Figure 4. Loss of aplnr results in a delay in mesp gene expression and the attenuation of Sqt and Cyc activity in a non-cell autonomous manner by aplnr.

(A) Animal view of mespaa expression at 50% epiboly (5.25 hpf) in embryos treated with 10 μM of SB505124 from 4–5.25 hpf. Animal cap view with dorsal to the bottom. (B) Animal view of mespaa expression at 50% epiboly (5.25 hpf) in embryos in which cells expressing 4 pg of sqt RNA were transplanted into the animal cap. Animal cap view with dorsal to the bottom. (C–J) Expression of mespaa and mespab at 50% epiboly (5.3 hpf) (C,D,G,H) and the shield stage (6 hpf) (E,F,I,J) in WT and aplnra/b morphant embryos when examined by WISH. Embryos are viewed from the animal pole with dorsal at the bottom. (K) Animal cap transplant of GFP expressing control cells detected by WISH. (L–T) Animal cap transplants of sqt or cyc overexpressing cells into WT (L–O) or aplnra/b morphant embryos (P–S) at 5.5 hpf. gsc and ntl expression is displayed in blue and gfp expressing donor cells are marked in brown. Both donor cells and hosts are of the same background (WT into WT or morphant into morphant). Embryos are viewed from the animal pole with dorsal at the bottom. Data are represented as means ± SEM. (U–X) Animal cap transplants of cells expressing high levels of sqt RNA at 5.5 hpf. ntl expression is visualized in blue and gfp expressing donor cells are brown. Four different combinations of donor/host cells were examined, WT into WT (donor into host) (U), aplnra/b morphant into WT (V), WT into aplnra/b morphant (W) and aplnra/b morphant into morphant (X). Donor cells are circled in white. Embryos are viewed from the animal pole with dorsal at the bottom.

DOI: http://dx.doi.org/10.7554/eLife.13758.010

Figure 4—figure supplement 1. mespaa and mespab are Nodal target genes and Nodal ligand expression is not affected in aplnra/b morphant embryos.

(A) Animal view of mespab expression at 50% epiboly (5.25 hpf) in embryos treated with 10 μM of SB505124 from 4–5.25 hpf. Animal cap view with dorsal to the bottom. (B) Animal view of mespab expression at 50% epiboly (5.25 hpf) in embryos in which cells expressing 4 pg of sqt RNA were transplanted into the animal cap. Animal cap view with dorsal to the bottom. (C–N) Lateral view of the Nodal ligands sqt (C–H) and cyc (I–N) expression at sphere (4 hpf), 30% epiboly (4.7 hpf) and 50% epiboly (5.25 hpf) in WT and aplnra/b morphant embryos.

DOI: http://dx.doi.org/10.7554/eLife.13758.012

Figure 4—figure supplement 2. Aplnr is required to enhance Nodal signaling for proper cardiac development.

(A–B) Model for the Nodal-mediated nature of the Aplnr phenotype. In WT embryos, Aplnr is required for the appropriate Nodal threshold to be reached in order to initiate the expression of the downstream transcriptional program to drive the ingression of cardiac progenitors and their migration towards the anterior of the embryo. Threshold refers to the integrated level of Nodal signaling that a cell is required to receive in order to activate this program. In aplnr mutant embryos it takes a longer period of time in order for this threshold to be reached and results in a delay in gene expression and ingression of the mesendoderm. As a result cardiac progenitors are unable to migrate all the way to the heart forming regions. Red circle denotes the location of cardiac progenitors in the embryo. (C–D) Model for the autonomy of Aplnr function in the context of regulating Nodal signaling. Aplnr appears to be required not in Nodal secreting cells or the cardiac progenitor cells receiving the signal, but instead in the surrounding environment. This role is important for the activation of the transcriptional program required for development of cardiac progenitor cells (CPCs). In the absence of Aplnr, the Nodal signal received by the cardiac progenitor is diminished (demonstrated by a reduction in the colour of the green Nodal ligands). This may reflect improper ligand processing or activity.

DOI: http://dx.doi.org/10.7554/eLife.13758.013