Tbx16 is essential for Fgf4-driven upregulation of tbx16 and suppression of tbxta. Embryos from a tbx16+/− incross injected with 150 pg fgf4 mRNA or control. (A) By 24 hpf, Fgf4-injected embryos have disorganised heads and, although lacking obvious trunk or tail, some contain twitching muscle. (B) ISH for col1a2 for dermomyotome/connective tissue and myhz1 for skeletal muscle revealed muscle in fgf4-injected sibs, but not in tbx16−/− mutants. Boxed areas are magnified to show the alternating pattern of aggregated muscle and connective tissue in Fgf4-injected siblings, but the reduced col1a2 and absent myhz1 mRNA in Fgf4-injected mutants. Note the aggregation of posterior mesoderm cells into strands around the yolk. (C) ISH for tbx16 mRNA in embryos from a tbx16+/− incross at around 90% epiboly. Nonsense-mediated decay of the mutant transcript is apparent (arrow). fgf4 RNA injection increases tbx16 mRNA in paraxial mesoderm, widens dorsal axial notochord domain (asterisks) and causes aggregation of paraxial cells in siblings, but suppresses residual tbx16 transcript in mutants. (D) Immunodetection of Tbxt protein and tbx16 mRNA in Fgf4-injected and control embryos from a tbx16+/− incross. The Fgf4-injected tbx16−/− mutant (bottom) reveals nuclear Tbxt protein in the entire posterior mesoderm. Residual tbx16 mRNA in the prechordal region (arrows) but absence in posterior mesoderm demonstrates the genotype. (E) Widespread upregulation of shha mRNA reveals the notochord-like character of posterior mesoderm in Fgf4-injected tbx16−/− mutant. Scale bars: 100 µm.
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