Embryos used for β-galactosidase staining, in situ hybridizations and phenotypic analysis were dissected from their deciduas in PBS at room temperature prior to staining/hybridization. Embryos were stained for β-galactosidase activity or used for in situ hybridizations using established protocols [67] (T. Yamaguchi, personal communication). Probes used for in situ RNA hybridizations were labeled with DIG according to standard procedures [67]. Plasmids or PCR products used for probe synthesis were as follows T, Lhx1, GATA6, Lefty1, FGF4, FGF8, HesX1, Hhex, Cripto, BMP4, Otx2, Nodal, Cer, Foxa2, Erb2, Fgfr2, Mash1, JunB, VEGF and Flk1. In situ signals were detected using BM Purple AP Substrate according to manufactures instructions (Roche). Embryos selected for sectioning were post fixed in 4% PFA overnight at 4°C, dehydrated in alcohol and embedded in paraffin and sectioned after β-galactosidase staining. Sectioned embryos were obtained using standard techniques [67]. Embryos genotyped following β-galactosidase staining or in situ hybridization were prepared post staining by digesting the embryo proper overnight at 55°C in 10 to 20 µl PBS, 0.1% tween 20, 2 mg/ml PCR grade protenase K (Roche). Proteinase K was heat inactivated at 100°C for 15 min and 2 µl was subsequently used for PCR genotyping.
