Julia E. Dallman - US grants

The long-term goal of this research is to understand the mechanisms that regulate the temporal and tissue specific expression of the peripheral nervous system specific Na channel, PN1. Na channels, expressed in the axons and cell bodies of peripheral nervous system (PNS) neurons, are in large part responsible for making these cells electrically excitable. The PN1 Na channel has the following unique properties: first, it is one of the only genes expressed predominantly in the PNS; and second, PN1 is rapidly and dramatically upregulated upon brief (1 min.) exposure to nerve growth factor (NGF) and interferon gamma (IFNgamma) by an as yet unknown mechanism. Our research plan is to: 1) Use genetic and biochemical techniques to identify PN1 regulatory DNA elements that confer the PN1 expression pattern on reporter genes transfected into several cell lines. 2) Determine the mechanism-immediate early genes and response elements-by which PN1 is upregulated by NGF and IFNgamma. The yeast one hybrid screen developed In the Pls lab will be used to isolate immediate early genes. 3) Test the constructs isolated in 1 and 2 for their ability to drive expression of a reporter gene in vivo by creating transgenic mice expressing the different constructs. Results from these experiments should be applicable in an experimental or therapeutic context to drive gene expression specifically PNS.

DESCRIPTION (provided by applicant): The applicant's goal is to obtain a position as Assistant Professor at an academic institution. The conventional route to this goal has been thwarted by childbirth, followed by a debilitating series of treatments for cancer. The proposed project provides a strong component of new training for the study of a zebrafish motility mutant, shocked, which compliments and extends skills acquired from graduate student and post-doctoral training. Over one hundred zebrafish mutations exist that affect the ability of a young fish to swim. Several of these mutations have already proved relevant to human myasthenic syndromes. The shocked mutation is unusual in that the motility defect improves with age. Shocked fish are initially paralyzed but acquire the ability to swim over the course of several days. Preliminary data suggests that excessive electrical coupling underlies the defective swimming phenotype in shocked fish. Although the vertebrate neuromuscular junction is the best studied of any synapse and electrical coupling is a feature of all immature vertebrate muscle, the significance of electrical coupling among muscle cells has not been addressed. The proposed research includes three aims: 1) To pursue the functional consequences of gap junctions for muscle synaptic physiology in wild type and shocked fish; 2) to identify the gene that underlies the shocked phenotype by parallel approaches of positional cloning and sequencing likely candidates; and 3) to understand the mechanisms that underlie the initial paralysis in shocked fish as well as their subsequent recovery. This work will be carried out in the laboratory of Dr. Paul Brehm, an expert in development of the vertebrate neuromuscular synapse and ion channel function. More recently his laboratory has focused on analyzing zebrafish motility mutants and has made great progress in a short time, discovering the molecular basis for several motility mutants including sofa potato (acetylcholine receptor delta subunit), relaxed (dihydropyridine receptor), and twitch once (rapsyn). Moreover, these studies have yielded new insight into the roles of the receptor and rapsyn in structuring the synapse. Dr. Paul Brehm's laboratory is part of a tight knit group of five independent researchers with distinct but related interests and shared microscopy and molecular facilities that create an environment with ample resources to carry out the proposed research.

DESCRIPTION (provided by applicant): The applicant's goal is to obtain a position as Assistant Professor at an academic institution. The conventional route to this goal has been thwarted by childbirth, followed by a debilitating series of treatments for cancer. The proposed project provides a strong component of new training for the study of a zebrafish motility mutant, shocked, which compliments and extends skills acquired from graduate student and post-doctoral training. Over one hundred zebrafish mutations exist that affect the ability of a young fish to swim. Several of these mutations have already proved relevant to human myasthenic syndromes. The shocked mutation is unusual in that the motility defect improves with age. Shocked fish are initially paralyzed but acquire the ability to swim over the course of several days. Preliminary data suggests that excessive electrical coupling underlies the defective swimming phenotype in shocked fish. Although the vertebrate neuromuscular junction is the best studied of any synapse and electrical coupling is a feature of all immature vertebrate muscle, the significance of electrical coupling among muscle cells has not been addressed. The proposed research includes three aims: 1) To pursue the functional consequences of gap junctions for muscle synaptic physiology in wild type and shocked fish; 2) to identify the gene that underlies the shocked phenotype by parallel approaches of positional cloning and sequencing likely candidates; and 3) to understand the mechanisms that underlie the initial paralysis in shocked fish as well as their subsequent recovery. This work will be carried out in the laboratory of Dr. Paul Brehm, an expert in development of the vertebrate neuromuscular synapse and ion channel function. More recently his laboratory has focused on analyzing zebrafish motility mutants and has made great progress in a short time, discovering the molecular basis for several motility mutants including sofa potato (acetylcholine receptor delta subunit), relaxed (dihydropyridine receptor), and twitch once (rapsyn). Moreover, these studies have yielded new insight into the roles of the receptor and rapsyn in structuring the synapse. Dr. Paul Brehm's laboratory is part of a tight knit group of five independent researchers with distinct but related interests and shared microscopy and molecular facilities that create an environment with ample resources to carry out the proposed research.

DESCRIPTION (provided by applicant): Autism Spectrum Disorders (ASDs) are currently estimated to impact 1-2.6% of children world-wide, representing a steep rise in ASD prevalence with annual costs to the United States alone calculated at $126 billion (2012). Because known therapies are less effective with increasing age of diagnosis, addressing outstanding questions about ASD etiology is a research priority. In the more common mammalian models however, embryonic stages are inaccessible therefore embryogenesis presents a major gap in our understanding of ASD etiology. To address this gap, we propose to generate zebrafish ASD models to focus explicitly on functional consequences in embryos of mutations known to cause ASD. Rather than investigate social behaviors typically used to define ASD, we focus on internal phenotypes (endophenotypes) of neuroanatomy and physiology. Following this strategy, our preliminary data from morpholino knockdown experiments demonstrate common phenotypes when either of two distinct ASD-linked genes, SHANK3 and SYNGAP1, is knocked down in zebrafish. Common phenotypes include developmental delay and seizure-like behaviors. These seizure-like behaviors are likely explained by dramatic reductions in the numbers of inhibitory GABAergic neurons in both morphant models. Developmental delay, seizures, and reduced markers of GABAergic signaling are also characteristic of individuals with ASD. To follow up on these preliminary studies we propose to generate stable gene knock-outs of zebrafish shank3 and syngap1. By creating and analyzing stable mutant lines with respect to the development of GABAergic brain circuits, our goal is to determine the developmental mechanisms that underlie GABA deficits. In the long-term, these zebrafish ASD models can also serve as the basis for the discovery of therapeutic targets and environmental risk factors.

The digestive distress that commonly accompanies Autism Spectrum Disorders (ASDs) significantly degrades the quality of life of those affected and their families. ASDs are currently estimated to impact 1 in 88 children, and yet the co-occurring gastrointestinal (GI) distress is understudied, having only recently been recognized by the medical establishment; in fact, there are currently no unified treatment strategies for ASD-linked GI distress. Our goal is to identify the mechanisms that underlie GI distress in ASD as a means to suggest effective therapeutic strategies. To do this, we have developed zebrafish models of one of the most prevalent genetic forms of ASD, Phelan McDermid Syndrome. This syndrome's GI symptoms include diarrhea, reflux, and cyclical vomiting. Phelan McDermid Syndrome is known to be caused by loss of one copy of the SHANK3 gene, a condition our team has been able to replicate in zebrafish. Our zebrafish model provides an innovative way to determine the mechanisms by which shank3 mutations are related to the symptoms of GI distress. Zebrafish have two unique characteristics that make them an ideal model for this work: their larvae are transparent, allowing us to see their GI function in process, while they are still alive; and we can study the development of regulatory circuits to identify those shank3 mutant tissues that produce GI distress. In addition, zebrafish and human nervous systems and GI tracts are remarkably similar, suggesting that the mechanisms we discover will be applicable to both. The zebrafish's small size and aquatic habitat also support targeted pharmacological screens to test therapeutic strategies. In aim one, we test how the shank3 mutations that cause Phelan McDermid Syndrome affect the development of tissues known to regulate GI function. In aim two, we test whether hormones and neurotransmitters known to regulate GI function can improve digestion in the shank3 mutant fish. There is a tremendous need to address GI distress in ASD. This project will improve our understanding of this problem and will pave the way for developing solutions.