Lucy E. O'Brien, Ph.D. - US grants

DESCRIPTION (provided by applicant): Tissue self renewal is essential to the viability of the adult organism. In the intestine, the renewal process has built-in flexibility to adapt to external influences by altering the proliferation/death equilibrium. However, the signals that trigger adaptation and the mechanisms that re-equilibrate homeostatic balance are virtually unexplored. With the recent discovery that multipotent stem cells renew the intestinal lining in the adult Drosophila midgut, the powerful advantages of the Drosophila system-superior genetic tools, sophisticated cellular analysis, and high experimental tractability-can be applied to the outstanding problem of how tissue homeostasis is dynamically regulated. The ultimate goals of this research are to understand how death and proliferation are coordinated within tissues to achieve homeostasis and to uncover the mechanisms that enable homeostatic flexibility. This proposal focuses on nutrient-driven mucosal remodeling, a paradigm of intestinal adaptation. Combined genetic and cellular approaches will be used to examine the hypothesis that distinct systemic and local mechanisms regulate nutrient-driven adaptation. Aim 1 will characterize how nutrients alter the spatial and temporal profile of the proliferaton [sic]/death balance in intestinal homeostasis. Aim 2 will investigate the role of systemic, nutrient-sensitive endocrine signals, particularly insulin and a Drosophila neuropeptide Y homolog, in homeostatic remodeling. Aim 3 will investigate the role of local cellular interactions by determining how enterocytes act through tissue structure to control the proliferation of nearby stem cells, culminating in a genetic screen to identify novel genes, both nutrient-sensitive and -insensitive, that promote short-range homeostatic control. The results from these studies will shed new light on the pathways that underlie intestinal adaptation. Under the mentorship of Dr. David Bilder and co-mentorship of Dr. John Forte, both leaders in the irrespective fields, the candidate will gain expertise in newer areas of GI physiology, stem cell biology, and genetic screening while pursuing her long-standing interest in epithelial tissue dynamics. This research and training plan will faciliate [sic] the candidate's progression to autonomy by helping her establish an independent and complementary research project within the UC Berkeley Department of Molecular and Cell Biology. PUBLIC HEALTH RELEVANCE: The ability of the intestine to renew itself can be overwhelmed by injury and disease, leading to impaired nutrient absorption and long-term intravenous feeding. To develop better therapies, we need to know more about the basic genes and processes that control intestinal renewal. Studying intestinal renewal in fruit flies, a simple animal model, will generate new leads that can be explored further in mammals.

DESCRIPTION (provided by applicant): Renewal of the intestinal epithelium requires a balance between progenitor cell divisions and differentiated cell loss. Maintaining division-loss balance is essential for digestive health, while its disruption characterizes numerous intestinal pathologies. Division-loss balance involves feedback signals that act within the epithelium; however, the molecular nature of these signals is largely unknown. Our long-range goal is to understand the processes that give rise to the spatiotemporal dynamics of intestinal cells in vivo. Supporting this goal, the objective of this proposal is to investigate mechano-sensitive mechanisms that coordinate stem cell divisions with the epithelium's need for new cells. These studies will exploit the tractability of the adult Drosophila intestine, whose simple stem cell lineage and advanced genetic tools enable precise mechanistic investigation. We have previously shown that the Drosophila intestine exhibits a stem cell driven, reversible growth response to increased dietary load (O'Brien et al., Cell 2011). Preliminary evidence from our lab suggests a correlation between stem cell division rate and the degree of intestinal distention. This correlation is reminiscent of density-sensitive proliferation in epithelial culture, a collectve cell behavior controlled by mechanotransduction through the adhesion receptor E-cadherin and the transcription factor YAP. Here, we will examine the hypothesis that analogous mechano-sensitive signals stimulate intestinal stem cell divisions when enterocytes are sparse. Specifically, we will (1) determine the mechano-sensitivity of E-cadherin and YAP in niche and non-niche cells during intestinal distention, and (2) elucidate the niche- and non-niche roles of E-cadherin and YAP in density-sensitive division control. Accomplishment of these aims may identify a mechano-sensitive pathway that links enterocyte density to stem cell divisions, providing basic insight into homeostatic control of intestinal renewal. Our proposed work is significant because knowledge of how cell loss and production are coordinated may engender future therapeutic strategies to enhance intestinal repair and regeneration. Our approach is innovative because it draws a novel conceptual link between density-sensitive proliferation and homeostatic tissue renewal, and because it exploits the unique attributes of an emerging experimental system. Finally, data from these studies will provide the foundation for a detailed, R01-level investigation of the mechanobiology of intestinal renewal and remodeling.

? DESCRIPTION (provided by applicant): Effective and safe stem cell therapies must build upon knowledge of how stem cells generate precise numbers of differentiated cells to meet the body's needs. In adult organ renewal, each stem cell division triggers a pivotal decision between asymmetric, symmetric-stem, and symmetric-terminal fates. To sustain constant numbers of stem and differentiated cells, these three fate outcomes must be collectively balanced. Conversely, dysplasia or degeneration arises if fate balance is lost. Yet in contrast to the well-studied pathways that execute fate outcomes, the upstream events that decide between fate outcomes are virtually unknown. Our long-term goal is to understand the mechanisms that arbitrate the organ-wide balance of division fates. Toward this goal, here we probe the cellular basis of symmetric and asymmetric fate decisions-in vivo and in real time-by combining live imaging with the versatile genetic tools of Drosophila. Using the adult Drosophila midgut, we have made a path breaking innovation by developing long-term imaging of epithelial renewal at high cellular resolution in live animals. Our methodology enables individual stem cell divisions to be captured in their native context and fate decisions to be visualized in real time. We will investigate three fundamental questions about the cellular and molecular nature of fate decisions. In Aim 1, we ask whether fate decisions are made by the dividing mother stem cell, by equipotent daughter cells, or a combination. Using live imaging, we will directly test the mother-control mechanisms of oriented cell division and fate determinant partitioning, and the daughter-control mechanism of Notch-mediated lateral inhibition. We will evaluate whether different mechanisms bias toward different fates, and examine whether initial fate decisions can be overturned by later-acting mechanisms. Aim 2 builds upon exciting preliminary data those stem cells are motile, which provokes the question of whether motility influences fates by altering proximity to spatially localized signals. We will determine how motility impacts fate decisions, probe the interplay between motility and fate outcomes, and identify the cytoskeletal regulators that instigate motility. In Aim 3, we turn to the adhesion junctions that define epithelal architecture and ask how this multicellular adhesive network integrates into fate decisions. We will separately perturb basal, lateral, and apical adhesion receptors on stem, daughter, and differentiated neighbor cells and parse how distinct receptors on different cell surfaces influence fate decision mechanisms and outcomes. Because epithelial stem cell biology and architecture are broadly conserved, the fate decision mechanisms uncovered here will potentially extend to epithelial organs in vertebrates, including humans. Ultimately, understanding the basic mechanisms that decide between division fates will open new therapeutic avenues to combat stem cell pathologies and promote organ regeneration.

PROJECT SUMMARY Many adult-onset pathologies, including aging-associated cancers, fibrosis, and degenerative diseases, start with transient cellular dysfunction that, over long timescales, progresses to organ-scale, physiological dys- function. To develop effective and precise treatments, we must identify the cellular events that drive disease progress and pinpoint where and when these events occur. Live in vivo imaging is uniquely suited to study cell and tissue dynamics in their native context and in real time. However for internal organs, most imaging meth- odologies fall short of providing a continuous view of the cellular origins of disease; they track either discrete events over minutes to hours or longitudinal processes over weeks to months?but not both. ?Bellymount?, our new imaging platform for adult Drosophila, is poised to fill this gap. Bellymount?s sim- ple design enables individual cells to be visualized within all major abdominal organs in adult Drosophila. Since imaging is non-invasive, the same cells and organs can be visualized repeatedly over periods up to four weeks. This innovative technology enables the first longitudinal analyses of slow physiological phenomena in intact Drosophila adults. In the proposed work, we will develop a new long-term (?12 hour) time-lapse capability to augment Bellymount?s existing longitudinal capability, thus enabling multi-hour cellular events and multi-week physiologi- cal outcomes to be correlated directly in the same individuals. At present, animal viability limits Bellymount im- aging sessions to <2 hours, which is too short to fully capture multi-hour events and informative numbers of infrequent, minutes-long events. In Aim 1, we will extend imaging sessions toward a goal of ?12 hours. To prolong viability, we will incorporate microfluidic delivery of liquid nutrients and pulsed, attenuated anesthesia into the Bellymount platform. We will determine the duration of long-term time-lapse imaging that is compatible with viability and assess the frequency with which long-term imaging can be applied in the course of a longitu- dinal experiment. In Aim 2, we will provide proof-of-principle for our combined long-term and longitudinal meth- odology by constructing a full timeline of cellular events during acute intestinal injury and subsequent regenera- tion. We will record intestinal injury during acute ingestion of chemical toxins in real-time. Using the same ani- mals, we will longitudinally monitor subsequent regeneration for 2 weeks or until it is complete. Finally, we will compare the responses of young and old adults to gain insight into the key sources of ageing-associated re- generative decline. Overall, successful completion of these Aims will provide first-of-its-kind methodology for ultra-long-term imaging of cellular, organ-scale, and inter-organ physiology in adult Drosophila.

PROJECT SUMMARY Many adult organs--for instance, intestine, mammary gland, skeletal muscle, skin?respond to reduced levels of functional demand by shrinking their physical size. In these organs, cells are lost faster than they are made, leading to a reduction in total cell number. The intestine is a broadly conserved exemplar of demand- driven organ shrinkage. In wild animals, cyclic periods of starvation cause intestinal size to shrink by 60-75%. Humans also undergo healthy intestinal shrinkage, but excessive or dysregulated cell loss can quickly become pathological, as seen in enteropathies like celiac sprue, endotoxemia, and giardiasis. Yet?unlike the mechanisms that balance cell division/loss during everyday turnover?the mechanisms that tune cell imbalance for physiological shrinkage are virtually unknown. The roadblock to mechanistic investigation of intestinal shrinkage has been the lack of a tractable laboratory model, which must allow cells (and their dynamic behaviors) to be monitored across time and must possess cell-specific markers and other tools to facilitate mechanistic studies. Historically, studies used rodents, but modern research protocols cannot replicate natural famine/feast cycles. My lab has developed a new invertebrate model of intestinal shrinkage that is both tractable and genetically manipulable: the Drosophila adult midgut, akin to the vertebrate small intestine. We demonstrate that intestinal shrinkage is conserved in Drosophila, and we document that its underlying basis is the massive squeezing-out of now-superfluous enterocytes through active extrusion. Here, we investigate intestinal shrinkage from both sides of the equation for net cellular balance: mature cell loss (Aim 1) and stem cell capacity (Aim 2). Our studies leverage the midgut?s superlative toolkit of cell- specific genetic reporters and our own pioneering innovations for real-time and longitudinal imaging of functioning midguts inside live animals. In Aim 1, we ask how the gut senses loss of ingested food? mechanical compression, lack of nutrients, or both. We test if two known regulators of extrusion, the transcriptional co-activator YAP/Yorkie and intercellular Ca2+ waves, function during shrinking to increase extrusions. Third, we probe whether a shrinking gut regulates cell extrusions at the organ scale or at the level of individual cells. In Aim 2, we seek the mechanisms that cause a 75% culling of the stem cell pool during shrinkage?even as stem cell mitoses paradoxically increase. We will test if stem cells initiate non-self- renewing divisions, adopt terminal fates directly, and/or activate apoptosis. The fly gut?s digestive physiology, stem cell lineages, and molecular regulation are similar to humans. Hence by elucidating the cell-to-organ scale mechanisms that operate at this frontier of tissue biology, this project may yield leads for therapies to treat cellular imbalances in human disease.

PROJECT SUMMARY Adult stem cells are the agents of organ renewal, remodeling and repair. Their hallmark ability to concomitantly self-renew and produce terminal progeny enables lifelong maintenance of organ form and function. At any given point in time, stem cells receive an ever-changing panoply of local and systemic signals that reinforce stemness, activate division, or direct cellular fate as needed to respond to the tissue?s evolving needs. These signals are deployed across space and time to marshal diverse stem cell behaviors for coordinated, organ-scale outputs such as tissue homeostasis. Such emergent properties are fundamental to the biology of adult tissues and essential for human health?yet, our grasp of their workings is rudimentary. My lab seeks to uncover the cellular mechanisms that underlie the robustness and flexibility of adult organ maintenance. The goal of our MIRA program is to build a comprehensive framework for understanding how each individual cell is guided by local and systemic signals for a net result of cellular equilibrium at the organ scale. Our model system is the adult Drosophila midgut, a stem cell-based, tubular epithelial organ that is functionally equivalent to the vertebrate stomach and small intestine. Our approach leverages unique live imaging capabilities?pioneered in our lab?and precision genetic tools to illuminate real-time cell dynamics in vivo and to probe the mechanisms that tune these dynamics. Here, we focus on three questions with broad significance to stem cell-based epithelial organs: 1) How does the spatial distribution of stem cells??which we find is non-random, due to autonomous stem cell motility? ?impact the efficiency and robustness of organ turnover? 2) What are the real-time spatial kinetics of the EGF feedback signals that equilibrate stem cell divisions and differentiated cell death, and does ectopic manipulation of these kinetics support or negate a point-source model for organ size control? 3) How do new, differentiating cells, which are born outside of the epithelium?s sealed network of occluding junctions, integrate seamlessly into the organ as they differentiate? These studies build upon and expand our R01-funded work on organ-scale stem cell dynamics. Since the cellular life cycle is a universal feature of self-renewing organs, the tunable, population-level mechanisms that we uncover in the Drosophila midgut will provide a template for thinking about more complex organs, including those in humans.