NRG1/ErbB signalling controls the dialogue between macrophages and neural crest-derived cells during zebrafish fin regeneration

AbstractFish species, such as zebrafish (Danio rerio), can regenerate their appendages after amputation through the formation of a heterogeneous cellular structure named blastema. Here, by combining live imaging of triple transgenic zebrafish embryos and single-cell RNA sequencing we established a detailed cell atlas of the regenerating caudal fin in zebrafish larvae. We confirmed the presence of macrophage subsets that govern zebrafish fin regeneration, and identified a foxd3-positive cell population within the regenerating fin. Genetic depletion of these foxd3-positive neural crest-derived cells (NCdC) showed that they are involved in blastema formation and caudal fin regeneration. Finally, chemical inhibition and transcriptomic analysis demonstrated that these foxd3-positive cells regulate macrophage recruitment and polarization through the NRG1/ErbB pathway. Here, we show the diversity of the cells required for blastema formation, identify a discrete foxd3-positive NCdC population, and reveal the critical function of the NRG1/ErbB pathway in controlling the dialogue between macrophages and NCdC.

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TNF signaling and macrophages govern fin regeneration in zebrafish larvae

Cell Death and Disease ◽

10.1038/cddis.2017.374 ◽

2017 ◽

Vol 8 (8) ◽

pp. e2979-e2979 ◽

Cited By ~ 47

Author(s):

Mai Nguyen-Chi ◽

Béryl Laplace-Builhé ◽

Jana Travnickova ◽

Patricia Luz-Crawford ◽

Gautier Tejedor ◽

...

Keyword(s):

Cell Activation ◽

Molecular Mechanisms ◽

Blastema Cell ◽

Direct Role ◽

Caudal Fin ◽

Fin Regeneration ◽

Blastema Formation ◽

Zebrafish Larvae ◽

Macrophage Subset ◽

Inflammatory Macrophages

Abstract Macrophages are essential for appendage regeneration after amputation in regenerative species. The molecular mechanisms through which macrophages orchestrate blastema formation and regeneration are still unclear. Here, we use the genetically tractable and transparent zebrafish larvae to study the functions of polarized macrophage subsets during caudal fin regeneration. After caudal fin amputation, we show an early and transient accumulation of pro-inflammatory macrophages concomitant with the accumulation of non-inflammatory macrophages which, in contrast to pro-inflammatory macrophages, remain associated to the fin until the end of the regeneration. Chemical and genetic depletion of macrophages suggested that early recruited macrophages that express TNFα are critical for blastema formation. Combining parabiosis and morpholino knockdown strategies, we show that TNFα/TNFR1 signaling pathway is required for the fin regeneration. Our study reveals that TNFR1 has a necessary and direct role in blastema cell activation suggesting that macrophage subset balance provides the accurate TNFα signal to prime regeneration in zebrafish.

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Two Different Transgenes to Study Gene Silencing and Re-Expression During Zebrafish Caudal Fin and Retinal Regeneration

The Scientific World JOURNAL ◽

10.1100/tsw.2006.328 ◽

2006 ◽

Vol 6 ◽

pp. 65-81 ◽

Cited By ~ 34

Author(s):

Ryan Thummel ◽

Christopher T. Burket ◽

David R. Hyde

Keyword(s):

Tissue Regeneration ◽

Transgene Expression ◽

Egfp Expression ◽

Transgenic Zebrafish ◽

Retinal Regeneration ◽

Caudal Fin ◽

Fin Regeneration ◽

Differentiated Cells ◽

Neuronal Progenitors ◽

Wound Epithelium

We used the 500-bpXenopusef1-α promoter and the 2-kb zebrafish histone2A.F/Zpromoter to generate several independent transgenic zebrafish lines expressing EGFP. While both promoters drive ubiquitous EGFP expression in early zebrafish development, they are systematically silenced in several adult tissues, including the retina and caudal fin. However, EGFP expression is temporarily renewed in the adult during either caudal fin or retinal regeneration. In the Tg(H2A.F/Z:EGFP)ntline, EGFP is moderately expressed in both the wound epithelium and blastema of the regenerating caudal fin. In the Tg(ef1-α:EGFP)ntline, EGFP expression is reinitiated and restricted to the blastema of the regenerating caudal fin and colabels with BrdU, PCNA, andmsxc-positive cells. Thus, these two ubiquitous promoters drive EGFP transgene expression in different cell populations during caudal fin regeneration. We further analyzed the ability of theef1-α:EGFPtransgene to label nonterminally differentiated cells during adult tissue regeneration. First, we demonstrated that the transgene is highly methylated in adult zebrafish caudal fin tissue, but not during fin regeneration, implicating methylation as a potential means of transgene silencing in this line. Next, we determined that theef1-α:EGFPtransgene is also re-expressed during adult retinal regeneration. Specifically, theef1-α:EGFPtransgene colabels with PCNA in the Müglia, a specialized cell that is the source of neuronal progenitors during zebrafish retinal regeneration. Thus, we concluded that Tg(ef1-α:EGFP)nt line visually marks nonterminally differentiated cells in multiple adult regeneration environments and may prove to be a useful marker in tissue regeneration studies in zebrafish.

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Inhibitory effects of polystyrene microplastics on caudal fin regeneration in zebrafish larvae

Environmental Pollution ◽

10.1016/j.envpol.2020.114664 ◽

2020 ◽

Vol 266 ◽

pp. 114664

Author(s):

Linqi Gu ◽

Li Tian ◽

Gan Gao ◽

Shaohong Peng ◽

Jieyu Zhang ◽

...

Keyword(s):

Inhibitory Effects ◽

Caudal Fin ◽

Fin Regeneration ◽

Zebrafish Larvae

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Inhibition of BMP and FGF signaling prior to wound epithelium formation leads to an aberrant regenerative response in teleost fish Poecilia latipinna

10.1101/2021.01.27.428424 ◽

2021 ◽

Author(s):

Isha Ranadive ◽

Sonam Patel ◽

Siddharth Pai ◽

Kashmira Khaire ◽

Suresh Balakrishnan

Keyword(s):

Cell Death ◽

Teleost Fish ◽

The Other ◽

Fgf Signaling ◽

Caudal Fin ◽

Fin Regeneration ◽

Blastema Formation ◽

Messenger Molecules ◽

Wound Epithelium

The BMP and FGF pathways play a pivotal role in the successful regeneration of caudal fin of teleost fish. Individual inhibition of these pathways led to impaired caudal fin regeneration until the pharmacologic inhibitor of FGF (SU5402) and BMP (LDN193189) were metabolized off. Therefore, in the current study both these pathways were inhibited collectively wherein inhibition of BMP and FGF during the wound epithelium formation led to stalling of the process by bringing down the established levels of shh and runx2. In members of the treatment group, it was observed that, each blastema grows crouched rather than linear and the regrown lepidotrichia therefore remain tilted down. Amongst the other irregularities observed, the transition from epithelial to mesenchymal cells was found hindered due to down-regulation of snail and twist, brought about by BMP and FGF inhibition. Compromised expression of Snail and twist deranged the normal levels of cadherins causing disruption in the transition of cells. Lastly, blocking BMP and FGF delayed blastema formation and proliferation due to diminished levels of fgf2, fgf8, fgf10 and bmp6, while casp3 and casp9 levels remained heightened causing accelerated cell death. This study not only highlights the axial role of BMP and FGF pathways in regeneration but also accentuates the collaboration amongst the two. This ingenious coordination of signalling further reinforces the involvement of relaying messenger molecules between these crucial pathways.

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Effects of 17α‑ethinylestradiol on caudal fin regeneration in zebrafish larvae

The Science of The Total Environment ◽

10.1016/j.scitotenv.2018.10.275 ◽

2019 ◽

Vol 653 ◽

pp. 10-22 ◽

Cited By ~ 1

Author(s):

Liwei Sun ◽

Linqi Gu ◽

Hana Tan ◽

Pan Liu ◽

Gan Gao ◽

...

Keyword(s):

Caudal Fin ◽

Fin Regeneration ◽

Zebrafish Larvae

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A proliferation gradient between proximal and msxb-expressing distal blastema directs zebrafish fin regeneration

Development ◽

10.1242/dev.129.11.2607 ◽

2002 ◽

Vol 129 (11) ◽

pp. 2607-2617 ◽

Cited By ~ 9

Author(s):

Alex Nechiporuk ◽

Mark T. Keating

Keyword(s):

High Rate ◽

Proliferating Cells ◽

Homogeneous Population ◽

Fin Regeneration ◽

New Model ◽

Blastema Formation ◽

Phosphohistone H3 ◽

Slow Cycling ◽

Early Regeneration ◽

Regeneration Blastema

Previous studies of zebrafish fin regeneration led to the notion that the regeneration blastema is a homogeneous population of proliferating cells. Here, we show that the blastema consists of two components with markedly distinct proliferation properties. During early blastema formation, proliferating cells are evenly distributed. At the onset of regenerative outgrowth, however, blastemal cells are partitioned into two domains. Proximal blastemal cells proliferate at a high rate, shifting from a median G2 of more than 6 hours to approximately 1 hour. By contrast, the most distal blastemal cells do not proliferate. There is a gradient of proliferation between these extremes. Using bromodeoxyuridine incorporation and anti-phosphohistone H3 labeling, we find a 50-fold difference in proliferation across the gradient that extends approximately 50 μm, or ten cell diameters. We show that during early regeneration, proliferating blastemal cells express msxb, a homeodomain transcriptional repressor. While msxb is widely expressed among proliferating cells during blastema formation, its expression becomes restricted to a small number of non-proliferating, distal blastemal cells during regenerative outgrowth. Bromodeoxyuridine pulse-chase experiments show that distal and proximal blastemal cells are formed from proliferating, msxb-positive blastemal cells, not from preexisting slow-cycling cells. These data support the idea that blastema formation results from dedifferentiation of intraray mesenchymal cells. Based on these findings, we propose a new model of zebrafish fin regeneration in which the function of non-proliferating, msxb-expressing, distal blastemal cells is to specify the boundary of proliferation and provide direction for regenerative outgrowth.

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Vitamin C supplementation improves growth performance and caudal fin regeneration in zebrafish ( Danio rerio )

Aquaculture Nutrition ◽

10.1111/anu.13407 ◽

2021 ◽

Author(s):

Pedro Luiz Pucci Figueiredo Carvalho ◽

Pedro Henrique Ventura Almeida ◽

William dos Santos Xavier ◽

Igor Simões Tiagua Vicente ◽

Matheus Gardim Guimarães ◽

...

Keyword(s):

Vitamin C ◽

Growth Performance ◽

Danio Rerio ◽

Caudal Fin ◽

Fin Regeneration ◽

Vitamin C Supplementation

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Regrowth of zebrafish caudal fin regeneration is determined by the amputated length

Scientific Reports ◽

10.1038/s41598-020-57533-6 ◽

2020 ◽

Vol 10 (1) ◽

Cited By ~ 3

Author(s):

Toshiaki Uemoto ◽

Gembu Abe ◽

Koji Tamura

Keyword(s):

Caudal Fin ◽

Fin Regeneration

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P0053A METHOD FOR THE ISOLATION OF FLUORESCENT GLOMERULI FROM ZEBRAFISH LARVAE

Nephrology Dialysis Transplantation ◽

10.1093/ndt/gfaa142.p0053 ◽

2020 ◽

Vol 35 (Supplement_3) ◽

Author(s):

Anna Iervolino ◽

Tim Lange ◽

Florian Siegerist ◽

Maximilian Schindler ◽

Giovambattista Capasso ◽

...

Keyword(s):

Glomerular Filtration ◽

Fluorescent Protein ◽

Transgenic Zebrafish ◽

Renal Tubules ◽

Glomerular Filtration Barrier ◽

Podocyte Injury ◽

Zebrafish Larvae ◽

Filtration Barrier ◽

Glomerular Damage

Abstract Background and Aims The zebrafish is a powerful animal model to study the glomerular morphology and the function of the permselectivity of the glomerular filtration barrier. Since zebrafish larvae develop quickly and can be bred to transparency, in vivo observation of these animals is possible. At 48 hours post fertilization (dpf), zebrafish develop a single filtering glomerulus which is attached to a pair of renal tubules. Like in mammals, the glomerular filtration barrier consists of a fenestrated endothelium, the glomerular basement membrane (GBM) and interdigitating podocyte foot processes bridged by a molecularly conserved slit diaphragm. By the use of genetically modified zebrafish strains with fluorescently labeled podocytes, it is possible to study alterations of the glomerulus during the development of renal disease directly in vivo and in vitro. As an injury model we used the nitroreductase/metronidazole (NTR/MTZ) zebrafish line to induce podocyte apoptosis and detachment from the GBM. Moreover, treatment of these larvae with MTZ induces glomerular injury that mimics focal segmental glomerulosclerosis (FSGS). The aim of our study was to establish a glomeruli isolation method which allows us to identify deregulation of miRNAs and mRNAs in the injured glomeruli by sequencing. Method The transgenic zebrafish strain Cherry (Tg(nphs2:Eco.nfsB-mCherry); mitfaw2/w2; mpv17a9/a9) which expresses the prokaryotic enzyme nitroreductase (NTR) fused to mCherry, a red fluorescent protein, under the control of the podocyte-specific podocin (nphs2) promoter in a transparent zebrafish strain, was used. The NTR/MTZ is a model of cell ablation to mimic podocyte injury. The prodrug MTZ (80 µM) is converted into a cytotoxin by NTR leading to a dose-dependent apoptosis exclusively in NTR-expressing podocytes. To induce podocyte injury, we treated Cherry larvae at 4 days post fertilization with MTZ (80 µM) freshly dissolved in 0.1% DMSO-E3 medium for 48 hours. Control larvae were treated with 0.1% DMSO-E3 medium. The treatment was stopped by a MTZ washout at 6 dpf. In order to perform the miRNA and mRNA sequencing on glomeruli isolated from MTZ-treated and control larvae we tried to establish a method to obtain total RNA samples of good quality. For this purpose, three different approaches were tested and validated: 1) Sieving method, 2) Fluorescence-Activated Cell Sorting method (FACS), and 3) manual isolation of glomeruli by using a micropipette. Results Zebrafish larvae developed a glomerular damage similar to FSGS after MTZ-treatment. MTZ-treated larvae showed severe pericardial edema, a reduction of the nephrin and podocin expression, proteinuria and an increased mortality rate at 8 dpf. After many tests we showed that glomeruli isolation using the sieving method and FACS were not efficient due to contaminations with other organs (sieving) and a loss of a large amount of cells per sample (FACS), respectively. Samples of the required quality for sequencing resulted only from the manual glomeruli isolation. Conclusion Here we describe methods to isolate fluorescent glomeruli from transgenic zebrafish larvae. For our studies, we used the NTZ/MTR kidney disease model in order to identify mRNAs and miRNAs regulated in response to glomerular damage. This technique will further allow to screen for healing drugs in high-throughput experiments.

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