[A comparison of the knockout efficiencies of two codon-optimized Cas9 coding sequences in zebrafish embryos].

Recent years have witnessed the rapid development of the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein(CRISPR/Cas9)system. In order to realize gene knockout with high efficiency and specificity in zebrafish, several labs have synthesized distinct Cas9 cDNA sequences which were cloned into different vectors. In this study, we chose two commonly used zebrafish-codon-optimized Cas9 coding sequences (zCas9_bz, zCas9_wc) from two different labs, and utilized them to knockout seven genes in zebrafish embryos, including the exogenous egfp and six endogenous genes (chd, hbegfa, th, eef1a1b, tyr and tcf7l1a). We compared the knockout efficiencies resulting from the two zCas9 coding sequences, by direct sequencing of PCR products, colony sequencing and phenotypic analysis. The results showed that the knockout efficiency of zCas9_wc was higher than that of zCas9_bz in all conditions.

Transcriptional factors smad1 and smad9 act redundantly to mediate zebrafish ventral specification downstream of smad5.

Bone morphogenetic proteins (BMPs) are multifunctional growth factors that play crucial roles during embryonic development and cell fate determination. Nuclear transduction of BMP signals requires the receptor type Smad proteins, Smad1, Smad5, and Smad9. However, how these Smad proteins cooperate in vivo to regulate various developmental processes is largely unknown. In zebrafish, it was widely believed that the maternally expressed smad5 is essential for dorso-ventral (DV) patterning, and the zygotically transcribed smad1 is not required for normal DV axis establishment. In the present study, we have identified zygotically expressed smad9, which cooperates with smad1 downstream of smad5, to mediate zebrafish early DV patterning in a functional redundant manner. Although knockdown of smad1 or smad9 alone does not lead to visible dorsalization, double knockdown strongly dorsalizes zebrafish embryos, which cannot be efficiently rescued by smad5 overexpression, whereas the dorsalization induced by smad5 knockdown can be fully rescued by overexpression of smad1 or smad9. We have further revealed that the transcription initiations of smad1 and smad9 are repressed by each other, that they are direct transcriptional targets of Smad5, and that smad9, like smad1, is required for myelopoiesis. In conclusion, our study uncovers that smad1 and smad9 act redundantly to each other downstream of smad5 to mediate ventral specification and to regulate embryonic myelopoiesis.

Cross-species cloning: influence of cytoplasmic factors on development.

It is widely accepted that the crosstalk between naive nucleus and maternal factors deposited in the egg cytoplasm before zygotic genome activation is crucial for early development. This crosstalk may also exert some influence on later development. It is interesting to clarify the relative roles of the zygotic genome and the cytoplasmic factors in development. Cross-species nuclear transfer (NT) between two distantly related species provides a unique system to study the relative role and crosstalk between egg cytoplasm and zygotic nucleus in development. In this review, we will summarize the recent progress of cross-species NT, with emphasis on the cross-species NT in fish and the influence of cytoplasmic factors on development. Finally, we conclude that the developmental process and its evolution should be interpreted in a systemic way, rather than in a way that solely focuses on the role of the nuclear genome.

[A brief history of zebrafish research--toward biomedicine].

Ever since George Streisinger pioneered his research using zebrafish (Danio rerio), at the University of Oregon in 1972, the zebrafish not only has become a unique animal model in basic research, due to its fine embryonic and (molecular) genetics technique/tool developed globally, but it is also a favorite model of choice in the biomedical research, i.e., used for establishing human disease models and discovering lead drug/small chemical in the past decade. In this review, we will briefly describe the history of zebrafish research, emphasizing the well-recognized milestones, and stress how the models of leukemia, melanoma, immunity/infectious diseases and neuronal defects/neuro-degeneration diseases have been established and how pharmaceutical industry and research scientists make use of zebrafish to obtain potential therapeutic drugs. We believe that this direction of zebrafish research will lead to a better understanding of some nasty human diseases and their pathogenic mechanisms, and eventually help to achieve a better health of human beings.

[Nuclear transfer and reprogramming in fish].

As an important sub-field in the study of animal cloning, fish nuclear transfer was first established in the early 1960s by Chinese embryologists. Due to its advantages, zebrafish has become a unique animal model to study the mystery of reprogramming in nuclear transfer. This article summarizes the history and current situation in fish nuclear transfer technology and discusses the factors that may influence the development of the cloned embryos. A comprehensive understand-ing of the mechanism for epigenetic modification following nuclear transfer, such as genomic DNA methylation and histone acetylation and/or methylation, will likely increase the success rate and eventually lead to the future freedom of cloning technique.

[Cloning and promoter analysis of gonadotrophin-releasing hormone â ¡ in the orange-spotted grouper (Epinephelus coioides)].

Gonadotrophin-releasing hormone (GnRH) is a key regulator of reproduction in all vertebrates. We first cloned the cDNA and genomic DNA sequences coding for GnRHⅡ gene in the orange-spotted grouper (Epinephelis coioides), an economically important marine fish, and then cloned its promoter sequence. The region responsible for the cell-specific expression of GnRHⅡ was located between -2005 bp to -956 bp from the translation start site. GnRHⅡ promoter driven EGFP expression in transgenic zebrafish showed that GnRHⅡ-positive neurons were primarily located in the midbrain and in the eyes. Our results provide an improved understanding of the regulatory mechanism and function of GnRHⅡ of E. coioides.

[TALE nuclease engineering and targeted genome modification].

Artificial designer nucleases targeting specific DNA sequences open up a new field for reverse genetics study. The rapid development of engineered endonucleases (EENs) enables targeted genome modification theoretically in any species. The construction of transcription activator-like effector nucleases (TALENs) is simpler with higher specificity and less toxicity than zinc-finger nucleases (ZFNs). Here, we summarized the recent progresses and prospects of TALEN technology, with an emphasis on its structure, function, and construction strategies, as well as a collection of species and genes that have been successfully modified by TALENs, especially the application in zebrafish.

[Detection of dynamic expression of microRNAs in vivo using a dual-fluorescence reporter system/miRNA Tracer in zebrafish].

microRNAs (miRNAs) are short noncoding RNAs that have been found in a wide variety of organisms and many have been shown to play essential roles by regulating the stability and translation of target messenger RNAs (mRNAs) in animals and plants. Temporal and spatial expression is critical for the regulatory function of miRNAs. To analyze the dynamic expression of particular miRNA in vivo, we constructed a dual-fluorescence reporter system based on Tol2 transposon, in which two reporter genes, enhanced green fluorescent protein (eGFP) and monomeric red fluorescent protein 1 (mRFP1), were driven by the heat shock promoter (hsp) from zebrafish hsp70 gene in an opposite orientation. To sense the existence of a particular miRNA, the complementary DNA sequence of the corresponding miRNA was inserted into the 3'-UTR region of one of the two reporter genes. By injecting the corresponding plasmid DNA into zebrafish embryos, we were able to monitor the abundance and dynamics of miRNA miR-206 in live embryos. To further evaluate this method, we made a collection of transgenic zebrafish with stable integration of dual-fluorescence reporter plasmids targeting different miRNAs, including miR-206 and miR-219. Our results showed that this dual-fluorescence reporter system, which is also called miRNA Tracer, could faithfully monitor the appearance and disappearance of target miRNAs in defined cell lineages during zebrafish development in these fish lines. Our dual-fluorescence reporter/Tracer system provides an important tool for further in-depth studies on miRNAs in zebrafish.

Activation of GH signaling and GH-independent stimulation of growth in zebrafish by introduction of a constitutively activated GHR construct.

Growth hormone (GH) gene transfer can markedly increase growth in transgenic fish. In the present study we have developed a transcriptional assay to evaluate GH-signal activation (GHSA) in zebrafish embryos. By analyzing the transcription of c-fos and igf1, and the promoter activity of spi2.1, in zebrafish embryos injected with different constructs, we found that overexpression of either GH or growth hormone receptor (GHR) resulted in GHSA, while a synergetic overexpression of GH and GHR gave greater activation. Conversely, overexpression of a C-terminal truncated dominant-negative GHR (ΔC-GHR) efficiently blocked GHSA epistatic to GH overexpression, demonstrating the requirement for a full GHR homodimer in signaling. In view of the importance of signal-competent GHR dimerization by extracellular GH, we introduced into zebrafish embryos a constitutively activated GHR (CA-GHR) construct, which protein products constitutively dimerize the GHR productively by Jun-zippers to activate downstream signaling in vitro. Importantly, overexpression of CA-GHR led to markedly higher level of GHSA than the synergetic overexpression of GH and GHR. CA-GHR transgenic zebrafish were then studied in a growth trial. The transgenic zebrafish showed higher growth rate than the control fish, which was not achievable by GH transgenesis in these zebrafish. Our study demonstrates GH-independent growth by CA-GHR in vivo which bypasses normal IGF-1 feedback control of GH secretion. This provides a novel means of producing growth enhanced transgenic animals based on molecular protein design.

Comparative expression of zebrafish lats1 and lats2 and their implication in gastrulation movements.

Large tumor suppressor (Lats) is a Ser/Thr kinase, and it presents an important function in tumor suppression. lats was originally identified in Drosophila and recently in mammals. In mammals, it contains two homologues, lats1 and lats2. In the present study, lats1 and lats2 were characterized from zebrafish (Danio rerio), which is the first report of lats in a nonmammalian vertebrate. The primary structure, genomic organization, and phylogenesis of lats from different species were studied, and the results suggest that lats1 is the direct descendant of invertebrate lats, whereas lats2 is formed by genome duplication. In zebrafish, both lats genes are maternally expressed, while they show distinctly different expression profiles during gastrulation. lats1 is almost ubiquitously expressed through development, and lats2 is more prominently expressed in the non-neural ectoderm region of zebrafish gastrula. Most intriguingly, as revealed by cell tracing and gene expression analysis, morpholino-mediated knockdown of either lats1 or lats2 led to obvious defects of cell migration in gastrulation, indicating the functional significance of lats in gastrulation movements.

Identification of a novel gene K23 over-expressed in fish cross-subfamily cloned embryos.

A novel gene-K23, differentially expressed in cross-subfamily cloned embryos, was isolated by RACE-PCR technique. It had 2580 base pairs (bp) in length, with a 1,425 bp open reading frame (ORF) encoding a putative protein of 474 amino acids (aa). Bioinformatic analysis indicated that K23 had 22 phosphorylation sites, but it had no signal peptides. Developmental expression analysis in zebrafish showed that K23 transcripts were maternally expressed in ovum and the amount of K23 transcripts increased gradually from zygote to pharyngula period. Subcellular localization analysis revealed that K23 protein was homogeneously distributed both in nuclei and cytoplasm. Taken together, our findings indicate that K23 gene is a novel gene differentially expressed in fish cross-subfamily cloned embryos.

[The growth promoting activity of two different kinds of reconstructed terminators in transgenic common carp].

The transgenic promoter directly affects the activity of transgenic gene. Choice of terminator has been proved to affect the activity of transgenic gene. Since the relevant theoretical research is rare, using the terminator of transgenic gene or the promoter gene to construct the transgenic vector has not come to the conclusion. In order to construct a better transgenic vector to nurture "all fish" growth hormone transgenic fish which has a rapid growth character, two different transgenic vectors were constructed separately by the terminator of beta-actin gene and the terminator of growth hormone gene. After micro-injection, we obtained the P0 generation of "all fish" transgenic fish farming groups. Comparing the activities of the two different transgenic vectors, the weight distribution of the breeding groups from growth hormone gene terminator transgenic vector felt into a normal distribution; however, the weight distribution of the breeding groups originated from beta-actin gene terminator exhibited a non-normal mode and in a rightist trend. The average weight of GH (Growth hormone) gene terminator transgenic groups was significantly higher than that of the beta-actin gene terminator transgenic groups. Interestingly, a fast-growth transgenic carp was found from a mixed farming group, which was positive for the growth hormone gene terminator. This result showed that "all fish" GH gene transgenic carp could grow fast, and the transgenic gene could inherit from generation to generation. Our results suggest that the growth hormone gene terminator transgenic vector has stronger growth-promoting activity than the beta-actin gene terminator transgenic vector.

Single-cell analyses identify distinct and intermediate states of zebrafish pancreatic islet development.

Pancreatic endocrine islets are vital for glucose homeostasis. However, the islet developmental trajectory and its regulatory network are not well understood. To define the features of these specification and differentiation processes, we isolated individual islet cells from TgBAC(neurod1:EGFP) transgenic zebrafish and analyzed islet developmental dynamics across four different embryonic stages using a single-cell RNA-seq strategy. We identified proliferative endocrine progenitors, which could be further categorized by different cell cycle phases with the G1/S subpopulation displaying a distinct differentiation potential. We identified endocrine precursors, a heterogeneous intermediate-state population consisting of lineage-primed alpha, beta and delta cells that were characterized by the expression of lineage-specific transcription factors and relatively low expression of terminally differentiation markers. The terminally differentiated alpha, beta, and delta cells displayed stage-dependent differentiation states, which were related to their functional maturation. Our data unveiled distinct states, events and molecular features during the islet developmental transition, and provided resources to comprehensively understand the lineage hierarchy of islet development at the single-cell level.

Identification and characterization of a novel gene differentially expressed in zebrafish cross-subfamily cloned embryos.

BACKGROUND: Cross-species nuclear transfer has been shown to be a potent approach to retain the genetic viability of a certain species near extinction. However, most embryos produced by cross-species nuclear transfer were compromised because that they were unable to develop to later stages. Gene expression analysis of cross-species cloned embryos will yield new insights into the regulatory mechanisms involved in cross-species nuclear transfer and embryonic development. RESULTS: A novel gene, K31, was identified as an up-regulated gene in fish cross-subfamily cloned embryos using SSH approach and RACE method. K31 complete cDNA sequence is 1106 base pairs (bp) in length, with a 342 bp open reading frame (ORF) encoding a putative protein of 113 amino acids (aa). Comparative analysis revealed no homologous known gene in zebrafish and other species database. K31 protein contains a putative transmembrane helix and five putative phosphorylation sites but without a signal peptide. Expression pattern analysis by real time RT-PCR and whole-mount in situ hybridization (WISH) shows that it has the characteristics of constitutively expressed gene. Sub-cellular localization assay shows that K31 protein can not penetrate the nuclei. Interestingly, over-expression of K31 gene can cause lethality in the epithelioma papulosum cyprinid (EPC) cells in cell culture, which gave hint to the inefficient reprogramming events occurred in cloned embryos. CONCLUSION: Taken together, our findings indicated that K31 gene is a novel gene differentially expressed in fish cross-subfamily cloned embryos and over-expression of K31 gene can cause lethality of cultured fish cells. To our knowledge, this is the first report on the determination of novel genes involved in nucleo-cytoplasmic interaction of fish cross-subfamily cloned embryos.

Site-directed gene integration in transgenic zebrafish mediated by cre recombinase using a combination of mutant lox sites.

With current gene-transfer techniques in fish, insertion of DNA into the genome occurs randomly and in many instances at multiple sites. Associated position effects, copy number differences, and multiple gene interactions make gene expression experiments difficult to interpret and fish phenotype less predictable. To meet different fish engineering needs, we describe here a gene targeting model in zebrafish. At first, four target zebrafish lines, each harboring a single genomic lox71 target site, were generated by zebrafish transgenesis. The zygotes of transgenic zebrafish lines were coinjected with capped Cre mRNA and a knockin vector pZklox66RFP. Site-specific integration event happened from one target zebrafish line. In this line two integrant zebrafish were obtained from more than 80,000 targeted embryos (integrating efficiency about 10(-4) to 10(-5)) and confirmed to have a sole copy of the integrating DNA at the target genome site. Genomic polymerase chain reaction analysis and DNA sequencing verified the correct gene target events where lox71 and lox66 have accurately recombined into double mutant lox72 and wild-type loxP. Each integrant zebrafish chosen for analysis harbored the transgene rfp at the designated egfp concatenates. Although the Cre-mediated recombination is site specific, it is dependent on a randomly placed target site. That is, a genomic target cannot be preselected for integration based solely on its sequence. Conclusively, an rfp reporter gene was successfully inserted into the egfp target locus of zebrafish genome by Cre-lox-mediated recombination. This site-directed knockin system using the lox71/lox66 combination should be a promising gene-targeting platform serving various purposes in fish genetic engineering.

Cloning and characterization of cytochrome c oxidase subunit I (COXI) in Gobiocypris rarus.

In study of gene expression profile in cloned embryos which derived from D. rerio embryonic nuclei and G. rarus enucleated eggs, cytochrome c oxidase subunit I (COXI) of G. rarus, exhibiting difference at expression level between cloned embryos and zebrafish embryo, was cloned. Its full cDNA length is 1654 bp and contains a 1551 bp open reading frame, encoding a 5.64 kDa protein of 516 amino acids. The alignment result shows that mitochondrion tRNAser is co-transcripted with COXI, which just was the 3'-UTR of COXI. Molecular phylogenic analysis based on COXI indicates G. rarus should belong to Gobioninae, which was not in agreement with previous study according to morphological taxonomy. Comparison of DNA with cDNA shows that RNA editing phenomenon does not occur in the COXI of G. rarus.

An NGS-based approach for the identification of sex-specific markers in snakehead (Channa argus).

We described a next generation sequencing (NGS)-based approach to identify sex-specific markers and subsequently determine whether a species has male or female heterogamety. To test the accuracy of this technique, we examined the snakehead (Channa argus), which is economically important freshwater fish in China. Males grow faster than females, and there is significant interest in developing methods to skew breeding towards all-males to increase biomass yields. NGS was conducted on DNAs of individual female and male, the male reads were spitted into 60 bp K-mers and aligned to the female reference genome assembled by female reads, unaligned male K-mers-60 were kept in next filter process. Meanwhile, DNA sample of 48 females was pooled and sequenced, this data was further used to filter out the previous unaligned male K-mers-60. Hence, numbers of candidate Y chromosome-specific sequences were screened out, their sex-specificity were validated in wild snakeheads through PCR amplification. Finally, three Y chromosome-specific fragments (Contig-275834, Contig-359642, and Contig-418354) were identified, and specific primers were obtained to distinguish the sex of snakehead. Additionally, a pair of primers of Contig-275834 (275834X/Y-F and 275834X/Y-R) was exploited to distinguish XX females, XY males, and YY super-males, whose amplification products of different lengths were produced for different sexes. Therefore, our work demonstrated the ability of NGS data in identification of sex-specific markers, and the pipeline adopted in our study could be applied in any species of sex differentiation. Furthermore, the sex-specific markers have tremendous potential for improving the efficiency of all-male breeding practices in snakehead.

Upstream regulatory region of zebrafish lunatic fringe: isolation and promoter analysis.

Lunatic fringe (Lfng), one modulator of Notch signaling, plays an essential part in demarcation of tissues boundaries during animal early development, especially somitogenesis. To characterize the promoter of zebrafish lfng and generate somite-specific transgenic zebrafish, we isolated the upstream regulatory region of zebrafish lfng by blast search at the Ensembl genome database ( http://www.ensembl.org ) and analyzed the promoter activity using green fluorescent protein (GFP) as a reporter. Promoter activity assay in zebrafish shows that the 0.2-kb fragment containing GC-box, CAAT-box, and TATA-box can direct tissue-specific GFP expression, while the 0.4-kb and 1.2-kb fragments with further upstream sequence included drive GFP expression more efficiently. We produced lfngEGFP-transgenic founders showing somite-specific expression of GFP and consequently generated a hemizygous lfngEGFP-transgenic line. The eggs from lfngEGFP-transgenic female zebrafish show strong GFP expression, which is consistent to the reverse-transcription polymerase chain reaction PCR (RT-PCR) detection of lfng transcripts in the fertilized eggs. This reveals that zebrafish lfng is a maternal factor existing in matured eggs, suggesting that fish somitogenesis may be influenced by maternal factors.