Amy M. Bejsovec - US grants

This proposal explores the mechanisms by which Wingless (Wg) signaling promotes cell fate decisions in the Drosophila embryonic epidermis. Wg and its vertebrate Wnt homologues are essential for generating pattern in both invertebrate and vertebrate embryos. Inappropriate activation of the Wnt signaling pathway has been implicated in mammary tumors in mice, and melanomas and colon carcinomas in humans. Therefore, understanding how these molecules influence cell fate decisions is relevant to both fetal and adult human health issues. The project utilizes the easily scored larval pattern as an assay system for Wg-mediated cellular decisions. Drosophila embryos secrete a tough cuticular layer that displays a rich pattern of structures, indicative of discrete cell identities in the underlying epidermis. Wg signaling is required for generating the diverse array of cuticular structures observed in the wild- type animal. Aim 1 of this proposal discusses a mutant Wg molecule that specifies some, but not all, of the cell fates normally found in wild-type pattern. The biochemical basis for this limited signaling activity will be examined with respect to putative Wg receptor molecules and processing steps known to be essential for Wg function. Aim 2 tests other components of the Wg signal transduction cascade to determine their precise roles in the distinct Wg-mediated cell fate decisions. Germ line clone analysis of the recently identified transcription factor, dTCF, will be undertaken to determine whether the subtle effects of its zygotic loss are due to maternal contribution, or reflect participation in only a subset of the Wg-mediated cellular responses. Aim 3 examines other genes that influence the diversity of cell fates specified by Wg activity, particularly concentrating on a newly identified gene that specifically influences the diversity generated by the mutant Wg molecule described in Aim 1. This specificity may indicate a novel function in the Wg pathway. Aim 4 describes a plan for the molecular cloning of this new gene and for the subsequent characterization of its gene product.

Project Summary
The long term goals of the Bejsovec laboratory are to understand how signaling molecules are distributed within developing embryos and how that distribution specifies positional information within a tissue. The lab focuses on the Wnt family of secreted growth factors, which play a central role in specifying cell fates during the development of all animal embryos. These molecules undergo lipid-modification but are capable of promoting long-range signaling events in populations of cells distant from the source cells. In embryos, these events include patterning of the nervous system, limbs and internal organs; in adults, Wnt signaling promotes cell proliferation in stem cell populations and inappropriate activation of the pathway is associated with human cancers, particularly colorectal cancer. The powerful genetics of Drosophila make it an excellent model system for exploring the molecular and cellular mechanisms of Wnt distribution and signaling activity.

In Drosophila embryos, the Wnt homolog, Wingless (Wg), is produced in a single row of cells in each segment of the epidermis but its activity is required in all cells of the segment for proper patterning. Previous work in the Bejsovec lab has shown that Wg can be mutated to disrupt its movement without destroying its intrinsic signaling activity. This suggests a cellular mechanism for Wg distribution that is independent of its signal transduction pathway. To identify the cellular machinery responsible for Wg protein distribution, second site mutations that suppress the movement-defective mutant phenotype have been isolated: these mutations show phenotypes suggesting that they influence the Wg transport process. Aim 1 of this application proposes to characterize the genes identified by their suppressor phenotypes and Aim 2 proposes gain of function techniques for identifying other components required for transport. These experimental approaches will reveal details of the machinery involved in processing and propagating the Wg/Wnt signal.

Intellectual Merit: In the field of signal transduction, most work has focussed on understanding receptor activation and the subsequent intracellular events triggered by ligand-receptor interaction. Much less is known about how ligands arrive at distant target cells to trigger response, yet this is a critical problem in embryonic development where many signaling molecules have long-range effects on patterning. This project, if funded, will define the cellular machinery required for ligand transport. Identifying and characterizing the proteins responsible for ligand movement is a crucial first step in understanding this important phenomenon.

Broader Impacts: Ligand distribution may play a part in tumorigenesis and metastasis, in addition to its fundamental role in regulating embryonic pattern formation. This work in Drosophila will identify cellular components required for ligand transport which may have homologs that play a similar role in vertebrates. These may reveal new targets of mutation during oncogenesis, and understanding their activity will provide clues for therapeutic intervention in Wnt-associated cancers. The genetic screens proposed are ideal for introducing high school students and undergraduates to basic research. This laboratory is committed to providing students, particularly women and under-represented minorities, with hands-on experience in a research laboratory. Past students have co-authored papers, demonstrating their deep involvement in the research effort.

DESCRIPTION (provided by applicant): Wnt signaling determines cell fates during embryonic development in all animals, including humans, and is required later in life to maintain stem cell populations. Excess Wnt activity is associated with human cancers, particularly colon cancer. To understand how Wnt signaling causes cancer, the factors that normally control Wnt pathway activity must be fully characterized. The long term objective of the Bejsovec laboratory is to dissect the mechanics of this important signaling pathway, using both the Drosophila genetic model system and cultured human cells. This lab has identified by mutational analysis many important pathway components, such as Tcf, the transcription factor that activates Wnt target genes, and Apc2, a human tumor suppressor homolog that negatively regulates the Wnt pathway. This proposal focuses on another negative regulatory gene identified in the same mutational screen: tumbleweed (tum) is a homolog of the human RacGap1, a GTPase activating protein (GAP). Surprisingly, the GAP activity of this gene is essential for cell division, but its GAP activity is not required for its role in Wnt regulation. Two Tum-interacting molecules, identified in yeast two- hybrid screens, strongly modulate Wnt pathway activity in human cells. Pavarotti (Pav), a kinesin-like protein, acts as a repressor in a similar fashion to Tum, while the other interactor, a novel fly protein tentatively named Sills, enhances Wnt-induced target gene expression. This application proposes three aims to determine the mechanism of action for Tum and its binding partners. Both Tum and Pav play a Wnt-independent role in organizing the cytoskeleton during cell division, so a primary issue is whether their Wnt- dependent role also occurs in the cytoplasm. Aim 1 will determine the subcellular locations relevant to their Wnt-modulating activity. Aim 2 will address whether Tum and Pav interact directly with other Wnt pathway components or with the transcription complex that forms on Wnt target gene promoters. Aim 3 will characterize the cellular function of Sills and its human homolog, using both fly embryos and cultured cells to determine how it enhances Wnt activity. These experiments will help formulate a clearer picture of how living tissue modulates the response to Wnt signal. PUBLIC HEALTH RELEVANCE: Wnt signaling is essential for normal development in all animals, but abnormal Wnt signaling is associated with various forms of cancer, particularly colorectal cancer. Experiments proposed here will use both the fruitfly model system and cultured human cells to study newly-discovered genetic factors that regulate Wnt signaling.

? DESCRIPTION (provided by applicant): A broad underlying cause of human disease is the incorrect spatial or temporal control of gene expression. The nuclear pore complex (NPC), which was long regarded simply as a gateway through which molecules move in and out of the nucleus, has emerged as an important modulator of expression. A novel link between the NPC and developmental signaling pathways, another key regulator of gene transcription, has been discovered in Drosophila, which motivates the proposed research. Megator, the fly homolog of the Tpr nuclear pore protein, was identified as a binding partner for the Wg/Wnt inhibitor, Tum. Loss of Megator function in the developing fly wing disrupted expression of Wg target genes without disrupting production of Wg itself, indicating that Megator is required for proper response to Wg/Wnt signaling. Two other nucleoporins (Nups) that were identified subsequently also appear to regulate developmental pathways. Loss of Nup154/155 or Nup214 function drastically disrupted the developing wing and leg without affecting other developing tissues. The unique pattern defects caused by Megator, Nup 154/155 or Nup214 disruption suggest that they are involved in cellular processes beyond just structural integrity of the NPC. They may target the nuclear import of key signaling pathway components, modulate signaling pathways independently of the NPC, or organize chromatin within the nucleus in ways that differentially influence the target genes of specific signaling pathways. The goal of this project is to distinguish among these hypotheses for the tissue-specific nature of the Nup activities, using the powerful molecular and genetic tools available in the Drosophila model system. Cell to cell signaling is required for embryonic patterning in all animals. In humans, loss of proper signaling leads to birth defects, whereas hyperactivity of some developmental pathways is associated with a variety of childhood and adult cancers. Because signaling pathways in Drosophila are highly conserved with humans, what is learned in the fly will be directly applicable to human cell biology. By studying these pathways and their interaction with the NPC, this project aims to provide new insight into disparate human disease states, and may reveal that they share similar cellular underpinnings. The results obtained in these exploratory experiments will guide future investigation into the relationship between signal transduction and the NPC. This work has the potential to blaze a new trail in understanding how nuclear architecture influences normal development and disease processes.

The fruitfly, Drosophila, is easy to manipulate in the laboratory and can be used to discover genes that control the growth and shaping of animal embryos. Flies are fed a chemical that causes mutations in the DNA, the progeny are bred and then examined for altered development of the fly. A mutation that breaks an essential gene will cause dramatic changes in the body pattern. Many of the fly genes identified this way have human counterparts, which perform very similar functions during human development. The Bejsovec laboratory uses this strategy to study the inner workings of a genetic pathway that is shared between flies and humans. This pathway, triggered by the Wg/Wnt growth factor, tells cells where they are in the body and what special structure to become; inappropriate re-activation of the Wnt pathway in adult tissues is associated with human cancers. The Bejsovec lab has found that three genes already known to work together to control cell division, have a second unexpected function in controlling the Wnt pathway. This project will determine how these genes exert control over the pathway, and how their action disrupts the fly body plan. Broader impacts of the project include deeper understanding of developmental mechanisms in all animal species, and a hands-on research experience for undergraduate and high school students in the Bejsovec laboratory and for undergraduates in an existing laboratory course.

Although the Wg/Wnt pathway plays a central role in development and tissue homeostasis, the sequence of events, from Wg/Wnt binding at the cell surface to the downstream activation of target genes, is still not completely understood. Tumbleweed (Tum), Pavarotti (Pav) and Pebble (Pbl) repress Wg/Wnt pathway activity in cultured human cells and in fly embryos. These genes may represent a crucial missing link, perhaps missed in earlier screens for Wg/Wnt pathway components because all three genes have essential functions in cell division. In cytokinesis, Tum and Pav position Pbl-RhoGEF at the equator to activate the G protein, Rho, which organizes the actin contractile ring. Work proposed here will determine whether (and how) Tum, Pav, and Pbl make contact with known Wg/Wnt components, and will test whether GTPase activation is involved. In addition, reducing Tum or Pbl function in adult precursor cells creates a novel homeotic transformation which may result from the simultaneous disruption of cell signaling and cell division. Genetic and molecular tools available in Drosophila will be used to determine how misregulation of signaling and/or cytokinesis might alter segmental identity. The resulting data will provide insight into Wg/Wnt pathway control and tissue patterning, and may reveal new connections between cell division and cell fate specification during development. The unique appearance of the transformed flies will appeal to undergraduate and high school students, engaging them in the scientific enterprise and helping to recruit a diverse group, including women, socioeconomically disadvantaged and underrepresented minority students, into the workforce.