Our goal is to identify cellular, molecular and genetic mechanisms that determine how neurons are made in the appropriate number and location in the neural plate. In order to identify such mechanisms we have identified zebrafish mutants with an aberrant pattern of early neurons. Analysis of these mutants is beginning to define mechanisms that are essential for patterning early neurogenesis in the zebrafish embryo. The headless (hdl) mutant was identified by its expanded domain of trigeminal neurons. Expansion of trigeminal neurons in this mutant is accompanied by an expansion of the neural domain at the midbrain-hindbrain boundary (MHB), adjacent to which trigeminal neurons are normally generated. Expansion of neural domains associated with the MHB is at the cost of anterior neural structures like eyes, forebrain and most of the midbrain, which are lost in this mutant. Ectopic activation of Wnt/Wingless signaling during early gastrulation has previously been shown to have similar effects on head development suggesting that there is loss of a mechanism that inhibits Wnt signaling in hdl mutants. Genetic mapping of the mutation revealed that hdl encodes a member of the Tcf/Lef family, Tcf3, a transcription factor that acts as a repressor of Wnt target genes through its association with co-repressors, or as an activator through its association with beta-catenin. The hdl mutant phenotype is rescued by microinjection of synthetic RNA encoding wild-type Tcf3. The mutant phenotype is also rescued by a deletion of Tcf3 that lacks the beta-catenin binding domain, and by a chimaeric repressor, Engrailed-Tcf3. Conversely, a chimaeric activator, VP16-Tcf3, generates a headless phenotype in wild-type embryos. Our results suggest that basal repression of Wnt targets by Tcf3 during early gastrulation is essential for expression of genes responsible for determining anterior neural fate. Previous studies have suggested a critical role for secreted Wnt antagonists as head inducers. Our studies suggest that these Wnt antagonists act as head inducers by maintaining repression of Wnt target genes provided by Tcf3. In the absence of this repression the Wnt antagonists are ineffective head inducers. The neurogenic mutant, mind bomb (mib), is characterized by an over-production of early neurons. Functional analysis suggests these mutants have a defect in the Notch signaling pathway. Previous studies have shown that Notch signaling mediates lateral inhibition and normally limits the number of neurons produced in the embryo. We showed that activation of Notch signaling reduces the neurogenic phenotype and ectopic expression of genes that inhibit Notch signaling mimic the mib phenotype. Positional cloning has helped identify a strong candidate for mib. We are in the process of determining if mutations in this candidate are responsible for the mutant phenotype in mib mutants. We are also investigating the role of Notch in neural plate formation. Notch3 is expressed during gastrulation in the neurectoderm. Later its expression appears to be restricted to neuroblasts within the neural plate and neural tube. We find that ectopic expression of RNA encoding an activated form of Notch3 in the ectoderm increases the size of the neurectoderm. However, persistent expression of a constitutively active form of Notch3 during gastrulation, induced by expressing it under control of a heat shock promoter, appears to inhibit cells from being incorporated in the neural tube. These observations suggest that Notch signaling initially promotes the formation of the neurectoderm but later it limits the number of cells that adopt a neural fate within the neurectoderm.Together these studies reveal critical roles played by the Wnt and Notch signaling pathways in patterning early neurogenesis. By providing genetic evidence for the critical role played by Tcf3 in Wnt signaling these studies illustrate how zebrafish mutants are deepening our understanding of signaling pathways that are widely used during vertebrate development.
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