John Y. Kuwada, Ph.D. - US grants

The long term objective of this research is to understand the cellular mechanisms underlying the embryonic development of the stereotyped cellular pattern in the central nervous system. The complexity of trying to understand the processes of cell determination during the development of the vertebrate central nervous system in terms of specific cell lineages and cell interactions, makes the reductionist approach of choosing a simple system quite attractive. It is hoped that this approach will reveal fundamental principles common to all organisms, but might be masked and are certainly much more difficult to study in more complex species. The early embryonic development of the insect central nervous system is in many respects remarkably similar to vertebrate neurogenesis. The grasshopper embryo in particular is an attractive preparation in which to study these question because the neuroepithelium is very thin and highly transparent; the cells are relatively large, highly accessible, and can be individually identified from birth to maturation; and the pattern of precursors and their progeny is relatively simple and highly stereotyped. A variety of cellular techniques will be used to study the roles of cell lineage and cell interaction in the determination of both individual neuronal precursor cells, and their individual neuronal progeny. In particular, the study will focus on the spatial and temporal pattern of regulation versus cell autonomy. The neuroepithelial cells along the dorsal midline (a relatively simple two dimensional sheet of five precursor cells and their progeny) will be manipulated using laser ablations, the embryos grown in culture, and the cells assayed using a variety of lineage and morphology markers via intracellular injections, immunocytochemistry using serum and monoclonal antibodies, and electron microscopy.

The objective is to understand how neurons in the vertebrate central nervous system (CNS) make proper connections with each other. This is one of the fundamental problems in development neurobiology. Answers to this problem should have important implications for many developmental diseases of the nervous system and problems concerning regeneration following injury to the CNS. One important aspect of how neurons become properly wired is how their growth cones navigate through the developing embryo to find their correct target neurons. However, the mechanisms which guide neuronal growth cones to their targets in the vertebrate CNS have been difficult to study primarily due to the complexity and large number of neurons found in the CNS of most vertebrates. I propose to study this problem in the embryonic spinal cord of the fish, the simplest part of the CNS of a relatively simple vertebrate. The early embryonic spinal cord of fish is an excellent preparation for pursuing this question since it contains a relatively small number of neurons which are easily visualized in the living embryo, can be identified as individuals or members of a small pool of homogeneous neurons, and studied with methods allowing analysis of single neurons. The aims of this proposal are to describe the behavior of single identified growth cones and to delineate the cellular mechanisms which guide these growth cones to their targets. This will be accomplished by intracellular injection of a variety of dyes into individual embryonic neurons, analyzing their growth cones and the substrates of these growth cones with light and electron microscopy, and manipulating the environment of these growth cones by selectively ablating individual cells with a laser microbeam.

The long term goal of this research project is to understand how neuronal growth cones reach their correct target sites in the vertebrate central nervous system (CNS). This is a fundamental problem in neuroscience. The delineation of how this is done normally should be important for understanding what goes wrong during developmental diseases of the nervous system and why regeneration following injury to the CNS is normally so poor. Evidence, from research funded by a grant to which this proposal is a continuation demonstrated that: 1) the early spinal cord of fish embryos is extremely simple consisting of a small number of identified neurons and non-neuronal cells; 2) growth cones of the five earliest neuronal classes reach their destinations in the CNS by following stereotyped, cell-specific pathways; 3) the behaviors of identified growth cones at specific sties suggest that identified cells may attract, inhibit, and direct their cell- specific turns. This proposal will test a number of hypotheses generated by this and other studies by laser ablating specific cells in the fish embryonic cord and assaying their effect by a combination of methods. This includes labeling single cells by intracellular dye injections, labeling all neurons or a specific subset of neurons with monoclonal antibodies, and electron microscopy. 1) Do growth cones receive directional cues from specific cells in their environment? 2) Do growth cones become sensitive to certain environmental cues only after interactions with other cells? 3) Can some cells specifically attract certain growth cones? 4) Can some cells specifically inhibit certain growth cones?

The long term goal of this research project is to understand how neuronal growth cones reach their target sites in the vertebrate central nervous system (CNS). This is a fundamental problem in neuroscience. The delineation of how this is done should be important for understanding what goes wrong during developmental diseases of the nervous system and why regeneration following injury or disease to the CNS is normally so poor. Growth cones reach their targets by selecting appropriate pathways and by extending in the correct direction on these pathways. This proposal seeks to analyze how growth cones know what direction they should extend in the vertebrate brain. Although many experiments have demonstrated that growth cones can distinguish appropriate pathways from inappropriate ones relatively little is known about the nature of directionality cues. Past experiments suggest that directional information is provided by extrinsic polarity cues that are likely encoded as a gradient of molecules distributed along an axis. However, it is not known whether a system of widely distributed and orthogonally arrayed (dorsal/ventral and anterior/posterior) polarity cues specify direction for all growth cones in the vertebrate CNS. The zebrafish embryo may be especially useful for analysis of these issues since it is a vertebrate embryo that can be analyzed with a combination of cellular, molecular, and genetic techniques. This proposal seeks to systematically examine how growth cones know direction by transplanting neurons to ectopic sites and by time lapse analysis of their growth cones. The proposed experiments take advantage of the ability to precisely manipulate even single cells with the ability to observe living growth cones in zebrafish embryos. These experiments should determine whether polarity cues guide growth cones, how the cues are distributed, how many cues exist, how growth cones behave under the action of polarity cues, and possibly identify sources of polarity cues. In the future molecular analysis and the emerging genetics of zebrafish may identify candidate polarity genes for growth cones in the zebrafish. A detailed understanding of polarity cues at the cellular level should then be invaluable for the functional analysis of potential growth cone polarity genes in the zebrafish.

nucleic acid sequence; growth cones; gene expression; neurogenesis; developmental genetics; zebrafish; embryo /fetus tissue /cell culture; genetically modified animals; computer assisted sequence analysis;

This proposal will examine the role of netrins and semaphorins for axon guidance in the zebrafish. The discovery of semaphorins and netrins has lead to an appreciation of chemoattractive and repulsive mechanisms for axon guidance. However, our understanding of the in vivo action of these molecules is still incomplete. 1. The semaphorins are a large family of molecules, but aspects of their function is only known for 3 semaphorins. This proposal will examine the in vivo action of 2 more semaphorins. 2. It is clear that several mechanisms work coordinately to guide axons. We now have some understanding of how individual netrins and semaphorins work, but do not yet know how these molecules work together to guide specific growth cones. This proposal will see how these molecules work in concert to guide spinal commissural axons. 3. Commissural growth cones first extend toward the midline and then away from the midline in the CNS. How they do this despite the presence of bilaterally symmetric guidance cues, such as chemoattractive netrins expressed at the ventral midline of the CNS, is not well understood. This proposal tests a mechanism that accounts for this behavior. Based upon our analysis of netrins and semaphorins in zebrafish and the actions of these molecules in other organisms, a model that incorporates 2 netrins and 2 semaphorins was generated to account for pathfinding by spinal commissural axons. The critical feature of the model is that commissural growth cones change their responses to these molecules at the ventral midline, perhaps as a result of interactions with the floor plate. Commissural growth cones extend ventrally on the ipsilateral side of the cord because they are repulsed by Sema Z2 secreted by the roof plate and attracted by Netrin-1a and Netrin-1b secreted by the ventral half of the cord and floor plate, respectively; pause at the floor plate to reprogram themselves; and then extend ventrally on the contralateral side because they are insensitive to or attracted by Sema Z2, insensitive to or repulsed by Netrin-1b, and insensitive to or remain attracted to Netrin-1a. Furthermore, Sema Z7 which is now expressed by the dorsal cord repulses the growth cones so that they now turn to extend longitudinally in the dorsal cord. Our basic strategy will be to manipulate semaphorins and netrins and determine how this affects the behavior of growth cones in zebrafish embryos. In addition, we will test the redundancy proposed in the model by examining commissural growth cones in spinal cords missing both the roof and floor plate so that they are devoid of molecules including Sema Z2 and Netrin-1b derived from the roof and floor plate.

DESCRIPTION (provided by applicant): Semaphorins are a large family of molecules that repulse growth cones, but in some cases also attract them. The analyses of semaphorins and their neuropilin/plexin receptors have made important advances in our understanding of how growth cones are guided to their targets. However, our understanding of the function of semaphorins is still incomplete. First, the functional analysis of vertebrate semaphorins has been mostly of the secreted, Class 3 semaphorins. Our understanding of the function of the large transmembrane, Class 4 semaphorins is limited to Sema4D/CD100which plays a vital role in the immune system, but no nervous system function is known for any Class 4 semaphorin. This application will examine how Sema4E, a novel Class 4 semaphorin recently cloned by our lab, guides growth cones in the zebrafish embryo. Second, most vertebrate semaphorins that affect neurons exhibit a repulsive effect on axons, although in certain circumstances semaphorins can attract growth cones in vitro. Nevertheless, there are no examples of attractive actions on axons by a vertebrate semaphorin in vivo. This application will examine whether Sema3D, whose function is unknown in any organism, attracts and/or induces branching by axons in vivo in zebrafish embryos. The accessibility, manipulability, and transparency of the embryo have led to high resolution, cellular analyses of how specific growth cones find their targets in zebrafish. Furthermore, our laboratory has generated transgenic zebrafish in which several semaphorins can be induced, shown that transgenes can be activated in individual cells by focusing a laser microbeam onto cells, "knocked down" semaphorins by injection of morpholino antisense oligos and applied dominant negative strategies. Preliminary evidence suggests that Sema4E and Sema3Al may restrict branchiomotor axons to their normal pathway, while Sema3D may attract or induce branching within the pharyngeal arch targets of these axons. This application will use these transgenic lines and generate new ones in order to assay both gain- and loss-of-function phenotypes for Sema4E, Sema3Al, and Sema3D to clarify their roles for guidance of the branchiomotor growth cones to and within the pharyngeal arches that they innervate.

Very often, experimentally induced mutation of a gene does not lead to a detectable physiological deficit because remaining components of the system are able to compensate for the loss of function. Occasionally, experimental mutation leads to a dramatic deficit, thereby providing insight into fundamental organizational principles. Mutation of the TRMP-7 gene results in a loss of motility in zebrafish. This loss appears to be due to a deficit in the ability of sensory neurons to respond to touch stimuli. The TRMP-7 gene encodes a protein that forms part of an ion channel whose failure to operate results in a dramatic behavioral phenotype.

This project will focus upon understanding how this channel works to ensure proper development and operation of motor circuits by combining molecular biological and electrophysiological techniques. Specific aims include identification of neurons that have the TRMP7 protein, analysis of differences in neural circuitry between normal and mutant zebrafish, and functional analysis of different domains of the TRMP-7 gene. Studying the mechanisms underlying the dramatic behavioral changes caused by mutation of this single gene in the relatively simple nervous system of the zebrafish will very likely provide insight into fundamental genetic and neuronal mechanism found throughout the animal kingdom.

An integral part of the project will be the involvement and training of undergraduates including those from under-represented groups, that will be recruited from the PI's courses and from the Summer Research Opportunity Program at the University of Michigan that is open to all American students. These students will work directly with the PI and be exposed to genetics, molecular biology, genomic databases, and electrophysiology. The goal will be to spark an interest in research and encourage students to pursue a Ph.D. program. Additionally, a new undergraduate course, 'Genes, Brain and Behavior', will be developed that will incorporate the methods and research findings from zebrafish mutants. This course will stress the genetic analysis of brain and behavior with particular attention to genetic model systems such as Drosophila, C. elegans, and zebrafish in addition to rodents. The course will incorporate the findings from analyses of zebrafish behavioral mutations including those derived from the PI's lab.

PROJECT SUMMARY The molecular process regulating the localization of acetylcholine receptors (AChRs) at the neuromuscular junction (NMJ) requires agrin, a factor secreted by motor neurons. This process involves various other molecules, but the signaling process in muscles initiated by agrin is poorly understood. We are examining this process in zebrafish which are vertebrates amenable to genetic analysis for the identification of genes important for a biological process and analysis of the in vivo function of these genes. We generated the zebrafish ennui mutation in which AChRs are mislocalized and identified the ennui gene as one encoding for LRP4 that in mammals is required for proper clustering of AChRs. We propose to use the ennui mutants to better understand the in vivo role of LRP4 for the formation of the vertebrate NMJ. We isolated the viable ennui mutation that showed a decreased electrophysiological response at the NMJ. The reduced response was due to a dramatic decrease in synaptic AChRs and high levels of AChRs mislocalized to the ends of muscles. The mutant phenotype is cell autonomous, and exogenous agrin induced AChR clusters in wildtype muscles but not in ennui muscles. These results suggested that the ennui gene encoded for a muscle factor required for agrin-induced localization of AChRs to the NMJ. The ennui gene was identified as lrp4 by a combination of genetic mapping of the mutation and genomic analysis. LRP4 is a member of the low-density lipoprotein receptor family and is expressed by early stage muscles. Although lrp4 was recently found to be critical for proper localization of AChRs in mice, there is little known about how LRP4 may mediate agrin signaling and how it might interact with other well studied components of the agrin-initiated signaling pathway. We propose to explore these issues with experiments that utilize the advantages of zebrafish for examining in vivo gene function. Aim 1: We will examine how LRP4 is distributed in muscle by generating antibodies and/or expression of fluorescently labeled LRP4 and see if LRP4 co-localizes with other known NMJ components. Aim 2: We will analyze how LRP4 regulates aneural AChR clusters that form prior to innervation by a combination of antisense knockdowns and expression of specific forms of LRP4. Aim 3: We will establish whether LRP4 is an aggregation factor and see how LRP4 and MuSK, a component of the agrin receptor complex, are functionally related. Aim 4: We will assay how the interaction of LRP4 and MuSK affects the in vivo development of the NMJ.

DESCRIPTION (provided by applicant): Contractions of skeletal muscles are regulated by a process called excitation-contraction (EC) coupling and defects in EC coupling are associated with numerous human muscle diseases. Motor neurons activate skeletal muscles by releasing neurotransmitter that causes the voltage across the muscle membrane to change. EC coupling is the process by which the change in muscle voltage is converted to a release of calcium ions from a specialized intracellular organelle called the sarcoplasmic recticulum (SR) in muscles. The increase in calcium ions in turn initiates contraction by activating the contractile proteins. EC coupling occurs at triadic junctions of the transverse tubules that are infoldings of the muscle membrane and outpocketings of the SR. The two main molecular components responsible for EC coupling are the dihydropyridine receptor (DHPR), a voltage dependent protein in the triadic transverse tubule membrane, and the ryanodine receptor (RYR), a calcium ion release channel located in the triadic SR membrane. These two proteins face each other in the triad and are thought to directly interact during EC coupling. The voltage changes across the muscle membrane are detected by DHPRs that in turn directly activate RYRs to release calcium ions from the SR. EC coupling requires a complex of proteins including DHPR and RYR localized to triads. Although much is known about the role of DHPR and RYR, relatively little is known about the identities and functions of other components of the triadic molecular complex. We identified a zebrafish mutation that is deficient in motor behaviors and found that the causative gene encodes a novel muscle adaptor protein that we found is a key regulator of EC coupling. The adaptor protein localizes to triads, binds to the DHPR-RYR1 complex and is required for proper release of calcium ions by the SR and contraction by skeletal muscles. We further found that the gene encoding this adaptor protein in humans is the basis for a debilitating congenital myopathy in which 36% of individuals afflicted die by age 18. Finally our evidence suggests that mutations of this gene lead to a decrease in DHPR in muscle by improper trafficking of DHPR to triads once they are synthesized. We propose to take advantage of the identification of this novel protein as a key regulator of EC coupling and a new causative gene for congenital myopathy to analyze how this protein regulates EC coupling and how a defect in the protein leads to congenital myopathy. For this we will take advantage of the ability to readily generate transgenic zebrafish and the unique ability to examine cellular processes in living zebrafish embryos. We propose to examine how trafficking of DHPRs are affected by mutations in this gene by generating transgenic zebrafish in which DHPRs are tagged with a fluorescent protein. We further found that the adaptor protein binds a subunit of the DHPR so will identify the sequences in the adaptor protein and DHPR subunit required for binding and examine the consequences of a loss of this binding. We also generated adaptor protein gene knockout mice to extend our analysis to mammalian muscles. This knowledge should help us better understand the biology of myopathies and could potentially lead to therapeutic agents for congenital myopathies.