Deficiency of Notch signaling in pericytes results in arteriovenous malformations.

Arteriovenous malformations (AVMs) are high-flow lesions directly connecting arteries and veins. In the brain, AVM rupture can cause seizures, stroke, and death. Patients with AVMs exhibit reduced coverage of the vessels by pericytes, the mural cells of microvascular capillaries; however, the mechanism underlying this pericyte reduction and its association with AVM pathogenesis remains unknown. Notch signaling has been proposed to regulate critical pericyte functions. We hypothesized that Notch signaling in pericytes is crucial to maintain pericyte homeostasis and prevent AVM formation. We inhibited Notch signaling specifically in perivascular cells and analyzed the vasculature of these mice. The retinal vessels of mice with deficient perivascular Notch signaling developed severe AVMs, together with a significant reduction in pericytes and vascular smooth muscle cells (vSMC) in the arteries, while vSMCs were increased in the veins. Vascular malformations and pericyte loss were also observed in the forebrain of embryonic mice deficient for perivascular Notch signaling. Moreover, the loss of Notch signaling in pericytes downregulated Pdgfrb levels and increased pericyte apoptosis, pointing to a critical role for Notch in pericyte survival. Overall, our findings reveal a mechanism of AVM formation and highlight the Notch signaling pathway as an essential mediator in this process.

Dual Role of Rbpj in the Maintenance of Neural Progenitor Cells and Neuronal Migration in Cortical Development.

The development of the cerebral cortex is directed by a series of methodically precise events, including progenitor cell proliferation, neural differentiation, and cell positioning. Over the past decade, many studies have demonstrated the critical contributions of Notch signaling in neurogenesis, including that in the developing telencephalon. However, in vivo evidence for the role of Notch signaling in cortical development still remains limited partly due to the redundant functions of four mammalian Notch paralogues and embryonic lethality of the knockout mice. Here, we utilized the conditional deletion and in vivo gene manipulation of Rbpj, a transcription factor that mediates signaling by all four Notch receptors, to overcome these challenges and examined the specific roles of Rbpj in cortical development. We report severe structural abnormalities in the embryonic and postnatal cerebral cortex in Rbpj conditional knockout mice, which provide strong in vivo corroboration of previously reported functions of Notch signaling in neural development. Our results also provide evidence for a novel dual role of Rbpj in cell type-specific regulation of two key developmental events in the cerebral cortex: the maintenance of the undifferentiated state of neural progenitor cells, and the radial and tangential allocation of neurons, possibly through stage-dependent differential regulation of Ngn1.

Myeloid-specific targeting of Notch ameliorates murine renal fibrosis via reduced infiltration and activation of bone marrow-derived macrophage.

Macrophages play critical roles in renal fibrosis. However, macrophages exhibit ontogenic and functional heterogeneities, and which population of macrophages contributes to renal fibrosis and the underlying mechanisms remain unclear. In this study, we genetically targeted Notch signaling by disrupting the transcription factor recombination signal binding protein-Jκ (RBP-J), to reveal its role in regulation of macrophages during the unilateral ureteral obstruction (UUO)-induced murine renal fibrosis. Myeloid-specific disruption of RBP-J attenuated renal fibrosis with reduced extracellular matrix deposition and myofibroblast activation, as well as attenuated epithelial-mesenchymal transition, likely owing to the reduced expression of TGF-ß. Meanwhile, RBP-J deletion significantly hampered macrophage infiltration and activation in fibrotic kidney, although their proliferation appeared unaltered. By using macrophage clearance experiment, we found that kidney resident macrophages made negligible contribution, but bone marrow (BM)-derived macrophages played a major role in renal fibrogenesis. Further mechanistic analyses showed that Notch blockade reduced monocyte emigration from BM by down-regulating CCR2 expression. Finally, we found that myeloid-specific Notch activation aggravated renal fibrosis, which was mediated by CCR2+ macrophages infiltration. In summary, our data have unveiled that myeloid-specific targeting of Notch could ameliorate renal fibrosis by regulating BM-derived macrophages recruitment and activation, providing a novel strategy for intervention of this disease.

Transcription factor Ptf1a in development, diseases and reprogramming.

The transcription factor Ptf1a is a crucial helix-loop-helix (bHLH) protein selectively expressed in the pancreas, retina, spinal cord, brain, and enteric nervous system. Ptf1a is preferably assembled into a transcription trimeric complex PTF1 with an E protein and Rbpj (or Rbpjl). In pancreatic development, Ptf1a is indispensable in controlling the expansion of multipotent progenitor cells as well as the specification and maintenance of the acinar cells. In neural tissues, Ptf1a is transiently expressed in the post-mitotic cells and specifies the inhibitory neuronal cell fates, mostly mediated by downstream genes such as Tfap2a/b and Prdm13. Mutations in the coding and non-coding regulatory sequences resulting in Ptf1a gain- or loss-of-function are associated with genetic diseases such as pancreatic and cerebellar agenesis in the rodent and human. Surprisingly, Ptf1a alone is sufficient to reprogram mouse or human fibroblasts into tripotential neural stem cells. Its pleiotropic functions in many biological processes remain to be deciphered in the future.

The Notch Family Transcription Factor, RBPJκ, Modulates Glucose Transporter and Ovarian Steroid Hormone Receptor Expression During Decidualization.

During decidualization, endometrial stromal cells differentiate into a secretory phenotype to modulate the uterine microenvironment and promote embryo implantation. This highly metabolic process relies on ovarian steroid receptors and glucose transporters. Canonical Notch signaling is mediated by the transcription factor Recombination Signal Binding Protein for Immunoglobulin Kappa J Region (RBPJ). Loss of RBPJ in the mouse uterus (Pgrcre/+Rbpjflox/flox; Rbpj c-KO) results in subfertility in part due to an abnormal uterine-embryonic axis during implantation and, as described herein, decidualization failure. Induced in vivo decidualization in Rbpj c-KO mice was impaired with the downregulation of decidual markers and decreased progesterone receptor (Pgr) signaling. Consistent with in vivo mouse data, RBPJ knockdown during in vitro Human uterine fibroblast (HuF) cell decidualization results in the reduced expression of decidual marker genes along with PGR. Expression of the glucose transporter, SLC2A1, was decreased in the RBPJ-silenced HuF cells, which corresponded to decreased Slc2a1 in the secondary decidual zone of Rbpj c-KO mouse uteri. Exogenous administration of pyruvate, which bypasses the need for glucose, rescues PRL expression in RBPJ-deficient HuF cells. In summary, Notch signaling through RBPJ controls both ovarian steroid receptor PGR and glucose transporter SLC2A1 expression during decidualization, and this dysregulation likely contributes to embryo implantation failure.

Neural crest Notch/Rbpj signaling regulates olfactory gliogenesis and neuronal migration.

The neural crest-derived ensheathing glial cells of the olfactory nerve (OECs) are unique in spanning both the peripheral and central nervous systems: they ensheathe bundles of axons projecting from olfactory receptor neurons in the nasal epithelium to their targets in the olfactory bulb. OECs are clinically relevant as a promising autologous cell transplantation therapy for promoting central nervous system repair. They are also important for fertility, being required for the migration of embryonic gonadotropin-releasing hormone (GnRH) neurons from the olfactory placode along terminal nerve axons to the medial forebrain, which they enter caudal to the olfactory bulbs. Like Schwann cell precursors, OEC precursors associated with the developing olfactory nerve express the glial marker myelin protein zero and the key peripheral glial transcription factor Sox10. The transition from Schwann cell precursors to immature Schwann cells is accelerated by canonical Notch signaling via the Rbpj transcription factor. Here, we aimed to test the role of Notch/Rbpj signaling in developing OECs by blocking the pathway in both chicken and mouse. Our results suggest that Notch/Rbpj signaling prevents the cranial neural crest cells that colonize the olfactory nerve from differentiating as neurons, and at later stages contributes to the guidance of GnRH neurons.

Blockade of RBP-J-Mediated Notch Signaling Pathway Exacerbates Cardiac Remodeling after Infarction by Increasing Apoptosis in Mice.

BACKGROUND: Ischemic heart disease (IHD) is the major cause of death in patients with cardiovascular disease. Cardiac remodeling is a common pathological change following myocardial infarction (MI), and cardiomyocyte apoptosis plays a key role in this change. Transcription factor recombination signal-binding protein-J (RBP-J)-mediated Notch signaling pathway has been implicated in several inherited cardiovascular diseases, including aortic valve diseases, but whether the RBP-J-mediated Notch signaling pathway plays a role in cardiomyocyte apoptosis after MI is unclear. METHOD: We crossed RBP-Jfl/fl mice and Myh6-Cre/Esr1 transgenic mice to delete RBP-J in vivo and to partly inhibit the canonical Notch signaling pathway. MI was induced in mice by permanent ligation of the left anterior descending coronary artery followed by the knockout of RBP-J. Cardiac function and morphology were assessed by echocardiography and histological analysis 4 weeks after infarction. In addition, the expression and regulation of apoptosis-related molecules were examined by real time PCR and western blot. RESULTS: RBP-J knockout decreased the survival rate and deteriorated post-MI remodeling and function in mice, and this effect was associated with increased cardiomyocyte apoptosis. The potential mechanisms might be related to the downregulated expression of bcl-2, upregulated expression of bax, and cleaved-caspase 3 to exacerbate cardiomyocyte apoptosis. CONCLUSION: These findings show that the RBP-J-mediated Notch signaling pathway in cardiomyocytes limits ventricular remodeling and improves cardiac function after MI. The RBP-J-mediated Notch signaling pathway has a protective role in cardiomyocyte apoptosis following cardiac injury.

Canonical Notch Signaling Directs the Fate of Differentiating Neurocompetent Progenitors in the Mammalian Olfactory Epithelium.

The adult olfactory epithelium (OE) has the remarkable capacity to regenerate fully both neurosensory and non-neuronal cell types after severe epithelial injury. Lifelong persistence of two stem cell populations supports OE regeneration when damaged: the horizontal basal cells (HBCs), dormant and held in reserve; and globose basal cells, a heterogeneous population most of which are actively dividing. Both populations regenerate all cell types of the OE after injury, but the mechanisms underlying neuronal versus non-neuronal lineage commitment after recruitment of the stem cell pools remains unknown. We used both retroviral transduction and mouse lines that permit conditional cell-specific genetic manipulation as well as the tracing of progeny to study the role of canonical Notch signaling in the determination of neuronal versus non-neuronal lineages in the regenerating adult OE. Excision of either Notch1 or Notch2 genes alone in HBCs did not alter progenitor fate during recovery from epithelial injury, whereas conditional knock-out of both Notch1 and Notch2 together, retroviral transduction of progenitors with a dominant-negative form of MAML (mastermind-like), or excision of the downstream cofactor RBPJ caused progeny to adopt a neuronal fate exclusively. Conversely, we show that overexpressing the Notch1-intracellular domain (N1ICD) either genetically or by transduction blocks neuronal differentiation completely. However, N1ICD overexpression requires both alleles of the canonical cofactor RBPJ to specify downstream lineage. Together, our results suggest that canonical RBPJ-dependent Notch signaling through redundant Notch1 and Notch2 receptors is both necessary and sufficient for determining neuronal versus non-neuronal differentiation in the regenerating adult OE.SIGNIFICANCE STATEMENT Despite the substantial reconstitution of the olfactory epithelium and its population of sensory neurons after injury, disruption and exhaustion of neurogenesis is a consequence of aging and a cause of olfactory dysfunction. Understanding the mechanisms underlying the generation of replacement neurons and non-neuronal cells is critical to any therapeutic strategy aimed at rebuilding a functional neuroepithelium. The results shown here demonstrate that canonical Notch signaling determines the balance between neurons and non-neuronal cells during restoration of the epithelium after injury. Moreover, the complexities of the multiple Notch pathways impinging on that decision are dissected in detail. Finally, RBPJ, the canonical Notch transcriptional cofactor, exhibits a heretofore unreported haploinsufficiency in setting the balance among the regenerating populations.

Endothelial notch signaling is essential to prevent hepatic vascular malformations in mice.

UNLABELLED: Liver vasculature is crucial for adequate hepatic functions. Global deletion of Notch signaling in mice results in liver vascular pathologies. However, whether Notch in endothelium is essential for hepatic vascular structure and function remains unknown. To uncover the function of endothelial Notch in the liver, we deleted Rbpj, a transcription factor mediating all canonical Notch signaling, or Notch1 from the endothelium of postnatal mice. We investigated the hepatic vascular defects in these mutants. The liver was severely affected within 2 weeks of endothelial deletion of Rbpj from birth. Two-week old mutant mice had enlarged vessels on the liver surface, abnormal vascular architecture, and dilated sinusoids. Vascular casting and fluorosphere passage experiments indicated the presence of porto-systemic shunts. These mutant mice presented with severely necrotic liver parenchyma and significantly larger hypoxic areas, likely resulting from vascular shunts. We also found elevated levels of VEGF receptor 3 together with reduced levels of ephrin-B2, suggesting a possible contribution of these factors to the generation of hepatic vascular abnormalities. Deletion of Rbpj from the adult endothelium also led to dilated sinusoids, vascular shunts, and necrosis, albeit milder than that observed in mice with deletion from birth. Similar to deletion of Rbpj, loss of endothelial Notch1 from birth led to similar hepatic vascular malformations within 2 weeks. CONCLUSIONS: Endothelial Notch signaling is essential for the development and maintenance of proper hepatic vascular architecture and function. These findings may elucidate the molecular pathogenesis of hepatic vascular malformation and the safety of therapeutics inhibiting Notch. (Hepatology 2016;64:1302-1316).

Notch-independent RBPJ controls angiogenesis in the adult heart.

Increasing angiogenesis has long been considered a therapeutic target for improving heart function after injury such as acute myocardial infarction. However, gene, protein and cell therapies to increase microvascularization have not been successful, most likely because the studies failed to achieve regulated and concerted expression of pro-angiogenic and angiostatic factors needed to produce functional microvasculature. Here, we report that the transcription factor RBPJ is a homoeostatic repressor of multiple pro-angiogenic and angiostatic factor genes in cardiomyocytes. RBPJ controls angiogenic factor gene expression independently of Notch by antagonizing the activity of hypoxia-inducible factors (HIFs). In contrast to previous strategies, the cardiomyocyte-specific deletion of Rbpj increased microvascularization of the heart without adversely affecting cardiac structure or function even into old age. Furthermore, the loss of RBPJ in cardiomyocytes increased hypoxia tolerance, improved heart function and decreased pathological remodelling after myocardial infarction, suggesting that inhibiting RBPJ might be therapeutic for ischaemic injury.

RBPJ Controls Development of Pathogenic Th17 Cells by Regulating IL-23 Receptor Expression.

Interleukin-17 (IL-17)-producing helper T cells (Th17 cells) play an important role in autoimmune diseases. However, not all Th17 cells induce tissue inflammation or autoimmunity. Th17 cells require IL-23 receptor (IL-23R) signaling to become pathogenic. The transcriptional mechanisms controlling the pathogenicity of Th17 cells and IL-23R expression are unknown. Here, we demonstrate that the canonical Notch signaling mediator RBPJ is a key driver of IL-23R expression. In the absence of RBPJ, Th17 cells fail to upregulate IL-23R, lack stability, and do not induce autoimmune tissue inflammation in vivo, whereas overexpression of IL-23R rescues this defect and promotes pathogenicity of RBPJ-deficient Th17 cells. RBPJ binds and trans-activates the Il23r promoter and induces IL-23R expression and represses anti-inflammatory IL-10 production in Th17 cells. We thus find that Notch signaling influences the development of pathogenic and non-pathogenic Th17 cells by reciprocally regulating IL-23R and IL-10 expression.

CSL regulates AKT to mediate androgen independence in prostate cancer progression.

BACKGROUND: Aberrant signaling pathways leads to cancer initiation and progression. Both Notch and PI3K/AKT signaling pathways are believed to be involved in prostate cancer. How the interaction between the two pathways contributes to prostate cancer progression to androgen independence is still elusive. METHODS: Prostate cancer cells were grown in RPMI 1,640 supplemented with 10% heat-inactivated fetal bovine serum (FBS) or 10% charcoal-stripped heat-inactivated fetal bovine serum (FCS), 1% penicillin-streptomycin in 75 cm2 polystyrene flasks, and maintained at 37 °C in a humidified atmosphere with 5% CO2 . Cell proliferation, invasion were performed with cell counting, matrigel assay in vitro. Dual luciferase assays were performed using reporter plasmids with ARE (Androgen Response Element, ARE). RNA interference was applied to gene silence. Tumorigenicity of cancer cells was evaluated by mouse xenograft in vivo. RESULTS: A subpopulation of casodex resistant prostate cancer cells were identified with an overexpressed androgen receptor (AR) and aggressive phenotypes, characterized with high proliferation, invasion in vitro and enhanced tumorigenesis in vivo. Gene profiling for androgen-dependent LNCaP and androgen-independent LNCaP-CR revealed that both CSL and AKT gave the similar expressional pattern upon casodex treatment. Immunoblot demonstrated that CSL and AKT were dramatically suppressed in androgen dependent LNCaP cells, but slightly inhibited in LNCaP-CR cells as well as other androgen independent prostate cancer cells. Further studies indicated that CSL regulates AKT, and subsequently AR in prostate cancer cells. AKT mediates casodex resistance and androgen independence through regulation of cyclin D1. CONCLUSION: CSL-AKT-AR axis might play an important role in prostate cancer progression. Targeting CSL depleted the casodex resistant population through inhibition of the AKT, suggesting a more effective therapeutic strategy for abrogating casodex resistance in advanced prostate cancer.

Myeloid-Specific Blockade of Notch Signaling by RBP-J Knockout Attenuates Spinal Cord Injury Accompanied by Compromised Inflammation Response in Mice.

The outcome of spinal cord injury (SCI) is determined by both neural cell-intrinsic survival pathways and tissue microenvironment-derived signals. Macrophages dominating the inflammatory responses in SCI possess both destructive and reparative potentials, according to their activation status. Notch signaling is involved in both cell survival and macrophage-mediated inflammation, but a comprehensive role of Notch signaling in SCI has been elusive. In this study, we compared the effects of general Notch blockade by a pharmaceutical γ-secretase inhibitor (GSI) and myeloid-specific Notch signal disruption by recombination signal binding protein Jκ (RBP-J) knockout on SCI. The administration of Notch signal inhibitor GSI resulted in worsened hind limb locomotion and exacerbated inflammation. However, mice lacking RBP-J, the critical transcription factor mediating signals from all four mammalian Notch receptors, in myeloid lineage displayed promoted functional recovery, attenuated glial scar formation, improved neuronal survival and axon regrowth, and mitigated inflammatory response after SCI. These benefits were accompanied by enhanced AKT activation in the lesion area after SCI. These findings demonstrate that abrogating Notch signal in myeloid cells ameliorates inflammation response post-SCI and promotes functional recovery, but general pharmaceutical Notch interception has opposite effects. Therefore, clinical intervention of Notch signaling in SCI needs to pinpoint myeloid lineage to avoid the counteractive effects of global inhibition.

RBP-J imposes a requirement for ITAM-mediated costimulation of osteoclastogenesis.

Osteoclastogenesis requires activation of RANK signaling as well as costimulatory signals from immunoreceptor tyrosine-based activation motif-containing (ITAM-containing) receptors/adaptors, predominantly tyrosine kinase-binding proteins DAP12 and FcRγ, in osteoclast precursors. It is not well understood how costimulatory signals are regulated and integrated with RANK signaling. Here, we found that osteopetrotic bone phenotypes in mice lacking DAP12 or DAP12 and FcRγ are mediated by the transcription factor RBP-J, as deletion of Rbpj in these mice substantially rescued the defects of bone remodeling. Using a TNF-α-induced model of inflammatory bone resorption, we determined that RBP-J deficiency enables TNF-α to induce osteoclast formation and bone resorption in DAP12-deficient animals. Thus, RBP-J imposes a requirement for ITAM-mediated costimulation of RANKL or TNF-α-induced osteoclastogenesis. Mechanistically, RBP-J suppressed induction of key osteoclastogenic factors NFATc1, BLIMP1, and c-FOS by inhibiting ITAM-mediated expression and function of PLCγ2 and activation of downstream calcium-CaMKK/PYK2 signaling. Moreover, RBP-J suppressed Plcg2 expression and downstream calcium oscillations indirectly by a TGF-ß/PLCγ2/calcium axis. Together, our findings indicate that RBP-J suppresses ITAM-mediated costimulation, thereby limiting crosstalk between ITAM and RANK/TNFR signaling and allowing fine tuning of osteoclastogenesis during bone homeostasis and under inflammatory conditions. Furthermore, these data suggest that environmental cues that regulate RBP-J expression/function potentially modulate the requirement for costimulatory signaling for osteoclast differentiation and bone remodeling.

Blocking Notch in endothelial cells prevents arteriovenous fistula failure despite CKD.

Neointima formation causes the failure of 60% of arteriovenous fistulas (AVFs) within 2 years. Neointima-forming mechanisms are controversial but possibly linked to excess proinflammatory responses and dysregulated Notch signaling. To identify how AVFs fail, we anastomosed the carotid artery to the internal jugular vein in normal and uremic mice and compared these findings with those in failed AVFs from patients with ESRD. Endothelial cells (ECs) of AVFs in uremic mice or patients expressed mesenchymal markers (FSP-1 and/or α-SMA) and exhibited increased expression and nuclear localization of Notch intracellular domain compared with ECs of AVFs in pair-fed control mice. Furthermore, expression of VE-Cadherin decreased, whereas expression of Notch1 and -4, Notch ligands, the downstream transcription factor of Notch, RBP-Jκ, and Notch target genes increased in ECs of AVFs in uremic mice. In cultured ECs, ectopic expression of Notch ligand or treatment with TGF-ß1 triggered the expression of mesenchymal markers and induced endothelial cell barrier dysfunction, both of which were blocked by Notch inhibition or RBP-Jκ knockout. Furthermore, Notch-induced defects in barrier function, invasion of inflammatory cells, and neointima formation were suppressed in mice with heterozygous knockdown of endothelial-specific RBP-Jκ. These results suggest that increased TGF-ß1, a complication of uremia, activates Notch in endothelial cells of AVFs, leading to accelerated neointima formation and AVF failure. Suppression of Notch activation could be a strategy for improving AFV function in uremia.

RBP-Jκ-dependent Notch signaling is required for murine articular cartilage and joint maintenance.

OBJECTIVE: Osteoarthritis (OA) is a degenerative disease resulting in severe joint cartilage destruction and disability. While the mechanisms underlying the development and progression of OA are poorly understood, gene mutations have been identified within cartilage-related signaling molecules, implicating impaired cell signaling in OA and joint disease. The Notch pathway has recently been identified as a crucial regulator of growth plate cartilage development, and components are expressed in joint tissue. This study was undertaken to investigate a novel role for Notch signaling in joint cartilage development, maintenance, and the pathogenesis of joint disease in a mouse model. METHODS: We performed the first mouse gene study in which the core Notch signaling component, RBP-Jκ, was tissue specifically deleted within joints. The Prx1Cre transgene removed Rbpjk loxP-flanked alleles in mesenchymal joint precursor cells, while the Col2Cre(ERT2) transgene specifically deleted Rbpjk in postnatal chondrocytes. Murine articular chondrocyte cultures were also used to examine Notch regulation of gene expression. RESULTS: Loss of Notch signaling in mesenchymal joint precursor cells did not affect embryonic joint development in mice, but rather, resulted in an early, progressive OA-like pathology. Additionally, partial loss of Notch signaling in murine postnatal cartilage resulted in progressive joint cartilage degeneration and an age-related OA-like pathology. Inhibition of Notch signaling altered the expression of the extracellular matrix (ECM)-related factors type II collagen (COL2A1), proteoglycan 4, COL10A1, matrix metalloproteinase 13, and ADAMTS. CONCLUSION: Our findings indicate that the RBP-Jκ-dependent Notch pathway is a novel pathway involved in joint maintenance and articular cartilage homeostasis, a critical regulator of articular cartilage ECM-related molecules, and a potentially important therapeutic target for OA-like joint disease.

Regulation of dendritic cell differentiation in bone marrow during emergency myelopoiesis.

Although accumulation of dendritic cell (DC) precursors occurs in bone marrow, the terminal differentiation of these cells takes place outside bone marrow. The signaling, regulating this process, remains poorly understood. We demonstrated that this process could be differentially regulated by Notch ligands: Jagged-1 (Jag1) and Delta-like ligand 1 (Dll1). In contrast to Dll1, Jag1, in vitro and during induced myelopoiesis in vivo, prevented DC differentiation by promoting the accumulation of their precursors. Although both ligands activated Notch in hematopoietic progenitor cells, they had an opposite effect on Wnt signaling. Dll1 activated Wnt pathways, whereas Jag1 inhibited it via downregulation of the expression of the Wnt receptors Frizzled (Fzd). Jag1 suppressed fzd expression by retaining histone deacetylase 1 in the complex with the transcription factor CSL/CBF-1 on the fzd promoter. Our results suggest that DC differentiation, during induced myelopoiesis, can be regulated by the nature of the Notch ligand expressed on adjacent stroma cells.

Wnt signaling regulates left-right axis formation in the node of mouse embryos.

The node triggers formation of the left-right axis in mouse embryos by establishing local asymmetry of Nodal and Cerl2 expression. We found that Wnt3 is expressed in perinodal crown cells preferentially on the left side. The enhancer responsible for Wnt3 expression was identified and found to be regulated by Foxa2 and Rbpj under the control of Notch signaling. Rbpj binding sites suppress enhancer activity in pit cells of the node, thereby ensuring crown cell-specific expression. In addition, we found that the expression of Gdf1 and Cerl2 is also regulated by Notch signaling, suggesting that such signaling may induce the expression of genes related to left-right asymmetry as a set. Furthermore, Cerl2 expression became symmetric in response to inhibition of Wnt-ß-catenin signaling. Our results suggest that Wnt signaling regulates the asymmetry of Cerl2 expression, which likely generates a left-right difference in Nodal activity at the node for further amplification in lateral plate mesoderm.

Notch signaling regulates tubular morphogenesis during repair from biliary damage in mice.

BACKGROUND & AIMS: Repair from biliary damages requires the biliary specification of hepatic progenitor cells and the remodeling of ductular reactive structures into branching biliary tubules. We hypothesized that the morphogenetic role of Notch signaling is maintained during the repair process and have addressed this hypothesis using pharmacologic and genetic models of defective Notch signaling. METHODS: Treatment with DDC (3,5-diethoxycarbonyl-1,4-dihydrocollidine) or ANIT (alpha-naphthyl-isothiocyanate) was used to induce biliary damage in wild type mice and in mice with a liver specific defect in the Notch-2 receptor (Notch-2-cKO) or in RPB-Jk. Hepatic progenitor cells, ductular reaction, and mature ductules were quantified using K19 and SOX-9. RESULTS: In DDC treated wild type mice, pharmacologic Notch inhibition with dibenzazepine decreased the number of both ductular reaction and hepatic progenitor cells. Notch-2-cKO mice treated with DDC or ANIT accumulated hepatic progenitor cells that failed to progress into mature ducts. In RBP-Jk-cKO mice, mature ducts and hepatic progenitor cells were both significantly reduced with respect to similarly treated wild type mice. The mouse progenitor cell line BMOL cultured on matrigel, formed a tubular network allowing the study of tubule formation in vitro; γ-secretase inhibitor treatment and siRNAs silencing of Notch-1, Notch-2 or Jagged-1 significantly reduced both the length and number of tubular branches. CONCLUSIONS: These data demonstrate that Notch signaling plays an essential role in biliary repair. Lack of Notch-2 prevents biliary tubule formation, both in vivo and in vitro. Lack of RBP-Jk inhibits the generation of biliary-committed precursors and tubule formation.