Significance Super-enhancers (SEs) hold stronger power than regular enhancers to direct gene expression in the regulation of stem cell pluripotency. To dissect how pluripotency-associated SEs have evolved in mammals, we performed a systematic comparison of SEs among pigs, humans, and mice. Our analysis allowed the identification of three pluripotency-associated SEs (SE-SOX2, SE-PIM1, and SE-FGFR1) that are conserved in Placentalia (accounting for 94% of mammals) as well as many species-specific SEs. All three SEs were sufficient to direct pluripotency-dependent gene expression, and disruption of each conserved SE caused the loss of stem cell pluripotency. Our work highlights a small number of conserved SEs essential for the maintenance of pluripotency.

Super-enhancers conserved within placental mammals maintain stem cell pluripotency

Abstract Liver development is a complex process that is regulated by a series of signaling pathways. Three-dimensional (3D) chromatin architecture plays an important role in transcriptional regulation; nonetheless, its dynamics and role in the rapid transition of core liver functions during development and obesity-induced metabolic stress remain largely unexplored. To investigate the dynamic chromatin architecture during liver development and under metabolic stress, we generated high-resolution maps of chromatin architecture for porcine livers across six major developmental stages (from embryonic day 38 to the adult stage) and under a high-fat diet-induced obesity. The characteristically loose chromatin architecture supports a highly plastic genome organization during early liver development, which fundamentally contributes to the rapid functional transitions in the liver after birth. We reveal the multi-scale reorganization of chromatin architecture and its influence on transcriptional regulation of critical signaling processes during liver development, and show its close association with transition in hepatic functions (i.e., from hematopoiesis in the fetus to metabolism and immunity after birth). The limited changes in chromatin structure help explain the observed metabolic adaptation to excessive energy intake in pigs. These results provide a global overview of chromatin architecture dynamics associated with the transition of physiological liver functions between prenatal development and postnatal maturation, and a foundational resource that allows for future in-depth functional characterization. 

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Dynamic 3D genome reorganization during development and metabolic stress of the porcine liver

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Pluripotent stem cells (PSCs) have unlimited self-renewal and multifunctional development potential in vitro. Porcine PSCs are highly desirable due to the conserved characteristics between pigs and humans. Extended PSCs (EPSCs) are additionally capable of differentiating into embryonic (Em) and extraembryonic (E×Em) parts. Here, we employed the LCDM culture system (consisting of human LIF, CHIR99021, (S)-(+)-dimethindene maleate, and minocycline hydrochloride), which can establish EPSCs from humans and mice, to derive and maintain stable porcine PSCs (pLCDM) from in vivo blastocysts. Transcriptome analysis revealed the unique molecular characteristics of pLCDMs compared with early-stage embryos. Meanwhile, the parallels and differences in the transcriptome features among pLCDMs, human EPSCs, and mouse EPSCs were carefully analyzed and evaluated. Most noteworthy, the trophoblast lineage differentiation tendency of pLCDMs was clarified by inducing trophoblast-like cells and trophoblast stem cells (TSCs) in vitro. Further research found that 2 of the small molecules in LCDM culture system, (S)-(+)-dimethindene maleate (DiM) and minocycline hydrochloride (MiH), probably play a crucial role in promoting trophoblast lineage differentiation potential of pLCDMs. 

OUP accepted manuscript

In brief Trophectoderm is the first tissue to differentiate in the early mammalian embryo and is essential for hatching, implantation, and placentation. This review article discusses the roles of Ras homolog family members (RHO) and RHO-associated coiled-coil containing protein kinases (ROCK) in the molecular and cellular regulation of trophectoderm formation. Abstract The trophectoderm (TE) is the first tissue to differentiate during the preimplantation development of placental mammals. It constitutes the outer epithelial layer of the blastocyst and is responsible for hatching, uterine attachment, and placentation. Thus, its formation is the key initial step that enables the viviparity of mammals. Here, we first describe the general features of TE formation at the morphological and molecular levels. Prospective TE cells form an epithelial layer enclosing an expanding fluid-filled cavity by establishing the apical-basal cell polarity, intercellular junctions, microlumen, and osmotic gradient. A unique set of genes is expressed in TE that encode the transcription factors essential for the development of trophoblasts of the placenta upon implantation. TE-specific gene expressions are driven by the inhibition of HIPPO signaling, which is dependent on the prior establishment of the apical-basal polarity. We then discuss the specific roles of RHO and ROCK as essential regulators of TE formation. RHO and ROCK modulate the actomyosin cytoskeleton, apical-basal polarity, intercellular junctions, and HIPPO signaling, thereby orchestrating the epithelialization and gene expressions in TE. Knowledge of the molecular mechanisms underlying TE formation is crucial for assisted reproductive technologies in human and farm animals, as it provides foundation to help improve procedures for embryo handling and selection to achieve better reproductive outcomes. 

Trophectoderm formation: regulation of morphogenesis and gene expressions by RHO, ROCK, cell polarity, and HIPPO signaling

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.

Xianya Huang

Papers

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