Elucidation of the pluripotent potential of bovine embryonic lineages facilitates the establishment of formative stem cell lines.

The establishment of epiblast-derived pluripotent stem cells (PSCs) from cattle, which are important domestic animals that provide humans with milk and meat while also serving as bioreactors for producing valuable proteins, poses a challenge due to the unclear molecular signaling required for embryonic epiblast development and maintenance of PSC self-renewal. Here, we selected six key stages of bovine embryo development (E5, E6, E7, E10, E12, and E14) to track changes in pluripotency and the dependence on signaling pathways via modified single-cell transcription sequencing technology. The remarkable similarity of the gene expression patterns between cattle and pigs during embryonic lineage development contributed to the successful establishment of bovine epiblast stem cells (bEpiSCs) using 3i/LAF (WNTi, GSK3ßi, SRCi, LIF, Activin A, and FGF2) culture system. The generated bEpiSCs exhibited consistent expression patterns of formative epiblast pluripotency genes and maintained clonal morphology, normal karyotypes, and proliferative capacity for more than 112 passages. Moreover, these cells exhibited high-efficiency teratoma formation as well as the ability to differentiate into various cell lineages. The potential of bEpiSCs for myogenic differentiation, primordial germ cell like cells (PGCLCs) induction, and as donor cells for cell nuclear transfer was also assessed, indicating their promise in advancing cell-cultured meat production, gene editing, and animal breeding.

MAT2A is essential for zygotic genome activation by maintaining of histone methylation in porcine embryos.

Methionine adenosyltransferase 2A (MAT2A) is an essential enzyme in the methionine cycle that generates S-adenosylmethionine (SAM) by reacting with methionine and ATP. SAM acts as a methyl donors for histone and DNA methylation, which plays key roles in zygotic genome activation (ZGA). However, the effects of MAT2A on porcine ZGA remain unclear. To investigate the function of MAT2A and its underlying mechanism in porcine ZGA, MAT2A was knocked down by double-stranded RNA injection at the 1-cell stage. MAT2A is highly expressed at every stage of porcine embryo development. The percentages of four-cell-stage embryos and blastocysts were lower in the MAT2A-knockdown (KD) group than in the control group. Notably, depletion of MAT2A decreased the levels of H3K4me2, H3K9me2/3, and H3K27me3 at the four-cell stage, whereas MAT2A KD reduced the transcriptional activity of ZGA genes. MAT2A KD decreased embryonic ectoderm development (EED) and enhancer of zeste homolog 2 (EZH2) expression. Exogenous SAM supplementation rescued histone methylation levels and developmental arrest induced by MAT2A KD. Additionally, MAT2A KD significantly increased DNA damage and apoptosis. In conclusion, MAT2A is involved in regulating transcriptional activity and is essential for regulating histone methylation during porcine ZGA.

Mesenchymal Osr1+ cells regulate embryonic lymphatic vessel formation.

The lymphatic system is formed during embryonic development by the commitment of specialized lymphatic endothelial cells (LECs) and their subsequent assembly in primary lymphatic vessels. Although lymphatic cells are in continuous contact with mesenchymal cells during development and in adult tissues, the role of mesenchymal cells in lymphatic vasculature development remains poorly characterized. Here, we show that a subpopulation of mesenchymal cells expressing the transcription factor Osr1 are in close association with migrating LECs and established lymphatic vessels in mice. Lineage tracing experiments revealed that Osr1+ cells precede LEC arrival during lymphatic vasculature assembly in the back of the embryo. Using Osr1-deficient embryos and functional in vitro assays, we show that Osr1 acts in a non-cell-autonomous manner controlling proliferation and early migration of LECs to peripheral tissues. Thereby, mesenchymal Osr1+ cells control, in a bimodal manner, the production of extracellular matrix scaffold components and signal ligands crucial for lymphatic vessel formation.

LncRNAs in the Dlk1-Dio3 Domain Are Essential for Mid-Embryonic Heart Development.

The Dlk1-Dio3 domain is important for normal embryonic growth and development. The heart is the earliest developing and functioning organ of the embryo. In this study, we constructed a transcriptional termination model by inserting termination sequences and clarified that the lack of long non-coding RNA (lncRNA) expression in the Dlk1-Dio3 domain caused the death of maternal insertion mutant (MKI) and homozygous mutant (HOMO) mice starting from E13.5. Parental insertion mutants (PKI) can be born and grow normally. Macroscopically, dying MKI and HOMO embryos showed phenomena such as embryonic edema and reduced heart rate. Hematoxylin and eosin (H.E.) staining showed thinning of the myocardium in MKI and HOMO embryos. In situ hybridization (IHC) and quantitative reverse-transcription polymerase chain reaction (qRT-PCR) showed downregulation of lncGtl2, Rian, and Mirg expression in MKI and HOMO hearts. The results of single-cell RNA sequencing (scRNA-Seq) analysis indicated that the lack of lncRNA expression in the Dlk1-Dio3 domain led to reduced proliferation of epicardial cells and may be an important cause of cardiac dysplasia. In conclusion, this study demonstrates that Dlk1-Dio3 domain lncRNAs play an integral role in ventricular development.

An essential role for Cmtr2 in mammalian embryonic development.

CMTR2 is an mRNA cap methyltransferase with poorly understood physiological functions. It catalyzes 2'-O-ribose methylation of the second transcribed nucleotide of mRNAs, potentially serving to mark RNAs as "self" to evade the cellular innate immune response. Here we analyze the consequences of Cmtr2 deficiency in mice. We discover that constitutive deletion of Cmtr2 results in mouse embryos that die during mid-gestation, exhibiting defects in embryo size, placental malformation and yolk sac vascularization. Endothelial cell deletion of Cmtr2 in mice results in vascular and hematopoietic defects, and perinatal lethality. Detailed characterization of the constitutive Cmtr2 KO phenotype shows an activation of the p53 pathway and decreased proliferation, but no evidence of interferon pathway activation. In summary, our study reveals the essential roles of Cmtr2 in mammalian cells beyond its immunoregulatory function.

Transcriptome Profiling of Mouse Embryonic Fibroblast Spontaneous Immortalization: A Comparative Analysis.

Cell immortalization, a hallmark of cancer development, is a process that cells can undergo on their path to carcinogenesis. Spontaneously immortalized mouse embryonic fibroblasts (MEFs) have been used for decades; however, changes in the global transcriptome during this process have been poorly described. In our research, we characterized the poly-A RNA transcriptome changes after spontaneous immortalization. To this end, differentially expressed genes (DEGs) were screened using DESeq2 and characterized by gene ontology enrichment analysis and protein-protein interaction (PPI) network analysis to identify the potential hub genes. In our study, we identified changes in the expression of genes involved in proliferation regulation, cell adhesion, immune response and transcriptional regulation in immortalized MEFs. In addition, we performed a comparative analysis with previously reported MEF immortalization data, where we propose a predicted gene regulatory network model in immortalized MEFs based on the altered expression of Mapk11, Cdh1, Chl1, Zic1, Hoxd10 and the novel hub genes Il6 and Itgb2.

Mechanisms underlying the direct programming of mouse embryonic fibroblasts to thymic epithelial cells by FOXN1.

Thymic epithelial cells (TECs) are crucial to the ability of the thymus to generate T cells for the adaptive immune system in vertebrates. However, no in vitro system for studying TEC function exists. Overexpressing the transcription factor FOXN1 initiates transdifferentiation of fibroblasts into TEC-like cells (iTECs) that support T-cell differentiation in culture or after transplant. In this study, we have characterized iTEC programming at the cellular and molecular level in mouse to determine how it proceeds, and have identified mechanisms that can be targeted for improving this process. These data show that iTEC programming consists of discrete gene expression changes that differ early and late in the process, and that iTECs upregulate markers of both cortical and medullary TEC (cTEC and mTEC) lineages. We demonstrate that promoting proliferation enhances iTEC generation, and that Notch inhibition allows the induction of mTEC differentiation. Finally, we show that MHCII expression is the major difference between iTECs and fetal TECs. MHCII expression was improved by co-culturing iTECs with fetal double-positive T-cells. This study supports future efforts to improve iTEC generation for both research and translational uses.

Erythropoiesis in the mammalian embryo.

Red blood cells (RBCs) comprise a critical component of the cardiovascular network, which constitutes the first functional organ system of the developing mammalian embryo. Examination of circulating blood cells in mammalian embryos revealed two distinct types of erythroid cells: large, nucleated "primitive" erythroblasts followed by smaller, enucleated "definitive" erythrocytes. This review describes the current understanding of primitive and definitive erythropoiesis gleaned from studies of mouse and human embryos and induced pluripotent stem cells (iPSCs). Primitive erythropoiesis in the mouse embryo comprises a transient wave of committed primitive erythroid progenitors (primitive erythroid colony-forming cells, EryP-CFC) in the early yolk sac that generates a robust cohort of precursors that mature in the bloodstream and enucleate. In contrast, definitive erythropoiesis has two distinct developmental origins. The first comprises a transient wave of definitive erythroid progenitors (burst-forming units erythroid, BFU-E) that emerge in the yolk sac and seed the fetal liver where they terminally mature to provide the first definitive RBCs. The second comprises hematopoietic stem cell (HSC)-derived BFU-E that terminally mature at sites colonized by HSCs particularly the fetal liver and subsequently the bone marrow. Primitive and definitive erythropoiesis are derived from endothelial identity precursors with distinct developmental origins. Although they share prototypical transcriptional regulation, primitive and definitive erythropoiesis are also characterized by distinct lineage-specific factors. The exquisitely timed, sequential production of primitive and definitive erythroid cells is necessary for the survival and growth of the mammalian embryo.

Hippo pathway inactivation through subcellular localization of NF2/merlin in outer cells of mouse embryos.

The Hippo pathway plays a crucial role in cell proliferation and differentiation during tumorigenesis, tissue homeostasis and early embryogenesis. Scaffold proteins from the ezrin-radixin-moesin (ERM) family, including neurofibromin 2 (NF2; Merlin), regulate the Hippo pathway through cell polarity. However, the mechanisms underlying Hippo pathway regulation via cell polarity in establishing outer cells remain unclear. In this study, we generated artificial Nf2 mutants in the N-terminal FERM domain (L64P) and examined Hippo pathway activity by assessing the subcellular localization of YAP1 in early embryos expressing these mutant mRNAs. The L64P-Nf2 mutant inhibited NF2 localization around the cell membrane, resulting in YAP1 cytoplasmic translocation in the polar cells. L64P-Nf2 expression also disrupted the apical centralization of both large tumor suppressor 2 (LATS2) and ezrin in the polar cells. Furthermore, Lats2 mutants in the FERM binding domain (L83K) inhibited YAP1 nuclear translocation. These findings demonstrate that NF2 subcellular localization mediates cell polarity establishment involving ezrin centralization. This study provides previously unreported insights into how the orchestration of the cell-surface components, including NF2, LATS2 and ezrin, modulates the Hippo pathway during cell polarization.

Transition of signal requirement in hematopoietic stem cell development from hemogenic endothelial cells.

Hematopoietic stem cells (HSCs) develop from hemogenic endothelial cells (HECs) in vivo during mouse embryogenesis. When cultured in vitro, cells from the embryo phenotypically defined as pre-HSC-I and pre-HSC-II have the potential to differentiate into HSCs. However, minimal factors required for HSC induction from HECs have not yet been determined. In this study, we demonstrated that stem cell factor (SCF) and thrombopoietin (TPO) induced engrafting HSCs from embryonic day (E) 11.5 pre-HSC-I in a serum-free and feeder-free culture condition. In contrast, E10.5 pre-HSC-I and HECs required an endothelial cell layer in addition to SCF and TPO to differentiate into HSCs. A single-cell RNA sequencing analysis of E10.5 to 11.5 dorsal aortae with surrounding tissues and fetal livers detected TPO expression confined in hepatoblasts, while SCF was expressed in various tissues, including endothelial cells and hepatoblasts. Our results suggest a transition of signal requirement during HSC development from HECs. The differentiation of E10.5 HECs to E11.5 pre-HSC-I in the aorta-gonad-mesonephros region depends on SCF and endothelial cell-derived factors. Subsequently, SCF and TPO drive the differentiation of E11.5 pre-HSC-I to pre-HSC-II/HSCs in the fetal liver. The culture system established in this study provides a beneficial tool for exploring the molecular mechanisms underlying the development of HSCs from HECs.

Involvement of METTL3-mediated m6A methylation in the early development of porcine cloned embryos.

METTL3-mediated N6-methyladenosine (m6A) modification is critical for gametogenesis and early embryonic development. However, the function of METTL3-mediated m6A modification in the early development of somatic nuclear transfer embryos (SCNT) remains unclear. Here, we found that METTL3 mRNA and protein levels exhibit dynamic changes during the early development of porcine SCNT embryos. The levels of METTL3 mRNA and protein in SCNT embryos at specific developmental stages differ from those in parthenogenetic activation (PA) counterparts. SiRNA injection effectively reduced the levels of METTL3 mRNA and protein in 4-cell embryos and blastocysts. METTL3 knockdown significantly reduced the cleavage and blastocyst rates of SCNT embryos. METTL3 knockdown significantly reduced the number of total cells and trophectoderm (TE) cells in the resulting blastocysts and perturbed cell lineage allocation. In addition, METTL3 knockdown reduced the levels of m6A modification in 4-cell embryos and blastocysts. Importantly, METTL3 knockdown decreased the expression levels of CDX2, GATA3, NANOG and YAP, and increased the expression levels of SOX2 and OCT4. Taken together, these results demonstrate that METTL3-mediated m6A modification regulates early development and lineage differentiation of porcine SCNT embryos.

Osteopontin expression in dromedary camel's conceptuses during the peri-implantation period.

The reproductive efficiency of dromedary camels is hindered by challenges such as early embryonic mortality, which may be attributed to a lack of synchronization between conceptus signalling and uterine receptivity. Understanding the intricate biological processes involved in feto-maternal interactions during implantation is crucial to address these limitations. Osteopontin (OPN) is a protein involved in cell signalling and adhesion, playing a crucial role in embryonic implantation. Previous studies have shown the presence of OPN in the uterine endometrium of various mammalian species including dromedary camels. However, the expression pattern of OPN in dromedary conceptuses remains unexplored. Thus, the current study aimed, for the first time, to investigate the temporospatial expression of OPN in dromedary conceptuses during the peri-implantation period at Days 8, 10, and 12 of pregnancy. Twelve conceptuses were recovered non-surgically from pregnant females on Days 8, 10, 12 of pregnancy. Quantitative real-time PCR (qrt-PCR), immunohistochemistry (IHC), and immunofluorescence (IF) were employed for analysis of the expression of OPN mRNA and protein. The results revealed significant increases in both OPN mRNA and protein expression started on Day 10 and peaked at Day 12 of pregnancy. Immuno-localization confirmed the presence of OPN protein in the trophectoderm and endoderm of dromedary conceptuses. In conclusion, the expression and localization of OPN during the peri-implantation period in dromedary conceptuses imply its involvement as a crucial reproductive factor and its upregulation during this period, with a pronounced increase close to attachment time (Day 12 of pregnancy) further supports its role in embryo adhesion, implantation, and placentation.

SANTO: a coarse-to-fine alignment and stitching method for spatial omics.

With the flourishing of spatial omics technologies, alignment and stitching of slices becomes indispensable to decipher a holistic view of 3D molecular profile. However, existing alignment and stitching methods are unpractical to process large-scale and image-based spatial omics dataset due to extreme time consumption and unsatisfactory accuracy. Here we propose SANTO, a coarse-to-fine method targeting alignment and stitching tasks for spatial omics. SANTO firstly rapidly supplies reasonable spatial positions of two slices and identifies the overlap region. Then, SANTO refines the positions of two slices by considering spatial and omics patterns. Comprehensive experiments demonstrate the superior performance of SANTO over existing methods. Specifically, SANTO stitches cross-platform slices for breast cancer samples, enabling integration of complementary features to synergistically explore tumor microenvironment. SANTO is then applied to 3D-to-3D spatiotemporal alignment to study development of mouse embryo. Furthermore, SANTO enables cross-modality alignment of spatial transcriptomic and epigenomic data to understand complementary interactions.

Evidence for metabolism of creatine by the conceptus, placenta, and uterus for production of adenosine triphosphate during conceptus development in pigs†.

In pigs, the majority of embryonic mortality occurs when free-floating conceptuses (embryos/fetuses and associated placental membranes) elongate, and the uterine-placental interface undergoes folding and develops areolae. Both periods involve proliferation, migration, and changes in morphology of cells that require adenosine triphosphate (ATP). We hypothesize that insufficient ATP in conceptus and uterine tissues contributes to conceptus loss in pigs. Creatine is stored in cells as phosphocreatine for ATP regeneration through the creatine-creatine kinase- phosphocreatine pathway. However, the expression of components of this pathway in pigs has not been examined throughout gestation. Results of qPCR analyses indicated increases in AGAT, GAMT, CKM, CKB, and SLC6A8 mRNAs in elongating porcine conceptuses, and immunofluorescence microscopy localized guanidinoacetate N-methyltransferase, creatine kinase M, and creatine kinase B proteins to the trophectoderm of elongating conceptuses, to the columnar chorionic epithelial cells at the bottom of chorioallantoic troughs, and to endometrial luminal epithelium at the tops of the endometrial ridges of uterine-placental folds on Days 40, 60, and 90 of gestation. Guanidinoacetate N-methyltransferase protein is expressed in endometrial luminal epithelium at the uterine-placental interface, but immunostaining is more intense in luminal epithelium at the bottoms of the endometrial ridges. Results of this study indicate that key elements of the pathway for creatine metabolism are expressed in cells of the conceptus, placenta, and uterus for potential production of ATP during two timepoints in pregnancy with a high demand for energy; elongation of the conceptus for implantation and development of uterine-placental folding during placentation.

Extraembryonic gut endoderm cells undergo programmed cell death during development.

Despite a distinct developmental origin, extraembryonic cells in mice contribute to gut endoderm and converge to transcriptionally resemble their embryonic counterparts. Notably, all extraembryonic progenitors share a non-canonical epigenome, raising several pertinent questions, including whether this landscape is reset to match the embryonic regulation and if extraembryonic cells persist into later development. Here we developed a two-colour lineage-tracing strategy to track and isolate extraembryonic cells over time. We find that extraembryonic gut cells display substantial memory of their developmental origin including retention of the original DNA methylation landscape and resulting transcriptional signatures. Furthermore, we show that extraembryonic gut cells undergo programmed cell death and neighbouring embryonic cells clear their remnants via non-professional phagocytosis. By midgestation, we no longer detect extraembryonic cells in the wild-type gut, whereas they persist and differentiate further in p53-mutant embryos. Our study provides key insights into the molecular and developmental fate of extraembryonic cells inside the embryo.

LEUTX regulates porcine embryonic genome activation in somatic cell nuclear transfer embryos.

Emerging evidence highlights the regulatory role of paired-like (PRD-like) homeobox transcription factors (TFs) in embryonic genome activation (EGA). However, the majority of PRD-like genes are lost in rodents, thus prompting an investigation into PRD-like TFs in other mammals. Here, we showed that PRD-like TFs were transiently expressed during EGA in human, monkey, and porcine fertilized embryos, yet they exhibited inadequate expression in their cloned embryos. This study, using pig as the research model, identified LEUTX as a key PRD-like activator of porcine EGA through genomic profiling and found that LEUTX overexpression restored EGA failure and improved preimplantation development and cloning efficiency in porcine cloned embryos. Mechanistically, LEUTX opened EGA-related genomic regions and established histone acetylation via recruiting acetyltransferases p300 and KAT2A. These findings reveal the regulatory mechanism of LEUTX to govern EGA in pigs, which may provide valuable insights into the study of early embryo development for other non-rodent mammals.

Association of embryonic inositol status with susceptibility to neural tube defects, metabolite profile, and maternal inositol intake.

Maternal nutrition contributes to gene-environment interactions that influence susceptibility to common congenital anomalies such as neural tube defects (NTDs). Supplemental myo-inositol (MI) can prevent NTDs in some mouse models and shows potential for prevention of human NTDs. We investigated effects of maternal MI intake on embryonic MI status and metabolism in curly tail mice, which are genetically predisposed to NTDs that are inositol-responsive but folic acid resistant. Dietary MI deficiency caused diminished MI in maternal plasma and embryos, showing that de novo synthesis is insufficient to maintain MI levels in either adult or embryonic mice. Under normal maternal dietary conditions, curly tail embryos that developed cranial NTDs had significantly lower MI content than unaffected embryos, revealing an association between diminished MI status and failure of cranial neurulation. Expression of inositol-3-phosphate synthase 1, required for inositol biosynthesis, was less abundant in the cranial neural tube than at other axial levels. Supplemental MI or d-chiro-inositol (DCI) have previously been found to prevent NTDs in curly tail embryos. Here, we investigated the metabolic effects of MI and DCI treatments by mass spectrometry-based metabolome analysis. Among inositol-responsive metabolites, we noted a disproportionate effect on nucleotides, especially purines. We also found altered proportions of 5-methyltetrahydrolate and tetrahydrofolate in MI-treated embryos suggesting altered folate metabolism. Treatment with nucleotides or the one-carbon donor formate has also been found to prevent NTDs in curly tail embryos. Together, these findings suggest that the protective effect of inositol may be mediated through the enhanced supply of nucleotides during neural tube closure.

Comparative transcriptome profile of embryos at different developmental stages derived from somatic cell nuclear transfer (SCNT) and in-vitro fertilization (IVF) in riverine buffalo (Bubalus bubalis).

Somatic cell nuclear transfer (SCNT) is a very important reproductive technology with many diverse applications, such as fast multiplication of elite animals, the production of transgenic animals and embryonic stem (ES) cells. However, low cloning efficiency, a low live birth rate and the abnormally high incidence of abnormalities in the offspring born are attributed to incomplete or aberrant nuclear reprogramming. In SCNT embryos, the aberrant expression pattern of the genes throughout embryonic development is responsible for the incomplete nuclear reprogramming. The present study was carried out to identify the differential gene expression (DEGs) profile and molecular pathways of the SCNT and IVF embryos at different developmental stages (2 cell, 8 cell and blastocyst stages). In the present study, 1164 (2 cell), 1004 (8 cell) and 530 (blastocyst stage) DEGs were identified in the SCNT embryos as compared to IVF embryos. In addition, several genes such as ZEB1, GDF1, HSF5, PDE3B, VIM, TNNC, HSD3B1, TAGLN, ITGA4 and AGMAT were affecting the development of SCNT embryos as compared to IVF embryos. Further, Gene Ontology (GO) and molecular pathways analysis suggested, SCNT embryos exhibit variations compared to their IVF counterparts and affected the development of embryos throughout the different developmental stages. Apart from this, q-PCR analysis of the GDF1, TMEM114, and IGSF22 genes were utilized to validate the RNA-seq data. These findings contribute valuable insights about the different genes and molecular pathways underlying SCNT embryo development and offer crucial information for improving SCNT efficiency.

Regulation of Myc transcription by an enhancer cluster dedicated to pluripotency and early embryonic expression.

MYC plays various roles in pluripotent stem cells, including the promotion of somatic cell reprogramming to pluripotency, the regulation of cell competition and the control of embryonic diapause. However, how Myc expression is regulated in this context remains unknown. The Myc gene lies within a ~ 3-megabase gene desert with multiple cis-regulatory elements. Here we use genomic rearrangements, transgenesis and targeted mutation to analyse Myc regulation in early mouse embryos and pluripotent stem cells. We identify a topologically-associated region that homes enhancers dedicated to Myc transcriptional regulation in stem cells of the pre-implantation and early post-implantation embryo. Within this region, we identify elements exclusively dedicated to Myc regulation in pluripotent cells, with distinct enhancers that sequentially activate during naive and formative pluripotency. Deletion of pluripotency-specific enhancers dampens embryonic stem cell competitive ability. These results identify a topologically defined enhancer cluster dedicated to early embryonic expression and uncover a modular mechanism for the regulation of Myc expression in different states of pluripotency.

TET activity safeguards pluripotency throughout embryonic dormancy.

Dormancy is an essential biological process for the propagation of many life forms through generations and stressful conditions. Early embryos of many mammals are preservable for weeks to months within the uterus in a dormant state called diapause, which can be induced in vitro through mTOR inhibition. Cellular strategies that safeguard original cell identity within the silent genomic landscape of dormancy are not known. Here we show that the protection of cis-regulatory elements from silencing is key to maintaining pluripotency in the dormant state. We reveal a TET-transcription factor axis, in which TET-mediated DNA demethylation and recruitment of methylation-sensitive transcription factor TFE3 drive transcriptionally inert chromatin adaptations during dormancy transition. Perturbation of TET activity compromises pluripotency and survival of mouse embryos under dormancy, whereas its enhancement improves survival rates. Our results reveal an essential mechanism for propagating the cellular identity of dormant cells, with implications for regeneration and disease.