Notch–Delta Signaling is Required for Mueller Glia Differentiation in the Zebrafish Retina

Abstract

Abstract: : Purpose: Notch–Delta signaling is involved in cell fate decisions. Notch is a Type I transmembrane receptor that binds to Delta, a transmembrane ligand. Ligand–receptor binding triggers intramembrane proteolysis, releasing the intracellular domains (ICD) of both receptor and ligand, which translocate to the nucleus to alter gene transcription. Two models of Notch–Delta signaling in the nervous system have been proposed: lateral inhibition in which Delta ICD promotes neuron differentiation and Notch ICD promotes continued proliferation, and an instructive mechanism in which Notch ICD promotes gliogenesis. The goal of the experiments presented here was to clarify the role of Notch–Delta signaling during retinal neurogenesis and gliogenesis. Methods: We used gamma–secretase inhibitors that prevent intramembrane proteolysis of both Delta and Notch ICD, which were applied to zebrafish embryos beginning at 9 hours post–fertilization (hpf) and ending at different time points. The effects on retinal development were assayed by immunocytochemistry and in situ hybridization. Results: After blocking Notch–Delta signaling from 9 to 65 hpf, neuronal markers for photoreceptors and ganglion cells were expressed, but retinal lamination was disrupted. In contrast, Mueller glia (MG) were absent from the retina, whereas GFAP–labeled astrocytes were present, but reduced, in the brain. When embryos were removed from the drug at 48 hpf and continued developing in drug–free embryo media until 96 hpf, retinogenesis recovered, and laminated retina, including MG, was generated by retinal progenitors at the periphery. However, in embryos that remained in the drug from 9 to 65 hpf and were then transferred to embryo media to develop to 96 hpf, no recovery was observed and no MG were produced. Mitotic activity was similar in control and drug–treated embryos, with numerous mitotic cells present at 48hpf and a steady decrease in the number of mitotic cells as development continued. TUNEL–labeling revealed a significant increase in apoptosis in the retina of drug–treated embryos compared to controls at 65hpf. Conclusions: Notch–Delta signaling in the zebrafish retina has an instructive role and is essential for MG differentiation but is not required for retinal neurons to differentiate. We further propose that the lack of MG in the drug–treated zebrafish embryos may result in the absence of laminar organization, suggesting that MG are present in the developing retina at an earlier time than generally believed.

Keywords: Muller cells • retinal development • apoptosis/cell death