A zebrafish gene trap line expresses GFP recapturing expression pattern of foxjlb

Foxj1 has been found to play an important role in cilia formation and function in vertebrates.The zebrafish or Xenopus genome expresses two Foxj1 genes, foxjla/FoxJ1 and foxj1b/FoxJ1.2. In this study, we have generated a zebrafish transgenic line T2BGSZ10 by Tol2 transposon-based gene trapping approach. T2BGSZ10 transgenic fish carry an insertion of the transposon genome into the first intron of thefoxjl1b locus. This insertion results in GFP expression in the forebrain, otic vesicles, floorplate, pronephric ducts and other domains during embryogenesis, which recaptures the expression pattern offoxjlb. Although normal expression offoxjlb is dramatically reduced,T2BGSZ10 homozygous embryos develop normally and grow to adulthood without detectable defects, which may be due to the incomplete interruption of foxj1b expression. Nevertheless, this transgenic line may serve as a useful model for dynamic observation of GFP-labeled tissues and organs and for isolation of GFP-labeled cells.     

A zebrafish gene trap line expresses GFP recapturing expression pattern of foxjlb

Foxj1 has been found to play an important role in cilia formation and function in vertebrates.The zebrafish or Xenopus genome expresses two Foxj1 genes, foxjla/FoxJ1 and foxj1b/FoxJ1.2. In this study, we have generated a zebrafish transgenic line T2BGSZ10 by Tol2 transposon-based gene trapping approach. T2BGSZ10 transgenic fish carry an insertion of the transposon genome into the first intron of thefoxjl1b locus. This insertion results in GFP expression in the forebrain, otic vesicles, floorplate, pronephric ducts and other domains during embryogenesis, which recaptures the expression pattern offoxjlb. Although normal expression offoxjlb is dramatically reduced,T2BGSZ10 homozygous embryos develop normally and grow to adulthood without detectable defects, which may be due to the incomplete interruption of foxj1b expression. Nevertheless, this transgenic line may serve as a useful model for dynamic observation of GFP-labeled tissues and organs and for isolation of GFP-labeled cells.     

Somatic mutagenesis with a Sleeping Beauty transposon system leads to solid tumor formation in zebrafish

Large-scale sequencing of human cancer genomes and mouse transposon-induced tumors has identified a vast number of genes mutated in different cancers. One of the outstanding challenges in this field is to determine which genes, when mutated, contribute to cellular transformation and tumor progression. To identify new and conserved genes that drive tumorigenesis we have developed a novel cancer model in a distantly related vertebrate species, the zebrafish, Danio rerio. The Sleeping Beauty (SB) T2/Onc transposon system was adapted for somatic mutagenesis in zebrafish. The carp ß-actin promoter was cloned into T2/Onc to create T2/OncZ. Two transgenic zebrafish lines that contain large concatemers of T2/OncZ were isolated by injection of linear DNA into the zebrafish embryo. The T2/OncZ transposons were mobilized throughout the zebrafish genome from the transgene array by injecting SB11 transposase RNA at the 1-cell stage. Alternatively, the T2/OncZ zebrafish were crossed to a transgenic line that constitutively expresses SB11 transposase. T2/OncZ transposon integration sites were cloned by ligation-mediated PCR and sequenced on a Genome Analyzer II. Between 700–6800 unique integration events in individual fish were mapped to the zebrafish genome. The data show that introduction of transposase by transgene expression or RNA injection results in an even distribution of transposon re-integration events across the zebrafish genome. SB11 mRNA injection resulted in neoplasms in 10% of adult fish at ∼10 months of age. T2/OncZ-induced zebrafish tumors contain many mutated genes in common with human and mouse cancer genes. These analyses validate our mutagenesis approach and provide additional support for the involvement of these genes in human cancers. The zebrafish T2/OncZ cancer model will be useful for identifying novel and conserved genetic drivers of human cancers.     

Foxj1 regulates asymmetric gene expression during left-right axis patterning in mice

Mice with a targeted mutation of the foxj1 gene demonstrate either D- or L-looping of the embryonic cardiac tube. Foxj1 is expressed in ventral cells of the embryonic node prior to asymmetric, left-right expression of other genes. Despite an absence of 9+2 cilia in foxj1(-/-) mice, 9+0 cilia are present in the node of foxj1(-/-) embryos. In foxj1(-/-) embryos, the patterns of expression of the TGF-beta family member nodal and the homeobox family member pitx2 are randomized. No expression of the TGF-beta family member lefty-2 is observed in any foxj1(-/-) early somite stage embryos. Foxj1 thus acts early in left-right axis patterning and regulates asymmetric gene expression. This regulation does not appear to be the result of a direct interaction between Foxj1 and the genes examined.     

An enhancer trap zebrafish line for lateral line development and regulation of six2b expression

Zebrafish lateral line system which is derived from neurogenic placodes has become a popular model for developmental biology since its formation involves cell migration, pattern formation, organogenesis, and hair cell regeneration. Transgenic lines play a crucial role in lateral line system study. Here, we identified an enhancer trap transgenic zebrafish line Et(gata2a:EGFP)189b (ET189b for short), which expressed enhanced green fluorescent protein (EGFP) in the pituitary, otic, and lateral line placodes and their derivatives. Especially, in neuromast, the accessory cells rather than hair cells were labeled by EGFP. Furthermore, we found the Tol2 transposon construct is integrated at the proximal upstream region of six2b gene locus. And EGFP expression of ET189b closely reflects the expression of endogenous six2b during development and after dkk1b over-expression. Taken together, our results indicated that ET189b is an ideal line for research on lateral line development and regulation of six2b expression.     

Generation and Characterization of a Transgenic Zebrafish Expressing the Reverse Tetracycline Transactivator

Conditional expression of a target gene during zebrafish development is a powerful approach to elucidate gene functions. The tetracycline-controlled systems have been successfully used in the modulation of gene expression in mammalian cells, but few lines of zebrafish carrying these systems are currently available. In this study, we had generated a stable transgenic zebrafish line that ubiquitously expressed the second-generation of reverse Tet transactivator （rtTA-M2）. Southern blotting analysis and high-throughput genome sequencing verifed that a single copy of rtTA-M2 gene had stably integrated into the zebrafish genome. After induction with doxycycline （Dox）, a strong green fluorescent protein （GFP） was seen in rtTA-transgenic eggs injected with pTRE--EGFP plasmids. The fluorescent signal gradually decreased after the withdrawal of Dox and disappeared. However, leaky expression of GFP was undetectable before Dox- induction. Additionally, transgenic embryos expressing rtTA-M2 exhibited no obvious defects in morphological phenotypes, hatching behavior and expression patterns of developmental marker genes, suggesting that rtTA-M2 had little effect on the development of transgenic zebrafish. Moreover, expressed Dickkopf-1 （DKK1） in pTRE-DKKl-injected embryos led to alterations in the expression of marker genes associated with Wnt signaling. Thus, this rtTA-transgenic zebrafish can be utilized to dissect functions of genes in a temporal manner.     

Expression and Functional Characterization of Smyd1a in Myofibril Organization of Skeletal Muscles

Background

Smyd1, the founding member of the Smyd family including Smyd-1, 2, 3, 4 and 5, is a SET and MYND domain containing protein that plays a key role in myofibril assembly in skeletal and cardiac muscles. Bioinformatic analysis revealed that zebrafish genome contains two highly related smyd1 genes, smyd1a and smyd1b. Although Smyd1b function is well characterized in skeletal and cardiac muscles, the function of Smyd1a is, however, unknown.

Methodology/Principal Findings

To investigate the function of Smyd1a in muscle development, we isolated smyd1a from zebrafish, and characterized its expression and function during muscle development via gene knockdown and transgenic expression approaches. The results showed that smyd1a was strongly expressed in skeletal muscles of zebrafish embryos. Functional analysis revealed that knockdown of smyd1a alone had no significant effect on myofibril assembly in zebrafish skeletal muscles. However, knockdown of smyd1a and smyd1b together resulted in a complete disruption of myofibril organization in skeletal muscles, a phenotype stronger than knockdown of smyd1a or smyd1b alone. Moreover, ectopic expression of zebrafish smyd1a or mouse Smyd1 transgene could rescue the myofibril defects from the smyd1b knockdown in zebrafish embryos.

Conclusion/Significance

Collectively, these data indicate that Smyd1a and Smyd1b share similar biological activity in myofibril assembly in zebrafish embryos. However, Smyd1b appears to play a major role in this process.     

Faithful expression of a tagged Fugu WT1 protein from a genomic transgene in zebrafish: efficient splicing of pufferfish genes in zebrafish but not mice

The teleost fish are widely used as model organisms in vertebrate biology. The compact genome of the pufferfish, Fugu rubripes, has proven a valuable tool in comparative genome analyses, aiding the annotation of mammalian genomes and the identification of conserved regulatory elements, whilst the zebrafish is particularly suited to genetic and developmental studies. We demonstrate that a pufferfish WT1 transgene can be expressed and spliced appropriately in transgenic zebrafish, contrasting with the situation in transgenic mice. By creating both transgenic mice and transgenic zebrafish with the same construct, we show that Fugu RNA is processed correctly in zebrafish but not in mice. Furthermore, we show for the first time that a Fugu genomic construct can produce protein in transgenic zebrafish: a full-length Fugu WT1 transgene with a C-terminal β-galactosidase fusion is spliced and translated correctly in zebrafish, mimicking the expression of the endogenous WT1 gene. These data demonstrate that the zebrafish:Fugu system is a powerful and convenient tool for dissecting both vertebrate gene regulation and gene function in vivo.     

BDNF-TrkB Axis Regulates Migration of the Lateral Line Primordium and Modulates the Maintenance of Mechanoreceptor Progenitors

BDNF and its specialized receptor TrkB are expressed in the developing lateral line system of zebrafish, but their role in this organ is unknown. To tackle this problem in vivo, we used transgenic animals expressing fluorescent markers in different cell types of the lateral line and combined a BDNF gain-of-function approach by BDNF mRNA overexpression and by soaking embryos in a solution of BDNF, with a loss-of-function approach by injecting the antisence ntrk2b-morpholino and treating embryos with the specific Trk inhibitor K252a. Subsequent analysis demonstrated that the BDNF-TrkB axis regulates migration of the lateral line primordium. In particular, BDNF-TrkB influences the expression level of components of chemokine signaling including Cxcr4b, and the generation of progenitors of mechanoreceptors, at the level of expression of Atoh1a-Atp2b1a.     

The Nogo-C2/Nogo Receptor Complex Regulates the Morphogenesis of Zebrafish Lateral Line Primordium through Modulating the Expression of dkk1b,a Wnt Signal Inhibitor

The fish lateral line (LL) is a mechanosensory system closely related to the hearing system of higher vertebrates, and it is composed of several neuromasts located on the surface of the fish. These neuromasts can detect changes in external water flow, to assist fish in maintaining a stationary position in a stream. In the present study, we identified a novel function of Nogo/Nogo receptor signaling in the formation of zebrafish neuromasts. Nogo signaling in zebrafish, like that in mammals, involves three ligands and four receptors, as well as three co-receptors (TROY, p75, and LINGO-1). We first demonstrated that Nogo-C2, NgRH1a, p75, and TROY are able to form a Nogo-C2 complex, and that disintegration of this complex causes defective neuromast formation in zebrafish. Time-lapse recording of the CldnB::lynEGFP transgenic line revealed that functional obstruction of the Nogo-C2 complex causes disordered morphogenesis, and reduces rosette formation in the posterior LL (PLL) primordium during migration. Consistent with these findings, hair-cell progenitors were lost from the PLL primordium in p75, TROY, and Nogo-C2/NgRH1a morphants. Notably, the expression levels of pea3, a downstream marker of Fgf signaling, and dkk1b, a Wnt signaling inhibitor, were both decreased in p75, TROY, and Nogo-C2/NgRH1a morphants; moreover, dkk1b mRNA injection could rescue the defects in neuromast formation resulting from knockdown of p75 or TROY. We thus suggest that a novel Nogo-C2 complex, consisting of Nogo-C2, NgRH1a, p75, and TROY, regulates Fgf signaling and dkk1b expression, thereby ensuring stable organization of the PLL primordium.     

荧光转基因斑马鱼Tg(ttn.2:EGFP)的构建

为了建立一种用于研究肌肉和心脏发育及其相关疾病的绿色荧光蛋白(enhanced green fluorescent protein,EGFP)转基因斑马鱼品系,本研究使用斑马鱼ttn.2基因编码区上游启动子序列和绿色荧光蛋白基因编码序列构建了重组表达载体,并将该载体和Tol2转座酶的加帽mRNA显微共注射入斑马鱼1-细胞期胚胎,通过荧光检测、遗传杂交筛选和分子鉴定等方法,成功建立了能稳定遗传的Tg(ttn.2:EGFP)转基因斑马鱼品系。荧光表达分析及原位杂交分析结果表明,绿色荧光信号在斑马鱼肌肉和心脏组织中特异表达模式与ttn.2基因的mRNA表达一致。通过反向PCR鉴定转基因表达载体在F1代斑马鱼品系中的随机整合位点,结果表明:No.33转基因品系的EGFP基因整合在斑马鱼的4号和11号染色体上,No.34转基因品系则整合在1号染色体上。该荧光转基因斑马鱼品系Tg(ttn.2:EGFP)的成功构建为肌肉和心脏发育以及相关疾病研究提供了一个新的理想实验模型。此外,绿色荧光强烈表达的斑马鱼品系还可以作为一种新的观赏鱼。     

Multiplex Conditional Mutagenesis Using Transgenic Expression of Cas9 and sgRNAs

Determining the mechanism of gene function is greatly enhanced using conditional mutagenesis. However, generating engineered conditional alleles is inefficient and has only been widely used in mice. Importantly, multiplex conditional mutagenesis requires extensive breeding. Here we demonstrate a system for one-generation multiplex conditional mutagenesis in zebrafish (Danio rerio) using transgenic expression of both cas9 and multiple single guide RNAs (sgRNAs). We describe five distinct zebrafish U6 promoters for sgRNA expression and demonstrate efficient multiplex biallelic inactivation of tyrosinase and insulin receptor a and b, resulting in defects in pigmentation and glucose homeostasis. Furthermore, we demonstrate temporal and tissue-specific mutagenesis using transgenic expression of Cas9. Heat-shock-inducible expression of cas9 allows temporal control of tyr mutagenesis. Liver-specific expression of cas9 disrupts insulin receptor a and b, causing fasting hypoglycemia and postprandial hyperglycemia. We also show that delivery of sgRNAs targeting ascl1a into the eye leads to impaired damage-induced photoreceptor regeneration. Our findings suggest that CRISPR/Cas9-based conditional mutagenesis in zebrafish is not only feasible but rapid and straightforward.     

inka1b expression in the head mesoderm is dispensable for facial cartilage development

Inka box actin regulator 1 (Inka1) is a novel protein identified in Xenopus and is found in vertebrates. While Inka1 is required for facial skeletal development in Xenopus and zebrafish, it is dispensable in mice despite its conserved expression in the cranial neural crest, indicating that Inka1 function in facial skeletal development is not conserved among vertebrates. Zebrafish bears two paralogs of inka1 (inka1a and inka1b) in the genome, with the biological roles of inka1b barely known. Here, we analyzed the expression and function of inka1b during facial skeletal development in zebrafish. inka1b was expressed sequentially in the head mesoderm adjacent to the pharyngeal pouches essential for facial skeletal development at the stage of arch segmentation. However, a loss-of-function mutation in inka1b displayed normal head development, including the pouches and facial cartilages. The normal head of inka1b mutant fish was unlikely a result of the genetic redundancy of inka1b with inka1a, given the distinct expression of inka1a and inka1b in the cranial neural crest and head mesoderm, respectively, during craniofacial development. Our findings suggest that the inka1b expression in the head mesoderm might not be essential for head development in zebrafish.