Xenopus laevis provides a unique animal model, alternative to mouse, to study immunology. Even though, several methodologies have been developed for the generation of transgenic Xenopus, to date none have been adapted for the Xenopus laevis/gilli (LG) isogenetic clones that are essential for immunological studies. Since LG clones are generated via gynogenesis, transgenic methods using transgene integration into the sperm nuclei are not suited. Therefore, we have tested three alternative methods for LG transgenesis: the phiC31 integrase, the Sleeping Beauty transposase and the I-SceI meganuclease. All three techniques produced transgenic LG clones; however, the I-SceI meganuclease was most effective. It resulted in high transgenesis efficiency (35–50%), bright non-mosaic GFP expression as well as stable germline transmission with 100% of the progeny carrying the transgene. Production of transgenic LG clones will allow us to modulate immune gene expression and further strengthen Xenopus laevis as a biomedical model.
Amphibian; Xenopus laevis; immunology; transgenesis; phiC31 integrase; Sleeping Beauty transposase
Two main classes of models address the earliest steps of left-right patterning: those postulating that asymmetry is initiated via cilia-driven fluid flow in a multicellular tissue at gastrulation, and those postulating that asymmetry is amplified from intrinsic chirality of individual cells at very early embryonic stages. A recent study revealed that cultured human cells have consistent left-right (LR) biases that are dependent on apical-basal polarity machinery. The ability of single cells to set up asymmetry suggests that cellular chirality could be converted to embryonic laterality by cilia-independent polarity mechanisms in cell fields. To examine the link between cellular polarity and LR patterning in a vertebrate model organism, we probed the roles of apical-basal and planar polarity proteins in the orientation of the LR axis in Xenopus. Molecular loss-of-function targeting these polarity pathways specifically randomizes organ situs independently of contribution to the ciliated organ. Alterations in cell polarity also disrupt tight junction integrity, localization of the LR signaling molecule serotonin, the normally left-sided expression of Xnr-1, and the LR instruction occurring between native and ectopic organizers. We propose that well-conserved polarity complexes are required for LR asymmetry and that cell polarity signals establish the flow of laterality information across the early blastoderm independently of later ciliary functions.
left-right asymmetry; Vangl2; Par6; conjoined twins; heterotaxia; ion flux
Epigenetic regulation defines the commitment and potential of cells, including the limitations in their competence to respond to inducing signals. This review discusses the developmental origins of chromatin state in Xenopus and other vertebrate species and provides an overview of its use in genome annotation. In most metazoans the embryonic genome is transcriptionally quiescent after fertilization. This involves nucleosome-dense chromatin, repressors and a temporal deficiency in the transcription machinery. Active histone modifications such as H3K4me3 appear in pluripotent blastula embryos, whereas repressive marks such as H3K27me3 show a major increase in enrichment during late blastula and gastrula stages. The H3K27me3 modification set by Polycomb restricts ectopic lineage-specific gene expression. Pluripotent chromatin in Xenopus embryos is relatively unconstrained, whereas the pluripotent cell lineage in mammalian embryos harbors a more enforced type of pluripotent chromatin.
chromatin; pluripotent; lineage specification; polycomb; mid-blastula transition; zygotic gene activation
Ciliated epithelia are important in a wide variety of biological contexts where they generate directed fluid flow. Here we address the fundamental advances in understanding ciliated epithelia that have been achieved using Xenopus as a model system. Xenopus embryos are covered with a ciliated epithelium that propels fluid unidirectionally across their surface. The external nature of this tissue, coupled with the molecular tools available in Xenopus and the ease of microscopic analysis on intact animals has thrust Xenopus to the forefront of ciliated epithelia biology. We discuss advances in understanding the molecular regulators of ciliated epithelia cell fate as well as basic aspects of ciliated epithelia cell biology including ciliogenesis and cell polarity.
Xenopus; cilia; ciliogenesis; planar cell polarity; cell fate specification
Transgenesis is an essential, powerful tool for investigating gene function and the activities of enhancers, promoters and transcription factors in the chromatin environment. In Xenopus, current methods generate germ-line transgenics by random insertion, often resulting in mosaicism, position-dependent variations in expression, and lab-to-lab differences in efficiency. We have developed and tested a Xenopus FLP-FRT Recombinase Mediated Transgenesis (X-FRMT) method. We demonstrate transgenesis of Xenopus laevis by FLP-catalyzed recombination of donor plasmid cassettes into F1 tadpoles with host cassette transgenes. X-FRMT provides a new method for generating transgenic Xenopus. Once Xenopus lines harboring single host cassettes are generated, X-FRMT should allow for the targeting of transgenes to well characterized integration site(s), requiring no more special reagents or training than that already common to most Xenopus labs.
Flippase; X-FRMT; recombinase-mediated cassette exchange; FLP-FRT recombination
Defining the regulatory molecular networks involved in patterning the developing anterior endoderm is essential to understanding how the pancreas, liver, stomach and duodenum are discretely specified from each other. In this study, we analyzed the expression and function of the double-stranded RNA-binding protein Staufen2 in Xenopus laevis endoderm. We found that staufen2 was broadly expressed within the developing endoderm beginning at gastrulation becoming localized to the anterior endoderm at later stages. Through morpholino-mediated knockdown, we demonstrate that Staufen2 function is required for proper formation of the stomach, liver and pancreas. We define that its function is required during gastrulation for proper patterning of the dorsal-ventral axis and that it acts to regulate expression of BMP signaling components.
Pancreas; RNA binding protein; Organizer; BMP; morpholino
Promoting ectopic development of pancreatic beta cells from other cell types is one of the strategies being pursued for the treatment of diabetes. To achieve this, a detailed outline of the molecular lineage that operates in pancreatic progenitor cells to generate beta cells over other endocrine cell types is necessary. Here, we demonstrate that early transient expression of the endocrine progenitor bHLH protein Neurogenin 3 (Ngn3) favors the promotion of pancreatic beta and delta cell fates over an alpha cell fate, while later transient expression promotes ectopic development of all three endocrine cell fates. We found that short-term activation of Ngn3 in Xenopus laevis endoderm just after gastrulation was sufficient to promote both early and ectopic development of beta and delta cells. By examining gene expression changes four hours after Ngn3 activation we identified several new downstream targets of Ngn3. We show that several of these are required for the promotion of ectopic beta cells by Ngn3 as well as for normal beta cell development. These results provide new detail regarding the Ngn3 transcriptional network operating in endocrine progenitor cells to specify a beta cell phenotype and should help define new approaches to promote ectopic development of beta cells for diabetes therapy.
Diabetes; Reprogramming; Endoderm Progenitor; Microarray; Endocrine; Transcription Factor; Hormone inducible
Using a combination of deep sequencing and bioinformatics approach, we for the first time identify miRNAs and their relative abundance in mature, metaphase II arrested eggs in X. laevis. We characterize 115 miRNAs that have been described either in X. tropicalis (85), X. laevis (9) or other vertebrate species (21) that also map to known Xenopus pre-miRNAs and to the X. tropicalis genome. Additionally, 72 new X. laevis putative candidate miRNAs are identified based on mapping to X. tropicalis genome within regions that have the propensity to form hairpin loops. These data expand on the availability of genetic information in X. laevis and identifies target miRNAs for future functional studies.
Deep sequencing; bioinformatics; miRBase; genetics; amphibian
A decade after the human genome sequence, most vertebrate gene functions remain poorly understood, limiting benefits to human health from rapidly advancing genomic technologies. Systematic in vivo functional analysis is ideally suited to the experimentally accessible Xenopus embryo, which combines embryological accessibility with a broad range of transgenic, biochemical and gain-of-function assays. The diploid X. tropicalis adds loss-of-function genetics and enhanced genomics to this repertoire. In the last decade diverse phenotypes have been recovered from genetic screens, mutations have been cloned, and reverse genetics in the form of TILLING and targeted gene editing have been established. Simple haploid genetics and gynogenesis and the very large number of embryos produced streamline screening and mapping. Improved genomic resources and the revolution in high-throughput sequencing are transforming mutation cloning and reverse genetic approaches. The combination of loss-of-function mutant backgrounds with the diverse array of conventional Xenopus assays offers a uniquely flexible platform for analysis of gene function in vertebrate development.
amphibian; early development; genetics; organogenesis; Xenopus; tropicalis
Dentin matrix protein1 (DMP1), highly conserved in humans and mice, is highly expressed in teeth, the skeleton, and to a lesser extent in nonskeletal tissues such as brain, kidney, and salivary gland. Pathologically, DMP1 is associated with several forms of cancers and with tumor-induced osteomalacia. Conventional disruption of the murine Dmp1 gene results in defects in dentin in teeth and in the skeleton, including hypophosphatemic rickets, and abnormalities in phosphate homeostasis. Human DMP1 mutations are responsible for the condition known as autosomal recessive hypophosphatemic rickets. For better understanding of the roles of DMP1 in different tissues at different stages of development and in pathological conditions, we generated Dmp1 floxed mice in which loxP sites flank exon 6 that encodes for over 80% of DMP1 protein. We demonstrate that Cre-mediated recombination using Sox2-Cre, a Cre line expressed in epiblast during early embryogenesis, results in early deletion of the gene and protein. These homozygous Cre-recombined null mice display an identical phenotype to conventional null mice. This animal model will be useful to reveal distinct roles of DMP1 in different tissues at different ages.
DMP1; Cre-loxP; gene targeting; mouse; bone
The spinal cord is the first site of temporal and spatial integration of nociceptive signals in the pain pathway. Neuroplastic changes occurring at this site contribute critically to various chronic pain syndromes. Gene targeting in mice has generated important insights into these processes. However, the analysis of constitutive (global) gene-deficient mice is often hampered by confounding effects arising from supraspinal sites. Here, we describe a novel Cre mouse line which expresses the Cre recombinase under the transcriptional control of the Hoxb8 gene. Within the neural axis of these mice, Hoxb8-Cre expression is found in spinal cord neurons and glial cells, and in virtually all neurons of the dorsal root ganglia, but spares the brain apart from a few cells in the spinal trigeminal nucleus. The Hoxb8-Cre mouse line should be a valuable new tool for the in vivo analysis of peripheral and spinal gene functions in pain pathways.
spinal cord; dorsal root ganglia; astrocytes; pain; Cre-loxP system; Hox genes; brain-sparing gene-deletion; glycine transporter type 1
Outer hair cells (OHCs) in the cochlea are crucial for the remarkable hearing sensitivity and frequency tuning. To understand OHC physiology and pathology, it is imperative to use mouse genetic tools to manipulate gene expression specifically in OHCs. Here, we generated 2 prestin knockin mouse lines: 1) the prestin-CreERT2 line, with an internal ribosome entry site (IRES)-CreERT2-FRT-Neo-FRT cassette inserted into the prestin locus after the stop codon, and 2) the prestin-CreERT2-NN line, with the FRT-Neo-FRT removed subsequently. We characterized the inducible Cre activity of both lines by crossing them with the reporter lines CAG-eGFP and Ai6. Cre activity was induced with tamoxifen at various postnatal ages and only detected in OHCs, resembling the endogenous prestin expression pattern. Moreover, prestin-CreERT2 +/− (heterozygotes) and +/+ (homozygotes) as well as prestin-CreERT2-NN +/− mice displayed normal hearing. These 2 prestin-CreERT2 mouse lines are therefore useful tools to analyze gene function in OHCs in vivo.
Prestin; Cre recombinase; inner ear; outer hair cells
Cre-mediated apoptosis has been observed in many contexts in mice expressing Cre-recombinase, and can confound the analysis of genetically engineered conditional mutant or transgenic alleles. Several mechanisms have been proposed to explain this phenomenon. We find that the degree of apoptosis induced correlates roughly with the copy number of loxP sites present in the genome and that some level of increased apoptosis accompanies the presence of even only a few loxP sites, as occurs in conditional floxed alleles. Cre-induced apoptosis in this context is completely p53-dependent, suggesting that the apoptosis is stimulated by p53 activation in response to DNA damage incurred during the process of Cre-mediated recombination.
transgenic mice; loxP sites; Cre recombinase; apoptosis
The p75NTR neurotrophin receptor has been implicated in multiple biological and pathological processes. While significant advances have recently been made in understanding the physiologic role of p75NTR, many details and aspects remain to be determined. This is in part because the two existing knockout mouse models (exons 3 or 4 deleted, respectively), both display features that defy definitive conclusions. Here we describe the generation of mice that carry a conditional p75NTR (p75NTR-FX) allele made by flanking exons 4–6, which encode the transmembrane and all cytoplasmic domains, by loxP sites. To validate this novel conditional allele, both neural crest-specific p75NTR/Wnt1-Cre mutants and conventional p75NTR null mutants were generated. Both mutants displayed abnormal hind limb reflexes, implying that loss of p75NTR in neural crest-derived cells causes a peripheral neuropathy similar to that seen in conventional p75NTR mutants. This novel conditional p75NTR allele will offer new opportunities to investigate the role of p75NTR in specific tissues and cells.
nerve growth factor receptor; signaling; mouse mutant; growth retardation; nerve abnormalities
Wnt/β-catenin signaling initiates taste papilla development in mouse embryos, however, its involvement in taste cell turnover in adult mice has not been explored. Here we used the BATGAL reporter mouse model, which carries an engineered allele in which the LacZ gene is expressed in the presence of activated β-catenin, to determine the responsiveness of adult taste bud cells to canonical Wnt signaling. Double immunostaining with markers of differentiated taste cells revealed that a subset of type I, II and III taste cells express β-galactosidase. Using in situ hybridization, we showed that β-catenin activates the transcription of the LacZ gene mainly in intragemmal basal cells that are immature taste cells, identified by their expression of Sonic Hedgehog (Shh). Finally, we showed that β-catenin activity is significantly reduced in taste buds of 25 week-old mice compared to 10 week-old animals. Our data suggest that Wnt/β-catenin signaling may influence taste cell turnover by regulating cell differentiation. Reduced canonical Wnt signaling in older mice could explain in part the loss of taste sensitivity with aging, implicating a possible deficiency in the rate of taste cell renewal. More investigations are now necessary to understand if and how Wnt signaling regulates adult taste cell turnover.
Taste cell; Wnt/β-catenin; Sonic Hedgehog (Shh); cell renewal; BATGAL; aging
The ovaries of early embryos (40 days after fertilization) of the bat Carollia perspicillata contain numerous germ-line cysts, which are composed of 10 to 12 sister germ cells (cystocytes). The variability in the number of cystocytes within the cyst and between the cysts (that defies the Giardina rule) indicates that the mitotic divisions of the cystoblast are asynchronous in this bat species. The serial section analysis showed that the cystocytes are interconnected via intercellular bridges that are atypical, strongly elongated, short-lived, and rich in microtubule bundles and microfilaments. During the later stages of embryonic development (44–46 days after fertilization), the somatic cells penetrate the cyst, and their cytoplasmic projections separate individual oocytes. Separated oocytes surrounded by the single layer of somatic cells constitute the primordial ovarian follicles. The oocytes of C. perspicillata are similar to mouse oocytes and are asymmetric. In both species, this asymmetry is clearly recognizable in the localization of the Golgi complexes. The presence of germ-line cysts and intercellular bridges (although non-canonical) in the fetal ovaries of C. perspicillata indicate that the formation of germ-line cysts is an evolutionarily conserved phase in the development of the female gametes throughout the animal kingdom.
ovary; germ-line cyst; intercellular bridge; Chiroptera
Live-imaging is an essential tool to visualize live cells and monitor their behaviors during development. This technology demands a variety of mouse reporter lines, each uniquely expressing a fluorescent protein. Here, we developed an R26R-RG reporter mouse line that conditionally and simultaneously expresses mCherry and EGFP in nuclei and plasma membranes, respectively, from the Rosa26 locus. The intensity and resolution of mCherry nuclear localization and EGFP membrane localization were demonstrated to be sufficient for live-imaging with embryos that express RG (mCherry and EGFP) ubiquitously and specifically in fetal Sertoli cells. The conditional R26R-RG reporter mouse line should be a useful tool for labeling nuclei and membranes simultaneously in distinct cell populations.
Rosa26 locus; conditional cell labeling; dual cell labeling; nuclear labeling; membrane labeling
Transgenic mice are vital tools in both basic and applied research. Unfortunately, the transgenesis process as well as many other assisted reproductive techniques involving embryo transfer rely on vasectomized males to induce pseudopregnancy in surrogate mothers. Vasectomy is a surgical procedure associated with moderate pain and must be carried out under full anaesthesia by qualified personnel. Eliminating the need for vasectomy would be beneficial from the economic and animal welfare point of view. Our aim was to develop a transgene-based alternative to the surgical vasectomy procedure. We generated several transgenic mouse lines expressing a Protamine-1 (Prm1) EGFP fusion protein under the transcriptional and translational regulatory control of Prm1. Male mice from lines showing moderate transgene expression were fully fertile whereas strong overexpression of the Prm1-EGFP fusion protein resulted in complete and dominant male sterility without affecting the ability to mate and to produce copulatory plugs. Sterility was due to impaired spermatid maturation affecting sperm viability and motility. Furthermore, sperm having high Prm1-EGFP levels failed to support preimplantation embryonic development following Intracytoplasmic Sperm Injection (ICSI). The “genetic vasectomy system” was further improved by genetically linking the dominant male sterility to ubiquitous EGFP expression in the soma as an easy phenotypic marker enabling rapid genotyping of transgenic males and females. This double transgenic approach represents a reliable and cost-effective “genetic vasectomy” procedure making the conventional surgical vasectomy methodology obsolete.
vasectomy; transgenesis; protamine; spermatozoa; fertilization
Because of their capacity to give rise to various types of cells in vitro, embryonic stem and embryonal carcinoma (EC) cells have been used as convenient models to study the mechanisms of cell differentiation in mammalian embryos. In this study, we explored the mouse P19 EC cell line as an effective tool to investigate the factors that may play essential roles in mesoderm formation and axial elongation morphogenesis. We first demonstrated that aggregated P19 cells not only exhibited gene expression patterns characteristic of mesoderm formation but also displayed elongation morphogenesis with a distinct anterior–posterior body axis as in the embryo. We then showed by RNA interference that these processes were controlled by various regulators of Wnt signaling pathways, namely β-catenin, Wnt3, Wnt3a, and Wnt5a, in a manner similar to normal embryo development. We further showed by inhibitor treatments that the axial elongation morphogenesis was dependent on the activity of Rho-associated kinase. Because of the convenience of these experimental manipulations, we propose that P19 cells can be used as a simple and efficient screening tool to assess the potential functions of specific molecules in mesoderm formation and axial elongation morphogenesis.
Wnt3; Wnt3a; Wnt5a; β-catenin; epiblast; convergent extension; ROCK
The Bmp2 3'untranslated region (UTR) sequence bears a sequence conserved between mammals and fishes that can post-transcriptionally activate or repress protein synthesis. We developed a map of embryonic cells in the mouse where this potent Bmp2 regulatory sequence functions by using a lacZ reporter transgene with a 3’UTR bearing two loxP sites flanking the ultra-conserved sequence. Cre-recombinase-mediated deletion of the ultra-conserved sequence caused strong ectopic expression in proepicardium, epicardium and epicardium-derived cells (EPDC) and in tissues with known epicardial contributions (coronary vessels and valves). Transient transfections of reporters in the epicardial/mesothelial cell (EMC) line confirmed this repression. Ectopic expression of the recombined transgene also occurred in the aorta, outlet septum, posterior cardiac plexus, cardiac and extra-cardiac nerves and neural ganglia. Bmp2 is dynamically regulated in the developing heart. 3’UTR-mediated mechanisms that restrain BMP2 synthesis may be relevant to congenital heart and vasculature malformations and to adult diseases involving aberrant BMP2 synthesis.
mRNA; post-transcriptional; gene regulation; transgenic mice; aorta; coronary vasculature; epicardial cells; neural crest
With the intention to modulate gene expression in vascular mural cells of remodeling vessels, we generated and characterized transgenic mouse lines with Cre recombinase under the control of the platelet-derived growth factor receptor-β promoter, referred to as Tg(Pdgfrb-Cre)35Vli. Transgenic mice were crossed with the Gt(ROSA)26Sortm1Sor strain and examined for Cre activation by β-galactosidase activity, which was compared with endogenous Pdgfrb expression. In addition, Pdgfrb-Cre mice were used to drive expression of a conditional myc-tagged Cthrc1 transgene. There was good overlap of β-galactosidase activity with endogenous Pdgfrb immunoreactivity. However, dedifferentiation of vascular mural cells induced by carotid artery ligation revealed a dramatic discrepancy between ROSA26 reporter activity and Pdgfrb promoter driven Cre dependent myc-tagged Cthrc1 transgene expression. Our studies demonstrate the capability of the Pdgfrb-Cre mouse to drive conditional transgene expression as a result of prior Cre mediated recombination in tissues known to express endogenous Pdgfrb. In addition, the study shows that ROSA26 promoter driven reporter mice are not suitable for lineage marking of smooth muscle in remodeling blood vessels.
vascular smooth muscle; dedifferentiation; Cthrc1
A transgenic mouse line named iUBC-KikGR was generated which expresses the photoconvertible fluorescent protein Kikume Green-Red (KikGR) under the control of the human Ubiquitin C promoter. KikGR is natively a green fluorophore which can be converted into a red fluorophore upon exposure to UV light. KikGR is expressed broadly throughout transgenic embryos from the 2-cell stage onward, and in the adult. Specificity of photoconversion can range from the entire embryo, to a region of an organ, to a few individual cells, depending on the needs of the experimenter. Cell movements, tissue reorganization, and migration can then be observed in real time by culturing the tissue of interest as an explant on the microscope stage. The iUBC-KikGR transgenic line represents a singular genetic reagent which can be used for fate mapping, lineage tracing, and live visualization of cell behaviors and tissue movements in multiple organs at multiple time points.
fate mapping; lineage tracing; photoconvertible fluorescent protein; KikGR; explant culture
We have characterized a transgenic mouse line in which enhanced green fluorescent protein (EGFP) is expressed under the control of multimerized LEF-1 responsive elements. In embryos, EGFP was detected in known sites of Wnt activation, including the primitive streak, mesoderm, neural tube, somites, heart, limb buds, mammary placodes, and whisker follicles. In vitro cultured transgenic embryonic fibroblasts upregulated EGFP expression in response to activation of Wnt signaling by GSK3β inhibition. Mammary tumor cell lines derived from female LEF-EGFP transgenic mice treated with the carcinogen 7, 12-dimethylbenz[a]anthracene (DMBA) also express EGFP. Thus, this transgenic line is useful for ex vivo and in vitro studies of Wnt signaling in development and cancer.
Wnt reporter; EGFP; embryo; β-catenin; transgenic mouse; breast cancer
Time-lapse imaging is often the only way to appreciate fully the many dynamic cell movements critical to neural development. Zebrafish possess many advantages that make them the best vertebrate model organism for live imaging of dynamic development events. This review will discuss technical considerations of time-lapse imaging experiments in zebrafish, describe selected examples of imaging studies in zebrafish that revealed new features or principles of neural development, and consider the promise and challenges of future time-lapse studies of neural development in zebrafish embryos and adults.
zebrafish; imaging; time-lapse; neural development; neurons
To facilitate dynamic imaging of neural crest (NC) lineages and discrimination of individual cells in the enteric nervous system (ENS) where close juxtaposition often complicates viewing, we generated a mouse BAC transgenic line that drives a Histone2BVenus (H2BVenus) reporter from Sox10 regulatory regions. This strategy does not alter the endogenous Sox10 locus and thus facilitates analysis of normal NC development. Our Sox10-H2BVenus BAC transgene exhibits temporal, spatial, and cell-type specific expression that reflects endogenous Sox10 patterns. Individual cells exhibiting nuclear–localized fluorescence of the H2BVenus reporter are readily visualized in both fixed and living tissue and are amenable to isolation by fluorescence activated cell sorting (FACS). FACS-isolated H2BVenus+ enteric NC-derived progenitors (ENPs) exhibit multi-potency, readily form neurospheres, self-renew in vitro and express a variety of stem cell genes. Dynamic live imaging as H2BVenus+ ENPs migrate down the fetal gut reveals cell fragmentation suggesting that apoptosis occurs at a low frequency during normal development of the ENS. Confocal imaging both during population of the fetal intestine and in post-natal gut muscle strips revealed differential expression between individual cells consistent with down-regulation of the transgene as progression towards non-glial fates occurs. The expression of the Sox10-H2BVenus transgene in multiple regions of the peripheral nervous system will facilitate future studies of NC lineage segregation as this tool is expressed in early NC progenitors and maintained in enteric glia.
Neural Crest; Sox10 Transcription Factor; Enteric Nervous System; Bacterial Artificial Chromosome (BAC); Live Imaging; Lineage Restriction