6 research outputs found
Molecular Characterization of Notch1 Positive Progenitor Cells in the Developing Retina
<div><p>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<sup>+</sup>) 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<sup>+</sup> cells at embryonic day 14 (E14) and postnatal day 0 (P0). To isolate Notch1<sup>+</sup> 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<sup>+</sup> 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<sup>+</sup> cells compared to whole retinal cell populations. At P0, Notch1, Hes5, and Dll1 expression was significantly higher in Notch1<sup>+</sup> cells than in whole retinas. Otx2 expression was more than thirty times higher than Atoh7 expression in Notch1<sup>+</sup> 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<sup>+</sup> progenitors—since they heavily express both pro-ganglion cell (Atoh7) and pro-photoreceptor cell (Otx2) activators—can differentiate into either ganglion cells or photoreceptors.</p></div
Notch3 deficiency results in reduced ganglion cell numbers in the retina.
<p><b>A)</b> Confocal images of flat-mounted retinas from Notch3 knockout (Notch3KO) animals and wild type (WT) littermates were collected. RGCs were labeled with beta III Tubulin antibodies for counting. <b>B)</b> Numbers of RGCs were compared between Notch3KO and WT animals. Values are means ± SEM (*P < 0.05, n = 10 eyes).</p
Differential gene expression was confirmed by qRT-PCR and demonstrated a consistent correlation with microarray data for a group of selected genes.
<p>For each gene, results are expressed as percentages ± SEM of the corresponding values in the Notch1<sup>+</sup> cells isolated from E14 developing retinas (*P < 0.05).</p
Immunomagnetic separation proved to be an effective method for positive selection of Notch1-specific cells from developing retinas.
<p><b>A)</b> The purity of isolated cells was tested using anti-Notch1 antibody (green). Propidium iodide (PI, red) was used to visualize the nucleus of the cell. Bar is 50 μm. <b>B)</b> The levels of Notch1 expression in isolated cells were higher compared to expression of Notch1 in whole retinas. Gene expression was measured by qRT-PCR. Results are expressed as a percentage of the corresponding value in the Notch1<sup>+</sup> cells isolated from embryonic day 14 developing retinas ± SEM (*P < 0.05).</p
Functional annotation for the selected genes revealed by One-Way ANOVA.
<p>Functional annotation for the selected genes revealed by One-Way ANOVA.</p
Hierarchical clustering of 6,301 differentially expressed genes showed that Notch1<sup>+</sup> cells grouped near the same developmental stage retina cluster.
<p>Cluster analysis (hierarchical clustering) was performed using Gene Cluster 3.0 software.</p