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Proc Natl Acad Sci U S A. Sep 18, 2012; 109(38): 15383–15388.
Published online Sep 4, 2012. doi:  10.1073/pnas.1207580109
PMCID: PMC3458357
Evolution

Molecular analysis of the amphioxus frontal eye unravels the evolutionary origin of the retina and pigment cells of the vertebrate eye

Abstract

The origin of vertebrate eyes is still enigmatic. The “frontal eye” of amphioxus, our most primitive chordate relative, has long been recognized as a candidate precursor to the vertebrate eyes. However, the amphioxus frontal eye is composed of simple ciliated cells, unlike vertebrate rods and cones, which display more elaborate, surface-extended cilia. So far, the only evidence that the frontal eye indeed might be sensitive to light has been the presence of a ciliated putative sensory cell in the close vicinity of dark pigment cells. We set out to characterize the cell types of the amphioxus frontal eye molecularly, to test their possible relatedness to the cell types of vertebrate eyes. We show that the cells of the frontal eye specifically coexpress a combination of transcription factors and opsins typical of the vertebrate eye photoreceptors and an inhibitory Gi-type alpha subunit of the G protein, indicating an off-responding phototransductory cascade. Furthermore, the pigmented cells match the retinal pigmented epithelium in melanin content and regulatory signature. Finally, we reveal axonal projections of the frontal eye that resemble the basic photosensory-motor circuit of the vertebrate forebrain. These results support homology of the amphioxus frontal eye and the vertebrate eyes and yield insights into their evolutionary origin.

Keywords: evolution, vision, cephalochordate

The evolutionary origin of vertebrate eyes is enigmatic. Charles Darwin appreciated the conceptual difficulty in accepting that an organ as complex as the vertebrate eye could have evolved through natural selection (1). Part of the problem lies in the paucity of extant phyla with useful gradations that occurred during eye evolution, thus providing a scenario that led to the emergence of the vertebrate eye. For example, the eye of the adult lamprey (a jawless vertebrate) is remarkably similar to the eye of jawed vertebrates in the overall design, retina cell types, and multiple classes of opsins (2). Given these similarities, it is likely that the last common ancestor of jawless and jawed vertebrates already possessed an elaborate camera-type lens eye. To understand the seemingly sudden origin of the vertebrate eye, its evolutionary precursor must be identified within the nonvertebrate chordates lacking elaborated eye structures. Because of its basal phylogenetic position within the chordates (3), its slowly evolving genome (4), and its ancestral morphology, the cephalochordate amphioxus represents a traditional model organism for understanding the origin of vertebrate organs. Extensive electron microscopy studies of the cerebral vesicle of the basal chordate amphioxus revealed several putative photoreceptive organs—dorsal ocelli, Joseph cells, lamellar body, and the unpaired “frontal eye” (5). The pigmented dorsal ocelli and the Joseph cells are morphologically and molecularly related to invertebrate eye photoreceptors (6, 7), whereas the frontal eye and the lamellar body traditionally have been homologized to the vertebrate eyes and pineal gland, respectively, based on their position and morphological features. However, these statements of homology have been a matter of debate (8), and the lack of adequate comparative molecular evidence has not allowed any firm conclusion to be drawn.

In this work we focus specifically on the functional molecules (c-opsin, melanin, and serotonin) and on transcription factors (Rx, Otx, Pax4/6, Mitf) playing crucial roles during vertebrate eye development. Unlike the majority of published amphioxus expression studies, we use a set of amphioxus-specific antibodies in combination with confocal microscopy to gain cellular resolution and coexpression information and to track axonal projections of the frontal eye. Our data provide evidence that the amphioxus frontal eye is an opsin-based photoreceptive organ and that the frontal eye photoreceptors and pigment cells are homologous to rods/cones and pigment cells of the vertebrate eyes, respectively.

Results

Developmental Patterning of the Amphioxus Frontal Eye.

Vertebrates have two separate sets of eyes, the lateral visual eyes and the dorso-medial pineal and parietal “eyes” that play a role in the detection of ambient light and, in some groups, convey a fast response to predator shadows (911). Previous studies have revealed a small set of transcription factors that specify photoreceptor cells in both retina and pineal gland. Expression of retinal homeobox (Rx) is an early marker for the developing retina and pineal gland and is required for eye vesicle morphogenesis (1214). We cloned the amphioxus rx gene (Fig. S1 for the phylogenetic tree) and determined its expression in the developing cerebral vesicle. We found that rx demarcates the anterior end of the cerebral vesicle from the 24-hours postfertilization (hpf) stage onwards (Fig. 1 AC). This location is where the frontal eye will start to differentiate at later stages, as evidenced by the presence of cells with long dendrites and cilia that exit the neuropore (15) and by the presence of a spot of dark pigment (Fig. 1 D and E). However, rx expression has not been detected in the area of the lamellar body (Fig. 1 D and F), the previously proposed homolog of the vertebrate pineal gland (16). During differentiation stages rx expression becomes restricted to the cells lying behind the most anterior tip of the cerebral vesicle.

Fig. 1.
Developmental expression of amphioxus rx. (A) The earliest trace of rx expression was detected in late neurula (24 hpf) in the anterior part of the cerebral vesicle (arrowhead). (B) In early larva (30 hpf), the expression becomes stronger and demarcates ...

To determine further whether any of the differentiating cells of amphioxus frontal eye would resemble the vertebrate photoreceptors molecularly, we produced antibodies against the amphioxus Otx, Pax4/6, and Rx transcription factors (Table S1). The antibodies showed the ability to recognize their respective antigens (Fig. S2 AC and Fig. S3), recapitulated the RNA in situ expression patterns, and provided robust signal clearly distinguishable from nonspecific epidermal signal that we attribute to endogenous GFP expression (17) and secondary antibody trapping (Fig. 2E).

Fig. 2.
Expression of otx, pax4/6, and rx in the amphioxus cerebral vesicle. (A) Overview of the anterior part of a larva at the 2.5–gill-slit stage typically used in this study. (B) Detailed view of the most anterior part of the larva (defined by red ...

Expression of the single amphioxus otx and pax4/6 orthologs has been detected previously in the anterior portion of the amphioxus cerebral vesicle (18, 19), but whole-mount RNA in situ hybridization analysis has not provided cellular resolution. Fluorescent confocal immunohistochemistry of amphioxus larvae with antibodies directed against amphioxus Otx and Pax4/6 proteins revealed colabeling of a single row of cells in the very anterior of the frontal eye (Fig. 2 CF), termed “Row 1” by Lacalli et al (15). These cells are adjacent to the cells containing the dark pigment and thus are the most likely candidates for photoreceptor cells (15). Pax4/6 protein expression also was detected more posteriorly in cells scattered in the floor of the cerebral vesicle (Fig. 2 F) in a pattern similar to that of Rx (Fig. 2 GI and Fig. S4). Interestingly, in addition to the differentiated cells bearing the apical extension, a small subset of Rx-positive cells buried deeper in the cerebral vesicle floor retained a rounded shape (Fig. 2H), suggesting a possible undifferentiated state.

Row1 Cells of the Frontal Eye Express C-Opsin Genes and the Gi-Alpha Protein Subunit.

To challenge the possible photosensitive nature of Row1 cells in the amphioxus frontal eye (15), we set out to identify cells expressing the amphioxus c-opsin genes. Sixteen opsins have been detected in the amphioxus genome, four of which are related to the vertebrate rod, cone, and pineal opsins (20, 21). Phylogenetic analysis revealed that ancestral chordates possessed one c-opsin gene that by repeated and independent duplications gave rise to four paralogs in amphioxus and to numerous paralogs in the vertebrate lineage (20). We could not detect expression of any of the amphioxus c-opsin genes by RNA whole-mount in situ hybridization and subsequent RT-PCR analysis revealed a low mRNA expression level of these opsins, suggesting a low mRNA expression level; therefore we produced antibodies against all four c-opsin proteins (Table S1). Antibody staining indeed revealed specific expression of c-opsin1 and c-opsin3 in the Row1 (15, 22) cells of the amphioxus frontal eye (Fig. 3 A and B). The specificity of each antibody was confirmed by the loss of specific signal after preadsorption with the respective antigen (Fig. S3). We further noted that the c-opsin1 and c-opsin3 antibodies labeled morphologically distinct cells within Row 1 (compare Fig. 3 A and B and Fig. S5), consistent with possible differential responses to distinct wavelengths. None of the other rows of the frontal eye was positive for any of the other c-opsins. To characterize phototransduction in the amphioxus frontal eye further, we cloned the proteins of the amphioxus G-alpha subunit see Fig. S6 for the phylogenetic tree). The proteins of the G-alpha subunit are specific for distinct phototransductory cascades in vertebrates and invertebrates (23). In vertebrate rods and cones, transducin signals to phosphodiesterase that hydrolyses cGMP and shuts down the dark current, mediating an “off response” to light (23). The activity of such phosphodiesterase is stimulated by transducins, which arose by gene duplication of a more ancestral Gi gene encoding the inhibitory Gi-alpha subunit (23). Because the amphioxus genome predates the duplication events that generated transducins later in evolution, we investigated the expression of their more ancestral counterpart Gi. The only amphioxus Gi gene that we found is expressed in the most anterior cells of the amphioxus frontal eye (Fig. 3C). We also investigated expression of the Go-alpha subunit, which is active in the photoreceptors of vertebrate pineal eyes (11) and in the ciliary photoreceptors of mollusks (24). The amphioxus Go gene is expressed more broadly in the amphioxus cerebral vesicle (Fig. 3D); however, the most anterior portion of the frontal eye appeared to be specifically excluded from the Go expression domain, indicating that it is unlikely that the Row1 cells also signal via the Go-alpha subunit. Notably, the Gq-alpha subunit characteristic for invertebrate rhabdomeric photoreceptor cells has not been detected in the amphioxus frontal eye (6). This result suggests that the Row1 cells of the frontal eye couple to an inhibitory G-alpha subunit protein mediating the off response.

Fig. 3.
Expression of amphioxus c-opsins and G-alpha subunits in the cerebral vesicle. Amphioxus larvae were stained with mouse polyclonal sera raised against the C-terminal portion of amphioxus c-opsins. For easy orientation, the bright-field (BF) images of ...

Pigmented Cells of the Amphioxus Frontal Eye.

Cells of the vertebrate retinal pigmented epithelium (RPE) are specified by the Mitf transcription factor and use melanin as a shading pigment (25). We investigated expression of amphioxus mitf within the cerebral vesicle and found it restricted to the pigment cells of the frontal eye (Fig. 4 A and B). In addition, the vertebrate Otx2 paralog acts in tight cooperation with Mitf during RPE development and differentiation (26). This might also be the case in amphioxus, where mitf is expressed concomitantly with otx in the pigment cells of the frontal eye (Fig. 2 DF). Furthermore, we exposed developing amphioxus embryos and larvae to phenylthiourea (PTU), a specific inhibitor of melanin synthesis causing the absence of melanin pigment in the vertebrate eye (27). After PTU exposure, the dark pigment of the frontal eye was abolished completely (Fig. 4 CE), indicating that melanin is indeed the only dark pigment of the frontal eye.

Fig. 4.
Characterization of the frontal eye pigment cells. The Lower panels in A and B include the brightfield channel to visualize the pigment spot. (A) The specificity of Mitf antibody has been confirmed by specific nuclear staining (yellow arrowhead) in the ...

Projections.

Finally, we analyzed axonal projections from frontal eye cells. Previous transmission electronic microscopy studies revealed only short projections to the laterally adjacent frontal nerves (22). We took advantage of the serotoninergic nature of the Row2 cells of frontal eye, which are in direct contact with the Row1 cells (22, 28), and detected long basal axonal projections from the frontal eye Row2 cells toward the posterior cerebral vesicle (Fig. 5 A and B), where we observed massive serotonin varicosities within the tegmental neuropil (22).

Fig. 5.
The projections of the frontal eye region. (A) Serotonergic Row2 cells project axons to the neuropil (dashed outline), where the axons terminate by many varicosities (white arrowheads). A few varicosities are also encountered along the axons (yellow arrows). ...

Discussion

The thorough immunohistochemical study presented in this work defines, at least in part, a molecular fingerprint for the amphioxus frontal eye at cellular resolution (summarized in Fig. 5C) and thus provides important insight into the evolutionary origin of the vertebrate eyes. The expression profile of vertebrate eye-specific regulatory (Rx, Otx, Pax4/6, and Mitf) genes and differentiation markers (c-opsins, Gi, melanin) in the amphioxus cerebral vesicle strongly supports the homology of photoreceptor and pigmented cells of the amphioxus frontal eye and the corresponding cell types in the vertebrate retina and retinal pigmented epithelium, respectively.

Regulatory Signature.

The early developmental patterning of the amphioxus frontal eye is performed by the same set of transcription factors (Otx, Rx, and Pax4/6) as the vertebrate retina. In vertebrates, Otx2 controls the development of both the retina and the pineal gland (29) and another vertebrate paralogue of Otx, cone rod homeobox (Crx) transcription factor, is crucial for the terminal differentiation of the rods and cones (30). Likewise, vertebrate Pax6 is necessary for proper eye development (31, 32), and the expression of pax4 is characteristic for differentiated rods and cones (33, 34). During later stages, otx and pax4/6 remain expressed in the differentiated Row1 photoreceptors, albeit at lower levels that suffice for maintenance of the differentiated state of the given cell type. This situation exemplifies the division of labor of the chordate single-copy orthologs (such as amphioxus pax4/6 and otx) after gene duplication in the vertebrates, where Pax6 and Otx2 are active during early eye/pineal gland development, and their paralogs Pax4 and Crx act during the terminal differentiation stages. Although the transient expression of rx in the precursors of ciliary photoreceptors (Fig. 1B) seems to be evolutionarily conserved (35), the absence of rx in the differentiated photoreceptors of Row1 cells contrasts with its expression in vertebrate differentiated photoreceptors and its involvement in the regulation of phototransduction genes (36). The overlapping expression of rx and ci-opsin1 in the ciliary photoreceptor of tunicates (37, 38) suggests either the acquisition of rx for the direct regulation of photoreceptor genes such as opsins at the base of Olfactores or amphioxus-specific loss of rx role for maintaining the differentiated ciliary photoreceptor program. The small population of rx-positive cells lacking the apical cilium might represent, as in vertebrates (3941), a progenitor subpopulation needed for further growth of the frontal eye later in development. The absence of expression of otx and rx in the lamellar organ challenges its proposed homology with the vertebrate pineal gland, but the currently available data are too sparse to allow any conclusions. To resolve this issue, further molecular characterization of the lamellar organ will be rewarding.

Evolutionary Precursor of the Vertebrate Phototransduction Cascade.

The expression of two ciliary opsins and the proteins of the Gi-alpha subunit in the amphioxus frontal eye provides molecular evidence of the photosensory nature of Row1 cells and corroborates the homology of these amphioxus cells and vertebrate rods, cones, and/or pinealocytes. Because c-opsin and Gi-alpha also are coexpressed in tunicate ciliary photoreceptors (38, 42), the amphioxus frontal eye photoreceptors using ciliary opsin coupled to Gi-alpha represent the ancestral chordate condition and an evolutionary forerunner of more sophisticated vertebrate visual photoreceptors. In early vertebrate evolution the two rounds of genome duplication giving rise to the vertebrate visual opsin subclass (by duplicating ancestral chordate ciliary opsin genes) and a new subclass of Gi-derived Gt-alpha protein subunits provided enough genetic material to allow biochemical evolution (43) to provide the highly efficient phototransductory system operating in today’s vertebrate rods and cones.

Frontal Eye Pigment Cells Are Homologous to the Cells of the Vertebrate RPE.

Unlike previous observations based on morphological and chemical properties (44, 45), our data provide biochemical evidence that the only shielding pigment of amphioxus pigment cells is melanin. The molecular fingerprint of the amphioxus frontal eye pigment cells, expressing mitf, otx, and pax2/5/8 (46, 47), resembles the fingerprint of the vertebrate retinal pigmented epithelium (48). In both amphioxus and vertebrates, the pigmented cells are located directly adjacent to the ciliary photoreceptor cells (Row1 cells in amphioxus and rods and cones in the vertebrates), further corroborating the homology of amphioxus and vertebrate eyes.

Neural Circuitry of the Frontal Eye.

The Row2 cells projecting axons to the tegmental neuropil provide further evidence for the homology of the frontal eye and the vertebrate retina. As projection neurons, Row2 cells would correspond to retinal ganglion cells [and to horizontal and amacrine cells, the presumed sister cell types of the ganglion cells (7)]. However, more information about Row2-specific expression will be needed to substantiate this issue and to test for any relationship to the rhabdomeric photoreceptor lineage (7). The tegmental neuropil has been compared with locomotor control regions of the vertebrate hypothalamus, where paracrine release modulates locomotor patterns such as feeding and swimming (49). Consistent with this idea, FoxD (50) and Brn1/2/4 (51), whose vertebrate orthologs are expressed in the hypothalamus, also are expressed in the tissue apposed the tegmental neuropil in amphioxus. In addition, the retinohypothalamic tract in vertebrates projects to the anterior hypothalamic area involved in the control of basic behaviors (52) and also the pineal fibers connect bilaterally to the rostral hypothalamus (53). The amphioxus tegmental neuropil in the posterior cerebral vesicle receives not only projections from the frontal eye area but also bilateral afferents from the giant cells located in the primary motor center (PMC) (49). The PMC, positioned immediately beyond the caudal end of the cerebral vesicle, likewise is connected to the frontal eye region via an asymmetrical lateral dendrite of one of the above-mentioned giant cells (49). Given that the PMC cells lie in the gbx expression region (54), and some of them also are positive for pax4/6 (Fig. 2F), their molecular identity resembles that of the vertebrate interpeduncular nucleus located in the hindbrain and involved in locomotor control (55). Taken together, these findings indicate that in amphioxus the frontal eye projects to the neurosecretory/tegmental neuropil and to the locomotor center, like the eye and the pineal gland in vertebrates.

The study highlights the advantage of cellular resolution, gene coexpression, and structural analyses using molecular markers to define the neuronal circuitry in the amphioxus cerebral vesicle. Our data reveal direct innervation and indicate paracrine release of serotonin from Row2 cells in the tegmental neuropil in the posterior cerebral vesicle, reminiscent of retinohypothalamic projections in the vertebrates. Also, the frontal eye directly innervates locomotor control regions in the primary motor center, again reminiscent of more complex vertebrate circuits of the retina and pineal. The amphioxus frontal eye circuit thus represents a very simple precursor circuit that, by expansion, duplication, and divergence, might have given rise to photosensory-locomotor circuits as found in the extant vertebrate brain.

Experimental Procedures

Animals.

Branchiostoma floridae larvae were obtained in Tampa Bay (Florida), during the spawning season in August 2010. At the 2.5–gill-slit stage, the animals were fixed with 3-(N-morpholino)propanesulfonic acid (MOPS) fixative (0.1 M MOPS, 2 mM MgSO4, 1 mM EGTA, 0.5M NaCl, pH 7.5) for 30 min at room temperature and then were transferred to 100% methanol. Larvae for RNA in situ hybridization were kindly provided by Linda Z. Holland (Scripps Institution of Oceanography, University of California at San Diego, La Jolla, CA). B. lanceolatum larvae for the PTU experiment were provided by the amphioxus facility of the Arendt group, European Molecular Biology Laboratory, Heidelberg.

Immunohistochemistry.

If not otherwise stated, all incubation steps were carried out at room temperature. Specimens were transferred to 1× PBS, 0.1% (vol/vol) Tween 20 (PBT) through 50% (vol/vol) and 25% (vol/vol) methanol in PBS. Specimens were washed three times (20 min each washing) in PBT, blocked in block solution [10% (wt/wt) BSA in PBS] for 1 h, and incubated with preadsorbed sera (dilutions are given in Table S1) overnight at 4 °C. On the next day, specimens were washed three or four times in PBT (20 min each washing) and were incubated with secondary antibodies for 2 h. Secondary antibodies were washed away with three washings in PBT (20 min each washing). Nuclear counterstaining was carried out by incubation with 1 μg/mL DAPI in PBS and washing three times (5 min each washing). For FM4-64FX (Invitrogen) staining, the larvae were incubated in the dye (10 μg/mL) for 10 min in PBS and were washed twice with PBS.

For fluorescence/confocal microscopy, the specimens were mounted in VECTASHIELD (Vector Laboratories, Inc.) using small coverslips as spacers between the coverslip and the slide. The confocal images were taken using a Leica SP5 confocal microscope and were processed (contrast, brightness, and histogram adjustment) with FIJI free image analysis software (http://fiji.sc).

RNA in Situ Hybridization.

Whole-mount RNA in situ hybridization to amphioxus larvae was performed according to a standard protocol (56). The only modification was the omission of levamisole in washes on day 3 (step vi).

PTU Treatment.

B. lanceolatum embryos raised at 20 °C were treated in the dark with 0.22 mM PTU (stock solution 100 mM PTU in 96% EtOH) either from the 18-hpf stage onwards or from the 42-hpf stage onwards. Ethanol at the same dilution was used as a negative control. The drug was changed every 24 h. At 66 hpf the specimens were fixed and documented.

Supplementary Material

Supporting Information:

Acknowledgments

We thank Nicholas and Linda Holland for providing fixed animals for whole-mount in situ hybridization and help with RNA in situ hybridization, Jitka Lachova for immunization of mice, and Cestmir Vlcek for excellent help in sequencing. This study was supported by the Grant Agency of the Czech Republic Grants P305/10/2141 and P305/10/J064 (to Z.K.) and DFG AR 387/2-1 (to D.A.) and the Grant Agency of the Charles University, Grant GAUK 91808 (to P.V.) and by IMG institutional support RVO 68378050.

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