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. 2020 Apr 9;29(10):1648–1657. doi: 10.1093/hmg/ddaa064

Otx2b mutant zebrafish have pituitary, eye and mandible defects that model mammalian disease

Hironori Bando 1, Peter Gergics 1,2, Brenda L Bohnsack 2, Kevin P Toolan 1, Catherine E Richter 3, Jordan A Shavit 3, Sally A Camper 1,✉
PMCID: PMC7322578  PMID: 32277752

Abstract

Combined pituitary hormone deficiency (CPHD) is a genetically heterogeneous disorder caused by mutations in over 30 genes. The loss-of-function mutations in many of these genes, including orthodenticle homeobox 2 (OTX2), can present with a broad range of clinical symptoms, which provides a challenge for predicting phenotype from genotype. Another challenge in human genetics is functional evaluation of rare genetic variants that are predicted to be deleterious. Zebrafish are an excellent vertebrate model for evaluating gene function and disease pathogenesis, especially because large numbers of progeny can be obtained, overcoming the challenge of individual variation. To clarify the utility of zebrafish for the analysis of CPHD-related genes, we analyzed the effect of OTX2 loss of function in zebrafish. The otx2b gene is expressed in the developing hypothalamus, and otx2bhu3625/hu3625 fish exhibit multiple defects in the development of head structures and are not viable past 10 days post fertilization (dpf). Otx2bhu3625/hu3625 fish have a small hypothalamus and low expression of pituitary growth hormone and prolactin (prl). The gills of otx2bhu3625/hu3625 fish have weak sodium influx, consistent with the role of prolactin in osmoregulation. The otx2bhu3625/hu3625 eyes are microphthalmic with colobomas, which may underlie the inability of the mutant fish to find food. The small pituitary and eyes are associated with reduced cell proliferation and increased apoptosis evident at 3 and 5 dpf, respectively. These observations establish the zebrafish as a useful tool for the analysis of CPHD genes with variable and complex phenotypes.

Introduction

Orthodenticle homeobox 2 (Otx2) is a homeobox gene with an essential role in the development of several craniofacial structures including the eye, jaw, and pituitary gland. It is expressed in the epiblast, anterior visceral endoderm, anterior definitive endoderm, and anterior neuroectoderm before and during gastrulation, and is involved in the patterning of the midbrain and forebrain (1). In humans, whole gene deletions and heterozygous loss-of-function mutations in OTX2 cause variable anomalies with incomplete penetrance. Most cases present with abnormalities in craniofacial (2) and ocular development (3) and hypopituitarism (4), including either isolated or combined pituitary hormone deficiency (CPHD) (5). Otx2 heterozygous mutant mice (Otx2+/−) have poor viability. At birth, Otx2+/− mice have variable features that mimic that of human patients. The phenotype ranges from acephaly, micrognathia, anophthalmia, microphthalmia to apparently normal development. Several genetic modifiers have been mapped, but not identified, in mice (6). Thus, the incomplete penetrance of OTX2 loss-of-function mutations in humans is likely caused by variation in other genes that enhance or suppress the phenotype (7).

Rodent models are costly for the analysis of genes like Otx2 that exhibit incomplete penetrance and highly variable phenotype. This is especially true with Otx2 because neonatal lethality necessitates the utilization of large numbers of pregnant females to obtain sufficient numbers of embryos for quantitative analysis. Conversely, hundreds of fertilized eggs can be obtained from one zebrafish female in a single spawning, facilitating the analysis of variable phenotypes by increasing the sample size. A few zebrafish studies have been reported with human CPHD-related gene mutants and morphants. These include fibroblast growth factor 3 (fgf3) (8), shh (9), gli1/2 (10), pitx3 (11) and pit1 (12). In some cases, pituitary and eye function were incompletely characterized, leaving it unclear whether the zebrafish is a reliable model for human CPHD-disease pathogenesis. Morpholino oligonucleotide (MO)-mediated knockdown of otx2b had variable, modest effects in zebrafish, although there was evidence that decreased otx2b exacerbated the effects of knockdown of other genes involved in head development such as pgap1, msx1 and prrx1 (2). Studies utilizing MO knockdown can be problematic because of off-target effects, the hypomorphic nature of the model, the retention of maternal mRNA, and the variability associated with microinjection (13). The use of genetically modified zebrafish can overcome these problems.

Here we report defects in the pituitary glands, mandibles and eyes of otx2b mutant fish that model the features of patients with OTX2 mutations. The results suggest that otx2b deficiency causes reduced cell proliferation and increased apoptosis, resulting in organ hypoplasia. In addition, the eye and pituitary hormone deficiencies likely cause the early death of otx2b mutant zebrafish.

Results

otx2b hu3625/hu3625 fish die between 11 and 12 days post fertilization (dpf)

In zebrafish, like the majority of teleosts, 30–40% of the genome is duplicated. Genome duplication during evolution results in 2 orthologs of the otx2 gene: otx2a and otx2b. These genes encode proteins with only 70% identical amino acids. Human and mouse OTX2 are identical, and they have 90% amino acid conservation with otx2b, but otx2a is more divergent. These data suggest that otx2b is the primary ortholog of mammalian OTX2. Therefore, we analyzed otx2b mutant fish.

Otx2bhu3625 fish, which were generated by ethyl-N-nitrosourea mutagenesis through the Zebrafish Mutation Project (Wellcome Sanger Institute) (14), carry an A to C point mutation in the essential splice acceptor site of intron 2 (c.250-2A > C) that is predicted to ablate splicing from exon 2 to exon 3 (Fig. 1A). Failure to splice is predicted to produce mRNA that would encode a truncated version of the protein, p.W85ter22, which is normally 289 amino acids. This A > C change generates a novel Msp1 site that is useful for genotyping WT, heterozygous and homozygous mutant fish (Fig. 1B). The Mendelian distribution of genotypes was as expected (1:2:1) through 10 dpf. However, there was an absence of otx2bhu3625/hu3625 fish at 13 dpf (Table 1) indicating that the homozygous mutants died between 11 and 12 dpf. The analysis of otx2b transcripts by reverse transcription-polymerase chain reaction (RT-PCR) in 5 dpf fish revealed reduction in transcripts spliced from exon 1 to exon 2 in otx2bhu3625/hu3625 fish in contrast to otx2bhu3625/+ and otx2b+/+ fish (Fig. 1C). Further, although transcripts spliced from exon 2 to exon 3 were readily detectable in otx2bhu3625/+ and otx2b+/+ fish, none were detected in otx2bhu3625/hu3625, suggesting that disruption of the splice site caused nonsense-mediated mRNA decay, as expected based on the position of the premature termination codon. Mutant mRNA degradation may be associated with the upregulation of paralogous genes as transcriptional genetic compensation (15). RT-PCR analysis revealed no change in the expression of otx2a, paralogue of otx2b, in either in otx2bhu3625/+ or otx2bhu3625/hu3625 (Fig. 1C).

Figure 1.

Figure 1

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The otx2bhu3625/hu3625 mutant fish have no functional otx2b transcripts. (A) otx2bhu3625 variant is an A to C mutation in an invariant part of the splice acceptor site upstream of exon 3. (B) The otx2bhu3625 mutation introduces an MspI digestion site within the mutant allele. The uncut WT product measures 315 bp, while the otx2bhu3625 product is cleaved by MspI into 217 bp and 98 bp fragments. (C) Otx2bhu3625/hu3625 homozygous fish had no detectable otx2b cDNA transcripts prior to the mutated splice site (exon 1–2) or containing the mutated splice site (exon 2–3). otx2a cDNA transcripts Genomic DNA produced the expected size bands for WT (+/+), heterozygotes (+/hu3625) and homozygotes (hu3625/hu3625). The detection of ef1a was used as an internal control. (D and E) Representative images and median measurement of body length at 10 dpf demonstrated no difference between +/+, heterozygotes (+/hu3625) and homozygotes (hu3625/hu3625). Sample numbers are 14 WT, 27 heterozygotes and 8 homozygotes.

Table 1.

otx2bhu3625/hu3625 zebrafish show early death before 13 dpf. Genotype distribution from the intercross of heterozygous otx2b+/hu3625 zebrafish matings

Age of assessment Analyzed number Number [+/+]−[+/hu3625]−[hu3625/hu3625] % [+/+]−[+/hu3625]−[hu3625/hu3625]
1 dpf 269 69–136—64 25.7–50.6—23.8
2 dpf 125 30–58—37 24.0–46.4—29.6
3 dpf 306 70–161—75 22.9–52.6—24.5
5 dpf 528 125–257—146 23.7–48.7—27.7
10 dpf 614 167–312—135 27.2–50.8—22.0
13 dpf 86 27–59—0 31.4–68.6—0
2 months 167 66–101—0 39.5–60.5—0
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dpf = days post fertilization.

otx2b hu3625/hu3625 have diminished pituitary hormone expression

Human OTX2 variants can be associated with GH deficiency and CPHD while heterozygous mouse mutants show missing or dysmorphic pituitary glands (16, 17). In zebrafish, pituitary progenitor cells in the hypophyseal placode are present by 19 h post fertilization (hpf) (18) and differentiate into prolactin (prl)-expressing cells by 1 dpf and growth hormone (gh)-expressing cells by 2 dpf. At 1 dpf, otx2bhu3625/hu3625 embryos had decreased the expression of lim homeobox protein 3 (lim3) (65.6 ± 29.2% in contrast to wild type (WT), Fig. 2A and B), an early marker for pituitary progenitor cells suggesting poor commitment to pituitary cell fate. Further, differentiation into prl and gh-positive cells was assessed by in situ hybridization and quantification of the area of pituitary hormone expression. Hormone-expressing cells are clustered together in the zebrafish pituitary, and the gh- and prl-expressing cells have only 1 cluster (19). Thus, differentiation into these cell types can be assessed by carrying out in situ hybridization for pituitary hormone transcripts and quantifying the area of pituitary hormone expressing cells. The prl-expressing area in otx2bhu3625/hu3625 mutants was significantly smaller in contrast WT and otx2bhu3625/+ heterozygotes at 5 (73.8 ± 14.8% in contrast to WT) and 10 dpf (71.6 ± 12.4% in contrast to WT), but not at 1 and 3 dpf (Fig. 2C and D, Supplementary Fig. S1A–F). However, at these earlier time points small differences between genotypes may be obscured by the high intensity of prl expression. prl has an important role in osmoregulation via regulation of transcription of sodium transporters in the gills (20), and consistently 7 dpf otx2bhu3625/hu3625 mutants showed weaker sodium accumulation in the gills than WT and heterozygotes (Fig. 2I and J). In situ hybridization also demonstrated that the gh-expressing area was significantly smaller at 3 (70.3 ± 18.7% in contrast to WT), 5 (72.1 ± 12.1% in contrast to WT) and 10 dpf (62.1 ± 14.1% in contrast to WT, Fig. 2E and F, Supplementary Fig. S1G–J). Despite the decrease in gh-expressing area, there was no difference in body length of the otx2bhu3625/hu3625 larvae at 10 dpf (Fig. 1D and E). Thus, while prl deficiency in otx2bhu3625/hu3625 mutants likely affects osmotic pressure homeostasis, decreased gh had no effect on larval growth prior to early lethality.

Figure 2.

Figure 2

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otx2bhu3625/hu3625 have decreased areas of pituitary hormone expression. (A and B) Whole-mount in situ hybridization (A) and median area of distribution (B) at 1 dpf of lim3 demonstrated decreased expression in homozygotes (hu3625/hu3625) in contrast to WT (+/+) and heterozygotes (+/hu3625). Sample numbers are 12 WT, 20 heterozygotes and 13 homozygotes. (C–F) Whole-mount in situ hybridization and median area of distribution at 10 dpf of prl (C and D) and gh (E and F) demonstrated decreased expression in homozygotes. Sample numbers of prl and gh are 9 WT, 11 heterozygotes and 9 homozygotes and 11 WT, 10 heterozygotes and 9 homozygotes, respectively. (G and H) Whole-mount in situ hybridization (G) and median area of distribution at 1 dpf (H) of nkx2.4b demonstrated decreased expression in homozygotes. Sample numbers are 13 WT, 18 heterozygotes and 11 homozygotes. (I and J) Homozygotes showed a higher percentage of larvae in contrast to WT and heterozygotes with decreased sodium accumulation within the gills (J), which was graded positive, weak positive and negative (I). Sample numbers are 14 WT, 30 heterozygotes and 9 homozygotes. (Scale:10 μm).

In mammals, Otx2 is expressed in the forebrain, midbrain, cerebellum and retina. Further, expression is detected in the developing hypothalamus, but there is little or no expression within the pituitary (7, 21). In zebrafish, in situ hybridization showed that otx2b was expressed at the forebrain to midbrain-hindbrain boundary but not in the pituitary between 1 and 10 dpf (Supplementary Fig. S2A). Thus, the reduced commitment to pituitary fate and decreased prl and gh expression in otx2bhu3625/hu3625 mutants must be indirect. Signals from the hypothalamus, such as nk2 homeobox 4b (nkx2.4b) and fgf3, are important for pituitary placode growth, and 1 dpf otx2bhu3625/hu3625 mutants showed a smaller region of nkx2.4b-expression (78.9 ± 9.7% compared to WT) and slightly shorter length of the fgf3-positive region in the hypothalamus (86.6 ± 15.5% compared to WT) (Fig. 2G and H; Supplementary Fig. S2B). This suggests that otx2b expression in the hypothalamus is indirectly responsible for pituitary development and function.

otx2b hu3625/hu3625 have mandible and eye defects

In humans, whole gene deletions and heterozygous mutations of OTX2 are associated with mandibular anomalies including micrognathia and agnathia and ocular defects such as microphthalmia, anophthalmia and coloboma. Using the jaw index, which is the ratio of jaw to head size (22), the 10 dpf otx2bhu3625/hu3625 mutant larvae demonstrated a modest, but statistically significant reduction in mandible size in contrast to other genotypes (only 3.4% reduction from WT, Fig. 3A and B). In addition, 10 dpf otx2bhu3625/hu3625 mutant larvae showed small eyes (67.6 ± 11.7% size of WT, Fig. 3C and D, Supplementary Fig. S3A–C). Methylacrylate sections at 3 and 10 dpf confirmed a microphthalmic eye that showed disorganization of the neural layers of the retina in otx2bhu3625/hu3625 mutant larvae (Fig. 3E and F) (23). However, optokinetic reflex response to evaluate oculomotor and visual function demonstrated no differences between genotypes, indicating that the mutant larvae can at least detect gross motion (data not shown). We hypothesized that decreased vision contributed to early death of otx2bhu3625/hu3625 larvae because visual acuity is important for finding food after nutrition from the yolk is exhausted. The requirement for food intake coincides with lethality of otx2bhu3625/hu3625 larvae at ~11 dpf. Intake of fluorescent food at 7 and 10 dpf was scored qualitatively as ‘positive’, in which the entire stomach was fluorescent, ‘minimal’, in which only a trace of food was in the stomach, and ‘negative’. At 10 dpf, the majority of otx2bhu3625/hu3625 mutant larvae was negative for food intake, 16.7% had a trace of food, and none was positive (Fig. 3G and H). To control for the possibility that otx2bhu3625/hu3625 are not well enough to eat food attributable to other physiological problems, we performed the food intake experiment with 7 dpf fish (Supplementary Fig. S3D and E). Seven dpf otx2bhu3625/hu3625 larvae were also negative for food accumulation, indicating that the mutants had impaired vision and did not have other physiologic problems that prevented food intake.

Figure 3.

Figure 3

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otx2bhu3625/hu3625 displayed mandible and ocular defects similar to human variants. (A and B) Alcian blue cartilage staining at 10 dpf. (A) demonstrated smaller mandibles as calculated by the median jaw index [B, (left jaw length (red line) + right jaw length)/2)/head transverse diameter (yellow line) × 100] in the homozygotes (hu3625/hu3625) in contrast to WT (+/+) and heterozygotes (+/hu3625). Sample numbers are 20 WT, 22 heterozygotes and 20 homozygotes. (C and D) External images of 10 dpf larvae (C) demonstrate decreased anterior-posterior and dorsal-ventral eye size and reduced median eye volume (D) in homozygotes (hu3625/hu3625) in contrast to WT (+/+) and heterozygotes (+/hu3625). Sample numbers are 23 WT, 64 heterozygotes and 10 homozygotes. (E and F) Methylacrylate sections at 3 dpf (E) demonstrated impaired differentiation and disorganization of the neural retina in homozygotes in contrast to WT and heterozygotes. At 10 dpf (F) some homozygotes showed the loss of retinal architecture and integrity. (G) food intake was graded ‘positive’, ‘minimal’ and ‘negative’. (H) Homozygotes showed a significantly lower percentage of larvae at 10 dpf in contrast to WT and heterozygotes with poor food intake. Sample numbers are 7 WT, 15 heterozygotes and 12 homozygotes.

Reduced cell proliferation and elevated apoptosis in the developing head of otx2b deficient zebrafish

Since otx2bhu3625/hu3625 mutants have small pituitary, mandible and eyes, we hypothesized that decreased cell proliferation and increased apoptosis underlie the hypoplasia of these organs. In situ hybridization for proliferating cell nuclear antigen (PCNA) showed decreased staining in the head and eye region in otx2bhu3625/hu3625 at 3 dpf (Fig. 4A), but not 1 dpf (data not shown). Acridine orange staining to assess for apoptosis demonstrated accumulation in the heads of otx2bhu3625/hu3625 mutants but not in WT or otx2bhu3625/+ at 5 dpf (Fig. 4B). No differences were noted between genotypes at either 1 or 3 dpf (data not shown). Thus, organ hypoplasia in the otx2bhu3625/hu3625 mutants was associated with decreased cell proliferation and increased apoptosis in the forebrain and midbrain.

Figure 4.

Figure 4

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otx2bhu3625/hu3625 shows low cell proliferation and high apoptosis in the head. (A) Whole-mount in situ hybridization of PCNA demonstrated decreased cell proliferation in 3 dpf homozygotes (hu3625/hu3625) in contrast to WT (+/+) and heterozygotes (+/hu3625). (B) Acridine orange staining at 5 dpf revealed increased apoptosis within the head region in homozygotes (hu3625/hu3625) in contrast to WT (+/+) and heterozygotes (+/hu3625).

Discussion

Homozygous otx2b mutant zebrafish have striking defects in pituitary and eye development and modest changes in mandible development. These structures are also affected in humans and rodents with heterozygous loss-of-function alleles, indicating the conservation of gene function across vertebrates. However, heterozygous otx2b zebrafish are phenotypically normal, indicating a difference in dosage sensitivity between fish and mammals. This could be attributable to otx2a, the paralogue of otx2b, which may partially compensate the loss of otx2b function. In the current studies, otx2b mutant fish were analyzed as (i) otx2b morphants exacerbate the morphant phenotype of other genes that regulate eye and jaw development (2), (ii) a functional study with fugu fish (Takifugu niphobles) showed only otx2b functions as a head organizer gene (24), (iii) otx2b has a higher degree of homology with the mammalian Otx2 gene in contrast to otx2a and (iv) the Otx2b protein was registered as a homologous to the OTX2 protein in other species. Thus, despite the differences between mammals and zebrafish, the mutant zebrafish are a useful tool for analyzing the functional significance of rare, likely deleterious mutations in OTX2 that are identified in human patients.

In the current studies, otx2bhu3625/hu3625 mutant larvae showed microphthalmic eyes with disorganization of the neural retina. This resulted in abnormal vision, which likely led to difficulty in finding food after the yolk sac is depleted (~6–7 dpf) and eventual starvation and early mortality around 11 dpf. In humans, OTX2 mutations are associated with the microphthalmia-anophthalmia-coloboma spectrum and can lead to varying degrees of visual impairment and in some cases complete blindness. In mice, in vitro gene expression analysis suggests that Otx2, in coordination with Sox2 (25), is a direct upstream regulator of Rax. Like OTX2, human mutations in retina and anterior neural fold homeobox (Rax) or its homolog in mice (Rx1) and zebrafish (rx3) are also associated with microphthlamia, anophthalmia and coloboma, indicting an important signaling pathway in eye development.

In mammals, OTX2 is a known regulator of pituitary development and its function. Similarly, in the current studies, we found that in zebrafish otx2b was required for anterior pituitary development. However, since otx2b expression was not detected in the pituitary itself, the gene acted indirectly through the hypothalamus, which was found to be small with reduced nkx2.4b and fgf3 expression in otx2bhu3625/hu3625 mutants. This parallels the mechanistic findings in mice, in which selective disruption of Otx2 in the mouse neural ectoderm and subsequent ventral diencephalon causes anterior pituitary hypoplasia with poor development of the infundibulum and stalk and decreased FGF signaling (8). In mouse neural crest development, OTX2 protein is translocated to cells that do not express it (26). We cannot exclude the possibility that Otx2b protein translocates from non-pituitary tissue to the developing pituitary gland.

Otx2b was also required for pituitary function as otx2bhu3625/hu3625 mutants showed decreased expression of lim3, zebrafish orthologue of Lhx3 and reduced expression of prl and gh. Reduced prl, which controls several ion transporters and regulates osmotic homeostasis in zebrafish (20), led to decreased ion intake, without obvious edema, in otx2bhu3625/hu3625 mutants. More drastic reductions in prl action, such as prl mutant zebrafish or MO-knockdown of prl receptors, causes a more profound phenotype including dysregulation of sodium, potassium and chloride homeostasis and obvious edema and death between 6 and 16 dpf. Taken together, this suggests that the reduced expression of prl alters osmoregulation and may contribute to the early death of the otx2bhu3625/hu3625 mutant larvae (Fig. 5).

Figure 5.

Figure 5

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Proposed mechanism of small pituitary, eye and mandibular and early death of otx2bhu3625/hu3625.

The otx2bhu3625/hu3625 mutant zebrafish exhibited both high apoptosis and low cell proliferation in the developing head, which contributed to the hypoplasia of multiple craniofacial structures. In different mouse models, Otx2 knockout also demonstrated alterations in proliferation and apoptosis, although not within the same tissue. For example, hypothalamus-specific Otx2 knockout mice exhibited reduced proliferation, but no obvious cell death (7), whereas photoreceptor-specific Otx2 knockout mice had increased apoptosis but not reduced cell proliferation (27). Further, human induced pluripotent stem cells carrying a clinically significant OTX2 variant showed increased apoptosis when differentiated into a pituitary cell fate (28). The differences in cell death and proliferation may be tissue and species dependent, but also may be attributable to the timing of experiments related to development and animal death.

In conclusion, we demonstrate that homozygous loss of otx2b in zebrafish affects development of the hypothalamus, pituitary gland, mandible and eye. Because otx2b is expressed in the neural ectoderm, and not the oral ectoderm, the hypopituitarism of otx2b mutant zebrafish is secondary to hypothalamic hypoplasia. We show the utility of mutant zebrafish for the functional analysis of OTX2 and other human CPHD-related genes that are associated with a broad variety of phenotypes.

Materials and Methods

Zebrafish

All the procedures were performed under the University of Michigan University Committee on the Use and Care of Animals guidelines. Zebrafish were maintained under standard temperature (28.5°C) and light cycle conditions (14 h on, 10 h off). The supplementation of larval fish food started at 5 dpf. Otx2b mutant fish (otx2bhu3625) on an AB background were generated by the Zebrafish Mutation Project at the Wellcome Sanger Institute using ethyl-N-nitrosourea mutagenesis and commercially obtained from Zebrafish International Resource Center (ZIRC, University of Oregon, Eugene, OR, USA).

Genotyping

DNA was extracted from caudal fin tissue amputated from fish anesthetized with tricaine (Sigma-Aldrich) by using TNES buffer (10 mm Tris pH 7.5, 400 mm NaCl, 100 mm ethylenediaminetetraacetic acid (EDTA) and 0.6% sodium dodecyl sulfate (SDS)) with proteinase K (PK), followed by co-precipitation with 6 M NaCl, precipitation with 100% EtOH, and re-suspension with TE buffer. DNA extraction from larvae after taking the photos for blinded experiments were extracted by the following solution; 50 mm Tris–HCl pH 8.0, 0.5% Tween-20, and 800 μg/ml PK. After overnight incubation, samples were incubated at 95°C for 15 min and added as PCR template. A 315 base pair (bp) fragment containing the mutation-induced MspI cleavage site was amplified by PCR using the primer pair (forward/reverse): 5′-cgtgtacaggtacgcatctttcaactc-3′/5′-gctcttctttttggcaggtcgcacc-3′ and the following amplification parameters: 95°C for 3 min; 35 cycles of 95°C for 30 s, 57°C for 30 s and 72°C for 30 s; and 72°C for 4 min. The PCR products were incubated at 37°C with MspI for 1 h and then separated by gel electrophoresis. The PCR product for the WT allele was 315 bp, whereas the restriction enzyme digestion of the hu3625 allele resulted in 217 bp and 98 bp fragments (Fig. 1B).

Measurement of larval size

Five and 10 dpf zebrafish larvae were anesthetized in tricaine and then mounted in 3% methyl cellulose for live imaging on a stereo microscope, which was calibrated with a stage micrometer slide. The genotypes of larvae were blinded until after larval size data were collected.

RNA extraction and RT-PCR

Total RNAs were extracted from 5 dpf sample with TRIzol Reagent (Invitrogen) following standard protocols (29). Reverse transcription was performed with SuperScript First-Strand Synthesis System for RT-PCR (Invitrogen). Primer sets were as follows (forward/reverse): ef1a (5′-caagcccatgtgtgtggaga-3′/5′-atcaagaagagtagtaccgctagcattac-3′), otx2b exon 1–2 (5′-acggtcaacgggctaagttt-3′/5′-acctcctcgcgcatgaaaat-3′), otx2b exon 2–3(5′-aatcaacttgccggagtccc-3′/5′- tttccatgaggacgcttggt-3′) and otx2a (5′-atacctcaagcaggccccgta-3′/5′-gcaaattaatcttcagggcgacc-3′).

Whole-mount in situ hybridization

Whole-mount RNA in situ hybridization was performed following standard protocols (30). Briefly, embryos/larvae were fixed with 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) and hydrated by 100% methanol at −20°C. After bringing samples to room temperature, samples were incubated in 50% methanol and washed with PBS with 0.1% Tween (PBST). Samples older than 24 hpf were bleached with 3% hydrogen peroxide (H2O2) and 0.5% potassium hydroxide (KOH). PK digestion in Tris–EDTA was performed, following blocking of endogenous alkaline phosphatase (AP) with 0.1 M triethanolamine in 0.25% acetic anhydride. Samples were pre-hybridized for 1 h and then incited with the denatured anti-sense RNA probes overnight at 56°C. Samples were washed with serial solutions; 50% formamide with 1X saline–sodium citrate buffer (SSC), 2X SSC, 0.2X SSC, 0.1X SSC with PBST and PBST. Samples were incubated with blocking reagent (Roche) and then anti-DIG-AP (1:5000, Roche) overnight at 4°C. Samples were washed several times with PBST and then developed with 5-bromo-4-chloro-3-indolyl-phosphate/nitro blue tetrazolium (Roche) in 0.1 M TRIS pH 9.5/0.1 M NaCl2 and 0.05 M MgCl2 overnight. Samples were washed with PBS-T and 4% PFA-PBS and stored in 100% glycerol. Images were obtained on a concave slide, followed by genotyping. The pictures of each median data are shown in the figures. In situ hybridization probes were a gift of Dr Matthias Hammerschmidt. The PCNA probes were kindly provided by Dr Alessandro Cellerino (31).

Acridine orange staining

Acridine orange staining for detecting apoptosis was performed as previously reported (32, 33). Briefly, larvae were incubated in the dark for 30 min at room temperature in acridine orange solution (2 μg/ml, Sigma, A9231), followed by washing 4 times for 5 min each in fish water. Larvae were anesthetized, mounted in 3% methylcellulose and visualized with a fluorescence stereo microscope.

Eye histology

Zebrafish larvae were fixed in 2% PFA/1.5% glutaraldehyde overnight at 4°C and then embedded in methylacrylate. Blocks were section at 5 μm and mounted on slides. Staining with Lee’s stain was performed using standard techniques (34). Permanent coverslips were placed using mounting medium (CytoSeal; Richard-Allan Scientific, Kalamazoo, MI).

Cartilage and bone staining

Cartilage and bone staining were performed as previously reported with modification (35). Briefly, after fixation with formaldehyde/PBS, cartilage was stained with alcian blue solution (70 μl 0.4% alcian blue/70% ethanol + 925 μl 95% ethanol + 55 μl 1 M MgCl2) for 4 h. After discarding the alcian blue solution, samples were washed with 95% ethanol. Bone was stained by alizarin red solution (10 μl 0.5% alizarin red/water + 990 μl 95% ethanol) for 2 days. Following cartilage and bone staining, samples were bleached with 1.5% H2O2 + 1% KOH. Bleached samples were cleared by 20% glycerol with 0.25% KOH and store in 50% glycerol and 0.1% KOH at 4°C. The jaw index was calculated as previously reported (22); Jaw index = [(left jaw length + right jaw length)/2]/head transverse diameter × 100.

Food intake test

Food intake test were performed as previously reported with a small modification (36). Briefly, fertilized eggs were collected and housed in about 50 ml of embryo medium in 10 cm Petri dishes. Larvae were trained to feed by sprinkling the medium in each dish with 2 mg of powdered larval fish food from 4 to 9 dpf. The larvae were allowed to feed for 3–4 h and then transferred to a clean Petri dish containing fresh embryo medium. Fluorescent food is a dried mixture of 150 μl of yellow–green (505/515) fluorospheres (F8827, Invitrogen, Carlsbad, CA), 50 μl of deionized water, and 100 mg of powdered larval food. On 7 or 10 dpf, powdered fluorescent food was fed to the larvae in the same manner as during the training period. The larvae were allowed to feed on fluorescent food for 2 h, mounted in 3% methyl cellulose, and imaged with a fluorescent stereomicroscope. We classified the food intake level as enough, by chance level and none. After imaging, the larvae were harvested for DNA extraction and genotyping.

Sodium influx analysis

Sodium influx was analyzed using sodium green staining (37). Briefly, 7 dpf larvae were incubated in 10 μM sodium green tetraacetate (S6901, Invitrogen) in fish water for 1 h. Subsequently, larvae were washed with fish water. Larvae were anesthetized with tricaine, mounted in 3% methyl cellulose, and the accumulation of fluorescent sodium in the gills was visualized using a fluorescence stereomicroscope. Larvae were then harvested for DNA extraction and genotyping. We divided the level of sodium influx into three categories: strong positive, weak positive and negative.

Image analysis

Images of cartilage and bone staining, eye size, food intake test, acridine orange staining and sodium influx analysis were obtained with a DFC310 FX Digital Color Camera (Leica Microsystems, Wetzlar, Germany) and were processed using LasX software (Leica Microsystems). The smaller sized eyes were used for calculation of the eyes. Images of whole mount in situ hybridization were obtained with DS-Ri1 (Nikon, Tokyo, Japan) and were processed using ViewNX 2 (Nikon). Data were analyzed using Fiji software (38).

Statistical analysis

Statistical analyses were performed using JMP Statistical Database Software version 12.2.0 (SAS Institute, Cary, NC). Analysis of variance, Wilcoxon signed-rank test and Fisher’s exact test were used as appropriate. A P-value of < 0.05 was considered statistically significant.

Supplementary Material

Supplementary_Figure1_ddaa064
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Supplementary_Figure2_ddaa064
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Supplementary_Figure3_ddaa064
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Acknowledgment

We thank Natasha Golovchenko, Sierra S. Nishizaki and Anthony Antonellis (Department of Human Genetics, University of Michigan Medical School), and Queena Zhao, Allison Ferguson and Hao Hao Pontius (Division of Pediatric Hematology/Oncology, Department of Pediatrics, University of Michigan) for managing the fish system. We thank Brad Nelson for tissue processing, sectioning and staining of the methyl acrylate ocular sections. We thank Antionette L. Williams and Phillip E. Kish (Department of Ophthalmology and Visual Sciences, Kellogg Eye Center, University of Michigan) and laboratory members for their fruitful suggestions.

Conflict of Interest statement: None declared.

Funding

National Institutes of Health (R01 30428 to S.A.C.; R01 HL124232, and R01 HL125774 to J.A.S.); the Japan Society for the Promotion of Science Overseas Research Fellowship, the Japan Society (to H.B.); Vision Research Core (P30 EY007003 to B.N.) from the National Eye Institute, Department of Ophthalmology and Visual Sciences, Kellogg Eye Center, University of Michigan.

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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_Figure1_ddaa064
Click here for additional data file. (765.1KB, png)
Supplementary_Figure2_ddaa064
Click here for additional data file. (41.8MB, png)
Supplementary_Figure3_ddaa064
Click here for additional data file. (135.4KB, png)

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