Eduardo R. Macagno - US grants

The long-range goal of this work is to understand certain fundamental mechanisms of development, particularly regarding neurogenesis, differentiation and selective synapse formation. This goal is addressed by studying how individual regions of the central nervous system, and nerve cells within those regions, become morphologically and physiologically differentiated from one another during development in order to perform specialized functions. The segmented nervous system of the medicinal leech was chosen for these experiments because of its great simplicity, the possibility of studying individual identified neurons repeatedly, and the ready accessibility of embryonic and postembryonic stages to experimental manipulation. There are two parts to this project. The first part concerns the very basic question of how the generation of central neurons may be regulated by peripheral organs. It comprises the study of an interaction between the male genitalia and the two central ganglia that innervate it. This interaction is mediated by an inductive signal that triggers the birth and differentiation of a special population of neurons in these ganglia. The experiments proposed are designed to yield answers to the questions (1) when exactly does the interaction take place?, (2) is the time of interaction defined by the nervous system or by the target tissue?, and (3) which cells are the source of the inductive signal, which carry it to the central nervous system, and which cells respond to the signal? Identification of the cellular elements involved in this interaction is a necessary prerequisite for the future exploration of the molecular mechanisms of this interaction. The second part of this project addresses another basic question: how selectivity in the formation of synaptic connections is achieved. It consists of a comprehensive examination of the interactions between an identified motor neuron and peripheral tissues that result in the formation of neuromuscular junctions with the neuron's target, the male genitalia. These experiments are carried out, in a complementary manner, both in the animal and in isolated cells in culture. A multi-faceted approach, involving embryological perturbations and electrophysiological, anatomical and immunocytological techniques, will be employed in this project. Further detailed study of these fundamental problems in the simple nervous system of the medicinal leech will provide valuable insights applicable to the study of both normal and abnormal development of more complex systems.

The long-term objective of this project is the elucidation of the mechanisms that guide neurons through the process of selecting particular synaptic targets and control the features of these connections. Our previous studies of the visual system of Daphnia magna have centered on the selection process. Our specific aim for the period of this proposal is to examine, in quantitative detail, the structural features of synaptic connections between specific neurons in order to answer several important questions: To what degree are the fine structural features of the synaptic connections between identified neurons the same among animals with the same genetic background and raised under identical conditions? How are these features affected by changing environmental conditions? How do they change with the age of the animal? Does variation or constancy of synaptic features correlate with variation or constancy of behavior? Daphnia are simple enough to permit the detailed quantitative studies needed for answering these questions, yet complex enough to have sophisticated optomotor behaviors. Our approach is multidisciplinary, employing anatomical, behavioral and physiological techniques. The quantitative anatomical measurements will be carried out using the techniques for computer-aided reconstruction of nerve cells from serial electron micrographs which we have developed. Properties we will measure include number of synaptic sites, their area and distribution. In the behavioral studies we will use video recording to measure eye movements produced by various patterns of visual stimulation. The physiological studies will include recording from photoreceptors and interneurons involved in the optomotor behaviors. We expect to obtain answers to basic questions about the structure, function and development of synaptic connections which will contribute significantly to the understanding of how abnormalities arise in the structure and function of the nervous system.

Funds are requested for the purchase of a STEM/SEM attachment for a JEOL JEM-1200EX electron microscope and for a computer system to be coupled to the electron microscope for use in direct image analysis. The Computer-Coupled Electron Microscope will be used by several investigators in the Columbia University Department of Biological Sciences whose scientific interest lie primarily in the areas of Cellular and Developmental Neurobiology. The projects that will require the use of this instrument include studies of: (1) the molecular structure of acetylcholine receptors, (2) the ultrastructure of developing rat neuromuscular junctions, (3) the effects of specific mutations on the ultrastructure of sensory cells in the nematode Caenorhabditis elegans, (4) the action of androgens in the sexual differentiation of neurons and muscles in Xenopus, and (5) the synaptic circuitry underlying processing of sensory information in arthropods and fish. An important aspect of this work will be the computer-aided analysis of serial electron images. The 3-D reconstruction of neurons and their synaptic connections will be carried out using existing hardware and software developed for this purpose in the Department's Computer Graphics Facility.

It has been recently demonstrated that there is direct DNA sequence homology between several Drosophila homeotic genes. This region has been called the homeo box and has been shown to code for a conserved, about 60 amino acid residue sequence. Cross-hybridization of genomic DNA from various other species with probes containing the Drosophila homeo box has suggested that regions homologous to the homeo box are evolutionarily conserved. Cross-hybridization has also been useful in the isolation of some of the genes that contain this region in frogs, mice and humans. Because the Drosophila genes that contain the homeo box affect developmental processes, in particular segmentation and segmental differentiation, it has been proposed that the homeo box could also be a marker for such genes in other species. A group of animals in which a test of this idea would be particularly appropriate are leeches. Leeches, like all other annelids and like the insects, display an obvious segmental organization of their bodies. Current evolutionary theory has the insects descending from an annelid ancestor, suggesting the possibility that the insect homeotic genes have evolved from corresponding annelid genes. Furthermore, our preliminary observations indicate that leeches also have genes that contain regions which cross-hybridize with the fly homeo box. The aim of the project proposed here is to identify and isolate such genes in a few species of leeches, and to determine whether their spatial and temporal patterns of expression are similar to those of the Drosophila genes that contain the homeo box. We will pursue this goal by first determining the approximate number of homeo box cross-hybridizing regions in the leech by Southern blot analysis. We will then determine the degree of homology with the homeo box by sequencing the regions of leech DNA of interest and will characterize their transcripts by Northern analysis. Finally, we will do in situ hybridization on serial tissue sections of leech embryos to assess the spatial and temporal patterns of expression of the identified leech genes. In particular, we will study the expression of these genes at the level of individual, identified neurons in the leech CNS, to determine whether differential gene expression correlates with the segmental differentiation of neuronal phenotypes that we have characterized. The results of this investigation should have a significant bearing on our understanding of the evolution of genes that control early development and of the role of such genes in the genesis of phenotypic heterogeneity in the CNS.

Neuromuscular junctions in newborn rats differ from adult junctions in several ways. First, the postsynaptic membrane is smooth in neonates but has extensive secondary folds in adults. Second, subneural acetylcholine receptors (AChR's) have a longer mean channel open time and a lower conductance in neonates than in adults. Finally, newborn endplates are multiply innervated but mature junctions are singly innervated. Rat soleus muscles develop postsynaptic folds, convert AChR gating properties, and eliminate excess synaptic inputs during the first three weeks after birth. This project focuses on the two postsynaptic events, particularly the shift in AChR channel properties. The single-channel recording technique will be used to characterize more fully the ways in which the gating properties of neonatal and adult AChR's differ. The mechanism behind these changes will be explored in several ways. First, an in vitro system will be used to see if phosphorylation of AChR's affects their gating properties. Second, a particular myasthenic antiserum will be tested for its ability to specifically bind to and label neonatoal-type AChR's. Third, the role of active cellular processes and of certain enzymes in mediating channel conversion will be tested by treating muscles with inhibitors. Finally, the role of the nerve in regulating postsynaptic maturation will be examined. When soleus muscles are denervated shortly after birth, both channel conversion and the formation of postsynaptic folds are inhibited. To determine if this reflects loss of a trophic substance or lack of muscle activity, we will see if direct, chronic electrical stimulation of muscles can substitute for innervation in promoting endplate maturation. The results should lead to a better understanding of synapse maturation, of AChR gating properties, and of the interactions between nerve and muscle during development.

This computer resource facility is currently completing two major core projects which we believe will, during the next year, make a major contribution to the research community in general and our collaborative research in particular. FASTRUN, an attached processor computing device for which the original design was done at the facility, is now being constructed at the Brookhaven National Laboratory as a joint project. This processor, which is a long pipeline to compute the non-bonded pairwise forces, will be completed and delivered to Columbia within the next two months. Combined with a STAR-100 array processor, now running at the facility and a microvax as host, the system which MERCURY (Molecular Energy Refinement at Columbia University) is expected to have a computing speed somewhat greater than a CRAY-2 but only for molecular mechanics of large molecules; dynamics energy minimization, free energy of binding, etc. The system will be used in efforts to compute the conformations of molecules whose amino acid sequence is known and are homologous to structures which have been solved crystallographically. It will also be used to compute the ionic conductance through a protein which forms a small ion channel as the amino acid sequence of the protein is changed by site-directed mutagenesis. The second major project is the development of a computing system for a SUN 160/3 with image processing boards for automatic extraction of cell and fiber outlines from serial section micrographs. The system now works well enough so that it can reduce the time and tedium of manual nerve tracing by about a factor of ten. It will be available for neuroanatomical users in the near future, and will be used initially for reconstruction of the cerebellum in developing mice in connection with experiments which study gene expression by means of in situ hybridization. The programs will be improved substantially so that the system will run for long periods without any investigator intervention. The verification and decision making which must be done by the human observer, will be carried out in one interactive process after the automatic system has operated on previously stored images. As the hardware and the software for this system is developed, it will be available for use by many investigators both local and from other institutions. As new hardware becomes available in the second year of the proposed grant, we expect to convert the manual CARTOS system on the LOANER, so that it can carry out automatic nerve tracing and be available to outside users.

Many cells in advanced metazoans, particularly neurons, acquire unique, position-specific identities during development. Work with the fruit fly, Drosophila melanogaster, has led to the identification of many of the genes involved in this fundamental process. About 30 of these have been shown to have direct DNA sequence homology in a region called the homeo box that has been shown to code for a DNA-binding, conserved sequence of 60 amino acids. Cross-hybridization of genomic DNA from various other species with Drosophila probes has shown that the homeo box is conserved in evolution and has led to the isolation of homologous genes in frogs, mice and humans. Because most of the Drosophila homeo box genes affect developmental processes, in particular segmentation and segmental differentiation, it has been proposed that the homeo box could also be a marker of such genes in other species. Recent work also shows high levels of expression in the nervous system and suggests a role for these genes in the determination of neuronal fate and in differentiation. A group of animals in which a test of these ideas is particularly appropriate is the leeches. Leeches, like other annelids and arthropods, display an obvious segmental organization of their bodies. Current evolutionary theory has the arthropods and the annelids descending from a common, unsegmented ancestor, suggesting the possibility that the two groups may have cognate genes with similar or related functions. Our previous observations have demonstrated the existence of leech homeo box genes and their expression, particularly in the central nervous system, in spatiotemporal patterns like those found for homeo box genes in vertebrates and for homeotic genes in the fly. Nucleotide sequence similarities indicate that these may be leech cognates of fly homeotic genes and are, therefore, excellent candidates to be among the genes that specify segmental identity in the leech. The central aim of this project is to characterize leech genes of this class and to explore their functions by interfering with their expression. In particular, we will exploit the ability to work with identified leech neurons to determine which express these genes and to search for phenotypic differences that correlate with differential expression patterns. The techniques to be employed in this work include standard molecular cloning, in situ hybridization, construction of fusion proteins and raising antibodies, antisense RNA injection, immunohistochemistry, intracellular dye injection and electrophysiological recording, and computer-aided structural analysis. The results of this investigation should have a significant bearing on our understanding of the evolution of genes that control early development and how these genes may control neuronal phenotype.

DESCRIPTION: The central aim of this project is to explore in vivo the mechanisms underlying the formation of axonal terminal arborizations and the innervation of peripheral targets by central neurons. Specific leech central neurons (P neurons) extend axons early in development that pioneer the paths to the targets and are the first to establish terminal fields. In the process of generating these fields, homologous P cells inhibit each other from growing extensively in each other's territory. Later-growing neurons (AP cells) use these pioneers as substrates for growth to and branching at the target area; in fact, the pioneer is both necessary and sufficient for the generation of normal Ap cell arbors. These results indicate that a series of enhancing and/or inhibiting interactions modulate the extension, retraction and branching of neurites during the establishment of peripheral arbors by leech central neurons. The working hypotheses of this project are that these interactions (a) modulate neurite growth by affecting the assembly of cytoskeletal components, and (b) are mediated at least in part by changes in intracellular calcium levels. The experiments proposed in this grant are designed to test these hypotheses and to provide the bases for future examination of the mechanisms involved in the signaling and responses of these cells the principal experimental approaches will be (1) to study the kinematics of arbor growth using time-lapse analysis of dye-filled embryonic neurons in intact animals, (2) to determine the arrangement of cytoskeletal elements at various stages of growth and after experimental manipulations by injecting tagged actin and tubuli subunits into individual growing neurons, and by electron microscopy, and (3) to search for relations between changes in cytoskeletal arrangements and intracellular calcium concentration. These studies will provide further insight into production and selective retraction of axonal projections, a common and important feature of the innervation of peripheral tissues.

DESCRIPTION (provided by applicant): A key aspect of the development of many types of cells, particularly neurons, is that they must extend projections over long distances and varied terrains in order to interact with appropriate targets. In order to achieve this goal, projections carry on their surfaces molecules that allow them to detect and respond appropriately to external cues. The work proposed here seeks to understand how one particular kind of molecule, a receptor phosphatase we have identified and named HmLAR2, performs this function. Receptor phosphatases have been implicated, in systems from flies to rats, as key components of the signaling pathways that allow cells to respond in specific ways to environmental factors, both soluble and bound to other cells or extracellular matrices. We plan to examine how the dynamics of cell growth are affected when HmLAR2 is experimentally perturbed or deleted, or expressed ectopically in cells that normally do not express the corresponding gene. Having already identified a relevant external signal as well as a putative internal substrate that could mediate how this molecule affects cell growth, we now seek to understand further its mechanisms of action by using a preparation, the medicinal leech, that allows very refined live imaging of individual cells in the intact living embryo with sophisticated microscopes. In experiments up to this point we have learned that this molecule is critical for growth and the maintenance of migrating structures in one particular kind of cell that expresses it. We also seek to establish the generality of this observation by studying, in the same system, other cells that also express this molecule. Our long-term objective is to obtained detailed knowledge of the molecular pathways of which HmLAR2 is a member, to understand how this molecule is regulated by the cell, and to explain how, in molecular terms, it achieves its function as a transducer of external signals into internal responses. This detailed level of knowledge will then allow us to understand how pathological states that affect the function of this important class of surface receptor may lead to a breakdown in the development of tissues and organs, including nerve and muscle tissues, that is representative of some types human disease conditions.

ABSTRACT E. Macagno, PI Proposal # IOB-0446346

This project will address an important question about the development of the nervous system, specifically, what is the role of certain types of interactions among nerve cells in defining how the nervous system is assembled. Members of the laboratory have identified and are characterizing the group of molecules that are thought to mediate these types of interactions, and will use cutting-edge techniques to affect whether these molecules are present at the right time and place where they are thought to function. The ideas they will test relate to the formation of specific patterns of connections that are critical for proper function of neuronal circuits The neuroscientists will use a very simple nervous system to answer these questions, that of the medicinal leech. In this animal, nerve cells are large and easily identified, which greatly facilitates these experiments. Since the same types of molecules are thought to exist in and affect how human nerve cells interact as they develop, the findings expected from this research project with non-human tissues will have a strong impact on our understanding of the normal assembly of the both invertebrate and vertebrate nervous systems.

The proposed research will serve as an excellent training experience for undergraduates, graduate students, and postdoctoral researchers. The leech embryo and nervous system are easy for beginners to master and the laboratory will provide training in biology at various levels, including molecular biology, cellular biology, and physiology. UC San Diego serves a diverse student population, from which researchers will be recruited.

Intellectual Merit: Through collaboration between its Divisions of Physical and Biological Sciences and Division of Student Affairs, UCSD is providing scholarships for low-income undergraduates majoring in quantitative or interdisciplinary science. Thirty qualifying students will receive $3,000 annually over four years or until graduation beginning in fall, 2006; 12-15 upper-division students will receive $3,000 annually for two years effective fall, 2008. Upper-division students who have completed a summer research program or internship may receive an additional scholarship of $1,850. Each Scholar will have opportunities for enrichment activities involving faculty, industrial partners and peers through mentoring and collaborative learning experiences.

Broader impact: Recruiting is initiated through personalized letters from PIs to potentially eligible students. To encourage applications from women, underrepresented students and those with disabilities, notices are posted in specialized campus locations such as the Women's and Cross-Cultural Centers. Primary objectives are to support timely degree completion; increase research and internship opportunities; and increase numbers of low-income, underrepresented and first generation college students, women, and persons with disabilities entering quantitative and interdisciplinary science graduate programs.

Eduardo Macagno
IOS 0745134: Receptor Phosphatase Roles in Self-Avoidance and Arbor Tiling

As an embryo develops, groups of 'like' cells, particularly some types of nerve cells, become organized in functionally important ways, dividing and sharing space in developing tissues and organs. A good example of this process is the growth of the sensory "arbors" of cells that detect mechanical stimuli, which expand until they run into each other and stop growing, thus achieving complete and non-redundant coverage of the skin, much as tiles cover a floor. This "tiling" process is critical for an animal to be able to detect where stimuli are coming from on their surfaces and thus to respond appropriately - leaving uncovered areas would lead to dangerous "blind spots." Tiling requires not only that branches of cells of the same type recognize and avoid each other (mutual exclusion) but that branches of the same cell do so as well (self-avoidance). This project is designed to yield information on the mechanisms responsible for mutual exclusion and self-avoidance, an important step towards a general understanding the assembling of complex cellular ensembles. The proposed studies will be carried out on a specific set of cells of the medicinal leech, a uniquely advantageous model system that allows the detailed characterization of cellular interactions as they occur in the living, intact embryo. Previous work from this laboratory identified a membrane receptor on the surfaces of these cells that, the researchers have proposed, creates a signal to stop growing and retract when it meets with other copies of itself on other parts of a cell. The proposed work will focus on understanding exactly how this class of receptor regulates cellular growth and arbor extension. The Broader Impacts of this proposal are twofold. First, the work will provide valuable insights into the mechanistic underpinnings of arbor tiling and the genesis of multicellular structures. And second, this work will provide a multitude of training opportunities for undergraduate students, including those from underrepresented minorities.

The University of California at San Diego has received a grant to develop computational tools to support the study of the development and repair of nervous system development at the molecular level. Knowledge of the tissue distribution of essential molecules is necessary for understanding how biological systems function, how they grow, and how they repair themselves following trauma or disease. In this project, a multidisciplinary team of investigators will design, test and implement new tools to analyze data obtained by means of the recently developed technique of mass spectrometry imaging applied to the mapping of peptides and proteins in biological tissues. Application of these new methods will yield detailed maps of the temporal and spatial distributions of thousands of individual molecules and the capacity to examine patterns of expression as well as correlations in expression within ensembles of molecules. These new methods will be developed and tested first in simple model organisms, to characterize and compare the molecular components in the embryonic, adult and regenerating nervous system. Later, they will be applied in studies of mammalian nervous system slices in order to answer, among other questions, how stem cells are intercalated into and how they mature in adult nervous systems, during normal replacement or artificial replacement following cell loss due to disease or aging. All computational and bioinformatic tools developed in the course of this project will be made available openly to other scientists. The project will train a group of scientists at multiple levels, from undergraduates to postdoctoral fellows, in this exciting new area of basic and applied research. Addiitonal information may be found at http://genomes.ucsd.edu/leechmaster/.

This project will generate new information on how an important family of proteins, the GAP Junction proteins, partakes in the assembly of a functional brain from its component parts, neurons and glial cells. These proteins join together to form electrical synapses, a major means of communication between nerve cells. During brain development, they help to define which cells can join together in functional circuits. Mutations in gap junction proteins can lead to devastating abnormalities in brain function. Experiments will be carried out in a simple animal with a simple brain, the medicinal leech, where interactions between small numbers of well-characterized nerve cells can be explored in greater depth, with diverse experimental approaches and tools. Since critical molecular mechanisms are highly conserved throughout evolution, these studies will advance the general understanding of how nervous systems are built, including those of more complex species, such as mammals. Additionally, leech neurobiology is ideally suited to undergraduate training, offering a strong foundation in molecular and cellular biology and electrophysiology. Student recruitment for this project will focus on Community College transfer students, particularly those from underrepresented groups, during the summer before they enter UCSD, to provide an early exposure to scientific research. Continuing their research experiences during their undergraduate careers, they will contribute to the badly needed increase of well-trained graduates in STEM fields.

The proposed studies will be based on recent findings that indicate that different types and distributions of gap junction proteins can regulate the shape and connectivity of individual leech neurons. Gap junction proteins belong to large gene families that form inter-cellular channels and allow direct exchange of ions and small molecules. Each channel is comprised of six protein subunits, and since many neurons express unique complements of multiple gap junction proteins that can form the channels, a cell's expression profile might function like lock-and-key recognition factors. This project will test how different gap junction proteins can affect the characteristic morphology and connectivity of identified neurons in the leech central nervous system. The expression and function of gap junction proteins will be modulated by gene knockdown and transgene expression in individual developing neurons, and morphological changes will be assayed by digitizing neuronal arbors using confocal microscopy and quantifying branch distribution, number and length. Intracellular dye-tracer injections and electrical recordings will detect changes in their connectivity.