[Show abstract][Hide abstract] ABSTRACT: Notch signaling is required for many developmental processes, yet differences in the signaling abilities of various Notch ligands are poorly understood. Here, we have isolated a splice variant of the zebrafish Notch ligand deltaC in which the inclusion of the last intron leads to a truncation of the C-terminal 39 amino acids (deltaC(tv2)). We show that, unlike deltaC(tv1), deltaC(tv2) cannot function effectively in somitogenesis but has an enhanced ability to signal during midline development. Additionally, over-expression of deltaC(tv2) preferentially affects anterior midline development, while another Notch ligand, deltaD, shows a posterior bias. Using chimeric Deltas we show that the intracellular domain is responsible for the strength of signal in midline development, while the extracellular domain influences the anterior-posterior bias of the effect. Together our data show that different deltas can signal in biologically distinct ways in both midline formation and somitogenesis. Moreover, it illustrates the importance of cell-type-dependent modifiers of Notch signaling in providing ligand specificity.
[Show abstract][Hide abstract] ABSTRACT: Zebrafish somitogenesis is governed by a segmentation clock that generates oscillations in expression of several Notch pathway genes, including her1, her7 and deltaC. Using a combination of pharmacological inhibition and Mendelian genetics, we show that DeltaD and DeltaC, two Notch ligands, represent functionally distinct signals within the segmentation clock. Using high-resolution fluorescent in situ hybridization, the oscillations were divided into phases based on eight distinct subcellular patterns of mRNA localization for 140,000 cells. her1, her7 and deltaC expression was examined in wild-type, deltaD(-/-) and deltaC(-/-) embryos. We identified areas within the tailbud where the clock is set up in the progenitor cells (priming), where the clock starts running (initiation), and where the clocks of neighbouring cells are entrained (synchronization). We find that the clocks of motile cells are primed by deltaD in a progenitor zone in the posterior tailbud and that deltaD is required for cells to initiate oscillations on exiting this zone. Oscillations of adjacent cells are synchronized and amplified by deltaC in the posterior presomitic mesoderm as cell movement subsides and cells maintain stable neighbour relationships.
[Show abstract][Hide abstract] ABSTRACT: The Tübingen large-scale zebrafish genetic screen completed in 1996 identified a set of five genes required for orderly somite segmentation. Four of them have been molecularly identified and three were found to code for components of the Notch pathway, which are required for the coordinated oscillation of gene expression, known as the segmentation clock, in the presomitic mesoderm (PSM). Here, we show that the final member of the group, beamter (bea), codes for the Notch ligand DeltaC, and we present and characterize two new alleles, including one allele encoding for a protein truncated in the 7th EGF repeat and an allele deleting only the DSL domain which was previously shown to be necessary for ligand function. Interestingly however, when we over-express any of the mutant deltaC mRNAs, we observe antimorphic effects on both hindbrain neurogenesis and hypochord formation. Expression of bea/deltaC oscillates in the PSM, and a triple fluorescent in situ analysis of its oscillation in relation to that of other oscillating genes in the PSM reveals differences in subcellular localization of the oscillating mRNAs in individual cells in different oscillation phases. Mutations in aei/deltaD and bea/deltaC differ in the way they disrupt the oscillating expression of her1 and deltaC. Furthermore, we find that the double mutants have significantly stronger defects in hypochord formation but not in somitogenesis or hindbrain neurogenesis, indicating genetically that the two delta's may function either semi-redundantly or distinctly, depending upon context.