Semaphorin 2a secreted by oenocytes signals through plexin B and plexin A to guide sensory axons in the Drosophila embryo

The semaphorin gene family has been shown to play important roles in axonal guidance in both vertebrates and invertebrates. Both transmembrane (Sema1a, Sema1b, Sema5c) and secreted (Sema2a, Sema2b) forms of semaphorins exist in Drosophila. Two Sema receptors, plexins (Plex) A and B, have also been identified. Many questions remain concerning the axon guidance functions of the secreted semaphorins, including the identity of their receptors. We have used the well-characterized sensory system of the Drosophila embryo to address these problems. We find novel sensory axon defects in sema2a loss-of-function mutants in which particular axons misproject and follow inappropriate pathways to the CNS. plexB loss-of-function mutants show similar phenotypes to sema2a mutants and sema2a interacts genetically with plexB, supporting the hypothesis that Sema2a signals through PlexB receptors. Sema2a protein is expressed by larval oenocytes, a cluster of secretory cells in the lateral region of the embryo and the sema2a mutant phenotype can be rescued by driving Sema2a in these cells. Ablation of oenocytes results in sensory axon defects similar to the sema2a mutant phenotype. These data support a model in which Sema2a, while being secreted from oenocytes, acts in a highly localized fashion: It represses axon extension from the sensory neuron cell body, but only in regions in direct contact with oenocytes.     

Localized repressors delineate the neurogenic ectoderm in the early Drosophila embryo

The Dorsal gradient produces sequential patterns of gene expression across the dorsoventral axis of early embryos, thereby establishing the presumptive mesoderm, neuroectoderm, and dorsal ectoderm. Spatially localized repressors such as Snail and Vnd exclude the expression of neurogenic genes in the mesoderm and ventral neuroectoderm, respectively. However, no repressors have been identified that establish the dorsal limits of neurogenic gene expression. To investigate this issue, we have conducted an analysis of the ind gene, which is selectively expressed in lateral regions of the presumptive nerve cord. A novel silencer element was identified within the ind enhancer that is essential for eliminating expression in the dorsal ectoderm. Evidence is presented that the associated repressor can function over long distances to silence neighboring enhancers. The ind enhancer also contains a variety of known activator and repressor elements. We propose a model whereby Dorsal and EGF signaling, together with the localized Schnurri repressor, define a broad domain of ind expression throughout the entire presumptive neuroectoderm. The ventral limits of gene expression are defined by the Snail and Vnd repressors, while the dorsal border is established by the newly defined silencer element.     

Primordium specific requirement of the homeotic gene fork head in the developing gut of the Drosophila embryo

Summary The homeotic gene fork head (fkh) of Drosophila melanogaster promotes terminal as opposed to segmental development in the ectodermal parts of the gut. Molecular analysis revealed that fkh expression is not restricted to the ectodermal parts of the gut, but is detectable in a variety of other tissues. Therefore, the phenotype of fkh mutant embryos was re-examined using molecular probes as tissue specific markers. With the exception of the nervous system, which was not studied, phenotypic effects were found in all tissues expressing fkh protein in the wild-type. Particularly, these tissues include all components of the gut in the Drosophila embryo: the foregut and hindgut, the midgut and the yolk nuclei. The defects observed in the gut of fkh mutant embryos are primordium specific.     

Terminal versus segmental development in the Drosophila embryo: the role of the homeotic gene fork head

Summary Mutations of the homeotic gene fork head (fkh) of Drosophila transform the non-segmented terminal regions of the embryonic ectoderm into segmental derivatives: Pre-oral head structures and the foregut are replaced by post-oral head structures which are occasionally associated with thoracic structures. Posterior tail structures including the hindgut and the Malpighian tubules are replaced by post-oral head structures associated with anterior tail structures. The fkh gene shows no maternal effect and is required only during embryogenesis. The phenotypes of double mutants indicate that fkh acts independently of other homeotic genes (ANT-C, BX-C, spalt) and caudal. In addition, the fkh domains are not expanded in Polycomb (Pc) group mutant embryos. Ectopic expression of the homeotic selector genes of the ANT-C and BX-C in Pc group mutant embryos causes segmental transformations in terminal regions of the embryo only in the absence of fkh gene activity. Thus, fkh is a region-specific homeotic rather than a selector gene, which promotes terminal as opposed to segmental development. Offprint requests to: Institut für Biologie II (Genetik), Universität Tübingen, Auf der Morgenstelle 28, D-7400 Tübingen, Federal Republic of Germany     

Non-neutral processes drive the nucleotide composition of non-coding sequences in Drosophila

The nature of the forces affecting base composition is a key question in genome evolution. There is uncertainty as to whether differences in the GC contents of non-coding sequences reflect differences in mutational bias, or in the intensity of selection or biased gene conversion. We have used a polymorphism dataset for non-coding sequences on the X chromosome of Drosophila simulans to examine this question. The proportion of GC-->AT versus AT-->GC polymorphic mutations in a locus is correlated with its GC content. This implies the action of forces that favour GC over AT base pairs, which are apparently strongest in GC-rich sequences.     

The small GTPase Rac plays multiple roles in epithelial sheet fusion--dynamic studies of Drosophila dorsal closure

The coordinated migration and fusion of epithelial sheets is a crucial morphogenetic tool used on numerous occasions during the normal development of an embryo and re-activated as part of the wound healing response. Drosophila dorsal closure, whereby a hole in the embryonic epithelium is zipped closed late in embryogenesis, serves as an excellent, genetically tractable model for epithelial migration. Using live confocal imaging, we have dissected multiple roles for the small GTPase Rac in this process. We show that constitutive activation of Rac1 leads to excessive assembly of lamellipodia and precocious halting of epithelial sweeping, possibly through premature activation of contact-inhibition machinery. Conversely, blocking Rac activity, either by loss-of-function mutations or expression of dominant negative Rac1, disables the assembly of both actin cable and protrusions by epithelial cells. Movies of mutant embryos show that continued contraction of the amnioserosa is sufficient to draw the epithelial edges towards one another, allowing the zipper machinery to bypass non-functioning regions of leading edge. In addition to illustrating the key role of Rac in organization of leading edge actin, loss-of-function mutants also provide substantive proof that Rac acts upstream in the Jun N-terminal kinase (JNK) cascade to direct epithelial cell shape changes during dorsal closure.     

Mummy encodes an UDP-N-acetylglucosamine-dipohosphorylase and is required during Drosophila dorsal closure and nervous system development

Throughout development cell-cell interactions are of pivotal importance. Cells bind to each other or share information via secreted signaling molecules. To a large degree, these processes are modulated by post-translational modifications of membrane proteins. Glycan-chains are frequently added to membrane proteins and assist their exact function at the cell surface. In addition, the glycosylation pathway is required to generate GPI-linkage in the endoplasmatic reticulum. Here, we describe the analysis of the cabrio/mummy gene, which encodes an UDP-N-acetylglucosamine diphosphorylase. This is a well-conserved and central enzyme in the glycosylation pathway. As expected from this central role in glycosylation, cabrio/mummy mutants show many phenotypic traits ranging from CNS fasciculation defects to defects in dorsal closure and eye development. These phenotypes correlate well with specific glycosylation and GPI-anchorage defects in mummy mutants.     

The Female Post-Mating Response Requires Genes Expressed in the Secondary Cells of the Male Accessory Gland in Drosophila melanogaster

Seminal proteins from the Drosophila male accessory gland induce post-mating responses (PMR) in females. The PMR comprise behavioral and physiological changes that include increased egg laying, decreased receptivity to courting males, and changes in the storage and use of sperm. Many of these changes are induced by a “sex peptide” (SP) and are maintained by SP’s binding to, and slow release from, sperm. The accessory gland contains two secretory cell types with distinct morphological and developmental characteristics. Products of these “main” and “secondary” cells work interdependently to induce and maintain the PMR. To identify individual genes needed for the morphology and function of secondary cells, we studied iab-6^cocu males, whose secondary cells have abnormal morphology and fail to provide products to maintain the PMR. By RNA-seq, we identified 77 genes that are downregulated by a factor of >5× in iab-6^cocu males. By functional assays and microscopy, we tested 20 candidate genes and found that at least 9 are required for normal storage and release of SP in mated females. Knockdown of each of these 9 genes consequently leads to a reduction in egg laying and an increase in receptivity over time, confirming a role for the secondary cells in maintaining the long-term PMR. Interestingly, only 1 of the 9 genes, CG3349, encodes a previously reported seminal fluid protein (Sfp), suggesting that secondary cells may perform essential functions beyond the production and modification of known Sfps. At least 3 of the 9 genes also regulate the size and/or abundance of secondary cell vacuoles, suggesting that the vacuoles’ contents may be important for the machinery used to maintain the PMR.     

Interaction between Sleep and Metabolism in Drosophila with Altered Octopamine Signaling

Sleep length and metabolic dysfunction are correlated, but the causal relationship between these processes is unclear. Octopamine promotes wakefulness in the fly by acting through the insulin-producing cells (IPCs) in the fly brain. To determine if insulin signaling mediates the effects of octopamine on sleep:wake behavior, we assayed flies in which insulin signaling activity was genetically altered. We found that increasing insulin signaling does not promote wake, nor does insulin appear to mediate the wake-promoting effects of octopamine. Octopamine also affects metabolism in invertebrate species, including, as we show here, Drosophila melanogaster. Triglycerides are decreased in mutants with compromised octopamine signaling and elevated in flies with increased activity of octopaminergic neurons. Interestingly, this effect is mediated at least partially by insulin, suggesting that effects of octopamine on metabolism are independent of its effects on sleep. We further investigated the relative contribution of metabolic and sleep phenotypes to the starvation response of flies with altered octopamine signaling. Hyperactivity (indicative of foraging) induced by starvation was elevated in octopamine receptor mutants, despite their high propensity for sleep, indicating that their metabolic state dictates their behavioral response under these conditions. Moreover, flies with increased octopamine signaling do not suppress sleep in response to starvation, even though they are normally hyper-aroused, most likely because of their high triglyceride levels. Together, these data suggest that observed correlations between sleep and metabolic phenotypes can result from shared molecular pathways rather than causality, and environmental conditions can lead to the dominance of one phenotype over the other.     

Testing limits to adaptation along altitudinal gradients in rainforest Drosophila

Given that evolution can generate rapid and dramatic shifts in the ecological tolerance of a species, what prevents populations adapting to expand into new habitat at the edge of their distributions? Recent population genetic models have focused on the relative costs and benefits of migration between populations. On the one hand, migration may limit adaptive divergence by preventing local populations from matching their local selective optima. On the other hand, migration may also contribute to the genetic variance necessary to allow populations to track these changing optima. Empirical evidence for these contrasting effects of gene flow in natural situations are lacking, largely because it remains difficult to acquire. Here, we develop a way to explore theoretical models by estimating genetic divergence in traits that confer stress resistance along similar ecological gradients in rainforest Drosophila. This approach allows testing for the coupling of clinal divergence with local density, and the effects of genetic variance and the rate of change of the optimum on the response to selection. In support of a swamping effect of migration on phenotypic divergence, our data show no evidence for a cline in stress-related traits where the altitudinal gradient is steep, but significant clinal divergence where it is shallow. However, where clinal divergence is detected, sites showing trait means closer to the presumed local optimum have more genetic variation than sites with trait means distant from their local optimum. This pattern suggests that gene flow also aids a sustained response to selection.     

Modulation of the Suppressor of fused protein regulates the Hedgehog signaling pathway in Drosophila embryo and imaginal discs

The Suppressor of fused (Su(fu)) protein is known to be a negative regulator of Hedgehog (Hh) signal transduction in Drosophila imaginal discs and embryonic development. It is antagonized by the kinase Fused (Fu) since Su(fu) null mutations fully suppress the lack of Fu kinase activity. In this study, we overexpressed the Su(fu) gene in imaginal discs and observed opposing effects depending on the position of the cells, namely a repression of Hh target genes in cells receiving Hh and their ectopic expression in cells not receiving Hh. These effects were all enhanced in a fu mutant context and were suppressed by cubitus interruptus (Ci) overexpression. We also show that the Su(fu) protein is poly-phosphorylated during embryonic development and these phosphorylation events are altered in fu mutants. This study thus reveals an unexpected role for Su(fu) as an activator of Hh target gene expression in absence of Hh signal. Both negative and positive roles of Su(fu) are antagonized by Fused. Based on these results, we propose a model in which Su(fu) protein levels and isoforms are crucial for the modulation of the different Ci states that control Hh target gene expression.