Kryn Stankunas, Ph.D. - US grants

DESCRIPTION (provided by applicant): Hearts defects are the most common congenital defect and a major contributor to heart disease. Frequently, congenital heart diseases are diagnosed late in their progression, when treatments become temporary and have significant side effects. The applicant's long term goal is to establish an independent laboratory leveraging discoveries about the molecular basis of heart development into improved diagnostics and therapies for heart disease. During the remainder of his postdoctoral training at Stanford University, coursework in lab management, study of cardiovascular biology, laboratory research, and publication of manuscripts will support the applicant's transition to independency. Mentored and independent research will focus on the applicant's discovery that endocardial BAF chromatin remodeling complexes have remarkably specific roles in two heart regions. In the ventricles, the BAF complex determines the extracellular matrix required for morphogenesis of muscle cells into trabeculae by repressing transcription of a matrix protease, ADAMTS1. This regulation appears to be dynamic, as later in development ADAMTS1 expression increases to prevent excessive trabeculation. At the endocardial cushions that develop into valves, the BAF complex regulates an endocardial-to-mesenchymal transformation (EMT) that gives rise to cushion-populating cells. I hypothesize that endocardial BAF complexes establish regulatory "switches" at key loci to control developmental events in different regions of the heart. In the ventricles, I propose the BAF complex is recruited to ADAMTS1 by specific cooperating factors to induce dynamic changes in nucleosome organization to repress transcription. In the cushions, I hypothesize the BAF complex regulates transcription of secreted regulators of Wnt signaling. Misexpression of these factors in the absence of the BAF complex induces a premature and ectopic activation of Wnt that blocks EMT. These hypotheses will be pursued using two Specific Aims: 1) Determine if microenvironment changes regulate cell signaling in the ventricles. Describe cis-and Trans acting factors that recruit the BAF complex to ADAMTS1. Delineate nucleosome modifications that cooperate with the BAF complex to repress ADAMTS1. 2) Determine if inhibiting Wnt signaling restores EMT in embryos lacking endocardial BAF complexes. Describe the expression of Wnt regulating transcripts as potential targets of the BAF complex in endocardial cushions.

DESCRIPTION (provided by applicant): Semilunar valve (SLV) diseases, including bicuspid aortic valves (BAV), are remarkably common and yet their genetic and developmental origins are poorly understood. Likewise, it remains unclear how disrupted embryonic valve development progresses into overt valve disease. Our long-term goal is to understand how gene regulation drives sequential developmental processes that ultimately produce complex, patterned valves and how these processes go awry in SLV disease. These gene regulatory events require transcription factors to interface with a chromatinized genome, suggesting that chromatin regulators are key components of SLV developmental networks. One important event is an endocardial-to-mesenchymal transformation (EMT) that occurs early in valve development to populate endocardial cushions (ECs), including the proximal outflow tract (pOFT) cushions that contribute tissue to SLVs. Our objectives are to 1) understand how chromatin remodeling integrates with cell signaling during EMT, and 2) determine mechanisms by which disruptions of valve development progress into diseased SLVs. Our central hypothesis is that endocardial Brg1-associated factor (BAF) chromatin remodeling complexes interact with Wnt signaling effectors to promote pOFT EMT. As a result, when endocardial Brg1 is deleted a subtype of OFT mesenchyme is depleted. Without these cells, cusp overgrowth and fusion results in thickened and malpatterned SLVs, including BAV. The rationale for our efforts is that defining chromatin remodeling roles during EMT will shed light on how SLV disease originates. Further, our mouse models of SLV disease will enable an understanding of the cellular and molecular progression of valve disease. Our specific aims are: 1) Determine the molecular networks that the BAF complex interfaces with to direct EMT and 2) Determine mechanisms of SLV disease progression in mice lacking endocardial- lineage Brg1. In pursuit of the first Aim, we will compare cellular and molecular pOFT defects seen in unpublished genetic models disrupting Brg1 and Wnt signaling. We will apply a transformative new TU-tagging technology to define dynamic, endocardial transcriptomes dependent on each pathway. Using new cell culture approaches, we will test biochemical interactions between BAF, Wnt effectors, and chromatin in EC cells. For the second Aim, we will use genetic lineage tracing to determine contributions of EMT-derived cells to distinct SLV regions, define interactions between SLV mesenchyme sub-populations, characterize misexpressed transcripts that may drive SLV disease progression, and describe a new mouse model of adult SLV disease of potential utility in preclinical trials. Our proposed research uses novel technological and paradigmatic approaches to pursue unresolved questions of SLV development and disease. These contributions will be significant as they will shed light on the human genetics of SLV disease and inform regenerative medicine approaches. Our newly identified transcripts associated with a BAV model may represent biomarkers for disease diagnostics or therapeutic targets to prevent congenitally abnormal valves from becoming diseased.

PROJECT SUMMARY:  Organs  and  other  complex  tissues  ?know?  when  and  how  to  stop  growing  to  arrive  at  the  correct  size  and  shape.  Disruption  of  organ  size  control  mechanisms  can  lead  to  congenital  abnormalities,  poor  organ  homeostasis  and  tissue  repair,  and  tumors.  Adult  zebrafish  caudal  fins,  including  their  complex  skeleton  and  other  tissues,  perfectly  regenerate  to  their  original  size  and  shape  regardless  of  the  nature  or  position  of  the  injury.  Therefore,  zebrafish  fin  regeneration  provides  a  compelling  and  genetically  tractable  vertebrate  model  to interrogate organ size control mechanisms. Prevailing models for robust fin size regeneration speculate that  fin  cells  maintain  a  multitude  of  ?positional  identities?  that  somehow  instruct  different  degrees  of  outgrowth.  We  propose  a  distinct and straightforward model  that  neatly  explains  how  fin  size  and  shape  is  restored  without  invoking  molecularly  encoded  positional  information.  A  key  cell  population  at  the  distal  end  of  the  regenerating  fin  that  we  term  the  ?niche?  produces  Wnt  signals  that  promote  fin  outgrowth  by  sustaining  progenitor  cells.  We  identify  Dachsund  transcription  factors  as  novel  niche  markers  and  show  that  the  niche  uniquely  forms  from  intra-­??ray  mesenchyme  that  populates  the  inside  of  the  cylindrical,  differentially  sized,  and  progressively  tapered  fin  rays.  We  show  that  the  niche,  and  therefore  Wnt,  steadily  dissipates  as  regeneration unfolds; once exhausted, growth stops. As such, regenerated fin size is dictated by the amount of  niche  formed  upon  damage  ?  which  is  simply  dependent  on  the  availability  of  intra-­??ray  mesenchyme  and  hence  bone  width  at  the  damage  site.  This  ?transpositional  scaling?  model  suggests  that  macro-­??scale  fin  size  and  shape  is  determined  by  mesenchyme-­??niche  state  transitions  and  self-­??restoring  bone  geometry  rather  than  unique positional identities of individual cells. We will explore this paradigm and uncover underlying cell and  molecular  mechanisms  for  size  control  during  fin  regeneration  by  three  Specific  Aims:  1.  Define  intra-­??ray  mesenchyme / distal niche lineage cell states, transitions, and fates, 2. Determine signaling and transcriptional  mechanisms  maintaining  niche  state  and  function,  and  3.  Determine  niche  ?countdown  timer?  mechanisms  using longfint2 zebrafish ? which we show have a broken timer due to misexpression of the kcnh2a ion channel.  This insight suggests ion channels and Ca2+ signaling govern niche cell self-­??renewal. Our program will support  a  potentially  broadly  applicable  ?transpositional  scaling?  concept  with  exemplary  mechanisms  for  how  organ  size  and  shape  are  determined  by  dynamic  populations  of  tissue-­??resident  niche  cells.  Our  study  will  have  additional  human  health  impacts  since  1)  understanding  bone  regeneration  in  zebrafish  may  inform  regenerative medicine approaches for human bone disease, and 2) kcnh2a is the zebrafish orthologue of kcnh2,  which  is  commonly  mutated  in  long  QT  syndrome  and  encodes  a  protein  that  is  a  notorious  therapeutic  ?off-­?? target?. Our paradigmatic and diverse technological innovations will open up new directions and inspire other  scientists, broadening our project?s impact on both fundamental research and regenerative medicine.    

PROJECT SUMMARY The University of Oregon's long-standing predoctoral Developmental Biology Training Program's goal is to train rigorous, skilled, and innovative developmental biologists. Our trainees develop abilities to lead research programs of their own, communicate discoveries to other scientists and the public, and teach future generations of scientists. Our multi-faceted training equips students to become leading academic and non- academic scientists or achieve other influential research-related careers. Individualized research training within one of many active and diverse laboratories is the core of our program. Trainees' thesis research builds upon a continuously innovating curricular foundation. Core graduate-level courses are Molecular Genetics and Developmental Genetics and one of Developmental Neurobiology, Stem Cells & Regeneration, and Genomics Approaches. Additionally, all trainees take Advanced Biological Statistics. We surround research and coursework with a wealth of enhancing and broadening experiences. Examples include first year rotations, required teaching, a dissertation advisory committee, journal clubs, a monthly interest group, annual student research reports, and interactions with visiting speakers. We offer various career development activities especially to support students interested in non-academic careers. A unique highlight is our annual trainee-organized Developmental Biology Training Program symposium, where trainees host leading scientists to share their research on a topic of the students' choosing. We request continued support for seven predoctoral positions within a program that includes 22 highly collaborative, productive, and well-funded labs dedicated to graduate training. We have expanded the reach and vitality of our research and training with six new Assistant Professors, all of whom are top recruits and represent diverse areas of developmental biology research. They join the DBTP's internationally respected senior faculty to position us to maintain our outsized record of innovative research and training. The DBTP unites faculty and trainees from two Departments (Biology or Chemistry & Biochemistry) and four Institutes (Institute of Molecular Biology, Institute of Neuroscience, Institute of Ecology and Evolution, Oregon Institute of Marine Biology). Trainees are exposed to research across Institutes due to a rich tradition of collaboration, common training activities, the close proximity of most labs, and outstanding core facilities. As such, our program fosters interdisciplinary training that bridges genetics, genomics, molecular biology, cell biology, computational biology, neuroscience, and evolutionary biology. This breadth complements the focused project- oriented training students receive in their thesis labs, producing creative and confident scientists empowered to direct impactful research programs or assume other science leadership roles.