Recent developments in stem cell biology have enabled the study of cell fate decisions in early human development that are impossible to study in vivo. However, understanding how development varies across individuals and, in particular, the influence of common genetic variants during this process has not been characterised. Here, we exploit human iPS cell lines from 125 donors, a pooled experimental design, and single-cell RNA-sequencing to study population variation of endoderm differentiation. We identify molecular markers that are predictive of differentiation efficiency of individual lines, and utilise heterogeneity in the genetic background across individuals to map hundreds of expression quantitative trait loci that influence expression dynamically during differentiation and across cellular contexts.There are amendments to this paper

SUMMARYThe genomic regulatory programs that underlie human organogenesis are poorly understood. Pancreas development, in particular, has pivotal implications for pancreatic regeneration, cancer, and diabetes. We have now characterized the regulatory landscape of embryonic multipotent progenitor cells that give rise to all pancreatic epithelial lineages. Using human embryonic pancreas and embryonic stem cell-derived progenitors we identify stage-specific transcripts and associated enhancers, many of which are co-occupied by transcription factors that are essential for pancreas development. We further show that TEAD1, a Hippo signaling effector, is an integral component of the transcription factor combinatorial code of pancreatic progenitor enhancers. TEAD and its coactivator YAP activate key pancreatic signaling mediators and transcription factors, and regulate the expansion of pancreatic progenitors. This work therefore uncovers a central role of TEAD and YAP as signal-responsive regulators of multipotent pancreatic progenitors, and provides a resource for the study of embryonic development of the human pancreas.

28Recent developments in stem cell biology have enabled the study of cell fate decisions in early 29 human development that are impossible to study in vivo. However, understanding how 30 development varies across individuals and, in particular, the influence of common genetic 31 variants during this process has not been characterised. Here, we exploit human iPS cell lines 32 from 125 donors, a pooled experimental design, and single-cell RNA-sequencing to study 33 population variation of endoderm differentiation. We identify molecular markers that are 34 predictive of differentiation efficiency, and utilise heterogeneity in the genetic background 35 across individuals to map hundreds of expression quantitative trait loci that influence 36 expression dynamically during differentiation and across cellular contexts. 37Population-scale single-cell profiling of differentiating iPS cells 63 We considered a panel of well-characterized human iPSC lines derived from 125 unrelated 64 donors from the Human Induced Pluripotent Stem Cell initiative (HipSci) collection [1]. In order 65to increase throughput and mitigate the effects of batch variation, we exploited a novel pooled 66 differentiation assay, combining sets of four to six lines in one well prior to differentiation (28 67 differentiation experiments performed in total; hereon "experiments"; Fig. 1A, S1, S2). Cells 68 were collected at four differentiation time points (iPSC; one, two and three days post initiation 69 -hereon day0, day1, day2 and day3) and their transcriptomes were assayed using full-length 70RNA-sequencing (Smart-Seq2 [7]) alongside the expression of selected cell surface markers 71using FACS (TRA-1-60, CXCR4; Fig. S3, S4; Methods). Following quality control (QC), 72 36,044 cells were retained for downstream analysis, across which 11,231 genes were 73 expressed ( Fig. S5; Methods). Exploiting that each cell line's genotype acts as a unique 74 barcode, we demultiplexed the pooled cell populations, enabling identification of the cell line 75 of origin for each cell (similar to [8]; Methods). At each time point, cells from between 104 and 76 112 donors were captured, with each donor being represented by an average of 286 cells 77 (after QC, Fig. S2; Tables S1, S2; Methods). The success of the differentiation protocol was 78 validated using canonical cell-surface marker expression: consistent with previous studies [9], 79 an average of 72% cells were TRA-1-60(+) in the undifferentiated state (day0) and an average 80 of 49% of cells were CXCR4(+) three days post differentiation (day3; Fig. S3). 81 82Variance component analysis across all genes (using a linear mixed model; Methods) 83 revealed the time point of collection as the main source of variation, followed by the cell line 84 of origin and the experimental batch (Fig. 1B). Consistent with this, the first Principal 85 Component (PC) was strongly associated with differentiation time (Fig. 1C, S6; Methods), 86 motivating its use to order cells by their differentiation status (hereafter "pseudotime" ...

ADP is the cognate agonist of the P2Y 1 , P2Y 12 , and P2Y 13 receptors. With the goal of identifying a high potency agonist that selectively activates the P2Y 1 receptor, we examined the pharmacological selectivity of the conformationally constrained non-the three ADP-activated receptors. Each P2Y receptor was expressed transiently in COS-7 cells, and inositol lipid hydrolysis was quantified as a measure of receptor activity. In the case of the G i -linked P2Y 12 and P2Y 13 receptors, a chimeric G protein, G␣ q/i , was coexpressed to confer a capacity of these G i -linked receptors to activate phospholipase C. 2MeSADP (2-methylthio-ADP) was a potent agonist at all three receptors exhibiting EC 50 values in the sub to low nanomolar range. In contrast, whereas (N)-methanocarba-2MeSADP was an extremely potent (EC 50 ϭ 1.2 Ϯ 0.2 nM) agonist at the P2Y 1 receptor, this non-nucleotide analog exhibited no agonist activity at the P2Y 12 receptor and very low activity at the P2Y 13 receptor. (N)-Methanocarba-2MeSADP also failed to block the action of 2MeSADP at the P2Y 12 and P2Y 13 receptors, indicating that the (N)-methanocarba analog is not an antagonist at these receptors. The P2Y 1 receptor selectivity of (N)-methanocarba-2MeSADP was confirmed in human platelets where it induced the shape change promoted by P2Y 1 receptor activation without inducing the sustained platelet aggregation that requires simultaneous activation of the P2Y 12 receptor. These results provide the first demonstration of a high-affinity agonist that discriminates among the three ADP-activated P2Y receptors, and therefore, introduce a potentially important new pharmacological tool for delineation of the relative biological action of these three signaling proteins.The G protein-coupled P2Y receptor family is comprised of at least eight different human receptors that are activated by nucleoside diphosphates, nucleoside triphosphates, or nucleotide sugars to regulate a broad range of physiological responses including neurotransmission, muscle contraction, ion secretion, and platelet aggregation (Dubyak and ElMoatassim, 1993;Harden et al., 1998;Ralevic and Burnstock, 1998). A complex set of ectoenzymes that metabolize extracellular nucleotides has complicated the study of these receptors Zimmermann, 2000). This difficulty is exacerbated by the lack of selective agonists or antagonists for most of the P2Y receptors.

SummaryRho-family GTPases are activated by the exchange of bound GDP for GTP, a process that is catalyzed by Dbl-family guanine nucleotide exchange factors (GEFs). The catalytic unit of Dbl-family GEFs consists of a Dbl-homology (DH) domain followed almost invariantly by a pleckstrin-homology (PH) domain. The majority of the catalytic interface forms between the switch regions of the GTPase and the DH domain, but full catalytic activity often requires the associated PH domain. Although PH domains are usually characterized as lipid binding regions, they also participate in protein-protein interactions. For example, the DH-associated PH domain of Dbs must contact its cognate GTPases for efficient exchange. Similarly, the N-terminal DH/PH fragment of Trio, which catalyzes exchange on both Rac1 and RhoG, is four-fold more active in vitro than the isolated DH domain. Given continued uncertainty regarding functional roles of DH-associated PH domains, we have undertaken structural and functional analyses of the N-terminal DH/PH cassette of Trio. The crystal structure of this fragment of Trio bound to nucleotide-depleted Rac1 highlights the engagement of the PH domain with Rac1 and substitution of residues involved in this interface substantially diminishes activation of Rac1 and RhoG. Also, these mutations significantly reduce the ability of full-length Trio to induce neurite outgrowth dependent on RhoG activation in PC-12 cells. Overall, these studies substantiate a general role for DH-associated PH domains in directly engaging Rho GTPases for efficient guanine nucleotide exchange and support a parsimonious explanation for the essentially invariant linkage between DH and PH domains.