Genetic defects of GDF6 in the zebrafish out of sight mutant and in human eye developmental anomalies

Abstract

Background: The size of the vertebrate eye and the retina is likely to be controlled at several stages of embryogenesis by mechanisms that affect cell cycle length as well as cell survival. A mutation in the zebrafish out of sight (out) locus results in a particularly severe reduction of eye size. The goal of this study is to characterize the outm233 mutant, and to determine whether mutations in the out gene cause microphthalmia in humans.

Results: In this study, we show that the severe reduction of eye size in the outm233 mutant is caused by a mutation in the zebrafish gdf6a gene. Despite the small eye size, the overall retinal architecture appears largely intact, and immunohistochemical studies confirm that all major cell types are present in outm233 retinae. Subtle cell fate and patterning changes are present predominantly in amacrine interneurons. Acridine orange and TUNEL staining reveal that the levels of apoptosis are abnormally high in outm233 mutant eyes during early neurogenesis. Mutation analysis of the GDF6 gene in 200 patients with microphthalmia revealed amino acid substitutions in four of them. In two patients additional skeletal defects were observed.

Conclusions: This study confirms the essential role of GDF6 in the regulation of vertebrate eye size. The reduced eye size in the zebrafish outm233 mutant is likely to be caused by a transient wave of apoptosis at the onset of neurogenesis. Amino acid substitutions in GDF6 were detected in 4 (2%) of 200 patients with microphthalmia. In two patients different skeletal defects were also observed, suggesting pleitrophic effects of GDF6 variants. Parents carrying these variants are asymptomatic, suggesting that GDF6 sequence alterations are likely to contribute to the phenotype, but are not the sole cause of the disease. Variable expressivity and penetrance suggest a complex non-Mendelian inheritance pattern where other genetic factors may influence the outcome of the phenotype.

Figure 1

Phenotype of the zebrafish out of sight mutant. (A - B) Lateral views of wild-type zebrafish (A) and of the out^m233 mutant (B) at 36 hpf, 48 hpf (a1 and b1), 72 hpf (a2 and b2) and 96 hpf (a3 and b3). The out^m233 mutant is characterized by a severe reduction in eye size. No obvious defects are observed in other organs. (C - D) Transverse plastic sections of wild-type (C) and out^m233 mutant (D) retinae at 36 hpf, 4 dpf (c1 and d1) and 5 dpf (c2 and d2). Although the retina and lens are grossly reduced in size in out^m233 mutants, their overall architectures appear largely intact. (E - F) An anophthalmic out^m233 homozygous mutant at ca. 2 months of age. An enlargement of the head region is shown in (F). (G - H) A wild-type individual (G) and its microphthalmic out^m233 sibling (H) at 2.5 months of age. In A, B, and E-H, anterior is to the left.

Figure 2

Positional cloning of the zebrafish out of sight mutation. (A) The out locus maps to chromosome 16 between markers Z6293 and Z8819. Sequence analysis of the gdf6a gene from this interval identified a mutation affecting its start codon (c.1A > G, p. Met1Val) in mutant (C) but not wild-type (B) individuals. SP = signal peptide, ASD = active signaling domain. (D, E) The overexpression of wild-type (E) but not mutant (D) gdf6a causes ventral eye defects. Shown are lateral (left panels) and ventro-lateral (right panels) views of zebrafish larvae at 3 dpf. Anterior is to the left, asterisks indicate the lens. (F) The frequency of ventral eye defects, following the overexpression of wild-type or mutant gdf6a in four independent experiments, as indicated on the horizontal axis (1 through 4). Numbers above the bar graph indicate sample sizes.

Figure 3

The number of cells in wild-type and out^m233mutant retinae. (A - B) Hoechst staining of 4-μm plastic sections through the retina of wild-type (A) and out^m233 (B) embryos at 36 hpf. (C) Diagram showing the number of nuclei that were counted in the dorsal, ventral, and entire retina. At 36 hpf, cell numbers in mutant embryos are not significantly different from these in wild-type siblings. Cells were counted on sections from 7 and 4 embryos for wild-type and mutant, respectively. (D - E) Hoechst staining of 4-μm plastic sections through the retina of wild-type (D) and out^m233 (E) embryos at 96 hpf. (F) Diagram showing the number of nuclei that were counted in the ganglion cell layer (GCL), in the inner nuclear layer (INL) and in the photoreceptor layer (PRCL). By 96 hpf, cell numbers in all these three layers are reduced in mutant embryos, compared to the wild type (for GCL, p < 0.01; for INL and PRCL, p < 0.001; t-test). For both wild-type and mutant, cells were counted on sections from 6 embryos. Asterisks indicate the lens, and arrowheads the optic nerve.

Figure 4

Cell fate in wild-type and out^m233 retinae. (A-B) Transverse sections through the retina stained with anti-neurolin antibody (red) in wild-type (A) and out^m233 (B) embryos. The ganglion cell layer and the optic nerve display grossly normal appearance at 56 hpf. Sections were counterstained with YoPro (green). (C-D). Anti-parvalbumin antibody (red) stains a subset of amacrine cells on transverse sections through the retinae of wild-type (C) and out^m233 mutant (D) embryos. A displacement of some amacrine cells towards and occasionally into the photoreceptor cell layer is observed in approximately 50% of out^m233 embryos (arrowhead in D). (E - F) Anti-paravalbumin and Zpr1 double immunostaining (both in green) in wild-type (E) and in out^m233 mutant (F) embryos. In approximately 50% of the embryos, Zpr1-positive cells are absent in the dorsal and/or ventral regions of the photoreceptor layer (arrowheads in F). (G - H) Images of whole embryos at 42.5 hpf. Ganglion cells are visualized with brn3c-GFP transgene in wild-type (G) and out^m233 mutant (H) embryos. Anterior is to the left. (I -J) The Zpr1 antibody stains red and green cones (green signal) while anti-serotonin antibody labels serotonin-positive amacrine cells (red) in wild-type (I) and out^m233 mutant (J) embryos. (K-L) Anti-carbonic anhydrase antibody (red) stains Müller glia in wild-type (K) and out^m233 mutant (L) embryos. Arrowhead in the inset indicates the apical processes of the Muller glia, which terminate at the outer limiting membrane. (M - N) Anti-GABA antibody (green) stains a subset of amacrine cells in wild-type (M) and out^m233 mutant (N) embryos. On the majority of sections, the number of GABA-positive neurons is increased in the retinal periphery of out^m233 homozygotes (arrowheads). (O) Graph showing the frequency of GABA-positive cells in the central retina. The number of cells per an arbitrary unit of distance if provided. Fewer GABA-positive cells are observed in the mutant, compared to the wild type (p < 0.01, t-test). Blue dots represent sections. As some sections have the same frequency of GABA-positive cells, the number of dots does not equal the number of sections. n ≥ 4 embryos for both wild-type and mutant. Asterisks mark the lens. (C-F) and (I-N) show retinae at 5 dpf.

Figure 5

Cell proliferation in the out of sight retina. (A - B) Immunolabeling of transverse cryosections through the retinae of wild-type (A) and out^m233 mutant (B) embryos with anti-phospho-histone H3 (red) antibodies at 36 hpf. Sections were counterstained with YoPro (green). (C) Graph illustrating the numbers of phospho-H3 histone-positive nuclei on sections through wild-type and out^m233 mutant retinae as indicated. (D) Graph showing the ratio of phospho-H3-positive cells to all cells. No differences between wild-type and out^m233 embryos were identified. In (C - D), black dots represent sections. As some sections have the same number of phospho-H3-positive cells, the number of dots does not equal the number of sections. n ≥ 10 embryos and ≥ 15 sections for both wild-type and mutant samples. Asterisks indicate the lens, and arrowheads the optic nerve. Dorsal is up.

Figure 6

Apoptosis in out^m233 mutant embryos. (A - H) Transverse cryosections through the zebrafish eye. Apoptosis is detected using the TUNEL assay (red signal). Levels of apoptosis are abnormally high in out^m233 mutant (E-H) eyes during early neurogenesis. At 30 hpf, there is an increase of apoptosis in the lens and the retina of mutants (E) compared to the wild-type (A). Apoptosis persists in and around the lens region at 48 hpf (F, compare to B). By 76 hpf, hardly any apoptosis is visible in the mutant eye (G), and a significantly increased amount of apoptosis occurs in the wild-type (C) retina, compared to the mutant. At 5 dpf, hardly any apoptotic cells are found in both the wild type (D) and the mutant (H). (I) The amount of cell death is quantitated. The average number of apoptotic cells per section is provided. Arrowheads point to the lens. n ≥ 9 sections from at least 3 embryos for each time point both for wild-type and mutant samples.

Figure 7

GDF6 analysis in patients with microphthalmia, anophthalmia and coloboma. (A) Two different heterozygous amino acid changes, p.Ala249Glu and p.Ala319Thr, were detected in four unrelated individuals. (B) The sequence of the heterozygous p.Ala249Glu variant in patient 792-535. (C) The affected half-sib of patient 792-535 does not carry the p.Ala249Glu variant, while it was detected in the unaffected father.