Hedgehog (Hh) plays crucial roles in tissue-patterning and activates signaling in Patched (Ptc)-expressing cells. Paracrine signaling requires release and transport over many cell diameters away by a process that requires interaction with heparan sulfate proteoglycans (HSPGs). Here, we examine the organization of functional, fluorescently tagged variants in living cells by using optical imaging, FRET microscopy, and mutational studies guided by bioinformatics prediction. We find that cell-surface Hh forms suboptical oligomers, further concentrated in visible clusters colocalized with HSPGs. Mutation of a conserved Lys in a predicted Hh-protomer interaction interface results in an autocrine signaling-competent Hh isoform--incapable of forming dense nanoscale oligomers, interacting with HSPGs, or paracrine signaling. Thus, Hh exhibits a hierarchical organization from the nanoscale to visible clusters with distinct functions.

All imaginal discs in Drosophila are made up of a layer of columnar epithelium or the disc proper and a layer of squamous epithelium called the peripodial membrane. Although the developmental and molecular events in columnar epithelium or the disc proper are well understood, the peripodial membrane has gained attention only recently. Using the technique of lineage tracing, we show that peripodial and disc proper cells arise from a common set of precursors cells in the embryo, and that these cells diverge in the early larval stages. However, peripodial and disc proper cells maintain a spatial relationship even after the separation of their lineages. The peripodial membrane plays a significant role during the regional subdivision of the wing disc into presumptive wing, notum and hinge. The Egfr/Ras pathway mediates this function of the peripodial membrane. These results on signaling between squamous and columnar epithelia are particularly significant in the context of in vitro studies using human cell lines that suggest a role for the Egfr/Ras pathway in metastasis and tumour progression.

Hox proteins are transcription factors and key regulators of segmental identity along the anterior posterior axis across all bilaterian animals. Despite decades of research, the mechanisms by which Hox proteins select and regulate their targets remain elusive. We have carried out whole-genome ChIP-chip experiments to identify direct targets of Hox protein Ultrabithorax (Ubx) during haltere development in Drosophila. Direct targets identified include upstream regulators or cofactors of Ubx. Homothorax, a cofactor of Ubx during embryonic development, is one such target and is required for normal specification of haltere. Although Ubx bound sequences are conserved amongst various insect genomes, no consensus Ubx-specific motif was detected. Surprisingly, binding motifs for certain transcription factors that function either upstream or downstream to Ubx are enriched in these sequences suggesting complex regulatory loops governing Ubx function. Our data supports the hypothesis that specificity during Hox target selection is achieved by associating with other transcription factors.

In the fruitfly Drosophila melanogaster, the differential development of wing and haltere is dependent on the function of the Hox protein Ultrabithorax (Ubx). Here we compare Ubx-mediated regulation of wing patterning genes between the honeybee, Apis mellifera, the silkmoth, Bombyx mori and Drosophila. Orthologues of Ubx are expressed in the third thoracic segment of Apis and Bombyx, although they make functional hindwings. When over-expressed in transgenic Drosophila, Ubx derived from Apis or Bombyx could suppress wing development, suggesting evolutionary changes at the level of co-factors and/or targets of Ubx. To gain further insights into such events, we identified direct targets of Ubx from Apis and Bombyx by ChIP-seq and compared them with those of Drosophila. While majority of the putative targets of Ubx are species-specific, a considerable number of wing-patterning genes are retained, over the past 300 millions years, as targets in all the three species. Interestingly, many of these are differentially expressed only between wing and haltere in Drosophila but not between forewing and hindwing in Apis or Bombyx. Detailed bioinformatics and experimental validation of enhancer sequences suggest that, perhaps along with other factors, changes in the cis-regulatory sequences of earlier targets contribute to diversity in Ubx function.

We present a model for muscle development in the second thoracic segment whereby mesodermal properties are specified entirely by induction, in contrast to muscle development in other segments, where autonomous function for homeotic selector genes is also required.

No abstract

We report cloning and characterization of coro, which codes for the Drosophila homologue of the F-actin binding protein coronin. Viable alleles of coro produce a variety of phenotypes in leg, wing and eye development, which are similar to the phenotypes observed as a result of mutations in genes associated with the actin cytoskeleton and/or membrane trafficking. Homozygous lethal mutations in coro results in the disruption of the actin cytoskeleton in wing imaginal discs. Formation of both basolateral septate junctions and apical adherens junctions are also adversely affected in epithelial cells. Both viable and lethal alleles of coro show genetic interactions with syntaxin1A, a gene required for membrane trafficking. They also show enhanced response to over-expression of Decapentaplegic (Dpp) and its receptor Thick vein. Tracing of Dpp morphogen using a Dpp::GFP fusion construct suggested defects in the endocytic pathway, which resulted in uniform distribution of Dpp along the AP axis rather than a gradient from the AP boundary. Our results provide a genetic link between endocytosis/exocytosis events involving F actin-coated vesicles and the establishment of morphogen gradient.

In the third thoracic segment of Drosophila, wing development is suppressed by the homeotic selector gene Ultrabithorax (Ubx) in order to mediate haltere development. Previously, we have shown that Ubx represses dorsoventral (DV) signaling to specify haltere fate. Here we examine the mechanism of Ubx-mediated downregulation of DV signaling. We show that Wingless (Wg) and Vestigial (Vg) are differentially regulated in wing and haltere discs.