Cellular, Molecular And Genetic Analysis Of Neural Fate

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Our goal is to determine how neurons are made in the appropriate number and location during early neurogenesis. Previous studies have identified remarkable similarities in mechanisms that determine neural fate in the fruit fly, Drosophila, and vertebrates during early development. In zebrafish, expression of neurogenin1 (ngn1), a homolog of the Drosophila proneural gene, atonal, defines proneuronal domains where cells have the potential to become neurons in the neural plate. Mechanisms that determine early expression of ngn1 in the neuroectoderm, however, remain poorly defined. Iroquois (Iro) genes encode an atypical class of homeodomain proteins that were originally identified in Drosophila for their role in determining formation of sensory bristles. Analysis of their function in Drosophila revealed an early role for Iro genes in defining the identity of large territories in the embryo where later they have a role in determining expression of proneural genes. We have identified a novel Iro gene, iro7 in zebrafish. Iro7 is expressed during gastrulation along with iro1 in a compartment of the dorsal ectoderm that includes the prospective midbrain-hindbrain boundary (MHB), the adjacent neural crest and the trigeminal sensory neurons. The iro1 and iro7 expression domain is expanded in headless (hdl) and masterblind (mbl) mutants that are characterized by expansion of the MHB domain and adjacent tissues including the domain of ngn1 expression where trigeminal sensory neurons are formed. Expansion of iro1 and iro7 expression in hdl and mbl mutants is due to the loss of mechanisms that repress Wnt target genes during gastrulation suggesting that the territory of iro1 and iro7 expression is determined by Wnt signaling during early development. A knock-down of iro7 function with anti-sense morpholino oligos revealed that iro7 is essential for expression of ngn1 in the domain where trigeminal sensory neurons are formed. A knock-down of both iro1 and iro7 revealed additional roles in neural crest development and establishment of the isthmic organizer at the MHB. Together, these results suggest that iro1 and iro7 are required for establishment of tissues that define the identity of a dorsal compartment of the ectoderm where these Iro genes are expressed early in development. The neurogenic mutant, mind bomb (mib), is characterized by an over-production of early neurons and functional analysis suggests these mutants have a defect in lateral inhibition mediated by Notch signaling, which normally limits the number of cells permitted to become neurons within proneuronal domains. We found that inhibition of Notch1a and Notch5 function leads to a phenotype very similar to that seen in mib mutants where the increase in the number of early neurons within proneuronal domains is accompanied by reduced expression of HER4, a gene whose expression is induced by Notch signaling. Positional cloning revealed mib is a novel gene whose function had not been determined in other model organisms. It encodes a protein with RING domains identifying it as a potential ubiquitin ligase. Analysis of five mutant alleles revealed point mutations that are expected to result in truncated proteins or in critical amino acid substitutions in the RING domain. Ubiquitin ligases facilitate addition of ubiquitin to specific substrate proteins, typically targeting them for destruction in proteosomes and/or modifying their function in the cells. We are currently investigating how function of mib as a ubiquitin ligase contributes to the efficiency of Notch signaling during early neurogenesis. Our analysis of mib mutants has also revealed that expression of another atonal homolog, zath1, and neurogenic genes like DeltaA and Notch5 play a role in defining cells that become sensory hair cells in the lateral line system. The ability to examine progressive selection of sensory hair cells in this easily accessible part of the peripheral nervous system makes it attractive to study mechanisms involved in determining the distribution of early neurons. Together these studies illustrate how a combination of cellular, molecular and genetic approaches in zebrafish are revealing fundamental mechanisms involved in patterning early neurogenesis in the vertebrate embryo.