Dr. Bruce A. Edgar

The development of an animal or plant from a single egg cell involves a highly regulated program of cell growth and proliferation. Dr. Edgar's research group has focused on the genetic logic underlying Drosophila's cell proliferation program, aiming to characterize the different types of cell cycles that occur during development, identify the critical regulatory factors, and understand how these factors are in turn affected by developmental programming and environmental conditions. Recent studies have characterized the genes and pathways that control cell growth, delved into how cell growth interfaces with the cell cycle control apparatus, and addressed how developmental signaling and nutrition regulate both cell growth and cell cycle progression. We make extensive use of classical and molecular genetics, mosaic analysis, quantitative cytological assays of growth and proliferation, gene expression profiling, proteomics, and forward genetic screens. We seek to define new genes and mechanisms involved in growth control that will impact general paradigms in cell and developmental biology, and which have relevance to medical conditions involving cell and tissue growth including cancer diagnosis and therapy, stem cell biology and regeneration, wound healing, and metabolic diseases such as diabetes and obesity.

Theme 1: Developmental Control of Cell Growth

Objectives: This project has three long-term objectives: 1) To understand the cell-intrinsic mechanisms that control cell mass increase (growth); 2) To determine how cell cycle progression is coordinated with growth; and 3) To learn how patterned growth is regulated during organ morphogenesis.

Overview: This is a long-term project that has used genetic screens and candidate gene studies to identify and then characterize genes that regulate cell growth. Past studies have characterized the cellular and developmental functions of the Drosophila genes myc, pi3k, Tor, Rheb, Cyclin D, E2F, Ras, wg (a WNT), dpp (a BMP), and others. One current study is focused on Ack1, a non-receptor tyrosine kinase that we identified in a genetic screen for growth effectors. We find that Ack-1 is a dose dependent regulator of growth in many cell types, that it acts synergistically with Ras/MapK signaling, and that it promotes rampant stem cell expansion in the Drosophila gut, generating a condition similar to colon cancer. Our immediate objectives are to determine how this interesting gene controls cell growth, and to understand how it's activity is regulated during normal development in vivo. A second current study is focused on TIF-IA, a limiting regulator of rRNA synthesis that we find to be a critical growth-regulatory target of TOR signaling. Our immediate objective is to determine how nutritional signals acting via TOR regulate the function of the RNA polymerase I complex in general, and TIF-IA in particular. For both TIF-IA and Ack1, a combination of genetic and proteomic approaches are being used. Another aspect of this project is to determine how cell growth is coupled to cell cycle progression in developing Drosophila tissues. To this end we have developed a model for the control of endocycles, which are widely used in invertebrates and plants to allow cell growth. We are testing the hypothesis that growth regulators such as Myc, Tor, Ack1, and TIF-IA control rates of endocycle progression by affecting the translation of Cyclin E and E2F1, which are limiting regulators of the initiation of DNA synthesis.

Potential Impact: This work will define new genes and mechanisms involved in growth control and should impact general paradigms in cell and developmental biology. It has relevance to medical conditions involving cell and tissue growth including cancer diagnosis and therapy, regeneration, wound healing, diabetes and other metabolic diseases.

Theme 2: Growth regulatory targets of the TSC1/2 tumor suppressor complex

Objective: To identify molecular pathways via which the TSC1/2 tumor suppressor complex inhibits cell growth.

Overview: Tuberous sclerosis (TS) is an autosomal dominant disease involving widespread benign tumors and a predisposition to metastatic cancer. Most cases are caused by loss-of-heterozygosity in one of two genes, TSC1 or TSC2, which encode a protein complex. The TSC1/2 complex is thought to suppress cell growth by inhibiting Rheb, a small GTPase that is an essential activator of the Target-Of-Rapamycin (TOR) kinase. TOR in turn controls diverse metabolic processes required for cell growth including protein synthesis, nutrient import, autophagy, cytoskeletal organization, and transcription. TOR has two well-characterized targets in metazoans, S6K and 4EBP, but genetic analysis in mice and Drosophila indicate that these cannot account for the striking overgrowth phenotypes that occur when TSC function is lost. Moreover, it is unclear that all of the effects of TSC mutation are mediated by Rheb and/or TOR. Hence the identification and characterization of additional effectors of the TSC1/2 complex is required to advance our understanding of the molecular basis of this disease. This project will: 1) Identify and characterize new gene products required for TSC and Rheb function, and 2) Evaluate the hypothesis that the TSC1/2 complex mediates all of its effects via Rheb and TOR. Specific Aims are: 1) To use heavy isotope labeling and quantitative mass spectrometry (MS) to comprehensively characterize the effects of loss-of-TSC function, and gain-of-Rheb function, on the expressed proteome in Drosophila (S2) and human embryonic kidney (HEK293) cells; 2) To screen the Drosophila genome for modifiers of eye hypertrophy caused by loss of the TSC1/2 complex, or overexpression of Rheb; 3) To functionally validate potential TSC-effectors identified in Aims 1 and 2 in human cells.

Potential Impact: TSC-effector genes and pathways identified in this way are expected to be essential for the deregulation of cell growth that causes the benign tumors that plague TS patients. As such, some of these genes and pathways may constitute targets for diagnosis and treatment of the disease.

Theme 3: Growth-Regulatory targets of Cyclin D/Cdk4

Objective: To define the molecular mechanism targetted by Cyclin D/Cyclin dependent kinase 4 complexes (CycD/Cdk4) to drive cellular growth.

Overview: Deregulation of at least one component of the Ink4/Cyclin D1/retinoblastoma pathway is a common feature of most of human cancers, but the mechanism by which these genes contribute to tumorigenesis is poorly understood. Using Drosophila we discovered that Cyclin D/Cdk4 complexes, in addition to their widely appreciated function as promoters of cell cycle progression, also regulate rates of cell mass increase (growth). We discovered that CycD/Cdk4 does this by controling mitochondrial activity, via unknown targets distinct from those in the well-studied pRb/E2F pathway. We are using Drosophila genetics to identify and characterize these novel growth-regulatory targets, which we believe may be important for understanding CycD/Cdk4 function during normal and neoplastic development. Specific aims of this project comprise: 1) Identification and characterization of growth regulatory targets of CycD/Cdk4 using genome-wide screens for mutations that dominantly suppress overgrowth of the eye caused by ectopic CycD/Cdk4; 2) Identification of CycD/Cdk4 targets by gene expression profiling; 3) Characterizing the Hph/Hif-1/VHL and JAK/STAT pathways as mediators of CycD/Cdk4-driven growth; and 5) Tests to determine how CycD/Cdk4 effects mitochondrial function. Findings from Drosophila are also being validated in human cells.

Potential Impact: This project aims to elucidate how CycD/Cdk4 alters cell physiology to promote growth, and should contribute to molecular paradigms explaining how cell growth and cell division are coupled. It should also allow identification of novel Cyclin D and Ink4 targets in humans, and thus help to reveal how growth of our own cells is controlled during normal and neoplastic development. Some of the identified genes could be useful targets for cancer diagnosis or anti-cancer chemotherapeutics.

Theme 4: Developmental Control of Cell Cycle Exit

Objective: To define the molecular/genetic mechanism used to terminate cell cycle progression upon cell differentiation in the fly wing and eye.

Overview: One of the unsolved mysteries of development concerns how growth and form are controlled. The regulation of cell proliferation is central to this problem, and the mechanisms controlling cell cycle exit upon cell differentiation are particularly relevant. While cell cycle exit has been studied in cell culture and in vivo in model organisms, the mechanisms that couple differentiation signals to the cell cycle control apparatus are still poorly understood. This project aims to determine the mechanism of cell cycle exit at differentiation in the Drosophila wing and eye. Work to date has revealed that differentiation signals dominantly suppress the transcription of cell cycle control genes, including cyclin E and cdc25, via an E2F/RB-independent mechanism. Hence a current aim of this project is to analyze the transcriptional regulatory regions of these two critical cell cycle control genes to identify the cis-acting elements that mediate their silencing at differentiation. Following this, we will employ a DNA binding/quantitative proteomics approach to identify the trans-acting factors that mediate silencing of these genes at differentiation. A parallel, ongoing aim is to perform unbiased, forward genetic screens in the fly to identify novel genes involved in cell cycle exit. These approaches should identify new gene products that mediate cell cycle exit, and provide a paradigm for future studies of how differentiation signals interface with the cell cycle control apparatus to restrain cell proliferation.

Potential Impact: Cell cycle exit is critical in carcinogenesis, where it is bypassed, and in wound healing and regeneration, where the process is reversed to allow renewed proliferation. Because the genetic networks that orchestrate patterning, differentiation, and cell cycle control are conserved between Drosophila and mammals, the results obtained herein should be generally relevant to human development and disease.

Theme 5: Intestinal Stem Cell Homeostasis

Objectives: To define the mechanisms controling the growth and proliferation of Drosophila adult gut stem cells, and use these cells to test models of human colon cancer.

Overview: The recent discovery of proliferative stem cells in the gut of adult Drosophila has provided an opportunity to study intestinal stem cell biology in a genetically tractable system. We and other labs have characterized the development of this stem cell population from its larval progenitors, and assessed the roles of known signaling pathways and cell cycle control genes in stem cell expansion. We find that Ras/MAPK signaling is a critical regulator of gut stem cell expansion during larval development, as well as during gut self-renewal in adults. Gut stem cell proliferation is also regulated by the Ack1 non-receptor tyrosine kinase mentioned above, by the JAK/STAT pathway, and by Cyclin E and the Cyclin E-specific inhibitor, P27/dacapo. Future studies will aim to profile gene expression patterns in the adult gut stem cells to determine the factors that allow their infinite renewal, and to further define the signals that regulate their growth and proliferation. Forward and reverse genetic screens will be undertaken to define genes that, like Ack1, deregulate stem cell expansion and gut hyperplasia. The functional homology between mammalian and insect gut homeostasis will be assessed, and novel genes found to regulate gut stem cell proliferation in Drosophila will be tested for similar roles in mammals.

Potential Impact: This project will develop a system for genetic studies of gut stem cell biology, and may identify novel genes important in stem cell regulation. After validation, the human orthologs of such genes may be useful targets for diagnosing and treating colon and other stem cell-based cancers.