Masakazu Hashimoto

Planar polarization of node cells determines the rotational axis of node cilia

Rotational movement of the node cilia generates a leftward fluid flow in the mouse embryo because the cilia are posteriorly tilted. However, it is not known how anterior-posterior information is translated into the posterior tilt of the node cilia. Here, we show that the basal body of node cilia is initially positioned centrally but then gradually shifts toward the posterior side of the node cells. Positioning of the basal body and unidirectional flow were found to be impaired in compound mutant mice lacking Dvl genes. Whereas the basal body was normally positioned in the node cells of Wnt3a−/− embryos, inhibition of Rac1, a component of the noncanonical Wnt signalling pathway, impaired the polarized localization of the basal body in wild-type embryos. Dvl2 and Dvl3 proteins were found to be localized to the apical side of the node cells, and their location was polarized to the posterior side of the cells before the posterior positioning of the basal body. These results suggest that posterior positioning of the basal body, which provides the posterior tilt to node cilia, is determined by planar polarization mediated by noncanonical Wnt signalling.

Electroporation enables the efficient mRNA delivery into the mouse zygotes and facilitates CRISPR/Cas9-based genome editing

Recent use of the CRISPR/Cas9 system has dramatically reduced the time required to produce mutant mice, but the involvement of a time-consuming microinjection step still hampers its application for high-throughput genetic analysis. Here we developed a simple, highly efficient, and large-scale genome editing method, in which the RNAs for the CRISPR/Cas9 system are electroporated into zygotes rather than microinjected. We used this method to perform single-stranded oligodeoxynucleotide (ssODN)-mediated knock-in in mouse embryos. This method facilitates large-scale genetic analysis in the mouse.

Electroporation of Cas9 protein/sgRNA into early pronuclear zygotes generates non-mosaic mutants in the mouse

The CRISPR/Cas9 system is a powerful tool for elucidating the roles of genes in a wide variety of organisms including mice. To obtain genetically modified embryos or mice by this method, Cas9 mRNA and sgRNA are usually introduced into zygotes by microinjection or electroporation. However, most mutants generated with this method are genetically mosaic, composed of several types of cells carrying different mutations, which complicates phenotype analysis in founder embryos or mice. To simplify the analysis and to elucidate the roles of genes involved in developmental processes, a method for producing non-mosaic mutants is needed. Here, we established a method for generating non-mosaic mouse mutant embryos. We introduced Cas9 protein and sgRNA into in vitro fertilized (IVF) zygotes by electroporation, which enabled the genome editing to occur before the first replication of the mouse genome. As a result, all of the cells in the mutant carried the same set of mutations. This method solves the problem of mosaicism/allele complexity in founder mutant embryos or mice generated by the CRIPSR/Cas9 system.

Translation of anterior–posterior polarity into left–right polarity in the mouse embryo

The breaking of left-right symmetry in the mouse involves unidirectional fluid flow. Rotational movement of the node cilia generates leftward flow because the cilia are posteriorly tilted. However, it is unknown how anterior-posterior (A-P) information is translated into the posterior tilt of the node cilia. The tilt is determined by the position of the basal body of node cilia. Some of the planar cell polarity (PCP) core proteins such as Dvl are asymmetrically distributed in the node cells, and positioning of the basal body is impaired in mutant mice lacking Dvl genes. Therefore, posterior positioning of the basal body is determined by planar polarization involving noncanonical Wnt signaling. However, the identity of initial A-P information remains unknown.

A Wnt5 activity asymmetry and intercellular signaling via PCP proteins polarize node cells for left-right symmetry breaking.

Polarization of node cells along the anterior-posterior axis of mouse embryos is responsible for left-right symmetry breaking. How node cells become polarized has remained unknown, however. Wnt5a and Wnt5b are expressed posteriorly relative to the node, whereas genes for Sfrp inhibitors of Wnt signaling are expressed anteriorly. Here we show that polarization of node cells is impaired in Wnt5a-/-Wnt5b-/- and Sfrp mutant embryos, and also in the presence of a uniform distribution of Wnt5a or Sfrp1, suggesting that Wnt5 and Sfrp proteins act as instructive signals in this process. The absence of planar cell polarity (PCP) core proteins Prickle1 and Prickle2 in individual cells or local forced expression of Wnt5a perturbed polarization of neighboring wild-type cells. Our results suggest that opposing gradients of Wnt5a and Wnt5b and of their Sfrp inhibitors, together with intercellular signaling via PCP proteins, polarize node cells along the anterior-posterior axis for breaking of left-right symmetry.

Somatic cell reprogramming-free generation of genetically modified pigs

A new and highly efficient method for generating mutant pigs by electroporating the CRISPR/Cas9 system into zygotes. Genetically modified pigs for biomedical applications have been mainly generated using the somatic cell nuclear transfer technique; however, this approach requires complex micromanipulation techniques and sometimes increases the risks of both prenatal and postnatal death by faulty epigenetic reprogramming of a donor somatic cell nucleus. As a result, the production of genetically modified pigs has not been widely applied. We provide a simple method for CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 gene editing in pigs that involves the introduction of Cas9 protein and single-guide RNA into in vitro fertilized zygotes by electroporation. The use of gene editing by electroporation of Cas9 protein (GEEP) resulted in highly efficient targeted gene disruption and was validated by the efficient production of Myostatin mutant pigs. Because GEEP does not require the complex methods associated with micromanipulation for somatic reprogramming, it has the potential for facilitating the genetic modification of pigs.

GFRA2 Identifies Cardiac Progenitors and Mediates Cardiomyocyte Differentiation in a RET-Independent Signaling Pathway

Summary A surface marker that distinctly identifies cardiac progenitors (CPs) is essential for the robust isolation of these cells, circumventing the necessity of genetic modification. Here, we demonstrate that a Glycosylphosphatidylinositol-anchor containing neurotrophic factor receptor, Glial cell line-derived neurotrophic factor receptor alpha 2 (Gfra2), specifically marks CPs. GFRA2 expression facilitates the isolation of CPs by fluorescence activated cell sorting from differentiating mouse and human pluripotent stem cells. Gfra2 mutants reveal an important role for GFRA2 in cardiomyocyte differentiation and development both in vitro and in vivo. Mechanistically, the cardiac GFRA2 signaling pathway is distinct from the canonical pathway dependent on the RET tyrosine kinase and its established ligands. Collectively, our findings establish a platform for investigating the biology of CPs as a foundation for future development of CP transplantation for treating heart failure.

Making practical use of IPv6 anycasting: mobile IPv6 based approach

Anycast is a new IPv6 function in which an anycast address can be assigned to multiple nodes that provide the same service. The anycast packet is transmitted to one appropriate node selected out of all the nodes assigned to the same anycast address. Although it is expected that anycast will be used in various ways, the use of anycast is quite limited today. This is because anycast functionalities are still unclear, and it is difficult to realize a global anycast that requires existing routing protocols to be radically changed. In this paper, we clarify and classify some essential anycast issues and propose a new and practical communication architecture, based on mobile IPv6 architecture, that overcomes the anycast difficulties.

Epiblast formation by Tead-Yap-dependent expression of pluripotency factors and competitive elimination of unspecified cells

The epiblast is a pluripotent cell population first formed in preimplantation embryos and its quality is important for proper development. Here, we examined the mechanisms of epiblast formation and found that the Hippo pathway transcription factor Tead and its coactivator Yap regulate expression of pluripotency factors. After specification of the inner cell mass, Yap accumulates in the nuclei and activates Tead. Tead activity is required for strong expression of pluripotency factors and is variable in the forming epiblast. Cells showing low Tead activity are eliminated from the epiblast through cell competition. Pluripotency factor expression and Myc control cell competition downstream of Tead activity. Cell competition eliminates unspecified cells and is required for proper organization of the epiblast. These results suggest that induction of pluripotency factors by Tead activity and elimination of unspecified cells via cell competition ensure the production of an epiblast with naïve pluripotency.

16-P018 Cell polarity in the node for basal body positioning and nodal flow

Then they differentiate into ciliated cells by intercalating radially into the outer layer during mid neurulae stages. We show that dystroglycan (DG), a transmembrane receptor linking the extracellular matrix to the cytoskeleton, is expressed in cells of the inner layer. The mRNA and proteins are expressed when skin epithelialisation begins and disappeared at tail bud stage when the larval skin is fully differentiated suggesting DG unexplored roles in the specification and/or formation of this tissue. In order to study the functions of DG during skin formation, lossof-function experiments were performed using DG antisense morpholinos. Depletion of DG gives rise to a skin surface depleted in ciliated cells. In vitro and in vivo analyses of this phenotype show that ciliated cells precursors differentiate since they expressed alpha-tubulin. But they do not intercalate in the outer layer and do not undergo ciliogenesis. We propose that DG is required for ciliated cell intercalation during skin formation. The mechanisms implicated in this process are still under study.