CELLULAR, MOLECULAR AND GENETIC ANALYSIS OF NEURAL FATE IN ZEBRAFISH EMBRYOS

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Our goal is to identify cellular, molecular and genetic mechanisms that determine how neurons are made in the appropriate number and location in the zebrafish neural plate. We are investigating a neurogenic mutant, mindbomb (mib), which is characterized by an over-production of early neurons. Previous observations suggest that this mutant has a defect in the neurogenic gene pathway which mediates lateral inhibition and normally limits the number of neurons produced in the embryo. Our linkage analysis, however, shows that mib does not appear to be linked to many of the known genes in the Notch signaling pathway. In order to identify this gene we identified a genetic marker within 0.4 cM of mib and used it to initiate a chromosomal walk with YACs and BACs. We have now covered a little under half the distance and are within 0.25 cM of mib. We expect the walk to define a BAC on which mib is located soon which should allow us to identify mib within the next year. We are also examining the function of zebrafish homologues of the neurogenic gene Notch in early neurogenesis. Specifically, we are investigating the role of Notch3 in neural plate formation. Notch3 is expressed during gastrulation in a pattern that anticipates formation of the anlage of the neural plate. Lineage analysis shows that at 90% epiboly the dorso-lateral neurectoderm contains a mixed population of cells that could give rise to either epidermis, neural crest or neural tissue. To investigate the possibility that Notch activation inhibits neural fate and helps select cells that adopt a neural fate we have examined the effects of Notch activation in both zebrafish and Xenopus embryos. We find that in both organisms, ectopic expression of an activated form of Notch3 in the ectoderm leads to what looks like a wider caudal neural plate and a loss of anterior neural tissue. Similar observations had been made previously by others when an activated form of Xotch was expressed in Xenopus and these observations, in part, had been used to conclude that Notch leads to an expansion of neural tissue. However, we find that ectopic expression of activated Notch3 in the ectoderm leads to a reduction of expression of sox2, a neural marker in Xenopus embryos. Furthermore,cells in which Notch3 has been ectopically expressed tend not to associate with surrounding uninjected cells in the neural tissue. In addition, expression of activated Notch leads to aberrant behavior of cells during convergence and extension leaving them with a relatively lateral and caudal distribution. These effects of Notch are consistent with Notch activation suppressing acquisition of neural fate and behavior as characterized by expression of specific neural markers and cell movements characteristic of neural cells during gastrulation and neurulation. Decreased convergence extension movements during gastrulation provide an alternative explanation for the apparently wider neural plate observed after ectopic expression of activated Notch. We are continuing to investigate the potential physiological role of Notch in selection of a neural fate in the neurectoderm.Early neurons are distributed in three longitudinal domains of the neural plate. Using early neuronal markers to visualize the early pattern of neurons we carried out an in situ hybridization based genetic screen to identify genes that influence the pattern of early neurons. We identified three classes of mutants characterized by an aberrant pattern of early sensory neurons. The first contains a single mutant narrowminded, characterized by loss of RB neurons, the second class also contains one mutant, necklace, characterized by loss of anterior brain structures and by the extension of the Trigeminal neurons around the rostral end of the neural plate. The third class consists of seven mutant lines. In each of these the lateral longitudinal domain, which contains Rohon-Beard (RB) sensory neurons, extends caudally in mutants creating an ectopic band of neurons that skirts the caudal neural plate. These lines are accompanied by variable tail defects and have been collectively called skirt mutants. Examination of dorso-ventral patterning in skirt mutants shows that most are accompanied by a mild degree of dorsalization. Consistent with this observation, mutants in one of the five complementation groups identified were found to have mutations intolloid, ametalloprotease that cleaves chordinand normally promotes ventral fates. Expression of neurogen in 1 during gastrulation shows that the longitudinal domain where RB cells form is oriented dorso-ventrally during early development and a dorsalized phenotype leads to the ventral extension of this domain around the circumference of the embryo. Later, convergent extension movements re-orient this proneuronal domain along the antero- posterior axis leading to the skirt phenotype characterized by what appears to be a caudal extension of the domain of RB neurons. Together, the narrowminded, necklace and skirt mutants help define how the early pattern of sensory neurons is generated in the zebrafish embryo. These studies will lead to insights about human diseases and birth disorders characterized by an aberrant distribution of neurons. Molecular mechanisms that determine the spatial distribution of cells in the nervous system are also used in other organ systems. Understanding how a simple pattern of neurons is generated in zebrafish will help us learn how cell-cell interactions lead to the self-organization of tissue heterogeneity in the developing embryo.