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Methods for profiling genes at the single-cell level have revolutionized our ability to study several biological processes and systems including development, differentiation, response programmes and disease progression. In many of these studies, cells are profiled over time in order to infer dynamic changes in cell states and types, sets of expressed genes, active pathways and key regulators. However, time-series single-cell RNA sequencing (scRNA-seq) also raises several new analysis and modelling issues. These issues range from determining when and how deep to profile cells, linking cells within and between time points, learning continuous trajectories, and integrating bulk and single-cell data for reconstructing models of dynamic networks. In this Review, we discuss several approaches for the analysis and modelling of time-series scRNA-seq, highlighting their steps, key assumptions, and the types of data and biological questions they are most appropriate for.

The “Four Empires” scheme appears in literature from around the ancient Near East, as well as in the biblical book of Daniel. Daniel’s scheme was adopted in subsequent Jewish literature as a basic division of world history. In addition, the book of Daniel appears to have had a prominent place in the Qumran library. Scholars have identified, or suggested, the existence of the “Four Empires” scheme in two texts found among the Qumran scrolls, the “New Jerusalem” text (4Q554), and, especially, in the so-called “Four Kingdoms”(!) text (4Q552–553). This paper will examine these texts, will argue that the “four empires” scheme is not attested in the Qumran scrolls (apart from Daniel), and will suggest alternative understandings of those two texts.

The spatial architecture of the islets of Langerhans is vitally important for their correct function, and alterations in islet morphogenesis often result in diabetes mellitus. We have previously reported that Roundabout (Robo) receptors are required for proper islet morphogenesis. As part of the Slit-Robo signaling pathway, Robo receptors function in conjunction with Slit ligands to mediate axon guidance, cell migration, and cell positioning in development. However, the role of Slit ligands in islet morphogenesis has not yet been determined. Here, we report that Slit ligands are expressed in overlapping and distinct patterns in both endocrine and nonendocrine tissues in late pancreas development. We show that the function of either Slit2 or Slit3, which are predominantly expressed in the pancreatic mesenchyme, is required and sufficient for islet morphogenesis, while Slit1, which is predominantly expressed in the β cells, is dispensable for islet morphogenesis. We further show that Slit functions as a repellent signal to β cells. These data suggest that clustering of endocrine cells during islet morphogenesis is guided, at least in part, by repelling Slit2/3 signals from the pancreatic mesenchyme.

The spatial architecture of the islets of Langerhans is vitally important for their correct function, and alterations in islet morphogenesis often result in diabetes mellitus. We have previously reported that Roundabout (Robo) receptors are required for proper islet morphogenesis. As part of the Slit-Robo signaling pathway, Robo receptors work in conjunction with Slit ligands to mediate axon guidance, cell migration, and cell positioning in development. However, the role of Slit ligands in islet morphogenesis has not yet been determined. Here we report that Slit ligands are expressed in overlapping and distinct patterns in both endocrine and non-endocrine tissues in late pancreas development. We show that function of either Slit2 or Slit3, which are predominantly expressed in the pancreatic mesenchyme, is required and sufficient for islet morphogenesis, while Slit1, which is predominantly expressed in the β cells, is dispensable for islet morphogenesis. We further show that Slit functions as a repellent signal to β cells. These data suggest that clustering of endocrine cells during islet morphogenesis is guided, at least in part, by repelling Slit2/3 signals from the pancreatic mesenchyme.

Summary: In vitro differentiation of pluripotent cells into β cells is a promising alternative to cadaveric-islet transplantation as a cure for type 1 diabetes (T1D). During the directed differentiation of human embryonic stem cells (hESCS) by exogenous factors, numerous genes that affect the differentiation process are turned on and off autonomously. Manipulating these reactions could increase the efficiency of differentiation and provide a more complete control over the final composition of cell populations. To uncover in vitro autonomous responses, we performed single-cell RNA sequencing on hESCs as they differentiate in spherical clusters. We observed that endocrine cells and their progenitors exist beside one another in separate compartments that activate distinct genetic pathways. WNT pathway inhibition in the endocrine domain of the differentiating clusters reveals a necessary role for the WNT inhibitor APC during islet formation in vivo. Accordingly, WNT inhibition in vitro causes an increase in the proportion of differentiated endocrine cells. : In vitro differentiation of pluripotent cells into β cells is a promising alternative to cadaveric islet transplantation as a cure for type 1 diabetes. Sharon et al. use scRNA-seq to identify the cell populations that form during the process and uncover a role for WNT pathway inhibition during endocrine differentiation. Keywords: Single-cell RNA sequencing, human embryonic stem cells, hESCs, in vitro differentiation, β cells, insulin, diabetes, pancreas development, WNT pathway, APC

Summary The pancreatic islets of Langerhans regulate glucose homeostasis. The loss of insulin-producing β cells within islets results in diabetes, and islet transplantation from cadaveric donors can cure the disease. In vitro production of whole islets, not just β cells, will benefit from a better understanding of endocrine differentiation and islet morphogenesis. We used single-cell mRNA sequencing to obtain a detailed description of pancreatic islet development. Contrary to the prevailing dogma, we find islet morphology and endocrine differentiation to be directly related. As endocrine progenitors differentiate, they migrate in cohesion and form bud-like islet precursors, or “peninsulas” (literally “almost islands”). α cells, the first to develop, constitute the peninsular outer layer, and β cells form later, beneath them. This spatiotemporal collinearity leads to the typical core-mantle architecture of the mature, spherical islet. Finally, we induce peninsula-like structures in differentiating human embryonic stem cells, laying the ground for the generation of entire islets in vitro.

The title Ethnarch appears in Second Temple sources in reference to four Judean rulers: Simon the Hasmonean, John Hyrcanus, Hyrcanus II, and Archelaus, son of Herod. This evidence is usually taken for granted. However, a meticulous analysis of the sources shows that we should not rely on the evidence pertaining to the early Hasmoneans (Simon and John Hyrcanus), and it rather seems that the title was first employed only by the Romans (probably Julius Caesar) for Hyrcanus II. The paper further asserts that this title exemplifies a unique perception of the Jewish people by the Romans. Additionally, the paper notes some ramifications that this understanding of the title Ethnarch and the view which it exemplifies may have on certain issues of the Second Temple period.*

Human ESCs (HESCs) are self-renewing pluripotent cell lines that are derived from the inner cell mass of blastocyst-stage embryos. These cells can produce terminally differentiated cells representing the three embryonic germ layers. We thus hypothesized that during the course of in vitro differentiation of HESCs, progenitor-like cells are transiently formed. We demonstrated that LEFTY proteins, which are known to play a major role during mouse gastrulation, are transiently expressed during HESC differentiation. Moreover, LEFTY proteins seemed to be exclusively expressed by a certain population of cells in the early human embryoid bodies that does not overlap with the population expressing the ESC marker OCT4. We also showed that LEFTY expression is regulated at the cellular transcription level by molecular labeling of LEFTY-positive cells. A DNA microarray analysis of LEFTY-overexpressing cells revealed a signature of cell surface markers such as CADHERIN 2 and 11. Expression of LEFTY controlled by NODAL appears to have a substantial role in mesodermal origin cell population establishment, since inhibition of NODAL activity downregulated expression not only of LEFTY A and LEFTY B but also of BRACHYURY, an early mesodermal marker. In addition, other mesodermal lineage-related genes were downregulated, and this was accompanied by an upregulation in ectoderm-related genes. We propose that during the initial step of HESC differentiation, mesoderm progenitor-like cells appear via activation of the NODAL pathway. Our analysis suggests that in vitro differentiation of HESCs can model early events in human development.

The author draws attention to a somewhat neglected series of events that he believe set the stage for that survival and for some of those post-70 developments.The author refers to the events of 67-37 BCE. He considers the notion that the period of the loss of independence and the inception of Roman rule in Judea is when the seeds of these developments were sown, or was at least a major factor in shaping them. This chapter offers preliminary steps in this direction. First, the case will be made that the early Roman period marked the first fundamental separation between the state and religious authority in Judea. It discusses institutional innovations in this period. Finally, the chapter discusses the possible impact of developments of this period on the centrality of the Jerusalem Temple, along with possible effects that impact may have had on the development of another institution, namely, the synagogue. Keywords:Judea; religious authority; Roman; sovereignty

Human embryonic stem cells differentiate into gastrula organizer cells that express typical markers and induce secondary axes when injected into frog embryos. Here, we report that these human organizer cells express DUXO (DUX of the Organizer), a novel member of the double-homeobox (DUX) family of transcription factors, a group of genes unique to placental mammals. Both of DUXO's homeodomains share high similarity with those of Siamois and Twin, the initial inducers of the amphibian gastrula organizer. DUXO overexpression in human embryoid bodies induces organizer related genes, whereas its knock down hampers formation of the organizer and its derivatives. Finally, we show that DUXO regulates GOOSECOID, the canonical organizer marker, in a direct manner, suggesting that DUXO is a major regulator of human organizer formation.

In the mammalian embryo, the developing pancreas is surrounded by mesoderm compartments, which are important for generating the signals and mechanical constraints that induce the formation of the pancreatic bud and its differentiation. Most protocols of pancreatic organoid generation do not include this mesoderm niche and have partial success in capturing the full repertoire of the pancreas. This work aims to generate a pancreatic organoid by differentiating mouse embryonic stem cells (mESCs) under controlled conditions to mesoderm in parallel to pancreas identity, without the use of poorly defined extracellular matrix such as Matrigel. The first step in our work focused on mESCs differentiation in adherent culture. We were able to obtain a higher differentiation efficacy to definitive endoderm and specifically to pancreas progenitors (PPs) by first differentiating mESCs to epiblast stem cells (EpiSCs), a primed pluripotent state. Next, we built a three-dimension (3D) culture, by aggregating PPs with EpiSC-derived mesoderm progenitors (MPs), recapitulating cellular interactions occurring during embryonic pancreas development. Culture of these MP-PP organoids, referred here as pancreatic organoids, generated the different pancreatic cell types, i.e. endocrine, acinar and ductal cells, as well as blood vessel-like networks. Furthermore, single-cell RNA sequencing analysis of the pancreatic organoids revealed gene expression patterns similar to those of the mouse embryonic pancreas at E12, E14 and E17, primarily in the endocrine differentiation lineage.

This report describes a case of esophageal duplication cyst presenting as a densely calcified lesion in the lower posterior mediastinum.