Analysis on molecular mechanisms of neuralization and early patterning in the formation of the presumptive midbrain-hindbrain boundary region

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To explore the molecular mechanism of neuralization and early pattern formation in vertebrate embryos, we investigated the following two subjects.1) Functional analysis of XHR1 : ESR1 is a bHLH transcriptional repressor gene, and was identified as a candidate for XHR1 target genes. We performed (1) gene activation analysis using a XRH1-VP16-GR construct and a protein synthesis inhibitor, (2) gain-of-function analysis of XHR1 and ESR1, and (3) loss-of-function analysis using antisense-morpholino oligos for XHR1. We showed that ESR is a direct target of XHR1 ; that XHR1 is necessary and sufficient for suppression of ESR1 expression; and that overexpression of ESR1 represses the MHB gene Pax2. Furthermore, in contrast to ESR1, XHR1 was not activated by Notch signaling, and XHR1 directly repressed neuronal genes, neurogenin and delta. These data suggest that XHR1 functions as a prepattern gene for MHB formation as well as for proneural cluster formation by repressing neuronal differentiati … More on2) Analysis of XHR1 gene regulation : We cloned and analyzed a Xenopus tropicalis HR1 (XtHR1) promoter, which faithfully recapitulated the endogenous XHR1 expression. Surprisingly, transgenic and animal cap assays revealed distinct enhancers, named the early and late enhancer modules (the EEM and LEM), that govern XHR1 expression in pre-MHB and MHB regions, respectively. Furthermore, the EEM was found to integrate Noggin, Wnt, and FGF signals. Gain-of-function and dominant-negative approaches in vivo showed that neural inducers activate XHR1 ; Wnt signaling confines XHR1 expression; and moderate and strong FGF signaling enhances and suppresses XHR1 expression, respectively. These data suggest that the XHR1 expression of the pre-MHB region is induced through the EEM by signaling molecules from the Spemann organizer.Thus, we have shown the molecular mechanism of XHR1 induction in the pre-MHB region and a molecular cascade mediated by XHR1 from the pre-MHB to the MHB region, an organizing center of brain formation. Less