Serotonergic modulation of vigilance states in zebrafish and mice.

Vigilance refers to being alertly watchful or paying sustained attention to avoid potential threats. Animals in vigilance states reduce locomotion and have an enhanced sensitivity to aversive stimuli so as to react quickly to dangers. Here we report that an unconventional 5-HT driven mechanism operating at neural circuit level which shapes the internal state underlying vigilance behavior in zebrafish and male mice. The neural signature of internal vigilance state was characterized by persistent low-frequency high-amplitude neuronal synchrony in zebrafish dorsal pallium and mice prefrontal cortex. The neuronal synchronization underlying vigilance was dependent on intense release of 5-HT induced by persistent activation of either DRN 5-HT neuron or local 5-HT axon terminals in related brain regions via activation of 5-HTR7. Thus, we identify a mechanism of vigilance behavior across species that illustrates the interplay between neuromodulators and neural circuits necessary to shape behavior states.

De novo establishment of circuit modules restores locomotion after spinal cord injury in adult zebrafish.

Mechanisms underlying spontaneous locomotor recovery after spinal cord injury (SCI) remain unclear. Using adult zebrafish with complete SCI, we show that V2a interneurons regrow their axon to bridge the lesioned spinal segments in a subclass-specific and chronological order. Early after SCI, reestablishment of a unitary high-rhythm locomotor circuit is driven merely by axon-regrown fast V2a interneurons. Later, the reestablished intraspinal de novo circuit is organized into a modular design by axon-regrown fast and slow V2a interneurons rostral to the lesion, selectively driving caudal fast V2a/motor neurons and slow V2a/motor neurons, respectively. This orderly circuitry reestablishment determines the stepwise restoration of locomotor repertoire and recapitulates developmental processes. This progress can be interrupted by ablation of calretinin, a fast module-related protein, and accelerated by physical training. These findings suggest that promotion of axon regrowth of propriospinal V2a interneurons and establishment of de novo intraspinal circuits underpin the effectiveness of physical training in patients after SCI.

miR-731 modulates the zebrafish heart morphogenesis via targeting Calcineurin/Nfatc3a pathway.

BACKGROUND: Zebrafish miR-731 is orthologous of human miR-425, which has been demonstrated to have cardio-protective roles by a variety of mechanisms. The miR-731 morphants show pericardium enlargement, and many DEGs (differentially expressed genes) are enriched in 'Cardiac muscle contraction' and 'Calcium signaling pathway', implying that miR-731 plays a potential role in heart function and development. However，the in vivo physiological role of miR-731 in the heart needs to be fully defined. METHODS: Zebrafish miR-731 morphants were generated by morpholino knockdown, and miR-731 knockout zebrafish was generated by CRISRP/Cas9. We observed cardiac morphogenesis based on whole-mount in situ hybridization. Furthermore, RNA-seq and qRT-PCR were used to elucidate the molecular mechanism and analyze the gene expression. Double luciferase verification and Western blot were used to verify the target gene. RESULTS: The depletion of miR-731 in zebrafish embryos caused the deficiency of cardiac development and function, which was associated with reduced heart rate, ventricular enlargement and heart looping disorder. In addition, mechanistic study demonstrated that Calcineurin/Nfatc3a signaling involved in miR-731 depletion induced abnormal cardiac function and developmental defects. CONCLUSION: MiR-731 regulates cardiac function and morphogenesis through Calcineurin/Nfatc3a signaling. GENERAL SIGNIFICANCE: Our studies highlight the potential importance of miR-731 in cardiac development.

An injury-induced serotonergic neuron subpopulation contributes to axon regrowth and function restoration after spinal cord injury in zebrafish.

Spinal cord injury (SCI) interrupts long-projecting descending spinal neurons and disrupts the spinal central pattern generator (CPG) that controls locomotion. The intrinsic mechanisms underlying re-wiring of spinal neural circuits and recovery of locomotion after SCI are unclear. Zebrafish shows axonal regeneration and functional recovery after SCI making it a robust model to study mechanisms of regeneration. Here, we use a two-cut SCI model to investigate whether recovery of locomotion can occur independently of supraspinal connections. Using this injury model, we show that injury induces the localization of a specialized group of intraspinal serotonergic neurons (ISNs), with distinctive molecular and cellular properties, at the injury site. This subpopulation of ISNs have hyperactive terminal varicosities constantly releasing serotonin activating 5-HT1B receptors, resulting in axonal regrowth of spinal interneurons. Axon regrowth of excitatory interneurons is more pronounced compared to inhibitory interneurons. Knock-out of htr1b prevents axon regrowth of spinal excitatory interneurons, negatively affecting coordination of rostral-caudal body movements and restoration of locomotor function. On the other hand, treatment with 5-HT1B receptor agonizts promotes functional recovery following SCI. In summary, our data show an intraspinal mechanism where a subpopulation of ISNs stimulates axonal regrowth resulting in improved recovery of locomotor functions following SCI in zebrafish.

A specialized spinal circuit for command amplification and directionality during escape behavior.

In vertebrates, action selection often involves higher cognition entailing an evaluative process. However, urgent tasks, such as defensive escape, require an immediate implementation of the directionality of escape trajectory, necessitating local circuits. Here we reveal a specialized spinal circuit for the execution of escape direction in adult zebrafish. A central component of this circuit is a unique class of segmentally repeating cholinergic V2a interneurons expressing the transcription factor Chx10. These interneurons amplify brainstem-initiated escape commands and rapidly deliver the excitation via a feedforward circuit to all fast motor neurons and commissural interneurons to direct the escape maneuver. The information transfer within this circuit relies on fast and reliable axo-axonic synaptic connections, bypassing soma and dendrites. Unilateral ablation of cholinergic V2a interneurons eliminated escape command propagation. Thus, in vertebrates, local spinal circuits can implement directionality of urgent motor actions vital for survival.

A neuronal circuit that generates the temporal motor sequence for the defensive response in zebrafish larvae.

Animals use a precisely timed motor sequence to escape predators. This requires the nervous system to coordinate several motor behaviors and execute them in a temporal and smooth manner. We here describe a neuronal circuit that faithfully generates a defensive motor sequence in zebrafish larvae. The temporally specific defensive motor sequence consists of an initial escape and a subsequent swim behavior and can be initiated by unilateral stimulation of a single Mauthner cell (M-cell). The smooth transition from escape behavior to swim behavior is achieved by activating a neuronal chain circuit, which permits an M-cell to drive descending neurons in bilateral nucleus of medial longitudinal fascicle (nMLF) via activation of an intermediate excitatory circuit formed by interconnected hindbrain cranial relay neurons. The sequential activation of M-cells and neurons in bilateral nMLF via activation of hindbrain cranial relay neurons ensures the smooth execution of escape and swim behaviors in a timely manner. We propose an existence of a serial model that executes a temporal motor sequence involving three different brain regions that initiates the escape behavior and triggers a subsequent swim. This model has general implications regarding the neural control of complex motor sequences.

Zebrafish miR-462-731 is required for digestive organ development.

MicroRNAs (miRNAs), as important regulators of post-transcriptional gene expression, play important roles in the occurrence and function of organs. In this study, morpholino (MO) knockdown of miR-462/miR-731 was used to investigate the potential mechanisms of the miR-462-731 cluster during zebrafish liver development. The results showed significant reduction of digestive organs, especially liver and exocrine pancreas after the miR-462/miR-731 knockdown, and those phenotypes could be partially rescued by corresponding miRNA duplex. Acinar cells of the exocrine pancreas were also severely affected with pancreatic insufficiency. In particular, knockdown of miR-462 caused pancreas morphogenesis abnormity with specific bilateral exocrine pancreas. Additionally, it was found that miR-731 played a role in liver and exocrine pancreas development by directly targeting dkk3b, instead of the down-regulation of Wnt/ß-catenin signaling. These findings contribute significantly to our understanding of molecular mechanisms of miR-462-731 cluster in development of digestive organs.

Zebrafish miR-462-731 regulates hematopoietic specification and pu.1-dependent primitive myelopoiesis.

MicroRNAs (miRNAs) play significant roles in both embryonic hematopoiesis and hematological malignancy. Zebrafish miR-462-731 cluster is orthologous of miR-191-425 in human which regulates proliferation and tumorigenesis. In our previous work, miR-462-731 was found highly and ubiquitously expressed during early embryogenesis. In this study, by loss-of-function analysis (morpholino knockdown combined with CRISRP/Cas9 knockout) and mRNA profiling, we suggest that miR-462-731 is required for normal embryonic development by regulating cell survival. We found that loss of miR-462/miR-731 caused a remarkable decrease in the number of erythroid cells as well as an ectopic myeloid cell expansion at 48 hpf, suggesting a skewing of myeloid-erythroid lineage differentiation. Mechanistically, miR-462-731 provides an instructive input for pu.1-dependent primitive myelopoiesis through regulating etsrp/scl signaling combined with a novel pu.1/miR-462-731 feedback loop. On the other hand, morpholino (MO) knockdown of miR-462/miR-731 resulted in an expansion of posterior blood islands at 24 hpf, which is a mild ventralization phenotype resulted from elevation of BMP signaling. Rescue experiments with both BMP type I receptor inhibitor dorsomorphin and alk8 MO indicate that miR-462-731 acts upstream of alk8 within the BMP/Smad signaling pathway and functions as a novel endogenous BMP antagonist. Besides, an impairment of angiogenesis was observed in miR-462/miR-731 morphants. The specification of arteries and veins was also perturbed, as characterized by the irregular patterning of efnb2a and flt4 expression. Our study unveils a previously unrecognized role of miR-462-731 in BMP/Smad signaling mediated hematopoietic specification of mesodermal progenitors and demonstrates a miR-462-731 mediated regulatory mechanism driving primitive myelopoiesis in the ALPM. We also show a requirement for miR-462-731 in regulating arterial-venous specification and definitive hematopoietic stem cell (HSC) production. The current findings might provide further insights into the molecular mechanistic basis of miRNA regulation of embryonic hematopoiesis and hematological malignancy.

The molecular characterization, expression pattern and alternative initiation of Megalobrama amblycephala Hif prolyl hydroxylase Phd1.

HIF prolyl hydroxylase 1 (PHD1) functions in prolyl hydroxylation on mammal hypoxia-inducible factors (HIF), important transcription factors involved in hypoxia, however the roles of Phd1 in fish remain unclear. In this study, the full-length cDNA and promoter sequences of blunt snout bream (Megalobrama amblycephala) phd1 gene were isolated by a modified RACE strategy. The phd1 cDNA was 2672 bp for encoding 481 amino acid residues. In silico assays indicated that phd1 had 5 exons, and a 348 bp CpG island in the exon1, and several transcription factor binding sites (CAAT box, HRE, ARNT, FOX, etc) were also found on the promoter. The quantitative real-time PCR results suggested that phd1 mRNA was constitutively expressed in all detected tissues, and higher in the blood, brain and heart in normoxia, but significantly decreased after hypoxia in all detected tissues except for gill. Western blot assays indicated that two Phd1 isoforms were generated by alternative translation initiation. Moreover, these two isoforms were both localized in the nucleus, therein only the senior isoform promoted cell proliferation. Taken together, the present study firstly describes the functions of M. amblycephala two Phd1 isoforms in hypoxia and cell proliferation.

The zebrafish miR-125c is induced under hypoxic stress via hypoxia-inducible factor 1α and functions in cellular adaptations and embryogenesis.

Hypoxia is a unique environmental stress. Hypoxia inducible factor-lα (HIF-lα) is a major transcriptional regulator of cellular adaptations to hypoxic stress. MicroRNAs (miRNAs) as posttranscriptional gene expression regulators occupy a crucial role in cell survival under low-oxygen environment. Previous evidences suggested that miR-125c is involved in hypoxia adaptation, but its precise biological roles and the regulatory mechanism underlying hypoxic responses remain unknown. The present study showed that zebrafish miR-125c is upregulated by hypoxia in a Hif-lα-mediated manner in vitro and in vivo. Dual-luciferase assay revealed that cdc25a is a novel target of miR-125c. An inverse correlation between miR-125c and cdc25a was further confirmed in vivo, suggesting miR-125c as a crucial physiological inhibitor of cdc25a which responds to cellular hypoxia. Overexpression of miR-125c suppressed cell proliferation, led to cell cycle arrest at the G1 phase in ZF4 cells and induced apoptotic responses during embryo development. More importantly, miR-125c overexpression resulted in severe malformation and reduction of motility during zebrafish embryonic development. Taken together, we conclude that miR-125c plays a pivotal role in cellular adaptations to hypoxic stress at least in part through the Hif-1α/miR-125c/cdc25a signaling and has great impact on zebrafish early embryonic development.

Effect of Low Temperature on Globin Expression, Respiratory Metabolic Enzyme Activities, and Gill Structure of Litopenaeus vannamei.

Low temperature frequently influences growth, development, and even survival of aquatic animals. In the present study, physiological and molecular responses to low temperature in Litopenaeus vannamei were investigated. The cDNA sequences of two oxygen-carrying proteins, cytoglobin (Cygb) and neuroglobin (Ngb), were isolated. Protein structure analysis revealed that both proteins share a globin superfamily domain. Real-time PCR analysis indicated that Cygb and Ngb mRNA levels gradually increased during decrease in temperatures from 25 to 15°C and then decreased at 10°C in muscle, brain, stomach, and heart, except for a continuing increase in gills, whereas they showed a different expression trend in the hepatopancreas. Hemocyanin concentration gradually reduced as the temperature decreased. Moreover, the activities of respiratory metabolic enzymes including lactate dehydrogenase (LDH) and succinate dehydrogenase (SDH) were measured, and it was found that LDH activity gradually increased while SDH activity decreased after low-temperature treatment. Finally, damage to gill structure at low temperature was also observed, and this intensified with further decrease in temperature. Taken together, these results show that low temperature has an adverse influence in L. vannamei, which contributes to systematic understanding of the adaptation mechanisms of shrimp at low temperature.

Effects of Acute Hypoxia and Reoxygenation on Physiological and Immune Responses and Redox Balance of Wuchang Bream (Megalobrama amblycephala Yih, 1955).

To study Megalobrama amblycephala adaption to water hypoxia, the changes in physiological levels, innate immune responses, redox balance of M.amblycephala during hypoxia were investigated in the present study. When M. amblycephala were exposed to different dissolved oxygen (DO) including control (DO: 5.5 mg/L) and acute hypoxia (DO: 3.5 and 1.0 mg/L, respectively), hemoglobin (Hb), methemoglobin (MetHb), glucose, Na+, succinatedehydrogenase (SDH), lactate, interferon alpha (IFNα), and lysozyme (LYZ), except hepatic glycogen and albumin gradually increased with the decrease of DO level. When M. amblycephala were exposed to different hypoxia time including 0.5 and 6 h (DO: 3.5 mg/L), and then reoxygenation for 24 h after 6 h hypoxia, Hb, MetHb, glucose, lactate, and IFNα, except Na+, SDH, hepatic glycogen, albumin, and LYZ increased with the extension of hypoxia time, while the above investigated indexes (except albumin, IFNα, and LYZ) decreased after reoxygenation. On the other hand, the liver SOD, CAT, hydrogen peroxide (H2O2), and total ROS were all remained at lower levels under hypoxia stress. Finally, Hif-1α protein in the liver, spleen, and gill were increased with the decrease of oxygen concentration and prolongation of hypoxia time. Interestingly, one Hsp70 isoforms mediated by internal ribozyme entry site (IRES) named junior Hsp70 was only detected in liver, spleen and gill. Taken together, these results suggest that hypoxia affects M. amblycephala physiology and reduces liver oxidative stress. Hypoxia-reoxygenation stimulates M. amblycephala immune parameter expressions, while Hsp70 response to hypoxia is tissue-specific.

Involvement of the miR-462/731 cluster in hypoxia response in Megalobrama amblycephala.

MicroRNAs (miRNAs) are non-coding small RNAs showing both evolutionarily conserved and unique features and are involved in nearly all biological processes. In the present study, the role played by miR-462/731 cluster miRNAs in hypoxia response in Megalobrama amblycephala, an important freshwater fish, was investigated. The M. amblycephala miR-462/731 cluster locus and their 5' flanking sequences were sequenced and analyzed. In M. amblycephala and other teleost fish species, the mature sequences of miR-462 and miR-731 were identical and hypoxia-responsive elements (HREs) were identified upstream of the miR-462/731 loci. The two miRNAs were significantly induced in the liver, spleen, gill, muscle, and brain after hypoxia treatment. The expression of both miRNAs was also upregulated in cells that received treatment which mimicked hypoxia. Furthermore, reporter assay revealed that M. amblycephala HREs can be activated by hypoxia. Taken together, the 462/731 cluster may play a role in the regulation of the hypoxia response in M. amblycephala.

Zebrafish let-7b acts downstream of hypoxia-inducible factor-1α to assist in hypoxia-mediated cell proliferation and cell cycle regulation.

AIMS: Hypoxia-inducible factor-1α (HIF-1α) is a transcriptional regulator of cellular responses to hypoxic stress. MicroRNAs (miRNAs) play an essential role in hypoxia-mediated cellular responses. Previous studies have identified some let-7 family members as hypoxia-regulated miRNAs (HRMs). In the present study, we aimed to investigate whether zebrafish let-7b/7f contribute cellular hypoxic response in a Hif-1α-dependent manner. MAIN METHODS: Stable suppression of zebrafish hif-1α was achieved by microinjection of an optimized short-hairpin RNA (shRNA) expression vector. Next-generation sequencing was conducted to characterize miRNA and mRNA expression profiles. MiRNA promoter analysis and target detection was performed by dual-luciferase assay. Quantitative real-time PCR (qRT-PCR) and western blot were used to determine the expression of let-7b/7f, Hif-1α and Foxh1. Proliferation of ZF4 cells was examined using Cell Counting Kit-8 (CCK-8) and cell cycle progression was analyzed by flow cytometry assay. KEY FINDINGS: Correlation between 7 miRNAs and 76 putative targets was identified based on integrated analysis of miRNA-mRNA profiles. Let-7b and let-7f were further considered as potential HRMs, with let-7b further validated as Hif-1α up-regulated. In addition, Forkhead-box H1 (Foxh1) was confirmed as a bona fide downstream target of let-7b. Furthermore, overexpression of both let-7b and let-7f repressed cell proliferation through blocking cell cycle progression of the G1-S transition. SIGNIFICANCE: Our findings for the first time suggest zebrafish let-7b acts downstream of Hif-1α to assist in hypoxia-mediated cell proliferation and cell cycle regulation at least in part through the downregulation of foxh1. We also identified 4 novel potential HIF-1α-regulated miRNAs in zebrafish.

The zebrafish miR-462/miR-731 cluster is induced under hypoxic stress via hypoxia-inducible factor 1α and functions in cellular adaptations.

Hypoxia, a unique and essential environmental stress, evokes highly coordinated cellular responses, and hypoxia-inducible factor (HIF) 1 in the hypoxia signaling pathway, an evolutionarily conserved cellular signaling pathway, acts as a master regulator of the transcriptional response to hypoxic stress. MicroRNAs (miRNAs), a major class of posttranscriptional gene expression regulators, also play pivotal roles in orchestrating hypoxia-mediated cellular adaptations. Here, global miRNA expression profiling and quantitative real-time PCR indicated that the up-regulation of the miR-462/miR-731 cluster in zebrafish larvae is induced by hypoxia. It was further validated that miR-462 and miR-731 are up-regulated in a Hif-1α-mediated manner under hypoxia and specifically target ddx5 and ppm1da, respectively. Overexpression of miR-462 and miR-731 represses cell proliferation through blocking cell cycle progress of DNA replication, and induces apoptosis. In situ detection revealed that the miR-462/miR-731 cluster is highly expressed in a consistent and ubiquitous manner throughout the early developmental stages. Additionally, the transcripts become restricted to the notochord, pharyngeal arch, liver, and gut regions from postfertilization d 3 to 5. These data highlight a previously unidentified role of the miR-462/miR-731 cluster as a crucial signaling mediator for hypoxia-mediated cellular adaptations and provide some insights into the potential function of the cluster during embryonic development.

Study on the immune response to recombinant Hsp70 protein from Megalobrama amblycephala.

The expression of heat shock protein 70 (Hsp70) is induced in response to many factors including high temperature, infection, metal pollutants and toxic chemicals. In this study, Megalobrama amblycephala HSP70 promoter was cloned, and characteristic heat shock elements (HSEs) were identified in the promoter region. The recombinant M. amblycephala Hsp70 protein (rMaHsp70) was expressed and purified from Escherichia coli BL21 (DE3). To evaluate in vivo immune response of rMaHsp70, we administered intraperitoneal (IP) injection, and demonstrated that rMaHsp70 stimulated M. amblycephala immune activity by inducing the expression of HSP70, HIF-1α, HSC70, CXCR4b, TNF-α and IL-1ß mRNAs in liver, headkidney, spleen and gill, as well as SOD, glutathione, lysozyme and interferon alpha proteins in serum and liver. The effect of rMaHsp70 as adjuvant against Aeromonas hydrophila was assessed by injecting a mixed vaccine of rMaHsp70 and A. hydrophila (A. hydrophila/Hsp70) into M. amblycephala, and the relative percent survival (RPS) in the A. hydrophila/Hsp70 group was 75% compared to 50% in the A. hydrophila/PBS group. Furthermore, rMaHsp70 also promoted the proliferation and suppressed apoptosis in M. amblycephala fin cells (MAF) in a dose-dependent manner. Taken together, these results suggest that rMaHsp70 can induce organic immune response and improve environmental tolerance.

Growth differentiation factor 9 of Megalobrama amblycephala: molecular characterization and expression analysis during the development of early embryos and growing ovaries.

Growth differentiation factor 9 (GDF9) is a member of the transforming growth factorß superfamily and plays an essential role during follicle maturation in mammals. In the present study, the full-length complementary DNA (cDNA) of gdf9 was obtained from Megalobrama amblycephala. The cDNA sequence is 2,061 bp in length with an open reading frame of 1,287 bp encoding 428 amino acid residues. The deduced amino acid sequence shared identities of about 42-86 % with the homologues of other vertebrates. During the early development of embryos, the gdf9 mRNA was detected in zygote with significantly high level and declined sharply by 47 and 87 % at 4 hours post-fertilization (hpf) and 6 hpf and even to an undetectable level through advancing stages. Expression analysis based on quantitative real-time PCR revealed that gdf9 mRNA was mainly expressed in ovary, but much lower levels were also found in some nonovarian tissues. Within the follicle, gdf9 mRNA was localized both in the oocytes and the follicle layer cells by in situ hybridization. During the ovarian cycle, gdf9 mRNA significantly decreased after the previtellogenic stage and became to increase again after the fully grown stage. The results imply that Gdf9 may play critical physiological functions in M. amblycephala early embryonic development and reproduction.

Megalobrama amblycephala cardiac troponin T variants: molecular cloning, expression and response to nitrite.

Cardiac troponin T (TNNT2), as a member of troponin superfamily, plays important roles during early cardiogenesis, and contraction and relaxation of myocardial cells. In this study, two alternatively spliced variants of Megalobrama amblycephala TNNT2 were identified showing a difference of 19 amino acids in the N-terminal hypervariable region. The longer cDNA (TNNT2-1) was 1,118 bp, encoding 284 amino acid residues, contained conserved central tropomyosin-binding region, cardiac specific signal and C-terminal segments except the N-terminal hypervariable region. The TNNT2 transcripts first appeared at 16 hours post-fertilization (hpf) peaking at 28 hpf (onset of heartbeat). In addition, strong expression of TNNT2 was found in the cardiac muscle. After nitrite exposure, the increased TNNT2 expression levels in the heart indicated that nitrite might induce cardiac injury. Results of semi-quantitative RT-PCR indicated that the two alternatively spliced variants existed in early development stages since their first appearance at 16 hpf and heart, spleen, headkiney of M. amblycephala. The shorter transcript (TNNT2-2) was proved to be dominant in the embryos and heart of M. amblycephala, furthermore, the increase of TNNT2 expression level in the heart after nitrite exposure was mainly caused by TNNT2-2.