Comparative Analyses of Single-Cell Transcriptomic Profiles between In Vitro Totipotent Blastomere-like Cells and In Vivo Early Mouse Embryonic Cells

The developmental potential within pluripotent cells in the canonical model is restricted to embryonic tissues, whereas totipotent cells can differentiate into both embryonic and extraembryonic tissues. Currently, the ability to culture in vitro totipotent cells possessing molecular and functional features like those of an early embryo in vivo has been a challenge. Recently, it was reported that treatment with a single spliceosome inhibitor, pladienolide B (plaB), can successfully reprogram mouse pluripotent stem cells into totipotent blastomere-like cells (TBLCs) in vitro. The TBLCs exhibited totipotency transcriptionally and acquired expanded developmental potential with the ability to yield various embryonic and extraembryonic tissues that may be employed as novel mouse developmental cell models. However, it is disputed whether TBLCs are ‘true’ totipotent stem cells equivalent to in vivo two-cell stage embryos. To address this question, single-cell RNA sequencing was applied to TBLCs and cells from early mouse embryonic developmental stages and the data were integrated using canonical correlation analyses. Differential expression analyses were performed between TBLCs and multi-embryonic cell stages to identify differentially expressed genes. Remarkably, a subpopulation within the TBLCs population expressed a high level of the totipotent-related genes Zscan4s and displayed transcriptomic features similar to mouse two-cell stage embryonic cells. This study underscores the subtle differences between in vitro derived TBLCs and in vivo mouse early developmental cell stages at the single-cell transcriptomic level. Our study has identified a new experimental model for stem cell biology, namely ‘cluster 3’, as a subpopulation of TBLCs that can be molecularly defined as near totipotent cells.

Unveiling Complexity and Multipotentiality of Early Heart Fields

Rationale: Extraembryonic tissues, including the yolk sac and placenta, and the heart within the embryo, work to provide crucial nutrients to the embryo. The association of congenital heart defects (CHDs) with extraembryonic tissue defects further supports the potential developmental relationship between the heart and extraembryonic tissues. Although the development of early cardiac lineages has been well-studied, the developmental relationship between cardiac lineages, including epicardium, and extraembryonic mesoderm remains to be defined. Objective: To explore the developmental relationships between cardiac and extraembryonic lineages. Methods and Results: Through high-resolution single cell and genetic lineage/clonal analyses, we show an unsuspected clonal relationship between extraembryonic mesoderm and cardiac lineages. Single-cell transcriptomics and trajectory analyses uncovered two mesodermal progenitor sources contributing to left ventricle cardiomyocytes, one embryonic and the other with an extraembryonic gene expression signature. Additional lineage-tracing studies revealed that the extraembryonic-related progenitors reside at the embryonic-extraembryonic interface in gastrulating embryos, and produce distinct cell types forming the pericardium, septum transversum, epicardium, dorsolateral regions of the left ventricle and atrioventricular canal myocardium, and extraembryonic mesoderm. Clonal analyses demonstrated that these progenitors are multipotent, giving rise to not only cardiomyocytes and serosal mesothelial cell types but also, remarkably, extraembryonic mesoderm. Conclusions: Overall, our results reveal the location of previously unknown multipotent cardiovascular progenitors at the embryonic-extraembryonic interface, and define the earliest embryonic origins of serosal mesothelial lineages, including the epicardium, which contributes fibroblasts and vascular support cells to the heart. The shared lineage relationship between embryonic cardiovascular lineages and extraembryonic mesoderm revealed by our studies underscores an underappreciated blurring of boundaries between embryonic and extraembryonic mesoderm. Our findings suggest unexpected underpinnings of the association between congenital heart disease and placental insufficiency anomalies, and the potential utility of extraembryonic cells for generating cardiovascular cell types for heart repair.

Leptin Distribution in Rat Foetal and Extraembryonic Tissues in Late Gestation: A Physiological View of Amniotic Fluid Leptin

Prenatal leptin is key to regulating foetal growth and early metabolic programming. The presence of intact leptin in rat foetal (at late gestation) and neonatal (immediately after birth) stomach content and mucosa has been previously described, suggesting that it may act as a regulatory nutrient for the neonate rats, be internalised by the stomach, and play a physiological role early in life, which requires to be further investigated, including its origin. We aimed to study the ontogeny of the presence of leptin in the foetal stomach and key extraembryonic tissues in rats at late gestation (days 18–21). Leptin concentration was determined by enzyme-linked immunosorbent assay, and placental leptin immunolocalisation was analysed by immunohistochemistry. Leptin showed a sudden appearance in the amniotic fluid (AF) at day 20 of gestation, gastric content (swallowed AF), stomach, and umbilical cord, significantly increasing at day 21. Leptin levels in these fluids and tissues were positively correlated. In the placenta, leptin was detectable at all the studied days, but its localisation changed from widespread throughout the placenta at day 18 to well-defined in the labyrinth zone from day 19 onwards. The results support a possible internalisation of AF leptin by the immature stomach of near-term foetuses and suggest that changes in placental leptin localisation might help to explain the sudden appearance of leptin in AF at gestational day 20, with potential physiological significance regarding short-term feeding control and metabolic programming in the developing offspring.

Zebrafish embryonic explants undergo genetically encoded self-assembly

Embryonic stem cell cultures are thought to self-organize into embryoid bodies, able to undergo symmetry-breaking, germ layer specification and even morphogenesis. Yet, it is unclear how to reconcile this remarkable self-organization capacity with classical experiments demonstrating key roles for extrinsic biases by maternal factors and/or extraembryonic tissues in embryogenesis. Here, we show that zebrafish embryonic tissue explants, prepared prior to germ layer induction and lacking extraembryonic tissues, can specify all germ layers and form a seemingly complete mesendoderm anlage. Importantly, explant organization requires polarized inheritance of maternal factors from dorsal-marginal regions of the blastoderm. Moreover, induction of endoderm and head-mesoderm, which require peak Nodal-signaling levels, is highly variable in explants, reminiscent of embryos with reduced Nodal signals from the extraembryonic tissues. Together, these data suggest that zebrafish explants do not undergo bona fide self-organization, but rather display features of genetically encoded self-assembly, where intrinsic genetic programs control the emergence of order.

Fine-tuned adaptation of embryo-endometrium pairs at implantation revealed by gene regulatory networks Tailored conceptus-maternal communication at implantation

ABSTRACTInteractions between embryo and endometrium at implantation are critical for the progression and the issue of pregnancy. These reciprocal actions involve exchange of paracrine signals that govern implantation and placentation. However, it remains unknown how these interactions between the conceptus and the endometrium are coordinated at the level of an individual pregnancy. Under the hypothesis that gene expression of endometrium is dependent on gene expression of extraembryonic tissues, we performed an integrative analysis of transcriptome profiles of paired conceptuses and endometria obtained from pregnancies initiated by artificial insemination. We quantified strong dependence (|r|>0.95, eFDR<0.01) in transcript abundance of genes expressed in the extraembryonic tissues and genes expressed in the endometrium. The profiles of connectivity revealed distinct co-expression patterns of extraembryonic tissues with caruncular and intercaruncular areas of the endometrium. Notably, a subset of highly co-expressed genes between conceptus (n=229) and caruncular areas of the endometrium (n=218, r>0.9999, eFDR<0.001) revealed a blueprint of gene expression specific to each pregnancy. Functional analyses of genes co-expressed between conceptus and endometrium revealed significantly enriched functional modules with critical contribution for implantation and placentation, including “in utero embryonic development”, “placenta development” and “regulation of transcription”. Functional modules were remarkably specific to caruncular or intercaruncular areas of the endometrium. The quantitative and functional association between genes expressed in conceptus and endometrium emphasize a coordinated communication between these two entities in mammals. To our knowledge, we provide first evidence that implantation in mammalian pregnancy relies on the ability of the conceptus and the endometrium to develop a fine-tuned adaptive response characteristic of each pregnancy.

What makes the maternal X chromosome resistant to undergoing imprinted X inactivation?

In the mouse, while either X chromosome is chosen for inactivation in a random fashion in the embryonic tissue, the paternally derived X chromosome is preferentially inactivated in the extraembryonic tissues. It has been shown that the maternal X chromosome is imprinted so as not to undergo inactivation in the extraembryonic tissues. X-linked noncoding Xist RNA becomes upregulated on the X chromosome that is to be inactivated. An antisense noncoding RNA, Tsix , which occurs at the Xist locus and has been shown to negatively regulate Xist expression in cis, is imprinted to be expressed from the maternal X in the extraembryonic tissues. Although Tsix appears to be responsible for the imprint laid on the maternal X, those who disagree with this idea would point out the fact that Tsix has not yet been expressed from the maternal X when Xist becomes upregulated on the paternal but not the maternal X at the onset of imprinted X-inactivation in preimplantation embryos. Recent studies have demonstrated, however, that there is a prominent difference in the chromatin structure at the Xist locus depending on the parental origin, which I suggest might account for the repression of maternal Xist in the absence of maternal Tsix at the preimplantation stages. This article is part of the themed issue ‘X-chromosome inactivation: a tribute to Mary Lyon’.

Variable phenotypic penetrance of thrombosis in adult mice after tissue-selective and temporally controlled Thbd gene inactivation

Key Points Thrombomodulin deficiency in adult mice induces a fatal coagulopathy caused by the lack of cofactor function for activation of protein C. The severity of thrombosis after birth is modulated by in utero thrombomodulin expression in extraembryonic tissues.