Haploid mouse germ cell precursors from embryonic stem cells reveal Xist activation from a single X chromosome.

Mammalian haploid cells have applications for genetic screening and substituting gametic genomes. Here, we characterize a culture system for obtaining haploid primordial germ cell-like cells (PGCLCs) from haploid mouse embryonic stem cells (ESCs). We find that haploid cells show predisposition for PGCLCs, whereas a large fraction of somatic cells becomes diploid. Characterization of the differentiating haploid ESCs (haESCs) reveals that Xist is activated from and colocalizes with the single X chromosome. This observation suggests that X chromosome inactivation (XCI) is initiated in haploid cells consistent with a model where autosomal blocking factors set a threshold for X-linked activators. We further find that Xist expression is lost at later timepoints in differentiation, which likely reflects the loss of X-linked activators. In vitro differentiation of haploid PGCLCs can be a useful approach for future studies of potential X-linked activators of Xist.

Microbial communities in floodplain ecosystems in relation to altered flow regimes and experimental flooding.

River floodplains are spatially diverse ecosystems that respond quickly to flow variations and disturbance. However, it remains unclear how flow alteration and hydrological disturbance impacts the structure and biodiversity of complex microbial communities in these ecosystems. Here, we examined the spatial and seasonal dynamics of microbial communities in aquatic (benthic) and terrestrial habitats of three hydrologically contrasting (natural flow, residual flow, hydropeaking flow) floodplain systems. Microbial communities (alpha and beta diversity) differed more among floodplain habitats than between riverine floodplains. Microbial communities in all systems displayed congruent seasonal effects. In the residual and hydropeaking systems, an experimental flood was released from a reservoir to mimic a natural high flow event causing hydromorphological disturbance. The experimental flood caused a temporary shift in microbial communities by releasing microbes from the reservoir as well as redistributing communities among floodplain habitats. The flood-mediated shift in community structures had only a transient impact as pelagic bacteria did not persist within floodplain habitats over time after the flood. More frequent pulse disturbances might lead to an alternate structure of bacterial communities in floodplains over time.

Environmental and Microbial Interactions Shape Methane-Oxidizing Bacterial Communities in a Stratified Lake.

In stratified lakes, methane-oxidizing bacteria (MOB) are strongly mitigating methane fluxes to the atmosphere by consuming methane entering the water column from the sediments. MOB communities in lakes are diverse and vertically structured, but their spatio-temporal dynamics along the water column as well as physico-chemical parameters and interactions with other bacterial species that drive the community assembly have so far not been explored in depth. Here, we present a detailed investigation of the MOB and bacterial community composition and a large set of physico-chemical parameters in a shallow, seasonally stratified, and sub-alpine lake. Four highly resolved vertical profiles were sampled in three different years and during various stages of development of the stratified water column. Non-randomly assembled MOB communities were detected in all compartments. We could identify methane and oxygen gradients and physico-chemical parameters like pH, light, available copper and iron, and total dissolved nitrogen as important drivers of the MOB community structure. In addition, MOB were well-integrated into a bacterial-environmental network. Partial redundancy analysis of the relevance network of physico-chemical variables and bacteria explained up to 84% of the MOB abundances. Spatio-temporal MOB community changes were 51% congruent with shifts in the total bacterial community and 22% of variance in MOB abundances could be explained exclusively by the bacterial community composition. Our results show that microbial interactions may play an important role in structuring the MOB community along the depth gradient of stratified lakes.

Polyploidy of semi-cloned embryos generated from parthenogenetic haploid embryonic stem cells.

In mammals, the fusion of two gametes, an oocyte and a spermatozoon, during fertilization forms a totipotent zygote. There has been no reported case of adult mammal development by natural parthenogenesis, in which embryos develop from unfertilized oocytes. The genome and epigenetic information of haploid gametes are crucial for mammalian development. Haploid embryonic stem cells (haESCs) can be established from uniparental blastocysts and possess only one set of chromosomes. Previous studies have shown that sperm or oocyte genome can be replaced by haESCs with or without manipulation of genomic imprinting for generation of mice. Recently, these remarkable semi-cloning methods have been applied for screening of key factors of mouse embryonic development. While haESCs have been applied as substitutes of gametic genomes, the fundamental mechanism how haESCs contribute to the genome of totipotent embryos is unclear. Here, we show the generation of fertile semi-cloned mice by injection of parthenogenetic haESCs (phaESCs) into oocytes after deletion of two differentially methylated regions (DMRs), the IG-DMR and H19-DMR. For characterizing the genome of semi-cloned embryos further, we establish ESC lines from semi-cloned blastocysts. We report that polyploid karyotypes are observed in semi-cloned ESCs (scESCs). Our results confirm that mitotically arrested phaESCs yield semi-cloned embryos and mice when the IG-DMR and H19-DMR are deleted. In addition, we highlight the occurrence of polyploidy that needs to be considered for further improving the development of semi-cloned embryos derived by haESC injection.

Gaining Insights into the Function of Post-Translational Protein Modification Using Genome Engineering and Molecular Cell Biology.

Modifications by kinases are a fast and reversible mechanism to diversify the function of the targeted proteins. The OCT4 transcription factor is essential for preimplantation development and pluripotency of embryonic stem cells (ESC), and its activity is tightly regulated by post-transcriptional modifications. Several phosphorylation sites have been identified by systemic approaches and their functions proposed. Here, we combined molecular and cellular biology with CRISPR/Cas9-mediated genome engineering to pinpoint the function of serine 12 of OCT4 in ESCs. Using chemical inhibitors and an antibody specific to OCT4 phosphorylated on S12, we identified cyclin-dependent kinase (CDK) 7 as upstream kinase. Surprisingly, generation of isogenic mESCs that endogenously ablate S12 revealed no effects on pluripotency and self-renewal, potentially due to compensation by other phosphorylation events. Our approach reveals that modification of distinct amino acids by precise genome engineering can help to clarify the functions of post-translational modifications on proteins encoded by essential gene in an endogenous context.

Derivation of Haploid Neural Stem Cell Lines by Selection for a Pax6-GFP Reporter.

Haploid cells facilitate genetic screening of recessive mutations for a single set of chromosomes. Haploid embryonic stem cells (haESCs) have been achieved in several species and widely utilized in genetic screens. The fact that haESCs undergo substantial diploidization during differentiation has limited the screening to other haploid cell types. In this study, we report a method to establish haploid neural stem cells (haNSCs) by selection for a Pax6 reporter. We inserted a green fluorescence protein (GFP) marker gene by homologous recombination into the Pax6 locus of an haESC line. GFP-positive haploid cells could be sorted and further cultured in the NSC medium for more than 30 passages. The established haNSCs expressed neural lineage markers and could differentiate into neurons, oligodendroglia, and astrocytes. Our study shows the feasibility of deriving haploid proliferative somatic cell lines using a genetically encoded reporter that suggest a system for genetic screening of neural and retinal development.

A fast and efficient size separation method for haploid embryonic stem cells.

Hemizygous mutations introduced in haploid genomes can directly expose a phenotype, thus facilitating gene function analysis and forward genetic screening. Recently, mammalian haploid cells could be derived from mouse, rat, monkey, and human embryos and have been applied to screens of cellular mechanisms including cell signaling, pathogen host factors, and developmental pathways. Notably, haploid cell cultures have an intrinsic tendency for diploidization and, thus, require periodic cell sorting. Here, we report a method for rapid purification of haploid mouse embryonic stem cells from mixed cell populations with high viability and yield. Our method uses membranes with micrometer pores for force-free separation and facilitates enrichment of haploid cells without flow cytometry. The separation method simplifies maintaining haploid cell cultures and has further applications in establishing haploid cell lines from embryos and isolating cell cycle phases of mammalian cells.

Flow cytometry combined with viSNE for the analysis of microbial biofilms and detection of microplastics.

Biofilms serve essential ecosystem functions and are used in different technical applications. Studies from stream ecology and waste-water treatment have shown that biofilm functionality depends to a great extent on community structure. Here we present a fast and easy-to-use method for individual cell-based analysis of stream biofilms, based on stain-free flow cytometry and visualization of the high-dimensional data by viSNE. The method allows the combined assessment of community structure, decay of phototrophic organisms and presence of abiotic particles. In laboratory experiments, it allows quantification of cellular decay and detection of survival of larger cells after temperature stress, while in the field it enables detection of community structure changes that correlate with known environmental drivers (flow conditions, dissolved organic carbon, calcium) and detection of microplastic contamination. The method can potentially be applied to other biofilm types, for example, for inferring community structure for environmental and industrial research and monitoring.

Hydrologic linkages drive spatial structuring of bacterial assemblages and functioning in alpine floodplains.

Microbial community assembly and microbial functions are affected by a number of different but coupled drivers such as local habitat characteristics, dispersal rates, and species interactions. In groundwater systems, hydrological flow can introduce spatial structure and directional dependencies among these drivers. We examined the importance of hydrology in structuring bacterial communities and their function within two alpine floodplains during different hydrological states. Piezometers were installed in stream sediments and surrounding riparian zones to assess hydrological flows and also were used as incubation chambers to examine bacterial community structures and enzymatic functions along hydrological flow paths. Spatial eigenvector models in conjunction with models based on physico-chemical groundwater characteristics were used to evaluate the importance of hydrologically-driven processes influencing bacterial assemblages and their enzymatic activities. Our results suggest a strong influence (up to 40% explained variation) of hydrological connectivity on enzymatic activities. The effect of hydrology on bacterial community structure was considerably less strong, suggesting that assemblages demonstrate large functional plasticity/redundancy. Effect size varied between hydrological periods but flow-related mechanisms always had the most power in explaining both bacterial structure and functioning. Changes in hydrology should be considered in models predicting ecosystem functioning and integrated into ecosystem management strategies for floodplains.

Identification of Spen as a Crucial Factor for Xist Function through Forward Genetic Screening in Haploid Embryonic Stem Cells.

In mammals, the noncoding Xist RNA triggers transcriptional silencing of one of the two X chromosomes in female cells. Here, we report a genetic screen for silencing factors in X chromosome inactivation using haploid mouse embryonic stem cells (ESCs) that carry an engineered selectable reporter system. This system was able to identify several candidate factors that are genetically required for chromosomal repression by Xist. Among the list of candidates, we identify the RNA-binding protein Spen, the homolog of split ends. Independent validation through gene deletion in ESCs confirms that Spen is required for gene repression by Xist. However, Spen is not required for Xist RNA localization and the recruitment of chromatin modifications, including Polycomb protein Ezh2. The identification of Spen opens avenues for further investigation into the gene-silencing pathway of Xist and shows the usefulness of haploid ESCs for genetic screening of epigenetic pathways.

Spatio-temporal patterns of major bacterial groups in alpine waters.

Glacial alpine landscapes are undergoing rapid transformation due to changes in climate. The loss of glacial ice mass has directly influenced hydrologic characteristics of alpine floodplains. Consequently, hyporheic sediment conditions are likely to change in the future as surface waters fed by glacial water (kryal) become groundwater dominated (krenal). Such environmental shifts may subsequently change bacterial community structure and thus potential ecosystem functioning. We quantitatively investigated the structure of major bacterial groups in glacial and groundwater-fed streams in three alpine floodplains during different hydrologic periods. Our results show the importance of several physico-chemical variables that reflect local geological characteristics as well as water source in structuring bacterial groups. For instance, Alpha-, Betaproteobacteria and Cytophaga-Flavobacteria were influenced by pH, conductivity and temperature as well as by inorganic and organic carbon compounds, whereas phosphorous compounds and nitrate showed specific influence on single bacterial groups. These results can be used to predict future bacterial group shifts, and potential ecosystem functioning, in alpine landscapes under environmental transformation.

Bacterial structures and ecosystem functions in glaciated floodplains: contemporary states and potential future shifts.

Glaciated alpine floodplains are responding quickly to climate change through shrinking ice masses. Given the expected future changes in their physicochemical environment, we anticipated variable shifts in structure and ecosystem functioning of hyporheic microbial communities in proglacial alpine streams, depending on present community characteristics and landscape structures. We examined microbial structure and functioning during different hydrologic periods in glacial (kryal) streams and, as contrasting systems, groundwater-fed (krenal) streams. Three catchments were chosen to cover an array of landscape features, including interconnected lakes, differences in local geology and degree of deglaciation. Community structure was assessed by automated ribosomal intergenic spacer analysis and microbial function by potential enzyme activities. We found each catchment to contain a distinct bacterial community structure and different degrees of separation in structure and functioning that were linked to the physicochemical properties of the waters within each catchment. Bacterial communities showed high functional plasticity, although achieved by different strategies in each system. Typical kryal communities showed a strong linkage of structure and function that indicated a major prevalence of specialists, whereas krenal sediments were dominated by generalists. With the rapid retreat of glaciers and therefore altered ecohydrological characteristics, lotic microbial structure and functioning are likely to change substantially in proglacial floodplains in the future. The trajectory of these changes will vary depending on contemporary bacterial community characteristics and landscape structures that ultimately determine the sustainability of ecosystem functioning.