Fig. 5

gdf3 Is Downstream of tbx1 during Specification of nkx2.5⁺ Pharyngeal Progenitors

(A) Bar graph showing the relative expression levels of gdf3, nkx2.5, and tbx1 transcripts in 6 somite stage (ss) control (CTRL) and tbx1 mutant embryos measured by qPCR. Three biological replicates were analyzed. Error bars show SD. ^∗p < 0.05 as determined by an unpaired two-tailed t test.

(B and C) Bright-field images of 6 ss CTRL (B; n = 40) and tbx1 mutant (C; n = 12) embryos processed by in situ hybridization with a gdf3 riboprobe. Dorsal views, anterior up.

(D–I) Confocal z-stacks of the tbx1, gdf3, and nkx2.5 expression domains on the right side of a 6 ss wild-type zebrafish embryo (n = 7) processed by RNAscope triple-fluorescent in situ hybridization with tbx1 (pseudocolored cyan), gdf3 (pseudocolored magenta), and nkx2.5 (pseudocolored yellow) riboprobes. Merged triple- (D), double- (E and F), and single- (G–I) channel images are shown.

(J) Projection of selected z-stack images from the boxed region in (D) is shown at higher magnification.

(K) A single confocal section from (J) is shown in (K) (arrowheads are shown to indicate regions of overlap) with DAPI nuclei counterstaining.

(L) Schematic diagrams showing the domain structures of the predicted protein products of wild-type (WT) gdf3 and a null allele of gdf3, gdf3^fb20, generated by CRISPR/Cas9-mediated genome editing. The asterisk highlights the location of a frameshifting 10-bp deletion in the gdf3^fb20 mRNA, which causes the translation of six ectopic amino acids (white) before a premature stop codon truncates the protein upstream of the mature ligand domain.

(M–O) Bright-field z-stacks of live 28 hr post-fertilization (hpf) CTRL (M; n = 25), zygotic gdf3 (N; Zgdf3, n = 25), and maternal-zygotic gdf3 (O; MZgdf3, n = 40) zebrafish embryos. Lateral views, anterior left.

In all cases, little to no variation was observed between animals in each experimental group. Scale bars, 25 μm.