Histone H3.3 maintains genome integrity during mammalian development.

Histone H3.3 is a highly conserved histone H3 replacement variant in metazoans and has been implicated in many important biological processes, including cell differentiation and reprogramming. Germline and somatic mutations in H3.3 genomic incorporation pathway components or in H3.3 encoding genes have been associated with human congenital diseases and cancers, respectively. However, the role of H3.3 in mammalian development remains unclear. To address this question, we generated H3.3-null mouse models through classical genetic approaches. We found that H3.3 plays an essential role in mouse development. Complete depletion of H3.3 leads to developmental retardation and early embryonic lethality. At the cellular level, H3.3 loss triggers cell cycle suppression and cell death. Surprisingly, H3.3 depletion does not dramatically disrupt gene regulation in the developing embryo. Instead, H3.3 depletion causes dysfunction of heterochromatin structures at telomeres, centromeres, and pericentromeric regions of chromosomes, leading to mitotic defects. The resulting karyotypical abnormalities and DNA damage lead to p53 pathway activation. In summary, our results reveal that an important function of H3.3 is to support chromosomal heterochromatic structures, thus maintaining genome integrity during mammalian development.

PiggyBac Transposon-Mediated Mutagenesis in Rats Reveals a Crucial Role of Bbx in Growth and Male Fertility.

Bobby sox homolog (Bbx) is an evolutionally conserved gene, but its biological function remains elusive. Here, we characterized defects of Bbx mutant rats that were created by PiggyBac-mediated insertional mutagenesis. Smaller body size and male infertility were the two major phenotypes of homozygous Bbx mutants. Bbx expression profile analysis showed that Bbx was more highly expressed in the testis and pituitary gland than in other organs. Histology and hormonal gene expression analysis of control and Bbx-null pituitary glands showed that loss of Bbx appeared to be dispensable for pituitary histogenesis and the expression of major hormones. BBX was localized in the nuclei of postmeiotic spermatids and Sertoli cells in wild-type testes, but absent in mutant testes. An increased presence of aberrant multinuclear giant cells and apoptotic cells was observed in mutant seminiferous tubules. TUNEL-positive cells costained with CREM (round spermatid marker), but not PLZF (spermatogonia marker), gammaH2Ax (meiotic spermatocyte marker), or GATA4 (Sertoli cell marker). Finally, there were drastically reduced numbers and motility of epididymal sperm from Bbx-null rats. These results suggest that loss of BBX induces apoptosis of postmeiotic spermatids and results in spermiogenesis defects and infertility.

Ikbkap/Elp1 deficiency causes male infertility by disrupting meiotic progression.

Mouse Ikbkap gene encodes IKAP--one of the core subunits of Elongator--and is thought to be involved in transcription. However, the biological function of IKAP, particularly within the context of an animal model, remains poorly characterized. We used a loss-of-function approach in mice to demonstrate that Ikbkap is essential for meiosis during spermatogenesis. Absence of Ikbkap results in defects in synapsis and meiotic recombination, both of which result in increased apoptosis and complete arrest of gametogenesis. In Ikbkap-mutant testes, a few meiotic genes are down-regulated, suggesting IKAP's role in transcriptional regulation. In addition, Ikbkap-mutant testes exhibit defects in wobble uridine tRNA modification, supporting a conserved tRNA modification function from yeast to mammals. Thus, our study not only reveals a novel function of IKAP in meiosis, but also suggests that IKAP contributes to this process partly by exerting its effect on transcription and tRNA modification.

Generation of knock-in mice that express nuclear enhanced green fluorescent protein and tamoxifen-inducible Cre recombinase in the notochord from Foxa2 and T loci.

The node and the notochord are important embryonic signaling centers that control embryonic pattern formation. Notochord progenitor cells present in the node and later in the posterior end of the notochord move anteriorly to generate the notochord. To understand the dynamics of cell movement during notochord development and the molecular mechanisms controlling this event, analyses of cell movements using time-lapse imaging and conditional manipulation of gene activities are required. To achieve this goal, we generated two knock-in mouse lines that simultaneously express nuclear enhanced green fluorescent protein (EGFP) and tamoxifen-inducible Cre, CreER(T2) , from two notochord gene loci, Foxa2 and T (Brachury). In Foxa2(nEGFP-CreERT2/+) and T(nEGFP-CreERT2/+) embryos, nuclei of the Foxa2 or T-expressing cells, which include the node, notochord, and endoderm (Foxa2) or wide range of posterior mesoderm (T), were labeled with EGFP at intensities that can be used for live imaging. Cre activity was also induced in cells expressing Foxa2 and T 1 day after tamoxifen administration. These mice are expected to be useful tools for analyzing the mechanisms of notochord development.

Pronephric tubulogenesis requires Daam1-mediated planar cell polarity signaling.

Canonical ß-catenin-mediated Wnt signaling is essential for the induction of nephron development. Noncanonical Wnt/planar cell polarity (PCP) pathways contribute to processes such as cell polarization and cytoskeletal modulation in several tissues. Although PCP components likely establish the plane of polarization in kidney tubulogenesis, whether PCP effectors directly modulate the actin cytoskeleton in tubulogenesis is unknown. Here, we investigated the roles of Wnt PCP components in cytoskeletal assembly during kidney tubule morphogenesis in Xenopus laevis and zebrafish. We found that during tubulogenesis, the developing pronephric anlagen expresses Daam1 and its interacting Rho-GEF (WGEF), which compose one PCP/noncanonical Wnt pathway branch. Knockdown of Daam1 resulted in reduced expression of late pronephric epithelial markers with no apparent effect upon early markers of patterning and determination. Inhibiting various points in the Daam1 signaling pathway significantly reduced pronephric tubulogenesis. These data indicate that pronephric tubulogenesis requires the Daam1/WGEF/Rho PCP pathway.

Generation and characterization of iUBC-KikGR photoconvertible transgenic mice for live time-lapse imaging during development.

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 two-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.

A mouse reporter line to conditionally mark nuclei and cell membranes for in vivo live-imaging.

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.

Sertoli cell behaviors in developing testis cords and postnatal seminiferous tubules of the mouse.

Sertoli cells are the primary structural component of the fetal testis cords and postnatal seminiferous tubules. Live imaging technologies facilitate the visualization of cell morphologies and behaviors through developmental processes. A transgenic mouse line was generated using a fragment of the rat Gata4 gene to direct the expression of a dual-color fluorescent protein reporter in fetal and adult Sertoli cells. The reporter encoded a red fluorescent protein, monomeric Cherry (mCherry), fused to histone 2B and enhanced green fluorescent protein (EGFP) fused to a glycosylphosphatidylinositol sequence, with a self-cleaving 2A polypeptide separating the two fusion proteins. After translation, the red and green fluorescent proteins translocated to the nucleus and plasma membrane, respectively, of Sertoli cells. Transgene expression in testes was first detected by fluorescent microscopy around Embryonic Day 12.0. Sertoli cell division and migration were visualized during testis cord formation in organ culture. Initially, the Sertoli cells had mesenchyme-like morphologies and behaviors, but later, the cells migrated to the periphery of the testis cords to become epithelialized. In postnatal seminiferous tubules, Sertoli nuclei were evenly spaced when viewed from the external surface of tubules, and Sertoli cytoplasm and membranes were associated with germ cells basally in a rosette pattern. This mouse line was bred to previously described transgenic mouse lines expressing EGFP in Sertoli cytoplasm or a nuclear cyan fluorescent protein (Cerulean) and mCherry in plasma membranes of germ cells. This revealed the physical relationship between Sertoli and germ cells in developing testis cords and provided a novel perspective on Sertoli cell development.

TGF-beta induces apoptosis through Smad-mediated expression of DAP-kinase.

Transforming growth factor-beta (TGF-beta) and TGF-beta-related factors induce apoptosis in a variety of tissues; however, the mechanism underlying this induction is largely unknown. Here, we demonstrate that TGF-beta induces the expression of the death-associated protein kinase (DAP-kinase) as an immediate early response in cells that undergo apoptosis in response to TGF-beta. DAP-kinase is a positive mediator of apoptosis induced by certain cytokines and oncogenes. We show that the DAP-kinase promoter is activated by TGF-beta through the action of Smad2, Smad3 and Smad4. Overexpression of DAP-kinase triggers apoptosis in the absence of TGF-beta, whereas inhibition of DAP-kinase activity protects cells from TGF-beta-induced apoptosis, blocks TGF-beta-induced release of cytochrome c from mitochondria and prevents TGF-beta-induced dissipation of the mitochondrial membrane potential. Our findings indicate that DAP-kinase mediates TGF-beta-dependent apoptosis by linking Smads to mitochondrial-based pro-apoptotic events.

Ephrin A4-ephrin receptor A10 signaling promotes cell migration and spheroid formation by upregulating NANOG expression in oral squamous cell carcinoma cells.

Ephrin type-A receptor 10 (EPHA10) has been implicated as a potential target for breast and prostate cancer therapy. However, its involvement in oral squamous cell carcinoma (OSCC) remains unclear. We demonstrated that EPHA10 supports in vivo tumor growth and lymphatic metastasis of OSCC cells. OSCC cell migration, epithelial mesenchymal transition (EMT), and sphere formation were found to be regulated by EPHA10, and EPHA10 was found to drive expression of some EMT- and stemness-associated transcription factors. Among EPHA10 ligands, exogenous ephrin A4 (EFNA4) induced the most OSCC cell migration and sphere formation, as well as up-regulation of SNAIL, NANOG, and OCT4. These effects were abolished by extracellular signal-regulated kinase (ERK) inhibition and NANOG knockdown. Also, EPHA10 was required for EFNA4-induced cell migration, sphere formation, and expression of NANOG and OCT4 mRNA. Our microarray dataset revealed that EFNA4 mRNA expression was associated with expression of NANOG and OCT4 mRNA, and OSCC patients showing high co-expression of EFNA4 with NANOG or OCT4 mRNA demonstrated poor recurrence-free survival rates. Targeting forward signaling of the EFNA4-EPHA10 axis may be a promising therapeutic approach for oral malignancies, and the combination of EFNA4 mRNA and downstream gene expression may be a useful prognostic biomarker for OSCC.

Generation of aggrecan-CreERT2 knockin mice for inducible Cre activity in adult cartilage.

The function of cartilage in the adult is dependent on a host of regulatory molecules such as growth factors, extracellular matrix, enzymes, signaling molecules, and transcription factors. However, germline mutations in some genes that are expressed in adult cartilage lead to embryonic or perinatal lethality. To examine the function of these and other genes postnatally, we have generated a targeted mouse by homologous recombination that "knocks in" the inducible Cre recombinase construct, CreERT2, in the 3' untranslated region of the endogenous mouse aggrecan gene (Agc1(tm(IRES-creERT2))). The properties and efficiency of the inducible cre recombinase were tested by examining X-gal staining of tissues from embryos as well as growing and adult Agc1(tm(IRES-creERT2)/+);Rosa 26R mice. These mice were injected with the inducer, tamoxifen, at different time points during embryonic development and postnatally up to 6 months of age. Strong X-gal staining was observed in growth plate and articular cartilage as well as the fibrocartilage of meniscus, trachea, and intervertebral discs reproducing the pattern of endogenous aggrecan gene expression. In conclusion, we have generated a mouse model in which genes implicated in cartilage degenerative diseases can be inactivated in a spatial and temporal fashion in postnatal and adult mice.

Dual fluorescent protein reporters for studying cell behaviors in vivo.

Fluorescent proteins (FPs) are useful tools for visualizing live cells and their behaviors. Protein domains that mediate subcellular localization have been fused to FPs to highlight cellular structures. FPs fused with histone H2B incorporate into chromatin allowing visualization of nuclear events. FPs fused to a glycosylphosphatidylinositol anchor signal sequence label the plasma membrane, highlighting cellular shape. Thus, a reporter gene containing both types of FP fusions would allow for effective monitoring of cell shape, movement, mitotic stage, apoptosis, and other cellular activities. Here, we report a binary color-coding system using four differently colored FP reporters that generates 16 distinct color codes to label the nuclei and plasma membranes of live cells in culture and in transgenic mice. As an initial test of this system in vivo, the promoter of the human Ubiquitin C (UBC) gene was used to widely express one of the color-code reporters. Widespread expression of the reporter was attained in embryos; however, both male and female transgenic mice were infertile. In contrast, the promoter of the mouse Oct4/Pou5f1 gene linked to two different color-code reporters specifically labeled blastocysts, primordial germ cells, and postnatal germ cells, and these mice were fertile. Time-lapse movies of fluorescently-labeled primordial germs cells demonstrate the utility of the color-code system to visualize cell behaviors. This set of new FP reporters should be a useful tool for labeling distinct cell populations and studying their behaviors in complex tissues in vivo.

Interferon-stimulated gene 15 modulates cell migration by interacting with Rac1 and contributes to lymph node metastasis of oral squamous cell carcinoma cells.

In an effort to understand the underlying mechanisms of lymph node metastasis in oral squamous cell carcinoma (OSCC), through in vivo selection, LN1-1 cells were previously established from OEC-M1 cells and showed enhanced lymphangiogenesis and lymphatic metastasis capabilities. In the current study, we use a stable isotope labeling with amino acids in cell culture (SILAC) and liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based proteomic platform to compare LN1-1 to OEC-M1 cells. Interferon-stimulated gene 15 (ISG15) was found highly expressed in LN1-1 cells. Immunohistochemical analysis and meta-analysis of publicly available microarray datasets revealed that the ISG15 level was increased in human OSCC tissues and associated with poor disease outcome. Knockdown of ISG15 had minimal effects on tumor growth but did decrease tumor lymphangiogenesis and lymphatic metastasis of LN1-1 cells. Consistent with the in vivo assay, ISG15 knockdown did not impair cell growth but diminished cell migration, invasion, and transendothelial migration in vitro. ISG15-induced cell migration was independent of ISGylation and associated with membrane protrusion. Ectopic expression of ISG15 increased Rac1 activity and knockdown of Rac1 impaired ISG15-enhanced migration. Furthermore, Rac1 colocalized with ISG15 to a region of membrane protrusion and ISG15 coimmunoprecipitated with Rac1, especially with the Rac1-GDP form. Importantly, as shown by proximity ligation assays, ISG15 and Rac1 physically interacted with each other. Our results indicated that ISG15 affects cell migration by interacting with Rac1 and regulating Rac1 activity and contributes to lymphatic metastasis in OSCC.

ERK Activation Modulates Cancer Stemness and Motility of a Novel Mouse Oral Squamous Cell Carcinoma Cell Line.

We established the NHRI-HN1 cell line from a mouse tongue tumor induced by 4-nitroquinoline 1-oxide (4-NQO)/arecoline, with further selection for cell stemness via in vitro sphere culture, to evaluate potential immunotherapies for oral squamous cell carcinoma (OSCC) in East and Southeast Asia. In vivo and in vitro phenotypic characterization, including tumor growth, immune modulator administration, gene expression, morphology, migration, invasion, and sphere formation assays, were conducted. NHRI-HN1 cells are capable of generating orthotopic tumors in syngeneic mice. Interestingly, immune stimulation via CpG oligodeoxynucleotide (CpG-ODN) dramatically reduced the tumor growth in NHRI-HN1 cell-injected syngeneic mice. The pathways enriched in genes that were differentially expressed in NHRI-HN1 cells when compared to non-tumorigenic cells were similar to those that were identified when comparing human OSCC and non-tumorous tissues. NHRI-HN1 cells have characteristics of epithelial-mesenchymal transition (EMT), including enhanced migration and invasion. NHRI-HN1 cells showed aggressive cell growth and sphere formation. The blockage of extracellular signal-regulated kinase (ERK) activation suppressed cell migration and reduced stemness characteristics in NHRI-HN1 cells, similar to human OSCC cell lines. Our data suggest that NHRI-HN1 cells, showing tumorigenic characteristics of EMT, cancer stemness, and ERK activation, are sufficient in modeling human OSCC and also competent for use in investigating oral cancer immunotherapies.

A novel selection marker for efficient DNA cloning and recombineering in E. coli.

Production of recombinant DNA in bacterial cells is an essential technique in molecular biology. Plasmids are usually maintained in an E. coli host by antibiotic selection. However, there are only a few antibiotic-resistance markers available in common use. Here we report the adoption of a novel selection marker, mfabI (mutant fabI) for plasmid propagation in E. coli. mfabI expands the limited repertoire of selection markers and allows for more efficient molecular manipulation and plasmid propagation in E. coli. We show that mfabI is not only an efficient plasmid selection marker, but it also possesses unique activity that may facilitate molecular manipulation of unstable sequences. Furthermore, we have incorporated mfabI in the recombineering tool kit for generating mouse gene targeting vectors and demonstrate the advantage of using mfabI-containing recombineering vectors.

Insertional mutagenesis by a hybrid piggyBac and sleeping beauty transposon in the rat.

A hybrid piggyBac/Sleeping Beauty transposon-based insertional mutagenesis system that can be mobilized by simple breeding was established in the rat. These transposons were engineered to include gene trap sequences and a tyrosinase (Tyr) pigmentation reporter to rescue the albinism of the genetic background used in the mutagenesis strategy. Single-copy transposon insertions were transposed into the rat genome by co-injection of plasmids carrying the transposon and RNA encoding piggyBac transposase into zygotes. The levels of transgenic Tyr expression were influenced by chromosomal context, leading to transgenic rats with different pigmentation that enabled visual genotyping. Transgenic rats designed to ubiquitously express either piggyBac or Sleeping Beauty transposase were generated by standard zygote injection also on an albino background. Bigenic rats carrying single-copy transposons at known loci and transposase transgenes exhibited coat color mosaicism, indicating somatic transposition. PiggyBac or Sleeping Beauty transposase bigenic rats bred with wild-type albino rats yielded offspring with pigmentation distinct from the initial transposon insertions as a consequence of germline transposition to new loci. The germline transposition frequency for Sleeping Beauty and piggyBac was ∼10% or about one new insertion per litter. Approximately 50% of the insertions occurred in introns. Chimeric transcripts containing endogenous and gene trap sequences were identified in Gabrb1 mutant rats. This mutagenesis system based on simple crosses and visual genotyping can be used to generate a collection of single-gene mutations in the rat.

R26R-GR: a Cre-activable dual fluorescent protein reporter mouse.

Green fluorescent protein (GFP) and its derivatives are the most widely used molecular reporters for live cell imagining. The development of organelle-specific fusion fluorescent proteins improves the labeling resolution to a higher level. Here we generate a R26 dual fluorescent protein reporter mouse, activated by Cre-mediated DNA recombination, labeling target cells with a chromatin-specific enhanced green fluorescence protein (EGFP) and a plasma membrane-anchored monomeric cherry fluorescent protein (mCherry). This dual labeling allows the visualization of mitotic events, cell shapes and intracellular vesicle behaviors. We expect this reporter mouse to have a wide application in developmental biology studies, transplantation experiments as well as cancer/stem cell lineage tracing.

Bmp4-directed nuclear cyan fluorescent protein provides a tool for live imaging and reveals cellular resolution of Bmp4 expression patterns during embryogenesis.

The genesis of hair follicles in mammals involves multiple inductive and suppressive signaling interactions between epithelial cells and the underlying mesenchymal tissue. Bmp4 (Bone Morphogenetic Protein 4) is expressed in the mesenchymal tissue surrounding developing hair follicles. The BMP signaling pathway is suggested to play a suppressive role in hair follicle induction. In addition, it is also found to be important for specification of different cell types in mature hair follicles. Knowledge of the precise expression pattern of Bmp4 during hair follicle differentiation should provide insights into how these suppressive and differentiative roles regulate hair follicle development. However, in situ hybridization studies do not provide sufficient cellular resolution, and three-dimensional reconstructions of serial sections are tedious. We have targeted a nuclear-localized cyan fluorescent protein (CFP) reporter into the endogenous Bmp4 locus. Nuclear CFP expression was detected in embryonic tissues in a Bmp4-specific pattern, including lateral mesoderm, limb bud, dorsal optic cup, lung bud epithelium, heart outflow tract, otocyst, branchial arches, nasal pits, mammary buds, vibrassa and hair follicles. In developing hair follicles, the nuclear CFP reporter provided precise cellular resolution of Bmp4 expression patterns. These mice provide a novel tool for visualizing Bmp4 expression patterns in live and fixed tissues with cellular resolution. In addition, these studies reveal a novel view of the arrangement and dynamic changes of Bmp4-expressing cells of different stage hair follicles.

PiggyBac transposon-mediated, reversible gene transfer in human embryonic stem cells.

Permanent and reversible genetic modifications are important approaches to study gene function in different cell types. They are also important for stem cell researchers to explore and test the therapeutic potential of stem cells. The piggyBac transposon from insects is a rising nonviral system that efficiently mutagenizes and mediates gene transfer into the mammalian genome. It is also characterized by its precise excision, leaving no trace sequence behind so that the genomic integrity of the mutated cell can be restored. Here, we use an optimized piggyBac transposon system to mediate gene transfer and expression of a bifunctional fluorescent reporter in human embryonic stem (ES) cells. We provide molecular evidence for transposase-mediated piggyBac integration events and functional evidence for successful expression of a transferred fluorescent protein genes in human ES cells and their in vitro differentiated derivatives. We also demonstrate that the integrated piggyBac transposon can be removed and an undisrupted insertion site can be restored, which implies potential applications for its use in gene therapy and genetics studies.

Wnt signaling maintains the notochord fate for progenitor cells and supports the posterior extension of the notochord.

The notochord develops from notochord progenitor cells (NPCs) and functions as a major signaling center to regulate trunk and tail development. NPCs are initially specified in the node by Wnt and Nodal signals at the gastrula stage. However, the underlying mechanism that maintains the NPCs throughout embryogenesis to contribute to the posterior extension of the notochord remains unclear. Here, we demonstrate that Wnt signaling in the NPCs is essential for posterior extension of the notochord. Genetic labeling revealed that the Noto-expressing cells in the ventral node contribute the NPCs that reside in the tail bud. Robust Wnt signaling in the NPCs was observed during posterior notochord extension. Genetic attenuation of the Wnt signal via notochord-specific beta-catenin gene ablation resulted in posterior truncation of the notochord. In the NPCs of such mutant embryos, the expression of notochord-specific genes was down-regulated, and an endodermal marker, E-cadherin, was observed. No significant alteration of cell proliferation or apoptosis of the NPCs was detected. Taken together, our data indicate that the NPCs are derived from Noto-positive node cells, and are not fully committed to a notochordal fate. Sustained Wnt signaling is required to maintain the NPCs' notochordal fate.