Control of lens development by Lhx2-regulated neuroretinal FGFs

Abstract

Fibroblast growth factor (FGF) signaling is an essential regulator of lens epithelial cell proliferation and survival, as well as lens fiber cell differentiation. However, the identities of these FGF factors, their source tissue and the genes that regulate their synthesis are unknown. We have found that Chx10-Cre;Lhx2^lox/lox mice, which selectively lack Lhx2 expression in neuroretina from E10.5, showed an early arrest in lens fiber development along with severe microphthalmia. These mutant animals showed reduced expression of multiple neuroretina-expressed FGFs and canonical FGF-regulated genes in neuroretina. When FGF expression was genetically restored in Lhx2-deficient neuroretina of Chx10-Cre;Lhx2^lox/lox mice, we observed a partial but nonetheless substantial rescue of the defects in lens cell proliferation, survival and fiber differentiation. These data demonstrate that neuroretinal expression of Lhx2 and neuroretina-derived FGF factors are crucial for lens fiber development in vivo.

Keywords: FGF; Genetics; Lens; Mouse; Retina; Transcription factor.

Fig. 1.

Loss of function of Lhx2 in neuroretina led to microphthalmia and lens development defects. (A,B) Lateral view of control (A) and Chx10-Cre;Lhx2^lox/lox animals (B) indicating microphthalmia in Chx10-Cre;Lhx2^lox/lox animals. (C-F) Hematoxylin and Eosin staining of eye sections from E13.5 (C,D) and P0.5 (E,F) of control (C,E) and Chx10-Cre;Lhx2^lox/lox (D,F) mice. Dotted green and black outlines mark the retinas and lenses, respectively (C-F) and insets (D,F) are digital zooms of the boxed regions. (G) Graph indicating average neuroretinal area and lens area in sections of control and Chx10-Cre;Lhx2^lox/lox mice at P0.5. Data were analyzed using unpaired two-tailed t-test; n=7 for Chx10-Cre;Lhx2^lox/+; n=11 for Chx10-Cre;Lhx2^lox/lox; ****P<0.0001. Scale bars: 5 mm in A,B; 100 µm in C-F.

Fig. 2.

Fgf3, Fgf9 and Fgf15 were downregulated in Chx10-Cre;Lhx2^lox/lox retinas. (A) Real-time quantitative PCR analysis of Fgf3, Fgf9 and Fgf15 mRNA expression levels in Chx10-Cre;Lhx2^lox/lox retinas compared with Chx10-Cre;Lhx2^lox/+ controls at E13.5. Data represent mean normalized to Gapdh values±s.e.m. Data were analyzed using unpaired two-tailed t-test; n=3; *P<0.05; ***P<0.001; ****P<0.0001. (B-G) In situ hybridization of Fgf3, Fgf9 and Fgf15 mRNA expression levels in Chx10-Cre;Lhx2^lox/+ (B,D,F) and Chx10-Cre;Lhx2^lox/lox (C,E,G) eyes at E13.5. (C,E) Dotted red circles mark the lenses shown at higher magnification in the left-hand insets. (B-G) Lens Lhx2 expression in adjacent sections is shown in right-hand insets. Lhx2 expression in RPE is maintained and outlines the neural retina in unpigmented animals (right-hand insets in C,E). Scale bar: 100 µm.

Fig. 3.

Cre-mediated induction of Fgf10 expression in Chx10-Cre;Lhx2^lox/lox;pMes-Fgf10 retinas restored expression of FGF-regulated genes in lens. (A-D) Mouse genetics and anticipated retinal-derived FGF expression for (A) Chx10-Cre;Lhx2^lox/+, (B) Chx10-Cre;Lhx2^lox/+; pMes-Fgf10, (C) Chx10-Cre;Lhx2^lox/lox and (D) Chx10-Cre;Lhx2^lox/lox;pMes-Fgf10 eyes. (E-T) In situ hybridization demonstrates Fgf10 induction in neuroretina (E-H), and induction of expression of FGF-regulated genes, including Etv5 (I-L), Etv1 (M-P) and Spry2 (Q-T). Right-hand insets indicate Lhx2 expression in adjacent sections. Lhx2 expression in RPE is maintained and outlines the neural retina in unpigmented animals (insets in G,K,O,S,H,T). Dotted red circles mark the lenses shown at higher magnification in the left-hand insets (G-T). Scale bar: 100 µm.

Fig. 4.

Overexpression of Fgf10 in Chx10-Cre;Lhx2^lox/lox;pMes-Fgf10 animals rescued lens fiber development. Developmental time-course of immunohistochemical staining for Prox1 (green) and β-crystallin (red) in lenses at E11.5 (A-D), E12.5 (E-H), E13.5 (I-L), E15.5 (M-P), E18.5 (Q-T) and P0.5 (U-X) of Chx10-Cre;Lhx2^lox/+ (A,E,I,M,Q,U), Chx10-Cre;Lhx2^lox/+;pMes-Fgf10 (B,F,J,N,R,V), Chx10-Cre;Lhx2^lox/lox (C,G,K,O,S,W) and Chx10-Cre;Lhx2^lox/lox; pMes-Fgf10 (D,H,L,P,T,X) animals . Nuclei are counterstained with DAPI (blue). Scale bars: 100 µm.

Fig. 5.

Chx10-Cre;Lhx2^lox/lox;pMes-Fgf10 animals expressed the mature lens fiber marker Aquaporin-0. Developmental time-course of immunohistochemistry for Prox1 (green) and Aquaporin-0 (red) expression in lenses at E11.5 (A-D), E12.5 (E-H), E13.5 (I-L), E15.5 (M-P), E18.5 (Q-T) and P0.5 (U-X) of Chx10-Cre;Lhx2^lox/+ (A,E,I,M,Q,U), Chx10-Cre;Lhx2^lox/+;pMes-Fgf10 (B,F,J,N,R,V), Chx10-Cre;Lhx2^lox/lox (C,G,K,O,S,W) and Chx10-Cre;Lhx2^lox/lox;pMes-Fgf10 (D,H,L,P,T,X) animals. Nuclei are counterstained with DAPI (blue). Scale bars: in N, 100 µm for M,N; in L, 100 µm for A-L; in P, 100 µm for O,P; in X, 100 µm for Q-X.

Fig. 6.

Fgf10 overexpression rescued defects in lens cell proliferation and cell death. (A-D) Immunostaining of E12.5 eye sections for Ki67 (green) and activated caspase 3 (c-Caspase3, red). White dotted circles mark the lenses. Scale bars: 100 µm. (E) Graph indicating the percentage of Ki67-positive cells relative to all DAPI-positive cells in the lenses. (F) Graph indicating the percentage of c-Caspase3-positive cells relative to all DAPI-positive cells in the lenses. Data were analyzed using one-way ANOVA followed by Sidak's test; n=6 for Chx10-Cre;Lhx2^lox/+ and Chx10-Cre;Lhx2^lox/+;pMes-Fgf10; n=18 for Chx10-Cre;Lhx2^lox/lox and Chx10-Cre;Lhx2^lox/lox;pMes-Fgf10, *P<0.05; ****P<0.0001. Error bars indicate s.e.m. (G) An diagram of the findings of this study.

Fig. 7.

Selective deletion of Lhx2 in neuroretina led to downregulation of BMP signaling in the neuroretina and lens. (A-L) In situ hybridization of Bmp4 (A-D), Bmp7 (E-H) and Lhx2 (I-L) mRNA expression levels in Chx10-Cre;Lhx2^lox/+ (A,E,I), Chx10-Cre;Lhx2^lox/+; pMes-Fgf10 (B,F,J), Chx10-Cre;Lhx2^lox/lox (C,G,K) and Chx10-Cre;Lhx2^lox/lox;pMes-Fgf10 (D,H,L) eyes at E13.5. Dotted red circles mark the lenses (C,D,G,H,L). (M-P) Immunohistochemical staining for Pax6 (green) and pSmad1/5/9 (red) in lenses of Chx10-Cre;Lhx2^lox/+ (M), Chx10-Cre;Lhx2^lox/+;pMes-Fgf10 (N), Chx10-Cre;Lhx2^lox/lox (O) and Chx10-Cre;Lhx2^lox/lox;pMes-Fgf10 animals (P) at E13.5. Nuclei are counterstained with DAPI (blue). The images of pSmad1/5/9 staining in single channel and at higher magnification are included underneath. Dotted white circles mark the lenses (O,P). Scale bars: 100 µm.