Using the Tg(nrd:egfp)/albino zebrafish line to characterize in vivo expression of neurod

In this study, we used a newly-created transgenic zebrafish, Tg(nrd:egfp)/albino, to further characterize the expression of neurod in the developing and adult retina and to determine neurod expression during adult photoreceptor regeneration. We also provide observations regarding the expression of neurod in a variety of other tissues. In this line, EGFP is found in cells of the developing and adult retina, pineal gland, cerebellum, olfactory bulbs, midbrain, hindbrain, neural tube, lateral line, inner ear, pancreas, gut, and fin. Using immunohistochemistry and in situ hybridization, we compare the expression of the nrd:egfp transgene to that of endogenous neurod and to known retinal cell types. Consistent with previous data based on in situ hybridizations, we show that during retinal development, the nrd:egfp transgene is not expressed in proliferating retinal neuroepithelium, and is expressed in a subset of retinal neurons. In contrast to previous studies, nrd:egfp is gradually re-expressed in all rod photoreceptors. During photoreceptor regeneration in adult zebrafish, in situ hybridization reveals that neurod is not expressed in Müller glial-derived neuronal progenitors, but is expressed in photoreceptor progenitors as they migrate to the outer nuclear layer and differentiate into new rod photoreceptors. During photoreceptor regeneration, expression of the nrd:egfp matches that of neurod. We conclude that Tg(nrd:egfp)/albino is a good representation of endogenous neurod expression, is a useful tool to visualize neurod expression in a variety of tissues and will aid investigating the fundamental processes that govern photoreceptor regeneration in adults.     

Fate restriction in the growing and regenerating zebrafish fin

We use transposon-based clonal analysis to identify the lineage classes that make the adult zebrafish caudal fin. We identify nine distinct lineage classes, including epidermis, melanocyte/xanthophore, iridophore, intraray glia, lateral line, osteoblast, dermal fibroblast, vascular endothelium, and resident blood. These lineage classes argue for distinct progenitors, or organ founding stem cells (FSCs), for each lineage, which retain fate restriction throughout growth of the fin. Thus, distinct FSCs exist for the four neuroectoderm lineages, and dermal fibroblasts are not progenitors for fin ray osteoblasts; however, artery and vein cells derive from a shared lineage in the fin. Transdifferentiation of cells or lineages in the regeneration blastema is often postulated. However, our studies of single progenitors or FSCs reveal no transfating or transdifferentiation between these lineages in the regenerating fin. This result shows that, the same as in growth, lineages retain fate restriction when passed through the regeneration blastema.     

Divergent requirements for fibroblast growth factor signaling in zebrafish maxillary barbel and caudal fin regeneration

The zebrafish maxillary barbel is an integumentary organ containing skin, glands, pigment cells, taste buds, nerves, and endothelial vessels. The maxillary barbel can regenerate (LeClair & Topczewski 2010); however, little is known about its molecular regulation. We have studied fibroblast growth factor (FGF) pathway molecules during barbel regeneration, comparing this system to a well‐known regenerating appendage, the zebrafish caudal fin. Multiple FGF ligands (fgf20a, fgf24), receptors (fgfr1‐4) and downstream targets (pea3, il17d) are expressed in normal and regenerating barbel tissue, confirming FGF activation. To test if specific FGF pathways were required for barbel regeneration, we performed simultaneous barbel and caudal fin amputations in two temperature‐dependent zebrafish lines. Zebrafish homozygous for a point mutation in fgf20a, a factor essential for caudal fin blastema formation, regrew maxillary barbels normally, indicating that the requirement for this ligand is appendage‐specific. Global overexpression of a dominant negative FGF receptor, Tg(hsp70l:dn‐fgfr1:EGFP)^pd1 completely blocked fin outgrowth but only partially inhibited barbel outgrowth, suggesting reduced requirements for FGFs in barbel tissue. Maxillary barbels expressing dn‐fgfr1 regenerated peripheral nerves, dermal connective tissue, endothelial tubes, and a glandular epithelium; in contrast to a recent report in which dn‐fgfr1 overexpression blocks pharyngeal taste bud formation in zebrafish larvae (Kapsimali et al. 2011), we observed robust formation of calretinin‐positive tastebuds. These are the first experiments to explore the molecular mechanisms of maxillary barbel regeneration. Our results suggest heterogeneous requirements for FGF signaling in the regeneration of different zebrafish appendages (caudal fin versus maxillary barbel) and taste buds of different embryonic origin (pharyngeal endoderm versus barbel ectoderm).     

Colored medaka and zebrafish: Transgenics with ubiquitous and strong transgene expression driven by the medaka β‐actin promoter

Conditional cell labeling, cell tracing, and genetic manipulation approaches are becoming increasingly important in developmental and regenerative biology. Such approaches in zebrafish research are hampered by the lack of an ubiquitous transgene driver element that is active at all developmental stages. Here, we report the isolation and characterization of the medaka fish (Oryzias latipes) β‐actin (Olactb) promoter, which drives constitutive transgene expression during all developmental stages, and the analysis of adult organs except blood cell types. Taking advantage of the compact medaka promoter, we succeeded in generating a zebrafish transgenic (Tg) line with unprecedentedly strong and widespread transgene expression from embryonic to adult stages. Moreover, the Tg carries a pair of loxP sites, which enables the reporter fluorophore to switch from DsRed2 to enhanced green fluorescent protein (EGFP). We induced Cre/loxP recombination with Tg(hsp70l: mCherry‐t2a‐Cre^ERt2) in the double Tg embryo and generated a Tg line that constitutively expresses EGFP. We further demonstrate the powerful application of Olactb‐driven Tgs for cell lineage tracing using transplantation experiments with embryonic cells at the shield stage and adult cells of regenerating fin. Thus, the use of promoter elements from medaka is an alternative approach to generate Tgs with stronger and even novel expression patterns in zebrafish. The Olactb promoter and the Tg lines presented here represent an important advancement for the broader use of Cre/loxP‐based Tg applications in zebrafish.     

F-spondin/spon1b expression patterns in developing and adult zebrafish

F-spondin, an extracellular matrix protein, is an important player in embryonic morphogenesis and CNS development, but its presence and role later in life remains largely unknown. We generated a transgenic zebrafish in which GFP is expressed under the control of the F-spondin (spon1b) promoter, and used it in combination with complementary techniques to undertake a detailed characterization of the expression patterns of F-spondin in developing and adult brain and periphery. We found that F-spondin is often associated with structures forming long neuronal tracts, including retinal ganglion cells, the olfactory bulb, the habenula, and the nucleus of the medial longitudinal fasciculus (nMLF). F-spondin expression coincides with zones of adult neurogenesis and is abundant in CSF-contacting secretory neurons, especially those in the hypothalamus. Use of this new transgenic model also revealed F-spondin expression patterns in the peripheral CNS, notably in enteric neurons, and in peripheral tissues involved in active patterning or proliferation in adults, including the endoskeleton of zebrafish fins and the continuously regenerating pharyngeal teeth. Moreover, patterning of the regenerating caudal fin following fin amputation in adult zebrafish was associated with F-spondin expression in the blastema, a proliferative region critical for tissue reconstitution. Together, these findings suggest major roles for F-spondin in the CNS and periphery of the developing and adult vertebrate.     

Comparative Expression Profiling Reveals an Essential Role for Raldh2 in Epimorphic Regeneration

Zebrafish have the remarkable ability to regenerate body parts including the heart and fins by a process referred to as epimorphic regeneration. Recent studies have illustrated that similar to adult zebrafish, early life stage larvae also possess the ability to regenerate the caudal fin. A comparative microarray analysis was used to determine the degree of conservation in gene expression among the regenerating adult caudal fin, adult heart, and larval fin. Results indicate that these tissues respond to amputation/injury with strikingly similar genomic responses. Comparative analysis revealed raldh2, a rate-limiting enzyme for the synthesis of retinoic acid, as one of the most highly induced genes across the three regeneration platforms. In situ localization and functional studies indicate that raldh2 expression is critical for the formation of wound epithelium and blastema. Patterning during regenerative outgrowth was considered to be the primary function of retinoic acid signaling; however, our results suggest that it is also required for early stages of tissue regeneration. Expression of raldh2 is regulated by Wnt and fibroblast growth factor/ERK signaling.     

Limited dedifferentiation provides replacement tissue during zebrafish fin regeneration

Unlike humans, some vertebrate animals are able to completely regenerate damaged appendages and other organs. For example, adult zebrafish will regenerate the complex structure of an amputated caudal fin to a degree that the original and replacement fins are indistinguishable. The blastema, a mass of cells that uniquely forms following appendage amputation in regenerating animals, is the major source of regenerated tissue. However, the cell lineage(s) that contribute to the blastema and their ultimate contribution(s) to the regenerated fin have not been definitively characterized. It has been suggested that cells near the amputation site dedifferentiate forming multipotent progenitors that populate the blastema and then give rise to multiple cell types of the regenerated fin. Other studies propose that blastema cells are non-uniform populations that remain restricted in their potential to contribute to different cell lineages. We tested these models by using inducible Cre-lox technology to generate adult zebrafish with distinct, isolated groups of genetically labeled cells within the caudal fin. We then tracked populations of several cell types over the entire course of fin regeneration in individual animals. We found no evidence for the existence of multipotent progenitors. Instead, multiple cell types, including epidermal cells, intra-ray fibroblasts, and osteoblasts, contribute to the newly regenerated tissue while remaining highly restricted with respect to their developmental identity. Our studies further demonstrate that the regenerating fin consists of many repeating blastema "units" dedicated to each fin ray. These blastemas each have an organized structure of lineage restricted, dedifferentiated cells that cooperate to regenerate the caudal fin.     

Temperature-Sensitive Mutations That Cause Stage-Specific Defects in Zebrafish Fin Regeneration

When amputated, the fins of adult zebrafish rapidly regenerate the missing tissue. Fin regeneration proceeds through several stages, including wound healing, establishment of the wound epithelium, recruitment of the blastema from mesenchymal cells underlying the wound epithelium, and differentiation and outgrowth of the regenerate. We screened for temperature-sensitive mutations that affect the regeneration of the fin. Seven mutations were identified, including five that fail to regenerate their fins, one that causes slow growth during regeneration, and one that causes dysmorphic bumps or tumors to develop in the regenerating fin. reg5 mutants fail to regenerate their caudal fins, whereas reg6 mutants develop dysmorphic bumps in their regenerates at the restrictive temperature. Temperature-shift experiments indicate that reg5 and reg6 affect different stages of regeneration. The critical period for reg5 occurs during the early stages of regeneration before or during establishment of the blastema, resulting in defects in subsequent growth of the blastema and failure to differentiate bone-forming cells. The critical period for reg6 occurs after the onset of bone differentiation and during early stages of regenerative outgrowth. Both reg5 and reg6 also show temperature-sensitive defects in embryonic development or in ontogenetic outgrowth of the juvenile fin.     

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.     

Positional cloning of a temperature-sensitive mutant emmental reveals a role for sly1 during cell proliferation in zebrafish fin regeneration

Here, we used classical genetics in zebrafish to identify temperature-sensitive mutants in caudal fin regeneration. Gross morphological, histological, and molecular analyses revealed that one of these strains, emmental (emm), failed to form a functional regeneration blastema. Inhibition of emm function by heat treatment during regenerative outgrowth rapidly blocked regeneration. This block was associated with reduced proliferation in the proximal blastema and expansion of the nonproliferative distal blastemal zone. Positional cloning revealed that the emm phenotype is caused by a mutation in the orthologue of yeast sly1, a gene product involved in protein trafficking. sly1 is upregulated in the newly formed blastema as well as during regenerative outgrowth. Thus, sly1 is essential for blastemal organization and proliferation during two stages of fin regeneration.     

Proteomic analysis of zebrafish caudal fin regeneration

The epimorphic regeneration of zebrafish caudal fin is rapid and complete. We have analyzed the biomechanism of zebrafish caudal fin regeneration at various time points based on differential proteomics approaches. The spectrum of proteome changes caused by regeneration were analyzed among controls (0 h) and 1, 12, 24, 48, and 72 h postamputation involving quantitative differential proteomics analysis based on two-dimensional gel electrophoresis matrix-assisted laser desorption/ionization and differential in-gel electrophoresis Orbitrap analysis. A total of 96 proteins were found differentially regulated between the control nonregenerating and regenerating tissues of different time points for having at least 1.5-fold changes. 90 proteins were identified as differentially regulated for regeneration based on differential in-gel electrophoresis analysis between the control and regenerating tissues. 35 proteins were characterized for its expression in all of the five regenerating time points against the control samples. The proteins identified and associated with regeneration were found to be directly allied with various molecular, biological, and cellular functions. Based on network pathway analysis, the identified proteome data set for regeneration was majorly associated in maintaining cellular structure and architecture. Also the proteins were found associated for the cytoskeleton remodeling pathway and cellular immune defense mechanism. The major proteins that were found differentially regulated during zebrafish caudal fin regeneration includes keratin and its 10 isoforms, cofilin 2, annexin a1, skeletal α1 actin, and structural proteins. Annexin A1 was found to be exclusively undergoing phosphorylation during regeneration. The obtained differential proteome and the direct association of the various proteins might lead to a new understanding of the regeneration mechanism.     

The regenerative capacity of the zebrafish caudal fin is not affected by repeated amputations

Background

The zebrafish has the capacity to regenerate many tissues and organs. The caudal fin is one of the most convenient tissues to approach experimentally due to its accessibility, simple structure and fast regeneration. In this work we investigate how the regenerative capacity is affected by recurrent fin amputations and by experimental manipulations that block regeneration.

Methodology/Principal Findings

We show that consecutive repeated amputations of zebrafish caudal fin do not reduce its regeneration capacity and do not compromise any of the successive regeneration steps: wound healing, blastema formation and regenerative outgrowth. Interfering with Wnt/ß-catenin signalling using heat-shock-mediated overexpression of Dickkopf1 completely blocks fin regeneration. Notably, if these fins were re-amputated at the non-inhibitory temperature, the regenerated caudal fin reached the original length, even after several rounds of consecutive Wnt/ß-catenin signalling inhibition and re-amputation.

Conclusions/Significance

We show that the caudal fin has an almost unlimited capacity to regenerate. Even after inhibition of regeneration caused by the loss of Wnt/ß-catenin signalling, a new amputation resets the regeneration capacity within the caudal fin, suggesting that blastema formation does not depend on a pool of stem/progenitor cells that require Wnt/ß-catenin signalling for their survival.     

Mps1 defines a proximal blastemal proliferative compartment essential for zebrafish fin regeneration

One possible reason why regeneration remains enigmatic is that the dominant organisms used for studying regeneration are not amenable to genetic approaches. We mutagenized zebrafish and screened for temperature-sensitive defects in adult fin regeneration. The nightcap mutant showed a defect in fin regeneration that was first apparent at the onset of regenerative outgrowth. Positional cloning revealed that nightcap encodes the zebrafish orthologue of mps1, a kinase required for the mitotic checkpoint. mps1 expression was specifically induced in the proximal regeneration blastema, a group of cells that normally proliferate intensely during outgrowth. The nightcap mutation caused severe defects in these cells. However, msxb-expressing blastemal cells immediately distal to this proliferative region did not induce mps1 and were retained in mutants. These results indicate that the proximal blastema comprises an essential subpopulation of the fin regenerate defined by the induction and function of Mps1. Furthermore, we show that molecular mechanisms of complex tissue regeneration can now be dissected using zebrafish genetics.     

Zebrafish keratin 8 is expressed at high levels in the epidermis of regenerating caudal fin

Cytokeratins are structural proteins of the intermediate filament family and are mainly expressed in epithelial cells. In several vertebrates it has been shown that keratin 8 is expressed in simple epithelial tissues, some non-epithelial tissue and in hyper-proliferative tissues during development and tumor transformation. We previously cloned and characterised the zebrafish (Danio rerio) homologous cytokeratin 8 cDNA (zfk8) which was described as an epidermal marker during zebrafish development. It has been found that the zfk8 gene is normally expressed in simple epithelia in embryonic and mature zebrafish. Using whole-mount in situ hybridisation, we show in this report that expression of zfk8 is tightly linked to the regeneration of caudal fin and exclusively observed in epidermal cells. It is strongly expressed in the epidermis overlaying the inter-rays zone of regenerating caudal fin. Our results indicate that in zebrafish, cytokeratin 8 is a suitable epidermal marker during regeneration.     

Establishment of a transgenic zebrafish EF1α:Kaede for monitoring cell proliferation during regeneration

Zebrafish is considered as a versatile experimental animal for various research models from development to diseases. In this study, we report the development of transgenic zebrafish line named as Tg(EF1α:Kaede) that expresses translation elongation factor 1 subunit alpha (EF1α) promoter linked to a fluorescent protein (FP), Kaede for monitoring proliferating cells in during regeneration. It was revealed that about 1.4 kb 5′-flanking region of the EF1α was sufficient for its promoter activity. Expression of Kaede with a property of photo-conversion from green to red was detected in different embryonic stages as well as various organs such as brain, heart, pancreas, intestine, ovary, and fins of adult fish. Cell proliferation pattern during fin regeneration was monitored after amputation of Tg(EF1α:Kaede) caudal fin and results shown that this system is simple and efficient method for detecting proliferating cells during tissue regeneration. Developed Tg(EF1α:Kaede) line has potential to investigate the cell proliferation, regeneration, wound healing capacities after tissue damage and evaluate the therapeutic power of wound healing drugs.