Award Date

5-1-2019

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Life Sciences

First Committee Member

Laurel Raftery

Second Committee Member

Andrew Andres

Third Committee Member

Kelly Ai-Sun Tseng

Fourth Committee Member

Mo Weng

Fifth Committee Member

Gary Kleiger

Number of Pages

335

Abstract

All multicellular organisms initially start out as a single cell. This cell must use the genetic information encoded in its DNA to multiply in number and build itself into a complex multicellular organism. How this process occurs is the focus of developmental biology, a field that seeks to understand how a combination of genetic information and environmental conditions shape a cell from its beginnings as a zygote all the way to maturity. A fundamental part of this process is the ability of cells to work together in order to build complex tissues and organs. Cells achieve this coordination by using signaling: The process of sending information from one cell to another. These signals are essential in providing cells with their identity and position within the tissue, and are not only used throughout development, but also during the maintenance of the tissue long after it is built.

Due to the constraints involved in studying cell signaling and development in humans, much of what we now know about these processes has been discovered from the study of model organisms. In my dissertation I used the common fruit fly, Drosophila melanogaster, to investigate how cells work together to migrate through a tissue and reorganize their shape. As evolution has resulted in the conservation of biological mechanisms between species, by studying morphogenesis in Drosophila, we can learn more about how other organisms use derivatives of these same processes to form their own tissues and organs. The signals that cells use to communicate and work together are encoded in their DNA, requiring genetic techniques to understand what information each signal conveys. As many techniques designed to manipulate the genome of Drosophila melanogaster have been pioneered over the past decades, this model organism has become a powerhouse for understanding how genetic regulation links to downstream changes in cell behavior and morphology.

This dissertation focuses on Drosophila oogenesis, a developmental process during which cells are constantly migrating, changing shape, and reorganizing to support the growth and development of an oocyte. Chapter 1 of this dissertation reviews how follicle cells are initially part of an epithelium which consists of a simple sheet of cells, and how this group of cells develops over time to form a complex tissue. During this time, cells within this epithelium are responding to environmental cues, internal genetic regulation, and external cellular signals in order to receive information and carry out their instructed roles. As a goal of our laboratory is to investigate how these instructive signals result in downstream changes to cell shape and morphology, Drosophila oogenesis serves as an excellent model system for this purpose.

Previous work in our lab found that during egg chamber development, an intriguing group of cells send and respond to signals known as Bone Morphogenetic Proteins (BMP). These cells, known as “centripetally migrating follicle cells”, undergo a concerted migration after they receive this signal, involving changes to their shapes, positions, and behaviors. The migration of these cells is required to enclose the anterior portion of the oocyte with an eggshell at the end of oogenesis, a process which protects it from a premature death by desiccation once it is fertilized and deposited into the environment. A goal of my dissertation research was to understand if the morphological changes observed in these centripetally migrating cells could be in response to the BMP signal they were receiving.

To determine if disruptions to BMP signaling could affect centripetal migration, substantial technical development was necessary to mark, watch, and record these cells as they migrated under a microscope. While previous protocols existed for the ex vivo culture of egg chambers at different points in their development, none were adapted for the study of this specific migration during stage 10B. Chapter 2 discusses the adaptations and optimizations that were necessary to create a protocol to time-lapse image egg chambers undergoing centripetal migration. This approach ultimately allowed me to culture egg chambers ex vivo for over 6 hours while simultaneously capturing the migratory dynamics of the centripetally migrating cells.

As a prerequisite to determining what aspects of centripetal migration might be regulated by BMP signaling, I first had to delineate how this migration normally occurred, work that is described in Chapter 3. While centripetal migration is known to be essential to the formation of a viable egg, to date no research has directly characterized the morphogenesis of these cells during egg chamber development. I found that centripetal migration appears to occur in two phases that exhibit unique characteristics compared to other cell migrations: First, leading centripetal cells elongate inward apically, while reducing their basal surface area. Second, following cells rapidly move inward, collectively migrating into the interior of the egg chamber to enclose the anterior face of the oocyte. To create a timeline delineating the normal progression of events in centripetal migration, I identified eight distinct morphological milestones that centripetal cells undergo during this process. I then tested the utility of this framework by using it to investigate cell-cell adhesion requirements between follicle cells and underlying germ cells during migration. This framework for centripetal migration facilitates the future study of this model system to investigate the links between genetic regulation and collective cell migration.

In Chapter 4, I describe my work in investigating if BMP signaling is required contemporaneously for the regulation of centripetal migration during late-stage oogenesis. I showed that type II BMP receptor known as Wishful thinking was expressed in a domain of cells that coincided with the centripetally migrating follicle cells, and that BMP signaling was also active in a similar region. This receptor was of particular interest due to its ability to regulate cellular dynamics in a manner that did not require changes to gene transcription, possibly serving as a fast-acting signal to initiate the start of centripetal migration. After I found that this receptor appeared to be largely dispensable for normal centripetal migration and a direct response to BMP signaling, I inhibited the activity of type I BMP receptors with the use of a chemical inhibitor known as DMH1. In doing so, I did not observe any discernable defects in centripetal migration but found abnormalities in a subsequent process known as nurse cell dumping, potentially revealing a role for BMP signaling in the regulation of this process. Whether or not concurrent BMP signaling is required to regulate centripetal migration is an ongoing question.

Altogether, the data presented in this dissertation constitute the first study of centripetal migration using live time-lapse imaging techniques and establish a framework that delineates its normal progression. Chapter 5 summarizes the important conclusions that arose out of my work and discusses the most pressing questions that remain on the horizon regarding the further characterization and understanding of centripetal migration. Supplemental Movies 3.1 - 3.6 pertaining to Chapter 3, as well as Supplemental Movies 4.1 - 4.4 pertaining to Chapter 4 are included as separate files.

Keywords

BMP; Centripetal; Drosophila; Migration; Oogenesis; Signaling

Disciplines

Cell Biology | Developmental Biology | Molecular Biology

Language

English

Movie 3_1.mp4 (2228 kB)
Movie 3_2.mp4 (4464 kB)
Movie 3_3.mp4 (4382 kB)
Movie 3_4.mp4 (4305 kB)
Movie 3_5.mp4 (2824 kB)
Movie 3_6.mp4 (4236 kB)
Movie 4_1.mp4 (1003 kB)
Movie 4_2.mp4 (1014 kB)
Movie 4_3.mp4 (634 kB)
Movie 4_4.mp4 (600 kB)

Available for download on Sunday, May 15, 2022


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