of the Rdh10flox allele to Cre recombinase caused excision of the loxP-flanked exon 2 thereby generating Rdh10delta allele. Embryos heterozygous for the Rdh10bgeo gene trap alleles were assessed for expression of the reporter bgeo transcript by staining for b-galactosidase activity. In these embryos b-galactosidase activity was very weak but the Rdh10bgeo gene trap transcript could be detected in a pattern similar to that of the endogenous Rdh10 transcript (Fig. 2 B–C). Both the endogenous Rdh10 mRNA, detected by RNA in situ hybridization and the Rdh10bgeo gene trap reporter are present in the posterior pharyngeal region and the lateral plate mesoderm between the fore- and hind-limb buds. Expression of Rdh10 mRNA and the Rdh10bgeo gene trap reporter are also present in the isthmus of the midbrain and in the dorsal somites. These sites correspond to the previously reported expression domains for Rdh10 [23,32]. Rdh10 gene trap allele causes severe retinoid deficiency phenotype Animals heterozygous for the Rdh10bgeo allele are viable and fertile, with no overt abnormalities. Homozygous Rdh10bgeo/bgeo embryos, however, exhibit all the hallmarks of extreme retinoid deficiency, with a phenotype remarkably similar to embryos lacking Aldh1a2. At E9.5, Rdh10bgeo/bgeo embryos display defects in embryo growth and axial turning (Fig. 3A–B). Posterior pharyn- geal arches are absent. Otic vesicles are present, but reduced in size. Somites are small and extension of the body axis is impaired. Hearts are unlooped and edemic. The parallels between the Rdh10bgeo/bgeo phenotype and phenotypes of Aldh1a22/2 mutants indicate that RDH10 deficiency is as deleterious to embryonic growth and patterning as is ALDH1A2 deficiency. Mice carrying the Rdh10bgeo allele were crossed with Rdh10trex to generate compound heterozygote Rdh10bgeo/trex embryos. The compound heterozygous embryos displayed a range of phenotypes (Fig. 3 C–D). The most severely affected resembled Rdh10bgeo/bgeo Figure 3. Phenotype of Rdh10 bgeo gene trap allele. (A) Wild type and (B) homozygous Rdh10bgeo/bgeo embryos were collected at E9.5 (Rdh10bgeo/bgeo n = 9). Wild type (C) and Rdh10bgeo/trex compound heterozygous (D) embryos were collected at E9.75 (n = 6). 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. doi:10.1371/journal.pone.0030698.g003 RDH10 Control of RA Synthesis embryos and the mildest were similar to Rdh10trex/trex embryos. Failure of the Rdh10bgeo allele to complement the Rdh10trex mutation provides genetic confirmation that the Rdh10 conditional construct is correctly targeted to the Rdh10 locus. Rdh10 deletion allele causes severe retinoid deficiency phenotype Similar to the Rdh10bgeo mice, animals heterozygous for the Rdh10delta mutation are also viable and fertile with no obvious defects. Rdh10delta/delta embryos can be recovered at the expected Mendelian frequency at E9.5 and E10.5 but viable homozygous embryos are not recovered at later stages (Rdh10delta/delta genotype: E9.5 n = 12/47, E10.5 n = 4/10, E11.75–E12.5 n = 0/32). In the homozygous state, the Rdh10delta allele produces all the classic hallmark phenotypes of severe retinoid deficiency (Fig. 4 A–D). Rdh10delta/delta mutants have defects in growth and axial turning. Axial turning defects are most apparent at E9.5, when many Rdh10delta/delta embryos appear to be caught in an abnormal leftward ‘‘J’’ shape or, occasionally, a rightward ‘‘L’’ body shape when viewed from the front (Fig. 4 B). Some embryos have a milder phenotype in which the tailbud is positioned in front of the embryo, indicating that axial turning is complete. The growth defect in Rdh10delta/delta mutants is most obvious at E10.5. For wild type embryos, overall embryo size increases substantially between E9.5 and E10.5 (Fig. 4 A, C). Rdh10delta/delta embryos, in contrast, appear to cease growth and axis extension around E9.25. Rdh10delta/delta embryos have abnormalities in many tissues and structures affected by retinoid deficiency including the heart, otic vesicle, pharyngeal arches and forelimb buds. Cardiac defects are observed in all Rdh10delta/delta embryos. Hearts fail to undergo normal looping and chamber formation, remaining, instead, simple tubes oriented vertically and enlarged by edema. Otic vesicles are present, but are tiny and occasionally duplicated. Pharyngeal arch defects are also apparent in Rdh10delta/delta embryos. First arches are small and malformed, while arches posterior to the first are absent altogether in most embryos. Forelimb buds are reduced in size or absent. The Rdh10delta construct was designed to cause a mutation via deletion of exon 2, generating a stop codon that should disrupt the open reading frame of the Rdh10 mRNA and target the transcript for nonsense mediated decay. In order to verify that the Rdh10delta transcript encodes a premature stop codon, we characterized Rdh10 cDNA from wild type and Rdh10delta/delta mutant embryos. The wild type Rdh10 mRNA encodes an open reading frame that spans the 6 spliced exons of the transcript (Fig. 4 E). Amplification of Rdh10 cDNA from Rdh10delta/delta embryos produced a distinct product 235 bp shorter than the wild type cDNA. Direct sequencing of the mutant Rdh10 cDNA revealed that exon 2 is absent and exon 1 is spliced directly to exon 3, producing a frame shift and generating a stop codon within the open reading frame in the third codon of exon 3 (Fig. 4 E). Protein produced from the Rdh10delta transcript would be encoded by exon 1 only. Quantitative RT- PCR was performed to assess transcript levels of the Rdh10delta transcript. Transcript from Rdh10delta/delta embryos was detected at ,50% the level produced by wild type embryos, suggesting that the Rdh10delta transcript is targeted for degradation by nonsense mediated decay (Fig. 4 F). Loss of Rdh10 or Aldha2 reduces RA production RDH10 and RALDH2 play sequential roles in synthesis of RA. In order to assess the extent to which the new Rdh10delta and Aldh1a2gri mutations disrupt RA synthesis, we utilized the RARE- lacZ reporter transgene, which allows the level and distribution of RA signaling to be visualized by staining for b-galactosidase PLoS ONE | www.plosone.org 5 February 2012 | Volume 7 | Issue 2 | e30698