Studies of gliogenesis in the central nervous system of the zebrafish (Danio rerio)

Oligodendrocytes (the myelinating cells of the central nervous system, CNS) and astrocytes (a heterogeneous class of cells with several different functions) constitute the two major classes of glial cells in the CNS. Both are found widely distributed in the white and grey matter of the adult mammalian brain. Glial cells, like neurons, develop from neuroepithelial precursor (stem) cells in the ventricular zone (VZ) of the brain and spinal cord. In the spinal cord, most oligodendrocyte progenitors (OLPs) originate from a restricted region of the neuroepithelium close to the floor plate (pMN domain) that earlier generates motor neurons, suggesting a common precursor for these cells. OLPs migrate out from this region throughout the CNS where they proliferate and differentiate, differentiating and creating myelin mainly in the white matter. Astrocytes are thought to arise from multiple locations along the dorsal-ventral axis of the VZ, distributing into both grey and white matter. Amongst other characteristics, the embryonic transparency, high fecundity and possibility of performing a large-scale mutant screens make zebrafish an attractive model for studying gene function and identifying new components of the genetic pathways regulating developmental processes. The primary goal of this project was to investigate the possibility of using zebrafish as a model for glial development in higher vertebrates including mammals, focusing primarily on oligodendrocyte development. I have examined the development of oligodendrocytes in immature and adult zebrafish spinal cord by investigating the expression of the zebrafish homologues of oligodendrocytes lineage: oligi, olig2, sox10, pdgf'rOL, nk2.2, and myelin markers: mbp, pip and p0. From these investigations I show that oligodendrocyte development in zebrafish is very similar to that of higher veitebrates. The characterization of these oligodendrocyte lineage markers, establishes a baseline to screen for mutants with defects in myelin. One mutant isolated in a pilot screen, otter, was characterized and demonstrated a defect in oligodendrocyte maturation. One difference between zebrafish and higher vertebrates is the absence of pdgfrCL expression in OLPs of zebrafish. I have used a transient transgenic approach to determine whether the as human regulatory function of the elements that direct expression of the human PDGFRot gene in OLPs and non-CNS tissues are conserved in fish. Two vectors were used: Human PDGFRa 2.2kb-/Z fragment (2.2kb-lacZ) containing non- CNS and human PDGFROC HOkb BAC containing both non-CNS and OLP specific cis- regulatory elements. 2.2kb-lacZ established the correct spatio-temporal expression in mesoderm and neural crest derived tissues, similar to the endogenous expression in zebrafish and the expression described in transgenic mouse (Zhang et al., 1998). Similarly, the HOkb BAC transgene gave a similar expression to non-CNS tissues, but it was also upregulated in scattered cells in the zebrafish CNS. However, due to technique limitations it was not possible to demonstrate that these cells were oligodendrocytes. Supplementary work included in this thesis studied homologues of genes known to be involved in astrocyte development in mammals including: fibroblast growth factor receptor 3 (JgfrJ), glutamine synthetase (gs) and glial fibrillary acid protein igjap). The patterns of expression were investigated in developing and adult zebrafish and Xenopus.