Neurogliogenesis and visual system development in "Drosophila" : genetic/genomic analysis of the "Glial cells missing" and "Egghead" genes

Complex nervous systems are made up by two major cell types, neuronal and glial cell types. A general observation that has been made by lineage analysis of neurogliogenesis in vertebrates and invertebrates is that neurons and glia often share common progenitors. Therefore an interesting question in cellular neurobiology is how neuronal versus glial cell fate is established. In Drosophila, glial cells missing (gcm) is a key control gene of gliogenesis. In gcm mutants, presumptive glial cells are transformed into neurons and, conversely, when gcm is ectopically misexpressed, presumptive neurons become glia. Since gcm encodes a transcription factor, it is proposed that a set of downstream genes are regulated by GCM that in turn execute the glial differentiation program.
In the first set of experiments carried out in this thesis, genome-wide oligonucleotide arrays were used to identify gcm downstream genes in a comprehensive manner. Differential gene expression was analyzed in wild type embryos and compared to embryos in which gcm was misexpressed throughout the neuroectoderm. We found hundreds of genes that were differentially expressed following gcm misexpression. They thus are potentially involved in aspects of glial development. This study is one of the first genome-wide analyses of gene expression events downstream of a key developmental transcription factor and represents a novel level of insight into the repertoire of genes that initiate and maintain cell fate choices in the development of the central nervous system.
Microarrays are powerful and efficient tools to quantify and compare gene expression on a large scale. However, as with all large-scale experiments, microarray experiments can be influenced by inherent biological factors. In vivo analysis suggests a low level of validation of the initial microarray data we obtained for gcm downstream genes. One of the main reasons accounting for this low verification rate appears to be the complexity of the tissue used for the microarray experiments. In the second microarray analysis of gcm gene action in neurogliogenesis performed in this thesis, tissue heterogeneity was reduced by using the technique of magnetic cell separation (MACS) to isolate neuroectoderm cells from Drosophila embryos. Validation studies by in situ hybridization of genes identified as differentially expressed in the sorted cell-based microarray experiments revealed high rates of verification. This suggests that reduction
of cell heterogeneity increases the ability of microarrays to reveal differential gene expression in the developing nervous system.
The subsequent major part of this thesis addresses the role of the egghead (egh) gene, a putative gcm downstream target that was identified by microarray analyses, in visual system development of Drosophila. It is known that the correct targeting of photoreceptor neurons (R-cells) in the developing Drosophila visual system requires multiple guidance systems in the eye-brain complex as well as the precise organization of the target area. Although the molecular mechanisms that underlie the targeting of R-cell axons have been studied intensively in the photoreceptor neurons of the developing eye, and to a lesser degree in the developing lamina and medulla, little is known about the possible role of the lobula complex which transiently abuts the lamina and medulla in the developing larval brain. In our study, we find that the egh gene, encoding a glycosyltransferase, is required for a compartment boundary between lamina glia and lobula cortex, which is necessary for appropriate retinal innervation of the lamina. In the absence of egh, perturbation of sheath-like glial processes occurs at the boundary region delimiting lamina glia and lobula cortex, and inappropriate invasion of lobula cortex cells across the boundary region disrupts the pattern of lamina glia resulting in inappropriate R1-R6 axonal projections. Further genetic analysis involving mosaics demonstrates that the requirement of egh is restricted to the lobula complex primordium. This study thus uncovers a novel role of egh gene function in the developing Drosophila visual system and underscores the unexpected role of the lamina/lobula compartment boundary in R1-R6 axon targeting.