Epidermis-restricted expression of zebrafish cytokeratin II is controlled by a -141/+85 minimal promoter, and cassette -141/-111 is essential for driving the tissue specificity

We isolated a 2.3 kb DNA segment from the upstream region of the zebrafish cytokeratin II (zfCKII) gene. Transgenic embryos, produced by using a series of 5' deletions linked to the red fluorescent protein (RFP) reporter, showed that the -141/+85 segment of zfCKII directed RFP expression in epidermal cells, whereas the -111/+85 segment did not. When -141/-111 was deleted from -355/+85 and microinjected into one-celled embryos, no fluorescence was observed at later stages, indicating that the -141/-111 segment is required for green fluorescent protein expression in epidermal cells. Furthermore, when a putative KLF-binding site at -119/-117 was mutated, RFP expression rates and intensities were reduced dramatically, although still observed, suggesting that -119/-117 within -141/-111 is a key cis-element for controlling epidermis-specific expression of the zfCKII gene. Finally, we generated a zebrafish transgenic line, Tg(zfCKII(2.3):RFP), which carries an upstream 2.3 kb regulatory region of the zfCKII gene fused with RFP. The expression pattern in the epidermal cells of Tg(zfCKII(2.3):RFP) fish recapitulated that of the endogenous gene. F2 embryos derived from Tg(zfCKII(2.3):RFP) males crossed with wild-type females revealed that the earliest onset of RFP expression was at the sphere stage, indicating that this transgenic approach can be used for studying zygotic expression of maternally inherited genes.     

Transgenic zebrafish line with over-expression of Hedgehog on the skin: a useful tool to screen Hedgehog-inhibiting compounds

We generated a transgenic line Tg(k18:shh:RFP) with overexpression of Sonic hedgehog in the skin epidermis. By 5 day-post-fertilization (dpf), many epidermal lesions were clearly observed, including a swollen yolk sac, epidermis growth malformation around the eyes and at the basement of the pectoral fins. Skin histology revealed embryos derived from Tg(k18:shh:RFP) displayed an elevated Nuclear/Cytoplasmic ratio and pleomorphic nuclei compared to their wild type littermates, suggesting the abnormal growth pattern on the epidermis of Tg(k18:shh:RFP) embryos were dysplasia. Later (by 7 dpf), Tg(k18:shh:RFP) embryos displayed broader pectoral fins which are similar to the polydactyly phenotypes of Nevoid basal cell carcinoma syndrome (NBCCS)/Gorlin patients and polydactylous mice. In addition, treatment with cyclopamine is able to enhance and prolong the survival rates and survival durations of Tg(k18:shh:RFP) embryos. In conclusion, this unique Tg(k18:shh:RFP) fish line, should be an excellent experimental animal for screening for a lower toxicity level of the new Hh-inhibitor and can even be used as a new anti-cancer drug-screening platform.     

An Apo-14 promoter-driven transgenic zebrafish that marks liver organogenesis

Several transgenic zebrafish lines for liver development studies had been obtained in the first decade of this century, but not any transgenic GFP zebrafish lines that mark the through liver development and organogenesis were reported. In this study, we analyzed expression pattern of endogenous Apo-14 in zebrafish embryogenesis by whole-mount in situ hybridization, and revealed its expression in liver primordium and in the following liver development. Subsequently, we isolated zebrafish Apo-14 promoter of 1763 bp 5'-flanking sequence, and developed an Apo-14 promoter-driven transgenic zebrafish Tg(Apo14: GFP). And, maternal expression and post-fertilization translocation of Apo-14 promoter-driven GFP were observed in the transgenic zebrafish line. Moreover, we traced onset expression of Apo-14 promoter-driven GFP and developmental behavior of the expressed cells in early heterozygous embryos by out-crossing the Tg(Apo14: GFP) male to the wild type female. Significantly, the Apo-14 promoter-driven GFP is initially expressed around YSL beneath the embryo body at 10 hpf when the embryos develop to tail bud prominence. In about 14-somite embryos at 16-17 hpf, a typical "salt-and-pepper" expression pattern is clearly observed in YSL around the yolk sac. Then, a green fluorescence dot begins to appear between the notochord and the yolk sac adjacent to otic vesicle at about 20 hpf, which is later demonstrated to be liver primordium that gives rise to liver. Furthermore, we investigated dynamic progression of liver organogenesis in the Tg(Apo14: GFP) zebrafish, because the Apo-14 promoter-driven GFP is sustainably expressed from hepatoblasts and liver progenitor cells in liver primordium to hepatocytes in the larval and adult liver. Additionally, we observed similar morphology between the liver progenitor cells and the GFP-positive nuclei on the YSL, suggesting that they might originate from the same progenitor cells in early embryos. Overall, the current study provides a transgenic zebrafish line that marks the through liver organogenesis.     

Inducible Sterilization of Zebrafish by Disruption of Primordial Germ Cell Migration

During zebrafish development, a gradient of stromal-derived factor 1a (Sdf1a) provides the directional cue that guides the migration of the primordial germ cells (PGCs) to the gonadal tissue. Here we describe a method to produce large numbers of infertile fish by inducing ubiquitous expression of Sdf1a in zebrafish embryos resulting in disruption of the normal PGC migration pattern. A transgenic line of zebrafish, Tg(hsp70:sdf1a-nanos3, EGFP), was generated that expresses Sdf1a under the control of the heat-shock protein 70 (hsp70) promoter and nanos3 3?UTR. To better visualize the PGCs, the Tg(hsp70:sdf1a-nanos3, EGFP) fish were crossed with another transgenic line, Tg(kop:DsRed-nanos3), that expresses DsRed driven by the PGC-specific kop promoter. Heat treatment of the transgenic embryos caused an induction of Sdf1a expression throughout the embryo resulting in the disruption of their normal migration. Optimal embryo survival and disruption of PGC migration was achieved when transgenic embryos at the 4- to 8-cell stage were incubated at 34.5°C for 18 hours. Under these conditions, disruption of PGC migration was observed in 100% of the embryos. Sixty-four adult fish were developed from three separate batches of heat-treated embryos and all were found to be infertile males. When each male was paired with a wild-type female, only unfertilized eggs were produced and histological examination revealed that each of the adult male fish possessed severely under-developed gonads that lacked gametes. The results demonstrate that inducible Sdf1a expression is an efficient and reliable strategy to produce infertile fish. This approach makes it convenient to generate large numbers of infertile adult fish while also providing the capability to maintain a fertile brood stock.     

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。     

Mouse in red: red fluorescent protein expression in mouse ES cells, embryos, and adult animals

Spectral variants of green fluorescent protein are widely used in live samples for a broad range of applications: from visualization of protein interactions, through following gene expression, to marking particular cells in complex tissues. Higher wavelength emissions (such as red) are preferred due to the lower background-autofluorescence in tissues (Miyawaka et al., Nat Cell Biol Suppl S1-7, 2003). Until now, however, red fluorescent proteins (RFP) have displayed toxicity in murine embryos, which has hampered its application in this model. Here we report strong expression of a recently developed RFP variant, DsRed.T3, in mouse ES cells, embryos, and adult mice. Our results show that the red fluorescent wavelength has a superior tissue penetrance compared with spectral variants of lower wavelength. Furthermore, we have generated an ES cell line and a corresponding transgenic mouse line in which red fluorescence is activated upon Cre excision. Finally, we introduced cell type-specifically expressed Cre transgenes into this Cre recombinase reporter cell line, and by using the tetraploid embryo complementation assay, we could directly verify the Cre recombinase specificity on ES cell-derived embryos/animals.     

Two different transgenes to study gene silencing and re-expression during zebrafish caudal fin and retinal regeneration

We used the 500-bp Xenopus ef1-alpha promoter and the 2-kb zebrafish histone 2A.F/Z promoter to generate several independent transgenic zebrafish lines expressing EGFP. While both promoters drive ubiquitous EGFP expression in early zebrafish development, they are systematically silenced in several adult tissues, including the retina and caudal fin. However, EGFP expression is temporarily renewed in the adult during either caudal fin or retinal regeneration. In the Tg(H2A.F/Z:EGFP)nt line, EGFP is moderately expressed in both the wound epithelium and blastema of the regenerating caudal fin. In the Tg(ef1-alpha:EGFP)nt line, EGFP expression is reinitiated and restricted to the blastema of the regenerating caudal fin and colabels with BrdU, PCNA, and msxc-positive cells. Thus, these two ubiquitous promoters drive EGFP transgene expression in different cell populations during caudal fin regeneration. We further analyzed the ability of the ef1-alpha:EGFP transgene to label nonterminally differentiated cells during adult tissue regeneration. First, we demonstrated that the transgene is highly methylated in adult zebrafish caudal fin tissue, but not during fin regeneration, implicating methylation as a potential means of transgene silencing in this line. Next, we determined that the ef1-alpha:EGFP transgene is also re-expressed during adult retinal regeneration. Specifically, the ef1-alpha:EGFP transgene colabels with PCNA in the Müller glia, a specialized cell that is the source of neuronal progenitors during zebrafish retinal regeneration. Thus, we concluded that Tg(ef1-alpha:EGFP)nt line visually marks nonterminally differentiated cells in multiple adult regeneration environments and may prove to be a useful marker in tissue regeneration studies in zebrafish.     

Zebrafish Embryonic Slow Muscle Is a Rapid System for Genetic Analysis of Sarcomere Organization by CRISPR/Cas9, but Not NgAgo

Zebrafish embryonic slow muscle cells, with their superficial localization and clear sarcomere organization, provide a useful model system for genetic analysis of muscle cell differentiation and sarcomere assembly. To develop a quick assay for testing CRISPR-mediated gene editing in slow muscles of zebrafish embryos, we targeted a red fluorescence protein (RFP) reporter gene specifically expressed in slow muscles of myomesin-3-RFP (Myom3-RFP) zebrafish embryos. We demonstrated that microinjection of RFP-sgRNA with Cas9 protein or Cas9 mRNA resulted in a mosaic pattern in loss of RFP expression in slow muscle fibers of the injected zebrafish embryos. To uncover gene functions in sarcomere organization, we targeted two endogenous genes, slow myosin heavy chain-1 (smyhc1) and heat shock protein 90 α1 (hsp90α1), which are specifically expressed in zebrafish muscle cells. We demonstrated that injection of Cas9 protein or mRNA with respective sgRNAs targeted to smyhc1 or hsp90a1 resulted in a mosaic pattern of myosin thick filament disruption in slow myofibers of the injected zebrafish embryos. Moreover, Myom3-RFP expression and M-line localization were also abolished in these defective myofibers. Given that zebrafish embryonic slow muscles are a rapid in vivo system for testing genome editing and uncovering gene functions in muscle cell differentiation, we investigated whether microinjection of Natronobacterium gregoryi Argonaute (NgAgo) system could induce genetic mutations and muscle defects in zebrafish embryos. Single-strand guide DNAs targeted to RFP, Smyhc1, or Hsp90α1 were injected with NgAgo mRNA into Myom3-RFP zebrafish embryos. Myom3-RFP expression was analyzed in the injected embryos. The results showed that, in contrast to the CRISPR/Cas9 system, injection of the NgAgo-gDNA system did not affect Myom3-RFP expression and sarcomere organization in myofibers of the injected embryos. Sequence analysis failed to detect genetic mutations at the target genes. Together, our studies demonstrate that zebrafish embryonic slow muscle is a rapid model for testing gene editing technologies in vivo and uncovering gene functions in muscle cell differentiation.     

Differential regulation of keratin 8 and 18 messenger RNAs in differentiating F9 cells

F9 embryonal carcinoma cells (F9EC) can be induced to differentiate in vitro into epithelial cells expressing keratin 8 (K8) and keratin 18 (K18). cDNAs corresponding to K8 and K18 mRNAs were cloned and used to study the change in the abundance of these mRNAs during differentiation of F9 cells into parietal endoderm-like cells by treatment with retinoic acid (RA) or with RA and dibutyryl cAMP (Bt₂cAMP). Using an RNase protection assay, it was determined that K8 mRNA was induced slightly before K18 mRNA and that it accumulated to a greater extent than K18 mRNA. Furthermore, differentiation in presence of Bt₂cAMP plus RA resulted in an earlier induction of the two mRNAs and a higher level of expression of K8 mRNA. These results indicate that K8 and K18 mRNAs are regulated differently in F9 cells.     

Integration of Double-Fluorescence Expression Vectors into Zebrafish Genome for the Selection of Site-Directed Knockout/Knockin

Production of zebrafish by modifying endogenous growth hormone (GH) gene through homologous recombination is described here. We first constructed the targeting vectors pGHT1.7k and pGHT2.8k, which were used for the knockout/knockin of the endogenous GH gene of zebrafish, and injected these two vectors into the embryos of zebrafish. Overall, the rate of targeted integration with the characteristic of germ line transmission in zebrafish was 1.7×10⁻⁶. In one experimental patch, the integrating efficiency of pGHT2.8k was higher than that of pGHT1.7k, but the lethal effect of pGHT2.8k was stronger than that of pGHT1.7k. The clones with the correct integration of target genes were identified by a simple screening procedure based on green fluorescent protein (GFP) and RFP dual selection, which corresponded to homologous recombination and random insertion, respectively. The potential homologous recombination zebrafish was further bred to produce a heterozygous F₁ generation, selected based on the presence of GFP. The potential targeted integration of exogenous GH genes into a zebrafish genome at the P₀ generation was further verified by polymerase chain reaction and Southern blot analysis. Approximately 2.5% of potential founder knockout and knockin zebrafish had the characteristic of germ line transmission. In this study, we developed an efficient method for producing the targeted gene modification in zebrafish for future studies on genetic modifications and gene functions using this model organism. ^★Equal contributions to this article.     

Differential regulation of keratin 8 and 18 messenger RNAs in differentiating F9 cells.

F9 embryonal carcinoma cells (F9EC) can be induced to differentiate in vitro into epithelial cells expressing keratin 8 (K8) and keratin 18 (K18). cDNAs corresponding to K8 and K18 mRNAs were cloned and used to study the change in the abundance of these mRNAs during differentiation of F9 cells into parietal endoderm-like cells by treatment with retinoic acid (RA) or with RA and dibutyryl cAMP (Bt2cAMP). Using an RNase protection assay, it was determined that K8 mRNA was induced slightly before K18 mRNA and that it accumulated to a greater extent than K18 mRNA. Furthermore, differentiation in presence of Bt2cAMP plus RA resulted in an earlier induction of the two mRNAs and a higher level of expression of K8 mRNA. These results indicate that K8 and K18 mRNAs are regulated differently in F9 cells.     

Generation of Two-color Transgenic Zebrafish Using the Green and Red Fluorescent Protein Reporter Genes gfp and rfp

Two tissue-specific promoters were used to express both green fluorescent protein (GFP) and red fluorescent protein (RFP) in transgenic zebrafish embryos. One promoter (CK), derived from a cytokeratin gene, is active specifically in skin epithelia in embryos, and the other promoter (MLC) from a muscle-specific gene encodes a myosin light chain 2 polypeptide. When the 2 promoters drove the 2 reporter genes to express in the same embryos, both genes were faithfully expressed in the respective tissues, skin or muscle. When the 2 fluorescent proteins were expressed in the same skin or muscle cells under the same promoter, GFP fluorescence appeared earlier than RFP fluorescence in both skin and muscle tissues, probably owing to a higher detection sensitivity of GFP. However, RFP appeared to be more stable as its fluorescence steadily increased during development. Finally, F₁ transgenic offspring were obtained expressing GFP in skin cells under the CK promoter and RFP in muscle cells under the MLC promoter. Our study demonstrates the feasibility of monitoring expression of multiple genes in different tissues in the same transgenic organism.     

Global analysis of hematopoietic and vascular endothelial gene expression by tissue specific microarray profiling in zebrafish

In this study, we utilize fluorescent activated cell sorting (FACS) of cells from transgenic zebrafish coupled with microarray analysis to globally analyze expression of cell type specific genes. We find that it is possible to isolate cell populations from Tg(fli1:egfp)(y1) zebrafish embryos that are enriched in vascular, hematopoietic and pharyngeal arch cell types. Microarray analysis of GFP+ versus GFP- cells isolated from Tg(fli1:egfp)(y1) embryos identifies genes expressed in hematopoietic, vascular and pharyngeal arch tissue, consistent with the expression of the fli1:egfp transgene in these cell types. Comparison of expression profiles from GFP+ cells isolated from embryos at two different time points reveals that genes expressed in different fli1+ cell types display distinct temporal expression profiles. We also demonstrate the utility of this approach for gene discovery by identifying numerous previously uncharacterized genes that we find are expressed in fli1:egfp-positive cells, including new markers of blood, endothelial and pharyngeal arch cell types. In parallel, we have developed a database to allow easy access to both our microarray and in situ results. Our results demonstrate that this is a robust approach for identification of cell type specific genes as well as for global analysis of cell type specific gene expression in zebrafish embryos.     

HuC:Kaede, a useful tool to label neural morphologies in networks in vivo

A simple and efficient procedure for labeling neurons is a prerequisite for investigating the development of neural networks in zebrafish. To label neurons we used Kaede, a fluorescent protein with a photoconversion property allowing conversion from green to red fluorescence following irradiation with UV or violet light. We established a zebrafish stable transgenic line, Tg(HuC:Kaede), expressing Kaede in neurons under the control of the HuC promoter. This transgenic line was used to label a small number of neurons in the trigeminal ganglion. Also, using embryos injected with the transgene, we labeled peripheral axon arbors of a Rohon-Beard neuron at 4 days postfertilization and observed the dendrite development of a tectal neuron for 3 days. These data indicate that Kaede is a useful tool to selectively label neural networks in zebrafish.     

Modified bacterial artificial chromosomes for zebrafish transgenesis

Transgenesis using bacterial artificial chromosomes (BAC) offers greater fidelity in directing desirable expression of foreign genes. Application of this technology in the optically transparent zebrafish with fluorescent protein reporters enables unparalleled visual analysis of regulation of gene expression in a living organism. Here we describe a streamlined procedure of direct selecting multiple BAC clones based on public sequence databases followed by rapid modification with GFP or RFP for transgenic analysis in zebrafish. Experimental procedures for BAC DNA preparation, microinjection of zebrafish embryos and screening of transgenic zebrafish carrying GFP/RFP modified BAC clones are detailed.