expression patterns during eye development in medaka embryos, and compared its expression pattern to that of aldh1a2 and aldh1a3 in zebrafish embryos. In zebrafish, aldh1a2 and aldh1a3 are expressed in the neural retina in two sectors along the dorso-ventral axis in 1.5 day post-fertilization (dpf) embryos (Figure 6B). The aldh1a2 gene is strongly expressed dorsally and aldh1a3 is expressed ventrally (Figure 6B), confirming earlier results [65,84,86,87]. Low signal of aldh1a2 ventral expression was still observed remaining from earlier stages before optic cup invagination is completed, but the signal disappeared at later stages. We found that in medaka embryos, after optic cup invagination, aldh1a2 was expressed strongly both dorsally and ventrally, a pattern that recapitulates the sum of the expression domains of aldh1a2 and aldh1a3 genes in zebrafish (Figure 6B). The ventral aldh1a2 expression domain does not disappear at later stages in medaka (data not shown), in contrast to zebrafish or mouse, in which aldh1a2 is down-regulated by the time that optic cup formation is completed [88]. Thus, our findings support hypothesis 2, in which medaka Aldh1a2 provides an alternative ventral source of RA in the absence of aldh1a3, in contrast to zebrafish in which ventral RA is supplied by Aldh1a3. This situation could have arisen by the evolutionary gain of the ability to express aldh1a2 at high levels in the ventral domain of the developing eye after optic cup invagination in the medaka lineage. Discussion This work illustrates how comparative analysis of whole genomes is important for functional connectivities between humans and model organisms. Analysis of conserved syntenies related to individual gene families helps identify lineage-specific gene gains and losses that can translate to evolving developmental mechanisms. Using the evolution of the Aldh1a family as a case study, we sought to probe the general mechanisms underlying the impact of gene loss on the functional fate of surviving paralogs after genome duplications while preserving unaltered ancestral developmental programs. Gene Loss in Teleosts Followed Expansion of the Aldh1a Family in Stem Vertebrates Retinoic acid plays important morphogenetic roles in chordate embryonic development. The recent identification of components of the RA genetic machinery in non-chordate deuterostomes and in protostomes opens the possibility that expansion and reduction in RA-related gene families could have played a role in the developmental diversification of bilaterians [51,52]. The Aldh1a gene family, which encodes enzymes that synthesize RA, has expanded independently several times during the evolution of the three chordate subphyla, the Cephalochordata, Urochordata and Vertebrata [59]. Within vertebrates, the expansion of the Aldh1a family generated three main paralogs - Aldh1a1, Aldh1a2 and Aldh1a3 - but the phylogenetic relationships and origins of these genes remained uncertain [59,65]. To identify gene gains and losses, one must first reconstruct the evolutionary genomic history of a gene family. We undertook a combination of phylogenetic and comparative genomic analyses of conserved syntenies that clarified the evolutionary history of the Aldh1a family. Phylogenetic results showed that Aldh1a1 and Aldh1a2 form sister clades and Aldh1a3 occupies a basal position in the phylogenetic tree rooted on cephalochordate Aldh1a genes (Figure 1). This analysis breaks the trichotomy observed in one previous analysis [59] and is opposite to the topology rooted on the far more distant Aldh2 gene family in another analysis [65]. Further support for the new understanding of Aldh1a family member relationships ((Aldh1a1, Aldh1a2) Aldh1a3) comes from Gene Loss and Evolution of Surviving Paralogs comparative genomic analyses of conserved syntenies in the genomic neighborhoods of Aldh1a paralogs in human, mouse, zebrafish, stickleback and medaka, which showed extensive conservation of syntenies between Aldh1a1 and Aldh1a2 genetic neighborhoods (Figure 2B). The congruency of the inferred historical relationships that arise from the new phylogeny and conserved syntenies, which are independent datasets, forces the conclusion that Aldh1a1 and Aldh1a2 are sisters and both are cousins to the Aldh1a3 gene. Based on results obtained from the analysis of synteny conservation of the Aldh1a1 genomic neighborhoods across human and model organism genomes, we infer an evolutionary model that reconstructs the genomic history of the Aldh1a family, and integrates previous work by Nakatani et al., (2007) [26] that had reconstructed the re-organization of the ancestral chromosomes (named A to J) of the last common ancestor of vertebrates through R1, R2 and R3 genome duplications (Figure 7). Because Aldh1a2 and Aldh1a3 are syntenic (on the same chromosome) in human, zebrafish, and stickleback genomes, we conclude that this was the state in their last common ancestor (Figure 7 step 1). According to Nakatani’s reconstruction, Hsa15 mostly derives from the post-R2 ancestral chromosome ‘‘A4’’, which allows us to infer that Aldh1a2 and Aldh1a3 were syntenic in the ancestral chromosome A4 (Figure 7 step 1). After our comparative analysis of synteny conservation between human and mouse, which ruled out the possibility of reciprocal Aldh1a3 paralog losses (Figure 3) and showed that Aldh1a3 genes are actual orthologs (Figure 1), we conclude that the Aldh1a3-ogm was already absent in the last common ancestor of tetrapods and teleosts (Figure 7 step 1). If Aldh1a2 and Aldh1a3 were syntenic in the ancestral state, we reason that a chromosomal translocation might have occurred during the evolution of the rodent lineage to separate them into different chromosomes (e.g Mmu9 and Mmu7 in Figure 7 step 2). Because the fourth Aldh1a paralog of rodents (i.e. Aldh1a7 in mouse) is adjacent and oppositely oriented to Aldh1a1, separated only by 0.5 Mb with no intervening genes, we conclude that the fourth Aldh1a rodent paralog originated by a rodent-specific tandem gene duplication associated with a local inversion (Figure 7 step 2) that was probably followed by subsequent amino acid sequence changes that destroyed its ability to synthesize RA [64]. In contrast to tetrapods, teleosts lack an Aldh1a1 ortholog, and whether this is due to a gene loss in teleosts, or a gene gain by tetrapods was previously unknown. Our whole-genome compar- isons of conserved synteny answer this question by identifying genomic neighborhoods orthologous to the human ALDH1A1 genomic neighborhood in zebrafish, stickleback and medaka (Figure 5). This finding is consistent with the new Aldh1a phylogeny (Figure 1) and provides strong evidence supporting the conclusion that Aldh1a1 was present in the last common ancestor before the tetrapod and teleost lineages split (Figure 7 step 1). Thus, we conclude that the absence of Aldh1a1 in teleosts is due to gene loss, probably in stem teleosts or ealier in stem actinopterygians (Figure 7 step 3), and discards the hypothesis that Aldh1a1 is a tetrapod innovation. This finding illustrates the power of comparative genomics to discern cases of gene losses from cases of gene gains, even in situations in which no proper outgroup is available. In human and mouse, Aldh1a1 and Aldh1a3-ogm genomic neighborhoods are not syntenic (e.g. on Hsa9 and Hsa5, respectively). Interestingly, however, in zebrafish, stickleback and medaka, genomic neighborhoods orthologous to those of human ALDH1A1 and ALDH1A3-ogm are syntenic (e.g., on Dre5, GacXIII and Ola9, see Figure 4A–C and right panels of Figure 7). Thus, just as Aldh1a2 and Aldh1a3 were syntenic after R2, it is likely that PLoS Genetics | www.plosgenetics.org 11 May 2009 | Volume 5 | Issue 5 | e1000496