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Fig. 3. Loss of Fgf8 signaling disrupts Erm expression and Isl1 production in the anterior heart field. (A-F) Whole-mount (right lateral) 9-10ss embryos after in situ hybridization with an Erm antisense riboprobe. Genotypes are listed above each column; littermate controls processed with mutants are shown. Black arrowheads indicate the domains of normal Erm expression; red arrowheads indicate regions of decreased expression. (A'-F') Upper row sections are anterior through the developing OFT/RV; bottom sections are at the level of the atrium (AT). (B',C') Two Fgf8^C/-;MesP1Cre^/+ mutants; the mild mutant (B') has relatively normal Erm expression, but the severely affected mutant (C') has decreased Erm in SM, OFT and ventral endoderm. (D',E') Erm expression is comparable in Fgf8^C/+;Isl1^+/+ and Fgf8^C/+;Isl1^Cre/+ embryos. (F') Severe Fgf8^C/-;Isl1^Cre/+ mutant with decreased Erm expression in ventral endoderm, SM and OFT. Note that endodermal expression is more severely affected than in the Fgf8^C/-;MesP1Cre^/+ mutants and that ectodermal expression remains intact. (G-I) Isl1 mRNA in the 10ss MesP1Cre series; decreased Isl1 expression is apparent in the mutant (red arrowheads). (J-L) Isl1 mRNA in the 7ss Isl1Cre series; Isl1 expression is markedly decreased in Fgf8^C/-;Isl1^Cre/+ mutants (L, red arrowheads) relative to Fgf8 ^C/+;Isl1^Cre/+ controls (K). (M-P'') Triple fluorescent immunohistochemistry detects Isl1 protein (green), apoptosis (TUNEL, red) and nuclei (Hoechst, blue). (M,M') Sections at the level of the OFT (M) and atrium (M') of a 10ss Fgf8^C/+;MesP1Cre^/+ control; endoderm, splanchnic and ventral pharyngeal mesoderm, and myocardial cells accumulating to the OFT stain intensely with anti-Isl1 antibody (green arrowheads). (N,N') Same planes of section as above in an Fgf8;MesP1Cre mutant; the OFT is narrow and midline. The intensity of the Isl1 signal and the number of Isl1-positive (green) cells in the OFT and contiguous SM are decreased (red arrowheads). (M'',N'') Another 10ss control and mutant at the level of the OFT; this mutant has no OFT, no Isl1-positive cells in the heart tube (red arrowheads, HT) and excess apoptosis in the Isl1-positive endoderm and adjacent OFT (white arrowheads). (O,O') 8ss Fgf8^C/+;Isl1^Cre/+ control; at this stage, the OFT is just beginning to accrue and stains with Isl1 (green arrowheads). (P,P') Fgf8^C/-;Isl1^Cre/+ mutant. Note the excess apoptosis in the Isl1-positive endoderm (white arrowhead). The OFT is abnormally short and few Isl1-positive cells are present in the SM, HT or atrium (red arrowheads). (O'',P'') The alterations in Isl1 protein are reproducible in another 8ss Fgf8^C/+;Isl1^Cre/+ control and Fgf8^C/-;Isl1^Cre/+ mutant; the mutant has few Isl1-positive cells in the HT (red arrowheads) and excess apoptosis in endoderm (white arrowhead). (Q-V') Right lateral views of the pharynx/heart of embryos assayed for Isl1 mRNA. Genotypes are listed above, somite stages at the left. The level of Isl1 mRNA (red arrowheads) correlates with the severity of the cardiac/OFT phenotype in mutants.

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