FGF4 Independent Derivation of Trophoblast Stem Cells from the Common Vole

The derivation of stable multipotent trophoblast stem (TS) cell lines from preimplantation, and early postimplantation mouse embryos has been reported previously. FGF4, and its receptor FGFR2, have been identified as embryonic signaling factors responsible for the maintenance of the undifferentiated state of multipotent TS cells. Here we report the derivation of stable TS-like cell lines from the vole M. rossiaemeridionalis, in the absence of FGF4 and heparin. Vole TS-like cells are similar to murine TS cells with respect to their morphology, transcription factor gene expression and differentiation in vitro into derivatives of the trophectoderm lineage, and with respect to their ability to invade and erode host tissues, forming haemorrhagic tumours after subcutaneous injection into nude mice. Moreover, vole TS-like cells carry an inactive paternal X chromosome, indicating that they have undergone imprinted X inactivation, which is characteristic of the trophoblast lineage. Our results indicate that an alternative signaling pathway may be responsible for the establishment and stable proliferation of vole TS-like cells

Introducing an expanded CAG tract into the huntingtin gene causes a wide spectrum of ultrastructural defects in cultured human cells.

Modeling of neurodegenerative diseases in vitro holds great promise for biomedical research. Human cell lines harboring a mutations in disease-causing genes are thought to recapitulate early stages of the development an inherited disease. Modern genome-editing tools allow researchers to create isogenic cell clones with an identical genetic background providing an adequate "healthy" control for biomedical and pharmacological experiments. Here, we generated isogenic mutant cell clones with 150 CAG repeats in the first exon of the huntingtin (HTT) gene using the CRISPR/Cas9 system and performed ultrastructural and morphometric analyses of the internal organization of the mutant cells. Electron microscopy showed that deletion of three CAG triplets or an HTT gene knockout had no significant influence on the cell structure. The insertion of 150 CAG repeats led to substantial changes in quantitative and morphological parameters of mitochondria and increased the association of mitochondria with the smooth and rough endoplasmic reticulum while causing accumulation of small autolysosomes in the cytoplasm. Our data indicate for the first time that expansion of the CAG repeat tract in HTT introduced via the CRISPR/Cas9 technology into a human cell line initiates numerous ultrastructural defects that are typical for Huntington's disease

Dynamics of the Two Heterochromatin Types during Imprinted X Chromosome Inactivation in Vole <i>Microtus levis</i>

<div><p>In rodent female mammals, there are two forms of X-inactivation – imprinted and random which take place in extraembryonic and embryonic tissues, respectively. The inactive X-chromosome during random X-inactivation was shown to contain two types of facultative heterochromatin that alternate and do not overlap. However, chromatin structure of the inactive X-chromosome during imprinted X-inactivation, especially at early stages, is still not well understood. In this work, we studied chromatin modifications associated with the inactive X-chromosome at different stages of imprinted X-inactivation in a rodent, <i>Microtus levis</i>. It has been found that imprinted X-inactivation in vole occurs in a species-specific manner in two steps. The inactive X-chromosome at early stages of imprinted X-inactivation is characterized by accumulation of H3K9me3, HP1, H4K20me3, and uH2A, resembling to some extent the pattern of repressive chromatin modifications of meiotic sex chromatin. Later, the inactive X-chromosome recruits trimethylated H3K27 and acquires the two types of heterochromatin associated with random X-inactivation.</p></div

The Y-chromosome chromatin in vole trophoblast stem cells.

<p>Immunofluorescence (uH2A, green) combined with DNA FISH (MS4 repeat, red). Metaphase spreads were counterstained with DAPI (blue). X-chromosome (X) and Y-chromosome (Y) are indicated by arrows.</p

Primary and secondary antibodies used in immunofluorescence assay.

<p>Primary and secondary antibodies used in immunofluorescence assay.</p

The XY-body chromatin modifications at the meiotic and postmeiotic stages in vole spermatogenesis.

<p>(<b>A</b>) Co-immunostaining of H3K9me3 (red), HP1 (green), uH2A (green) with antibodies to SCP3 (red) (marker of pachytene stage) was used to distinguish between spermatocytes I and round spermatids; (<b>B</b>) Immunostaining (H3K9me3, green) combined with DNA FISH (MS4 repeat, red); (<b>C</b>) Co-immunostaining of H3K9me3 (green) and H4K20me3 (red). Nuclei were counterstained with DAPI (blue).</p

Dynamics of repressive chromatin modification during vole TS cell differentiation.

<p>(<b>A</b>) Localization of Eed and H3K27me3 on the inactive X-chromosome at different stages of TS cell differentiation; (<b>B</b>) H3K27me3 accumulation on Xi is accompanied by a significant decrease in HP1. Immunostaining with antibodies to HP1 (green) and H3K27me3 (red) at different stages of TS cell differentiation.</p

Accumulation and redistribution of repressive chromatin modifications during vole TS cell differentiation.

<p>(<b>A</b>) uH2A forms distinct bands on Xi and is excluded from heterochromatic regions of Xi and other chromosomes. Immunostaining (uH2A, red and H3K9me3, green) combined with DNA FISH (MS4 repeat); (<b>B</b>) H3K27me3 accumulation on Xi. Immunostaining with antibodies to H3K27me3 (red) and HP1 (green) combined with DNA FISH (MS4 repeat). Metaphase spreads were counterstained with DAPI (blue). Active (Xa) and inactive (Xi) X-chromosomes are indicated by arrows.</p

Repressive chromatin modifications at early stages of imprinted XCI during vole preimplantation and postimplantation development.

<p>(<b>A</b>) Immunofluorescence (H3K9me3, green and H3K27me3, grey) combined with RNA FISH (<i>Xist</i> RNA, red) at the blastocyst stage. (<b>B</b>) Immunofluorescence (H4K20me3, green) combined with RNA FISH (<i>Xist</i> RNA, red) at the blastocyst stage. (<b>C</b>) Immunostaining (H3K27me3, red and HP1, green) of extraembryonic ectoderm of 7,5-day cryosectioned embryos.</p

Enrichment and genome sequence of the group I.1a ammonia-oxidizing Archaeon "Ca. Nitrosotenuis uzonensis" representing a clade globally distributed in thermal habitats.

The discovery of ammonia-oxidizing archaea (AOA) of the phylum Thaumarchaeota and the high abundance of archaeal ammonia monooxygenase subunit A encoding gene sequences in many environments have extended our perception of nitrifying microbial communities. Moreover, AOA are the only aerobic ammonia oxidizers known to be active in geothermal environments. Molecular data indicate that in many globally distributed terrestrial high-temperature habits a thaumarchaeotal lineage within the Nitrosopumilus cluster (also called "marine" group I.1a) thrives, but these microbes have neither been isolated from these systems nor functionally characterized in situ yet. In this study, we report on the enrichment and genomic characterization of a representative of this lineage from a thermal spring in Kamchatka. This thaumarchaeote, provisionally classified as "Candidatus Nitrosotenuis uzonensis", is a moderately thermophilic, non-halophilic, chemolithoautotrophic ammonia oxidizer. The nearly complete genome sequence (assembled into a single scaffold) of this AOA confirmed the presence of the typical thaumarchaeotal pathways for ammonia oxidation and carbon fixation, and indicated its ability to produce coenzyme F420 and to chemotactically react to its environment. Interestingly, like members of the genus Nitrosoarchaeum, "Candidatus N. uzonensis" also possesses a putative artubulin-encoding gene. Genome comparisons to related AOA with available genome sequences confirmed that the newly cultured AOA has an average nucleotide identity far below the species threshold and revealed a substantial degree of genomic plasticity with unique genomic regions in "Ca. N. uzonensis", which potentially include genetic determinants of ecological niche differentiation