MOB-NDR Kinase Signaling Components are Required for Epithelial Tube Formation in the Drosophila Follicular Epithelium

A major goal of developmental biology is to understand how a single fertilized cell can give rise to the many functional tissues and organs, of specific sizes and shapes, that make up the adult body plan. Over the last 25 years, developmental geneticists have uncovered much concerning the cell-to-cell communication systems that are necessary to build complex tissues and organs. For example, throughout development, cells communicate with their neighbors using specialized signaling molecules. These signals are instructive and provide “signal-receiving” cells with information about space and time. That is, signal-receiving cells “learn” precisely where they are located, and, how far along in development they are; they then use this information to guide their own development. For example, receipt of instructive signals leads to altered cellular behaviors in the signal-receiving cells. These behaviors are essential to the development of multicellular organisms such as fruit flies and humans, and include: cell division or cell death, changes in cell shape, and tissue reorganization driven by the movement or migration of cells. However, it remains unclear how signal-receiving cells translate instructive signals into the appropriate cellular behaviors that build and sculpt tissues and organs. One approach to tackle this question is to study the genes whose products are required, in the signal-receiving cells, to bring about changes in cellular behavior. The research presented in this dissertation focuses on a set of genes that promote the development of a simple sheet of cells, an epithelium, into a three- dimensional epithelial tube. Specifically, I used a genetics-based approach to investigate epithelial tube formation in the follicular epithelium of the common fruit fly, Drosophila melanogaster. The follicular epithelium is a major component of an organ-like structure, the egg chamber. In chapter 1 of this dissertation, my co-authors and I present a detailed literature review of the egg chamber as an experimental system. The egg chamber consists of an internal cluster of 16 interconnected germ cells that are enveloped by a simple sheet of cells, the follicular epithelium. As egg chambers develop, the follicular epithelium undergoes multiple rounds of patterning—that is, in response to cell- signaling, the follicular epithelium is subdivided into discrete groups of cells that express different sets of genes. Each of these groups of cells then participate in distinct cellular behaviors that ultimately transform the overall architecture of the follicular epithelium. Chapter 1 of this dissertation provides an in-depth discussion of each of these cellular behaviors. Previous studies carried out in the Raftery laboratory identified a genetic tool, known as an enhancer trap, as a powerful reagent to investigate how cells communicate with one another in order to pattern the follicular epithelium. Using this genetic tool, I identified the gene mob2, which encodes a Mob-family protein as a candidate regulator of epithelial remodeling (refer to chapter 4). Mob-family proteins are small, non-catalytic, regulatory proteins. Their best characterized function is that of essential activators of nuclear Dbf2-related (NDR) kinases. In chapter 2 of this dissertation, I provide a detailed literature review of NDR kinases regulation by Mob- family proteins—focusing primarily on Drosophila melanogaster. In chapters 4 and 5, I investigate the function of genes encoding Mob- and NDR-family proteins in the context of follicular epithelial remodeling. In Drosophila, there are four genes encoding Mob- family proteins (mob1, mob2, mob3 and mob4) and two genes encoding NDR kinases (warts and tricornered). To investigate the function of mob2, I generated targeted loss of function alleles (detailed in chapter 4) that eliminate Mob2-dependent regulation of NDR-family kinases. Using these newly generated alleles, I found that Mob2-dependent regulation of NDR- family kinases is not essential. Homozygous mob2 mutants are viable, fertile, and appear morphologically normal. However, I found that mob2 function is required for normal epithelial tube formation in the Drosophila egg chamber. mob2 loss of function results in the formation of short and misshapen tubes. Using tissue-specific knock- down, I further showed that mob2 function is required in the follicle cells to promote normal epithelial tube formation. Having identified Mob2 as a regulator of epithelial tube formation, I next sought to determine whether either or both of the Drosophila NDR kinases were similarly required. In chapter 5 of this dissertation, I showed that tricornered knock-down in the follicle cells produces fully penetrant defects in epithelial tube formation. These defects are more severe than those associated with constitutive mob2 loss of function. Furthermore, the defects in epithelial tube formation associated with tricornered knock- down suggest that tricornered plays a role in the follicle cells to promote cellular behaviors that are essential for the earliest stages of tube formation. These results raised the possibility that other genes encoding Mob-family proteins may compensate for mob2 loss of function; or alternatively, that in the egg chamber, mob2 plays a minor role in epithelial tube formation. Consistent with the latter interpretation, follicle cell- specific knock-down of mob4 produces fully penetrant defects in epithelial tube formation that mirror tricornered knock-down (chapter 5). Taken together, the data I present in this dissertation shows that the NDR kinase, Tricornered, plays a major role in forming, shaping, and elongating an epithelial tube. Furthermore, I identified Mob2 and Mob4 as additional Mob-NDR signaling components that participate in epithelial tube formation. Based on the distinct phenotypes produced when Mob2 or Mob4 is disrupted, I propose a model whereby Tricornered and Mob4 function together to promote early tube formation and where Tricornered, Mob2, and Mob4 work together later in epithelial tube development to promote elongation and morphological refinement of the epithelial tube (discussed in chapter 6). The morphogenetic roles for the other Mob family proteins, Mob1 and Mob3, remain open questions