The lots-of-rods-junior locus regulates rod number and spatial patterning

In our previously published genetic screen [53], [59] to isolate loci that regulate rod development and spatial patterning in zebrafish, we identified a mutation, lots-of-rods-junior (ljr^p23ahub) that results in an increased number and uniform distribution of rods across the retina in otherwise normal appearing larvae. In wild-type (WT) larvae, rods are asymmetrically patterned along the dorsal-ventral axis of the eye, with the highest density in the ventral retina, few in a belt spanning the central retina, and sporadic, yet non-random labeling across the dorsal retina [60]. In homozygous ljr^p23ahub larvae, immunolabeling for rods results in a uniform distribution typical of the cone pattern (Fig 1A and 1B). For all mutant samples analyzed, conformity ratios (CR) were significantly different from random based on Cook’s criteria (p<0.05), and Nearest Neighbor Dispersion Analysis (NNDA) indicates the rods are arrange in a uniform pattern (p<0.05) [61]. Previously, we reported that mutations in tbx2b (lor^p25bbtl) result in an increased number and uniform distribution of rods through a cell fate switch of SWS1-cone precursors into rods [53] (Fig 1A). Mating of homozygous ljr^p23ahub adults to homozygous tbx2b^p25bbtl adults revealed that ljr^p23ahub complements tbx2b^p25bbtl; all larvae displayed a rod pattern and SWS1 cone number typical of WT larvae. Co-immunolabeled larvae from mating of the double heterozygous ljr^p23ahub/tbx2b^p25bbtl adults revealed that double homozygous larvae for ljr^p23ahub/tbx2b^p25bbtl displayed an additive number of rods and few UV-sensitive cones suggesting that these two genes regulate photoreceptor cell patterning through distinct mechanisms (Fig 1A, 1B and 1C). Furthermore, ljr^p23ahub is semi-dominant; heterozygous ljr^p23ahub larvae display a rod number between those of WT and homozygous ljr^p23ahub mutants (Fig 1D).

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Fig 1. ljr^p23ahub mutants display an increased number and uniform distribution of rod photoreceptors.

(A) Confocal immunofluorescent images labeled for rods (red) and UV-sensitive cones (green) from WT, ljr^p23ahub, lor^p25bbtl and homozygous lor^p25bbtl/ljr^p23ahub retinas at 4 days-post-fertilization (dpf). WT larvae show asymmetric rod distribution in central and dorsal retina and a uniform distribution of UV-sensitive cones across the entire retina. ljr^p23ahub and lor^p25bbtl mutants demonstrate an increased number and uniform arrangement of rods but fewer UV-sensitive cones labeled in lor^p25bbtl. Double-mutant retinas display higher rod labeling and few UV-sensitive cones. (B) Graph showing the average number of rods per unit area dorsal to the optic nerve (WT, n = 5; ljr^p23ahub, n = 5; lor^p25bbtl, n = 5; ljr^p23ahub/lor^p25bbtl, n = 5). Significantly-different means by one-way ANOVA with Tukey’s post-hoc test, b vs c, p<0.05, all other comparisons p<0.0001. (C) Graph showing the average number of UV-sensitive cones per unit area from samples used in 1B, a vs b, p<0.0001. (D) Graph showing the average number of rods per unit area (WT, n = 5; ljr^p23ahub/+, n = 6; ljr^p23ahub, n = 5); a vs b, p<0.05, all other comparisons p<0.0001. (E) Flat mount views of confocal immunofluorescent images labeled for red-sensitive (brighter signal) and green -sensitive (dimmer signal) cones from WT and ljr^p23ahub retinas at 4 dpf. ljr^p23ahub mutants maintain the alternating arrangement of red- and green-sensitive cones. (F) Graph showing the average number of red- and green-sensitive cones per unit area (WT, n = 6; ljr^p23ahub, n = 6). No significant differences are observed, Student’s t test, p>0.05. Error bars represent SD.

https://doi.org/10.1371/journal.pgen.1005968.g001

Whole mount ljr^p23ahub larvae and WT controls were immunolabeled for Zpr-1 monoclonal antibody, which recognizes Arr3a, a selective marker for the red- and green-sensitive cones [62]. In confocal images of flat mount WT retinas, Zpr-1-labeling appears as rows of cells with more intensely fluorescent red-sensitive cones alternating with less intense labeling of the green-sensitive cones (Fig 1E). No significant change in the number of Zpr-1 labeled cones in ljr^p23ahub mutant was observed when compared to WT (Fig 1F). Immunolabeling of serial sections of WT and ljr^p23ahub mutant larvae with polyclonal antisera to RH2, SWS2, and SWS1 revealed no differences in their expression levels (S1A Fig). No differences were observed for cell specific markers of amacrine, bipolar, ganglion or Muller cells in ljr^p23ahub compared to WT, suggesting a very specific retinal phenotype (S1B Fig).

ljr^p23ahub as a hypomorphic allele of six7

Genetic linkage analysis positioned the ljr^p23ahub locus to a 0.3 Mb interval on chromosome 7 encompassing seven genes (S2A Fig): Ras and Rab interactor 1 (Rin1), actin related protein (Arp2), six7, beta-1,4 glucoronyltransferase 1, prefoldin subunit 2, nitrilase, and chloride channel 3. Genes lacking eye specific expression (Zfin) or with housekeeping functions were excluded from further consideration leaving six7 as the most plausible candidate. In order to more comprehensively examine for molecular lesions associated with the ljr^p23ahub we performed whole-genome sequencing of DNA pooled from 118 homozygous ljr^p23ahub larvae. Genome-wide SNP frequencies of reads were calculated against the Tuebingen (zv9) reference genome to identify regions depleted of SNPs that were associated with the mutant genetic background. Large, contiguous depletions in SNPs were present on chromosomes 3, 7, 9, and 20. A 5-megabase depletion of SNPs present on chr7 was centered directly over the mapping interval based on genetic linkage analysis (Fig 2A). Further examination of this interval revealed two regions, 14 kb and 40 kb upstream of six7, devoid of uniquely-aligning reads that were not associated with assembly gaps. The region 40 kb upstream of six7, but not the region 14 kb upstream, was also associated with sequence conservation among six7 genes from 4 other fish species, but not with Six3 from frog, mouse, or human. This region also displayed ChIP-seq signal for H3K4me1 in 24 hours-post-fertilization (hpf) embryos [63]. This mark is associated with distal enhancers in mammals [64]. To verify that the deletion was associated with the ljr^p23ahub mutation, DNA samples from fin clips of homozygous ljr^p23ahub adults and wildtype AB and TL strains of zebrafish were subjected to PCR using primer pairs targeting a DNA sequence unique to the distal deletion or the first exon of six7 as a control. PCR confirmed the co-segregation of the genomic deletion upstream of six7 with ljr^p23ahub (Fig 2A); the upstream region was only amplified in the TL and AB DNA. six7 is a teleost specific member of the sine oculis homeobox family of transcription factors, which have important roles in eye and forebrain patterning [55], [65], [66], [67], [68]. In zebrafish, six7 expression co-localized with six3a/b transcripts in the anterior region of the forebrain and optic vesicles [55], [69] with no detectable expression by RT-PCR at 24 hpf [55]. However, we and others observed by in situ hybridization that by 48 hpf, six7 expression is detectable in the retina, specifically in neuroblasts and the differentiating outer nuclear layer (ONL) coincident with photoreceptor cell genesis [58] (Fig 2B). More precisely, between 48 hpf and 52 hpf more general labeling of the retinal neuroblasts gives way to robust six7 expression that follows the temporal and spatial wave of photoreceptor genesis spreading from the ventral to the nasal and temporal retina [70], [71]. qRT-PCR for six7 transcripts at developmental stages from 10 hpf to 52 hpf mirrored the in situ hybridization; in ljr^p23ahub mutant embryos, greater expression was observed at 10 hpf compared with WT, and expression was absent in ljr^p23ahub mutant embryos at 18 hpf and from both groups at 24 hpf. Lower levels in ljr^p23ahub mutant embryos at 52 hpf were observed (Fig 2C). Thus, the spatial and temporal pattern of six7 expression and the changes observed in ljr^p23ahub embryos are consistent with previously described roles in photoreceptor development [57], [58].

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Fig 2. six7 knockdown phenocopies the increase and uniform distribution of rods from ljr^p23ahub mutants.

(A) Frequency of SNPs across chromosome 7 relative to the TL reference genome (danRer7) calculated from whole-genome sequencing of a pool of DNA extracted from 118 ljr^p23ahub mutants where TL is the reference background. Below, 75 kb view of the SNP-depleted region on chromosome 7. A 2.4 kb region depleted of uniquely-aligning reads is highlighted in teal. Shown are tracks for H3K4me1 [63], read alignments from whole-genome sequencing of ljr^p23ahub mutants, and multiz-based sequence conservation UCSC genome browser tracks of six7 across four fish species and Six3 in frog, human, and mouse [72]. Gel electrophoresis of PCR products of six7 exon 1 and the upstream genomic region amplified from AB, TL and ljr^p23ahub genomic DNA. (B) Whole mount in situ hybridization shows six7 expression confined to retinal neuroblasts and differentiating ONL spatially and temporally with photoreceptor genesis. Dorsal is up. (C) Quantitative RT-PCR (qRT-PCR) performed on mRNA from control (WT) and ljr^p23ahub embryos at 10 hpf, 18 hpf, 24 hpf and 52 hpf reveal down regulation of six7 expression in ljr^p23ahub at 52 hpf. Relative transcript abundance was normalized to actin levels and is presented as the mean fold change in expression relative to 10 hpf controls (n = 30 embryos per group). All the real-time PCR experiments were carried out in triplicate. Significant differences observed at 18 hpf and 52 hpf, Student’s t test, *p<0.05. (D) Retinal cryosection from 4 dpf un-injected control WT and six7-MO1 injected embryos immunolabeled for rods (4C12, green). six7-morphants display an increase in the number of rods as detected in ljr^p23ahub. Note the lack of gaps in rod distribution in the central retina of six7-knockdown larvae. (E) Graph showing dosage dependent increase in the average number of rods per unit area dorsal to the optic nerve of WT and six7-MO1 injected embryos (WT un-injected, n = 4; six7-MO1, n = 6, each dose), One-way ANOVA with Tukey’s post-hoc test. a vs b, p<0.05, b vs c, p<0.001, a vs c, p<0.0001. Error bars represent SD. nb, neuroblast; vp, ventral patch; onl, outer nuclear layer; MO1, morpholino 1.

https://doi.org/10.1371/journal.pgen.1005968.g002

To test the candidacy of six7 as the mutated gene in ljr^p23ahub, two antisense morpholinos targeting either the 5’UTR region (MO1) [69] or the donor splice site in the first intron (MO3) of six7 were injected into one-cell-stage WT embryos (S2B Fig). Injection of either morpholino phenocopied ljr^p23ahub mutants (Fig 2D). Morphants showed increased rod immunolabeling in a dosage dependent manner (Fig 2E), and did not demonstrate any obvious morphological defects. To confirm the efficiency of the splice blocking morpholino, RNA was isolated from un-injected and MO3-injected embryos, and the region spanning exon 1 and exon 2 amplified by PCR from resulting cDNA. Sequence data revealed that the MO3-injections resulted in alternative splicing upstream of the initiation codon deleting the majority of the exon 1 of six7 mRNA including the SIX domain (S2C Fig).

Previous studies reported highly conserved roles for six3/six6 family members in patterning of the forebrain and eye field, and in zebrafish six3b and six7 appear to be functionally redundant [58], [73], [74]. To study the hypothesis that six3a/b could be a target of six7, the levels of expression of six3a/six3b were determined by qRT-PCR. No difference in expression levels for six3a/six3b transcripts were detected between WT and ljr^p23ahub mutant (S2D Fig), suggesting that six7 is not regulating the expression of six3a/six3b. Consistent with the expression of six7 in ljr^p23ahub during forebrain patterning, analysis of 128 embryos from inbreeding of double heterozygous adults for ljr^p23ahub and six3b^vu87 did not result in any embryos displaying a greatly reduced or absent eye phenotype as previously observed for six7-morpholino knockdown on the six3b mutant background or in the recently reported double mutant harboring deletions of six7 and six3b [69],[58]. Based upon the genetic analysis, whole genome sequencing and gene expression changes, we propose that ljr^p23ahub is a hypomorphic allele of six7 that affects a regulatory element controlling expression during photoreceptor genesis.

To further characterize the increased labeling for rods, cell counts from methylene blue stained plastic sections of 4-days-post-fertilization (dpf) six7-MO1 embryos revealed a modest yet significant increased number of cells in the ONL compared with WT retinas, consistent with the observed increased rod number (S2E Fig). However, no changes were detected in the number of nuclei in the inner nuclear layer (INL) or the ganglion cell layer (GCL), arguing against a general increase in neurogenesis across the retina (χ², p> 0.05). Additionally, at 6 dpf, electron microscopy of the ONL in the central retina, where the highest change in rod numbers were detected, showed that rods in six7-MO1 were characterized by an outer segment composed of stacks of discs enclosed within the plasma membrane, a vitread located nucleus and a single invaginating synapse at the terminal (S2F Fig). These results suggest that knockdown of six7 led to an increased number of retinal cells specifically in the ONL with gene expression and morphological characteristics consistent with rods.

six7 morphants show extended proliferation in the ONL

The increased cell number in the ONL and lack of changes in cone numbers open the possibility that six7 regulates mitosis during photoreceptor development. Proliferation was assayed by EdU incorporation or phospho-histone 3 (PH3) immunolabeling. In zebrafish, neurogenesis occurs in three distinct waves; postmitotic cells appear first in the GCL, followed by the INL, and finally the ONL. At 48 hpf, a time coincident with photoreceptor cell genesis [75], co-labeling for EdU incorporation and in situ hybridization for six7 showed complex patterns of labeling. In histological sections, EdU labeling was most abundant in wedged-shaped clusters of highly proliferative cells near to the dorsal and ventral ciliary marginal zone (CMZ), and to a lesser degree in the developing ONL. In contrast, six7 expression near the CMZ was opposite of the EdU labeling; highest proximal to the CMZ where the neuroblasts had taken on a more salt and pepper EdU-labeling pattern (arrows), and nearly absent from the highly proliferative CMZ. In the central retina, more robust labeling for six7 coincided with reduced EdU incorporation (Fig 3A). These data show that six7 is expressed in photoreceptor precursors at or near the time of terminal mitosis.

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Fig 3. Photoreceptor progenitor proliferation is regulated by six7.

(A) EdU (red) and six7-in situ labeling (blue) in a retinal cryosection from 48-hpf WT embryos. Note the expression of six7 is coincident with proliferating cells in the ONL. (B) EdU labeling in retinal cryosections of WT, six7-MO control and six7 morphants at 52 hpf. Note that EdU labeled cells persist in the central retina in six7 morphants. (C) Retinal cryosections of WT and six7 morphants labeled with EdU at 48 hpf and immunolabeled for rods (4C12, green) at 4 dpf. Proliferative cells are restricted to the ONL and differentiate as photoreceptors in WT and six7-knockdown retinas. Proliferative cells colabeled for rod markers in the central retina, (arrowheads, inset). (D) Retinal cryosections of WT and six7-morphants immunolabeled for phospho-histone 3 (PH3) at 48 hpf and (E) graph showing the number of PH3-positive cells in the INL and ONL per sections (WT, n = 5; and six7-MO1, n = 4; 3–4 sections/retina). Mitosis levels increased significantly in the ONL in six7-morphants. No significant changes were observed in the INL. (***p = 0.0007, Student’s t test). (F) Retinal cryosections of WT and ljr^p23ahub at 3 dpf co-labeled for TUNEL (red) and rods (4C12, green), nuclei counterstained with SYTO61. (G) Quantification of TUNEL positive cells in the ONL, INL and GCL of WT (n = 7) and ljr^p23ahub (n = 9); (*p<0.05, *****p<0.0001, Student’s t test calculated on log transformed data). (H) Transverse retinal sections from WT and six7-MO1 injected animals at 3-dpf co-labeled for TUNEL (red) and rods (4C12, green), nuclei counterstained with SYTO61. (I) Quantification of TUNEL positive cells in the ONL, INL and GCL of control (n = 7) and six7-MO1 injected embryos (n = 6); (*p<0.05, Student’s t test calculated on log transformed data). Error bars represent SD. Dorsal is up. ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer; CMZ, ciliary marginal zone.

https://doi.org/10.1371/journal.pgen.1005968.g003

In a second set of experiments, un-injected, six7 MO1-, and control morpholino-injected embryos were incubated with EdU at 48 or 52 hpf and immediately processed for labeling. At 48 hpf, all treatment groups showed EdU labeling in the ONL. However, at 52 hpf, six7 MO1-injected embryos showed nuclear EdU labeling in the ONL but none was observed in the un-injected and 5 base mismatch control morpholino-injected embryos (Fig 3B). To determine if the EdU-labeled cells in the six7-morphant retinas differentiate specifically as rods, embryos were labeled with EdU at 48 hpf and maintained until 72 hpf, then fixed and processed for immunolabeling with a rod specific marker. EdU positive cells in the central retina co-labeled preferentially with a rod marker in six7-morphant retinas. However, some EdU positive cells differentiated as cones, consistent with the coincident timing of rod and cone differentiation in un-injected embryos (Fig 3C). We next immunolabeled retinas with anti PH3 to verify that the increased number of cells labeled with EdU was reflected by changes in the level of mitosis (Fig 3D). At 48 hpf retinas from six7-morphant embryos showed significant increases in the PH3 immunolabeling of the ONL compared to un-injected embryos (Fig 3E; Student t-test; p<0.001). No significant changes were observed in labeling of the INL. To test if the six7-depleted cells are biased to differentiate as rods, genetic chimeras were generated by transplanting cells from six7 MO1-injected or WT donor embryos into WT hosts. In histological sections, six7-MO1 donors cells immunolabeled for a rod specific marker at a rate three times higher than WT transplanted cells (p<0.05; S3A Fig). The spatial and temporal appearance of the additional mitoses and bias to form rods led us to test the identity of the proliferative cells. WT and six7-knockdown retinas were labeled by in situ hybridizations with molecular markers for retinal progenitors, rx1 and pax6a [76], [77] or two transcription factors expressed by developing photoreceptors, crx and neurod [78],[79],[80]. By 48 hpf in the WT and morphant larvae, the expression of the retinal progenitor marker rx1 was restricted to the CMZ and the pax6a gene was expressed by neuroblasts of the CMZ and in neurons located in the GCL and the proximal portion of the INL [81]. None was observed in the ONL (S3B Fig). Probes for crx and neurod strongly label the forming ONL and to lesser extent cells of the INL, with no differences observed between WT and mutant larvae (S3B Fig). The data suggest that the mitotic cells are photoreceptor progenitors.

In lower vertebrates, such as zebrafish, cell death in the retina can trigger proliferation of Muller glia cells and photoreceptor regeneration [82], [83]. To exclude apoptosis-induced proliferation as the mechanism leading to the increase in rod number, sections from control, ljr^p23ahub, and six7-morphant retinas were subjected to transferase-mediated dUTP nick end labeling (TUNEL) assay. Few TUNEL positive cells were observed in WT and control-morpholino retinas. A modest increased in TUNEL positive cells was observed in the inner retina and GCL in the six7-MO1 retina or ljr^p23ahub compared with control embryos (Fig 3F–3I), however none was observed in the ONL ruling out cell death-induced regeneration as the mechanism triggering the increase in mitosis and rod number. Lastly, previous studies in chick embryos have shown that ablation of the dorsal retina results in expansion of ventral domain and increased rod number in the central retina [84]. Given the high number of rods in the ventral patch of the zebrafish retina, we tested for expansion of ventral markers and loss of dorsal markers in ljr^p23ahub mutants. However, in situ hybridization for the dorsal marker tbx2b, the midline marker cyp26c1, and the ventral marker vax2 showed no difference in labeling of WT and mutant embryos (S3C Fig), decreasing the likelihood that the increased rod number resulted from alteration of dorsal-ventral patterning of the optic cup.

Genome editing of six7 locus recapitulates the increased rod number in ljr^p23ahub mutant larvae

Based upon the sequencing data, morpholino phenotypes and gene expression, we designed TALENs (transcription activator-like effector nucleases) to target six7. The homeodomain and the SIX domain are two evolutionarily conserved domains in the SIX proteins involved in DNA-protein or protein-protein interactions respectively [85], and mutations in the SIX domain in SIX3 are associated with congenital brain and eye defects [86]. Therefore a TALENs pair was designed to target 18-bp and 20-bp flanking a 14-bp spacer sequence of the first exon of six7, which corresponds to the SIX domain (Fig 4A). mRNAs encoding for the TALENs pair were co-injected into one-cell stage zebrafish embryos. Surviving embryos were grown to adulthood and mated to WT adults. Disruption of the six7 locus in the F1 larvae was detected by the loss of the HaeIII restriction site in the spacer region (Fig 4B). Fifty-two percent of the founders transmitted TALEN-induced mutations to the F1 (Fig 4C). F1 progeny were grown to adults and heterozygous carriers identified by fin clip analysis. F2 carriers of the following alleles were used in subsequent studies: c. 217_229del CAGGTGGCCCGAG, p. (Q11Cfs*39), from now on (six7^fl4); p. (E10Ifs*50); p. (F7Lfs*44), (all predicted to result in frameshift mutations and premature termination of the Six7 protein). Approximately, one quarter of the F2 progeny from inter-crosses between carriers demonstrated an increased number (t-test, p<0.001) and uniform distribution of rods (Cook’s CR, p<0.05; NNDA, p<0.05) as observed in ljr^p23ahub mutants (Fig 4D). Genotyping of the F2 larvae revealed that one quarter of the embryos with the lots-of-rods phenotype were heterozygous for the six7^fl4 mutation, consistent with the semi-dominance previously observed in ljr^p23ahub mutants. In mating between carriers or homozygous mutant adults, six7^fl4 failed to complement ljr^p23ahub mutants (Figs 4D and S4A; One-way ANOVA, Tukey’s follow-up test, p<0.0001); no significant difference in rod number was observed between six7^fl4/six7^fl4, six7^fl4//ljr^p23ahub, and ljr^p23ahub/ljr^p23ahub larvae (One-way ANOVA, p>0.5) providing genetic evidence that ljr^23ahub is indeed an allele of six7, and ljr^p23ahub shall be referred to as six7^p23ahub. Lastly, as previously observed in six7-knockdown embryos, immunolabeling for PH3 showed significant difference in sections from six7^fl4 homozygous embryos and WT siblings (S4B Fig). But unlike the six7^p23ahub allele, six7^fl4 homozygous animals showed significantly reduced viability (χ², p<0.05), but the few adults recovered were fertile.

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Fig 4. TALENs-mediated knockout of six7 locus recapitulates ljr^p23ahub phenotype.

(A) Schematic representation of the Six7 protein domains. six7-TALENs target site and the HaeIII restriction enzyme site are highlighted. The left and right monomer binding sites are underlined. (B) RFLP of six7 locus by HaeIII. A new 170 bp DNA-fragment is detected in six7^fl4 carriers compared with controls. (C) Sequence analysis of the six7 target region shows recovery of multiple indel alleles of six7. The target region is highlighted in red. Dots represent deletions and lower case letters indicate insertions. The six7 sequence from WT is shown as a comparison. (D) Confocal immunofluorescent images labeled for rods (red) from WT and six7^fl4 knockout mutants at 4 dpf. six7^fl4 knockout mutants phenocopy ljr^p23ahub mutants. Sequencing chromatograms of WT and six7^fl4 mutants illustrated the c.217_229del CAGGTGGCCCGAG (del13) six7 mutation in homozygous zebrafish. The bar graph shows the average number of rods counted in 1–3 different areas per retina (WT (n = 8), six7^fl4 (n = 7), six7 ^fl4/p23ahub (n = 4); One-way ANOVA with Tukey’s post-hoc test. a vs b, p<0.0001. (E) Tangential views of confocal immunofluorescent images labeled for red-sensitive (brighter signal) and green-sensitive (dimmer signal) cones from WT, six7^p23ahub, and six7^fl4 retinas at 4 dpf. Graph showing the average number of red/green-sensitive cones per unit area of WT (n = 3), six7^p23ahub (n = 4), and six7^fl4 (n = 3); (ns p> 0.05; a vs b p ≤ 0.05; significant difference one-way ANOVA with Tukey’s post-hoc test). (F) Whole mount in situ hybridization for RH2 probe (green-sensitive cone opsin) in WT (n = 30), six7^p23ahub (n = 30) and six7^fl4 embryos (n = 30) at 4 dpf. WT and six7^p23ahub embryos often showed the same pattern and number of green-sensitive opsin cone labeling (Dorsal is up and nasal to the left), while six7^fl4 knockout showed no labeling or few cells labeling for green-sensitive cone opsins (inset). Graph showing the percentage of unlabeled and labeled embryos. Notice that 17% of the six7^p23ahub embryos were un-labeled for green-sensitive cone opsin. (G) Retinal cryosections of in situ hybridization for green-sensitive cone opsin in six7^p23ahub (n = 5) embryos immunolabeled with 1D1 (rods). Rods and green-sensitive cone opsin probes labeled different cells in the ONL of six7^p23ahub embryos. (H) Evidence of cell death. Flat mount confocal image of nuclei counterstained with DAPI and retinal cryosections from WT and six7^fl4 animals at 4dpf co-labeled for TUNEL (red) and rods (4C12, green). six7^fl4 mutants (n = 6, 1–2 sections/retina) showed an increase in apoptotic cells, especially in the ONL compared with WT (n = 3, 1–2 sections/retina), (arrows pointing to apoptotic cells in the ONL).

https://doi.org/10.1371/journal.pgen.1005968.g004

RH2 opsin expression and green-sensitive cone precursor survival are dependent upon six7 expression

Ogawa et al. (2015) recently reported altered cone opsin expression in six7 knock-out animals with RH2 expression nearly absent and SWS2 significantly reduced [58]. However, we observed few alterations with green-sensitive cone opsin expression in six7^p23ahub homozygous and six7 morphant larvae and no changes in SWS2 immunolabeling. Therefore, we tested for differences in cone photoreceptor cell phenotypes between the six7^p23ahub and six7^fl4 alleles. Immunolabeling and confocal analyses revealed a significant decline in the number of Zpr1/Arr3a positive photoreceptors in homozygous six7^fl4 larvae consistent with the lack of RH2 expression reported previously (Fig 4E). Immunolabeled cells were evenly distributed across the retina, but clearly isolated from their neighbors as opposed to the mosaic of alternating red- and green-sensitive cones observed in the WT and six7^p23ahub mutant larvae (Fig 4E). In situ hybridization using an RH2 probe or immunolabeling for green-sensitive opsin demonstrated that six7^fl4/fl4 larvae were mostly devoid of green-sensitive opsin expression with only a few pairs of labeled cells in either eye (Fig 4F and inset); immunolabeling of histological sections confirmed the phenotype (S4 Fig). By comparison, homozygous six7^p23ahub mutants show variable penetrance of the loss of green-sensitive opsin expression phenotype; 83% of six7^p23ahub homozygous larvae showed a WT labeling pattern and only 17% showed labeling similar to the six7^fl4 mutants (Fig 4F).Co-labeling with a rhodopsin antibody showed that all six7^p23ahub mutants retained the increased rod number regardless of the presence or absence of the green-sensitive opsin labeling (Fig 4G), and green-sensitive opsin positive photoreceptors do not co-label with several different rod markers, suggesting that the extra rods in the six7^p23ahub mutant retinas are not a rod-cone hybrid.

The similarities and differences in phenotypes of the hypomorphic and knock-out alleles from our lab and previously reported [58] suggest that six7 regulates two distinct processes in photoreceptor cell genesis: terminal mitosis and differentiation or survival of green-sensitive cones precursors. Consistent with our hypothesis, confocal images of DAPI-labeled retinas from homozygous six7^fl4 larvae showed gaps in the photoreceptor mosaic and small intensely labeled structures consistent with nuclei of dead or dying cells (Fig 4H). TUNEL was performed on six7^fl4 retinas at 56 hpf, a time when expression of the all opsin subtypes should be detected [70], [87], and at 96 hpf, when all of the cones are mature. Few apoptotic nuclei were detected in WT or mutant retinas at 56 hpf. However, as development progressed to 96 hpf, considerable labeling of TUNEL positive cells was observed in the ONL of six7^fl4 retinas (Fig 4H). Labeling was also observed in the INL and in fibers extending across both plexiform layers to the inner limiting membrane. The morphology and labeling pattern are consistent with that of Muller glial cells which have been shown to become TUNEL positive from phagocytosis of cellular debris following photoreceptor degeneration [88], [89], although we cannot rule out the possibility that a small population of cells in the inner retina is also dying. Together, the data show that six7 is essential for development of green-sensitive cone precursors in the zebrafish retina and in its absence the precursors die.

six7 functions cell autonomously to regulate rod number and green-sensitive cone precursor survival

Genetic chimeras were generated to further test the cell autonomy of six7 in photoreceptor biology. At blastula stage, cells were transplanted from rhodamine dextran-injected six7^fl4 mutant donors into equivalent stage WT hosts. As control, WT cells were transplanted into WT embryos at the same developmental stage. The fate of WT vs six7^fl4 donor cells showed statistically significant differences based upon co-immunolabeling host embryos for green-sensitive opsin and a rod marker (χ², p<0.0001). Of 96 six7^fl4 rhodamine positive donor cells in the ONL of 3 hosts, 49 cells (51%) co-labeled with a rod specific marker consistent with the data from the MO-injected genetic mosaics, but only 2 cells (2%) immunolabeled for green-sensitive opsin. However, the neighboring host cells frequently labeled for green-sensitive opsin (Fig 5A). In stark contrast, 16% of 106 rhodamine-labeled WT-donor cells co-labeled for the green-sensitive opsin, but only 5.6% for the rod-specific marker. These data are consistent with a cell-autonomous role of six7 in regulating rod number and green-sensitive cone precursor differentiation or survival.

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Fig 5. six7 acts cell autonomously.

(A) six7^fl4-donor cells or (B) WT-donor cells were labeled by the tracer rhodamine-dextran, transplanted into WT genetic background and allowed to develop until 4 dpf. Expression of rods (4C12, green) and green-sensitive opsin (pink) were detected by whole mount immunolabeling. Note that six7^fl4-transplanted cells frequently differentiate as rod photoreceptors (orange, arrows) and rarely immunolabeled for green-sensitive opsins while neighboring WT cells differentiate as green-sensitive cones. Significant difference was observed in the percent of six7^fl4-donor cells that differentiate into rods or cones compared to WT donor cells (χ², p<0.0001).

https://doi.org/10.1371/journal.pgen.1005968.g005

Zebrafish lines and maintenance

Zebrafish (Danio rerio) were reared, bred and staged according to standard methods [106]. ljr^p23ahub was isolated from a three-generation screening of N-ethyl-N-nitrosurea-mutagenized zebrafish immunolabeled for rods at 5 dpf [88]. Mutagenesis was performed at the University of Pennsylvania as previously described [107]. The lor^p25bbtl mutant was previously characterized [53]. The six3b^vu87 mutant was previously described [69] and was generously provided by Dr. Solnica-Krezel (Washington University, St. Louis, MO). All animal procedures were approved by the Florida State University (FSU) Institutional Animal Care and Use Committee, ACUC Protocol #1421. Animals were anesthetized using MS222 and euthanized in ice water.

Mapping and sequencing

ljr^p23ahub mutant embryos from mating heterozygous adults were identified by immunolabeling as previously described [53]. Linkage mapping was performed at the Zebrafish Mapping Facility at the University of Louisville from DNA isolated from 100 ljr^p23ahub mutant- and 100 WT sibling embryos using simple sequence-length polymorphism markers. Fine resolution mapping was performed with 463 ljr^p23ahub mutant embryos [108]. Genomic DNA from 118 ljr^p23ahub mutant embryos was isolated (DNeasy Blood 7 Tissue Kit; Quiagen, Valencia, CA, USA) and used for Illumina sequencing at the University of Texas Genomic Sequencing and Analysis Facility as previously described [109]. Reads were aligned to the zv9 Zebrafish genome assembly (ensembl) with BWA [110] using default parameters. Reads with alignment quality of at least 30 were used identify SNPs against the zv9 genome assembly using samtools mpileup and bcftools 0.1.19 [111]. SNP densities were calculated using bedtools2 [112]. Data was visualized using the UCSC genome Browser [113], [72]. Genomic DNA was isolated from tail-clip of adult zebrafish and the candidate deleted region was confirmed by PCR in: ljr^p23ahub mutants (n = 9) and WT embryos from: AB genetic background (n = 6), TL genetic background (n = 6) using the primers listed in S1 Table. PCR of six7 fragment was used as positive control using primers listed in S1 Table.

Morpholino injections

One of three different morpholinos (MO) were injected into one-cell stage WT embryos (Gene Tools, LLC, Philomath, OR): mispaired-control MO, 5’-CGAACGCCATTCCGAGTCTGACTAAC-3’; antisense nucleotide targeting six7 5’-UTR (MO1),5’-CCAACGGCATTCCAGTGTGAGTAAC-3’ [73]; and six7 splice-blocking MO (MO3), 5’-GTACTTTTTGGTCTCACCTTAAAGC-3’. Unless otherwise stated, embryos were injected with 0.87 ng of the indicated MO. To confirm the efficiency of MO3, RNA was isolated from un-injected and MO3-injected embryos and the region spanning from exon 1 to exon 2 of the six7-transcript was amplified by PCR using primers listed in S1 Table. The truncated six7-transcript was sequenced using Applied Biosystems 3730 Genetic Analyzer with Capillary Electrophoresis (Foster City, CA).

Genome editing by TALENs

TALEN expression vectors were constructed in the Mutation Generation and Detection Core, University of Utah to target the exon 1 of six7 transcript. DNA plasmids were linearized by NotI (Invitrogen, Carlsbad, CA) and used as templates for TALEN mRNA synthesis with SP6 mMESSAGE mMACHINE Kit (Ambion, Austin, TX). To target the six7 genomic sequence, 50–200 pg of the pair of TALEN mRNAs were injected in one-cell stage zebrafish embryos. Injected embryos were raised to adulthood and crossed to WT animals to generate the F1. DNA was extracted from either F1 embryos (groups of three to six embryos) from the outcross of founders or tail clips from adult F1 fish. To screen for insertions and deletions (indels), DNA was extracted and used as the PCR template to amplify the six7-TALENs targeted region using primers listed in S1 Table. The DNA fragment was subjected to restriction fragment length polymorphism (RFLP) assay. Indels were tracked by loss of HaeIII (Invitrogen) restriction enzyme site in the targeted region. PCR products were sequenced to characterize the indels. The F1 embryos of positive founders were intercrossed to generate the F2 generation. F1 and F2 embryos were fixed in 4% paraformaldehyde in 80% phosphate-buffered saline (PFA/PBS) and processed for whole-mount rod immunolabeling as described (see Immunohisto-and immunocytochemistry).

Real-time quantitative PCR

RNA extraction was performed in TRIzol (Invitrogen) from pool of whole embryos (n = 30) at 10 hpf, 18 hpf, 24 hpf and 52 hpf. Transcription into cDNA was performed using SuperScript™ II Reverse Transcriptase (Invitrogen). Real time quantitative PCR (RT-qPCR) was carried out using a 7500 Real-Time PCR Systems (Applied Biosystems) with SRBY-Green PCR Master Mix (Applied Biosystems) and the primers listed in S1 Table. Three biological replicates were performed for each developmental time and were duplicated for each cDNA sample for six7 qRT-PCR. The fold expression change was normalized to β-actin using the 2^-∆∆CT (Livak) method [114]. Student’s t test was applied for comparison between groups at each developmental time.

Immunohisto-and immunocytochemistry

Immunolabeling of larvae whole mount or cryosections (10 μm) was performed as previously described [88]. Sections and enucleated eyes from whole-mounted immunolabeled larvae were imaged using either a Zeiss Axiovert S100 fluorescent microscope (Carl Zeiss Inc., Thornwood, NY) or a LSM 510 or LSM 710 (Carl Zeiss) Laser Confocal equipped with a 40x C-Apochromat water immersion objective (N.A. 1.2). The following primary antibodies were used: monoclonal antibody 4C12 that labels rods (1:200, [115]), a monoclonal antibody zpr1 that labels double cone cells (arr3a) (1:20, [62]), a monoclonal antibody 1D1 against rhodopsin [115] a polyclonal antibody against zebrafish blue-, red-, green- or UV-sensitive cone opsin (1:200, [12]), Zn8 which recognizes ganglion cells (1/10, ZIRC), 5E11 that labels amacrine cells ([115], PKCα that labels bipolar cells (1/100, [57]), CAZ which recognizes Muller glia (1/100, [116]) and polyclonal PH3 antibody that labels mitosis marker phospho-Histone 3 (1:500; Cat. No. 06–570, Millipore, Billerica, MA). Host-specific, Alexa fluor-conjugated secondary antibodies (Invitrogen) were used at a dilution of 1:200. Sections were counterstained with 4′,6-diamidino-2-phenylindole, dihydrochloride (DAPI, 1:15000; Sigma-Aldrich).

Proliferation was assessed by incubation of 48 hpf and 52 hpf embryos in fish water with 1.5 mM EdU (5-ethynil-2’-dexyuridine) during 30 minutes and subsequently fixed in 4% paraformaldehyde (PFA/PBS). The EdU labeling was processed by the Click-iT EdU Alexa Fluor 546 Imaging kit (Invitrogen) following the manufacturer’s instructions. For lineage tracing cell experiments, the EdU was washed with fish water and the embryos were incubated until 4 dpf and subjected to immunohistochemistry.

Light and transmission electron microscopy

Larvae were euthanized with tricaine and processed according to the protocol by [117], with slight modifications. Briefly, larvae were fixed overnight with 1% glutaraldehyde and 1% osmium tetroxide in 0.1 M cacodylate buffer. The eyes were then washed 3 times and dehydrated through a graded (70%, 75%, 90%, 100%, 100%, 100%) acetone-water series. The tissue was infiltrated overnight at room temperature in a 1:1 mixture of epoxy resin and 100% acetone. Larvae were then embedded in epoxy resin and placed in a 60°C oven for 22 hours. 1μm sections were cut with a microtome and mounted on glass slides and stained with 1% methylene blue in 1% sodium borate. Photomicrographs were taken with a Zeiss Axiovert microscope, and images were captured by the Zeiss Axiocam Digital Camera and processed using the Axiovision software. For electron microscopy, sections were collected on copper grids and viewed with a FEM CM 120 transmission electron microscope. Electron micrographs were taken using a Tietz Tem-Cam F224 slow scan CCD camera, prior to being imported into Photoshop version 8.0 (Adobe Systems).

Quantitative analysis

Confocal images from whole eyes immunolabeled for UV-sensitive cones and rods were analyzed with the Scion Image Software (Scion Corp, Frederick, MA). Areas of 3500 μm² located dorsal to the optic nerve [53] were counted for rods and UV-sensitive opsin expressing cones in WT (n = 5); lor^p25bbtl (n = 5); ljr^p23ahub (n = 5); double mutant lor^{p25bbtl/ljrp23ahub} (n = 5) at 4 dpf. Quantification of rods was conducted similarly as for WT (n = 5); ljr^p23ahub (n = 5) and ljr^p23ahub/+ (n = 6) an independent experiment. In addition, rods were similarly counted in WT (n = 8); six7 ^fl4/fl4 (n = 7), six7 ^fl4/p23ahub (n = 4). The number of red-green-sensitive cones was quantified in WT (n = 6); ljr^p23ahub (n = 6); and in independent experiment WT (n = 3); six7 ^p23ahub (n = 6); six7 ^fl4 (n = 3). When possible two of 3500 μm² retinal areas were counted. six7-MO1 injected retinas (n = 5–6 retinas/each MO1 dose) were imaged and rods were quantified at 4 dpf for 3500 μm² area. Un-injected (n = 4) retinas were used as controls. The average number of UV-, Arr3a-positive or rod-positive cells per unit area and the standard deviation (SD) were reported. One-way ANOVA with Tukey’s post-hoc test was used to compare means of rods or UV-sensitive cones between different genotypes. Student t test was applied to compare two sample data.

The number of PH3 positive cells in the ONL and INL was quantified using 10-μm-thick-retina sections of 21 000 μm² area per section that excluded the CMZ. The following strains and number (n) of 48-hpf retinas were analyzed: WT (n = 5), six7-MO1 injected embryos (n = 4). A Student’s t-test was conducted to compare the number of PH3 positive cells in ONL and INL between WT and six7-knockdown retinas. Same procedure was used to count PH3 positive cells in the ONL and INL of six7 ^fl4/f+ (n = 5) and six7 ^fl4/fl4 (n = 11). Un-paired Student t test with Welch’s correction was used for statistical analysis.

Quantitative analysis of photoreceptor pattern was performed as described [59]. Nearest Neighbor Dispersion Analysis (NNDA) was determined using Biotas and the conformity ratio was calculated and analyzed for randomness using the Ready-Reckoner Chart of Cook [61]. Fluorescent structures were assigned (x, y) coordinate using ImageJ software (National Institutes of Health Windows version, (http://rsbweb.nih.gov/ij/index.html). For each point in the field Nearest Neighbor Distance (NND) was calculated using Biotas (Version 1.02; Ecological software Solutions).as previously described [59].

Whole-mount in situ hybridizations

Whole-mount in situ hybridizations were performed as previously described [118] using pools of 25 embryos at 28 hpf and between 46–52 hpf. The antisense riboprobes were: six7 (this study), vax2 (this study), cyp26c1 (this study) and tbx2b [53]. The plasmids containing the probes for neurod [80], crx [76], pax6a [119], rx1 [120] were kindly provided by A.C. Morris (University of Kentucky, Lexington, KY). To prepare a probe for six7, a 444 bp fragment of six7 gene was amplified from a cDNA fragment obtained from 10 hpf embryos, using primers listed in S1 Table and cloned into the vector PCR2.1-TOPO (Invitrogen). Antisense RNA probe was synthesized with a digoxigenin RNA-labeling kit (Roche, Indianapolis, IN) by in vitro transcription with T7 RNA polymerase, according to the manufacturer's instructions. A 620 bp of vax2-cDNA fragment and a 692 bp of cyp26c1 were amplified using primers listed in S1 Table. The antisense probes were prepared as described above. The hybridized probe was detected with alkaline phosphatase coupled with anti-digoxigenin antibodies and NBT/X-phosphate substrate (Roche). Labeled embryos were cleared in a graded series of glycerol and viewed on a Zeiss Axiovert S100 microscope. Images were captured by Carl Zeiss Axiocam Color Microscope camera and processed with Axiovision SE64 Rel 4.9.1 and Photoshop 5.5 (Adobe, Mountain View, CA) software.

TUNEL assay

Terminal deoxynucleotide transferase (TdT)-mediated dUTP nick labeling (TUNEL) was performed on 3 dpf retinal cryosections using the ApopTag Red In Situ Apoptosis Detection Kit (Millipore, Temecula, CA) according the manufacturer’s instructions and co-labeled for rods (4C12) to identify mutants. TUNEL positive cells were counted in the ONL, INL and GCL from: WT (n = 7); ljr (n = 9); control (n = 7) and six7-MO1 (n = 6), one section for individual embryo. TUNEL positive cell counts were transformed (log Y+1) before student t test was conducted. TUNEL assay was performed in six7^fl4 mutants and WT embryos at 56 hpf and 4 dpf. Tail-clip genotyping was used to identify mutants at 56 hpf. The following strains and number (n) of 56 hpf were analyzed: WT (n = 5), six7^fl4 (n = 7) and at 4 dpf: WT (n = 3), six7^fl4 (n = 6).

Cell transplantation

Genetic chimeras were generated as previously described [121]. Donor embryos were injected at the 1- and 2-cell stage with the lysine-fixable, dextran-conjugated Alexa Fluor 594 (Invitrogen). Donor blastulae cells were transferred to unlabeled host cells. At 4 dpf the chimeras were fixed with 4% PFA/PBS and immunolabeled for rods and green-opsin as described above. Imaging of the whole-dissected eyes was performed by confocal microscopy (WT into WT, n = 3; six7^fl4 into WT n = 3; WT into WT, n = 5; six7-MO1 into WT, n = 6). The number of rhodamine-dextran labeled cells, 4C12/dextran (rod from donor cells)-labeled and green-opsin/dextran labeled cells were quantified. The percentage number of donor cells differentiated as rod photoreceptor or green-sensitive cones were compared for six7^fl4 mutants vs WT transplants into WT background. Numbers of donor cells were counted across the retinal layers from retinal sections of six7-MO into WT. Statistical analysis was performed by chi-square test.