Haploid-genetic screening of trophectoderm specification identifies Dyrk1a as a repressor of totipotent-like status.

Trophectoderm (TE) and the inner cell mass are the first two lineages in murine embryogenesis and cannot naturally transit to each other. The barriers between them are unclear and fascinating. Embryonic stem cells (ESCs) and trophoblast stem cells (TSCs) retain the identities of inner cell mass and TE, respectively, and, thus, are ideal platforms to investigate these lineages in vitro. Here, we develop a loss-of-function genetic screening in haploid ESCs and reveal many mutations involved in the conversion of TSCs. The disruption of either Catip or Dyrk1a (candidates) in ESCs facilitates the conversion of TSCs. According to transcriptome analysis, we find that the repression of Dyrk1a activates totipotency, which is a possible reason for TE specification. Dyrk1a-null ESCs can contribute to embryonic and extraembryonic tissues in chimeras and can efficiently form blastocyst-like structures, indicating their totipotent developmental abilities. These findings provide insights into the mechanisms underlying cell fate alternation in embryogenesis.

[Imprinting genes modified parthenogenetic embryonic stem cells produce full-term mouse via tetraploid complementation].

Parthenogenetic embryonic stem cells (pESCs) derived from bi-maternal genomes do not have competency of tetraploid complementation, due to lacking of paternal imprinting genes. To make pESCs possess fully development potentials and similar pluripotency to zygote-derived ESCs, we knocked out one allelic gene of the two essential maternal imprinting genes (H19 and IG) in their differentially methylated regions (DMR) via CRISPR/Cas9 system and obtained double knock out (DKO) pESCs. Maternal pESCs had similar morphology, expression levels of pluripotent makers and in vitro neural differentiation potentials to zygotes-derived ESCs. Besides that, DKO pESCs could contribute to full-term fetuses through tetraploid complementation, proving that they held fully development potentials. Derivation of DKO pESCs provided a type of major histocompatibility complex (MHC) matched pluripotent stem cells, which would benefit research in regenerative medicine.

Derivation of Haploid Trophoblast Stem Cells via Conversion In Vitro.

Owing to their single genome, haploid cells are powerful to uncover unknown genes by performing genetic screening in mammals. However, no haploid cell line from an extraembryonic lineage has been achieved yet, which limits the application of haploid cells in placental genetic screening. Here, we show that overexpression of Cdx2 can convert haploid embryonic stem cells to trophoblast stem cells (TSCs). p53 deletion reduces diploidization during the conversion and guarantees the generation of haploid-induced TSCs (haiTSCs). haiTSCs not only share the same molecular characterization with trophoderm-derived TSCs but also possess multipotency to placental lineages in various procedures. In addition, haiTSCs can maintain haploidy in the long term, assisted by periodic sorting and with reliance on FGF4 and heparin. Finally, we perform piggyBac-mediated high-throughput mutation in haiTSCs and use them in trophoblast lineage genetic screening. Deep sequencing analysis and validation experiments prove that Htra1 is a blocker for spongiotrophoblast specification.

Overexpression of PSP1 enhances growth of transgenic Arabidopsis plants under ambient air conditions.

The importance of the phosphorylated pathway (PPSB) of L-serine (Ser) biosynthesis in plant growth and development has been demonstrated, but its specific role in leaves and interaction with photorespiration, the main leaf Ser biosynthetic pathway at daytime, are still unclear. To investigate whether changes in biosynthesis of Ser by the PPSB in leaves could have an impact on photorespiration and plant growth, we overexpressed PSP1, the last enzyme of this pathway, under control of the Cauliflower Mosaic Virus 35S promoter in Arabidopsis thaliana. Overexpressor plants grown in normal air displayed larger rosette diameter and leaf area as well as higher fresh and dry weight than the wild type. By contrast, no statistically significant differences to the wild type were observed when the overexpressor seedlings were transferred to elevated CO2, indicating a relationship between PSP1 overexpression and photorespiration. Additionally, the transgenic plants displayed higher photorespiration, an increase in CO2 net-uptake and stronger expression in the light of genes encoding enzymes involved in photorespiration. We further demonstrated that expression of many genes involved in nitrogen assimilation was also promoted in leaves of transgenic plants and that leaf nitrate reductase activity increased in the light, too, although not in the dark. Our results suggest a close correlation between the function of PPSB and photorespiration, and also nitrogen metabolism in leaves.