Molecular characterization of Notch1 positive progenitor cells in the developing retina

Abstract

The oscillatory expression of Notch signaling in neural progenitors suggests that both repressors and activators of neural fate specification are expressed in the same progenitors. Since Notch1 regulates photoreceptor differentiation and contributes (together with Notch3) to ganglion cell fate specification, we hypothesized that genes encoding photoreceptor and ganglion cell fate activators would be highly expressed in Notch1 receptor-bearing (Notch1 +) progenitors, directing these cells to differentiate into photoreceptors or into ganglion cells when Notch1 activity is diminished. To identify these genes, we used microarray analysis to study expression profiles of whole retinas and isolated from them Notch1 + cells at embryonic day 14 (E14) and postnatal day 0 (P0). To isolate Notch1 + cells, we utilized immunomagnetic cell separation. We also used Notch3 knockout (Notch3KO) animals to evaluate the contribution of Notch3 signaling in ganglion cell differentiation. Hierarchical clustering of 6,301 differentially expressed genes showed that Notch1 + cells grouped near the same developmental stage retina cluster. At E14, we found higher expression of repressors (Notch1, Hes5) and activators (Dll3, Atoh7, Otx2) of neuronal differentiation in Notch1 + cells compared to whole retinal cell populations. At P0, Notch1, Hes5, and Dll1 expression was significantly higher in Notch1 + cells than in whole retinas. Otx2 expression was more than thirty times higher than Atoh7 expression in Notch1 + cells at P0. We also observed that retinas of wild type animals had only 14% (P < 0.05) more ganglion cells compared to Notch3KO mice. Since this number is relatively small and Notch1 has been shown to contribute to ganglion cell fate specification, we suggested that Notch1 signaling may play a more significant role in RGC development than the Notch3 signaling cascade. Finally, our findings suggest that Notch1 + progenitors-since they heavily express both pro-ganglion cell (Atoh7) and pro-photoreceptor cell (Otx2) activators-can differentiate into either ganglion cells or photoreceptors.