Effect of PCDH19 missense mutations on cell-to-cell proximity and neuronal development under heterotypic conditions.

The mutation of the X-linked protocadherin (PCDH) 19 gene in heterozygous females causes epilepsy. However, because of the erosion of X-chromosome inactivation (XCI) in female human pluripotent stem cells, precise disease modeling often leads to failure. In this study, using a mathematical approach and induced pluripotent stem cells retaining XCI derived from patients with PCDH19 missense mutations, we found that heterotypic conditions, which are composed of wild-type and missense PCDH19, led to significant cell-to-cell proximity and impaired neuronal differentiation, accompanied by the aberrant accumulation of doublecortin, a microtubule-associated protein. Our findings suggest that ease of adhesion between cells expressing either wild-type or missense PCDH19 might lead to aberrant cell aggregation in early embryonic phases, causing poor neuronal development.

Nanosheet coating improves stability of human pluripotent stem cell culture on glass substrates.

High-resolution imaging analysis using various types of cells is an essential tool for dissecting cell functions. Generally, obtaining such images requires the cells to be cultured on glass substrates; however, it often results in the unstable status of cells. In this study, we report that coating the glass substrate using nanosheet composed of hydrophobic polystyrene, with Matrigel, significantly improves the viability of human pluripotent stem cells (hPSCs). Moreover, the nanosheet coating does not affect the transcriptome status of hPSC and enables researchers to perform the high-resolution imaging assay. These results indicate that the nanosheet coating is beneficial to the cells vulnerable to glass substrate culture. Using the nanosheet coating, we revealed that the spreading status of lnc RNA XIST, essential for X-chromosome inactivation (XCI) in female cells, in the nuclei significantly differs in each hPSC line. Taken together, our study provides a novel method to investigate biological questions using high-resolution imaging techniques.

Potential UV-Protective Effect of Freestanding Biodegradable Nanosheet-Based Sunscreen Preparations in XPA-Deficient Mice.

Xeroderma pigmentosum (XP) is a rare autosomal recessive hereditary disorder. As patients with XP are deficient in nucleotide excision repair, they show severe photosensitivity symptoms. Although skin protection from ultraviolet (UV) radiation is essential to improve the life expectancy of such patients, the optimal protective effect is not achieved even with sunscreen application, owing to the low usability of the preparations. Nanosheets are two-dimensional nanostructures with a thickness in the nanometer range. The extremely large aspect ratios of the nanosheets result in high transparency, flexibility, and adhesiveness. Moreover, their high moisture permeability enables their application to any area of the skin for a long time. We fabricated preparations containing avobenzone (BMDBM) based on freestanding poly (L-lactic acid) (PLLA) nanosheets through a spin-coating process. Although monolayered PLLA nanosheets did not contain enough BMDBM to protect against UV radiation, the layered nanosheets, consisting of five discrete BMDBM nanosheets, showed high UV absorbance without lowering the adhesive strength against skin. Inflammatory reactions in XPA-deficient mice after UV radiation were completely suppressed by the application of BMDBM-layered nanosheets to the skin. Thus, the BMDBM layered nanosheet could serve as a potential sunscreen preparation to improve the quality of life of patients with XP.

De-erosion of X chromosome dosage compensation by the editing of XIST regulatory regions restores the differentiation potential in hPSCs.

Human pluripotent stem cells (hPSCs) regularly and irreversibly show the erosion of X chromosome inactivation (XCI) by long non-coding RNA (lncRNA) XIST silencing, causing challenges in various applications of female hPSCs. Here, we report reliable methods to reactivate XIST with monoallelic expression in female hPSCs. Surprisingly, we find that the editing of XIST regulatory regions by Cas9-mediated non-homologous end joining is sufficient for the reactivation of XIST by endogenous systems. Proliferated hPSCs with XIST reactivation show XCI from an eroded X chromosome, suggesting that hPSCs with normal dosage compensation might lead to a growth advantage. Furthermore, the use of targeting vectors, including the XIST regulatory region sequences and selection cassette, enables XIST reactivation in hPSCs with high efficiency. XIST-reactivated hPSCs can show the restoration of differentiation potential. Thus, our findings demonstrate that XIST re-expression is a beneficial method to maximize the use of female hPSCs in various applications, such as proper disease modeling.

De novo DNA methyltransferases DNMT3A and DNMT3B are essential for XIST silencing for erosion of dosage compensation in pluripotent stem cells.

Human pluripotent stem cells (hPSCs) have proven to be valuable tools for both drug discovery and the development of cell-based therapies. However, the long non-coding RNA XIST, which is essential for the establishment and maintenance of X chromosome inactivation, is repressed during culture, thereby causing erosion of dosage compensation in female hPSCs. Here, we report that the de novo DNA methyltransferases DNMT3A/3B are necessary for XIST repression in female hPSCs. We found that the deletion of both genes, but not the individual genes, inhibited XIST silencing, maintained the heterochromatin mark of H3K27me3, and did not cause global overdosage in X-linked genes. Meanwhile, DNMT3A/3B deletion after XIST repression failed to restore X chromosome inactivation. Our findings revealed that de novo DNA methyltransferases are primary factors responsible for initiating erosion of dosage compensation in female hPSCs, and XIST silencing is stably maintained in a de novo DNA-methylation-independent manner.

Deletion of lncRNA XACT does not change expression dosage of X-linked genes, but affects differentiation potential in hPSCs.

Female human pluripotent stem cells (hPSCs) regularly show erosion of X chromosome inactivation featured by the loss of the long non-coding (lnc) RNA XIST and the accumulation of lncXACT. Here, we report that a common mechanism for the initiation of erosion depends on XIST loss but not XACT accumulation on inactive X chromosomes. We further demonstrate that XACT deletion does not affect X-linked gene dosage in eroded hPSCs and that aberrant XIST RNA diffusion induced by the CRISPR activation system is independent of the presence of XACT RNA. In contrast, the deletion of XACT results in the upregulation of neuron-related genes, facilitating neural differentiation in both male and eroded female hPSCs. XACT RNA repression by CRIPSR inhibition results in the same phenotype. Our study finds that XACT is dispensable for maintaining the erosion of X-lined gene repression on inactive X chromosomes but affects neural differentiation in hPSCs.

Alopecia areata susceptibility variant in MHC region impacts expressions of genes contributing to hair keratinization and is involved in hair loss.

BACKGROUND: Alopecia areata (AA) is considered a highly heritable, T-cell-mediated autoimmune disease of the hair follicle. However, no convincing susceptibility gene has yet been pinpointed in the major histocompatibility complex (MHC), a genome region known to be associated with AA as compared to other regions. METHODS: We engineered mice carrying AA risk allele identified by haplotype sequencing for the MHC region using allele-specific genome editing with the CRISPR/Cas9 system. Finally, we performed functional evaluations in the mice and AA patients with and without the risk allele. FINDINGS: We identified a variant (rs142986308, p.Arg587Trp) in the coiled-coil alpha-helical rod protein 1 (CCHCR1) gene as the only non-synonymous variant in the AA risk haplotype. Furthermore, mice engineered to carry the risk allele displayed a hair loss phenotype. Transcriptomics further identified CCHCR1 as a novel component interacting with hair cortex keratin in hair shafts. Both, these alopecic mice and AA patients with the risk allele displayed morphologically impaired hair and comparable differential expression of hair-related genes, including hair keratin and keratin-associated proteins (KRTAPs). INTERPRETATION: Our results implicate CCHCR1 with the risk allele in a previously unidentified subtype of AA based on aberrant keratinization in addition to autoimmune events. FUNDING: This work was supported by JSPS KAKENHI (JP16K10177) and the NIHR UCLH Biomedical Research center (BRC84/CN/SB/5984).

Imprinted X-chromosome inactivation impacts primitive endoderm differentiation in mouse blastocysts.

Epigenetic and transcriptome alterations are essential for lineage specification, represented by imprinted X-chromosome inactivation (iXCI) in female mouse preimplantation embryos. However, how various factors affect transcriptome states and lineage commitment remains unclear. We found that in vitro culture duration strongly influences transcriptional variation compared to iXCI loss. Single-cell analysis of the inner cell mass (ICM) for major transcription and epigenomic factors revealed that sex-specific differences in expression are diminished by loss of iXCI in the primitive endoderm (PrE) but not in the epiblast. Females had a higher proportion of ICM compared to that in males, and PrE development was affected by iXCI states in female embryos. Our findings provide insight into sex differences and iXCI function in lineage specification.

Computer simulation of emerging asymmetry in the mouse blastocyst.

The mechanism of embryonic polarity establishment in mammals has long been controversial. Whereas some claim prepatterning in the egg, we recently presented evidence that mouse embryonic polarity is not established until blastocyst and proposed the mechanical constraint model. Here we apply computer simulation to clarify the minimal cellular properties required for this morphology. The simulation is based on three assumptions: (1) behavior of cell aggregates is simulated by a 3D vertex dynamics model; (2) all cells have equivalent mechanical properties; (3) an inner cavity with equivalent surface properties is gradually enlarged. However, an initial attempt reveals a requirement for an additional assumption: (4) the surface of the cavity is firmer than intercellular surfaces, suggesting the presence of a basement membrane lining the blastocyst cavity, which is indeed confirmed by published data. The simulation thus successfully produces a structure recapitulating the mouse blastocyst. The axis of the blastocyst, however, remains variable, leading us to an additional assumption: (5) the aggregate is enclosed by a capsule, equivalent to the zona pellucida in vivo. Whereas a spherical capsule does not stabilize the blastocyst axis, an ellipsoidal capsule eventually orients the axis in accordance with its longest diameter. These predictions are experimentally verified by time-lapse recordings of mouse embryos. During simulation, equivalent cells form two distinct populations composed of smaller inner cells and larger outer cells. These results reveal a unique feature of early mammalian development: an asymmetry may emerge autonomously in an equivalent population with no need for a priori intrinsic differences.

Dynamic rearrangement of surface proteins is essential for cytokinesis.

Cytokinesis is a complex process that involves dynamic cortical rearrangement. Our recent time-lapse recordings of the mouse egg unexpectedly revealed a high motility of the second polar body (2pb). Experiments to address its underlying mechanism show that neither mechanical compression by the zona pellucida nor the connection via the mid-body is required for the 2pb movement. Time-lapse recordings establish that the 2pb moves together with the cell membrane. These recordings, in which cell surface proteins are labeled with fluorescent latex-microbeads or monovalent antibodies against whole mouse proteins, indicate that the majority of the surface proteins dynamically accumulate in the cleavage furrow at every cell division. Comparable dynamics of the cell surface proteins, and specifically of E-cadherin, are also observed in cultured epithelial cells. The surface protein dynamics are closely correlated with, and dependent on, those of the underlying cortical actin. The cortical actin network may form a scaffold for membrane proteins and thereby transfer them during contractile ring formation toward the cleavage furrow. Immobilization of surface proteins by tetravalent lectin-mediated crosslinking results in the failure of cleavage, demonstrating that the observed protein dynamics are essential for cytokinesis. We propose that dynamic rearrangement of the cell surface proteins is a common feature of cytokinesis, playing a key role in modifying the mechanical properties of the cell membrane during cortical ingression.

Hypomethylation of paternal DNA in the late mouse zygote is not essential for development.

Global demethylation of DNA which marks the onset of development occurs asynchronously in the mouse; paternal DNA is demethylated at the the zygote stage, whereas maternal DNA is demethylated later in development. The biological function of such asymmetry and its underlying mechanisms are currently unknown. To test the hypothesis that the early demethylation of male DNA may be associated with protamine-histone exchange, we ,used round spermatids, whose DNA is still associated with histones, for artificial fertilization (round spermatid injection or ROSI), and compared the level of methylation of metaphase chromosomes in the resulting zygotes with the level of methylation in zygotes obtained after fertilization using mature sperm heads (intracytoplasmic sperm injection or ICSI). In contrast to ICSI-derived zygotes, ROSI-derived zygotes possessed only slightly demethylated paternal DNA. Both types of zygotes developed to term with similar rates which shows that hypomethylation of paternal DNA at the zygotic metaphase is not essential for full development in mice. Incorporation of exogenously expressed histone H2BYFP into paternal pronuclei was significantly higher in ICSI-derived zygotes than in ROSI-derived zygotes. Surprisingly, in the latter the incorporation of histone H2BYFP into the paternal pronucleus was still significantly higher than into the maternal pronucleus, suggesting that some exchange of chromatin-associated proteins occurs not only after ICSI but also after ROSI. This may explain why after ROSI, some transient demethylation of paternal DNA occurs early after fertilization, thus providing support for the hypothesis regarding the link between paternal DNA demethylation and protamine/histone exchange.

Diploidized eggs reprogram adult somatic cell nuclei to pluripotency in nuclear transfer in medaka fish (Oryzias latipes).

Reprogramming of adult somatic cell nuclei to pluripotency has been unsuccessful in non-mammalian animals, primarily because of chromosomal aberrations in nuclear transplants, which are considered to be caused by asynchrony between the cell cycles of the recipient egg and donor nucleus. In order to normalize the chromosomal status, we used diploidized eggs by retention of second polar body release, instead of enucleated eggs, as recipients in nuclear transfer of primary culture cells from the caudal fin of adult green fluorescent protein gene (GFP) transgenic medaka fish (Oryzias latipes). We found that 2.7% of the reconstructed embryos grew into adults that expressed GFP in various tissues in the same pattern as in the donor fish. Moreover, these fish were diploid, fertile and capable of passing the marker gene to the next generation in Mendelian fashion. We hesitate to call these fish 'clones' because we used non-enucleated eggs as recipients; in effect, they may be chimeras consisting of cells derived from diploid recipient nuclei and donor nuclei. In either case, fish adult somatic cell nuclei were reprogrammed to pluripotency and differentiated into a variety of cell types including germ cells via the use of diploidized recipient eggs.

Ploidy mosaicism in well-developed nuclear transplants produced by transfer of adult somatic cell nuclei to nonenucleated eggs of medaka (Oryzias latipes).

Chromosomal abnormalities such as ploidy mosaicism have constituted a major obstacle to the successful nuclear transfer of adult somatic cell nuclei in lower vertebrates to date. Euploid mosaicism has been reported previously in well-developed amphibian transplants. Here, we investigated ploidy mosaicisms in well-developed transplants of adult somatic cell nuclei in medaka fish (Oryzias latipes). Donor nuclei from primary cultured cells from the adult caudal fin of a transgenic strain carrying the green fluorescent protein gene (GFP) were transferred to recipient nonenucleated eggs of a wild-type strain to produce 662 transplants. While some of the transplants developed beyond the body formation stage and several hatched, all exhibited varying degrees of abnormal morphology, limited growth and subsequent death. Twenty-one transplants, 19 embryos and two larvae, were selected for chromosomal analysis; all were well-developed 6-day-old or later embryonic stages exhibiting slight morphological abnormalities and the same pattern of GFP expression as that of the donor strain. In addition, all exhibited various levels of euploid mosaicism with haploid-diploid, haploid-triploid or haploid-diploid-triploid chromosome sets. No visible chromosomal abnormalities were observed. Thus, euploid mosaicism similar to that observed in amphibians was confirmed in well-developed nuclear transplants of fish.

Space asymmetry directs preferential sperm entry in the absence of polarity in the mouse oocyte.

Knowledge about the mechanism that establishes embryonic polarity is fundamental in understanding mammalian development. In re-addressing several controversial claims, we recently proposed a model in which mouse embryonic polarity is not specified until the blastocyst stage. Before fertilization, the fully differentiated oocyte has been characterized as "polarized," and we indeed observed that the sperm preferentially enters the polar body half. Here we show that preferential sperm entry is not due to an intrinsic polarity of the oocyte, since fertilization takes place uniformly when the zona pellucida is removed. We suggest that the term "asymmetry" denotes morphological differences, whereas "polarity" in addition implies developmental consequences. Thus, the mouse oocyte can be considered "asymmetric" but "non-polarized." The penetration through the zona pellucida is also random, and a significant proportion of sperm binds to the oocyte membrane at a point distant from the zona penetration site. Time-lapse recordings confirmed that sperm swim around the perivitelline space before fertilization. Experimental enlargement of the perivitelline space in the non-polar body half increased the regional probability of fertilization. Based on these experiments, we propose a model in which the space asymmetry exerted by the first polar body and the zona pellucida directs sperm entry preferentially to the polar body half, with no need for oocyte polarity.

Polarity of the mouse embryo is established at blastocyst and is not prepatterned.

Polarity formation in mammalian preimplantation embryos has long been a subject of controversy. Mammalian embryos are highly regulative, which has led to the conclusion that polarity specification does not exist until the blastocyst stage; however, some recent reports have now suggested polarity predetermination in the egg. Our recent time-lapse recordings have demonstrated that the first cleavage plane is not predetermined in the mouse egg. Here we show that, in contrast to previous claims, two-cell blastomeres do not differ and their precise future contribution to the inner cell mass and/or the trophectoderm cannot be anticipated. Thus, all evidence so far strongly suggests the absence of predetermined axes in the mouse egg. We observe that the ellipsoidal zona pellucida exerts mechanical pressure and space constraints as the coalescing multiple cavities are restricted to one end of the long axis of the blastocyst. We propose that these mechanical cues, in conjunction with the epithelial seal in the outer cell layer, lead to specification of the embryonic-abembryonic axis, thus establishing first polarity in the mouse embryo.