Rebecca D. Burdine - US grants

Project Summary: All vertebrates have internal asymmetries along the left-right (LR) axis, such as the asymmetric placement of organs about the midline. Correct asymmetric placement of organs is critical for their function, as it allows for proper connectivity within the developing vasculature. While many genes implicated in LR patterning have been identified, we do not yet know how the LR axis is established or patterned, how the LR axis is aligned with the dorsal-ventral or anterior-posterior axes, or how organs obtain their final asymmetric positions. This proposal focuses on two zebrafish mutants, switch hitter (swt) and non-partisan (npt), with distinctive LR patterning phenotypes. swt mutants result in either wildtype or completely reversed organ patterning. Consistent with this phenotype, swt mutants often display right-sided expression of nodal components. We hypothesize that swt functions upstream of nodal to bias the directionality of the LR axis, npt mutants properly establish asymmetric nodal expression, but asymmetric organ morphogenesis is affected. We hypothesize that npt functions downstream of nodal to affect organ asymmetries. We will explore the potential roles for Swt and Npt in two mechanisms implicated in LR patterning: cilia formation and movement, and lateral plate mesoderm cell polarity. We will image heart morphogenesis in live wildtype and npt and swt mutant embryos to lay the foundations for understanding asymmetric organ positioning and the role of npt in this process. We will determine the subcellular localization for Swt and Npt and use mosaic analysis to determine their sites of function. Finally, we will use tandem affinity purification and two-hybrid analysis to identify proteins that interact with Swt and Npt. Relevance: It is estimated that as many as 1/5000 infants are born with LR patterning defects which are often manifested as congenital heart disease (Casey 2000). In this proposal, we plan to characterize the roles of two zebrafish mutations in LR patterning. Since many genes involved in LR patterning have conserved roles in humans, the proposed studies will enhance our understanding of LR patterning and the genetic basis of laterality defects that can result in congenital heart disease

DESCRIPTION (provided by applicant): Cilia driven flow is a key element in widely accepted models for left-right patterning. Yet we still do not know how cilia or cilia-driven flow give riseto the asymmetric expression of nodal. The long-term goal is to determine how the LR axis is established and how the asymmetric information generated is utilized to direct organ morphogenesis. The next step towards realizing this goal is to identify how signaling downstream of cilia establishes asymmetric nodal expression. The central hypothesis underlying this proposal is that cilia-driven fluid flow in Kupffer's vesicle (KV) signals through he PC1L1/PC2 channel complex to repress transcription of charon on the left side of the embryo. This hypothesis is based in part on preliminary data demonstrating that the zebrafish nodal gene southpaw (spaw) is not expressed in cells lining KV and is thus not the direct target of signaling downstream of flow. Instead, loss of PC2 activity affects the asymmetric expression of the Nodal inhibitor charon at KV. PC2 is thought to be the flow sensor in LR patterning, suggesting that charon is the target of flow-generated signaling. Thus, charon expression at higher levels on the right side of the embryo would inhibit Spaw signaling on the right, allowing Spaw to signal preferentially to the left side of the embryo. The rationale for this project is tha upon completion, this work will have provided the missing links between cilia and the control of asymmetric nodal expression. This information is crucial to fully understand how signaling downstream of cilia may participate LR patterning, disruptions in which cause congenital heart disease (CHD). The central hypothesis will be tested through three specific aims: 1) Determine how signals downstream of the non-specific cation channel PC2 regulate charon asymmetry. 2) Determine if PC1L1 signals are required to regulate charon asymmetry. 3) Identify the transcriptional mechanisms that generate asymmetric charon expression. In Aim 1, the localization and activity of PC2 required for generating charon asymmetry will be explored. Calcium imaging with KV specific promoters will be used to determine if PC2 is creating calcium signals in response to flow at the right place and time to affect charon expression. In Aim 2, the role for PC1L1 in zebrafish will be confirmed. Experiments to test whether the C-terminal tail is cleaved in response to flow in order to regulate transcription will be tested. In Aim 3, the smallest enhancer fragment capable of driving asymmetric expression of charon will be determined and used to identify transcription factors and signaling pathways involved in asymmetric charon expression. The proposed research is significant because it is the first step towards understanding how flow influences signaling that participates in establishment of left-right patterning. The approach is innovative, as it is a directed approach utilizing the tools and genetics available in the zebrafish system, to tackle a difficult but critical question in left-rigt patterning that is not currently being explored fully. Ultimately this work will identify signaling networks participating in the establishment of the LR axis and will provide new targets to investigate as factors underlying CHD. PUBLIC HEALTH RELEVANCE: The proposed research is relevant to public heath because our identification of the signaling pathways involved in establishing left-right patterning will provide important new targets to investigate as factors underlying congenital heart disease (CHD). As surgical intervention improves, there is a growing need to identify the important causative factors for CHD, so that adults born with CHD can receive appropriate health care and proper testing to determine their risk of having a child with CHD. Thus, the proposed research is relevant to the NIH's mission to develop the fundamental knowledge needed to extend healthy life and reduce the burdens of illness.

How cells are arranged in three dimensions to produce functional organs such as the heart is a fundamental question in biology that has not been fully addressed. Once cells are instructed to become heart tissue, they must coordinate their movements to organize into a multi-layered functional structure. One critical aspect of this process is how cells arrange differently on the left versus the right side of the heart, commonly referred to as left-right patterning. Previous work has determined that the FoxH1 transcription factor plays an important role in integrating multiple TGFbeta signals in heart cells so that they respond appropriately to left-right patterning information. Utilizing the powerful genetics available in the zebrafish system, the roles of FoxH1 in coordinating TGFbeta signaling and directing left-right cell migrations in the developing heart will be explored. The outcome of this work will be a better understanding of how the heart forms, especially with regards to left-right patterning. This information will have significant impact on stem cell studies aimed at producing functional organs for transplant. This project is well suited to be conducted by graduate students and undergraduates and will be the framework for recruiting women and underrepresented minorities into research through thesis work at Princeton and through the Summer Research Experience program conducted by the Department of Molecular Biology.

? DESCRIPTION (provided by applicant): Our work is designed to provide fundamental understanding of RASopathies, a large class of developmental abnormalities caused by the germline mutations in components of the RAS/MAPK pathway. Patients with these conditions display a broad spectrum of phenotypes, including heart defects, short stature, facial dysmorphisms, and neurocognitive delays. Whole genome sequencing of RASopathies provides new sequence variants in the well characterized components of the RAS pathway, but the functional consequences of these sequence variations is poorly understood. We will quantitatively characterize the functional and phenotypic consequences of the activating mutations in MEK, a core component of the RAS pathway. Focusing on the same group of mutations, we will first determine their effects on the regulation and enzymatic activity of MEK (Aim 1). This will be done using mass spectrometry-based kinetic assays with purified proteins. Next, we will quantify the consequences of activating mutations for the kinetics of RAS signaling in vivo (Aim 2). This will be done using a quantitative imaging approach, with which we will monitor RAS signaling in Drosophila embryos. Finally, we will determine how the same mutations influence RAS-dependent tissue morphogenesis (Aim 3). This will be done using live imaging experiments in zebrafish, focusing on heart development as a model of a morphogenetic event that is commonly affected in RASopathies. Our studies should provide mechanistic insights into the biochemical and morphogenetic effects of mutations identified in a large group of human developmental abnormalities and highlight the importance of using multiple models and quantitative approaches for answering a specific biological question.

? DESCRIPTION (provided by applicant): For the past 15 years, the Eunice Kennedy Shriver National Institute of Child Health & Human Development has organized ten Structural Birth Defects Meetings with the goal of bringing together investigators funded through the NICHD Birth Defects Initiative. The overriding goal of these meetings has been to foster a collaborative environment for the exchange of research findings to enhance the translation of basic knowledge and insights from functional genomic studies into the development of new, innovative, and efficacious strategies for the molecular diagnosis, treatment, and prevention of human structural birth defects. Funded investigators spanned multiple disciplines and included developmental biologists, cell biologists, biophysicists, clinicians, genomicists, geneticists, epidemiologists, and biostatisticians and bioinformaticians. At any given meeting, 50-60 investigators attended as well as relevant NIH staff. At the 10th Structural Birth Defects Meeting in December 2014, a decision was made to transition the planning of future meetings from NICHD staff to the structural birth defects research community opening the way for attendance by others from the research community and, in particular, trainees. This application seeks funding for the 11th Structural Birth Defects Meeting to be held April 3-5, 2017 in Bethesda, MD at the Federation of American Societies for Experimental Biology (FASEB) Conference Center under the sponsorship of the Society for Developmental Biology (SDB). The intent is to more than double the size of the meeting while maintaining the interactive, multidisciplinary nature of the presentations and discussions. There are no concurrently running sessions and all participants are expected to attend all the presentations. The format of the meeting consists predominately of short presentations of hypothesis driven findings grouped into several topic areas followed by discussion. As has become the tradition, a keynote speaker is invited from an area not necessarily mainstream to birth defects research but of high relevance to the field thus stimulating discussion and exchange of new ideas. A second speaker is invited to introduce a topic which will then be discussed more formally by a roundtable panel as well as all participants. For the first time poster sessions will be held to facilitate participation of as man trainees as possible. These will be held during extended lunch breaks on two days allowing for adequate participation by all. Informal evening activities will engage senior scientists and trainees to encourage mentoring and networking.

Abstract/Project Summary Idiopathic scoliosis (IS) affects 3% of children worldwide, yet the underlying cause(s) of this condition are poorly understood. Adolescent IS (AIS) commonly develops during the rapid growth phase in adolescence, leading to disfigurement, reduced pulmonary functions and chronic pain. We recently demonstrated that defects in cerebrospinal fluid (CSF) flow underlie the development of AIS in a zebrafish model. Defects in ptk7, mutations in which are causative of IS in humans, or in genes important in cilia motility, disrupt CSF flow in zebrafish. This results in the formation of scoliotic curves during a rapid growth phase in juvenile zebrafish, mimicking the onset of this disorder in humans. We demonstrate that prevention of scoliosis in this model can be achieved by expressing wildtype genes products only in motile ciliated lineages in zebrafish. Importantly, we have used a temperature sensitive cilia motility mutant to demonstrate that zebrafish can recover from the onset of scoliotic curves if cilia motility is restored in a critical time window. In this proposal, we will take the next logical step by investigating how CSF flow is sensed and how the fish responds to this information. We will investigate the role of ciliated CSF sensing neurons in the spinal canal in the development of scoliotic curves in our IS model. We will determine if the recently identified POC5 gene in human IS causes defects in CSF generation or sensation as our model would predict. We will characterize the spatial requirements for motile cilia in our model, and determine if the immune response we detect impacts spinal curve progression. At the conclusion of this work, we will have needed insights into the mechanism underlying the development and progression of spinal curvatures in response to altered CSF flow. This information is crucial for exploration of strategies to limit, or prevent, human AIS.

Summary Advances in molecular genetics have exacerbated the growing gap between clinical and basic genetics. A great deal of informative data is being generated through the exome and genome sequencing of patients with suspected genetic syndromes. However, without functional follow through or application in basic science labs, much of these data are wasted. Working to bridge the artificial divide between human mutations and basic developmental biology has the potential to significantly advance both disciplines. Informative patients can provide insight into new genes and variants that lead to structural birth defects, and expose novel pathways of normal and abnormal human development. Functional analysis of variants of uncertain significance generated by clinical testing can be the key to proving or disproving their pathogenicity. In this study we combine the unique training and resources of a medical geneticist with specialty training in pediatrics, clinical genetics, and molecular genetics (Bhoj), with the novel techniques and skills of a highly successful basic developmental biology researcher (Burdine). The foci of this collaboration are RASopathy spectrum syndromes, Noonan syndrome and related disorders. RASopathies are one of the leading single-gene causes of congenital cardiac and craniofacial malformations, and the genes involved have important roles in neurodevelopment, cancer predisposition, and cardiomyopathy. Despite their vital role in both the clinic and laboratory, about one third of patients with a clinical diagnosis of a RASopathy disorder have no mutations in known genes. There is a wealth of knowledge in gene discovery in these patients, functional analysis of new variants in known genes, and application of that knowledge to developmental biology. Our hypothesis is that sequencing of a highly informative patient cohort will lead to the discovery of novel RASopathy genes, and variants in these genes can be rapidly interrogated through a zebrafish system, which will lead to insights into basic developmental biology and the significance of clinical gene variants. We will utilize the infrastructure in place at the Children's Hospital of Philadelphia (CHOP) to perform exome sequencing on selected cohort of patients that have both craniofacial and heart defects, hallmarks of RASopathies. Indeed, two causative RASopathy genes, PPP1CB and RRAS2, have been identified with this approach by Dr. Bhoj and colleagues. Dr. Burdine's group at Princeton University have experience modeling RASoapthies in the zebrafish, and will use this expertise to evaluate any variants in known and new RASoapthy genes identified by Dr. Bhoj's group. By utilizing the recent advances in homologous recombination in zebrafish, new models will be generated to further explore the underlying biology of craniofacial and heart defects cause by identified genes. The researchers will meet frequently, and have developed a collaborative plan to enhance each other's knowledge base and develop additional collaborations between CHOP and Princeton University. At the end of the project period, we anticipate having generated sufficient preliminary data to support a joint R01 application to further these studies.