The murine cerebellum is a powerful model system in which to study the control of pattern during the formation of the CNS. The work to be performed during the next project period draws on techniques of quantitative morphology, mouse genetics and modern molecular biology to provide a unique approach to understanding the forces that guide the creation of this important brain region. The work is designed to test the hypothesis that the cerebellar anlage is """"""""compartmentalized"""""""" twice. Once at the early neural tube stage when the precursors for the entire cerebellar anlage are set aside, and a second time after neurogenesis is complete when the patterns of foliation and sagittal banding is established. The experiments proposed in this five year plan will explore the causes and consequences of both compartmentalization events. Chimeric mice will be made suing both Engrailed-1 and Engrailed-2 mutant embryos aggregated with genetically marked wild-type embryos. Both Engrailed genes are expressed in a band at the midbrain/hindbrain junction at embryonic day 8.5. An engineered mutation in Engrailed-1 results in the complete absence of the midbrain and cerebellum while a comparable loss of Engrailed-2 function leads to a 30 percent reduction in cell number and an alteration in the adult folial pattern. Animals whose brains are mosaics of normal and mutant cells will provide a great deal of important information on the roles of these two homoeobox genes in guiding the behavior of early cerebellar precursor cells, in particular their ability to regulate cell number and pattern. The second compartmentalization event is hypothesized to occur after the ventricular zone cells have permanently left the cell cycle and begun migration and differentiation. We will expand the description of the compartmental structure of the cerebellum by extending the analysis to the deep cerebellar nuclei. The molecular elements that regulate the pattern of sagittal bands will be sought, by analyzing the spatiotemporal expression pattern of the Zebrin II/aldolase C gene. Genomic constructs will be engineered in both plasmids and BACs and used to express marker proteins. Analysis will be done in both cerebellar slices and transgenic mice. The location of the cis-elements that govern Purkinje cell specificity and banding fidelity will be identified. Once this aspect of the project is completed we will search for the trans-acting proteins that interact with these elements and through their identity learn more about the genetic regulation of the second compartmentalization event.
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