Vertebrate features revealed in the rudimentary eye of the Pacific hagfish (Eptatretus stoutii)

Hagfish eyes are markedly basic compared to the eyes of other vertebrates, lacking a pigmented epithelium, a lens, and a retinal architecture built of three cell layers – the photoreceptors, interneurons & ganglion cells. Concomitant with hagfish belonging to the earliest-branching vertebrate group (the jawless Agnathans), this lack of derived characters has prompted competing interpretations that hagfish eyes represent either a transitional form in the early evolution of vertebrate vision, or a regression from a previously elaborate organ. Here we show the hagfish retina is not extensively degenerating during its ontogeny, but instead grows throughout life via a recognizable Pax6+ ciliary marginal zone. The retina has a distinct layer of photoreceptor cells that appear to homogeneously express a single opsin of the rh1 rod opsin class. The epithelium that encompasses these photoreceptors is striking because it lacks the melanin pigment that is universally associated with animal vision; notwithstanding, we suggest this epithelium is a homolog of gnathosome Retinal Pigment Epithelium (RPE) based on its robust expression of RPE65 and its engulfment of photoreceptor outer segments. We infer that the hagfish retina is not entirely rudimentary in its wiring, despite lacking a morphologically distinct layer of interneurons: multiple populations of cells exist in the hagfish inner retina that differentially express markers of vertebrate retinal interneurons. Overall, these data clarify Agnathan retinal homologies, reveal characters that now appear to be ubiquitous across the eyes of vertebrates, and refine interpretations of early vertebrate visual system evolution. HIGHLIGHTS Hagfish eyes are degenerate but not degenerating, i.e. rudimentary but growing Retinal interneurons discovered implying ancestral hagfish had derived retinas & vision Despite lacking pigment, a Retinal Pigmented Epithelium homolog functions in hagfish Revised synapomorphies illuminate the dimly lit origins of vertebrate eye evolution GRAPHICAL ABSTRACT

has prompted competing interpretations that hagfish eyes represent either a transitional 5 form in the early evolution of vertebrate vision, or a regression from a previously elaborate 6 organ. Here we show the hagfish retina is not extensively degenerating during its ontogeny, 7 but instead grows throughout life via a recognizable Pax6+ ciliary marginal zone. The retina 8 has a distinct layer of photoreceptor cells that appear to homogeneously express a single 9 opsin of the rh1 rod opsin class. The epithelium that encompasses these photoreceptors is 10 striking because it lacks the melanin pigment that is universally associated with animal vision; 11 notwithstanding, we suggest this epithelium is a homolog of gnathosome Retinal Pigment 12 Epithelium (RPE) based on its robust expression of RPE65 and its engulfment of 13 photoreceptor outer segments. We infer that the hagfish retina is not entirely rudimentary in 14 its wiring, despite lacking a morphologically distinct layer of interneurons: multiple 15 populations of cells exist in the hagfish inner retina that differentially express markers of 16 vertebrate retinal interneurons. Overall, these data clarify Agnathan retinal homologies, 17 reveal characters that now appear to be ubiquitous across the eyes of vertebrates, and refine 18 interpretations of early Vertebrate eyes are remarkably conserved and complex, appearing early in the 25 evolution of this group as the familiar single-chambered camera-style organ with advanced 26 optics at the anterior, and photoreceptors and retinal pigmented epithelium (RPE) lining the 27 posterior of the eye's globe. Across all vertebrate taxa, this morphology deviates little despite 28 diverse visual ecologies and is easily recognizable even in early-branching vertebrates 29 including the jawless lamprey. Homology of the eye across vertebrates is evident across 30 various levels -anatomy, development, neural architecture, synaptic wiring, visual cell 31 physiology, and molecular markers (Chow and Lang, 2001;Walls, 1942;Zuber, 2003). The eye 32 is key to the success of vertebrates, yet its evolutionary origins have remained obscured. information through their axons (the optic nerve) to the higher visual centres of the brain. A 43 richly melanized RPE lines the back of the eye chamber, enveloping the photoreceptors, that 44 functions to block stray photons and provide critical support for photoreceptor physiology 45 (Strauss, 2005). 46 It is difficult to overstate (or briefly summarize) the impressive degree of conservation 47 across vertebrate retinas at various levels of eye organization. While celebrated exceptions 48 exist, such as rod-rich deep sea tube eyes (Collin et al. 1997) and four-eye fish (Borwein and 49 Hollenberg, 1973), this vertebrate ocular bauplan is evident in lamprey, and thus has been 50 retained since early vertebrate evolution prior to the advent of jaws or paired fins (limbs). 51 Hagfish are amongst the very rare exceptions to this canonical eye description in 52 vertebrates and are particularly noteworthy because they populate an early branch on the 53 vertebrate tree. Hagfish eyes, across two genera, are thus remarkable compared to all other 54 vertebrates for the ocular characters they (seem to) lack, apparently concomitant with 55 hagfish being positioned at the base of the vertebrate tree. Hagfish eyes are small, 56 unpigmented and lack diagnostic jawed vertebrate features such as a duplex retina (with both  57 rods & cones), lens, cornea and extraocular musculature (Locket andJorgensen 1998, Lamb et 58 al., 2007). The eye-cup is masked by semi-transparent or non-transparent overlying 59 epidermis, the transparency of the skin varies across species (Fernholm and Holmberg, 1975). 60 In some species, such as the Atlantic hagfish (Myxine glutinosa), the eye is also buried under 61 body wall muscle (Fernholm, 1974). Though a lens and ocular musculature are absent, 62 photoreceptors are present, as are retinal ganglion cells (Holmberg, 1971;1970). Moreover, 63 the hagfish neural retina lacks lamination into three nuclear layers, which is suggestive of a 64 pineal-like architecture lacking interneurons, and of a simpler photoreceptive organ more  65  suitable for detecting circadian light rhythms than for resolving images or vision.  66   The phylogenetic position and starkly rudimentary eye in hagfish have historically  67 positioned them as a transitional form in early vertebrate visual system evolution; though an 68 alternative view is that hagfish eyes may be rudimentary due to loss of characters. 69 Delineating between these two explanations would fill a substantial gap in understanding 70 how the vertebrate eye and its 'inimitable contrivances' evolved. 71 Here, we revisit the rudimentary hagfish retina to better appreciate the earliest steps 72 in the history of vertebrate visual system evolution. Our objective was to explore retinal 73 structure and identify cell types by molecular markers in the hagfish with a focus on Pacific 74 hagfish (Eptatretus stouii) because its visual system seems somewhat less 75 degenerate/rudimentary, compared to other species where the eyes are concealed by muscle 76 (Fernholm and Holmberg, 1975;Jørgensen et al., 1998

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The Pacific hagfish retina is simple and unpigmented 88 The eyes of Pacific hagfish (E. stoutii) are found beneath a layer of translucent skin (Fig. 1A).

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They are small, embedded in the surrounding craniofacial muscle, and lack extra-ocular 90 musculature attachments (Fig. 1B,C). Most notably, the eyes completely lack pigmentation 91 (Fig 1C,D). No lens is apparent, though the vitreous of the eye is protein-dense, evident by the 92 high degree of hematoxylin staining (Fig. 1D). In accordance with an absent lens, no 93 intraocular musculature is identifiable within the eye. 94 In cross section, the typical vertebrate neural retina (exemplified here by zebrafish) is 95 stratified into three distinct cellular layers (ONL, INL GCL) divided by two synaptic layers (IPL 96 ,OPL) (Fig 1E). In contrast, the retina of E. stoutii is not morphologically separated into this 97 same layered organization (Fig 1F). While the precise identities of cells across the width of the 98 retina are a subject of the current investigation, photoreceptors are found at the scleral-most 99 region, while presumptive retinal ganglion cells are found basal to this, towards the vitreous 100 (Fig. 1F). Overall, the Pacific hagfish eye is rudimentary compared to exemplar eyes of other 101 vertebrate taxa (see Introduction), consistent with past reports in various hagfish species 102 (Fernholm and Holmberg, 1975;Lamb et al., 2007;Locket and Jørgensen, 1998). 103 to degenerating eyes of other vertebrates adapted to dim-light environs (e.g. cavefish in 108 which the eye diminishes significantly), we measured eye size in Pacific hagfish caught off the 109 West coast of Canada. Because the hatching and raising of hagfish embryos for experimental 110 purposes is rarely attainable, we collected wild-caught individuals and used fish size as a 111 proxy for age. The eye was found to increase in both length and mass during ontogeny, and 112 over a broad range of fish sizes (Fig. 2F- shaped nuclei distinct from other retinal cells in the central areas of the retina ( Fig. 2A). A 124 gradient of more elaborate cell morphologies is observed closer to the center of the retina, 125 with increasing resemblance to fully differentiated cells; this gradient is reminiscent of 126 observing more mature central retina in teleost fish ( Fig. 2A'-A''). The simpler retinal 127 neuroepithelium at the margins is observed to merge with a more distal (scleral) epithelium 128 (the presumptive RPE, described below). This merged retinal margin is positioned at the edge 129 of the eye cup, comparable to the gnathostome CMZ where it would be contiguous with 130 tissue forming the pupil. 131 In gnathostomes, the CMZ expresses PAX6 (Fischer et al., 2014). An ortholog of PAX6 132 highly conserved with that of other vertebrates, was found to be expressed in the hagfish 133 retina. PAX6 expression is most concentrated at the tip of the retinal marginal zone ( Fig. 2B-134 E), suggesting that not only do these cells appear undifferentiated by morphology, but that 135 they also express transcripts related to the maintenance of a multipotent cell state (Fischer et 136 al., 2014;Marquardt et al., 2001). 137

Interneurons are found despite the lack of vertebrate-typical retinal layering 138
Apart from photoreceptors and retinal ganglion cells, the cell identities within the hagfish 139 retina have remained obscured and a segregated interneuron layer in hagfish is not obvious. 140 This simple retinal composition noted in various hagfish species is interpreted by some to 141 reflect a primitive neural architecture more reminiscent of a the two-layered morphology and 142 physiology of the pineal organ (Lamb et al., 2007). We observed that the inner retinal cells 143 below the photoreceptor layer in Pacific hagfish are a diverse mix of cells that possess nuclei 144 that are not homogenous in shape or size, with diverse heterochromatin patterning (Supp. Fig  145  2). We hypothesized that additional cell types aside from retinal ganglion cells populate this 146 As a separate challenge to the hypothesis that hagfish retinas have a simple neural 155 architecture and lack interneurons, we characterized the distribution of the synapses. We 156 observed immunoreactivity for synapse marker SV2 (synaptic vesicle protein 2) to localize into 157 two layers of hagfish retina (Fig. 3C, Supp. Fig 3), one being associated with the basal end of 158 photoreceptors, and a second layer intermingled with nuclei closer to the vitreous. Staining of 159 F-actin (enriched in synapses across diverse animals) using fluorescently-labelled-phalloidin 160 also supported the existence of multiple synaptic layers (Supp. Fig 3). Though less precisely 161 organized, this synaptic architecture is reminiscent of jawed vertebrate retinas, where two 162 distinct plexiform layers demarcate the presence of interneurons connecting the 163 photoreceptor and ganglion cells. In jawed vertebrates, the retina is sustained by the retinal pigment epithelium (RPE), a 182 monostratified epithelium with dense melanin, located between the photoreceptor outer 183 segments and the choroid (a network of vasculature that supports the outer retina). This vital 184 partnership between the neural retina and RPE is unique to vertebrates. 185 We sought to determine if hagfish photoreceptors, which appear morphologically rod-186 like (Fig. 4E) Fig. 4). 198 In situ hybridization of hagfish RH1 using RNA probes designed from transcriptome 199 derived sequences showed highly specific staining in the photoreceptors in close association 200 with the nuclei (Fig. 4A-C). RH1 appears to be expressed in all photoreceptors, making the 201 presence of other ciliary photoreceptor types unlikely. Additionally, the photoreceptor outer 202 segments are readily immunolabelled by ZPR-3 (zebrafish rod opsin antibody) and 1D4 203 (monoclonal antibody raised against bovine rhodopsin C-terminus) and 4D2 (monoclonal 204 antibody raised against bovine RH1 N-terminus) (STAR methods, Fig. 4 D-E). The protein 205 exhibiting rod-opsin-immunoreactivity was localized to cellular compartments located distally 206 (scleral side) of the RH1 transcript localization, i.e. in the photoreceptor outer segments. This 207 location was also noted as being immediately apical of an actin-rich retinal strip (Supp. shown that these cells possess microvilli on their basal surface in close association with 215 nearby photoreceptor outer segments (Fernholm and Holmberg, 1975;Holmberg, 1971). This 216 tissue has a similar location and organization to the retinal pigment epithelium (RPE) found in 217 other vertebrates, but is strikingly disparate (even macroscopically, Fig. 1, S1) due to its 218 complete lack of melanin pigment. Melanin pigment found in the RPE of vertebrates is key to 219 sustaining healthy function in the eye. Curiously, though the eye is unpigmented, we have 220 found via RNA-Seq that pigment-related genes are expressed in the eye, including tyrosinase 221 (necessary for melanin synthesis) and PMEL (pre-melanosome protein necessary for stable 222 melanin deposition in the melanosome) (Fig. 5A). Thus, we have termed this non-pigmented 223 RPE equivalent the "Retinal (non)Pigment Epithelium" or RnPE. 224 A major function of the RPE is to maintain the integrity of the photoreceptor outer 225 segments by phagocytosing shed photoreceptor discs. This process renews the 226 photoreceptors and in part prevents oxidative stress. As previously documented (Holmberg,227 1971), we have found ultrastructural evidence of phagosome-like organelles in the hagfish 228 RnPE, which contain what appears to be pieces of photoreceptor outer segments (Fig. 5C). 229 The RPE also serves a highly necessary role in retinoid cycling -specifically, the production of 230 11-cis retinal, a chromophore associated with visual opsins that is required for visual function. 231 Phototransduction cannot be sustained without the renewal of 11-cis retinal by the RPE. binding protein (IRBP) are also involved in the transport of highly hydrophobic retinoids 238 between the RPE and photoreceptors (Strauss, 2005). Analysis of our transcriptome revealed 239 that homologs of RPE65, LRAT, RDH5, CRALBP and CRBP are expressed in the hagfish eye ( Fig.  240  5A). IRBP could not be identified. Further, we found that the expression of RPE65 is highly 241 specific and robustly expressed within all cells of the RnPE (Fig. 5B). In sum, the presence of 242 phagosomes, the interdigitiation with polarized ciliary photoreceptors, the presence of 243 melanin synthesis machinery, and the localized expression of conserved markers of RPE cells 244 support the RnPE being a homolog of gnathostome RPE that was pigmented in the last 245 common ancestor of hagfish and jawed vertebrates. 246  It is less parsimonious to speculate these striking similarities arose via convergent evolution, 294 and so the last common ancestor of hagfish, lampreys and gnathostomes most likely had a 295 retina with neural architecture (and concomitant visual ability) similar to modern jawed 296 vertebrates. At least in this aspect, the hagfish retina appears to have regressed over 297 evolutionary time (perhaps associated with its deep-sea dim light habitats). 298 Hagfish photoreceptors appear simpler than those of other vertebrates, found either 299 as a whorled structure in Myxine species, or shortened and non-tapered in Eptatretus species 300 (Holmberg, 1971 Our work elevates and expands upon studies characterizing hagfish photoreceptors as rods by 310 their rod-typical spectral sensitivity, and electrophysiological response to low light stimuli 311 (Kobayashi, 1964;Steven, 1955). 312 The RPE provides a number of services to the photoreceptors including retinoid 313 cycling, nutrient and metabolite exchange between vasculature and the retina, and 314 phagocytosis of sloughed photoreceptor outer segments. These two interdigitated tissues co-315 exist in a highly intertwined relationship which, when disrupted, results in malfunction and 316 disease (Strauss, 2005 should strive to identify lingering cone networks, if any exist. Leveraging genomic studies will 380 be particularly necessary in interrogating the full extent of opsin gene loss. 381 The absence of pigment is one of the most striking features in the hagfish eye. Fossil 382 data for hagfish suggests that the eyes of at least one basal hagfish species possessed 383 pigment implying a pattern of regression in extant species (Gabbott et al., 2016). Oddly, 384 though melanin is lost from the RPE, the machinery for producing melanin-rich tissues is 385 obviously intact and functional in hagfish skin (except overlying the eye). Further, we have 386 shown that the retina expresses pigment related transcripts including PMEL (required for 387 melanin deposition) and tyrosinase (necessary for melanin synthesis) (Julien et al., 2007; 388 Oetting and King, 1999). Melanin loss in the RnPE may have had an additional accelerating 389 role in the degeneration of other eye tissues by leaving them prone to bleaching and 390 oxidative stress which may explain the small and sparse photoreceptors of hagfish (Lamb,391 2013). In models of albinism, eye function is seriously impacted in connection with an 392 absence of melanin (Schraermeyer et al., 2006). 393 Regression of visual cycle machinery at the RPE could also account for the simple 394 complement and small size of hagfish photoreceptors-for example IRBP was the only retinoid 395 cycling constituent not found in the hagfish transcriptome, though it remains to be 396 determined if it is lost from the genome. IRBP is a conserved carrier protein that transports 397 retinoids between photoreceptors and RPE and its loss in mice IRBP slows, but does not 398 eliminate, the recovery of photosensitivity after photobleaching, suggesting that it may not 399 be necessary for retinoid cycling, though it does facilitate a faster restoration of 400 photoreceptor sensitivity (Palczewski et al., 1999;Ripps et al., 2000). Interestingly, IRBP -/-401 mice have shortened photoreceptors (Ripps et al., 2000), a feature also seen in hagfish. 402 403

Conclusions and limitations 404
We document that at least one species of extant Hagfish possesses several previously hidden 405 eye synapomorphies; this clarifies the evolutionary origins of the vertebrate eye. Thus, the 406 last common ancestor of agnathans and gnathostomes is inferred to have exhibited the 407 following key characters for vertebrate expansion into diverse photic niches: i) continued 408 retinal growth late into ontogeny supported by a stem cell niche at the retinal margin; ii) a 409 pigmented RPE supporting a forest of ciliary visual photoreceptors; iii) a complex retinal 410 wiring able to compute and decant the photoreceptor outputs into a representation of the 411 visual scene. 412 As we do not yet have access to embryos of this species, we cannot know if changes to 413 development are responsible for the absence of lens, pigment, and/or cone photoreceptors in 414 adults. It is additionally possible that regression over evolutionary time has eliminated these 415 structures altogether. Other species of hagfish may give us greater insight into the 416 evolutionary history of degeneration in this lineage. For example, the Atlantic hagfish which 417 belongs to a different genus (Myxine), is in some ways even more rudimentary than that of 418 the E. stoutii and could provide additional perspective on character absence (Fernholm and 419 Holmberg, 1975). Further work is warranted in appreciating the evolutionary and 420 developmental history of visual photoreceptors of hagfish: particularly as they stand in 421 contrast to the cones and cone-like rods found in several species of lamprey (Dickson and 422 Graves, 1979; Morshedian and Fain, 2015). 423 The results of this study reiterate the great potential for photoreceptive function in 424 hagfish retinas and strengthen the argument for a common origin of vertebrate eyes from a 425 single ancestor that predated the divergence of the agnathans from the gnathostomes.

Sequence homology searching, transcript alignment and tree making 572
Following de novo transcriptome assembly, transcript annotation with 7 functional databases 573 was performed (NR, NT, GO, COG, KEGG, Swissprot, Interpro). To confirm annotations of 574 transcripts of interest, known protein coding sequences from a representative cross section 575 of vertebrates (including zebrafish, mouse, chicken, and frog) were used to search the hagfish 576 eye transcriptome using BLAST 2.6.0 (Altschul et al., 1997) and HMMER 3.1b1 (Eddy, 2011). 577 The top three highly matched sequences were retained, and aligned to homologs using 578 MAFFT under default settings (Katoh et al., 2002;Pearson, 2013). For genes that are part of 579 large and closely related protein families, transcript identity was further confirmed by 580 phylogenetic analysis conducted using IQ-TREE (Nguyen et al., 2015). Branch support was 581 determined using ultrafast bootstrap (1000 bootstrap replicates) (