IDENTIFICATION OF THE GENE(S) RESPONSIBLE FOR ALAGILLE S

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Jagged1 (JAG1) gene encodes a ligand for a Notch receptor, and mutations in the JAG1 are responsible for Alagille syndrome (AGS). AGS is a developmental disorder characterized by the paucity of bile ducts, and affecting multiple other organ systems including heart, eyes, vertebrae and face. Mutations in many other members of the Notch pathway have also been found to be associated with human diseases and mouse phenotypes. Zebrafish is an excellent model for vertebrate development, and therefore we have initiated efforts to explore the role of jagged genes in zebrafish development, and in developmental diseases like Alagille syndrome. As a part of this effort, we have isolated and characterized Jagged homologous genes from zebrafish. Unlike in humans where two Jaggeds (Jagged 1 and 2) have been identified, we find threehomologs (termed Jagged 1, 2 and 3) in zebrafish. We isolated full-length cDNAs for all three Jagged genes, mapped their chromosomal location and studied their expression during early embryonic development. We find that Jaggeds 1 and 3 are similar to each other and appear to have originated as a result of genomic duplication in zebrafish. Ectopic expression of jagged RNAs leads to a reduction of neurons in mib mutant embryos in which reduced lateral inhibition permits too many neural progenitors to differentiate into neurons. Positional cloning revealed that the mib is a novel gene in the Notch pathway, which encodes a RING Ubiquitin ligase that is essential for efficient activation of Notch by Delta. Antisense oligonucleotides (Morpholino derivatives) generated for Jaggeds 1, 2 and 3 are being used to explore their role in liver development. Combinations of Jagged and Notch gene knockdowns alter zebrafish biliary development in a manner compatible with an AGS phenocopy. The Notch pathway plays critical roles in many developmental processes, and we plan explore the specific roles of the Notch receptors and their ligands, jaggeds and delta, using antisense oligonucleotides.