Investigation of functional Notch signal sending cells in developing mouse retina

Abstract

The vertebrate neural cell development is coordinated process of neural stem cell (NSC) and finally produce mature layer and structure through diverse specification and differentiation. To preserve vertebrate neural progenitor cell (NPC), Notch signaling in neural progenitor cells is activated by ligands expressed in adjacent cells. During retinal development, retinal progenitor cells (RPCs) generate seven major cell types, two types of photoreceptors (Rod cells, Cone cells), interneurons (bipolar cells, horizontal cells, astrocytes), and M$\ddot{u}$ller glia, in chronological sequence. To identify sources of functionally active Notch ligands for RPCs, I negatively regulated Notch ligand activities through the specific elimination of an ubiquitin E3-ligase Mindbomb1 (Mib1) in RPCs, post-mitotic retinal neurons (PMNs), and retinal pigment epithelium (RPE), respectively. The Mib1-deficient RPCs failed to induce Notch activation in intra-lineage RPCs, which prematurely differentiated into retinal neurons. However, the Mib1-deficient PMNs shows no significant difference between wild-type which means Notch signaling between PMNs and RPCs is dispensable. Lastly, I found that Mib1 in RPE was crucial for Notch activation in RPC. I discover a novel function of embryonic RPE that expresses Notch ligands at the apical membrane that contact with RPC apical membrane to trigger Notch activation in a future RPC. To confirm this, we conducted rescue experiments using LSL-Dll1 (loxP-Stop-loxP) line which expresses Dll1 (one of Notch ligand in RPE) under Cre expression. As a result, Mib1-deficient RPE with ectopic expression of Dll1 rescues the abnormal retinal development phenotypes which showed in Mib1-deficient RPE mice. Together, I propose a model that a RPC daughter receives additional Notch signals from RPE to acquire RPC fate competitively while the other sister cell only receives a sub-threshold inter-RPC Notch signaling to differentiate to retinal neuron.