Onecut-dependent Nkx6.2 transcription factor expression is required for proper formation and activity of spinal locomotor circuits.

In the developing spinal cord, Onecut transcription factors control the diversification of motor neurons into distinct neuronal subsets by ensuring the maintenance of Isl1 expression during differentiation. However, other genes downstream of the Onecut proteins and involved in motor neuron diversification have remained unidentified. In the present study, we generated conditional mutant embryos carrying specific inactivation of Onecut genes in the developing motor neurons, performed RNA-sequencing to identify factors downstream of Onecut proteins in this neuron population, and employed additional transgenic mouse models to assess the role of one specific Onecut-downstream target, the transcription factor Nkx6.2. Nkx6.2 expression was up-regulated in Onecut-deficient motor neurons, but strongly downregulated in Onecut-deficient V2a interneurons, indicating an opposite regulation of Nkx6.2 by Onecut factors in distinct spinal neuron populations. Nkx6.2-null embryos, neonates and adult mice exhibited alterations of locomotor pattern and spinal locomotor network activity, likely resulting from defective survival of a subset of limb-innervating motor neurons and abnormal migration of V2a interneurons. Taken together, our results indicate that Nkx6.2 regulates the development of spinal neuronal populations and the formation of the spinal locomotor circuits downstream of the Onecut transcription factors

Notch Signaling Regulates Bile Duct Morphogenesis in Mice

BACKGROUND: Alagille syndrome is a developmental disorder caused predominantly by mutations in the Jagged1 (JAG1) gene, which encodes a ligand for Notch family receptors. A characteristic feature of Alagille syndrome is intrahepatic bile duct paucity. We described previously that mice doubly heterozygous for Jag1 and Notch2 mutations are an excellent model for Alagille syndrome. However, our previous study did not establish whether bile duct paucity in Jag1/Notch2 double heterozygous mice resulted from impaired differentiation of bile duct precursor cells, or from defects in bile duct morphogenesis. METHODOLOGY/PRINCIPAL FINDINGS: Here we characterize embryonic biliary tract formation in our previously described Jag1/Notch2 double heterozygous Alagille syndrome model, and describe another mouse model of bile duct paucity resulting from liver-specific deletion of the Notch2 gene. CONCLUSIONS/SIGNIFICANCE: Our data support a model in which bile duct paucity in Notch pathway loss of function mutant mice results from defects in bile duct morphogenesis rather than cell fate specification

HNF1α inhibition triggers epithelial-mesenchymal transition in human liver cancer cell lines

<p>Abstract</p> <p>Background</p> <p>Hepatocyte Nuclear Factor 1α (HNF1α) is an atypical homeodomain-containing transcription factor that transactivates liver-specific genes including albumin, α-1-antitrypsin and α- and β-fibrinogen. Biallelic inactivating mutations of <it>HNF1A </it>have been frequently identified in hepatocellular adenomas (HCA), rare benign liver tumors usually developed in women under oral contraceptives, and in rare cases of hepatocellular carcinomas developed in non-cirrhotic liver. HNF1α-mutated HCA (H-HCA) are characterized by a marked steatosis and show activation of glycolysis, lipogenesis, translational machinery and mTOR pathway. We studied the consequences of HNF1α silencing in hepatic cell lines, HepG2 and Hep3B and we reproduced most of the deregulations identified in H-HCA.</p> <p>Methods</p> <p>We transfected hepatoma cell lines HepG2 and Hep3B with siRNA targeting HNF1α and obtained a strong inhibition of HNF1α expression. We then looked at the phenotypic changes by microscopy and studied changes in gene expression using qRT-PCR and Western Blot.</p> <p>Results</p> <p>Hepatocytes transfected with HNF1α siRNA underwent severe phenotypic changes with loss of cell-cell contacts and development of migration structures. In HNF1α-inhibited cells, hepatocyte and epithelial markers were diminished and mesenchymal markers were over-expressed. This epithelial-mesenchymal transition (EMT) was related to the up regulation of several EMT transcription factors, in particular <it>SNAIL </it>and <it>SLUG</it>. We also found an overexpression of TGFβ1, an EMT initiator, in both cells transfected with HNF1α siRNA and H-HCA. Moreover, TGFβ1 expression is strongly correlated to HNF1α expression in cell models, suggesting regulation of TGFβ1 expression by HNF1α.</p> <p>Conclusion</p> <p>Our results suggest that HNF1α is not only important for hepatocyte differentiation, but has also a role in the maintenance of epithelial phenotype in hepatocytes.</p

Cholangiocytes derived from human induced pluripotent stem cells for disease modeling and drug validation.

The study of biliary disease has been constrained by a lack of primary human cholangiocytes. Here we present an efficient, serum-free protocol for directed differentiation of human induced pluripotent stem cells into cholangiocyte-like cells (CLCs). CLCs show functional characteristics of cholangiocytes, including bile acids transfer, alkaline phosphatase activity, γ-glutamyl-transpeptidase activity and physiological responses to secretin, somatostatin and vascular endothelial growth factor. We use CLCs to model in vitro key features of Alagille syndrome, polycystic liver disease and cystic fibrosis (CF)-associated cholangiopathy. Furthermore, we use CLCs generated from healthy individuals and patients with polycystic liver disease to reproduce the effects of the drugs verapamil and octreotide, and we show that the experimental CF drug VX809 rescues the disease phenotype of CF cholangiopathy in vitro. Our differentiation protocol will facilitate the study of biological mechanisms controlling biliary development, as well as disease modeling and drug screening.This work was funded by ERC starting grant Relieve IMDs (L.V., N.H.), the Cambridge Hospitals National Institute for Health Research Biomedical Research Center (L.V., N.H., F.S.), the Evelyn trust (N.H.) and the EU Fp7 grant TissuGEN (M.CDB.). FS has been supported by an Addenbrooke’s Charitable Trust Clinical Research Training Fellowship and a joint MRC-Sparks Clinical Research Training Fellowship.This is the author accepted manuscript. The final version is available from Nature Publishing Group via http://dx.doi.org/10.1038/nbt.327

Macrophage-derived Wnt opposes Notch signaling to specify hepatic progenitor cell fate in chronic liver disease

During chronic injury a population of bipotent hepatic progenitor cells (HPCs) become activated to regenerate both cholangiocytes and hepatocytes. Here we show in human diseased liver and mouse models of the ductular reaction that Notch and Wnt signaling direct specification of HPCs via their interactions with activated myofibroblasts or macrophages. In particular, we found that during biliary regeneration, expression of Jagged 1 (a Notch ligand) by myofibroblasts promoted Notch signaling in HPCs and thus their biliary specification to cholangiocytes. Alternatively, during hepatocyte regeneration, macrophage engulfment of hepatocyte debris induced Wnt3a expression. This resulted in canonical Wnt signaling in nearby HPCs, thus maintaining expression of Numb (a cell fate determinant) within these cells and the promotion of their specification to hepatocytes. By these two pathways adult parenchymal regeneration during chronic liver injury is promoted

sox9b Is a Key Regulator of Pancreaticobiliary Ductal System Development

The pancreaticobiliary ductal system connects the liver and pancreas to the intestine. It is composed of the hepatopancreatic ductal (HPD) system as well as the intrahepatic biliary ducts and the intrapancreatic ducts. Despite its physiological importance, the development of the pancreaticobiliary ductal system remains poorly understood. The SRY-related transcription factor SOX9 is expressed in the mammalian pancreaticobiliary ductal system, but the perinatal lethality of Sox9 heterozygous mice makes loss-of-function analyses challenging. We turned to the zebrafish to assess the role of SOX9 in pancreaticobiliary ductal system development. We first show that zebrafish sox9b recapitulates the expression pattern of mouse Sox9 in the pancreaticobiliary ductal system and use a nonsense allele of sox9b, sox9bfh313, to dissect its function in the morphogenesis of this structure. Strikingly, sox9bfh313 homozygous mutants survive to adulthood and exhibit cholestasis associated with hepatic and pancreatic duct proliferation, cyst formation, and fibrosis. Analysis of sox9bfh313 mutant embryos and larvae reveals that the HPD cells appear to mis-differentiate towards hepatic and/or pancreatic fates, resulting in a dysmorphic structure. The intrahepatic biliary cells are specified but fail to assemble into a functional network. Similarly, intrapancreatic duct formation is severely impaired in sox9bfh313 mutants, while the embryonic endocrine and acinar compartments appear unaffected. The defects in the intrahepatic and intrapancreatic ducts of sox9bfh313 mutants worsen during larval and juvenile stages, prompting the adult phenotype. We further show that Sox9b interacts with Notch signaling to regulate intrahepatic biliary network formation: sox9b expression is positively regulated by Notch signaling, while Sox9b function is required to maintain Notch signaling in the intrahepatic biliary cells. Together, these data reveal key roles for SOX9 in the morphogenesis of the pancreaticobiliary ductal system, and they cast human Sox9 as a candidate gene for pancreaticobiliary duct malformation-related pathologies

Comparison of Hepatic-like Cell Production from Human Embryonic Stem Cells and Adult Liver Progenitor Cells: CAR Transduction Activates a Battery of Detoxification Genes

In vitro production of human hepatocytes is of primary importance in basic research, pharmacotoxicology and biotherapy of liver diseases. We have developed a protocol of differentiation of human embryonic stem cells (ES) towards hepatocyte-like cells (ES-Hep). Using a set of human adult markers including CAAT/enhancer binding protein (C/EBPalpha), hepatocyte nuclear factor 4/7 ratio (HNF4alpha1/HNF4alpha7), cytochrome P450 7A1 (CYP7A1), CYP3A4 and constitutive androstane receptor (CAR), and fetal markers including alpha-fetoprotein, CYP3A7 and glutathione S-transferase P1, we analyzed the expression of a panel of 41 genes in ES-Hep comparatively with human adult primary hepatocytes, adult and fetal liver. The data revealed that after 21 days of differentiation, ES-Hep are representative of fetal hepatocytes at less than 20 weeks of gestation. The glucocorticoid receptor pathway was functional in ES-Hep. Extending protocols of differentiation to 4 weeks did not improve cell maturation. When compared with hepatocyte-like cells derived from adult liver non parenchymal epithelial (NPE) cells (NPE-Hep), ES-Hep expressed several adult and fetal liver makers at much greater levels (at least one order of magnitude), consistent with greater expression of liver-enriched transcription factors Forkhead box A2, C/EBPalpha, HNF4alpha and HNF6. It therefore seems that ES-Hep reach a better level of differentiation than NPE-Hep and that these cells use different lineage pathways towards the hepatic phenotype. Finally we showed that lentivirus-mediated expression of xenoreceptor CAR in ES-Hep induced the expression of several detoxification genes including CYP2B6, CYP2C9, CYP3A4, UDP-glycosyltransferase 1A1, solute carriers 21A6, as well as biotransformation of midazolam, a CYP3A4-specific substrate

In Vitro Differentiation of Embryonic and Adult Stem Cells into Hepatocytes: State of the Art

Stem cells are a unique source of self-renewing cells within the human body. Before the end of the last millennium, adult stem cells, in contrast to their embryonic counterparts, were considered to be lineage-restricted cells or incapable of crossing lineage boundaries. However, the unique breakthrough of muscle and liver regeneration by adult bone marrow stem cells at the end of the 1990s ended this long-standing paradigm. Since then, the number of articles reporting the existence of multipotent stem cells in skin, neuronal tissue, adipose tissue, and bone marrow has escalated, giving rise, both in vivo and in vitro, to cell types other than their tissue of origin. The phenomenon of fate reprogrammation and phenotypic diversification remains, though, an enigmatic and rare process. Understanding how to control both proliferation and differentiation of stem cells and their progeny is a challenge in many fields, going from preclinical drug discovery and development to clinical therapy. In this review, we focus on current strategies to differentiate embryonic, mesenchymal(-like), and liver stem/progenitor cells into hepatocytes in vitro. Special attention is paid to intracellular and extracellular signaling, genetic modification, and cell-cell and cell-matrix interactions. In addition, some recommendations are proposed to standardize, optimize, and enrich the in vitro production of hepatocyte-like cells out of stem/progenitor cells

A morphogenetic EphB/EphrinB code controls hepatopancreatic duct formation

© 2019 The Authors. Published by Springer. This is an open access article available under a Creative Commons licence. The published version can be accessed at the following link on the publisher’s website: https://doi.org/10.1038/s41467-019-13149-7The hepatopancreatic ductal (HPD) system connects the intrahepatic and intrapancreatic ducts to the intestine and ensures the afferent transport of the bile and pancreatic enzymes. Yet the molecular and cellular mechanisms controlling their differentiation and morphogenesis into a functional ductal system are poorly understood. Here, we characterize HPD system morphogenesis by high-resolution microscopy in zebrafish. The HPD system differentiates from a rod of unpolarized cells into mature ducts by de novo lumen formation in a dynamic multi-step process. The remodeling step from multiple nascent lumina into a single lumen requires active cell intercalation and myosin contractility. We identify key functions for EphB/EphrinB signaling in this dynamic remodeling step. Two EphrinB ligands, EphrinB1 and EphrinB2a, and two EphB receptors, EphB3b and EphB4a, control HPD morphogenesis by remodeling individual ductal compartments, and thereby coordinate the morphogenesis of this multi-compartment ductal system.This work was funded by the Novo Nordisk Foundation (NNF17CC0027852) and Danish National Research Foundation (DNRF116). J.C. and D.G.W. were supported by the Francis Crick Institute, which receives its core funding from Cancer Research UK (FC001217), the UK Medical Research Council (FC001217), and the Wellcome Trust (FC001217). S.C. was supported by an SNSF Early Postdoc Mobility fellowship (P2ZHP3_164840) and a Long Term EMBO Postdoc fellowship (ALTF 511-2016), and L.S. and J.B.A. by the Independent Research Fund Denmark (DFF; Sapere Aude2 4183-00118B).Published versio