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Fig. 2. FoxH1 represses Nodal target genes at blastula and gastrula stages. (A) FoxH1, but not FoxH1A6, represses expression of endogenous mesodermal and Nodal genes. Embryos were injected in the vegetal pole with mRNA encoding myc-FoxH1 or myc-FoxH1A6 (250 pg). cDNA was prepared from pooled whole embryos collected at blastula stage (for Xnr5 and Xnr6) and gastrula stage (for Xbra, Chd and Gsc). qPCR was performed for the mesodermal marker Brachyury, the organizer genes Chordin and Goosecoid, and the Nodal ligands Xnr1, Xnr5 and Xnr6. The data presented are the combined results of three independent biological replicates, and have been normalized to expression of a housekeeping gene, EF1α. Error bars represent standard error. * indicates p<0.05 by Student's t-test. (B) FoxH1, but not FoxH1A6, inhibits expression of Xnr5 and Xnr6 in the blastula vegetal pole. Embryos injected in the vegetal pole with mRNA encoding myc-FoxH1 or myc-FoxH1A6 (250 pg) were collected at the late blastula stage and subjected to in situ hybridization for either Xnr5 or Xnr6. (C) FoxH1, but not FoxH1A6, blocks expression of Xbra and Chd in the gastrula. Embryos were injected in the vegetal pole with mRNA encoding myc-FoxH1 or myc-FoxH1A6 (250 pg) and collected at the mid-gastrula stage. In situ hybridization was performed to analyze expression of Xbra and Chd. 100 embryos were analyzed for each condition, and the percentage of embryos displaying normal (dark gray bar) or reduced (light gray bar) expression was quantified in the bar graphs to the right. Expression of FoxH1 reduced the expression of Xnr5, Xnr6, Xbra and Chd. |
