grhl2b is a predicted downstream target of fgf8 signalling. (A-L) Downregulation of fgf8 causes loss of cerebellum and enhanced apoptosis (ace phenotype; A-C), a defect that is recapitulated when fgf8 and grhl2b morpholinos are co-injected at individual sub-phenotypic doses (D-F). More severely affected fgf8-grhl2b double knockdown embryos display enhanced apoptosis and loss of MHB folding in a dose-dependent manner (G-I). No phenotypes or increased apoptosis are seen when fgf8 and control morpholinos are co-injected at sub-phenotypic doses (J-L). Arrows and brackets in all panels show position of the MHB; B,E,H,K show TUNEL staining of the relevant phenotype. (M) Luciferase activity from pGL3-eng2a-PROM is significantly decreased following co-injection of sub-phenotypic doses of MO:fgf8 and MO:grhl2b, but not when either MO is injected at sub-phenotypic doses together with MO:control.(N) spec1 expression is lost in fgf8 morphants at the formative stages of MHB morphogenesis. (O) Putative model of the predicted functional pathways of fgf8/erk-mediated regulation of grhl2b in MHB maintenance, neural apoptosis and MHB folding.
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