Date of Award

2021

Document Type

Open Access Dissertation

Degree Name

Doctor of Philosophy in Biochemistry and Molecular Biology (PhD)

Administrative Home Department

Department of Biological Sciences

Advisor 1

Thomas Werner

Committee Member 1

Chandrashekhar Joshi

Committee Member 2

Stephen Techtmann

Committee Member 3

Hairong Wei

Abstract

Toolkit genes are set of genes that orchestrate the development of basic body plan of animals, and they are highly conserved in all animals. The co-option of the toolkit genes into the pigmentation pathway has led to the evolution of novel species. This study focuses on understanding how the complex color patterns in animals develop by using the Drosophila species in the quinaria group as models. We developed an mRNA in situ hybridization (ISH) protocol, which allowed us to study gene expression patterns in the abdomen of developing pupae of non-model Drosophila species (Chapter 2). Through ISH, we found that the pigmentation gene y foreshadowed the adult D. guttifera abdominal pattern. Following the discovery of the wingless gene’s (a toolkit gene) expression on the wings of D. guttifera by my advisor, Dr. Thomas Werner, we used ISH to screen 110 putative toolkit genes in the abdomen of D. guttifera to identify putative upstream activators of the pigmentation gene y. We identified five toolkit genes, wingless (wg), hedgehog (hh), abdominal-A (abd-A), decapentaplegic (dpp), and zenknullt (zen) that may collectively orchestrate the patterning in the abdomen of D. guttifera. Using the transgenic technique for modifying non-model Drosophila species (Chapter 1), Dr. Raja Komal (a former Ph.D. student in our lab) deployed the reporter assay technique to investigate the cis-regulatory elements (CREs) that control y gene expression. Dr. Raja showed that only one CRE (gut y spot CRE) controls all six rows of spots, and that this CRE contains a stripe-inducing CRE at its core. In an attempt to provide direct genetic evidence for the roles of toolkit genes in complex pattern formation, we ectopically expressed the cDNAs of the toolkit genes, using the gut y stripe CRE as a driver for gene overexpression. Our intention was to manipulate the adult color pattern on the D. guttifera abdomen to change it into a stripe pattern. Unfortunately, the gut y stripe CRE could not drive the toolkit genes’ expression at a time when it matters for the induction of pigmentation. Unfortunately, no earlier acting abdominal CRE is available for this species to ectopically express our toolkit genes. Furthermore, we performed RNAi knockdown experiments of the toolkit genes, which resulted in gross developmental abnormalities in the fruit flies, causing them to die before the pigmentation patterns could be observed. We suggest that this outcome may be due to the vital roles that these developmental genes play at every stage of Drosophila development.

To understand how novel animal color patterns evolved, it is essential to query how the formation of color patterns varies among closely related species. Therefore, we compared the expression patterns of terminal pigmentation genes (Dopa decarboxylase (Ddc), tan (t), and yellow (y)) in three fruit fly species in the quinaria species group – D. guttifera, D. palustris, and D. subpalustris. Our results show that the genes y, t, and Ddc are co-expressed in modular and identical patterns in the pupal abdomens in each species, which correlate with the adult abdominal pigmentation pattern.

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