( A) Whole-mount in situ hybridization for  vgll4l,  yap,  taz,  vgll4b and  tead3a at gastrula and at the 6-somite stage. Vgll4l is expressed in DFCs at 60% (dorsal view), 80% (lateral view) and 90% epiboly (vegetal pole view) but is not expressed in the KV at the 6-somite stage (vegetal pole view). Yap and Taz are shown at 80% epiboly and at the 6-somite stage in vegetal pole view, Vgll4b at gastrula stage and Tead3a at the 6-somite stage in vegetal pole views. Tead1a that is constitutively expressed is not presented. White arrowheads point to DFCs, blue arrowheads point to the KV. ( B) Cardiac jogging analyzed at 25 hr post fertilization (hpf). Graphs indicate the percentage of embryos with normal Left jog (L jog - yellow), Right jog (R jog – dark blue) or no jog (light blue), visualized by in situ hybridization (top, h: heart) with  a myosin light chain 7 (myl7) probed at 25 hpf in: wild-type (WT) embryos; embryos injected with standard (std) MO or with Vgll4l, Vgll4b, Yap, Taz, Yap and Taz, Tead1a or Tead3a MOs; embryos injected with ASO; rescue experiments of morphant phenotypes by injection of MO insensitive RNA; incubation with 2.5 µM of Verteporfin, a Yap inhibitor. For each experiment the name of gene, name and amount of MO and/or RNA injected are indicated on the left. For double Yap/Taz MO-KD, 4 ng Yap MOsp and 4 ng Taz MOsp2 have been injected. DFC ‘name of the gene’ MO indicates DFC-targeted knockdown experiment (Wang et al., 2013). (MO + RNA) stands for rescue experiment of the indicated MO together with 100 ng of the corresponding, MO insensitive, mRNA. ( C) Schematic of functional domains present in WT and in Vgll4l, Vgll4b, Yap, Taz and Tead3a mutants. nls: nuclear localization signal, PDZ: PDZ-binding motif, TA: transcription activation domain, TB: TEAD binding domain, TcoF-BD: transcription cofactor binding domain, TEA: DNA-binding TEA/ATTS domain, TDU: TONDU domain, WW: WW domain. Numbers indicate the position of the last amino-acid of each peptide. ( D) Laterality defects of homozygous mutant embryos and of embryos homozygous mutant for Taz, heterozygous for Yap, analyzed as described in ( B) for their cardiac jogging at 25 hpf. Numerical data for ( B) and ( D) are provided in Figure 1—source data 1.

(A) Yap protein is present in nuclei of dorsal marginal cells at 75% epiboly. (B) Double labeling for Yap and Sox17 identifying these cells to be DFCs. (C) Sox17 immunolabeling revealing the DFCs.

Graphs indicate the percentage of embryos with normal expression of lft1 in the left heart primordium and the left dorsal diencephalon (yellow), with abnormal bilateral lft1 expression in the two heart primordia and in left and right dorsal diencephalon (light blue) and with situs inversus with lft1 expression in the right heart primordium and in the right dorsal diencephalon (dark blue). n: number of embryos analyzed. Numerical data for (B) and (D) are provided in Figure 1—figure supplement 2—source data 1.

10.7554/eLife.45241.005Figure 1—figure supplement 2—source data 1.

(A–F) Side view of live WT embryo (A) and of embryos homozygous (-/-) mutant for vgll4l (B), vgll4b (C), yap (D), taz (E) and tead3a (F) at 2 days of development showing that the global morphology of the embryo is not affected by loss of function of these TFs/TcoFs. (G–L) Cardiac jogging at 25 hr post fertilization (hpf) in (G) WT (L jog: left jog) and in homozygous mutants visualized by in situ hybridization with myosin light chain 7 (myl7) probed at 25 hpf for vgll4l (H), vgll4b (I), yap (J), taz (K) and tead3a (L) mutants. Embryos with abnormal laterality (R jog - right jog or no jog) are presented. Embryos are in front view except in (L), R jog) presented in dorsal view.

( A–D) Illustration of the strong decrease in the size of the KV at the 12-somite stage in loss-of-function conditions shown in brightfield for ( A) a control embryo (Ctrl) and for ( B) a TEAD1a morphant embryo (Tead1a-MO) and by in situ hybridization using a  dand5 probe in ( C) Ctrl and in ( D) Vgll4b morphant embryo (Vgll4b-MO). ( E–H) Effect of Vgll4l (V4l), Vgll4b (V4b), Tead1a (T1a), Tead3a (T3a) loss of function and of Yap/Taz (Y/T) double loss of function on: ( E) the size of the KV (expressed as the area of the planar projection of its lumen), ( F) the number of DFCs present at early gastrula stage (60% epiboly) and at the end of gastrulation (bud stage), ( G) the proliferation of the DFCs measured as their mitotic index at 75% of epiboly, ( H) the survival of DFCs measured as their apoptotic index at 90% epiboly. In all cases control (Ctrl) embryos were injected with 8 ng of Standard MO. Graph indicates the mean of each experiment, error bars indicate standard deviation and dots indicate the individual measurement for DFC groups or individual KV in control and loss of function conditions. Statistical significance between controls and the different loss-of-function conditions: two-tailed unpaired t-test. *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001. ns: not significant. Numerical data for ( E–H) and details of statistical analysis are provided in Figure 2—source data 1.

(A–F) Dorsal view of the KV of the Tg(sox17:GFP)^s870 line. KV cilia (red) are detected by immunolabelling using an anti-acetylated tubulin antibody in (A) WT embryo of the Tg(sox17:GFP)^s870 line and in homozyous mutant (-/-) embryos of (B) vgll4l^va1/va1; s870Tg, (C) vgll4b^va2/va2; s870Tg, (D) yap^va3/va3; s870Tg, (E) taz/wwtr1^va4/va4 and (G) tead3a^{sa14593/sa14593}; s870Tg lines. Scale bars: 20 µm.

(A, B) Brightfield view of the KV in (A) WT and (B) an embryo overexpressing Yap in DFCs at the 12-somite stage (12 s). (C, D) Visualization of the KV (green) and of KV cilia (red – immunolabeling using an anti-acetylated tubulin antibody) at the 12-somite stage in (C) WT embryos of the Tg(sox17:GFP)^s870 line and (D) embryo of the same line overexpressing Yap in DFCs. Scale bars 20 µm. (E–F) lateral view of (E) a WT embryo and (F) an embryo overexpressing Yap in the DFCs at the 18-somite stage (18 s). Arrows point to the KV. (G) Quantification of the size of the KV (expressed as the area of the planar projection of its lumen) in WT and in Yap gain of function. (H) Quantification of the number of cells present in the DFCs at early gastrula stage and in the KV at the 12-somite stage. Numerical data for (G–H) and details of statistical analysis are provided in Figure 2—figure supplements 2—source data 1.

10.7554/eLife.45241.011Figure 2—figure supplement 2—source data 1.

(A–C) Visualization of KV cilia at the 10-somite stage using an anti-acetylated tubulin antibody in (A) control embryos of the WT Tg(dusp6:GFP) line showing both KV cells (green) and cilia (red) or (B) only cilia. (C) cilia in Vgll4b morphant. Scale bar: 40 µm. (D) Length of KV cilia in Control (Ctrl) embryos and in Vgll4l, Vgll4b, Yap/Taz, Tead1a and Tead3a morphant embryos. Control (Ctrl) embryos were injected with 8 ng of Standard MO. Graph indicates the mean of cilia length, error bars the standard deviation. Statistical significance between controls and different loss-of-function conditions: two-tailed unpaired t-test. ****p≤0.0001. Numerical data and details of statistical analysis are provided in Figure 3—source data 1.

10.7554/eLife.45241.014Figure 3—source data 1.

Whole-mount in situ hybridization for genes that are expressed in DFCs at 70–90% epiboly in control (Ctrl) embryos and that are strongly downregulated in Vgll4l MO knockdown. Embryos are in dorsal view, animal pole to the top (A–L), in vegetal pole view dorsal to the right (M–W) and in lateral view anterior to the top dorsal to the right (X). Name of the genes probed is indicated in between control (top) and Vgll4l loss of function embryos (bottom).

DFC specific MO knockdown for Vgll4l (Vgll4l^DFC-MO, 8 ng Vgll4l MOsp), Yap (Yap^DFC-MO, 8 ng of Yap MOsp) and DFC targeted injection of control morpholino (ctrl, 8 ng std MO) analyzed by in situ hybridization for the expression of DFC specific genes. The name of the genes probed is indicated in the lower left corner of each panel. Embryos are in dorsal view animal pole to the top except dnmt3bb.1 and mbd3b that are in a vegetal pole view dorsal to the right.

ZFIN is incorporating published figure images and captions as part of an ongoing project. Figures from some publications have not yet been curated, or are not available for display because of copyright restrictions.

ZFIN is incorporating published figure images and captions as part of an ongoing project. Figures from some publications have not yet been curated, or are not available for display because of copyright restrictions.

(A–F) Immunolabeling of DFC nuclei of embryos of the Tg(Sox17:GFP) strain with antibodies to five methyl Cytosine (5meC) in (A–C) WT and (D–F) vgll4l homozygous mutant. Dotted lines delimit the DFC clusters. (G) Quantification of DNA methylation measured by immunofluorescence intensity. The values on the graph correspond to the mean of 5meC fluorescence intensity per nuclei (FI/nuclei) quantified with the ImageJ software on all nuclei of DFC clusters in morphants for Vgll4l and Dnmt3bb.1 and in embryos from a cross between two heterozygous Vgll4l mutants that have been individually genotyped after measurement of FI/nuclei of their DFCs. Fluorescence intensity per nuclei is expressed in relative units (R.U.). Statistical significance between controls (standard MO or vgll4l +/+) and loss of function conditions: two-tailed unpaired t-test. ns, not significant (p>0.05), *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001. Numerical data and details of statistical analysis for (G) are provided in Figure 10—source data 1.

10.7554/eLife.45241.025Figure 10—source data 1.