Cellular and molecular analysis of motor neuron development in the zebrafish hindbrain

Proper development of the nervous system depends upon the production of neurons in the correct numbers, and in the correct locations. Many cell fate decisions in the developing nervous system depend on intercellular signaling mediated by the transmembrane proteins Notch and Delta. Failure of Notch signaling in zebrafish mind bomb (mib) mutants generates a neurogenic phenotype in the hindbrain characterized by the overproduction of some neuronal cell types, and accompanied by a significant loss of motor neurons and neuroepithelial cells. Genetic mosaic analysis indicates that hindbrain motor neuron patterning and fusion defects in mib mutants arise non-cell autonomously, partly due to the differentiation of midline neuroepithelial cells into neurons. These results highlight the importance of non-neuronal cells to rhombomere boundaries formation and midline tissues which are the sources of signals for numerous patterning mechanisms within the developing brain. Additional signaling pathways act on these neuronal precursors to induce the formation of specific neuronal types. For example, motor neurons are induced by secreted proteins encoded by the hedgehog gene family. Mutations in shh alone lead to reduced induction of hindbrain motor neurons, while reduction of twhh expression does not affect motor neuron number. However, when both functions of twhh and shh are disrupted, there is a complete failure to induce hindbrain motor neurons. These results demonstrate that shh and twhh play a cooperative role in zebrafish hindbrain motor neuron induction, and suggest that hindbrain motor neuron induction in zebrafish may be dependent on the concentration of total hedgehog activity, rather than different hedgehog proteins. A subset of motor neurons migrates tangentially into more caudal hindbrain locations following induction in anterior hindbrain regions in the wild-type zebrafish but fail to do so in the trilobite mutant. Genetic mosaic analysis revealed that the neuronal migration defect in tri mutants is due to the loss of cell autonomous and non-cell autonomous tri function. In vivo time-lapse and molecular analyses demonstrate that mutations in the zebrafish gene trilobite (tri) specifically affect the ability of motor neurons in the hindbrain to polarize their protrusions, leading to the elimination of tangential motor neuron migration. We now know that tri encodes the Planar Cell Polarity gene, Strabismus, previously unidentified for its role in mediating neuronal cell movements. tri therefore represents an important genetic tool in understanding the mechanisms underlying tangential neuronal migration.