Myomaker and Myomixer Characterization in Gilthead Sea Bream Under Different Myogenesis Conditions.

The skeletal muscle is formed by multinucleated myofibers originated by waves of hyperplasia and hypertrophy during myogenesis. Tissue damage triggers a regeneration process including new myogenesis and muscular remodeling. During myogenesis, the fusion of myoblasts is a key step that requires different genes’ expression, including the fusogens Myomaker and Myomixer. The present work aimed to characterize these proteins in gilthead sea bream and their possible role in in vitro myogenesis, at different fish ages and during muscle regeneration after induced tissue injury. Myomaker is a transmembrane protein highly conserved among vertebrates; whereas Myomixer is a micropeptide that is moderately conserved but maintains its crucial AxLyCxL motif. myomaker expression is restricted to skeletal muscle, while the expression of myomixer is more ubiquitous. In primary myocytes culture, myomaker and myomixer expression peaked at day 6 and day 8, respectively. During regeneration, the expression of both fusogens and all the myogenic regulatory factors showed a peak after 16 days post-injury. Moreover, myomaker and myomixer were present at different ages, but in fingerlings there were significantly higher transcript levels than in juveniles or adult fish. Overall, Myomaker and Myomixer are valuable markers of muscle growth that together with other regulatory molecules, can provide a deeper understanding of myogenesis regulation in fish.

Herbal Preparation (Bromelain, Papain, Curcuma, Black Pepper) Enhances Mineralization and Reduces Glucocorticoid-Induced Osteoporosis in Zebrafish

Natural foods with antioxidant properties, such as curcuma, papain, bromelain and black pepper, have been indicated as a potential natural therapeutic approach against osteoporosis. Zebrafish are an excellent animal model to study the effects of herbal preparations on osteogenesis and bone metabolism, both in physiological and in pathological conditions. Our study was aimed at evaluating whether curcuma-bromelain-papain-pepper herbal preparation (CHP) administered in embryos and adult fish is capable of promoting bone wellness in physiological and osteoporotic conditions. The effect of CHP has been studied in embryonic osteogenesis and glucocorticoid-induced osteoporosis (GIOP) in an adult fish model in which drug treatment induces a bone-loss phenotype in adult scales very similar to that which characterizes the bones of human patients. CHP prevented the onset of the osteoporotic phenotype in the scales of GIOP in adult zebrafish, with the osteoblastic and osteoclastic metabolic activity maintaining unaltered. CHP is also able to attenuate an already established GIOP phenotype, even if the alteration is in an advanced phase, partially restoring the normal balance of the bone markers alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRAP) and stimulating anabolic reparative processes. The results obtained indicated CHP as a potential integrative antioxidant therapy in human bone-loss diseases.

​Molecular Mechanism of Extreme Hypoxia Tolerance Difference between Male and Female Adult Fish and Juvenile Fish of Acrossocheilus fasciatus by Transcriptomics

Background: Dissolved oxygen in water is an important limiting factor for fish. In this study, the suffocation point and transcriptomes analysis of Acrossocheilus fasciatus with different age and gender are helpful to analyze the effects of gender and age on extreme hypoxia tolerance. Methods: First of all, we compared the difference in tolerance to extreme hypoxia among 15 fish from male adult fish, female adult fish and juvenile fish of Acrossocheilus fasciatus in each group by asphyxiation point experiment. Then, we analyzed the molecular mechanism of extreme hypoxia tolerance difference between male and female adult fish and juvenile fish by transcriptomics. Result: Female adult fish of Acrossocheilus fasciatus showed the strongest tolerance to extreme hypoxia in the asphyxiation point experiment. In transcriptomes experiments in all samples, we found that the expression of ncoa4 and facl4 was significantly down regulated and the expression of jnk, gpx4 and jip-1 was significantly increased in females adult fish.

Mosmo Is Required for Zebrafish Craniofacial Formation

Hedgehog (Hh) signaling is a highly regulated molecular pathway implicated in many developmental and homeostatic events. Mutations in genes encoding primary components or regulators of the pathway cause an array of congenital malformations or postnatal pathologies, the extent of which is not yet fully defined. Mosmo (Modulator of Smoothened) is a modulator of the Hh pathway, which encodes a membrane tetraspan protein. Studies in cell lines have shown that Mosmo promotes the internalization and degradation of the Hh signaling transducer Smoothened (Smo), thereby down-modulating pathway activation. Whether this modulation is essential for vertebrate embryonic development remains poorly explored. Here, we have addressed this question and show that in zebrafish embryos, the two mosmo paralogs, mosmoa and mosmob, are expressed in the head mesenchyme and along the entire ventral neural tube. At the cellular level, Mosmoa localizes at the plasma membrane, cytoplasmic vesicles and primary cilium in both zebrafish and chick embryos. CRISPR/Cas9 mediated inactivation of both mosmoa and mosmob in zebrafish causes frontonasal hypoplasia and craniofacial skeleton defects, which become evident in the adult fish. We thus suggest that MOSMO is a candidate to explain uncharacterized forms of human congenital craniofacial malformations, such as those present in the 16p12.1 chromosomal deletion syndrome encompassing the MOSMO locus.

Cod’s Journey Back Home—From Birth to Nursery

Adult cod swim hundreds of kilometers away from home to release their eggs into the ocean water. After some days, tiny larvae hatch from the eggs. At first, the larvae have a small food reserve to sustain them during their first days of life. Soon after emptying the yolk-sac, the larvae must find food on their own. Both eggs and larvae are carried by the ocean currents and they experience large changes in conditions as they drift back to the nursery areas where their parents came from, where the larvae grow up to become adult fish. Our research on cod indicates that the number and location of cod larvae are associated with the size of the spawning adults; and that the number of larvae influences how many cod will grow to be recruits. So, it is important to also know the living conditions of the parents before they spawn, which will be important for the survival of their offspring.

Effects of Zinc Ions Released from Ti-NW-Zn Surface on Osteogenesis and Angiogenesis in Vitro and in an in Vivo Zebrafish Model

Objective: Zinc-modified titanium materials have been widely applied in oral implants, among them, our previous studies have also successfully prepared a novel acid-etched micro-structured titanium surface modified with zinc-containing nanowires (Ti-NW-Zn). However, there are very few reports concerned about the biological safeties of zinc ions released from material surface and the appropriate concentration range of released zinc ions which was more conducive to angiogenesis and bone regeneration. Danio rerio (Zebrafish) represents a powerful alternative in vivo model to study both angiogenesis and osteogenesis. Then, the aim of this study is to investigate the effects of zinc ions released from Ti-NW-Zn surfaces on angiogenesis and osteogenesis via zebrafish model, and further revealed the relationship between angiogenesis and osteogenesis via HUVECs and MC3T3-E1s in vitro models.Materials and methods: Tg (Fli‐1:EGFP)y1 zebrafish embryos were used to investigate the in vivo angiogenesis exposure to zinc ions, Wild-type Zebrafish (Danio rerio) adult fish and their embryos were used to investigate the in vivo bone regeneration and survival rate. Cell experiments including proliferation, adhesion, osteogenic differentiation and signaling pathway experiments were carried out by CCK-8, Laser scanning confocal microscope and Western blot, etc. Zinc ion release assay was detected by a Zinc assay kit.Results: The zinc ions released from Ti-NW-Zn surfaces were far lower than median lethal concentrations (LCs) of both embryos and adult fish, which was beneficial to osteogenesis and angiogenesis promotion of zebrafish model. Moreover, the appropriate concentration range of zinc ions was 1-2 ppm in vitro models, which could induce HUVECs proliferations, and the conditioned medium (CM) collected from the supernatant of 1-2 ppm zinc ions-induced HUVECs medium could obviously promote MC3T3-E1 osteoblasts behaviors via activated the MAPK/ERK signaling pathway.Conclusion: Zinc ions released from Ti-NW-Zn surfaces was at a biological safe and appropriate concentration, which could promote the angiogenesis and osteogenesis in vivo and vitro. The positive effects of the appropriate concentration of zinc ions on osteoblast behaviors might be regulated by activating the MAPK/ERK signaling pathway.

Acute Hypoxia Elevates Arginase 2 and Induces Polyamine Stress Response in Zebrafish via Evolutionary-conserved Mechanism

Living organisms repeatedly encounter stressful events and apply various strategies to survive. Polyamines are omnipresent bioactive molecules with multiple functions. Their transient synthesis, inducible by numerous stressful stimuli, is termed the polyamine stress response. Animals developed evolutionary-conserved strategies to cope with stresses. The urea cycle is an ancient attribute that deals with ammonia excess in terrestrial species. Remarkably, most fish retain the urea cycle genes fully expressed during the early stages of development and silenced in adult animals. Environmental challenges instigate urea synthesis in fish despite substantial energetic costs, which poses a question of the urea cycle's evolutionary significance. Arginase plays a critical role in oxidative stress-dependent reactions being the final urea cycle' enzyme. Its unique subcellular localization, high inducibility, several regulation levels provide a supreme ability to control the polyamine synthesis rate. Of note, oxidative stress instigates the arginase-1 activity in mammals. Arginase is also dysregulated in aging organisms' brain and muscle tissues, indicating its role in the pathogenesis of age-associated diseases. We designed a study to investigate the levels of the urea cycle and polyamine synthesis-related enzymes in a fish model of acute hypoxia. We evidence synchronized elevation of arginase-2 and ornithine decarboxylase following oxidative stress in adult fish and aging animals that underlines the specific function of arginase-2 in fish. Moreover, we demonstrate oxidative stress-associated polyamine synthesis' induction and urea cycle' arrest in adult fish. The subcellular arginase localization found in the fish seems to correspond to its possible evolutionary roles.

Effect of Cypermethrin 25% EC, an Endocrine Disrupting Chemical and pH on Reproductive Health of Puntius javanicus

In the present study an attempt has been made to investigate the combined effect of sub-lethal dose of Cypermethrin 25% EC and pH on histoarchitecture of gonads and GnRH hormone of Puntius javanicus. 96h LC50 value was determined by method of Karber and it was found to be 3.65 µg/L for Puntius javanicus. Adult fish were exposed to sub-lethal concentration (0.365 µg/L) of Cypermethrin 25% EC in different pH of water, i.e. pH 5, pH 6, pH 7, pH 8.5 and pH 9.5 for 15 days. Significant histopathological alterations were found in testis and ovary of the fish subjected to sub-lethal concentration of cypermethrin 25% EC. Significant decrease (p?0.001) of GnRH concentration was recorded in pesticide exposed fishes. Significantly more histopathological alterations of gonads and decrease of GnRH concentration were recorded when fish were exposed to sub-lethal concentration of cypermethrin 25% EC in pH 5, pH 6 and pH 9.5, compared to fish exposed to sub-lethal concentration of cypermethrin 25% EC in pH 7 water. A significant increase of GnRH concentration (p?0.05) was documented in fishes exposed to sub-lethal concentration of cypermethrin in pH 8.5, compared to fishes exposed to sub-lethal concentration of cypermethrin in pH 7 and the concentration is found to be almost equal to the control group (pH 7, without toxicant). At this medium histoarchitecture of testis and ovary retained almost normal histology with least alterations.

Abortive intussusceptive angiogenesis causes multi-cavernous vascular malformations

Mosaic inactivation of CCM2 in humans causes cerebral cavernous malformations (CCMs) containing adjacent dilated blood-filled multi-cavernous lesions. We used CRISPR-Cas9 mutagenesis to induce mosaic inactivation of zebrafish ccm2 resulting in a novel lethal multi-cavernous lesion in the embryonic caudal venous plexus (CVP) caused by obstruction of blood flow by intraluminal pillars. These pillars mimic those that mediate intussusceptive angiogenesis; however, in contrast to the normal process, the pillars failed to fuse to split the pre-existing vessel in two. Abortive intussusceptive angiogenesis stemmed from mosaic inactivation of ccm2 leading to patchy klf2a over-expression and resultant aberrant flow signaling. Surviving adult fish manifested histologically-typical hemorrhagic CCM. Formation of mammalian CCM requires the flow-regulated transcription factor KLF2; fish CCM and the embryonic CVP lesion failed to form in klf2a null fish indicating a common pathogenesis with the mammalian lesion. These studies describe a zebrafish CCM model and establish a mechanism that can explain the formation of characteristic multi-cavernous lesions.