Tag: Kryn Stankunas

Determining roles of Wnt signaling in mammalian heart valve development

Presenter: Andrew Mckay, Biology

Poster: C-2

Mentor: Kryn Stankunas, Institute of Molecular Biology

Heart defects occur in 2% of live births, and of these, valve defects are the most common. By studying normal heart valve development we hope to find the genetic causes of these defects. Our primary question is: what signals direct the remodeling of embryonic heart valves into thin, elongated leaflets? We hypothesize that local Wnt signals direct heart valve remodeling by regulating cell proliferation and morphogenesis through activation of the NFATc1 transcription factor. To accomplish this, we use transgenic mice that allow us to block Wnt signaling during narrow time windows of embryogenesis. We inject transgenic mice with doxycycline, a molecule that causes the transgenes to temporarily express the Wnt antagonist Dkk-1 and inhibit Wnt signaling in particular cell types. I helped determined which of three transgenic lines best inhibits Wnt signaling and produces the most consistent and robust phenotype for valve defects. This line will be used for future experiments in which we will stain the sections for various cell type markers, indicators of proliferation, and localization of the transcription factor NFATc1. We will then use these results to determine if Wnt signaling affects cell proliferation and NFATc1 localization in targeted cell types in the developing valve.

The Role of Chromatin Remodeling Complex in Valve Development

Presenter: Maithri Sarangam

Mentor: Kryn Stankunas

AM Poster Presentation

Poster 43

Congenital heart diseases are one of the most common types of birth defects. Studying the development of this complex organ may provide insight into the causes of such defects. It has been shown that chromatin remodeling complexes play a role in directing developmental processes. They affect expression of genes by modifying chromatin, the DNA-protein complex in the nucleus. One particular chromatin remodeling complex, the BAF complex, has been shown to be important in heart development. We believe the BAF complex is required during the remodeling and elongation phase of aortic valve development. The aortic valve controls blood flow from the heart through the aorta. We used mouse models and complex genetic techniques to study the role of the BAF complex in mammalian heart valve development. Using a crelox system, we induced a loss of function of the BAF complex in endocardial cells, which make up the lining the heart, by knocking out Brg1, the key ATP-ase required for the complex to function. We then used various histological and immunofluorescence stains to study the resulting phenotype at different time points during the elongation and remodeling phase. The aortic valve phenotypes in the mutant embryos deviated from that of their wild type litter-mates, suggesting that the BAF complex does have a role in this phase of valve development.

The Role of the BAF Chromatin Remodeling Complex during Heart Valve Development

Presenter : Maithri Sarangam

Mentor : Kryn Stankunas

Major : Biology

Poster 56

Human heart valves are remarkable structures that open and close billions of time during a lifetime. Like any structure under constant mechanical strain, their shape and molecular composition are finely tuned to maximize efficiency and longevity. The development of these valves must also be finely tuned to produce proper the shape and composition, or dangerous health consequences may arise. We are studying the developmental processes of valve formation. Particularly, we are interested in the role of the BAF chromatin remodel- ing complex. Chromatin refers to the complex of DNA and histone proteins. Cell nuclei contain a series of cylindrical histone com- plexes, around which DNA is wrapped. The structure resembles a single thread wrapped around a series of spools with approximately
2 loops per spool. The BAF complex regulates gene expression by altering nucleosome positioning. We believe the BAF complex is required for the proper development of the aortic and pulmonic valves. We used mouse models and complex mouse genetic techniques to study the role of the BAF complex on mammalian heart valve development. Using a crelox system, we caused a loss of function of the BAF complex by knocking out Brg1, the key ATP-ase required for the complex to function. We then used various staining methods to study the resulting phenotype at different time points during the later stages of development. The results suggest that the BAF complex is required for proper organization of the valve shape and molecular composition.

Tracing the Endocardial Cell Lineage of Developing Heart Valves Using MADMMatter Pools?

Presenter : Amy Jones

Mentor : Kryn Stankunas

Major : Biology, Human Physiology

Poster 15

Heart valve development is a complex, multi-step process. During development, endocardial cushions form at specific locations in
the early heart tube. These cushions are populated by endocardial cells that delaminate in a process known as epithelial-mesenchymal transformation (EMT). In the mouse, EMT takes place between embryonic day 9.5-10.5. It is a vital process to understand since the heart valves are derived from this cardiac cushion tissue. Current models of cushion EMT suggest that a large number of endocardial cells undergo EMT. An alternative hypothesis is that only a few individual cells initially populate the cushions that then proliferate to expand cushion mesenchyme.To better understand how endocardial-derived cells contribute to heart valves, I am using a novel mouse genetic system termed MADM (mosaic analysis with double markers). MADM uses the Cre-lox system to permanently label specified cells by fluorescent protein expression. For my studies, I use the Tie2 promoter to direct Cre expression and instruct the MADM system to specifically trace the endocardial cell lineage. The low frequency by which labeled cells are generated allows me to exquisitely moni- tor contributions of clonally-related endocardial cells to developing valves. By gaining a complete understanding of the contribution of endothelial-lineage cells to developing valves, we can identify when embryonic heart malformations originate, supporting the development of therapeutics to prevent defective valves from progressing to a diseased state.

Opposing Roles of Wnt and BMP signaling in Zebrafish caudal fin regeneration

Presenter: Alan Gomez (Biology)

Mentor: Kryn Stankunas

Oral Presentation

Panel B: “Vertebrate Expression” Walnut Room

Concurrent Session 3: 1:45-3:00pm

Facilitator: Chris Moe

Zebrafish and many other vertebrates possess an incredible ability to regenerate damaged or amputated body parts including the retina, spinal chord, fins and limbs. The zebrafish caudal fin is a widely studied system because of its simple anatomy, accessibility, and robust ability to completely and consistently regenerate after amputation in under two weeks. Regeneration of the zebrafish caudal fin is dependent upon the formation of a proliferating mass of cells that grow at the amputation site called the blastema. The cells that line the bone rays in the mature zebrafish contribute to the blastema along with other cell types and are responsible for rebuilding the lost bone. These osteoblasts become activated after a fin injury through dramatic changes in their genetic program. Two signaling pathways called Wnt and BMP are particularly important for inducing these genetic changes and for organizing the spatial localization of osteoblasts in the blastema. BMP and Wnt are proteins that cause changes in gene expression programs of the cells that they physically interact with at the cell surface. Proper expression of Wnt and BMP in the blastema is essential for regeneration to occur. By examining the genes activated by Wnt and BMP during regeneration we have concluded that the gene expression programs activated by BMP work to counteract those of Wnt signaling. Investigating this feedback between BMP and Wnt has helped to characterize the specific roles that these signaling pathways play during the process of bone regeneration.

Vascular Endothelial Growth Factor (VEGF) Signaling Contributions to Heart Ventricle Development

Presenter: Justine Nguyen

Faculty Mentor: Kryn Stankunas, Kate Karfilis

Presentation Type: Poster 79

Primary Research Area: Science

Major: Human Physiology

Cardiomyopathies are congenital heart diseases that affect the heart musculature, which could cause the heart to become weaker and pump less blood efficiently. The purpose of my research is to study the developmental programs that underlie ventricular trabeculation and the role vascular endothelial growth factor (VEGF) plays in regulating this process. VEGF plays a distinct role in direct signaling of angiogenesis along with the cardiac muscle formation and trabeculation in the ventricles. If the gene pathways for the development of trabeculation in the heart are understood, then in a disease state, appropriate remedies could be determined based on where the genes are expressed incorrectly. Currently, two possible hypotheses could explain VEGF signaling and its role in trabecular development. One hypothesis is that VEGF signaling is directly turning on a gene that directs VEGF signaling while the other hypothesis is that the two cell types (endocardial and myocardial cells) are directly interacting with each other due to VEGF signaling. In order to study trabecular development, pregnant mice are dissected when the embryos are developing the trabeculations. Embryos are processed so that their hearts are examined through various cellular biology techniques. A specific small molecule inhibitor, Cabozantinib is used in order to inhibit VEGF signaling, disrupting the formation of the trabeculae. A VEGF inhibited sample can be compared to an untreated wildtype sample to compare the differences in the trabeculation development.

Investigating the Fuction of a Novel Gene in Heart Valve Development

Presenter: Thomas Forman

Faculty Mentor: Fernanda M. Bosada, Kryn Stankunas

Presentation Type: Poster 62

Primary Research Area: Science

Major: Biology, Human Physiology

Funding Source: Huestis-McLean Memorial Scholarship Recipient, $450; Apex Scholarship, University of Oregon, $1000 per term

Congenital valve disease affects at least two percent of the world’s population, a remarkable frequency that underlines the urgent need to understand the etiology of these common birth defects. These underlying abnormalities may originate from disruption of embryonic valve development. Primordial heart valves proceed through complex signaling events to form thin, elongated leaflets/cusps. However, the roles of many of these signaling pathways in valve development remain incompletely understood. Previous research indicates that Receptor Tyrosine Kinase (RTK) signaling, commonly involved in cell proliferation, is activated during valve mesenchyme expansion. We found that Lrig1, an RTK negative regulator, is dynamically expressed in the valves throughout development. We observed high expression in the endocardium of the atrioventricular canal (AVC) during the first steps of valve development. During valve mesenchyme expansion and elongation, we detected Lrig1 at the distal end of both the AVC and semilunar (SL) valves. Using a transgenic mouse line to knock out Lrig1 function, we demonstrated that homozygous Lrig1 embryos have hypertrophic AVC valves, although we observed no such defect in the SL valves. Interestingly, mutant AVC valves are not overproliferative at 13.5 days post-fertilization. We hypothesize that Lrig1 negatively regulates endocardial-to-mesenchymal transformation (EMT) in early valve development. Fate-mapping of Lrig1+ cells will further elucidate the roles of Lrig1. Our findings are the first ever to describe a function for Lrig1 in the heart valves.

Investigating the Role of EZH2 in Heart Development

Presenter(s): Diana Nguyen − Biology, Human Physiology

Faculty Mentor(s): Gabriel Yette, Kryn Stankunas

Poster 48

Research Area: Genetics, Heart Development, Biology

Funding: O’Day Fellowship in Biological Sciences and the Office of the Vice President for Research and Innovation

Nearly 1% of individuals are born with a congenital heart defect (CHD), making CHDs the most common birth defect. Understanding the genetic and epigenetic underpinnings of heart development has the potential to aid in developing tools to diagnose and treat CHD. It is increasingly evident that chromatin structure and histone modifications play essentiaL roles in heart development and homeostasis. A histone modification of interest is the tri-methylation of lysine 27 of histone H3 (H3K27me3), which is associated with gene repression. This modification is catalyzed by EZH2, the methyltransferase component of Polycomb Repressive Complex 2 (PRC2), and can be removed by Kdm6 family of demethylases. Recent work in the Stankunas lab shows that disruption of Kdm6ba and Kdm6bb in zebrafish result in smaller, poorly trabeculated ventricles of the heart. Interestingly, hearts of mice with Cre/lox dependent knockdown of Ezh2 exhibit hyper-trabeculation, ventricular septation, thinning of the ventricular wall, and aberrant skeletal muscle gene activation. These studies highlight the proper maintenance of H3K27me3 necessary for proper development. Yet, the effects of EZH2 on heart development in zebrafish has not been well explored. Zebrafish are ideal for investigating the early stages of heart development since they are transparent, and develop outside the mother, allowing for easy observation of the heart during this crucial period; elements that evades the Cre/lox system in mice. We generated an ezh2-null allele allowing us to conduct heart development studies. We hypothesize that PRC2/EZH2 is necessary to establish and maintain cardiomyocyte cellular identity by repressing developmental pathways of similar tissue types. For this project, we aim to: 1) Establish when and where EZH2 is expressed in the heart during development, and 2) Determine morphological and functional changes in developing hearts of ezh2 null zebrafish.

Short Range Sonic Hedgehog Signaling Promotes Heterotypic Cell Interactions Underlying Branching Morphogenesis of the Zebrafish Fin Skeleton

Presenter(s): Joshua Braunstein – Biology

Faculty Mentor(s): Kryn Stankunas, Scott Stewart

Poster 83

Research Area: Developmental Biology

Funding: ESPRIT
IMB Summer Scholarship Award
Alden Award

Zebrafish remarkably regenerate severed fins, perfectly restoring their original size and branched skeletal pattern. Sonic hedgehog a (Shha)-expressing epidermal cells mediate ray branching during regeneration by guiding localization of the pre-osteoblasts (pObs) while migrating and splitting into two populations. However, mechanisms of shha induction, the splitting of shha+ epidermal cells, and the mechanisms underlying epidermal to pObs interactions remain unresolved. Towards answering these questions, we explored if Hh/Smo signaling and epidermal dynamics also underlie developmental ray branching. We found that shha is expressed initially in basal epidermal cells along the entire length of forming fin bones in juvenile fish. As bones progressively mature, shha becomes distally restricted to epidermal cells neighboring Runx2+ pObs. We used TgBAC(ptch2:Kaede) fish and photoconversion to show Hh/Smo signaling is restricted to these pObs and immediately adjacent epidermal cells. shha+ epidermal cells split into two groups immediately preceding ray branching. By live imaging, we found these basal epidermal cells migrate distally over the pObs, cease Hh/Smo signaling, and are then shed. Small molecule inhibition of Hh/Smo using BMS-833923 increased epidermal migration speed, suggesting Hh/Smo signaling typically restricts the rate of migration by adhering epidermal cells to the pObs. Additional small molecule trials show the pathway is largely dedicated to ray branching during fin development. We conclude that instructive shha+ epidermal movements and Shh/Smo-promoted adhesion between epidermal cells and pObs direct branching morphogenesis to pattern the fin skeleton during both development and regeneration.

Mutational Analysis of dach Genes During Zebrafish Fin Regeneration

Presenter(s): George Deardorff

Co Presenter(s): Bryson Ramona

Faculty Mentor(s): Kryn Stankunas & Scott Stewart

Poster 44

Session: Sciences

Following amputation, zebrafish fins, comprised of intricate skeletal rays and other tissues, perfectly regenerate to their original size and shape regardless of the nature or position of injury. A cell population observed in the regenerating fin, termed “niche”, produces Wnt signals that promote fin outgrowth. As a known transcriptional regulator of the niche, dach plays a role in maintaining proper regeneration. Depending upon the extent of regenerative demand, dach becomes enriched at the distal region of the regenerating fin, and is eventually downregulated once the fin has stopped regenerating. A mechanistic explanation for dach induction, in addition to a thorough understanding of how it regulates the niche, is lacking. To explore the role of dach we used CRISPR/Cas9 gene editing to mutate two isoforms of the dach gene, dachc and dacha. By utilizing high-resolution fin imaging, our data showed that dachc mutants display improper morphology, including: abnormal joint segmentation, fusion of rays, and trident-shaped branching patterns. Surprisingly, this observation was not seen during development, but rather only after amputation and subsequent regeneration. Further, while dacha single mutants exhibited normal regeneration, dachc;dacha double mutants died off at an appreciable rate during development. Our data shows a novel role that dach has in promoting correct branching patterns, in addition to its unique regulation in development versus regeneration. Demonstrating how the misregulation of certain genes like dach can lead to the disruption of growth control mechanisms is critical for understanding the basis of a range of diseases.