Abstract
Purpose :
Retinal progenitor cell (RPC) specification into appropriate retinal cell types is temporally and spacially coordinated by well-characterized intrinsic signals endowing RPCs with neurogenic or gliogenic potential. The cellular communication network 2 (CCN2), also known as connective tissue growth factor or CTGF, is a secreted extracellular matrix protein with regulatory functions in vasoproliferative and fibrotic diseases. Although CCN2 expression extends to developmental processes in the central nervous system, the role of such extrinsic signal in retinogenesis is unknown. Herein, we used mouse genetics and omic approaches to determine the functional importance of CCN2 in retinogenesis.
Methods :
A CCN2-green fluorescent protein bacterial artificial chromosome transgenic mouse was used as a proxy to analyze CCN2 expression pattern during retinogenesis. Moreover, mice with global deletion of CCN2 were generated by crossing CCN2 floxed and cytomegalovirus promoter-driven Cre mice. RPC growth and differentiation were analyzed in transverse cryosections of retinas by immunohistochemical methods at embryonic stages E11, E14, E16, and E18. Transcriptomic changes were determined by RNA-sequencing analysis of retinas from CCN2-deficient and control littermate mice at E18.
Results :
At E11, CCN2 was expressed in the outer neuroblastic layer, which consists of RPCs and differentiating photoreceptors (PRs). At E14, 45 and 23% of CCN2 producing cells were positive for the RPC markers Sox2 and CHX10, respectively. From P1 onwards, CCN2 expression was restricted to Müller glia and vascular cells. Meanwhile, CCN2 deficiency in mice resulted in retinal hypocellularity with reduced RPC pool and impaired competence of remaining RPCs to generate immediate-early and late- born retinal cell types. CCN2 deficiency induced a dramatic reduction of Sox2 to Pax6 ratio, which is key to the maintenance of RPC neurogenic competence and multipotency. CCN2 gene inactivation caused down-regulation of marker and transcription regulator genes of retinal ganglion cells, PRs, and Müller glia.
Conclusions :
CCN2 expression is required for the generation of neuronal and glial diversity in the retina. Understanding the regulation and function of CCN2 in retinal neurogenesis is of key importance toward optimization of cellular replacement therapy in congenital retinal diseases.
This is a 2021 ARVO Annual Meeting abstract.