Stephen DiNardo - US grants

A major goal of embryogenesis is the establishment of body form. To accomplish this, each cell must differentiate appropriately for its position. Several questions about "positional information" arise. How is it generated? Of what is it constituted? How is it read accurately by each cell? And, once read, how do cells execute commands to differentiate properly? For all their complexity, these issues have become tractable as a consequence of molecular genetic analysis of embryogenesis in the fruit fly. This proposal addresses the nature of positional information and how specific cells read that information. At the cellular blastoderm in embryogenesis, pair-rule proteins cooperate to activate the expression of identity-determining genes in specific cells. In this manner some cells are given their positional identities directly. The genes specifying identity are members of the segment polarity gene class. Subsequently, other cells have their identities established by virtue of cell communication circuits that are controlled by the segment polarity genes. The pair-rule gene oddpaired plays a central role in activating three segment polarity genes, wingless, engrailed and gooseberry, each of which has distinct roles in cell patterning. This proposal addresses the mechanism by which oddpaired activates these cell patterning genes. The mechanisms revealed by these studies are likely to be at work in other organisms where cellular interactions and inductive events predominate during development.

Stephen DiNardo IBN 9418271 Cells within certain lineages must be continually replenished throughout the life of an organism. This is accomplished through the establishment and maintenance of stem cells. The mechanisms that instruct stem cell fate and that regulate the proliferation of their daughter cells are poorly understood. The overall objective of this proposal is to investigate how stem cells are established and maintained, using the model of Drosophila spermatogenesis that provides a unique opportunity to combine experimental and genetic approaches. The experimental manipulations first proposed will attempt to answer two questions: does cell signaling instruct stem cell fate? and are the number of daughter cell divisions internally programmed or controlled by neighboring cells? The first will be addressed by ablating the male hub cells, using a diptheria toxin inducible system, to determine whether the maintenance of the germline stem cell fate is governed by the signals emanating from these somatic cells. Heterospecific transplantation experiments, using two species that differ in the number of stem cell divisions, will determine if counting is intrinsic to daughter germ cells, or imposed by signals from the two somatic cells that surround them. The genetic approaches proposed will identify further mutations that interfere with these decisions. To identify genes that cause under- or over-proliferation of germ cells, or that cause discrete changes in the number of mitotic divisions, a powerful genetic screen, using the FLP/FRT recombinase system, is proposed. Molecular markers will be used to determine whether the primary defect in the mutants selected is in the production, maintenace or division of stem cells, or, alternatively, in the capacity of the daughter cells to divide. ***

The incidence of testicular cancers and of male infertility is increasing. Insight into the cellular causes of this can only come with a systematic understanding of the regulatory pathways governing spermatogenesis. In order to generate functional sperm, several diverse cellular processes are coordinated during spermatogenesis. These processes include the growth and maturation of spermatocytes, which occurs in close association with surrounding somatic cells, suggesting that soma-germline communication is involved. This proposal focuses on spermatogenesis in Drosophila, which is similar to that in mammals, and in which genetic analysis provides a way to systematically dissect the signals necessary for spermatocyte development. The transcriptional activator Eyes absent (Eya) is expressed in somatic cyst cells where it is required for the maintenance of encysted spermatocytes. Sine oculis (So), a homeodomain protein that appears to act in a transcriptional regulatory complex with Eya, is also required for spermatocyte maintenance. The hypothesis to be tested is that an Eya/So-dependent signal from cyst cells is required for spermatocyte development. The role of Eya/So in cyst cell fate and behavior will be assessed. In addition, the stage at which Eya and So affect spermatocyte development will be defined. Next, the ectopic expression of Eya and So will test whether the Eya/So-dependent signal is permissive or instructive. Complementary approaches, including the analysis of 40 spermatocyte degeneration mutants and a genetic screen for suppressors, will attempt to identify the signal from somatic cells, or components of its response in spermatocytes.

DESCRIPTION (Applicant's Abstract): The long-term focus is the control over pattern as exersized by Organizers. In the Drosophila embryo, adjacent rows of cells flanking each parasegment secrete two organizing signals, Wingless and Hedgehog. These two signals are responsible for assigning cell fates across the remainder of the parasegment, and these signals act asymmetrically from the organizing center. The aims of this proposal focus on genes involved in Wingless signaling from the organizer. Both cell culture and transgenic approaches are outlined for arrow, which encodes a novel Wg signal transducer. The experiments will determine whether the Arrow transmembrane protein binds directly to Wingless, or complexes with the Frizzled proteins as a co-receptor. Intracellularly, yeast two-hybrid studies, as well as cell culture and in vitro analysis are proposed to explore how Arrow transduces the Wingless signal. Analogous two hybrid and in vivo studies will determine how lines, which encodes a stage-specific factor regulating the response to Wg, interacts with known or novel Wg signal transducers. Response elements in defined Wingless target genes will be studied to identify how signal integration is achieved as the organizer operates. Finally, two genetic screens are proposed to identify novel components that either interact with Arrow or Lines, or control organizer asymmetry.

The Wnt gene family encodes secreted glycoproteins that play diverse roles during vertebrate and invertebrate development and have been associated with tumor formation in mice and humans. Analyses of Wnt signaling in multiple tissues and species have led to the hypothesis that Wnt proteins use similar signaling mechanisms. However, a critical question for understanding Wnt activity is how specificity between different family members is achieved. This application proposes to address this issue by examining the signaling pathways of two Wnt proteins during Drosophila development. Four Wnt genes have been identified in Drosophila, but only wingless, the ortholog of Wnt-1, has been well characterized. A second Wnt gene, DWnt-4, has been shown to be expressed in a partially overlapping pattern with wingless and the two genes influence some of the same processes but in distinct ways. Three specific aims are presented to test the hypothesis that Wnt genes elicit specific effects by interacting with both shared and unique signaling components and to identify signaling components that are unique to DWnt-4. The first specific aim is to determine the function of DWnt-r through generation of loss-of-function mutations and phenotypic analysis. The second specific aim is to identify genes that specifically interact with D Wnt-4 using genetic enhancement and suppression screens. The third specific aim uses in vitro experiments involving transient transfection of tissue culture cells to ask whether previously identified components of the wingless signaling pathway can mediate Dwnt-4 signaling.

? DESCRIPTION (provided by applicant): There is intense interest in the circuits that guide stem cell behavior. In many of our tissues, these stem cell regulatory circuits are controlled by the niches that house the stem cells. it is not understood how the niche is specified and assembled in a tissue, and then how it executes control over the stem cell pool. Understanding these interactions will be crucial to the use of stem cells in regenerative medicine. This proposal utilizes one of the most well-understood stem cell-niche systems, the Drosophila testis. Here, a small group of cells (hub cells) act as part of the niche, leading to the activation of signaling pathways in adjacent cells. In this way, nearby somatic cells take on cyst stem cell fate (CySC), while nearby germline cells, intermingled with these CySCs, take on germline stem cell fate (GSC). Importantly, these two distinct stem cell types must coordinate their production of daughter cells for spermatogenesis to occur properly. Over the last funding cycle, we made fundamental breakthroughs in live- imaging both niche assembly and niche function. Here, we capitalize on this unprecedented level of resolution to tackle the mechanisms that underlie niche assembly and function. Hub formation, and the attendant attachment of stem cells, is the major architectural event of gonadogenesis. The specification and placement of hub cells among somatic gonadal precursors (SGPs) generates an anteriorly-anchored proliferation center that will drive spermatogenesis in a polarized manner. To generate that polarity, a subset of pre-hub cells must respond to positioning cues. However, neither those cues nor their source are currently known. This proposal seeks to define those cues and how they work. In many of our tissues, niches house multiple stem cell types, just as does the Drosophila testis niche. Thus, to maintain tissues the behavior of the multiple resident stem cell types must be coordinated. However, in no case do we understand how. This proposal will address this essential question.

DESCRIPTION (provided by applicant): The Drosophila embryonic epidermis has been an exemplary model system for understanding how transcription factor networks and cell signaling modules are coordinated to pattern tissues and embryos. Recently, it has begun to reveal how those circuits impact the underlying cell biology to drive morphogenesis. Thus, the study of this epithelium holds the promise of a complete understanding of the circuit between developmental cell signaling and the effector proteins that control cellular morphogenesis. This is the overarching focus of this proposal. The remodeling of cell sheets underlies most of morphogenesis during development. For instance, it is the key to extending the axis of the vertebrate body plan, gastrulation, neurulation, and organ formation, in general. When these processes go awry, severe birth defects can result. Surprisingly, it is not understood how specific cell interfaces are selected out for remodeling. Within cell sheets, individual cells often can discern with high fidelity one of their edges from all others, and this is known as tissue polarity. In fact, tissue polarity is likely what assists in the ability of cells to select out a specific edge for remodeling. Tissue polarity is a conserved feature, operating in most if not all epithelia from the invertebrate, Drosophila, through to mammals, and a similarly conserved set of genes is at the center of assigning that polarity. Still, it is unclear how these genes confer polarity, what the effector mechanisms are, and how the polarity circuit is linked to developmental cell signaling. This proposal focuses on two key aspects of tissue cellular morphogenesis: the remodeling of cell sheets;and the assignment of polarity within the plane of the epithelium (so-called "tissue-polarity"). Finally, this work attempts to identify the cell biological mechanisms at the heart of these rearrangements. PUBLIC HEALTH RELEVANCE: Cell polarity is a driving force that shapes embryos and organ systems. Defects in polarity underlie several syndromes, such as ciliary diseases, and deafness. The genes that coordinate polarity were first identified in Drosophila, and their continued study in fruitflies is necessary to understand how they work.

? DESCRIPTION (provided by applicant): Our tissues inexorably decline during aging. A cause of this is intrinsic changes to the stem cells responsible for tissue homeostasis, or changes in the niche regulating stem cell activity. To explore potential links between cytoskeletal function and aging, there can be great advantage to studying a tissue that has already contributed fundamentally to our understanding of the impact of aging on stem cell function. The Drosophila testis is such a tissue, as germline stem cells (GSCs) have been shown to age, and both intrinsic and extrinsic regulators have been defined. To maintain tissue homeostasis, stem cells must exercise tight spatial and temporal control over daughter cell production. Thus, we have focused on abscission, the last step of cytokinesis, involving the complete separation of daughter cells. Abscission is a key point of regulation generally during cytokinesis, and microtubule and f-actin cytoskeletal dynamics play seminal roles during this process. Abscission has been difficult to image in vivo, but we have been successful using simultaneous imaging of Actin and Myosin II. Our analysis shows that abscission is dramatically delayed in GSCs, even after the contractile ring and the midbody microtubules are disassembled. Surprisingly, a new, filamentous actin-enriched structure succeeds the contractile ring, and serves to stabilize the midbody. We define a regulatory circuit controlling this novel cytoskeletal feature, as well as controls by Aurora B Kinase. Lastly, we find that aging significantly affects abscission, opening up this system to the study of aging and the cytoskeleton. Due to the ease of genetic and pharmacological manipulations, along with the well-characterized behavior of GSCs at steady state and upon aging, the Drosophila testis provides an ideal system in which to study the temporal dynamics, genetic control and roles for cytoskeletal components in abscission events. Here, we test for potential causative links between cytoskeletal control over abscission and aging within this adult stem cell population.

Stem cells are necessary for tissue homeostasis, and are often localized to specialized niches that control their function. In this manner, niches control virtually all aspects of stem cell dynamics, and these properties of stem cells are essential to tissue maintenance. Recent work has shown that precise cellular architecture is important in order for a niche to communicate with fidelity to the stem cells it controls. A major issue is that the field does not understand how niches are initially formed in a tissue, nor the key cell biological steps that constitute stem cell dynamics., and how these are regulated by niche signals. Our lab made significant advances on these questions over the past five years of GM funding. First, after identifying key signals and transcriptional regulators of niche cell specification, we revealed the dynamics of niche formation by live-imaging. This enabled us to suggest mechanisms underlying niche morphogenesis that are so central to its function. We pursue the underlying mechanisms in this proposal, capitalizing on our knowledge of cytoskeletal control over cell-cell organization, and in particular the involvement of actomyosin contractility and RhoA signaling. Second, we discovered that the niche imposes precise control over fundamental cellular processes such as abscission and midbody inheritance in order to regulate stem cell dynamics during tissue homeostasis and upon stresses, such as aging. We found that dysregulations of either of these fundamental cell biological processes compromises tissue function. Furthermore, the control of midbody inheritance as detailed in the proposal should have an impact generally on our understanding of how normal and cancer cells deal with defects in centrosome number. To address our hyptheses, we combine the powerful genetic and molecalur approaches available in using Drosohila, with state-of-the-art assays in cellualr mechanics, which include high spatial and temporal live-imaging, FRAP and laser ablation. Collectively, our approaches should reveal the undelying mechnics that establish a niche properly, and reveal important cellular features that allow stem cells to maintain tissues. !