RDH10 Control of RA Synthesis Figure 1. Rdh10trex and new Aldh1a2gri mutant phenotype comparison. Wild type (A,D), Rdh10trex/trex mutant (B,E), and Aldh1a2gri/gri mutant (C,F) embryos were collected at E9.5 (A–C) and E10.5 (D–F). Formalin-fixed embryos were stained with DAPI and imaged by confocal microscopy. For each embryo, a Z-stack of confocal slices was collapsed to form a ‘‘pseudo-SEM’’ image. (A–F) For each mutant phenotype at each indicated stage n.5 embryos. (G) Sequence of Aldh1a2 gene at junction between Exon 4 and Intron 4–5 with grimace mutation single base change indicated beneath. Exon 4 genomic DNA sequence is indicated by yellow highlight, intron 4–5 sequence indicated by pink highlight. (H) Histogram representing relative levels of Aldh1a2 mRNA from wild-type, heterozygous Aldh1a2gri/+ and homozygous Aldh1a2gri/gri embryos, as assessed by quantitative RT-PCR, demonstrating loss of Aldh1a2 transcript in homozygous mutants. fl, forelimb bud; h, heart; ov, otic vesicle; ppa, posterior pharyngeal arches. doi:10.1371/journal.pone.0030698.g001 named ‘‘grimace’’[29] that exhibited interesting abnormalities in formation of the pharyngeal arches (Fig. 1C). Mapping, cloning and sequence analysis were used to identify the grimace mutation, which was determined to be a single base change within the Aldh1a2 locus. We therefore henceforth refer to this mutation as Aldh1a2gri. Sequence analysis of genomic DNA reveals that the Aldh1a2gri mutation alters the 59 splice consensus sequence at the boundary of exon 4 and intron 4–5, changing the second intronic nucleotide from a conserved T to a C (Fig. 1G). The altered splice consensus sequence disrupts splicing of Aldh1a2 mRNA and leads to a reduction in Aldh1a2 transcript level. Quantitative RT-PCR reveals that Aldh1a2 transcript in homozygous Aldh1a2gri/gri mutants is reduced to less than 1% of that of wild type embryos (Fig. 1H). We therefore interpret the new Aldh1a2gri mutation is either a allele of Aldh1a2 or an extreme hypomorph. Consistent with the loss of Aldh1a2 transcript, we observe the PLoS ONE | www.plosone.org 3 February 2012 | Volume 7 | Issue 2 | e30698