Browsing by Author "Poss, Kenneth D"
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Item Open Access A Clonal Analysis of Zebrafish Heart Morphogenesis and Regeneration(2014) Gupta, VikasAs vertebrate embryos grow and develop into adults, their organs must acquire mass and mature tissue architecture to maintain proper homeostasis. While juvenile growth encompasses a significant portion of life, relatively little is known about how individual cells proliferate, with respect to one another, to orchestrate this final maturation. For its simplicity and ease of genetic manipulations, the teleost zebrafish (Danio rerio) was used to understand how the proliferative outputs of individual cells generate an organ from embryogenesis into adulthood.
To define the proliferative outputs of individual cells, a multicolor clonal labeling approach was taken that visualized a large number of cardiomyocyte clones within the zebrafish heart. This Brainbow technique utilizes Cre-loxP mediated recombination to assign cells upwards of ~90 unique genetic tags. These tags are comprised of the differential expression of 3 fluorescent proteins, which combine to give rise to spectrally distinct colors that represent these genetic tags. Tagging of individual cardiomyocytes was induced early in development, when the wall of the cardiac ventricle is a single myocyte thick. Single cell cardiomyocyte clones within this layer expanded laterally in a developmentally plastic manner into patches of variable shapes and sizes as animals grew into juveniles. As maturation continued into adulthood, a new lineage of cortical muscle appeared at the base of the ventricle and enveloped the ventricle in a wave of proliferation that fortified the wall to make it several myocytes thick. This outer cortical layer was formed from a small number (~8) of dominant cortical myocyte clones that originated from trabecular myocytes. These trabecular myocytes were found to gain access to the ventricular surface through rare breaches within the single cell thick ventricular wall, before proliferating over the surface of the ventricle.
These results demonstrated an unappreciated dynamic juvenile remodeling event that generated the adult ventricular wall. During adult zebrafish heart regeneration, the primary source of regenerating cardiomyocytes stems from this outer wall of muscle. Regenerating cardiomyocytes within this outer layer of muscle are specifically marked by the cardiac transcription factor gene gata4, which they continue to express as they proliferate into the wound area.
Using heart regeneration to guide investigation of juvenile cortical layer formation, we found that both processes shared similar molecular and tissue specific responses including expression and requirement of gata4. Additional markers suggested that juvenile hearts were under stress and that this stress could play a role to initiate cortical morphogenesis. Indeed, experimental injury or a physiologic increase in stress to juvenile hearts caused the ectopic appearance of cortical muscle, demonstrating that injury could trigger premature morphogenesis.
These studies detail the cardiomyocyte proliferative events that shape the heart and identify molecular parallels that exist between regeneration and cortical layer formation. They show that adult zebrafish heart regeneration utilizes an injury/stress responsive program that was first used to remodel the heart during juvenile growth.
Item Open Access Cardiac Mitogen Signaling During Zebrafish Heart Regeneration(2020) Shoffner, AdamAbstract
Adult zebrafish demonstrate a remarkable capacity to regenerate heart tissue following injury, and thus have served as a valuable model for developing our understanding of cardiac repair and regeneration. Recent work has identified and characterized multiple cardiac mitogens all of which can drive cardiomyocyte (CM) division in the absence of injury. Despite these impressive responses, little is known regarding the shared specific molecular mechanisms of CM proliferation that lie downstream of these unique ligand-receptor interactions. Here, we found that the tumor suppressor p53 was significantly suppressed during regeneration which correlated with increases in the transcription of p53’s primary negative regulator Mdm2. Using established and newly generated transgenic lines we demonstrated that experimentally altering cellular p53 levels affects CM proliferation. Inducible overexpression of the cardiac mitogens Nrg1 and Vegfaa demonstrated similar findings with increased mdm2 transcription and p53 suppression during regeneration along with augmented CM proliferation with loss of p53. Furthermore, we observed significant overlap between gata4 and mdm2 gene expression domains during development, following heart injury, and with mitogen stimulation suggesting potential interactions between these two genes. Our findings indicate a novel injury and mitogen-induced function of Mdm2 to repress p53 during zebrafish heart regeneration. Here we also investigated the presence of additional cardiac mitogens, specifically HB-EGF, an ErbB ligand. We found that both HB-EGF paralogs are both present in the zebrafish heart and are both transcriptionally upregulated near the site of injury. A newly generated set of novel HB-EGF transgenic reporters, knock-outs, and overexpression lines will further investigate the importance of these early findings and HB-EGF signaling which will add to our understanding of heart regeneration.
Item Open Access Cellular and Molecular Mechanisms of Cardiac Chamber Maturation in Zebrafish(2018) Foglia, MatthewThe formation of the heart is a critical part of development that, if defective, can lead to congenital malformations incompatible with life. An improved understanding of the cellular and molecular processes that build the heart is essential to elucidate the causes of congenital defects and to design appropriate therapies. Relatively little is known about how the cardiac chambers adopt distinct forms to follow their specialized functions. Here, I have used a multicolor genetic labeling system to trace the progeny of zebrafish atrial cardiomyocytes as they expand to form the mature atrial myocardium. By comparing the observed cellular dynamics to those previously mapped in the ventricle, I identified characteristics of chamber development, including wall thickening, wall composition, and internal muscle formation, that contribute to the structural divergence of the chambers. As coronary vessel formation is one such chamber-specific morphogenetic process, I then explored the effect of a chamber-specific growth factor on cardiac development and homeostasis. Using a transgenic reporter and an inducible overexpression tools, I found ectopic expression of this growth factor stimulates cardiomyocyte proliferation. However, overexpression also blocks regeneration, possibly due to the abolition of an endogenous gradient localized to the site of injury. These findings not only provide new details for how the cardiac chambers form, but also demonstrate how understanding developmental phenomena can provide insights into important concepts of regenerative medicine.
Item Open Access Clonal Analysis of the Zebrafish Fin Regeneration Blastema(2016) Tornini, Valerie AngelaRegeneration is a remarkable feat of developmental regrowth and patterning. The blastema is a mass of progenitor cells that enables complete regeneration of amputated salamander limbs or fish fins. Despite years of study, methodologies to identify and track blastemal cell progenies have been deficient, restricting our understanding of appendage regeneration at a cellular and molecular level. To bridge this knowledge gap, gene expression analysis, the generation of transgenic and mutant zebrafish, qualitative and quantitative analyses, morphological measurements, and chemical treatments were used to assess molecular and cellular processes involved in fin regeneration. Two main findings arose from these methods. The first provides evidence that connective tissue progenitors are rapidly organized into a scalable blueprint of lost structures, and that amputation stimulates resident cells to reset proximodistal positional information. The second identifies a fibroblast subpopulation near uninjured fin joints that contributes to the blastemal progenitor population. These findings reveal insights on cellular and molecular mechanisms of appendage regeneration and provide a basis for work exploring how cells in an adult vertebrate bone appendage coordinately rebuild a new structure.
Item Open Access Epigenetic Profiling of Zebrafish Fin Regeneration(2020) Thompson, John DaylandThe ability to regenerate after injury is quite astonishing, yet not all organisms share this ability. Mammalian genomes likely encode all gene products required to regenerate an amputated limb, yet they lack the correct instructions for strategically modulating those gene products to accomplish limb regeneration. While the catalogue of defined cell dynamics and molecular factors in tissue regeneration is expanding, we know comparatively little of how genes involved in regenerative events are engaged upon injury, despite decades of research. Certain non-mammalian vertebrates like salamanders and zebrafish possess these instructions, which exist as cis-regulatory elements that can direct expression of their target genes during regeneration. To identify candidate tissue regeneration enhancer elements (TREEs) important for zebrafish fin regeneration, we performed ATAC-seq from bulk tissue or purified fibroblasts of uninjured and regenerating caudal fins. We identified tens of thousands of DNA regions from each sample type with dynamic accessibility during regeneration, and assigned these regions to proximal genes with corresponding changes in expression by RNA-seq. To determine the extent to which these profiles reveal bona fide TREEs, we tested the sufficiency and requirements of several sequences in stable transgenic lines and mutant lines with homozygous deletions. Our study generates a new resource for dissecting the regulatory mechanisms of appendage generation and reveals a range of requirements for individual TREEs in control of regeneration programs.
Item Open Access Gene regulatory networks controlling an epithelial-mesenchymal transition(2007-05-03T18:54:08Z) Wu, Shu-YuEpithelial-mesenchymal transitions (EMTs) are fundamental and indispensable to embryonic morphogenesis throughout the animal kingdom. At the onset of gastrulation in the sea urchin embryo, micromere-derived primary mesenchyme cells (PMCs) undergo an EMT process to ingress into the blastocoel, and these cells later become the larval skeleton. Much has been learned about PMC specification in sea urchin embryos. However, much less is known about how states of the sequentially progressing PMC gene regulatory network (GRN) controls the EMT process during PMC ingression. Transcriptional regulators such as Snail and Twist have emerged as important molecules for controlling EMTs in many model systems. Sea urchin snail and twist genes were cloned from Lytechinus variegates, and each has been experimentally connected to the PMC regulatory network; these experiments demonstrate several requirements for PMC ingression, and in doing so, begin to illustrate how a gene regulatory network state controls morphogenesis. Functional knockdown analyses of Snail with morpholino-substituted antisense oligonucleotides (MASO) in whole embryos and chimeras demonstrated that Snail is required in micromeres for PMC ingression. Investigations also show that Snail downregulates cadherin expression as an evolutionarily conserved mechanism, and Snail positively regulates a required endocytic clearance of epithelial membrane molecules during EMT. Perturbation experiments indicate that Twist has accessory roles in regulating PMC ingression, and possibly plays a maintenance role in PMC specification network state. In addition, Twist also functions in the post-EMT network state, particularly in directing PMC differentiation and skeletogenesis. The recently annotated sea urchin genome accelerates the discovery of new genes and holds strong promise of mapping out a complete canvas of the micromere-PMC gene regulatory network. Using the genome resources we successfully cloned several newly identified PMC genes, and found most of them to be expressed in micromeres just prior to ingression of the nascent PMCs. Current experiments focus on the roles of these genes in preparing for, executing, and/or controlling the mesenchymal behavior following PMC ingression. The functions and inter-relationships of these genes will greatly augment our understanding of how a gene regulatory network state controls a crucial morphogenetic event.Item Open Access Investigating Dynamics of Tissue Regeneration via Live Imaging of Zebrafish Scales(2019) Cox, BenRegeneration occurs throughout the animal kingdom and is a well-studied
feature of many model organisms, yet the field lacks a fundamental understanding of
the real-time dynamics of cell behavior during regeneration. I discuss how existing
knowledge of regeneration may be used to inform efforts to translate these remarkable
feats of animals to human regeneration and present research that uses live imaging to
improve understanding of cell origins and diversification during regeneration in the
scale, focusing specifically on osteoblasts the matrix-depositing cells that divide and heal
bone injuries. I developed an imaging platform to monitor and quantify individual and
collective behaviors of osteoblasts in adult zebrafish scales. I show that a founder pool
of osteoblasts emerges through de novo differentiation within one day of scale plucking,
then diversifies across the primordium by two days after injury, with region-specific
changes in proliferation, cell shape, and cell death rates coincident with acquisition of
mature scale morphology. I also demonstrate a role for Fgf signaling in scale
regeneration and present tools for high resolution imaging studies of basal epidermal
cells during skin and scale injury. These findings demonstrate the value of live imaging
in revealing novel biology and gaining a more complete picture of the many complex
processes that must be elegantly choreographed to achieve tissue regeneration.
Item Open Access JNK Signaling Mediates Glial Proliferation in the Regenerating Zebrafish Spinal Cord(2023) Becker, Clayton JZebrafish possess the remarkable capacity to regenerate from spinal cord injuries that would leave mammals such as humans permanently paralyzed. Much research into zebrafish spinal cord regeneration has focused on identifying extracellular growth factors and matrix components which create a pro-regenerative environment; however, it is just as important to identify and understand the transcription factors which control pro-regenerative transcriptional responses within the resident stem cell population of the spinal cord, and the signaling cascades which translate the known extracellular ligands into cellular responses. Using CRISPR/Cas9, we generated two novel transcription factor knockout zebrafish lines which we tested for spinal cord regeneration defects and found no difference in regenerative capacity. Using a chemical screen, we identify JNK signaling as a necessary regulator of glial cell cycling and tissue bridging during spinal cord regeneration in larval zebrafish. With a kinase translocation reporter, we visualize and quantify JNK signaling dynamics at single-cell resolution in glial cell populations in developing larvae and during injury-induced regeneration. Glial JNK signaling is patterned in time and space during development and regeneration, decreasing globally as the tissue matures and increasing in the rostral cord stump upon transection injury. Thus, we present a tool to visualize signaling activity in the larval zebrafish spinal cord and demonstrate that dynamic JNK activity after spinal cord injury directs a proliferative response of glial cells during spinal cord regeneration.
Item Open Access Mechanisms that drive cardiomyocyte proliferation during zebrafish heart regeneration(2014) Gemberling, Matthew PHeart disease is the leading cause of death in the developed world. Adult mammals cannot replace lost cardiac tissue after injury, leading to reduced quality of life and increased instances of future cardiac issues. Zebrafish possess the ability to regenerate lost cardiac muscle after injury. Upon injury, the zebrafish heart responds in a coordinated fashion resulting in activation of the epicardium and endocardium, cardiomyocyte proliferation, and subsequent vascularization and innervation of the newly formed muscle. Thus zebrafish represent an ideal genetic model to dissect the mechanisms of heart regeneration. Previously, it was discovered that regulatory sequences of the cardiac transcription factor, gata4, become active in the ventricular wall following injury and that these gata4+ cardiomyocytes proliferate and contribute the majority of new muscle to the regenerate. We uncovered that gata4 function is required for cardiomyocyte proliferation and regeneration after injury. Cardiomyocyte proliferation is required to achieve proper regeneration and lack of cardiomyocyte proliferation is a hallmark of failed regeneration in the mammalian system. Therefore, understanding the signals that induce mature cardiomyocyte division is of great scientific and clinical relevance. Utilizing transgenic approaches, we have found that gata4 function and Nrg1 signaling are critical regulators of cardiomyocyte proliferation. We found that Nrg1 was expressed following injury in the zebrafish heart and that inhibition of nrg1-erbb signaling blunted cardiomyocyte proliferation. Using transgenic over-expression of Nrg1, we found that Nrg1 was capable of increasing injury-induced cardiomyocyte proliferation. Furthermore we found that activation of Nrg1 in the uninjured adult heart induces cardiomyocyte proliferation and hallmarks of the regenerative program. Long-term nrg1 expression leads to patterned hyperplastic expansion of the zebrafish ventricle. To our knowledge, this is the first description of a single factor that is sufficient to induce such a dramatic hyperplastic response in an adult heart.
Item Open Access Mechanisms Underlying Bone Cell Recovery During Zebrafish Fin Regeneration(2013) Singh, Sumeet PalZebrafish regenerate amputated caudal fins, restoring the size and shape of the original appendage. Regeneration requires generation of diverse cell types comprising the adult fin tissue. Knowledge of the cellular source of new cells and the molecules involved is fundamental to our understanding of regenerative responses. In this dissertation, the contribution made by the bone cells towards fin regeneration is investigated. Fate mapping of osteoblasts revealed that spared osteoblasts contribute only to regenerating osteoblasts and not to other cell types, thereby suggesting lineage restriction during fin regeneration. The functional significance of osteoblast contribution to fin regeneration is tested by developing an osteoblast ablation tool capable of drug induced loss of bone cells. Normal fin regeneration in the absence of resident osteoblast population suggests that the osteoblast contribution is dispensable and provides evidence for cellular plasticity during fin regeneration. To uncover the genes involved in proliferation of osteoblasts within the fin regenerate, a candidate in-situ screen was carried out and revealed bone specific expression of fgfr4 and twist3. Transgenic tools for visualization of gene expression confirmed the screen results. Knockdown of twist3 by morpholino antisense technology impedes fin regeneration. Mutant heterozygotes for twist3 were generated using genome editing reagents, which will enable loss-of-function study in future.
Item Open Access Nrg1 is an injury-induced cardiomyocyte mitogen for the endogenous heart regeneration program in zebrafish.(Elife, 2015-04-01) Gemberling, Matthew; Karra, Ravi; Dickson, Amy L; Poss, Kenneth DHeart regeneration is limited in adult mammals but occurs naturally in adult zebrafish through the activation of cardiomyocyte division. Several components of the cardiac injury microenvironment have been identified, yet no factor on its own is known to stimulate overt myocardial hyperplasia in a mature, uninjured animal. In this study, we find evidence that Neuregulin1 (Nrg1), previously shown to have mitogenic effects on mammalian cardiomyocytes, is sharply induced in perivascular cells after injury to the adult zebrafish heart. Inhibition of Erbb2, an Nrg1 co-receptor, disrupts cardiomyocyte proliferation in response to injury, whereas myocardial Nrg1 overexpression enhances this proliferation. In uninjured zebrafish, the reactivation of Nrg1 expression induces cardiomyocyte dedifferentiation, overt muscle hyperplasia, epicardial activation, increased vascularization, and causes cardiomegaly through persistent addition of wall myocardium. Our findings identify Nrg1 as a potent, induced mitogen for the endogenous adult heart regeneration program.Item Open Access Organ-Level Communication During Heart Regeneration In Zebrafish.(2022) Sun, FeiTissue regeneration has been primarily investigated as local remodeling events in response to tissue damage or loss. Recent studies, however, indicate that uninjured structures can respond to distant tissue trauma and, in some cases, regulate tissue regeneration. One of the key questions that haven’t been answered in the field is how animals simultaneously exert customized control of local and remote injury responses during regeneration. Taking advantage of the genetic cardiomyocyte ablation system developed in adult zebrafish, we explored uninjured brain and kidney responses to heart regeneration. This dissertation identified a transcription factor gene, cebpd, through transcriptomic profiling of the uninjured brain and kidney during zebrafish heart regeneration. The expression of cebpd is induced both locally in the epicardial tissue of regenerating hearts and distantly in the brain ependymal layer and renal tubules. Knocking out cebpd using the CRISPR system, we found that cebpd is required for tissue repair adjacent to an injury event, as well as in the physiological sequelae of fluid regulation encompassing remote tissues. By profiling and molecular genetics in zebrafish, we identified a novel class of remote tissue regenerative enhancer elements (r-TREEs) responsible for remote gene activation during tissue regeneration. Interestingly, removing cebpd associated enhancer element CEN only abolished gene activation in remote uninjured brain and kidney but not local regenerating hearts. We further demonstrated that corticosteroid receptor activities are sufficient and required for CEN-dependent regulation of gene expression in remote tissues during regeneration. Loss of CEN perturbed fluid regulation in zebrafish during heart regeneration. My findings suggest a novel concept in tissue regeneration, in which r-TREEs segregate local and remote responses and stratify regeneration and physiological functions of key regulatory genes to achieve whole-organism coordination during regeneration.
Item Open Access Ras controls melanocyte expansion during zebrafish fin stripe regeneration.(Dis Model Mech, 2010-07) Lee, Yoonsung; Nachtrab, Gregory; Klinsawat, Pai W; Hami, Danyal; Poss, Kenneth DRegenerative medicine for complex tissues like limbs will require the provision or activation of precursors for different cell types, in the correct number, and with the appropriate instructions. These strategies can be guided by what is learned from spectacular events of natural limb or fin regeneration in urodele amphibians and teleost fish. Following zebrafish fin amputation, melanocyte stripes faithfully regenerate in tandem with complex fin structures. Distinct populations of melanocyte precursors emerge and differentiate to pigment regenerating fins, yet the regulation of their proliferation and patterning is incompletely understood. Here, we found that transgenic increases in active Ras dose-dependently hyperpigmented regenerating zebrafish fins. Lineage tracing and marker analysis indicated that increases in active Ras stimulated the in situ amplification of undifferentiated melanocyte precursors expressing mitfa and kita. Active Ras also hyperpigmented early fin regenerates of kita mutants, which are normally devoid of primary regeneration melanocytes, suppressing defects in precursor function and survival. By contrast, this protocol had no noticeable impact on pigmentation by secondary regulatory melanocyte precursors in late-stage kita regenerates. Our results provide evidence that Ras activity levels control the repopulation and expansion of adult melanocyte precursors after tissue loss, enabling the recovery of patterned melanocyte stripes during zebrafish appendage regeneration.Item Open Access Regulation of Progenitor Cell Proliferation During Zebrafish Fin Regeneration(2009) Lee, YoonsungVertebrates like urodele and teleost have an enhanced capacity for regeneration, when compared to mammals. Recently, the teleost zebrafish (Danio rerio) has become a popular model for studying regenerative events, due to the ability to regenerate multiple organs such as the fin and the heart, and the diverse genetic approaches available for functional studies. In my thesis studies, I have used the zebrafish caudal fin as a model system to understand molecular and cellular mechanism of appendage regeneration.
Pharmacological and genetic studies have revealed that Fgf signaling is important for appendage regeneration. To dissect the mechanism of Fgfs during zebrafish fin regeneration, lab colleagues and I have generated and utilized transgenic animals in which Fgf signaling can be experimentally increased or decreased. Through these transgenic studies, I found that position-dependent Fgf signaling directs regenerative growth and blastemal proliferation. Proximally-amputated fin regenerates grow at higher rates than the distally-amputated, owing to position-dependent amounts of Fgf activity. Further studies using new transgenics have provided an understanding of mechanisms by which Fgfs influence epidermal regulation of the blastema. Loss- and gain-of-function studies of Fgfs reveal that Fgf signaling both positively and negatively regulated shh expression in the epidermis to maintain blastemal function.
During the fin regeneration process, pigmentation pattern is re-established as along with bone structures and connective tissues. While the lineage of the blastema is not precisely clear, pigment cells in the fin regenerates are thought to be derived from melanocyte stem cells. Therefore, melanocyte regeneration is an informative system to understand the mechanism underlying regulation of adult stem cells during regeneration. As part of my thesis studies, we generated transgenic animals in which ectopic Ras expression can be experimentally induced. Transgenic studies, combined with pharmacological approaches, have revealed that Ras controls self-renewal of melanocyte stem cells during fin pigment regeneration.
Item Open Access Regulation of Tissue Regeneration in Zebrafish by Vitamin D Signaling(2020) Chen, AnzhiAdult mammals have limited regenerative capacities. Lost limbs are replaced by scar tissues. Heart attacks lead to massive cardiomyocyte death without robust proliferative response at the injury sites, resulting in permanently impaired cardiac function. While current treatments mainly rely on drug, tissue transplantation, cell therapy and tissue engineering, there remains an urgent need for new strategies to promote regeneration due to limitations in efficiency and specificity in existing approaches. Previous research in our lab identified vitamin D as a potent pro-regenerative factor in zebrafish. To further determine the role of vitamin D signaling in regeneration, we applied drug treatments, generated transgenic zebrafish, and performed proliferation and regeneration essays to examine the necessity and sufficiency of the pathway in regulating fin and heart regeneration. In addition, we designed a novel system to allow spatial and temporal sensitization of tissue’s response to vitamin D signaling utilizing the tissue regeneration enhancer elements (TREEs) and tested this for its ability to promote regeneration in a tissue-specific way. Key findings from this study are: 1) vitamin D signaling is required for zebrafish fin and heart regeneration; 2) enhanced vitamin D signaling promotes fin and heart regeneration; 3) Temporal overexpression of vitamin D receptor at injury sites regulated by TREEs enables locally sensitized response to vitamin D signaling and improves fin regeneration without global effects in zebrafish.
Item Open Access Requirements for Regenerative Mechanisms in Tissue Growth and Homeostasis in Adult Zebrafish(2009) Wills, Airon AleaseThe teleost zebrafish (danio rerio) has a highly elevated regenerative capacity compared to mammals, with the ability to quickly and correctly regenerate complex organs such as the fin and the heart following amputation. Studies in other highly regenerative systems suggest that regenerative capacity is directly related to the homeostatic demands of a given tissue, such as high basal levels of cell turnover or the ability to modify tissue size in response to homeostatic changes. However, it is not known if this relationship is present in vertebrate tissues with blastema-based regeneration. To test this idea, we investigated whether markers associated with regeneration are expressed in uninjured zebrafish tissues, and if treatments that block regeneration also lead to homeostatic defects over long periods.
We found that regenerative capacity is generally required for homeostasis in the fin, as multiple genetic treatments that block regeneration also led to a degenerative loss of distal fin tissue in uninjured animals. In addition, we found that there is extensive cell turnover in the distal fin tissues, accompanied by expression of critical effectors of blastemal regeneration. Both cell proliferation and gene expression were sensitive to changes in Fgf signaling, a factor that is critical for fin regeneration.
In the heart, we found that although there is little cell turnover in uninjured adult animals, the zebrafish heart can undergo rapid, dramatic cardiogenesis in response to animal growth. These growth conditions induce cardiomyocyte hyperplasia similar to regeneration, and induce gene expression changes in the epicardium, a tissue that is critical for cardiac regeneration. We find that the epicardium continually contributes cells to the uninjured heart, even in the absence of cardiac growth. If this contribution is prevented via a long-term block of Fgf signals, scarring can result, indicating that continual activity of epicardium derived cells (EPDCs) is critical for cardiac homeostasis. We have generated reagents that allow us to visualize EPDCs, and find that they contribute cardiac fibroblasts and perivascular cells during rapid cardiac growth. Uncovering the fate of EPDCs during cardiac homeostasis and regeneration will allow us to better understand their function, and may lead to the development of regenerative therapies for human cardiovascular diseases.
Item Open Access Understanding Positional Information During Zebrafish Fin Regeneration(2013) Nachtrab, GregoryRegeneration is a remarkable feat that can only be accomplished by a small number of animals. The regeneration of vertebrate limbs is one such case as certain salamanders and fish regenerate robustly while mammalian ability to regenerate is extremely limited. Successful regeneration requires not just cell proliferation after injury but also the patterning of the new tissue into a suitable replacement structure. The process by which this patterning happens is referred to as positional memory. Identification of factors responsible for positional memory in vertebrate appendage regeneration has remained elusive. This dissertation establishes zebrafish pectoral fins as a model system for studying and defining positional memory factors. This has been accomplished through careful morphological measurements, gene expression profiling, construction of transgenic zebrafish strains, and the use of various chemical reagents. Two stunning examples of positional information in the pectoral fin have been discovered. First is the region-specific defect in male pectoral fin regeneration governed by an androgen's influence on GSK3 activity. The second is the role for hand2 in maintaining restricted vitamin D signaling and thus small bones in the posterior region of the pectoral fin. hand2 is the first defined positional memory factor in a zebrafish fin. However, in spite of this success the tools required for further dissection of positional memory are not available and thus the potential for meaningful future work is slight.
Item Open Access Vacuole Formation Guides the Regenerative Path of the Zebrafish Notochord(2021) Garcia, JamieThe notochord is a defining feature of our phylum Chordata and has critical roles in human development that are highly conserved in vertebrates. The notochord functions as a hydrostatic scaffold to provide structural rigidity needed for anterior-posterior axis elongation and later for proper spine development. The notochord’s mechanical properties depend on its unique structure. In zebrafish, the notochord consists of a core of giant vacuolated cells surrounded by an epithelial -like sheath. Previous research from our lab has shown that during early development, the notochord vacuole rapidly accumulates fluid and expands within the inelastic notochord sheath. In this work we first investigated the molecular processes by which large vacuolated cells of the notochord maintain integrity while being subjected to a significant amount of stress. We determined that caveolae play a mechanoprotective role in the zebrafish notochord and are crucial in preserving notochord integrity. Upon loss of caveolae, the vacuolated cell collapses at discrete positions under the mechanical strain of locomotion then sheath cells invade the inner notochord and differentiate into vacuolated cells thereby restoring notochord function and allowing normal spine development. Findings from our caveolae work next allowed us to investigate the arrangement of vacuolated cells within the zebrafish notochord. During notochord morphogenesis, the vacuolated cells in wild-type zebrafish arrange themselves in a staircase pattern. However, in both caveolae and vacuole mutants, this pattern is disrupted. We investigated the basis of this pattern and found that it can be described by simple physical principles. We modeled the arrangement of vacuolated cells using a system composed of silicone tubing and sodium polyacrylate jelly beads demonstrating that what we observe in vivo can be described by the theory developed for the packing of spheres in cylinders. We determined that the organization of vacuolated cells within the zebrafish notochord is controlled by the density of fluid filled vacuoles and the diameter of the notochord tube. Lastly, based on our finding that sheath cells of the notochord can form de novo vacuoles, we wanted to identify key factors contributing to notochord vacuole biogenesis and integrity. We used a two-pronged transcriptomics and proteomics approach to identify proteins involved in de novo vacuole formation. We find that loss of a protein previously linked to lysosome related organelle function, Lyst, leads to fragmentation of notochord vacuoles and impaired axis elongation. Interestingly, upon injury of the notochord, sheath cells fail to form a fully inflated vacuole and continue to grow outside of notochord boundaries, forming a tumor-like mass. The tumor-like mass appears very similar to a rare tumor type called chordoma, which is characterized by overgrowth of intervertebral disc tissue. This work suggests that Lyst is important for notochord vacuole biogenesis in zebrafish and may play an important role in chordoma formation. Our work has elucidated novel mechanisms of cell surface integrity and has shown how proper vacuolated cell inflation leads to a structurally intact notochord. Additionally, we have demonstrated the remarkable regenerative capacity of the zebrafish notochord and identified potential regulators of both vacuole biogenesis and chordoma formation.