Inhibition of Notch2 by Numb/Numblike controls myocardial compaction in the heart

Aims The ventricular wall of the heart is composed of trabeculated and compact layers, which are separated by yet unknown processes during embryonic development. Here, we wanted to explore the role of Notch2 and Numb/Numblike for myocardial trabeculation and compaction. Methods and results We found that Notch2 activity is specifically down-regulated in the compact layer during cardiac development in the mouse. The biological role of Notch2 down-regulation was investigated by the expression of constitutively active Notch2 in the myocardium of transgenic mice, resulting in hypertrabeculation, reduced compaction, and ventricular septum defects. To disclose the mechanism that inhibited Notch2 activity during the formation of myocardial layers, we analysed potential suppressors of Notch signalling. We unveiled that concomitant but not separate ablation of Numb and Numblike in the developing heart leads to increased Notch2 activity along with hypertrabeculation, reduced compaction, and ventricular septum defects, phenocopying effects gained by overexpression of constitutively active Notch2. Expression profiling revealed a strong up-regulation of Bmp10 in Numb/Numblike mutant hearts, which might also interfere with trabeculation and compaction. Conclusion This study identified potential novel roles of Numb/Numblike in regulating trabeculation and compaction by inhibiting Notch2 and Bmp10 signallin

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

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

A Modified RMCE-Compatible Rosa26 Locus for the Expression of Transgenes from Exogenous Promoters

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

Notch2 signaling in development and cancer

Notch signaling via cell to cell interaction is a key mechanism in regulating proliferation, cell fate decisions and survival of various cell types in both invertebrates and vertebrates. Among the four Notch receptors (Notch1-4) described in mammals, most studies focused on Notch1, while Notch2 functions are poorly understood. In humans, Notch2 mutations are associated with a variety of developmental disorders and tumor formation in several tissues. In the liver, Notch2 deletion was shown to cause Alagille syndrome (AGS), which is characterized by impaired intrahepatic bile duct (IHBD) development. In glioblastoma multiforme (GBM), the most aggressive form of CNS tumors, Notch2 is amplified and high Notch2 levels correlate with poor prognosis. However, the exact role of Notch2 in these human pathologies is not established. In order to investigate the function of Notch2 in AGS and GBM at the level of cells, tissues and organs, I generated transgenic mice that allow for tissue-specific expression of activated Notch2 (Notch2ICD), which mimics ligand-induced activation of Notch2 signaling. To address the function of Notch2 signaling in AGS and IHBD development, Notch2ICD expression was induced in hepatoblasts. Hepatoblasts are the bipotential progenitors in the liver that give rise to either hepatocytes or biliary epithelial cells (BECs) that undergo tubulogenesis to form IHBDs. I observed that ectopic Notch2ICD expression in hepatoblasts induces biliary epithelial cell (BEC) differentiation, tubulogenesis of IHBDs, and BEC survival. These findings shed light on the role for Notch2 in AGS, since they provide an explanation why AGS patients with Notch2 mutations suffer from impaired IHBD development. It is believed that GBM originates from glioma stem cells (GSCs) which can derive from developmentally stalled neural stem cells (NSCs). Thus, I addressed whether Notch2 plays a role in regulating NSC proliferation and differentiation, possibly predisposing NSCs to become GSCs and eventually GBMs. Therefore, I generated mice that ectopically express activated Notch2 in NSCs and compared the induced molecular alterations to those in GSCs from GBM cell lines and primary GBM biopsies. I show that key features of GSCs, such as increased proliferation and astrocytic lineage commitment, are induced by ectopic Notch2 signaling in NSCs. Aberrant Notch2 expression may therefore predispose NSCs to become GSCs that give rise to GBMs. Moreover, Notch2 signaling enhanced survival of GBM cells, possibly explaining the increased aggressiveness of GBMs with high Notch2 levels. Therefore, blockade of Notch2 signaling may interfere with GBM cell survival, and the formation and proliferation of GSCs and thus be of therapeutic benefit for the treatment of GBMs, for which no cure is available yet

Constitutive activation of Notch2 signalling confers chemoresistance to neural stem cells via transactivation of fibroblast growth factor receptor-1

Notch signalling regulates neural stem cell (NSC) proliferation, differentiation and survival for the correct development and functioning of the central nervous system. Overactive Notch2 signalling has been associated with poor prognosis of aggressive brain tumours, such as glioblastoma multiforme (GBM). We recently reported that constitutive expression of the Notch2 intracellular domain (N2ICD) enhances proliferation and gliogenesis in NSCs. Here, we investigated the mechanism by which Notch2 promotes resistance to apoptosis of NSCs to cytotoxic insults. We performed ex vivo studies using NSC cultures from transgenic mice constitutively expressing N2ICD. These NSCs expressed increased levels of pro-survival factors and lack an apoptotic response to the topoisomerase inhibitor etoposide, not showing neither mitochondrial damage nor caspase activation. Interestingly, Notch2 signalling also regulated chemoresistance of human GBM cells to etoposide. We also identified a signalling crosstalk with FGF signalling pathway involved in this resistance to apoptosis of NSCs. Aberrant Notch2 expression enhances fibroblast growth factor receptor-1 (FGFR1) activity to specifically target the AKT-GSK3 signalling pathway to block apoptosis. These results have implications for understanding molecular changes involved in both tumorigenesis and therapy resistance. Keywords: Chemoresistance, FGFR1, Glioma, Neural stem cells, Notch

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

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

Activation of Yap Directed Transcription by Knock-down of Conserved Cellular Functions

The Yap-Hippo pathway has a significant role in regulating cell proliferation and growth, thus controlling organ size and regeneration. The Hippo pathway regulates two highly conserved, transcription co-activators YAP and TAZ. The upstream regulators of the Yap-Hippo pathway have not been fully characterized. The aim of this study was to use a siRNA screen, in a liver biliary cell line, to identify regulators of the Yap-Hippo pathway that allow activation of the YAP transcription co-activator at high cell density. Activation of the Yap transcription co-activator was monitored using a high content, image based assay that measured the intracellular localization of native YAP protein. Active siRNAs were identified and further validated by quantification of CYR61 mRNA levels (a known Yap target gene). The effect of compounds targeting the putative gene targets identified as hits was also used for further validation. A number of validated hits reveal basic aspects of Yap-Hippo biology; such as components of the nuclear pore, by which YAP cytoplasmic/nuclear shuttling occurs, or how proteasomal degradation regulates intracellular YAP concentrations, which then alter YAP localization and transcription. Such results highlight how targeting conserved cellular functions can lead to validated activity in phenotypic assays

Activation of Yap Directed Transcription by Knock-down of Conserved Cellular Functions

The Yap-Hippo pathway has been shown to have a significant role in regulating cell proliferation and growth and hence controlling organ size and regeneration, as well as the differentiation of stem cells. These cell fate decisions are regulated by the Hippo pathway through the action of two, highly conserved, transcription co-activators YAP and TAZ. However, the upstream regulators of the Yap-Hippo pathway have not been fully characterized and may vary in different cell types and organs. The aim of this study was to use a siRNA screen, in a liver biliary cell line, to identify regulators of the Yap-Hippo pathway that when knocked-down allow activation of the YAP transcription co-activator at high cell density. This project used a commercially available library of siRNAs purchased from Qiagen that consisted of four siRNAs targeting seven thousand genes of the “druggable” genome. The library was screened using a reverse transfection protocol with a biliary derived cell line which was grown to high cell density. Activation of the Yap transcription co-activator was monitored using a high content, image based assay that measured the intracellular localization of native YAP protein. Active siRNAs were identified and further validated by quantification of CYR61 mRNA levels (a known Yap target gene) following knock-down and the effect of compounds targeting the putative gene targets identified as hits was also used for further validation. A number of validated hits reveal basic aspects of Yap-Hippo biology; such as components of the nuclear pore, by which YAP cytoplasmic/nuclear shuttling occurs, or how proteasomal degradation regulates intracellular YAP concentrations, which then alter YAP localization and transcription. Such results highlight how targeting conserved cellular functions can lead to validated activity in phenotypic assays

Inhibition of Notch2 by Numb/Numblike controls myocardial compaction in the heart

The ventricular wall of the heart is composed of trabeculated and compact layers, which are separated by yet unknown processes during embryonic development. Here, we wanted to explore the role of Notch2 and Numb/Numblike for myocardial trabeculation and compaction.; We found that Notch2 activity is specifically down-regulated in the compact layer during cardiac development in the mouse. The biological role of Notch2 down-regulation was investigated by the expression of constitutively active Notch2 in the myocardium of transgenic mice, resulting in hypertrabeculation, reduced compaction, and ventricular septum defects. To disclose the mechanism that inhibited Notch2 activity during the formation of myocardial layers, we analysed potential suppressors of Notch signalling. We unveiled that concomitant but not separate ablation of Numb and Numblike in the developing heart leads to increased Notch2 activity along with hypertrabeculation, reduced compaction, and ventricular septum defects, phenocopying effects gained by overexpression of constitutively active Notch2. Expression profiling revealed a strong up-regulation of Bmp10 in Numb/Numblike mutant hearts, which might also interfere with trabeculation and compaction.; This study identified potential novel roles of Numb/Numblike in regulating trabeculation and compaction by inhibiting Notch2 and Bmp10 signalling

Combined deletion of Lgr4 and Lgr5 impairs embryonic mouse development

Lgr4 and Lgr5 proteins are known markers of adult and embryonic tissue stem cells in various organs. However, the role of these proteins in propagating and maintaining individual tissue stem cell compartments is still controversial. While it was reported that Lgr4 is dispensable for normal embryonic gut development, Lgr4 deletion functionally impaired maintenance of the postnatal and adult intestinal crypt stem cell compartment. Furthermore, concomitant deletion of Lgr4 in Lgr5-null embryos was able to rescue their perinatal lethality, whereas combined deletion of Lgr4 and Lgr5 in adult mice exacerbated the latter phenotype, suggesting antagonistic or complementary functions of both receptors, respectively. While the effects of Lgr4 deletion during embryonic skin and kidney development have been reported, combined deletion of Lgr4 and Lgr5 has not been studied to date. To elucidate the functions of Lgr4 and Lgr5 during intestinal crypt development and to study their role in developing kidney and skin, we generated homozygous mice lacking either Lgr4 (Lgr4KO), Lgr5 (Lgr5KO) or both receptors (Lgr4/5dKO). Lgr4 deletion resulted in loss of Lgr5+ intestinal stem cells and impaired proliferation in the developing gut of E16.5 mice, a phenotype that was not further increased nor ameliorated by combined deletion of Lgr4 and Lgr5 (Lgr4/5dKO). In skin, E16.5 Lgr4KO and Lgr4/5dKO mice displayed impaired proliferation of basal cell progenitors accompanied by reduced epidermal thickness and reduced numbers of hair follicles. In contrast to E16.5 Lgr4KO mice, Lgr4/5dkO mice did neither show dilated kidney tubules nor cysts. However, E16.5 Lgr4/5dKO mice showed impaired kidney cell proliferation which was not observed in Lgr4KO mice. In summary, our data show that combined deletion of Lgr4 and Lgr5 impairs embryonic development with a dominant role of Lgr4 and support a complementary rather than an antagonistic function for both receptors