beamter/deltaC and the role of Notch ligands in the zebrafish somite segmentation, hindbrain neurogenesis and hypochord differentiation

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.

Automated gene oscillation phase classification for zebrafish presomitic mesoderm cells

“…Fluorescent in situ hybridizations were performed by using methods described in Ref. 7. The embryos were flat-mounted in glycerol, and confocal stacks were collected on a BioRad 1,024 confocal microscope using a Zeiss Neofluor 253 objective (8).…”

“…The selected optical sections were generally $20 lm apart and had an optical thickness of \3 lm. As shown in Figure 1, the red channel, referenced as nuclei channel, records the nuclei information by staining cell nuclei with propidium iodide, while the green channel, referenced as mRNA channel, records the information of mRNA subcellular distribution using highresolution fluorescent in situ hybridization (6)(7)(8). A mask image and a lookup table were also supplied for each zebrafish PSM image in the dataset, which provide manual annotation and classification result for cells in the corresponding zebrafish PSM image (8).…”

“…THE zebrafish (Danio rerio) has recently emerged as an important vertebrate model organism in developmental biology, functional genomics, disease modeling, and drug discovery (1)(2)(3)(4)(5)(6)(7)(8). The rapid embryonic development and the transparency of zebrafish embryo render it possible to visualize their early developmental events in vivo.…”

“…The segmentation clock causes cells to undergo repeated cycles of transcriptional activation and repression. By fluorescent in situ hybridization (6,7), these oscillations can be visualized. Figure 1 shows two examples of zebrafish presomitic mesoderm (PSM) images; the red blobs are cell nuclei and the green fluorescent stain appears in or around nuclei showing the mRNA localization which reveals different oscillation phases.…”

Coupling cellular oscillators: A mechanism that maintains synchrony against developmental noise in the segmentation clock

“…To determine the phase of individual oscillators with a higher time resolution, a recently developed fluorescent high-resolution ISH has been shown to be a very powerful tool (Kosman et al, 2004;Julich et al, 2005). An example of the usefulness of this new technique can be seen in Figure 1c.…”