Expression pattern of Runt-related transcription factor (RUNX) family members and the role of RUNX1 during kidney development.

Runt-related transcription factor (RUNX) family members play critical roles in the development of multiple organs. Mammalian RUNX family members, consisting of RUNX1, RUNX2, and RUNX3, have distinct tissue-specific expression and function. In this study, we examined the spatiotemporal expression patterns of RUNX family members in developing kidneys and analyzed the role of RUNX1 during kidney development.

Null mutation of exocyst complex component 3-like does not affect vascular development in mice.

Exocyst is an octameric protein complex implicated in exocytosis. The exocyst complex is highly conserved among mammalian species, but the physiological function of each subunit in exocyst remains unclear. Previously, we identified exocyst complex component 3-like (Exoc3l) as a gene abundantly expressed in embryonic endothelial cells and implicated in the process of angiogenesis in human umbilical cord endothelial cells.

Induction of fetal meiotic oocytes from embryonic stem cells in cynomolgus monkeys.

Human in vitro oogenesis provides a framework for clarifying the mechanism of human oogenesis. To create its benchmark, it is vital to promote in vitro oogenesis using a model physiologically close to humans. Here, we establish a foundation for in vitro oogenesis in cynomolgus (cy) monkeys (Macaca fascicularis): cy female embryonic stem cells harboring one active and one inactive X chromosome (Xa and Xi, respectively) differentiate robustly into primordial germ cell-like cells, which in xenogeneic reconstituted ovaries develop efficiently into oogonia and, remarkably, further into meiotic oocytes at the zygotene stage.

Essential Roles of on Cardiovascular Development in Mice.

Angiogenesis is a process to generate new blood vessels from pre-existing vessels and to maintain vessels, and plays critical roles in normal development and disease. However, the molecular mechanisms underlying angiogenesis are not fully understood. This study examined the roles of during development in mice.

Conformational change of RNA-helicase DHX30 by ALS/FTD-linked FUS induces mitochondrial dysfunction and cytosolic aggregates.

Genetic mutations in fused in sarcoma (FUS) cause amyotrophic lateral sclerosis (ALS). Although mitochondrial dysfunction and stress granule have been crucially implicated in FUS proteinopathy, the molecular basis remains unclear. Here, we show that DHX30, a component of mitochondrial RNA granules required for mitochondrial ribosome assembly, interacts with FUS, and plays a crucial role in ALS-FUS.

Ex vivo reconstitution of fetal oocyte development in humans and cynomolgus monkeys.

In vitro oogenesis is key to elucidating the mechanism of human female germ-cell development and its anomalies. Accordingly, pluripotent stem cells have been induced into primordial germ cell-like cells and into oogonia with epigenetic reprogramming, yet further reconstitutions remain a challenge. Here, we demonstrate ex vivo reconstitution of fetal oocyte development in both humans and cynomolgus monkeys (Macaca fascicularis).

Chromosomal-scale de novo genome assemblies of Cynomolgus Macaque and Common Marmoset.

Cynomolgus macaque (Macaca fascicularis) and common marmoset (Callithrix jacchus) have been widely used in human biomedical research. Long-standing primate genome assemblies used the human genome as a reference for ordering and orienting the assembled fragments into chromosomes. Here we performed de novo genome assembly of these two species without any human genome-based bias observed in the genome assemblies released earlier.

Non-human primates as a model for human development.

Human development has been studied for over a century, but the molecular mechanisms underlying human embryogenesis remain largely unknown due to technical difficulties and ethical issues. Accordingly, mice have been used as a model for mammalian development and studied extensively to infer human biology based on the conservation of fundamental processes between the two species. As research has progressed, however, species-specific differences in characteristics between rodents and primates have become apparent.

GATA transcription factors, SOX17 and TFAP2C, drive the human germ-cell specification program.

The in vitro reconstitution of human germ-cell development provides a robust framework for clarifying key underlying mechanisms. Here, we explored transcription factors (TFs) that engender the germ-cell fate in their pluripotent precursors. Unexpectedly, , , and , which act under the BMP signaling and are indispensable for human primordial germ-cell-like cell (hPGCLC) specification, failed to induce hPGCLCs.

Generation of Transgenic Cynomolgus Monkeys Overexpressing the Gene for Amyloid-β Precursor Protein.

Alzheimer's disease (AD) is the most common cause of dementia and understanding its pathogenesis should lead to improved therapeutic and diagnostic methods. Although several groups have developed transgenic mouse models overexpressing the human amyloid-β precursor protein (APP) gene with AD mutations, with and without presenilin mutations, as well as APP gene knock-in mouse models, these animals display amyloid pathology but do not show neurofibrillary tangles or neuronal loss. This presumably is due to differences between the etiology of the aged-related human disease and the mouse models.

Monkeys mutant for PKD1 recapitulate human autosomal dominant polycystic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD) caused by PKD1 mutations is one of the most common hereditary disorders. However, the key pathological processes underlying cyst development and exacerbation in pre-symptomatic stages remain unknown, because rodent models do not recapitulate critical disease phenotypes, including disease onset in heterozygotes. Here, using CRISPR/Cas9, we generate ADPKD models with PKD1 mutations in cynomolgus monkeys.

Generation of an OCT3/4 reporter cynomolgus monkey ES cell line using CRISPR/Cas9.

Cynomolgus monkey ES (Cyn ES) cells can be generated in a similar manner as human ES cells. However, Cyn ES cells are difficult to maintain in an undifferentiated state by untrained researchers. For easier culture, we generated an OCT3/4-P2A tdTomato IRES Zeocin Cyn ES cell line using CRISPR/Cas9 genome editing technology.

Comprehensive evaluation of ubiquitous promoters suitable for the generation of transgenic cynomolgus monkeys†.

Nonhuman primates (NHPs) are considered to be the most valuable models for human transgenic (Tg) research into disease because human pathology is more closely recapitulated in NHPs than rodents. Previous studies have reported the generation of Tg NHPs that ubiquitously overexpress a transgene using various promoters, but it is not yet clear which promoter is most suitable for the generation of NHPs overexpressing a transgene ubiquitously and persistently in various tissues. To clarify this issue, we evaluated four putative ubiquitous promoters, cytomegalovirus (CMV) immediate-early enhancer and chicken beta-actin (CAG), elongation factor 1α (EF1α), ubiquitin C (UbC), and CMV, using an in vitro differentiation system of cynomolgus monkey embryonic stem cells (ESCs).

Generating Vegfr3 reporter transgenic mouse expressing membrane-tagged Venus for visualization of VEGFR3 expression in vascular and lymphatic endothelial cells.

Vascular endothelial growth factor receptor 3 (Vegfr3) has been widely used as a marker for lymphatic and vascular endothelial cells during mouse embryonic development and in adult mouse, making it valuable for studying angiogenesis and lymphangiogenesis under normal and pathological conditions. Here, we report the generation of a novel transgenic (Tg) mouse that expresses a membrane-localized fluorescent reporter protein, Gap43-Venus, under the control of the Vegfr3 regulatory sequence. Vegfr3-Gap43-Venus BAC Tg recapitulated endogenous Vegfr3 expression in vascular and lymphatic endothelial cells during embryonic development and tumor development.

Flexible adaptation of male germ cells from female iPSCs of endangered .

In mammals, the Y chromosome strictly influences the maintenance of male germ cells. Almost all mammalian species require genetic contributors to generate testes. An endangered species, , has a unique sex chromosome composition XO/XO, and genetic differences between males and females have not been confirmed.

Derivation of Induced Trophoblast Cell Lines in Cattle by Doxycycline-Inducible piggyBac Vectors.

Trophectoderm lineage specification is one of the earliest differentiation events in mammalian development. The trophoblast lineage, which is derived from the trophectoderm, mediates implantation and placental formation. However, the processes involved in trophoblastic differentiation and placental formation in cattle remain unclear due to interspecies differences when compared with other model systems and the small repertoire of available trophoblast cell lines.

Generation of transgenic cynomolgus monkeys that express green fluorescent protein throughout the whole body.

Nonhuman primates are valuable for human disease modelling, because rodents poorly recapitulate some human diseases such as Parkinson's disease and Alzheimer's disease amongst others. Here, we report for the first time, the generation of green fluorescent protein (GFP) transgenic cynomolgus monkeys by lentivirus infection. Our data show that the use of a human cytomegalovirus immediate-early enhancer and chicken beta actin promoter (CAG) directed the ubiquitous expression of the transgene in cynomolgus monkeys.

Generation of Naïve Bovine Induced Pluripotent Stem Cells Using PiggyBac Transposition of Doxycycline-Inducible Transcription Factors.

Generation of pluripotent stem cells (PSCs) in large domestic animals has achieved only limited success; most of the PSCs obtained to date have been classified as primed PSCs, which possess very little capacity to produce chimeric offspring. By contrast, mouse PSCs have been classified as naïve PSCs that can contribute to most of the tissues of chimeras, including germ cells. Here, we describe the generation of two different types of bovine induced pluripotent stem cells (biPSCs) from amnion cells, achieved through introduction of piggyBac vectors containing doxycycline-inducible transcription factors (Oct3/4, Sox2, Klf4, and c-Myc).

A hyperactive piggyBac transposon system is an easy-to-implement method for introducing foreign genes into mouse preimplantation embryos.

Transgenic mice are important tools for genetic analysis. A current prominent method for producing transgenic mice involves pronuclear microinjection into 1-cell embryos. However, the total transgenic efficiency obtained using this method is less than 10%.

Establishment of trophoblast stem cells under defined culture conditions in mice.

The inner cell mass (ICM) and trophoblast cell lineages duet early embryonic development in mammals. After implantation, the ICM forms the embryo proper as well as some extraembryonic tissues, whereas the trophoectoderm (TE) exclusively forms the fetal portion of the placenta and the trophoblast giant cells. Although embryonic stem (ES) cells can be derived from ICM in cultures of mouse blastocysts in the presence of LIF and/or combinations of small-molecule chemical compounds, and the undifferentiated pluripotent state can be stably maintained without use of serum and feeder cells, defined culture conditions for derivation and maintenance of undifferentiated trophoblast stem (TS) cells have not been established.

A modified EpiSC culture condition containing a GSK3 inhibitor can support germline-competent pluripotency in mice.

Embryonic stem cells (ESCs) can contribute to the tissues of chimeric animals, including the germline. By contrast, epiblast stem cells (EpiSCs) barely contribute to chimeras. These two types of cells are established and maintained under different culture conditions.