Nuclease-mediated gene manipulation of factors implicated in zebrafish neurogenesis

Complex and differential gene expression programs give rise to several cell types that constitute the different parts of the organism. This cell fate determination is controlled by a group of proteins, named transcription regulators (TRs). Our group investigates the molecular mechanisms underlying neurogenesis using the zebrafish as a model system. To that purpose, a genome-wide analysis of TR gene expression was performed in our laboratory, and hundreds of these regulators were identified. On the basis of these initial studies, a number of TRs were selected for further characterization. For this project, two model systems for zebrafish neurogenesis were chosen: The embryonic spinal cord and the adult telencephalon. The spinal cord is considered as relatively simple and is used to understand the neural differentiation and function in vertebrates during development. Based on morpholinos knockdown experiments, two closely related genes sox1a and sox1b encoding transcription factors, were shown to play a role in the specification of a newly observed sub-type of interneurons, named V2c, in the ventral spinal cord of the zebrafish. Nevertheless, the epistatic relationship between these genes has still to be investigated. On the other hand, in the adult brain, new neurons are continuously generated from neural progenitor cells. The zebrafish brain contains many more progenitor zones compared to mammals, reflecting its great capacity of neuronal proliferation and regeneration. Focusing on the telencephalic zones, because of its similarities with the mammalian brain, several factors were identified with a potential role in adult neurogenesis. Among those regulators, Id1 that acts as a dominant negative factor for basic helix-loop-helix (bHLH) transcription factors was chosen for further investigations because of promising previous observations using morpholino knockdown and protein overexpression methods. However, in order to perform loss-of-function studies and to deepen our understanding about the role of chosen TR genes and their molecular mechanisms controlling neurogenesis and adult brain regeneration, newly developed tools for gene manipulation were used: The transcription activator-like effector nucleases (TALENs) and the Type II clustered regulatory interspaced short palindromic repeats (CRISPR). Both methods use the nuclease activity to create double-stranded-breaks (DSBs) that are repaired via an error-prone pathway, non-homologous end-joining (NHEJ), creating mismatches in the DNA, or via a homologous recombination (HR) pathway allowing incorporation of a DNA sequence in a specific location of the genome. After implementation of these two methods shortly after their development, I could show the efficiency of both in inducing mutations in the zebrafish genome, thus by using the gene no-tail (ntl) as a proof of principle. I then created with TALENs heritable mutations in the gene id1 and assessed three generations of progeny in order to select a knockout line for the cited gene. Mutations in id3 were also induced with both TALENs and CRISPR and other id genes were selected in order to continue the study concerning their possible redundant activities and their implication in adult neurogenesis and brain regeneration in the zebrafish. In parallel, I used the CRISPR system to simultaneously mutate the genes sox1a and sox1b in order to confirm their role in the specification of interneurons in the zebrafish spinal cord. Considering the long-lasting lack of site-specific mutagenesis in zebrafish and the controversies in the last few years about the specificity of the morpholino antisense technology, the new genetic tools characterized and applied in this PhD work open the door to a wide range of investigations