Browsing by Subject "Zebrafish"

As 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.

Mycobacterial infection leads to the formation of characteristic immune cell aggregates called granulomas. In humans and animal models, tuberculous granuloma formation is accompanied by dramatic remodeling of host vasculature which ultimately benefits the infecting mycobacteria, suggesting the bacteria may actively drive this host process. First, we sought to identify bacterial factors that promote granuloma vascularization. Using Mycobacterium marinum transposon mutants in a zebrafish infection model, we revealed the enzyme Proximal Cyclopropane Synthase of alpha-Mycolates (PcaA) as an important bacterial determinant of host angiogenesis. We found that PcaA-modified trehalose dimycolate, an abundant glycolipid in the mycobacterial cell wall, drives activation of host VEGF signaling and subsequent granuloma vascularization. To facilitate our continuing investigation of granuloma dynamics, we next sought to expand and improve upon the transgenic tools for studying macrophages in the zebrafish model. I describe two such tools: i) the macrophage-specific zebrafish mfap4 promoter, which allows long-term in vivo visualization and manipulation of macrophages during mycobacterial infection, and ii) the first zebrafish transgenic line with constitutive, ubiquitous Cas9 expression, as well as a transgene design capable of generating sgRNAs using macrophage-specific promoters. These tools allow CRISPR/Cas9 gene editing in vivo in the zebrafish in a macrophage-restricted manner.

The 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.

The formation of a single lumen during tubulogenesis is crucial for the development and function of many organs. Although 3D cell culture models have identified molecular mechanisms controlling lumen formation in vitro, their function during vertebrate organogenesis is poorly understood. In this work we used the zebrafish gut as a model to investigate single lumen formation during tubulogenesis. Previous work has shown that multiple small lumens enlarge through fluid accumulation and coalesce into a single lumen. However, since lumen formation occurs in the absence of apoptosis, other cellular processes are necessary to facilitate single lumen formation.

Using light sheet microscopy and genetic approaches we identified a distinct intermediate stage in lumen formation, characterized by two adjacent un-fused lumens. These lumens are separated by cell contacts that contain basolateral adhesion proteins. We observed that lumens arise independently from each other along the length of the gut and do not share a continuous apical surface. Resolution of this intermediate phenotype into a single, continuous lumen requires the remodeling of basolateral contacts between adjacent lumens and subsequent lumen fusion.

Furthermore, we provide insight into the genetic mechanisms regulating lumen formation through the analysis of the Hedgehog pathway. We show that lumen resolution, but not lumen opening, is impaired in smoothened (smo) mutants, indicating that fluid-driven lumen enlargement and resolution are two distinct processes. We also show that smo mutants exhibit perturbations in the Rab11 trafficking pathway, which led us to demonstrate that Rab11-mediated recycling, but not degradation, is necessary for single lumen formation. Taken together, this work demonstrates that lumen resolution is a distinct genetically-controlled process, requiring cellular rearrangement and lumen fusion events, to create a single, continuous lumen in the zebrafish gut.

The means by which cells, tissues, and organisms undergo morphogenesis are variable and highly regulated, and the mechanisms that govern cellular changes in response to signaling cues are poorly understood. This study seeks to address the role of a newly characterized protein in zebrafish in translating signaling cues into physical changes within a cell.

The Cordon–bleu (Cobl) gene is widely conserved in vertebrates, with developmentally regulated axial and epithelial expression in mouse and chick embryos. In vitro, Cobl can bind monomeric actin and nucleate formation of unbranched actin filaments, while in cultured cells it can modulate the actin cytoskeleton. However, an essential role for Cobl in vivo has yet to be determined. We have identified the zebrafish cobl ortholog and have used zebrafish as a model to assess the requirements for Cobl in embryogenesis. We find that cobl shows enriched expression in ciliated epithelial tissues during zebrafish organogenesis. The utilization of antibodies developed against Cobl shows that the protein is concentrated along the apical domain of ciliated cells, in close proximity to the apical actin cap.

Reduction of cobl by antisense morpholinos reveals an essential role in embryonic morphogenesis and organ development. A requirement for Cobl was shown for the proper function of various and ciliated epithelial organs. Cobl appears to direct the elongation of motile cilia in organs such as Kupffer’s vesicle and the pronephros. In Kupffer’s vesicle, the reduction in Cobl coincides with a reduction in the amount of apical F-actin. Additionally, Cobl may play a role during gastrulation cell movements and convergence and extension morphogenesis during early embryonic development. Thus, Cobl may represent a molecular activity that couples developmental patterning signals with local intracellular cytoskeletal dynamics to support elongation of motile cilia and tissue morphogenesis.

Mycobacterium tuberculosis (Mtb) is the causative agent of the disease tuberculosis, which kills more people worldwide than any other infectious disease. In 2017, nearly 2 million people died of tuberculosis. Despite the advent of antibiotics targeting Mtb, the global spread of tuberculosis continues. The development of antibiotic resistance within the bacteria has further complicated the already long and difficult course of treatment for the disease. New therapeutics are necessary to combat tuberculosis. A novel treatment strategy is the use of host-directed therapies, which provide an orthogonal approach to killing intracellular pathogens. Rather than directly targeting bacterial pathways, which may lead to the development of mutations that result in resistance to the drug, host directed therapies (HDTs) target the host immune response to the disease. To uncover these host directed therapies, we have utilized the zebrafish-Mycobacterium marinum model system. Using zebrafish infected with their natural pathogen, Mycobacterium marinum, a close genetic relative to Mtb, we show that we can enhance the ability of the host immune response to kill intracellular bacteria.

In Chapter 1, I introduce tuberculosis as a disease and discuss the past, present and future of treating the disease. I discuss potential host targets for immune modulating therapies, including autophagy, inflammation, and inflammasomes. I highlight the role of calcium signaling in immune cells, specifically neutrophils and macrophages. I briefly describe zebrafish as a model system, emphasizing their use to study immune responses and host-pathogen interactions. In Chapter 2, we show calcium is required for immune cell activity and motility in neutrophils. Calcium is a signal that leads neutrophils not only to wound sites but also to sites of infection and inflammation. We then enhance calcium signaling through potentiation of the membrane channel P2RX7 with the small molecule clemastine, an FDA-approved over-the-counter antihistamine in Chapter 3. We show that clemastine treatment reduces bacterial burden in a P2RX7 –dependent manner in zebrafish larvae. P2RX7 activation leads to assembly of inflammasomes in macrophages, a key immune cell of mycobacterial infection. In human mycobacterial disease, many of the bacteria are contained within structures called granulomas, in which host macrophages and other immune cells have formed a cuff around the bacteria, creating a space that is recalcitrant to treatment with frontline antibiotics. Clemastine is effective in these established infection structures, indicating that it may be a feasible strategy to treat human tuberculosis. We discuss how mycobacteria evade the host immune response and demonstrate how a small molecule can overcome these evasion strategies for improved host outcome.

The intestine is a multifunctional organ that must perform dichotomous roles in order to maintain health. While it is the primary site of absorption of dietary nutrients, it must also serve as a barrier to both the multitude of microorganisms which reside in the intestinal lumen (the microbiota) and foreign compounds (xenobiotics) which can be toxic to the host. Moreover, the microbiota are required for normal physiology, regulating immunological development, metabolism and behavior. Understanding how the intestine maintains homeostasis and responds to insult in the face of a chemically and microbially complex and dynamic environment is not only a fundamental question of biology, but has important implications for human health. We used zebrafish in order to better understand how the intestine responds to xenobiotics (Chapter 2) and transduces signals from the microbiota to the immune system (Chapter 3).

In Chapter 1, I introduce the complex and reciprocal interactions between xenobiotics, the microbiota, and the host. I highlight examples whereby the microbiota modulates the activity and toxicity of pharmaceuticals, with relevance to diseases of different organ systems. I also describe mechanisms by which the intestine responds to xenobiotic toxicity, and finally advocate for the use of novel model organisms to improve our understanding of these complex interactions.

In Chapter 2, I present our work using the NSAID Glafenine to explore how the intestine responds to xenobiotic challenge. Using transgenic zebrafish and high resolution in vivo imaging, we demonstrate loss epithelial cells in a live animal following xenobiotic challenge. Moreover, Glafenine causes intestinal inflammation, which is potentiated by microbial dysbiosis. We also show that Glafenine can directly alter microbiota composition. Glafenine treatment resulted in activation of the unfolded protein response (UPR), and while pharmacological inhibition of the UPR sensor Ire1a suppressed Glafenine-induced IEC loss, this was associated with increased inflammation and mortality. Ultimately, we demonstrate that Glafenine-induced intestinal toxicity is likely due to off-target inhibition of multidrug resistance (MDR) efflux pumps, as other MDR inhibitors were able to elicit similar phenotypes. Collectively, our findings revealed that (i) MDRs serve an evolutionarily conserved role in maintenance of intestinal homeostasis and (ii) IEC delamination is a protective mechanism which serves to limit inflammation and promote animal survival.

While studies in gnotobiotic mice and zebrafish have demonstrated that the microbiota are required for normal development of the innate immune system, the underlying host and microbial signals which mediate these effects remain largely unknown. We had previously demonstrated that motility of gut commensal bacteria in zebrafish was important for successful colonization of some strains and stimulation of the normal host innate immune response to colonization. In Chapter 3, we describe how microbiota colonization is associated with changes in the PMN transcriptome in addition to promoting systemic abundance and distribution of myeloid cells. Intriguingly, the only pattern recognition receptors found to be differentially expressed in PMNs were the Flagellin receptors tlr5a and tlr5b. Colonization of zebrafish larvae with bacteria lacking Flagellin resulted in attenuated PMN transcriptional activation compared to larvae colonized with isogenic wild type (WT) bacteria. We subsequently demonstrated that direct exposure to purified Flagellin can potently induce transcriptional activation in zebrafish PMNs. These findings identify how the presence of the microbe associated molecular pattern (MAMP) Flagellin serves as a bacterial cue from the microbiota which promotes PMN activation. In Chapter 4, I offer perspectives as to how the Glafenine-zebrafish model system can be used to more deeply investigate host-microbiota-xenobiotic interactions, and genetic, biochemical and computational analyses can help delineate mechanisms by which MDR efflux pumps function in the maintenance of intestinal homeostasis. Moreover, I propose the use of bacterial screens as well as inflammatory and infectious challenge assays in order to better understand the functional outcomes of PMN transcriptional activation elicited by microbiota-derived signals such as Flagellin.

Regeneration 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.