Calcineurin controls proximodistal blastema polarity in zebrafish fin regeneration

Planarian flatworms regenerate their heads and tails from anterior or posterior wounds and this regenerative blastema polarity is controlled by Wnt/β-catenin signaling. It is well known that a regeneration blastema of appendages of vertebrates such as fish and amphibians grows distally. However, it remains unclear whether a regeneration blastema in vertebrate appendages can grow proximally. Here, we show that a regeneration blastema in zebrafish fins can grow proximally along the proximodistal axis by calcineurin inhibition. We used fin excavation in adult zebrafish to observe unidirectional regeneration from the anterior cut edge (ACE) to the posterior cut edge (PCE) of the cavity and this unidirectional regeneration polarity occurs as the PCE fails to build blastemas. Furthermore, we found that calcineurin activities in the ACE were greater than in the PCE. Calcineurin inhibition induced PCE blastemas, and calcineurin hyperactivation suppressed fin regeneration. Collectively, these findings identify calcineurin as a molecular switch to specify the PCE blastema of the proximodistal axis and regeneration polarity in zebrafish fin.

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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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Macrophages modulate adult zebrafish tail fin regeneration

Development ◽

10.1242/dev.120642 ◽

2015 ◽

Vol 142 (2) ◽

pp. 406-406 ◽

Cited By ~ 14

Author(s):

T. A. Petrie ◽

N. S. Strand ◽

C.-T. Yang ◽

J. S. Rabinowitz ◽

R. T. Moon

Keyword(s):

Adult Zebrafish ◽

Fin Regeneration

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

Development ◽

10.1242/dev.129.22.5141 ◽

2002 ◽

Vol 129 (22) ◽

pp. 5141-5149

Author(s):

Kenneth D. Poss ◽

Alex Nechiporuk ◽

Ann M. Hillam ◽

Stephen L. Johnson ◽

Mark T. Keating

Keyword(s):

Tissue Regeneration ◽

Positional Cloning ◽

Molecular Mechanisms ◽

Mitotic Checkpoint ◽

Temperature Sensitive ◽

Fin Regeneration ◽

And Function ◽

Regeneration Blastema

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 nightcapencodes 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 mps1and 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.

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Zebrafish kit mutation reveals primary and secondary regulation of melanocyte development during fin stripe regeneration

Development ◽

10.1242/dev.127.17.3715 ◽

2000 ◽

Vol 127 (17) ◽

pp. 3715-3724 ◽

Cited By ~ 10

Author(s):

J.F. Rawls ◽

S.L. Johnson

Keyword(s):

Receptor Tyrosine Kinase ◽

Mutational Analysis ◽

Adult Zebrafish ◽

Wild Type ◽

Kit Mutation ◽

Fin Regeneration ◽

Normal Melanocyte ◽

Kit Receptor ◽

Early Regeneration ◽

Secondary Mode

Fin regeneration in adult zebrafish is accompanied by re-establishment of the pigment stripes. To understand the mechanisms underlying fin stripe regeneration and regulation of normal melanocyte stripe morphology, we investigated the origins of melanocytes in the regenerating fin and their requirement for the kit receptor tyrosine kinase. Using pre-existing melanin as a lineage tracer, we show that most fin regeneration melanocytes develop from undifferentiated precursors, rather than from differentiated melanocytes. Mutational analysis reveals two distinct classes of regeneration melanocytes. First, an early regeneration class develops dependent on kit function. In the absence of kit function and kit-dependent melanocytes, a second class of melanocytes develops at later stages of regeneration. This late kit-independent class of regeneration melanocytes has little or no role in wild-type fin stripe development, thus revealing a secondary mode for regulation of fin stripes. Expression of melanocyte markers in regenerating kit mutant fins suggests that kit normally acts after mitf and before dct to promote development of the primary kit-dependent melanocytes. kit-dependent and kit-independent melanocytes are also present during fin stripe ontogeny in patterns similar to those observed during regeneration.

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Notch regulates blastema proliferation and prevents differentiation during adult zebrafish fin regeneration

Development ◽

10.1242/dev.087346 ◽

2013 ◽

Vol 140 (7) ◽

pp. 1402-1411 ◽

Cited By ~ 49

Author(s):

J. Munch ◽

A. Gonzalez-Rajal ◽

J. L. de la Pompa

Keyword(s):

Adult Zebrafish ◽

Fin Regeneration

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Impact of hypoxia induced VEGF and its signaling during caudal fin regeneration in Zebrafish

10.1101/105767 ◽

2017 ◽

Cited By ~ 1

Author(s):

sagayaraj.R Vivek ◽

R. Malathi

Keyword(s):

Wound Healing ◽

Blood Vessel ◽

Cobalt Chloride ◽

Adult Zebrafish ◽

Caudal Fin ◽

Fin Regeneration ◽

Blood Vessel Formation ◽

Vessel Formation ◽

Vegf Signaling ◽

Fin Length

ABSTRACT:Hypoxia is known to play important role during various cellular process, including regeneration. Regeneration is a complex process involving wound healing and tissue repair. We propose that hypoxia might mediate regeneration through angiogenesis involving angiogenic factors such as VEGF, VEGF-R2, NRP1a during the wound healing process. We have chosen Zebrafish model to study the role of hypoxia induced regeneration. Unlike mammals Zebrafish has the ability to regenerate. Hypoxic condition was mimicked using inorganic salt cobalt chloride to study caudal fin regeneration in adult Zebrafish. Intense blood vessel formation, with increased tail fin length experimented at various time points have been observed when adult zebrafish caudal fin partially amputated were exposed to 1% CoCl2. Regeneration is enhanced under hypoxia, with increased VEGF expression. To study the significance of VEGF signaling during wound healing and tissue regeneration, sunitinib well known inhibitor of VEGF receptor is used against CoCl2-induced caudal fin regeneration. Diminished fin length, lowering of blood vessel formation was documented using angioquant software, reduction in mRNA level of hypoxia inducible factors, VEGF and other pro-angiogenic genes such as VEGF, VEGF-R2, NRP1A, FGFR2, ANGPT1 were observed, while reduction in VEGF protein was demonstrated using western blot analysis. Genistein inhibitor of HIF-1α completely arrested regeneration, with suppression of VEGF highlighting the significance of hypoxia induced VEGF signaling during fin regeneration. Our results suggest that hypoxia through HIF-1α might lead to angiogenesis involving VEGF signaling during wound healing and this might throw light on therapeutic efficacy of cobalt chloride during regeneration.

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ScreenCube: A 3D Printed System For Rapid and Cost-Effective Chemical Screening in Adult Zebrafish

10.1101/163469 ◽

2017 ◽

Author(s):

Adrian T. Monstad-Rios ◽

Claire J. Watson ◽

Ronald Y. Kwon

Keyword(s):

Small Molecule ◽

Cost Effective ◽

Adult Zebrafish ◽

Fin Regeneration ◽

Drug Dosing ◽

Chemical Screening ◽

Models Of Development ◽

Dosing Regimens ◽

3D Printed ◽

Fish Survival

ABSTRACTPhenotype-based small molecule screens in zebrafish embryos and larvae have been successful in accelerating pathway and therapeutic discovery for diverse biological processes. Yet, the application of chemical screens to adult physiologies has been relatively limited due to additional demands on cost, space, and labor associated with screens in adult animals. Here, we present a 3D printed system and methods for intermittent drug dosing that enable rapid and cost-effective chemical administration in adult zebrafish. Using pre-filled screening plates, the system enables dosing of 96 fish in ˜3min, with a tenfold-reduction in drug quantity compared to that used in previous chemical screens in adult zebrafish. We characterize water quality kinetics during immersion in the system, and use these kinetics to rationally design intermittent dosing regimens that result in 100% fish survival. As a demonstration of system fidelity, we show the potential to identify two known chemical inhibitors of adult tail fin regeneration, cyclopamine and dorsomorphin. By developing methods for rapid and cost-effective chemical administration in adult zebrafish, this study expands the potential for small molecule discovery in post-embryonic models of development, disease, and regeneration.

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An early Shh-H2O2 feedback loop controls the progression of the regenerative program during adult zebrafish fin regeneration

10.1101/2021.08.19.456615 ◽

2021 ◽

Author(s):

Marion Thauvin ◽

Rodolphe Matias de Sousa ◽

Marine Alves ◽

Michel Volovitch ◽

Sophie Vriz ◽

...

Keyword(s):

Reactive Oxygen Species ◽

Sonic Hedgehog ◽

Feedback Loop ◽

Ros Production ◽

Oxygen Species ◽

Adult Zebrafish ◽

Regenerative Processes ◽

Fin Regeneration ◽

Spatiotemporal Regulation ◽

New Findings

AbstractReactive oxygen species (ROS), originally classified as toxic molecules, have attracted increasing interest given their actions in cell signaling. Among these molecules, Hydrogen peroxide (H2O2) is the major ROS produced by cells and acts as a second messenger to modify redox-sensitive proteins or lipids. After amputation, tight spatiotemporal regulation of ROS is required first for wound healing and later to initiate the regenerative program. However, the mechanisms carrying out this sustained ROS production and their integration with signaling pathways are still poorly understood. We focused on the early dialog between H2O2 and Sonic Hedgehog (Shh) during fin regeneration. We demonstrate that H2O2 controls Shh expression and that Shh in turn regulates the H2O2 level via a canonical pathway. Moreover, this tightly controlled feedback loop changes during the successive phases of the regenerative program. Dysregulation of the Hedgehog pathway has been implicated in several developmental syndromes, diabetes and cancer. These data support the existence of a very early feedback loop between Shh and H2O2 that might be more generally involved in various physiological or pathological processes. These new findings pave the way to improve regenerative processes, particularly in vertebrates.

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