Prox1 and fibroblast growth factor receptors form a novel regulatory loop controlling lens fiber differentiation and gene expression

Abstract

Lens epithelial cells differentiate into lens fibers (LFs) in response to a fibroblast growth factor (FGF) gradient. This cell fate decision requires the transcription factor Prox1, which has been hypothesized to promote cell cycle exit in differentiating LF cells. However, we find that conditional deletion of Prox1 from mouse lenses results in a failure in LF differentiation despite maintenance of normal cell cycle exit. Instead, RNA-seq demonstrated that Prox1 functions as a global regulator of LF cell gene expression. Intriguingly, Prox1 also controls the expression of fibroblast growth factor receptors (FGFRs) and can bind to their promoters, correlating with decreased downstream signaling through MAPK and AKT in Prox1 mutant lenses. Further, culturing rat lens explants in FGF increased their expression of Prox1, and this was attenuated by the addition of inhibitors of MAPK. Together, these results describe a novel feedback loop required for lens differentiation and morphogenesis, whereby Prox1 and FGFR signaling interact to mediate LF differentiation in response to FGF.

Keywords: Lens; Morphogenesis; Regulatory loop.

Fig. 1.

Prox1 deletion from the early lens arrests its development at the LV. (A-F) Mouse eye sections at E12.5 (A,D), E13.5 (B,E) and E15.5 (C,F) stained with Hematoxylin and Eosin. In WT, primary lens fibers (LFs; pink) were evident by E12.5 (A), with secondary fibers produced at E13.5 (B) and E15.5 (C). In Prox1 cKO lenses, the posterior-most cells never elongate into eosinophilic primary (D) or secondary fibers (E,F). (G-L) Immunofluorescence staining for Prox1. Prox1 is expressed in WT primary LFs at E12.5 (G) and in elongating secondary LFs at E13.5 (H) and E15.5 (I). Prox1 protein levels are reduced in Prox1 cKO by E12.5 (J), and Prox1 immunoreactivity is absent from Prox1 cKO lenses by E13.5 (K,L). (A-F) Blue, Hematoxylin; pink, Eosin. (G-L) Blue, Draq5 (DNA); red, Prox1. a, anterior; p, posterior; r, retina; e, lens epithelium; f, LFs. Scale bars: 200 µm in A-F; 100 µm in G-L.

Fig. 2.

Posterior cells of the Prox1 cKO LV exit the cell cycle. (A,A′) E13.5 WT lenses exhibit EduClick (EduC)-positive cells (red) in the epithelium (arrows), but they were absent from the transition zone and from LFs. (B,B′) E13.5 Prox1 cKO lenses maintained cell proliferation in the anterior aspect of the lens (arrows), while no EduC labeling was detected at the lens posterior. (C,C′) Immunofluorescence staining of WT E13.5 lenses showed expression of the cell cycle inhibitor p57^Kip2 (red) in differentiating LFs. (D,D′) Similarly, p57^Kip2 was still expressed in the most posterior cells of the Prox1 cKO lens. (E-F′) TUNEL assays. Programmed cell death was not observed in WT lenses or the tunica vasculosa lentis at E13.5 (E,E′). Isolated TUNEL-positive nuclei were seen in the posterior LV of Prox1 cKO at E13.5 (F,F′, arrows); however, robust TUNEL staining was consistently observed in the tunica vasculosa lentis of Prox1 cKO at E13.5 (F,F′, arrows). Boxed regions in A-F are shown at higher magnification in A′-F′. The dashed line delineates the lens capsule. (A-F′) Blue, Draq5 (DNA). (A-B′) Red, EduC. (C-D′) Red, p57^Kip2. (E-F′) Green, TUNEL. a, anterior eye; p, posterior eye; r, retina; c, cornea; e, lens epithelium; tvl, tunica vasculosa lentis; f, LFs. Scale bars: 100 µm.

Fig. 3.

LF marker expression decreases in Prox1 cKO. (A) qRT-PCR of E14.5 Prox1 cKO lenses compared with WT. *P≤0.01 by nested ANOVA. αA-crystallin (Cryaa; P=0.007), βA1-crystallin (Cryba1; P=0.001), βB1-crystallin (Crybb1; P=0.002), γC-crystallin (Crygc; P=0.0001) and aquaporin 0 (P=0.004) mRNA levels are reduced in Prox1 cKO lenses. Error bars indicate s.d.; n=3. (B,E) β-crystallin protein (brown) localizes to WT LFs at E13.5 (B), but this is reduced in Prox1 cKO lenses (E). (C,F) γ-Crystallin protein (green) is found in elongated LFs in the E13.5 WT lens (C), but its levels are reduced in the Prox1 cKO lens (F). (D,G) Aquaporin 0 protein (red) is found in E13.5 WT LFs (D), whereas little is detected in Prox1 cKO lenses (G). (B,E) Blue, Hematoxylin. (C,D,F,G) Blue, Draq5 (DNA). a, anterior eye; p, posterior eye; r, retina; e, lens epithelium; f, LFs. Scale bars: 200 µm in B,E; 100 µm in C,D,F,G.

Fig. 4.

Prox1 cKO lenses have a normal distribution of epithelial markers. (A,B) E-cadherin (red) is expressed in the epithelium and absent at the transition zone of both WT (A) and Prox1 cKO (B) E13.5 lenses. (C,D) The transcription factor Pax6 (red) is preferentially localized to the lens epithelium and reduced at the transition zones of both WT (C) and Prox1 cKO (D) lenses at E14.5. Blue, Draq5 (DNA). a, anterior eye; p, posterior eye; e, lens epithelium; f, LFs. Scale bar: 100 µm.

Fig. 5.

Prox1 deletion results in the misregulation of genes that are highly enriched in expression in the developing lens. RNA-seq was performed on E13.5 Prox1 cKO and WT lenses immediately following the loss of Prox1 protein. (A,B) The DAVID bioinformatics resource was used to group/cluster the downregulated (downDEGs) (A) and upregulated (upDEGs) (B) genes into functionally related gene ontology (GOs) categories with cluster enrichment scores representing their likelihood of affecting biological function. (C-H) The relative lens-enrichment score of each DEG [FC, fold change from the whole body (WB) control] at E10.5 (C), E11.5 (D), E12.5 (E), E16.5 (F), E19.5 (G) and P0 (H) was determined using iSyTE. The values obtained were plotted in a dot plot with Prox1 cKO DEGs [fold change (FC) of gene expression in Prox1 cKO lens compared with WT control] on the x-axis. The iSyTE lens enrichment in expressed as FC compared with the WB reference dataset (as described by Lachke et al., 2012) on the y-axis. As shown in the key, the size of the solid circles (downregulated in Prox1 cKO) or triangles (upregulated in Prox1 cKO) represents the lens expression levels in microarray fluorescence intensity units (data obtained from iSyTE and other publically available datasets). The color of the circles or triangles represents lens-enrichment scores in FC (red, lens-enriched genes; green, genes expressed at higher levels in the WB reference). As the lens progresses from E12.5 to P0, the vast majority of DEGs in the downDEGs category fall into the lens-enriched (upper left quadrant) category compared with upDEGs (upper right quadrant), which was significant by a χ² test for goodness of fit (P<0.00001). Notably, χ² values increased between the LV and the onset of LF differentiation (see Materials and Methods). (I) The fraction of the top 500 or top 100 lens-enriched genes at various developmental stages that are downregulated in Prox1 cKO. Error bars indicate s.e.m.; n=3. Red dotted line indicates the percentage of genes expected to be downregulated in a random sample.

Fig. 6.

Prox1 cKO lenses exhibit a decrease in the expression of three FGFRs and their downstream signaling. (A) qRT-PCR revealed significant decreases in the expression of Fgfr3 (P=0.01), the atypical receptor Fgfrl1 (P=0.04), and the FGF co-receptor Lctl (P=0.03) in E14.5 Prox1 cKO lenses; *P<0.05 by nested ANOVA. Error bars indicate s.d.; n=3. (B) By immunohistochemistry pErk1/2 (brown) was detected at the transition zones of WT lenses (B) and was absent from the equator of Prox1 cKO lenses (C) at E13.5. Similarly, robust pAKT staining (brown) was observed at the transition zones of WT lenses at E13.5 (D), but was absent in the posterior LFs of Prox1 cKO lenses (E). Blue, Hematoxylin. a, anterior; p, posterior, r, retina; e, epithelium; f, LFs. Scale bar: 100 µm.

Fig. 7.

Prox1 binds to the putative FGFR3, FGFRL1 and LCTL promoters as assayed by ChIP. (A-C) The regions upstream of the transcriptional start site (TSS) of Fgfr3, Fgfrl1 and Lctl were screened for matches to the Prox1 consensus binding sequences: (A or C) AAG(N)(not A) or CA(not A)(N)(N)(C or G)(C or T) (Chen et al., 2008). Putative Prox1 binding sites (bold) were identified in the Fgfr3 promoters of human, mouse and chicken (A), which matched the previously characterized Prox1 binding sites in the human FGFR3 promoter (Shin et al., 2006) and are numbered relative to the TSS. Predicted Prox1 binding sites (bold) are present upstream of human, mouse and chicken Fgfrl1 (B) and Lctl (C) putative TSSs. (D-F) ChIP was performed on embryonic chicken lenses, pulling down chromatin complexed with Prox1 protein. DNA fragments corresponding to the regions upstream of FGFR3 (D), FGFRL1 (E) and LCTL (F) were recovered at an enriched frequency when compared with regions 5 kb downstream of each TSS, as assayed by qRT-PCR followed by two-way ANOVA with *P≤0.05. Error bars indicate s.d.; n=3.

Fig. 8.

Prox1 expression is regulated by FGFR-mediated MAPK signaling. (A-C) Explants cultured without FGF2 maintained their cuboidal epithelial morphology (B, cell outlines) and did not upregulate Prox1 (C). (D-F) Explants cultured in a high dose (100 ng/ml) of FGF2 displayed cell multi-layering and cell elongation (E, cell outlines), features of LF differentiation in vitro, and exhibited elevated levels of nuclear Prox1 (F). (G-I) When cultured in the presence of the FGFR antagonist SU5402, explants maintained their cuboidal epithelial morphology (H, cell outlines) and did not increase their nuclear Prox1 expression (I) in response to FGF2, similar to controls not treated with FGF2 (C). (J-L) When cultured in the presence of an inhibitor of the MAPK pathway kinase Mek1 (UO126), explants maintained their cuboidal epithelial morphology (K, cell outlines) and slightly increased their nuclear Prox1 expression (L) in response to FGF2, as compared with controls not treated with FGF2 (C); however, these explants did not recapitulate the upregulation of nuclear Prox1 observed in non-inhibited samples (F). (M,N) Explants cultured with or without (100 ng/ml) FGF2 and with or without inhibitors were analyzed by western blot for Prox1 and Gapdh. Blots were quantified by densitometry using ANOVA followed by a Šídák multiple comparison post-test and are presented with s.e.m. Induction of LF differentiation with FGF2 resulted in a significant upregulation of Prox1 that was blocked by addition of the MAPK inhibitor UO126 but not significantly blocked by addition of the PI3K/AKT inhibitor LY294002. Blue, Hoechst; red, β-catenin; green, Prox1. Scale bars: 100 µm.

Fig. 9.

Model of Prox1 crosstalk with FGFRs. In normal lenses, FGFs interact with the canonical FGFR1-4, or with FGFRL1, to drive intracellular signaling. LCTL facilitates the interaction of FGFR1,2,4 with endocrine FGFs. Activation of FGFRs promotes signaling via the MAPK pathway, upregulating Prox1 expression in response to FGF stimulation. Further, Prox1 upregulates Fgfr3, Fgfrl1 and Lctl expression through direct promoter interactions, sensitizing these cells to respond to FGFs, driving LF differentiation. LEC, lens epithelial cell; LFC, lens fiber cell.