Figure 2

chch represses mesodermal markers. Mesodermal markers were examined by RNA in situ hybridization and real-time PCR in embryos microinjected with chch-EnR mRNA. Inhibition of chch does not alter expression of dorsal mesodermal genes chd (A, B) or flh (C, D). Expression of mesodermal markers at shield stage, including spt (E-F) and ntl (G-H) are expanded slightly in chch-inhibited embryos. Overexpression of chch mRNA does not alter no-tail expression at shield stage (I-J). chch inhibition results in a decrease in Sip1 gene expression at bud stage (M-N), while chch activation results in Sip1 induction at shield stage (K-L). Real-time PCR analysis of mesodermal markers in chch-inhibited embryos (O). The fold change in transcript levels (y-axis) is graphed relative to control embryos following overexpression of chch-EnR mRNA, chch-ATGMO and chch mRNA. This analysis reveals that the domains of mesodermal genes are expanded at shield stage while dorsal mesodermal genes are unaffected. Conversely, overexpression of chch mRNA does not result in alteration of early mesodermal gene expression. The induction of ntl expression following chch inhibition with the ATGMO can be rescued by co-expression of chch mRNA (P). Real-time PCR analysis of Sip1 mRNA in chch, chch-EnR and chch-ATGMO treated embryos (Q). Induction of chch results in a 50% increase in Sip1 expression, conversely, inhibition of chch results in a 70% reduction of Sip1 expression. The fold change in Sip1 transcript levels (y-axis) is graphed relative to control embryos. Views: A-B, K-L are animal pole; C-D, M-N are dorsal; E-J are lateral.