Impaired GSH biosynthesis disrupts eye development, lens morphogenesis and PAX6 function

Abstract

Purpose: The purpose of this study was to elucidate the role and molecular consequences of impaired glutathione (GSH) biosynthesis on eye development.

Methods: GSH biosynthesis was impaired in surface ectoderm-derived ocular tissues by crossing Gclc^f/f mice with hemizygous Le-Cre transgenic mice to produce Gclc^f/f/Le-Cre^Tg/- (KO) mice. Control mice included Gclc^f/fand Gclc^wt/wt/Le-Cre^Tg/- mice (CRE). Eyes from all mice (at various stages of eye development) were subjected to histological, immunohistochemical, Western blot, RT-qPCR, RNA-seq, and subsequent Gene Ontology, Ingenuity Pathway Analysis and TRANSFAC analyses. PAX6 transactivation activity was studied using a luciferase reporter assay in HEK293T cells depleted of GSH using buthionine sulfoximine (BSO).

Results: Deletion of Gclc diminished GSH levels, increased reactive oxygen species (ROS), and caused an overt microphthalmia phenotype characterized by malformation of the cornea, iris, lens, and retina that is distinct from and much more profound than the one observed in CRE mice. In addition, only the lenses of KO mice displayed reduced crystallin (α, β), PITX3 and Foxe3 expression. RNA-seq analyses at postnatal day 1 revealed 1552 differentially expressed genes (DEGs) in the lenses of KO mice relative to those from Gclc^f/f mice, with Crystallin and lens fiber cell identity genes being downregulated while lens epithelial cell identity and immune response genes were upregulated. Bioinformatic analysis of the DEGs implicated PAX6 as a key upstream regulator. PAX6 transactivation activity was impaired in BSO-treated HEK293T cells.

Conclusions: These data suggest that impaired ocular GSH biosynthesis may disrupt eye development and PAX6 function.

Keywords: Glutathione; Lens development; Lens transcriptome; Microphthalmia; PAX6.

Figure 1.. Surface-ectoderm derived tissue-specific deletion of Gclc reduces ocular GSH content.

(A) Agarose gel (2%) separation of genomic DNA obtained from ear tissue of P14-aged Gclc^f/f/Le-Cre^−/− (CON), Gclc^wt/f/Le-Cre^Tg/- (HET), and Gclc^f/f/Le-Cre^Tg/- (KO) mice by PCR amplification of the Gclc wild-type (Gclc(wt)) or Gclc floxed (Gclc (f)) alleles, or the Le-Cre transgene (Cre(Tg)). Two PCR reactions were needed to genotype mice to detect the presence of Gclc wt and Cre transgene alleles (left lane for each mouse) and the Gclc f allele (right lane for each mouse). (B) Western blot analysis of GCLC and α-crystallin proteins in the lens and retina of P1-aged CON, HET and KO mice. (C) Immunohistochemical staining of GCLC in CON and KO mice aged E14.5. Lens germinative zone and newly differentiated fiber cells (black box) are shown at higher magnification (bottom row). Black arrow indicates high expression of GCLC in the germinative zone. (D) GSH levels in the whole eyes of P21 CON (white bar) and KO (black bar) mice. Data are presented as the mean and associated s.e.m. from 3–4 mice. * P < 0.05, two-tailed Mann-Whitney test. (E) ROS levels in the lenses of P21 CON (black bar), KO (grey bar) and CRE (white bar). Data are presented as the mean and associated s.d. from 3 mice. **** P < 0.0001, one-way ANOVA, with Tukey’s correction, compared to other group (as indicated). (F) Representative photographs (A’-D’) and slit-lamp microscopy images (E’-F’) of eyes from P21-aged CON and KO mice. The images are representative of results obtained in all 3 mice per genotype.

Figure 2.. Gclc deletion causes severe ocular malformations.

(A-P’) Representative images of hematoxylin & eosin-stained eyes from CON (A-L) and KO (A’- P’) mice aged E14.5 (A-C, A’-D’), P1 (D-F, E’-H’), P20 (G-I, I’-L’) or P50 (J-L, M’-P’). The images are reflective of results obtained in all 3 mice per genotype. Lens (black box) in A, B’, D, E’, I’ and M’ are shown at higher magnification in B, B’, E, G’, J’, and N’, respectively. Retina (dashed black box) in A, A’, F’, I’ and M’ are shown at higher magnification in C, D’, H’, L’ and P’, respectively. Cornea (yellow box) in G, J’ and M’ is shown at higher magnification in H, L’, and P’. Blood vessels are indicated by a black arrowhead (C, D’, F, F’) and malformed lens epithelium is indicated by a yellow arrowhead (J’, N’). Abbreviations: AC, anterior chamber; C, cornea; I, Iris; L, lens; R, retina; GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; ONL, outer nuclear layer.

Figure 3.. Transcriptomic differences between lenses of P1-aged CON and KO mice.

(A) Volcano plot illustrating differentially expressed genes (DEGs) in the lenses of P1-aged CON and KO mice. Expression levels in the lenses of KO mice are shown as log₂fold change (log2FC) and -log10Padj relative to lenses of CON mice (pooled samples from 3 mice). DEGs were classified as: |log₂FC| ≥ 1; Benjamini-Hochberg adjusted P (Padj) < 0.05. Colors: Blue, expression in KO mice significantly downregulated; Grey, no significant change in expression; Red, expression in KO mice significantly upregulated. (B-C) Gene ontology of upregulated (B) and downregulated (C) genes as determined by David Bioinformatics Resources. The ten most highly significant biological process (BP) terms and the number of genes in each term are shown. P values were calculated by Benjamini-Hochberg method (B) or Fischer’s exact test (C). Fischer’s exact test was used because the Benjamini-Hochberg method limited our ability to detect BP terms in the downregulated genes.

Figure 4.. Altered expression of crystallin and other lens cell identity genes in KO lenses of P1-aged KO mice.

(A-C) RNA-seq box plot and heatmaps indicating the expression of key lens cell identity genes in the lens at P1-aged KO mice (pooled samples from 3 mice). (A) RNA-seq box plots indicating variance stabilizing transformed (VST) normalized count data for differentially-expressed crystallin genes in the lens of KO mice. VST count data are shown as mean (thin horizontal bar) ± standard deviation (error bar). P values were calculated by Benjamini-Hochberg method. Colors: red, CON; blue, KO. (B-C) RNA-seq expression heatmap showing differentially-expressed lens epithelial cell (B) and fiber cell (C) identity genes in the lenses of KO mice. Data are shown as VST count data and z-score (standard deviations from mean). Gclc control sample is indicated by the prefix CON and each Gclc knockout sample is indicated by the prefix KO. Dot represents one pooled sample. (D) Pearson correlation (coefficient of determination) of lens gene expression levels (with gene expression shown as log₂fold change (log2FC) in the lenses of KO mice relative to the lenses of CON mice) between Pax6 microarray and Gclc KO RNA-seq datasets restricted to DEGs in Pax6 KO lens (upper left corner) or restricted to only differentially-expressed lens identity genes (orange points; grey box). Each point represents one gene. Blue line indicates linear trendline.

Figure 5.. Impaired PAX6 function is predicted to cause a lens disorder and is influenced by intracellular GSH concentration.

(A) Volcano plot indicating the expression of PAX6-regulated genes that were differentially expressed in the lenses of Gclc KO mice relative to CON mice. PAX6-regulated genes are highlighted. Differentially expressed genes were classified as |log₂FC| ≥ 1; Benjamini-Hochberg adjusted P (Padj) < 0.05. Colors: blue, expression downregulated; grey, no significant change in expression; red; expression upregulated. (B) Changes in PAX6-regulated genes predicts impaired PAX6 function. Colors: blue, predicted reduced activity; green, reduced gene expression; orange, predicted lens disorder; red, increased gene expression. Shapes: described by key located to the right of the figure. Diagram created using Ingenuity Pathway Analysis software (Qiagen, Hilgen, Germany). (C-D) Representative images of immunohistochemical staining of ocular PAX6 expression in E14.5 (C) and P3 (D) -aged CON and KO mice. Whole eye (top row) and lens (middle and bottom rows) expression of PAX6 are shown as brown stain. Images are representative of results obtained in all 3 mice per genotype. Regions contained in boxes are shown at higher magnification, 400X (bottom row). Abbreviations: L, lens; R, retina. (E) Impact of BSO treatment on PAX6 transactivation activity in HEK293T cells transfected with a PAX6 luciferase reporter construct. Data are presented as the mean of the ratio of measured firefly luciferase (FLuc) to renilla luciferase (RLuc) and associated s.d. (n ≥ 6 wells from at least three independent plates). * P < 0.05, one-way ANOVA with post-hoc Dunnett’s test, compared to 0 µM BSO.