Gene Loss and Evolution of Surviving Paralogs Figure 6. Conserved syntenies provide evidence that aldh1a3 was secondarily lost in medaka. (A) Comparative syntenic analysis of ALDH1A3 genomic neighborhoods in human, stickleback, and medaka. These results show that aldh1a3 was lost in the medaka lineage. ALDH1A3 orthologs are highlighted in red, and ALDH1A3 nearest neighbors are labeled in purple. The presence of one LTR-flanked retrotransposon including an ORF2 reverse transcriptase (in brown) in the putative locus of the lost aldh1a3 gene suggests the hypothesis that the insertion of the retrotransposon was related to the aldh1a3 loss. (B) Comparative analysis by in situ hybridization of the expression of aldh1a gene family members in the developing eye of zebrafish and medaka reveals that the medaka aldh1a2 gene recapitulates both the dorsal expression of aldh1a2 and the ventral expression of aldh1a3 in zebrafish. This result suggests that in medaka, aldh1a2 provides a ventral RA source after the loss of aldh1a3. doi:10.1371/journal.pgen.1000496.g006 fugu and zebrafish [74–76]. BLAST comparisons against the medaka genome revealed the presence of the same type of ORF2 associated with the same type of LTR sequences in other genomic locations where LTR retrotransposons have been predicted, such as the MHC Class I Region (GenBank accession number BA000027). The identification of an LTR retrotransposon at a site orthologous to the location of aldh1a3 genes in other teleosts and in humans (Figure 6A) is exactly what would be expected under the hypothesis that the insertion of an LTR retrotransposon disrupted or silenced the aldh1a3 gene in the medaka lineage after it diverged from the stickleback lineage. This molecular event may have led to the loss of aldh1a3 in medaka. According to the results of the dotplot (Figure 4C), scaffold572 is likely to assemble eventually into medaka Ola3. Further sequencing of this genomic location or genetic mapping of a marker in this scaffold is necessary to corroborate this prediction. Consequences of gene loss for the function of surviving paralogs. The apparent loss of aldh1a3 in medaka made us wonder about the functional consequences of this loss and its potential effect on evolution in this lineage. Aldh1a3 plays a crucial role in the synthesis of retinoic acid, and it is especially important for the development of the ventral part of the eye, where a strongly conserved Aldh1a3 expression domain has been described in all vertebrates studied so far, including mouse, chicken, Xenopus and zebrafish [58,65,77–84]. In mouse, for instance, homozygous Aldh1a3 mutant embryos fail to complete the formation of the ventral cup of the eye and the closure of the chorioid fissure, resembling aberrations observed in colobomas in human retinas of patients with cat eye syndrome [82,85]. In medaka, two alternative hypotheses might explain the effects of aldh1a3 gene loss in the development of the eye: Under hypothesis 1, the development of the ventral part of the eye in medaka may have become independent of RA; under hypothesis 2, medaka might have an alternative mechanism to supply RA ventrally during eye development. To test these hypotheses, we cloned medaka aldh1a2 cDNA (submitted to GenBank FJ516380), used it as probe to study PLoS Genetics | www.plosgenetics.org 10 May 2009 | Volume 5 | Issue 5 | e1000496