Dynamic remodeling of the extra cellular matrix during zebrafish fin regeneration

Extracellular matrix plays a dynamic role during the process of wound healing, embryogenesis and tissue regeneration. Caudal fin regeneration in zebrafish is an excellent model to study tissue and skeletal regeneration. We have analyzed the expression pattern of some of the well characterized ECM proteins during the process of caudal fin regeneration in zebrafish. Our results show that a transitional matrix analogous to the one formed during newt skeletal and heart muscle regeneration is synthesized during fin regeneration. Here we demonstrate that a provisional matrix rich in hyaluronic acid, tenascin C, and fibronectin is synthesized following amputation. Additionally, we observed that the link protein Hapln1a dependent ECM, consisting of Hapln1a, hyaluronan and proteoglycan aggrecan, is upregulated during fin regeneration. Laminin, the protein characteristic of differentiated tissues, showed only modest change in the expression pattern. Our findings on zebrafish fin regeneration implicates that changes in the extracellular milieu represent an evolutionarily conserved mechanism that proceeds during tissue regeneration, yet with distinct players depending on the type of tissue that is involved.     

Fibroblast growth factor pathway component expression in the regenerating zebrafish fin

Adult zebrafish regenerate their appendages (fins) after amputation including the regeneration of bone structures (fin rays). Fibroblast growth factor (Fgf) signaling, which is involved in morphogenetic processes during development, has been shown to be essential for the process of fin regeneration. Moreover, mutations in Fgf pathway component genes lead to abnormal skeletal growth in teleosts and mammals, including humans, illustrating the importance of Fgf signaling in the growth control of tissues. Here, we revisited Fgf signaling pathway component expression by RNA in situ hybridization to test for the expression of about half of the ligands and all receptors of the pathway in the regenerating zebrafish fin. Expression patterns of fgf7, fgf10b, fgf12b, fgf17b and fgfr1b have not been reported in the literature before. We summarize and discuss known and novel localization of expression and find that all five Fgf receptors (fgfr1a, fgfr1b, fgfr2, fgfr3 and fgfr4) and most of the tested ligands are expressed in specific regions of the regenerate. Our work provides a basis to study domain specific functions of Fgf signaling in the regenerating teleost appendage.     

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration of adult tissues, such as the heart¹, retina², spinal cord³, optic nerve⁴, sensory hair cells⁵, and fins⁶.The zebrafish fin is a relatively simple appendage that is easily manipulated to study multiple stages in epimorphic regeneration. Classically, fin regeneration was characterized by three distinct stages: wound healing, blastema formation, and fin outgrowth. After amputating part of the fin, the surrounding epithelium proliferates and migrates over the wound. At 33 °C, this process occurs within six hours post-amputation (hpa, Figure 1B)^6,7. Next, underlying cells from different lineages (ex. bone, blood, glia, fibroblast) re-enter the cell cycle to form a proliferative blastema, while the overlying epidermis continues to proliferate (Figure 1D)⁸. Outgrowth occurs as cells proximal to the blastema re-differentiate into their respective lineages to form new tissue (Figure 1E)⁸. Depending on the level of the amputation, full regeneration is completed in a week to a month.The expression of a large number of gene families, including wnt, hox, fgf, msx, retinoic acid, shh, notch, bmp, and activin-betaA genes, is up-regulated during specific stages of fin regeneration^9-16. However, the roles of these genes and their encoded proteins during regeneration have been difficult to assess, unless a specific inhibitor for the protein exists¹³, a temperature-sensitive mutant exists or a transgenic animal (either overexpressing the wild-type protein or a dominant-negative protein) was generated^7,12. We developed a reverse genetic technique to quickly and easily test the function of any gene during fin regeneration.Morpholino oligonucleotides are widely used to study loss of specific proteins during zebrafish, Xenopus, chick, and mouse development^17-19. Morpholinos basepair with a complementary RNA sequence to either block pre-mRNA splicing or mRNA translation. We describe a method to efficiently introduce fluorescein-tagged antisense morpholinos into regenerating zebrafish fins to knockdown expression of the target protein. The morpholino is micro-injected into each blastema of the regenerating zebrafish tail fin and electroporated into the surrounding cells. Fluorescein provides the charge to electroporate the morpholino and to visualize the morpholino in the fin tissue.This protocol permits conditional protein knockdown to examine the role of specific proteins during regenerative fin outgrowth. In the Discussion, we describe how this approach can be adapted to study the role of specific proteins during wound healing or blastema formation, as well as a potential marker of cell migration during blastema formation.     

Transient reduction of 5-methylcytosine and 5-hydroxymethylcytosine is associated with active DNA demethylation during regeneration of zebrafish fin

Although dedifferentiation, transformation of differentiated cells into progenitor cells, is a critical step in the regeneration of amphibians and fish, the molecular mechanisms underlying this process, including epigenetic changes, remain unclear. Dot blot assays and immunohistochemical analyses revealed that, during regeneration of zebrafish fin, the levels of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) are transiently reduced in blastema cells and cells adjacent to the amputation plane at 30 h post-amputation (hpa), and the level of 5mC, but not 5hmC, is almost restored by 72 hpa. We observed that the dedifferentiated cells showed reduced levels of 5mC and 5hmC independent of cell proliferation by 24 hpa. Furthermore, expressions of the proposed demethylation- and DNA repair-related genes were detected during fin regeneration. Taken together, our findings illustrate that the transient reduction of 5mC and 5hmC in dedifferentiated cells is associated with active demethylation during regeneration of zebrafish fin.     

The chemokine SDF-1 regulates blastema formation during zebrafish fin regeneration

The work presented in this study focuses on blastema formation in epimorphic regeneration. We describe the expression pattern of Sdf1a and Sdf1b (the chemokines stromal-cell-derived factor-1a and 1b) and their two receptors Cxcr4a and Cxcr4b during zebrafish fin regeneration. We demonstrate that Sdf1a/Cxcr4a plays a critical role in fin regeneration and more precisely in epidermal cell proliferation, an important process for blastema formation. In mammals, a single cxcr4 gene is involved both in chemotaxis and cell proliferation and survival; we discuss in this study a possible functional division of the two cxcr4 zebrafish genes.     

Gene expression analysis of zebrafish retinal ganglion cells during optic nerve regeneration identifies KLF6a and KLF7a as important regulators of axon regeneration

Unlike mammals, teleost fish are able to mount an efficient and robust regenerative response following optic nerve injury. Although it is clear that changes in gene expression accompany axonal regeneration, the extent of this genomic response is not known. To identify genes involved in successful nerve regeneration, we analyzed gene expression in zebrafish retinal ganglion cells (RGCs) regenerating their axons following optic nerve injury. Microarray analysis of RNA isolated by laser capture microdissection from uninjured and 3-day post-optic nerve injured RGCs identified 347 up-regulated and 29 down-regulated genes. Quantitative RT-PCR and in situ hybridization were used to verify the change in expression of 19 genes in this set. Gene ontological analysis of the data set suggests regenerating neurons up-regulate genes associated with RGC development. However, not all regeneration-associated genes are expressed in differentiating RGCs indicating the regeneration is not simply a recapitulation of development. Knockdown of six highly induced regeneration-associated genes identified two, KLF6a and KLF7a, that together were necessary for robust RGC axon re-growth. These results implicate KLF6a and KLF7a as important mediators of optic nerve regeneration and suggest that not all induced genes are essential to mount a regenerative response.     

斑马鱼Gfi1.1基因的克隆及其体外转录

目的:克隆斑马鱼Gfi1.1基因的全长cDNA,运用T7 RNA聚合酶对含有Gfi1.1基因的ORF区进行体外转录,在体外合成5端带有帽子结构的Gfi1.1 mRNA分子,为后续研究斑马鱼Gfi1.1基因的功能打下基础。方法:应用RT-PCR从斑马鱼组织中扩增出Gfi1.1 cDNA片段,经回收纯化与pGM-T载体连接并转化感受态细菌DH-5α,通过蓝白筛选酶切鉴定阳性菌落,小量提取质粒,Nde I限制性内切酶线性化pGM-T-Gfi1.1质粒,运用T7 RNA聚合酶对Gfi1.1基因进行体外转录及加帽。经凝胶电泳对目的片段进行鉴定。结果:RT-PCR扩增获得约1.2 kb的DNA片段,DNA序列分析的结果与GenBank上的序列(NM_001020776)一致,酶切线性化及体外转录加帽pGM-T-Gfi1.1,凝胶电泳鉴定RNA分子大小与预期完全一致。结论:成功克隆斑马鱼Gfi1.1基因并体外转录及加帽pGM-T-Gfi1.1。     

A Zebrafish Model of Diabetes Mellitus and Metabolic Memory

Diabetes mellitus currently affects 346 million individuals and this is projected to increase to 400 million by 2030. Evidence from both the laboratory and large scale clinical trials has revealed that diabetic complications progress unimpeded via the phenomenon of metabolic memory even when glycemic control is pharmaceutically achieved. Gene expression can be stably altered through epigenetic changes which not only allow cells and organisms to quickly respond to changing environmental stimuli but also confer the ability of the cell to "memorize" these encounters once the stimulus is removed. As such, the roles that these mechanisms play in the metabolic memory phenomenon are currently being examined.We have recently reported the development of a zebrafish model of type I diabetes mellitus and characterized this model to show that diabetic zebrafish not only display the known secondary complications including the changes associated with diabetic retinopathy, diabetic nephropathy and impaired wound healing but also exhibit impaired caudal fin regeneration. This model is unique in that the zebrafish is capable to regenerate its damaged pancreas and restore a euglycemic state similar to what would be expected in post-transplant human patients. Moreover, multiple rounds of caudal fin amputation allow for the separation and study of pure epigenetic effects in an in vivo system without potential complicating factors from the previous diabetic state. Although euglycemia is achieved following pancreatic regeneration, the diabetic secondary complication of fin regeneration and skin wound healing persists indefinitely. In the case of impaired fin regeneration, this pathology is retained even after multiple rounds of fin regeneration in the daughter fin tissues. These observations point to an underlying epigenetic process existing in the metabolic memory state. Here we present the methods needed to successfully generate the diabetic and metabolic memory groups of fish and discuss the advantages of this model.     

GH indirectly enhances the regeneration of transgenic zebrafish fins through IGF2a and IGF2b

The somatotropic axis, composed essentially of the growth hormone (GH) and insulin-like growth factors (IGFs), is the main regulator of somatic growth in vertebrates. However, these protein hormones are also involved in various other major physiological processes. Although the importance of IGFs in mechanisms involving tissue regeneration has already been established, little is known regarding the direct effects of GH in these processes. In this study, we used a transgenic zebrafish (Danio rerio) model, which overexpresses GH from the beta-actin constitutive promoter. The regenerative ability of the caudal fin was assessed after repeated amputations, as well as the expression of genes related to the GH/IGF axis. The results revealed that GH overexpression increased the regenerated area of the caudal fin in transgenic fish after the second amputation. Transgenic fish also presented a decrease in gene expression of the GH receptor (ghrb), in opposition to the increased expression of the IGF1 receptors (igf1ra and igf1rb). These results suggest that transgenic fish have a higher sensitivity to IGFs than to GH during fin regeneration. With respect to the different IGFs produced locally, a decrease in igf1a expression and a significant increase in both igf2a and igf2b expression was observed, suggesting that igf1a is not directly involved in fin regeneration. Overall, the results revealed that excess GH enhances fin regeneration in zebrafish through igf2a and igf2b expression, acting indirectly on this major physiological process.     

Emerging roles for retinoids in regeneration and differentiation in normal and disease states

The vitamin A (retinol) metabolite, all-trans retinoic acid (RA), is a signaling molecule that plays key roles in the development of the body plan and induces the differentiation of many types of cells. In this review the physiological and pathophysiological roles of retinoids (retinol and related metabolites) in mature animals are discussed. Both in the developing embryo and in the adult, RA signaling via combinatorial Hox gene expression is important for cell positional memory. The genes that require RA for the maturation/differentiation of T cells are only beginning to be cataloged, but it is clear that retinoids play a major role in expression of key genes in the immune system. An exciting, recent publication in regeneration research shows that ALDH1a2 (RALDH2), which is the rate-limiting enzyme in the production of RA from retinaldehyde, is highly induced shortly after amputation in the regenerating heart, adult fin, and larval fin in zebrafish. Thus, local generation of RA presumably plays a key role in fin formation during both embryogenesis and in fin regeneration. HIV transgenic mice and human patients with HIV-associated kidney disease exhibit a profound reduction in the level of RARβ protein in the glomeruli, and HIV transgenic mice show reduced retinol dehydrogenase levels, concomitant with a greater than 3-fold reduction in endogenous RA levels in the glomeruli. Levels of endogenous retinoids (those synthesized from retinol within cells) are altered in many different diseases in the lung, kidney, and central nervous system, contributing to pathophysiology. This article is part of a Special Issue entitled Retinoid and Lipid Metabolism.     

Differential regulation of msx genes in the development of the gonopodium,an intromittent organ,and of the "sword," a sexually selected trait of swordtail fishes (Xiphophorus)

The possession of a conspicuous extension of colored ventral rays of the caudal fin in male fish of swordtails (genus Xiphophorus) is a prominent example for a trait that evolved by sexual selection. To understand the evolutionary history of this so-called sword molecularly, it is of interest to unravel the developmental pathways responsible for extended growth of sword rays during development of swordtail males. We isolated two msx genes and showed that they are differentially regulated during sword outgrowth. During sword growth in juvenile males, as well as during testosterone-induced sword development and fin ray regeneration in the sword after amputation, expression of msxC is markedly up-regulated in the sword forming fin rays. In contrast, msxE/1 is not differentially expressed in ventral and dorsal male fin rays, suggesting a link between the development of male secondary sexual characters in fins and up-regulation of msxC expression. In addition, we showed that msx gene expression patterns differ significantly between Xiphophorus and zebrafish. We also included in our study the gonopodium, a testosterone-dependent anal fin modification that serves as a fertilization organ in males of live-bearing fishes. Our finding that increased levels of msxC expression are associated with the testosterone-induced outgrowth of the gonopodium might suggest either that at least parts of the signaling pathways that pattern the evolutionary older gonopodium have been coopted to evolve a sexually selected innovation such as the sword or that increased msxC expression may be inherent to the growth process of long fin rays in general.