A monoclonal antibody raised in rat (10C9.1) labels zebrafish UV cone outer segments, allowing all cone subtypes to be simultaneously labeled by immunohistochemistry.

<p>Antibody 10C9.1 specifically labels the outer segments of a class of short single cones in the adult zebrafish retina (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092991#pone.0092991.s001" target="_blank">Figure S1</a> panel A). 10C9.1 specificity is supported (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092991#pone.0092991.s001" target="_blank">Figure S1</a> panels B–D), including by a dramatic decrease in number of cells labeled when 10C9.1 is applied to retinas from zebrafish mutants (<i>tbx2b^lor/lor</i>) that have a paucity of UV cones. A. The population of single cones labeled by 10C9.1 is the UV cones, because established antibodies against the other single cone class, the blue cones, labels a distinct cone population (A′). 10C9.1 enables an unprecedented combination of antibodies raised in different species that simultaneously label and distinguish all cone photoreceptor subtypes (A”). E. Further evidence that 10C9.1 labels UV cone outer segments comes from its co-localization with UV cones filled with green fluorescent protein (GFP), and its exclusion from blue cones filled with mCherry (mCh) in transgenic zebrafish (<i>Tg(-5.5opn1sw1:EGFP)kj9;Tg(-3.5opn1sw2:mCherry)ua3011</i>). Panel B is available as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092991#pone.0092991.s004" target="_blank">Movie S1</a>. Scale bars 30 μm.</p

<i>gdf6a</i> modulates <i>tbx2b</i> regulation of UV cone and rod development.

<p><b>A, B</b>. When [<i>gdf6a^+/s327;tbx2b^+/lor</i>] compound heterozygous mutants are in-crossed (inx), a disproportionate fraction of microphthalmic offspring exhibit the <i>lots-of-rods</i> phenotype compared to normophthalmic siblings, <i>tbx2b^+/lo</i>^r in-crosses, and to predicted Mendelian ratios of the recessive <i>lots-of-rods</i> phenotype (<i>X</i>² ***p<0.001; 3 replicates of n = 17, 19, 35 microphthalmics; 6dpf). UV cones and rods were labeled using antibodies 10C9.1 and 4C12 displayed in magenta and green, respectively. A portion of microphthalmic larvae with the <i>lots-of-rods</i> phenotype has a <i>tbx2b^+/lor</i> genotype (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092991#pone-0092991-t001" target="_blank">Table 1</a>). <b>C, D</b>. When [<i>gdf6a^+/s327</i>;<i>tbx2b^+/fby</i>] compound heterozygous mutants are in- crossed, the <i>lots-of-rods</i> phenotype is again observed at higher rates in microphthlamic eyes compared to normophthalmic eyes (<i>X</i>² *p = 0.007; 1 replicate, n = 39 microphthalmics, 6 dpf). Panel D shows rod opsin in situ hybridization (red). Scale bars are all 50 μm. <b>E</b>. Genotyping for the <i>lor</i> mutation was performed via linkage analysis using an A/T synonymous SNP located before the DNA binding domain of <i>tbx2b</i> in <i>lor</i> and non-<i>lor</i> alleles, respectively. <i>gdf6a^s327/s327</i> mutants with a corresponding SNP of T were used in crossing of the mutant lines.</p

<i>gdf6a</i> positively modulates the abundance of <i>tbx2b</i> transcript during stages of retinal development when photoreceptors differentiate.

<p>All panels show in situ hybridization using <i>tbx2b</i> riboprobe. <i>gdf6a^s327/s327</i> mutants have less tbx2b expression at 3 days post-fertilization (dpf) compared to normophthalmic siblings, akin to tbx2blor/lor mutants. Fractions represent proportion of clutch represented by image shown. Scale bars 100 μm; inl, inner nuclear layer.</p

Disruption of <i>tbx2b</i> does not modify the <i>gdf6a</i> microphthalmic phenotype.

<p><b>A</b>. <i>gdf6a^s327/s327</i> mutants exhibit microphthalmia (observed at 3dpf) but <i>tbx2b^−/−</i> mutants (<i>lor</i> and <i>fby</i>) do not, indicating that disruption of <i>tbx2b</i> does not interfere with identical pathways as <i>gdf6a</i> in early eye development. <b>B</b>. Microphthalmia is rarely observed in <i>tbx2b</i> mutant in-crosses (inx) alone (<i>tbx2b^+/lor</i> in-cross shown, n = 220) compared to in-crosses of <i>gdf6a^+/s327</i>, which yield 25% with microphthalmia (following Mendelian ratios of inheritance and recessive phenotype). When [<i>gdf6a^+/s327;tbx2b^+/lor</i>] or [<i>gdf6a^+/s327;tbx2b^+/fby</i>] compound heterozygous mutants are in-crossed (n = 121 and 195 respectively, both at 6dpf), rates of microphthalmia do not increase significantly from rates expected of in-crosses of <i>gdf6a^+/s327</i> alone (<i>X</i>² p = 0.873 and p = 0.137, respectively). <b>C</b>. The eye size compared to body length (shown as ratio) of a [<i>gdf6a^+/s327;tbx2b^+/lor</i>] in-cross does not reveal a subset of intermediate eye sizes, but remains bimodal, with the normophthalmic curve (right curve) showing a normal distribution (Shapiro-Wilk Normality test, W = 0.9888, p = 0.6532) (n = 118, 4dpf).</p

Primers used to identify single nucleotide polymorphisms (SNPs) for <i>tbx2b</i> genotyping.¹

1<p>SNP names from <a href="http://www.ncbi.nlm.nih.gov/snp/" target="_blank">http://www.ncbi.nlm.nih.gov/snp/</a>, SNP ultimately used for genotyping in this study is in bold.</p

Identification of compound [<i>gdf6a^s327/s327</i>; <i>tbx2b</i>] mutants with mismatched phenotype and genotype regarding <i>tbx2b</i>.

<p>Identification of compound [<i>gdf6a^s327/s327</i>; <i>tbx2b</i>] mutants with mismatched phenotype and genotype regarding <i>tbx2b</i>.</p

Mutation in <i>gdf6a</i> does not disrupt tbx2b function in UV-versus-rod photoreceptor specification, but <i>gdf6a</i> rather plays a role in blue cone specification.

<p>A,B. <i>tbx2b^lor/lor</i> mutants have fewer UV cones and more rods than wildtype fish (the <i>lots-of-rods</i> phenotype) (Kruskall-Wallis ANOVA, **p<0.005), but <i>gdf6a^s327/s327</i> mutants have a normal abundance ratio and distribution of UV cones and rods (n = 10 wildtype, 8 <i>tbx2b^lor/lor</i>, and 7 <i>gdf6a^s327/s327</i>; UV cones expressing GFP and rods were labeled with antibody 4C12). Scale bars 30 μm and 80 μm, respectively. C. Larval <i>gdf6a^s327/s327</i> mutants have a unique cone photoreceptor phenotype in which there are significantly fewer blue cones relative to UV cones at all ages examined (which is not observed in <i>tbx2b^lor/lor</i> or <i>tbx2b^fby/fby</i> mutants- not shown) (Kruskall-Wallis ANOVA, ***p<0.001) Sample sizes at 3 days post-fertilization (dpf) are n = 17 larvae per genotype quantifying cells visualized via opsin in situ hybridization (Panel D); at 4 dpf data are from n = 9 wild type and n = 13 mutants assessed via GFP and mCherry transgene expression in cones; at 6dpf data are from 2 replicates of n = 4+7 wild type and n = 5+6 mutants assessed via transgene expression in cones (Panel E). D. UV and blue cones identified in 3 dpf by in situ hybridization against their respective opsins (Scale bars are 100 μm). E. UV and blue cones identified in transgenic lines at 6dpf by expression of GFP and mCherry, respectively (Scale bars are 60 μm and 40 μm in sibling and mutants, respectively).</p

<i>gdf6a</i> and <i>tbx2b</i> mutants do not share the microphthalmic phenotype, despite a shared pathway in early eye development.

<p>A. <i>gdf6a^s327/s327</i> mutants (labeled <i>gdf6a^−/−</i> in figures) exhibit microphthalmia to varying degrees of severity during development and throughout adulthood, unlike their wild type and heterozygous siblings. <i>tbx2b</i> mutants do not exhibit microphthalmia, and their eyes develop normally. Scale bars 2 mm. B, C, D. Coronal sections of adult zebrafish heads, comparing microphthalmic <i>gdf6a^s327/s327</i> (B) and wildtype fish (B′). Microphthalmia and anophthalmia present variably in <i>gdf6a^s327/s327</i> fish (e.g. right and left eyes in B, respectively) and eyes are often noted to possess a lens (L), though in this instance the right eye is inverted such that the anterior segment is oriented towards the midline. RPE (r) and a thin layer of photoreceptors (p) are discernable in <i>gdf6a^s327/s327</i> fish (C′), though other retinal layers are not recognizable due to multiple tissue infoldings. In panel D, the lens was presumably displaced away from the iris during dissection/fixation. Note C is at higher magnification compared to D. Scale bar in B 1 mm; C, D is .5 mm; C′, D′ is .1 mm. L, lens; v, vitreous; r, RPE layer; p, photoreceptor layer.</p

Regeneration of Cone Photoreceptors when Cell Ablation Is Primarily Restricted to a Particular Cone Subtype

<div><p>We sought to characterize the regenerated cells, if any, when photoreceptor ablation was mostly limited to a particular cone subtype. This allowed us to uniquely assess whether the remaining cells influence specification of regenerating photoreceptors. The ability to replace lost photoreceptors <em>via</em> stem cell therapy holds promise for treating many retinal degenerative diseases. Zebrafish are potent for modelling this because they have robust regenerative capacity emanating from endogenous stem cells, and abundant cone photoreceptors including multiple spectral subtypes similar to human fovea. We ablated the homolog of the human S-cones, the ultraviolet-sensitive (UV) cones, and tested the hypothesis that the photoreceptors regenerating in their place take on identities matching those expected from normal cone mosaic development. We created transgenic fish wherein UV cones can be ablated by addition of a prodrug. Thus photoreceptors developed normally and only the UV cones expressed nitroreductase; the latter converts the prodrug metronidazole to a cell-autonomous neurotoxin. A significant increase in proliferation of progenitor cell populations (p<0.01) was observed when cell ablation was primarily limited to UV cones. In control fish, we found that BrdU primarily incorporated into rod photoreceptors, as expected. However the majority of regenerating photoreceptors became cones when retinal cell ablation was predominantly restricted to UV cones: a 2-fold increase in the relative abundance of cones (p = 0.008) was mirrored by a 35% decrease in rods. By primarily ablating only a single photoreceptor type, we show that the subsequent regeneration is biased towards restoring the cognate photoreceptor type. We discuss the hypothesis that, after cone death, the microenvironment formed by the remaining retinal cells may be influential in determining the identity of regenerating photoreceptors, though other interpretations are plausible. Our novel animal model provides control of ablation that will assist in identifying mechanisms required to replace cone photoreceptors clinically to restore daytime vision.</p> </div

Regeneration investigated via a cell-specific ablation method.

<p>We engineered fish where a subset of ultraviolet-sensitive (UV) cone photoreceptors exclusively express the NTR-mCherry transgene (A). Upon treatment with a metronidazole (MTZ) prodrug solution (trapezoid), nitroreductase (nfsb gene, NTR protein) converts MTZ into a cell-autonomous cytotoxin (B, C, represented as a triangle), resulting in DNA crosslinking and ablation of only the cones expressing NTR (D), without disruption to neighbouring cells. Following cell death, BrdU is applied and it incorporates into DNA of proliferating cells (magenta nuclei). Regeneration of the targeted photoreceptors occurs once the MTZ treatment is removed (E).</p