Fig. 3. Ablation of Frs2 compromises expansion of SHF progenitor cells to the OFT myocardium. (A) Sections from Frs2^f/f (a) Frs2^cn/Nkx (b) and Frs2^cn/Mef (c) mouse embryos immunostained with anti-ISL1 (green) and anti-phosphorylated histone H3 (red) antibodies. Nuclei were counterstained with To-Pro3 (blue). The total and proliferating ISL1⁺ cell numbers in the OFT and SM from four individuals are shown in d,e (mean ±s.d.). (B) Reduced OFT length and SHF lineage in Frs2 mutants (a-c). The OFT lengths at E9.5 as measured from six individuals are presented in d (mean ±s.d.). (e,f) X-Gal staining. The blue staining represents cells from SHF progenitors in which the R26R reporter was activated. The outline of the X-Gal-stained area in the control (e) has been superimposed on the mutant (f) to better illustrate the difference. Insets are sections from the same tissues demonstrating OFT myocardium derived from the Mef2c^Cre lineage (black arrows). (C) Compromised ERK1/2, but not AKT, phosphorylation in the Frs2^cn/Nkx OFT and SM. Embryo sections were immunostained with anti-phosphorylated ERK1/2 (a-c) or phosphorylated AKT (d-f) antibodies (green). Nuclei were counterstained with To-Pro3 (red). (D) Inhibition of the MAP kinase, but not the PI3K/AKT, pathway reduces the contribution of ISL1⁺ cells to the OFT. (a-c) Short-term (24 hour) cultures of E8.5 embryos were sectioned and stained by anti-ISL1 antibody (green) and To-Pro3 (red). Insets are high-magnification views of the same sections. (d) Cultured E8.5 embryos were treated with ERK1/2 or PI3K inhibitors as indicated. Adjacent sections from the same embryos were immunostained with anti-phosphorylated ERK1/2 or AKT (green) and with To-Pro3 (red), demonstrating the specificity and efficacy of the ERK1/2 and PI3K inhibitors. w, wild-type allele.