Vertebrate ancestors had only cone-like photoreceptors. The duplex retina evolved in jawless vertebrates with the advent of highly photosensitive rod-like photoreceptors. Despite cones being the arbiters of high-resolution color vision, rods emerged as the dominant photoreceptor in mammals during a nocturnal phase early in their evolution. We investigated the evolutionary and developmental origins of rods in two divergent vertebrate retinas. In mice, we discovered genetic and epigenetic vestiges of short-wavelength cones in developing rods, and cell-lineage tracing validated the genesis of rods from S cones. Curiously, rods did not derive from S cones in zebrafish. Our study illuminates several questions regarding the evolution of duplex retina and supports the hypothesis that, in mammals, the S-cone lineage was recruited via the Maf-family transcription factor NRL to augment rod photoreceptors. We propose that this developmental mechanism allowed the adaptive exploitation of scotopic niches during the nocturnal bottleneck early in mammalian evolution.

A typical mammalian retina accommodates a great abundance of rods with a paucity of cones; few exceptions include rare cone-dominant retinas of a subset of diurnal species and highly specialized regions (such as fovea) in primate retinas (). On the contrary, most non-mammalian vertebrates possess abundant cone photoreceptors in multiple spectral subclasses. This stark difference in retinal composition may reflect a “nocturnal bottleneck” hypothesized to have occurred early in the evolutionary history of mammals, wherein early mammals adapted to novel scotopic (dim light) niches (). We were curious as to how a rod-dominant duplex retina evolved in a relatively short time span. We hypothesized that the abundance of rod photoreceptors in the mammalian retina originated from, and at the expense of, S cones with the co-emergence of an NRL-centered, rod-specific, regulatory network and that developing rods would therefore carry vestiges (“footprints”) of S cones ( Figure 1 B). Here, we provide multiple lines of evidence that developing rods in mouse retinas show a well-defined molecular footprint of S cones, whereas rods in zebrafish, representing an outgroup to tetrapods and having a cone-rich retina, do not. While alternative explanations are possible, our studies argue in favor of the recruitment of S cones to augment rods in mammalian retinas and suggest a developmental mechanism involving NRL that facilitated the adaptation to scotopic ecological niches during a “nocturnal bottleneck” experienced by the ancestors of extant mammals.

In the mammalian retina, the generation of rods and cones is controlled by the combined actions of two transcription factors, Maf-family neural retina leucine zipper protein (NRL) and thyroid hormone receptor β2 (TRβ2) (), and S cones are proposed to be the default fate of post-mitotic photoreceptor precursors (). In mice, loss of NRL results in a retina with predominantly S cones in place of rods (), and ectopic expression of NRL in cone precursors is sufficient to induce rod differentiation (). NRL is expressed in rod photoreceptors shortly after the final mitosis, as indicated by GFP expression directed by a 2.5-kb Nrl promoter (), consistent with antibody immunostaining studies (). Furthermore, mice lacking TRβ2 have S cones but no medium-wavelength-sensitive (M) cones (), and replacement of Nrl by Trb2 produces M cones instead of rods (). Transcriptional control of rod photoreceptor birth is less well resolved in cone-dominant vertebrates such as teleosts (), especially with respect to nrl, whose expression is not limited to rods in zebrafish (). Notably, the loss of T-box transcription factor tbx2b in zebrafish results in rod generation from UV cone precursors (). In addition, thyroid hormone treatment affects photoreceptor development in trout (), and thrb2 modulates the generation of different cone subtypes in zebrafish ().

gdf6a is required for cone photoreceptor subtype differentiation and for the actions of tbx2b in determining rod versus cone photoreceptor fate.

Short-wavelength-sensitive (S) and long-wavelength-sensitive (L) cone opsins were present in the last common ancestor of jawed and jawless vertebrates (), while rod visual pigment Rh1 (rhodopsin) and other cone opsin classes emerged subsequently by duplication of an ancestral, likely short-wavelength, opsin gene (). In concordance, rods first appear as an intermediate form in agnathan species (i.e., jawless vertebrates) such as lampreys and become more evident in gnathostomes (i.e., jawed vertebrates) () ( Figure 1 A ). Despite functional specialization, rod morphology and phototransduction machinery are similar to those of cones (), and rod signals piggyback on cone pathways in retinal circuitry (). On the basis of phylogenetic and anatomical analyses, rod photoreceptors were proposed to have originated from ancestral cone-like photoreceptors ().

(B) Hypothesis being tested in the present study. Green and purple curves represent cumulative rod and cone number, respectively. The respective shades indicate opsin expression. x Axis indicates developmental stages of mouse retina. E, embryonic day; B, birth; P, postnatal day.

(A) Co-emergence of rod photoreceptors and Nrl during vertebrate evolution. True functional rod photoreceptors exist only among jawed vertebrates in the evolutionary tree of Chordata (), but Nrl-like Maf gene is encoded in the lamprey genome ( Figure 7 B) and may be expressed in RhA (rod visual pigment) expressing rod-like cone photoreceptors (). Red bar indicates the time period that corresponds to nocturnal bottleneck during mammalian evolution. The tapering down and thickening of second and third rows of the table indicates a decrease in cone abundance and an increase in rod abundance observed in most mammals, respectively. Time-calibrated tree was redrawn from. mya, million years ago; Monotremes, egg-laying mammals; Marsupials, pouched mammals; Eutherians, placental mammals.

Molecular evidence for dim-light vision in the last common ancestor of the vertebrates.

The duplex retina in vertebrates consists of specialized cone and rod photoreceptors (). Cones mediate non-quenching, rapid responses to photons with high acuity in daylight, whereas rods allow maximum sensitivity and energy conservation at the expense of spatial and temporal resolution. Cone photoreceptors also enable color discrimination by combining outputs of visual pigments (opsins) having distinct peak wavelength sensitivity (). How and when the duplex retina evolved, however, remains a long-standing mystery ().

We also evaluated NRL and other Maf protein sequences across vertebrate species ( Figure S7 ). Transactivation and DNA binding domains were highly conserved in all vertebrate species including zebrafish and mouse. Protein sequence comparison revealed a high degree of conservation between all chicken long MAFs and vertebrate NRLs in both transactivation and DNA binding domains ( Figure S7 ). To test for functional conservation, we introduced the chicken MAFA gene, which is expressed in avian rods and a few other retinal neuronal types but not in cones (), in cone-only Nrlmouse retina () ( Figure 8 ). Chicken MAFA, but not GFP empty vector, induced the expression of rhodopsin ( Figure 8 B) and characteristic single synaptic ribbon observed in rods (), as opposed to cone opsin expression and multiple ribbons present in cones ( Figures 8 C and 8D). Chicken MAFA-induced cone-to-rod transformation was confirmed by expression of rod-specific genes including Nr2e3, Gnat1, and Cnga1, but not cone genes such as Arr3 and Gnat2, in MAFA-expressing Nrlmouse retina ( Figure 8 E). Thus, chicken MAFA induces rod development akin to the actions of mouse Nrl when expressed ectopically in Nrlretinas.

(E) qRT-PCR analysis of the transfected retina. Expression of selected cone-specific (Arr3 and Gnat2) and rod-specific (Rho, Nr2e3, Gnat1, and Cnga1) genes in chicken MAFA-expressing Nrl −/− retina was assayed by qRT-PCR and compared with the respective expression in Nrl −/− retina transfected with an empty control vector. Error bars indicate SEM.

(D) Bar graph summarizing the number of ribbons per chicken MAFA-expressing cell (indicated by GFP). Error bars indicate SEM.

(C) GFP signal (green) and RIBEYE immunoreactivity (red) in electroporated Nrl −/− retina at P21. DAPI counterstains all nuclei. Insets in each micrograph show a high-magnification image of synaptic area of the transfected cells as outlined with the dotted line. Merged image is shown in the rightmost micrograph. Scale bar, 20 μm.

(B) GFP signal (green; marks electroporated cells) and rhodopsin (RHO) immunoreactivity (red) of electroporated Nrl −/− retina at P21. DAPI counterstains all nuclei. Merged images are shown in the rightmost column. Scale bars, 50 μm.

(A) Chicken MAFA expression vector design. GFP or FLAG-tagged chicken MAFA-T2A-GFP was placed under cytomegalovirus promoter. MAFA expression vector or empty control vector was introduced by in vivo electroporation into dividing progenitor cells in P2 Nrl −/− mouse retina.

Next, we explored the phylogeny of NRL and related Maf proteins from a range of vertebrate genomes including jawless agnathans, cartilaginous and bony fishes, terrestrial vertebrates, and representatives of each major lineage of mammals ( Figure 7 B). Our results show that NRL and other closely related Maf paralogy classes have each originated among the jawed vertebrates, with a single distant ortholog being present in the earlier branching agnathan (Petromyzon) genome. Furthermore, each of these Maf paralogy classes, including NRL, demonstrates dynamic histories of loss among taxa. Strikingly, while we infer that NRL has been lost from several non-mammalian taxa, NRL loci are retained in all mammalian genomes included, in contrast to each of the other paralogy classes. Furthermore, the mammalian NRL genes are separated from non-mammalian paralogs by the longest internal branch in our tree, indicating a burst of molecular evolution. Together, our results suggest an evolutionary history for mammalian NRL whereby rapid molecular innovations in the lineage leading to mammals are followed by strong conservation and gene retention.

Our data describe a novel developmental origin of rod abundance in mouse (a representative placental mammal) compared with zebrafish (an earlier branching, non-amniotic vertebrate with a cone-dominant retina), encouraging us to determine a mechanistic explanation. We first compared genomic sequences spanning the Nrl gene in selected vertebrates. DNA sequences in the coding region of Nrl are highly conserved in all vertebrates ( Figure 7 A ). Strikingly, however, we observed extensive conservation of genomic sequences upstream and downstream of Nrl in mammals as well as in the intronic regions and UTRs of selected loci ( Figure 7 A). We also noted the gradual evolution of conserved genomic sequences spanning Nrl from the egg-laying platypus to the pouched and placental mammals, consistent with the appearance of rod-dominant duplex retina and the pivotal role of NRL in mammalian rod cell fate determination.

(B) Phylogenomic analysis of Nrl and related Maf genes among vertebrates. A single homolog is present in the genome of the agnathan Petromyzon while other Maf paralogs, including Nrl, originated in the lineage leading to gnathostomes. Mammalian Nrl loci are shaded in gray. Colored circles indicate bootstrap support for nodes greater than 50%. Inset: species relationships of the 30 vertebrate genomes included in this analysis.

(A) Comparison of genomic sequences within and flanking areas of Nrl. Yellow shading highlights a high degree of DNA sequence homology across all vertebrate species in the protein-coding region of Nrl, and purple shading indicates additional conserved domains including upstream regulatory sequences and UTRs.

Advent of Nocturnal Mammals Coincides with the Acquisition of Novel Regulatory Elements and Rod-Specific Expression of NRL

Figure 7 Advent of Nocturnal Mammals Coincides with the Acquisition of Novel Regulatory Elements and Rod-Specific Expression of NRL

Advent of Nocturnal Mammals Coincides with the Acquisition of Novel Regulatory Elements, Rod-Specific Expression, and Deep Conservation of NRL

Next we examined whether rods in a cone-dominated, teleost retina were also specified from a cone lineage. Teleosts branched earlier than mammals in the evolutionary history of vertebrates. To address this, we engineered a lineage-tracing zebrafish line with two genetic constructs that constituted a positive-feedback mechanism (“Kaloop” technology, Figure 6 A ). We used this line to permanently label cells with the history of expression of UV cone opsin sws1, a zebrafish homolog of mouse Opn1sw. We characterized 4,024 cells that had expressed sws1 opsin at some point in development from ten retinas of 4-dpf (days post fertilization) zebrafish larvae. The vast majority of lineage-traced cells (4,021 of 4,024; 99.92%) unambiguously lacked immunolabeling by 4C12 (a rod marker), whereas three cells (0.08%) found among two individuals were ambiguously labeled (i.e., they probably were not rods, though we cannot formally exclude this possibility) ( Figure 6 B). Lineage tracing of an mCherry transgene in each cell, where image resolution permitted assessment, was tightly associated with immunolabeling by the UV cone marker 10C9.1 antibody ( Figure 6 B). Although sws1 is the closest homolog of mouse Opn1sw, we also traced the lineage of zebrafish blue cone opsin sws2 to exclude the possibility that rods may have derived from the other S-cone class. As with sws1 lineage-traced cells, we did not find any rods with sws2 lineage ( Figure S5 ). Therefore, the cells with a history of sws1 or sws2 expression exclusively differentiate into UV or blue-sensitive cones, respectively.

(B) Lineage tracing of sws1-expressing cells using sws1:KalTA4; UAS:nfsB-mCherry-V2A-KalTA4 zebrafish line. The UV-sensitive cones (cyan, 10C9.1) and rods (green, 4C12) of 4-dpf zebrafish were labeled using immunohistochemistry. Representative images from 1 of 10 left eyes. D, dorsal; V, ventral; N, nasal; T, temporal. Scale bar, 50 μm in low-magnification images and 20 μm in high-magnification image (bottom right).

(A) Lineage tracing strategy in zebrafish. Lineage tracing in zebrafish was accomplished using a feedforward system that has been previously designated as “Kaloop” () (see Figure S6 ). Two constructs, Tg[sws1:KalTA4] and Tg[UAS:nfsB-mCherry-V2A-KalTA4] (ua3139 and ua3137, respectively), were independently inserted to generate the transgenic zebrafish. In ua3137, a fluorescent reporter protein mCherry is fused in-frame to a bacterial nitroreductase gene (nfsB), and the fusion protein is connected via the labile linker peptide V2A to a second copy of the KalTA4 transcription factor.

To further test whether rod photoreceptors in mice originated from S cones, we performed lineage tracing using the Opn1swp-Cre mouse () by breeding it to ROSA26-iAP reporter line () ( Figure 5 A ). Here, active Opn1sw promoters permanently tag the cell by inducing alkaline phosphatase (AP) expression ( Figure 5 A), allowing inference of lineage history. Cell bodies of most, but not all, rods and cones in the outer nuclear layer showed heavy AP activity in the P28 retinas ( Figures 5 B and S4 ), demonstrating the expression of S-opsin in rods at an early stage in their development. Immunostaining with antibodies against markers of mature photoreceptors revealed stronger AP staining in S cones and further confirmed the history of S-opsin promoter activity in rods ( Figures S4 D and S4E). Similar experiments using Opn1mwp-Cre mouse, which expresses Cre recombinase under the control of M-opsin promoter (), demonstrated no overlap between rod and M-cone lineage ( Figures 5 C and S4 C). Occasional ganglion cell staining was detected in both Opn1sw and Opn1mw lineage tracing experiments ( Figure S4 C). Detection of the history of Opn1sw expression in most mature mouse rods was further validated by an independent lineage tracing experiment using BAC transgenic Opn1swp-Cre and Z/EG fluorescence reporter lines ( Figure 5 D). In accordance with the results above, GFP lineage tracer was detected in both cones and most rods in mature retinas even though Cre and S-opsin immunoreactivity was observed only in mature cones ( Figures 5 E and 5F). Robust lineage tracer labeling of S cones in most but not all rods, and not of any other retinal neuron, in the BAC transgenic Opn1swp-Cre mice strongly argues in favor of the derivation of rods from S-cone lineage.

(E and F) Lineage tracing of S-opsin-expressing cells using Opn1swp-Cre (BAC tg); Z/EG mouse line. (E) CRE (green) and S-opsin (magenta) immunoreactivity in the outer portion of ONL. Note that S-opsin signal is strong in outer segments, and only weak S-opsin immunoreactivity can be detected in inner segments and cell bodies (merged image). (F) GFP + cells (green) are found both in outer and inner layers of ONL (i.e., cone and rod nuclei layers, the upper and lower half of ONL in the micrograph, respectively), while S-opsin + cones (red) are located only in the outer portion of ONL. OS, outer segment; IS, inner segment; ONL, outer nuclear layer; Cone, outer portion of ONL where cone nuclei are located; Rod, inner ONL with rod nuclei. Scale bar, 20 μm.

(D) Lineage tracing strategy using BAC transgenic mouse line. Opn1swp-Cre BAC transgenic mouse line was crossed with Z/EG reporter line. Cells with active Opn1sw promoter express Cre recombinase, which permanently activates GFP expression in all progeny of the cells that once expressed Opn1sw.

(B and C) Lineage tracing of S-opsin-expressing cells using Opn1swp-Cre; ROSA26-iAP (B) or tracing of M-opsin lineage cells using Opn1mwp-Cre; ROSA26-iAP mouse lines (C). Shown is AP staining (purple) in the photoreceptor layer of adult (P28) retina of respective genotype. OS, outer segment; IS, inner segment; ONL, outer nuclear layer; OPL, outer plexiform layer; Cone, outer portion of ONL where cone nuclei are located; Rod, inner ONL with rod nuclei. Scale bar, 20 μm.

(A) Lineage tracing strategy in mouse. Opn1swp-Cre mouse line was crossed with ROSA26-iAP line. In this line, cells with active Opn1sw promoter express Cre recombinase, which brings alkaline phosphatase (AP) gene back to the correct configuration by inverting the DNA piece surrounded by two loxP sites in head-to-head orientation. Once recombined, a constitutive promoter in the ROSA locus drives AP reporter gene expression even when cells no longer have Opn1sw promoter activity.

Distinct roles of transcription factors brn3a and brn3b in controlling the development, morphology, and function of retinal ganglion cells.

Our hypothesis that mammalian rods derive from cells fated as S cones further predicts that developing rods would exhibit a chromatin state concordant with S-cone characteristics. To address this, we performed reduced representation bisulfite sequencing (RRBS) and histone modification chromatin immunoprecipitation sequencing (ChIP-seq) using flow-sorted rod photoreceptors at three key stages of development: P2, P10, and P28 (see Figure 2 ). We also analyzed DNase I hypersensitivity sequencing (DNase-seq) data from developing mouse retinas (). In P2 rods, chromatin state was unfavorable for active Rho transcription as indicated by the high degree of DNA methylation (meDNA), limited enrichment of active histone mark H3 lysine 4 trimethylation (H3K4me3) at the promoter, and low chromatin accessibility (indicated by lack of DNase-seq signal) ( Figure 4 ). In contrast, Opn1sw promoter exhibited hallmarks of active chromatin state in P2 rods; these include the absence of meDNA, accumulation of H3K4me3, chromatin accessibility, and a low level of repressive H3K27me3 marks ( Figure 4 ). Rod-specific epigenetic architecture was acquired by P10 and maintained thereafter with concurrent accumulation of repressive chromatin marks in Opn1sw ( Figure 4 ). The observed chromatin dynamics were specific to opsin gene loci and other rod- and cone-specific genes (data not shown). The promoters of photoreceptor-specific transcription factors including Nrl ( Figure 4 ) and of constitutively expressed genes possessed active chromatin architecture at all stages of rod development, whereas genes specifically expressed in non-neuronal tissues exhibited repressed chromatin state (data not shown). Overall, the state of chromatin in newly post-mitotic rods, but not in mature rods, is favorable for expression of S-opsin and other cone genes.

Epigenetic state on Rho, Opn1sw, and Nrl genes were revealed by reduced representation bisulfite sequencing (RRBS; DNA methylation profiling), H3K4me3 and H3K27me3 ChIP-seq, and DNase I hypersensitivity sequencing (DNase-seq) at indicated developmental stages. Data were generated using flow-sorted rod photoreceptors from Nrlp-GFP mouse retina, except for retina DNase-seq data that were downloaded from the mouse ENCODE project (). Percentage ± SEM of methylated cytosine in the promoter regions is indicated as bar graphs. Aligned sequencing reads for mRNA expression (dark blue), H3K4me3 (green, active mark) and H3K27me3 (red, repressive mark) enrichment, and DNase hypersensitivity sites (orange) are shown as histograms. Promoter of each gene, defined as −1 kb to +1 kb from the transcription start site, is highlighted with gray shading.

Taken together, our findings exclude the possibility of S-opsin + rods being an artifact of the transgenic mouse line. Our data thus demonstrate the expression of S-opsin and other cone phototransduction genes in mouse rod photoreceptors during early stages of development.

To further validate the preceding results, we performed FACS analysis of photoreceptors from an independent Opn1swp-Venus transgenic mouse line where the S-opsin promoter was used to drive the expression of the Venus reporter gene. Consistent with the above data, NRL immunostaining was detectable in 85.6% of Venuscells at P2, and Venus and NRL double-positive cells accounted for 12% of NRLcells in the developing retina; however, the number of double-positive cells was negligible in the P28 retina ( Figure 3 E).

We then examined the expression of S-opsin in dissociated Nrlp-GFPretinal cells at P2 and P28 ( Figures 3 A and S3 ). Consistent with the RNA-seq and previously published P0 immunohistochemistry data (), S-opsin immunostaining was clearly detectable in GFProds at P2 but not at P28. Quantification by FACS analysis demonstrated that 13.8% of GFPcells were also positive for S-opsin at P2, but double-positive cells were negligible at P28 ( Figure 3 B). In addition, FACS analysis of NRL and S-opsin double-immunostained wild-type retinal cells revealed 26.4% NRLcells being S-opsinat P2, while double-positive cells decreased to almost none by P28 ( Figure 3 C). We then estimated the number of S-opsinand S-opsin/GFP double-positive cells at different developmental stages. P2 retina contained a greater number of total S-opsincells compared with P28 retina ( Figure 3 D). A majority (72%) of S-opsincells at P2 (on an average ∼141,000) was also positive for GFP; however, the number of S-opsin/GFP double-positive cells was negligible by P28. The number of cells that were positive for only S-opsin remained remarkably constant ( Figure 3 D).

(E) Quantification of NRL-expressing S cones at indicated time points by flow cytometry of Opn1swp-Venus mouse retina. Proportion of cells in each subpopulation is indicated as a percentage relative to total cells, and percentage of Venus + cells among NRL + rods is shown in parentheses.

(D) Quantification of S-opsin + and GFP/S-opsin double-positive photoreceptors per retina. Percentage of each cell population determined by flow cytometry of Nrlp-GFP retina was multiplied by total retinal cell number at the corresponding stage. y Axis indicates mean cell number (×10 5 ) ± SEM.

(C) Quantification of S-opsin and NRL double-positive cells by immunostaining followed by flow cytometry. Percentage of cells in each subpopulation is indicated. Percentage of S-opsin + cells among NRL + population is shown in parentheses.

(B) Quantification of S-opsin + rod photoreceptors by flow cytometry. Percentage of cells in each quadrant relative to total counted cells is indicated in the dot plots, and percentage of S-opsin + cells among GFP + rods is shown in parentheses.

(A) Immunocytochemistry of dissociated Nrlp-GFP retinal cells. S-opsin (purple) and GFP (green) double-positive cells were detected at P2 but not P28.

To validate RNA-seq results, we performed qPCR analysis using RNA purified from 50 manually collected Nrlp-GFPcells each at P2 (peak of newborn rods) and P28 (mature rods). As predicted, rhodopsin expression increased dramatically in P28 rods compared with P2 rods, whereas S-opsin expression was undetectable in mature rods ( Figure 2 C).

To test our hypothesis, we examined whether rod photoreceptors showed a signature of gene expression similar to S cones during development. We took advantage of an Nrlp-GFP transgene to obtain pure populations of rods from wild-type mouse retinas and of S-cone-like photoreceptors from Nrlretinas (). Transcriptome profiling, using RNA sequencing (RNA-seq) of fluorescence-activated cell sorting (FACS)-purified rods from Nrlp-GFP retinas revealed a progressive increase in the expression of rod-specific phototransduction genes, including rhodopsin (Rho), as the rods reached anatomical and functional maturity ( Figure 2 A ). As predicted, absolutely no known rod-specific gene was expressed in Nrlp-GFP photoreceptors from the rod-less Nrlretinas ( Figure 2 A). Interestingly, we noted strong expression of S-opsin (Opn1sw), but not M-opsin (encoded by Opn1mw), and of many cone-specific phototransduction genes, well above the median gene-expression values, in the developing rods from wild-type Nrlp-GFP retinas ( Figure 2 B). The expression of cone genes diminished with rod maturation in wild-type Nrlp-GFP retinas but not in photoreceptors of Nrlretinas ( Figure 2 B). Expression of several progenitor genes was also detected in rods at postnatal day2 (P2) and P4, but rapidly declined as rod birth was completed and maturation began ( Figure S1 ), reflecting the likely contribution of GFProds that just exited terminal mitosis (). Genes specifically expressed in other retinal cell types (ganglion, bipolar, amacrine, and horizontal cells) were barely detectable in the rod transcriptome data ( Figure S1 ). As a low level of opsin expression was recently reported in cultured human epidermal skin cells (), we examined S-opsin expression in ENCODE datasets () to ensure the specificity of Opn1sw expression as an S-cone marker. S-opsin expression was not detected in any non-retinal human/mouse tissue or cell type (including epidermal cells) for which expression data were available ( Figure S2 ).

(C) qRT-PCR validation of Rho and Opn1sw expression from 50 manually collected GFP + rods. Data are normalized to P2 expression level and presented as mean of fold change (FC) ± SEM.

(A and B) Gene-expression profiles of selected (A) rod-specific and (B) cone-specific phototransduction genes in the rod photoreceptors purified from Nrlp-GFP mouse retina. Developmental time points are indicated on the x axis; y axis shows FPKM ± SEM from RNA-seq data. Gene expression in photoreceptors from Nrlp-GFP;Nrl −/− retina is shown as mean by dark gray lines and range of five indicated genes as gray shading. The six time points selected for RNA-seq analyses represent key stages of mouse rod development: P2, peak of post-mitotic rod generation; P4, robust increase in phototransduction gene expression; P6, rod outer segment discs beginning to form; P10, outer segment elongation and synaptogenesis; P14, eye opening and outer segment/synapse formation completed; P28, rods fully mature.

Discussion

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et al. Targeting of GFP to newborn rods by Nrl promoter and temporal expression profiling of flow-sorted photoreceptors. + cells, where no contamination is allowed, confirmed high Opn1sw expression in P2 rods compared with P28 rods (see Multiple lines of evidence validate the expression of Opn1sw and other cone genes in developing rods. Though not emphasized as such, Nrlp-GFP cells in P0 mouse retina are documented to express S-opsin (). Both transcriptome and epigenome analyses demonstrate the robustness of co-expression of Nrl and Opn1sw in isolated mouse rod photoreceptors. We used flow-sorted rods of purity of over 96% (>99% for most samples) for all next-generation sequencing (NGS) experiments; thus, S-cone contamination in purified rod samples cannot explain the expression values (fragments per kilobase of exon per million fragments mapped [FPKM]) in our samples. We note that the genes associated with inner retina show no to negligible expression in flow-sorted rods (see Figure S1 ). Although some progenitor genes were detected in early developing rods (P2 and P4), it is not surprising given that Nrl expression commences during or immediately after the final mitosis (). Furthermore, qRT-PCR analysis of manually picked GFPcells, where no contamination is allowed, confirmed high Opn1sw expression in P2 rods compared with P28 rods (see Figure 2 C). Multiple independent experiments, including immunocytochemistry and flow cytometry using distinct combinations of reporter mouse lines (see Figure 3 ), point to Opn1sw expression in immature rods. Lineage-tracing experiments using two different S-opsin Cre lines and phylogenetic analysis provide additional strong support of our hypothesis.

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Sharlin D.S.

Swaroop A.

Forrest D. Two transcription factors can direct three photoreceptor outcomes from rod precursor cells in mouse retinal development. Oh et al., 2007 Oh E.C.

Khan N.

Novelli E.

Khanna H.

Strettoi E.

Swaroop A. Transformation of cone precursors to functional rod photoreceptors by bZIP transcription factor NRL. Jacobs, 2013 Jacobs G.H. Losses of functional opsin genes, short-wavelength cone photopigments, and color vision–a significant trend in the evolution of mammalian vision. Developmental plasticity can support the evolution of novel traits by providing the raw materials (i.e., phenotypic variation) necessary for rapid adaptation (), and vertebrate photoreceptors are known to demonstrate such plasticity. In humans, opsin expression is plastic over the course of development. S-opsin expression precedes L-/M-opsin expression, and the number of cones expressing both increases during development, followed by their decrease at later stages (). Similarly, mouse genetic studies have demonstrated the developmental plasticity of post-mitotic photoreceptor precursors (). Thus, it is reasonable to predict that the recruitment of S cones to augment rod photoreceptors in mammalian retinas via the plastic redeployment of NRL enabled the rapid adaptation of early mammals to the nocturnal niche. Notably, loss of Sws2 and Rh2 as well as a shift in spectral sensitivity of Sws1 also coincided with this critical period (). Thus, multiple lines of evidence suggest a fundamental reorganization of the mammalian retina during the nocturnal bottleneck.

Whitney et al., 2011 Whitney I.E.

Raven M.A.

Lu L.

Williams R.W.

Reese B.E. A QTL on chromosome 10 modulates cone photoreceptor number in the mouse retina. We recognize that our proposed mechanism for the transition from cone-dominant to rod-dominant retinas in early mammals does not provide a complete explanation for the developmental transformation of mammalian retinas that took place during the nocturnal bottleneck. In addition to their cellular composition, rod-dominant retinas also have a greatly increased proportion of photoreceptors. Thus, an explanation is also required for the increase in progenitor proliferation and cell number. How cell number is modulated in developing retinas is largely unclear, although the myoblast oncogene Myb has been implicated in determining cone photoreceptor number ().

In conclusion, we propose that the evolutionary origin of the majority of mammalian rods occurred by their developmental recruitment from the S-cone lineage, a phenomenon not observed in non-mammalian vertebrates. We further hypothesize that this process was predicated on the origins of novel regulatory sequences of Nrl that restrict its expression to rod photoreceptors in mammals. We show that the origins of novel Nrl regulatory sequences coincide with a period of punctuated visual system evolution early in mammalian evolution known as the nocturnal bottleneck, followed by strong conservation and stasis in extant mammalian species. Our study provides critical mechanistic details underlying the rapid adaption to the scotopic light environment that was the driving force behind the profound cone-to-rod transformation that occurred during the nocturnal bottleneck, and reveals how the evolutionary history of a neuronal cell type can be revealed by comparative molecular and ontogenetic analysis.