Analysis of developmental and regenerative spinal motor neuron generation in zebrafish larvae

Abstract

In contrast to mammals, adult zebrafish are able to regenerate motor neurons and regain swimming ability within 6 weeks after a spinal cord injury. During this regenerative process, a range of developmental signals such as dopamine and serotonin are found to be re-deployed. This makes the research of embryonic signals become essential for the promotion of regeneration in the future. In my research, I am interested in identifying genes that are important for motor neuron development and motor axon differentiation. I also aimed to study the ability of zebrafish larvae to regenerate spinal motor neurons, and whether they can be used to study the essential developmental cues and the mechanisms underlying successful functional recovery. Motor axons grow out of the spinal cord in a motor neuron subtype specific manner and innervate different muscle groups to facilitate locomotor movements. To find genes and important pathways involved in motor neuron generation and axon development in zebrafish, we conducted an ENU-induced mutagenesis screen in islet-1:GFP transgenic zebrafish, in which a subset of dorsally projecting motor neurons are labelled. We have discovered 6 mutants displaying delayed or inhibited appearance of secondary motor neurons and/or motor axon deficits among 111 F2 families screened. Through subsequent mutant phenotypical analysis, I focused my study in two mutant lines manifesting a lack of islet-1:GFP motor neurons, and an absence of islet-1:GFP motor axons. I used various molecular markers to characterise the mutant phenotypes and observed several additional anatomical defects. I also initiated the study of causative mutation analysis based on the candidate gene list generated from Next Generation Sequencing (NGS). To gain an insight of the genes’ role in motor neuron development and axonal differentiation, I started functional analyses in order to confirm genes that are responsible for the observed motor neuron/axon phenotypes, and I have achieved some promising preliminary results. Motor neurons are generated from the motor neuron progenitor domain (pMN). This neurogenesis process sharply declines at 48 hours post-fertilisation (hpf), while pMN progenitor cells continue to proliferate to produce oligodendrocytes. By inflicting a mechanical lesion in the spinal cord of zebrafish larvae, we demonstrated that they are capable of regenerate new motor neurons and achieve full functional recovery within 48 hours following the injury, sharing similar mechanisms to that of the adult zebrafish. I further studied oligodendrocyte generation and found that pMN domain is able to switch from oligodendrogenesis to motor neuron generation after a spinal lesion. This demonstrates the high plasticity of the pMN domain. Interestingly, the generation of dorsal Pax2-positive interneurons was not altered after the lesion, suggesting that the regenerative potential differs in different progenitor domains. This study showed that the motor neuron regenerative process in zebrafish larvae is robust and they can be used for studying motor neuron regeneration. Taken together, the discovery of the genes from our screen will provide insights to the developmental cues that are involved in motor neuron generation and axon growth. Furthermore, spinal cord lesion in larval zebrafish larvae is established as a regenerative model that can be utilized to dissect the roles and mechanisms of these signals and pathways in the promotion of motor neuron regeneration.