RNA in situ hybridization and pseudo-SEM imaging of whole mouse embryos Whole mount in situ hybridization for Rdh10 was performed as in [23]. Embryo morphology was visualized by staining whole embryos with DAPI to label all cell nuclei and imaging fluorescent signal. Embryos were fixed in 4% formalin overnight at 4uC then stained with 2 ug/ml DAPI in PBS and imaged by confocal microscopy using an LSM5 Pascal confocal microscope. For each embryo, a Z-stack of confocal slices was collapsed to form a ‘‘pseudo-SEM’’ image. RA signaling was visualized by staining embryos carrying the RARE-lacZ transgene for b-galactosidase activity. A subset of the brightfield embryo images (Fig. 2D, Fig. 5 G–J, 6B) were processed with Helicon Focus image processing software to compile and render the focused regions of multiple focal planes as a single in-focus image. A minimum of 5 embryos were used for each parameter analysed unless otherwise stated. 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