Figure 5.
Hex is required for normal cardiogenesis and ectopic heart induction by Dkk-1. (A) Twenty nanograms of Hex antisense (HexMo), control (ControlMo) morpholino oligodeoxynucleotides, or an mRNA encoding a constitutively active Hex protein (HexVP16) was injected into two ventral or dorsal blastomeres of 4-cell-stage embryos (Materials and Methods). For ventral injections, Dkk-1 mRNA was included to induce ectopic cardiogenesis. VMZ or DMZ explants were prepared from ventrally or dorsally injected embryos, respectively, at the onset of gastrulation (stage 10.25-10.5) and maintained in culture to approximately stage 30, when they were processed for in situ hybridization to TnIc or MHCα. Note that antisense HexMo, but not ControlMo, attenuated cardiac marker expression in both DMZ and Dkk-1-injected VMZ, indicating that Hex is specifically required for normal and ectopic cardiogenesis. Hex has been characterized as both a transcriptional repressor and activator. HexVP16 mRNA also attenuated cardiogenesis and mimicked the Hex morpholino oligo-mediated depletion, indicating that the repressive function of Hex is required for cardiogenesis. Coinjection of an mRNA encoding a mouse Hex transcript, which is not recognized by the morpholino sequence, rescued both MHCα and TnIc expression, showing that the morpholino effect is specific to Hex. (B) Examples of attenuation of both ectopic Dkk-1-induced cardiogenesis in VMZ explants (panels a-d,a′-d′) and normal cardiogenesis in DMZ explants (panels e-i,e′-i′). Explants stained for TnIc (panels a-i) or MHCα (panesl a′-i′). Uninjected DMZs (panels e,e′), ControlMo-injected DMZs (panels f,f′), and VMZs injected with DKK-1 either alone or with ControlMO (panels a,a′,b,b′) express cardiac markers in the heart region (arrowheads), whereas HexMo (panels c,c′,g,g′) or HexVP16 mRNA (panels d,d′,h,h′) injected explants frequently eliminated staining. Coinjection of mouse Hex mRNA rescued the cardiac deficit of Hex morpholino-injected DMZ explants (panels i,i′), sometimes in the absence of recognizable head structures (panel i′). cg denotes the highly pigmented cement glands that are present in DMZ explants and induced ectopically by Dkk-1 in VMZ tissues.