Zebrafish Caudal Fin Regeneration Research Articles

A novel method to assess photobiomodulation in stimulating regenerative capacity and vascularization in zebrafish.

Photobiomodulation (PBM) therapy is a continuously growing approach to stimulating healing and reducing inflammation and pain. However, its effects in the fields of regenerative medicine and tissue engineering are still under investigation. Studying PBM effects on the regenerative capacity of zebrafish can allow the application of novel clinical approaches where the impact of PBM will be cross-linked with the stem-cell therapeutic approaches. This study was done to establish an in-vivo experimental setup for studying the effects of laser and ultraviolet therapy on zebrafish caudal-fin regeneration and vascularization. Thirty zebrafish were randomly and equally allocated into three groups. The caudal-fins of all zebrafish were amputated under anaesthesia. In the first control group, the caudal-fin was only monitored until fully regenerated. In the second group, the amputated-fin was irradiated with ultraviolet. Finally, in the third group, the amputated-fin was irradiated with laser. Caudal-fin regeneration and vascularization were assessed at days 0, 3, 6, 9, 12, and 15 in all fish. In terms of regeneration, the results indicated that it is possible to discriminate the regenerative effect of laser with the experimental setup as laser therapy showed a statistically significant difference when compared to control-group. It was also found that regenerative stimulation of the group that received ultraviolet therapy showed significant difference when compared to the control group. In terms of vascularization, there was a statistically significant difference in all groups of the study, which may suggest that laser as well as ultraviolet have limited effects in terms of improving vascularization. This study presented a novel, simple and inexpensive method for the assessment of PBM effects on zebrafish. Laser and ultraviolet therapy appeared to act as regenerative stimulators for caudal-fin regeneration of zebrafish. However, laser therapy results were, to some extent, better than ultraviolet therapy. This novel in-vivo design of the experiment led to more rapid and reproducible results than in-vitro experiments.

Ethyl butyrate inhibits caudal fin regeneration in adult zebrafish by disrupting extracellular matrix remodeling

Wound healing and tissue regeneration are influenced by a variety of factors. Adverse lifestyle habits, such as excessive alcohol consumption, delay wound healing and increase the risk of secondary infections. Ethyl butyrate is a common food additive widely used to enhance the aroma of alcoholic beverages. This additive is generally considered harmless to human health in both industrial and domestic settings. However, the ecotoxicity and its effects on wound healing have not been elucidated. In this study, we used zebrafish as the experimental animal, and the caudal fins were amputated to explore the effects of ethyl butyrate on wound healing and tissue regeneration. The effect of ethyl butyrate on blastema and bone regeneration and its impact on the transcriptional levels of regeneration-related genes and inflammation-related genes were evaluated. RNA-seq was conducted to determine the differentially expressed genes (DEGs) between the treatment and the control groups. KEGG and GO analysis was conducted to explore the functions of DEGs. Significantly enriched GO terms and KEGG pathways were identified to explore the molecular mechanism underlying the inhibition of zebrafish caudal fin regeneration by ethyl butyrate. The results demonstrated that ethyl butyrate significantly inhibited the regeneration of zebrafish caudal fins, including blastema and bone regeneration. Ethyl butyrate exposure significantly downregulated the expression of genes associated with bone and blastema regeneration and inflammation response. KEGG and GO functional analyses revealed that the DEGs were associated with significant enrichment of extracellular matrix-receptor interactions. Ethyl butyrate treatment downregulated the expression of most extracellular matrix-related genes. These findings indicate that ethyl butyrate potentially modulates pathways associated with the structure, adhesion, modification, and degradation of the extracellular matrix, thereby disrupting extracellular matrix remodeling, inhibiting wound inflammation, impairing blastema and bone regeneration and ultimately hindering caudal fin regeneration. In summary, the findings demonstrate that ethyl butyrate disrupts extracellular matrix remodeling and inhibits the regeneration of zebrafish caudal fins. These results provide valuable insights into the rational use of ethyl butyrate and further investigation of wound healing mechanisms.

Quantitative Characterization of Zebrafish Caudal Fin Regeneration Based on Mueller Matrix OCT In Vivo.

Zebrafish serves as a valuable model for studying tissue regeneration due to their comprehensive regenerative abilities, particularly in bone tissue. In this study, a Mueller matrix optical coherence tomography (OCT) system was applied to monitor the regenerative processes of zebrafish caudal fins in vivo. The analysis focused on evaluating the thickness of the caudal fin tip and the distribution of internal bone tissue during the regenerative process. Subsequently, the effect of ectoine solution on the regeneration process was observed and discussed. Our findings revealed that the caudal fin blastema did not exhibit phase-induced polarization characteristics in the Mueller matrix OCT images. Statistical analyses indicated that the caudal fins did not fully regenerate to their original state within 21 days. Furthermore, the results suggested that ectoine solution could enhance tissue regeneration. This approach provides a method for quantifying zebrafish caudal fin regeneration and advances observation techniques for biomedical and clinical applications.

Exploring the wound healing potential of Lobostemon fruticosus using in vitro and in vivo bioassays

Ethnopharmacological relevanceLobostemon fruticosus (L.) H.Buek is a perennial and woody shrub of the Boraginaceae family, found in the Cape region of South Africa. The leaves and twigs are used to treat dermatological conditions such as wounds, burns, ringworm, erysipelas and eczema. Anti-inflammatory, antibacterial, antiviral and anti-proliferative activities of L. fruticosus have been reported. However, there is a void in research which reports on the wound healing properties of this plant. Aim of the studyAligned with the traditional use of L. fruticosus, our study aimed to use in vitro and in vivo bioassays to confirm the wound healing potential of the plant. Materials and methodsAn aqueous methanol extract (80% v/v) of L. fruticosus was prepared using a sample collected from the Western Cape Province of South Africa and chromatographically profiled by ultra-performance liquid chromatography coupled to mass spectrometry (UPLC-MS). The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cytotoxicity assay was performed to determine the non-toxic concentrations of the extract for subsequent use in the in vitro scratch assay. Both the human keratinocyte (HaCaT) and fibroblast (BJ-5ta) cell lines were employed in the in vitro scratch assay. The in vivo caudal fin amputation assay was used to assess the wound healing potential of L. fruticosus, by monitoring fin regeneration in zebrafish larvae treated with the plant extract at various concentrations. ResultsSix major compounds were tentatively identified in the L. fruticosus extract namely; globoidnan A, globoidnan B, rutin, rabdosiin, sagerinic acid and rosmarinic acid. The potentially toxic pyrrolizidine alkaloids were also identified and quantitatively confirmed to be present at a low concentration of 119.58 ppm (m/m). Treatment of HaCaT and BJ-5ta cells with the plant extract in the scratch assay resulted in an increase in cell migration, which translates to accelerated wound closure. After 24 hr treatment with 100 μg/mL of extract, wound closure was recorded to be 91.1 ± 5.7% and 94.1 ± 1.3% for the HaCaT and BJ-5ta cells, respectively, while the untreated (medium) controls showed 72.3 ± 3.3% and 73.0 ± 4.3% for the two cell lines, respectively. Complete wound closure was observed between 24 and 36 hr, while the untreated control group did not achieve 100% wound closure by the end of the observation period (48 hr). In vivo, the crude extract at 100 μg/mL accelerated zebrafish caudal fin regeneration achieving 100.5 ± 3.8% regeneration compared to 68.3 ± 6.6% in the untreated control at two days post amputation. ConclusionsThe study affirms the wound healing properties, as well as low toxicity of L. fruticosus using both in vitro and in vivo assays, which supports the traditional medicinal use. Other in vitro assays that target different mechanisms involved in wound healing should be investigated to support the current findings.

Radix Rehmanniae Praeparata promoted zebrafish fin regeneration through aryl hydrocarbon receptor-dependent autophagy

Headings ethnopharmacological relevanceRehmanniae Radix Praeparata (RRP), a staple in traditional Chinese medicine, is derived from Rehmannia glutinosa Libosch and is renowned for its wound-healing properties. Despite its clinical prevalence, the molecular mechanisms underlying RRP's wound-healing effects have not been fully elucidated. Aim of the studyThis research endeavored to delineate the molecular and cellular mechanisms underlying the beneficial effects of RRP on wound healing, utilizing a zebrafish model. Materials and methodsZebrafish larvae at 3 days post-fertilization were amputated at the fin and subsequently treated with RRP. The pro-wound healing and regenerative effects of RRP were evaluated through morphological analysis, assessment of cell proliferation and apoptosis, Additionally, mechanistic insights were gained through a comprehensive approach encompassing network pharmacology analysis, cell tracing, RNA-sequencing, CRISPR/Cas9 gene editing, and pharmacological inhibition. ResultsOur findings demonstrate that RRP significantly accelerates caudal fin regeneration in zebrafish following injury by suppressing cell apoptosis, promoting cell proliferation, and upregulating the expression of regenerative-related genes. Furthermore, RRP triggers autophagy signals during the regenerative process, which is attenuated by the autophagy inhibitor chloroquine (CQ). Notably, the administration of RRP enhances the expression of ahr1 and ahr2 in the regenerating fin. Genetic knockout of ahr1a, ahr1b, or ahr2 using CRISPR/Cas9, or pharmacological blockade of AHR signals with the antagonist CH-223191, diminishes the regenerative potential of RRP. Remarkably, zebrafish lacking ahr2 completely lose their fin regeneration ability. Additionally, inhibition of AHR signaling suppresses autophagy signaling during fin regeneration. ConclusionsThis study uncovers that RRP stimulates fin regeneration in zebrafish by inducing AHR signals and, at least partially, activating the autophagy process. These findings provide novel insights into the molecular mechanisms underlying the wound-healing effects of RRP and may pave the way for the development of novel therapeutic strategies.

ERK-activated CK-2 triggers blastema formation during appendage regeneration.

Appendage regeneration relies on the formation of blastema, a heterogeneous cellular structure formed at the injury site. However, little is known about the early injury-activated signaling pathways that trigger blastema formation during appendage regeneration. Here, we provide compelling evidence that the extracellular signal-regulated kinase (ERK)-activated casein kinase 2 (CK-2), which has not been previously implicated in appendage regeneration, triggers blastema formation during leg regeneration in the American cockroach, Periplaneta americana. After amputation, CK-2 undergoes rapid activation through ERK-induced phosphorylation within blastema cells. RNAi knockdown of CK-2 severely impairs blastema formation by repressing cell proliferation through down-regulating mitosis-related genes. Evolutionarily, the regenerative role of CK-2 is conserved in zebrafish caudal fin regeneration via promoting blastema cell proliferation. Together, we find and demonstrate that the ERK-activated CK-2 triggers blastema formation in both cockroach and zebrafish, helping explore initiation factors during appendage regeneration.

DESIGNING, SYNTHESIS AND BIOLOGICAL EVALUATION OF PYRAZOLINE DERIVATIVES TARGETING PANCREATIC CANCER CELLS

In continuation of the previous work based upon pyrazoline derivatives having cytotoxic activity, twenty-one 1,3,5-substituted pyrazoline derivatives were designed taking into consideration the important functional groups of methisazone, sorafenib and chalcone. the designed derivatives were screened using a preliminary molecular docking simulation study for evaluation of their binding interactions with receptor-2 of vascular endothelial growth factor, i.e., pDb ID: 3WZD. the synthesized derivatives were biologically evaluated for in vivo anti-angiogenic activity using adult zebrafish, its embryo, and in vitro anti-proliferative activity against pancreatic cancer mIA-pA-CA-2 cell line using the sulforhodamine b assay. Compound 5b emerged as a promising hit molecule as it manifested moderate in vitro cytotoxic activity. besides, its ability to inhibit zebrafish caudal fin regeneration with less phenotypical changes in zebrafish embryos suggests its promising potential against pancreatic cancer by VeGFR-2 inhibition as a mode of action.

Topiramate promotes osteogenic differentiation through AMPK-dependent phosphorylation of Smad1/5/9

Topiramate [2,3:4,5-bis-o-(1-methylethylidene) β-D-fructo-pyranose sulfamate; TPM] is one of the most used new-generation antiepileptic drugs. It has been reported to regulate the differentiation of human bone cells. However, the molecular mechanism of TPM in osteoblast differentiation is not fully elucidated. In the present study, we examined the effect of TPM on osteogenic differentiation of C3H10T1/2, MC3T3-E1, primary mouse calvarial cells, and primary bone marrow stem cells (BMSCs). Primary cells were isolated from mice calvaria and bone marrow respectively. Expression of the osteogenic gene was determined by RT-PCR. The osteogenic protein levels were measured by Western blot analysis. Alkaline phosphatase (ALP) staining experiment was performed to evaluate ALP activity. Alizarin red s (ARS) staining was performed to measure zebrafish caudal fin regeneration. Treatment of TPM up-regulated the osteogenic genes including distal-less homeobox 5 (Dlx5) and runt-related transcription factor 2 (Runx2). In addition, TPM also increased the Dlx5 and Runx2 protein levels, Smad1/5/9 phosphorylation, and alkaline phosphatase (ALP) activity. Furthermore, TPM activated AMPK, and inhibition of AMPK decreased TPM-induced osteogenic differentiation. In the zebrafish model, osteogenic effect of TPM was identified. TPM was increased amputated caudal fin rays of zebrafish. These results demonstrate that TPM enhances osteogenic differentiation via AMPK-mediated Smad1/5/9 phosphorylation.

Duox is the primary NADPH oxidase responsible for ROS production during adult caudal fin regeneration in zebrafish

Sustained elevated levels of reactive oxygen species (ROS) have been shown to be essential for regeneration in many organisms. This has been shown primarily via the use of pharmacological inhibitors targeting the family of NADPH oxidases (NOXes). To identify the specific NOXes involved in ROS production during adult caudal fin regeneration in zebrafish, we generated nox mutants for duox, nox5 and cyba (a key subunit of NOXes 1-4) and crossed these lines with a transgenic line ubiquitously expressing HyPer, which permits the measurement of ROS levels. Homozygous duox mutants had the greatest effect on ROS levels and rate of fin regeneration among the single mutants. However, duox:cyba double mutants showed a greater effecton fin regeneration than the single duox mutants, suggesting that Nox1-4 also play a role during regeneration. This work also serendipitously found that ROS levels in amputated adult zebrafish fins oscillate with a circadian rhythm.

Effect of Mango Leaf Shoot Extract (Mangifera indica L.) on Zebrafish (Danio rerio) Cell Regeneration Induced by Hyperglicemia

Abstract. In many cases, diabetics often have difficulty in wound healing and lead to amputation. Hyperglycemia is an early symptom of diabetic which is characterized by high blood glucose levels. Cell regeneration is one of part by wound healing process. Mango plants have benefits as medicinal plants because they have secondary metabolites that can help heal wounds. The purpose of this study was to determine the effect of mango leaf shoot extract on blood glucose levels of zebrafish (Danio rerio) hyperglycemia due to alloxan and glucose induction and role of mango leaf shoot extract on zebrafish caudal fin regeneration. This study used an experimental method with 5 group treatments: control group, hyperglycemia, metformin 0,75 w/v, extract 10 µg/ml and extract 20 µg/ml. Parameters observed include blood glucose level, fin length and survival rate. Hyperglycemia was measured by blood glucose level, caudal fin regeneration was analayzed using fin length, also environmental factors and survival rate were measured. Result showed that mango leaf shoot extract 20 µg/ml was efective in reducing blood glucose levels, reaching 68,31% and aproaching metformin 0,75 w/v treatment with a decrease in blood glucose levels reaching 70,1%. Mango leaf shoot extract with concentration 20 µg/ml significantly different (p < 0,05) from hyperglycemia group and extract 10 µg/ml. Extract 20 µg/ml was effective in assisting the regeneration process with an increase caudal fin length reaching 0.94 cm during the treatment period.

Regulatory T cells regulate blastemal proliferation during zebrafish caudal fin regeneration.

The role of T cells in appendage regeneration remains unclear. In this study, we revealed an important role for regulatory T cells (Tregs), a subset of T cells that regulate tolerance and tissue repair, in the epimorphic regeneration of zebrafish caudal fin tissue. Upon amputation, fin tissue-resident Tregs infiltrate into the blastema, a population of progenitor cells that produce new fin tissues. Conditional genetic ablation of Tregs attenuates blastemal cell proliferation during fin regeneration. Blastema-infiltrating Tregs upregulate the expression of igf2a and igf2b, and pharmacological activation of IGF signaling restores blastemal proliferation in Treg-ablated zebrafish. These findings further extend our understandings of Treg function in tissue regeneration and repair.

Single-cell resolution of MET- and EMT-like programs in osteoblasts during zebrafish fin regeneration

SummaryZebrafish regenerate fin rays following amputation through epimorphic regeneration, a process that has been proposed to involve the epithelial-to-mesenchymal transition (EMT). We performed single-cell RNA sequencing (scRNA-seq) to elucidate osteoblastic transcriptional programs during zebrafish caudal fin regeneration. We show that osteoprogenitors are enriched with components associated with EMT and its reverse, mesenchymal-to-epithelial transition (MET), and provide evidence that the EMT markers cdh11 and twist2 are co-expressed in dedifferentiating cells at the amputation stump at 1 dpa, and in differentiating osteoblastic cells in the regenerate, the latter of which are enriched in EMT signatures. We also show that esrp1, a regulator of alternative splicing in epithelial cells that is associated with MET, is expressed in a subset of osteoprogenitors during outgrowth. This study provides a single cell resource for the study of osteoblastic cells during zebrafish fin regeneration, and supports the contribution of MET- and EMT-associated components to this process.

Propolis Can Improve Caudal Fin Regeneration in Zebrafish (Danio rerio) Induced by The Combined Administration of Alloxan and Glucose.

Hyperglycemia, a primary symptom in diabetes mellitus, is associated with difficulties in wound healing and regeneration. This condition is due to the length of the inflammatory phase and free radicals. Furthermore, there is evidence that molecular pathogenesis is involved in impaired wound healing in diabetics. As an animal model, zebrafish have many shared orthologous genes with human that are involved in protein regulation of wound healing and regeneration. Little is known about natural drugs that may be used to treat complications of wound healing in diabetes. Propolis, however, is known to consist of various organic compounds such as phenols and flavonoids with antioxidant and anti-inflammatory activities. This research aims to study propolis' effect on caudal fin regeneration and relative expression of several genes belonging to Hedgehog, bone morphogenetic protein (BMP), and Wnt signaling hyperglycemic (HG) zebrafish. GC-MS analysis and antioxidant activity testing were performed on ethanolic extract of propolis (EEP). Caudal fin regeneration was analyzed using ImageJ; blood glucose levels were measured; and relative gene expression analysis of shha, igf2a, bmp2b, and col1a2 was performed by the real-time polymerase chain reaction method with the β-actin housekeeping gene. Impairment of caudal fin regeneration in zebrafish hyperglycemia was characterized by a low percentage of regeneration and decreased relative gene expression. EEP at 15 ppm could increase the percentage of caudal fin regeneration and the expression of shha, igf2a, bmp2b, and col1a2. Based on the results, it appears that phenols and flavonoids from the EEP can improve the caudal fin regeneration of HG zebrafish.

Basal epidermis collective migration and local Sonic hedgehog signaling promote skeletal branching morphogenesis in zebrafish fins

Teleost fish fins, like all vertebrate limbs, comprise a series of bones laid out in characteristic pattern. Each fin's distal bony rays typically branch to elaborate skeletal networks providing form and function. Zebrafish caudal fin regeneration studies suggest basal epidermal-expressed Sonic hedgehog (Shh) promotes ray branching by partitioning pools of adjacent pre-osteoblasts. This Shh role is distinct from its well-studied Zone of Polarizing Activity role establishing paired limb positional information. Therefore, we investigated if and how Shh signaling similarly functions during developmental ray branching of both paired and unpaired fins while resolving cellular dynamics of branching by live imaging. We found shha is expressed uniquely by basal epidermal cells overlying pre-osteoblast pools at the distal aspect of outgrowing juvenile fins. Lateral splitting of each shha-expressing epidermal domain followed by the pre-osteoblast pools precedes overt ray branching. We use ptch2:Kaede fish and Kaede photoconversion to identify short stretches of shha+basal epidermis and juxtaposed pre-osteoblasts as the Shh/Smoothened (Smo) active zone. Basal epidermal distal collective movements continuously replenish each shha+domain with individual cells transiently expressing and responding to Shh. In contrast, pre-osteoblasts maintain Shh/Smo activity until differentiating. The Smo inhibitor BMS-833923 prevents branching in all fins, paired and unpaired, with surprisingly minimal effects on caudal fin initial skeletal patterning, ray outgrowth or bone differentiation. Staggered BMS-833923 addition indicates Shh/Smo signaling acts throughout the branching process. We use live cell tracking to find Shh/Smo restrains the distal movement of basal epidermal cells by apparent ‘tethering’ to pre-osteoblasts. We propose short-range Shh/Smo signaling promotes these heterotypic associations to couple instructive basal epidermal collective movements to pre-osteoblast repositioning as a unique mode of branching morphogenesis.

Single-Cell Resolution of MET- and EMT-Like Programs in Osteoblasts During Zebrafish Fin Regeneration

While humans have limited potential for limb regeneration, some vertebrates can regenerate bony appendages following amputation. During zebrafish fin regeneration, mature osteoblasts at the amputation stump dedifferentiate and migrate to the blastema, where they re-enter the cell cycle and then re-differentiate to form new bone. Osteoblastic cells differentially exhibit mesenchymal and epithelial characteristics during fin regeneration, however little is known about how these transitions occur. Here, we performed single-cell RNA-sequencing (scRNA-seq) studies to elucidate osteoblastic transcriptional programs during zebrafish caudal fin regeneration. In contrast to existing scRNA-seq resources, our dataset captures a large number of osteoblastic cells, allowing for their in-depth analysis. We found osteoprogenitors are enriched with components associated with the epithelial-to-mesenchymal transition (EMT) and its reverse, mesenchymal-to-epithelial transition (MET). In trajectory analyses, osteoblastic cells solely expressed EMT components, or transiently expressed MET components prior to expressing those for EMT. We found that cdh11, a cancer EMT marker, is expressed during osteoblast dedifferentiation. We also found that esrp1, a regulator of alternative splicing in epithelial cells whose expression is important for MET, is expressed in a subset of osteoprogenitors during outgrowth. This study provides a valuable single cell resource for the study of osteoblastic cells during zebrafish fin regeneration, and identifies MET- and EMT-associated components which may be important for successful appendage regeneration.

Expression analysis of Hsp90α and cytokines in zebrafish caudal fin regeneration

Zebrafish (Danio rerio) is an ideal model organism for exploring the ability and mechanism of tissue regeneration in the vertebrate. However, the specific cellular and molecular mechanism of caudal fin regeneration in zebrafish remains largely unclear. Therefore, we first confirmed the crucial period of fin regeneration in adult zebrafish by morphological and histological analysis. Then we performed RNA-Seq analysis of the caudal fin regeneration at three key stages, which provided some clues for exploring the mechanism of caudal fin regeneration. Moreover, we also determined the expressions of inflammatory cytokines IL-1β, IL-6, IL-8, IL-10, TGF-β, and the immune-related pathway JAK2α and STAT1b in the caudal fin of zebrafish following fin amputation by quantitative real time PCR (qPCR). Particularly, Hsp90α expression at mRNA and protein level determined by qPCR and Western blotting, respectively, and whole-mount in situ hybridization of Hsp90α were also performed in this study. The results showed that inflammatory cytokines were mainly expressed in the early period of caudal fin regeneration (1–3 days post amputation, dpa), indicating that fish immune system was involved in the fin regeneration. Furthermore, the high expression of Hsp90α in the vicinity of blastema and blood vessels of the regenerating fin suggests that Hsp90α may play a role in the initiation and promotion of caudal fin regeneration. Overall, our results provide a framework for further understanding the cellular and molecular mechanism in caudal fin regeneration.

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

Microplastic pollution is pervasive in aquatic environments, but the potential effects of microplastics on aquatic organisms are still under debate. Given that tissue damage is unavoidable in fish and the available data mostly concentrate on healthy fish, there is a large chance that the ecotoxicological risk of microplastic pollution is underrated. Therefore, in this study, the effects of microplastics on the regenerative capacity of injured fish were investigated using a zebrafish caudal fin regeneration model. After fin amputation at 72 h post fertilization, the larvae were exposed to polystyrene microplastics (0.1–10 mg/L) with diameters of 50 or 500 nm. Microplastic exposure significantly inhibited fin regeneration, both morphologically and functionally. Furthermore, the signaling networks that regulate fin regeneration, as well as reactive oxygen species signaling and the immune response, both of which are essential for tissue repair and regeneration, were altered. Transcriptomic analyses of the regenerating fin confirmed that genes related to fin regeneration were transcriptionally modulated in response to microplastic exposure and that metabolic pathways were also extensively involved. In conclusion, this study demonstrated for the first time that microplastic exposure could disrupt the regenerative capacity of fish and might eventually impair their fitness in the wild.

Functional role of annexins in zebrafish caudal fin regeneration – A gene knockdown approach in regenerating tissue

Regeneration is an adaptive phenomenon with wide biological implications spread heterogeneously in almost all the organism including human beings. The ability of regeneration varies from species to species for its complexity. Epimorphic regeneration of zebrafish caudal fin tissue is the most widely studied regeneration mechanism for its discrete and rapid regenerative capability. Several genes and proteins were found to be associated with regenerative mechanisms of zebrafish caudal fin tissue. In this study we have evaluated the functional role of Annexin 2a and 2b genes during zebrafish caudal fin tissue regeneration using inventive electroporation techniques for targeting the gene involving CRISPR-Cas9 technology. The electroporation of the CRISPR was performed on the adult zebrafish caudal fin tissue post amputation. We report retarded growth during the regeneration of caudal fin tissue when Annexin 2a and 2b genes were knocked down, which was validated through gene expression & sequencing analysis and further supported by high-throughput quantitative proteomic analysis of the fin tissue. Annexin family genes such as ANXA13, ANXA1a, ANXA5b were also found to be repressed with their expression. Knocking down of ANXA2a and 2b in regenerating caudal fin tissue compromises regenerating capacity as these genes are involved in cell to cell communication and extracellular matrix growth. This study proves that ANXA2a and 2b plays a significant role in epimorphic regeneration of zebrafish caudal fin tissue.

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

Fish have a high ability to regenerate fins, including the caudal fin. After caudal fin amputation, original bi-lobed morphology is reconstructed during its rapid regrowth. It is still controversial whether positional memory in the blastema cells regulates reconstruction of fin morphology as in amphibian limb regeneration, in which limb blastema cells located at the same proximal-distal level have the same positional identity. We investigated growth period and growth rate in zebrafish caudal fin regeneration. We found that both the growth period and growth rate differed for fin rays that were amputated at the same proximal-distal level, indicating that it takes different periods of time for fin rays to restore their original lengths after straight amputation. We also show that more proximal amputation takes longer period to reconstruct the original morphology/size than more distal amputation. Statistical analysis suggested that both the growth period/rate are determined by amputated length (depth) regardless of the fin ray identity along dorsal-ventral axis. In addition, we suggest the possibility that the structural/physical condition such as width of the fin ray at the amputation site (niche at the stump) may determine the growth period/rate.

Yap induces osteoblast differentiation by modulating Bmp signalling during zebrafish caudal fin regeneration.

Osteoblast differentiation is a key process for bone homeostasis and repair. Multiple signalling pathways have been associated with osteoblast differentiation, yet much remains unknown on how this process is regulated in vivo Previous studies have proposed that the Hippo pathway transcriptional co-activators YAP and TAZ (also known as YAP1 and WWTR1, respectively) maintain progenitor stemness and inhibit terminal differentiation of osteoblasts, whereas others suggest they potentiate osteoblast differentiation and bone formation. Here, we use zebrafish caudal fin regeneration as a model to clarify how the Hippo pathway regulates de novo bone formation and osteoblast differentiation. We demonstrate that Yap inhibition leads to accumulation of osteoprogenitors and prevents osteoblast differentiation in a cell non-autonomous manner. This effect correlates with a severe impairment of Bmp signalling in osteoblasts, likely by suppressing the expression of the ligand bmp2a in the surrounding mesenchymal cells. Overall, our findings provide a new mechanism of bone formation through the Hippo-Yap pathway, integrating Yap in the signalling cascade that governs osteoprogenitor maintenance and subsequent differentiation during zebrafish caudal fin regeneration.