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

Montalta-He, Haiqiong. Functional genomic analysis of developmental control gene action in the embryonic nervous system of "Drosophila". 2003, Doctoral Thesis, University of Basel, Faculty of Science.

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Official URL: http://edoc.unibas.ch/diss/DissB_6580

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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.
Advisors:Reichert, Heinrich
Committee Members:Stöckli, Esther T.
Faculties and Departments:05 Faculty of Science > Departement Biozentrum > Former Organization Units Biozentrum > Molecular Zoology (Reichert)
UniBasel Contributors:Reichert, Heinrich
Item Type:Thesis
Thesis Subtype:Doctoral Thesis
Thesis no:6580
Thesis status:Complete
Bibsysno:Link to catalogue
Number of Pages:164
Language:English
Identification Number:
Last Modified:22 Jan 2018 15:50
Deposited On:13 Feb 2009 14:48

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