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. 2026 Sep 28;534(10):e70206. doi: 10.1002/cne.70206

Patterns of Proliferation in the Pacific Hagfish (Eptatretus stoutii) Retina Suggest an Ancient Origin for Adult Neurogenesis

Sarah N Bradshaw 1, William T Allison 1,✉
PMCID: PMC13618028  PMID: 42803506

ABSTRACT

Comparative morphology has revealed fascinating structural and organizational differences in the eyes of cyclostomes (jawless vertebrates) compared to gnathostomes (jawed vertebrates). This work has helped us to better understand the evolutionary origin of the vertebrate retina, but certain questions remain unresolved. Here we complement that approach by examining retinal development. We sought to characterize post‐larval (“adult”) retinogenesis in a representative cyclostome, the Pacific hagfish (Eptatretus stoutii). Knowledge of hagfish neurodevelopment is limited by poor access to embryos, but evidence suggests continued retinal neurogenesis occurs late into their ontogeny. A brief pulse of EdU was used to detect proliferating cells, if any, in the retina. This was followed by in situ hybridization to test whether homologs of gnathostome genes might have conserved expression patterns that direct retinogenesis in a jawless vertebrate. EdU+ cells were detected at the retinal periphery of the hagfish (a region reminiscent of the ciliary marginal zone of gnathostomes) and within the central retina. In addition, hagfish eyes were found to express several key genes required for vertebrate retinal neurogenesis. At least two of these genes, OtxA and Rx (retinal homeobox), were enriched within the proliferative retinal periphery. These findings support adult retinogenesis as an ancient and deeply conserved trait within the vertebrate lineage.

Keywords: agnatha, ciliary marginal zone, evo‐devo, visual system development

The hagfish, a jawless early‐branching vertebrate, has very rudimentary eyes that are diminutive and oddly devoid of melanin pigment. Yet, the degenerate hagfish eye grows throughout life and expresses canonical genes of vertebrate retinal development.

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1. Introduction

The vertebrate eye and retina are highly conserved sensory structures that are remarkable for their morphological complexity, their capacity for image formation, and their mysterious evolutionary origins. Paired eyes and a sophisticated retina arose early in evolution and are present in all extant vertebrates (Bradshaw and Allison 2022). The light sensitive organs in the closest outgroup, the non‐vertebrate chordates, are relatively simple clusters of photoreceptor and pigment cells that do not resemble eyes and are only capable of light detection and not image formation (Lamb 2013). To understand how the eyes of vertebrates gained such complexity, previous work has utilized fossils and comparative morphology (Collin et al. 2009; Collin 2010; Davies et al. 2012; Lamb 2013; Gabbott et al. 2016). These studies have revealed that the eyes of the early‐branching jawless vertebrates (cyclostomes) contain striking differences in structure and organization from jawed vertebrates (gnanthostomes). Visual system researchers long debated if these differences are due to retention of ancestral traits or secondary loss over evolutionary time (Fernholm and Holmberg 1975; Collin et al. 2009; Lamb 2013; reviewed in Bradshaw and Allison 2022). More recent arguments convince us of the latter, that the eyes of hagfish regressed over evolutionary time. These include the monophyly of hagfish with visually‐adept lampreys (Mallatt and Sullivan 1998; Kuraku et al. 1999; Heimberg et al. 2010; Ota et al. 2011; Miyashita et al. 2019), alongside works identifying hidden features in hagfish eyes (Dong and Allison 2021), and consistent with regression of various cephalic traits in the hagfish lineage (Oisi et al. 2013).

When compared to other vertebrates, the hagfish eye is strikingly small and rudimentary. These organisms are deep‐sea marine scavengers, and little is known about the development of their visual system (or their development in general) (Locket and Jørgensen 1998; Martini 1998). The eye is buried under a translucent layer of skin (or even concealed beneath muscle in certain species) (Fernholm and Holmberg 1975) (Figure 1A). Remarkably, the hagfish eye lacks both pigment and a lens (Figure 1B,C). Based on morphology, the retinal layers appear poorly laminated (Dong and Allison 2021) (Figure 1D,E). However, this rudimentary retina is a remnant following the evolutionary regression of a once elaborate organ (Dong and Allison 2021; Bradshaw and Allison 2022). Molecular markers revealed the presence of photoreceptors, interneurons, and retinal ganglion cells within the hagfish retina (Dong and Allison 2021). Moreover, although the retinal pigment epithelium layer is non‐pigmented, it expresses gene products required to complete the retinoid cycle, suggesting the hagfish retina has the potential to be more functional (and share a greater affinity with gnathostome eyes) than previously thought (Dong and Allison 2021).

FIGURE 1.

FIGURE 1

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Many components of the hagfish eye and retina are rudimentary/disorganized compared to other vertebrates, but the hagfish eye appears to contain a proliferative zone (ciliary marginal zone, CMZ). (A) In adult hagfish, the diminutive eye (black arrow) is buried under a translucent patch of skin. (B) Under the skin patch the hagfish eye is small and unpigmented. (C) The hagfish eye has a layered retina but no lens (indicated by the white arrow). (D, E) Compared to the retina of jawed vertebrates (i.e., zebrafish (D)) which are highly laminated, the hagfish retina (E) has poor organization. The hagfish retinal epithelium is unpigmented (unique compared to other vertebrates) and will be referred to as RnPE (retinal non‐pigmented epithelium). While the RnPE and a photoreceptor layer can be identified, interneurons and retinal ganglion cells are not clearly delineated into separate layers. (F, G) The retinae of many gnathostomes (i.e., zebrafish (F)) contain a proliferative peripheral region known as the ciliary marginal zone (indicated by the red arrow). The hagfish retinal periphery (G) contains a region with morphological similarity to the gnathostome CMZ. Ant. = anterior, post. = posterior, RPE = retinal pigmented epithelium, and RnPE = retinal non‐pigmented epithelium.

We have begun to explore retinogenesis in the Pacific hagfish (Eptatretus stoutii). Retinal development in some cyclostomes, lampreys, has been described (Suzuki and Grillner 2018), yet hagfish diverged from other vertebrates over 600 million years ago (Blair and Hedges 2005), such that hagfish eye development could reveal informative deep homologies or divergent mechanisms. Moreover, obtaining hagfish in embryonic or larval stages is extremely difficult (Gorbman 1997; Holland 2007; Kuratani and Ota 2008; Ota and Kuratani 2006, 2008), and so identifying a proliferative zone that generates a retina in “adult” (post‐larval)1 hagfish would enable evo‐devo experiments that seek to compare retinal development in the well‐studied jawed vertebrates (Ganthostomes).

In fish and amphibians, adult retinogenesis recapitulates many aspects of retinal embryonic development (Perron et al. 1998; Xu et al. 2020). This occurs at the ciliary marginal zone (CMZ), a stem cell niche found at the retinal periphery in teleost fish and amphibians (Raymond et al. 2006). At the edge of this region are undifferentiated cells that express stem cell/progenitor cell markers (i.e., Pax6). Moving centrally, cells of the CMZ become more differentiated and begin to express neuronal (and later mature retinal cell) markers (i.e., NeuroD, Atoh7, Otx2, and Crx). Cell morphology also changes throughout the CMZ, with more mature cells located towards the retinal interior. In the hagfish retina, Pax6 is expressed at the retinal periphery (Dong and Allison 2021). Photoreceptor morphology also appears to differ across the retina, with mature outer segments present in centrally located photoreceptor cells but absent at the cells of the retinal periphery (Dong and Allison 2021).

Evidence of a proliferative region in the hagfish retina is surprising given the rudimentary nature of the eye. In addition, limited information from lamprey suggests that the only other living cyclostome group does not have adult retinal neurogenesis (Villar‐Cheda et al. 2008). If the retina of adult hagfish has a CMZ, this would provide support for the early origin of conserved developmental mechanisms driving retinal growth in cyclostomes and gnathostomes. Here, we use proliferative markers and gene expression to ask if post‐larval (“adult”) hagfish retina, despite its degenerate appearance, has ongoing proliferation akin to jawed vertebrates.

2. Materials and Methods

2.1. Animal Collection and Care

This study was conducted under the approval of the BioSciences Animal Care and Use Committee at the University of Alberta (Animal Use Protocol number: AUP00000077), which adheres to the Canadian Council for Animal Care guidelines. Animals were caught and held according to a Department of Fisheries and Oceans Canada collection permit (XR 225 2021) and the animal use protocol approved by the Bamfield Marine Sciences Center (BMSC) (RS‐21‐06). Hagfish were acquired near BMSC in Barkley Sound on the west coast of Vancouver Island, British Columbia, Canada. BMSC staff caught the hagfish using a Korean cone trap baited with dead fish (BMSC Animal Care Standard Operating Procedure, Hagfish (Eptatretus stoutii), v.3 (Bartlett and Janusson 2019)). The traps were deployed for 8–12 hours and then brought to the surface. Hagfish were held in large tanks with circulating seawater until tissue was collected (1‐3 days). Hagfish were euthanized by administration of excess MS‐222 (tricaine methanesulfonate). Eye and brain tissues were collected from euthanized specimens and were preserved in 4% paraformaldehyde/5% sucrose//0.1 M phosphate buffer (referred to as 4% PFA from here on) or RNAlater (Invitrogen, Cat. No. AM7020).

2.2. EdU Administration and Tissue Collection

Ten hagfish were anesthetized via MS‐222 administration until they no longer responded to physical stimuli (i.e., touching, poking). The animals ranged in size from 27 to 110 g. An intraperitoneal injection was completed by inserting a needle into the ventral side of the hagfish abdomen with the skin held off the wall of the abdomen. 20 ul of 10 mM EdU (5‐ethynyl‐2’‐deoxyuridine) (EdU in vivo kit, baseclick GmbH, Cat. No. BCK488‐IV‐IM‐S) diluted in PBS (phosphate buffered saline, pH 7.4) was injected per gram of body weight (Zhang et al. 2014). Fish were then allowed to recover in a bucket with circulating seawater. After 24 hours the fish were sacrificed, and eye and brain tissues were collected and preserved in 4% PFA as described previously.

2.3. Cryopreservation and Cryo‐Sectioning of Fixed Tissue

After fixation with 4% PFA, hagfish eye samples were cryopreserved in a series of sucrose washes. The eyes were washed for 1 h in 12.5% sucrose//0.1 M phosphate buffer (room temperature), 1 h in 20% sucrose//0.1 M phosphate buffer (room temperature), and then overnight (at 4°C) in 30% sucrose//phosphate buffer. The following day, the eyes were placed in a 1:1 solution of 30% sucrose//phosphate buffer and OCT (optimal cutting temperature) embedding compound (hereafter referred to as OCT) (VWR Premium Frozen Section Compound, Cat. No. 95057–838) for 1 h (at 4°C). The samples were then flash frozen in a mold (made by taking the bottom half of a 1.5 mL centrifuge tube and fixing it to a glass slide with nail polish) filled with OCT using dry ice. The frozen samples in the molds (‘cryostat chucks’) were stored at ‐80°C at least overnight prior to sectioning. The samples were sectioned at 10 µM thickness using a Leica CM 1900 UV cryostat (chamber temperature: ‐20°C). The sections were placed onto Superfrost Plus microscope slides (ThermoFisher Scientific, Cat. No. 12‐550‐15) and kept at ‐80°C at least overnight until use.

2.4. EdU Detection Assay

An EdU cell proliferation detection kit (EdU in vivo kit, baseclick GmbH, Cat. No. BCK488‐IV‐IM‐S or BCK647‐IV‐IM‐S) was used to detect EdU+ cells in retinal sections from six of the ten hagfish individuals treated with EdU. Sectioned eye tissue was thawed and fixed in 4% PFA for 15 min prior to the EdU detection assay. The samples were then washed thrice for 5 min in 3% bovine serum albumin in PBS (pH 7.4) (hereafter 3% BSA) and re‐hydrated with PBS + 0.1% Tween (PBS‐T) for 30 min. Following this, the sections were permeabilized by adding a solution of PBS with 0.1% Triton X‐100 onto the samples for 20 min (and then rinsed 3 × 5 min in 3% BSA). The EdU detection cocktail was prepared according to the manufacturer's instructions (EdU in vivo kit, baseclick GmbH, Cat. No. BCK488‐IV‐IM‐S or BCK647‐IV‐IM‐S) and applied to the slides for 30 min. The samples were kept in the dark during this period and gently rocked/agitated every 5 min. Once the detection solution was removed, the samples were rinsed twice for 5 min in 3% BSA and 2 × 5 min in PBS‐T. Nuclei were stained using 1ug/ml DAPI (4',6‐diamidino‐2‐phenylindole, dihydrochloride) (Invitrogen, Cat. No. D1306) diluted 1:1000 in PBS‐T. The nuclear stain was applied for 10 min. The DAPI solution was then removed, and the samples washed 3 × 15 min in PBS. The completed slides were covered with 80% glycerol and sealed with nail polish. The slides were imaged using an LSM 710 confocal microscope (Zeiss) or an Olympus FV3000 confocal microscope. Of the samples from the six individuals imaged, the sections from three individuals (EdU 5, EdU 6, and EdU 8) were intact enough to be used for quantification.

2.5. EdU Cell Quantification and Statistical Analysis

To determine if EdU + cells were occurring in specific regions of the hagfish retina, the length of the RnPE layer of each retinal section was measured in ImageJ (Schneider et al. 2012). This length was then used to divide the retinal sections into six equal regions (regions 1–6). Region 1 was the most peripheral, whereas region 6 was the most central. The number of EdU+ cells was then quantified in each region for each of the three individuals. This data was analyzed by a 2‐way ANOVA followed by a Tukey's multiple comparisons test (GraphPad Prism version 9.5.1 for Windows, GraphPad Software, San Diego, California, USA, www.graphpad.com, RRID:SCR_002798). In a second analysis, the same retinal section images were divided into a peripheral “CMZ‐like” zone (based on the hagfish retinal morphology) and a “central” zone (the remainder of the retina). The number of EdU+ cells was then counted in the two regions. This data was also analyzed by a 2‐way ANOVA.

2.6. DAPI and Phalloidin Labeling

Sectioned zebrafish (n = 1 eye) and hagfish (n = 1 eye) tissues were thawed to room temperature and blocked in a mixture of 10% donkey serum (Sigma‐Aldrich, Cat. No. D9663‐10ML)//PBS‐ T for 1 h. The samples were then labeled with 1:1000 DAPI and 1:500 Phalloidin‐546 (Invitrogen, Cat. No. A22283)//PBS‐T for 30 min. The samples were washed 3 × 15 min in PBS‐T and then covered with 80% glycerol and sealed with nail polish. The slides were imaged using an LSM 710 confocal microscope (Zeiss).

2.7. BLAST Searches and Synteny Analysis

tBLASTn (translated nucleotide BLAST) searches of representative vertebrate retinogenesis genes against the hagfish genome were completed through the Ensembl BLAST search interface, whereas searches against the RNA sequencing dataset were performed with the BLAST function available in Geneious Prime (v. 2022.0, Biomatters, RRID:SCR_010519). Reverse BLAST searches were completed by querying E. stoutii RNA transcript sequences against the NCBI Nucleotide collection (nr/nt) database to further support sequence homology.

Two approaches were used to visualize the syntenic relationships between the three gnathostome Otx paralogs (Otx1, Otx2, and Otx5/Crx) and the four cyclostome paralogs (OtxA, OtxB, OtxC, and OtxD). First, Genomicus (v.106) (Muffato et al. 2010; Nguyen et al. 2022) was used to visualize the relationships between the Otx genes across representative chordate species (mouse [Mus musculus], Western clawed frog [Xenopus tropicalis], spotted gar [Lepisosteus oculatus], coelacanth [Latimeria chalumnae], elephant shark [Callorhinchus milii], sea lamprey [Petromyzon marinus], and inshore hagfish [Eptatretus burgeri]). The results generated were compared to genomic data in the National Center for Biotechnology Information (NCBI) Genome Data Viewer database (Sayers et al. 2022) (when this data was available—the NCBI database does not contain an annotated hagfish genome).

For synteny analysis using Simple Synteny (Veltri et al. 2016) a tBLASTn search with a mouse query sequence (the proteins of a mouse Otx gene and the 10 genes directly to either side) was used to identify syntenic genomic regions surrounding each Otx paralog (the 1 M base pairs directly adjacent on either side of each Otx gene) for the lamprey (P. marinus) and hagfish (E. burgeri). This was repeated using the proteins for each of the three mouse Otx paralogs (Otx1, Otx2, and Crx) and their adjacent genes as query sequences. The e‐value threshold used was 0.0001 and the minimum query coverage cut‐off was left at one. The results generated were compared to the NCBI Genome Data Viewer database and hits that were not supported by both the database and the Simple Synteny analysis were excluded (for hagfish an annotated genome assembly is not available on NCBI, so only the syntenic analysis was completed). Query and genomic sequences were retrieved from NCBI for most species examined. Genomic sequences for the hagfish (E. burgeri) were retrieved from the Ensembl database (Ensembl Genome Browser v. 108, Cunningham et al. 2022).

2.8. Phylogenetic Analysis

A protein alignment of Otx paralogs (from representative chordates) was generated using Geneious Prime (Geneious Prime v. 2022.0.1, Biomatters Ltd., RRID:SCR_010519) with the Geneious alignment setting and inputted into the IQ‐tree webserver (Nguyen et al. 2015; Trifinopoulos et al. 2016). A maximum‐likelihood tree was generated using IQ‐tree default settings (and ultrafast bootstrap‐1000 iterations) (Hoang et al. 2018). Protein sequences were retrieved from NCBI (most organisms) or Ensembl (hagfish).

2.9. CDNA Production

Hagfish RNA previously extracted by Emily Dong was used to generate cDNA for riboprobe production. cDNA was made as per the manufacturer's instructions (Quantabio, Cat. No. 95048–100).

2.10. Riboprobe Production

An antisense riboprobe to label the E. stoutii homolog of Rx was produced from a plasmid template using published methods (Barthel and Raymond 1993; Barratt and Arkell 2020, Dong and Allison 2021). The template was generated by creating a gene block (IDT—Integrated DNA Technologies) based on the sequence from CL3100.Contig2_All (Rx) (Supplemental Table 1). Restriction enzyme sites were added to either end of the gene block to facilitate ligation into a PCS2+ plasmid. The plasmid was linearized with BamHI and used in a transcription reaction overnight with a T3 RNA polymerase (Roche, Cat. No.11031171001) and DIG (digoxigenin) conjugated dNTPs (Roche, Cat. No. 11277073910) to generate an antisense Rx probe. The RNA from the reaction was precipitated and the probe was suspended in 50 uL of RNase free water to assess concentration and quality via a Nanodrop 2000 (Thermo Scientific, Cat. No. ND‐2000). The probe was diluted to its final concentration in Hauptmann's hybridization solution and kept at ‐80°C before use.

A riboprobe to label the homolog of hagfish (E. stoutii) OtxA was generated from a template created via a PCR reaction on hagfish eye cDNA (Phusion high‐fidelity polymerase kit [ThermoFisher Scientific, Cat. No. F530S]) (Barthel and Raymond 1993; Barratt and Arkell 2020, Dong and Allison 2021). Primers were designed to amplify E. stoutii OtxA with a T7 RNA polymerase site added to the 5’ end of the reverse primer (Supplemental Table 2). The amplicon underwent a transcription reaction similar to the plasmid derived probes (except using a T7 RNA polymerase (Roche, Cat. No. 10881767001)) and the RNA was then precipitated, and quantified using the same methods as above. The final Rx riboprobe was 1206 bp long and spanned three exons of the gene (Supplemental Table 1). The OtxA riboprobe was 591 bp long and spanned three exons of the gene (Supplemental Table 2).

2.11. In Situ Hybridization

In situ hybridization was completed as per Dong and Allison 2021. Slides were thawed to room temperature and fixed with 4% PFA for 10 min. After removing the PFA and rinsing the slides with RNase free 2X SSC (saline‐sodium citrate buffer), sections were treated with proteinase K (Roche, Cat. No. 3115836001) for 2–10 min (probe dependent—see Supplemental Table 3). They were then rinsed with RNase free 2X SSC and fixed again in PFA for an additional 10 min. After removing PFA and rinsing with 2X SSC, the slides were placed into a mixture of 0.3% acetic anhydride and 0.1 M TEA (triethanolamine) for 10 min before being dehydrated through a series of ethanol washes (50%, 75%, 95%, 100% ethanol diluted in DEPC treated water). The slides were then dried for an hour at room temperature. The slides were incubated with Hauptmann's hybridization solution for an hour at room temperature before the appropriate riboprobe (diluted in Hauptmann's solution) was applied (the concentration used was probe dependent—see Supplemental Table 3). For negative control slides, Hauptmann's hybridization solution without an added probe was used. The sections were then placed in a hybridization chamber overnight at 55°C.

The following day the probe solution was removed and the slides underwent a series of post hybridization washes at 55°C—10 min in 2/3 post‐hybridization buffer (1:1 2X SSC:formamide): 1/3 2X SSC, 5 min in 1/3 post‐hybridization buffer: 2/3 2X SSC, 5 min in 2X SSC, 20 min in 0.2X SSC+0.1% Tween, and 2×20 min in 0.1X SSC+0.1% Tween. The slides were blocked in maleate buffer for an hour before being incubated with an alkaline phosphatase conjugated anti‐DIG antibody (Roche, Cat. No. 11093274910) diluted 1:1000 in the maleate block overnight (4°C). The slides were then stained with a solution made using nitro blue tetrazolium (NBT)/ 5‐Bromo‐4‐chloro‐3‐indolyl phosphate (BCIP) ready‐to‐use tablets (Roche, Cat. No.11697471001) following the manufacturer's instructions to visualize the bound riboprobe. For fluorescent in situ hybridization (FISH), after the post‐hybridization washes, the slides were incubated in an alkaline phosphatase conjugated anti‐DIG POD (peroxidase) conjugated antibody (Roche, Cat. No. 11207733910) (antibody diluted 1:100 in the maleate block). The slides were incubated with the antibody solution in the dark overnight (4°C). The following day the slides were rinsed 3×15 min with PBS‐T. The slides were then incubated in an Alexa Fluor 488 tyramide detection solution (length of incubation was signal dependent) prepared according to the manufacturer's instructions (Alexa Fluor 488 Tyramide SuperBoost Kit, Invitrogen, B40943). The reaction was stopped with application of a 1X stop reaction solution (Alexa Fluor 488 Tyramide SuperBoost Kit, Invitrogen, B40943) for 10 min. Slides were rinsed 3 X with PBS‐T to remove residual detection and stop solutions. The slides were then stained for DAPI as previously described and sealed with 80% glycerol and nail polish. The slides were imaged on an LSM 710 Confocal (Zeiss) or an FV3000 Confocal (Olympus).

3. Results

3.1. The Post‐Embryonic Hagfish Retina Continues to Proliferate

Recent work suggests the hagfish eye may continue to grow late into ontogeny (Dong and Allison 2021), including increases in eye size with body size (and presumably age) of hagfish. In addition, the peripheral region of the hagfish retina has morphological similarities with the gnathostome ciliary marginal zone (CMZ) (Figure 1F,G).

To assess whether the adult hagfish retina contains proliferating cells, several adult hagfish were treated with a brief 24 h pulse of EdU (a thymidine analog incorporated into DNA of proliferating cells (Chehrehasa et al. 2009)) and sacrificed the next day. Sectioned eye tissue from those individuals underwent a ClickIt EdU detection reaction to label EdU positive (newly born) cells. EdU‐positive (EdU+) retinal cells were detected in the eyes of all EdU‐treated animals examined (six individuals). The majority of retinal sections contained one or more cells positively labeled for EdU (Figures 2, 3). The EdU+ cells often occurred in clusters at the peripheral edges of the retina (Figure 2A,B). Some hagfish retina sections also contained several EdU+ cells in the more central portions of the retina. No such labeling was observed in hagfish that did not receive EdU (Figure 2C).

FIGURE 2.

FIGURE 2

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EdU (5‐ethynyl‐2’‐deoxyuridine) incorporation reveals proliferating cells in the post‐embryonic hagfish retina. An abundance of EdU‐positive (EdU+) cells at the retinal margins clarifies that ongoing proliferation contributes to adult retinal growth in this otherwise very rudimentary tissue. (A) an overview of a hagfish retinal section from an adult hagfish treated with EdU 24 h prior to sacrifice. The white arrows indicate cells that are EdU+. Nuclei are stained with DAPI. (A’) the same image with only EdU labeling shown. (B) a closer view of the periphery of the retinal section in A (boxed region). White arrows indicate there are multiple EdU+ cells in this region. (B') the same image as panel B with only EdU labeling shown. (C) A retinal section from an animal not treated with EdU (negative control). No EdU+ cells are observed. (C’) The same image as panel C with only EdU labeling shown. Vitreous = vitreous side of retina. CMZ = putative ciliary marginal zone.

FIGURE 3.

FIGURE 3

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Quantification of EdU positive (EdU+) cells in the hagfish retina suggests proliferating cells are concentrated at the peripheral retina. (A) Division of the retina for EdU+ cell quantification. Region 6 is most central and region 1 most peripheral. (B) Number of EdU+ cells per region across 21 retinal sections (from three EdU treated individuals—EdU 5, EdU 6, and EdU 8). The average number of EdU + cells per individual and per each region is plotted, along with maximum and minimum (whiskers). The number of EdU cells in region 1 was statistically different from the number found in the other 5 regions (P < 0.001). (C) Proportion of total number of EdU+ cells found in each retinal region. (D) The average number of EdU + cells found in the ‘CMZ’ (defined by morphology) zone compared to the rest of the retina for each individual. The overall number of EdU + cells (across all three individuals) located in the CMZ zone does not differ significantly from the number of cells located in the more central retina. Error bars represent standard error. Panels B‐D created in GraphPad Prism version 9.5.1 for Windows.

Qualitatively, the abundance of EdU+ cells was concentrated at the peripheral margins of the retina, nearest to the iris (Figure 2A, B), consistent with the location of a CMZ in teleosts and other species (Raymond et al. 2006). To support this qualitative observation, we counted the EdU positive cells in various retinal regions from three individuals: images of hagfish retina were divided into 6 equal sections distributed across the peripheral to central retina, and EdU+ cells were enumerated (Figure 3A). The majority of EdU+ cells were peripherally located (based on 21 retinal sections from three EdU treated individuals—EdU 5, EdU 6, and EdU 8) (Figure 3B). Over half (57%) of the EdU positive cells occurred in the most peripheral region (region 1) in the three individuals we examined (Figure 3C). A two‐way ANOVA showed the retinal region had a significant effect on the number of EdU+ cells observed (p < 0.0001) whereas the individual retinal samples were taken from did not (p = 0.06). There was a significant interaction between the effects of individual and region (F (10, 108) = 6.133, p < 0.0001). The number of EdU+ cells in region 1 was significantly higher than the number of cells occurring in any of the other five regions (Tukey's multiple comparisons test, p < 0.0001). In one individual, denoted “Edu 6”, less EdU was detected throughout the retina, and no concentration of Edu labeling at the periphery was apparent. To reiterate, while the statistical results are under‐powered by small sample size, our intent with this statistical analysis was to provide the reader with additional support to our qualitative observation that, when the EdU labeling was robust, the hagfish retinal periphery has a notable enrichment of proliferating cells.

Next, we separated the analysis into the small peripheral region of the retina that morphologically resembles the gnathostome CMZ (the area between the site where the ONL and INL layers merge and the edge of the retina (Raymond et al. 2006); see Figure 1F,G) compared to the larger central retina. In this analysis (two‐way ANOVA for region (“CMZ” vs. central retina) and individual), there was no significant difference between the number of cells observed in the CMZ region and the rest of the retina (p = 0.73) (Figure 3D). Many proliferative cells occur in the smaller hagfish retinal CMZ‐like zone, and a similar substantial number of EdU+ cells were observed in the larger central parts of the retina as well.

3.2. Bioinformatic and Phylogenetic Analysis Supports Homology of Hagfish Retinal Transcripts With Critical Retinal Development Genes in Lamprey and Jawed Vertebrates

To identify potential markers of hagfish retinal development, we mined transcriptomics data and publicly available genomes. To consider gene homology, we used sequence identity, reciprocal BLAST, and gene synteny analyses. The identity of these homologs was initially identified via tBLASTn searches of the hagfish genome (E. burgeri genome assembly available on Ensembl v. 108) and an available RNAseq dataset of E. stoutii (generated by Emily Dong; Dong and Allison 2021) with query sequences from representative gnathostomes (mouse, spotted gar, and elephant shark) (Supplemental Tables 4–5). These analyses reveal that hagfish have homologs for several genes known to be required for gnathostome retinal development. We prioritized bioinformatic analysis of Rx and OtxA as known markers of stem cells in the vertebrate CMZ. These genes appear to be homologous to gnathostome genes based on gene synteny (Supplemental Tables 4–8, Supplemental Figures 1–8). The homology of hagfish and gnathostome Rx is also supported by a synteny analysis from Kon and Furukawa (2020).

3.3. The Hagfish Retina Expresses Markers for Retinal Proliferation and Neurogenesis

In situ hybridization on hagfish retinal sections reveals expression of OtxA throughout the hagfish retina (Figure 4). Several cell populations appear to label for the OtxA probe, including cells in the RnPE (retinal non‐pigmented epithelium), the retinal periphery (CMZ), the inner nuclear layer, and possibly the photoreceptor layer. Expression in the RnPE is particularly strong. In situ hybridization performed for the Rx probe reveals Rx expression within cells of the inner nuclear layer and perhaps the retinal periphery (Figure 5).

FIGURE 4.

FIGURE 4

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OtxA is expressed throughout the hagfish (Eptatretus stoutii) retina. In situ hybridization probes were visualized with colorimetric detection (panels A–C) or fluorescent detection (panels D–G). (A) Overview of the retinal section labeled with OtxA. (A’) Magnified image of black box in panel A showing OtxA labeled cells in the RnPE. (A″) Magnified image of blue box in panel A showing OtxA labeled cells in the inner nuclear layer. (A"’) Magnified image of red box in panel A showing OtxA labeled cells in the CMZ region. (B) OtxA labeled cells in the photoreceptor layer and inner nuclear layer of the E. stoutii retina (not from section in panel A). (C) Negative control section with no probe applied. (D) Retinal section fluorescently labeled with OtxA and stained with DAPI. (E) same image as panel D but only showing OtxA label. (F, G) Negative control section with no riboprobe applied, with and without DAPI respectively. RnPE = retinal non‐pigmented epithelium, vitreous = vitreous side of the hagfish retina, CMZ = ciliary marginal zone. Red arrows = CMZ. Blue arrows = inner nuclear layer. Purple arrows = photoreceptors. Black arrows = RnPE.

FIGURE 5.

FIGURE 5

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Rx is expressed in the inner nuclear layer and (perhaps) the periphery of the hagfish (Eptatretus stoutii) retina. In situ hybridization probes were visualized with NBT/BCIP detection of alkaline phosphatase. (A) Overview of retinal section labeled with Rx. (B) Magnified image of blue box in panel A showing Rx labeled cells in the inner nuclear layer. (C) Magnified image of red box in panel A showing Rx labeled cells in the CMZ. RnPE = retinal non‐pigmented epithelium, vitreous = vitreous side of the hagfish retina, CMZ = ciliary marginal zone. Red arrow = CMZ. Blue arrow = inner nuclear layer.

4. Discussion

4.1. The Adult Hagfish Retina Continues to Proliferate Despite Its Rudimentary Features

Although the hagfish retina is small and has reduced organization compared to other vertebrates, EdU cell labeling reveals that the adult hagfish retina contains actively proliferating cells (Figures 2, 3). Retinal sections examined from EdU treated animals consistently contained EdU+ cells, with over 50% of these cells being located at the retinal margin (Figure 3). This finding mirrors a retinal stem cell niche found in other vertebrates, the ciliary marginal zone (CMZ) (Raymond et al. 2006). Other data also supports the hagfish retina growing throughout ontogeny. Eye size increases with hagfish body size, suggesting continuous retinal growth through the animal's lifespan (Dong and Allison 2021). In addition, Dong and Allison (2021) demonstrated that the periphery of the hagfish retina expresses Pax6, a marker for retinal progenitor cells and the CMZ. The peripheral hagfish retina has conserved epithelial morphology with gnathostomes, and a shared feature of central retinal cells being more differentiated compared to the simple peripheral epithelium. Together with this study's findings that the hagfish retinal periphery expresses OtxA and Rx (markers of developing retinal neurons and neuronal progenitors respectively—see below), these results support the EdU+ cells at the hagfish retinal periphery may be part of a CMZ‐like structure (Dong and Allison 2021).

The vertebrate CMZ is a stem cell niche that is best known from studies on teleost fish and amphibians (Raymond et al. 2006). The niche is less prominent in adult birds and is essentially absent in mammals, and recently identified in sharks (Fischer et al. 2014; Hernández‐Núñez et al. 2021). One paper suggests the niche may also be absent in the lamprey retina (Villar‐Cheda et al. 2008). Therefore, finding active growth of the retina in adult hagfish is surprising, given that the hagfish eye is so rudimentary in form and the possible absence of this structure in the other extant cyclostome taxa. Why maintain and actively grow a tissue that cannot be used for vision? This raises the possibility that there are other functions for the hagfish retina (e.g., regulation of circadian rhythm) or that continued proliferation could be a paedomorphic trait where developmental retinal growth continues into adulthood.

In addition to reporting an enrichment of proliferating cells at the CMZ, we also noted EdU+ cells in the central mature retina. The identity of these cells is currently unknown, but their presence is consistent with proliferating cells in the mature retina of teleost fish. It is possible these cells could be a Müller glia‐like cell, a retinal glial cell that can de‐differentiate to produce new retinal neurons. Müller glia proliferation can be activated in response to retinal injury (Raymond et al. 2006). In teleosts, these glia also proliferate under normal physiological conditions to produce certain retinal cell types (i.e., rod photoreceptors) (Lenkowski and Raymond 2014). For this study, collecting hagfish from their natural environment involved the capture from a depth of 80 m, and abrupt movement of traps to the surface. This likely exposed the fish to brighter light than they were accustomed to and could have induced retinal injury. Indeed, there is one report of Müller glia in a species of lamprey based on immunohistochemistry markers, but no studies have examined the hagfish retina for the presence of Müller glia as of yet. Alternatively, the unidentified EdU+ cells could represent other cell types, such as microglia (Silverman and Wong 2018) or blood cells. Further work is needed to confirm the identity of the centrally located EdU+ cells. Similarly, completing EdU labeling in tandem with in situ hybridization for genes such as Notch1 or Olig2 (highly conserved neuronal progenitor markers) could help to determine the nature of the retinal periphery in hagfish (Perron and Harris 2000; Nakamura et al. 2006; Dvoriantchikova et al. 2015).

4.2. Hagfish Possess Homologs of Genes Critical for Gnathostome Retinal Development

Examination of hagfish genomic and RNAseq datasets revealed the hagfish eye expresses transcripts of several genes critical for vertebrate eye/retinal development. Some genes of particular interest as markers of retinogenesis include the retinal homeobox (Rx) gene and the Otx genes (hagfish have 4 paralogs—OtxA, OtxB, OtxC, and OtxD) (Supplemental Figures 1–2, Supplemental Tables 4–5). In gnathostomes these genes are required for proliferation and maintenance of retinal progenitor cells (Rx (Nelson et al. 2009)) and differentiation of several neuronal cell types (Otx genes—photoreceptor and bipolar cell specification (Nishida et al. 2003; Viczian et al. 2003)).

4.3. Hagfish Homologs of Otx2 and Rx Are Expressed at the Retinal Periphery and in the Inner Nuclear Layer

In situ hybridization confirmed that several putative retinogenesis genes identified via genomic and RNA database searches are expressed in the hagfish (E. stoutii) retina. The expression of OtxA was particularly prominent (Figure 4). OtxA labeling in several cell populations is particularly relevant when comparing to the gnathostome Otx2 retina expression profile. The RnPE (retinal non‐pigmented epithelium) cellular layer of the hagfish retina was heavily labeled by the OtxA probe (Figure 4A’). This expression pattern matches closely with the Otx2 expression in mammalian retinae at the RPE (retinal pigmented epithelium), where it is vital for RPE cell specification and maintenance (Béby and Lamonerie 2013; Rath et al. 2007). OtxA was also expressed in other areas of the hagfish retina, including directly adjacent to the RnPE (potentially the photoreceptor layer), the inner nuclear layer, and the retinal periphery (Figure 4A“,A’”,B, D, E). Expression in the photoreceptor layer corresponds to the gnathostome Otx2 expression in photoreceptors. Otx2 is critical for photoreceptor specification, and in some vertebrates, Otx2 expression is maintained in the photoreceptors and is required for photoreceptors to remain healthy (Nishida et al. 2003). Otx2 is also utilized by gnathostomes to specify bipolar cells (Yamamoto et al. 2020). If the inner nuclear layer cells labeled by OtxA correspond to hagfish bipolar cells, this again would match the gnathostome expression profile. Performing a double labeling in situ hybridization experiment with the OtxA probe and a marker for hagfish bipolar cells (such as PKC‐ɑ) will be a vital next step to confirm this (Dong and Allison 2021). Although the similar expression profiles of OtxA and Otx2 are suggestive that these genes may serve similar functions in hagfish and gnathostomes, respectively, we cannot conclusively state based on the expression data that the hagfish OtxA is promoting retinal development/retinogenesis. As mentioned, in other vertebrates, Otx2 functions in the maintenance of mature retinal neurons in addition to the specification of developing neurons. However, the expression of OtxA at the retinal periphery (the putative CMZ zone) suggests that at least some of the OtxA expression is occurring in newly forming retinal neurons.

There is one other study that demonstrated OtxA expression in the retina of a cyclostome. Yamamoto et al. (2020) showed that OtxA is present in the retina of the Japanese lamprey (Lethenteron camtschaticum). However, in the adult lamprey, OtxA was only observed in the photoreceptor layer. The authors suggest that Otx2 may have taken on its bipolar specification role only in the gnathostomes, and in cyclostomes it contributes solely to photoreceptor formation. However, the hagfish OtxA in situ hybridization data suggests otherwise. In adult hagfish, OtxA expression occurred in multiple retinal layers. Therefore, the absence of OtxA in lamprey bipolar cells compared to jawed vertebrates is likely not generalizable to cyclostomes overall.

As Rx is a marker of retinal progenitor cells, expression of this gene was expected at the hagfish retinal periphery, but a stronger Rx signal was detected in the inner nuclear layer (INL) (Figure 5). Rx is a gene required for the maintenance of retinal progenitors in gnathostomes (Nelson et al. 2009). As a result, during early development this gene has a broad expression pattern that gets restricted to specific areas (e.g., CMZ) once the retinal layers are established (Chuang et al. 1999). In adult zebrafish, Rx1 and Rx2 are expressed in the CMZ (and cone photoreceptors), and Rx3 is expressed in cells in the inner nuclear layer (Chuang et al. 1999). The hagfish In situ hybridization data showed the greatest Rx expression occurs across the INL layer (Figure 5A,B) which is comparable to the expression pattern of zebrafish Rx3. The expression was not consistent across the whole layer but rather occurred in scattered cells. There was some expression near the CMZ on one side of the retina (Figure 5C). However, the expression was unclear on the opposite side.

5. Conclusion

The hagfish is a poorly understood early‐branching vertebrate with a bizarre, yet fascinating, eye morphology. Investigating the development of this organism may reveal previously hidden clues to the evolutionary origins of the vertebrate eye and retina. One of the greatest limitations to studying hagfish is the logistical difficulty in acquiring embryos. However, examination of the hagfish retina in this study, together with a previous study (Dong and Allison 2021), has revealed the presence of a proliferative zone that we interpret as a CMZ‐like structure. This creates an avenue to explore retinal neurogenesis in hagfish. The presence of a CMZ also suggests post‐embryonic neurogenesis may have a more ancient origin in the vertebrate lineage than previously believed.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Supplementary Materials: cne70206‐sup‐0001‐SuppMat.docx

CNE-534-e70206-s001.docx (4MB, docx)

Acknowledgments

We thank the staff at the Bamfield Marine Sciences Center (BMSC) who collected and housed the live hagfish for this study. A special thanks to Junho Eom (UBC) who assisted with the administration of the intraperitoneal EdU injections. We also appreciate the help offered by Dr. Zachary Hall who provided advice for the EdU labeling protocol. We are grateful for Guobin Sun and Kacie Norton who assisted with the microscopy. Finally, we thank Emily Dong who assisted with generating the RNA sequencing dataset and the hagfish RNA used in this project. This work was funded by an NSERC Discovery grant to WTA and an NSERC Studentship to SNB.

Endnotes

1

We did not determine if the animals we are studying had reached sexual maturity, which is often a requirement in the definition of ‘adult’. Yet, the use of terms like “post‐larval” fails to adequately capture that the large animals we examined are beyond their first year of life, and well beyond the early developmental stages where CNS neurogenesis typically ceases in tetrapods.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Materials: cne70206‐sup‐0001‐SuppMat.docx

CNE-534-e70206-s001.docx (4MB, docx)

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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