Gene Expression Profiling Of Notch1 Positive Progenitor Cells Isolated From Developing Retina

Abstract

Purpose: The dynamic (oscillatory) expression of the Notch signaling cascade in neural progenitors suggests that transcripts of both repressors and activators of neural fate specification should be expressed by the same progenitors. Since Notch1 regulates photoreceptor differentiation and contributes (together with Notch3) to ganglion cell fate specification in developing retinas, we hypothesized that the 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 ganglion cells when Notch1 activity is diminished.

Methods: To identify these genes, we used MEEBO microarrays to study the expression profiles of whole (heterogeneous) retinas and isolated from retinas Notch1+ cell populations at embryonic day 14 (E14) and postnatal day 0 (P0). To isolate Notch1+ cells, we employed an immunomagnetic cell separation approach. ANOVA and hierarchical clustering were applied for statistical analysis. We also used Notch3 knockout (Notch3KO) animals to evaluate the contribution of the Notch3 signaling in ganglion cell differentiation.

Results: Our microarray data revealed that Notch1+ cells represent separate populations of progenitors at E14 and P0. At E14, we found high expression of repressors (such as Notch1 and Hes5) and activators (such as Dll3, Atoh7 and Otx2) of neuronal differentiation in Notch1+ cells compared to whole retinal cell populations. Meanwhile, Notch1, Hes5, and Dll1 expression at P0 was significantly higher in Notch1+ cells than in whole retinas. Otx2 expression was more than thirty times higher compared to Atoh7 expression in Notch1+ cells at P0. Our data indicated that Notch3+ cells represent a population distinct from the Notch1+ cell population, and that Notch3+ cells can therefore independently regulate ganglion cell fate specification. However, retinas of wild type animals had only 14% more ganglion cells compared to Notch3KO mice.

Conclusions: Our findings suggest that Notch1+ progenitors can differentiate into either ganglion cells or photoreceptors, since both pro-ganglion cell (Atoh7) and pro-photoreceptor cell (Otx2) activators are highly expressed in these cells. While Notch3 signaling can also contribute to ganglion cell fate specification, our data suggest that the role of Notch1 is more significant.