Fig. 1. Histochemical and fluorescence in situ hybridizations identify the molecular boundaries of the AHF/SpM in the craniofacial mesoderm at cardiac crescent stages. The molecular identities of distinct cell populations in the head were determined, using histochemical in situ hybridization (ISH) and a double-fluorescence ISH method in St. 8+ chick embryos. AHF/SpM cells are delineated by dashed ellipses. (A-D) Whole-mount ISH for the indicated genes. Dashed lines indicate the plane of the stained transverse sections (A'-D'). Nkx2.5 and Isl1 are expressed in a broader portion of the SpM (defined as the AHF/SpM), whereas C-actin and Gata4 are expressed in the more-differentiated SpM. (E-E'') Fluorescent ISH for Isl1 (E) and Nkx2.5 (E'), and their overlay (E''), showing that both genes are expressed in both the differentiated SpM and AHF/SpM. (F-F'') Fgf10 (F), Nkx2.5 (F') and overlay (F''). (G-G'') The expression of CPM marker Cyp26c1 (G) and the AHF/SpM marker Nkx2.5 (G') defining the boundary between the CPM and the AHF/SpM (G''). (H-H'') Expression of the differentiated SpM marker Gata4 (H) and Nkx2.5 (H'). The merged image (H'') demonstrates that the AHF can be molecularly identified as comprising Nkx2.5⁺ and Gata4^- cells. (I-I'') Tbx20 expression (I) marking both differentiated SpM and AHF/SpM. Tbx5 expression (I') labeled only the differentiated SpM. The merged image (I'') shows that the AHF can be molecularly identified as comprising Tbx20⁺/Tbx5^- cells. nt, neural tube; no, notochord; CPM, cranial paraxial mesoderm; AHF/SpM, anterior heart field/splanchnic mesoderm.