Functional genomic analysis of developmental control gene action in the embryonic nervous system of "Drosophila"

Abstract

In the past two decades, developmental genetic analysis of the molecular control elements involved in early embryonic brain patterning has uncovered the existence of structurally and functionally homologous genes that have comparable, and indeed interchangeable functions in vertebrates and invertebrates. The cephalic gap gene family orthodenticle(otd)/Otx is expressed in the anterior brain of Drosophila and mouse. These genes play an important role during the formation of the anterior brain since mutation of otd/Otx2 causes the loss of entire rostral brain in both phyla. Reciprocal gene replacement experiments have demonstrated the functional equivalence of otd and Otx genes. The homeotic genes are expressed in a virtually co-linear anteroposterior pattern in the developing posterior brain of Drosophila and mouse, where they are required for the patterning of the region and the specification of segmental neuronal identity. These findings indicate the evolutionary conservation of cephalic gap gene and homeotic gene action in embryonic brain development and propose the conserved genetic network composed of genes controlled by these genes. In order to gain more information about the molecular basis of the genetic network underlying the observation of evolutionary conservation of key developmental control gene action, it is interesting and important to investigate the downstream targets of these control genes. To this end, this thesis takes advantage of the sequenced genome of Drosophila and the availability of high-density oligonucleotide array techniques to identify downstream genes at a genome wide level: As an initial part of this thesis, microarray analysis of diffe rential gene expression after heat shock revealed substantial changes in gene expression level for known heat -shock genes and identified numerous heat shock-inducible genes. These results demonstrated that high-density oligonucleotide arrays are sensitive, efficient, and quantitative instruments for the analysis of large -scale gene expression in Drosophila embryos. Based on this, in two subsequent parts of this thesis, this functional genomic approach was used to probe for candidate target genes of otd and labial(lab). In a first part, microarray experiments focused on the lab gene. High-density oligonucleotide arrays with probe sets representing 1,513 identified and sequenced genes were used to analyze differential gene expression following lab overexpre ssion in Drosophila embryos. A number of novel candidate downstream target genes for lab were identified, suggesting that LAB differentially regulates a limited and distinct set of embryonically expressed Drosophila genes. This provides preliminary information for further mechanism-orientated experiments. In a second part, microarray experiments focused on otd/Otx genes. In order to understand the functional equivalence of the Drosophila otd gene and the vertebrate Otx gene and gain insights into potential downstream genes of otd gene in the fly, a first genome wide quantitative transcript imaging experiment was carried out. This experiment was designed to study differential gene expression in flies in which either the Drosophila otd gene or the human Otx2 gene was overexpressed under the control of heat shock. These experiments indicated that 93 genes, approximately one third of the otd-regulated transcripts, also respond to overexpression of the human Otx2 gene in Drosophila. We postulate that these transcripts are common downstream targets of the fly otd gene and the human Otx2 gene in Drosophila which are likely to represent the molecular basis of the functional equivalence of otd and Otx2 gene action in Drosophila. A final part of the thesis was aimed at reducing false positive results of microarray experiments. For this, methods were developed using the magnetic cell sorting technique to isolate specific cell population from Drosophila embryos for specific expression profiling. These methods were the n applied to identify new candidate downstream genes of the gene glial cells missing (gcm) which is a key regulator during gliogenesis. The GAL4-UAS system was used to direct expression of a transmembrane protein, mCD8-GFP, exclusively to the neuroectoderm of stage 11 embryos, which permitted a high rate of purification of viable cells from the neuroectoderm as assayed by both cellular and molecular methods. Based on the sorted neuroectodermal cells, differential gene expression was analyzed in wildtype embryos versus embryos in which gcm was misexpressed throughout the neuroectoderm. Follow-up validation studies of genes identified as differentially expressed by in situ hybridization revealed a rate of confirmation for the sorted cellbased microarray experiments of more than 80%. This strongly contrasts to the high false positive rate revealed by microarray experiments based on wholemount embryos. Our results strongly suggest that reduction of cell heterogeneity through cell sorting techniques leads to a marked increase in the ability of microarrays to reveal differential gene expression in the developing nervous system.