XB-ART-2382Genes Dev. February 1, 2005; 19 (3): 387-96.
Inhibition of canonical Wnt/beta-catenin signaling by Dickkopf-1 (Dkk-1) or Crescent initiates cardiogenesis in vertebrate embryos. However, nearly nothing is known about the downstream effectors of these secreted Wnt antagonists or the mechanism by which they activate heart formation. Here we show that Wnt antagonists in Xenopus stimulate cardiogenesis non-cell-autonomously, up to several cells away from those in which canonical Wnt/beta-catenin signaling is blocked, indicative of an indirect role in heart induction. A screen for downstream mediators revealed that Dkk-1 and other inhibitors of the canonical Wnt pathway induce the homeodomain transcription factor Hex, which is normally expressed in endoderm underlying the presumptive cardiac mesoderm in amphibian, bird, and mammalian embryos. Loss of Hex function blocks both endogenous heart development and ectopic heart induction by Dkk-1. As with the canonical Wnt pathway antagonists, ectopic Hex induces expression of cardiac markers non-cell-autonomously. Thus, to initiate cardiogenesis, Wnt antagonists act on endoderm to up-regulate Hex, which, in turn, controls production of a diffusible heart-inducing factor. This novel function for Hex suggests an etiology for the cardiac malformations in Hex mutant mice and will make possible the isolation of factors that induce heart directly in the mesoderm.
PubMed ID: 15687261
PMC ID: PMC546516
Article link: Genes Dev.
Grant support: F32 HL69595 NHLBI NIH HHS , R01 HL59502 NHLBI NIH HHS , R01 HL67079 NHLBI NIH HHS , R01 HL059502 NHLBI NIH HHS , R01 HL067079 NHLBI NIH HHS , F32 HL069595 NHLBI NIH HHS
Genes referenced: dkk1 frzb2 gsk3b hhex mapk8 nkx2-5 nodal nodal1 tbx5 tnni3
Morpholinos referenced: hhex MO1
Article Images: [+] show captions
|Figure 1. Non-cell-autonomous induction of Nkx2.5 and Tbx5 by Gsk3β and dnTCF3. (A) Embryos were injected into one ventral blastomere at the 16-32-cell stage with mRNAs encoding Gsk3β or dnTCF3 along with Texas Red lysinated dextran (TRLDx, 10,000 MW) as a lineage label. The normally noncardiogenic VMZ mesendoderm was explanted at the onset of gastrulation (stage 10.25-10.5), cultured until cardiac tissue expressed Nkx2.5 and Tbx5 (stage 23-25), then stained for gene expression by in situ hybridization (blue). (B-E) Examples of non-cell-autonomous gene induction on histological section. Injection of GSK3β or dnTCF3 mRNAs (with TRLDx) induced Tbx5 (B,D) and Nkx2.5 (C,E). In situ hybridization is shown in the left panels, TRLDx fluorescence in the middle panels, and merged images on the right panels. Note that Tbx5- and Nkx2.5-positive cells were localized at a distance from lineage-labeled cells. Bar in B represents 20 μM.|
|Figure 2. Non-cell-autonomous induction of late cardiac markers by Gsk3β and dnTCF3. (A-D) Examples of induction of non-cell-autonomous induction of MHCα (A,B) and TnIc (C,D) at stage 35 induced by dnTCF3 (A,C) and Gsk3β (B,D). Lineage label (Alexa Fluor 546 Dextran [AD546])-positive cells were never found to overlap expression of the induced cardiac genes and were seen only rarely in adjoining cells; rather, they were displaced distantly in the explant during morphogenetic tissue movements that characterize gastrulation in intact embryos. Insets show that each explant contains robustly lineage-labeled cells in sections distant to those with cardiac gene expression. Bars in D represent 20 μM.|
|Figure 3. Induction of Hex mRNA by Gsk3β and dnTCF3. (A) Embryos were injected with synthetic mRNA for Gsk3β and dnTCF into one ventral blastomere of a 4-8-cell-stage embryo. Noncardiogenic VMZ explants were dissected at the onset of gastrulation (stage 10.25-10.5) then analyzed for mRNA levels by quantitative RT-PCR (mean of four experiments ± standard error). The asterisk indicates a statistically significant difference (t-test) from control β-gal-injected samples for VMZ explants expressing Gsk3β (P ≤ 0.031) and dnTCF3 (P ≤ 0.032). (B-D) Examples of induction visualized by in situ hybridization and staining for β-galactosidase. Control injection of nls-β-gal did not induce Hex (B), whereas Gsk3β (C) or dnTCF3 (D) both induced Hex. In contrast to induction of cardiac markers, the spatial domain of Hex-positive cells largely overlapped the lineage-labeled cells. Note that some lineage-labeled cells in centers of explants do not express Hex, suggesting that induction is spatially constrained. (E-G′) Examples of induction visualized by in situ hybridization and AD546. Control injections of AD546 do not induce Hex (E), whereas injection of both GSK3b (F,F′) and dnTCF3 (G,G′) induce Hex. In situ hybridization staining is shown in F and G, and merged in situ hybridization staining and AD546 fluorescence (F′,G′) show nearly perfect overlap between cells expressing Hex and the lineage label. Bar in B represents 20 μM.|
|Figure 4. Non-cell-autonomous induction of Nkx2.5 and Tbx5 by Hex. (A,B) Hex mRNA was injected with a fluorescent lineage tracer (TRLDx, 10,000 MW) with Hex into one ventral blastomere at the 16-32-cell stage as in Figure 1A. VMZ mesendoderm was explanted at stage 10.25-10.5, cultured to stage 23-25, and assayed for Tbx5 (A) or Nkx2.5 (B). In situ hybridization is shown in the left panels, TRLDx fluorescence in the middle panels, and merged images on the right panels. Note gene induction at a distance from the region labeled with the lineage label. Bar in B represents 20 μM.|
|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.|
|Figure 6. Molecular relay for heart induction. Inhibitors of canonical Wnt/β-catenin signaling produced by the Organizer act on endoderm to induce Hex, which, in turn, controls the production of a diffusible factor that acts back on mesoderm to induce early cardiac genes (e.g., Nkx2.5 and Tbx5). Hex functions as a transcriptional repressor and might induce production of a diffusible activator by repression of a repressor (shown as ×). Not depicted is the alternate scenario that Hex directly suppresses expression of a diffusible repressor of cardiogenesis. The finding that Hex induced early cardiac markers, whereas Gsk3β and dnTCF3 promoted progression to expression of myocardial markers (MHCα and TnIc), suggests that parallel pathways, perhaps involving activation of JNK and/or PK-C, regulate Hex-independent diffusible factors from the endoderm. Nodal related proteins (XNr) and noncanonical Wnts might complement Hex-dependent heart induction, but it is not yet clear whether they act on endoderm or directly on cardiac mesoderm.|