Regeneration in the Zebrafish Model System
Mammalian tissues achieve remarkable feats of regeneration. After removal of more than two-thirds of its mass, the liver rapidly regenerates within several days by hepatocyte proliferation. Multipotent hematopoietic stem cells replenish red and white blood cells, and skin, muscle, and intestine are repaired by tissue-specific stem cells. However, this regenerative capacity is distributed unequally among mammalian organs: limbs, brain, spinal cord, and heart display minimal regeneration after tissue damage or loss. How and why tissue regeneration does (or does not) occur are critical questions; the answers have the potential to impact the clinical outcomes of the many diseases of organ damage, including myocardial infarction, Alzheimer's disease, and diabetes.
It has been known for centuries that certain nonmammalian vertebrates, such as urodele amphibians and teleost fish, regenerate complex tissues much more effectively than mammals. Salamanders have long been the central characters employed in vertebrate regeneration studies. Two features make the teleost zebrafish a powerful, complementary model system to study organ regeneration. First, they are highly regenerative, equipped to regrow amputated fins, injured retinae, transected optic nerves and spinal cord, and resected heart muscle. Second, unlike salamanders, they are amenable to both forward and reverse genetic approaches. As is customary with genetic model systems, a wide array of community resources is available for gene discovery and molecular characterization in zebrafish, including mutagenesis screens, transgenesis, microarrays, developmental markers, and genome sequence information.
My laboratory is investigating the biology of spectacular regenerative events in zebrafish to discover new cellular mechanisms, and we are also developing new tools to interrogate regeneration deeply at the molecular level. Over the next several years, we will pursue fundamental aspects of organ regeneration—most importantly, how tissue renewal is stimulated by injury, and how newly created cells recognize position and functionally incorporate into existing tissue.
There is little or no natural regeneration of the major structural cells of the adult mammalian heart, the cardiomyocytes, after experimental injury paradigms. This regenerative deficiency is highly relevant to human disease, given that the number one cause of morbidity and mortality in the United States is ischemic myocardial infarction and scarring. As a postdoctoral fellow, I found that zebrafish regenerate cardiac muscle after removal of 20 percent of the ventricle, with little or no scarring. This unique model of cardiac injury and regeneration puts my laboratory in a position to address an important question: How are new cardiac cells created and functionally integrated into an injured, contracting, adult heart?
It is a primary goal of our work to define the origin and developmental potential of cellular contributors to regenerated cardiac tissue. Toward this goal, we are developing myriad tools for lineage tracing in zebrafish. These technologies will also advance our field's ability to ask questions about gene function during heart regeneration at high spatiotemporal resolution.
My laboratory recently discovered a new biological relevance for the epicardium, a thin epithelial layer enveloping the cardiac chambers. We have found that the adult zebrafish epicardium responds rapidly and robustly to injury, proliferating and inducing markers of the embryonic epicardium within 1–2 days of resection of the ventricular apex. Both cardiac chambers are enveloped by this activated epicardium, which covers the injured apex within 7–14 days of amputation. A subpopulation of cells from the overlying epicardium then invades and helps neovascularize the regenerating tissue, a process highly reminiscent of epicardial behavior during embryonic heart development. We also showed that epicardial cell incorporation into the wound and neovascularization during regeneration are dependent on the fibroblast growth factor signaling pathway. We are exploring several fascinating questions regarding how epicardial cells, and generally the nonmuscle cells that comprise the cardiac environment, respond to injury and facilitate regeneration. These avenues should yield clues to altering the regenerative capacity of the injured mammalian heart.
Zebrafish fins are external, transparent, nonvital, and highly organized structures, making them ideal for asking fundamental, high-resolution questions about complex tissue regeneration. Within two weeks after amputation of the caudal fin, a series of healing, proliferation, and patterning events replaces bone, epidermis, blood vessels, nerves, and connective tissue mesenchyme. We use forward and reverse genetic approaches to identify new regulatory mechanisms critical for appendage regeneration.
A hallmark of limb or fin regeneration is formation of the blastema, a proliferative mass of mesenchymal cells that is maintained as a zone of progenitor tissue for new structures. The central questions are these: (1) What cells give rise to the blastema, and how is its formation activated by injury? (2) How is blastemal activity maintained appropriately throughout regeneration? (3) How is the completion of regeneration remembered and enacted? An obvious strength of the zebrafish model system is the opportunity for mutagenesis screens. In work as a postdoctoral fellow, I and others performed the first screen for temperature-sensitive defects in fin regeneration, efforts that led to identification of four new genes required for regeneration. In my lab, we are currently screening to identify more regeneration genes, and we have expanded and modified our approach to identify greater numbers of interesting mutants.
During appendage regeneration in urodeles and teleosts, tissue replacement is meticulously regulated such that only the appropriate structures are recovered, a phenomenon referred to as positional memory. It is believed that there exists, or is quickly established after amputation, a gradient of positional information along the axes of the appendage that assigns region-specific instructions to injured tissue. We are identifying new candidate genes for maintaining positional memory, and we are using this information to direct reverse genetic approaches that will functionally define new regeneration and homeostasis genes.