Zebrafish Fin Research Articles

Expression of meis and hoxa11 in dipnoan and teleost fins provides new insights into the evolution of vertebrate appendages

BackgroundThe concerted activity of Meis and Hoxa11 transcription factors is essential for the subdivision of tetrapod limbs into proximo-distal (PD) domains; however, little is know about the evolution of this patterning mechanism. Here, we aim to study the expression of meis and hoxa11 orthologues in the median and paired rayed fins of zebrafish and in the lobed fins of the Australian lungfish.ResultsFirst, a late phase of expression of meis1.1 and hoxa11b in zebrafish dorsal and anal fins relates with segmentation of endochondral elements in proximal and distal radials. Second, our zebrafish in situ hybridization results reveal spatial and temporal changes between pectoral and pelvic fins. Third, in situ analysis of meis1, meis3 and hoxa11 genes in Neoceratodus pectoral fins identifies decoupled domains of expression along the PD axis.ConclusionsOur data raise the possibility that the origin of stylopod and zeugopod lies much deeper in gnathostome evolution and that variation in meis and hoxa11 expression has played a substantial role in the transformation of appendage anatomy. Moreover, these observations provide evidence that the Meis/Hoxa11 profile considered a hallmark of stylopod/zeugopod patterning is present in Neoceratodus.

Heterogeneous fates and dynamic rearrangement of regenerative epidermis-derived cells during zebrafish fin regeneration.

The regenerative epidermis (RE) is a specialized tissue that plays an essential role in tissue regeneration. However, the fate of the RE during and after regeneration is unknown. In this study, we performed Cre-loxP-mediated cell fate tracking and revealed the fates of a major population of the RE cells that express fibronectin 1b (fn1b) during zebrafish fin regeneration. Our study showed that these RE cells are mainly recruited from the inter-ray epidermis, and that they follow heterogeneous cell fates. Early recruited cells contribute to initial wound healing and soon disappear by apoptosis, while the later recruited cells contribute to the regenerated epidermis. Intriguingly, many of these cells are also expelled from the regenerated tissue by a dynamic caudal movement of the epidermis over time, and in turn the loss of epidermal cells is replenished by a global self-replication of basal and suprabasal cells in fin. De-differentiation of non-basal epidermal cells into the basal epidermal cells did not occur during regeneration. Overall, our study reveals the heterogeneous fates of RE cells and a dynamic rearrangement of the epidermis during and after regeneration.

Comparative anatomy and development of zebrafish fin muscles: basis for functional, developmental, and macroevolutionary studies

Dario rerio is a model organism extensively used in numerous evo‐devo studies devoted to the fin skeletal anatomy and fin‐limb transition. While the skeleton of the paired and median fins of the zebrafish have been described in great detail, little is known about the anatomy and development of the appendicular musculature of this fish. In order to provide a basis for myological, comparative and evolutionary studies, we described for the first time the development, identity, overall configuration, and attachments of all appendicular muscles of D. rerio. We show that the muscle pattern of the caudal fin differs from patterns seen in other zebrafish fins but it is very similar among teleosts in general. This finding supports the idea that developmentally and evolutionary this fin is very peculiar. We describe for the first time interhypural muscles present in the caudal fin of young fishes and absent in adults. The musculature of the adult zebrafish pectoral and pelvic fins is very similar in general, but we note marked differences concerning the development of these muscles. We therefore discuss whether the pectoral appendage might be evolutionary closer to the head than to the pelvic appendage. We provide the anatomical and developmental description of the dorsal and anal fin muscles in zebrafish and describe homologies between the appendicular structures of zebrafish and other fish taxa. We discuss evolutionary trends within these clades to investigate whether the zebrafish is a good model for teleosts in particular and actinopterygians in general. Lastly, we discuss examples of phylo‐devo parallelism that links evolution and development. Our data on the adult phenotype and myogenesis in the zebrafish provide a basis for more integrative developmental, functional, evolutionary, and pathological studies.Support or Funding InformationThis work was supported by Russian Foundation for Basic Research, project nos. 14‐04‐00590 and 17‐04‐01617. The research was done using equipment of the Core Centrum of Insitute of Developmental Biology RAS.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abnormal development of the paired and median fins in the hyperthyroidism case series of the zebrafish (Danio rerio)

Hypo‐ and hyperthyroidism are severe medical conditions frequently found in humans living all over the globe. These diseases are caused by the dysfunction of the thyroid gland and lead to the abnormal embryonic development, various neurological problems, and brain damage. In order to better understand these two conditions in humans, we used zebrafish (Danio rerio) as a model to study the variability of effects of thyroid hormones. We compared normal development of zebrafish fins with pathological series of fishes extensively treated with triiodothyronine (T3) hormone.Analysis of the fish hyperthyroidism case series revealed certain symptoms similar to those present in human patients. Namely, we observed malformations of the appendicular skeleton and fusion of bones, likely occurred due to the late ossification, as well as significant underdevelopment of the appendicular musculature. Surprisingly, T3 hormone treatment induced excessive myogenesis in the dorsal and anal fin folds where muscles do not normally develop. In addition to various anatomical defects, hyperthyroidism resulted into accelerated growth of fishes in general and led to heterochrony. Interestingly, defects found in our pathological series seem to be similar to defects caused in zebrafishes by stress of the environmental change. These similarities between defects caused by completely different conditions support Pere Alberch's idea of ‘logic of monsters', according to which evolution is so constrained that even when it goes ‘wrong’ ‐ e.g. in cases of severe defects ‐ the resulting phenotypes mirror those seen in other abnormal conditions as well as those seen in the natural variations and/or normal phenotype of other taxa.These results have crucial implications for both Evo‐Devo research and evolutionary developmental medicine, or ‘Evo‐Devo‐Path’. Specifically, because hyper‐ and hypothyroidism are two disorders frequently found in humans, and because such conditions are deeply connected with heterochrony and thus with micro‐ and macroevolution.Support or Funding InformationThis work was supported by Russian Foundation for Basic Research, project nos. 14‐04‐00590 and 17‐04‐01617. The research was done using equipment of the Core Centrum of Institute of Developmental Biology RAS.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Structural basis of thalidomide enantiomer binding to cereblon

Thalidomide possesses two optical isomers which have been reported to exhibit different pharmacological and toxicological activities. However, the precise mechanism by which the two isomers exert their different activities remains poorly understood. Here, we present structural and biochemical studies of (S)- and (R)-enantiomers bound to the primary target of thalidomide, cereblon (CRBN). Our biochemical studies employed deuterium-substituted thalidomides to suppress optical isomer conversion, and established that the (S)-enantiomer exhibited ~10-fold stronger binding to CRBN and inhibition of self-ubiquitylation compared to the (R)-enantiomer. The crystal structures of the thalidomide-binding domain of CRBN bound to each enantiomer show that both enantiomers bind the tri-Trp pocket, although the bound form of the (S)-enantiomer exhibited a more relaxed glutarimide ring conformation. The (S)-enantiomer induced greater teratogenic effects on fins of zebrafish compared to the (R)-enantiomer. This study has established a mechanism by which thalidomide exerts its effects in a stereospecific manner at the atomic level.

Inhibition of Epithelial-Mesenchymal Transition and Tissue Regeneration by Waterborne Titanium Dioxide Nanoparticles.

Titanium dioxide nanoparticles (TiO2NPs) are among the most widely manufactured nanomaterials with broad applications in food industry, cosmetics, and medicine. Although the toxicity of TiO2NPs at high doses has been extensively explored, the potential health risks of TiO2NPs exposure at nontoxic concentrations remain poorly understood. Epithelial-mesenchymal transition (EMT) plays pivotal roles in a diversity of physiological and pathological processes, including tissue regeneration and cancer metastasis. In this study, we find that the cellular uptake of TiO2NPs inhibits EMT-mediated cell remodeling and cell migration without exhibiting cytotoxicity. Further investigation reveals that TiO2NPs suppress the process of EMT through the blockade of transforming growth factor-β (TGFβ) signaling. Particularly, TiO2NPs interact with the TGFβ receptor TβRI/II complex, induce its lysosomal degradation, and thereby downregulate expression of TGFβ target genes. Moreover, we show that waterborne TiO2NPs do not elicit toxicity in healthy tissues but hamper EMT-mediated wound healing in two animal models. Long-term exposure of TiO2NPs in environmental water and drinking water impede the regeneration of amputated fin in zebrafish and the recovery of intestinal mucosal damage in colitic mice. Our results reveal the previously unknown effects of TiO2NPs during tissue remodeling and repair, which have significant implications in their risk assessment and management.

Estimation of Hidden Chemoattractant Field from Observed Cell Migration Patterns

Neutrophilic chemotaxis is essential to immune system response to external threats. During this process cells alternate between directed motion towards the higher concentration of external stimuli and correlated random walk. An individual neutrophil migration can thus be characterised as a stochastic dynamical process driven by an external chemotactic environment that is typically not measured. This introduces the problem of estimating spatially-varying chemoattractant concentration field from the observed migration patterns of cell populations. We propose a solution to this estimation problem in a statistical inference framework. The framework has measured cell positions in the field as inputs and employs the expectation-maximisation algorithm for joint estimation of full cell states and parameters of the chemoattractant field decomposed with cubic B-splines. The performance of the developed algorithm is accessed via process in vivo measurements of cell positions in the injured tail fin of zebrafish. Estimation results for different injury types evidence that the proposed estimation algorithm provides a rigorous connection between mathematical modelling and experimental data.

A regulatory pathway involving retinoic acid and calcineurin demarcates and maintains joint cells and osteoblasts in regenerating fin.

During zebrafish fin regeneration, blastema cells lining the epidermis differentiate into osteoblasts and joint cells to reconstruct the segmented bony rays. We show that osteoblasts and joint cells originate from a common cell lineage, but are committed to different cell fates. Pre-osteoblasts expressing runx2a/b commit to the osteoblast lineage upon expressing sp7, whereas the strong upregulation of hoxa13a correlates with a commitment to a joint cell type. In the distal regenerate, hoxa13a, evx1 and pthlha are sequentially upregulated at regular intervals to define the newly identified presumptive joint cells. Presumptive joint cells mature into joint-forming cells, a distinct cell cluster that maintains the expression of these factors. Analysis of evx1 null mutants reveals that evx1 is acting upstream of pthlha and downstream of or in parallel with hoxa13a Calcineurin activity, potentially through the inhibition of retinoic acid signaling, regulates evx1, pthlha and hoxa13a expression during joint formation. Furthermore, retinoic acid treatment induces osteoblast differentiation in mature joint cells, leading to ectopic bone deposition in joint regions. Overall, our data reveal a novel regulatory pathway essential for joint formation in the regenerating fin.

COMPARATIVE EFFECT OF CRUDE EXTRACT OF PARKIA BIGLANDULOSA AND ITS ISOLATE ON REGENERATIVE ANGIOGENESIS IN ADULT ZEBRAFISH MODEL

As cancer continues to be one of the biggest threats worldwide and causes maximum deaths after cardiovascular diseases, a greater emphasis must be given for newer strategies for the development of anti-cancer drugs. Cancer treatment strategies target various mechanisms such as apoptosis, metastasis, angiogenesis, etc. Angiogenesis, which is the process of generation of new capillary blood vessels, can be an excellent target for anti-cancer therapy, as a tumor cannot grow further without it. The present study aims to evaluate and compare anti-angiogenic activity of ethanolic extract of Parkia biglandulosa (EEPB) and its isolate β-sitosterol by regenerative angiogenesis assay using caudal fin of adult zebrafish. One of its active constituents that is β-sitosterol, which was expected to show anti-cancer activity, was isolated by using flash chromatography and its activity was checked along with crude ethanolic extract of Parkia biglandulosa (EEPB). Results of regenerative angiogenesis compared by one way ANOVA followed by Tukey’s test showed significant inhibition of fin growth when compared with control group which signified anti-angiogenic effect of test compounds.

Novel Heparin Receptor Transmembrane Protein 184a Regulates Angiogenesis in the Adult Zebrafish Caudal Fin.

Transmembrane protein 184A (TMEM184A) was recently identified as the heparin receptor in vascular cells. Heparin binds specifically to TMEM184A and induces anti-proliferative signaling in vitro. Though it is highly conserved, the physiological function of TMEM184A remains unknown. The objective of this study was to investigate the expression and effects on vascular regeneration of TMEM184A using the adult zebrafish regenerating caudal fin as an in vivo model. Here, we show that Tmem184a is expressed in vascular endothelial cells (ECs) of mature and regenerating zebrafish fins. Transient morpholino (MO)-mediated knockdown of Tmem184a using two validated MOs results in tangled regenerating vessels that do not grow outward and limit normal overall fin regeneration. A significant increase in EC proliferation is observed. Consistent with in vitro work with tissue culture vascular cells, heparin has the opposite effect and decreases EC proliferation which also hinders overall fin regeneration. Collectively, our study suggests that Tmem184a is a novel regulator of angiogenesis.

Differential oxidation of protein-tyrosine phosphatases during zebrafish caudal fin regeneration

Zebrafish have the capacity to regenerate lost tissues and organs. Amputation of the caudal fin results in a rapid, transient increase in H2O2 levels emanating from the wound margin, which is essential for regeneration, because quenching of reactive oxygen species blocks regeneration. Protein-tyrosine phosphatases (PTPs) have a central role in cell signalling and are susceptible to oxidation, which results in transient inactivation of their catalytic activity. We hypothesized that PTPs may become oxidized in response to amputation of the caudal fin. Using the oxidized PTP-specific (ox-PTP) antibody and liquid chromatography-mass spectrometry, we identified 33 PTPs in adult zebrafish fin clips of the total of 44 PTPs that can theoretically be detected based on sequence conservation. Of these 33 PTPs, 8 were significantly more oxidized 40 min after caudal fin amputation. Surprisingly, Shp2, one of the PTPs that were oxidized in response to caudal fin amputation, was required for caudal fin regeneration. In contrast, Rptpα, which was not oxidized upon amputation, was dispensable for caudal fin regeneration. Our results demonstrate that PTPs are differentially oxidized in response to caudal fin amputation and that there is a differential requirement for PTPs in regeneration.

Live fate-mapping of joint-associated fibroblasts visualizes expansion of cell contributions during zebrafish fin regeneration.

The blastema is a mass of progenitor cells responsible for regeneration of amputated salamander limbs and fish fins. Previous studies have indicated that resident cell sources producing the blastema contribute lineage-restricted progeny to regenerating tissue. However, these studies have labeled general cell types rather than granular cell subpopulations, and they do not explain the developmental transitions that must occur for distal structures to arise from cells with proximal identities in the appendage stump. Here, we find that regulatory sequences of tph1b, which encodes an enzyme that synthesizes serotonin, mark a subpopulation of fibroblast-like cells restricted to the joints of uninjured adult zebrafish fins. Amputation stimulates serotonin production in regenerating fin fibroblasts, yet targeted tph1b mutations abrogating this response do not disrupt fin regeneration. In uninjured animals, tph1b-expressing cells contribute fibroblast progeny that remain restricted to joints throughout life. By contrast, upon amputation, tph1b+ joint cells give rise to fibroblasts that distribute across the entire lengths of regenerating fin rays. Our experiments visualize and quantify how incorporation into an appendage blastema broadens the progeny contributions of a cellular subpopulation that normally has proximodistal restrictions.

Dynamics of actinotrichia regeneration in the adult zebrafish fin

The skeleton of adult zebrafish fins comprises lepidotrichia, which are dermal bones of the rays, and actinotrichia, which are non-mineralized spicules at the distal margin of the appendage. Little is known about the regenerative dynamics of the actinotrichia-specific structural proteins called Actinodins. Here, we used immunofluorescence analysis to determine the contribution of two paralogous Actinodin proteins, And1/2, in regenerating fins. Both proteins were detected in the secretory organelles in the mesenchymal cells of the blastema, but only And1 was detected in the epithelial cells of the wound epithelium. The analysis of whole mount fins throughout the entire regenerative process and longitudinal sections revealed that And1-positive fibers are complementary to the lepidotrichia. The analysis of another longfin fish, a gain-of-function mutation in the potassium channel kcnk5b, revealed that the long-fin phenotype is associated with an extended size of actinotrichia during homeostasis and regeneration. Finally, we investigated the role of several signaling pathways in actinotrichia formation and maintenance. This revealed that the pulse-inhibition of either TGFβ/Activin-βA or FGF are sufficient to impair deposition of Actinodin during regeneration. Thus, the dynamic turnover of Actinodin during fin regeneration is regulated by multiple factors, including the osteoblasts, growth rate in a potassium channel mutant, and instructive signaling networks between the epithelium and the blastema of the regenerating fin.

Harmless effects of argon plasma on caudal fin regeneration and embryogenesis of zebrafish: novel biological approaches for safe medical applications of bioplasma

The argon plasma jet (Ar-PJ) is widely used in medical fields such as dermatology and dentistry, and it is considered a promising tool for cancer therapy. However, the in vivo effects of Ar-PJ for medical uses have not yet been investigated, and there are no biological tools to determine the appropriate clinical dosages of Ar-PJ. In this study, we used the caudal fin and embryo of zebrafish as novel in vivo tools to evaluate the biosafety of Ar-PJ. Typically, Ar-PJ is known to induce cell death in two-dimensional (2D) cell culture systems. By contrast, no detrimental effects of Ar-PJ were shown in our 3D zebrafish systems composed of 2D cells. The Ar-PJ-treated caudal fins grew by an average length of 0.7 mm, similar to the length of the normally regenerating fins. Remarkably, Ar-PJ did not affect the expression patterns of Wnt8a and β-Catenin, which play important roles in fin regeneration. In the embryo system, 85% of the Ar-PJ-treated embryos hatched, and the lateral length of these embryos was ~3.3 mm, which are equivalent to the lengths of normal embryos. In particular, vasculogenesis, which is the main cellular process during tissue regeneration and embryogenesis, occurred normally under the Ar-PJ dose used in this study. Therefore, our biosafety evaluation tools that use living model systems can be used to provide an experimental guideline to determine the clinically safe dosage of Ar-PJ.

Regulation of the proprotein convertases expression and activity during regenerative angiogenesis: Role of hypoxia-inducible factor (HIF)

Proprotein convertases (PCs) are involved in various physiological and pathological processes ranging from embryogenesis to carcinogenesis. Here, using the zebrafish fin regeneration model, we report induced expression of furin and PC5 but not PACE4 and PC7 during fin regeneration that is associated with increased PC activity. Stabilization of HIF by cobalt chloride (CoCl2) further increases these processes. The use of the general PC-inhibitor decanoyl-RVKR-cholromethyl ketone (CMK) inhibited control and CoCl2-induced PC activity. CoCl2 inhibits embryonic zebrafish ZF4 cell proliferation and caudal fin regeneration that is associated with the expression of the anti-proliferative genes P21, P16, PC3 and P53 in ZF4 cells and in non-regenerating stump tissues. In contrast, during fin regeneration, HIF stabilization failed to promote the expression of these anti-proliferative genes and maintained high expression of cyclin D. Further analysis revealed that CoCl2 maintained the formation of immature regenerating vasculature that was associated with amplified expression of OCT4 and various angiogenic factors reported to be PC substrates and/or downstream effectors. These findings revealed that while furin and PC5 expression/activity and their substrates/effectors are regulated during fin regeneration, HIF stabilization by CoCl2 has the potential to modulate these processes and impact on the regenerative process and vessels organization.

Fin-fold development in paddlefish and catshark and implications for the evolution of the autopod.

The evolutionary origin of the autopod involved a loss of the fin-fold and associated dermal skeleton with a concomitant elaboration of the distal endoskeleton to form a wrist and digits. Developmental studies, primarily from teleosts and amniotes, suggest a model for appendage evolution in which a delay in the AER-to-fin-fold conversion fuelled endoskeletal expansion by prolonging the function of AER-mediated regulatory networks. Here, we characterize aspects of paired fin development in the paddlefish Polyodon spathula (a non-teleost actinopterygian) and catshark Scyliorhinus canicula (chondrichthyan) to explore aspects of this model in a broader phylogenetic context. Our data demonstrate that in basal gnathostomes, the autopod marker HoxA13 co-localizes with the dermoskeleton component And1 to mark the position of the fin-fold, supporting recent work demonstrating a role for HoxA13 in zebrafish fin ray development. Additionally, we show that in paddlefish, the proximal fin and fin-fold mesenchyme share a common mesodermal origin, and that components of the Shh/LIM/Gremlin/Fgf transcriptional network critical to limb bud outgrowth and patterning are expressed in the fin-fold with a profile similar to that of tetrapods. Together these data draw contrast with hypotheses of AER heterochrony and suggest that limb-specific morphologies arose through evolutionary changes in the differentiation outcome of conserved early distal patterning compartments.

The careg element reveals a common regulation of regeneration in the zebrafish myocardium and fin

The existence of common mechanisms regulating organ regeneration is an intriguing concept. Here we report on a regulatory element that is transiently activated during heart and fin regeneration in zebrafish. This element contains a ctgfa upstream sequence, called careg, which is induced by TGFβ/Activin-β signalling in the peri-injury zone of the myocardium and the fin mesenchyme. In addition, this reporter demarcates a primordial cardiac layer and intraray osteoblasts. Using genetic fate mapping, we show the regenerative competence of careg-expressing cells. The analysis of the heart reveals that the primordial cardiac layer is incompletely restored after cryoinjury, whereas trabecular and cortical cardiomyocytes contribute to myocardial regrowth. In regenerating fins, the activated mesenchyme of the stump gives rise to the blastema. Our findings provide evidence of a common regenerative programme in cardiomyocytes and mesenchyme that opens the possibility to further explore conserved mechanisms of the cellular plasticity in diverse vertebrate organs.

Transient inflammatory response mediated by interleukin-1β is required for proper regeneration in zebrafish fin fold.

Cellular responses to injury are crucial for complete tissue regeneration, but their underlying processes remain incompletely elucidated. We have previously reported that myeloid-defective zebrafish mutants display apoptosis of regenerative cells during fin fold regeneration. Here, we found that the apoptosis phenotype is induced by prolonged expression of interleukin 1 beta (il1b). Myeloid cells are considered to be the principal source of Il1b, but we show that epithelial cells express il1b in response to tissue injury and initiate the inflammatory response, and that its resolution by macrophages is necessary for survival of regenerative cells. We further show that Il1b plays an essential role in normal fin fold regeneration by regulating expression of regeneration-induced genes. Our study reveals that proper levels of Il1b signaling and tissue inflammation, which are tuned by macrophages, play a crucial role in tissue regeneration.

Transcriptomic, proteomic, and metabolomic landscape of positional memory in the caudal fin of zebrafish

Regeneration requires cells to regulate proliferation and patterning according to their spatial position. Positional memory is a property that enables regenerating cells to recall spatial information from the uninjured tissue. Positional memory is hypothesized to rely on gradients of molecules, few of which have been identified. Here, we quantified the global abundance of transcripts, proteins, and metabolites along the proximodistal axis of caudal fins of uninjured and regenerating adult zebrafish. Using this approach, we uncovered complex overlapping expression patterns for hundreds of molecules involved in diverse cellular functions, including development, bioelectric signaling, and amino acid and lipid metabolism. Moreover, 32 genes differentially expressed at the RNA level had concomitant differential expression of the encoded proteins. Thus, the identification of proximodistal differences in levels of RNAs, proteins, and metabolites will facilitate future functional studies of positional memory during appendage regeneration.

In vivo quantification of mechanical properties of caudal fins in adult zebrafish.

ABSTRACTThe caudal fins of adult zebrafish are supported by multiple bony rays that are laterally interconnected by soft interray tissue. Little is known about the fin's mechanical properties that influence bending in response to hydrodynamic forces during swimming. Here, we developed an experimental setup to measure the elastic properties of caudal fins in vivo by applying micro-Newton forces to obtain bending stiffness and a tensional modulus. We detected overall bending moments of 1.5×10−9–4×10−9 N m2 along the proximal–distal axis of the appendage showing a non-monotonous pattern that was not due to the geometry of the fin itself. Surgical disruption of the interray tissues along the proximal–distal axis revealed no significant changes to the overall bending stiffness, which we confirmed by determining a tensional modulus of the interray tissue. Thus, the biophysical values suggest that the flexibility of the fin during its hydrodynamic performance predominantly relies on the mechanical properties of the rays.