Inhibition of HDAC activity directly reprograms murine embryonic stem cells to trophoblast stem cells.

Embryonic stem cells (ESCs) can differentiate into all cell types of the embryonic germ layers. ESCs can also generate totipotent 2C-like cells and trophectodermal cells. However, these latter transitions occur at low frequency due to epigenetic barriers, the nature of which is not fully understood. Here, we show that treating mouse ESCs with sodium butyrate (NaB) increases the population of 2C-like cells and enables direct reprogramming of ESCs into trophoblast stem cells (TSCs) without a transition through a 2C-like state. Mechanistically, NaB inhibits histone deacetylase activities in the LSD1-HDAC1/2 corepressor complex. This increases acetylation levels in the regulatory regions of both 2C- and TSC-specific genes, promoting their expression. In addition, NaB-treated cells acquire the capacity to generate blastocyst-like structures that can develop beyond the implantation stage in vitro and form deciduae in vivo. These results identify how epigenetics restrict the totipotent and trophectoderm fate in mouse ESCs.

A new Prdm1-Cre line is suitable for studying the second heart field development.

The second heart field (SHF) plays a pivotal role in heart development, particularly in outflow tract (OFT) morphogenesis and septation, as well as in the expansion of the right ventricle (RV). Two mouse Cre lines, the Mef2c-AHF-Cre (Mef2c-Cre) and Isl1-Cre, have been widely used to study the SHF development. However, Cre activity is triggered not only in the SHF but also in the RV in the Mef2c-Cre mice, and in the Isl1-Cre mice, Cre activation is not SHF-specific. Therefore, a more suitable SHF-Cre line is desirable for better understanding SHF development. Here, we generated and characterized the Prdm1-Cre knock-in mice. In comparison with Mef2c-Cre mice, the Cre activity is similar in the pharyngeal and splanchnic mesoderm, and in the OFT of the Prdm1-Cre mice. Nonetheless, it was noticed that Cre expression is largely reduced in the RV of Prdm1-Cre mice compared to the Mef2c-Cre mice. Furthermore, we deleted Hand2, Nkx2-5, Pdk1 and Tbx20 using both Mef2c-Cre and Prdm1-Cre mice to study OFT morphogenesis and septation, making a comparison between these two Cre lines. New insights were obtained in understanding SHF development including differentiation into cardiomyocytes in the OFT using Prdm1-Cre mice. In conclusion, we found that Prdm1-Cre mouse line is a more appropriate tool to monitor SHF development, while the Mef2c-Cre mice are excellent in studying the role and function of the SHF in OFT morphogenesis and septation.

Surface roughness and gloss alteration of polished resin composites with various filler types after simulated toothbrush abrasion.

Background/purpose: Few studies have focused on the influence of simulated toothbrush abrasion on the surface qualities of novel nanofilled and nanohybrid composites. The aim of the study was to evaluate the surface roughness and gloss values of resin-based composite (RBC) materials with various filler types before and after simulated toothbrush abrasion. Materials and methods: One nanofilled (Filtek Z350 XT [FT3]), two nanohybrids (Harmonize [HM] and Clearfil Majesty [CM]) and one microhybrid (Filtek Z250 [FT2]) were evaluated. Twelve specimens of each material were made and polished with silicon carbide sandpapers. Initial surface roughness and gloss values were measured as negative controls. Then, all specimens were subjected to simulated toothbrush abrasion on a custom-made apparatus. After 2000, 4000 and 8000 cycles, the surface roughness and gloss values of all specimens were tested. One additional specimen from each group was selected for scanning electron microscope (SEM) analysis. Results: For FT3, Ra and GU values did not significantly change until after 8000 cycles during the process of toothbrushing (P > 0.05). For HM, CM and FT2, the Ra and GU values significantly decreased after 4000 and 8000 cycles of toothbrush abrasion (P < 0.05). After 8000 cycles of toothbrush abrasion, FT3 presented the lowest surface roughness and highest gloss values of all materials (P < 0.05). SEM images showed that surface textures and irregularities corresponded to the results of surface roughness and gloss. Conclusion: Surface roughness and gloss after simulated toothbrush abrasion were material dependent. Nanofilled resin composite presented the lowest Ra values and highest GU values.

Maternal mRNA deadenylation and allocation via Rbm14 condensates facilitate vertebrate blastula development.

Early embryonic development depends on proper utilization and clearance of maternal transcriptomes. How these processes are spatiotemporally regulated remains unclear. Here we show that nuclear RNA-binding protein Rbm14 and maternal mRNAs co-phase separate into cytoplasmic condensates to facilitate vertebrate blastula-to-gastrula development. In zebrafish, Rbm14 condensates were highly abundant in blastomeres and markedly reduced after prominent activation of zygotic transcription. They concentrated at spindle poles by associating with centrosomal γ-tubulin puncta and displayed mainly asymmetric divisions with a global symmetry across embryonic midline in 8- and 16-cell embryos. Their formation was dose-dependently stimulated by m6 A, but repressed by m5 C modification of the maternal mRNA. Furthermore, deadenylase Parn co-phase separated with these condensates, and this was required for deadenylation of the mRNAs in early blastomeres. Depletion of Rbm14 impaired embryonic cell differentiations and full activations of the zygotic genome in both zebrafish and mouse and resulted in developmental arrest at the blastula stage. Our results suggest that cytoplasmic Rbm14 condensate formation regulates early embryogenesis by facilitating deadenylation, protection, and mitotic allocation of m6 A-modified maternal mRNAs, and by releasing the poly(A)-less transcripts upon regulated disassembly to allow their re-polyadenylation and translation or clearance.

Anti-chronic myeloid leukemia activity and quantitative structure-activity relationship of novel thiazole aminobenzamide derivatives.

The anti-chronic myeloid leukemia activity of thiazole aminobenzamide derivatives in vitro was tested by a methanethiosulfonate (MTS)-based viability assay method, and the result showed that some compounds exhibited good inhibitory activities against human chronic myeloid leukemia cell line K562, imatinib-resistant strain K562/R and T135I mutant cell line BaF3-ABL-BCR-T315I. Comparative molecular field analysis (CoMFA) and comparative molecular similarity index analysis (CoMSIA) methods were used to analyze the relationship between the structure of thiazole aminobenzamide derivatives and the inhibition of K562/R cell activity. In CoMFA, Q2 was 0.899 and R2 was 0.963; in CoMSIA, Q2 and R2 were 0.840 and 0.903, respectively. These data indicated that the selected test set showed suitable external predictive ability. Combined with the contour map results, we further analyzed the three-dimensional quantitative structure (3D-QSAR) model. The results demonstrated that in the backbone of the thiazole aminobenzamide derivative, the substitution of a small group at R1 position, or the introduction of a hydrophilic group at R2 position, or the introduction of a large-volume amino acid at R3 position may be beneficial to improve the anti-CML activity of the compound.

Rabl2 GTP hydrolysis licenses BBSome-mediated export to fine-tune ciliary signaling.

Cilia of higher animals sense various environmental stimuli. Proper ciliary signaling requires appropriate extent of BBSome-mediated export of membrane receptors across ciliary barrier transition zone (TZ) through retrograde intraflagellar transport (IFT) machinery. How the barrier passage is controlled, however, remains unknown. Here, we show that small GTPase Rabl2 functions as a molecular switch for the outward TZ passage. Rabl2-GTP enters cilia by binding to IFT-B complex. Its GTP hydrolysis enables the outward TZ passage of the BBSome and its cargos with retrograde IFT machinery, whereas its persistent association leads to their shedding from IFT-B during the passing process and consequently ciliary retention. Rabl2 deficiency or expression of a GTP-locked mutant impairs the ciliary hedgehog signaling without interfering with ciliation and respectively results in different spectrums of mouse developmental disorders. We propose that the switch role of Rabl2 ensures proper turnover of the BBSome and ciliary membrane receptors to fine-tune cilia-dependent signaling for normal embryonic development and organismic homeostasis.

PHF7 is a novel histone H2A E3 ligase prior to histone-to-protamine exchange during spermiogenesis.

Epigenetic regulation, including histone-to-protamine exchanges, controls spermiogenesis. However, the underlying mechanisms of this regulation are largely unknown. Here, we report that PHF7, a testis-specific PHD and RING finger domain-containing protein, is essential for histone-to-protamine exchange in mice. PHF7 is specifically expressed during spermiogenesis. PHF7 deletion results in male infertility due to aberrant histone retention and impaired protamine replacement in elongated spermatids. Mechanistically, PHF7 can simultaneously bind histone H2A and H3; its PHD domain, a histone code reader, can specifically bind H3K4me3/me2, and its RING domain, a histone writer, can ubiquitylate H2A. Thus, our study reveals that PHF7 is a novel E3 ligase that can specifically ubiquitylate H2A through binding H3K4me3/me2 prior to histone-to-protamine exchange.

Author Correction: CRISPR-Cas9-mediated base-editing screening in mice identifies DND1 amino acids that are critical for primordial germ cell development.

In Fig. 2a of this Technical Report originally published, the authors inadvertently used the same set of images for the 4B2N1 and 4B2N3 cells when preparing the figure. The three images (bright field, Oct4-EGFP and pCAG-mRFP) of 4B2N3 cells have now been replaced with the correct versions. The source data for the four cell lines in Fig. 2a, captured in the three independent experiments, have been deposited to Figshare (https://doi.org/10.6084/m9.figshare.7387607.v1), and the figure legends and Methods section have been amended to reflect this. Additionally, the unprocessed blots in Supplementary Fig. 7 corresponding to the top right 'WCL IB: Flag' panel of Fig. 7e were mistakenly duplicates of the unprocessed blots for the bottom left 'IP Flag IB: HA' panel of Fig. 7e, and all unprocessed blots for Supplementary Fig. 6 were mislabelled as blots corresponding to Supplementary Fig. 7. Supplementary Fig. 7 has now been updated to show the correct unprocessed blots for the bottom left 'IP Flag IB: HA' panel of Fig. 7e and to correct the labelling of the unprocessed blots corresponding to Supplementary Fig. 6.

CRISPR-Cas9-mediated base-editing screening in mice identifies DND1 amino acids that are critical for primordial germ cell development.

CRISPR-mediated base editing can introduce single-nucleotide changes in the DNA of living cells. One intriguing application of base editing is to screen pivotal amino acids for protein function in vivo; however, it has not been achieved. Here, we report an enhanced third-generation base-editing system with extra nuclear localization sequences that can efficiently introduce a homozygous base mutation in embryonic stem cells. Meanwhile, we establish a strategy to generate base-mutant mice by injection of haploid embryonic stem cells carrying a constitutively expressed enhanced third-generation base-editing system (4B2N1) and single guide RNA into oocytes. Moreover, transfection of 4B2N1 cells with a single guide RNA library targeting the Dnd1 gene allows one-step generation of mutant mice with a base mutation. This enables the identification of four missense mutations that completely deplete primordial germ cells through disruption of DND1 protein stability and protein-protein interaction. Thus, our strategy provides an effective tool for in vivo screening of amino acids that are crucial for protein function.

Single-cell RNA-seq uncovers dynamic processes and critical regulators in mouse spermatogenesis.

A systematic interrogation of male germ cells is key to complete understanding of molecular mechanisms governing spermatogenesis and the development of new strategies for infertility therapies and male contraception. Here we develop an approach to purify all types of homogeneous spermatogenic cells by combining transgenic labeling and synchronization of the cycle of the seminiferous epithelium, and subsequent single-cell RNA-sequencing. We reveal extensive and previously uncharacterized dynamic processes and molecular signatures in gene expression, as well as specific patterns of alternative splicing, and novel regulators for specific stages of male germ cell development. Our transcriptomics analyses led us to discover discriminative markers for isolating round spermatids at specific stages, and different embryo developmental potentials between early and late stage spermatids, providing evidence that maturation of round spermatids impacts on embryo development. This work provides valuable insights into mammalian spermatogenesis, and a comprehensive resource for future studies towards the complete elucidation of gametogenesis.

Haploid embryonic stem cells can be enriched and maintained by simple filtration.

Mammalian haploid embryonic stem cells (haESCs) serve as a powerful tool for genetic analyses at both the cellular and organismal levels. However, spontaneous diploidization of haESCs limits their use in these analyses. Addition of small molecules to the culture medium to control the cell cycle can slow down diploidization, but cell-sorting methods such as FACS are still required to enrich haploid cells for long-term maintenance in vitro Here, acting on our observation that haploid and diploidized cells differ in diameter, we developed a simplified filtration method to enrich haploid cells from cultured haESCs. We found that regular cell filtration with this system reliably maintained the haploidy of mouse haESCs for over 30 passages. Importantly, CRISPR/Cas9-mediated knockout and knockin were successfully achieved in the filtered cells, leading to stable haploid cell lines carrying the desired gene modifications. Of note, by injecting haESCs into metaphase II oocytes, we efficiently obtained live mice with the expected genetic traits, indicating that regular filtration maintained the functional integrity of haESCs. Moreover, this filtration system was also feasible for derivation of mouse haESCs from parthenogenetic haploid blastocysts and for human haESC maintenance. In conclusion, we have identified a reliable, efficient, and easy-to-handle technique for countering diploidization of haploid cells, a major obstacle in haESC applications.

CRISPR-Cas9-Mediated Genetic Screening in Mice with Haploid Embryonic Stem Cells Carrying a Guide RNA Library.

Mouse androgenetic haploid embryonic stem cells (AG-haESCs) can support full-term development of semi-cloned (SC) embryos upon injection into MII oocytes and thus have potential applications in genetic modifications. However, the very low birth rate of SC pups limits practical use of this approach. Here, we show that AG-haESCs carrying deletions in the DMRs (differentially DNA methylated regions) controlling two paternally repressed imprinted genes, H19 and Gtl2, can efficiently support the generation of SC pups. Genetic manipulation of these DKO-AG-haESCs in vitro using CRISPR-Cas9 can produce SC mice carrying multiple modifications with high efficiency. Moreover, transfection of DKO-AG-haESCs with a constitutively expressed sgRNA library and Cas9 allows functional mutagenic screening. DKO-AG-haESCs are therefore an effective tool for the introduction of organism-wide mutations in mice in a single generation.

[A spectral unmixing method of estimating main minerals abundance of lunar soils].

Estimating minerals abundance from reflectance spectra is one of the fundamental goals of remote sensing lunar exploration, and the main difficulties are the complicated mixing law of minerals spectrum and spectral features being sensitive to several kinds of factors such as topography, particle size and roughness etc. A method based on spectral unmixing was put forward and tested in the present paper. Before spectra are unmixed the spectral continuum is removed for clarifying and strengthening spectral features. The absorption features and reflectance features (the upward curving parts of spectra between absorption features) are integrated for unmixing to improve the unmixing performance. The Hapke model was used to correct unmixing error due to nonlinear mixing of minerals spectra. Forty three mixed spectra of olivine, clinopyroxene, hypersthene and plagioclase were used to validate the above method. The four minerals abundance was estimated under the conditions of being unaware of endmember spectra used to mix, granularity and chemical composition of minerals. Residual error, abundance error and correlation coefficient between retrieved and true abundance were 5.0 Vol%, 14.4 Vol% and 0.92 respectively. The method and result of this paper could be referred in the lunar minerals mapping of imaging spectrometer data such as M3.