The blastocyst (the early mammalian embryo) forms all embryonic and extra-embryonic tissues, including the placenta. It consists of a spherical thin-walled layer, known as the trophectoderm, that surrounds a fluid-filled cavity sheltering the embryonic cells . From mouse blastocysts, it is possible to derive both trophoblast and embryonic stem-cell lines , which are in vitro analogues of the trophectoderm and embryonic compartments, respectively. Here we report that trophoblast and embryonic stem cells cooperate in vitro to form structures that morphologically and transcriptionally resemble embryonic day 3.5 blastocysts, termed blastoids. Like blastocysts, blastoids form from inductive signals that originate from the inner embryonic cells and drive the development of the outer trophectoderm. The nature and function of these signals have been largely unexplored. Genetically and physically uncoupling the embryonic and trophectoderm compartments, along with single-cell transcriptomics, reveals the extensive inventory of embryonic inductions. We specifically show that the embryonic cells maintain trophoblast proliferation and self-renewal, while fine-tuning trophoblast epithelial morphogenesis in part via a BMP4/Nodal-KLF6 axis. Although blastoids do not support the development of bona fide embryos, we demonstrate that embryonic inductions are crucial to form a trophectoderm state that robustly implants and triggers decidualization in utero. Thus, at this stage, the nascent embryo fuels trophectoderm development and implantation.

The adult mouse subependymal zone provides a niche for mammalian neural stem cells (NSCs). However, the molecular signature, self-renewal potential, and fate behavior of NSCs remain poorly defined. Here we propose a model in which the fate of active NSCs is coupled to the total number of neighboring NSCs in a shared niche. Using knock-in reporter alleles and single-cell RNA sequencing, we show that the Wnt target Troy identifies both active and quiescent NSCs. Quantitative analysis of genetic lineage tracing of individual NSCs under homeostasis or in response to injury reveals rapid expansion of stem-cell number before some return to quiescence. This behavior is best explained by stochastic fate decisions, where stem-cell number within a shared niche fluctuates over time. Fate mapping proliferating cells using a Ki67 allele confirms that active NSCs reversibly return to quiescence, achieving long-term self-renewal. Our findings suggest a niche-based mechanism for the regulation of NSC fate and number.

Chronic pancreatitis and pancreatic ductal adenocarcinoma (PDAC) are characterized by extensive fibrosis. Importantly, in PDAC, this results in poor vascularization and impaired drug delivery to the cancer cells. Therefore, the combined targeting of pancreatic tumor stroma and chemotherapy should enhance response rates, but the negative outcome of a recent phase III clinical trial for the combination of chemotherapy and hedgehog pathway inhibition suggests that other means also need to be considered. Emerging data indicate that elimination of cancer stem cells as the root of the cancer is of pivotal importance for efficient treatment of pancreatic cancer. Recently, we demonstrated in a highly relevant preclinical mouse model for primary pancreatic cancers that the combination of cancer stem cell-targeting strategies in combination with a stroma-targeting agent, such as a hedgehog pathway inhibitor and chemotherapy, results in significantly enhanced long-term and progression-free survival. In the present study, we demonstrate mechanistically that Nodal-expressing pancreatic stellate cells are an important component of the tumor stroma for creating a paracrine niche for pancreatic cancer stem cells. Secretion of the embryonic morphogens Nodal/Activin by pancreatic stellate cells promoted in vitro sphere formation and invasiveness of pancreatic cancer stem cells in an Alk4-dependent manner. These data imply that the pancreatic cancer stem cell phenotype is promoted by paracrine Nodal/Activin signaling at the tumor-stroma interface. Therefore, targeting the tumor microenvironment is not only able to improve drug delivery but, even more importantly, destroys the cancer stem cell niche and, therefore, should be an integral part of cancer stem cell-based treatment strategies.

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The early mammalian conceptus (blastocyst) comprises an outer trophoblast globe that forms an axis originating from the inner embryonic cells. From the mouse conceptus, Trophoblast stem cells (TSCs) are derived, which are in vitro analogues of early trophoblasts. Here, we show that TSCs contain plastic subpopulations reflecting developmental states ranging from pre-to post-implantation trophoblasts. However, upon exposure to a specific combination of embryonic inductive signals, TSCs globally acquire properties of pre-implantation polar trophoblasts (gene expression, self-renewal) juxtaposing the inner embryonic cells, and an enhanced, homogeneous epithelial phenotype. These lines of polar-like TSCs (pTSCs) represent a transcriptionally earlier state that more efficiently forms blastoids, whose inner embryonic cells then induce the patterning of gene expression along the embryonic-abembryonic axis. Altogether, delineating the requirements and properties of polar trophoblasts and blastocyst axis formation in vitro provides a foundation for the precise description and dissection of early development.

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17During germ cell development, cells undergo a drastic switch from mitosis to meiosis to 18 form haploid germ cells. Sequencing and computational technologies now allow studying 19 development at the single-cell level. Here we developed a multiplexed trajectory 20 reconstruction to create a high-resolution developmental map of spermatogonia and 21 prophase-I spermatocytes from testes of a Dazl-GFP reporter mouse. We identified three 22 main transitions in the meiotic prophase-I: meiotic entry, the meiotic sex chromosome 23 inactivation (MSCI), and concomitant pachytene activation. We validated the key features 24 of these transitions in vivo using single molecule FISH. Focusing on MSCI, we found that 25 34% of sex chromosomal genes are induced shortly before MSCI, that silencing time is 26 diverse and correlates with specific gene functions. These highlight a previously 27 underappreciated level of regulation of MSCI. Finally, we found that spermatozoal genes 28 in pachytene are activated in a temporal pattern reflecting the future anatomic and 29 functional order of the sperm cell. Altogether we highlighted how precise and sequential 30 changes in gene expression regulate cellular states in meiotic prophase-I. 31 54 (Baarends et al., 2005) are well described, the transcriptional control of these regulators 55 and the dynamics of gene expression silencing are still to be identified. 56 Another feature of male meiosis is a massive wave of gene expression at the pachytene 57 stage (Fallahi et al., 2010), when mid-meiosis cells express a remarkable array of post-58 meiotic genes involved in spermiogenesis (da Cruz et al., 2016). The dynamics of the 59 activation of this transcriptional program, and whether sex chromosomal genes can 60escape MSCI are open questions in the field, which we analyzed in detail. 61 We thus set out to draw a temporally resolved, transcriptome-wide gene expression atlas 62 from meiotic commitment until prophase-I, in order to unravel transcriptional changes that 63 coordinate germ cell differentiation and meiotic transition. We sequenced mRNA from 64 3 1880 single cells, multiplex reconstructed their temporal order in silico, which we validated 65 via microscopy. This allowed us to describe the precise sequence of transcriptome-wide 66 changes upon MSCI and pachytene activation. 67 Results 68 1. Single-cell transcriptomics of Dazl+ germ cells identifies stem cells along 69 with 4 stages of prophase-I 70Dazl is expressed from spermatogonial to early pachytene stage (Kim, Cooke and Rhee, 71 2012). We thus used Dazl-GFP reporter mice to isolate cells during 72 meiotic entry and prophase from testes of adult mice (2-4 month) ( Fig. 1A). We applied 73 single-cell mRNA sequencing using the SORT-seq protocol , and 74 sequenced 1880 single cells from 3 mice. After trimming and sequence alignment to the 75 mm10 reference transcriptome (Online Methods), we filtered low quality cells with fewer 76 than 3000 unique (UMI corrected) transcripts and selected 1274 high-quality germ cells. 77 ...