Lhx2 Is an Essential Factor for Retinal Gliogenesis and Notch Signaling

Abstract

Müller glia (MG) are the only glial cell type produced by the neuroepithelial progenitor cells that generate the vertebrate retina. MG are required to maintain retinal homeostasis and support the survival of retinal neurons. Furthermore, in certain vertebrate classes, MG function as adult stem cells, mediating retinal regeneration in response to injury. However, the mechanisms that regulate MG development are poorly understood because there is considerable overlap in gene expression between retinal progenitor cells and differentiated MG. We show that the LIM homeodomain transcription factor Lhx2 is required for the development of MG in the mouse retina. Temporally controlled knock-out studies reveal a requirement for Lhx2 during all stages of MG development, ranging from the proliferation of gliocompetent retinal progenitors, activation of Müller-specific gene expression, and terminal differentiation of MG morphological features. We show that Lhx2 regulates gliogenesis in part by regulating directly the expression of Notch pathway genes including Notch1, Dll1, and Dll3 and gliogenic transcription factors such as Hes1, Hes5, Sox8, and Rax. Conditional knock-out of Lhx2 resulted in a rapid downregulation of Notch pathway genes and loss of Notch signaling. We further demonstrate that Müller gliogenesis induced by misexpression of the potently gliogenic Notch pathway transcriptional effector Hes5 requires Lhx2 expression. These results indicate that Lhx2 not only directly regulates expression of Notch signaling pathway components, but also acts together with the gliogenic Notch pathway to drive MG specification and differentiation.

Keywords: Lhx2; Müller glia; Notch; differentiation; retina.

Figure 1.

Expression pattern of endogenous Lhx2 and the Pdgfrα-Cre transgene during retinal development. a–j, Immunohistochemistry demonstrating expression of Lhx2 during retinal development. a, b, Lhx2 is expressed throughout the retinal neuroepithelium and the retinal pigment epithelium at E10 and E12, but not in the lens or extraocular mesenchyme. The weak fluorescence in the lens at E12 represents nonspecific and extra-nuclear background staining. c, Expression of Lhx2 is downregulated from newly generated retinal ganglion cells and amacrine cells by E14. d, e, Lhx2 expression continues to be downregulated by newborn neurons as retinogenesis progresses from E16 to E18. f, g, k, l, In postnatal retina, Lhx2 is expressed in remaining mitotic progenitors (f, g, yellow arrowheads) that coexpress Vsx2 and Ki67 (k, l) and by a subset of amacrine cells (f, g, white arrows). h–j, m–o, By P7, Lhx2 is restricted to Glul, P27^Kip1, and Sox9 expressing MG (h–j, red arrowheads, m–o) and a small subset of amacrine cells (h–j, white arrows). p, q, The Pdgfrα-Cre transgene is expressed in a subset of retinal progenitor cells at P0 and P4 in the retina. r–u, Pdgfrα-Cre; R26-stop-YFP labels MG, which express the MG markers Lhx2, P27^Kip1, Glul, and Rlbp1 at P21. v, 97.6% (SE = 0.33%; n = 4) of P27^Kip1 expressing MG are colabeled with YFP, demonstrating Pdgfrα-Cre activation in MG. GCL, Ganglion cell layer; INL, inner nuclear layer; NBL, neuroblastic layer; ONL, outer nuclear layer; E, embryonic day. 1.5× digital enlargements without DAPI labeling are included for P21 data. Scale bars, 150 μm (a), 175 μm (b), 200 μm (c), 50 μm (d–u).

Figure 2.

Retinal loss of function of Lhx2 disrupts MG development. a–h, Pdgfrα-Cre-mediated Lhx2 loss of function results in the loss of YFP⁺ MG and severe retinal dysplasia by P30. b, d, f, arrows, Staining of the MG markers P27^Kip1, Glul, and Rlbp1 is lost among the remaining YFP-labeled cells in the medial INL in the Pdgfrα-Cre;R26-stop-YFP;Lhx2^lox/lox animals, whereas Sox9 expression is reduced but not completely lost (h, arrows, asterisk). i, j, Identification of regions of normal histology correlates with failed Lhx2 loss of function and persistent Lhx2 expression. k, Immunohistochemistry showing YFP and phosphohistone H3 (PHH3) colabeling at P0 in Pdgfrα-Cre;R26-stop-YFP;Lhx2^+/+ and Pdgfrα-Cre;R26-stop-YFP;Lhx2^lox/lox animals. l, There is a significant (p < 0.05) reduction of YFP-labeled cells in Pdgfrα-Cre;R26-stop-YFP;Lhx2^lox/lox animals, 24.2% (SE = 2.45%, n = 4) compared with controls Pdgfrα-Cre;R26-stop-YFP;Lhx2^+/+, 45.7% (SE = 1.15%, n = 4) at P0. m, Retinal dissociates showing YFP and Ki67 colabeling at P0 in Pdgfrα-Cre;R26-stop-YFP;Lhx2^+/+ and Pdgfrα-Cre;R26-stop-YFP;Lhx2^lox/lox animals. n, There is a significant (p < 0.05) reduction of YFP-labeled cells coexpressing Ki67 in Pdgfrα-Cre;R26-stop-YFP;Lhx2^lox/lox animals, 24.2% (SE = 1.81%, n = 4) compared with 42% (SE = 4.28%, n = 4) in controls at P0. No significant proportional change in coexpression of Ki67 in YFP-ve cells in conditional knock-outs compared with controls was seen (o). The number of TUNEL/YFP-labeled apoptotic cells in Pdgfrα-Cre;R26YFP;Lhx2^lox/lox animals is elevated at P0 but not statistically significant (n > 0.05) (n). *Significant change by two-tailed Student's t test. 1.5× digital enlargements without DAPI labeling are included for b, d, f, and h. Scale bars: 50 μm (a, c, e, g, i–k), 100 μm (m).

Figure 3.

Mosaic loss of function of Lhx2 by electroporation disrupts MG development. a–h. Electroporation of pCAG-Cre into Lhx2^lox/lox mice at P0 disrupts the development of MG as shown by multiple molecular markers. Regions of high electroporation efficiency show ONL dysplasia (b, d, f, h, white arrows). i, The proportion of electroporated cells expressing the MG markers P27^Kip1, Glul, Rlbp1, and Sox9 is significantly (p < 0.05, n = 6) decreased from 4.9%, 5.6%, 4.4%, and 5%, respectively, in Lhx2^+/+ to 0.98%, 0.82%, 1%, and 1.5%, respectively, in Lhx2^lox/lox retinas. j, No change was detected in the proportion of MG generated among nonelectroporated cells (P27^Kip1 or Glul⁺GFP⁺/DAPI). k, We detected no significant increase in cell death by activated caspase-3 labeling (p > 0.05, n = 3). l–p, There was no significant change in the fraction of bipolar cells (identified by Vsx2 expression), photoreceptors (identified by morphology), amacrine cells (identified by Pax6 expression and morphology), or the glycinergic amacrine cell subset (identified by glycine expression and morphology) after electroporation of pCAG-Cre into control or Lhx2^lox/lox retinas. *Statistical significance. 1.5× digital enlargements without DAPI labeling are included for a–h. Scale bars, 50 μm (a, c, e, g, l–o).

Figure 4.

Conditional loss of function of Lhx2 in MG results in specific deficits in MG differentiation and/or reactive gliosis. a, Conditional activation of the Rax-CreER^T2 transgene by intraperitoneal administration of tamoxifen from P1 to P3 shows that Cre expression is restricted to MG precursors. b, Activation of the Glast-CreER^T2 transgene by intraperitoneal administration of tamoxifen from P4–P8 showing that Cre expression is restricted to MG. c, Glast-CreER^T2-mediated Lhx2 loss of function resulted in upregulation of Gfap expression but no changes in morphology. d, Rax-CreER^T2-mediated Lhx2 loss of function in MG precursors results in dysmorphic MG at P14, upregulation of Gfap, and retinal dysplasia (d, arrows). e–h, Rax-CreER^T2-mediated Lhx2 loss of function results in decreased expression of the MG markers Rlbp1 (e, f, arrows) and Glul (g, h, arrows, asterisks). 1.5× digital enlargements without DAPI labeling are included for a, b, and e–h. Scale bars, 50 μm (all panels).

Figure 5.

Conditional loss of function of Lhx2 results in disruption of the MG component of the apical outer limiting. a, Rax-CreER^T2;Lhx2^lox/lox MG-like cells do not terminate at the apical outer limiting membrane (white arrows) and do not colabel Ctnnb1 (also known as β-catenin). b, c, Glast-CreER^T2;Lhx2^lox/lox MG form normal apical termini that colabel with Ctnnb1 (white arrows). 4-OHTx, 4-hydroxytamoxifen. Scale bars, 50 μm (all panels).

Figure 6.

Lhx2 loss of function perturbs Notch signaling and blocks Hes5-mediated gliogenesis. a, b, Co-electroporation of pCAG-Cre with pCBFRE-GFP at P0. Expression of the Notch reporter is significantly (p < 0.05; n = 5) decreased among electroporated cells at P3 in Lhx2^lox/lox mice. c, d, f, h–j, l, Electroporation of Hes5 at P0 significantly induces the formation of MG by P14 (p < 0.05; n = 6 for all of p27^Kip1; Glul; Rlbp; Sox9). d–l, Electroporation of pCAG-Cre with pCAGIG-Hes5 into Lhx2^lox/lox vs Lhx2^+/+ mice shows that the effect of Hes5 is blocked by concurrent Lhx2 loss of function (p < 0.05; n = 6; for all of p27^Kip1; Glul; Rlbp; Sox9). 1.5-fold digital enlargements without DAPI labeling are included for d–k. ^ Significant increase; *significant decrease. Scale bars, 50 μm (all panels).

Figure 7.

Retinal cell class enrichment and gene ontology analysis of RNA-Seq data generated from Pdgfrα-Cre-mediated Lhx2 knock-outs. a, b, Retinal cellular enrichment analysis for downregulated and upregulated genes using a cutoff of p < 0.05 and fold change >1.6 or <−1.6. x-axis represents the proportion of genes falling into a retinal cell type class. Gene expression may occur in more than one cell type, so proportions do not sum to 1. c, Gene ontology enrichment for downregulated genes identified by RNA-Seq ranked by p-value. Hyp/EmThal, Hypothalamus/ eminentia thalami; GC, ganglion cells; AC, amacrine cells; BC, bipolar cells; HC, horizontal cells.

Figure 8.

Expression of Notch pathway, gliogenic, RPC-enriched, and hypothalamic-enriched genes in the Lhx2-deficient retina. a, qRT-PCR analysis of P0.5 Pdgfrα-Cre;Lhx2^lox/lox and control Lhx2^lox/lox mice. Bars on the graph represent the mean relative quantity (RQ) of expression of the gene target, with error bars representing the minimum and maximum value of RQ observed in the study. *Statistical significance. b–m, In situ hybridization performed on P0.5 Pdgfrα-Cre;Lhx2^lox/lox and control Lhx2^lox/lox mice. Arrows indicate regions of loss or gain of expression in Pdgfrα-Cre;Lhx2^lox/lox mice. Scale bars, 500 μm (5× low magnification), 200 μm (20× high magnification).

Figure 9.

ChIP analysis of Lhx2 in the developing retina. a, Relative percentages of input recovery for the H3K27Ac and isotype control fractions in the retina at P2. Bars represent SEM (n = 3, minimum degrees of freedom = 9). a′, Fold enrichment for the H3K27Acetylated IP on indicated promoter sites. The target regions are all significantly enriched (p < 0.0001) by t test assuming unequal variances between Lhx2 ChIP and Ig control fractions. b, c, Lhx2 ChIP was performed on retinal tissue collected at P2 and P8. Graphs show the mean percentages of input recovery for the immunoprecipitated fractions and the isotype controls compared by two-tailed t test. *Statistical significance (p < 0.05). Error bars represent the SE (n > = 3). Target regions were inferred from a computational analysis of Lhx2 consensus sequences near the genes of interest. d, Ratio of Lhx2 occupancy at target promoters, P2 versus P8. P2 and P8 occupancy levels were normalized to isotypic controls before ratio calculation.