promotes skeletal muscle differentiation. J. Biol. Chem. 277(50): 48889-98. 12376544

Majumdar, A., Nagaraj, R. and Banerjee, U. (1997). strawberry notch encodes a conserved nuclear protein that functions downstream of Notch and regulates gene expression along the developing wing margin in Drosophila. Genes Dev. 11: 1341-1353. PubMed Citation: 9171377

Martín-Castellanos, C. and Edgar, B. A. (2002). A characterization of the effects of Dpp signaling on cell growth and proliferation in the Drosophila wing. Development 129: 1003-1013. 11861483

Maves, L. and Schubiger, G. (1998). A molecular basis for transdetermination in Drosophila imaginal discs: interactions between wingless and decapentaplegic signaling. Development 125(1): 115-124. PubMed Citation: 9389669

Mohit, P., et al. (2005). Modulation of AP and DV signaling pathways by the homeotic gene Ultrabithorax during haltere development in Drosophila. Dev. Biol. 291(2): 356-67. 16414040

Mou, X., Duncan, D. M., Baehrecke, E. H. and Duncan, I. (2012). Control of target gene specificity during metamorphosis by the steroid response gene E93. Proc Natl Acad Sci U S A 109: 2949-2954. PubMed ID: 22308414

Nagaraj, R., et al. (1999). Role of the EGF receptor pathway in growth and patterning of the Drosophila wing through the regulation of vestigial. Development 126: 975-985. PubMed Citation: 9927598

Neumann, C. J. and Cohen, S. M. (1996). A heirarchy of cross-regulation involving Notch, wingless, vestigial and cut organizes the dorsal/ventral axis of the Drosophila wing. Development 122, 3477-3485. PubMed Citation: 8951063

Neumann, C. J. and Cohen, S. M. (1997). Long-range action of Wingless organizes the dorsal-ventral axis of the Drosophila wing. Development 124: 871-880. PubMed Citation: 9043068

Neumann, C. J. and Cohen, S. M. (1998). Boundary formation in Drosophila wing: Notch activity attenuated by the POU protein Nubbin. Science 281(5375): 409-413. PubMed Citation: 9665883

Nicholson, S. C., Nicolay, B. N., Frolov, M. V. and Moberg, K. H. (2011). Notch-dependent expression of the archipelago ubiquitin ligase subunit in the Drosophila eye. Development 138(2): 251-60. PubMed Citation: 21148181

Nussbaumer, U., et al. (2000). Expression of the blistered/DSRF gene is controlled by different morphogens during Drosophila trachea and wing development. Mech. Dev. 96: 27-36. PubMed Citation: 10940622

Okulski, H., Druck, B., Bhalerao, S. and Ringrose, L. (2011). Quantitative analysis of polycomb response elements (PREs) at identical genomic locations distinguishes contributions of PRE sequence and genomic environment. Epigenetics Chromatin 4: 4. PubMed Citation: 21410956

Paumard-Rigal, S., et al. (1998). Specific interactions between vestigial and scalloped are required to promote wing tissue proliferation in Drosophila melanogaster. Dev. Genes Evol. 208(8): 440-446. PubMed Citation: 9799424

Prasad, M., Bajpai, R. and Shashidhara, L. S. (2003). Regulation of Wingless and Vestigial expression in wing and haltere discs of Drosophila. Development 130: 1537-1547. 12620980

Rabinow, L. and Birchler, J. A. (1990). Interactions of vestigial and scabrous with the Notch locus of Drosophila melanogaster. Genetics 125: 41-50. PubMed Citation: 2160402

Rodriguez, D. d. A., et al. (2002). Different mechanisms initiate and maintain wingless expression in the Drosophila wing hinge. Development 129: 3995-4004. 12163403

Schoborg, T., Kuruganti, S., Rickels, R. and Labrador, M. (2013). The Drosophila gypsy insulator supports transvection in the presence of the vestigial enhancer. PLoS One 8: e81331. PubMed ID: 24236213

Shashidhara LS., et al. (1999). Negative regulation of dorsoventral signaling by the homeotic gene Ultrabithorax during haltere development in Drosophila. Dev. Biol. 212(2): 491-502. PubMed ID: 10433837

Silber, J., et al. (1993). The vestigial locus of Drosophila melanogaster is involved in resistance to inhibitors of dTMP synthesis. Mol Gen Genet 241: 42-8

Simmonds, A., Hughes, S., Tse, J., Cocquyt, S. and Bell, J. (1997). The effect of dominant vestigial alleles upon vestigial-mediated wing patterning during development of Drosophila melanogaster. Mech. Dev. 67(1): 17-33

Simmonds, A. J., et al. (1998a). Molecular interactions between Vestigial and Scalloped promote wing formation in Drosophila. Genes Dev. 12(24): 3815-3820

Simmonds, A. J. and Bell, J. B. (1998b). A genetic and molecular analysis of an invectedDominant mutation in Drosophila melanogaster. Genome 1998 Jun;41(3):381-90

Srivastava, A., MacKay, J. O, Bell, J. B. (2002). A Vestigial:Scalloped TEA domain chimera rescues the wing phenotype of a scalloped mutation in Drosophila melanogaster. Genesis 33(1): 40-7. 12001068

Srivastava, A. J. and Bell, J. B. (2003). Further developmental roles of the Vestigial/Scalloped transcription complex during wing development in Drosophila melanogaster. Mech. Dev. 120: 587-596. 12782275

Sudarsan, V., et al. (2001). Myoblast diversification and ectodermal signaling in Drosophila. Dev. Cell 1: 829-839. 11740944

Sui, L., Pflugfelder, G. O. and Shen, J. (2012). The Dorsocross T-box transcription factors promote tissue morphogenesis in the Drosophila wing imaginal disc. Development 139(15): 2773-82. PubMed Citation: 22782723

Sustar, A. and Schubiger, G. (2005). A transient cell cycle shift in Drosophila imaginal disc cells precedes multipotency. Cell 120: 383-393. 15707896

Takanaka, Y. and Courey, A. J. (2005). SUMO enhances Vestigial function during wing morphogenesis. Mech. Dev. 122: 1130-1137. 16026969

Torres-Vazquez, J., Warrior, R. and Arora, K. (2000). schnurri is required for dpp-dependent patterning of the Drosophila wing. Dev. Bio. 227: 388-402.

Varadarajan, S. and VijayRaghavan, K. (1999). scalloped functions in a regulatory loop with vestigial and wingless to pattern the Drosophila wing. Dev. Genes Evol. 209(1): 10-17

Vaudin, P., et al. (1999). TONDU (TDU), a novel human protein related to the product of vestigial (vg) gene of Drosophila melanogaster interacts with vertebrate TEF factors and substitutes for Vg function in wing formation. Development 126: 4807-4816

Wang, S.-H., Simcox, A. and Campbell, G. (2000). Dual role for Drosophila epidermal growth factor receptor signaling in early wing disc development. Genes Dev. 14: 2271-2276.

Weatherbee, S. D., et al. (1998). Ultrabithorax regulates genes at several levels of the wing-patterning hierarchy to shape the development of the Drosophila haltere. Genes Dev. 12(10): 1474-1482. Medline abstract: 9585507

Whited, J. L., Cassell, A., Brouillette, M. and Garrity, P. A. (2004). Dynactin is required to maintain nuclear position within postmitotic Drosophila photoreceptor neurons. Development 131: 4677-4686. Medline abstract: 15329347

Whitworth, A. J. and Russell, S. (2003). Temporally dynamic response to Wingless directs the sequential elaboration of the proximodistal axis of the Drosophila wing, Dev. Bio. 254: 277-288. 12591247

Williams, J. A., Bell, J. B. and Carroll, S. B. (1991). Control of Drosophila wing and haltere development by the nuclear vestigial gene product. Genes Dev 5: 2481-95. PubMed Citation: 1752439

Williams, J. A., Paddock, S.W. and Carroll, S.B. (1993). Pattern formation in a secondary field: a hierarchy of regulatory genes subdivides the developing Drosophila wing disc into discrete subregions. Development 117: 571-584. PubMed Citation: 8330528

Williams, J. A., et al. (1994). Organization of wing formation and induction of a wing-patterning gene at the dorsal/ventral compartment boundary. Nature 368: 299-305. PubMed Citation: 8127364

Zecca, M., Basler, K. and Struhl, G. (1996). Direct and long-range action of a Wingless morphogen gradient. Cell 87: 833-844. PubMed citation: 8945511

Zecca, M. and Struhl, G. (2007a). Recruitment of cells into the Drosophila wing primordium by a feed-forward circuit of vestigial autoregulation. Development 134(16): 3001-10. PubMed citation: 17634192

Zecca, M. and Struhl, G. (2007b). Control of Drosophila wing growth by the vestigial quadrant enhancer. Development 134(16): 3011-20. PubMed citation: 17634191

Zecca, M. and Struhl, G. (2010). A feed-forward circuit linking wingless, fat-dachsous signaling, and the warts-hippo pathway to Drosophila wing growth. PLoS Biol. 8(6): e1000386. PubMed Citation: 20532238

Zider, A., et al. (1996). vestigial gene expression in Drosophila melanogaster is modulated by the dTMP pool. Mol. Gen. Genet. 251: 91-98. PubMed Citation: 8628252

Zirin, J. D. and Mann, R. S. (2004). Differing strategies for the establishment and maintenance of teashirt and homothorax repression in the Drosophila wing. Development 131: 5683-5693. 15509768


vestigial: Biological Overview | Evolutionary Homologs | Regulation | Targets of Activity | Developmental Biology | Effects of Mutation

date revised: 10 October 2014

Home page: The Interactive Fly © 1997 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.