Abstract
Purpose :
Extrinsic signals are important regulators of retinal development, though their identity is still largely unknown. Single-cell RNA-seq analysis of developing mouse and human retinas performed in our lab has identified a number of strong candidate factors. These include the neuropeptide somatostatin, which is expressed by retinal ganglion cells in the developing retina while its receptor is expressed at the same time by neurogenic retinal progenitor cells (RPCs). I tested the hypothesis that somatostatin acts as a quorum signal that serves to promote or inhibit RPC proliferation, neurogenesis, and/or production of different retinal cell types using an in vitro model of mouse embryonic retinal explants.
Methods :
Retinal explants were grown from embryonic day 14 to postnatal day 0. Explants were grown in the presence of either the Somatostatin receptor 2 (Sstr2) agonist (1R,1'S,3'R/1R,1'R,3'S)-L-054,264 or the Sstr2 antagonist CYN 154806 at a concentration 3 orders of magnitude above its Ki/IC50, respectively, or in the absence of a small-molecule compound as a control.
The explants for each condition were consolidated into three samples. qPCR was used to determine the expression level of 8 cell-type specific marker genes and 3 extrinsic signal genes relative to the internal control Gapdh for each sample.
A One-way ANOVA of gene expression vs condition was used to calculate a p-value for the difference in expression between a treatment and its control for each gene for both treatments.
Results :
The explants treated with the Sstr2 antagonist showed a 20% average decrease (p=0.01) in expression of the general retinal progenitor cell marker Ccnd1 compared to controls. They also showed a 16% decrease (p=0.08) in the neurogenic progenitor cell marker Atoh7. Since these are cell-type specific marker genes, this indicates a decrease in the overall proportion of retinal progenitor cells in the explant relative to the control.
The explants treated with Sstr2 agonist showed an 18% decrease (p=0.015) in the photoreceptor cell marker Crx, indicating a decrease in the proportion of photoreceptor cells relative to control.
Conclusions :
Somatostatin appears to act as a quorum signal generated from retinal ganglion cells to promote retinal progenitor cell proliferation and neurogenesis and inhibit production of photoreceptor cell types.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.