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Multiple intrinsic factors act in concert with Lhx2 to direct retinal gliogenesis

View Article: PubMed Central - PubMed

ABSTRACT

Müller glia (MG) are the principal glial cell type in the vertebrate retina. Recent work has identified the LIM homeodomain factor encoding gene Lhx2 as necessary for both Notch signaling and MG differentiation in late-stage retinal progenitor cells (RPCs). However, the extent to which Lhx2 interacts with other intrinsic regulators of MG differentiation is unclear. We investigated this question by investigating the effects of overexpression of multiple transcriptional regulators that are either known or hypothesized to control MG formation, in both wildtype and Lhx2-deficient RPCs. We observe that constitutively elevated Notch signaling, induced by N1ICD electroporation, inhibited gliogenesis in wildtype animals, but rescued MG development in Lhx2-deficient retinas. Electroporation of Nfia promoted the formation of cells with MG-like radial morphology, but did not drive expression of MG molecular markers. Plagl1 and Sox9 did not induce gliogenesis in wildtype animals, but nonetheless activated expression of the Müller marker P27Kip1 in Lhx2-deficient cells. Finally, Sox2, Sox8, and Sox9 promoted amacrine cell formation in Lhx2-deficient cells, but not in wildtype retinas. These findings demonstrate that overexpression of individual gliogenic factors typically regulates only a subset of characteristic MG markers, and that these effects are differentially modulated by Lhx2.

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Electroporation of N1ICD maintains radial RPCs and is sufficient to rescue loss of MG development resulting from Lhx2 loss of function.(a–f) Lhx2+/+ retinas electroporated with Cre/GFP/N1ICD. (a–c) Fluorescent immunohistochemical labeling of electroporated retinas with GFP and the proliferation markers PHH3 and KI67, or the progenitor/amacrine/ganglion cell marker PAX6. Arrows indicate co-labeled cells. (d–f) fluorescent co-labeling with the MG markers P27Kip1, GLUL, and RLBP1. (g,h) Lhx2lox/lox retinas electroporated with Cre/GFP/N1ICD and analyzed by fluorescent immunohistochemical labeling with P27Kip1 and GLUL. (i,j) Quantification of GFP/P27Kip1 and GFP/GLUL co-labeled cells in Lhx2+/+ and Lhx2lox/lox mice following Cre/GFP or Cre/GFP/N1ICD. (k,l) Quantification of radial cells in Lhx2+/+ or Lhx2lox/lox mice following Cre/GFP or Cre/GFP/N1ICD electroporation. *Indicates significant decrease while ^ indicates significant increases (P < 0.05, N = 6 for marker counts, N = 12 for radial morphology counts). ONL, outer nuclear layer; INL inner nuclear layer; GCL, ganglion cell layer. Scale bars: 50 μm (c,f,g,h).
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f1: Electroporation of N1ICD maintains radial RPCs and is sufficient to rescue loss of MG development resulting from Lhx2 loss of function.(a–f) Lhx2+/+ retinas electroporated with Cre/GFP/N1ICD. (a–c) Fluorescent immunohistochemical labeling of electroporated retinas with GFP and the proliferation markers PHH3 and KI67, or the progenitor/amacrine/ganglion cell marker PAX6. Arrows indicate co-labeled cells. (d–f) fluorescent co-labeling with the MG markers P27Kip1, GLUL, and RLBP1. (g,h) Lhx2lox/lox retinas electroporated with Cre/GFP/N1ICD and analyzed by fluorescent immunohistochemical labeling with P27Kip1 and GLUL. (i,j) Quantification of GFP/P27Kip1 and GFP/GLUL co-labeled cells in Lhx2+/+ and Lhx2lox/lox mice following Cre/GFP or Cre/GFP/N1ICD. (k,l) Quantification of radial cells in Lhx2+/+ or Lhx2lox/lox mice following Cre/GFP or Cre/GFP/N1ICD electroporation. *Indicates significant decrease while ^ indicates significant increases (P < 0.05, N = 6 for marker counts, N = 12 for radial morphology counts). ONL, outer nuclear layer; INL inner nuclear layer; GCL, ganglion cell layer. Scale bars: 50 μm (c,f,g,h).

Mentions: We electroporated neonatal mouse retinas with control pCAG-Cre/pCALNL-GFP (Cre) or pCAGGS-N1ICD/pCAG-Cre/pCALNL-GFP (N1ICD/Cre) constructs and analyzed the electroporated retinas at postnatal day (P)14. The pCAG-Cre plasmid constitutively expresses Cre recombinase, while pCALNL-GFP expresses the GFP fluorescent reporter following Cre mediated excision of a transcriptional stop site. The pCAGGS-N1ICD plasmid constitutively expresses the N1ICD domain. In wildtype (WT) retinas electroporated with Cre, we observed that approximately 5% of electroporated cells expressed the MG markers P27Kip1, GLUL, or displayed radial morphology characteristic of MG, where cell processes extended from the basal inner limiting membrane to the apical outer limiting membrane (4.9% P27Kip1 +ve; 5.6% GLUL +ve; 4.9% MG-like radial morphology) (Fig. 1a–f,i,k). Electroporation of N1ICD dramatically reduced the number of P27Kip1 and GLUL-expressing cells to 0.4 and 0.7% respectively (Fig. 1d,e,i). Furthermore, co-labeling with the MG marker RLBP1 was not detected (Fig. 1f). Conversely, the proportion of cells exhibiting radial morphology significantly increased to 7.7% (Fig. 1k).


Multiple intrinsic factors act in concert with Lhx2 to direct retinal gliogenesis
Electroporation of N1ICD maintains radial RPCs and is sufficient to rescue loss of MG development resulting from Lhx2 loss of function.(a–f) Lhx2+/+ retinas electroporated with Cre/GFP/N1ICD. (a–c) Fluorescent immunohistochemical labeling of electroporated retinas with GFP and the proliferation markers PHH3 and KI67, or the progenitor/amacrine/ganglion cell marker PAX6. Arrows indicate co-labeled cells. (d–f) fluorescent co-labeling with the MG markers P27Kip1, GLUL, and RLBP1. (g,h) Lhx2lox/lox retinas electroporated with Cre/GFP/N1ICD and analyzed by fluorescent immunohistochemical labeling with P27Kip1 and GLUL. (i,j) Quantification of GFP/P27Kip1 and GFP/GLUL co-labeled cells in Lhx2+/+ and Lhx2lox/lox mice following Cre/GFP or Cre/GFP/N1ICD. (k,l) Quantification of radial cells in Lhx2+/+ or Lhx2lox/lox mice following Cre/GFP or Cre/GFP/N1ICD electroporation. *Indicates significant decrease while ^ indicates significant increases (P < 0.05, N = 6 for marker counts, N = 12 for radial morphology counts). ONL, outer nuclear layer; INL inner nuclear layer; GCL, ganglion cell layer. Scale bars: 50 μm (c,f,g,h).
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f1: Electroporation of N1ICD maintains radial RPCs and is sufficient to rescue loss of MG development resulting from Lhx2 loss of function.(a–f) Lhx2+/+ retinas electroporated with Cre/GFP/N1ICD. (a–c) Fluorescent immunohistochemical labeling of electroporated retinas with GFP and the proliferation markers PHH3 and KI67, or the progenitor/amacrine/ganglion cell marker PAX6. Arrows indicate co-labeled cells. (d–f) fluorescent co-labeling with the MG markers P27Kip1, GLUL, and RLBP1. (g,h) Lhx2lox/lox retinas electroporated with Cre/GFP/N1ICD and analyzed by fluorescent immunohistochemical labeling with P27Kip1 and GLUL. (i,j) Quantification of GFP/P27Kip1 and GFP/GLUL co-labeled cells in Lhx2+/+ and Lhx2lox/lox mice following Cre/GFP or Cre/GFP/N1ICD. (k,l) Quantification of radial cells in Lhx2+/+ or Lhx2lox/lox mice following Cre/GFP or Cre/GFP/N1ICD electroporation. *Indicates significant decrease while ^ indicates significant increases (P < 0.05, N = 6 for marker counts, N = 12 for radial morphology counts). ONL, outer nuclear layer; INL inner nuclear layer; GCL, ganglion cell layer. Scale bars: 50 μm (c,f,g,h).
Mentions: We electroporated neonatal mouse retinas with control pCAG-Cre/pCALNL-GFP (Cre) or pCAGGS-N1ICD/pCAG-Cre/pCALNL-GFP (N1ICD/Cre) constructs and analyzed the electroporated retinas at postnatal day (P)14. The pCAG-Cre plasmid constitutively expresses Cre recombinase, while pCALNL-GFP expresses the GFP fluorescent reporter following Cre mediated excision of a transcriptional stop site. The pCAGGS-N1ICD plasmid constitutively expresses the N1ICD domain. In wildtype (WT) retinas electroporated with Cre, we observed that approximately 5% of electroporated cells expressed the MG markers P27Kip1, GLUL, or displayed radial morphology characteristic of MG, where cell processes extended from the basal inner limiting membrane to the apical outer limiting membrane (4.9% P27Kip1 +ve; 5.6% GLUL +ve; 4.9% MG-like radial morphology) (Fig. 1a–f,i,k). Electroporation of N1ICD dramatically reduced the number of P27Kip1 and GLUL-expressing cells to 0.4 and 0.7% respectively (Fig. 1d,e,i). Furthermore, co-labeling with the MG marker RLBP1 was not detected (Fig. 1f). Conversely, the proportion of cells exhibiting radial morphology significantly increased to 7.7% (Fig. 1k).

View Article: PubMed Central - PubMed

ABSTRACT

M&uuml;ller glia (MG) are the principal glial cell type in the vertebrate retina. Recent work has identified the LIM homeodomain factor encoding gene Lhx2 as necessary for both Notch signaling and MG differentiation in late-stage retinal progenitor cells (RPCs). However, the extent to which Lhx2 interacts with other intrinsic regulators of MG differentiation is unclear. We investigated this question by investigating the effects of overexpression of multiple transcriptional regulators that are either known or hypothesized to control MG formation, in both wildtype and Lhx2-deficient RPCs. We observe that constitutively elevated Notch signaling, induced by N1ICD electroporation, inhibited gliogenesis in wildtype animals, but rescued MG development in Lhx2-deficient retinas. Electroporation of Nfia promoted the formation of cells with MG-like radial morphology, but did not drive expression of MG molecular markers. Plagl1 and Sox9 did not induce gliogenesis in wildtype animals, but nonetheless activated expression of the M&uuml;ller marker P27Kip1 in Lhx2-deficient cells. Finally, Sox2, Sox8, and Sox9 promoted amacrine cell formation in Lhx2-deficient cells, but not in wildtype retinas. These findings demonstrate that overexpression of individual gliogenic factors typically regulates only a subset of characteristic MG markers, and that these effects are differentially modulated by Lhx2.

No MeSH data available.


Related in: MedlinePlus