Guo Y , Dorn T , Kühl SJ , Linnemann A , Rothe M , Pfister AS , Vainio S , Laugwitz KL , Moretti A , Kühl M .

             cardiac cell development

	Fig. 1. Isl1 positively regulates Dkk1 expression. A, B. Isl1OE ES line at day 4 of cardiac differentiation (A) and Isl1-overexpressing endodermal NFPE cells (B) show increased Dkk1 expression as determined by real-time RT-PCR. Dkk1 gene expression levels were normalized to Hprt (A) or Gapdh (B); n = 3. C. Schematic representation of the 1.3 kb promoter region upstream of the Dkk1 transcription site containing two predicted Isl1-binding sites. Deletion constructs generated are given. D. Bar graph illustrating the luciferase activity. NFPE cells were transiently transfected with a pGL3-basic luciferase reporter or with the same vector encompassing the 1.3 kb Dkk1 promoter fragment, together with an expression vector containing GFP control or Isl1. In addition, modified pGL3-basic-Dkk1-promoter constructs were used, in which either one or both Isl1-binding sites were deleted. The luciferase levels were normalized for the β-galactosidase activity of a co-transfected RSV-βGal and shown as luciferase activity relative to pGL3-basic plus GFP control (RLU, relative light units). n = 6–8. For all panels: Mean values with standard errors are given. *p < 0.05, **p < 0.01 and ***p < 0.001 with Student’ T-test.
	Fig. 2. Expression of dkk1 and isl1 and their regulation in early development of Xenopus laevis. A. WMISH shows the spatial expression of isl1 and dkk1 in early cardiogenesis. Both isl1 and dkk1 are present in the cardiac crescent at stage 15 (white arrowhead). At stage 20 (white arrowhead) and stage 24 (black arrowhead), dkk1 is expressed in the middle line where isl1 is absent. At stage 28, dkk1 is expressed in a small expression domain at the ventral midline, whereas isl1 covers a broader expression domain (black arrowheads). White dotted lines indicate the cement gland. Black dotted lines indicate the orientation of the sections shown in B. B. Parasaggital and cross sections reveal co-expression of dkk1 and isl1 in the border of ventral mesoderm (bvm, red arrowhead) and cardiac mesoderm (cm) (green arrowhead). At stages 24 and 28, dkk1 is expressed in the overlying endoderm (yellow arrowhead) in close proximity to the cardiac mesoderm. bvm: border of ventral mesoderm, cg: cement gland, cm: cardiac mesoderm, hm: head mesenchyme, oe: oral evagination, pc: prosencephalon, sha: stomodeal-hypophyseal anlage. C. Isl1 deletion leads to a reduced dkk1 expression at stage 20 (black arrow). Dotted lines indicate the cement gland. Quantification is presented in D. E. At stage 28 dkk1 expression is reduced at the ventral midline upon Isl1 knockdown (white and black arrowheads). Quantitative presentation is shown in F. Mean values with standard errors are given. n = number of independent experiments, N = total number of embryos analyzed. *p < 0.05 with Mann-Whitney rank sum test.
	Fig. 3. Dkk1 depletion results in heart malformation and cardiac defects in Xenopus laevis. A. The sequence of Xenopus dkk1 and human DKK1 at the MO binding site. The start codon is marked in green. Red stars indicate the bases in the human DKK1 RNA that differ from Xenopus dkk1. B. Unilaterally injected Dkk1 MO but not Control MO blocked translation of the dkk1MO-GFP fusion construct. Dkk1 MO did not block translation of the hDKK1MO-GFP construct. C. Knocking down Dkk1 bilaterally with Dkk1 MO leads to deformed heart (red arrowhead in lateral view, red dotted line in front view) and cardiac edema (white arrowhead in front view) at stage 42. D. Quantitative presentation of the Dkk1 MO injected embryos with cardiac edema shown in C. E. Heart rate was significantly reduced in embryos with bilateral injection of Dkk1 MO. F-J. Analysis of the heart morphology in control embryos and Dkk1 depleted morphants. F. Representative images of stage 42 embryos showing normal heart morphology in one Control MO injected embryo and heart defects in two Dkk1 MO injected embryos. From left to right: ventral view of the embryos and heart stained for cardiac troponin T, close-up view of the hearts, sections through the hearts. G. Representative images of hearts isolated from bilaterally MO injected embryos. Atrial (a) width, ventricular (v) width, a-v length and outflow tract as illustrated in H were measured and presented in I and J, respectively. a: atrium, oft: outflow tract,v: ventricle. Mean values with standard errors are given. n = number of independent experiments, N = total number of embryos analyzed. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 with Mann-Whitney rank sum test.
	Fig. 4. Reduced number of terminally differentiated cardiomyocytes upon loss of Dkk1. A. Sections of embryos are shown stained for cardiac troponin T (red) to label cardiomyocytes and phosho-histone H3 (PH3, green) lo mark proliferating cells and counterstained with DAPI (blue) to highlight nuclei at the stages indicated. Yellow arrowheads indicate proliferating cardiomyocytes positive for cardiac troponin and phosho-histone H3. B. Cardiac troponin T positive cardiomyocytes were quantified on continuous serial sections of stained embryos. Embryos bilaterally injected with Dkk1 MO showed reduced number of cardiomyocytes at the stages indicated. C. The mitotic index (MI) as defined by the percentage of phosho-histone H3 positive cardiomyocytes is not affected upon Dkk1 MO injection. N = number of independent embryos counted. *p < 0.05, **p < 0.01, with Mann-Whitney rank sum test. Scale bar, 100 μm.
	Fig. 5. The expression of differentiation cardiac markers is downregulated upon loss of Dkk1 and can be rescued by co-injection of Dkk1 MO and human DKK1. A. Front view embryos of stage 28 showing reduced expression of cardiac marker genes on MO injected side (black arrow). Control MO or Dkk1 MO was injected unilaterally at the dorsal-vegetal site in embryos at stage 4. Un-injected side served as internal control. Data quantification is shown in B. C. Co-injection of Dkk1 MO and human DKK1 restore the reduced expression (black arrow) of markers as shown in stage 28 embryos of front view. Data quantification is presented in D. White dotted lines indicate the cement gland. Mean values with standard errors are given. n = number of independent experiments, N = total number of embryos analyzed. *p < 0.05, **p < 0.01 with Mann-Whitney rank sum test.
	Fig. 6. The expression of differentiation cardiac markers is delayed upon loss of Dkk1. Heart-enriched explants as illustrated on the top (marked in pink) were isolated from stage 28 and 34 embryos bilaterally injected with either Control MO or Dkk1 MO. Expression of cardiac marker genes in these explants was analyzed by real-time RT-PCR and presented as relative expression to gapdh. Median values are given. The whiskers indicate the maximum and minimum values. n = number of independent experiments (n = 5–10). *p < 0.05, **p < 0.01, n.s. not significant with Mann-Whitney rank sum test.
	Fig. 7. Wnt/β-catenin activity is increased in the developing heart and inhibition of Wnt/β-catenin signaling rescues loss of Dkk1 phenotype. A. Wnt/β-catenin activity was monitored in the developing heart tissue (marked in pink) isolated from embryos at indicated stages. B. Schematic representation of EnR-LefδN-GR755A construct. Upon injection of this construct into embryos, the expression of LefδN can be induced to inhibit Wnt/β-catenin signaling by adding dexamethasone. C. Expression of cardiac differentiation markers was analyzed in embryos at stage 28 (black arrow). Dotted lines indicate the cement gland. Inhibition of Wnt/β-catenin signaling from stage 20 rescued loss of Dkk1 phenotype in unilaterally injected embryos. n = number of independent experiments, N = total number of embryos analyzed. Mean values with standard errors are given. *p < 0.05, **p < 0.01 with Mann-Whitney rank sum test.
	Fig. 8. Wnt11a signaling acts downstream of Dkk1 during early cardiogenesis. A. Front view of stage 28 embryos showing the reduced expression of wnt11a and alcam upon unilateral Dkk1 knockdown (black arrow). White dotted lines indicate the cement gland. B. Co-injection of Dkk1 MO with alcam restored the expression of cardiac marker genes (black arrow) at stage 28. C. Co-injection of Dkk1 MO with wnt11a rescued the expression of cardiac marker genes (black arrow) at stage 28. D. Reduced expression of wnt11a by Dkk1 MO injection is restored by inhibiting Wnt/β-catenin signaling. Quantifications are shown next to the image of the embryos. Black dotted lines indicate the cement gland. n = number of independent experiments, N = total number of embryos analyzed. Mean values with standard errors are given. *p < 0.05, **p < 0.01 with Mann-Whitney rank sum test.
	Fig. 9. Cardiomyocytes detach from the myocardium upon loss of Dkk1. Sections of embryos are shown stained for cardiac troponin T (red) to label cardiomyocytes and counterstained with DAPI (blue) to highlight nuclei at the stages indicated. Upon loss of Dkk1 ectopic troponin T positive cells (white arrowheads) were found in the indicated number of cases. Embryos shown are the same as analyzed in Fig. 4. Scale bar, 100 μm.
	Supplement Fig. 1: Serial sections through a heart of a Control MO injected embryo derived from OPT image stacks at stage 43.
	Supplement Fig. 2: Serial sections through a heart of a Dkk1 MO injected embryo derived from OPT image stacks at stage 43
	Supplement Fig. 3: Loss of Dkk1 does not result in apoptosis A,B. Apoptotic cells were stained at stage 28 by TUNEL assay. Only few apoptotic cells were detected on sections as indicated by the yellow arrowhead. No difference between Control MO and Dkk1 MO injected embryos was observed. C-F. Apoptotic cells were detected by immunofluorescence staining against cleaved caspase 3 (C-Csp3) followed by serial sections. Cardiomyocytes were detected by cardiac troponin T staining (Tnnt2). Nuclei were counterstained with DAPI. Only few apoptotic cells were detected at stage 33 (C,D) or 37 (E,F) as labelled by the yellow arrowhead. Scale bar, 100 µm.
	Supplement Fig. 4 Mesoderm formation and cardiac specification are not dependent on Dkk1. A. Expression of mesoderm marker tbxt is not affected upon Dkk1 knockdown. tbxt expression was analyzed at stage 10 embryos injected with Dkk1 MO or Control MO at stage 4. Red dotted circles indicate the location of the cardiac progenitor cell population induced at the onset of gastrulation. Quantification is shown in B. C. Expression of early cardiac markers is not altered upon Dkk1 depletion. Gene expression was analyzed at stage 13 embryos injected with Dkk1 MO or Control MO at stage 4. Quantification is shown in D. Mean values with standard errors are given. n = number of independent experiments, N = total number of embryos analyzed.
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