Kpna6 deficiency causes infertility in male mice by disrupting spermatogenesis.

Spermatogenesis is driven by an ordered series of events, which rely on trafficking of specific proteins between nucleus and cytoplasm. The karyopherin α family of proteins mediates movement of specific cargo proteins when bound to karyopherin ß. Karyopherin α genes have distinct expression patterns in mouse testis, implying they may have unique roles during mammalian spermatogenesis. Here, we use a loss-of-function approach to determine specifically the role of Kpna6 in spermatogenesis and male fertility. We show that ablation of Kpna6 in male mice leads to infertility and has multiple cumulative effects on both germ cells and Sertoli cells. Kpna6-deficient mice exhibit impaired Sertoli cell function, including loss of Sertoli cells and a compromised nuclear localization of the androgen receptor. Furthermore, our data demonstrate devastating defects on spermiogenesis, including incomplete sperm maturation and a massive reduction in sperm number, accompanied by disturbed histone-protamine exchange, differential localization of the transcriptional regulator BRWD1 and altered expression of RFX2 target genes. Our work uncovers an essential role of Kpna6 in spermatogenesis and, hence, in male fertility.

Frizzled3 inhibits Vangl2-Prickle3 association to establish planar cell polarity in the vertebrate neural plate.

The orientation of epithelial cells in the plane of the tissue, known as planar cell polarity (PCP), is regulated by interactions of asymmetrically localized PCP protein complexes. In the Xenopus neural plate, Van Gogh-like2 (Vangl2) and Prickle3 (Pk3) proteins form a complex at the anterior cell boundaries, but how this complex is regulated in vivo remains largely unknown. Here, we use proximity biotinylation and crosslinking approaches to show that Vangl2-Pk3 association is inhibited by Frizzled3 (Fz3, also known as Fzd3), a core PCP protein that is specifically expressed in the neuroectoderm and is essential for the establishment of PCP in this tissue. This inhibition required Fz3-dependent Vangl2 phosphorylaton. Consistent with our observations, the complex of Pk3 with nonphosphorylatable Vangl2 did not polarize in the neural plate. These findings provide evidence for in vivo regulation of Vangl2-Pk3 complex formation and localization by a Frizzled receptor.

Vangl2 cooperates with Rab11 and Myosin V to regulate apical constriction during vertebrate gastrulation.

Core planar cell polarity (PCP) proteins are well known to regulate polarity in Drosophila and vertebrate epithelia; however, their functions in vertebrate morphogenesis remain poorly understood. In this study, we describe a role for PCP signaling in the process of apical constriction during Xenopus gastrulation. The core PCP protein Vangl2 is detected at the apical surfaces of cells at the blastopore lip, and it functions during blastopore formation and closure. Further experiments show that Vangl2, as well as Daam1 and Rho-associated kinase (Rock), regulate apical constriction of bottle cells at the blastopore and ectopic constriction of ectoderm cells triggered by the actin-binding protein Shroom3. At the blastopore lip, Vangl2 is required for the apical accumulation of the recycling endosome marker Rab11. We also show that Rab11 and the associated motor protein Myosin V play essential roles in both endogenous and ectopic apical constriction, and might be involved in Vangl2 trafficking to the cell surface. Overexpression of Rab11 RNA was sufficient to partly restore normal blastopore formation in Vangl2-deficient embryos. These observations suggest that Vangl2 affects Rab11 to regulate apical constriction during blastopore formation.

Activation of the PTHRP/adenylate cyclase pathway promotes differentiation of rat XEN cells into parietal endoderm, whereas Wnt/ß-catenin signaling promotes differentiation into visceral endoderm.

During early mammalian development, primitive endoderm (PrE) is specified and segregated away from the pluripotent epiblast. At a later developmental stage, PrE forms motile parietal endoderm (PE) lying proximal to the trophectoderm, and visceral endoderm (VE) that contacts the developing epiblast and extraembryonic ectoderm. Mouse extraembryonic endoderm (XEN) cells were isolated and became widely used to study signals governing lineage specification. Rat XEN cell lines have also been derived, but were distinguished from mouse by expression of SSEA1 and Oct4. We showed here that rat XEN cells grown in the presence of a GSK3 inhibitor or overexpressing ß-catenin exhibited enhanced formation of cell contacts and decreased motility. Rat XEN cells treated with BMP4 revealed similar morphological changes. Furthermore, we observed that rat XEN cells cultured with GSK3 inhibitor formed adhesion and tight junctions, and acquired bottom-top polarity, indicating the formation of VE cells. In contrast, forskolin, an activator of the cAMP pathway, induced the disruption of cell contacts in rat XEN cells. Treatment with forskolin induced PE formation and epithelial-mesenchymal transition (EMT) in rat XEN cells. Using microarray and real-time PCR assays, we found that VE versus PE formation of rat XEN cells was correlated with change in expression levels of VE or PE marker genes. Similar to forskolin, EMT was prompted upon treatment of rat XEN cells with recombinant parathyroid hormone related peptide (PTHRP), an activator of the cAMP pathway in vivo. Taken together, our data suggest that rat XEN cells are PrE-like cells. The activation of Wnt or BMP4 pathways in rat XEN cells leads to the acquisition of VE characteristics, whereas the activation of the PTHRP/cAMP pathway leads to EMT and the formation of PE.

Analysis of Planar Cell Polarity Complexes by Proximity Biotinylation in Xenopus Embryos.

Understanding signaling processes operating in cells during development and disease requires extensive knowledge of protein interactions. Proximity-dependent biotinylation mediated by a promiscuous bacterial biotin ligase is a sensitive approach for evaluating protein interactions under physiological conditions. This technique allows for assessing protein association when conventional pull-down assays are not applicable due to high background or transient nature of the interaction. In contrast to many studies of proximity biotinylation in cultured cells, this protocol has been adapted to detect protein interactions in Xenopus embryos. Here, we apply this technique to evaluate planar cell polarity (PCP) complexes formed by Prickle3 and Vangl2, and show that Prickle3 fused to the N-terminal fragment of the biotin ligase from Aquifex aeolicus efficiently biotinylates Vangl2 in vivo. We present our step-by-step proximity biotinylation protocol that provides a reliable semiquantitative assay for protein interactions and highlights the use of Xenopus embryos as a model for biochemical studies.

Par3 interacts with Prickle3 to generate apical PCP complexes in the vertebrate neural plate.

Vertebrate neural tube formation depends on the coordinated orientation of cells in the tissue known as planar cell polarity (PCP). In the Xenopus neural plate, PCP is marked by the enrichment of the conserved proteins Prickle3 and Vangl2 at anterior cell boundaries. Here we show that the apical determinant Par3 is also planar polarized in the neuroepithelium, suggesting a role for Par3 in PCP. Consistent with this hypothesis, interference with Par3 activity inhibited asymmetric distribution of PCP junctional complexes and caused neural tube defects. Importantly, Par3 physically associated with Prickle3 and promoted its apical localization, whereas overexpression of a Prickle3-binding Par3 fragment disrupted PCP in the neural plate. We also adapted proximity biotinylation assay for use in Xenopus embryos and show that Par3 functions by enhancing the formation of the anterior apical PCP complex. These findings describe a mechanistic link between the apical localization of PCP components and morphogenetic movements underlying neurulation.

G1 checkpoint is compromised in mouse ESCs due to functional uncoupling of p53-p21Waf1 signaling.

Mouse embryonic stem cells (mESCs) lack of G1 checkpoint despite that irradiation (IR) activates ATM/ATR-mediated DDR signaling pathway. The IR-induced p53 localizes in the nuclei and up-regulates p21/Waf1 transcription but that does not lead to accumulation of p21/Waf1 protein. The negative control of the p21Waf1 expression appears to occur at 2 levels of regulation. First, both p21/Waf1 gene transcription and the p21/Waf1 protein content increase in mESCs treated with histone-deacetylase inhibitors, implying its epigenetic regulation. Second, proteasome inhibitors cause the p21/Waf1 accumulation, indicating that the protein is a subject of proteasome-dependent degradation in ESСs. Then, the dynamics of IR-induced p21Waf1 protein show its accumulation at long-term time points (3 and 5 days) that coincides with an increase in the proportion of G1-phase cells, down-regulation of Oct4 and Nanog pluripotent gene transcription and activation of endoderm-specific genes sox17 and afp. In addition, nutlin-dependent stabilization of p53 in mESC was also accompanied by the accumulation of p21/Waf1 as well as restoration of G1 checkpoint and an onset of differentiation. Thus, the lack of functional p21/Waf1 is indispensable for maintaining self-renewal and pluripotency of mESCs.

Derivation, characterization, and stable transfection of induced pluripotent stem cells from Fischer344 rats.

The rat represents an important animal model that, in many respects, is superior to the mouse for dissecting behavioral, cardiovascular and other physiological pathologies relevant to humans. Derivation of induced pluripotent stem cells from rats (riPS) opens the opportunity for gene targeting in specific rat strains, as well as for the development of new protocols for the treatment of different degenerative diseases. Here, we report an improved lentivirus-based hit-and-run riPS derivation protocol that makes use of small inhibitors of MEK and GSK3. We demonstrate that the excision of proviruses does not affect either the karyotype or the differentiation ability of these cells. We show that the established riPS cells are readily amenable to genetic manipulations such as stable electroporation. Finally, we propose a genetic tool for an improvement of riPS cell quality in culture. These data may prompt iPS cell-based gene targeting in rat as well as the development of iPS cell-based therapies using disease models established in this species.

Characterization of trophoblast and extraembryonic endoderm cell lineages derived from rat preimplantation embryos.

BACKGROUND: Previous attempts to isolate pluripotent cell lines from rat preimplantation embryo in mouse embryonic stem (ES) cell culture conditions (serum and LIF) were unsuccessful, however the resulting cells exhibited the expression of such traditional pluripotency markers as SSEA-1 and alkaline phosphatase. We addressed the question, which kind of cell lineages are produced from rat preimplantation embryo under "classical" mouse ES conditions. RESULTS: We characterized two cell lines (C5 and B10) which were obtained from rat blastocysts in medium with serum and LIF. In the B10 cell line we found the expression of genes known to be expressed in trophoblast, Cdx-2, cytokeratin-7, and Hand-1. Also, B10 cells invaded the trophectodermal layer upon injection into rat blastocysts. In contrast to mouse Trophoblast Stem (TS) cells proliferation of B10 cells occurred independently of FGF4. Cells of the C5 line expressed traditional markers of extraembryonic-endoderm (XEN) cells, in particular, GATA-4, but also the pluripotency markers SSEA-1 and Oct-4. C5 cell proliferation exhibited dependence on LIF, which is not known to be required by mouse XEN cells. CONCLUSIONS: Our results confirm and extend previous findings about differences between blastocyst-derived cell lines of rat and mice. Our data show, that the B10 cell line represents a population of FGF4-independent rat TS-like cells. C5 cells show features that have recently become known as characteristic of rat XEN cells. Early passages of C5 and B10 cells contained both, TS and XEN cells. We speculate, that mechanisms maintaining self-renewal of cell lineages in rat preimplantation embryo and their in vitro counterparts, including ES, TS and XEN cells are different than in respective mouse lineages.

Activation of DNA damage response signaling in mouse embryonic stem cells.

Mouse embryonic stem cells (mESC) are characterized by high proliferation activity. mESC are highly sensitive to genotoxic stresses and do not undergo G(1)/S checkpoint upon DNA-damage. mESC are supposed to develop sensitive mechanisms to maintain genomic integrity provided by either DNA damage repair or elimination of defected cells by apoptosis. The issue of how mESC recognize the damages and execute DNA repair remains to be studied. We analyzed the kinetics of DNA repair foci marked by antibodies to phosphorylated ATM kinase and histone H2AX (gammaH2AX). We showed that mESC display non-induced DNA single-strand breaks (SSBs), as revealed by comet-assay, and a noticeable background of gammaH2AX staining. Exposure of mESC to gamma-irradiation induced the accumulation of phosphorylated ATM-kinase in the nucleus as well as the formation of additional gammaH2AX foci, which disappeared thereafter. To decrease the background of gammaH2AX staining in control non-irradiated cells, we pre-synchronized mESC at the G(2)/M by low concentration of nocodazol for a short time (6 h). The cells were then irradiated and stained for gammaH2AX. Irradiation induced the formation of gammaH2AX foci both in G(2)-phase and mitotic cells, which evidenced for the active state of DNA-damage signaling at these stages of the cell cycle in mESC. Due to the G(1)/S checkpoint is compromised in mESCs, we checked, whether wild-type p53, a target for ATM kinase, was phosphorylated in response to gamma-irradiation. The p53 was barely phosphorylated in response to irradiation, which correlated with a very low expression of p53-target p21/Waf1 gene. Thus, in spite of the dysfunction of the p53/Waf1 pathway and the lack of cell cycle checkpoints, the mESC are capable of activating ATM and inducing gammaH2AX foci formation, which are necessary for the activation of DNA damage response.