Phantom, a cytochrome P450 enzyme essential for ecdysone biosynthesis, plays a critical role in the control of border cell migration in Drosophila

AbstractThe border cells of Drosophila are a model system for coordinated cell migration. Ecdysone signaling has been shown to act as the timing signal to initiate the migration process. Here we find that mutations in phantom (phm), encoding an enzyme in the ecdysone biosynthesis pathway, block border cell migration when the entire follicular epithelium of an egg chamber is mutant, even when the associated germline cells (nurse cells and oocyte) are wild-type. Conversely, mutant germline cells survive and do not affect border cell migration, as long as the surrounding follicle cells are wild-type. Interestingly, even small patches of wild-type follicle cells in a mosaic epithelium are sufficient to allow the production of above-threshold levels of ecdysone to promote border cell migration. The same phenotype is observed with mutations in shade (shd), encoding the last enzyme in the pathway that converts ecdysone to the active 20-hydroxyecdysone. Administration of high 20-hydroxyecdysone titers in the medium can also rescue the border cell migration phenotype in cultured egg chambers with an entirely phm mutant follicular epithelium. These results indicate that in normal oogenesis, the follicle cell epithelium of each individual egg chamber must supply sufficient ecdysone precursors, leading ultimately to high enough levels of mature 20-hydroxyecdysone to the border cells to initiate their migration. Neither the germline, nor the neighboring egg chambers, nor the surrounding hemolymph appear to provide threshold amounts of 20-hydroxyecdysone to do so. This “egg chamber autonomous” ecdysone synthesis constitutes a useful way to regulate the individual maturation of the asynchronous egg chambers present in the Drosophila ovary

Semaphorin6A acts as a gate keeper between the central and the peripheral nervous system

<p>Abstract</p> <p>Background</p> <p>During spinal cord development, expression of chicken SEMAPHORIN6A (SEMA6A) is almost exclusively found in the boundary caps at the ventral motor axon exit point and at the dorsal root entry site. The boundary cap cells are derived from a population of late migrating neural crest cells. They form a transient structure at the transition zone between the peripheral nervous system (PNS) and the central nervous system (CNS). Ablation of the boundary cap resulted in emigration of motoneurons from the ventral spinal cord along the ventral roots. Based on its very restricted expression in boundary cap cells, we tested for a role of Sema6A as a gate keeper between the CNS and the PNS.</p> <p>Results</p> <p>Downregulation of Sema6A in boundary cap cells by <it>in ovo </it>RNA interference resulted in motoneurons streaming out of the spinal cord along the ventral roots, and in the failure of dorsal roots to form and segregate properly. PlexinAs interact with class 6 semaphorins and are expressed by both motoneurons and sensory neurons. Knockdown of PlexinA1 reproduced the phenotype seen after loss of Sema6A function both at the ventral motor exit point and at the dorsal root entry site of the lumbosacral spinal cord. Loss of either PlexinA4 or Sema6D function had an effect only at the dorsal root entry site but not at the ventral motor axon exit point.</p> <p>Conclusion</p> <p>Sema6A acts as a gate keeper between the PNS and the CNS both ventrally and dorsally. It is required for the clustering of boundary cap cells at the PNS/CNS interface and, thus, prevents motoneurons from streaming out of the ventral spinal cord. At the dorsal root entry site it organizes the segregation of dorsal roots.</p

Search for the receptor and target genes of sex-peptide

Bei Drosophila, wie auch bei vielen anderen Insekten, wird die Physiologie und das Verhalten der Weibchen durch eine Begattung beträchtlich verändert (Leopold, 1976). Während virginelle Weibchen nur ein paar wenige Eier legen, wird die Eilegerate in begatteten Weibchen bedeutend erhöht. Virginelle Weibchen akzeptieren werbende Männchen, nach einer Begattung werden sie jedoch abgewiesen (Chen, 1984). Obwohl viele Funktionen des SP gut charakterisiert wurden, sind der Rezeptor und die Reaktionskaskade weitgehend nicht bekannt. Das Ziel meiner Dissertation war es einige mögliche Kandidaten für den SP Rezeptor genauer zu charakterisieren, den SP Rezeptor zu identifizieren und weitere Komponenten der SP Reaktionskaskade im Weibchen zu finden. Die Versuche wurden unternommen mittels Microarrays und der "DUALmembrane system"-Technik einen SP Rezeptor zu identifizieren. Aus den Resultaten des Microarrays ergab sich, dass der gonadotropin-releasing hormone receptor (GRHR) sich als der beste Kandidat für einen SP Rezeptor erwies. Eine Analyse von GRHR Gen zeigte aber, dass dieses Gen nicht in die BR involviert ist, d.h. es handelt sich nicht um den SP Rezeptor. Deshalb wurde in der Folge das "DUALmembrane system" vom DualSystems Biotech für ein library screen von SP-bindenden Proteinen eingesetzt. Zwei cDNA libraries wurden aus Köpfen von 3-tägigen Fliegen hergestellt. Als Resultat ergab sich eine Interaktion zwischen SP und der off-track Rezeptor Tyrosin Kinase (Gen: otk). Weitere Arbeiten sind aber nötig, um nachzuweisen, dass es sich bei otk um den SP Rezeptor handelt. Um SP-regulierte Gene zu identifizieren die im Kopf und im Abdomen der Weibchen exprimiert werden, wurde eine "Genom-weite" Microarray-Analyse der entsprechenden Transkripte durchgeführt. Es konnte gezeigt werden, dass SP die Gene im Kopf und Abdomen differentiell reguliert. Zusätzlich konnte gezeigt werden, dass der N- terminale Teil für die Genregulation keine Rolle spielt. Der C-terminale Teil von SP ist verantwortlich für die Genexpression die zu den BR führt, und auch für die Aktivierung der Immun-Antwort im Abdomen der Weibchen. In Drosophila, as well as in many other insects, mating significantly changes the physiology and the behaviour of females (Leopold, 1976). Whereas virgin females lay only a few eggs, the egg laying rate is markedly increased in mated females. Furthermore, while virgin females readily accept courting males, they reject courting males after mating (Chen, 1984). Sex-Peptide (SP) is the major player in the stimulation of egg-laying and rejection behaviour in females (the two post-mating responses = PMR). Despite extensive knowledge about the many functions of SP, the receptor and the molecular mechanism of the SP response cascade are mainly unknown. The aim of my PhD thesis was to identify the receptor of SP, and to find components of the SP response cascade in the female. The attempts to identify the receptor of SP were performed by involvement of microarray and "DUALmembrane system" approaches. Gonadotropin-releasing hormone receptor (GRHR) resulted as the most promising candidate gene from the microarray approach. Further analysis of GRHR showed that this gene is not involved in the establishment of the PMR, and as a result, it does not encode a SP receptor. Therefore, the "DUALmembrane system" assay of DualSystems Biotech was used to find SP binding proteins by screening two cDNA libraries prepared from the heads of 3 d old females. As a result of the screening, an interaction was observed for SP with the off-track receptor tyrosine kinase (gene: otk). To determine genes which are regulated by SP in the head and in the abdomen to serve the two PMR, I performed a genome-wide expression analysis aimed at identifying all transcripts from the D. melanogaster head and abdomen that exhibit SP- dependent expression patterns. I found that SP differentially regulates gene expression in the heads and in the abdomen of Drosophila females. Furthermore, it was shown that the N-terminal part of SP is not participating in the gene regulation, and that the C-terminal part of SP from aa 8 to 36 is responsible for the regulation of gene expression leading to the two PMR and the activation of the immune response in the abdomen

Calsyntenin 1-mediated trafficking of axon guidance receptors regulates the switch in axonal responsiveness at a choice point

Axon guidance at choice points depends on the precise regulation of guidance receptors on the growth cone surface. Upon arrival at the intermediate target or choice point, a switch from attraction to repulsion is required for the axon to move on. Dorsal commissural (dI1) axons crossing the ventral midline of the spinal cord in the floor plate represent a convenient model for the analysis of the molecular mechanism underlying the switch in axonal behavior. We identified in chick a role for calsyntenin 1 in the regulation of vesicular trafficking of guidance receptors in dI1 axons at choice points. In cooperation with RabGDI, calsyntenin 1 shuttles Rab11-positive vesicles containing Robo1 to the growth cone surface in a precisely regulated manner. By contrast, calsyntenin 1-mediated trafficking of frizzled 3, a guidance receptor in the Wnt pathway, is independent of RabGDI. Thus, tightly regulated insertion of guidance receptors, which is required for midline crossing and the subsequent turn into the longitudinal axis, is achieved by specific trafficking

The effect of mating on immunity can be masked by experimental injection in female Drosophila melanogaster

Mating and immunity are two major components of fitness and links between them have been demonstrated in a number of recent investigations. In Drosophila melanogaster, a seminal fluid protein, sex-peptide (SP), up-regulates a number of antimicrobial peptide (AMP) genes in females after mating but the resulting effect on pathogen resistance is unclear. In this study, we tested (1) whether SP-induced changes in gene expression affect the ability of females to kill injected non-pathogenic bacteria and (2) how the injection process per se affects the expression of AMP genes relative to SP. The ability of virgin females and females mated to SP lacking or control males to clear bacteria was assayed using an established technique in which Escherichia coli are injected directly into the fly body and the rate of clearance of the injected bacteria is determined. We found no repeatable differences in clearance rates between virgin females and females mated to SP producing or SP lacking males. However, we found that the piercing of the integument, as occurs during injection, up-regulates AMP gene expression much more strongly than SP. Thus, assays that involve piercing, which are commonly used in immunity studies, can mask more subtle and biologically relevant changes in immunity, such as those induced by mating

Downregulation of Sema6A in BCCs results in translocation of motoneurons out of the spinal cord

<p><b>Copyright information:</b></p><p>Taken from "Semaphorin6A acts as a gate keeper between the central and the peripheral nervous system"</p><p>http://www.neuraldevelopment.com/content/2/1/28</p><p>Neural Development 2007;2():28-28.</p><p>Published online 18 Dec 2007</p><p>PMCID:PMC2238753.</p><p></p> In the absence of Sema6A from BCCs, motoneurons stream out of the ventral spinal cord and migrate along the ventral roots (arrows). The open arrow points to a motoneuron that is located in the ventral funiculus. In control-treated embryos motoneurons along ventral roots or in the ventral funiculus were rarely seen. Motoneurons were identified by Isl-1 (red). An EGFP plasmid was co-injected with the dsRNA derived from SEMA6A. Axons were stained with an antibody against neurofilament (blue). Note that sensory neurons in the DRG (asterisk in (a, b)) are also stained by Isl-1. Perturbation of Sema6B or Sema6D did not enhance the number of motoneurons in the periphery compared to control-treated embryos injected only with the plasmid encoding EGFP. Three asterisks indicate < 0.0001 for the comparison between dsS6A and all other treatment groups. Values are given as mean ± standard error of mean. Bar: 50 μm

Downregulation of PlexinA1 results in the same phenotype as seen in the absence of Sema6A

<p><b>Copyright information:</b></p><p>Taken from "Semaphorin6A acts as a gate keeper between the central and the peripheral nervous system"</p><p>http://www.neuraldevelopment.com/content/2/1/28</p><p>Neural Development 2007;2():28-28.</p><p>Published online 18 Dec 2007</p><p>PMCID:PMC2238753.</p><p></p> Motoneurons streaming out of the ventral spinal cord identified by Isl-1 staining were only found after downregulation of PlexinA1 (arrows). The open arrow points to a motoneuron that is located in the ventral funiculus. Note that sensory neurons in the DRG (asterisk) are also stained by Isl-1. Lack of none of the other PlexinAs enhanced the number of motoneurons found along the ventral roots compared to control-treated embryos (= 0.0001 for the comparison between dsPA1 and all other treatment groups (indicated by three asterisks); values are given as mean ± standard error of the mean; see Figure 2b). The phenotype seen after downregulation of Sema6A in dorsal BCCs was mimicked by both lack of PlexinA1 (c) and PlexinA4 (d). The effects of PlexinA downregulation were qualitatively different, however. In the absence of PlexinAs, the arrangement of DRGs, and not only the arrangement of their roots, was disorganized. A phenotype was seen in 83% of embryos lacking PlexinA1 and in 67% of the embryos lacking PlexinA4. Bar 50 μm in (a, b); 200 μm in (c, d)

Lack of Sema6A and Sema6D in dorsal BCCs results in aberrant segregation of dorsal roots

<p><b>Copyright information:</b></p><p>Taken from "Semaphorin6A acts as a gate keeper between the central and the peripheral nervous system"</p><p>http://www.neuraldevelopment.com/content/2/1/28</p><p>Neural Development 2007;2():28-28.</p><p>Published online 18 Dec 2007</p><p>PMCID:PMC2238753.</p><p></p> In control embryos axon bundles from each dorsal root ganglion extend to the DREZ in a well organized manner. Roots from adjacent DRGs are segregated and they are all of the same length (dashed bars). In contrast, in embryos lacking Sema6A, roots from adjacent DRGs are no longer segregated (arrowheads). The arrangement of roots arising from individual DRGs is strongly disorganized and roots are often formed by fibers from two adjacent DRGs (arrowheads in (b)). Similarly, roots are disorganized in embryos lacking Sema6D (arrowheads). In addition the length of the roots varied more in the absence of Sema6D (compare dashed bars in (c)). Strong phenotypes were seen in 71% of the embryos lacking Sema6A and in 68% of the embryos lacking Sema6D. Only 13% of the embryos injected with an EGFP plasmid had a comparable phenotype. Downregulation of Sema6B resulted in aberrant DRG shapes and root arrangement in 30% of the embryos. The shapes of DRGs were classified as arc-like when the distance between the most anterior and the most posterior fiber emanating from the DRG was the same as the anteroposterior diameter of the DRG; as bell-shaped when the fibers spread an anteroposterior length that was bigger than the diameter of the DRG; and as mushroom-like when the fibers entered the dorsal spinal cord in a segment that was shorter than the diameter of the DRG. Note that the diameter of the mushroom-like DRGs was smaller than the diameter of arc-like or bell-shaped DRGs. Bar: 200 μm

Ectopic expression of the Sema6A ectodomain or full-length Sema6A in motoneurons competes with BCC-derived Sema6A binding to motoneurons

<p><b>Copyright information:</b></p><p>Taken from "Semaphorin6A acts as a gate keeper between the central and the peripheral nervous system"</p><p>http://www.neuraldevelopment.com/content/2/1/28</p><p>Neural Development 2007;2():28-28.</p><p>Published online 18 Dec 2007</p><p>PMCID:PMC2238753.</p><p></p> The AP-tagged ectodomain of Sema6A binds to axons expressing PlexinAs. Both commissural axons (open arrowhead) and motor axons (arrowhead) express PlexinAs [20] and bind the Sema6A ectodomain. No binding of the AP-tag alone was detectable. Ectopic expression of both the ectodomain of Sema6A (not shown) and the full-length myc-tagged form resulted in motoneurons streaming out of the spinal cord along the ventral roots (arrows). Staining of the myc tag demonstrates expression of Sema6A in motor axons (arrowhead), consistent with a competitive role of motor axon-derived Sema6A with BCC-derived Sema6A in the periphery. As seen after downregulation of either Sema6A in BCCs (compare to Figure 7f) or PlexinA1 in motoneurons (compare to Figure 7g), ectopic expression of Sema6A resulted in the aberrant formation of BCC clusters. Bar: 100 μm