Fig. 7

Hoxb5b Acts Nonautonomously to Restrict Atrial Cell Number

(A–D) Experimental design for transplantation of WT donor cells into morphant host embryos. (A) WT donor embryos carrying Tg(cmlc2:DsRed2-nuc) were injected with fluorescein dextran at the one-cell stage. (B and C) At sphere stage, donor blastomeres were transplanted into the margin of hoxb5a or hoxb5b morphant host embryos. (D) At 48 hpf, cardiomyocyte progeny of donor-derived cells (arrows) were detected on the basis of their nuclear DsRed localization. Fixation destroys the fluorescence of the fluorescein dextran lineage tracer, allowing for clean visualization of Amhc (green).

(E) Example of donor cells (arrows) contributing to both the atrium and the ventricle in a hoxb5a morphant host.

(F) Example of donor cells (arrows) contributing to the atrium in a hoxb5b morphant host.

(G) Frequency of donor cell contributions to each cardiac chamber in hoxb5a and hoxb5b morphant hosts.

(H–K) Experimental design for transplantation of hoxb5b morphant or mRNA-injected donor cells into morphant host embryos. (H) WT donor embryos were injected with hoxb5b MO or hyperactive hoxb5b mRNA along with fluorescein dextran at the one-cell stage. (I and J) At sphere stage, donor blastomeres were transplanted into the margin of hoxb5b morphant host embryos carrying the transgene Tg(cmlc2:DsRed2-nuc). (K) At 48 hpf, we selected host embryos in which donor-derived cells contributed to anterior mesoderm lineages other than the heart and counted the number of cardiomyocytes in each of these hosts.

(L and M) Donor-derived cells expressing hyperactive hoxb5b nonautonomously reduce the number of atrial cells in hoxb5b morphant embryos. Asterisks represent statistically significant differences between the pair of values indicated by each bar. Error bars represent SD.

(N–P) Model of interactions between the forelimb field and heart field that restrict the number of CPs. (N) RA signaling acts on the forelimb field to promote formation of FPs (blue) and expression of RA-responsive genes, including hoxb5b (yellow oval). This indirectly results in production of two hypothesized repressive signals that limit formation of (1) APs (green) and (2) VPs (red). (O) In the absence of RA signaling, the expanded AP and VP populations occupy the space created by the loss of FPs. (P) Without hoxb5b, hypothesized to regulate repressive signal 1, FPs are intact and still restrict VP formation, but the AP population is expanded.