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

Jimmy De Melo,Brian S Clark,Seth Blackshaw

doi:10.1038/srep32757

Jimmy De Melo, Brian S Clark + Show 1 more

Open Access

https://doi.org/10.1038/srep32757

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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.

Highlights

Müller glia (MG) are adult radial glial cells that function as the primary physiological support cells within the retina
In wildtype (WT) retinas electroporated with Cre, we observed that approximately 5% of electroporated cells expressed the MG markers P27Kip[1], 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 P27Kip[1] and GLUL-expressing cells to 0.4 and 0.7% respectively (Fig. 1d,e,i)

Summary

Introduction

Müller glia (MG) are adult radial glial cells that function as the primary physiological support cells within the retina. MG are specified from the progenitor cells of the retinal neuroepithelium (RPCs), which generate retinal neurons, and share many morphological and molecular features with RPCs1–3 Both cell types feature radial processes that delimit the apical and basal surfaces of the retina[2,4] and both co-express many transcription factors, neurofilament proteins, and signaling pathway molecules[3,5,6]. Several studies show that TFs in the retina that control gliogenesis are often required for RPC maintenance, survival, proliferation, and/or progression through stages of developmental competence[16,17,18,19,20,21] This shared gene expression makes discrimination between immature MG precursors and RPCs difficult. More recent analysis indicates that N1ICD is less directly instructive, instead functioning to maintain undifferentiated RPCs in a slowly proliferative state while blocking activation of neurogenic genes[36,37]

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