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Fig. 1. Differential activity of MesP1Cre, Isl1Cre and Mef2cAHFCre relative to Fgf8 expression domains in cardiac crescent and early pharynx. (A-G) Anti-GFP immunohistochemistry in Fgf8^GFP/+;HPRT^Cre/+ embryos reveals the onset and location of all Fgf8 expression in the cardiac crescent and pharynx. (A-C) Whole-mount preparations show Fgf8GFP is already broadly expressed in the cardiac crescent (cc) by the LHF stage, and in the heart tube (HT) and AHF/SM at 5ss. (D,E) 6ss; transverse sections, anterior to posterior through the heart tube. Cells in the HT (white arrowheads), AHF/SM (yellow arrowheads), foregut (FG), first pouch endoderm (E, red arrowheads) and surface ectoderm (EC, blue arrowheads) are GFP positive. (F,G) 10ss (combined DIC/epifluorescence); Fgf8GFP is present in pharyngeal epithelia, SM and RV/OFT (yellow arrowheads), but not in the left ventricle (LV, white arrowhead). (A1-G1) Fgf8GFP expression in Fgf8^GFP/+;MesP1Cre^/+ embryos. (A1-C1) Whole-mounts demonstrate Fgf8GFP broadly in the cardiac crescent at LHF and 1ss, and in the HT at 5ss. (D1,E1) 5ss transverse sections; Fgf8GFP is in the HT and SM; endoderm is GFP-negative (gray arrowhead). (F1,G1) 12ss transverse sections; the LV and atrium are now Fgf8GFP negative (white arrowheads). (A2-G2) Fgf8GFP expression in Fgf8^GFP/+;Isl1^Cre/+ embryos. (A2-C2) Isl1Cre is not active in the cardiac crescent at 0ss (A2), but Fgf8GFP is present at 2ss (B2) and is widely expressed at 5ss (C2). (D2,E2) Pouch endoderm, SM, and ventral HT cells in 5ss embryos are Fgf8GFP positive. (F2,G2) 11ss; transverse sections reveal Fgf8GFP in pharyngeal epithelia, SM and OFT/RV but not in the LV or atrium (A). (A3-G3) Fgf8^GFP/+;Mef2cAHFCre embryos. Mef2cAHFCre is not active at 0ss and little GFP is detected in AHF mesoderm until 3ss (B3). Note the lack of expression in any ventral HT cells (D3); expression is restricted to the AHF/SM (E3) and RV/OFT (F3). (H-O2) Comparison of MesP1Cre and Isl1Cre recombination using the Rosa26R-Cre reporter and ß-galactosidase staining. (H-J) Whole mounts MesP1Cre-recombined at 0, 7ss (ventral views) and 34ss (right lateral). (K-K2) 7ss transverse sections of a MesP1Cre/Rosa26R embryo; recombination is evident throughout the head mesoderm (HM), SM and HT, and is restricted to mesoderm. (L-N) Whole mounts of Isl1Cre/Rosa26R embryos. At 1ss (L), few crescent cells are labeled. (M) 6ss, ventral view; black lines depict plane of sections shown in panels O-O2. (N) 24ss, right lateral view; pharyngeal arches and entire RV/OFT are labeled. (O-O2) 6ss transverse sectioned Isl1Cre/Rosa26R embryo; recombination is evident in the foregut (FG), endoderm (E), SM and many HT cells; scattered ventral myocardial cells that are unstained are indicated by the black arrowhead. Rare pharyngeal ectodermal (EC) cells are stained. (P-S) Schematics comparing the timing and location of Fgf8 ablation by different Cre drivers. (P,Q) Whole mounts at LHF-1ss and 2-3ss; Fgf8 is ablated in all myocardial precursors by MesP1Cre (blue); the onset of Isl1Cre is later and in fewer cells (purple); followed by Mef2c-AHFCre only in the AHF at the 3ss (yellow indicates that all three Cre drivers active). Note that the dorsoventral location of these populations is not depicted. (R) Transverse section at 5ss; Isl1Cre is active in pouch endoderm and surface ectoderm (red); all three drivers recombine the SM/AHF and dorsal HT. MesP1Cre ablates Fgf8 in more ventral HT cells, suggesting that Fgf8 is expressed in both heart fields. (S) Transverse section at 10-12ss; all three drivers ablate Fgf8 in SM and AHF-derived RV/OFT (yellow); Fgf8 is no longer expressed in the LV (gray).

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