Lineage regulators TFAP2C and NR5A2 function as bipotency activators in totipotent embryos.

During the first lineage segregation, a mammalian totipotent embryo differentiates into the inner cell mass (ICM) and trophectoderm (TE). However, how transcription factors (TFs) regulate this earliest cell-fate decision in vivo remains elusive, with their regulomes primarily inferred from cultured cells. Here, we investigated the TF regulomes during the first lineage specification in early mouse embryos, spanning the pre-initiation, initiation, commitment, and maintenance phases. Unexpectedly, we found that TFAP2C, a trophoblast regulator, bound and activated both early TE and inner cell mass (ICM) genes at the totipotent (two- to eight-cell) stages ('bipotency activation'). Tfap2c deficiency caused downregulation of early ICM genes, including Nanog, Nr5a2, and Tdgf1, and early TE genes, including Tfeb and Itgb5, in eight-cell embryos. Transcription defects in both ICM and TE lineages were also found in blastocysts, accompanied by increased apoptosis and reduced cell numbers in ICMs. Upon trophoblast commitment, TFAP2C left early ICM genes but acquired binding to late TE genes in blastocysts, where it co-bound with CDX2, and later to extra-embryonic ectoderm (ExE) genes, where it cooperatively co-occupied with the former ICM regulator SOX2. Finally, 'bipotency activation' in totipotent embryos also applied to a pluripotency regulator NR5A2, which similarly bound and activated both ICM and TE lineage genes at the eight-cell stage. These data reveal a unique transcription circuity of totipotency underpinned by highly adaptable lineage regulators.

Multifaceted SOX2-chromatin interaction underpins pluripotency progression in early embryos.

Pioneer transcription factors (TFs), such as OCT4 and SOX2, play crucial roles in pluripotency regulation. However, the master TF-governed pluripotency regulatory circuitry was largely inferred from cultured cells. In this work, we investigated SOX2 binding from embryonic day 3.5 (E3.5) to E7.5 in the mouse. In E3.5 inner cell mass (ICM), SOX2 regulates the ICM-trophectoderm program but is dispensable for opening global enhancers. Instead, SOX2 occupies preaccessible enhancers in part opened by early-stage expressing TFs TFAP2C and NR5A2. SOX2 then widely redistributes when cells adopt naive and formative pluripotency by opening enhancers or poising them for rapid future activation. Hence, multifaceted pioneer TF-enhancer interaction underpins pluripotency progression in embryos, including a distinctive state in E3.5 ICM that bridges totipotency and pluripotency.

NR5A2 connects zygotic genome activation to the first lineage segregation in totipotent embryos.

Zygotic genome activation (ZGA) marks the beginning of the embryonic program for a totipotent embryo, which gives rise to the inner cell mass (ICM) where pluripotent epiblast arises, and extraembryonic trophectoderm. However, how ZGA is connected to the first lineage segregation in mammalian embryos remains elusive. Here, we investigated the role of nuclear receptor (NR) transcription factors (TFs), whose motifs are highly enriched and accessible from the 2-cell (2C) to 8-cell (8C) stages in mouse embryos. We found that NR5A2, an NR TF strongly induced upon ZGA, was required for this connection. Upon Nr5a2 knockdown or knockout, embryos developed beyond 2C normally with the zygotic genome largely activated. However, 4-8C-specific gene activation was substantially impaired and Nr5a2-deficient embryos subsequently arrested at the morula stage. Genome-wide chromatin binding analysis showed that NR5A2-bound cis-regulatory elements in both 2C and 8C embryos are strongly enriched for B1 elements where its binding motif is embedded. NR5A2 was not required for the global opening of its binding sites in 2C embryos but was essential to the opening of its 8C-specific binding sites. These 8C-specific, but not 2C-specific, binding sites are enriched near genes involved in blastocyst and stem cell regulation, and are often bound by master pluripotency TFs in blastocysts and embryonic stem cells (ESCs). Importantly, NR5A2 regulated key pluripotency genes Nanog and Pou5f1/Oct4, and primitive endoderm regulatory genes including Gata6 among many early ICM genes, as well as key trophectoderm regulatory genes including Tead4 and Gata3 at the 8C stage. By contrast, master pluripotency TFs NANOG, SOX2, and OCT4 targeted both early and late ICM genes in mouse ESCs. Taken together, these data identify NR5A2 as a key regulator in totipotent embryos that bridges ZGA to the first lineage segregation during mouse early development.

OBOX regulates mouse zygotic genome activation and early development.

Zygotic genome activation (ZGA) activates the quiescent genome to enable the maternal-to-zygotic transition1,2. However, the identity of transcription factors that underlie mammalian ZGA in vivo remains elusive. Here we show that OBOX, a PRD-like homeobox domain transcription factor family (OBOX1-OBOX8)3-5, are key regulators of mouse ZGA. Mice deficient for maternally transcribed Obox1/2/5/7 and zygotically expressed Obox3/4 had a two-cell to four-cell arrest, accompanied by impaired ZGA. The Obox knockout defects could be rescued by restoring either maternal and zygotic OBOX, which suggests that maternal and zygotic OBOX redundantly support embryonic development. Chromatin-binding analysis showed that Obox knockout preferentially affected OBOX-binding targets. Mechanistically, OBOX facilitated the 'preconfiguration' of RNA polymerase II, as the polymerase relocated from the initial one-cell binding targets to ZGA gene promoters and distal enhancers. Impaired polymerase II preconfiguration in Obox mutants was accompanied by defective ZGA and chromatin accessibility transition, as well as aberrant activation of one-cell polymerase II targets. Finally, ectopic expression of OBOX activated ZGA genes and MERVL repeats in mouse embryonic stem cells. These data thus demonstrate that OBOX regulates mouse ZGA and early embryogenesis.

Translatome and transcriptome co-profiling reveals a role of TPRXs in human zygotic genome activation.

Translational regulation plays a critical role during the oocyte-to-embryo transition (OET) and zygotic genome activation (ZGA). Here, we integrated ultra-low-input ribosome profiling (Ribo-lite) with messenger RNA sequencing to co-profile the translatome and transcriptome in human oocytes and early embryos. Comparison with mouse counterparts identified widespread differentially translated gene functioning in epigenetic reprogramming, transposon defense, and small RNA biogenesis, in part driven by species-specific regulatory elements in 3' untranslated regions. Moreover, PRD-like homeobox transcription factors, including TPRXL, TPRX1, and TPRX2, are highly translated around ZGA. TPRX1/2/L knockdown leads to defective ZGA and preimplantation development. Ectopically expressed TPRXs bind and activate key ZGA genes in human embryonic stem cells. These data reveal the conservation and divergence of translation landscapes during OET and identify critical regulators of human ZGA.

Ultrasensitive Ribo-seq reveals translational landscapes during mammalian oocyte-to-embryo transition and pre-implantation development.

In mammals, translational control plays critical roles during oocyte-to-embryo transition (OET) when transcription ceases. However, the underlying regulatory mechanisms remain challenging to study. Here, using low-input Ribo-seq (Ribo-lite), we investigated translational landscapes during OET using 30-150 mouse oocytes or embryos per stage. Ribo-lite can also accommodate single oocytes. Combining PAIso-seq to interrogate poly(A) tail lengths, we found a global switch of translatome that closely parallels changes of poly(A) tails upon meiotic resumption. Translation activation correlates with polyadenylation and is supported by polyadenylation signal proximal cytoplasmic polyadenylation elements (papCPEs) in 3' untranslated regions. By contrast, translation repression parallels global de-adenylation. The latter includes transcripts containing no CPEs or non-papCPEs, which encode many transcription regulators that are preferentially re-activated before zygotic genome activation. CCR4-NOT, the major de-adenylation complex, and its key adaptor protein BTG4 regulate translation downregulation often independent of RNA decay. BTG4 is not essential for global de-adenylation but is required for selective gene de-adenylation and production of very short-tailed transcripts. In sum, our data reveal intimate interplays among translation, RNA stability and poly(A) tail length regulation underlying mammalian OET.

Effects of low molecular weight heparin combined with hyperbaric oxygen on neurologic function and coagulation factors in patients with intracranial venous thrombosis.

OBJECTIVE: To investigate the effects of low molecular weight heparin (LMWH) combined with hyperbaric oxygen (HBO) on the neurologic function and coagulation factors of patients with intracranial venous thrombosis (ICVT). METHODS: The clinical data of 80 patients with ICVT admitted to the No. 2 Hospital of Baoding from February 2020 to January 2021 were retrospectively analyzed. Patients were assigned to a control group (n=32) and a research group (n=48) according to different treatment methods. The neurological function score, and the levels of D-dimer (D-D), fibrinogen (FIB), tumor necrosis factor-α (TNF-α), and C-reactive protein (CRP) were compared between the two groups. The two groups were also compared regarding the curative effect, toxic and side effects, as well as quality of life (QoL). RESULTS: After treatment, the National Institutes of Health Stroke Scale (NIHSS) score was significantly lower in the research group compared to the control group. At 1, 2 and 3 weeks after treatment, the levels of D-D and FIB, as well as inflammatory factors TNF-α and CRP were lower in the research group compared to the control group. The overall response rate was significantly higher in the research group compared to the control group, while there was no significant difference in the total incidence of toxic and adverse effects between the two groups. After treatment, the QoL of patients assessed by the Generic Quality of Life Inventory-74 (GQOLI-74) from the domains of physical, social, and psychological function as well as material life status was significantly better in the research group. CONCLUSIONS: LMWH combined with HBO can effectively improve the clinical efficacy and neurologic function of patients with ICVT and reduce the levels of coagulation factors and inflammatory factors.

Generation and characterization of stable pig pregastrulation epiblast stem cell lines.

Pig epiblast-derived pluripotent stem cells are considered to have great potential and broad prospects for human therapeutic model development and livestock breeding. Despite ongoing attempts since the 1990s, no stably defined pig epiblast-derived stem cell line has been established. Here, guided by insights from a large-scale single-cell transcriptome analysis of pig embryos from embryonic day (E) 0 to E14, specifically, the tracing of pluripotency changes during epiblast development, we developed an in vitro culture medium for establishing and maintaining stable pluripotent stem cell lines from pig E10 pregastrulation epiblasts (pgEpiSCs). Enabled by chemical inhibition of WNT-related signaling in combination with growth factors in the FGF/ERK, JAK/STAT3, and Activin/Nodal pathways, pgEpiSCs maintain their pluripotency transcriptome features, similar to those of E10 epiblast cells, and normal karyotypes after more than 240 passages and have the potential to differentiate into three germ layers. Strikingly, ultradeep in situ Hi-C analysis revealed functional impacts of chromatin 3D-spatial associations on the transcriptional regulation of pluripotency marker genes in pgEpiSCs. In practice, we confirmed that pgEpiSCs readily tolerate at least three rounds of successive gene editing and generated cloned gene-edited live piglets. Our findings deliver on the long-anticipated promise of pig pluripotent stem cells and open new avenues for biological research, animal husbandry, and regenerative biomedicine.

Methylome inheritance and enhancer dememorization reset an epigenetic gate safeguarding embryonic programs.

Marked epigenetic reprogramming is essential to convert terminally differentiated gametes to totipotent embryos. It remains puzzling why postfertilization global DNA reprogramming occurs in mammals but not in nonmammalian vertebrates. In zebrafish, global methylome inheritance is however accompanied by extensive enhancer "dememorization" as they become fully methylated. By depleting maternal dnmt1 using oocyte microinjection, we eliminated DNA methylation in early embryos, which died around gastrulation with severe differentiation defects. Notably, methylation deficiency leads to derepression of adult tissue­specific genes and CG-rich enhancers, which acquire ectopic transcription factor binding and, unexpectedly, histone H3 lysine 4 trimethylation (H3K4me3). By contrast, embryonic enhancers are generally CG-poor and evade DNA methylation repression. Hence, global DNA hypermethylation inheritance coupled with enhancer dememorization installs an epigenetic gate that safeguards embryonic programs and ensures temporally ordered gene expression. We propose that "enhancer dememorization" underlies and unifies distinct epigenetic reprogramming modes in early development between mammals and nonmammals.

Clinical Efficacy of Conventional Heparin Anticoagulation Combined with Apixaban in the Treatment of Patients with Cerebral Venous Thrombosis and Its Effect on Serum D-Dimer and FIB Expression.

OBJECTIVE: The aim of this study was to explore the clinical efficacy of conventional heparin anticoagulation in combination with apixaban in the treatment of patients with cerebral venous thrombosis (CVT) and its influence on serum D-dimer (D-D) and fibrinogen (FIB). METHODS: One hundred and fifty-seven consecutive CVT patients admitted to our hospital from January 1, 2006, to December 31, 2013, were allocated into two groups according to the different treatment methods, of which 95 cases received standard anticoagulation therapy (standard group (SG)) and the remaining 62 cases were given apixaban therapy (research group (RG)). The curative effects and the changes of coagulation function during the treatment, as well as the incidence of adverse reactions, were analyzed in the two groups. The changes of D-D and FIB levels before treatment and at days 1, 4, and 7 posttreatment were detected. RESULTS: In treatment efficacy, RG was superior to SG. No evident difference was observed in the incidence of adverse events or coagulation function between the two groups. At day 1 posttreatment, D-D level was increased largely in both SG and RG, but the increase was much more significant in RG. However, D-D level was decreased gradually with time in both groups, and the reduction was more notable in RG. The FIB level in SG declined gradually with time after treatment and was higher than that in RG at the same time point. In RG, FIB was decreased gradually at day 1 and day 4 posttreatment, and its level at day 7 posttreatment showed no difference compared with that at day 4 posttreatment. Spearman's analysis identified that the higher the D-D level or the lower the FIB level at day 1 posttreatment was, the better the treatment efficacy was. After seven-day treatment, the lower the level of D-D and FIB was, the better the therapeutic effect was. Logistic analysis indicated that age, time of diagnosis, deep vein thrombosis (DVT), Glasgow Coma Scale (GCS) score, infection, Apixaban, D-D, and FIB all independently affect the treatment effect of patients. CONCLUSIONS: The combined use of Apixaban with heparin is high-performing and safe in the treatment of CVT. The changes of D-D and FIB levels during the treatment are strongly linked to the therapeutic effect, which can be used as plausible evaluation indexes for the efficacy of CVT.

The landscape of RNA Pol II binding reveals a stepwise transition during ZGA.

Zygotic genome activation (ZGA) is the first transcription event in life1. However, it is unclear how RNA polymerase is engaged in initiating ZGA in mammals. Here, by developing small-scale Tn5-assisted chromatin cleavage with sequencing (Stacc-seq), we investigated the landscapes of RNA polymerase II (Pol II) binding in mouse embryos. We found that Pol II undergoes 'loading', 'pre-configuration', and 'production' during the transition from minor ZGA to major ZGA. After fertilization, Pol II is preferentially loaded to CG-rich promoters and accessible distal regions in one-cell embryos (loading), in part shaped by the inherited parental epigenome. Pol II then initiates relocation to future gene targets before genome activation (pre-configuration), where it later engages in full transcription elongation upon major ZGA (production). Pol II also maintains low poising at inactive promoters after major ZGA until the blastocyst stage, coinciding with the loss of promoter epigenetic silencing factors. Notably, inhibition of minor ZGA impairs the Pol II pre-configuration and embryonic development, accompanied by aberrant retention of Pol II and ectopic expression of one-cell targets upon major ZGA. Hence, stepwise transition of Pol II occurs when mammalian life begins, and minor ZGA has a key role in the pre-configuration of transcription machinery and chromatin for genome activation.

High-mobility group nucleosomal binding domain 2 protects against microcephaly by maintaining global chromatin accessibility during corticogenesis.

The surface area of the human cerebral cortex undergoes dramatic expansion during late fetal development, leading to cortical folding, an evolutionary feature not present in rodents. Microcephaly is a neurodevelopmental disorder defined by an abnormally small brain, and many gene mutations have been found to be associated with primary microcephaly. However, mouse models generated by ablating primary microcephaly-associated genes often fail to recapitulate the severe loss of cortical surface area observed in individuals with this pathology. Here, we show that a mouse model with deficient expression of high-mobility group nucleosomal binding domain 2 (HMGN2) manifests microcephaly with reduced cortical surface area and almost normal radial corticogenesis, with a pattern of incomplete penetrance. We revealed that altered cleavage plane and mitotic delay of ventricular radial glia may explain the rising ratio of intermediate progenitor cells to radial glia and the displacement of neural progenitor cells in microcephalic mutant mice. These led to decreased self-renewal of the radial glia and reduction in lateral expansion. Furthermore, we found that HMGN2 protected corticogenesis by maintaining global chromatin accessibility mainly at promoter regions, thereby ensuring the correct regulation of the transcriptome. Our findings underscore the importance of the regulation of chromatin structure in cortical development and highlight a mouse model with critical insights into the etiology of microcephaly.

Epigenomic analysis of gastrulation identifies a unique chromatin state for primed pluripotency.

Around implantation, the epiblast (Epi) transits from naïve to primed pluripotency, before giving rise to the three germ layers. How chromatin is reconfigured during this developmental window remains poorly understood. We performed a genome-wide investigation of chromatin landscapes during this period. We find that enhancers in ectoderm are already pre-accessible in embryonic day 6.5 (E6.5) Epi when cells enter a primed pluripotent state. Unexpectedly, strong trimethylation of histone H3 at lysine 4 (H3K4me3) emerges at developmental gene promoters in E6.5 Epi and positively correlates with H3K27me3, thus establishing bivalency. These genes also show enhanced spatial interactions. Both the strong bivalency and spatial clustering are virtually absent in preimplantation embryos and are markedly reduced in fate-committed lineages. Finally, we show that KMT2B is essential for establishing bivalent H3K4me3 at E6.5 but becomes partially dispensable later. Its deficiency leads to impaired activation of developmental genes and subsequent embryonic lethality. Thus, our data characterize lineage-specific chromatin reconfiguration and a unique chromatin state for primed pluripotency.

H3K18ac Primes Mesendodermal Differentiation upon Nodal Signaling.

Cellular responses to transforming growth factor ß (TGF-ß) depend on cell context. Here, we explored how TGF-ß/nodal signaling crosstalks with the epigenome to promote mesendodermal differentiation. We find that expression of a group of mesendodermal genes depends on both TRIM33 and nodal signaling in embryoid bodies (EBs) but not in embryonic stem cells (ESCs). Only in EBs, TRIM33 binds these genes in the presence of expanded H3K18ac marks. Furthermore, the H3K18ac landscape at mesendodermal genes promotes TRIM33 recruitment. We reveal that HDAC1 binds to active gene promoters and interferes with TRIM33 recruitment to mesendodermal gene promoters. However, the TRIM33-interacting protein p300 deposits H3K18ac and further enhances TRIM33 recruitment. ATAC-seq data demonstrate that TRIM33 primes mesendodermal genes for activation by maintaining chromatin accessibility at their regulatory regions. Altogether, our study suggests that HDAC1 and p300 are key factors linking the epigenome through TRIM33 to the cell context-dependent nodal response during mesendodermal differentiation.

Resetting histone modifications during human parental-to-zygotic transition.

Histone modifications regulate gene expression and development. To address how they are reprogrammed in human early development, we investigated key histone marks in human oocytes and early embryos. Unlike that in mouse oocytes, the permissive mark trimethylated histone H3 lysine 4 (H3K4me3) largely exhibits canonical patterns at promoters in human oocytes. After fertilization, prezygotic genome activation (pre-ZGA) embryos acquire permissive chromatin and widespread H3K4me3 in CpG-rich regulatory regions. By contrast, the repressive mark H3K27me3 undergoes global depletion. CpG-rich regulatory regions then resolve to either active or repressed states upon ZGA, followed by subsequent restoration of H3K27me3 at developmental genes. Finally, by combining chromatin and transcriptome maps, we revealed transcription circuitry and asymmetric H3K27me3 patterning during early lineage specification. Collectively, our data unveil a priming phase connecting human parental-to-zygotic epigenetic transition.

Relations of neuropeptide Y and heme oxygenase-1 expressions with fetal brain injury in rats with intrahepatic cholestasis of pregnancy.

PURPOSE: To investigate the relations of neuropeptide Y (NPY) and heme oxygenase-1 (HO-1) expressions with fetal brain injury in rats with intrahepatic cholestasis of pregnancy (ICP). METHODS: Sixty rats pregnant for 15 days were randomly divided into experimental and control groups. The ICP model was established in experimental group. On the 21st day, the blood biochemical test, histopathological examination of pregnant rat liver and fetal brain tissues and immunohistochemical analysis of fetal rat brain tissues were performed. RESULTS: On the 21st day, the alanineaminotransferase, aspartate aminotransferase and total bile acid levels in experimental group were significantly higher than control group (P<0.01). Compared with control group, there was obvious vacuolar degeneration in pregnant rat liver tissue and fetal brain tissue in experimental group. NPY expression in fetal brain tissue was negative in control group and positive in experimental group. HO-1 expression in fetal brain tissue was strongly positive in control group and positive in experimental group. There was significant difference of immunohistochemical staining optical density between two groups (P<0.01). CONCLUSION: In fetal brain of ICP rats, the NPY expression is increased, and the HO-1 expression is decreased, which may be related to the fetal brain injury.

Relations of neuropeptide Y and heme oxygenase-1 expressions with fetal brain injury in rats with intrahepatic cholestasis of pregnancy

Abstract Purpose: To investigate the relations of neuropeptide Y (NPY) and heme oxygenase-1 (HO-1) expressions with fetal brain injury in rats with intrahepatic cholestasis of pregnancy (ICP). Methods: Sixty rats pregnant for 15 days were randomly divided into experimental and control groups. The ICP model was established in experimental group. On the 21st day, the blood biochemical test, histopathological examination of pregnant rat liver and fetal brain tissues and immunohistochemical analysis of fetal rat brain tissues were performed. Results: On the 21st day, the alanineaminotransferase, aspartate aminotransferase and total bile acid levels in experimental group were significantly higher than control group (P<0.01). Compared with control group, there was obvious vacuolar degeneration in pregnant rat liver tissue and fetal brain tissue in experimental group. NPY expression in fetal brain tissue was negative in control group and positive in experimental group. HO-1 expression in fetal brain tissue was strongly positive in control group and positive in experimental group. There was significant difference of immunohistochemical staining optical density between two groups (P<0.01). Conclusion: In fetal brain of ICP rats, the NPY expression is increased, and the HO-1 expression is decreased, which may be related to the fetal brain injury.

Activin/Smad2 and Wnt/ß-catenin up-regulate HAS2 and ALDH3A2 to facilitate mesendoderm differentiation of human embryonic stem cells.

Activin and Wnt signaling are necessary and sufficient for mesendoderm (ME) differentiation of human embryonic stem cells (ESCs). In this study, we report that during ME differentiation induced by Activin and Wnt, Activin/Smad2 induces a decrease of the repressive histone modification of H3K27me3 by promoting the proteasome-dependent degradation of enhancer of zeste 2 polycomb (EZH2)-repressive complex 2 subunit. As a result, recruitment of the forkhead protein FOXH1 on open chromatin regions integrates the signals of Activin/Smad2 and Wnt/ß-catenin to activate the expression of the ME genes including HAS2 and ALDH3A2 Consistently, H3K27me3 decrease is enriched on open chromatin around regulatory regions. Furthermore, knockdown of HAS2 or ALDH3A2 greatly attenuates ME differentiation. These findings unveil a pathway from extracellular signals to epigenetic modification-mediated gene activation during ME commitment.

A growth factor-free culture system underscores the coordination between Wnt and BMP signaling in Lgr5+ intestinal stem cell maintenance.

Lgr5+ intestinal stem cells (ISCs) drive the fast renewal of intestinal epithelium. Several signaling pathways have been shown to regulate ISC fates. However, it is unclear what are the essential signals to sustain the ISC self-renewal. Here we show that coordination between Wnt and BMP signaling activity is necessary and sufficient to maintain Lgr5+ ISCs self-renewal. The key function of R-spondin1 is to achieve a high activity of Wnt signaling in the organoid culture. Using the GSK3 inhibitor CHIR-99021 and the BMP type I receptor inhibitor LDN-193189, we can maintain Lgr5+ ISCs without growth factors in vitro. Our results define the basic signaling pathways sustaining Lgr5+ ISCs and set up a convenient and economical culture system for their in vitro expansion. This work also set up an example for growth factor-free culture of other adult stem cells.

Publisher Correction: Chromatin analysis in human early development reveals epigenetic transition during ZGA.

In this Letter, the 'Open chromatin' label in Fig. 4a should have been centred above the first three columns, and the black horizontal line underneath the label should have been removed. In addition, there should have been a vertical black line between the last two sets of panels for consistency. Minor changes have also been made to Fig. 1 and to the legend of Fig. 3. These errrors have been corrected online, and see Supplementary Information to the accompanying Amendment for the original Fig. 4.