The early mammalian embryo (blastocyst) contains three co-developing types of stem cells. Two supporting extraembryonic types -the trophectoderm and the primitive endoderm -encase and guide the pluripotent epiblast that eventually forms all body tissues. Unlike embryos, stem cell-based models of the embryo can be generated in large numbers and subjected to high-content screens as a basis for basic and biomedical discoveries (Rivron et al., 2018a;Vrij et al., 2016b). Here, we show that aggregates of naive Embryonic stem cells cultured in chemically-defined conditions and exposed to combinatorial screens of signaling molecules, rapidly (48 hours) and efficiently (80%) set apart PrE-like cells. These cells produce a basal lamina, generate progenitors resembling both visceral and parietal endoderm, and polarize co-developing Epiblast cells to form a proamniotic cavity. In blastoids, stem cell-based models of the early blastocyst (Rivron et al., 2018b), this combination of signals increases the ratio and number of Gata6+/Nanog+ cells and promotes the survival, growth and morphogenesis of a post-implantation-like Epiblast in vitro. Modeling early embryonic development in chemically-defined in vitro conditions shows that the primitive endoderm forms via a specific combination of signaling pathways and sufficient to drive the development of the Epiblast. Hamazaki et al., 2004). However, EBs are formed in chemically-undefined conditions and PrE is not generated efficiently, which occlude studying underlying mechanisms. Recently, we reported the generation of blastoids (Rivron et al., 2018b), embryonic stem cell (ESC) and trophoblast stem cell (TSC)-based structures resembling the E3.5 blastocyst. Building on previous observations (Gardner, 2000), this model proposed a range of inductive signals originating from the embryo and regulating trophectoderm development and implantation. Here, we investigate the role of signaling pathways forming the PrE under chemically-defined conditions, and its influence on post-implantation development of blastoids. Results Naïve pluripotency increase PrE differentiation.We used a high-content screening platform of non-adherent hydrogel microwells in 96 well-plates (Vrij et al., 2016a) to reproducibly aggregate small number of ESCs into EBs (Figure 1A). ESCs seeded into microwells were normally distributed across the 430 microwells within each well (7-12 cells per microwell) and formed EBs within 24 hours (Figure 1B, S1). We quantified PrE differentiation via in situ imaging of fluorescent reporters Pdgfrα (ESCs Pdgfrα-h2b-gfp/+ ) (Artus et al., 2010;Plusa et al., 2008) or Gata6 ESCs Gata6-h2b-venus/+ ). Leukemia inhibitory factor (Lif) appeared essential for the viability of EBs when formed with low cell numbers in serum-free B27N2 medium. However, EBs did not proliferate and formed only few PrE cells (1% and 4% of Pdgfrα+ EBs for 2D expansion in B27N2/2i/Lif and serum/Lif, respectively (Figure 1C). In contrast, serum/Lif induced proliferation and expression of Pdgfrα+ EBs (44%, Figure 1C, ...

Proper defense against microbial infection depends on the controlled activation of the immune system. This is particularly important for the innate immune receptors that recognize viral double-stranded RNA (dsRNA) and initiate antiviral immune responses with the potential of triggering systemic inflammation and immunopathology. How the functions of the dsRNA receptors and their downstream effector molecules are coordinately regulated to avoid excessive immune response is poorly understood. We here demonstrate that stress granules (SGs), biomolecular condensates that form in response to various stresses including viral dsRNA1,2, play key roles in regulating dsRNA-triggered immune response. Upon dsRNA stimulation, SGs recruit many innate immune molecules, including RIG-I-like receptors (RLRs), protein kinase R (PKR) and oligoadenylate synthases (OASes), target these molecules and dsRNA for autophagy and limit their functions through sequestration. In the absence of SGs, dsRNA stimulation results in hyperactivation of inflammatory signaling pathways, global translational arrest and bulk RNA degradation, altogether compromising the cellular capacity to restore homeostasis and triggering cell death. In contrast to most dsRNA-induced immune signaling pathways that are hyperactivated in the absence of SGs, a sub-branch of the RLR pathway (IRF3-dependent type I interferon signaling) shows time-dependent changes, where the initial spike in signaling is followed by a significant drop due to increased caspase-dependent negative feedback regulation. This highlights the role of SGs in regulating the delicate balance between the type I interferon pathway and cell death. Altogether, our data suggest that cells utilize SGs as shock absorbers to moderate antiviral innate immune response, thereby allowing cells to guard against its own immune system as well as viruses.

Objective: To evaluate the technical feasibility of an integrated ultrafast head magnetic resonance (MR) protocol using a sensitivity encoding (SENSE) technique for depicting parenchymal ischaemia and vascular compromise in patients with suspected recent stroke. Methods: 23 patients were evaluated with the ultrafast MR protocol using T2, T1, fluid attenuated inversion recovery (FLAIR), 3D time of flight magnetic resonance angiography (MRA), and diffusion weighted imaging (DWI) sequences. These were compared with routine conventional MR sequences. Results: One patient could not tolerate conventional imaging, although imaging using the three minute head SENSE protocol was diagnostic. Both conventional and ultrafast protocols were of similar diagnostic yield in the remaining patients. There were no significant differences in clinical diagnostic quality for the T1, T2, FLAIR, and DWI sequences. One MRA examination was of better quality when SENSE was used, owing to reduced motion artefacts and shorter imaging time. Conclusions: It is possible to undertake a comprehensive MR examination in stroke patients in approximately three to five minutes. Ultrafast imaging may become a useful triage tool before thrombolytic therapy. It may be of particular benefit in patients unable to tolerate longer sequences. Further work is necessary to confirm these findings in hyperacute stroke.

Essential genes are the central hubs of cellular networks. Despite their importance, the lack of high-throughput methods for titrating their expression has limited our understanding of the fitness landscapes against which essential gene expression levels are optimized. We developed a modified CRISPRi system leveraging the predictable reduction in efficacy of imperfectly matched sgRNAs to generate specific levels of CRISPRi activity and demonstrate its broad applicability in bacteria. Using libraries of mismatched sgRNAs, we characterized the expression-fitness relationships of essential genes in Escherichia coli and Bacillus subtilis. Remarkably, these relationships co-vary by pathway and are predominantly conserved between E. coli and B. subtilis despite ~ 2 billion years of evolutionary separation, suggesting that deeply conserved tradeoffs underlie bacterial homeostasis.One Sentence Summary: Bacterial essential genes have varying responses to CRISPRi knockdown that are largely conserved across ~2 billion years of evolution.

8 These authors contributed equally *email: m.m.maurice@umcutrecht.nl 2 Wnt/β-catenin signalling is a primary pathway for stem cell maintenance during tissue renewal and a frequent target for mutations in cancer. Impaired Wnt receptor endocytosis due to loss of the ubiquitin ligase RNF43 gives rise to Wnt-hypersensitive tumours that are susceptible to anti-Wnt-based therapy. Contrary to this paradigm, we identify a class of RNF43 truncating cancer mutations that strongly induce β-cateninmediated transcription, despite exhibiting retained Wnt receptor downregulating activity. Mechanistically, these RNF43 mutants trap Casein Kinase (CK)1 at the plasma membrane, which prevents β-catenin turnover and propels ligand-independent Wnt target gene transcription. When introduced in human colon stem cells, these oncogenic RNF43 mutants cooperate with p53 loss to drive a niche-independent program for selfrenewal and proliferation. Importantly, onco-RNF43 mutations, unlike conventional LOF RNF43 mutations, confer resistance to anti-Wnt-based therapy. Our data demonstrate the relevance of studying patient-derived mutations for understanding disease mechanisms and improved applications of precision medicine.Aberrant activation of Wnt/-catenin signalling is a key oncogenic event that confers an undifferentiated state and allows cancer cells to thrive outside their native niche constraint 1-3 .In adult stem cells, Wnt signalling is curbed by the negative feedback regulators RNF43 and ZNRF3, two homologous transmembrane ubiquitin ligases that induce removal of the Wnt receptors FZD and LRP6 from the cell surface via ubiquitin-mediated endocytosis and lysosomal degradation 4,5 . Within the stem cell niche, the activity of RNF43/ZNRF3 is counterbalanced by secreted proteins of the R-spondin (Rspo) family that form a complex with Leucine-rich repeat-containing G-protein coupled receptor 4/5 (Lgr4/5) to mediate Netherlands) for TP53 gRNA.