Search

Aneuploidy, an abnormal number of chromosomes, has previously been considered irremediable. Here, we report findings that euploid cells increased among cultured aneuploid cells after exposure to the protein ZSCAN4, encoded by a mammalian-specific gene that is ordinarily expressed in preimplantation embryos and occasionally in stem cells. For footprint-free delivery of ZSCAN4 to cells, we developed ZSCAN4 synthetic mRNAs and Sendai virus vectors that encode human ZSCAN4. Applying the ZSCAN4 biologics to established cultures of mouse embryonic stem cells, most of which had become aneuploid and polyploid, dramatically increased the number of euploid cells within a few days. We then tested the biologics on non-immortalized primary human fibroblast cells derived from four individuals with Down syndrome—the most frequent autosomal trisomy of chromosome 21. Within weeks after ZSCAN4 application to the cells in culture, fluorescent in situ hybridization with a chromosome 21-specific probe detected the emergence of up to 24% of cells with only two rather than three copies. High-resolution G-banded chromosomes further showed up to 40% of cells with a normal karyotype. These findings were confirmed by whole-exome sequencing. Similar results were obtained for cells with the trisomy 18 of Edwards syndrome. Thus a direct, efficient correction of aneuploidy in human fibroblast cells seems possible in vitro using human ZSCAN4.

Recent evidence suggests that ovarian high-grade serous carcinoma (HGSC) originates from the epithelium of the fallopian tube. However, most mouse models are based on the previous prevailing view that ovarian cancer develops from the transformation of the ovarian surface epithelium. Here, we report the extensive histological and molecular characterization of the mogp-TAg transgenic mouse, which expresses the SV40 large T-antigen (TAg) under the control of the mouse mullerian-specific Ovgp-1 promoter. Histological analysis of the fallopian tubes of mogp-TAg mice identified a variety of neoplastic lesions analogous to those described as precursors to ovarian HGSC. We identified areas of normal-appearing p53-positive epithelium that are similar to 'p53 signatures' in the human fallopian tube. More advanced proliferative lesions with nuclear atypia and epithelial stratification were also identified that were morphologically and immunohistochemically reminiscent of human serous tubal intraepithelial carcinoma (STIC), a potential precursor of ovarian HGSC. Beside these non-invasive precursor lesions, we also identified invasive adenocarcinoma in the ovaries of 56% of the mice. Microarray analysis revealed several genes differentially expressed between the fallopian tube of mogp-TAg and wild-type (WT) C57BL/6. One of these genes, Top2a, which encodes topoisomerase IIα, was shown by immunohistochemistry to be concurrently expressed with elevated p53 and was specifically elevated in mouse STICs but not in the surrounding tissues. TOP2A protein was also found elevated in human STICs, low-grade and high-grade serous carcinoma. The mouse model reported here displays a progression from normal tubal epithelium to invasive HGSC in the ovary, and therefore closely simulates the current emerging model of human ovarian HGSC pathogenesis. This mouse therefore has the potential to be a very useful new model for elucidating the mechanisms of serous ovarian tumourigenesis, as well as for developing novel approaches for the prevention, diagnosis and therapy of this disease.

Retinoic acid (RA) is one of the most potent inducers of differentiation of mouse embryonic stem cells (ESCs). However, previous studies show that RA treatment of cells cultured in the presence of a leukemia inhibitory factor (LIF) also result in the upregulation of a gene called Zscan4, whose transient expression is a marker for undifferentiated ESCs. We explored the balance between these two seemingly antagonistic effects of RA. ESCs indeed differentiated in the presence of LIF after RA treatment, but colonies of undifferentiated ESCs eventually emerged from these differentiated cells - even in the presence of RA. These colonies, named secondary colonies, consist of three cell types: typical undifferentiated ESCs expressing pluripotency genes such as Pou5f1, Sox2, and Nanog; cells expressing Zscan4; and endodermal-like cells located at the periphery of the colony. The capacity to form secondary colonies was confirmed for all eight tested ESC lines. Cells from the secondary colonies - after transfer to the standard ESC medium - retained pluripotency, judged by their strong alkaline phosphatase (ALP) staining, typical colony morphology, gene expression profile, stable karyotype, capacity to differentiate into all three germ layers in embryoid body formation assays, and successful contribution to chimeras after injection into blastocysts. Based on flow cytometry analysis (FACS), the proportion of Zscan4-positive cells in secondary colonies was higher than in standard ESC colonies, which may explain the capacity of ESCs to resist the differentiating effects of RA and instead form secondary colonies of undifferentiated ESCs. This hypothesis is supported by cell-lineage tracing analysis, which showed that most cells in the secondary colonies were descendents of cells transiently expressing Zscan4.

The efficiency of embryonic stem (ES) cell derivation relies on an optimized culture medium and techniques for treating preimplantation stage embryos. Recently, ES cell derivation from the preblastocyst developmental stage was reported by removing the zona pellucida from embryos of the most efficient strain for ES cell derivation (129Sv) during early preimplantation. Here, we showed that ES cells can be efficiently derived and maintained in a modified medium (MEMα), from preblastocysts of a low-efficiency mouse strain (a hybrid consisting of 50% B6, 25% CBA, and 25% DBA). Preblastocyst-derived ES cell lines were normal in terms of pluripotency-related protein expression, and chromosome number. Also, preblastocyst-derived ES cell lines from various culture conditions showed pluripotency in vivo through teratoma analysis. Interestingly, ES cell lines produced from preblastocysts and blastocysts, regardless of the derivation culture conditions, are nearly indistinguishable by their global gene expression profiles.

Induced pluripotent stem cells (iPSCs) generated by forced expression of four transcription factors offer promises for regenerative and therapeutic uses in human diseases. However, it is necessary to overcome the risk of tumorigenicity caused by the use of potent oncogenes and the use of randomly integrating vectors before the iPSC technology can be applied to human medicine. Stem cells and cancer cells share many features in common, implying that there are similar underlying mechanisms in their development. Small molecules have been used to induce cell reprogramming for lineage trans-differentiation and for maintaining pluripotency of stem cells. In this study, we investigated the possibility of replacing all reprogramming viral factors with small molecules. To this end, we evaluated the effects of carcinogens at nongenotoxic levels on somatic cells. We identified 16 candidate chemicals through biology-oriented in silico high-throughput screening with commercially available inventories from Sigma-Aldrich for cancer research, and established a reprogramming protocol of 16-day treatment followed by 5 days of recovery. This protocol was applied to B6/129 mouse embryonic fibroblasts (MEFs) at passage 3. From recovery day 2, colonies appeared at an efficiency of 0.02%. These colonies were positive for both alkaline phosphatase and surface specific embryonic antigen-1 (SSEA-1) at a comparable level to those of mouse embryonic stem cells (ESCs). Global gene expression analysis with a 38K gene MEEBO microarray revealed that the colonies had 564 (1.5%) differentially expressed genes compared to MEFs at day 0 of treatment, and these genes were enriched in "neuromuscular differentiation." Moreover, 122 differentially expressed genes in the colonies were ESC-enriched, including downregulated somatic markers and upregulated stem cell markers. In conclusion, combined chemical treatment of MEFs herein might have caused these cells to transverse to an intermediate state within the mesodermal lineages.

Tetraploid (4N) complementation assay is regard as the most stringent characterization test for the pluripotency of embryonic stem (ES) cells. The technology can generate mice fully derived from the injected ES cell (ES-4N) with 4N placentas. However, it remains a very inefficient procedure owing to a lack of information on the optimal conditions for ES incorporation into the 4N embryos. In the present study, we injected ES cells from embryos of natural fertilization (fES) and somatic cell nuclear transfer (ntES) into 4N embryos at various stages of development to determine the optimal stage of ES cells integration by comparing the efficiency of full-term ES-4N mouse generation. Our results demonstrate that fES/ntES cells can be incorporated into 4N embryos at 2-cell, 4-cell and blastocyst stages and full-term mice can be generated. Interestingly, ntES cells injected into the 4-cell group resulted in the lowest efficiency (5.6%) compared to the 2-cell (13.8%, P > 0.05) and blastocyst (16.7%, P < 0.05) stages. Because 4N embryos start to form compacted morulae at the 4-cell stage, we investigated whether the lower efficiency at this stage was due to early compaction by injecting ntES cells into artificially de-compacted embryos treated with calcium free medium. Although the treatment changed the embryonic morphology, it did not increase the efficiency of ES-4N mice generation. Immunochemistry of the cytoskeleton displayed microtubule and microfilament polarization at the late 4-cell stage in 4N embryos, which suggests that de-compaction treatment cannot reverse the polarization process. Taken together, we show here that a wide developmental range of 4N embryos can be used for 4N complementation and embryo polarization and compaction may restrict incorporation of ES cells into 4N embryos.

Deriving histocompatible embryonic stem (ES) cells by somatic cell nuclear transfer (SCNT) and parthenogenetic activation (PA) requires fresh oocytes, which prevents their applications in humans. Here, we evaluated the efficiency of deriving ES cells from mature metaphase II (MII) and immature metaphase I (MI) vitrified oocytes, by PA or SCNT, in a mouse model. We successfully generated ES cell lines from PA (MII and MI) and SCNT (MII and MI) blastocysts. These cell lines expressed genes and antigens characteristic of pluripotent ES cells and produced full-term pups upon tetraploid embryo complementation. This study established an animal model for efficient generation of patient-specific ES cell lines using cryopreserved oocytes. This is a major step forward in the application of therapeutic cloning and parthenogenetic technology in human regenerative medicine and will serve as an important alternative to the iPS cell technology in countries/regions where these technologies are permitted.

Although the first mouse embryonic stem (ES) cell lines were derived 2 decades ago, and standard protocols for ES cell derivation are widely used today, the technical difficulty of these protocols still pose a challenge for many investigators attempting to produce large numbers of ES cell lines, and are limited to only a few mouse strains. Recently, glucose concentration was shown to have a significant effect on the efficiency of ES cell derivation, but the mechanism(s) mediating this effect are still the subject of debate. In this report, we investigated the effect of glucose concentration on ES cell derivation efficiency from blastocysts in the context of a new medium, Minimum Essential Medium alpha (MEMalpha). Furthermore, we propose novel methods to improve mouse ES cell derivation efficiency using in vitro epigenetic modifications during early passages, combined with detection of Oct4-expressing cells. Based on the results reported here, modified MEMalpha containing high glucose improves the efficiency of ES cell derivation remarkably, compared with Knockout Dulbecco's-Modified Eagle Media (KDMEM). Epigenetic modifications are able to improve the efficiency even further.

Somatic cell nuclear transfer enables the generation of embryonic stem cells (ESCs) that genetically match the donor and can be used to treat disease through cell replacement therapies or to recapitulate patient-specific disease via in vitro differentiation. We performed a "proof-of-principle" study using tail tip fibroblasts from a mouse model of Parkinson's disease (Aphakia) as the donor cell nuclei for nuclear transfer and derived "customized" ESCs for in vitro analysis. Aphakia mice contain deletions in the pitx3 gene and show selective loss of dopamine neurons of the substantia nigra, specifically the neuron population susceptible to degeneration in Parkinson's disease. Using electrofusion nuclear transfer, we produced cloned Aphakia oocytes at rates similar to those for control, cloned oocytes. Aphakia ESCs were isolated and live mice were generated using tetraploid embryo complementation. In vitro differentiation of Aphakia ESCs to dopaminergic neurons revealed significantly fewer TH+ neurons that expressed MAP2, DAT, synaptophysin, VMAT2, and AHD2 compared to control nuclear transfer ESC cultures, supporting a role for Pitx3 in mesodiencephalic dopamine neuron maturation. Taken together, our studies define a customized in vitro ESC culture system used to analyze gene-specific contribution to dopamine neuron generation, maturation, and susceptibility to degeneration.

TAZ (transcriptional coactivator with PDZ-binding motif), also called WWTR1 (WW domain containing transcription regulator 1), is a 14-3-3-binding molecule homologous to Yes-associated protein. TAZ acts as a coactivator for several transcription factors as well as a modulator of membrane-associated PDZ domain-containing proteins, but its (patho)physiological roles remain unknown. Here we show that gene inactivation of TAZ in mice resulted in pathological changes in the kidney and lung that resemble the common human diseases polycystic kidney disease and pulmonary emphysema. Taz-null/ lacZ knockin mutant homozygotes demonstrated renal cyst formation as early as embryonic day 15.5 with dilatation of Bowman's capsules and proximal tubules, followed by pelvic dilatation and hydronephrosis. After birth, only one-fifth of TAZ-deficient homozygotes grew to adulthood and demonstrated multicystic kidneys with severe urinary concentrating defects and polyuria. Furthermore, adult TAZ-deficient homozygotes exhibited diffuse emphysematous changes in the lung. Thus TAZ is essential for developmental mechanisms involved in kidney and lung organogenesis, whose disturbance may lead to the pathogenesis of common human diseases.

The endothelin (Edn) system comprises three ligands (Edn1, Edn2 and Edn3) and their G-protein-coupled type A (Ednra) and type B (Ednrb) receptors. During embryogenesis, the Edn1/Ednra signaling is thought to regulate the dorsoventral axis patterning of pharyngeal arches via Dlx5/Dlx6 upregulation. To further clarify the underlying mechanism, we have established mice in which gene cassettes can be efficiently knocked-in into the Ednra locus using recombinase-mediated cassette exchange (RMCE) based on the Cre-lox system. The first homologous recombination introducing mutant lox-flanked Neo resulted in homeotic transformation of the lower jaw to an upper jaw, as expected. Subsequent RMCE-mediated knock-in of lacZ targeted its expression to the cranial/cardiac neural crest derivatives as well as in mesoderm-derived head mesenchyme. Knock-in of Ednra cDNA resulted in a complete rescue of craniofacial defects of Ednra-null mutants. By contrast, Ednrb cDNA could not rescue them except for the most distal pharyngeal structures. At early stages, the expression of Dlx5, Dlx6 and their downstream genes was downregulated and apoptotic cells distributed distally in the mandible of Ednrb-knock-in embryos. These results, together with similarity in craniofacial defects between Ednrb-knock-in mice and neural-crest-specific Galpha(q)/Galpha(11)-deficient mice, indicate that the dorsoventral axis patterning of pharyngeal arches is regulated by the Ednra-selective, G(q)/G(11)-dependent signaling, while the formation of the distal pharyngeal region is under the control of a G(q)/G(11)-independent signaling, which can be substituted by Ednrb. This RMCE-mediated knock-in system can serve as a useful tool for studies on gene functions in craniofacial development.

In polytocous animals, blastocysts are evenly distributed along each uterine horn and implant. The molecular mechanisms underlying these precise events remain elusive. We recently showed that lysophosphatidic acid (LPA) has critical roles in the establishment of early pregnancy by affecting embryo spacing and subsequent implantation through its receptor, LPA3. Targeted deletion of Lpa3 in mice resulted in delayed implantation and embryo crowding, which is associated with a dramatic decrease in the prostaglandins and prostaglandin-endoperoxide synthase 2 expression levels. Exogenous administration of prostaglandins rescued the delayed implantation but did not rescue the defects in embryo spacing, suggesting the role of prostaglandins in implantation downstream of LPA3 signaling. In the present study, to know how LPA3 signaling regulates the embryo spacing, we determined the time course distribution of blastocysts during the preimplantation period. In wild-type (WT) uteri, blastocysts were distributed evenly along the uterine horns at Embryonic Day 3.8 (E3.8), whereas in the Lpa3-deficient uteri, they were clustered in the vicinity of the cervix, suggesting that the mislocalization and resulting crowding of the embryos are the cause of the delayed implantation. However, embryos transferred singly into E2.5 pseudopregnant Lpa3-deficient uterine horns still showed delayed implantation but on-time implantation in WT uteri, indicating that embryo spacing and implantation timing are two segregated events. We also found that an LPA3-specific agonist induced rapid uterine contraction in WT mice but not in Lpa3-deficient mice. Because the uterine contraction is critical for embryo spacing, our results suggest that LPA3 signaling controls embryo spacing via uterine contraction around E3.5.

Defensins comprise a family of cationic antimicrobial peptides characterized by conserved cysteine residues. They are produced in various organs including skeletal muscle and are identified as key elements in the host defense system as potent effectors. At the same time, defensins have potential roles in the regulation of inflammation and, furthermore, can exert cytotoxic effects on several mammalian cells. Here, we developed transgenic mice overexpressing mouse β-defensin-6 to explore the pathophysiological roles of the defensin family as a novel mediator of inflammatory tissue injury. Unexpectedly, the transgenic mice showed short lifespan, poor growth, and progressive myofiber degeneration with functional muscle impairment, predominant centronucleated myofibers, and elevated serum creatine kinase activity, as seen in human muscular dystrophy. Furthermore, some of the transgenic myofibers showed IκBα accumulation, which would be related to the myofiber apoptosis of limb-girdle muscular dystrophy type 2A. The present findings may unravel a concealed linkage between the innate immune system and the pathophysiology of degenerative diseases.

Full-term development occurred when nuclei from mouse embryonic stem (ES) cells, synchronized in metaphase with nocodazole, were fused with enucleated oocytes or nuclei of reconstituted eggs and again fused with the enucleated blastomeres of fertilized two-cell embryos using inactivated Sendai virus. Two surviving male mice were derived from undifferentiated ES cell nuclei, one from single nuclear transfer and another from serial nuclear transfer. Both were noticeably small and died within 24 hr of birth for unknown reasons. These findings demonstrate that nuclear transfer of ES cells using the fusion method produces young, as does the piezoelectric-actuated nuclear transfer. J. Exp. Zool. 289:139-145, 2001.

Although the induced pluripotent stem cell (iPSC) procedure has clearly become the method of choice because of its convenience and for ethical considerations, when generating pluripotent stem cells from somatic cells, it is still important to consider the number of advantageous features of the somatic cell nuclear transfer procedure. For example, it will be interesting to discover whether iPSC factors, which were originally identified as genes expressed specifically in embryonic stem cells, can account for all of the unknown reprogramming factors present in pre-implantation embryos. With this point in mind, in this chapter we will review each reprogramming factor that has been reported in mouse and human iPSC studies from the perspective of its expression pattern in pre-implantation embryos. We will also examine some of the candidate genes that are expressed in pre-implantation embryos but do not form part of the common iPSC factors due to their lack of or low expression in embryonic stem cells, because such factors may help to efficiently produce high-quality iPSCs.

Recent evidence suggests that ovarian high-grade serous carcinoma (HGSC) originates from the epithelium of the fallopian tube. However, most mouse models are based on the previous prevailing view that ovarian cancer develops from the transformation of the ovarian surface epithelium. Here, we report the extensive histological and molecular characterization of the mogp-TAg transgenic mouse, which expresses the SV40 large T-antigen (TAg) under the control of the mouse müllerian-specific Ovgp-1 promoter. Histological analysis of the fallopian tubes of mogp-TAg mice identified a variety of neoplastic lesions analogous to those described as precursors to ovarian HGSC. We identified areas of normal-appearing p53-positive epithelium that are similar to 'p53 signatures' in the human fallopian tube. More advanced proliferative lesions with nuclear atypia and epithelial stratification were also identified that were morphologically and immunohistochemically reminiscent of human serous tubal intraepithelial carcinoma (STIC), a potential precursor of ovarian HGSC. Beside these non-invasive precursor lesions, we also identified invasive adenocarcinoma in the ovaries of 56% of the mice. Microarray analysis revealed several genes differentially expressed between the fallopian tube of mogp-TAg and wild-type (WT) C57BL/6. One of these genes, Top2a, which encodes topoisomerase IIα, was shown by immunohistochemistry to be concurrently expressed with elevated p53 and was specifically elevated in mouse STICs but not in the surrounding tissues. TOP2A protein was also found elevated in human STICs, low-grade and high-grade serous carcinoma. The mouse model reported here displays a progression from normal tubal epithelium to invasive HGSC in the ovary, and therefore closely simulates the current emerging model of human ovarian HGSC pathogenesis. This mouse therefore has the potential to be a very useful new model for elucidating the mechanisms of serous ovarian tumourigenesis, as well as for developing novel approaches for the prevention, diagnosis and therapy of this disease.

The developmental potency of mouse embryonic stem (ES) cells, which is the ability to contribute to a whole embryo, is known to deteriorate during long-term cell culture. Previously, we have shown that ES cells oscillate between Zscan4(-) and Zscan4(+) states, and the transient activation of Zscan4 is required for the maintenance of telomeres and genome stability of ES cells. Here we show that increasing the frequency of Zscan4 activation in mouse ES cells restores and maintains their developmental potency in long-term cell culture. Injection of a single ES cell with such increased potency into a tetraploid blastocyst gives rise to an entire embryo with a higher success rate. These results not only provide a means to rejuvenate ES cells by manipulating Zscan4 expression, but also indicate the active roles of Zscan4 in the long-term maintenance of ES cell potency.

During female reproductive life, ovarian follicle reserve is reduced by maturation and atresia until menopause ensues. Foxo3 is required to maintain the ovarian reserve in mice. Here we show that overexpression of constitutively active FOXO3 can increase ovarian reproductive capacity in mice. We find increased follicle numbers and decreased gonadotropin levels in aging FOXO3 transgenic mice compared to wild-type littermates, suggesting maintenance of a greater ovarian reserve. Based on cumulative progeny in aging animals, we find 31 to 49% increased fertility in transgenic females. The gene expression profile of Foxo3−/− knockout ovaries appears more mature than that of wild-type littermates, and the transgene induces a younger-looking profile, restoring much of the wild-type transcriptome. This is the first gain-of-function model of augmented reproductive reserve in mice, thus emphasizing the role of Foxo3 as a guardian of the ovarian follicle pool in mammals and a potential determinant of the onset of menopause.

Networks of transcription factors (TFs) are thought to determine and maintain the identity of cells. Here we systematically repressed each of 100 TFs with shRNA and carried out global gene expression profiling in mouse embryonic stem (ES) cells. Unexpectedly, only the repression of a handful of TFs significantly affected transcriptomes, which changed in two directions/trajectories: one trajectory by the repression of either Pou5f1 or Sox2; the other trajectory by the repression of either Esrrb, Sall4, Nanog, or Tcfap4. The data suggest that the trajectories of gene expression change are already preconfigured by the gene regulatory network and roughly correspond to extraembryonic and embryonic fates of cell differentiation, respectively. These data also indicate the robustness of the pluripotency gene network, as the transient repression of most TFs did not alter the transcriptomes.

Chimeric hamsters were produced by the aggregation of eight-cell embryos obtained from two stains (golden and albino) of Syrian hamster. Eight-cell embryos were collected by flushing of oviduct and uterus using HEPES-HECM-3 medium. The aggregated embryos by PHA were cultured in HECM-3 medium for 20 h under 10%O2-10%CO2-80%N2 at 37.5°C. Of 354 aggregated embryos, 87 (25%) and 234 (66%) had developed into morulae and blastocysts, respectively. Total 90 aggregates (morulae and blastocysts) were transferred to 3 recipients at Day 3 of pseudopregnancy. Of 3 recipients, 2 became pregnant. Seven pups were born from the two recipients. Of 7 pups born, 6 were determined as chimeric hamsters by coat color (agouti). Of 6 chimeras, 5 were phenotypic males, and 1 was a phenotypic female. All chimeric hamsters had normal external genitalia.

Calpains are Ca2+-dependent modulator Cys proteases that have a variety of functions in almost all eukaryotes. There are more than 10 well-conserved mammalian calpains, among which eutherian calpain-6 (CAPN6) is unique in that it has amino acid substitutions at the active-site Cys residue (to Lys in humans), strongly suggesting a loss of proteolytic activity. CAPN6 is expressed predominantly in embryonic muscles, placenta, and several cultured cell lines. We previously reported that CAPN6 is involved in regulating microtubule dynamics and actin reorganization in cultured cells. The physiological functions of CAPN6, however, are still unclear. Here, to elucidate CAPN6's in vivo roles, we generated Capn6-deficient mice, in which a lacZ expression cassette was integrated into the Capn6 gene. These Capn6-deficient mouse embryos expressed lacZ predominantly in skeletal muscles, as well as in cartilage and the heart. Histological and biochemical analyses showed that the CAPN6 deficiency promoted the development of embryonic skeletal muscle. In primary cultured skeletal muscle cells that were induced to differentiate into myotubes, Capn6 expression was detected in skeletal myocytes, and Capn6-deficient cultures showed increased differentiation. Furthermore, we found that CAPN6 was expressed in the regenerating skeletal muscles of adult mice after cardiotoxin-induced degeneration. In this experimental system, Capn6-deficient mice exhibited more advanced skeletal-muscle regeneration than heterozygotes or wild-type mice at the same time point. These results collectively showed that a loss of CAPN6 promotes skeletal muscle differentiation during both development and regeneration, suggesting a novel physiological function of CAPN6 as a suppressor of skeletal muscle differentiation., Author Summary The calpains arose evolutionarily as a family of Ca2+-regulated intracellular proteases with divergent structures and functions. Unique among mammalian calpains, calpain-6 is “non-proteolytic”, and its physiological role has remained unknown. In this study, using Capn6 knock-out mice, we discovered that Calpain-6 is involved in skeletal muscle development and regeneration in vivo. Calpain-6 was predominantly expressed in embryonic muscles and bone cartilage. Capn6 knock-out promoted skeletal-muscle development as a result of increased progenitor cell differentiation. Furthermore, calpain-6 was detected in regenerating skeletal muscles, and suppressed this process. These results indicate that mammalian calpain-6 is a suppressive modulator for skeletal muscle differentiation and growth. Our findings indicate that calpain-6 might serve as a therapeutic target for muscular dystrophy/atrophy or as a useful tool in tissue engineering. For calpain studies, the physiological non-proteolytic role of calpain-6 may shed light on the elusive calpain functions that are independent of their well-studied proteolytic activities. In addition, as this structurally non-proteolytic feature is unique to the calpain-6 of placental mammals among vertebrates, our mutant mice might provide insight into the relationship between molecular and biological evolution.

Although leukemia inhibitory factor (LIF) maintains the ground state pluripotency of mouse embryonic stem cells and induced pluripotent stem cells (iPSCs) by activating the Janus kinase/signal transducer and activator of transcription 3 (Jak/Stat3) pathway, the mechanism remained unclear. Stat3 has only been shown to promote complete reprogramming of epiblast and neural stem cells and partially reprogrammed cells (pre-iPSCs). We investigated if and how Jak/Stat3 activation promotes reprogramming of terminally differentiated mouse embryonic fibroblasts (MEFs). We demonstrated that activated Stat3 not only promotes but also is essential for the pluripotency establishment of MEFs during reprogramming. We further demonstrated that during this process, inhibiting Jak/Stat3 activity blocks demethylation of Oct4 and Nanog regulatory elements in induced cells, which are marked by suppressed endogenous pluripotent gene expression. These are correlated with the significant upregulation of DNA methyltransferase (Dnmt) 1 and histone deacetylases (HDACs) expression as well as the increased expression of lysine-specific histone demethylase 2 and methyl CpG binding protein 2. Inhibiting Jak/Stat3 also blocks the expression of Dnmt3L, which is correlated with the failure of retroviral transgene silencing. Furthermore, Dnmt or HDAC inhibitor but not overexpression of Nanog significantly rescues the reprogramming arrested by Jak/Stat3 inhibition or LIF deprivation. Finally, we demonstrated that LIF/Stat3 signal also represents the prerequisite for complete reprogramming of pre-iPSCs. We conclude that Jak/Stat3 activity plays a fundamental role to promote pluripotency establishment at the epigenetic level, by facilitating DNA demethylation/de novo methylation, and open-chromatin formation during late-stage reprogramming.

The generation of induced pluripotent stem cells (iPSCs) by the forced expression of defined transcription factors in somatic cells holds great promise for the future of regenerative medicine. However, the initial reprogramming mechanism is still poorly understood. Here we show that Zscan4, expressed transiently in 2-cell embryos and embryonic stem cells (ESCs), efficiently produces iPSCs from mouse embryo fibroblasts when coexpressed with Klf4, Oct4, and Sox2. Interestingly, the forced expression of Zscan4 is required only for the first few days of iPSC formation. Microarray analysis revealed transient and early induction of preimplantation-specific genes in a Zscan4-dependent manner. Our work indicates that Zscan4 is a previously unidentified potent natural factor that facilitates the reprogramming process and reactivates early embryonic genes.

Background In addition to determining static states of gene expression (high vs. low), it is important to characterize their dynamic status. For example, genes with H3K27me3 chromatin marks are not only suppressed but also poised for activation. However, the responsiveness of genes to perturbations has never been studied systematically. To distinguish gene responses to specific factors from responsiveness in general, it is necessary to analyze gene expression profiles of cells responding to a large variety of disturbances, and such databases did not exist before. Results We estimated the responsiveness of all genes in mouse ES cells using our recently published database on expression change after controlled induction of 53 transcription factors (TFs) and other genes. Responsive genes (N = 4746), which were readily upregulated or downregulated depending on the kind of perturbation, mostly have regulatory functions and a propensity to become tissue-specific upon differentiation. Tissue-specific expression was evaluated on the basis of published (GNF) and our new data for 15 organs and tissues. Non-responsive genes (N = 9562), which did not change their expression much following any perturbation, were enriched in housekeeping functions. We found that TF-responsiveness in ES cells is the best predictor known for tissue-specificity in gene expression. Among genes with CpG islands, high responsiveness is associated with H3K27me3 chromatin marks, and low responsiveness is associated with H3K36me3 chromatin, stronger tri-methylation of H3K4, binding of E2F1, and GABP binding motifs in promoters. Conclusions We thus propose the responsiveness of expression to perturbations as a new way to define the dynamic status of genes, which brings new insights into mechanisms of regulation of gene expression and tissue specificity.

Tetraploid (4N) complementation assay is regard as the most stringent characterization test for the pluripotency of embryonic stem (ES) cells. The technology can generate mice fully derived from the injected ES cell (ES-4N) with 4N placentas. However, it remains a very inefficient procedure owing to a lack of information on the optimal conditions for ES incorporation into the 4N embryos. In the present study, we injected ES cells from embryos of natural fertilization (fES) and somatic cell nuclear transfer (ntES) into 4N embryos at various stages of development to determine the optimal stage of ES cells integration by comparing the efficiency of full-term ES-4N mouse generation. Our results demonstrate that fES/ntES cells can be incorporated into 4N embryos at 2-cell, 4-cell and blastocyst stages and full-term mice can be generated. Interestingly, ntES cells injected into the 4-cell group resulted in the lowest efficiency (5.6%) compared to the 2-cell (13.8%, P0.05) and blastocyst (16.7%, P0.05) stages. Because 4N embryos start to form compacted morulae at the 4-cell stage, we investigated whether the lower efficiency at this stage was due to early compaction by injecting ntES cells into artificially de-compacted embryos treated with calcium free medium. Although the treatment changed the embryonic morphology, it did not increase the efficiency of ES-4N mice generation. Immunochemistry of the cytoskeleton displayed microtubule and microfilament polarization at the late 4-cell stage in 4N embryos, which suggests that de-compaction treatment cannot reverse the polarization process. Taken together, we show here that a wide developmental range of 4N embryos can be used for 4N complementation and embryo polarization and compaction may restrict incorporation of ES cells into 4N embryos.

Sirtuins are a phylogenetically conserved NAD+-dependent protein deacetylase/ADP-ribosyltransferase family implicated in diverse biological processes. Several family members localize to mitochondria, the function of which is thought to determine the developmental potential of preimplantation embryos. We have therefore characterized the role of sirtuins in mouse preimplantation development under in vitro culture conditions. All sirtuin members were expressed in eggs, and their expression gradually decreased until the blastocyst stage. Treatment with sirtuin inhibitors resulted in increased intracellular ROS levels and decreased blastocyst formation. These effects were recapitulated by siRNA-induced knockdown of Sirt3, which is involved in mitochondrial energy metabolism, and in Sirt3–/– embryos. The antioxidant N-acetyl-L-cysteine and low-oxygen conditions rescued these adverse effects. When Sirt3-knockdown embryos were transferred to pseudopregnant mice after long-term culture, implantation and fetal growth rates were decreased, indicating that Sirt3-knockdown embryos were sensitive to in vitro conditions and that the effect was long lasting. Further experiments revealed that maternally derived Sirt3 was critical. Sirt3 inactivation increased mitochondrial ROS production, leading to p53 upregulation and changes in downstream gene expression. The inactivation of p53 improved the developmental outcome of Sirt3-knockdown embryos, indicating that the ROS-p53 pathway was responsible for the developmental defects. These results indicate that Sirt3 plays a protective role in preimplantation embryos against stress conditions during in vitro fertilization and culture.

The transcription factor Pitx3 is expressed exclusively by mesodiencephalic dopaminergic neurons; however, ablation of Pitx3 results in selective degeneration of primarily dopaminergic neurons of the substantia nigra pars compacta, the neuronal population that is most vulnerable in Parkinson's disease. Although the exact molecular mechanisms of the action of Pitx3 are unclear, roles in both terminal maturation and/or survival of substantia nigra dopaminergic neurons have been suggested. To investigate the connection between Pitx3 and selective neurodegeneration, we generated embryonic stem cells from a Pitx3-deficient mouse (aphakia) for in-vitro differentiation to dopaminergic neurons. This 'loss of function'in-vitro system allowed us to examine characteristic features in dopaminergic neuron development and to assess the role that Pitx3 plays in the differentiation/maturation process. We found that aphakia embryonic stem cells generated 50% fewer tyrosine hydroxylase-positive/microtubule-associated protein (Map)2-positive mature neurons compared with control cultures. The expression of dopamine transport regulators and vesicle release proteins was reduced and dopamine release was unregulated in the Pitx3-deficient tyrosine hydroxylase-positive neurons generated. Treatment of aphakia embryonic stem cell cultures with retinoic acid resulted in a significant increase in mesodiencephalic tyrosine hydroxylase-positive neurons, providing further support for the role of Pitx3 in dopaminergic neuron specification through the retinoic acid pathway. Our study, using Pitx3-deficient embryonic stem cells in an in-vitro differentiation culture system, allowed us to assess the role of Pitx3 in the specification and final maturation of dopaminergic neurons.

Somatic cells can be reprogrammed to a totipotent state through nuclear transfer or cloning, because it has been demonstrated that the oocyte has the ability to reprogramme an adult nucleus into an embryonic state that can initiate the development of a new organism. Therapeutic cloning, whereby nuclear transfer is used to derive patient-specific embryonic stem cells, embraces an entire new opportunity for regenerative medicine. However, a key obstacle for human therapeutic cloning is that the source of fresh human oocytes is extremely limited. In the present review, we propose prospective sources of human oocytes by using oocyte cryopreservation, such as an oocyte bank and immature oocytes. We also address some potential issues associated with nuclear transfer when using cryopreserved oocytes. In the future, if the efficacy and efficiency of cryopreserved oocytes are comparable to those of fresh oocytes in human therapeutic cloning, the use of cryopreserved oocytes would be invaluable and generate a great impact to regenerative medicine.

Dnm3os, a gene that is transcribed into a non-coding RNA (ncRNA), contains three micro RNAs (miRNAs), miR-199a, miR-199a*, and miR-214, whose functions remain unknown in mammals. In this study, we introduced the lacZ gene into the Dnm3os locus to recapitulate its expression pattern and disrupt its function. Dnm3os(+/lacZ) heterozygous embryos showed beta-galactosidase activity, which reflected the authentic expression pattern of Dnm3os RNA. Most of the Dnm3os(lacZ/lacZ) homozygous pups died within one month of birth. After birth, Dnm3os(lacZ/lacZ) mice exhibited several skeletal abnormalities, including craniofacial hypoplasia, defects in dorsal neural arches and spinous processes of the vertebrae, and osteopenia. Importantly, the expression of miR-199a, miR-199a*, and miR-214 was significantly down-regulated in Dnm3os(lacZ/lacZ) embryos, supporting the assumption that Dnm3os serves as a precursor of these three miRNAs. Thus, Dnm3os has emerged as an miRNA-encoding gene that is indispensable for normal skeletal development and body growth in mammals.

In polytocous animals, blastocysts are evenly distributed along each uterine horn and implant. The molecular mechanisms underlying these precise events remain elusive. We recently showed that lysophosphatidic acid (LPA) has critical roles in the establishment of early pregnancy by affecting embryo spacing and subsequent implantation through its receptor, LPA3. Targeted deletion of Lpa3 in mice resulted in delayed implantation and embryo crowding, which is associated with a dramatic decrease in the prostaglandins and prostaglandin-endoperoxide synthase 2 expression levels. Exogenous administration of prostaglandins rescued the delayed implantation but did not rescue the defects in embryo spacing, suggesting the role of prostaglandins in implantation downstream of LPA3 signaling. In the present study, to know how LPA3 signaling regulates the embryo spacing, we determined the time course distribution of blastocysts during the preimplantation period. In wild-type (WT) uteri, blastocysts were distributed evenly along the uterine horns at Embryonic Day 3.8 (E3.8), whereas in the Lpa3-deficient uteri, they were clustered in the vicinity of the cervix, suggesting that the mislocalization and resulting crowding of the embryos are the cause of the delayed implantation. However, embryos transferred singly into E2.5 pseudopregnant Lpa3-deficient uterine horns still showed delayed implantation but on-time implantation in WT uteri, indicating that embryo spacing and implantation timing are two segregated events. We also found that an LPA3-specific agonist induced rapid uterine contraction in WT mice but not in Lpa3-deficient mice. Because the uterine contraction is critical for embryo spacing, our results suggest that LPA3 signaling controls embryo spacing via uterine contraction around E3.5.

Reciprocal interactions between blastocysts and receptive uteri are essential for successful implantation. This process is regulated by the timely interplay of two ovarian hormones, progesterone and estrogen. However, the molecular targets of these hormones are largely unknown. We showed recently that a small bioactive lysophospholipid, lysophosphatidic acid, plays a pivotal role in the establishment of implantation via its cellular receptor, LPA(3). Here we demonstrate that LPA(3) expression is positively and negatively regulated by steroid hormones in mouse uteri. The LPA(3) mRNA level in the uteri increased during early pseudopregnancy, peaking around 3.5 days post coitus (3.5 d.p.c.), then, decreased to the basal level on 4.5 d.p.c. LPA(3) expression remained at a low level in ovariectomized mice, and administration of progesterone to ovariectomized mice up-regulated LPA(3) mRNA expression. In addition, simultaneous administration of estrogen counteracted the effect of progesterone. These results show that progesterone and estrogen cooperatively regulate LPA(3) expression, thereby contributing to the receptivity of uteri during early pregnancy.

The molecular mechanisms affecting female reproduction, particularly therapeutically tractable ones, are incompletely understood. So the identification of a new signalling mechanism affecting fertility via embryo implantation could be important. The compound involved is lysophosphatidic acid (LPA), acting through a G protein-coupled receptor. Targeted deletion of the receptor, called LPA3, produces mice that display delayed implantation, altered implantation spacing, hypertrophic placentas and embryonic death. G protein-coupled receptors are among the most common targets of drug action, raising the possibility of developing new medicines for the treatment of infertility by targeting the LPA3 receptor. Every successful pregnancy requires proper embryo implantation. Low implantation rate is a major problem during infertility treatments using assisted reproductive technologies1. Here we report a newly discovered molecular influence on implantation through the lysophosphatidic acid (LPA) receptor LPA3 (refs 2–4). Targeted deletion of LPA3 in mice resulted in significantly reduced litter size, which could be attributed to delayed implantation and altered embryo spacing. These two events led to delayed embryonic development, hypertrophic placentas shared by multiple embryos and embryonic death. An enzyme demonstrated to influence implantation, cyclooxygenase 2 (COX2) (ref. 5), was downregulated in LPA3-deficient uteri during pre-implantation. Downregulation of COX2 led to reduced levels of prostaglandins E2 and I2 (PGE2 and PGI2), which are critical for implantation1. Exogenous administration of PGE2 or carbaprostacyclin (a stable analogue of PGI2) into LPA3-deficient female mice rescued delayed implantation but did not rescue defects in embryo spacing. These data identify LPA3 receptor-mediated signalling as having an influence on implantation, and further indicate linkage between LPA signalling and prostaglandin biosynthesis.