Conventional two-dimensional differentiation from pluripotency fails to recapitulate cell interactions occurring during organogenesis. Three-dimensional organoids generate complex organ-like tissues; however, it is unclear how heterotypic interactions affect lineage identity. Here we use single-cell RNA sequencing to reconstruct hepatocyte-like lineage progression from pluripotency in two-dimensional culture. We then derive three-dimensional liver bud organoids by reconstituting hepatic, stromal, and endothelial interactions, and deconstruct heterogeneity during liver bud development. We find that liver bud hepatoblasts diverge from the two-dimensional lineage, and express epithelial migration signatures characteristic of organ budding. We benchmark three-dimensional liver buds against fetal and adult human liver single-cell RNA sequencing data, and find a striking correspondence between the three-dimensional liver bud and fetal liver cells. We use a receptor-ligand pairing analysis and a high-throughput inhibitor assay to interrogate signalling in liver buds, and show that vascular endothelial growth factor (VEGF) crosstalk potentiates endothelial network formation and hepatoblast differentiation. Our molecular dissection reveals interlineage communication regulating organoid development, and illuminates previously inaccessible aspects of human liver development.
Pharmacological modulators of host-microbial interactions can in principle be identified using highcontent screens. However, a severe limitation of this approach is the lack of insights into the mode of action of compounds selected during the primary screen. To overcome this problem, we developed a combined experimental and computational approach. We designed a quantitative multiparametric image-based assay to measure intracellular mycobacteria in primary human macrophages, screened a chemical library containing FDA-approved drugs, and validated three compounds for intracellular killing of M. tuberculosis. By integrating the multiparametric profiles of the chemicals with those of siRNAs from a genome-wide survey on endocytosis, we predicted and experimentally verified that two compounds modulate autophagy, whereas the third accelerates endosomal progression. Our findings demonstrate the value of integrating small molecules and genetic screens for identifying cellular mechanisms modulated by chemicals. Furthermore, selective pharmacological modulation of host trafficking pathways can be applied to intracellular pathogens beyond mycobacteria.
Cell death via apoptosis induced by tumor necrosis factor-␣ (TNF-␣) plays an important role in many physiological and pathological conditions. The signal transduction pathway activated by this cytokine is known to be regulated by several intracellular messengers. In particular, in many systems nitric oxide (NO) has been shown to protect cells from TNF-␣-induced apoptosis. However, whether NO can be generated by the cytokine to down-regulate its own apoptotic program has never been studied. We have addressed this question in HeLa Tet-off cell clones stably transfected with the endothelial NO synthase under a tetracycline-responsive promoter. Endothelial NO synthase, induced about 100-fold in these cells by removal of the antibiotic, retained the characteristics of the native enzyme of endothelial cells, both in terms of intracellular localization and functional activity. Expression of the endothelial NO synthase was sufficient to protect from TNF-␣-induced apoptosis. This protection was mediated by the generation of NO. TNF-␣ itself stimulated endothelial NO synthase activity to generate NO through a pathway involving its lipid messenger, ceramide. Our results identify a novel mechanism of regulation of a signal transduction pathway activated by death receptors and suggest that NO may constitute a built-in mechanism by which TNF-␣ controls its own apoptotic program.
Identifying key molecules that launch regeneration has been a long sought goal. Multiple regenerative animals show an initial wound-associated proliferative response that transits into sustained proliferation if a significant portion of the body part has been removed 1-3. In the axolotl, appendage amputation initiates a round of wound-associated cell cycle induction followed by continued proliferation that is dependent on nerve-derived signals 4,5. A wound-associated molecule that triggers the initial proliferative response to launch regeneration has remained obscure. Using an expression cloning strategy followed by in vivo gain- and loss-of-function assays, we identified axolotl MARCKS like Protein (MLP) as an extracellularly released factor that induces the initial cell cycle response during axolotl appendage regeneration.
Apoptosis triggered by death receptors proceeds after defined signal-transduction pathways. Whether signaling at the receptor level is regulated by intracellular messengers is still unknown. We have investigated the role of two messengers, ceramide and nitric oxide (NO), on the apoptotic pathway activated in human monocytic U937 cells by tumor necrosis factor-␣ (TNF-␣) working at its p55 receptor. Two transduction events, the receptor recruitment of the adapter protein, TRADD, and the activation of the initiator caspase, caspase 8, were investigated. When administered alone, neither of the messengers had any effect on these events. In combination with TNF-␣, however, ceramide potentiated, whereas NO inhibited, TNF-␣-induced TRADD recruitment and caspase 8 activity. The effect of NO, which was cGMP-dependent, was due to inhibition of the TNF-␣-induced generation of ceramide. Our results identify a mechanism of regulation of a signal-transduction pathway activated by death receptors.
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