Endogenous neural stem cell reprograming for neuronal regeneration

Romain Madeleine, Ph.D.
MDI Biological Laboratory
Assistant Professor

Investigator: Romain Madelaine, Ph.D., MDI Biological Laboratory

Project Summary

Contrary to humans, some animals have the powerful capacity to fully regenerate damaged tissue and organs, including the nervous system. Our research focuses on the function of the stem cell population during tissue regeneration using the zebrafish as model organism. We take advantage of cutting-edge techniques in functional genetic analysis, by combining Tol2 transgenesis, Cre-Lox system for cell lineage tracing, single cell RNA-sequencing and CRISPR-Cas-9 genome editing method to characterize the mechanisms of cellular regeneration. Our work aims to identify regenerative factors involved in the stem cell activation and proliferation to open new paths for tissue regeneration in human.

In humans, olfactory or optic nerve injuries and associated neuronal degenerative diseases are often followed by permanent loss of smell or vision respectively. For patients who have lost olfactory or retinal neurons, an important challenge is to identify safe and effective methods to replace sensory neurons and thereby restore neuronal functions. In contrast to mammals, the zebrafish has the capacity neurons and thereby restore neuronal functions. In contrast to mammals, the zebrafish has the capacity to fully regenerate entire parts of the nervous system after injury. This process is dependent of endogenous neural stem cells (NSCs). Following injury, NSCs re-enter the cell cycle to proliferate and generate new neurons. This project will identify instructive factors sufficient to reprogram endogenous neural stem cells of the retina and olfactory epithelium (OE) to promote the neuronal regeneration. To reach this objective, we will take advantage of a large set of new genetic tools that we have generated (6 CRISPR/Cas-9 mutants and 8 transgenic lines), and apply cutting edge techniques combining neuronal genetic ablation, cell lineage tracing, high resolution confocal microscopy and single cell RNA-sequencing. In a first aim we will characterize the role of a newly identified olfactory GFAP+ cell type during neuronal regeneration in the OE. The proliferation and the fate of the GFAP+ population after injury will be investigated by genetic lineage tracing (Cre-Lox method), and the genuine stem cell identity of the GFAP expressing cells will be evaluated by live imaging after injury. In a second aim, we will analyze the role of different transcription factors during olfactory and retinal neurogenesis. To characterize the function of Tlx, Onecut and Meis2 transcription factors during the developmental and regenerative neurogenesis, we will study newly generated CRISPR/Cas-9 mutants for these genes and test the capacity of these factors to activate/reprogram endogenous NSCs to promote the formation of new neurons without injury. In a last aim, we will perform single cell RNA-sequencing to identify new candidate genes for cellular reprogramming. Promising candidates differentially regulated after injury will be tested to determine their roles in NSCs activation and their instructive capacities to promote NSCs proliferation and the neuronal differentiation in the absence of injury. Finally, the comparison of transcriptome profiles between olfactory and retinal NSCs after neuronal ablation will reveal a common genetic program used by NSCs during the neuronal regeneration. This project will lead to the identification and characterization of molecular factors controlling NSC activation/reprogramming.

Madelaine Lab at MDI Biological Laboratory