Brachyury co-operates with polycomb protein RYBP to regulate gastrulation and axial elongation .

Early embryonic development is a complex process where undifferentiated cells lose their pluripotency and start to gastrulate. During gastrulation, three germ layers form, giving rise to different cell lineages and organs. This process is regulated by transcription factors and epigenetic regulators, including non-canonical polycomb repressive complex 1s (ncPRC1s).

Polycomb protein RYBP activates transcription factor during cardiac differentiation of mouse embryonic stem cells.

RING1 and YY1 binding protein (RYBP) is primarily known to function as a repressor being a core component of the non-canonical polycomb repressive complexes 1 (ncPRC1s). However, several ncPRC1-independent functions of RYBP have also been described. We previously reported that RYBP is essential for mouse embryonic development and that null mutant embryonic stem cells cannot form contractile cardiomyocytes (CMCs) .

RYBP regulates Pax6 during in vitro neural differentiation of mouse embryonic stem cells.

We have previously reported that RING1 and YY1 binding protein (RYBP) is important for central nervous system development in mice and that Rybp null mutant (Rybp) mouse embryonic stem (ES) cells form more progenitors and less terminally differentiated neural cells than the wild type cells in vitro. Accelerated progenitor formation coincided with a high level of Pax6 expression in the Rybp neural cultures. Since Pax6 is a retinoic acid (RA) inducible gene, we have analyzed whether altered RA signaling contributes to the accelerated progenitor formation and impaired differentiation ability of the Rybp cells.

RYBP is important for cardiac progenitor cell development and sarcomere formation.

We have previously established that epigenetic regulator RING1 and YY1 binding protein (RYBP) is required for the contractility of embryonic stem (ES) cell derived cardiomyocytes (CMCs), suggesting its essential role in contractility. In order to investigate the underlying molecular events of this phenotype, we compared the transcriptomic profile of the wild type and Rybp null mutant ES cells and CMCs differentiated from these cell lines. We identified genes related to ion homeostasis, cell adhesion and sarcomeric organization affected in the Rybp null mutant CMCs, by using hierarchical gene clustering and Gene Ontology analysis.

Evolving Role of RING1 and YY1 Binding Protein in the Regulation of Germ-Cell-Specific Transcription.

Separation of germline cells from somatic lineages is one of the earliest decisions of embryogenesis. Genes expressed in germline cells include apoptotic and meiotic factors, which are not transcribed in the soma normally, but a number of testis-specific genes are active in numerous cancer types. During germ cell development, germ-cell-specific genes can be regulated by specific transcription factors, retinoic acid signaling and multimeric protein complexes.

Absence of Rybp Compromises Neural Differentiation of Embryonic Stem Cells.

Rybp (Ring1 and Yy1 Binding Protein) is a transcriptional regulator and member of the noncanonical polycomb repressive complex 1 with essential role in early embryonic development. We have previously described that alteration of Rybp dosage in mouse models induced striking neural tube defects (NTDs), exencephaly, and disorganized neurocortex. In this study we further investigated the role of Rybp in neural differentiation by utilising wild type (rybp (+/+)) and rybp null mutant (rybp (-/-)) embryonic stem cells (ESCs) and tried to uncover underlying molecular events that are responsible for the observed phenotypic changes.

Lack of Rybp in Mouse Embryonic Stem Cells Impairs Cardiac Differentiation.

Ring1 and Yy1 binding protein (Rybp) has been implicated in transcriptional regulation, apoptotic signaling and as a member of the polycomb repressive complex 1, it has an important function in regulating pluripotency and differentiation of embryonic stem cells (ESCs). Earlier, we had proved that Rybp plays an essential role in mouse embryonic and central nervous system development. This work identifies Rybp, as a critical regulator of heart development.

Grafted murine induced pluripotent stem cells prevent death of injured rat motoneurons otherwise destined to die.

Human plexus injuries often include the avulsion of one or more ventral roots, resulting in debilitating conditions. In this study the effects of undifferentiated murine iPSCs on damaged motoneurons were investigated following avulsion of the lumbar 4 (L4) ventral root, an injury known to induce the death of the majority of the affected motoneurons. Avulsion and reimplantation of the L4 ventral root (AR procedure) were accompanied by the transplantation of murine iPSCs into the injured spinal cord segment in rats.

Nucleotide substitutions revealing specific functions of Polycomb group genes.

POLYCOMB group (PCG) proteins belong to the family of epigenetic regulators of genes playing important roles in differentiation and development. Mutants of PcG genes were isolated first in the fruit fly, Drosophila melanogaster, resulting in spectacular segmental transformations due to the ectopic expression of homeotic genes. Homologs of Drosophila PcG genes were also identified in plants and in vertebrates and subsequent experiments revealed the general role of PCG proteins in the maintenance of the repressed state of chromatin through cell divisions.

Slow turning lateral vessel bioreactor improves embryoid body formation and cardiogenic differentiation of mouse embryonic stem cells.

Embryonic stem cells (ESCs) have the ability to form aggregates, which are called embryoid bodies (EBs). EBs mimic early embryonic development and are commonly produced for cardiomyogenesis. Here, we describe a method of EB formation in hydrodynamic conditions using a slow-turning lateral vessel (STLV) bioreactor and the subsequent differentiation of EBs into cardiomyocytes.

Generation of mouse induced pluripotent stem cells by protein transduction.

Somatic cell reprogramming has generated enormous interest after the first report by Yamanaka and his coworkers in 2006 on the generation of induced pluripotent stem cells (iPSCs) from mouse fibroblasts. Here we report the generation of stable iPSCs from mouse fibroblasts by recombinant protein transduction (Klf4, Oct4, Sox2, and c-Myc), a procedure designed to circumvent the risks caused by integration of exogenous sequences in the target cell genome associated with gene delivery systems. The recombinant proteins were fused in the frame to the glutathione-S-transferase tag for affinity purification and to the transactivator transcription-nuclear localization signal polypeptide to facilitate membrane penetration and nuclear localization.

Conditional and constitutive expression of a Tbx1-GFP fusion protein in mice.

Background: Velo-cardio-facial syndrome/DiGeorge syndrome (VCFS/DGS) is caused by a 1.5-3 Mb microdeletion of chromosome 22q11.2, frequently referred to as 22q11.

Generation of neuronal progenitor cells and neurons from mouse sleeping beauty transposon-generated induced pluripotent stem cells.

Mouse embryonic stem cells (ESCs) and induced pluripotent stem (iPS) cells can be used as models of neuronal differentiation for the investigation of mammalian neurogenesis, pharmacological testing, and development of cell-based therapies. Recently, mouse iPS cell lines have been generated by Sleeping Beauty (SB) transposon-mediated transgenesis (SB-iPS). In this study, we determined for the first time the differentiation potential of mouse SB-iPS cells to form neuronal progenitor cells (NPCs) and neurons.

Generation of mouse induced pluripotent stem cells from different genetic backgrounds using Sleeping beauty transposon mediated gene transfer.

Induced pluripotent stem (iPS) cell technology involves reprogramming somatic cells to a pluripotent state. The original technology used to produce these cells requires viral gene transduction and results in the permanent integration of exogenous genes into the genome. This can lead to the development of abnormalities in the derived iPS cells.

Enhanced cardiac differentiation of mouse embryonic stem cells by use of the slow-turning, lateral vessel (STLV) bioreactor.

Embryoid body (EB) formation is a common intermediate during in vitro differentiation of pluripotent stem cells into specialized cell types. We have optimized the slow-turning, lateral vessel (STLV) for large scale and homogenous EB production from mouse embryonic stem cells. The effects of inoculating different cell numbers, time of EB adherence to gelatin-coated dishes, and rotation speed for optimal EB formation and cardiac differentiation were investigated.

Chromatin remodeling enzyme Brg1 is required for mouse lens fiber cell terminal differentiation and its denucleation.

Background: Brahma-related gene 1 (Brg1, also known as Smarca4 and Snf2β) encodes an adenosine-5'-triphosphate (ATP)-dependent catalytical subunit of the (switch/sucrose nonfermentable) (SWI/SNF) chromatin remodeling complexes. SWI/SNF complexes are recruited to chromatin through multiple mechanisms, including specific DNA-binding factors (for example, heat shock transcription factor 4 (Hsf4) and paired box gene 6 (Pax6)), chromatin structural proteins (for example, high-mobility group A1 (HMGA1)) and/or acetylated core histones. Previous studies have shown that a single amino acid substitution (K798R) in the Brg1 ATPase domain acts via a dominant-negative (dn) mechanism.

Tbx3: another important piece fitted into the pluripotent stem cell puzzle.

Induced pluripotent stem cells (iPSCs) are novel tools for biomedical research, with a promise for future regenerative medicine applications. Recently, Han and colleagues reported in Nature that T box gene 3 (Tbx3) can improve the quality of mouse iPSCs and increase their germline transmission efficacy. This observation contributes greatly to the improvement of iPSC technology and might be a step towards 'designer' reprogramming strategies by generating high quality iPSCs.

Germline competence of mouse ES and iPS cell lines: Chimera technologies and genetic background.

In mice, gene targeting by homologous recombination continues to play an essential role in the understanding of functional genomics. This strategy allows precise location of the site of transgene integration and is most commonly used to ablate gene expression ("knock-out"), or to introduce mutant or modified alleles at the locus of interest ("knock-in"). The efficacy of producing live, transgenic mice challenges our understanding of this complex process, and of the factors which influence germline competence of embryonic stem cell lines.

Embryoid body formation from embryonic and induced pluripotent stem cells: Benefits of bioreactors.

Embryonic stem (ES) cells have the ability to differentiate into all germ layers, holding great promise not only for a model of early embryonic development but also for a robust cell source for cell-replacement therapies and for drug screening. Embryoid body (EB) formation from ES cells is a common method for producing different cell lineages for further applications. However, conventional techniques such as hanging drop or static suspension culture are either inherently incapable of large scale production or exhibit limited control over cell aggregation during EB formation and subsequent EB aggregation.

Promoter analysis of the rabbit POU5F1 gene and its expression in preimplantation stage embryos.

Background: The POU5F1 gene encodes the octamer-binding transcription factor-4 (Oct4). It is crucial in the regulation of pluripotency during embryonic development and widely used as molecular marker of embryonic stem cells (ESCs). The objective of this study was to identify and to analyse the promoter region of rabbit POU5F1 gene; furthermore to examine its expression pattern in preimplantation stage rabbit embryos.

Rybp, a polycomb complex-associated protein, is required for mouse eye development.

Background: Rybp (Ring1 and YY1 binding protein) is a zinc finger protein which interacts with the members of the mammalian polycomb complexes. Previously we have shown that Rybp is critical for early embryogenesis and that haploinsufficiency of Rybp in a subset of embryos causes failure of neural tube closure. Here we investigated the requirement for Rybp in ocular development using four in vivo mouse models which resulted in either the ablation or overexpression of Rybp.

In vivo imaging and differential localization of lipid-modified GFP-variant fusions in embryonic stem cells and mice.

The visualization of live cell behaviors operating in situ combined with the power of mouse genetics represents a major step toward understanding the mechanisms regulating embryonic development, homeostasis, and disease progression in mammals. The availability of genetically encoded fluorescent protein reporters, combined with improved optical imaging modalities, have led to advances in our ability to examine cells in vivo. We developed a series of lipid-modified fluorescent protein fusions that are targeted to and label the secretory pathway and the plasma membrane, and that are amenable for use in mice.

Rybp/DEDAF is required for early postimplantation and for central nervous system development.

The Rybp/DEDAF protein has been implicated in both transcriptional regulation and apoptotic signaling, but its precise molecular function is unclear. To determine the physiological role of Rybp, we analyzed its expression during mouse development and generated mice carrying a targeted deletion of Rybp using homologous recombination in embryonic stem cells. Rybp was found to be broadly expressed during embryogenesis and was particularly abundant in extraembryonic tissues, including trophoblast giant cells.