Satoshi Kurosaka

Pluripotent Lineage Definition in Bovine Embryos by Oct4 Transcript Localization

Abstract The POU-domain transcription factor Pou5f1 (Oct4) is restricted to pluripotent embryonic cells and the germ line of the mouse and is required for the maintenance of pluripotency of cells within the inner cell mass of the mouse blastocyst. Despite highly conserved genomic organization and regulatory regions between the mouse Oct4 gene and its bovine orthologue, bovine Oct4 protein is not restricted to the inner cell mass of blastocyst-stage embryos, suggesting that Oct4 may not be a key regulator of pluripotency in the bovine. We analyze the temporal and spatial distribution of Oct4 transcript in bovine oocytes and preimplantation-stage embryos, and in contrast to protein distribution, we find strong conservation between bovine and mouse. Oct4 transcript is present at low levels in the bovine oocyte. Similar to mouse, bovine Oct4 transcription begins one to two cell cycles after zygotic genome activation, followed by a sharp increase in transcription subsequent to compaction. Oct4 transcript is ubiquitously present in all cells of embryos at the morula stage; however, in Day 7 bovine blastocysts, Oct4 signal is not visible in the trophectoderm by in situ hybridization, indicating that transcriptional downregulation of Oct4 on differentiation is similar to that observed in mouse and other mammals. These results indicate that in contrast to protein distribution, regulation of Oct4 transcription is conserved between mammalian species.

Cell Biology of Embryonic Migration

Cell migration is an evolutionarily conserved mechanism that underlies the development and functioning of uni- and multicellular organisms and takes place in normal and pathogenic processes, including various events of embryogenesis, wound healing, immune response, cancer metastases, and angiogenesis. Despite the differences in the cell types that take part in different migratory events, it is believed that all of these migrations occur by similar molecular mechanisms, whose major components have been functionally conserved in evolution and whose perturbation leads to severe developmental defects. These mechanisms involve intricate cytoskeleton-based molecular machines that can sense the environment, respond to signals, and modulate the entire cell behavior. A big question that has concerned the researchers for decades relates to the coordination of cell migration in situ and its relation to the intracellular aspects of the cell migratory mechanisms. Traditionally, this question has been addressed by researchers that considered the intra- and extracellular mechanisms driving migration in separate sets of studies. As more data accumulate researchers are now able to integrate all of the available information and consider the intracellular mechanisms of cell migration in the context of the developing organisms that contain additional levels of complexity provided by extracellular regulation. This review provides a broad summary of the existing and emerging data in the cell and developmental biology fields regarding cell migration during development.

Arginylation-Dependent Neural Crest Cell Migration Is Essential for Mouse Development

Coordinated cell migration during development is crucial for morphogenesis and largely relies on cells of the neural crest lineage that migrate over long distances to give rise to organs and tissues throughout the body. Recent studies of protein arginylation implicated this poorly understood posttranslational modification in the functioning of actin cytoskeleton and in cell migration in culture. Knockout of arginyltransferase (Ate1) in mice leads to embryonic lethality and severe heart defects that are reminiscent of cell migration–dependent phenotypes seen in other mouse models. To test the hypothesis that arginylation regulates cell migration during morphogenesis, we produced Wnt1-Cre Ate1 conditional knockout mice (Wnt1-Ate1), with Ate1 deletion in the neural crest cells driven by Wnt1 promoter. Wnt1-Ate1 mice die at birth and in the first 2–3 weeks after birth with severe breathing problems and with growth and behavioral retardation. Wnt1-Ate1 pups have prominent defects, including short palate and altered opening to the nasopharynx, and cranial defects that likely contribute to the abnormal breathing and early death. Analysis of neural crest cell movement patterns in situ and cell motility in culture shows an overall delay in the migration of Ate1 knockout cells that is likely regulated by intracellular mechanisms rather than extracellular signaling events. Taken together, our data suggest that arginylation plays a general role in the migration of the neural crest cells in development by regulating the molecular machinery that underlies cell migration through tissues and organs during morphogenesis.

Structural, super-resolution microscopy analysis of paraspeckle nuclear body organization

Paraspeckles are nuclear bodies built on the long noncoding RNA Neat1. Using structural illumination microscopy, West et al. analyze the organization of paraspeckles at the submicron scale and show that paraspeckle proteins are arranged around bundles of Neat1, forming core-shell spheroidal structures dependent on the RNA binding protein Fus.

Arginylation regulates myofibrils to maintain heart function and prevent dilated cardiomyopathy

Protein arginylation mediated by arginyltransferase (ATE1) is essential for heart formation during embryogenesis, however its cell-autonomous role in cardiomyocytes and the differentiated heart muscle has never been investigated. To address this question, we generated cardiac muscle-specific Ate1 knockout mice, in which Ate1 deletion was driven by α-myosin heavy chain promoter (αMHC-Ate1 mouse). These mice were initially viable, but developed severe cardiac contractility defects, dilated cardiomyopathy, and thrombosis over time, resulting in high rates of lethality after 6months of age. These symptoms were accompanied by severe ultrastructural defects in cardiac myofibrils, seen in the newborns and far preceding the onset of cardiomyopathy, suggesting that these defects were primary and likely underlay the development of the future heart defects. Several major sarcomeric proteins were arginylated in vivo. Moreover, Ate1 deletion in the hearts resulted in a significant reduction of active and passive myofibril forces, suggesting that arginylation is critical for both myofibril structural integrity and contractility. Thus, arginylation is essential for maintaining the heart function by regulation of the major myofibril proteins and myofibril forces, and its absence in the heart muscle leads to progressive heart failure through cardiomyocyte-specific defects.

Conditional Tek Promoter-Driven Deletion of Arginyltransferase in the Germ Line Causes Defects in Gametogenesis and Early Embryonic Lethality in Mice

Posttranslational protein arginylation mediated by Ate1 is essential for cardiovascular development, actin cytoskeleton functioning, and cell migration. Ate1 plays a role in the regulation of cytoskeleton and is essential for cardiovascular development and angiogenesis—capillary remodeling driven by in-tissue migration of endothelial cells. To address the role of Ate1 in cytoskeleton-dependent processes and endothelial cell function during development, we produced a conditional mouse knockout with Ate1 deletion driven by Tek endothelial receptor tyrosine kinase promoter expressed in the endothelium and in the germ line. Contrary to expectations, Tek-Ate1 mice were viable and had no visible angiogenesis-related phenotypes; however, these mice showed reproductive defects, with high rates of embryonic lethality in the second generation, at stages much earlier than the complete Ate1 knockout strain. While some of the early lethality originated from the subpopulation of embryos with homozygous Tek-Cre transgene—a problem that has not previously been reported for this commercial mouse strain—a distinct subpopulation of embryos had lethality at early post-implantation stages that could be explained only by a previously unknown defect in gametogenesis originating from Tek-driven Ate1 deletion in premeiotic germs cells. These results demonstrate a novel role of Ate1 in germ cell development.

Unusual semi‐extractability as a hallmark of nuclear body‐associated architectural noncoding RNAs

NEAT1_2 long noncoding RNA (lncRNA) is the molecular scaffold of paraspeckle nuclear bodies. Here, we report an improved RNA extraction method: extensive needle shearing or heating of cell lysate in RNA extraction reagent improved NEAT1_2 extraction by 20‐fold (a property we term “semi‐extractability”), whereas using a conventional method NEAT1_2 was trapped in the protein phase. The improved extraction method enabled us to estimate that approximately 50 NEAT1_2 molecules are present in a single paraspeckle. Another architectural lncRNA, IGS16, also exhibited similar semi‐extractability. A comparison of RNA‐seq data from needle‐sheared and control samples revealed the existence of multiple semi‐extractable RNAs, many of which were localized in subnuclear granule‐like structures. The semi‐extractability of NEAT1_2 correlated with its association with paraspeckle proteins and required the prion‐like domain of the RNA‐binding protein FUS. This observation suggests that tenacious RNA–protein and protein–protein interactions, which drive nuclear body formation, are responsible for semi‐extractability. Our findings provide a foundation for the discovery of the architectural RNAs that constitute nuclear bodies.

Contractility of myofibrils from the heart and diaphragm muscles measured with atomic force cantilevers: effects of heart-specific deletion of arginyl-tRNA-protein transferase.

BACKGROUND Contractile properties of myofibrils from the myocardium and diaphragm in chronic heart failure are not well understood. We investigated myofibrils in a knockout (KO) mouse model with cardiac-specific deletion of arginyl-tRNA-protein transferase (α-MHCAte1), which presents dilated cardiomyopathy and heart failure. OBJECTIVE The aim of this study was to test the hypothesis that chronic heart failure in α-MHCAte1 mice is associated with abnormal contractile properties of the heart and diaphragm. METHODS We used a newly developed system of atomic force cantilevers (AFC) to compare myofibrils from α-MHCAte1 and age-matched wild type mice (WT). Myofibrils from the myocardium and the diaphragm were attached to the AFC used for force measurements during activation/deactivation cycles at different sarcomere lengths. RESULTS In the heart, α-MHCAte1 myofibrils presented a reduced force during full activation (89±9 nN/μm(2)) when compared to WT (132±11 nN/μm(2)), and the decrease was not influenced by sarcomere length. These myofibrils presented similar kinetics of force development (K(act)), redevelopment (K(tr)), and relaxation (K(rel)). In the diaphragm, α-MHCAte1 myofibrils presented an increased force during full activation (209±31 nN/μm(2)) when compared to WT (123±20 nN/μm(2)). Diaphragm myofibrils of α-MHCAte1 and WT presented similar K(act), but α-MHCAte1 myofibrils presented a faster K(rel) (6.11±0.41s(-1) vs 4.63±0.41 s(-1)). CONCLUSION Contrary to our working hypothesis, diaphragm myofibrils from α-MHCAte1 mice produced an increased force compared to myofibrils from WT. These results suggest a potential compensatory mechanism by which the diaphragm works under loading conditions in the α-MHCAte1 chronic heart failure model.

Androgenetic Embryonic Stem Cells Form Neural Progenitor Cells In Vivo and In Vitro

Uniparental zygotes with two paternal (androgenetic [AG]) or two maternal (gynogenetic [GG]; parthenogenetic [PG]) genomes are not able to develop into viable offspring but can form blastocysts from which embryonic stem cells (ESCs) can be derived. Although some aspects of the in vitro and in vivo differentiation potential of PG and GG ESCs of several species have been studied, the developmental capacity of AG ESCs is much less clear. Here, we investigate the potential of murine AG ESCs to undergo neural differentiation. We observed that AG ESCs differentiate in vitro into pan‐neural progenitor cells (pnPCs) that further give rise to cells that express neuronal‐ and astroglial‐specific markers. Neural progeny of in vitro‐differentiated AG ESCs exhibited fidelity of expression of six imprinted genes analyzed, with the exception of Ube3a. Bisulfite sequencing for two imprinting control regions suggested that pnPCs predominantly maintained their methylation pattern. Following blastocyst injection of AG and biparental (normal fertilized [N]) ESCs, we found widespread and evenly distributed contribution of ESC‐derived cells in both AG and N chimeric early fetal brains. AG and N ESC‐derived cells isolated from chimeric fetal brains by fluorescence‐activated cell sorting exhibited similar neurosphere‐initiating cell frequencies and neural multilineage differentiation potential. Our results indicate that AG ESC‐derived neural progenitor/stem cells do not differ from N neural progenitor/stem cells in their self‐renewal and neural multilineage differentiation potential.

Arginyltransferase suppresses cell tumorigenic potential and inversely correlates with metastases in human cancers

Arginylation is an emerging post-translational modification mediated by arginyltransferase (ATE1) that is essential for mammalian embryogenesis and regulation of the cytoskeleton. Here, we discovered that Ate1-knockout (KO) embryonic fibroblasts exhibit tumorigenic properties, including abnormally rapid contact-independent growth, reduced ability to form cell–cell contacts and chromosomal aberrations. Ate1-KO fibroblasts can form large colonies in Matrigel and exhibit invasive behavior, unlike wild-type fibroblasts. Furthermore, Ate1-KO cells form tumors in subcutaneous xenograft assays in immunocompromised mice. Abnormal growth in these cells can be partially rescued by reintroduction of stably expressed specific Ate1 isoforms, which also reduce the ability of these cells to form tumors. Tumor array studies and bioinformatics analysis show that Ate1 is downregulated in several types of human cancer samples at the protein level, and that its transcription level inversely correlates with metastatic progression and patient survival. We conclude that Ate1-KO results in carcinogenic transformation of cultured fibroblasts, suggesting that in addition to its previously known activities Ate1 gene is essential for tumor suppression and also likely participates in suppression of metastatic growth.