Lateral inhibition, mediated by Notch signaling, leads to the selection of cells that are permitted to become neurons within domains defined by proneural gene expression. Reduced lateral inhibition in zebrafish mib mutant embryos permits too many neural progenitors to differentiate as neurons. Positional cloning of mib revealed that it is a gene in the Notch pathway that encodes a RING ubiquitin ligase. Mib interacts with the intracellular domain of Delta to promote its ubiquitylation and internalization. Cell transplantation studies suggest that mib function is essential in the signaling cell for efficient activation of Notch in neighboring cells. These observations support a model for Notch activation where the Delta-Notch interaction is followed by endocytosis of Delta and transendocytosis of the Notch extracellular domain by the signaling cell. This facilitates intramembranous cleavage of the remaining Notch receptor, release of the Notch intracellular fragment, and activation of target genes in neighboring cells.

In vertebrates with mutations in the Notch cell-cell communication pathway, segmentation fails: the boundaries demarcating somites, the segments of the embryonic body axis, are absent or irregular. This phenotype has prompted many investigations, but the role of Notch signalling in somitogenesis remains mysterious. Somite patterning is thought to be governed by a "clock-and-wavefront" mechanism: a biochemical oscillator (the segmentation clock) operates in the cells of the presomitic mesoderm, the immature tissue from which the somites are sequentially produced, and a wavefront of maturation sweeps back through this tissue, arresting oscillation and initiating somite differentiation. Cells arrested in different phases of their cycle express different genes, defining the spatially periodic pattern of somites and controlling the physical process of segmentation. Notch signalling, one might think, must be necessary for oscillation, or to organize subsequent events that create the somite boundaries. Here we analyse a set of zebrafish mutants and arrive at a different interpretation: the essential function of Notch signalling in somite segmentation is to keep the oscillations of neighbouring presomitic mesoderm cells synchronized.

BackgroundEpidermal ionocytes play essential roles in the transepithelial transportation of ions, water, and acid-base balance in fish embryos before their branchial counterparts are fully functional. However, the mechanism controlling epidermal ionocyte specification and differentiation remains unknown.Methodology/Principal FindingsIn zebrafish, we demonstrated that Delta-Notch-mediated lateral inhibition plays a vital role in singling out epidermal ionocyte progenitors from epidermal stem cells. The entire epidermal ionocyte domain of genetic mutants and morphants, which failed to transmit the DeltaC-Notch1a/Notch3 signal from sending cells (epidermal ionocytes) to receiving cells (epidermal stem cells), differentiates into epidermal ionocytes. The low Notch activity in epidermal ionocyte progenitors is permissive for activating winged helix/forkhead box transcription factors of foxi3a and foxi3b. Through gain- and loss-of-function assays, we show that the foxi3a-foxi3b regulatory loop functions as a master regulator to mediate a dual role of specifying epidermal ionocyte progenitors as well as of subsequently promoting differentiation of Na+,K+-ATPase-rich cells and H+-ATPase-rich cells in a concentration-dependent manner.Conclusions/SignificanceThis study provides a framework to show the molecular mechanism controlling epidermal ionocyte specification and differentiation in a low vertebrate for the first time. We propose that the positive regulatory loop between foxi3a and foxi3b not only drives early ionocyte differentiation but also prevents the complete blockage of ionocyte differentiation when the master regulator of foxi3 function is unilaterally compromised.

Mutations causing a visible phenotype in the adult serve as valuable visible genetic markers in multicellular genetic model organisms such as Drosophila melanogaster, Caenorhabditis elegans and Arabidopsis thaliana. In a large scale screen for mutations affecting early development of the zebrafish, we identified a number of mutations that are homozygous viable or semiviable. Here we describe viable mutations which produce visible phenotypes in the adult fish. These predominantly affect the fins and pigmentation, but also the eyes and body length of the adult. A number of dominant mutations caused visible phenotypes in the adult fish. Mutations in three genes, long fin, another long fin and wanda affected fin formation in the adult. Four mutations were found to cause a dominant reduction of the overall body length in the adult. The adult pigment pattern was found to be changed by dominant mutations in wanda, asterix, obelix, leopard, salz and pfeffer. Among the recessive mutations producing visible phenotypes in the homozygous adult, a group of mutations that failed to produce melanin was assayed for tyrosinase activity. Mutations in sandy produced embryos that failed to express tyrosinase activity. These are potentially useful for using tyrosinase as a marker for the generation of transgenic lines of zebrafish.

During segmentation of the vertebrate hindbrain, a distinct population of boundary cells forms at the interface between each segment. Little is known regarding mechanisms that regulate the formation or functions of these cells. We have investigated a potential role of Notch signaling and find that in the zebrafish hindbrain, radical fringe is expressed in boundary cells and delta genes are expressed adjacent to boundaries, consistent with a sustained activation of Notch in boundary cells. Mosaic expression experiments reveal that activation of the Notch/Su(H) pathway regulates cell affinity properties that segregate cells to boundaries. In addition, Notch signaling correlates with a delayed neurogenesis at hindbrain boundaries and is required to inhibit premature neuronal differentiation of boundary cells. These findings reveal that Notch activation couples the regulation of location and differentiation in hindbrain boundary cells. Such coupling may be important for these cells to act as a stable signaling center.