A novel transgenic line using the Cre–lox system to allow permanent lineage‐labeling of the zebrafish neural crest

Accurate lineage tracing is crucial to understanding of developmental and stem cell biology, but is particularly challenging for transient and highly dispersive cell‐types like the neural crest (NC). The authors report in this article a new zebrafish transgenic line Tg(‐4725sox10:Cre)^ba74. This line expresses Cre under the control of a well‐characterized portion of the sox10 promoter and, by crossing to a floxed‐reporter line, the authors show in this article that expression in this line is consistent with those described for GFP reporter lines using the same promoter. Reporter expression is readily detected in patterns consistent with the early expression domains. Thus, the authors see all major groups (pigment, neural, and skeletal) of NC‐derived cell‐types, as well as cell‐types derived from the known non‐NC sites of sox10 expression, including otic epithelium and oligodendrocytes. This line provides an invaluable tool for the further study of zebrafish NC development and NC‐derived stem cells as well as that of the otic vesicle and oligodendrocytes. genesis 50:750–757, 2012. © 2012 Wiley Periodicals, Inc.     

Photoconvertible fluorescent proteins: a versatile tool in zebrafish skeletal imaging

One of the most frequently applied techniques in zebrafish (Danio rerio) research is the visualisation or manipulation of specific cell populations using transgenic reporter lines. The generation of these transgenic zebrafish, displaying cell- or tissue-specific expression of frequently used fluorophores such as Green Fluorescent Protein (GFP) or mCherry, is relatively easy using modern techniques. Fluorophores with different emission wavelengths and driven by different promoters can be monitored simultaneously in the same animal. Photoconvertible fluorescent proteins (pcFPs) are different from these standard fluorophores because their emission spectrum is changed when exposed to UV light, a process called photoconversion. Here, the benefits and versatility of using pcFPs for both single and dual fluorochrome imaging in zebrafish skeletal research in a previously generated osx:Kaede transgenic line are illustrated. In this line, Kaede, which is expressed under control of the osterix, otherwise known as sp7, promoter thereby labelling immature osteoblasts, can switch from green to red fluorescence upon irradiation with UV light. First, this study demonstrates that osx:Kaede exhibits an expression pattern similar to a previously described osx:nuGFP transgenic line in both larval and adult stages, hereby validating the use of this line for the imaging of immature osteoblasts. More in-depth experiments highlight different applications for osx:Kaede, such as lineage tracing and its combined use with in vivo skeletal staining and other transgenic backgrounds. Mineral staining in combination with osx:Kaede confirms osteoblast-independent mineralisation of the notochord. Osteoblast lineage tracing reveals migration and dedifferentiation of scleroblasts during fin regeneration. Finally, this study shows that combining two transgenics, osx:Kaede and osc:GFP, with similar emission wavelengths is possible when using a pcFP such as Kaede.     

Demonstration of site-directed recombination in transgenic zebrafish using the Cre/loxP system

To test the Cre/loxP recombination system in zebrafish, a stable transgenic zebrafish line was developed by using a floxed (loxP flanked) gfp(green fluorescent protein) gene construct under the muscle-specific mylz2 promoter. Like our previous non-floxed gfp transgenic line under the same promoter, the new transgenic line expresses GFP reporter faithfully in fast skeletal muscles to the same intensity. To demonstrate the excision of floxed gfp transgene, in vitro synthesized Cre RNA was injected into embryos of floxed gfp transgenic zebrafish and we found a dramatic reduction of GFP expression. To confirm the excision, PCR was performed and a DNA fragment of correct size was amplified as predicted from the Cre/loxP mediated excision. Finally, we cloned the fragment and sequence information confirmed that the excision occurred at the precise site as predicted. Our experiments demonstrated that the Cre/loxP system can function efficiently and accurately in the zebrafish system.     

BAC transgenic zebrafish reveal hypothalamic enhancer activity around obesity associated SNP rs9939609 within the human FTO gene

Single Nucleotide Polymorphisms in FTO intron 1 have been associated with obesity risk, leading to the hypothesis that FTO is the obesity‐related gene. However, other studies have shown that the FTO gene is part of the regulatory domain of the neighboring IRX3 gene and that enhancers in FTO intron 1 regulate IRX3. While Irx3 activity was shown to be necessary in the hypothalamus for the metabolic function of Irx3 in mouse, no enhancers with hypothalamic activity have been demonstrated in the risk‐associated region within FTO. In order to identify potential enhancers at the human FTO locus in vivo, we tested regulatory activity in FTO intron 1 using BAC transgenesis in zebrafish. A minimal gata2 promoter‐GFP cassette was inserted 1.3 kb upstream of the obesity associated SNP rs9939609 in a human FTO BAC plasmid. In addition to the previously identified expression domains in notochord and kidney, human FTO BAC:GFP transgenic zebrafish larvae expressed GFP in the ventral posterior tuberculum, the posterior hypothalamus and the anterior brainstem, which are also expression domains of zebrafish irx3a. In contrast, an in‐frame insertion of a GFP cassette at the FTO start codon resulted in weak ubiquitous GFP expression indicating that the promoter of FTO does likely not react to enhancers located in the obesity risk‐associated region. genesis 53:640–651, 2015. © 2015 The Authors. genesis Published by Wiley Periodicals, Inc.     

503unc,a small and muscle‐specific zebrafish promoter

The muscle‐specific UNC‐45b assists in the folding of sarcomeric myosin. Analysis of the zebrafish unc‐45b upstream region revealed that unc‐45b promoter fragments reliably drive GFP expression after germline transmission. The muscle‐specific 503‐bp minimal promoter 503unc was identified to drive gene expression in the zebrafish musculature. In transgenic Tg(?503unc:GFP) zebrafish, GFP fluorescence was detected in the adaxial cells, their slow fiber descendants, and the fast muscle. At later stages, robust GFP fluorescence is eminent in the cardiac, cranial, fin, and trunk muscle, thereby recapitulating the unc‐45b expression pattern. We propose that the 503unc promoter is a small and muscle‐specific promoter that drives robust gene expression throughout the zebrafish musculature, making it a valuable tool for the exploration of zebrafish muscle. genesis 51:443–447. © 2013 Wiley Periodicals, Inc.     

Visualization of skeletons and intervertebral disks in live fish larvae by fluorescent calcein staining and disk specific GFP expression

Zebrafish and medaka have become popular models for studying skeletal development because of high fecundity, shorter generation period, and transparency of fish embryo. The first step to study skeletal development is visualizing bone and cartilage. Live animal staining with fluorescent calcein have several advantages over the standard skeletal staining protocol by using alizarin red and alcian blue for bone and cartilage. However, there is no detailed study examining skeletal development of live marine fish larvae by calcein staining. Here we applied calcein staining to examine skeletal development in red sea bream larvae. In addition, green fluorescent protein (GFP) reporter zebrafish was employed to trace lineage analysis of intervertebral disk cells in live fish larvae. Calcein staining of red sea bream larvae successfully visualized development of craniofacial skeletons as well as urinary calculus. Histochemical detection of alkaline phosphatase (ALP) activity revealed that abnormal segmentation of notochord induced by RA during vertebral development in zebrafish. Immunohistochemistry clearly revealed that GFP‐positive cells in intervertebral space was nucleus polposus like cell in twhh‐GFP transgenic zebrafish. It was demonstrated usefulness of calcein and ALP staining and twhh‐GFP transgenic zebrafish for studying skeletal development in live fish larvae.     

Development of a transgenic zebrafish model expressing GFP in the notochord,somite and liver directed by the <Emphasis Type="Italic">hfe2</Emphasis> gene promoter

Hemojuvelin, also known as RGMc, is encoded by hfe2 gene that plays an important role in iron homeostasis. hfe2 is specifically expressed in the notochord, developing somite and skeletal muscles during development. The molecular regulation of hfe2 expression is, however, not clear. We reported here the characterization of hfe2 gene expression and the regulation of its tissue-specific expression in zebrafish embryos. We demonstrated that the 6 kb 5′-flanking sequence upstream of the ATG start codon in the zebrafish hfe2 gene could direct GFP specific expression in the notochord, somites, and skeletal muscle of zebrafish embryos, recapitulating the expression pattern of the endogenous gene. However, the Tg(hfe2:gfp) transgene is also expressed in the liver of fish embryos, which did not mimic the expression of the endogenous hfe2 at the early stage. Nevertheless, the Tg(hfe2:gfp) transgenic zebrafish provides a useful model to study liver development. Treating Tg(hfe2:gfp) transgenic zebrafish embryos with valproic acid, a liver development inhibitor, significantly inhibited GFP expression in zebrafish. Together, these data indicate that the tissue specific expression of hfe2 in the notochord, somites and muscles is regulated by regulatory elements within the 6 kb 5′-flanking sequence of the hfe2 gene. Moreover, the Tg(hfe2:gfp) transgenic zebrafish line provides a useful model system for analyzing liver development in zebrafish.     

Faithful expression of green fluorescent protein (GFP) in transgenic zebrafish embryos under control of zebrafish gene promoters

Although the zebrafish has become a popular model organism for vertebrate developmental and genetic analyses, its use in transgenic studies still suffers from the scarcity of homologous gene promoters. In the present study, three different zebrafish cDNA clones were isolated and sequenced completely, and their expression patterns were characterized by whole‐mount in situ hybridization as well as by Northern blot hybridization. The first clone encodes a type II cytokeratin (CK), which is specifically expressed in skin epithelia in early embryos and prominently expressed in the adult skin tissue. The second clone is muscle specific and encodes a muscle creatine kinase (MCK). The third clone, expressed ubiquitously in all tissues, is derived from an acidic ribosomal phosphoprotein P0 (arp) gene. In order to test the fidelity of zebrafish embryos in transgenic expression, the promoters of the three genes were isolated using a rapid linker‐mediated PCR approach and subsequently ligated to a modified green fluorescent protein (gfp) reporter gene. When the three hybrid GFP constructs were introduced into zebrafish embryos by microinjection, the three promoters were activated faithfully in developing zebrafish embryos. The 2.2‐kb ck promoter was sufficient to direct GFP expression in skin epithelia, although a weak expression in muscle was also observed in a few embryos. This pattern of transgenic expression is consistent with the expression pattern of the endogenous cytokeratin gene. The 1.5‐kb mck promoter/gfp was expressed exclusively in skeletal muscles and not elsewhere. By contrast, the 0.8‐kb ubiquitous promoter plus the first intron of the arp gene were capable of expressing GFP in a variety of tissues, including the skin, muscle, lens, neurons, notochord, and circulating blood cells. Our experiments, therefore, further demonstrated that zebrafish embryos can faithfully express exogenously introduced genes under the control of zebrafish promoters. Dev. Genet. 25:158–167, 1999. © 1999 Wiley‐Liss, Inc.     

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.     

The Fugu tyrp1 promoter directs specific GFP expression in zebrafish: tools to study the RPE and the neural crest‐derived melanophores

In vertebrates, pigment cells account for a small percentage of the total cell population and they intermingle with other cell types. This makes it difficult to isolate them for analyzes of their functions in the context of development. To alleviate such difficulty, we generated two stable transgenic zebrafish lines (pt101 and pt102) that express green fluorescent protein (GFP) in melanophores under the control of the 1 kb Fugu tyrp1 promoter. In pt101, GFP is expressed in both retinal pigment epithelium (RPE) cells and the neural crest‐derived melanophores (NCDM), whereas in pt102, GFP is predominately expressed in the NCDM. Our results indicate that the Fugu tyrp1 promoter can direct transgene expression in a cell‐type‐specific manner in zebrafish. In addition, our findings provide evidence supporting differential regulations of melanin‐synthesizing genes in RPE cells and the NCDM in zebrafish. Utilizing the varying GFP expression levels in these fish, we have isolated melanophores via flow cytometry and revealed the capability of sorting the NCDM from RPE cells as well. Thus, these transgenic lines are useful tools to study melanophores 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.     

Analyzing notochord segmentation and intervertebral disc formation using the twhh:gfp transgenic zebrafish model

To characterize the process of vertebral segmentation and disc formation in living animals, we analyzed tiggy-winkle hedgehog (twhh):green fluorescent protein (gfp) and sonic hedgehog (shh):gfp transgenic zebrafish models that display notochord-specific GFP expression. We found that they showed distinct patterns of expression in the intervertebral discs of late stage fish larvae and adult zebrafish. A segmented pattern of GFP expression was detected in the intervertebral disc of twhh:gfp transgenic fish. In contrast, little GFP expression was found in the intervertebral disc of shh:gfp transgenic fish. Treating twhh:gfp transgenic zebrafish larvae with exogenous retinoic acid (RA), a teratogenic factor on normal development, resulted in disruption of notochord segmentation and formation of oversized vertebrae. Histological analysis revealed that the oversized vertebrae are likely due to vertebral fusion. These studies demonstrate that the twhh:gfp transgenic zebrafish is a useful model for studying vertebral segmentation and disc formation, and moreover, that RA signaling may play a role in this process.     

Generation and characterization of DSPP‐Cerulean/DMP1‐Cherry reporter mice

To gain a better understanding of the progression of progenitor cells in the odontoblast lineage, we have examined and characterized the expression of a series of GFP reporters during odontoblast differentiation. However, previously reported GFP reporters (pOBCol2.3‐GFP, pOBCol3.6‐GFP, and DMP1‐GFP), similar to the endogenous proteins, are also expressed by bone‐forming cells, which made it difficult to delineate the two cell types in various in vivo and in vitro studies. To overcome these difficulties we generated DSPP‐Cerulean/DMP1‐Cherry transgenic mice using a bacterial recombination strategy with the mouse BAC clone RP24‐258g7. We have analyzed the temporal and spatial expression of both transgenes in tooth and bone in vivo and in vitro. This transgenic animal enabled us to visualize the interactions between odontoblasts and surrounding tissues including dental pulp, ameloblasts and cementoblasts. Our studies showed that DMP1‐Cherry, similar to Dmp1, was expressed in functional and fully differentiated odontoblasts as well as osteoblasts, osteocytes and cementoblasts. Expression of DSPP‐Cerulean transgene was limited to functional and fully differentiated odontoblasts and correlated with the expression of Dspp. This transgenic animal can help in the identification and isolation of odontoblasts at later stages of differentiation and help in better understanding of developmental disorders in dentin and odontoblasts.     

A mart-1::Cre transgenic line induces recombination in melanocytes and retinal pigment epithelium

The number of transgenic mouse lines expressing Cre in either type of pigment cells (melanocytes and retinal pigment epithelium, RPE) is limited, and the available lines do not always offer sufficient specificity. In this study, we addressed this issue and we report on the generation of a MART‐1::Cre BAC transgenic mouse line, in which the expression of Cre recombinase is controlled by regulatory elements of the pigment cell‐specific gene MART‐1 (mlana). When MART‐1::Cre BAC transgenic mice were bred with the ROSA26‐R reporter line, ß‐galactosidase expression was observed in RPE from E12.5 onwards, and in melanocyte precursors from E17.5, indicating that the MART‐1::Cre line provides Cre recombinase activity in pigment‐producing cells rather than in a particular lineage. In addition, breeding of this mouse line to mice carrying a conditional allele of RBP‐Jκ corroborated the reported phenotypes in both pigment cell lineages, inducing hair greying and microphthalmia. Our results thus suggest, that the MART‐1::Cre line may serve as a novel and useful tool for functional studies in melanocytes and the RPE.genesis 49:403–409, 2011. © 2011 Wiley‐Liss, Inc.