Volker Hartenstein - US grants

Our long term objectives are to understand the genetic control of cell differentiation in the Drosophila nervous system. The general strategy is to focus on one early differentiative step in Drosophila neural development, the segregation of sensory neurons. During segregation, postmitotic sensory neurons constrict apically and move out of the epidermal layer in which they were born. Using a developmental and molecular-genetic approach, we want to characterize genes which have an impact on this process. The specific objectives of this proposal are (1) to analyze the cellular mechanisms which underly sensory neuron segregation in wildtype development, and (2) to study the faint sausage (fas) gene which appears to be essential for neuronal segregation, as sensory neurons fail to segregate in homozygous fas- embryos. With regard to the first objective the following experiments are proposed: By blocking the function of microtubules, microfilaments, and cell adhesion (Ca++-dependent; integrin mediated) in an in vivo system it will be tested whether these functions are essential for sensory neuron segregation. It will further be asked whether mutations in genes implied in cell adhesion mechanisms (mys, 1(2)gl, fat) affect sensory neuron development. The study of the fas gene involves the analysis of ultrastructural defects in sensory neuron segregation in fas-embryos, fas-embryos derived from homozygous fas-eggs (germline clones), and clones of homozygous fas-cells in the adult epidermis. Birthdates and lineage relationships of the sensillum cells in fas-embryos will be determined. The fas locus will be mapped by recombination and deficiency mapping. The fas gene will be cloned and characterized by sequence analysis, in situ hybridization studies, and antibodies raised against the fas gene product. As fas appears to encode an essential factor for sensory neuron segregation, the molecular analysis will provide insight into the genetic control of this process.

Our long term objectives are to understand the genetic control of cell differentiation in the Drosophila nervous system. The general strategy is to focus on one early differentiative step in Drosophila neural development, the segregation of sensory neurons. During segregation, postmitotic sensory neurons constrict apically and move out of the epidermal layer in which they were born. Using a developmental and molecular-genetic approach, we want to characterize genes which have an impact on this process. The specific objectives of this proposal are (1) to analyze the cellular mechanisms which underly sensory neuron segregation in wildtype development, and (2) to study the faint sausage (fas) gene which appears to be essential for neuronal segregation, as sensory neurons fail to segregate in homozygous fas- embryos. With regard to the first objective the following experiments are proposed: By blocking the function of microtubules, microfilaments, and cell adhesion (Ca++-dependent; integrin mediated) in an in vivo system it will be tested whether these functions are essential for sensory neuron segregation. It will further be asked whether mutations in genes implied in cell adhesion mechanisms (mys, 1(2)gl, fat) affect sensory neuron development. The study of the fas gene involves the analysis of ultrastructural defects in sensory neuron segregation in fas-embryos, fas-embryos derived from homozygous fas-eggs (germline clones), and clones of homozygous fas-cells in the adult epidermis. Birthdates and lineage relationships of the sensillum cells in fas-embryos will be determined. The fas locus will be mapped by recombination and deficiency mapping. The fas gene will be cloned and characterized by sequence analysis, in situ hybridization studies, and antibodies raised against the fas gene product. As fas appears to encode an essential factor for sensory neuron segregation, the molecular analysis will provide insight into the genetic control of this process.

The earliest phase of nervous system development is characterized by large scale movements of cells. First, these cells must separate from the outer layer of the early embryo; secondly, they must obtain specific positions within the embryo. To reach these positions, the cells in many cases must migrate considerable distances. Upon reaching their proper location, they become stationary and form stable contacts with each other. It is of great importance to understand on a molecular level how these cellular processes are controlled. This project will use the embryonic optic lobe as a model system to analyze the molecular basis of morphogenetic movements during neuronal development. Using a variety of markers, the position and movement of these cells can be readily followed throughout normal and mutant development. Experiments will focus on the phenotypic and molecular characterization of a gene called sine oculis (so), which is necessary for the development of the optic lobe. Mutations of so cause failure of optic lobe invagination. Most recent analyses show that so encodes a putative transcription factor, that is a protein that is capable of turning on or off the expression of other genes. It is anticipated that the analysis of so and its function during the development of the optic lobe will answer many important questions.

PI: Hartenstein, Volker
IBN 0110715

Abstract:

This project analyzes the partitioning of the eye field in the Drosophila embryo. The eye field in the anterior neural plate of vertebrates and the head of the Drosophila embryo exhibit a high degree of similarity regarding the fate map of the visual system, and the signaling pathways controlling this fatemap. Drosophila counterparts of Shh (Hh), BMB4 (Dpp) and EGF, as well as Pax6, Six6 and many other regulatory genes all function in the Drosophila embryonic head. Preliminary data show that Dpp is secreted in the dorsal midline and forms a dorso-ventral gradient that specifies the different domains within the eye field. Dpp is then down-regulated in the eye field, except for its posterior boundary from where it may form a posterior-anterior gradient that partitions the visual primordium into larval and adult eye, and optic lobe. Hh is secreted at the lateral boundary of the eye field and may form a gradient that antagonizes the early Dpp gradient.
Dr. Hartenstein and his research group will address the topology and morphogenesis of the Drosophila eye field and test their models of Dpp and Hh function in partitioning the eye field. The first aim is to reconstruct the details of the fate map, and to investigate the morphogenesis of the visual system. The second aim addresses the function of dpp in the eye field by investigating the dpp loss of function, heterotopic and heterochronic expression of dpp, activated Dpp receptor, and dominant negative Dpp receptor. In the third aim Dr. Hartenstein will analyze the role of Hh and its interaction with Dpp. The hypothesis will be tested that Hh negatively interacts with Dpp function. This would represent an interesting parallel to the vertebrate neural tube where ventral release of Shh also antagonizes BMPs secreted from dorsal tissue. By generating Dpp:hh double mutants and expressing mutant constructs the interaction of these genes in the embryonic head will be tested. In aim four Dr. Hartenstein will address the control of Dpp activity in the embryonic head. By establishing specific differences and similarities between early eye patterning in Drosophila and vertebrates, Dr. Hartenstein anticipates to furnish relevant information that helps interpreting Shh and BMP function in neural fate determination and to provide important insights in the evolution of the brain.

The visual system of the fruitfly Drosophila has proven to be an excellent model to identify conserved genes that control how the visual system of an animal is formed. Much has been discovered about the photoreceptors and the structure of the Drosophila eye. In contrast, the central (brain) neurons that process visual information have received less attention. This grant will allow the Hartenstein lab to investigate the structure and development of the simple central visual neuropile (the circuit, made by axons, dendrites and synapses) of the Drosophila larval brain. Experiments in aim #1 (anatomical circuitry) will address the pattern of neuronal connections in the visual neuropile and map the neurons that send axons from the brain to the ventral nerve cord. Aim 2 (function) addresses how larval behavior is influenced by light stimuli. A functional assay (head turning away from a local light source) will be used to analyze quantitatively the effect of directed light on movement. Aim 3 (development) is based on previous findings indicating that many elements of the central visual system are derived from the same embryonic field that also gives rise to the eye. This eye field expresses a network of early eye genes (called eyeless, eyes absent, sine oculis, and dachshund) whose function is well established for the eye. Dr Hartenstein and his colleagues will investigate the expression and function of the early eye genes in the developing central visual system. Aim 4 (gene discovery) will use a novel "gene trap" method to screen for genes that are expressed only in the visual neuropile. The neurons expressing these genes will be characterized. A long-term goal of the research is the characterization of genes that are preferentially expressed in the visual neuropile.

Broader Impact: Genetic screens and phenotypic analyses in Drosophila have revealed many aspects of developmental neurobiology. The findings resulting from this research are likely to provide insight into a highly conserved gene network, the early eye genes, that plays multiple roles in vertebrate development.

At the same time, this research program serves as a vehicle for undergraduate teaching. The PI is responsible for a large undergraduate class, Developmental Biology, taken yearly by 150-200 UCLA undergraduates. Many undergraduates (between 6 and 10 per year) rotate through the lab and conduct short (1-3 quarters) research projects. These students are teamed up with postdocs or graduate students and gain practical research experience. The preliminary work leading to several of the proposed aims was conducted together with undergraduate students. Among the undergraduates is a sizeable number of minority students. UCLA has a special program (CARE) that awards short term fellowships for minority students to conduct research. The PI's lab currently hosts one CARE student, Luis Garcia, who visualized the photoreceptor terminals in the larval optic neuropile (aim #1).

Digital 3D models play an increasingly important role in neuroscience. Representing three-dimensional scaffolds in which functional data and gene expression data are entered and displayed graphically, the digital models become analytical tools that allow one to address neural connectivity and function, as well as gene function and gene interactions. This grant application proposes to generate a series of standardized digital atlas models of the developing Drosophila brain, a system used by many to investigate the genetic mechanism controlling the formation and function of neuronal circuits. The fly brain is formed by an invariant set of neuroblast lineages which represent structural units in terms of cell body location, axonal projection, and (to an extent that will be addressed in this proposal) connectivity. Axonal and dendritic arborizations, establish morphologically distinct neuropile compartments that are visible from the late embryo towards the adult. Compartments and lineages form a stereotyped pattern that will be captured in the proposed digital models. Having in mind their usefulness for us and others, as well as feasibility, the following models are proposed: (1) early embryonic neuroblast map, (2) late embryonic primary lineages in relation to evolving neuropile, (3) late larval secondary lineages in relation to neuropile compartments, (4) evolving secondary tract systems and neuropile compartments of the pupa. These models represent an integrated series because the "genetic address" of each neuroblast, defined by the known sets of genes expressed in the early embryonic neuroblast map, will be linked to the population of neurons and their axons modeled for the late embryo, larva and pupa. The goal of this modeling project is to provide a tool shared with the community, allowing to exploit the Drosphila brain more efficiently for developmental-genetic and functional questions. Models of neural lineages will make it possible to phrase specific experiments and to interpret mutant phenotypes.

DESCRIPTION (provided by applicant): Brains contain large number of neurons whose connections, formed by axonal and dendritic processes, are the structural underpinning of electrical circuits that control behavior. The analysis of circuits is of great importance. All acts of fine motor control, memory formation and cognition can only be understood if the circuitry within the brain compartments dealing with these functions is known. Likewise, the insight into psychiatric disease mechanisms and their pharmacological treatment requires brain circuitry to be known in detail. For example, recent findings suggest that diseases like autism or schizophrenia can be understood in terms of abnormalities in the micro- circuitry of the prefrontal cortex. We propose in this grant to develop and utilize bioinformatics tools that enable us to address circuitry in the Drosophila brain. The Drosophila central brain is formed by a stereotyped set of approximately 100 paired lineages, each one derived from one neuroblast. Neurons of one lineage form processes that spread within discrete compartments of the brain. Lineages thereby represent the most appropriate structural/developmental units of brain macro-circuitry. Reconstructing the projection of all lineages means to have generated an accurate map of Drosophila brain circuitry at the level of neuron populations (macro-circuitry). We propose to generate this map, presented in a standardized electronic format that is accessible to the neurobiology community. In addition, we will reconstruct circuitry at the level of individual synapses (micro-circuitry), which requires electron microscopy (EM). We have developed the software required for the automated recording, registration and navigation of large EM image data sets. We will further improve and use these tools to generate a digital EM dataset that, for the first time, encompasses the entire brain of an animal with a sizeable number of structurally complex neurons. Our software allows us to efficiently reconstruct the neural networks encountered in different parts of the brain. We anticipate that we will be able to learn structural principles about neural network that have general application.

Most adult organs contain populations of slowly cycling, undifferentiated stem cells. Aside from maintaining their own numbers, stem cells produce progeny that differentiate into the various cell types of the organ they are located in. Adult stem cells are under intensive study with the goal in mind to use these cells in regenerative medicine. Furthermore, it has become clear that neoplastic growth typically takes off from stem cells or their proliferating progeny. One important population of adult stem cells are the epithelial stem cells of the intestine. We recently found in the Drosophila adult hindgut a stem cell system that shows a high degree of similarity to the mammalian intestinal stem cells, but at the same time is considerably simpler to analyze genetically and developmentally. Drosophila hindgut stem cells are confined to a short segment of the hindgut, the hindgut proliferation zone (HPZ). Within the HPZ, self renewal, proliferation and differentiation is controlled by local sources of four signaling pathways, the Wnt/Wingless, Hedgehog, Notch and Jak/Stat pathway. In the proposed project we will address some fundamental properties of the HPZ: what are the functional relationships between these pathways, and what aspect of stem cell behavior (cell cycle? adhesion? migration? pluripotency?) does each of them control? Furthermore, the rapid development and relative simplicity of the Drosophila intestinal tract gives us the opportunity to investigate when and how the HPZ with its special signaling properties during development. Finally, we want to screen for novel genes involved in adult stem cell proliferation. Our overall goal is to advance the knowledge of the fly HPZ system to a degree that makes it possible to formulate additional, specific and readily translatable research programs, using the fly hindgut stem cells as a model system.

? DESCRIPTION (provided by applicant): Brain function is based upon the precise connectivity of a large number of neurons. Connectivity in turn depends in large part on the genetically determined wiring properties of neurons, including their neurite projection, branching, and placement of synaptic contacts with specific partners. To understand and manipulate brain circuits one needs a detailed knowledge of how the genes expressed in a developing neuron control the wiring properties of this cell. For genetic studies, Drosophila offers many advantages, in that virtually every gene can be targeted for knock-out or activation in a cell type selective manner. More importantly in the context of studying neuronal circuitry, the Drosophila brain is composed of a manageable number of stereotyped neuronal lineages, groups of neurons descended from individual stem cells (neuroblasts) born in the embryo. During the course of its proliferation, each neuroblast expresses characteristic sets of genes (transcription factors) which are thought to specify the wiring properties of the neurons born from that particular neuroblast during a particular time interval. These neurons form a so called sublineage. To learn about the genetic control of brain circuitry we and others have taken the approach to document the structural properties of lineages and sublineages, and correlate them to the dynamic pattern of gene expression in the neuroblast. During the previous funding period we have generated detailed maps and 3D models of all lineages constituting the adult and larval brain. We here propose three aims that continue and extend this work. First, we will reconstruct the connectivity of a subset of larval brain lineages and their sublineages that form a particular, well characterized circuit. This reconstruction will be done at a so far unparalleled level of resolution, using a series of several thousand contiguous electron microscopic sections in conjunction with a specially developed software package that allows us to assign all synapses to specific neurons and their lineages. Secondly, we will link the structurally defined lineages mapped in the larval brain with the neuroblasts of the embryo, using a technique that systematically labels all transcription factors expressed in neuroblasts and then follows the expression of these genes from neuroblast to lineage. Thirdly, we will screen for and genetically characterize genes that play a role in directing lineages to their proper place in a circuit.

? DESCRIPTION (provided by applicant): The studies of this application ask how gene expression controls neuronal connectivity and, thereby, brain function. This question is of general importance if one wants to understand, and (therapeutically) manipulate brain circuitry. We use the model system Drosophila, where virtually every gene can be targeted for knock-out or activation in a cell type selective manner Drosophila also offers the advantage that its brain i composed of a relatively small number of stereotyped neuronal lineages, groups of neurons descended from individual embryonic stem cells, called neuroblasts. During the course of its proliferation, each neuroblast expresses characteristic sets of regulatory genes. These genes control the differentiation of the neurons born from that particular neuroblast during a particular time interval. Through this mechanism, a lineage, or smaller subdivision of a lineage called sublineage, develops into a specific class of neurons which share common wiring properties, including the projection of their axons, branching pattern, and placement of synapses. Several discrete neuronal classes/lineages are put together into a neuronal circuit. We have identified a circuit, called the anterior visual pathway (AVP), which conducts input form the eye to a brain center, the central complex, known to process and store visual information in order to control fly locomotion (walking, flight). The central part of this circuit is formed by three lineages, whose neurons form several classes of highly ordered parallel and sequential elements. In our first aim we will investigate the function of the neuronal classes of the AVP, by recording their activity in response to defined visual stimuli. We will also demonstrate experimentally that these classes of neurons are directly connected by synapses. The second aim addresses the question how the developmental history of a neuron (time of birth, placement within the spatial framework of the developing brain) relates to its later connectivity within the AVP circuit. Furthermore, by genetically ablating specific classes of AVP neurons and monitoring the response of their normal synaptic partners, we will obtain important clues towards the role of specific cell interactions ordering connectivity. Thirdly, using high throughput RNAseq, we will analyze the complete assortment of genes (transcriptome) expressed differentially in two particular AVP sublineages, R3 and R2. These two classes are distinguished from each other by very few structural criteria, and we will screen for and then analyze genes responsible for their differences in wiring. We expect to identify genes which play a general role in controlling pathway choices and connectivity in the nervous system.

Summary One of the most pressing research goals in neurobiology is to understand how brain circuits develop, and how these circuits control the behavior of an animal. This problem is of general importance if one wants to understand, and (therapeutically) manipulate, brain circuitry in a medical setting. A prerequisite to attain this goal is (1) the detailed mapping of complete neuron assemblies that embody specific circuits, and (2) the availability of precision tools for functional studies. Both of these conditions are now met for Drosophila. Complete connectomes (digital maps that contain all brain neurons and their synaptic connections) exist for both the larval stage (funded in part by this grant in previous years) and the adult. And in addition, genetic tools have been developed that allow one to manipulate (that is, silence, or activate) virtually every neuron, or at least neuron class, and test for the effect on specific behaviors that one is interested in. The strategy then is to extract from the connectome a wiring diagram of a specific circuit, develop hypotheses of how the different elements in the circuit interact, and use genetic tools to test these hypotheses. Studies of this proposal focus on a Drosophila brain circuit involved in navigation. Animals navigate in response to sensory stimuli in order to find food and mating partners, or avoid danger. Brain centers controlling navigation require processed, multimodal sensory input (smells, visual cues) which are integrated with proprioceptive input (feed back from muscles, joints etc) to calculate the commands required to steer the animal in the right direction. Our analysis of the larval connectome highlights a brain center called the lateral accessory lobe (LAL) as a focus of interest. We have identified the relevant LAL neuron classes and their connections, and are in the process to systematically screen for genetic constructs with which we can target these neuron classes to do functional studies. Larvae have a simple, highly quantifiable navigation behavior that allows them to find a food source (by odor) or avoid light. We will analyze how the LAL controls motor circuits that carry out this behavior. The second objective of the proposal is to study how the larval LAL neurons become modified and incorporated in the LAL of the adult. Adult flies have a new set of organs (e.g., wings, legs) with which to move, and receptors with which to sense; but according to our initial data, the larval neurons remain and have to adapt to cope with their new input and output. Using the connectome of the adult brain and our genetic tools we intend to identify the descendants of larval neurons in the LAL, and to address their function in adult navigation.