Genetic Dissection of Dual Roles for the Transcription Factor six7 in Photoreceptor Development and Patterning in Zebrafish.

The visual system of a particular species is highly adapted to convey detailed ecological and behavioral information essential for survival. The consequences of structural mutations of opsins upon spectral sensitivity and environmental adaptation have been studied in great detail, but lacking is knowledge of the potential influence of alterations in gene regulatory networks upon the diversity of cone subtypes and the variation in the ratio of rods and cones observed in numerous diurnal and nocturnal species. Exploiting photoreceptor patterning in cone-dominated zebrafish, we uncovered two independent mechanisms by which the sine oculis homeobox homolog 7 (six7) regulates photoreceptor development. In a genetic screen, we isolated the lots-of-rods-junior (ljrp23ahub) mutation that resulted in an increased number and uniform distribution of rods in otherwise normal appearing larvae. Sequence analysis, genome editing using TALENs and knockdown strategies confirm ljrp23ahub as a hypomorphic allele of six7, a teleost orthologue of six3, with known roles in forebrain patterning and expression of opsins. Based on the lack of predicted protein-coding changes and a deletion of a conserved element upstream of the transcription start site, a cis-regulatory mutation is proposed as the basis of the reduced expression of six7 in ljrp23ahub. Comparison of the phenotypes of the hypomorphic and knock-out alleles provides evidence of two independent roles in photoreceptor development. EdU and PH3 labeling show that the increase in rod number is associated with extended mitosis of photoreceptor progenitors, and TUNEL suggests that the lack of green-sensitive cones is the result of cell death of the cone precursor. These data add six7 to the small but growing list of essential genes for specification and patterning of photoreceptors in non-mammalian vertebrates, and highlight alterations in transcriptional regulation as a potential source of photoreceptor variation across species

TALENs-mediated knockout of <i>six7</i> locus recapitulates <i>ljr</i>^{<i>p23ahub</i>} phenotype.

<p>(A) Schematic representation of the Six7 protein domains. <i>six7</i>-TALENs target site and the Hae<i>III</i> restriction enzyme site are highlighted. The left and right monomer binding sites are underlined. (B) RFLP of <i>six7</i> locus by Hae<i>III</i>. A new 170 bp DNA-fragment is detected in <i>six7</i>^<i>fl4</i> carriers compared with controls. (C) Sequence analysis of the <i>six7</i> target region shows recovery of multiple indel alleles of <i>six7</i>. The target region is highlighted in red. Dots represent deletions and lower case letters indicate insertions. The <i>six7</i> sequence from WT is shown as a comparison. (D) Confocal immunofluorescent images labeled for rods (red) from WT and <i>six7</i>^<i>fl4</i> knockout mutants at 4 dpf. <i>six7</i>^<i>fl4</i> knockout mutants phenocopy <i>ljr</i>^{<i>p23ahub</i>} mutants. Sequencing chromatograms of WT and <i>six7</i>^<i>fl4</i> mutants illustrated the c.217_229del CAGGTGGCCCGAG (del13) <i>six7</i> mutation in homozygous zebrafish. The bar graph shows the average number of rods counted in 1–3 different areas per retina (WT (n = 8), <i>six7</i>^<i>fl4</i> (n = 7), <i>six7</i> ^{<i>fl4/p23ahub</i>} (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, <i>six7</i>^{<i>p23ahub</i>}, and <i>six7</i>^<i>fl4</i> retinas at 4 dpf. Graph showing the average number of red/green-sensitive cones per unit area of WT (n = 3), <i>six7</i>^{<i>p23ahub</i>} (n = 4), and <i>six7</i>^<i>fl4</i> (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 <i>in situ</i> hybridization for <i>RH2</i> probe (green-sensitive cone opsin) in WT (n = 30), <i>six7</i>^{<i>p23ahub</i>} (n = 30) and <i>six7</i>^<i>fl4</i> embryos (n = 30) at 4 dpf. WT and <i>six7</i>^{<i>p23ahub</i>} embryos often showed the same pattern and number of green-sensitive opsin cone labeling (Dorsal is up and nasal to the left), while <i>six7</i>^<i>fl4</i> 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 <i>six7</i>^{<i>p23ahub</i>} embryos were un-labeled for green-sensitive cone opsin. (G) Retinal cryosections of <i>in situ</i> hybridization for green-sensitive cone opsin in <i>six7</i>^{<i>p23ahub</i>} (n = 5) embryos immunolabeled with 1D1 (rods). Rods and green-sensitive cone opsin probes labeled different cells in the ONL of <i>six7</i>^{<i>p23ahub</i>} embryos. (H) Evidence of cell death. Flat mount confocal image of nuclei counterstained with DAPI and retinal cryosections from WT and <i>six7</i>^<i>fl4</i> animals at 4dpf co-labeled for TUNEL (red) and rods (4C12, green). <i>six7</i>^<i>fl4</i> 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).</p

<i>ljr</i>^{<i>p23ahub</i>} mutants display an increased number and uniform distribution of rod photoreceptors.

<p>(A) Confocal immunofluorescent images labeled for rods (red) and UV-sensitive cones (green) from WT, ljr^{<i>p23ahub</i>}, <i>lor</i>^{<i>p25bbtl</i>} and homozygous <i>lor</i>^{<i>p25bbtl</i>}/ljr^{<i>p23ahub</i>} 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^{<i>p23ahub</i>} and <i>lor</i>^{<i>p25bbtl</i>} mutants demonstrate an increased number and uniform arrangement of rods but fewer UV-sensitive cones labeled in <i>lor</i>^{<i>p25bbtl</i>}. 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; <i>ljr</i>^{<i>p23ahub</i>}, n = 5; <i>lor</i>^{<i>p25bbtl</i>}, n = 5; <i>ljr</i>^{<i>p23ahub</i>}<i>/lor</i>^{<i>p25bbtl</i>}, 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; <i>ljr</i>^{<i>p23ahub/+</i>}, n = 6; <i>ljr</i>^{<i>p23ahub</i>}, 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. <i>ljr</i>^{<i>p23ahub</i>} 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; <i>ljr</i>^{<i>p23ahub</i>}, n = 6). No significant differences are observed, Student’s <i>t</i> test, p>0.05. Error bars represent SD.</p

<i>six7</i> acts cell autonomously.

<p>(A) <i>six7</i>^<i>fl4</i>-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 <i>six7</i>^<i>fl4</i>-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 <i>six7</i>^<i>fl4</i>-donor cells that differentiate into rods or cones compared to WT donor cells (<i>χ</i>^<i>2</i>, p<0.0001).</p

Photoreceptor progenitor proliferation is regulated by <i>six7</i>.

<p>(A) EdU (red) and <i>six7-in situ</i> labeling (blue) in a retinal cryosection from 48-hpf WT embryos. Note the expression of <i>six7</i> is coincident with proliferating cells in the ONL. (B) EdU labeling in retinal cryosections of WT, <i>six7</i>-MO control and <i>six7</i> morphants at 52 hpf. Note that EdU labeled cells persist in the central retina in <i>six7</i> morphants. (C) Retinal cryosections of WT and <i>six7</i> 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 <i>six7</i>-knockdown retinas. Proliferative cells colabeled for rod markers in the central retina, (arrowheads, <i>inset</i>). (D) Retinal cryosections of WT and <i>six7</i>-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 <i>six7</i>-MO1, n = 4; 3–4 sections/retina). Mitosis levels increased significantly in the ONL in <i>six7</i>-morphants. No significant changes were observed in the INL. (***p = 0.0007, Student’s <i>t</i> test). (F) Retinal cryosections of WT and <i>ljr</i>^{<i>p23ahub</i>} 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 <i>ljr</i>^{<i>p23ahub</i>} (n = 9); (*p<0.05, *****p<0.0001, Student’s <i>t</i> test calculated on log transformed data). (H) Transverse retinal sections from WT and <i>six7</i>-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 <i>six7</i>-MO1 injected embryos (n = 6); (*p<0.05, Student’s <i>t</i> 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.</p

Novel Identity and Functional Markers for Human Corneal Endothelial Cells

PURPOSE: Human corneal endothelial cell (HCEC) density decreases with age, surgical complications, or disease, leading to vision impairment. Such endothelial dysfunction is an indication for corneal transplantation, although there is a worldwide shortage of transplant-grade tissue. To overcome the current poor donor availability, here we isolate, expand, and characterize HCECs in vitro as a step toward cell therapy. METHODS: Human corneal endothelial cells were isolated from cadaveric corneas and expanded in vitro. Cell identity was evaluated based on morphology and immunocytochemistry, and gene expression analysis and flow cytometry were used to identify novel HCEC-specific markers. The functional ability of HCEC to form barriers was assessed by transendothelial electrical resistance (TEER) assays. RESULTS: Cultured HCECs demonstrated canonical morphology for up to four passages and later underwent endothelial-to-mesenchymal transition (EnMT). Quality of donor tissue influenced cell measures in culture including proliferation rate. Cultured HCECs expressed identity markers, and microarray analysis revealed novel endothelial-specific markers that were validated by flow cytometry. Finally, canonical HCECs expressed higher levels of CD56, which correlated with higher TEER than fibroblastic HCECs. CONCLUSIONS: In vitro expansion of HCECs from cadaveric donor corneas yields functional cells identifiable by morphology and a panel of novel markers. Markers described correlated with function in culture, suggesting a basis for cell therapy for corneal endothelial dysfunction

<i>six7</i> knockdown phenocopies the increase and uniform distribution of rods from <i>ljr</i>^{<i>p23ahub</i>} mutants.

<p>(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 <i>ljr</i>^{<i>p23ahub</i>} 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 [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005968#pgen.1005968.ref063" target="_blank">63</a>], read alignments from whole-genome sequencing of <i>ljr</i>^{<i>p23ahub</i>} mutants, and multiz-based sequence conservation UCSC genome browser tracks of <i>six7</i> across four fish species and <i>Six3</i> in frog, human, and mouse [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005968#pgen.1005968.ref072" target="_blank">72</a>]. Gel electrophoresis of PCR products of <i>six7</i> exon 1 and the upstream genomic region amplified from AB, TL and <i>ljr</i>^{<i>p23ahub</i>} genomic DNA. (B) Whole mount <i>in situ</i> hybridization shows <i>six7</i> 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 <i>ljr</i>^{<i>p23ahub</i>} embryos at 10 hpf, 18 hpf, 24 hpf and 52 hpf reveal down regulation of <i>six7</i> expression in <i>ljr</i>^{<i>p23ahub</i>} at 52 hpf. Relative transcript abundance was normalized to <i>actin</i> 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 <i>t</i> test, *p<0.05. (D) Retinal cryosection from 4 dpf un-injected control WT and <i>six7</i>-MO1 injected embryos immunolabeled for rods (4C12, green). <i>six7</i>-morphants display an increase in the number of rods as detected in <i>ljr</i>^{<i>p23ahub</i>}. Note the lack of gaps in rod distribution in the central retina of <i>six7</i>-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 <i>six7</i>-MO1 injected embryos (WT un-injected, n = 4; <i>six7-</i>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.</p

Tbx2b is required for ultraviolet photoreceptor cell specification during zebrafish retinal development

The vertebrate rod and cone photoreceptors are highly specialized sensory neurons that transduce light into the chemical and electrical signals of the nervous system. Although the physiological properties of cones and rods are well known, only a handful of genes have been identified that regulate the specification of photoreceptor subtypes. Taking advantage of the mosaic organization of photoreceptors in zebrafish, we report the isolation of a mutation resulting in a unique change in photoreceptor cell fate. Mutation of the lots-of-rods (lor) locus results in a near one-for-one transformation of UV-cone precursors into rods. The transformed cells exhibit morphological characteristics and a gene-expression pattern typical of rods, but differentiate in a temporal and spatial pattern consistent with UV-cone development. In mutant larvae and adults, the highly ordered photoreceptor mosaic is maintained and degeneration is not observed, suggesting that lor functions after the specification of the other photoreceptor subtypes. In genetic chimeras, lor functions cell-autonomously in the specification of photoreceptor cell fate. Linkage analysis and genetic-complementation testing indicate that lor is an allele of tbx2b/fby (from beyond). fby was identified by a pineal complex phenotype, and carries a nonsense mutation in the T-box domain of the tbx2b transcription factor. Homozygous fby mutant larvae and lor/fby transheterozygotes also display the lots-of-rods phenotype. Based upon these data, we propose a previously undescribed function for tbx2b in photoreceptor cell precursors, to promote the UV cone fate by repressing the rod differentiation pathway