Patrick O'Farrell - US grants

The objective of this proposal is to investigate the molecular mechanisms underlying the determination of cell fate in embryonic development. In this study the engrailed gene of Drosophila, a gene that is essential for the determination of the anterior and posterior cell lineages of body segments, will be analyzed. Dr. O'Farrell has proposed to develop a new assay which will enable him to follow and analyze the fate of individual cells in early development. The second major specific aim is to investigate regulatory elements of the engrailed gene as defined by other genes that interact with engrailed. By applying both approaches this study will define when and where determination of patterns of engrailed expression occurs and will define events controlling pattern regulation. Identification of the developmental fate of the descendants of the cells that express the engrailed gene at the blastoderm stage of the Drosophila embryo is important for an understanding of the genetic regulation of segmentation. The method of marking cells will be a useful technique which will benefit all developmental biologists studying Drosophila.

Mutations in Drosophila have identified genes controlling major steps in development. The function of one of these genes, engrailed, is required to establish and maintain developmental compartments within each segment of the embryo and the adult. Molecular analysis has revealed that this gene is expressed in an exceedingly intricate spatial and temporal program. This program of expression appears to be the result of regulation in trans by a number of other developmental genes and in cis by about 65 kb of flanking sequences. It has been argued that a conserved protein domain that is found in the products of several Drosophila developmental genes might include a DNA binding domain. We found that a fusion protein that included the engrailed homeo domain behaved as a sequence specific DNA binding protein. We propose that the protein products of developmental genes having homeo domains are DNA binding proteins and regulators of transcription. Further, because these regulators function combinatorially we think that they interact. We propose experiments to test for the interaction of these regulators with DNA and with each other. We will directly measure physical interaction in vitro and design tests for functional interaction in vivo. We think that these interactions will prove fundamental because the elaborate process of pattern formation is presumably founded upon the regulatory interactions among the products of the developmental genes.

DESCRIPTION (provided by applicant): Orchestration of the dynamic events of development requires precise timing. In contrast to the much- studied spatial controls in development, we know little about temporal regulation. We are probing the intimate coordination in time between cell proliferation and embryonic development. We focus on the specialized cell cycles of the early Drosophila embryo, the characteristics of which are highly conserved across evolution. Embryogenesis begins with extremely fast, synchronous cell cycles, in the absence of gene expression. No growth accompanies these cycles, which divide the large egg cytoplasm into smaller cells. At cycle 9, the cycles begin to slow, initially very slightly but progressively, until mitosis 13 where the cell cycle abruptly lengthens. This shift in the character of the cell cycles culminates in a sudden and strong activation of transcription, and the onset of gastrulation - a transition called the Mid-Blastula Transition (MBT). All future divisions require gene expression and occur in intricate spatial patterns. We are probing the mechanisms that time and coordinate events for the faithful execution of the MBT, in particular the shift in cell cycle timing. Our first aim is to determine whether the cell cycle acts as a clock for the MBT or whether cell cycle and MBT are controlled by independent but synchronized clocks. At present, data suggests two different clocks but with a safety mechanism whereby continued cell cycles defer the MBT. We will manipulate cell cycle regulators to alter the cycle, and examine the effect on timing of MBT events. Our second aim is to investigate regulation of cell cycle duration. We recently found that the time required to replicate the genome dictates the duration of the early cycles. We will investigate the molecular mechanisms governing S phase length and determine the importance of S phase elongation in execution of the MBT. Finally, transition from the rapid cell cycle of the early embryo to the prolonged cycle of the post MBT embryo depends on the abrupt elimination of maternal Cdc25 at the beginning of cycle 14. Our third aim is to determine how the embryo down-regulates Cdc25 mRNA and protein to trigger the major changes at the MBT. For this we have a new antibody to follow the behavior of Cdc25 protein and we will identify the mechanisms involved in destabilizing the mRNA. These studies will probe the fundamental question of how time is measured in biological systems, yielding advances to understanding of development and cancer. PUBLIC HEALTH RELEVANCE: When an egg develops into an organism, the number of cells goes from one to many millions. We are probing how cells keep track of time so that they know when they should divide and when they should arrest the cell cycle, the factors that distinguish the creative growth in embryogenesis from the destructive growth of a tumor. The faithful regulation of proliferation we are studying is essential if organisms are to avoid birth defects and cancer, two issues of major importance in human health.

O'Farrell 9724706 To produce a body having coherent shape and structure, development must control the size of each body part. Detailed knowledge of development and cell cycle control in Drosophila provides unique advantages for an exploration of the mechanisms that coordinate development, cell proliferation and size. Dr. O'Farrell will examine this coordination in imaginal discs, the larval precursors to the adult cuticular structures. Imaginal cells grow exponentially during larval life but arrest when this disc reaches a mature size. Wingless (Wg) and Decapentaplegic (Dpp) are pattern regulators that are also synergistic activators of growth and proliferation in discs. Nitric oxide synthase (NOS) inhibits proliferation and its activity rises at about the time proliferation arrests in discs. He will explore the hierarchy of the inputs controlling proliferation by examining the effect of a perturbation (e.g. induction of Wg and Dpp) on the other processes (e.g., activation of NOS), and the ability of other treatments (e.g., induction of various cell cycle regulators) to override the perturbation. These epistasis like experiments should begin to outline the pathway of regulation used in size control and give us entries to begin a more detailed dissection of the controls. To provide probes for this pathway dissection Dr. O'Farrell will define the basis of cell cycle arrest upon termination of growth. Measurement of the levels of various cell cycle regulators and analysis of the ability of ectopic expression to bypass the arrest will define the limiting cell cycle regulators. Because control of growth (increase in mass regulates cell cycle, he will develop probes for growth. The rate of protein synthesis governs when cell achieve the size threshold required for division, and the translational initiation factor eIF4E has important inputs into cell cycle regulation. Using transgenes and a drug (rapamycin) that impairs eIF4E activity, he will explore its involvement in growth an d cell cycle quiescence. He will use similar techniques to alter cyclin D levels and activity, because it is implicated in the coupling of translation and cell cycle. As a measure of growth quiescence, he will use a newly developed in situ procedure to assess the level of nascent rRNA transcripts. Together these experiments should provide the ground work for detailed dissection of the controls of tissue and organ size.

[unreadable] DESCRIPTION (provided by applicant): The discovery of nitric oxide as a regulator of blood pressure, which was awarded the Nobel prize in 1995, launched intense investigation into its function in human health. Recognizing the diversity, complexity and conservation of nitric oxide actions, studies in model organisms appear relevant and needed. In our studies of Drosophila, nitric oxide induced behavioral and physiological changes consistent with a conserved role in adaptation to low oxygen (hypoxia). Seeking other parallels to its function in mammals, we found that nitric oxide activates innate immune responses in Drosophila. We developed robust assays in which tagged transgenes report immune induction in larvae or cultured cells (S2 cells) in response to nitric oxide, or to bacteria, or to hypoxia. The responses of S2 cells can be blocked by inactivation of specific genes by RNA interference (RNAi). To exploit this powerful avenue for genetic dissection, we constructed a library of 7,200 RNAs representing the conserved genes of Drosophila. We propose high-throughput RNAi screens for genes contributing to immune induction. In preliminary work, we identified the genes involved in the response to bacterial components and will do the same for genes involved in the responses to nitric oxide and hypoxia. We will further exploit our assays to define the sequence of gene action, thereby delineating distinctions and commonalities in the pathways transducing these signals. Using in vivo genetics and tests in culture, we will place the signaling pathways in their biological context. Localization of function will position gene action in a cascade that conveys immune responses from the site of infection to distant tissues. These studies will provide new models for the action of signals central to human physiology and health. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Hypoxia figures importantly in the biggest of health issues. It is responsible for the damage caused by the ischemia accompanying cardiac infarct and stroke, and it plays a central role in limiting tumor growth as well as blunting the actions of important chemotherapies. While we have a mechanistic understanding of hypoxic regulation of transcription by Hypoxia Inducible Factor (HIF), other modes of response to oxygen deprivation are still poorly understood. How does the mitochondrion, the major consumer of oxygen in the cell, adjust to shortfalls in oxygen supply, and how does it signal to the rest of the cell, demanding accommodations to these shortfalls? Reactive oxygen species produced by mitochondria have been ascribed the responsibility of communicating oxygen stress to the cell, but precise pathways of signal generation, transduction and action have not been established. Recognizing the complexity of the issues and the fundamental nature of the questions, it seems that the powerful genetics available in model organisms could make a major contribution. We found that nitric oxide (NO) mediates immediate responses to hypoxia in Drosophila, including an arrest of embryos in a reversible state of suspended animation. We have devoted ourselves to the development of a powerful and greatly simplified experimental context in which we can dissect the NO-signaling mechanisms. Our finding that NO regulates innate immune responses in Drosophila revealed a hypoxia-immunity connection, and gave us the simplification we sought. Immune reporters signal the action of NO, and the response to hypoxia can be recapitulated in cultured cells amenable to powerful RNAi screens. In this proposal, we will combine in vivo studies and analysis of cell culture models to investigate NO-mediated signaling. In vivo studies will probe how infection induces NOS, and how NO signaling contributes to the immune response. In Drosophila S2 cells, we will examine the basis of hypoxia-induced NO signaling that appears to originate in the mitochondria, and will trace the transformation and transport of this signal as it is conveyed to the cytoplasm. Finally, we will define the genes and pathways by which mitochondria sense hypoxia and signal the dramatic changes within the mitochondria, and will the coupling between mitochondrial changes and the signals emanating from the mitochondria that have such huge impacts on survival of cells and the well-being of man. These studies will give us a mechanistic understanding of a major component of the hypoxia response, contribute to the understanding of the biological diversity in tolerance to hypoxia, and give us approaches to manipulate hypoxia sensitivity to potentially benefit issues of important health concern. PUBLIC HEALTH RELEVANCE The most common life-threatening health problems, cardiac infarct and stroke, cause damage by interrupting oxygen supply, yet we understand little about the biological responses to the resulting acute hypoxia. We will dissect the mechanisms of response to hypoxia in a powerful model genetic system, Drosophila. Our findings have the potential to influence treatment of cardiac infarct and stroke, and could have an impact on areas as diverse as sustaining organs for transplantation, and cancer therapies. [unreadable] [unreadable] [unreadable] [unreadable]

ABSTRACT Our goal is to understand how mutations and inhibitors that disrupt general mitochondrial functions can cause syndromes with marked tissue specificity. We are developing new experimental models in which we can bring the powerful genetic tools in Drosophila to bear on this question. We will test whether the tissue specificity of genetic and chemical stressors occurs because they target interactions between general mitochondrial functions and tissue specific genes. A particular mutant of Drosophila Cytochrome oxidase subunit 1 is male sterile, and otherwise normal. We hypothesize that this highly specific phenotype is the result of a failure of this allele to work conjunction with a testis specific isoform of one of the other respiratory chain proteins. Indeed, ectopic expression of the somatic version of Cytochrome c in the testis suppresses the sterility phenotype. The proposed experiments will rigorously test whether this sterility is due to a specific deficit in the partnership of the mutant Cytochrome oxidase and the testis specific isoform of Cytochrome c. Additionally, we will engineer the fly eye as a biosensor for disruption of isoform-specific interactions of mitochondrial functions, and will apply it to identify mutations and chemicals interfering with these interactions. We will also explore tissue specificity resulting from a synergy of two defects, where a tissue specific defect sensitizes a tissue to diverse genetic and chemical stressors. Eye specific knockdown of E2F compromised growth to produce a slightly reduced eye. It also sensitized the eye to mitochondrial stress. A low dose of oligomycin that is without notable effect in other tissues, synergizes with E2F:RNAi in the eye to produce tissue transformations (e.g. antennae growing out of the eye) and hypertrophy. We hypothesize that this dysgenesis/hypertrophy relies on two inputs with a biologically universal relationship. Any mutation that inhibits growth of a specific tissue creates a selective environment favoring cells that can escape the growth limitation by transforming to another cell type (transdetermination). A second stress that destabilizes developmental fate would produce the fodder for this selection. Mitochondrial stress appears to provide this destabilizing input. We will test this model and screen for natural mutations and environmental chemicals contributing to the synergizing inputs. Since mammals express numerous proteins as tissue-specific isoforms, they carry many genes that can mutate to create a selection for transdetermination. Without synergizing input, these mutations would have little impact and could accumulate. Thus, we suspect that the human population has a large and insidious pool of polymorphisms that creates a diversity of chemical sensitivities. Recognition of sensitizing mutations should empower application of DNA sequencing to personalized health-care.

? DESCRIPTION (provided by applicant): Host management of the mitochondrial genome the small genome of the mitochondria has an influence out of proportion to its size. It is not just tha its few gene products are extremely important in energy metabolism. The apparent independence of the mitochondrial genome has required a complex administrative arrangement: the nucleus creates developmental programs that ensure the continued contribution of the mitochondrial genome to the host's needs. If the mitochondrial genome had free reign, it would evolve selfishly, enhance its replication to out compete its neighbors, and abandon production of electron transport functions, which do not naturally contribute to the replication or transmission of this genome. Nuclear management largely but not completely contains such unruly behaviors. This proposal explores the poorly understood regulatory interactions underlying the intergenomic relationship. For example, we found that mitochondrial DNA (mtDNA) is eliminated from mitochondria during spermatogenesis. This elimination enforces maternal-only inheritance, an inheritance pattern that limits mitochondrial genomes to a lineage. This limitation caps the evolutionary advantages of selfish behavior: for example, a super replicating genome might succeed within a lineage but it cannot spread to infect the whole breeding population. This proposal will examine the mechanism of this DNA elimination program and test the use of this DNA elimination pathway to control and limit mitochondrial genomes in somatic development. We will also use a bevy of new tools to study how different mitochondrial genomes within an organism compete for transmission, and how the nucleus oversees this competition. A specialized competition among mitochondrial genomes to populate the egg is important to the host. During this process, genomes functional in electron-transport out-replicate less functional genomes. This competition provides the purifying selection that eliminates mutations detrimental to the host. However, competition among mitochondrial genomes during growth and development is not based on function. During this stage, selfish interests dominate, and each mitochondrial genome strives to out-compete its neighbor, selecting for super replicating genomes. We created heteroplasmic lines with two mitochondrial genomes, one with a replicative advantage but functional deficit, and one with a functional edge but a replicative disadvantage. Retention of these two genomes depends on the strength of the selection in oogenesis versus selection during zygotic growth. We will examine the genetic changes in the mitochondrial genome and in the nuclear genome that influence this balance. This will identify the nuclear genes that modify competition to oversee the evolutionary directions taken by the mitochondrial genome, and the sites of their action on the mitochondrial genome. This approach will give us a molecular and genetic foothold on the mysterious processes controlling a central axis of regulation influencing metabolic disease, aging, metabolism and evolution.

Project Summary/Abstract We will address two fundamental aspects of biology in Drosophila. The first involves the mechanisms regulating the onset of differentiation of a naïve embryonic genome. Developmental time resolves progressive steps that introduce the specialized domains of chromatin structure. We found that the molecular hallmarks of heterochromatin emerge late, after heterochromatic behaviors that the marks were thought to specify. We will explore the mechanisms establishing the earlier specializations of heterochromatin domains. Clustered arrays of repeated sequences (satellites) become late replicating in the 14th cell cycle prior to histone methylation (H3K9me) and heterochromatin protein 1 (HP1) binding. We found that regulated recruitment of Rif1, a replication inhibitor, explains developmental onset of late replication, and that a temporal schedule of its dissociation directs a temporal program of sequential replication of the satellites. We will use powerful in vivo tools to understand how time is programmed. But what targets Rif1 to satellite sequences? We found that the satellite sequences are compacted even earlier, prior to Rif1 recruitment, but then what is the basis of compaction? Repetitiveness is the universal distinguishing feature of satellite sequences. Recently, our neighbor Sy Redding and the Rosen lab showed that in vitro assembled chromatin with nucleosomes periodically positioned on repetitive sequences autonomously condenses into a liquid-like phase. In collaboration with Sy Redding we will relate the simple physical observations and in vivo behavior of satellite repeats. The approaches taken here will define the progression of interactions that evolution selected to guide the initial formation of distinct genomic subdivisions that underlie much of complex metazoan biology. The second project examines the genetic independence of the mitochondrial (mt) genome. Despite its bacterial origins, the mt genome is viewed as well adapted; however, an independently transmitted genetic element always has a renegade option. It is cooperative only as long as it is advantageous. The distinct genetics of the multicopied maternally transmitted mt genome is usually learned as uncomplicated, but this is belied by complexities in the transmission of disease mutations and age dependent onset of phenotypes. Inadequate genetic tools have hidden important features of mt genome behaviors that we have accessed with new tools. We show that the ability of a mutant mt genome to compete with the pool of other genomes in a cell determines its fate. The nuclear genome manipulates this inter-mt genome competition to give beneficial outcomes, but fragile points in this nuclear management allow successful transmission of some mutant genomes and underlie an age-associated decline in mt genome integrity. We propose experiments that will dissect the basis of nuclear management of the mt genome. Identifying modulating nuclear activities will enhance predictability of mt disease severity and introduce new avenues for their therapeutic management.