Publications by authors named "Jan S Tchorz"

21 Publications

  • Page 1 of 1

ZNRF3 and RNF43 cooperate to safeguard metabolic liver zonation and hepatocyte proliferation.

Cell Stem Cell 2021 Jun 11. Epub 2021 Jun 11.

Novartis Institutes for BioMedical Research, Novartis Pharma AG, Basel, Switzerland. Electronic address:

AXIN2 and LGR5 mark intestinal stem cells (ISCs) that require WNT/β-Catenin signaling for constant homeostatic proliferation. In contrast, AXIN2/LGR5+ pericentral hepatocytes show low proliferation rates despite a WNT/β-Catenin activity gradient required for metabolic liver zonation. The mechanisms restricting proliferation in AXIN2+ hepatocytes and metabolic gene expression in AXIN2+ ISCs remained elusive. We now show that restricted chromatin accessibility in ISCs prevents the expression of β-Catenin-regulated metabolic enzymes, whereas fine-tuning of WNT/β-Catenin activity by ZNRF3 and RNF43 restricts proliferation in chromatin-permissive AXIN2+ hepatocytes, while preserving metabolic function. ZNRF3 deletion promotes hepatocyte proliferation, which in turn becomes limited by RNF43 upregulation. Concomitant deletion of RNF43 in ZNRF3 mutant mice results in metabolic reprogramming of periportal hepatocytes and induces clonal expansion in a subset of hepatocytes, ultimately promoting liver tumors. Together, ZNRF3 and RNF43 cooperate to safeguard liver homeostasis by spatially and temporally restricting WNT/β-Catenin activity, balancing metabolic function and hepatocyte proliferation.
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http://dx.doi.org/10.1016/j.stem.2021.05.013DOI Listing
June 2021

Proliferation tracing reveals regional hepatocyte generation in liver homeostasis and repair.

Science 2021 02;371(6532)

State Key Laboratory of Cell Biology, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai, China.

Organ homeostasis is orchestrated by time- and spatially restricted cell proliferation. Studies identifying cells with superior proliferative capacities often rely on the lineage tracing of a subset of cell populations, which introduces a potential selective bias. In this work, we developed a genetic system [proliferation tracer (ProTracer)] by incorporating dual recombinases to seamlessly record the proliferation events of entire cell populations over time in multiple organs. In the mouse liver, ProTracer revealed more hepatocyte proliferation in distinct zones during liver homeostasis, injury repair, and regrowth. Clonal analysis showed that most of the hepatocytes labeled by ProTracer had undergone cell division. By genetically recording proliferation events of entire cell populations, ProTracer enables the unbiased detection of specific cellular compartments with enhanced regenerative capacities.
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http://dx.doi.org/10.1126/science.abc4346DOI Listing
February 2021

Liver zonation-a journey through space and time.

Nat Metab 2021 Jan;3(1):7-8

Novartis Institutes for BioMedical Research, Novartis Pharma AG, Basel, Switzerland.

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http://dx.doi.org/10.1038/s42255-020-00333-zDOI Listing
January 2021

The Conundrum of the Pericentral Hepatic Niche: WNT/-Catenin Signaling, Metabolic Zonation, and Many Open Questions.

Authors:
Jan S Tchorz

Gene Expr 2020 11 22;20(2):119-124. Epub 2020 Sep 22.

Novartis Institutes for BioMedical Research, Novartis Pharma AGBaselSwitzerland.

WNT/-catenin signaling promotes stemness, proliferation, and cell fate decisions in various tissue stem cell compartments, which maintain organs with a high turnover of cells (e.g., skin, stomach, and gut). Thus, the -catenin target genes AXIN2 and LGR5 are widely considered as tissue stem cell markers. In contrast, AXIN2 and LGR5 are expressed in pericentral hepatocytes, which do not show overt proliferation during liver homeostasis. Given the low hepatocyte turnover, the liver does not require constant high rates of proliferation, whereas WNT/-catenin signaling is critical for metabolic zonation. Yet, WNT/-catenin pathway upregulation, including AXIN2 and LGR5 induction in hepatocytes throughout the liver, enables hepatocyte regeneration in response to various injuries. In this brief review, I discuss the role of WNT/-catenin signaling in controlling metabolic zonation and the conundrum around pericentral hepatocytes that have been proposed as liver stem cells.
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http://dx.doi.org/10.3727/105221620X16007982788168DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7650010PMC
November 2020

Prometheus revisited: liver homeostasis and repair.

Aging (Albany NY) 2020 03 23;12(6):4685-4687. Epub 2020 Mar 23.

Novartis Institutes for BioMedical Research, Novartis Pharma AG, Basel, Switzerland.

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http://dx.doi.org/10.18632/aging.102957DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7138553PMC
March 2020

Clinical translation of liver regeneration therapies: A conceptual road map.

Biochem Pharmacol 2020 05 5;175:113847. Epub 2020 Feb 5.

Novartis Institutes for BioMedical Research, Novartis Pharma AG, Basel, Switzerland. Electronic address:

The increasing incidence of severe liver diseases worldwide has resulted in a high demand for curative liver transplantation. Unfortunately, the need for transplants by far eclipses the availability of suitable grafts leaving many waitlisted patients to face liver failure and often death. Routine use of smaller grafts (for example left lobes, split livers) from living or deceased donors could increase the number of life-saving transplants but is often limited by the graft versus recipient weight ratio defining the safety margins that minimize the risk of small for size syndrome (SFSS). SFSS is a severe complication characterized by failure of a small liver graft to regenerate and occurs when a donor graft is insufficient to meet the metabolic demand of the recipient, leading to liver failure as a result of insufficient liver mass. SFSS is not limited to transplantation but can also occur in the setting of hepatic surgical resections, where life-saving large resections of tumors may be limited by concerns of post-surgical liver failure. There are, as yet no available pro-regenerative therapies to enable liver regrowth and thus prevent SFSS. However, there is optimism around targeting factors and pathways that have been identified as regulators of liver regeneration to induce regrowth in vivo and ex vivo for clinical use. In this commentary, we propose a roadmap for developing such pro-regenerative therapy and for bringing it into the clinic. We summarize the clinical indications, preclinical models, pro-regenerative pathways and safety considerations necessary for developing such a drug.
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http://dx.doi.org/10.1016/j.bcp.2020.113847DOI Listing
May 2020

AXIN2 Pericentral Hepatocytes Have Limited Contributions to Liver Homeostasis and Regeneration.

Cell Stem Cell 2020 01 19;26(1):97-107.e6. Epub 2019 Dec 19.

Novartis Institutes for BioMedical Research, Novartis Pharma AG, Basel, Switzerland. Electronic address:

The existence of specialized liver stem cell populations, including AXIN2 pericentral hepatocytes, that safeguard homeostasis and repair has been controversial. Here, using AXIN2 lineage tracing in BAC-transgenic mice, we confirm the regenerative potential of intestinal stem cells (ISCs) but find limited roles for pericentral hepatocytes in liver parenchyma homeostasis. Liver regrowth following partial hepatectomy is enabled by proliferation of hepatocytes throughout the liver, rather than by a pericentral population. Periportal hepatocyte injury triggers local repair as well as auxiliary proliferation in all liver zones. DTA-mediated ablation of AXIN2 pericentral hepatocytes transiently disrupts this zone, which is reestablished by conversion of pericentral vein-juxtaposed glutamine synthetase (GS) hepatocytes into GS hepatocytes and by compensatory proliferation of hepatocytes across liver zones. These findings show hepatocytes throughout the liver can upregulate AXIN2 and LGR5 after injury and contribute to liver regeneration on demand, without zonal dominance by a putative pericentral stem cell population.
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http://dx.doi.org/10.1016/j.stem.2019.10.011DOI Listing
January 2020

Hepatic ductular reaction: a double-edged sword.

Aging (Albany NY) 2019 10 23;11(21):9223-9224. Epub 2019 Oct 23.

Novartis Institutes for BioMedical Research, Novartis Pharma AG, Basel, Switzerland.

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http://dx.doi.org/10.18632/aging.102386DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6874463PMC
October 2019

USP7 inhibits Wnt/β-catenin signaling through promoting stabilization of Axin.

Nat Commun 2019 09 13;10(1):4184. Epub 2019 Sep 13.

Novartis Institutes for Biomedical Research, Novartis Pharma AG, Cambridge, MA, USA.

Axin is a key scaffolding protein responsible for the formation of the β-catenin destruction complex. Stability of Axin protein is regulated by the ubiquitin-proteasome system, and modulation of cellular concentration of Axin protein has a profound effect on Wnt/β-catenin signaling. Although E3s promoting Axin ubiquitination have been identified, the deubiquitinase responsible for Axin deubiquitination and stabilization remains unknown. Here, we identify USP7 as a potent negative regulator of Wnt/β-catenin signaling through CRISPR screens. Genetic ablation or pharmacological inhibition of USP7 robustly increases Wnt/β-catenin signaling in multiple cellular systems. USP7 directly interacts with Axin through its TRAF domain, and promotes deubiquitination and stabilization of Axin. Inhibition of USP7 regulates osteoblast differentiation and adipocyte differentiation through increasing Wnt/β-catenin signaling. Our study reveals a critical mechanism that prevents excessive degradation of Axin and identifies USP7 as a target for sensitizing cells to Wnt/β-catenin signaling.
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http://dx.doi.org/10.1038/s41467-019-12143-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6744515PMC
September 2019

YAP, but Not RSPO-LGR4/5, Signaling in Biliary Epithelial Cells Promotes a Ductular Reaction in Response to Liver Injury.

Cell Stem Cell 2019 07 9;25(1):39-53.e10. Epub 2019 May 9.

Novartis Institutes for BioMedical Research, Novartis Pharma AG, Basel, Switzerland.

Biliary epithelial cells (BECs) form bile ducts in the liver and are facultative liver stem cells that establish a ductular reaction (DR) to support liver regeneration following injury. Liver damage induces periportal LGR5+ putative liver stem cells that can form BEC-like organoids, suggesting that RSPO-LGR4/5-mediated WNT/β-catenin activity is important for a DR. We addressed the roles of this and other signaling pathways in a DR by performing a focused CRISPR-based loss-of-function screen in BEC-like organoids, followed by in vivo validation and single-cell RNA sequencing. We found that BECs lack and do not require LGR4/5-mediated WNT/β-catenin signaling during a DR, whereas YAP and mTORC1 signaling are required for this process. Upregulation of AXIN2 and LGR5 is required in hepatocytes to enable their regenerative capacity in response to injury. Together, these data highlight heterogeneity within the BEC pool, delineate signaling pathways involved in a DR, and clarify the identity and roles of injury-induced periportal LGR5+ cells.
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http://dx.doi.org/10.1016/j.stem.2019.04.005DOI Listing
July 2019

mTORC1 signaling suppresses Wnt/β-catenin signaling through DVL-dependent regulation of Wnt receptor FZD level.

Proc Natl Acad Sci U S A 2018 10 8;115(44):E10362-E10369. Epub 2018 Oct 8.

Chemical Biology and Therapeutics, Novartis Institutes for Biomedical Research, Novartis Pharma AG, Cambridge, MA 02139;

Wnt/β-catenin signaling plays pivotal roles in cell proliferation and tissue homeostasis by maintaining somatic stem cell functions. The mammalian target of rapamycin (mTOR) signaling functions as an integrative rheostat that orchestrates various cellular and metabolic activities that shape tissue homeostasis. Whether these two fundamental signaling pathways couple to exert physiological functions still remains mysterious. Using a genome-wide CRISPR-Cas9 screening, we discover that mTOR complex 1 (mTORC1) signaling suppresses canonical Wnt/β-catenin signaling. Deficiency in tuberous sclerosis complex 1/2 (TSC1/2), core negative regulators of mTORC1 activity, represses Wnt/β-catenin target gene expression, which can be rescued by RAD001. Mechanistically, mTORC1 signaling regulates the cell surface level of Wnt receptor Frizzled (FZD) in a Dishevelled (DVL)-dependent manner by influencing the association of DVL and clathrin AP-2 adaptor. Sustained mTORC1 activation impairs Wnt/β-catenin signaling and causes loss of stemness in intestinal organoids ex vivo and primitive intestinal progenitors in vivo. Wnt/β-catenin-dependent liver metabolic zonation gene expression program is also down-regulated by mTORC1 activation. Our study provides a paradigm that mTORC1 signaling cell autonomously regulates Wnt/β-catenin pathway to influence stem cell maintenance.
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http://dx.doi.org/10.1073/pnas.1808575115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6217415PMC
October 2018

MRI as Primary End Point for Pharmacologic Experiments of Liver Regeneration in a Murine Model of Partial Hepatectomy.

Acad Radiol 2016 11 29;23(11):1446-1453. Epub 2016 Aug 29.

Musculoskeletal Diseases Department, Novartis Institutes for BioMedical Research, Forum 1, Novartis Campus, Fabrikstr. 28.3.07, CH-4056 Basel, Switzerland. Electronic address:

Rationale And Objectives: The study aimed to validate magnetic resonance imaging (MRI)-based liver volumetry as a quantitative measure of hepatic regeneration in mice subjected to partial hepatectomy, in view of routine in vivo pharmacologic studies characterizing compounds aiming to accelerate liver regeneration.

Materials And Methods: Partial hepatectomy was performed in male B6 mice (n = 47). Images were acquired in 14.5 minutes from anesthetized and spontaneously respiring animals, without any gating and without administration of contrast material. Some of the mice (n = 6) were treated with 1, 4-bis [2-(3, 5-dichloropyridyloxy)] benzene (TCPOBOP), a synthetic agonist of mouse constitutive androstane receptor, or with the corresponding vehicle (n = 6). Postmortem analyses included total liver weight and histologic Ki67 expression.

Results: A highly significant correlation (R = 0.98, P = 1.5 × 10) was obtained between the MRI-derived liver volumes and the postmortem liver weights in hepatectomized, untreated mice. MRI reliably monitored enhanced murine liver regrowth following treatment with TCPOBOP, as confirmed by comparative hepatocyte proliferation (Ki67 expression) and liver weight analysis (R = 0.96, P = 2 × 10).

Conclusions: MRI-based monitoring of liver regrowth in mice without the requirement of euthanizing animals at several time points has been established. In comparison to terminal procedures, the number of hepatectomized mice needed to derive a liver (re)growth curve was reduced by a factor of 6. The feasibility of using this imaging approach in pharmacologic studies in the context of liver regeneration has been demonstrated.
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http://dx.doi.org/10.1016/j.acra.2016.07.008DOI Listing
November 2016

The RSPO-LGR4/5-ZNRF3/RNF43 module controls liver zonation and size.

Nat Cell Biol 2016 05 18;18(5):467-79. Epub 2016 Apr 18.

Division of Gastroenterology and Hepatology, University Hospital Basel, 4031 Basel, Switzerland.

LGR4/5 receptors and their cognate RSPO ligands potentiate Wnt/β-catenin signalling and promote proliferation and tissue homeostasis in epithelial stem cell compartments. In the liver, metabolic zonation requires a Wnt/β-catenin signalling gradient, but the instructive mechanism controlling its spatiotemporal regulation is not known. We have now identified the RSPO-LGR4/5-ZNRF3/RNF43 module as a master regulator of Wnt/β-catenin-mediated metabolic liver zonation. Liver-specific LGR4/5 loss of function (LOF) or RSPO blockade disrupted hepatic Wnt/β-catenin signalling and zonation. Conversely, pathway activation in ZNRF3/RNF43 LOF mice or with recombinant RSPO1 protein expanded the hepatic Wnt/β-catenin signalling gradient in a reversible and LGR4/5-dependent manner. Recombinant RSPO1 protein increased liver size and improved liver regeneration, whereas LGR4/5 LOF caused the opposite effects, resulting in hypoplastic livers. Furthermore, we show that LGR4(+) hepatocytes throughout the lobule contribute to liver homeostasis without zonal dominance. Taken together, our results indicate that the RSPO-LGR4/5-ZNRF3/RNF43 module controls metabolic liver zonation and is a hepatic growth/size rheostat during development, homeostasis and regeneration.
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http://dx.doi.org/10.1038/ncb3337DOI Listing
May 2016

YAP promotes proliferation, chemoresistance, and angiogenesis in human cholangiocarcinoma through TEAD transcription factors.

Hepatology 2015 Nov 25;62(5):1497-510. Epub 2015 Aug 25.

Novartis Institutes for Biomedical Research, Developmental and Molecular Pathways, Novartis Pharma AG, Basel, Switzerland.

Unlabelled: The Yes-associated protein (YAP)/Hippo pathway has been implicated in tissue development, regeneration, and tumorigenesis. However, its role in cholangiocarcinoma (CC) is not established. We show that YAP activation is a common feature in CC patient biopsies and human CC cell lines. Using microarray expression profiling of CC cells with overexpressed or down-regulated YAP, we show that YAP regulates genes involved in proliferation, apoptosis, and angiogenesis. YAP activity promotes CC growth in vitro and in vivo by functionally interacting with TEAD transcription factors (TEADs). YAP activity together with TEADs prevents apoptosis induced by cytotoxic drugs, whereas YAP knockdown sensitizes CC cells to drug-induced apoptosis. We further show that the proangiogenic microfibrillar-associated protein 5 (MFAP5) is a direct transcriptional target of YAP/TEAD in CC cells and that secreted MFAP5 promotes tube formation of human microvascular endothelial cells. High YAP activity in human CC xenografts and clinical samples correlates with increased MFAP5 expression and CD31(+) vasculature.

Conclusions: These findings establish YAP as a key regulator of proliferation and antiapoptotic mechanisms in CC and provide first evidence that YAP promotes angiogenesis by regulating the expression of secreted proangiogenic proteins.
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http://dx.doi.org/10.1002/hep.27992DOI Listing
November 2015

Functional roles of Lgr4 and Lgr5 in embryonic gut, kidney and skin development in mice.

Dev Biol 2014 Jun 26;390(2):181-90. Epub 2014 Mar 26.

Novartis Institutes for BioMedical Research, Novartis Pharma AG, Basel, Switzerland. Electronic address:

Lgr4 and Lgr5 are known markers of adult and embryonic tissue stem cells in various organs. However, whether Lgr4 and Lgr5 are important for embryonic development remains unclear. To study their functions during intestinal crypt, skin and kidney development we now generated mice lacking either Lgr4 (Lgr4KO), Lgr5 (Lgr5KO) or both receptors (Lgr4/5dKO). E16.5 Lgr4KO mice displayed complete loss of Lgr5+/Olfm4+intestinal stem cells, compromised Wnt signaling and impaired proliferation and differentiation of gut epithelium. Similarly, E16.5 Lgr4KO mice showed reduced basal cell proliferation and hair follicle numbers in the developing skin, as well as dilated kidney tubules and ectatic Bowman׳s spaces. Although Lgr4KO and Lgr5KO mice both died perinatally, Lgr5 deletion did not compromise embryonic development of gut, kidney or skin. Concomitant deletion of Lgr4 and Lgr5 did not prevent perinatal lethality, in contrast to a previous report that suggested rescue of Lgr5 KO perinatal lethality by a hypomorphic Lgr4 mutant. While the double deletion did not further promote the phenotypes observed in Lgr4KO intestines, impaired kidney cell proliferation, reduced epidermal thickness, loss of Lgr5+follicular epithelium and impaired hair follicle development were only observed in Lgr4/5dKO mice. This supports complementary functions of both receptors. Our findings clearly establish the importance of Lgr4 and Lgr5 during embryonic gut, skin and kidney development, with a dominant role of Lgr4.
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http://dx.doi.org/10.1016/j.ydbio.2014.03.009DOI Listing
June 2014

GABA suppresses neurogenesis in the adult hippocampus through GABAB receptors.

Development 2014 Jan 27;141(1):83-90. Epub 2013 Nov 27.

Embryology and Stem Cell Biology, Department of Biomedicine, University of Basel, Mattenstrasse 28, CH-4058 Basel, Switzerland.

Adult neurogenesis is tightly regulated through the interaction of neural stem/progenitor cells (NSCs) with their niche. Neurotransmitters, including GABA activation of GABAA receptor ion channels, are important niche signals. We show that adult mouse hippocampal NSCs and their progeny express metabotropic GABAB receptors. Pharmacological inhibition of GABAB receptors stimulated NSC proliferation and genetic deletion of GABAB1 receptor subunits increased NSC proliferation and differentiation of neuroblasts in vivo. Cell-specific conditional deletion of GABAB receptors supports a cell-autonomous role in newly generated cells. Our data indicate that signaling through GABAB receptors is an inhibitor of adult neurogenesis.
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http://dx.doi.org/10.1242/dev.102608DOI Listing
January 2014

Constitutive Notch2 signaling induces hepatic tumors in mice.

Hepatology 2013 Apr 14;57(4):1607-19. Epub 2013 Mar 14.

Department of Biomedicine, Hepatology Laboratory, University of Basel, Basel, Switzerland.

Unlabelled: Hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCC) are the most common liver tumors and a leading cause for cancer-related death in men. Notch2 regulates cellular differentiation in the developing and adult liver. Although aberrant Notch signaling is implicated in various cancers, it is still unclear whether Notch2 regulates proliferation and differentiation in liver carcinogenesis and thereby contributes to HCC and CCC formation. Here, we investigated the oncogenic potential of constitutive Notch2 signaling in the liver. We show that liver-specific expression of the intracellular domain of Notch2 (N2ICD) in mice is sufficient to induce HCC formation and biliary hyperplasia. Specifically, constitutive N2ICD signaling in the liver leads to up-regulation of pro-proliferative genes and proliferation of hepatocytes and biliary epithelial cells (BECs). Using the diethylnitrosamine (DEN) HCC carcinogenesis model, we further show that constitutive Notch2 signaling accelerates DEN-induced HCC formation. DEN-induced HCCs with constitutive Notch2 signaling (DEN(N2ICD) HCCs) exhibit a marked increase in size, proliferation, and expression of pro-proliferative genes when compared with HCCs from DEN-induced control mice (DEN(ctrl) HCCs). Moreover, DEN(N2ICD) HCCs exhibit increased Sox9 messenger RNA (mRNA) levels and reduced Albumin and Alpha-fetoprotein mRNA levels, indicating that they are less differentiated than DEN(ctrl) HCCs. Additionally, DEN(N2ICD) mice develop large hepatic cysts, dysplasia of the biliary epithelium, and eventually CCC. CCC formation in patients and DEN(N2ICD) mice is accompanied by re-expression of hepatocyte nuclear factor 4α(HNF4α), possibly indicating dedifferentiation of BECs.

Conclusion: Our data establish an oncogenic role for constitutive Notch2 signaling in liver cancer development.
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http://dx.doi.org/10.1002/hep.26165DOI Listing
April 2013

Homeostatic neurogenesis in the adult hippocampus does not involve amplification of Ascl1(high) intermediate progenitors.

Nat Commun 2012 Feb 14;3:670. Epub 2012 Feb 14.

Department of Molecular Embryology, Max Planck Institute of Immunobiology, Stubeweg 51, Freiburg D-79108, Germany.

Neural stem/progenitor cells generate neurons in the adult hippocampus. Neural stem cells produce transient intermediate progenitors (type-2 cells), which generate neuroblasts (type-3 cells) that exit the cell cycle, and differentiate into neurons. The precise dynamics of neuron production from the neural stem cells remains controversial. Here we lineage trace Notch-dependent neural stem cells in the dentate gyrus and show that over 7-21 days, the progeny of the neural stem cells progress through an Ascl1(high) intermediate stage (type-2a) to neuroblasts. However, contrary to predictions, this Ascl1(high) population is not an amplifying intermediate, but it differentiates into mitotic Tbr2(+) early neuroblasts, which in turn expand the lineage. After 100 days, the majority of the neural stem cell progeny are neuroblasts or postmitotic neurons. Hence, the neural stem cells require many weeks to generate differentiated neurons. On the basis of this temporal delay in differentiation and population expansion, we propose that the neural stem cell and early neuroblast divisions drive dentate gyrus neurogenesis and not the amplification of type-2a intermediate progenitors as was previously thought.
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http://dx.doi.org/10.1038/ncomms1670DOI Listing
February 2012

A modified RMCE-compatible Rosa26 locus for the expression of transgenes from exogenous promoters.

PLoS One 2012 13;7(1):e30011. Epub 2012 Jan 13.

Novartis Institute for Biomedical Research, Developmental and Molecular Pathways, Novartis Pharma AG, Basel, Switzerland.

Generation of gain-of-function transgenic mice by targeting the Rosa26 locus has been established as an alternative to classical transgenic mice produced by pronuclear microinjection. However, targeting transgenes to the endogenous Rosa26 promoter results in moderate ubiquitous expression and is not suitable for high expression levels. Therefore, we now generated a modified Rosa26 (modRosa26) locus that combines efficient targeted transgenesis using recombinase-mediated cassette exchange (RMCE) by Flipase (Flp-RMCE) or Cre recombinase (Cre-RMCE) with transgene expression from exogenous promoters. We silenced the endogenous Rosa26 promoter and characterized several ubiquitous (pCAG, EF1α and CMV) and tissue-specific (VeCad, αSMA) promoters in the modRosa26 locus in vivo. We demonstrate that the ubiquitous pCAG promoter in the modRosa26 locus now offers high transgene expression. While tissue-specific promoters were all active in their cognate tissues they additionally led to rare ectopic expression. To achieve high expression levels in a tissue-specific manner, we therefore combined Flp-RMCE for rapid ES cell targeting, the pCAG promoter for high transgene levels and Cre/LoxP conditional transgene activation using well-characterized Cre lines. Using this approach we generated a Cre/LoxP-inducible reporter mouse line with high EGFP expression levels that enables cell tracing in live cells. A second reporter line expressing luciferase permits efficient monitoring of Cre activity in live animals. Thus, targeting the modRosa26 locus by RMCE minimizes the effort required to target ES cells and generates a tool for the use exogenous promoters in combination with single-copy transgenes for predictable expression in mice.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0030011PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3258265PMC
May 2012

Notch2 signaling promotes biliary epithelial cell fate specification and tubulogenesis during bile duct development in mice.

Hepatology 2009 Sep;50(3):871-9

Department of Biomedicine, Institute of Physiology, University of Basel, Switzerland.

Unlabelled: Intrahepatic bile duct (IHBD) development begins with the differentiation of hepatoblasts into a single continuous biliary epithelial cell (BEC) layer, called the ductal plate. During ductal plate remodeling, tubular structures arise at distinct sites of the ductal plate, forming bile ducts that dilate into the biliary tree. Alagille syndrome patients, who suffer from bile duct paucity, carry Jagged1 and Notch2 mutations, indicating that Notch2 signaling is important for IHBD development. To clarify the role of Notch2 in BEC differentiation, tubulogenesis, and BEC survival, we developed a mouse model for conditional expression of activated Notch2 in the liver. We show that expression of the intracellular domain of Notch2 (Notch2ICD) differentiates hepatoblasts into BECs, which form additional bile ducts in periportal regions and ectopic ducts in lobular regions. Additional ducts in periportal regions are maintained into adulthood and connect to the biliary tight junction network, resulting in an increased number of bile ducts per portal tract. Remarkably, Notch2ICD-expressing ductal plate remnants were not eliminated during postnatal development, implicating Notch2 signaling in BEC survival. Ectopic ducts in lobular regions did not persist into adulthood, indicating that local signals in the portal environment are important for maintaining bile ducts.

Conclusion: Notch2 signaling regulates BEC differentiation, the induction of tubulogenesis during IHBD development, and BEC survival.
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http://dx.doi.org/10.1002/hep.23048DOI Listing
September 2009
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