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

Culture of Mouse Embryonic Stem Cells with Serum but without Exogenous Growth Factors Is Sufficient to Generate Functional Hepatocyte-Like Cells

Mouse embryonic stem cells (mESC) have been used to study lineage specification in vitro, including towards a hepatocyte-like fate, and such investigations guided lineage differentiation protocols for human (h)ESC. We recently described a four-step protocol to induce hepatocyte-like cells from hESC which also induced hepatocyte-like cell differentiation of mouse induced pluripotent stem cells. As ESC also spontaneously generate hepatocyte-like cells, we here tested whether the growth factors and serum used in this protocol are required to commit mESC and hESC to hepatocyte-like cells. Culture of mESC from two different mouse strains in the absence of serum and growth factors did not induce primitive streak/definitive endoderm genes but induced default differentiation to neuroectoderm on day 6. Although Activin-A and Wnt3 induced primitive streak/definitive endoderm transcripts most robustly in mESC, simple addition of serum also induced these transcripts. Expression of hepatoblast genes occurred earlier when growth factors were used for mESC differentiation. However, further maturation towards functional hepatocyte-like cells was similar in mESC progeny from cultures with serum, irrespective of the addition of growth factors, and irrespective of the mouse strain. This is in contrast to hESC, where growth factors are required for specification towards functional hepatocyte-like cells. Culture of mESC with serum but without growth factors did not induce preferential differentiation towards primitive endoderm or neuroectoderm. Thus, although induction of primitive streak/definitive endoderm specific genes and proteins is more robust when mESC are exposed to a combination of serum and exogenous growth factors, ultimate generation of hepatocyte-like cells from mESC occurs equally well in the presence or absence of exogenous growth factors. The latter is in contrast to what we observed for hESC. These results suggest that differences exist between lineage specific differentiation potential of mESC and hESC, requiring optimization of different protocols for ESC from either species

A Dedicated Promoter Drives Constitutive Expression of the Cell-Autonomous Immune Resistance GTPase, Irga6 (IIGP1) in Mouse Liver

Background: In general, immune effector molecules are induced by infection. Methodology and Principal Findings: However, strong constitutive expression of the cell-autonomous resistance GTPase, Irga6 (IIGP1), was found in mouse liver, contrasting with previous evidence that expression of this protein is exclusively dependent on induction by IFNc. Constitutive and IFNc-inducible expression of Irga6 in the liver were shown to be dependent on transcription initiated from two independent untranslated 59 exons, which splice alternatively into the long exon encoding the full-length protein sequence. Irga6 is expressed constitutively in freshly isolated hepatocytes and is competent in these cells to accumulate on the parasitophorous vacuole membrane of infecting Toxoplasma gondii tachyzoites. Conclusions and Significance: The role of constitutive hepatocyte expression of Irga6 in resistance to parasites invading from the gut via the hepatic portal system is discussed

Human Embryonic and Rat Adult Stem Cells with Primitive Endoderm-Like Phenotype Can Be Fated to Definitive Endoderm, and Finally Hepatocyte-Like Cells

Stem cell-derived hepatocytes may be an alternative cell source to treat liver diseases or to be used for pharmacological purposes. We developed a protocol that mimics mammalian liver development, to differentiate cells with pluripotent characteristics to hepatocyte-like cells. The protocol supports the stepwise differentiation of human embryonic stem cells (ESC) to cells with characteristics of primitive streak (PS)/mesendoderm (ME)/definitive endoderm (DE), hepatoblasts, and finally cells with phenotypic and functional characteristics of hepatocytes. Remarkably, the same protocol can also differentiate rat multipotent adult progenitor cells (rMAPCs) to hepatocyte-like cells, even though rMAPC are isolated clonally from cultured rat bone marrow (BM) and have characteristics of primitive endoderm cells. A fraction of rMAPCs can be fated to cells expressing genes consistent with a PS/ME/DE phenotype, preceding the acquisition of phenotypic and functional characteristics of hepatocytes. Although the hepatocyte-like progeny derived from both cell types is mixed, between 10–20% of cells are developmentally consistent with late fetal hepatocytes that have attained synthetic, storage and detoxifying functions near those of adult hepatocytes. This differentiation protocol will be useful for generating hepatocyte-like cells from rodent and human stem cells, and to gain insight into the early stages of liver development

Structure and evolution of the gorilla and orangutan growth hormone loci

In primates, the unigenic growth hormone (GH) locus of prosimians, expressed primarily in the anterior pituitary, evolved by gene duplications, independently in New World Monkeys (NWM) and Old World Monkeys (OWMs)/apes, to give complex clusters of genes expressed in the pituitary and placenta. In human and chimpanzee, the GH locus comprises five genes, GH-N being expressed as pituitary GH, whereas GH-V (placental GH) and CSHs (chorionic somatomammotropins) are expressed (in human and probably chimpanzee) in the placenta; the CSHs comprise CSH-A, CSH-B and the aberrant CSH-L (possibly a pseudogene) in human, and CSH-A1, CSH-A2 and CSH-B in chimpanzee. Here the GH locus in two additional great apes, gorilla (Gorilla gorilla gorilla) and orangutan (Pongo abelii), is shown to contain six and four GH-like genes respectively. The gorilla locus possesses six potentially expressed genes, gGH-N, gGH-V and four gCSHs, whereas the orangutan locus has just three functional genes, oGH-N, oGH-V and oCSH-B, plus a pseudogene, oCSH-L. Analysis of regulatory sequences, including promoter, enhancer and P-elements, shows significant variation; in particular the proximal Pit-1 element of GH-V genes differs markedly from that of other genes in the cluster. Phylogenetic analysis shows that the initial gene duplication led to distinct GH-like and CSH-like genes, and that a second duplication provided separate GH-N and GH-V. However, evolution of the CSH-like genes remains unclear. Rapid adaptive evolution gave rise to the distinct CSHs, after the first duplication, and to GH-V after the second duplication. Analysis of transcriptomic databases derived from gorilla tissues establishes that the gGH-N, gGH-V and several gCSH genes are expressed, but the significance of the many CSH genes in gorilla remains unclear

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

TFEB regulates murine liver cell fate during development and regeneration

It is well established that pluripotent stem cells in fetal and postnatal liver (LPCs) can differentiate into both hepatocytes and cholangiocytes. However, the signaling pathways implicated in the differentiation of LPCs are still incompletely understood. Transcription Factor EB (TFEB), a master regulator of lysosomal biogenesis and autophagy, is known to be involved in osteoblast and myeloid differentiation, but its role in lineage commitment in the liver has not been investigated. Here we show that during development and upon regeneration TFEB drives the differentiation status of murine LPCs into the progenitor/cholangiocyte lineage while inhibiting hepatocyte differentiation. Genetic interaction studies show that Sox9, a marker of precursor and biliary cells, is a direct transcriptional target of TFEB and a primary mediator of its effects on liver cell fate. In summary, our findings identify an unexplored pathway that controls liver cell lineage commitment and whose dysregulation may play a role in biliary cancer

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

New Approaches in the Differentiation of Human Embryonic Stem Cells and Induced Pluripotent Stem Cells toward Hepatocytes

Orthotropic liver transplantation is the only established treatment for end-stage liver diseases. Utilization of hepatocyte transplantation and bio-artificial liver devices as alternative therapeutic approaches requires an unlimited source of hepatocytes. Stem cells, especially embryonic stem cells, possessing the ability to produce functional hepatocytes for clinical applications and drug development, may provide the answer to this problem. New discoveries in the mechanisms of liver development and the emergence of induced pluripotent stem cells in 2006 have provided novel insights into hepatocyte differentiation and the use of stem cells for therapeutic applications. This review is aimed towards providing scientists and physicians with the latest advancements in this rapidly progressing field