depict weak expression of Aldh1a genes in a small part of each dorso- ventral sector or from earlier developmental stages prior to the complete invagination of the optic cup in Figure 8A. The rules of ancestral reconstruction imply that the retina of the last common vertebrate ancestor probably had a dorsal and a ventral sector, and the original Aldh1a1/2/3/3-ogm gene prior to the expansion of the Aldh1a family gene was likely expressed in both dorsal and ventral sectors (Figure 8A step 1). According to the evolutionary model proposed in Figure 7, the first expansion of the Aldh1a family occurred before R2 and generated Aldh1a1/2 and Aldh1a3/3-ogm. Because Aldh1a3 is the major paralog in the ventral sector of the retina in extant tetrapods and teleosts, and because Aldh1a1 or Aldh1a2 are the major paralogs in the dorsal sector of the retina in both tetrapods and teleosts, we infer that after the first duplication prior to R2, Aldh1a1/2 inherited the subfunction leading to expression in the dorsal sector of the retina, and Aldh1a3/3-ogm inherited the subfunction causing expression in the ventral sector (Figure 8A step 2). It is probable that this subfunctionalization event contributed to the preservation of both paralogs as expected under the duplication-degeneration-comple- mentation (DDC) model, in which the summation of the subfunctions that were split between gene duplicates equals the ancestral function prior the duplication event [3]. After R2, Aldh1a3-ogm was lost and Aldh1a3 became the main ventral source of RA in the retina. On the other hand, both Aldh1a1 and Aldh1a2 retained expression in the dorsal sector because it is preserved in frog, chicken and quail, but not in mouse. Thus we conclude that the absence of Aldh1a2 dorsal expression in mouse retina is due to a loss of an ancestral expression domain, which can be interpreted as an evolutionary innovation due to late subfunction partitioning [3], in which a function that was originally possessed by both Aldh1a1 and Aldh1a2 became partitioned exclusively to Aldh1a1 (Figure 8A step 3). Analysis of the ALDH1A2 expression pattern in the human retina will help elucidate whether the loss of the Aldh1a2 dorsal expression domain occurred before the split of lineages leading to human and rodents, or if it is a feature that has been acquired specifically in the rodent lineage. An important question is how gene loss can impact the evolution of gene regulation and gene function in surviving paralogs. After the loss of Aldh1a1 in teleosts, Aldh1a2 became the only source of RA in the dorsal retina, taking full responsibility for subfunctions originally shared with Aldh1a1. Natural selection would have gradually increased the strength of the ancestral dorsal domain of Aldh1a2 (Figure 8A step 4). Medaka lacks both aldh1a1 and aldh1a3 orthologs, and the only surviving Aldh1a gene is aldh1a2, which is expressed in both the dorsal and ventral domains of the retina (Figure 8A step 5). The fact that in zebrafish and mouse, Aldh1a2 is expressed early in the ventral retina prior to the closure of the optic cup and becomes progressively down-regulated until the completion of optic cup invagination (arrow in Figure 6B) [88], suggests that early expression followed by down-regulation of Aldh1a2 is an ancestral feature and that medaka evolved an innovative heterochronic mechanism to avoid the ventral down- regulation of aldh1a2 and to increase its ventral expression at later stages. Thus, it is likely that the dorsal and ventral paracrine sources of RA that have been suggested to regulate the development of perioptic mesenchimal derivative structures [56] is an ancestral feature that might be still preserved in teleosts. Comparative and functional analysis of the regulation of aldh1a paralogs during the development of the eye and other tissues in medaka, zebrafish and in other fishes, particularly outgroups, will be required to test this hypothesis. The evolution of functions among Aldh1a paralogs illustrates what may be a general phenomenon associated with evolution after Gene Loss and Evolution of Surviving Paralogs genome duplication: gene loss without altering developmental programs due to the preservation of functions in surviving paralogs. In our case study, the unaltered ancestral program provides both a dorsal and ventral supply of Aldh1a enzyme and hence dorsal and ventral sources of RA during retinal development. Comparative analysis shows that different paralogs can perform equivalent functions in different species. For instance, the ventral sector of the retina expresses aldh1a2 in medaka and aldh1a3 in zebrafish; and the dorsal sector of the retina expresses Aldh1a1 in mouse and aldh1a2 in zebrafish. Similar cases of what has been called function shuffling have been described for Hox genes [42]; Bmp genes [97], and Twist genes [43]. Gitelman (2007) proposed the term synfunctionalization to describe the process by which a paralog acquires the expression pattern of another paralog by gaining new regulatory elements, and thereby allowing losses of genes without changing the ancestral developmental program [43]. The acquisition of enhanced ventral expression by aldh1a2 in the face of aldh1a3 loss in medaka suggests several possible mechanisms for the apparent shuffling of functions between aldh1a3 and aldh1a2 that do not require the evolutionary gain of new regulatory elements (Figure 8B). Based on our findings, we propose a general mechanistic model to explain the loss of a paralog without altering the ancestral developmental program. After gene duplication from an ancestral gene a/b (Figure 8B Step 1), paralog b (e.g. aldh1a3) could lose the dorsal subfunction without penalty (Step 2) because it is covered by paralog a (e.g. aldh1a2). Next, mutations in negative regulatory elements or in upstream negative regulators that normally down-regulate paralog a expression in later developmental stages (e.g., after retina cup invagination) would facilitate an innovative heterochronic paralog a expression (Step 3). Finally, natural selection or genetic drift could act on natural variation that positively strengthens paralog a expression in the ventral domain (Step 3), while allowing relaxed selection for paralog b expression (Step 4), thereby facilitating the loss of paralog b (Step 5) without loss of the ancestral developmental program (Step 6). Overall, our results illustrate how comparative genomic analyses of conserved synteny, coupled with reconstruction of ancestral chromosomes, can provide a phylogenetic framework necessary for the identification of lineage-specific gene losses. Our analysis provides evidence for early subfunctionalization and late subfunc- tion-partitioning, and for the acquisition or modification of subfunctions by surviving paralogs that preserve unaltered ancestral developmental programs in the face of gene loss. Understanding the evolution of gene functions is fundamental for the proper interpretation of comparative analyses, especially when using model organisms to understand human gene functions. In the case of the Aldh1a family, although RA is important in human disease, we still know little about the spatio-temporal dynamics of the expression domains and functions of ALDH1A1, ALDH1A2 and ALDH1A3 genes during human development and adult organ homeostasis, other than RT-PCR studies [98], which do not provide enough resolution at the single cell level to understand how the sources of RA regulate physiological action. The evolutionary framework defined here provides information essential for the functional connectivity of human and model organism genomes, not only for RA signaling in eye development, but for the many organs in which RA plays important functions, including axis and limb development and cancer biology. Materials and Methods Ethics Statement All animals were handled in strict accordance with good animal practice as defined by the relevant animal welfare bodies, and all animal work was approved by the University of Oregon PLoS Genetics | www.plosgenetics.org 15 May 2009 | Volume 5 | Issue 5 | e1000496