mTOR mutation disrupts larval zebrafish tail fin regeneration via regulating proliferation of blastema cells and mitochondrial functions.

BACKGROUND: The larval zebrafish tail fin can completely regenerate in 3 days post amputation. mTOR, the main regulator of cell growth and metabolism, plays an essential role in regeneration. Lots of studies have documented the role of mTOR in regeneration. However, the mechanisms involved are still not fully elucidated. MATERIALS AND RESULTS: This study aimed to explore the role and mechanism of mTOR in the regeneration of larval zebrafish tail fins. Initially, the spatial and temporal expression of mTOR signaling in the larval fin was examined, revealing its activation following tail fin amputation. Subsequently, a mTOR knockout (mTOR-KO) zebrafish line was created using CRISPR/Cas9 gene editing technology. The investigation demonstrated that mTOR depletion diminished the proliferative capacity of epithelial and mesenchymal cells during fin regeneration, with no discernible impact on cell apoptosis. Insight from SMART-seq analysis uncovered alterations in the cell cycle, mitochondrial functions and metabolic pathways when mTOR signaling was suppressed during fin regeneration. Furthermore, mTOR was confirmed to enhance mitochondrial functions and Ca2 + activation following fin amputation. These findings suggest a potential role for mTOR in promoting mitochondrial fission to facilitate tail fin regeneration. CONCLUSION: In summary, our results demonstrated that mTOR played a key role in larval zebrafish tail fin regeneration, via promoting mitochondrial fission and proliferation of blastema cells.

longfin causes cis-ectopic expression of the kcnh2a ether-a-go-go K+ channel to autonomously prolong fin outgrowth.

Organs stop growing to achieve a characteristic size and shape in scale with the body of an animal. Likewise, regenerating organs sense injury extents to instruct appropriate replacement growth. Fish fins exemplify both phenomena through their tremendous diversity of form and remarkably robust regeneration. The classic zebrafish mutant longfint2 develops and regenerates dramatically elongated fins and underlying ray skeleton. We show longfint2 chromosome 2 overexpresses the ether-a-go-go-related voltage-gated potassium channel kcnh2a. Genetic disruption of kcnh2a in cis rescues longfint2, indicating longfint2 is a regulatory kcnh2a allele. We find longfint2 fin overgrowth originates from prolonged outgrowth periods by showing Kcnh2a chemical inhibition during late stage regeneration fully suppresses overgrowth. Cell transplantations demonstrate longfint2-ectopic kcnh2a acts tissue autonomously within the fin intra-ray mesenchymal lineage. Temporal inhibition of the Ca2+-dependent phosphatase calcineurin indicates it likewise entirely acts late in regeneration to attenuate fin outgrowth. Epistasis experiments suggest longfint2-expressed Kcnh2a inhibits calcineurin output to supersede growth cessation signals. We conclude ion signaling within the growth-determining mesenchyme lineage controls fin size by tuning outgrowth periods rather than altering positional information or cell-level growth potency.

Visualization of extracellular vesicles in the regenerating caudal fin blastema of zebrafish using in vivo electroporation.

Zebrafish have high regenerative ability in several organs including the fin. Although various mechanisms underlying fin regeneration have been revealed, some mechanisms remain to be elucidated. Recently, extracellular vesicles (EVs) have been the focus of research with regard to their role in cell-to-cell communication. It has been suggested that cells in regenerating tissues communicate using EVs. In this study, we examined the involvement of EVs in the caudal fin regeneration of zebrafish using an in vivo electroporation method. The process of regeneration appeared normal after in vivo electroporation, and the transferred plasmid showed mosaic expression in the blastema. We took advantage of this mosaic expression to observe the distribution of exosomal markers in the blastema. We transferred exosomal markers by in vivo electroporation and identified EVs in the regenerating caudal fin. The results suggest that blastemal cells communicate with other cells via EVs during caudal fin regeneration.

Potassium Channel-Associated Bioelectricity of the Dermomyotome Determines Fin Patterning in Zebrafish.

The roles of bioelectric signaling in developmental patterning remain largely unknown, although recent work has implicated bioelectric signals in cellular processes such as proliferation and migration. Here, we report a mutation in the inwardly rectifying potassium channel (kir) gene, kcnj13/kir7.1, that causes elongation of the fins in the zebrafish insertional mutant Dhi2059. A viral DNA insertion into the noncoding region of kcnj13 results in transient activation and ectopic expression of kcnj13 in the somite and dermomyotome, from which the fin ray progenitors originate. We made an allele-specific loss-of-function kcnj13 mutant by CRISPR (clustered regularly interspaced short palindromic repeats) and showed that it could reverse the long-finned phenotype, but only when located on the same chromosome as the Dhi2059 viral insertion. Also, we showed that ectopic expression of kcnj13 in the dermomyotome of transgenic zebrafish produces phenocopies of the Dhi2059 mutant in a gene dosage-sensitive manner. Finally, to determine whether this developmental function is specific to kcnj13, we ectopically expressed three additional potassium channel genes: kcnj1b, kcnj10a, and kcnk9 We found that all induce the long-finned phenotype, indicating that this function is conserved among potassium channel genes. Taken together, our results suggest that dermomyotome bioelectricity is a new fin-patterning mechanism, and we propose a two-stage bioelectricity model for zebrafish fin patterning. This ion channel-regulated bioelectric developmental patterning mechanism may provide with us new insight into vertebrate morphological evolution and human congenital malformations.

von Willebrand factor D and EGF domains is an evolutionarily conserved and required feature of blastemas capable of multitissue appendage regeneration.

Regenerative ability varies tremendously across species. A common feature of regeneration of appendages such as limbs, fins, antlers, and tails is the formation of a blastema-a transient structure that houses a pool of progenitor cells that can regenerate the missing tissue. We have identified the expression of von Willebrand factor D and EGF domains (vwde) as a common feature of blastemas capable of regenerating limbs and fins in a variety of highly regenerative species, including axolotl (Ambystoma mexicanum), lungfish (Lepidosiren paradoxa), and Polpyterus (Polypterus senegalus). Further, vwde expression is tightly linked to the ability to regenerate appendages in Xenopus laevis. Functional experiments demonstrate a requirement for vwde in regeneration and indicate that Vwde is a potent growth factor in the blastema. These data identify a key role for vwde in regenerating blastemas and underscore the power of an evolutionarily informed approach for identifying conserved genetic components of regeneration.

Low doses of ionizing radiation enhance angiogenesis and consequently accelerate post-embryonic development but not regeneration in zebrafish.

Low doses of ionizing radiation (LDIR) activate endothelial cells inducing angiogenesis. In zebrafish, LDIR induce vessel formation in the sub-intestinal vessels during post-embryonic development and enhance the inter-ray vessel density in adult fin regeneration. Since angiogenesis is a crucial process involved in both post-embryonic development and regeneration, herein we aimed to understand whether LDIR accelerate these physiological conditions. Our data show that LDIR upregulate the gene expression of several pro-angiogenic molecules, such as flt1, kdr, angpt2a, tgfb2, fgf2 and cyr61in sorted endothelial cells from zebrafish larvae and this effect was abrogated by using a vascular endothelial growth factor receptor (VEGFR)-2 tyrosine kinase inhibitor. Irradiated zebrafish present normal indicators of developmental progress but, importantly LDIR accelerate post-embryonic development in a VEGFR-2 dependent signaling. Furthermore, our data show that LDIR do not accelerate regeneration after caudal fin amputation and the gene expression of the early stages markers of regeneration are not modulated by LDIR. Even though regeneration is considered as a recapitulation of embryonic development and LDIR induce angiogenesis in both conditions, our findings show that LDIR accelerate post-embryonic development but not regeneration. This highlights the importance of the physiological context for a specific phenotype promoted by LDIR.

MicroRNA expression profiling of caudal fin cell of C. auratus gibelio upon cyprinid herpesvirus 2 infection.

As a member of the genus Cyprinivirus in the family Alloherpesviridae, Cyprinid herpesvirus 2 (CyHV-2) has caused great economic loss in the aquaculture industry, mainly in C. auratus gibelio and goldfish. However, the molecular mechanisms underlying the pathogenicity of CyHV-2 remain elusive. In this study, high-throughput sequencing technology was employed to explore the miRNA expression profiles of C. auratus gibelio (GiCF) caudal fin cells in response to Cyprinid Herpesvirus-2 (CyHV-2) infection. A total of 631 novel miRNAs and 409 known miRNAs were identified. The expression levels of 7 miRNAs were found as significantly modulated (5 down-regulation and 2 up-regulation; P < 0.01, |logFC|>1, TPM>10) in CyHV-2 infected cells. 7 miRNA and their potential mRNA targets were validated by Real-time PCR (qRT-PCR), respectively. Targets prediction and functional analysis of these 7 miRNAs revealed significant enrichment for several signaling pathways, including PPAR, p53 and FoxO pathways. These studies provided more valuable basis for further study on the roles of miRNAs in CyHV-2 replication and pathogenesis.

Recent advancements in understanding fin regeneration in zebrafish.

Zebrafish have the remarkable ability to fully regenerate a lost appendage, faithfully restoring its size, shape and tissue patterning. Studies over the past decades have identified mechanisms underlying the formation, spatial organization, and regenerative growth of the blastema, a pool of proliferative progenitor cells. The patterning of newly forming tissue is tightly regulated to ensure proper rebuilding of anatomy. Precise niche regulation of retinoic acid and sonic hedgehog signaling ensures adherence to ray-interray boundaries. The molecular underpinnings of systems underlying re-establishment of pre-amputation size and shape (positional information) are also slowly starting to emerge. Osteoblasts play an important role as a cellular source of regenerating skeletal elements, and in zebrafish both osteoblast dedifferentiation as well as de novo osteoblast formation occurs. Both dedifferentiation and proliferation are tightly controlled, which makes it interesting to compare it to tumorigenesis, and to identify potential players involved in these processes. This article is categorized under: Adult Stem Cells, Tissue Renewal, and Regeneration > Regeneration.

Wilms Tumor 1b Expression Defines a Pro-regenerative Macrophage Subtype and Is Required for Organ Regeneration in the Zebrafish.

Organ regeneration is preceded by the recruitment of innate immune cells, which play an active role during repair and regrowth. Here, we studied macrophage subtypes during organ regeneration in the zebrafish, an animal model with a high regenerative capacity. We identified a macrophage subpopulation expressing Wilms tumor 1b (wt1b), which accumulates within regenerating tissues. This wt1b+ macrophage population exhibited an overall pro-regenerative gene expression profile and different migratory behavior compared to the remainder of the macrophages. Functional studies showed that wt1b regulates macrophage migration and retention at the injury area. Furthermore, wt1b-null mutant zebrafish presented signs of impaired macrophage differentiation, delayed fin growth upon caudal fin amputation, and reduced cardiomyocyte proliferation following cardiac injury that correlated with altered macrophage recruitment to the regenerating areas. We describe a pro-regenerative macrophage subtype in the zebrafish and a role for wt1b in organ regeneration.

Deep evolutionary origin of limb and fin regeneration.

Salamanders and lungfishes are the only sarcopterygians (lobe-finned vertebrates) capable of paired appendage regeneration, regardless of the amputation level. Among actinopterygians (ray-finned fishes), regeneration after amputation at the fin endoskeleton has only been demonstrated in polypterid fishes (Cladistia). Whether this ability evolved independently in sarcopterygians and actinopterygians or has a common origin remains unknown. Here we combine fin regeneration assays and comparative RNA-sequencing (RNA-seq) analysis of Polypterus and axolotl blastemas to provide support for a common origin of paired appendage regeneration in Osteichthyes (bony vertebrates). We show that, in addition to polypterids, regeneration after fin endoskeleton amputation occurs in extant representatives of 2 other nonteleost actinopterygians: the American paddlefish (Chondrostei) and the spotted gar (Holostei). Furthermore, we assessed regeneration in 4 teleost species and show that, with the exception of the blue gourami (Anabantidae), 3 species were capable of regenerating fins after endoskeleton amputation: the white convict and the oscar (Cichlidae), and the goldfish (Cyprinidae). Our comparative RNA-seq analysis of regenerating blastemas of axolotl and Polypterus reveals the activation of common genetic pathways and expression profiles, consistent with a shared genetic program of appendage regeneration. Comparison of RNA-seq data from early Polypterus blastema to single-cell RNA-seq data from axolotl limb bud and limb regeneration stages shows that Polypterus and axolotl share a regeneration-specific genetic program. Collectively, our findings support a deep evolutionary origin of paired appendage regeneration in Osteichthyes and provide an evolutionary framework for studies on the genetic basis of appendage regeneration.

Development of anti-angiogenic erlotinib liposomal formulation for pulmonary hypertension: a QbD approach.

Pulmonary arterial hypertension (PAH) is the increase in mean pulmonary arterial pressure (> 25 mmHg). The development of the non-reversible plexiform lesions on the arterial walls of the pulmonary arteries has evolved as the reason to increase the pressure. The current treatments are directed towards the vasodilation of the pulmonary arteries via the endothelin, prostacyclin, and NO pathways which provides symptomatic relief. Deeper understanding of the disease leads to the various pathophysiological targets that play an important role in the development of PAH. Out of these, the angiogenetic mechanism of the pulmonary arterial smooth muscle cells has been proved to play an important role in PAH. Targeted therapies by anti-proliferative drugs may lead to the efficient treatment strategies to the root cause of PAH. Erlotinib, a receptor tyrosine kinase inhibitor, which acts on the epidermal growth factor receptor (EGFR), has shown promising results in clinical trials of PAH. The objective of the work has been the development of liposomal formulation of anti-proliferative drug, erlotinib HCl, via Quality by Design (QbD) approach. The liposomal formulation was developed using thin-film hydration technique and characterised for various physicochemical parameters, like particle size, % entrapment efficiency, DSC, FTIR, pXRD, and TEM. In the drug release study, the formulation showed sustained release of erlotinib over 24 h in simulated lung fluid pH 7.4. This developed formulation was evaluated in zebrafish tail fin regeneration assay for its anti-angiogenetic activity. The liposomal formulation inhibited the tail fin regeneration for 14 days indicating anti-angiogenetic activity.

Effects of 17α­ethinylestradiol on caudal fin regeneration in zebrafish larvae.

The ability to restore tissue function and morphology after injury is a key advantage of many fish for a greater chance of survival. The tissue regeneration process is regulated by multiple pathways, and it can therefore be hypothesized that environmental contaminants targeting components of these signaling pathways, may disrupt the fish's capability to repair or regenerate. This could lead to higher mortality and eventually even to a decline in populations. In this study, the effects of 17α­ethinylestradiol (EE2), a synthetic estrogen, were assessed on the regenerative capacity of larval zebrafish. Zebrafish aged 2 hour post fertilization (hpf) were exposed to 1, 10, or 100 ng/L EE2, and the caudal fins were amputated at 72 hpf. It was found that EE2 exposure significantly inhibited fin regeneration and changed locomotor behavior. The transcription levels for most of the genes involved in the signaling networks regulating the fin regeneration, such as axin2, fgfr1, bmp2b and igf2b, were down-regulated in the amputated fish in response to EE2 exposure, which was in contrast to their increased patterns in the vehicle-exposed control fish. Additionally, the mRNA levels of several immune-related genes, such as il-1ß, il-6, il-10 and nf-κb2, were significantly decreased after EE2 exposure, accompanied by a lower density of neutrophils migrated into the wound site. In conclusion, the present study indicated for the first time that estrogenic endocrine disrupting chemicals (EEDCs) could inhibit the regenerative capacity of zebrafish, and this effect was speculated to be mediated through the alteration in regeneration-related signaling pathways and immune competence. This work expands our knowledge of the potential effects of EEDCs on injured aquatic organisms, and highlights the ecotoxicological significance of relationships between regenerative process and endocrine system. This study also implies the potential application of fin regeneration assay for assessing immunotoxicity in ecotoxicological risk assessment.

Identification of myosin II as a cripto binding protein and regulator of cripto function in stem cells and tissue regeneration.

Cripto regulates stem cell function in normal and disease contexts via TGFbeta/activin/nodal, PI3K/Akt, MAPK and Wnt signaling. Still, the molecular mechanisms that govern these pleiotropic functions of Cripto remain poorly understood. We performed an unbiased screen for novel Cripto binding proteins using proteomics-based methods, and identified novel proteins including members of myosin II complexes, the actin cytoskeleton, the cellular stress response, and extracellular exosomes. We report that myosin II, and upstream ROCK1/2 activities are required for localization of Cripto to cytoplasm/membrane domains and its subsequent release into the conditioned media fraction of cultured cells. Functionally, we demonstrate that soluble Cripto (one-eyed pinhead in zebrafish) promotes proliferation in mesenchymal stem cells (MSCs) and stem cell-mediated wound healing in the zebrafish caudal fin model of regeneration. Notably, we demonstrate that both Cripto and myosin II inhibitors attenuated regeneration to a similar degree and in a non-additive manner. Taken together, our data present a novel role for myosin II function in regulating subcellular Cripto localization and function in stem cells and an important regulatory mechanism of tissue regeneration. Importantly, these insights may further the development of context-dependent Cripto agonists and antagonists for therapeutic benefit.

Bichirs employ similar genetic pathways for limb regeneration as are used in lungfish and salamanders.

Bichirs are a sister group to sarcopterygian and tetrapods that can fully regenerate their endochondral-skeleton-fins. Histological and transcriptomic comparison approaches have been used to investigate the morphology and genetic basis of bichir lobe-fin regeneration, with strong down-regulation of muscle-related genes and up-regulation of ECM-related genes and developmental genes being observed. Bichir limb regeneration involves similar cellular processes to those employed by lungfish and salamander, with MARCKS-like protein (MLP) that is known to be a putative regeneration-initiating molecule in salamander, also up-regulated in the early stages of bichir lobe-fin regeneration. These gene expression results suggest that limb regeneration pathways in these amphibians have a common ancestral inheritance, consistent with evolution from endochondral-skeleton-fin structures to endochondral-skeleton-limb structures of vertebrates.

Fin and body neuromuscular coordination changes during walking and swimming in Polypterus senegalus.

The ability to modulate the function of muscle is integral to an animal's ability to function effectively in the face of widely disparate challenges. This modulation of function can manifest through short-term changes in neuromuscular control, but also through long-term changes in force profiles, fatiguability and architecture. However, the relative extent to which shorter-term modulation and longer-term plasticity govern locomotor flexibility remains unclear. Here, we obtain simultaneously recorded kinematic and muscle activity data of fin and body musculature of an amphibious fish, Polypterus senegalus After examining swimming and walking behaviour in aquatically raised individuals, we show that walking behaviour is characterized by greater absolute duration of muscle activity in most muscles when compared with swimming, but that the magnitude of recruitment during walking is only increased in the secondary bursts of fin muscle and in the primary burst of the mid-body point. This localized increase in intensity suggests that walking in P. senegalus is powered in a few key locations on the fish, contrasting with the more distributed, low intensity muscle force that characterizes the stroke cycle during swimming. Finally, the increased intensity in secondary, but not primary, bursts of the fin muscles when walking probably underscores the importance of antagonistic muscle activity to prevent fin collapse, add stabilization and increase body support. Understanding the principles that underlie the flexibility of muscle function can provide key insights into the sources of animal functional and behavioural diversity.

Different Myosin Head Conformations in Bony Fish Muscles Put into Rigor at Different Sarcomere Lengths.

At a resting sarcomere length of approximately 2.2 µm bony fish muscles put into rigor in the presence of BDM (2,3-butanedione monoxime) to reduce rigor tension generation show the normal arrangement of myosin head interactions with actin filaments as monitored by low-angle X-ray diffraction. However, if the muscles are put into rigor using the same protocol but stretched to 2.5 µm sarcomere length, a markedly different structure is observed. The X-ray diffraction pattern is not just a weaker version of the pattern at full overlap, as might be expected, but it is quite different. It is compatible with the actin-attached myosin heads being in a different conformation on actin, with the average centre of cross-bridge mass at a higher radius than in normal rigor and the myosin lever arms conforming less to the actin filament geometry, probably pointing back to their origins on their parent myosin filaments. The possible nature of this new rigor cross-bridge conformation is discussed in terms of other well-known states such as the weak binding state and the 'roll and lock' mechanism; we speculate that we may have trapped most myosin heads in an early attached strong actin-binding state in the cross-bridge cycle on actin.

Tissue regeneration promotion effects of phenanthroimidazole derivatives through pro-inflammatory pathway activation.

A chemotherapeutic drug exerts favorable antitumor activity and simultaneously exhibits expectable inhibition on wound healing process. Phenanthroimidazole derivatives possess potent anticancer activity. However, only a few studies focused on the discovery of its potential effects on promoting tissue regeneration. In this study, four novel phenanthroimidazole derivatives were synthesized and characterized, and they exhibited evident inhibition on different tumor cells; compound 3 is the most active one. Moreover, 3 can promote wound healing of zebrafish in a dose-dependent manner. Further study demonstrated that 3 promoted the recruitment of inflammatory cells, formation of angiogenesis, and generation of reactive oxygen species and also influenced the motor behavior of zebrafish. Results indicated that 3 can accelerate the occurrence of pro-inflammation, angiogenesis, oxidative stress, and innervation, which play key roles in the facilitation of wound healing. Therefore, 3 can act as a bifunctional drug in inhibiting tumor and promoting tissue regeneration.

MicroRNA-223 Suppresses the Canonical NF-κB Pathway in Basal Keratinocytes to Dampen Neutrophilic Inflammation.

MicroRNA-223 is known as a myeloid-enriched anti-inflammatory microRNA that is dysregulated in numerous inflammatory conditions. Here, we report that neutrophilic inflammation (wound response) is augmented in miR-223-deficient zebrafish, due primarily to elevated activation of the canonical nuclear factor κB (NF-κB) pathway. NF-κB over-activation is restricted to the basal layer of the surface epithelium, although miR-223 is detected throughout the epithelium and in phagocytes. Not only phagocytes but also epithelial cells are involved in miR-223-mediated regulation of neutrophils' wound response and NF-κB activation. Cul1a/b, Traf6, and Tab1 are identified as direct targets of miR-223, and their levels rise in injured epithelium lacking miR-223. In addition, miR-223 is expressed in cultured human bronchial epithelial cells, where it also downregulates NF-κB signaling. Together, this direct connection between miR-223 and the canonical NF-κB pathway provides a mechanistic understanding of the multifaceted role of miR-223 and highlights the relevance of epithelial cells in dampening neutrophil activation.

Artificial lateral line based local sensing between two adjacent robotic fish.

The lateral line system (LLS) is a mechanoreceptive organ system with which fish and aquatic amphibians can effectively sense the surrounding flow field. The reverse Kármán vortex street (KVS), known to be a typical thrust-producing wake, is commonly observed in fish-like locomotion and is known to be generated by fish's tails. The vortex street generally reflects the motion information of the fish. A fish can use LLS to detect such vortex streets generated by its neighboring fish, thus sensing its own state and the states of its neighbors in a school of fish. Inspired by this typical biological phenomenon, we design a robotic fish with an onboard artificial lateral line system (ALLS) composed of pressure sensor arrays and use it to detect the reverse KVS-like vortex wake generated by its adjacent robotic fish. Specifically, the vortex wake results in hydrodynamic pressure variations (HPVs) in the flow field. By measuring the HPV using the ALLS and extracting meaningful information from the pressure sensor readings, the oscillating frequency/amplitude/offset of the adjacent robotic fish, the relative vertical distance and the relative yaw/pitch/roll angle between the robotic fish and its neighbor are sensed efficiently. This work investigates the hydrodynamic characteristics of the reverse KVS-like vortex wake using an ALLS. Furthermore, this work demonstrates the effectiveness and practicability of an artificial lateral line in local sensing for adjacent underwater robots, indicating the potential to improve close-range interaction and cooperation within a group of underwater vehicles through the application of ALLSs in the near future.

Phenotypic plasticity of muscle fiber type in the pectoral fins of Polypterus senegalus reared in a terrestrial environment.

Muscle fiber types in the pectoral fins of fishes have rarely been examined, despite their morphological and functional diversity. Here, we describe the distribution of fast and slow muscle fibers in the pectoral fins of Polypterus senegalus, an amphibious, basal actinopterygian. Each of the four muscle groups examined using mATPase staining showed distinct fiber-type regionalization. Comparison between fish raised in aquatic and terrestrial environments revealed terrestrially reared fish possess 28% more fast muscle compared with aquatically reared fish. The pattern of proximal-distal variation in the abductors differed, with a relative decrease in fast muscle fibers near the pectoral girdle in aquatic fish compared with an increase in terrestrial fish. Terrestrially reared fish also possess a greater proportion of very small diameter fibers, suggesting that they undergo more growth via hyperplasia. These observations may be a further example of adaptive plasticity in Polypterus, allowing for greater bursts of power during terrestrial locomotion.