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Characterization of a Drosophila Melanogaster Gene Regulatory Network by RNA-Interference and Cis-Regulatory Element Mutagenesis

Daniel, Emily Barbara
http://rave.ohiolink.edu/etdc/view?acc_num=dayton1714596785781399

Abstract Details

2024, Master of Science (M.S.), University of Dayton, Biology.
This thesis sought to advance the understanding of a gene regulatory network (GRN) that is responsible for the development of a male-specific (dimorphic) pattern of pigmentation on the 5th and 6th (A5 & A6) abdomen segments of the fruit fly species Drosophila (D.) melanogaster. The A5 and A6 protective cuticle tergites are fully black in color from the melanin pigment deposited from the underlying epidermal cells. These black tergites differ from the non-melanic A1-A4 tergites, and the A5 and A6 tergites of females. Previous studies identified 18 transcription factor genes whose functions are needed for this dimorphic pattern of tergite pigmentation. They also found the cis-regulatory elements (CREs) that control the epidermis expression of four “realizator” genes that encode enzymes for pigment metabolism (Ddc, tan, yellow, and ebony), and one CRE that controls expression of the bab paralog (bab1 and bab2) genes which encode key transcription factors that shape sex-specific patterns of pigmentation gene expression. Here, the activity of these CREs were assessed in genetic backgrounds in which RNA-interference (RNA-i) turned down the expression of these 18 genes one by one, and were imaged utilizing confocal microscopy. On the low end, the activity of the Ddc gene’s MEE1 CRE and the bab dimorphic element CRE were impacted by eight of the 18 transcription factors. On the high end, the e_AMS CRE of ebony and the t_MSE2 CRE for tan were impacted by 12 transcription factors. Among the nuances of these numerous CRE interactions were transcription factors like bab (bab1 and bab2) and grh whose expressions were required for the proper activity of the CREs for all five of the analyzed genes. In contrast, the transcription factor pdm3 was found to regulate the expression of a single gene, ebony. A second objective of this thesis was to see whether any of the regulatory relationships between transcription factors and CREs might involve direct physical interactions. To this end, the Ddc MEE1 CRE was surveyed for short sequences that match consensus binding motifs for seven of the eight transcription factors found to modulate the CRE’s activity. These potentially meaningful binding site sequences were mutated in the context of an otherwise normal CRE sequence. The mutant CRE alleles were then evaluated for their ability to activate the expression of a reporter gene in transgenic D. melanogaster. The results showed that the transcription factor proteins Sox102F, Vvl, Bab, and Exd may reign in the activity of this CRE in females through binding sites dispersed throughout the CRE. It was also shown that what seems to be a single binding site for the transcription factor Abd-B is absolutely necessary for the activity of this CRE in the pupal abdominal epidermis. The origin of this single site may have been a key change that brought the Ddc gene under the expression control of the Hox family transcription factor Abd-B. The thesis concludes with a discussion as to how further study in this GRN system could reveal how enhancer and silencer type CREs operate and evolve, and insights on this GRN’s interaction structure. This work is meritorious as the placement of traits at specific body plan coordinates, life stages, and sometimes on a specific sex are general features of animals, and that all traits have an evolutionary history. In spite of this importance, few GRNs have been well-characterized, and how traits evolve through the formation of new (derived) GRNs remains speculative. This thesis demonstrates the value of Drosophila tergite pigmentation as a model trait and D. melanogaster as a model species to study the evolution of GRNs. An understanding of how one trait and its GRN evolves will help the formation and testing of hypotheses for traits more broadly, including those for which genetic diseases are known to occur.
Thomas Williams (Advisor)
Yvonne Sun (Committee Chair)
Pothitos Pitychoutis (Committee Chair)
99 p.
Biology; Developmental Biology; Evolution and Development; Genetics
Gene Regulatory Network; RNA-interference; Cis-regulatory mutagenesis; Drosophila melanogaster pigmentation; Evolutionary developmental biology (Evo-Devo)

Recommended Citations

Citations

  • Daniel, E. B. (2024). Characterization of a Drosophila Melanogaster Gene Regulatory Network by RNA-Interference and Cis-Regulatory Element Mutagenesis [Master's thesis, University of Dayton]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=dayton1714596785781399

    APA Style (7th edition)

  • Daniel, Emily. Characterization of a Drosophila Melanogaster Gene Regulatory Network by RNA-Interference and Cis-Regulatory Element Mutagenesis. 2024. University of Dayton, Master's thesis. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=dayton1714596785781399.

    MLA Style (8th edition)

  • Daniel, Emily. "Characterization of a Drosophila Melanogaster Gene Regulatory Network by RNA-Interference and Cis-Regulatory Element Mutagenesis." Master's thesis, University of Dayton, 2024. http://rave.ohiolink.edu/etdc/view?acc_num=dayton1714596785781399

    Chicago Manual of Style (17th edition)

dayton1714596785781399
73
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This open access ETD is published by University of Dayton and OhioLINK.