The goal of the Unit on Vertebrate Neural Development is to understand how neurons are made in the appropriate number and location in the developing vertebrate nervous system. Toward that end, we use a combination of molecular, cellular, and genetic approaches to understand how a relatively simple pattern of early neurons is established in the zebrafish neural plate. Previous studies have shown remarkable similarities in the mechanisms by which cells are selected for a neural fate in the Drosophila neuroectoderm and for a neuronal fate in the vertebrate neural plate. Expression of neurogenin1 (ngn1), a vertebrate homolog of the Drosophila proneural gene atonal, defines domains in the neuroectoderm where cells have the potential to become neurons. Within each of these domains, lateral inhibition, mediated by Notch signaling, leads to the selection of cells that will become neurons. Over the past year, we have defined a role for Iroquois (Iro) genes in determining ngn1 expression in the neuroectoderm and discovered that mutations in a novel gene in the Notch pathway are responsible for the production of too many neurons in mind bomb (mib) mutants. We have also described the expression of proneural and neurogenic genes in the lateral line primordium and shown that this locus is an attractive system for the study of neuronal differentiation. These studies provide novel insights into factors that determine the fate of neural stem cells in the developing nervous system. In addition, identification of mib promises to reveal new ways of manipulating the Notch pathway, a signaling system that plays a critical role in normal development as well as in the development of cancer in a wide range of tissues.

iro1 and iro7 Define the Identity of an Anteroposterior Compartment in the Midbrain-Hindbrain Region Following Wnt Signaling
Itoh, Kim, Chitnis in collaboration with Kudoh^e and Dedakian^e
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 and expression of proneural genes. We 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 and masterblind 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 iro1and iro7 expression in these 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 knockdown of iro7 function with antisense morpholino oligos revealed that iro7 is essential for expression of ngn1 in the domain in which trigeminal sensory neurons are formed. A knockdown 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.

Identification of mib as a Novel Gene in the Notch Signaling Pathway
Kim, Palardy, Itoh, Yeo in collaboration with Chandrasekharappa,^a Oda,^a Jiang,^b Lewis,^b Kenworthy,^c Lippincott-Schwartz,^c Lorick,^d and Weissman^d
The neurogenic mutant, mind bomb (mib), is characterized by an overproduction of early neurons. Functional analysis suggests that the 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 in which 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 that 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. We are currently investigating how the function of mib as a ubiquitin ligase contributes to the efficiency of Notch signaling during early neurogenesis.

Expression of Proneural and Neurogenic Genes in the Zebrafish Lateral Line Primordium
Itoh, Chitnis
The lateral line primordium migrates under the skin, sequentially depositing groups of cells called neuromasts that contain sensory hair cells similar to those seen in the inner ear. We examined expression of zath1, a zebrafish atonal homolog, and found that such expression correlates with selection of hair cells within nascent neuromasts before their deposition by the migrating lateral line primordium. Expression of DeltaA and Notch3 (now called Notch5) in wild-type and mib mutant embryos suggested that events leading to selection of sensory neurons within the lateral line primordium are very similar to those that occur within the neural plate when cells are selected to become neurons.