Whole mount zebrafish embryos stained by in situ hybridization using gene-specific antisense RNA probes. Blue –violet staining indicates the presence of the cognate gene transcripts. (A–H) nkx2.5 staining at 12 (A), 13.5 (B), 18 (C), 19 (D), 20 (E), 22 (F), 24 (G), and 26 (H) hpf showing the progression from paired cardiac primordia (white arrows) to fused heart tube (ht). Noncardiac regions of staining are indicated (black arrows). (I-P) nkx2.7 at 10.5 (I), 13.5 (J), 18 (K), 19 (L), 20 (M), 24 (N), 36 (O), and 48 (P) hpf showing the earliest staining in the postgastrulation hypoblast (black arrows); white arrows indicate consolidations of apparent precardiac mesoderm. (Q –S) nkx2.3 at 24 (Q), 36 (R), and 48 (S) hpf. Specimens are viewed dorsolaterally with the rostral end of the embryo to the left and the dorsal axis up, except I, which is a head-on view (dorsal up), and A, C, M, O, and R, which are true dorsal projections. Brown pigment is endogenous melanin that appears after 24 hpf. Faint staining of neural structures was not reproducible. Labels: h, head; t, tailbud; y, yolk; ht, heart; g, gut; md, mandibular; hy, hyoid, and 1, 2, 3, 4, first through fourth branchial clefts; *, artifact. Numbers in lower left corner indicate hpf. Scale bars indicate 250 μm.

Sections of embryos stained by in situ hybridization using gene-specific antisense RNA probes for nkx2.5, nkx2.7, and nkx2.3 as labeled on each panel. Blue– violet staining indicates the presence of the cognate gene transcripts. (A–C) Transverse sections with nkx2.5 (A), nkx2.7 (B), and nkx2.3 (C) staining at 18, 19, and again 19 hpf, respectively, showing the relationship of the expressing cells to the fourth ventricle (IV) of the hindbrain, the yolk (y), the portion moyenne (pm), and the first somite (s). (D) Detail from a transverse section (right side only) of an 8 somite (13 hpf) embryo showing the position of very early nkx2.7-expressing hypoblast cells laterally between the neural keel (nk) and yolk (y). (E–G) Horizontal sections at 48 hpf with nkx2.5 (E), nkx2.7 (F), and nkx2.3 (G) staining, showing heart (ht) and/or overlying pharyngeal epithelium (pe) and mandibular arch epithelium (md). Brown pigment is melanin in the retina. (H) Detail of the cardiac tube from a horizontal section of a 36 hpf embryo stained with nkx2.7. The single layer of myocardial cells (mc) that express this gene surround poorly preserved endocardial cells (ec) that do not; the space between these layers is occupied in vivo by the cardiac jelly. (I–K) Transverse (I), angled (J, 45° to horizontal plane), and parasagittal (K) sections of 48 hpf embryos with nkx2.3, showing staining of pharyngeal epithelium both in the pouches and over the convexities of the arches, including the mandibular (md), hyoid (hy), and first through fourth gill (1,2,3,4) arches. Brown pigment as above. (L) Detail of an axial section of a 48 hpf embryo showing nkx2.3 staining of the gut epithelium (g) caudal to the pharynx; notochord (nc) and yolk (y) are indicated for purposes of orientation. Scale bars indicate 250 μm, except in H where the bar is 100 μm.