Zebrafish Her8a is activated by Su(H)-dependent Notch signaling and is essential for the inhibition of neurogenesis

Understanding how diversity of neural cells is generated is one of the main tasks of developmental biology. The Hairy/E(spl) family members are potential targets of Notch signaling, which has been shown to be fundamental to neural cell maintenance, cell fate decisions, and compartment boundary formation. However, their response to Notch signaling and their roles in neurogenesis are still not fully understood. In the present study, we isolated a zebrafish homologue of hairy/E(spl), her8a, and showed this gene is specifically expressed in the developing nervous system. her8a is positively regulated by Su(H)-dependent Notch signaling as revealed by a Notch-defective mutant and injection of variants of the Notch intracellular regulator, Su(H). Morpholino knockdown of Her8a resulted in upregulation of proneural and post-mitotic neuronal markers, indicating that Her8a is essential for the inhibition of neurogenesis. In addition, markers for glial precursors and mature glial cells were down-regulated in Her8a morphants, suggesting Her8a is required for gliogenesis. The role of Her8a and its response to Notch signaling is thus similar to mammalian HES1, however this is the converse of what is seen for the more closely related mammalian family member, HES6. This study not only provides further understanding of how the fundamental signaling pathway, Notch signaling, and its downstream genes mediate neural development and differentiation, but also reveals evolutionary diversity in the role of H/E(spl) genes.     

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.     

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.     

The HLH protein Extramacrochaetae is required for R7 cell and cone cell fates in the Drosophila eye

Notch signaling is one of the most important pathways in development and homeostasis, and is altered in multiple pathologies. Study of Drosophila eye development shows that Notch signaling depends on the HLH protein Extramacrochaetae. Null mutant clones show that extramacrochaetae is required for multiple aspects of eye development that depend on Notch signaling, including morphogenetic furrow progression, differentiation of R4, R7 and cone cell types, and rotation of ommatidial clusters. Detailed analysis of R7 and cone cell specification reveals that extramacrochaetae acts cell autonomously and epistatically to Notch, and is required for normal expression of bHLH genes encoded by the E(spl)-C which are effectors of most Notch signaling. A model is proposed in which Extramacrochaetae acts in parallel to or as a feed-forward regulator of the E(spl)-Complex to promote Notch signaling in particular cellular contexts.     

The two Tribolium E(spl) genes show evolutionarily conserved expression and function during embryonic neurogenesis

Tribolium castaneum is a well-characterised model insect, whose short germ-band mode of embryonic development is characteristic of many insect species and differs from the exhaustively studied Drosophila. Mechanisms of early neurogenesis, however, show significant conservation with Drosophila, as a characteristic pattern of neuroblasts arises from neuroectoderm proneural clusters in response to the bHLH activator Ash, a homologue of Achaete–Scute. Here we study the expression and function of two other bHLH proteins, the bHLH-O repressors E(spl)1 and E(spl)3. Their Drosophila homologues are expressed in response to Notch signalling and antagonize the activity of Achaete–Scute proteins, thus restricting the number of nascent neuroblasts. E(spl)1 and 3 are the only E(spl) homologues in Tribolium and both show expression in the cephalic and ventral neuroectoderm during embryonic neurogenesis, as well as a dynamic pattern of expression in other tissues. Their expression starts early, soon after Ash expression and is dependent on both Ash and Notch activities. They act redundantly, since a double E(spl) knockdown (but not single knockdowns) results in neurogenesis defects similar to those caused by Notch loss-of-function. A number of other activities have been evolutionarily conserved, most notably their ability to interact with proneural proteins Scute and Daughterless.