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theses

Organogenesis requires the spatiotemporal coordination of numerous cell signaling pathways. While many pathways have been investigated, the mechanism for ligand dispersal and quantitative analysis of signaling induction is largely unknown. The epidermal growth factor receptor (EGFR) signaling pathway is activated multiple times in tissues throughout development. In oogenesis, the TGF-α-like ligand, Gurken (GRK), is secreted from around the oocyte nucleus where it activates the EGFR in the overlying follicle cells. In addition to the multiple components of the pathway, the compartments of the egg chamber are changing along with the position of GRK. To study how signaling is regulated in time and space, we developed a mathematical model to recapitulate the dynamics of EGFR signaling in oogenesis. While some parameters were acquired from the literature, others were obtained through genetic perturbations and quantitative analysis. For example, we used CRISPR/Cas9 to generate a homozygous viable EGFR tagged with GFP. Using this fly, we followed the dynamic localization of GRK and EGFR. Importantly, using ELISA, we quantified the number of EGFR molecules per cell during stages 8-14 of oogenesis and stages 2 and 5 of embryogenesis.
While patterning of the follicular epithelium has been extensively studied, information about their regulation is mostly unknown. To better understand this regulation, we took advantage of the FlyLight collection that contains over 7,000 intergenic and intronic DNA fragments that can potentially drive the transcription factor GAL4. Cross listing the 84 genes known to be expressed during oogenesis with the 1200 genes in the FlyLight collection found 22 common genes that are represented by 281 fly lines. Of these lines, 61 show expression patterns in the follicle cells when crossed to a UAS-GFP reporter. Of the 61 lines, 19 recapitulate the full or partial pattern of the endogenous gene pattern. Mapping the distribution of all 61 lines, we found a significant enrichment of enhancers in the first intron in comparison to the 5’ proximal or distal regions of the gene model. Since all lines drive a GAL4 transcription factor, thus offering valuable resource for genetic manipulations. Our screen provides further evidence that complex gene-patterns are regulated combinatorially by enhancers controlling expression in simple domains.
 

ETD_9962

Ph.D.

Includes bibliographical references

Includes vita

doi:10.7282/t3-x06b-7t04

ETD doctoral

The author owns the copyright to this work.