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. 2020 Dec 18;29(20):3373-3387.
doi: 10.1093/hmg/ddaa228.

Nf2 fine-tunes proliferation and tissue alignment during closure of the optic fissure in the embryonic mouse eye

Wesley R Sun  1 , Sara Ramirez  1   2 , Kelly E Spiller  1 , Yan Zhao  1 , Sabine Fuhrmann  1   2
Affiliations

Nf2 fine-tunes proliferation and tissue alignment during closure of the optic fissure in the embryonic mouse eye

Wesley R Sun et al. Hum Mol Genet. .

Abstract

Uveal coloboma represents one of the most common congenital ocular malformations accounting for up to 10% of childhood blindness (~1 in 5000 live birth). Coloboma originates from defective fusion of the optic fissure (OF), a transient gap that forms during eye morphogenesis by asymmetric, ventral invagination. Genetic heterogeneity combined with the activity of developmentally regulated genes suggests multiple mechanisms regulating OF closure. The tumor suppressor and FERM domain protein Neurofibromin 2 (NF2) controls diverse processes in cancer, development and regeneration, via Hippo pathway and cytoskeleton regulation. In humans, NF2 mutations can cause ocular abnormalities, including coloboma, however, its actual role in OF closure is unknown. Using conditional inactivation in the embryonic mouse eye, our data indicate that loss of Nf2 function results in a novel underlying cause for coloboma. In particular, mutant eyes show substantially increased retinal pigmented epithelium (RPE) proliferation in the fissure region with concomitant acquisition of RPE cell fate. Cells lining the OF margin can maintain RPE fate ectopically and fail to transition from neuroepithelial to cuboidal shape. In the dorsal RPE of the optic cup, Nf2 inactivation leads to a robust increase in cell number, with local disorganization of the cytoskeleton components F-actin and pMLC2. We propose that RPE hyperproliferation is the primary cause for the observed defects causing insufficient alignment of the OF margins in Nf2 mutants and failure to fuse properly, resulting in persistent coloboma. Our findings indicate that limiting proliferation particularly in the RPE layer is a critical mechanism during OF closure.

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Figures

Figure 1
Figure 1
Nf2 disruption in ocular tissues causes coloboma in the embryonic mouse eye. (A) Control embryonic head at E13.5. (B) Nf2CKO embryos exhibit cleft lip and palate (arrowhead). (C) Higher magnification of E13.5 control eye. (D) In Nf2CKO embryos at E13.5, the ventral eye shows an OF closure defect with a narrow gap (arrowhead) and the pigment appears slightly darker. (E) Immunolabeling for the basement marker laminin in the E13.5 ventral optic cup of controls confirms absence of laminin and successful fusion of the OF region (arrowhead). Sagittal view with nasal orientation to the left. (F) Laminin is persistent in the ventral optic cup in E13.5 Nf2CKO embryos indicating failed OF fusion (arrowhead). (G) Brightfield image of the ventral optic cup in control showing pigmentation of the RPE in the fused OF (arrowhead). (H) In the ventral optic cup of E13.5 Nf2CKO embryos, pigmentation extends into the unfused OF (arrowhead) and the RPE layer appears slightly increased in height. Insets in G, H show magnified regions outlined in boxed area, revealing increased thickness in mutant RPE (H). (I) During OF closure at E11.5, YAP1 protein expression can be localized both to the cytoplasm and nucleus in the RPE (arrowheads point to RPE nuclei). (J) In Nf2CKO eyes, nuclear YAP1 expression can be increased in retina and RPE (arrowheads display nuclear Yap localization in RPE). RPE in I and J is indicated by dotted lines and sections were counterstained with 2-(4-amidinophenyl)-1H-indole-6-carboxamide (Dapi, blue). Insets in I and J show enlarged greyscale images of individual cells in the RPE (boxed area). I′ and J′ display the Dapi channel, I″ and J″ display the YAP1 channel. Individual nuclei in the insets are outlined by dotted lines. (K) YAP1 average integrated density per RPE nuclei shows a significant increase in Nf2CKO compared with control nuclei. (L, M) Cyclin D1 (CCND1)-labeled cells are significantly increased in Nf2CKO ventral RPE (M; arrowheads; N). Asterisks in I, J, L, M indicate OF. Data are shown as means ± SD, and P-values are indicated above the horizontal lines in each graph (significant < 0.05). Statistical analysis was performed using unpaired, two-tailed Student T-test (Welch’s correction included for K). Scale bars: 100 μm in E, L; 20 μm in I, J.
Figure 2
Figure 2
Analysis of tissue patterning in the Nf2CKO optic cup reveals persistence of RPE markers in the open OF. (A–J, M–P: E11.5; K, L: E13.5) A, B: Expression of the pan-ocular protein PAX6 is unaltered in Nf2CKO eyes (B). Asterisks label overlying OF. C, D: The retina-specific homeodomain protein VSX2 is normally expressed in Nf2CKO eyes (D). E, F: Analysis of TBX5 expression shows normal patterning of the dorsal optic cup in Nf2CKO embryos (F; arrow). G, H: In the ventral Nf2CKO optic cup, the key transcription factor PAX2 is normally localized (H). I, J: Expression of the RPE determination transcription factor OTX2 can be maintained in the open fissure of Nf2CKO eyes (J, arrows). K, L: At E13.5, coloboma is persistent in Nf2CKO embryos and the margins of the unclosed OF are lined with OTX2-positive cells (L, arrows). M, N: The bHLH transcription factor MITF is present in the RPE layer of the optic cup (M). In Nf2CKO embryos, MITF localization extends into the OF (N; arrows). O, P: Axin2 reporter expression, a readout for Wnt/β-catenin pathway activation, is not altered in the ventral RPE in Nf2CKO optic cups (arrows). Sagittal view with nasal orientation to the left. Scale bars: 100 μm in A, E, G.
Figure 3
Figure 3
Apicobasal polarity and intercellular junction assembly are maintained during OF closure in the ventral optic cup in Nf2CKO embryos. (A, B, E, F: E11.5; C, D: E11.0) A, B: Localization of pMLC2 on the apical surface of the RPE layer appears normal in Nf2CKO embryos (B; arrow). Asterisk marks the overlying OF. C, D: Apical distribution of the tight junction component ZO-1 is not affected in the Nf2CKO ventral optic cup (D; arrow). The cadherin associated protein α-catenin shows normal localization in the OF and apical space between retina and RPE in Nf2CKO eyes (F, arrows). Sagittal view with nasal orientation to the left. Scale bars: 20 μm in E, F.
Figure 4
Figure 4
Proliferation is upregulated in the RPE layer in the Nf2CKO optic cup. (A–D: E11.5; E–J: E11.0) A, B: Phospho-histone H3 (pHH3) labeling (red) of control (A) and Nf2CKO (B) detects ocular cells in G2/M phases of the cell cycle (red). Dapi is used as a nuclear label (blue). Quantification shows that the number of phospho-histone H3-labeled cells is not changed in the ventral optic cup in Nf2CKO eyes (C); however, there is a small significant increase in the ventral-most area that harbors the OF (V2; D). E-J: EdU labeling and quantification. EdU-labeled cells in control (E; green), with nuclear Dapi co-labeling (blue). In the RPE of the Nf2CKO ventral optic cup, the number of EdU-labeled cells is increased (F; arrowheads), in comparison with controls (E, arrowheads). Quantitative analysis of EdU incorporation reveals that the number of cells in S-phase is significantly upregulated specifically in the RPE but not in the retina (G, H). (RPE: control: 19.91% ± 1.74 SD; Nf2CKO: 29.07% ± 4.60 SD) The increase in EdU-labeled cell number is significant for the ventral RPE (I; control: 27.82% ± 5.66 SD; Nf2CKO: 49.86% ± 7.10 SD), while the dorsal RPE shows no change. Significant stimulation of proliferation in the ventral RPE applies to all regions in the ventral optic cup, including the V2 region harboring the OF (J). Data are shown as means ± SD, and P-values are indicated above the horizontal lines in each graph (significant < 0.05). Statistical analysis was performed using unpaired, two-tailed Student T-test or 2-way analysis of variance (ANOVA) with Sidak’s multiple comparison. Images show sagittal view with nasal orientation to the left. Scale bar: 100 μm in A, E.
Figure 5
Figure 5
Cell number in the RPE layer is increased in the Nf2CKO dorsal optic cup at E11.0. (A, B) MITF labeling reveals increased RPE layer thickness in Nf2CKO (B; arrowheads point to ectopic RPE cells located apically). (C, D) Quantification indicates a significant increase in total cell number in the dorsal RPE in Nf2CKO (C; see Supplementary Material, Figure S3C for subdivisions; control: 183.75 ± 15.13 SD; Nf2CKO: 271.00 ± 10.42 SD), while cell number is unchanged in the dorsal retina (D). Data are means ± SD, Student’s T-test was applied for statistical analysis, and P-values are indicated on the horizontal lines in each graph (significance < 0.05). (E) Phalloidin labeling in control demonstrates regular organization. (F) Phalloidin-labeling Nf2CKO reveals irregularities in localization in the dorsal RPE layer (arrowheads). (G) Normal localization of pMLC2 in control dorsal RPE. (H) In addition, mis-localization of pMLC2 is detectable in the dorsal RPE layer in Nf2CKO optic cups (arrowheads). (I, J) OTX2 labeling shows that supernumerary cells persist in the dorsal optic cup at E13.5 (J; arrowheads). Scale bars: 50 μm in A, I.
Figure 6
Figure 6
Rx3-Cre-mediated disruption of Nf2 results in defective alignment of OF margins during the closure process. (A–D) F-actin localization in the OF at E11.0 (A, B) and at E11.5 (C, D) in control (A, C) and Nf2CKO optic cups (B, D). A: In E11.0 control embryos, the OF margins become tightly aligned, shaped with a convex surface toward the basal space (arrowheads). (B) In the Nf2CKO OF, shape of the basal surface of the OF margins is convex, similar to controls, however, alignment of the margins is less advanced (B, arrowheads). At E11.5, the OF margins in controls are well aligned along most of the OF with flattened basal surfaces (C, arrowheads). The RPE layer consists of cuboidal cells (bracket). (D) In Nf2CKO eyes, the OF margins align only partially (arrowheads), exhibit convex shape, and OF cells continuous with the RPE layer show increased height in the apicobasal axis, thereby appearing columnar (bracket). (E) Quantification of aligned OF margins. Extent of attached margins was measured in the OF, as outlined in representative examples shown in C, D for aligned OF between arrowheads. In Nf2CKO, few margins show close alignment, which is often much reduced, compared with controls (lack of alignment defined = 0). Data are means ± SD and Student’s T-test was applied for statistical analysis. (F) Analysis of cellular height along the apicobasal axis of cells lining the ventral OF margins (see brackets in C, D; Supplementary Material, Figure S3G) reveals significantly increased cellular height in the temporal OF margin. Statistical analysis was performed using nested T-test, data are means (with minimal and maximal points). P-values are indicated on the horizontal lines in each graph , significance < 0.05 (E, F). (G) Proposed model summarizing our observations, for explanation, see text (left: control optic cup; right: Nf2CKO). The arrows point to potential effects of RPE hyperproliferation on the mechanodynamics of optic cup growth and apposition of OF margins. Sagittal view with nasal orientation to the left. (NR: neural retina, L: lens, N: nasal, T: temporal). Scale bars: 50 μm in A, C.
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References

    1. Morrison D., Fitz Patrick D., Hanson I., Williamson K., Heyningen V., Fleck B., Jones I., Chalmers J. and Campbell H. (2002) National study of microphthalmia, anophthalmia, and coloboma (MAC) in Scotland: investigation of genetic aetiology. J. Med. Genet., 39, 16–22. - PMC - PubMed
    1. Clementi M., Turolla L., Mammi I. and Tenconi R. (1992) Clinical anophthalmia: an epidemiological study in Northeast Italy based on 368,256 consecutive births. Teratology, 46, 551–553. - PubMed
    1. Williamson K.A. and Fitz Patrick D.R. (2014) The genetic architecture of microphthalmia, anophthalmia and coloboma. Eur. J. Med. Genet., 57, 369–380. - PubMed
    1. Shah S.P., Taylor A.E., Sowden J.C., Ragge N.K., Russell-Eggitt I., Rahi J.S., Gilbert C.E. and Surveillance of Eye Anomalies Special Interest Group (2011) Anophthalmos, microphthalmos, and typical coloboma in the United Kingdom: a prospective study of incidence and risk. Invest. Ophthalmol. Vis. Sci., 52, 558–564. - PubMed
    1. Maumenee I.H. and Mitchell T.N. (1990) Colobomatous malformations of the eye. Trans. Am. Ophthalmol. Soc., 88, 123–132 discussion 133-125. - PMC - PubMed

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