Identification and functional analysis of Zebrafish orthologs of genes

Cell motility and migration are integral to the complex mechanics of animal development, growth and maintenance. The precise and stereotypical orchestration of cell motility is controlled by a number of molecules, both within cells and in the extracellular environment. The phenomenon of axonal growth cone motility during neural circuit formation is a special case of cell movement, and has been studied extensively over the past several years. A number of receptor-ligand interactions have been described that regulate this process and one example involves the Roundabout (Robo) receptor and its extracellular, secreted ligand Slit. Both of these molecules are evolutionarily conserved from worms to humans and have been shown to play important roles in axon guidance. To gain a better understanding of Robo orthologs in vertebrates, we chose zebrafish as our model system for the many experimental advatages it offers. A PCR-based strategy was employed to find zebrafish orthologs of Robo. Two orthologs ( robo1 and robo3 ) were identified whose expression patterns in the nervous system and non-neural tissues suggested a role in axon guidance and cell motility. Further characterization of robo3 unveiled the presence of two distinct isoforms ( robo3a and robo3b ). The two isoforms exhibited spatially and temporally dynamic gene expression patterns. Functional analysis of robo3 isoforms using an antisense gene 'knock-down' strategy suggested that robo3b functions during gastrulation, whereas robo3a is required for motor axon pathfinding. This study uncovers a novel function of the Robo receptor family in cell movements during gastrulation, complementing an earlier study that suggests a role of zebrafish Slit orthologs in gastrulation. The implication of Robo-Slit signaling in cell movements during gastrulation underscores the conservation of molecular mechanisms in a variety of cell motility phenomena.