In situ hybridization for raldh2 and immunofluorescence staining assays were conducted as previously described (Kikuchi et al., 2011), and the results were imaged using a LeicaDM400 B LED microscope with an MC170 HD camera (Leica Microsystems, Wetzlar, Germany) or a Zeiss AXIO imager M1 microscope (Carl Zeiss AG, Oberkochen, Germany). Confocal images were captured using a Zeiss LSM 710 confocal microscope (Carl Zeiss AG, Oberkochen, Germany). In situ hybridization for shha was performed using RNAscope Reagent kits (Advanced Cell Diagnostics, Newark, CA), and the results were imaged using a LeicaDM400 B LED microscope and a Zeiss LSM 710 confocal microscope. EdU (8 mM) was intraperitoneally injected at 4, 5, and 6 dpi, and EdU incorporation was detected using a Click-iT EdU Alexa 647 Imaging Kit (Invitrogen, Carlsbad, CA).