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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.

No MeSH data available.


Electroporation of Rax both blocks MG development and fails to rescue the loss of MG resulting from Lhx2 loss of function, whereas Plagl1 blocks MG development but is sufficient to rescue P27Kip1 expression resulting from Lhx2 loss of function.(a–c) In situ hybridization of Plagl1 at P5, P7, and P14. Arrows show regions of Plagl1 RNA enrichment. (d–g) Electroporation of Lhx2+/+ and Lhx2lox/lox retinas with Cre/GFP/Rax and analyzed by immunohistochemical co-labeling of GFP with the MG markers P27Kip1 and GLUL. (h–k) Electroporation of Lhx2+/+ and Lhx2lox/lox retinas with Cre/GFP/Plagl1 and analyzed by immunohistochemical co-labeling of GFP with P27Kip1 and GLUL. (l,m) Quantification of GFP/P27Kip1 and GFP/GLUL co-labeled cells in Lhx2+/+ and Lhx2lox/lox mice following Cre/GFP, Cre/GFP/Rax or Cre/GFP/PlagL1 electroporation. (n,o) Quantification of radial cells in Lhx2+/+ or Lhx2lox/lox mice following Cre/GFP, Cre/GFP/Rax or Cre/GFP/PlagL1 electroporation. *Indicates significant decrease while ^ indicates significant increases (P < 0.05, N = 6 for marker counts, N = 12 for radial morphology counts). NBL, neuroblastic layer. Scale bars: 100 μm (a–c), 50 μm (d,h).
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f3: Electroporation of Rax both blocks MG development and fails to rescue the loss of MG resulting from Lhx2 loss of function, whereas Plagl1 blocks MG development but is sufficient to rescue P27Kip1 expression resulting from Lhx2 loss of function.(a–c) In situ hybridization of Plagl1 at P5, P7, and P14. Arrows show regions of Plagl1 RNA enrichment. (d–g) Electroporation of Lhx2+/+ and Lhx2lox/lox retinas with Cre/GFP/Rax and analyzed by immunohistochemical co-labeling of GFP with the MG markers P27Kip1 and GLUL. (h–k) Electroporation of Lhx2+/+ and Lhx2lox/lox retinas with Cre/GFP/Plagl1 and analyzed by immunohistochemical co-labeling of GFP with P27Kip1 and GLUL. (l,m) Quantification of GFP/P27Kip1 and GFP/GLUL co-labeled cells in Lhx2+/+ and Lhx2lox/lox mice following Cre/GFP, Cre/GFP/Rax or Cre/GFP/PlagL1 electroporation. (n,o) Quantification of radial cells in Lhx2+/+ or Lhx2lox/lox mice following Cre/GFP, Cre/GFP/Rax or Cre/GFP/PlagL1 electroporation. *Indicates significant decrease while ^ indicates significant increases (P < 0.05, N = 6 for marker counts, N = 12 for radial morphology counts). NBL, neuroblastic layer. Scale bars: 100 μm (a–c), 50 μm (d,h).

Mentions: The retinal expression pattern of Plagl1 in the mouse has not been reported. We performed in situ hybridization to detect Plagl1 RNA expression in the retina, and found expression consistent with MG localization (Fig. 3a–c). RNA expression was identified in the medial neuroblastic layer (NBL) at P5 (Fig. 3a), consistent with the location of differentiating MG. Plagl1 was similarly enriched in the medial INL, where MG are located at P7 (Fig. 3b). By P14, Plagl1 expression is less clearly concentrated in the medial INL, although expression remains generally enriched in the INL (Fig. 3c). Cumulatively, these results indicate that Plagl1 expression is enriched in developing MG.


Multiple intrinsic factors act in concert with Lhx2 to direct retinal gliogenesis
Electroporation of Rax both blocks MG development and fails to rescue the loss of MG resulting from Lhx2 loss of function, whereas Plagl1 blocks MG development but is sufficient to rescue P27Kip1 expression resulting from Lhx2 loss of function.(a–c) In situ hybridization of Plagl1 at P5, P7, and P14. Arrows show regions of Plagl1 RNA enrichment. (d–g) Electroporation of Lhx2+/+ and Lhx2lox/lox retinas with Cre/GFP/Rax and analyzed by immunohistochemical co-labeling of GFP with the MG markers P27Kip1 and GLUL. (h–k) Electroporation of Lhx2+/+ and Lhx2lox/lox retinas with Cre/GFP/Plagl1 and analyzed by immunohistochemical co-labeling of GFP with P27Kip1 and GLUL. (l,m) Quantification of GFP/P27Kip1 and GFP/GLUL co-labeled cells in Lhx2+/+ and Lhx2lox/lox mice following Cre/GFP, Cre/GFP/Rax or Cre/GFP/PlagL1 electroporation. (n,o) Quantification of radial cells in Lhx2+/+ or Lhx2lox/lox mice following Cre/GFP, Cre/GFP/Rax or Cre/GFP/PlagL1 electroporation. *Indicates significant decrease while ^ indicates significant increases (P < 0.05, N = 6 for marker counts, N = 12 for radial morphology counts). NBL, neuroblastic layer. Scale bars: 100 μm (a–c), 50 μm (d,h).
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f3: Electroporation of Rax both blocks MG development and fails to rescue the loss of MG resulting from Lhx2 loss of function, whereas Plagl1 blocks MG development but is sufficient to rescue P27Kip1 expression resulting from Lhx2 loss of function.(a–c) In situ hybridization of Plagl1 at P5, P7, and P14. Arrows show regions of Plagl1 RNA enrichment. (d–g) Electroporation of Lhx2+/+ and Lhx2lox/lox retinas with Cre/GFP/Rax and analyzed by immunohistochemical co-labeling of GFP with the MG markers P27Kip1 and GLUL. (h–k) Electroporation of Lhx2+/+ and Lhx2lox/lox retinas with Cre/GFP/Plagl1 and analyzed by immunohistochemical co-labeling of GFP with P27Kip1 and GLUL. (l,m) Quantification of GFP/P27Kip1 and GFP/GLUL co-labeled cells in Lhx2+/+ and Lhx2lox/lox mice following Cre/GFP, Cre/GFP/Rax or Cre/GFP/PlagL1 electroporation. (n,o) Quantification of radial cells in Lhx2+/+ or Lhx2lox/lox mice following Cre/GFP, Cre/GFP/Rax or Cre/GFP/PlagL1 electroporation. *Indicates significant decrease while ^ indicates significant increases (P < 0.05, N = 6 for marker counts, N = 12 for radial morphology counts). NBL, neuroblastic layer. Scale bars: 100 μm (a–c), 50 μm (d,h).
Mentions: The retinal expression pattern of Plagl1 in the mouse has not been reported. We performed in situ hybridization to detect Plagl1 RNA expression in the retina, and found expression consistent with MG localization (Fig. 3a–c). RNA expression was identified in the medial neuroblastic layer (NBL) at P5 (Fig. 3a), consistent with the location of differentiating MG. Plagl1 was similarly enriched in the medial INL, where MG are located at P7 (Fig. 3b). By P14, Plagl1 expression is less clearly concentrated in the medial INL, although expression remains generally enriched in the INL (Fig. 3c). Cumulatively, these results indicate that Plagl1 expression is enriched in developing MG.

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.