Notch signaling regulates neuroepithelial stem cell maintenance and neuroblast formation in Drosophila optic lobe development

Notch signaling mediates multiple developmental decisions in Drosophila. In this study, we have examined the role of Notch signaling in Drosophila larval optic lobe development. Loss of function in Notch or its ligand Delta leads to loss of the lamina and a smaller medulla. The neuroepithelial cells in the optic lobe in Notch or Delta mutant brains do not expand but instead differentiate prematurely into medulla neuroblasts, which lead to premature neurogenesis in the medulla. Clonal analyses of loss-of-function alleles for the pathway components, including N, Dl, Su(H), and E(spl)-C, indicate that the Delta/Notch/Su(H) pathway is required for both maintaining the neuroepithelial stem cells and inhibiting medulla neuroblast formation while E(spl)-C is only required for some aspects of the inhibition of medulla neuroblast formation. Conversely, Notch pathway overactivation promotes neuroepithelial cell expansion while suppressing medulla neuroblast formation and neurogenesis; numb loss of function mimics Notch overactivation, suggesting that Numb may inhibit Notch signaling activity in the optic lobe neuroepithelial cells. Thus, our results show that Notch signaling plays a dual role in optic lobe development, by maintaining the neuroepithelial stem cells and promoting their expansion while inhibiting their differentiation into medulla neuroblasts. These roles of Notch signaling are strikingly similar to those of the JAK/STAT pathway in optic lobe development, raising the possibility that these pathways may collaborate to control neuroepithelial stem cell maintenance and expansion, and their differentiation into the progenitor cells.     

The first deltex null mutant indicates tissue-specific deltex-dependent Notch signaling in Drosophila

Notch (N) is a single-pass transmembrane receptor. The N signaling pathway is an evolutionarily conserved mechanism that controls various cell-specification processes. Drosophila Deltex (Dx), a RING-domain E3 ubiquitin ligase, binds to the N intracellular domain, promotes N’s endocytic trafficking to late endosomes, and was proposed to activate Suppressor of Hairless [Su(H)]-independent N signaling. However, it has been difficult to evaluate the importance of dx, because no null mutant of a dx family gene has been available in any organism. Here, we report the first null mutant allele of Drosophila dx. We found that dx was involved only in the subsets of N signaling, but was not essential for it in any developmental context. A strong genetic interaction between dx and Su(H) suggested that dx might function in Su(H)-dependent N signaling. Our epistatic analyses suggested that dx functions downstream of the ligands and upstream of activated Su(H). We also uncovered a novel dx activity that suppressed N signaling downstream of N.     

Genetic regions that interact with loss- and gain-of-function phenotypes of <Emphasis Type="Italic">deltex</Emphasis> implicate novel genes in <Emphasis Type="Italic">Drosophila</Emphasis> Notch signaling

The Notch signaling pathway is an evolutionarily conserved mechanism that regulates many cell fate decisions. The deltex (dx) gene encodes an E3-ubiquitin ligase that binds to the intracellular domain of the Notch protein and regulates Notch signaling in a positive manner. However, it is still not clear how Dx does this. We generated a transgenic line, GMR-dx, which overexpresses dx in the developing Drosophila eye disc. The GMR-dx line showed a rough-eye phenotype, specific transformation of a photoreceptor cell (R3 to R4), and a rotation defect in the ommatidia. This phenotype was suppressed in combination with a dx loss-of-function mutant, indicating that it was due to a dx gain-of-function. We previously reported that overexpression of Dx results in the stabilization of Notch in late endosomes. Here, we found that three motifs in Dx, a region that binds to Notch, a proline-rich motif and a RING-H2 finger, were required for this stabilization, although the relative activity of these variants in this assay did not always correspond to the severity of the rough-eye phenotype. In an attempt to identify novel genes of the Notch pathway, we tested a large collection of chromosomal deficiencies for the ability to modify the eye phenotypes of the GMR-dx line. Twelve genomic segments that enhanced the rough-eye phenotype of GMR-dx were identified. To evaluate the specificity of these interactions, we then determined whether the deletions also interacted with the wing phenotypes associated with a loss-of-function mutation of dx, dx²⁴. Analyses based on whole-genome information allowed us to conclude that we have identified two novel loci that probably include uncharacterized genes involved in Dx-mediated Notch signaling.     

Down-regulation of Notch target gene expression by Suppressor of deltex

In Drosophila, Suppressor of deltex (Su(dx)) mutations display a wing vein gap phenotype resembling that of Notch gain of function alleles. The Su(dx) protein may therefore act as a negative regulator of Notch but its activity on actual Notch signalling levels has not been demonstrated. Here we show that Su(dx) does regulate the level of Notch signalling in vivo, upstream of Notch target genes and in different developmental contexts, including a previously unknown role in leg joint formation. Overexpression of Su(dx) was capable of blocking both the endogenous activity of Notch and the ectopic Notch signalling induced by the overexpression of Deltex, an intracellular Notch binding protein. In addition, using the conditional phenotype of the Su(dx)(sp) allele, we show that loss of Su(dx) activity is rapidly followed by an up-regulation of E(spl)mbeta expression, the immediate target of Notch signal activation during wing vein development. While Su(dx) adult wing vein phenotypes are quite mild, only affecting the distal tips of the veins, we show that the initial consequence of loss of Su(dx) activity is more severe than previously thought. Using a time-course experiment we show that the phenotype is buffered by feedback regulation illustrating how signalling networks can make development robust to perturbation.     

Notch信号途径中的Su(H)和E(spl)基因对果蝇天然免疫的影响

天然免疫系统是多细胞生物抵抗各种入侵微生物的第一道防线.Notch途径介导相邻细胞之间的相互作用,调节细胞、组织、器官的分化和发育.为了进一步探索Notch信号途径在果蝇天然免疫中的功能,利用Notch途径下游基因Su(H)和E(spl)的低表达突变体果蝇,通过体外注射病原体分析了生存率、血细胞的噬菌功能和抗菌肽的表达量以及突变体的血细胞数量.结果表明,革兰氏阴性细菌和真菌感染后果蝇E(spl)突变体的生存率、噬菌能力及抗菌肽的表达量明显降低,而且幼虫期血细胞出现异常增殖;Su(H)突变体只对真菌表现出敏感性,抗菌肽的表达量降低,但是对真菌的噬菌能力正常.此结果表明,Notch途径不仅影响个体的生长发育,而且在果蝇天然免疫中也起重要的调节作用.     

Perturbation of Notch/Suppressor of Hairless pathway disturbs migration of primordial germ cells in Xenopus embryo

Primordial germ cells (PGCs) in Xenopus embryo are specified in the endodermal cell mass and migrate dorsally toward the future gonads. The role of the signal mediated by Notch and Suppressor of Hairless [Su(H)] was analyzed on the migrating PGCs at the tailbud stage. X‐Notch‐1 and X‐Delta‐1 are expressed in the migrating PGCs and surrounding endodermal cells, whereas X‐Delta‐2 and X‐Serrate‐1 are expressed preferentially in the PGCs. Suppression and constitutive activation of the Notch/Su(H) signaling in the whole endoderm region or selectively in the PGCs resulted in an increase in ectopic PGCs located in lateral or ventral regions. Knocking down of the Notch ligands by morpholino oligonucleotides revealed that X‐Delta‐2 was indispensable for the correct PGC migration. The ectopic PGCs seemed to have lost their motility in the Notch/Su(H) signal‐manipulated embryos. Our results suggest that a cell‐to‐cell interaction via the Notch/Su(H) pathway has a significant role in the PGC migration by regulating cell motility.