Richard S. Mann - US grants

9506206 Mann The development of a multicellular animal requires that all cells have two types of information: where they are relative to their neighbors (positional information) and what structure they should generate (identity information). In Drosophila melanogaster, the homeotic selector genes link these two pieces of information: their large cis regulatory regions interpret positional information and their protein products control cellular identities by regulating the transcription of downstream target genes. These target genes are important to characterize to understand how homeotic genes determine identities. Therefore, homeotic target genes were isolated using a molecular screen. Among these targets is a putative transcription factor that is predicted to control neuroblast identities in the central nervous system and a potential component of the cytoskeleton. Interestingly, the human homologs of both genes are implicated in oncogenesis, suggesting that, in some contexts, they can control cellular differentiation and/or proliferation. To investigate their wild type functions during Drosophila development, loss- and gain-of-function mutations of these genes will be generated. The consequences these mutations have on both embryonic morphology and gene expression will be analyzed. Using in vivo misexpression assays, the functions of the wild type and oncogenic forms will be compared. In addition, the homeotic-responsive cis regulatory sequences that control the expression of these genes will be studied using in vivo reporter gene constructs and in vitro DNA-binding assays. Together, these experiments will provide an understanding of how positional information is translated into identity information by the homeotic genes. ***

[unreadable] DESCRIPTION (provided by applicant): [unreadable] [unreadable] The Hox family of transcriptional regulators are homeodomain proteins that play important roles in many aspects of animal development and disease. For these proteins to perform their functions, they need to achieve the correct functional specificities in vivo. Using the fruit fly, Drosophila melanogaster, as the model system, this project builds on earlier work to better understand how these transcriptional regulators function in vivo. In previous work, results were obtained suggesting that Hox proteins, in conjunction with cofactors of the Extradenticle (Exd, Pbx in vertebrates) and Homothorax (Hth; Meis in vertebrates) families, achieve DNA binding specificity by reading a sequence-dependent DNA structure using residues in their homeodomains and nearby linker regions. A second set of findings demonstrated that some Hox proteins interact with these cofactors via multiple, partially redundant interaction motifs. Third, results were obtained showing that one Hox protein in Drosophila, Ultrabithorax (Ubx), modifies appendage morphologies, in particular, appendage size, by altering the levels and mobilities of diffusible morphogens such as Decapentaplegic (Dpp). Ubx executes these modfications in part by transcriptionally regulating two genes, master of thickveins (mtv) and dally in the developing appendage. Building on these results, the aims of this project are to 1) extend and generalize the Exd-dependent Hox specificity model, 2) determine the role of multiple Exd interaction motifs that are present in some Hox proteins, and 3) determine which of the targets identified previously in the control of appendage morphology by Ubx are directly regulated by this Hox protein. The approaches for all of these aims rely heavily on using a combination of Drosophila genetic tools and in vitro protein-DNA interaction assays. X-ray crystallography, to determine the three dimensional structures of some Hox-Exd-DNA ternary complexes, will also be employed. [unreadable] [unreadable] Public Health Relevance: Although first analyzed in the context of anterior-posterior patterning, there are a wealth of critical functions in animal development from motor neuron specification to organogenesis to stem cell maintenance that are now appreciated to be controlled by Hox genes. Equally important and well studied are the roles that Hox genes and their cofactors play in human birth defects, such as limb malformations, and some cancers, such as leukemia. Thus, a mechanistic understanding of how these transcription factors regulate their target genes will impact our understanding of many aspects of developmental and disease biology. [unreadable] [unreadable] [unreadable]

During animal development, the various mechanisms that determine cell fates must be well coordinated. For example, long-range diffusible signaling molecules, short-range cell surface signaling molecules, and nuclear transcription factors must all function together as an integrated system. Further, these mechanisms must also operate in the context of a growing and continually changing tissue. The long-term goal of this project is to understand, in molecular terms, how such different mechanisms are integrated with each other, and with developmental time. The control of developmental pathways by the Hox genes provides an example of this problem. The Hox genes all encode DNA-binding homeodomain proteins that regulate the transcription of specific sets of downstream, target genes. Hox proteins, however, do not act alone: they bind DNA together with co-factor that increase both their binding site specificity and affinity. One well characterized family of Hox co-factors are encoded by the Drosophila extradenticle and vertebrate pbx genes. Unlike most homeodomain proteins, the Extradenticle protein is post-translationally regulated by controlling its cytoplasmic-to-nuclear translocation. Another homeodomain protein, encoded by the Drosophila homothorax gene, is required for Extradenticle's nuclear localization. In some cells, the nuclear localization of Extradenticle is controlled by diffusible signaling molecules, which, therefore, indirectly control Hox function. The work proposed is divided into four parts: the first investigates how Homothorax controls Extradenticle's nuclear localization; the second investigates Homothorax's role as a DNA-binding co-factor for the Hox proteins; and the third investigates a role for homothorax as a selector gene for antennal development. The four part proposes to use a genetic screen to identify genes that modulate Homothorax function. Parts one and two use cell biology to investigate the mechanism of Exd nuclear localization, and biochemistry to characterize critical protein-protein and protein-DNA interactions. The third and fourth parts use Drosophila genetics to investigate these problems. Characterizing these developmental mechanisms will shed light on how the vertebrate versions of these proteins, when altered, can cause cancer and human birth defects.

DESCRIPTION: (Applicant's Description) The long-term goal of this project is to characterize the genetic pathways controlled by dominantly acting oncogenes. To carry out these studies the model organism, Drosophila melanogaster, will be used because of the ability to carry out powerful genetic screens. The AML-ETO oncogene, which is responsible for a significant fraction of Acute Myeloid Leukemias, will be the focus of these studies. The immediate specific aims are to: 1) characterize the wild type function of the ETO ortholog in Drosophila, called nervy; 2) to characterize the gain-of-function phenotypes that are induced in Drosophila by expressing Nervy and AML-ETO proteins. In addition, the activities of chimeric proteins that are analogous to AML-ETO, but are constructed from the orthologous Drosophila genes, will also be characterized. 3) Once these data are in hand, genetic screens will be conducted in Drosophila to identify genes that interact with Nervy and AML-ETO in vivo. These screens will be identify components of the genetic pathways in which Nervy and AML-ETO function. Once additional components of these pathways have been identified, their relevance to leukemogenesis will be tested. If confirmed, these genes will provide additional targets for the development of anti-cancer drugs and other potentially novel therapies. Moreover, if successful, this approach may set a precedent for analyzing the genetic pathways in which other dominantly acting oncogenes function. The use of Drosophila genetics is proposed to help make connections between genetic functions that cannot be easily identified in mammalian experimental systems.

DESCRIPTION (provided by applicant): This project addresses fundamental questions in developmental biology using the developing leg of Drosophila melanogaster as the model system. In addition to having antero-posterior (AP) and dorso- ventral (DV) axes, the mature leg, as all appendages, also has a proximo-distal (PD) axis. Unlike the AP and DV axes, the PD axis must form de novo, within each thoracic hemisegment of a developing embryo. In the fly, the leg primordia are first identifiable as a cluster of ~30 cells that express the homeodomain-encoding Distalless (Dll) gene. By the end of larval development, the presumptive leg has ~20,000 cells and several defined domains along the PD axis. The goal of this project is understand how these cells obtain their positional information. Generally, a 'bottom-up' approach is used, in which cis-regulatory modules (CRMs) that drive the expression of PD genes are dissected in detail, to identify the upstream inputs that control their activities. In ths way, a network of CRM and gene activities will be generated. In the next funding period, particular attention will be given to 1) the most proximal PD domain, which co-expresses homothorax (hth), which encodes a homeodomain transcription factor, and teashirt (tsh), which encodes a Zn-finger nuclear factor and 2) the most distal PD domain, which is further elaborated due to the activities of the Epidermal Growth Factor Receptor pathway. In addition, the role of two 'ventral-identity' factors, encoded by the Drosophila homologs of vertebrate Sp8, dSp1 and buttonhead (btd), will be investigated, using both a CRM-centric and target gene approaches. Altogether, these experiments will lead to a better understanding of how positional information is generated in dividing cells, using transcription factors and signaling pathways that are conserved from flies to man.

[unreadable] DESCRIPTION (provided by applicant): The goal of this collaborative project is to identify transcriptional regulatory elements in the Drosophila melanogaster genome that are directly regulated by the Hox family of homeodomain proteins. To accomplish this goal, three experimental steps will be carried out. In the first, state-of-the art gene expression profiling methods will be used to analyze gene expression differences in embryos containing Hox gene mutations. The genotypes that will be used for gene profiling analysis incorporate redundant pain/vise comparisons to increase the probability that bona fide Hox-regulated genes will be identified. The second step will be to use one of two methods -- DamID or chromatin immunoprecipitation (ChIP) - to determine which genes in the Drosophila genome are directly bound by Hox proteins in vivo. Combined with the first step, these experiments will allow the identification of direct Hox target genes on a genome-wide scale. The third step in this analysis will be to use computational approaches to identify cis-regulatory elements that are shared between subsets of the directly regulated Hox target genes. Computationally-identified regulatory elements will be confirmed experimentally, both by in vitro protein-DNA binding experiments and by in vivo reporter gene analysis. Once these methods are worked out, they will be applicable to a wide variety of problems in biology. [unreadable] [unreadable]

The development of multicellular animals requires the coordinated activities of cell-cell signaling pathways and cell-type specific transcription factors. In this project, how these inputs are orchestrated during the development of animal appendages will be investigated. Using Drosophila melanogaster as the model system, the focus is how two signaling pathways, Wingless (Wg) and the BMP homolog Decapentaplegic (Dpp), create the proximo-distal (PD) axis of the leg. Previous work established that Wg and Dpp combine to activate the transcription of target genes at discreet positions along the PD axis. However, how these signals translate to transcriptional activation is not understood. Using reporter gene and DNA binding analyses, how PD genes are activated along the PD axis will be investigated. Second, a novel method to analyze the chromatin structure of PD gene regulatory sequences will be employed. Third, the role that cellular proliferation dynamics play in PD gene regulation will be investigated. These studies will impact public health in several ways. First, the mechanism by which secreted signals activate gene expression is common to many aspects of animal development and disease. Thus, a better understanding of these basic mechanisms is critical for deciphering how these pathways, when altered, contribute to diseases such as cancer. Second, many human birth defects are due to problems in the development of the appendages. As many of the transcription factors, signaling molecules, and underlying mechanisms controlling appendage development are conserved between Drosophila and humans, it is likely that a more comprehensive dissection of these processes in an experimentally powerful system such as Drosophila will provide important insights into these human birth defects.

PROJECT SUMMARY The morphological diversity among neurons is enormous and critical to their function, yet many gaps in our knowledge remain concerning how this diversity is genetically encoded. As with vertebrate motor neurons, the motor neurons of Drosophila melanogaster establish highly specific synapses on the correct muscle fibers in the appendages and also elaborate highly stereotyped dendritic arbors in the central nervous system (CNS). In the previous funding period, we characterized the individual progeny and transcription factor codes that govern neuronal identities for one of the largest neuroblast lineages that give rise to motor neurons and neuropil glia. We further described the role of Ig domain family members in establishing certain aspects of motor neuron identities. Live imaging provided unprecedented access to the relevant stages of motor neuron maturation during pupal stages. In the coming funding period we will extend these findings to understand how transcription factors in post- mitotic neurons regulate their target genes. We will carry out a series of whole genome and single cell studies to obtain this information on a genome-wide scale. Finally, we will analyze additional members of the Ig domain superfamily to assess what other roles, besides axon targeting, they play in motor neuron morphology during development.

DESCRIPTION (provided by applicant): Locomotion in all animal species relies on precise coordination: animals must synchronize a myriad of muscle flexion and extension events in a stereotyped and rhythmic manner. At the core of motor coordination are central pattern generators (CPGs), neural circuits that have the capacity to produce rhythmic outputs from relatively simple, non-rhythmic inputs. Although there exists a large amount of functional evidence for both locomotor and non-locomotor CPGs, the cellular components of CPGs that mediate coordinated locomotion in more complex systems remain largely undefined. Further, how locomotor CPG activities are integrated with each other and modified by descending and sensory inputs is also largely unknown at the cellular level. These gaps in our knowledge may be due not only to the complexity of the neural circuitry, but also a consequence of the complexity of the behaviors under investigation. To address these challenges, the long-term goal for this project is to complement and expand upon existing efforts in other systems to characterize locomotor neural circuits using the powerful genetic tools available in the fruit fly, Drosophila melanogaster. In this proposal, a novel, high-resolution assay that quantitatively measures dozens of walking parameters in the fruit fly model will be used to screen for specific mutant phenotypes that occur as a consequence of activating and/or suppressing neural activity in subsets of neurons. Follow-up experiments are proposed to identify the neurons that are responsible for these mutant phenotypes.

? DESCRIPTION (provided by applicant): The goal of the project team is to develop a robust, multi-lab research framework, enabled by large scale imaging, which will lead to principled integrative models of ethologically-relevant behaviors that incorporate a detailed knowledge of individual cell classes. The specific neurobiological question that the team will address is how the brain integrates sensory information in order to guide locomotion in a particular direction. Our strategy is to systematically map and functionally characterize the neural circuits that underlie goal-directed locomotion, using the fruit fly, Drosophila, in order to exploit the convergence of powerful genetic, optical, behavioral, and analytical tools that are available in this species. The proposal focuses primarily on refining functional imaging approaches to map the activity of small brain regions and populations of individual neurons in intact, behaving animals while they respond to a controlled panel of sensory stimuli. We have constructed a strategic plan consisting of seven interrelated research modules that create a flow for discovery that starts with functional imaging and ends with the development of integrative models for sensory-guided behavior. The goal of this proposal is to bring all research modules to the requisite level of maturity for future research. To achieve this goal this project will develop robust, quantitative and high throughput methods for: Functional 2-photon imaging using pan-neural drivers. ArcLight imaging using selected driver lines. Functional 2-photon imaging using pan-neural drivers. Circuit analysis of sensory motor pathways. And a plan for an integrative computational model of sensory-guided locomotion.

The common theme underlying the questions being addressed in this proposal is how gene expression is controlled by cis-regulatory modules (CRMs) and the transcription factors that they bind during animal development. Answers to this question are becoming increasingly important in the emerging era of Personalized Medicine, since we now have the ability to sequence thousands of genomes and to identify changes in DNA sequences that correlate with diseases ranging from epilepsy to obesity. However, when changes in DNA sequence map to the non-protein coding portion of the genome, we are nearly helpless in interpreting these changes. This project will help bridge this gap in our knowledge by establishing methods to assess how the Hox family of transcription factors function in vivo. In the next several years, one goal is to extend what has been learned about Hox transcription factor specificity to a more in vivo level, in particular, to break the sequence code that allows these TFs to select biologically relevant binding sites in vivo. A second major goal is to understand at a deeper level how multiple transcription factors and CRMs coordinate with each other to regulate genes in the right cells and at the correct developmental time. For this last set of experiments, the focus is on how the proximo-distal axis of the Drosophila leg is established during development.

A Brain Circuit Program for Understanding the Sensorimotor Basis of Behavior Abstract The Project team's long-term goal is to develop a comprehensive theory of animal behavior that explicitly incorporates neural processes operating across hierarchical levels ? from circuits that regulate the action of individual muscles to those that regulate behavioral sequences and decisions. Our innovative approach is guided by the notion that different brain regions are not linked within a single neuroanatomical tier, but rather constitute a series of hierarchically nested feedback loops. The effort is organized into four Research Projects, each focusing on a different processing stage related to: (1) muscle action, (2) motor patterns, (3) motion guidance, and (4) behavioral sequences. Demonstrating our commitment to team interaction, these Research Projects are not organized according to PIs laboratories, but rather each constitutes a collaborative multi- laboratory effort. The collective expertise of our research team spans the entire nervous system - from the sensory periphery to the motor periphery and was chosen to include experts in every experimental technique we require (molecular genetics, electrophysiology, optical imaging, biomechanics, quantitative behavioral analysis, control theory, and dynamic network theory). We will exploit mathematical approaches ? control theory and dynamic network theory in particular ? that are best suited to model feedback and the flow of information through and among different processing stages in the brain. The four complimentary and integrated Research Projects will focus on ethologically relevant natural behaviors, with an emphasis on recording methods that interrogate the functions of genetically identified neurons in intact, behaving animals ? a rigorous standard that is designed to have the broadest impact on systems neuroscience. Our research exploits a single, experimentally tractable model system (Drosophila melanogaster), in which we can easily study the functions of genetically identified cell classes in ethologically relevant behaviors. Our experiments emphasize methods that interrogate the functions of neurons in intact, behaving animals, a rigorous standard that is designed to have the broadest impact on systems neuroscience. Our research will be supported by an Instrumentation and Software Resource Core that will develop and support novel devices and software, so that we can continue to employ state-of-the-art experimental techniques and data analysis. Collectively, our research program constitutes a systematic attack on the neural basis of behavior that integrates vertically across phenomenological tiers. The result of our effort will be a new synthesis of how a fully embodied brain works to generate behavior.  

Project 2: The Neural Basis of Motor Pattern Loops Abstract The goal of Project 2 is to understand how motor pattern loops link high-level behavioral goals with low-level control of muscles. In the fly, such motor pattern loops are comprised of populations of descending neurons (DNs), which integrate dendritic signals in the central brain and project their axons to the ventral nerve cord (VNC). We will take advantage of cell-type specific Split-Gal4 lines to record and manipulate activity in DNs that target VNC circuits that control the fly legs and wings. We will combine optogenetic manipulation of DNs with fine-scale analysis of walking and flight behavior, and measure intracellular DN signals in behaving animals with whole whole-cell electrophysiology recordings. These efforts are divided into the following Specific Aims: Specific Aim 1: Determine how wing muscle action groups are recruited into functional actions by descending control. Specific Aim 2: Determine how leg muscle action groups are recruited into functional actions by descending control. Specific Aim 3: Characterize the activity patterns of identified descending neurons in behaving flies using in vivo whole-cell recordings.