Johann K. Eberhart, Ph.D. - US grants

DESCRIPTION (provided by applicant): A vast number of craniofacial dysmorphologies arise in humans; however, little is known about the normal development of the neurocranium and how these dysmorphologies occur. The research proposed here will broaden the understanding of neurocranial development using the zebrafish as a model system. First, cell-lineage tracing will be used to determine the origins of the neurocranium. This analysis provides a basic tool for understanding neurocranial development and gives inroads to the cellular etiology of dysmorphogenesis. Second, loss- and gain-of-function experiments will be carried out to determine the role of Shh in specifying the anterior cartilages of the neurocranium. Third, cell transplantation experiments will be performed to determine if mutations in Shh effector genes act cell autonomously or non-cell autonomously in development. Last, a forward genetic screen will be used to discover new mutations that affect the development of neurocranial cartilages.

[unreadable] DESCRIPTION (provided by applicant): There are hundreds of craniofacial diseases in humans and cleft palate is common among these. The goal of this proposal is to elucidate the signaling interactions and cellular behaviors underlying palatogenesis. The zebrafish provides a useful model system in which to study palatal development. Powerful genetic and cellular techniques are available in the zebrafish for studying gene function as well as cell and tissue signaling interactions. Additionally, the simplified palatal skeleton, consisting of far fewer neural crest palate progenitors than in mammals, and the optic clarity of the zebrafish embryo makes it ideal for analyzing cell behaviors occurring in palatogenesis. I propose to examine predictions of a reciprocal signaling hypothesis, in which signals from neural crest to the oral ectoderm and then back from the oral ectoderm to neural crest induce palatogenesis, and cause elongation of the palate through cell intercalations. In Specific Aim 1,1 examine the role of candidate genes for neural crest-derived signals and oral ectoderm response genes, turned on in the oral ectoderm. I use loss-of-function, gene expression, imaging, and genetic mosaic analyses to test the model that FgflO and Bmp4 signaling from the neural crest turns on pitx2 in the oral ectoderm, which, in turn, promotes palatogenesis. In Specific Aim 2,1 analyze the reciprocal signal, from oral ectoderm to neural crest. I use loss-of-function, imaging, and genetic mosaic analyses as well as construction of inducible transgenic zebrafish lines to test the prediction that Pdgf and ph/ephrin signaling from the oral ectoderm promotes palatogenesis. In Specific Aim 3,1 determine the cell behaviors that drive elongation of the palate. I use confocal time lapse analysis as well as cloning and characterization of novel zebrafish palate mutants to test the prediction that cell intercalations drive the extension of the zebrafish palate. The results I obtain during the course of these studies will shed light on the genetic and cellular causes of cleft palate. Additionally, two genes I propose to analyze, pitx2 and ephrin-B1, are known to be human craniofacial disease genes. Therefore, my analyses of these genes will provide direct insight into the cause of human disease. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Craniofacial diseases are some of the most common of human birth defects and can be extremely variable in their severity and extent. There are both genetic and environmental causes of craniofacial disease and it is likely that a large portion of disease variability is due to gene/environment interactions. It is our long-term objective to understand the mechanism of gene/environment interactions and how these interactions regulate disease severity. We have chosen to use Fetal Alcohol Syndrome (FAS) as a model of gene/environment interactions because FAS has variable craniofacial defects, has a known environmental cause (maternal alcohol consumption) and is clearly genetically regulated. However, we are lacking in our understanding of the genetic loci that control susceptibility to ethanol-induced craniofacial disease. We have utilized two innovative genetic screens to discover ethanol-interacting loci. Results from these screens demonstrate that the platelet-derived growth factor rector a (pdgfra) and ethanol-induced jaw hypoplasia (eih) loci interact synergistically with ethanol. While untreated pdgfra mutants have cleft palate, our first genetic screen demonstrated that ethanol-treated pdgfra mutants have profound and extensive craniofacial defects. Furthermore, ethanol-treatment causes palatal defects in pdgfra heterozygotes. We have shown that neural crest cells fail to migrate properly in untreated pdgfra mutants, but in ethanol treated pdgfra mutants and heterozygotes there is an increase in the amount of cell death. In a second genetic screen, we have found that ethanol-treated eih and Bone morphogenetic protein (Bmp) loss-of-function embryos have a jaw-loss phenotype similar to that in mutants that disrupt development of the anterior endoderm. Here, we determine the mechanisms for these interactions. In aim 1, we discover which Platelet-derived growth factor (Pdgf) family members regulate the severity and extent of craniofacial disease. In aim 2, we reveal the intracellular signaling events that are responsible for the separate migratory and protective roles that pdgfra plays in neural crest cells. In aim 3, we explore how eih interacts with the Bmp signaling pathway and we determine the extent to which ethanol disrupts endoderm development in eih and bmp morpholino injected embryos. Because of the conservation of gene function between zebrafish and humans, the results from our studies will provide key insights into the genetic loci that interact with the environment to modulate human craniofacial disease severity. )

? DESCRIPTION (provided by applicant): Ethanol is the most common teratogen and the leading cause of mental retardation. Fetal alcohol exposure can cause numerous birth defects, most commonly effecting the craniofacial skeleton and nervous system. Fetal Alcohol Spectrum Disorder describes the full range of potential ethanol-induced birth defects and has been estimated to have a prevalence of 10 in 1000 births. The timing and concentration of fetal alcohol exposure are important determinants of FASD phenotypes. There also appears to be genetic susceptibility to FASD, yet we know almost nothing about the nature of these susceptibility loci. The zebrafish embryo is particularly useful for genetic screening and, here, we propose genetic screens to identify and characterize loci that may underlie the facial and neural defects associated with FASD. In Aim 1, we screen zebrafish mutants ethanol-induced facial defects. In Aim 2, we determine the neural and behavioral outcomes of these gene- ethanol interactions. In Aim 3, we characterize the interaction between the mTOR and Hsp90 pathways in the etiology of ethanol-induced defects. Because of the conservation of gene function between zebrafish and humans, the results from our studies will provide key insights into the genetic loci that interact with ethanol to cause FASD.

? DESCRIPTION (provided by applicant): Ethanol is the most common teratogen and the leading cause of mental retardation. Fetal alcohol exposure can cause numerous birth defects, most commonly effecting the craniofacial skeleton and nervous system. Fetal Alcohol Spectrum Disorder describes the full range of potential ethanol-induced birth defects and has been estimated to have a prevalence of 10 in 1000 births. The timing and concentration of fetal alcohol exposure are important determinants of FASD phenotypes. There also appears to be genetic susceptibility to FASD, yet we know almost nothing about the nature of these susceptibility loci. The zebrafish embryo is particularly useful for genetic screening and, here, we propose genetic screens to identify and characterize loci that may underlie the facial and neural defects associated with FASD. In Aim 1, we screen zebrafish mutants ethanol-induced facial defects. In Aim 2, we determine the neural and behavioral outcomes of these gene- ethanol interactions. In Aim 3, we characterize the interaction between the mTOR and Hsp90 pathways in the etiology of ethanol-induced defects. Because of the conservation of gene function between zebrafish and humans, the results from our studies will provide key insights into the genetic loci that interact with ethanol to cause FASD.

Summary/Abstract Ethanol is the most common teratogen and the leading cause of mental retardation. Fetal alcohol exposure can cause numerous birth defects, most commonly effecting the craniofacial skeleton and nervous system. Fetal Alcohol Spectrum Disorder describes the full range of potential ethanol-induced birth defects and has been estimated to have a prevalence of 10 in 1000 births. The timing and concentration of fetal alcohol exposure are important determinants of FASD phenotypes. There also appears to be genetic and epigenetic factors underlying FASD, yet we know almost nothing about the interaction between these factors. The zebrafish embryo is particularly useful for these types of analyses. In Aim 1, we identify and characterize ethanol-sensitive gene modules and the ethanol-sensitive miRNAs that target them. In Aim 2, we develop tools to determine how miRNA activity alters cell behaviors. Because of the conservation of gene function between zebrafish and humans, the results from our studies will provide key insights into the interaction between genetic and epigenetic factors underlying FASD. !

Alcohol (ethanol) exposure during pregnancy is a well-recognized cause of birth defects and central nervous system disturbances that lead to cognitive and behavioral problems across the lifespan. Although clusters of physical features, in particular those involving the craniofacies, and neurobehavioral symptoms define fetal alcohol spectrum disorders (FASDs), there is considerable variation in the consequences of prenatal alcohol exposure. This variation impedes the accurate diagnosis of FASDs and confounds our complete understanding of the damage that can be caused by alcohol exposure. While some of the individual differences in the consequences of alcohol exposure are due to variations in the timing of exposure, genetic variability is a strong modifier of the effects of ethanol exposure. Elucidating the genetic factors that confer risk and resilience has been a slow process, usually accomplished by comparing ethanol?s effects among various strains of animals, or by candidate gene approaches. Here, we propose a cross-species genetic analysis, utilizing state-of-the-art whole transcriptomic sequencing (RNA-Seq), high-throughput CRISPR/Cas9 gene editing techniques, and genetic screening to drive the discovery of novel candidate genes that modify susceptibility to early gestational ethanol exposure. In Aim 1, RNA-Seq will be performed after ethanol or vehicle exposure in two closely related mouse strains that differ in their susceptibility to the teratogenic effects of ethanol. This experiment will reveal a number of genes that are differentially expressed in these ?at risk? and ?resilient? strains. Candidate genes are then refined and tested for significant associations with craniofacial and neuroanatomical dysmorphology, as well as neurobehavioral changes, using our zebrafish high-throughput screens, mouse MRI analysis (with Hammond) and mouse behavioral phenotyping. The dual species approach affords a highly conserved FASD model that is more relevant than studying either species alone. In Aim 2, we will perform an unbiased forward genetic screen in zebrafish to identify mutations that suppress the teratogenicity of ethanol. The roles of these genes will be tested in mice to identify conserved mechanisms of ethanol teratogenesis. These conserved genetic mechanisms of ethanol teratogenesis can then be tested by CIFASD members Foroud, Hammond, Mattson in human populations with prenatal ethanol exposure who vary in their craniofacial and neurobehavioral manifestations. Likewise, human whole-exome sequencing experiments proposed by Dr. Foroud will generate numerous candidate genes that will be tested and confirmed in our animal models for the purpose of identifying conserved teratogenic mechanisms. These highly translational studies will significantly contribute to our understanding of the genetic factors underlying the susceptibility to prenatal ethanol exposure, which may be used to improve diagnosis, treatment, and prevention of FASD, as well as provide insight into the teratogenic mechanisms of FASD.

Craniofacial dysmorphologies are some of the most common human birth defects and can be extremely variable in their severity and extent. Understanding, diagnosing and treating these dysmorphologies requires a detailed understanding of the normal genetic hierarchies, cell signaling and cellular interactions that drive the morphogenesis and integration of the multiple cell types within the craniofacial complex. Genetic and/or environmental perturbations that disrupt these normal processes can cause craniofacial dysmorphologies. Given that human craniofacial birth defects tend to be sporadic and non-syndromic, it is most likely that multifactorial perturbations are the most common cause of these defects. However, there are major gaps in our understanding of the normal processes in craniofacial development. Furthermore, we have very little understanding of how multifactorial interactions disrupt normal development. This R35 proposal will support the unique research program of Dr. Johann Eberhart?s lab. The signaling interactions mediating craniofacial morphogenesis are examined in the first program. The second program examines how craniofacial tissues integrate seamlessly with one another. The third program examines the environmental and gene-environment interactions that can disrupt craniofacial development. Dr. Eberhart is an extremely well trained and productive developmental biologist. He has been supported through various NIH-based mechanisms ever since he was an undergraduate student. He has authored 40 total publications, 28 since establishing his independent lab at UT Austin. He has established himself as a leader in the field of craniofacial development and has pioneering publications on muscle-tendon attachments, zebrafish palatal development and gene-environment interactions. Dr. Eberhart is routinely invited to present the work from his lab at national and international meetings. He is also regularly asked to provide scientific service, such as grant reviews and organizing/participating in scientific workshops. Dr. Eberhart is a tireless mentor. He not only ensures that his trainees research is top notch, but is also deeply engaged with their scientific careers. To date, every one of Dr. Eberhart?s trainees that have been eligible for fellowships have received at least one. Dr. Eberhart is also committed to increasing diversity in STEM fields and actively recruits promising young scientists from diverse backgrounds into the field.