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During normal development of the nervous system, extensive neuronal proliferation as well as death occurs. The extent of development death varies considerably between neuronal populations from little to almost 100%. Early born somatosensory neurons, known as Rohon-Beard cells, have served as an example of neurons that disappear during early developmental stages, presumably as their function is taken over by later developing dorsal root ganglion neurons. However, recent studies have raised questions about the extent to which zebrafish Rohon-Beard cells die during embryogenesis. While Rohon-Beard cells have distinguishing morphological features during embryonic stages development, they subsequently undergo substantial changes in their shape, size and position that hinder their unambiguous identification at later stages. To overcome this obstacle, we identify Rohon-Beard cells at one day, and using a combination of mosaic and stable transgenic labeling and repeated observation, follow them for 13–16 days post fertilization. We find that about 40% survive to late larval stages. Our studies also reveal that Rohon-Beard cells display an unusual repertoire of cell death properties. At one day, about 25% Rohon-Beard cells expose phosphatidyl serine at the surface membrane, but less than one Rohon-Beard cell/embryo expresses activated-caspase-3. Further, the temporal delay between detection of cell death markers and loss of the soma ranges from

The co-receptor CD4 plays an important role in distinguishing between helper T-cell (Th) and cytotoxic T lymphocyte (CTL). In the present study, we investigated the molecular features of CD4-2 cDNA to facilitate understanding of their roles in cobia (Rachycentron canadum). Two CD4-2 molecules have been identified and exhibited 16.10% amino acids identity with each other. The cDNA of CD4-2A consists of a 993 bp ORF encoding 330 aa with long intracytoplasmic tail containing conserved protein tyrosine kinase p56Lck binding (C-X-C) motif, a transmembrane region, and two extracellular Ig-like (Ig-like) domains are predicted. Comparatively, the cDNA of cobia CD4-2B consists of a 990 bp ORF encoding 329 aa without a transmembrane domain as well as C-X-C motif, and three Ig-like domains are present. Homology comparison showed that the CD4-2A aa sequence of cobia showed high similarity and similar structural features to CD4-2 from other species, while the deduced CD4-2B protein shares higher structural similarity to CD4-1 group. Phylogenetic analysis indicated that cobia CD4-2A was closer with CD4-2 molecules in other fish species, distant from the clade formed by fish CD4-1 and mammalian CD4 sequences. However, cobia CD4-2B grouped with other known teleost CD4-1 sequences. The expression pattern of CD4-2A and CD4-2B mRNA during the embryonic development followed the trend of an initial increase after fertilized, providing evidence of maternal transfer of CD4-2 homologues to the developing cobia embryos and larvae. All of these results are useful for better understanding of cell-mediated immunity of cobia. • The first report on the possible soluble CD4 in cobia. • Two CD4-2 molecules have been identified and exhibited 16.10% amino acids identity with each other. • The results confirmed a maternal transfer of CD4-2 homologues to cobia embryos and larvae. [ABSTRACT FROM AUTHOR]

Animal embryogenesis is initiated by maternal factors, but zygotic genome activation (ZGA) shifts regulatory control to the embryo during blastula stages. ZGA is thought to be mediated by maternally provided transcription factors (TFs), but few such TFs have been identified in vertebrates. Here we report that NF–Y and TALE TFs bind zebrafish genomic elements associated with developmental control genes already at ZGA. In particular, co-regulation by NF–Y and TALE is associated with broadly acting genes involved in transcriptional control, while regulation by either NF–Y or TALE defines genes in specific developmental processes, such that NF–Y controls a cilia gene expression program while TALE controls expression of hox genes. We also demonstrate that NF–Y and TALE-occupied genomic elements function as enhancers during embryogenesis. We conclude that combinatorial use of NF–Y and TALE at developmental enhancers permits the establishment of distinct gene expression programs at zebrafish ZGA. • NF–Y and TALE factors occupy genomic elements during zygotic genome activation. • Genomic elements defined by NF–Y and TALE occupancy act as enhancers in vivo. • NF–Y and TALE have both shared and independent transcriptional targets. • Combinatorial use of TALE and NF–Y factors controls distinct genetic programs. [ABSTRACT FROM AUTHOR]

Interleukin-1 receptor-associated kinase 1 (IRAK1) and IRAK4 are critical signalling mediators and play pivotal roles in the innate immune and inflammatory responses mediated by TLR/IL-1R. In the present study, two IRAK family members, On IRAK1 and On IRAK4, were identified in the Nile tilapia Oreochromis niloticus with a conserved N-terminal death domain and a protein kinase domain, similar to those of other fishes and mammals. The gene structures of On IRAK1 and On IRAK4 are organized into fifteen exons split by fourteen introns and ten exons split by nine introns. On IRAK1 and On IRAK4 were broadly expressed in all of the tissues tested, with the highest expression levels being observed in the blood and the lowest expression levels being observed in the liver. Both genes could be detected from 2 d post-fertilization (dpf) to 8 dpf during embryonic development. Moreover, the expression levels of On IRAK1 and On IRAK4 were clearly altered in all five tissues after Streptococcus agalactiae infection in vivo and could be induced by LPS, Poly I: C, S. agalactiae WC1535 and △CPS in Nile tilapia macrophages. The overexpression of On IRAK1 and On IRAK4 in 293T cells showed that they were both distributed in the cytoplasm and could significantly increase NF-κB activation. Interestingly, after transfection, On IRAK1 significantly upregulated On Myd88-induced NF-κB activation, while On IRAK4 had no effect on On Myd88-induced NF-κB activation. Co-immunoprecipitation (Co-IP) assays showed that On Myd88 did not interact with either On IRAK1 or On IRAK4 and that On IRAK1 did not interact with On IRAK4. Taken together, these findings suggest that On IRAK1 and On IRAK4 could play important roles in TLR/IL-1R signalling pathways and the immune response to pathogen invasion. • On IRAK1 and On IRAK4 gene were identified in Nile tilapia and were upregulated after S. agalactiae challenge. • On IRAK1 and On IRAK4 induced by LPS, Poly I: C, wild and mutant type S. agalactiae in in vitro. • On Myd88 did not interact with either On IRAK1 or On IRAK4, and On IRAK1 did not interact with On IRAK4. • Overexpression of On IRAK1 and On IRAK4 significantly increased NF-κB activation. • On IRAK1 significantly upregulated the NF-κB activity induced by On Myd88. [ABSTRACT FROM AUTHOR]

Post-transcriptional gene regulation through the recognition of specific elements in mRNAs is an important determinant of gene expression. The cis elements are recognised by RNA binding proteins (RBPs) and/or small non-coding RNAs, which then orchestrate a range of processes such as mRNA localization, translational control, and degradation. RNA regulation is critical for development and disruptions in regulatory mechanisms can cause disease. While mutations in numerous RBPs have been linked to diseases in humans, the contribution of mutations in RNA elements to disease manifestation is largely unknown. Danio rerio (zebrafish), a fish model is a widely used vertebrate system to study development and disease. Here, we describe how state-of-the-art genomics tools combined with in vivo functional studies in zebrafish have facilitated the discovery of RNA elements, many of which are functionally conserved. We also highlight the potential of zebrafish to model human diseases and for drug discovery. • RNA elements and RNA-binding proteins in Zebrafish. • RNA localization, translation and stability in zebrafish embryogenesis. • Zebrafish models of diseases linked to 3'UTR elements, RBP and RNP granules. [ABSTRACT FROM AUTHOR]

As molecular chaperones, heat shock proteins (HSPs) play essential roles in cells in response to stress conditions. Recent studies about immune functions of HSPs in fish have also been reported. In this study, based on the reported cDNA sequences of the four HSP genes, HSP70, HSC70, HSP90α and HSP90β, the temporal expression patterns of the four genes during embryonic development of dojo loach(Misgurnus anguillicaudatus) was assayed with qRT-PCR. All of the four genes were ubiquitously expressed in all detected embryonic developmental stages. Among of them, HSP70, HSC70 and HSP90β were highly expressed in the organ formation stage, while HSP90α was the highest expressed in myotome formation stage. Further, the immune responses of the four HSP genes were assayed when loach were infected with three different pathogens, bacterium (Flavobacterium cloumnare G4), parasite (Ichthyophthirius multifiliis) and fungus (Saprolegnia). All of the four genes were differentially expressed in four tissues such as skin, gills, spleen and kidney in response to the pathogenic invasion, but both HSP70 and HSP90α expressions were dramatically up-regulated. Further, the cellular responses of the loach skinand gill tissues were observed, in which the number of the skin goblet cells were significantly increased, and the gill lamellae became shorter and wider after infected. Thus, our work indicated that the HSPs may directly or indirectly involved in immune defense in fish, at least in the loach. • The expression patterns of HSP70, HSC70, HSP90α and HSP90β in different embryonic development stages of loach were analyzed. • The HSP proteins exhibited deferentially expressed in response to the different pathogenic invasion of loach. • Our work indicated that the HSPs may directly or indirectly involved in immune defense in fish, at least in the loach. [ABSTRACT FROM AUTHOR]

Coordination of iron acquisition and heme synthesis is required for effective erythropoiesis. The small teleost zebrafish (Danio rerio) is an ideal vertebrate animal model to replicate various aspects of human physiology and provides an efficient and cost-effective way to model human pathophysiology. Importantly, zebrafish erythropoiesis largely resembles mammalian erythropoiesis. Gene discovery by large-scale forward mutagenesis screening has identified key components in heme and iron metabolism. Reverse genetic screens, using morpholino-knockdown and CRISPR/Cas9, coupled with the genetic tractability of the developing embryo have further accelerated functional studies. Ultimately, the ex utero development of zebrafish embryos combined with their transparency and developmental plasticity could provide a deeper understanding of the role of iron and heme metabolism during early vertebrate embryonic development. [ABSTRACT FROM AUTHOR]

TCS [5-chloro-2-(2,4-dichlorophenoxy)phenol] caused a concentration dependent delay in embryonic development, delay and decline in hatching and reduction in length and weight of hatchlings along with an increase in abnormal/deformed embryos and larvae and percent mortality. These parameters varied in a species specific manner and increased with TCS residue in body. The 96 h LC 50 values of TCS for Cyprinus carpio , Ctenopharyngodon idella, Labeo rohita and Cirrhinus mrigala were estimated at 0.315, 0.116, 0.096 and 0.131 mg/L, respectively. Hatching got delayed by 16.33 h for C. carpio (0.47 and 0.50 mg/L TCS) and C. idella (0.20 mg/L TCS) but by 18.07 h for L. rohita (0.15 mg/L TCS) and by 19.33 h for C. mrigala (0.18 mg/L TCS). Spine malformations, oedema (yolk sac and cardiac) and deflated swim bladder were present in 100% larvae of C. carpio , C. idella , L. rohita and C. mrigala at 0.30, 0.08, 0.13 and 0.14 mg/L TCS, respectively. TCS also caused hemorrhage (all but C. idella, only 3.33%), albinism and deformed caudal fin (C. idella), hypopigmentation and rupturing of yolk sac (C. mrigala), gas bubble disease (C. mrigala and L. rohita), fusion of eyes (C. carpio) and degeneration of digestive tract (L. rohita) in 10–40% hatchlings. Exposed hatchlings were very weak and paralyzed, could not swim and remained settled at the bottom of jars. Embryonic development was observed to be an early indicator of the toxicity of TCS as oedema and bubbles in yolk were observed in 40–100% embryos/hatchlings at 0.08 mg/L TCS while 100% mortality was observed between 0.15 and 0.50 mg/L TCS. L. rohita was most sensitive and C. carpio was least sensitive to the stress of TCS. Accumulation of TCS in the hatchlings (1/10 of TCS in water) after 96 h exposure hints that even small quantities of TCS may change species diversity in natural waters. Image 1 • TCS is highly teratogenic to Cyprinus carpio, Ctenopharyngodon idella, Labeo rohita and Cirrhinus mrigala. • TCS accumulation in hatchlings corresponded with the developmental period/time for hatching time. • 96 h LC 50 value and percentage of abnormal larvae increased with accumulation of TCS in each species. • Embryonic development and growth are better indicators of the stress of TCS compared to mortality. • L. rohita was observed to be the most sensitive and C. carpio to be the least sensitive to the stress of TCS. [ABSTRACT FROM AUTHOR]

Haloperidol is a common butyrophenone-derivative antipsychotic drug that is used clinically to treat schizophrenia and to control Tourette disorder. Haloperidol has been shown to be an embryonic toxicant and to cause a variety of adverse effects that affect human embryonic development. However, the pathway impaired by haloperidol during the developmental stages remains unclear. To elucidate the innate toxicological pathway of haloperidol, we investigated the lethality of haloperidol during the embryonic development of zebrafish. We observed that haloperidol caused serious morphological changes, with an LD 50 of 9.7 x 10−6 ± 2.4 x 10−6 μg/L. Next, we established a systematic approach to perform metabolite profiling in embryonic zebrafish with various concentrations of haloperidol and analyzed the metabolites using ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF MS). A total of 304 metabolites were identified and 86 metabolites were chosen to predict potential pathways. Among the metabolites, we found through prediction that numerous metabolomics-biological pathways are associated with haloperidol, including peroxisome-proliferator-activated receptor (ppar), thromboxane, and mTOR signaling. Quantitative real time-qPCR was then used to validate the gene expression potentially associated with the thromboxane, which is a metabolic product of arachidonic acid and considered to be important for cell proliferation and the inflammatory response. To sum up, analysis of metabolites in the zebrafish model provides a system for mining biomarkers that reflect biological significance and highlight the therapeutic potency in humans. In addition, it may show potential for application to other pharmaceuticals to identify their various activities and clarify functional mechanisms in the future. • Metabolite profiling was established in haloperidol-treated embryonic zebrafish. • Toxicological pathway of haloperidol was elucidated by metabolomics-genomic network. • Thromboxane-associated signaling was affected by haloperidol treatment. [ABSTRACT FROM AUTHOR]

Peroxiredoxins (Prxs) are a widespread and greatly transcribed family of antioxidant proteins, which rapidly detoxify peroxynitrite, hydrogen peroxide and organic hydroperoxides. The Prxs family members also modulate various physiological functions, including cell growth, differentiation, embryonic development, immune response, apoptosis, lipid metabolism, and cellular homeostasis. In mammals, the physiological functions of Prxs have extensively been studied; however, the knowledge is scanty in their counterpart, aquatic invertebrates. In recent years, substantial progress has been made in our knowledge of Prxs physiological functions in aquatic invertebrates, which has raised interest in defining the contribution of immune responses and removal of reactive oxygen species. In this review, we describe the recent knowledge on the Prxs physiological function in immune responses and DNA protection activity in aquatic invertebrates. Image 1 [ABSTRACT FROM AUTHOR]

Abstract Protein enhancer of sevenless 2B, E(sev)2B, is a key adapter protein in the Ras/MAPK signaling pathway which has been reported to be involved in innate immunity. In this study, the gene that encodes As E(sev)2B was isolated from A. sinica. It was found to contain a 636 bp open reading frame encoding 211 amino acids with a calculated molecular mass of 24.357 kDa and a predicted isoelectric point of 5.39. The predicted protein contains a N-terminal Src homology 3 domain (SH3), a central Src homology 2 domain (SH2), and a C-terminal Src homology 3 domain (SH3). Homology analysis revealed that As E(sev)2B shares 49%–95% identity with E(sev)2B homologs from other species. In this study, the expression pattern and location of As E(sev)2B during different stages of embryonic development and bacterial challenge were investigated by means of real-time qPCR, Western blotting and immunohistochemistry. Results showed that the highest expression level of As E(sev)2B was at 0 h. After challenged by Gram-positive bacteria and Gram-negative bacteria, As E(sev)2B was remarkably upregulated at 106 cellsL−1 bacterial concentrations. These results suggested that As E(sev)2B plays a vital role during early embryonic development and in immune responses against bacterial challenge. Highlights • Open reading frame of E(sev)2B was isolated and characterized from Artemia sinica. • As E(sev)2B was expressed at all developmental stages using RT-qPCR and Western blotting. • The signals of As E(sev)2B were mainly detected in the cephalothorax by immunohistochemistry. • As E(sev)2B was remarkably upregulated under stimulated by Gram-positive and Gram-negative bacteria. [ABSTRACT FROM AUTHOR]

Abstract In order to test the delayed effect of radiation on the progeny of irradiated survivors, the human keratinocyte cell line HaCaT and the fish common bluegill embryonic cell line CHSE/F were exposed to low-dose high-LET α-radiation from Ra-226 or γ-rays. The clonogenic survival fraction, mitochondrial membrane polarization (MMP) and reproductive ability of the descendants of the surviving cells were measured. For progeny of irradiated HaCaT survivors, no delayed cell death occurred. On the contrary, progeny at about 47 cell doublings after Ra-226 irradiation and progeny at about 14 cell doublings after γ-irradiation showed increased clonogenic survival. However the total cell number was reduced for progeny of Ra-226-treated cells up to about 47 cell doublings after irradiation and for progeny of γ-irradiated cells up to about 28 doublings after irradiation, which means low reproductive ability had appeared. In addition, α-radiation from Ra-226 had greater impact on the MMP of the HaCaT progeny than γ-rays. MMP of progeny of Ra-226-treated cells decreased at 5 cell doublings after irradiation and increased dose-dependently at 19 cell doublings after treatment, and then decreased dose-dependently at 47 cell doublings, while there was no significant effect on MMP in progeny of γ irradiated cells. The progeny of Ra-226-irradiated CHSE/F survivors showed more serious damage than the offspring of γ-irradiated CHSE/F cells. Significant, dose-dependent delayed cell death occurred in progeny of surviving cells up to about 61 cell doublings after Ra-226 treatment, and the reproductive ability was also significantly reduced. But the MMP increased, which might be because of the increased removal of dead cells. For progeny of CHSE/F cells surviving γ-rays radiation, no significant change in clonogenic survival occurred, except for offspring of cells surviving low dose (0.1 Gy and 0.5 Gy) irradiation, which had higher survival than control up to about 28 cell doublings after irradiation. But the number of cells which were the progeny of γ-irradiated survivors decreased dose-dependently up to about 28 cell doublings after γ-irradiation. Highlights • Chronic low-dose high-LET α-radiation from Ra-226 is effective in inducing lethal mutations. • Decreased clonogenic survival did not occur in descendants of irradiated HaCaT survivors, and hormesis may be induced. • Mitochondrial membrane depolarization may be involved in the induction of lethal mutation. [ABSTRACT FROM AUTHOR]

Abstract The mannose receptor (MR) is a type I transmembrane protein. Its ectodomain has eight C-type lectin-like domains, which are able to recognize and mediate the phagocytosis of a wide range of pathogens. Comprehensive studies have revealed that mammalian MR is widely distributed in the mononuclear phagocyte system (MPS, previously known as the reticuloendothelial system) and play a key role both in the physiological clearance and cell activation. Hitherto, neither the MR distribution, nor the function of clearance and cell activation has been investigated in fish. In the previous study, we have reported the full-length cDNA of blunt snout bream MR, analyzed its structure and relative mRNA expression during embryogenesis and in the liver, head kidney, spleen and intestine of fish after stimulation with killed Aeromonas hydrophila. In the present study, we developed a rabbit polyclonal antibody against MR and undertook a systematic survey of the expression of MR at the protein level by immunohistochemistry. To get more information about MR function, the mRNA expression of MR, pro-inflammatory factor TNF-α and anti-inflammatory factor ARG2 genes was measured by qRT-PCR in the liver, head kidney, and spleen after A. hydrophila challenge. We first observed MR expression in the yolk sac at the fertilized egg stage and possibly MR was expressed by early macrophages. We also showed the MR distribution in head kidney, body kidney, spleen, liver, intestine, muscle, brain, heart, and gills. Following A. hydrophila challenge the MR immunoreactive cells became more widespread in head kidney and spleen, which are the major reticuloendothelial systems of fish. The quantitative studies at mRNA levels showed that there exists a high correlation between MR expression and immune cytokine expressions after bacteria challenge. Highlights • Distribution of fish MR from early embryogenesis through to adulthood in Megalobrama amblycephala. • Modulation of MR expression after challenge with Aeromonas hydrophila. • The expression of MR, TNF-α and ARG2 in the tissues of M. amblycephala after A. hydrophila challenge. [ABSTRACT FROM AUTHOR]

The augmented exposure of both environment and human being to electromagnetic waves and the concomitant lack of an unequivocal knowledge about biological consequences of these radiations, raised public interest on electromagnetic pollution. In this context, the present study aims to evaluate the biological effects on zebrafish (ZF) embryos of 100 MHz radiofrequency electromagnetic field (RF-EMF) exposure through a multidisciplinary protocol. Because of the shared synteny between human and ZF genomes that validated its use in biomedical research, toxicology and developmental biology studies, ZF was here selected as experimental model and a measurement protocol and biological analyses have been set up to clearly discriminate between RF-EMF biological and thermal effects. The results showed that a 100 MHz EMF was able to affect ZF embryonic development, from 24 to 72 h post fertilization (hpf) in all the analyzed pathways. Particularly, at the 48 hpf stage, a reduced growth, an increased transcription of oxidative stress genes, the onset of apoptotic/autophagic processes and a modification in cholesterol metabolism were detected. ZF embryos faced stress induced by EMF radiation by triggering detoxification mechanisms and at 72 hpf they partially recovered from stress reaching the hatching time in a comparable way respect to the control group. Data here obtained showed unequivocally the in vivo effects of RF-EMF on an animal model, excluding thermal outcomes and thus represents the starting point for more comprehensive studies on dose response effects of electromagnetic fields radiations consequences. [ABSTRACT FROM AUTHOR]

The process of gonadal development and mechanism involved in sex differentiation in eels are still unclear. The objectives were to investigate the gonadal development and expression pattern of sex-related genes during sex differentiation in the Japanese eel, Anguilla japonica . For control group, the elvers of 8–10 cm were reared for 8 months; and for feminization, estradiol-17β (E2) was orally administered to the elvers of 8–10 cm for 6 months. Only males were found in the control group, suggesting a possible role of environmental factors in eel sex determination. In contrast, all differentiated eels in E2-treated group were female. Gonad histology revealed that control male eels seem to differentiate through an intersexual stage, while female eels (E2-treated) would differentiate directly from an undifferentiated gonad. Tissue distribution and sex-related genes expression during gonadal development were analyzed by qPCR. The vasa , figla and sox3 transcripts in gonads were significantly increased during sex differentiation. High vasa expression occurred in males; figla and sox3 were related to ovarian differentiation. The transcripts of dmrt1 and sox9a were significantly increased in males during testicular differentiation and development. The c yp19a1 transcripts were significantly increased in differentiating and differentiated gonads, but did not show a differential expression between the control and E2-treated eels. This suggests that cyp19a1 is involved both in testicular differentiation and development in control males, and in the early stage of ovarian differentiation in E2-treated eels. Importantly, these results also reveal that cyp19a1 is not a direct target for E2 during gonad differentiation in the eel. [ABSTRACT FROM AUTHOR]

As a crucial component of Toll-like receptor (TLR)/interleukin-1 receptor (IL-1R) signaling pathways, IL-1R--associated kinase 4 (IRAK-4) plays a central role in innate immunity and embryonic development. Herein, we have characterized the full length cDNA of IRAK4 from Artemia sinica . Molecular characterization revealed that the sequence includes a 2550 bp open reading frame, encoding a predicted protein of 849 amino acids. The predicted protein contains a death domain in the N-terminus and two serine/threonine/tyrosine protein kinasedomains. Bioinformatics analysis showed that it belonged to a new member of the IRAK-4 family. The expression of As IRAK-4 was researched in various stages during embryonic development by several molecular biology methods including real time PCR, Western blotting and immunohistochemistry. The results showed that As IRAK-4 was constitutively expressed at all developmental stages from embryo to adult, and it was mainly expressed in the head and thorax at the early stages and on the surface of the alimentary canal at later stages. The highest expression level was at the 0 h, 15 h and 5 d stages of A. sinica . While challenged by Gram-positive and Gram-negative bacteria, AsIRAK-4 was remarkably upregulated with the rising concentration of bacteria. Our research revealed that As IRAK-4 plays a vital role in growth, development and innate immunity of A. sinica . [ABSTRACT FROM AUTHOR]

Sex determination pathways are extensively diverse across species, with the master sex-determinants being the most variable element. Despite this, there is a family of DM-domain transcription factors ( Dmrts ), which hold a highly conserved function in sexual development. This work is the first to describe a heterogametic sex-linked Dmrt in an invertebrate species, the Eastern spiny lobster, Sagmariasus verreauxi . We have termed the Y-linked, truncated paralogue of the autosomal iDmrt1, Sv-iDMY. Considering the master sex-determining function of both DMY in medaka and DM-W in frog, we hypothesised a similar function of Sv-iDMY. By conducting temporal expression analyses during embryogenesis we have identified a putative male sex-determining period during which iDMY > iDmrt1 . Employing a GAL4-transactivation assay we then demonstrate the dominant negative suppression of iDMY over its autosomal iDmrt1 paralogue, suggesting the mechanism with which iDMY determines sex. Comparative analyses of Sv-iDMY, DM-W and medaka DMY, highlight the C′-mediated features of oligomerisation and transactivation as central to the mechanism that each exerts. Indeed, these features may underpin the plasticity facilitating the convergent emergence of these three sporadic sex-linked master-Dmrts. [ABSTRACT FROM AUTHOR]

Intracellular Ca 2+ signaling regulates cellular activities during embryogenesis and in adult organisms. We generated stable Tg[βactin2:GCaMP6s] stl351 and Tg[ubi:GCaMP6s] stl352 transgenic lines that combine the ubiquitously-expressed Ca 2+ indicator GCaMP6s with the transparent characteristics of zebrafish embryos to achieve superior in vivo Ca 2+ imaging. Using the Tg[βactin2:GCaMP6s] stl351 line featuring strong GCaMP6s expression from cleavage through gastrula stages, we detected higher frequency of Ca 2+ transients in the superficial blastomeres during the blastula stages preceding the midblastula transition. Additionally, GCaMP6s also revealed that dorsal-biased Ca 2+ signaling that follows the midblastula transition persisted longer during gastrulation, compared with earlier studies. We observed that dorsal-biased Ca 2+ signaling is diminished in ventralized ichabod/β-catenin2 mutant embryos and ectopically induced in embryos dorsalized by excess β-catenin. During gastrulation, we directly visualized Ca 2+ signaling in the dorsal forerunner cells, which form in a Nodal signaling dependent manner and later give rise to the laterality organ. We found that excess Nodal increases the number and the duration of Ca 2+ transients specifically in the dorsal forerunner cells. The GCaMP6s transgenic lines described here enable unprecedented visualization of dynamic Ca 2+ events from embryogenesis through adulthood, augmenting the zebrafish toolbox. [ABSTRACT FROM AUTHOR]

The distribution and connectivity of marine populations are largely dependent on biophysical factors affecting pelagic larval dispersal between spawning at adult spawning sites and settlement to juvenile nursery habitats. Behaviour and swimming ability of pelagic larvae are increasingly understood to influence patterns of dispersal, but it is unclear which sensory cues are involved and when during ontogeny these abilities first develop. Here we studied the early ontogenetic development of responses to olfactory cues from coastal and estuarine waters in larvae of two temperate estuarine-associated fish species, Australian bass, Macquaria novemaculeata , and mulloway, Argyrosomus japonicus , to determine when olfaction begins to influence dispersal. Olfactory responses to habitat-associated cues were not present when larvae first transitioned from nonswimming to swimming (indicated by flexion of the notochord), but emerged after ca. 7 days in a species-specific manner that was consistent across different cohorts. Based on general additive models (GAMs), age (in days posthatch) best explained the ontogenetic pattern in both species. The emergence of chemotactic responses coincides with an exponential increase in swimming endurance reported for these species. This suggests the existence of ontogenetic milestones during larval development that, once reached, trigger active influence on dispersal. Salinity and pH did not influence choice behaviour after these ontogenetic milestones; however, the presence of cues generated by seagrass harvested from the estuary habitat elicited strong responses in fish larvae consistent with species-specific habitat preferences, indicating an important role for aquatic vegetation in driving these behaviours. [ABSTRACT FROM AUTHOR]

During skeletal muscle development, myocytes aggregate and fuse to form multinucleated muscle fibers. Inhibition of myocyte fusion is thought to significantly derail the differentiation of functional muscle fibers. Despite the purported importance of fusion in myogenesis, in vivo studies of this process in vertebrates are rather limited. Myomaker, a multipass transmembrane protein, has been shown to be the first muscle-specific fusion protein essential for myocyte fusion in the mouse. We have generated loss-of-function alleles in zebrafish myomaker , and found that fusion of myocytes into syncytial fast-twitch muscles was significantly compromised. However, mutant myocytes could be recruited to fuse with wild-type myocytes in chimeric embryos, albeit rather inefficiently. Conversely, overexpression of Myomaker was sufficient to induce hyperfusion among fast-twitch myocytes, and it also induced fusion among slow-twitch myocytes that are normally fusion-incompetent. In line with this, Myomaker overexpression also triggered fusion in another myocyte fusion mutant compromised in the function of the junctional cell adhesion molecule, Jam2a. We also provide evidence that Rac, a regulator of actin cytoskeleton, requires Myomaker activity to induce fusion, and that an approximately 3 kb of myomaker promoter sequence, with multiple E-box motifs, is sufficient to direct expression within the fast-twitch muscle lineage. Taken together, our findings underscore a conserved role for Myomaker in vertebrate myocyte fusion. Strikingly, and in contrast to the mouse, homozygous myomaker mutants are viable and do not exhibit discernible locomotory defects. Thus, in the zebrafish, myocyte fusion is not an absolute requirement for skeletal muscle morphogenesis and function. [ABSTRACT FROM AUTHOR]

The interbranchial lymphoid tissue (ILT) of Atlantic salmon originates from an embryological location that in higher vertebrates gives rise to both primary and secondary lymphoid tissues. Still much is unknown about the morphological and functional development of the ILT. In the present work a standardized method of organ volume determination was established to study its development in relation to its containing gill and the thymus. Based on morphological findings and gene transcription data, the ILT shows no signs of primary lymphoid function. In contrast to the thymus, an ILT-complex first became discernible after the yolk-sac period. After its appearance, the ILT-complex constitutes 3–7% of the total volume of the gill (excluding the gill arch) with the newly described distal ILT constituting a major part, and in adult fish it is approximately 13 times larger than the thymus. Confined regions of T-cell proliferation are present within the ILT. Communication with systemic circulation through the distal ILT is also highly plausible thus offering both internal and external recruitment of immune cells in the growing ILT. [ABSTRACT FROM AUTHOR]

Purpose To determine whether chronic low-dose α-particle radiation from Ra-226 over multiple cell generations can lead to an adaptive response in CHSE/F fish embryonic cells or HaCaT human epithelial cells receiving subsequent acute high-dose γ-ray radiation. Methods CHSE/F and HaCaT cells were exposed to very low doses of Ra-226 in medium for multiple generations prior to being challenged by a higher dose γ-ray radiation. The clonogenic assay was used to test the clonogenic survival of cells with or without being pretreated by radiation from Ra-226. Results In general, pretreatment with chronic radiation has no significant influence on the reaction of cells to the subsequent challenge radiation. Compared to unprimed cells, the change in clonogenic survival of primed cells after receiving challenge radiation is mainly due to the influence of the chronic exposure, and there's little adaptive response induced. However at several dose points, pretreatment of CHSE/F fish cells with chronic radiation resulted in a radiosensitive response to a challenge dose of γ-ray radiation, and pretreatment of HaCaT cells resulted in no effect except for a slightly radioresistant response to the challenge radiation which was not significant. Conclusion The results suggest that chronic low-dose radiation is not effective enough to induce adaptive response. There was a difference between human and fish cells and it may be important to consider results from multiple species before making conclusions about effects of chronic or low doses of radiation in the environment. The term “radiosensitive” or “adaptive” make no judgment about whether such responses are ultimately beneficial or harmful. [ABSTRACT FROM AUTHOR]

Insulin-like growth factors I and II (IGF-I and IGF-II) are important regulators of vertebrate growth and development. This study characterized the mRNA expressions of igf-i and igf-ii during turbot ( Scophthalmus maximus ) metamorphosis to elucidate the possible regulatory role of the IGF system in flatfish metamorphosis. Results showed that the mRNA levels of igf-i significantly increased at the early-metamorphosis stage and then gradually decreased until metamorphosis was completed. By contrast, mRNA levels of igf-ii significantly increased at the pre-metamorphosis stage and then substantially decreased during metamorphosis. Meanwhile, the whole-body thyroxine (T4) levels varied during larval metamorphosis, and the highest value was observed in the climax-metamorphosis. The mRNA levels of igf-i significantly increased and decreased by T4 and thiourea (TU, inhibitor of endogenous thyroid hormone) during metamorphosis, respectively. Conversely, the mRNA levels of igf-ii remained unchanged. Furthermore, TU significantly inhibited the T4-induced mRNA up-regulation of igf-i during metamorphosis. The whole-body thyroxine (T4) levels were significantly increased and decreased by T4 and TU during metamorphosis, respectively. These results suggested that igf-i and igf-ii may play different functional roles in larval development stages, and igf-i may have a crucial function in regulating the early metamorphic development of turbot. These findings may enhance our understanding of the potential roles of the IGF system to control flatfish metamorphosis and contribute to the improvement of broodstock management for larvae. [ABSTRACT FROM AUTHOR]

Larval turbot ( Scophthalmus maximus ) undergo metamorphosis, a late post-embryonic developmental event that precedes juvenile transition. Insulin-like growth factors (IGFs) are important endocrine/autocrine/paracrine factors that provide essential signals to control of the embryonic and postnatal development of vertebrate species, including fish. Accumulating evidence suggests that IGFs are involved in regulating the metamorphic development of flatfish. This mini review focus on the functions of all known IGFs (IGF-I and IGF-II) during the metamorphic development of turbot. Information about IGFs and insulin-like growth factors binding proteins (IGFBPs) from other teleosts is also included in this review to provide an overview of IGFs functions in the metamorphic development of turbot. These findings may enhance our understanding of the potential roles of the IGFs system in controlling of flatfish metamorphosis and contributing to the improvement of broodstock management strategies for larval turbot. [ABSTRACT FROM AUTHOR]

Lymphocyte antigen-6 (Ly6) superfamily is a large family of proteins and characterized by precisely spaced cysteine motifs, termed the three-finger fold. To date, a large number of members of the Ly6/uPAR family were identified among many species. In this study, we first report the identification and characterization of the secreted Ly2.1-3 proteins on the chromosome 2 in zebrafish and determine the expression pattern. Ly2.1-3 all possess a conserved LU domain and adopt similar three-finger structure with human CD59, SLURP1 and other Ly6 family members. Ly2.1-3 cluster on chromosome 2 and share high homology, possibly originated from chromosomal gene duplication. Ly2.1-3 exhibit distinct expression pattern in the endoderm, they were found abundantly and specifically in the digestive tract, liver and pancreas respectively. The differential expression pattern may suggest Ly2.1-3 acquire new function during gene duplication. The expression level of Ly2.1-3 were up-regulating challenged with LPS indicated that they have a role in innate immune responses of the digestive system during endotoxin challenge in early stage. [ABSTRACT FROM AUTHOR]

IgZ is considered to be the last immunoglobulin discovered in vertebrate species. In this study, the structure of secreted form of blunt snout bream ( Megalabrama amblycephala ) IgZ (sIgZ) heavy chain gene is VH-Cζ1-Cζ2-Cζ3-Cζ4, in which Cζ4 provides the specificity to the IgZ isotype. The deduced amino acid sequence of sIgZ shows highest similarity with that of Ctenopharyngodon idella (79%). The ontogeny of sIgZ gene expression showed a V-shape change pattern: decreased initially from unfertilized egg stage to 16-cell embryos and increased significantly from blastula stage, which exhibited maternal transmission effects. Compared with the juvenile fish, sIgZ mRNA level was higher in head kidney, spleen, trunk kidney, liver, intestine and gill of adult fish. In both juvenile and adult fish, sIgZ mRNA was detected in intestine and gill. Aeromonas hydrophila challenge experiment showed that sIgZ transcription significantly increased in skin, gill and intestine, indicating a prominent mucosal immune response. The results of Western blot also verified the protein alterations of sIgZ in mucosal organs in M. amblycephal . Our studies indicate a prominent role of IgZ in mucosa-associated lymphoid tissue immunity and further support the specialized role of IgZ in teleost mucosal immune responses. [ABSTRACT FROM AUTHOR]

Hemodynamic shear stress is sensed by the endocardial cells composing the inner cell layer of the heart, and plays a major role in cardiac morphogenesis. Yet, the underlying hemodynamics and the associated mechanical stimuli experienced by endocardial cells remains poorly understood. Progress in the field has been hampered by the need for high temporal resolution imaging allowing the flow profiles generated in the beating heart to be resolved. To fill this gap, we propose a method to analyze the wall dynamics, the flow field, and the wall shear stress of the developing zebrafish heart. This method combines live confocal imaging and computational fluid dynamics to overcome difficulties related to live imaging of blood flow in the developing heart. To provide an example of the applicability of the method, we discuss the hemodynamic frequency content sensed by endocardial cells at the onset of valve formation, and how the fundamental frequency of the wall shear stress represents a unique mechanical cue to endocardial, heart-valve precursors. [ABSTRACT FROM AUTHOR]

Follistatin-like 1 (Fstl1) peptides play important roles in inhibiting myoblast proliferation and differentiation. Here, we characterized and examined the expression patterns of fstl1a and -b in grass carp ( Ctenopharyngodon idellus ). These genes encode 314 aa and 310 aa peptides, respectively, sharing a sequence identity of 83%. Except for the existence of the follistatin-N-terminal (FOLN) and Kazal-type 2 serine protease inhibitor (Kazal 2) domains, grass carp Fstl1a and -b do not share amino acid sequence similarity with Fst1 and -b. Both fstl1a and -b mRNAs were widely expressed in adult tissues. During embryogenesis, grass carp fstl1a and -b mRNA was detected in the presomitic mesoderm and somites at 12 h post fertilization (hpf). At 24 hpf, fstl1a mRNA was expressed in the hindbrain, somites, notochord and tailbud, while fstl1b mRNA was only detected in the tailbud. At 36 hpf, fstl1a mRNA was detected in the hindbrain and notochord, and fstl1b was also expressed in the notochord. Furthermore, fstl1a and -b were downregulated in brain and liver tissue following injection with 10 or 50 μg hGH, while fstl1b was significantly up-regulated in muscle tissue after 10 μg hGH treatment. Both fstl1a and -b were significantly up-regulated at 2, 4 or 6 days of nutrient restriction, and fstl1a was still highly expressed in the liver and muscle after 3 days of refeeding, as was fstl1b in the brain and muscle. The expression of these genes returned to near control levels following 6 days of refeeding. Our findings suggest that the two fstl s play important but divergent roles in embryonic development and tissue growth regulation in grass carp. [ABSTRACT FROM AUTHOR]

Most fishes produce free-living embryos that are exposed to environmental stressors immediately following fertilization, including pathogenic microorganisms. Initial immune protection of embryos involves the chorion, as a protective barrier, and maternally-allocated antimicrobial compounds. At later developmental stages, host-genetic effects influence susceptibility and tolerance, suggesting a direct interaction between embryo genes and pathogens. So far, only a few host genes could be identified that correlate with embryonic survival under pathogen stress in salmonids. Here, we utilized high-throughput RNA-sequencing in order to describe the transcriptional response of a non-model fish, the Alpine whitefish Coregonus palaea , to infection, both in terms of host genes that are likely manipulated by the pathogen, and those involved in an early putative immune response. Embryos were produced in vitro , raised individually, and exposed at the late-eyed stage to a virulent strain of the opportunistic fish pathogen Pseudomonas fluorescens . The pseudomonad increased embryonic mortality and affected gene expression substantially. For example, essential, upregulated metabolic pathways in embryos under pathogen stress included ion binding pathways, aminoacyl-tRNA-biosynthesis, and the production of arginine and proline, most probably mediated by the pathogen for its proliferation. Most prominently downregulated transcripts comprised the biosynthesis of unsaturated fatty acids, the citrate cycle, and various isoforms of b-cell transcription factors. These factors have been shown to play a significant role in host blood cell differentiation and renewal. With regard to specific immune functions, differentially expressed transcripts mapped to the complement cascade, MHC class I and II, TNF-alpha, and T-cell differentiation proteins. The results of this study reveal insights into how P . fluorescens impairs the development of whitefish embryos and set a foundation for future studies investigating host pathogen interactions in fish embryos. [ABSTRACT FROM AUTHOR]

The epithelial to mesenchymal transition (EMT) is an essential process that occurs repeatedly during embryogenesis whereby stably adherent cells convert to an actively migrating state. While much is known about the factors and events that initiate the EMT, the steps that cells undergo to become directionally migratory are far less well understood. Zebrafish embryos lacking the transcription factors Tbx16/Spadetail and Mesogenin1 (Msgn1) are a valuable system for investigating the EMT. Mesodermal cells in these embryos are unable to perform the EMT necessary to leave the most posterior end of the body (the tailbud) and join the pre-somitic mesoderm, a process that is conserved in all vertebrates. It has previously been very difficult to study this EMT in vertebrates because of the multiple cell types in the tailbud and the morphogenetic changes the whole embryo undergoes. Here, we describe a novel tissue explant system for imaging the mesodermal cell EMT in vivo that allows us to investigate the requirements for cells to acquire migratory properties during the EMT with high spatio-temporal resolution. This method revealed that, despite the inability of tbx16;msgn1 -deficient cells to leave the tailbud, actin-based protrusions form surprisingly normally in these cells and they become highly motile. However, tbx16;msgn1 -deficient cells have specific cell-autonomous defects in the persistence and anterior direction of migration because the lamellipodia they form are not productive in driving anteriorward migration. Additionally, we show that mesoderm morphogenesis and differentiation are separable and that there is a migratory cue that directs mesodermal cell migration that is independent of Tbx16 and Msgn1. This work defines changes that cells undergo as they complete the EMT and provides new insight into the mechanisms required in vivo for cells to become mesenchymal. [ABSTRACT FROM AUTHOR]

Our understanding of the function of vitellogenin (Vg) in reproduction has undergone a transformation over the past decade in parallel with new insights into the role of Vg in immunity. Initially, Vg was regarded as a female-specific reproductive protein, which is cleaved into yolk proteins such as phosvitin (Pv) and lipovitellin (Lv), stored in egg, providing the nutrients for developing embryos. Recently, Vg is shown to be an immune-relevant molecule involved in the defense of the host against the microbes including bacterium and virus. Furthermore, Pv and Lv, that both are proteolytically cleaved products of Vg, play a defense role in developing embryos. Importantly, yolk protein-derived small peptides also display antimicrobial activity. These data together indicate that Vg, in addition to being involved in yolk protein formation, plays a non-reproductive role via functioning as an immune-relevant molecule in both parent fishes and their offspring. It also shows that yolk proteins and their degraded peptides are novel players in maternal immunity, opening a new avenue to study the functions of reproductive proteins. [ABSTRACT FROM AUTHOR]

Galectins are conserved family members with β-galactosides affinity that play multiple functions in embryogenesis, development and regulation of innate and adaptive immunity. However, little functional studies were reported in crustaceans. Here, a shrimp Litopenaeus vannamei galectin ( LvGal ) cDNA was identified with an open reading frame of 1017 bp, which encodes a putative protein of 338 amino acids. A carbohydrate recognition domain (CRD) and several amino acids residues involved in dimerization were found in Lv Gal. LvGal mRNA was mainly expressed in gills and hemocytes and upregulated post Vibrio anguillarum challenge. Recombinant Lv Gal (r Lv Gal) was expressed in Escherichia coli BL21 (DE3) and the purified r Lv Gal could strongly bind G − bacteria V. anguillarum and G + bacteria Micrococcus lysodeikticus . Besides, r Lv Gal exhibited strong activity to agglutinate V. anguillarum and weak activity to agglutinate M. lysodeikticus but no obvious antibacterial activity was found with selected bacteria. In addition, in vivo experiments showed r Lv Gal could promote phagocytosis of bacteria by hemocytes. Thus, through these collective data we predicted Lv Gal is involved in immune recognition and functions as a potential pattern recognition receptor. [ABSTRACT FROM AUTHOR]

Yellow catfish ( Pelteobagrus fulvidraco ) is an important freshwater fish species in China. In particular, an all-male population has been commercially produced for the males grow faster than females. However, the molecular mechanisms underlying sexual dimorphism of body size and sex differentiation are still unclear in yellow catfish. This study attempts to characterize and analyze the expression of Cytochrome P450 ( CYP ) family members that have been shown to play an important role in sex differentiation and metabolism in teleosts. A total of 25 CYP genes were identified from our transcriptomes by 454 pyrosequencing and Solexa sequencing, including 17 genes with complete open reading frame (ORF). Phylogenetic analyses were conducted to compare these genes with their counterparts from other teleosts. In the tissues of hypothalamic–pituitary–gonad (HPG) axis, most of the genes were expressed at uniform level in both sexes. However, multiple CYP genes displayed sexual dimorphic expression, such as cyp2AD , cyp4b , cyp8a , cyp11b2 , cyp17a and cyp27a expressed at higher level in testis than in ovary, whereas cyp2g , cyp7a , cyp8b , cyp19a1a and cyp26a expressed at higher level in ovary than in testis. The expression response of six CYP genes in ovary was also assessed after 17α-methyltestosterone (MT) treatment. Testis-biased expressed cyp11b2 and cyp17a were significantly up-regulated, while cyp11a and cyp19a1a were reduced in ovary after MT treatment. Our work is helpful for understanding molecular evolution of CYP genes in vertebrates and the mechanism of sexual dimorphism in teleosts. [ABSTRACT FROM AUTHOR]

Cigarette butts (CBs) littering is not just an unsightly nuisance but also a public health problem, because chemicals contained in cigarettes can leach into aquatic environments and pose a risk to the health of humans and wildlife. However, this risk is largely unrecognized or ignored by the public, and toxicological evidence of CBs is scarce. Therefore, we used medaka embryos ( Oryzias latipes ) to explore developmental toxicity of CBs. The embryos were exposed to various concentrations of leachates from smoked and unsmoked cigarette tobacco (ST and UST) and filters (SF and USF), and observed from 1 to 3 days post-fertilization. The images were recorded and several developmental endpoints analyzed. The values from these endpoints were then used to calculate the Integrated Biomarker Response and evaluate overall effects of the leachates. Some of the embryos were allowed to hatch, and the hatchlings were tested for anxiety-like behavior. Our results showed that low concentrations of the leachates from ST, UST, and SF raised the heart rate, accelerated development, and changed behavior, while high concentrations lowered the heart rate, suppressed development, and increased mortality. The lowest observed effect concentration for the leachates was ≤0.2 piece (pc)/L. The USF leachate had no effect at the concentration of 20 pc/L. Developmental toxicity of the leachates was ranked as: ST>UST>SF>USF. This study has demonstrated for the first time that CB leachates affect fish development, and provided toxicological evidence to better assess ecological impacts of CBs. [ABSTRACT FROM AUTHOR]

Toxicity of prometryne to early life stages of common carp was assessed. On the basis of accumulated mortality in the experimental groups lowest observed-effect concentration (LOEC) was estimated as 1100 µg/l; and no observed-effect concentration (NOEC) was 850 µg/l. Fulton's condition factor was significantly lower than in controls in fish exposed to 4000 µg/l after 7, 14, and 21 days. By day 14, fish exposed to 4000 µg/l prometryne showed significantly lower mass and total length compared to controls. Fish exposed the 1200 and 4000 µg/l showed delay in development, severe hyperaemia in gill, liver, and caudal and cranial kidney. Subchronic prometryne exposure of early-life stages of common carp at concentrations of 1200 and 4000 µg/l affected their survival, growth rate, early ontogeny, and histology. [ABSTRACT FROM AUTHOR]

Abstract: Extracellular signal-regulated kinase (ERK) is a serine/threonine-specific protein kinase, which participates in signaling transduction pathways that control intracellular events, including resumption of meiosis, embryogenesis, cell differentiation, cell proliferation, cell death and response to radiation. Some virus species evolved the ability to hijack the host cell ERK signaling transduction pathway for viral replications and gene expressions. To obtain a better understanding of ERK, we cloned a cDNA encoding ERK from the muscle of Fenneropenaeus chinensis (FcERK). The FcERK contained a 1098 bp open reading frame (ORF) encoding a protein of 365 amino acid residues with a conserved phosphorylation motif TEY in the kinase activation loop. Pair-wise and multiple sequence alignment revealed that ERK is highly conserved across taxa. The FcERK gene expressions in the hepatopancreas and gill were noticeably higher than the expression observed in the muscle. A challenge test was performed to reveal the responses of FcERK in different tissues to white spot syndrome virus (WSSV) infection. Post WSSV challenge, the FcERK expression in the gill significantly increased during the early stage of the viral infection, the FcERK expression in the muscle increased later than that in the gill, and the FcERK expression in the hepatopancreas significantly decreased. The FcERK gene expression profile accorded with the results that the virus primarily infects tissues originating from the ectoderm, with less infection of the tissues originating from the mesoderm, and hardly any infection in the tissues originating from the entoderm. Two single nucleotide polymorphisms (SNPs) were identified in the FcERK gene, involving C/T transition. The SNP genotypes of two groups of shrimps, respectively comprising 96 WSSV-resistant shrimps and 96 WSSV-susceptible shrimps were obtained using a high-resolution melting (HRM) method. In the two groups, the MAFs of both sites were greater than 0.05, and no site departed significantly (P < 0.05) from HWE. The genotype distributions of both mutation sites between the two groups were not significantly different. These results lead to a better understanding of the molecular mechanisms of the host–virus interaction and provide useful information for disease control. [Copyright &y& Elsevier]

Abstract: The zebrafish embryotoxicity test is a promising alternative assay for developmental toxicity. Classically, morphological assessment of the embryos is applied to evaluate the effects of compound exposure. However, by applying differential gene expression analysis the sensitivity and predictability of the test may be increased. For defining gene expression signatures of developmental toxicity, we explored the possibility of using gene expression signatures of compound exposures based on commonly expressed individual genes as well as based on regulated gene pathways. Four developmental toxic compounds were tested in concentration-response design, caffeine, carbamazepine, retinoic acid and valproic acid, and two non-embryotoxic compounds, d-mannitol and saccharin, were included. With transcriptomic analyses we were able to identify commonly expressed genes, which were mostly development related, after exposure to the embryotoxicants. We also identified gene pathways regulated by the embryotoxicants, suggestive of their modes of action. Furthermore, whereas pathways may be regulated by all compounds, individual gene expression within these pathways can differ for each compound. Overall, the present study suggests that the use of individual gene expression signatures as well as pathway regulation may be useful starting points for defining gene biomarkers for predicting embryotoxicity. [Copyright &y& Elsevier]

Highlights: [•] Two data sets cover (i) the complete reproductive cycle or (ii) the beginning of ovarian growth. [•] Long days (24L0D) either accelerate or stop the start of rapid ovarian growth. [•] Changes in pituitary Fsh, but not Lh, accompany entry into or block of ovarian growth. [•] Fsh receptor mRNA was stable, except during remodelling periods (atresia, ovulation). [•] Lh receptor mRNA increased with plasma estrogen and remained high after ovulation. [ABSTRACT FROM AUTHOR]

Abstract: The zinc-finger transcription factor insulinoma-associated 1 (Insm1, previously IA-1) is expressed in the developing nervous and neuroendocrine systems, and is required for cell type specific differentiation. Expression of Insm1 is largely absent in the adult, although it is present in neurogenic regions of the adult brain and zebrafish retina. While expression of Insm1 has also been observed in the embryonic retina of numerous vertebrate species, its function during retinal development has remained unexplored. Here, we demonstrate that in the developing zebrafish retina, insm1a is required for photoreceptor differentiation. Insm1a-deficient embryos were microphthalmic and displayed defects in rod and cone photoreceptor differentiation. Rod photoreceptor cells were more sensitive to loss of insm1a expression than were cone photoreceptor cells. Additionally, we provide evidence that insm1a regulates cell cycle progression of retinoblasts, and functions upstream of the bHLH transcription factors ath5/atoh7 and neurod, and the photoreceptor specification genes crx and nr2e3. Finally, we show that insm1a is negatively regulated by Notch-Delta signaling. Taken together, our data demonstrate that Insm1 influences neuronal subtype differentiation during retinal development. [Copyright &y& Elsevier]

Abstract: Zebrafish is considered as a versatile experimental animal for various research models from development to diseases. In this study, we report the development of transgenic zebrafish line named as Tg(EF1α:Kaede) that expresses translation elongation factor 1 subunit alpha (EF1α) promoter linked to a fluorescent protein (FP), Kaede for monitoring proliferating cells in during regeneration. It was revealed that about 1.4 kb 5′-flanking region of the EF1α was sufficient for its promoter activity. Expression of Kaede with a property of photo-conversion from green to red was detected in different embryonic stages as well as various organs such as brain, heart, pancreas, intestine, ovary, and fins of adult fish. Cell proliferation pattern during fin regeneration was monitored after amputation of Tg(EF1α:Kaede) caudal fin and results shown that this system is simple and efficient method for detecting proliferating cells during tissue regeneration. Developed Tg(EF1α:Kaede) line has potential to investigate the cell proliferation, regeneration, wound healing capacities after tissue damage and evaluate the therapeutic power of wound healing drugs. [Copyright &y& Elsevier]

Cytogenetic analysis methods were used to study the developing embryos of the following four Coregonidae species: Coregonus peled (Gmelin), Coregonus lavaretus (Gmelin), Coregonus nasus (Pallas) and Coregonus muksun (Pallas). Spawners of these species were selected from rivers and lakes of the Tyumen region (Russia) with different levels of pollution. The variability of the chromosomal aberration rate was analyzed during a monitoring period of more than 20 years. The level of chromosomal mutability in the embryonic cells of these fishes was found to have steadily increased during the observation period (1989–2011). The higher frequency of chromosomal mutations at different stages of embryogenesis in broad whitefish, C. nasus (Pallas), was noted in comparison with other species. The possible use of chromosomal mutation frequency as an indicator (biomarker) to characterize the state of a population is discussed. [Copyright &y& Elsevier]

Bilaterally symmetric organisms display mirror copies of their structures on both sides of the body, and the development of both sides is regulated by the same set of genes. Environmental variations can directly affect phenotype, and exposure to chemical contaminants at certain stages may modify embryonic development. The pesticide sodium pentachlorophenate (NaPCP) was used at the no-observable-effect concentration (NOEC) to determine the degree of susceptibility of zebrafish (Danio rerio) embryos in different developmentally susceptible windows (zygote, blastula, gastrula, segmentation, pharyngula and larva). Shape variation in the zebrafish viscerocranium and fluctuating asymmetry (FA), which increases in direct proportion to environmental stress, induced by exposure to NaPCP were measured with geometric morphometrics. Procrustes ANOVA was performed to estimate the shape variation around a symmetric consensus that accounted for the following factors: shape variation in individuals (I), variation by sides (S), the Individuals×Sides interaction (I×S), and the stages of exposure to the toxicant (Stages). Factors I, S and IxS accounted for most of the morphological variation (p<0.0001). Extensive deformities throughout the viscerocranium occurred during the window of exposure from gastrula to larva. Embryonic mortality occurred and was dependent on the stage of exposure. The NOEC concentration of NaPCP affected embryonic development in D. rerio and also induced lethal effects in embryos. FA was determined in both unexposed and NaPCP-exposed embryos and was greater in the control than in some exposure windows; besides, no correlation was found between FA and developmental stages, so our results do not support FA as a bioindicator of chemical stress but confirm its value in the study of morphological effects of toxicants. [Copyright &y& Elsevier]

Abstract: Ocular coloboma is a potentially blinding congenital eye malformation caused by failure of optic fissure closure during early embryogenesis. The optic fissure is a ventral groove that forms during optic cup morphogenesis, and through which hyaloid artery and vein enter and leave the developing eye, respectively. After hyaloid artery and vein formation, the optic fissure closes around them. The mechanisms underlying optic fissure closure are poorly understood, and whether and how this process is influenced by hyaloid vessel development is unknown. Here we show that a loss-of-function mutation in lmo2, a gene specifically required for hematopoiesis and vascular development, results in failure of optic fissure closure in zebrafish. Analysis of ocular blood vessels in lmo2 mutants reveals that some vessels are severely dilated, including the hyaloid vein. Remarkably, reducing vessel size leads to rescue of optic fissure phenotype. Our results reveal a new mechanism leading to coloboma, whereby malformed blood vessels interfere with eye morphogenesis. [Copyright &y& Elsevier]

Abstract: The Hedgehog (HH) signaling pathway is a central regulator of embryonic development, controlling the pattern and proliferation of a wide variety of organs. Previous studies have implicated the secreted protein, Scube2, in HH signal transduction in the zebrafish embryo () although the nature of the molecular function of Scube2 in this process has remained undefined. This analysis has been compounded by the fact that removal of Scube2 activity in the zebrafish embryo leads to only subtle defects in HH signal transduction in vivo (). Here we present the discovery of two additional scube genes in zebrafish, scube1 and scube3, and demonstrate their roles in facilitating HH signal transduction. Knocking down the function of all three scube genes simultaneously phenocopies a complete loss of HH signal transduction in the embryo, revealing that Scube signaling is essential for HH signal transduction in vivo. We further define the molecular role of scube2 in HH signaling. [Copyright &y& Elsevier]

Abstract: RING finger protein 11 (RNF11) is a novel regulator of immunity and cell survival via ubiquitination process in mammalian cells whereas its vertebrate embryonic roles are undefined. Here, we are reporting the isolation, expression and functional roles of an RNF11 orthologue, Rnf11-like in zebrafish embryos. Zebrafish Rnf11-like is composed of 154 amino acids containing RING-H2-finger domain in the C-terminal region and PY-motif. Spatiotemporal expression patterns of rnf11-like indicate that rnf11-like is expressed maternally and zygotically throughout embryogenesis. However, rnf11-like transcripts are present specifically in the presomatic mesoderm (PSM), and later in the brain and retina. Knock-down of Rnf11-like using rnf11-like-specific morpholino causes cell death and developmental defects in the posterior somites, elevating transcripts of NF-κB target gene, ikk1, a negative regulator of NF-κB signaling. All these findings indicate that Rnf11-like is an essential component of NF-κB signaling pathway for specification of the posterior somites in zebrafish embryos. [Copyright &y& Elsevier]

Abstract: Cardiomyocyte hypertrophy is a complex cellular behavior involving coordination of cell size expansion and myofibril content increase. Here, we investigate the contribution of cardiomyocyte hypertrophy to cardiac chamber emergence, the process during which the primitive heart tube transforms into morphologically distinct chambers and increases its contractile strength. Focusing on the emergence of the zebrafish ventricle, we observed trends toward increased cell surface area and myofibril content. To examine the extent to which these trends reflect coordinated hypertrophy of individual ventricular cardiomyocytes, we developed a method for tracking cell surface area changes and myofibril dynamics in live embryos. Our data reveal a previously unappreciated heterogeneity of ventricular cardiomyocyte behavior during chamber emergence: although cardiomyocyte hypertrophy was prevalent, many cells did not increase their surface area or myofibril content during the observed timeframe. Despite the heterogeneity of cell behavior, we often found hypertrophic cells neighboring each other. Next, we examined the impact of blood flow on the regulation of cardiomyocyte behavior during this phase of development. When blood flow through the ventricle was reduced, cell surface area expansion and myofibril content increase were both dampened, and the behavior of neighboring cells did not seem coordinated. Together, our studies suggest a model in which hemodynamic forces have multiple influences on cardiac chamber emergence: promoting both cardiomyocyte enlargement and myofibril maturation, enhancing the extent of cardiomyocyte hypertrophy, and facilitating the coordination of neighboring cell behaviors. [Copyright &y& Elsevier]

Abstract: In cranial skeletal development, the establishment of the ectomesenchymal lineage within the cranial neural crest is of great significance. Fgfs are polypeptide growth factors with diverse functions in development and metabolism. Fgf20b knockdown zebrafish embryos showed dysplastic neurocranial and pharyngeal cartilages. Ectomesenchymal cells from cranial neural crest cells were significantly decreased in Fgf20b knockdown embryos, but cranial neural crest cells with a non-ectomesnchymal fate were increased. However, the proliferation and apoptosis of cranial neural crest cells were essentially unchanged. Fgfr1 knockdown embryos also showed dysplastic neurocranial and pharyngeal cartilages. The present findings indicate that Fgf20b is required for ectomesenchymal fate establishment via the activation of Fgfr1 in zebrafish. [Copyright &y& Elsevier]

Abstract: In fish, asynchronous development of ovarian follicles, the simultaneous advance of vitellogenesis and oocyte maturation in one ovary, is a rational reproductive strategy to spawn consecutively in one spawning season. In this study, to clarify the mode of action of follicle stimulating hormone (FSH) and luteinizing hormone (LH) in asynchronous ovarian follicle development in daily egg production, we cloned cDNAs of the follicle-stimulating hormone receptor (FSHR) and luteinizing hormone receptor (LHR) in the bambooleaf wrasse (Pseudolabrus sieboldi), which exhibits clear diurnal spawning rhythms over 1month. In addition, different developmental stages of ovarian follicles were isolated from whole ovaries at various daily time points on 1day in the spawning season, and mRNA expression levels of FSHR and LHR were analyzed. Sequence analysis showed distinct differences in the number of putative leucine-rich repeats at the extracellular domain between FSHR and LHR, suggesting a difference in ligand-specificity. Real-time PCR analyses revealed that FSHR mRNA was highly expressed in early yolk-stage follicles but decreased at the end of vitellogenesis. In contrast, the expression of LHR mRNA was maintained at low levels in vitellogenic stage follicles but markedly elevated at the end of the vitellogenic and early migratory nucleus stages, thereafter markedly dropping in the late migratory nucleus stage. The present results suggest that co-regulation of vitellogenesis and oocyte maturation in one ovary is controlled by the stage-distinctive expression levels of FSHR and LHR mRNA in ovarian follicles, and daily switching of sensitivity from FSH to LH is required for daily egg production. [Copyright &y& Elsevier]

Abstract: In zebrafish, ovulated oocytes are loaded with maternal estrogen receptor 2a (esr2a) mRNA which is spread as granular and filamentous structures throughout the central ooplasm and is promptly relocated inside the blastodisc area at the 1-cell stage (0.2h post-fertilization, hpf), as shown by in situ hybridization. This transcript is available for translation until its sharp decline from 4 to 8 hpf, being replaced by low levels of zygotic esr2a mRNA mainly localized in the head region and around the yolk sac from 24 hpf until hatching at 48 hpf. To test the functional role of the maternal esr2a mRNA, 1- or 2-cell embryos were injected with 10.3ng each of morpholino (MO) to knockdown translation (MO2-esr2a) of both maternal and zygotic esr2a transcripts, with a missplicing MO (MO3-esr2a) to effectively block post-transcriptionally the zygotic transcript alone, and with a non-specific MO-control. Treatment with MO2-esr2a increased apoptosis in embryos, especially in the brain, and caused severe malformations in 63% of 1–5 dpf larvae, as compared to 10–11% in those treated with MO3-esr2a and MO-control. Defects included body growth delay with curved shape, persistent yolk sac with reduced sub-intestinal veins and swollen yolk extension, abnormal brain and splanchnocranium development, smaller eyes and otic vesicles, pericardial oedema, uninflated swim bladder and rudimentary caudal fin with aberrant circular swimming. Affected larvae could survive for only 12–14days. The MO2-esr2a phenotype was rescued with co-injection of 30pg/embryo of mutated zebrafish esr2a mRNA encoding the full length of Esr2a, but containing eight silent mutations in the region recognised by MO2-esr2a. A lower dosage (15pg) failed to recover mortality and abnormality. Raising the dosage to 60 and 90pg increased abnormality, but not mortality, whereas with 120pg both mortality and abnormality worsened, indicating a strict quantitative requirement of Esr2a. Co-injection of an anti-p53 MO failed to rescue the MO2-esr2a phenotype, eliminating the possibility of off-target effects. Pangenomic microarray analysis revealed that 240 and 219 significantly expressed transcripts were up- and down-regulated, respectively, by maternal Esr2a protein deficiency in 8-hpf MO2-esr2a embryos. Also at 48 hpf, 162 and 120 presumably zygotic transcripts were up- and down-regulated, respectively, but only 18 were in common with each of the 8-hpf sets. In total, the transcripts from 705 genes were affected by Esr2a knockdown. These findings suggest the involvement of maternal esr2a mRNA, presumably transactivated by maternal 17β-estradiol stored in the oocyte from enveloping granulosa cells, in the epigenetic programming of zebrafish development. [Copyright &y& Elsevier]

Abstract: Cypermethrin (CYP) is widely used for control of indoor and field pests. As a result, CYP is one of the most common contaminants in freshwater aquatic systems. In the present study, we investigated the effects of CYP exposure on the induction of apoptosis and immunotoxicity in zebrafish during the embryo developmental stage. The mRNA levels of some key genes including P53, Puma, Bax, Apaf1, Cas9 and Cas3 on the mitochondrial pathway of cell apoptosis were significantly up-regulated at the concentration of 3 and 10 μg/l CYP. Correspondingly, the activities of Cas3 and Cas9 increased significantly after exposure to 3 or 10 μg/l CYP. In addition, the mRNA levels of iNOS and the total content of NO were also up-regulated significantly after CYP exposure. Moreover, it was also observed that the mRNA levels of IFN, CXCL-Clc, CC-chem and C3, which are closely related to the innate immune system, were affected in newly hatched zebrafish when exposed to 3 and 10 μg/l CYP, exhibiting CYP’s prominent impacts on the innate immune system of zebrafish. Taken together, our results suggest that CYP has the potential to induce cell apoptosis and cause innate immune system disruption in zebrafish during the embryo stage. The information presented in this study will help elucidate the mechanism of CYP-induced toxicity in fish. [Copyright &y& Elsevier]