Foley AC , Korol O , Timmer AM , Mercola M .

F32 HL 69595 NHLBI NIH HHS , R01 HL 059502 NHLBI NIH HHS , R01 HL 067079 NHLBI NIH HHS , R01 HL059502-08 NHLBI NIH HHS , R01 HL067079-04 NHLBI NIH HHS , R01 HL083463-01A1 NHLBI NIH HHS , R21 HL071913-03 NHLBI NIH HHS , R01 HL059502 NHLBI NIH HHS , R01 HL067079 NHLBI NIH HHS , R01 HL083463 NHLBI NIH HHS , R21 HL071913 NHLBI NIH HHS , F32 HL069595 NHLBI NIH HHS

	Fig. 1. XNr1/Cripto and caAlk4 misexpression induce ectopic expression of cardiac genes Nkx2.5, Tbx5, TnIc and αMHC cell non-autonomously. (A) One ventral blastomere of 16–32 cell stage embryos were injected with mRNAs encoding XNr1 and Cripto or caAlk4 along with AD546 (red) as a lineage label. The normally non-cardiogenic 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) or Tnic and αMHC (stage 30), then stained for gene expression by in situ hybridization (blue). (B–G) Examples of cell non-autonomous gene induction on histological section. Injection of XNr1 and Cripto or caAlk4 mRNAs with AD 546 induced Nkx2.5 (B, F), Tbx5 (D) TnIc (E, G) and αMHC (C). Images represent a photo merging of brightfield and fluorescent images. Note that cardiac marker positive cells (blue) were localized at a distance from lineage-labeled cells (red). Scale bar in panels B, D–G represents 40 μM, scale bar in panel C represents 20 μM.
	Fig. 2. XNr1/Cripto and caAlk4 induce endogenous Cer mRNA. Embryos were injected with synthetic mRNA for XNr1 and Cripto or caAlk4 into one ventral blastomere of a 4–8 cell stage embryo. Non-cardiogenic VMZ explants were dissected at onset of gastrulation (stage 10.25–10.5) then either frozen immediately for subsequent RNA isolation or grown in culture and frozen when age matched siblings had reached stages 10.5, 11, 13 and 19 and analyzed for mRNA levels by quantitative RT-PCR (data presented represent the average of two technical replicates each from two of the 6 biological replicates performed). (A) Plot depicts the fold induction of Cer in injected cells relative to uninjected controls, after normalization to levels of the ubiquitously expressed Ef1α transcript. (B–I) Examples of Cer induction detected by in situ hybridization relative to the AD546 (B, C, G, H, I) or β-galactosidase (D–F) lineage labels that mark the progeny of injected cells. XNr1 and Cripto induced expression of Cerberus in mesendodermal cells in a manner that was largely cell autonomous (note nearly complete overlap of Cerberus with AD546 in merged image (C) and large number of purple stained nuclei in Cerberus-expressing tissue (D, E, green arrows)). Neighboring cells that did not express the injected mRNAs often expressed Cer, as can be seen in panel E by the presence of nuclei visualized by DAPI but without β-galactosidase lineage label stain (blue arrows). In contrast, cells in the deep endoderm (D, E, red arrows) and cells in the pigmented epithelium (F) never expressed Cer despite expressing the injected β-galactosidase lineage label. Injection of caAlk4 also induced expression of Cerberus (G–I); however, induction was almost entirely cell non-autonomous [note lack of overlap between injected cells (red) and Cer-expressing cells (blue)], suggestive that chronic misactivation of the Nodal pathway is incompatible with Cer expression. Scale bars in panels B–F represents 20 μM and in panels G–I 100 μM.
	Fig. 3. Cerberus is required for normal cardiogenesis and ectopic heart induction by XNr1 and Cripto but not for heart induction by the Wnt antagonist Dkk1. (A, B) Cerberus was co-injected into one ventral blastomere at the 4–8 cell stage with AD546. Explants of the non-cardiac VMZ were isolated at early gastrula stages 10.25 to 10.5 and grown until age-matched siblings had reached stage 23–35 and processed by in situ hybridization. Cerberus induced expression of both Nkx2.5 and Tbx5 (red arrowheads) cell non-autonomously. Scale bars represent 20 μM. (C) Morpholino mediated knockdown of Cerberus decreases the percentage of heart induction by XNr1 and Cripto but not by Dkk1. Asterisk indicates statistically significant (p < 0.001, Student's T-test) difference between treatment classes for greater than 3 experimental trials. (D–H′) Examples of explants showing effects of Cerberus morpholino-mediated knockdown on endogenous heart induction and heart induction by XNr1 and Cripto versus that by Dkk1. DMZ explants express the early and late cardiac markers Nkx2.5 and TnIC (red arrowheads panels D, E) whereas morpholino-injected explants lacked heart gene expression (D′, E′) without effecting head development and loss of Nkx2.5 was rescued by co-injection of a plasmid that directed expression of Cerberus protein (panel D′ inset). VMZ explants injected with XNr1 and Cripto expressed early and late cardiac markers Nkx2.5 and TnIc, respectively (red arrowheads G, G′). Morpholino-knockdown of Cerberus caused loss of heart markers without reduction in axial structures (F′, G′). Dkk1 injected either alone (H) or with a standard control morpholino (panel H insert) induced expression of TnIc (red arrowhead). Cer MO did not effect induction of TnIc by Dkk1 (panel H′ red arrowhead).
	Fig. 4. Cer-S inhibition of Nodal contributes to heart induction. (A–E) Embryos were injected into two dorsal/vegetal blastomeres at the 8 cell stage with either 2 pmol control MO (A), 1 pmol Hex MO (B), 2 pmol Cer MO (C) or combination of 2 pmol Cer MO and 1 pmol Hex MO (D). DMZ explants isolated at early gastrula stages (10–10.25) and grown until age-matched siblings had reached stage 23–25 when they were processed by in situ hybridization for expression of Nkx2.5 (arrowhead in panel A). Incidence of Nkx2.5 expression is shown in in panel E. Asterisks indicate statistical significance (p < 0.01, Student's T-test) between indicated conditions for greater than 3 independent experiments. (F–I) Embryos were injected into one ventral blastomere at the 4–8 cell stage with either the truncated form of Cerberus, Cer-S (F), the truncated version of the BMP receptor, tBR (G), or the two constructs together (H). VMZ explants were isolated at early gastrula stages 10.25 to 10.5 and grown until age-matched siblings had reached stage 23–25 and processed by in situ hybridization for expression of either Nkx2.5 (arrowhead in in panel H) or Tbx5. Neither Cer-S nor tBR alone induced expression of Nkx2,5 or Tbx5 whereas co-injection of Cer-S and tBR induced robust expression of both markers. The incidence of expression is plotted in the histogram (I). (J) The data indicate that two pathways for specification of the heart field in mesoderm are independent at the level of Hex and Cerberus. Dkk1 (and other canonical Wnt antagonists) act through the homeodomain transcriptional repressor Hex to induce an as yet unidentified diffusible intermediate whereas activation of the Nodal pathway results in production of the secreted factor Cerberus.

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