Kenneth D. Poss - US grants

DESCRIPTION: Myocardial infarction (Ml), the focal loss of cardiac muscle caused by ischemia, is the leading cause of death in this country. One reason why morbidity and mortality are intimately associated with Ml is that the human heart has an extremely limited ability to repair itself through regeneration of cardiomyocytes (CMs), the contractile cells of the heart. By contrast with mammals, the teleost zebrafish displays a highly efficient regenerative response to cardiac injury, able to renew up to 20% of resected ventricular myocardium. How new CMs originate during regeneration is poorly understood. The objective of this proposal is to exploit zebrafish as a model for understanding the cellular and molecular mechanisms of myocardial regeneration. Previous studies have documented how dedifferentiation, a programmed reduction in the specialized, functional components of a mature, differentiated cell, generates precursor cells during regeneration of the limb, tail, or lens in lower vertebrates. It is believed that the origin, proliferation, and patterning of these precursors are controlled by common embryonic signaling pathways. The overall hypothesis of our proposal is that myocardial damage in the zebrafish activates molecular programs that regulate a transition from contractile, non-proliferating cardiac muscle to regenerative CMs that proliferate and are patterned. To test this hypothesis, we propose three specific aims: 1) Define myocardial changes during regeneration This will include detailed studies of ultrastructural differentiation, gene expression, cellular proliferation, and cardiac function. We will also compare injury-induced myocardial gene expression between zebrafish and mice. 2) Determine the functions of Fibroblast growth factor (Fgf) signaling in myocardial regeneration. We will examine how experimental increases or decreases in Fgf signaling activity affect regenerative cardiogenesis, using new inducible transgenic lines that facilitate modulation of signaling through Fg receptors. 3) Identify the functions of retinoic acid (RA) signaling in myocardial regeneration. We wil generate and analyze transgenic lines that facilitate expression of a dominant-negative or constitutive! active RA receptor isotype. Our molecular genetic approach in zebrafish will provide a foundation fo understanding important regulatory mechanisms active during myocardial regeneration, and may lead t approaches for comprehending, and possibly enhancing, the limited regenerative response displayed b humans after Ml.

Adult teleost fish and urodele amphibians can regenerate entire amputated appendages. By contrast, regenerative healing of adult mammalian limbs is limited to the very tips of digits. One of the key challenges in developmental biology is to understand how and why tissue regeneration occurs. The hallmark of limb or fin regeneration is formation of a blastema, a mesenchymal structure that contains progenitor cells for new skeletal elements. As regeneration proceeds, blastemal cell proliferation and patterning are regulated such that lost tissues of correct size and shape are replaced. While the catalogue of defined cell dynamics and molecular factors in tissue regeneration is expanding, we know much less of how genes involved in regenerative events are engaged upon injury. That is, what are the DNA sequences that recruit activators (or repressors) of gene programs during tissue regeneration, how are these sequences distributed throughout the genomes of regeneration-competent animals, and what are the transcription factors that engage with these sequences? Recently, we identified a class of distal gene regulatory elements that preferentially activate gene expression during regeneration, can be engineered in simple constructs to express developmental factors that promote regeneration, and have the potential to be recognized by the transcriptional machinery of distant species. The overall goal of this proposal is to discover gene regulatory concepts and mechanisms that restore size and pattern to an amputated appendage. We will test the hypothesis that enhancer and silencer elements are crucial regulatory modules enabling appendage regeneration in zebrafish. This work will increase understanding of developmental regulation during vertebrate tissue regeneration, and provide important perspective for comprehending, and perhaps changing, the existing limitations in regenerative capacity of human tissues.

? DESCRIPTION (provided by applicant): Myocardial infarction (MI) is a common injury that causes permanent loss of hundreds of millions of cardiomyocytes (CMs), increasing susceptibility to heart failure and sudden death. Major goals of regenerative medicine are methodologies to enhance CM recovery after MI and to restore cardiac function to heart failure patients. Heart regeneration in its natural context builds muscle by proliferation of spared CMs, facilitated by influences of supporting cell types like epicardium, endocardium, vasculature, and inflammatory cells. The epicardial sheet covering the heart is activated by injury and aids muscle regeneration through paracrine effects and as a multipotent cell source, and has received much recent attention as a target in cardiac repair strategies. Yet, until recently, virtually nothing was known about epicardial homeostasis - whether and how these cells regenerate - in any species. This deficiency has presented a barrier to understanding the epicardium and developing it for therapeutic goals. In a new study, we established the capacity of the zebrafish epicardium to vigorously regenerate, and we identified influences on this process by the cardiac outflow tract and Hedgehog signaling. While this work broke new ground in adult epicardial biology, it simultaneously revealed new challenges. First, we understand little of the details by which epicardial cells tightly connected within a contiguous sheet respond to injury and/or mitogen sources. Second, our molecular understanding of epicardial regeneration, and how epicardial cells promote muscle regeneration, is primitive. Here, we will these address central issues in epicardial biology and heart regeneration, using cutting edge screening and analysis tools in zebrafish. 1) We will apply a new ex vivo model of epicardial regeneration and a panel of new transgenic reporters that visualize epicardial cell phases in real time, to define spatiotemporal proliferation dynamics of adult epicardial cells after cardiac injury. 2) We will harness single epicardial cell transcriptome data we have generated to define new epicardial markers, initiating our work with new reagents to define the role of the epicardial marker caveolin-1 in epicardial injury responses. 3) We will identify small molecule modulators of the epicardial injury response, using an ex vivo screening strategy and new transgenic tools to visualize and manipulate the epicardium. With these approaches, we will test the hypothesis that a vigorous epicardial injury response is critical for heart regeneration. By identifying key regulators of the dynamic epicardial injury response and its impact on muscle regeneration, our work will inform approaches for comprehending and enhancing the limited regenerative response displayed by humans after MI.

Myocardial infarction (MI) is a common injury that causes permanent loss of hundreds of millions of cardiac muscle cells, increasing susceptibility to heart failure and sudden death. Major goals of regenerative medicine are methodologies to enhance cardiomyocyte recovery after MI and to restore cardiac function to heart failure patients. Heart regeneration is limited in adult mammals, but occurs naturally in adult zebrafish and neonatal mice through the activation of cardiomyocyte division. Whereas the research community has identified several factors important for heart regeneration over the past decade, we still know little of the regulatory mechanisms needed to activate regeneration programs in injured cardiac tissue. In particular, questions of whether regulatory enhancer elements are employed, and how specific they are to regeneration, are virtually unexplored. This is a critical deficiency, as the identification and manipulation of such elements could both expand our understanding of regeneration and have applications in regenerative medicine. In a recent collaboration between our groups, we found evidence for tissue regeneration enhancer elements (TREEs) that trigger gene expression in injury sites and can be engineered to modulate the regenerative potential of vertebrate organs including the heart. Here, we propose a multi-PI project exploiting the strengths of the zebrafish and mouse model systems to delineate regulatory sequences that control regeneration programs, and to create TREE-based factor delivery constructs to optimize heart regeneration in higher and lower vertebrates. 1) We will define the cis-regulatory motifs and binding factors necessary for activity of a TREE that is linked to the zebrafish leptin b gene. 2) We will use this TREE to define effects of enhancer-delivered pro-regenerative factors after cardiac injury in zebrafish and mice, focusing initially on provision of mitogenic and angiogenic molecules. 3) We will use open chromatin profiling approaches to identify new TREEs that activate expression in endothelial and/or endocardial cell types during zebrafish heart regeneration. We will perform sequence comparisons in mice, and we will initiate a program to generate transgenic reporter and TREE deletion animals to test the sufficiency and requirements for these sequences in directing regeneration programs. With these approaches, we will test the hypothesis that cardiac injury activates regeneration enhancer elements to facilitate heart regeneration.

Primary and secondary damage caused by spinal cord (SC) injury permanently impairs sensory and motor functions. Developing means to treat and reverse SC injury remains a pressing need in regenerative medicine. Approximately 10,000 Americans suffer new SC injuries each year, requiring long-term therapeutic, rehabilitative, and psychological interventions. With an average age of 33.4 years at the time of injury, the population of SC injury patients is steadily increasing in the United States and around the world. By contrast with mammals, teleost fish are capable of efficient, natural recovery after SC damage. Following SC transection, adult zebrafish initiate a glial bridge that re-connects the SC. Bridging is a striking gial cell response that is thought to provide a natural scaffold for axon growth. Importantly, glial bridging occurs in zebrafish without the detrimental outcomes from reactive gliosis that is elicited in mammalian SC injury contexts. However, little is known about the cells that construct the bridge and the molecules that enact bridge formation. Here, we propose to: 1) investigate the signaling pathways that drive glial bridging and SC repair in zebrafish; and 2) define the subpopulation of glial cells that construct the bridge. This study will provide a mechanistic understanding of glial bridging during zebrafish SC regeneration, information that will guide approaches for comprehending and manipulating mammalian regenerative capacity.

ABSTRACT The adult mammalian heart shows little or no significant innate regeneration of cardiac muscle after injury, and instead heals by scarring. This regenerative deficiency has major socioeconomic consequences, given that ischemic myocardial infarction is a leading cause of morbidity and mortality in the United States and over 5 million Americans suffer from heart failure. Many years ago, we found that the teleost zebrafish displays a vigorous regenerative response after partial resection of the cardiac ventricle, involving creation of new cardiomyocytes with little or no scarring. This discovery pioneered the model system approach to heart regeneration that is employed by many laboratories today. For the past 15 years, the principal investigator has introduced new tools and strategies to reveal many key concepts and mechanisms underlying heart regeneration in zebrafish. Here, we describe an integrative program that defines at new resolution the molecular components of innate heart regeneration, as well as mechanisms by which these components are regulated. We will harness this information to innovate new methods for controlling the efficacy of heart regeneration across species, including attempts to stimulate regeneration in mammals through factor delivery and gene regulation using viral vectors. The discoveries we make will continue to inform the fundamental biology of heart regeneration, and they will directly relate to ideas and methods to enhance regeneration and reduce the incidence of heart failure in humans. They are also likely to extend beyond cardiovascular biology and have relevance to the repair of other tissues.

ABSTRACT Mammalian bone has the capacity throughout life to regenerate in response to fracture injury. However, there is a ceiling for this regenerative potential, with hurdles to regeneration after a major trauma like limb amputation. This has a significant socio-economic impact, as it is estimated that at least one in two Americans over age 50 is expected to have or be at risk of bone disease, and every year an estimated 1.5 million individuals suffer a fracture due to bone disease. We have developed imaging methods to study how osteoblasts drive bone regeneration in zebrafish, which display robust regeneration after major injury to bony structures like their fins, scales, and jaws. Our goal is to exploit this regenerative capacity, new imaging platforms we have created, and the molecular genetic approaches available in zebrafish to improve our ability to understand and manipulate the regenerative capacity of bone. We have recently identified Erk signaling waves as a mechanism for the control of osteoblast regeneration. This Supplement application extends our original proposal by testing the hypothesis that mechanical signals contribute to osteoblast regeneration by integrating inputs from the Erk pathway and Hippo/Yap-Taz pathway, another signaling pathway playing crucial roles in bone morphogenesis and regeneration.