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Different signaling pathways are deployed in specific developmental contexts to generate sexually dimorphic traits. Recently, Sexlethal (Sxl), the female determinant in Drosophila melanogaster, was shown to down-regulate Notch (N) signaling to accomplish sex-specific patterning. Paradoxically, however, both Sxl and N are ubiquitously expressed in all of the female cells. This raises a key question as to how, during monomorphic female development, N signaling escapes the negative impact of Sxl. Here, we uncover a regulatory loop involving Hrp48, an abundant. Drosophila hnRNP, Sxl and N, Phenotypic consequences of the partial loss of hrp48 resemble that of N but are more pronounced in females than in males. Likewise, N levels are drastically diminished only in females. Interestingly, monomorphic female tissues including wing, eye and antennal discs display considerable increase in Sxl amounts. Finally, female-specific attenuation of N signaling is rescued upon simultaneous removal of Sxl. Thus, our data demonstrate that in monomorphic contexts, Hrp48 functions as a moderator of Sxl expression to achieve adequate levels of N receptor production and signaling. We propose that it is critical to modulate the activities of the master determinant underling sexual dimorphism, to ensure that it does not function inappropriately in monomorphic tissues and disrupt their development. sex determination | dosage compensation doi/ 10.1073/pnas.0910570107

Many of the transcription factors and target genes that pattern the developing adult remain unknown. In the present stndy, we find that an ortholog of the poorly understood transcription factor, glucose transporter (GLUT4) enhancer factor (Glut4EF, GEF) [also known as the Huntington's disease gene regulatory region-binding protein (HDBP) 1], plays a critical role in specifying normal wing positioning in adult Drosophila. Glut4EF proteins are zinc-finger transcription factors named for their ability to regulate expression of GLUT4 but nothing is known of Glut4EF's in vivo physiological functions. Here, we identify a family of Glut4EF proteins that are well conserved from Drosophila to humans and find that mutations in Drosophila Glut4EF underlie the wing-positioning defects seen in stretch mutants. In addition, our results indicate that previously uncharacterized mutations in Glut4EF are present in at least 11 publicly available fly lines and on the widely used TM3 balancer chromosome. These results indicate that previous observations utilizing these common stocks may be complicated by the presence of Glut4EF mutations. For example, out results indicate that Glut4EF mutations are also present on the same chromosome as two gain-of-function mutations of the homeobox transcription factor Antennapedia (Antp) and underlie defects previously attributed to Antp. In fact, our results support a role for Glut4EF in the modulation of morphogenetic processes mediated by Antp, further highlighting the importance of Glut4EF transcription factors in patterning and morphogenesis.

The role of the transcription factor, Suppressor of Hairless in compartment formation during wing development in Drosophila embryo is studied.

The role of the calcineurin B2 gene in regulating the proper development of indirect flight muscle in the pupa of Drosophila is studied.

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

The Serrate and Delta genes of Drosophila encode two transmembrane proteins that act as ligands for the Notch receptor. Both have been shown to promote Notch activity during wing development and the specification of cell fate during neurogenesis. This observation has led to the suggestion that Serrate and Delta are functionally equivalent ligands for Notch. In this study we compare the activities of these proteins during wing development. Our results show that Serrate can activate or inactivate Notch in a concentration-dependent manner. While the inactivation is likely to be mediated by a dominant negative effect over Notch, the activation is similar to that elicited by Delta and requires the product of the Suppressor of Hairless gene. These results indicate that regulation of the concentration of Serrate during development must be an important way of regulating its activity.

The fringe (fng) gene codes for a putatively secreted dorsal to ventral signalling molecule that is crucial for wing formation in Drosophila. Placing cells with and without fng expression side by side activates wing margin development and distal wing outgrowth at the normal wing margin or at fng misexpression sites. The homeobox gene ap modulates the dorsal-specific expression of fng, indicating that ap participates in wing margin formation and growth through the transcriptional modulation of fng.

The activity of the Drosophila patterning system of anteroposterior subdivision of wing disc affects specification of the wing primordium in early second instar. The dorsoventral (DV) patterning system has no affect. The wingless gene (wg) has an important role in specifying the wing primordium. The gene controls the patterning activities of the DV compartment boundary. The DV system activates the wg and Vestigial proteins, and both the proteins are essential for the wing growth.

A variety of factors could influence how far developmental signals spread. For example, the Patched receptor limits the range of its ligand Hedgehog. Somehow, the Frizzled2 receptor has the opposite effect on its ligand. Increasing the level of Frizzled2 stabilizes Wingless and thus extends the Wingless gradient in Drosophila wing imaginal disks. Here we ask whether Frizzled or Frizzled2 affects the spread of Wingless in Drosophila embryos. We show that in the embryonic epidermis, the combined expression of both receptors is lowest in the engrailed domain. This is because expression of Frizzled is repressed by the Engrailed transcription factor, whereas that of Frizzled2 is repressed by Wingless signaling. Receptor downregulation correlates with an early asymmetry in Wingless distribution, characterized by the loss of Wingless staining in the engrailed domain. Raising the expression of either Frizzled or Frizzled2 in this domain prevents the early disappearance of Wingless-containing vesicles. Apparently, Wingless is captured, stabilized, and quickly internalized by either receptor. As far as we can tell, captured Wingless is not passed on to further cells and does not contribute to the spread of Wingless. Receptor downregulation in the posterior compartment may contribute to dampening the signal at the time when cuticular fates are specified. Key Words: Drosophila embryos; Wingless transport; Wingless stability; Engrailed; Frizzled; Frizzled2; epidermal pattern; signal modulation.

During Drosophila wing morphogenesis a series of cell-cell and cell-extracellular matrix interactiofis define the overall shape of the wing and placement of wing veins, decapentaplegic, a homolog of vertebrate BMP-4, is required for development of wing veinsand is expressed in the vein primordia during early pupal development. The BMP antagonist short gastrulation (sog) is expressed in a complementary pattern to dpp in intervein cells and opposes the vein-promoting activity of dpp. Regulated cleavage of Sog protein by Tolloid, a member of the astacin family of metalloproteases, has been hypothesized to generate forms with differential activities. We have assayed for the interaction of sog with components of the extracellular matrix and their receptors, based on the modification of vein phenotypes of enhancer piracy-sog lines in which sog is overexpressed. This analysis has revealed a genetic interaction between sog and integrins. This interaction involves specifically [Beta]PS and [Alpha]PS1 integrin subunits, in addition to the novel [Alpha]PS3 subunit. We also present evidence of a physical binding of a low-molecular-weight form of Sog to [Alpha]PS1. During pupal wing development, integrins perform the main role of regulating apposition between the dorsal and ventral surfaces of the wing. Our results suggest that, in addition to this role, integrins are important for the formation of wing veins by binding and possibly regulating the activity of Sog during pupal development.

Planar polarity signaling in the Drosophila wing regulates the proximal-to-distal orientation of hairs on the wing surface. The receptor Frizzled (Fz) and the cytoplasmic protein Dishevelled (Dsh) are mediators of this pathway. Fz and Dsh also function during canonical Wnt signaling in which Wnts act as activating ligands for Frizzleds. No Fz ligand has been identified for planar polarity signaling thus far, but given that Wnts bind Fz during canonical Wnt signaling, we hypothesized that Wnts may act as ligands during polarity signaling as well. Here, we show that overexpression of a GPI-linked Fz extracellular cysteine-rich domain (CRD) induces polarity defects in the wing. As the Fz CRD is sufficient for Wnt binding to Frizzleds, this result suggests that ligand binding may regulate Fz activity for polarity signaling and that this ligand may be a Wnt. To test whether Wnts can act as polarity ligands we overexpressed Wnts in wing tissues. One Wnt, DWnt-4, induces polarity defects and the phenotype is suppressed by the loss of Fz or Dsh. Several Wnts (including DWnt-4) are expressed at the wing margin during polarity signaling and overexpression of DWnt-4 in clones causes wing hairs to orient toward the Wnt source. In vitro binding studies between Fz CRD and different Drosophila Wnts show that the affinity of Fz CRD for DWnt-4 is higher than that for any other Wnt tested so far. These data show that Wnts are sufficient to induce polarization of wing cells and suggest that Wnts may bind to Fz to regulate cell polarity. We are currently testing the requirement for Wnts in this process.

Mechanisms to segregate cell populations play important roles in tissue patterning during animal development. Cell segregation can be driven by heritable states of gene expression that produce compartments that are separated by boundaries of lineage restriction. Cell populations can also be separated to produce adjacent domains that are globally stable in terms of gene expression, but that do not preclude cell mixing at the boundary. The mechanisms that direct segregation of cell populations are not well understood. Differences in cell affinity may be involved. I will present recent studies on two membrane proteins that are involved cell segregation in Drosophila wing development. These proteins are regulated by Apterous in the dorsal compartment and contribute to formation of the dorsal-ventral compartment boundary. Later they are regulated by Dpp and produce an affinity difference between medial and lateral regions that contributes to a secondary noncompartmental subdivision of the wing. Thus a common cellular mechanism may underlie two apparently different types of developmental subdivision.

The hiiragi (hrg) gene plays a key role in the development of the wing margin in Drosophila melanogaster. A mutation in the hrg gene resulted in a decrease in the level of the hrg transcript and was associated with a notched wing phenotype. We report here that the hrg gene encodes a poly(A) polymerase (PAP). The bovine cDNA for PAP type II reversed the phenotype due to mutation of the hrg gene, suggesting that hrg might encode a functional homolog of PAP. A mutation that reduced the enzymatic activity of Hrg failed to reverse the phenotype of hrg mutants, suggesting that the enzymatic activity of Hrg was required to rescue the wing phenotype. The levels of expression of wingless and cut at the presumptive wing margins were reduced in the late third-instar larvae of hrg mutants. These results suggest that the product of hrg is required for the normal expression of a series of genes in this region. Our results provide the first evidence that a PAP in Drosophila plays a key role in the early development of the wing margin, acting to regulate the specific expression of a series of genes via, perhaps, control of the processing of the 3' ends of transcripts. Key Words: poly(A) polymerase; wing development; Drosophila; Notch; cut; wingless.

neutralized (neu) represents one of the strong neurogenic mutants in Drosophila. Mutants of this class display, among other phenotypes, a strong overcommitment to neural fates at the expense of epidermal fates. We analyzed the role of neu during adult development by using mutant clonal analysis, misexpression of wild-type and truncated forms of Neu, and examination of genetic interactions with N-pathway mutations. We find that neu is required cell-autonomously for lateral inhibition during peripheral neurogenesis and for multiple asymmetric cell divisions in the sensory lineage. In contrast, neu is apparently dispensable for other N-mediated processes, including lateral inhibition during wing vein development and wing margin induction. Misexpression of wild-type Neu causes defects in both peripheral neurogenesis and wing vein development, while a truncated form lacking the RING finger is further capable of inhibiting formation of the wing margin. In addition, the phenotypes produced by misexpression of wild-type and truncated Neu proteins are sensitive to the dosage of several N-pathway components. Finally, using epitope-tagged Neu proteins, we localize Neu to the plasma membrane and reveal a novel morphology to the sensory organ precursor cells of wing imaginal discs. Collectively, these data indicate a key role for neu in the reception of the lateral inhibitory signal during peripheral neurogenesis. [C] 2001 Academic Press Key Words: neuralized; Notch signaling; Drosophila; sensory organ development; wing vein development.