Christine Beattie - US grants

The formation of appropriate connections between motoneurons and their target muscles is essential for animals to carry out their normal behavioral repertoire. Motoneuronal axons navigate through the developing embryo towards target muscles in a highly stereotyped manner making few navigational errors. The precision of this event is astonishing considering the complicated environment of a developing embryo and the distances many axons travel. Research over the past two decades has demonstrated that axon pathfinding is directed by interactions between the tip of the growing axon and guidance cues, either positive or negative, present in the environment. Although our knowledge of the cellular and molecular aspects of axonal guidance has increased, numerous questions remain unanswered. In particular, it is unclear how axons interact with different pathway environments and how axon pathways are established during development.
Using zebrafish as a model system, Dr. Beattie will address how a set of identified motoneuronal axons locate their correct target muscles. The zebrafish neuromuscular system offers a unique opportunity for studying this issue. Individually identifiable primary motoneurons pathfind along and innervate distinct muscle regions, thus enabling analysis of single motoneurons. Using a combination of mutational analysis, cell transplantation and molecular analysis, the experiments outlined in this proposal will reveal how axons interact with cells along their pathway, how motoneuronal pathways are established during development and how myotomes develop regions displaying unique properties with respect to growing axons. This investigation will impact the field by uncovering the events that control this essential component of axon guidance.

Abstract Sack
A Wide-Field Deconvolution Microscope for Cell and Developmental Biology

Deconvolution microscopy provides superior spatial resolution (especially at high magnification) that is ideal for co-localizing multiple fluorescent probes in fixed and living cells in three dimensions. A wide-field deconvolution microscopy facility will be established at Ohio State University. The system will be configured around a Zeiss Axiophot 2 microscope with motorized stage, a high-resolution cooled CCD camera, and integrated software for image acquisition and deconvolution.
Current core users will focus on the cell and developmental biology of plant, neurobiological and fungal material. Specific projects include: (1) the molecular functions of genes controlling cell proliferation, terminal differentiation, and axonal outgrowth in the Drosophila nervous systems, (2) the cell biology and regulation of plant vesicular trafficking and cytokinesis, (3) the functions and localization of g-tubulin in Arabidopsis and Aspergillus, (4) genes and mechanisms controlling stomatal patterning and development in Arabidopsis, (5) the molecular genetic regulation of axonal outgrowth in zebrafish, (6) functional interactions between plant nuclear matrix proteins, and (7) the differential spatial regulation of protein and mRNA localization within mouse Purkinje cells.
Deconvolution imaging is highly likely to advance the understanding of the function and localization of specific proteins in several model systems. This facility will enhance interactions between cell and developmental biologists in the College of Biological Sciences, the Plant Biotechnology Center and the Neurobiotechnology Center. It will also contribute substantively to the training of post-doctoral researchers, graduate students and undergraduate students.

DESCRIPTION (provided by applicant): Motoneuron diseases are devastating in that they rob individuals of the ability to move and are often fatal due to denervation of the respiratory system. Spinal muscular atrophy (SMA) is an autosomal recessive disease that causes motoneuron dysfunction leading to paralysis and in severe cases death making it a leading genetic cause of infant/toddler mortality. Analysis of SMA animal models reveals, motor axon defects, immature neuromuscular junctions (NMJs), and denervation suggesting that changes at the motor nerve terminal may initiate disease. The survival motor neuron (SMN) gene is the genetic cause of SMA and has a clearly defined role in assembling RNAs and proteins needed for mRNA splicing (snRNP assembly). Data from our lab and others, however, suggest that SMN may have other functions that are compromised when SMN levels are decreased. Using zebrafish as a model system, we have shown that SMN has an snRNP independent function important for normal motor axon outgrowth. Moreover, we have shown that plastin 3, an actin binding protein and the first identified modifier of human SMA, can rescue motor axon defects in zebrafish caused by low Smn levels. In addition, zebrafish smn mutants have severely reduced plastin 3 levels. In this proposal we will test the hypothesis that plastin 3 acts with SMN via an snRNP independent pathway to facilitate normal motoneuron development and function. To directly test this hypothesis, we will ask whether other SMA phenotypes are rescued by plastin 3 (Aim 1). This includes motoneuron and NMJ electrophysiology, SV2 protein at the NMJ, and survival. We will determine how plastin 3 is functioning with respect to SMN by performing a structure/function analysis (Aim 2). For these experiments we will use both plastin 3 and SMN mutants to define relevant domains. We will also test the hypothesis that plastin 3 is unique in its ability to modify SMA phenotypes by examining other actin binding proteins. We will test the hypothesis that the SMN plastin 3 interaction is independent of the snRNP function of SMN (Aim 3). Lastly, we will use live imaging to ask where SMN and plastin 3 proteins localize in motoneurons and does decreasing Smn change the levels and/or cellular localization of plastin 3 (Aim 4). Data derived from these Aims will directly address the relationship between SMN and plastin 3 as it relates to SMA using a combination of electrophysiology, molecular genetics, biochemistry, cell biology, and imaging. Moreover, it would establish an snRNP- independent mechanism of SMN that directly affects motoneuron function thus greatly advancing our understanding of this disease and revealing new therapeutic targets. Using zebrafish is a strength in that we can directly analyze motoneurons in vivo in SMA models that we have developed and easily generate novel transgenics to ask specific questions. This is a unique feature of this model system and thus these studies are highly relevant and will advance our understanding of how low Smn levels cause SMA. PUBLIC HEALTH RELEVANCE: Spinal muscular atrophy (SMA) is a motoneuron degenerative disease that is a leading cause of infant/toddler mortality. Low levels of the survival motor neuron (SMN) protein cause SMA, but how this happens is unclear. Recently a modifier of SMA, the actin binding protein plastin 3, was identified. Experiments in this proposal will directly test the hypothesis that SMN stabilized plastin 3 thus promoting normal motor axon outgrowth and synapses. The proposed experiments will elucidate the interaction and function of plastin 3 as it relates to SMN and has the potential to reveal novel therapeutic targets.

Movement is controlled by connections established during development between motor neurons and muscle cells. Motor neurons extend processes called axons out from their cell bodies, which leave the spinal cord and extend through the developing embryo to contact the appropriate muscle fibers. This process, referred to as axon guidance, is a fundamental component of development and is essential for survival. Axon guidance is highly precise with growth cones, the growing tip of axons making few navigational errors as they extend along stereotyped routes or pathways. While numerous examples of axon guidance events in vertebrates have been described at the cellular level, less is known about the molecules that control these events. The goal of this research is to elucidate the molecules and mechanisms that control motor axon guidance in vertebrates. The experiments outlined in this proposal address this using zebrafish as a model organism. Zebrafish is an excellent model system for studying vertebrate motor axon guidance due to its relatively simple nervous system, the ability to study embryos at early stages of development when axons are growing to their muscles, and the capability to induce, recover, and clone mutations. One approach is to study mutations that disrupt motor axon guidance in the embryonic zebrafish. The mutation, topped, dramatically and specifically affects the ability of ventral motor axons to reach their target muscles. Instead of progressing ventrally along their pathway, motor axons in topped mutants stall and fail to enter the ventral myotome at the normal time. Topped protein functions in the muscle to enable growth cones to extend along the ventromedial muscle suggesting that it is a cue on the muscle that directs ventrally extending axons. Using the zebrafish genetic map, a region of the genome was mapped that contains a protein, Semaphorin 5A, which is known to function in axon guidance in a different part of the nervous system. Using a series of genetic, cellular, and molecular approaches will elucidate whether semaphorin 5A is the gene disrupted in topped mutants. For example, preliminary evidence shows that decreasing Semaphorin 5A causes the same phenotype as the mutant suggesting that this protein is functioning in this process. The mechanism of Semaphorin 5A action will be addressed and the receptor this protein binds to will be isolated. Studying these mutations and proteins will provide novel insights into the genetic control of vertebrate motor axon guidance and will identify molecules that function in this essential developmental process.

The Beattie lab incorporates undergraduate students who perform experiments and participate in lab meetings. Active efforts to recruit graduate students who are members of under-represented minorities has led to the enrollment of one minority graduate student. In collaboration with local faculty, Dr. Beattie has developed a zebrafish lab course for students at Miami of Ohio, thus exposing them to a level of scientific practice that would normally be unavailable at this primarily undergraduate institution

DESCRIPTION (provided by applicant): The Ohio State University (OSU) Neuroscience Center provides support for studies aimed at analysis and treatment of neurological disorders with an emphasis on animal models. The Center is a collaborative effort of 43 neuroscientists (25 NINDS funded) from OSU and Nationwide Children's Hospital. The majority of these PIs work on aspects of nervous system disorders with four major focus areas being motoneuron disease, neuromuscular disease, CNS and PNS injury, and brain tumors. These PIs span numerous departments, centers, and institutes at OSU that support basic and translational neuroscience research making the Center a nucleus of neuroscience activity on campus. Center priorities align with and can leverage the Neuroscience Signature Program at OSU, a multi-million dollar effort supporting Neuroscience as one of five growth areas on campus. In addition, the Center aligns with the newly granted Clinical and Translational Science Award to the OSU Medical Center. To support the development, characterization and analysis of nervous system disease models, this proposal supports 5 research Cores. Core A (Administrative) will oversee the OSU Neuroscience Center. Core B (Genetics) will support the development of transgenic/knockout mice and the use of zebrafish with an emphasis on traditional and novel genetic approaches. In addition this Core will support a Genome Manipulation Facility focusing on zinc-finger nuclease and BAC recombineering techniques for the generation of new animal models. Core C (CNS/PNS Injury and Rodent Behavior) will provide the equipment and technical expertise to generate injury models and to analyze behavioral deficits associated with these models. Core D (Physiology) will provide equipment and technical assistance for both electrophysiology and muscle physiology to analyze these models. Core E (Imaging) will support both Confocal and MRI imaging with reduced costs and technical assistance. Core F (Xenograft) will provide equipment and technical assistance to generate mouse models of brain tumors. These unique Cores will provide PIs with access to centralized equipment and technical expertise unavailable in any single lab, thus substantially enhancing neuroscience research on the OSU campus.

DESCRIPTION (provided by applicant): Amyotrophic lateral sclerosis is an adult onset motoneuron degenerative disease with a lifetime risk of ~1/1000 that affects an estimated 30, 000 adults in the US at any one time with ~5000 new cases/year. Approximately 80% of the cases are fatal 5 yrs after diagnosis. There is no cure and only one FDA approved therapy that has a minor effect on the progression of the disease. Identifying drug targets for ALS will advance both our understanding of this disease and will reveal relevant targets for drug development. The most well characterized genetic form of ALS is caused by mutations in the SOD1 gene. Because SOD1 based ALS and sporadic ALS both cause the same disease, it is believed that drug targets will be shared. Mutations in SOD1 produce a dominant gain of function protein with unknown activity. Since SOD1 mutants do not cause ALS-like phenotypes in invertebrates, only rodent models of this disease exist thus limiting the type of experiments that can be performed. For example it is not possible to do genetic modifier screens, which are an excellent way to identify protein targets for drug intervention, in ALS mice. In addition, drug screens in mice are very expensive and require large numbers of animals. To generate another vertebrate model of ALS that can be used for genetic and drug screens, we generated transgenic zebrafish over expressing the well-characterized SODG93A and G85R mutations. When we generated the transgenic zebrafish, we incorporated a heat shock promoter (hsp70) driving the fluorescent protein, DsRed, to track our transgene. Upon identifying the transgenic lines, we found that the fish carrying the sod1 mutations turned on the heat shock response, as revealed by DsRed expression, independent of heat shock (referred to as sodmut hsp70 induction). This suggests that fish containing mutant Sod1 exhibit cellular stress starting at early larval stages. We propose to use this response as a read-out of mutant Sod1 gain-of-function toxicity. In this proposal we present preliminary data linking the sodmut hsp70 induction to ALS phenotypes. We then present Aims to develop and validate this read-out as an assay for screening. Lastly, we will use this in vivo assay to perform a pilot genetic modifier screen to identify novel targets for intervention. Development and validation of this unique in vivo, vertebrate assay of Sod1 mutant toxicity will allow future development of high-throughput screens and rational drug design for ALS. PUBLIC HEALTH RELEVANCE: Amyotrophic lateral sclerosis (ALS) is a fatal, neurodegenerative disease that has no cure. We have generated a zebrafish model of SOD1 ALS and find that it has a very early, easily scored, phenotype. In this proposal we will develop this phenotype into an assay for drug target identification and drug testing. This is a unique approach as it is a rapid, in vivo screen in a vertebrate and will serve as a basis for both genetic and drug screens to identify therapeutics for ALS.

DESCRIPTION (provided by applicant): Determining how combinations of genes interact as gene regulatory networks to produce cellular diversity is fundamental to understanding development. The neural crest (NC) has been studied extensively to elucidate mechanisms of cell diversification during development. The NC is a discrete and seemingly homogeneous undifferentiated stem cell-like ectodermal population of vertebrate embryonic precursor's cells that is the source of multiple different cell types including neurons and glia of the peripheral nervous system, pigment cells and major elements of the craniofacial skeleton, among others. Subsequent to the induction of the NC domain of the ectoderm during gastrulation, the fates of subsets of NC cells are specified as distinct sublineages that ultimately generate the complete cellular derivative repertoire of the progenitor population. How the fates of NC sublineages are specified during development is incompletely understood. Determining at the genetic level how differences between NC cells are established is essential to understanding how the NC generates such a vast array of different cell types. Studies in zebrafish and other vertebrates have indicated that several transcription factors are essential for the specification of distinct and overlapping subsets of NC sublineages, although none can individually account for NC cell diversification in its entirety. We found that in zebrafish foxd3; tfap2a double mutants all NC sublineages fail to be specified, indicating that foxd3 and tfap2a are synergistically and universally required for the initiation of NC diversification. Further, our studies indicate that the requirement for foxd3 and tfap2a for the initial specification of NC sublineages is due in part to their regulation of the NC expression of the SoxE family genes sox9a, sox9b and sox10. Together, these results have identified a framework gene regulatory network (GRN) that initiates NC diversification. Critically, however, the mechanisms by which framework GRN transcription factor interactions initiate NC diversification are not known. Equally important, the identified framework GRN cannot account for NC diversification in its entirety. Accordingly, we propose a research plan, based on the established framework GRN, to answer critical unresolved questions about the genetic regulation of the specification of NC sublineage fates which ultimately produces NC diversity. We will determine at the molecular level, employing a ChIP-based approach coupled with transgenic reporters, the mechanisms by which interactions between the frameworks GRN transcription factors specify NC cell fates. In addition, we will comprehensively identify additional foxd3- and tfap2adependent genes that, based on selection criteria, are candidates for the GRN controlling NC diversification using whole genome microarray expression profiling. We will then determine the functions of these candidates in regulating NC diversification using loss- and gain-of function approaches employing transgenic reporter wild type embryos and embryos singly or doubly mutant for genes comprising the framework GRN (foxd3, tfap2a, sox9a, sox9b and sox10) coupled with comprehensive phenotypic analysis of NC development. The results of our proposed studies will address critical deficiencies in the field by producing major fundamental advances in our understanding of the regulation of NC diversification. In addition, our results will generate applicable mechanistic paradigms for understanding cell diversification generally and provide a rich foundation for future comprehensive functional determination of the complete GRN controlling NC development. Lastly, given the high prevalence of clinically relevant conditions resulting from miscues during NC development, our results are likely to provide important insights for strategies to diagnose, treat and prevent human diseases such as neurocristopathies and cancers of NC origin.

? DESCRIPTION (provided by applicant): The goal of our work is to elucidate the function of the Survival Motor Neuron (SMN) protein in motoneuron development. This is an essential question because SMN is linked to the motoneuron disease Spinal Muscular Atrophy (SMA). SMA leads to paralysis and early death of infants/children. Work in SMA animal models and analysis of SMA patient autopsy tissue has revealed that motoneurons develop abnormally and fail to form axons correctly under disease conditions of low SMN. There is a critical need, therefore, to understand SMN function in motoneuron and motor axon development. Our previous work on SMN has lent insight into this process. We have shown in zebrafish genetic models that smn mutants have motor axon outgrowth defects including decreased motor axon filopodia, dramatically fewer axonal branches, and fewer neuromuscular junction synapses. We have also shown that SMN is localized to motor axons and its presence there is developmentally regulated. SMN binds a number of different mRNA binding proteins (RBPs) that are involved in localizing mRNAs into axons and growth cones. Localized mRNA translation functions in developing axons and controls axon guidance decisions, branching and outgrowth. In preliminary data we have validated that SMN binds the neuron specific RBP HuD in motoneurons and this interaction is developmentally regulated. We have also generated HuD mutants and find that they have motor axon defects. Based on these data we hypothesize that SMN localization to motor axons is critical for axonal development. We propose that SMN is a master regulator of RBP mRNA assembly to facilitate RNA localization to growing axons. To test this hypothesis, we will determine the complement of SMN:RBP complexes specifically in motoneurons and whether these complexes are developmentally regulated. We will use SMN variants (including a patient mutation) to elucidate relevant RBP binding domains. We will generate RBP mutants and characterize whether they have motor axon defects by analyzing developmental outgrowth and filopodial dynamics. We will determine whether motoneuron autonomous expression of these RBPs can rescue any motor axon defects. Lastly, we will test whether mRNA expression in motor axons is affected by SMN and RBP mutants. We will reveal whether SMN or SMN variants can rescue these defects and whether this is linked to rescue of motor axon outgrowth. Together these experiments will rigorously test the innovative concept that SMN is a master regulator of RBP complex assembly needed for localized mRNA transport to axons critical for developmental motor axon outgrowth. Knowledge gained from these key experiments will be impactful both towards our understanding of basic mechanisms in motor axon biology and towards our understanding of phenotypes associated with motoneuron disease.

Project Summary Understanding the mechanistic basis of motoneuron dysfunction and its role in motoneuron diseases would fill a major gap in neuroscience and advance new approaches for treating devastating diseases such as amyotrophic lateral sclerosis (ALS), hereditary motor neuropathy and spinal muscular atrophy (SMA). These diseases afflict over one hundred thousand adults, infants, and children per year in the US. ALS and SMA are particularly devastating diseases resulting in paralysis and death often within a few years of diagnosis. The genetics of these diseases indicates that motoneurons are particularly vulnerable to defects in proteins tasked with critical RNA processing functions. However, exactly why motoneurons are vulnerable to RNA processing defects is not understood. The scientific rationale for this project is to elucidate mechanistically how mishanding of RNAs can disrupt motoneuron function and lead to motoneuron death. Elucidating the motoneuron-specific RNA processing defects caused by these mutations is essential for understanding motoneurons in both normal and diseased conditions and will direct critically needed therapeutics. To tackle this issue, we focus on the ubiquitously expressed survival motor neuron (SMN) protein and the motoneuron disease SMA. SMA is a motoneuron disease that affects infants/children and is caused by low survival motor neuron (SMN) protein levels. SMN functions in many aspects of RNA metabolism. However, the critical RNA handling function of SMN in motoneurons is unresolved. Evidence supports that SMN interacts with various neuronal RNA binding proteins (RBPs) that stabilize and/or transport RNAs to axons and dendrites during development. Using unique zebrafish models that we have generated, we have shown that SMN is required for normal vertebrate motoneuron development including dendrite formation and motor axon outgrowth and arborization. This is a key finding and reveals that SMA is not a degenerative defect, but the motoneuron dysfunction is caused by poor motoneuron development leading to neuronal failure. We hypothesize that SMN associates with neuronal RBPs and their cargo RNAs in a developmentally regulated manner to direct motoneuron development including axon out growth and branching, dendrite formation, and synapse formation. To test this we will answer three essential questions: What SMN:RBP complexes are in developing motoneurons? How do defects in these RBPs affect motoneuron development? What RNAs are in these complexes, and how are they affected when SMN or the RBPs are missing or decreased? All of our experiments will be performed in vivo in motoneurons, the relevant cell type and use a broad range of experimental approaches such as biochemistry, mass spectrophotometry, RNAseq, single neuron imaging and genetics. Data from these experiments will have broad implications for understanding RNA involvement in normal motoneuron development, SMA, and other motoneuron diseases such as ALS. In addition, our approach will rigorously test the importance of SMN:RBP complexes and their associated RNAs revealing a fundamental molecular mechanism in motoneuron biology.