Specification of CNS macrophage subsets occurs postnatally in defined niches.

All tissue-resident macrophages of the central nervous system (CNS)-including parenchymal microglia, as well as CNS-associated macrophages (CAMs1) such as meningeal and perivascular macrophages2-7-are part of the CNS endogenous innate immune system that acts as the first line of defence during infections or trauma2,8-10. It has been suggested that microglia and all subsets of CAMs are derived from prenatal cellular sources in the yolk sac that were defined as early erythromyeloid progenitors11-15. However, the precise ontogenetic relationships, the underlying transcriptional programs and the molecular signals that drive the development of distinct CAM subsets in situ are poorly understood. Here we show, using fate-mapping systems, single-cell profiling and cell-specific mutants, that only meningeal macrophages and microglia share a common prenatal progenitor. By contrast, perivascular macrophages originate from perinatal meningeal macrophages only after birth in an integrin-dependent manner. The establishment of perivascular macrophages critically requires the presence of arterial vascular smooth muscle cells. Together, our data reveal a precisely timed process in distinct anatomical niches for the establishment of macrophage subsets in the CNS.

Single-Cell Gene-Regulatory Networks of Advanced Symptomatic Atherosclerosis.

BACKGROUND: While our understanding of the single-cell gene expression patterns underlying the transformation of vascular cell types during the progression of atherosclerosis is rapidly improving, the clinical and pathophysiological relevance of these changes remains poorly understood. METHODS: Single-cell RNA sequencing data generated with SmartSeq2 (≈8000 genes/cell) in 16 588 single cells isolated during atherosclerosis progression in Ldlr-/-Apob100/100 mice with human-like plasma lipoproteins and from humans with asymptomatic and symptomatic carotid plaques was clustered into multiple subtypes. For clinical and pathophysiological context, the advanced-stage and symptomatic subtype clusters were integrated with 135 tissue-specific (atherosclerotic aortic wall, mammary artery, liver, skeletal muscle, and visceral and subcutaneous, fat) gene-regulatory networks (GRNs) inferred from 600 coronary artery disease patients in the STARNET (Stockholm-Tartu Atherosclerosis Reverse Network Engineering Task) study. RESULTS: Advanced stages of atherosclerosis progression and symptomatic carotid plaques were largely characterized by 3 smooth muscle cells (SMCs), and 3 macrophage subtype clusters with extracellular matrix organization/osteogenic (SMC), and M1-type proinflammatory/Trem2-high lipid-associated (macrophage) phenotypes. Integrative analysis of these 6 clusters with STARNET revealed significant enrichments of 3 arterial wall GRNs: GRN33 (macrophage), GRN39 (SMC), and GRN122 (macrophage) with major contributions to coronary artery disease heritability and strong associations with clinical scores of coronary atherosclerosis severity. The presence and pathophysiological relevance of GRN39 were verified in 5 independent RNAseq data sets obtained from the human coronary and aortic artery, and primary SMCs and by targeting its top-key drivers, FRZB and ALCAM in cultured human coronary artery SMCs. CONCLUSIONS: By identifying and integrating the most gene-rich single-cell subclusters of atherosclerosis to date with a coronary artery disease framework of GRNs, GRN39 was identified and independently validated as being critical for the transformation of contractile SMCs into an osteogenic phenotype promoting advanced, symptomatic atherosclerosis.

Notch Signaling in Development, Tissue Homeostasis, and Disease.

Notch signaling is an evolutionarily highly conserved signaling mechanism, but in contrast to signaling pathways such as Wnt, Sonic Hedgehog, and BMP/TGF-ß, Notch signaling occurs via cell-cell communication, where transmembrane ligands on one cell activate transmembrane receptors on a juxtaposed cell. Originally discovered through mutations in Drosophila more than 100 yr ago, and with the first Notch gene cloned more than 30 yr ago, we are still gaining new insights into the broad effects of Notch signaling in organisms across the metazoan spectrum and its requirement for normal development of most organs in the body. In this review, we provide an overview of the Notch signaling mechanism at the molecular level and discuss how the pathway, which is architecturally quite simple, is able to engage in the control of cell fates in a broad variety of cell types. We discuss the current understanding of how Notch signaling can become derailed, either by direct mutations or by aberrant regulation, and the expanding spectrum of diseases and cancers that is a consequence of Notch dysregulation. Finally, we explore the emerging field of Notch in the control of tissue homeostasis, with examples from skin, liver, lung, intestine, and the vasculature.

Identification of a Notch transcriptomic signature for breast cancer.

BACKGROUND: Dysregulated Notch signalling contributes to breast cancer development and progression, but validated tools to measure the level of Notch signalling in breast cancer subtypes and in response to systemic therapy are largely lacking. A transcriptomic signature of Notch signalling would be warranted, for example to monitor the effects of future Notch-targeting therapies and to learn whether altered Notch signalling is an off-target effect of current breast cancer therapies. In this report, we have established such a classifier. METHODS: To generate the signature, we first identified Notch-regulated genes from six basal-like breast cancer cell lines subjected to elevated or reduced Notch signalling by culturing on immobilized Notch ligand Jagged1 or blockade of Notch by γ-secretase inhibitors, respectively. From this cadre of Notch-regulated genes, we developed candidate transcriptomic signatures that were trained on a breast cancer patient dataset (the TCGA-BRCA cohort) and a broader breast cancer cell line cohort and sought to validate in independent datasets. RESULTS: An optimal 20-gene transcriptomic signature was selected. We validated the signature on two independent patient datasets (METABRIC and Oslo2), and it showed an improved coherence score and tumour specificity compared with previously published signatures. Furthermore, the signature score was particularly high for basal-like breast cancer, indicating an enhanced level of Notch signalling in this subtype. The signature score was increased after neoadjuvant treatment in the PROMIX and BEAUTY patient cohorts, and a lower signature score generally correlated with better clinical outcome. CONCLUSIONS: The 20-gene transcriptional signature will be a valuable tool to evaluate the response of future Notch-targeting therapies for breast cancer, to learn about potential effects on Notch signalling from conventional breast cancer therapies and to better stratify patients for therapy considerations.

A molecular atlas of cell types and zonation in the brain vasculature.

Cerebrovascular disease is the third most common cause of death in developed countries, but our understanding of the cells that compose the cerebral vasculature is limited. Here, using vascular single-cell transcriptomics, we provide molecular definitions for the principal types of blood vascular and vessel-associated cells in the adult mouse brain. We uncover the transcriptional basis of the gradual phenotypic change (zonation) along the arteriovenous axis and reveal unexpected cell type differences: a seamless continuum for endothelial cells versus a punctuated continuum for mural cells. We also provide insight into pericyte organotypicity and define a population of perivascular fibroblast-like cells that are present on all vessel types except capillaries. Our work illustrates the power of single-cell transcriptomics to decode the higher organizational principles of a tissue and may provide the initial chapter in a molecular encyclopaedia of the mammalian vasculature.

Author Correction: A molecular atlas of cell types and zonation in the brain vasculature.

In Fig. 1b of this Article, 'Csf1r' was misspelt 'Csfr1'. In addition, in Extended Data Fig. 11b, owing to an error during figure formatting, the genes listed in the first column shifted down three rows below the first gene on the list, causing a mismatch between the gene names and their characteristics. These errors have been corrected online, and the original Extended Data Fig. 11b is provided as Supplementary Information to the accompanying Amendment.

Human Fis1 regulates mitochondrial dynamics through inhibition of the fusion machinery.

Mitochondrial dynamics is important for life. At center stage for mitochondrial dynamics, the balance between mitochondrial fission and fusion is a set of dynamin-related GTPases that drive mitochondrial fission and fusion. Fission is executed by the GTPases Drp1 and Dyn2, whereas the GTPases Mfn1, Mfn2, and OPA1 promote fusion. Recruitment of Drp1 to mitochondria is a critical step in fission. In yeast, Fis1p recruits the Drp1 homolog Dnm1p to mitochondria through Mdv1p and Caf4p, but whether human Fis1 (hFis1) promotes fission through a similar mechanism as in yeast is not established. Here, we show that hFis1-mediated mitochondrial fragmentation occurs in the absence of Drp1 and Dyn2, suggesting that they are dispensable for hFis1 function. hFis1 instead binds to Mfn1, Mfn2, and OPA1 and inhibits their GTPase activity, thus blocking the fusion machinery. Consistent with this, disruption of the fusion machinery in Drp1-/- cells phenocopies the fragmentation phenotype induced by hFis1 overexpression. In sum, our data suggest a novel role for hFis1 as an inhibitor of the fusion machinery, revealing an important functional evolutionary divergence between yeast and mammalian Fis1 proteins.

Notch signalling regulates epibranchial placode patterning and segregation.

Epibranchial placodes are the geniculate, petrosal and nodose placodes that generate parts of cranial nerves VII, IX and X, respectively. How the three spatially separated placodes are derived from the common posterior placodal area is poorly understood. Here, we reveal that the broad posterior placode area is first patterned into a Vgll2+/Irx5+ rostral domain and a Sox2+/Fgf3+/Etv5+ caudal domain relative to the first pharyngeal cleft. This initial rostral and caudal patterning is then sequentially repeated along each pharyngeal cleft for each epibranchial placode. The caudal domains give rise to the neuronal and non-neuronal cells in the placode, whereas the rostral domains are previously unrecognized structures, serving as spacers between the final placodes. Notch signalling regulates the balance between the rostral and caudal domains: high levels of Notch signalling expand the caudal domain at the expense of the rostral domain, whereas loss of Notch signalling produces the converse phenotype. Collectively, these data unravel a new patterning principle for the early phases of epibranchial placode development and a role for Notch signalling in orchestrating epibranchial placode segregation and differentiation.

PIM-induced phosphorylation of Notch3 promotes breast cancer tumorigenicity in a CSL-independent fashion.

Dysregulation of the developmentally important Notch signaling pathway is implicated in several types of cancer, including breast cancer. However, the specific roles and regulation of the four different Notch receptors have remained elusive. We have previously reported that the oncogenic PIM kinases phosphorylate Notch1 and Notch3. Phosphorylation of Notch1 within the second nuclear localization sequence of its intracellular domain (ICD) enhances its transcriptional activity and tumorigenicity. In this study, we analyzed Notch3 phosphorylation and its functional impact. Unexpectedly, we observed that the PIM target sites are not conserved between Notch1 and Notch3. Notch3 ICD (N3ICD) is phosphorylated within a domain, which is essential for formation of a transcriptionally active complex with the DNA-binding protein CSL. Through molecular modeling, X-ray crystallography, and isothermal titration calorimetry, we demonstrate that phosphorylation of N3ICD sterically hinders its interaction with CSL and thereby inhibits its CSL-dependent transcriptional activity. Surprisingly however, phosphorylated N3ICD still maintains tumorigenic potential in breast cancer cells under estrogenic conditions, which support PIM expression. Taken together, our data indicate that PIM kinases modulate the signaling output of different Notch paralogs by targeting distinct protein domains and thereby promote breast cancer tumorigenesis via both CSL-dependent and CSL-independent mechanisms.

Peri-arterial specification of vascular mural cells from naïve mesenchyme requires Notch signaling.

Mural cells (MCs) are essential for blood vessel stability and function; however, the mechanisms that regulate MC development remain incompletely understood, in particular those involved in MC specification. Here, we investigated the first steps of MC formation in zebrafish using transgenic reporters. Using pdgfrb and abcc9 reporters, we show that the onset of expression of abcc9, a pericyte marker in adult mice and zebrafish, occurs almost coincidentally with an increment in pdgfrb expression in peri-arterial mesenchymal cells, suggesting that these transcriptional changes mark the specification of MC lineage cells from naïve pdgfrblow mesenchymal cells. The emergence of peri-arterial pdgfrbhigh MCs required Notch signaling. We found that pdgfrb-positive cells express notch2 in addition to notch3, and although depletion of notch2 or notch3 failed to block MC emergence, embryos depleted of both notch2 and notch3 lost mesoderm- as well as neural crest-derived pdgfrbhigh MCs. Using reporters that read out Notch signaling and Notch2 receptor cleavage, we show that Notch activation in the mesenchyme precedes specification into pdgfrbhigh MCs. Taken together, these results show that Notch signaling is necessary for peri-arterial MC specification.

Triggering of a Dll4-Notch1 loop impairs wound healing in diabetes.

Diabetic foot ulcerations (DFUs) represent a major medical, social, and economic problem. Therapeutic options are restricted due to a poor understanding of the pathogenic mechanisms. The Notch pathway plays a pivotal role in cell differentiation, proliferation, and angiogenesis, processes that are profoundly disturbed in diabetic wounds. Notch signaling is activated upon interactions between membrane-bound Notch receptors (Notch 1-4) and ligands (Jagged 1-2 and Delta-like 1, 3, 4), resulting in cell-context-dependent outputs. Here, we report that Notch1 signaling is activated by hyperglycemia in diabetic skin and specifically impairs wound healing in diabetes. Local inhibition of Notch1 signaling in experimental wounds markedly improves healing exclusively in diabetic, but not in nondiabetic, animals. Mechanistically, high glucose levels activate a specific positive Delta-like 4 (Dll4)-Notch1 feedback loop. Using loss-of-function genetic approaches, we demonstrate that Notch1 inactivation in keratinocytes is sufficient to cancel the repressive effects of the Dll4-Notch1 loop on wound healing in diabetes, thus making Notch1 signaling an attractive locally therapeutic target for the treatment of DFUs.

MIEF1/2 orchestrate mitochondrial dynamics through direct engagement with both the fission and fusion machineries.

BACKGROUND: Mitochondrial dynamics is the result of a dynamic balance between fusion and fission events, which are driven via a set of mitochondria-shaping proteins. These proteins are generally considered to be binary components of either the fission or fusion machinery, but potential crosstalk between the fission and fusion machineries remains less explored. In the present work, we analyzed the roles of mitochondrial elongation factors 1 and 2 (MIEF1/2), core components of the fission machinery in mammals. RESULTS: We show that MIEFs (MIEF1/2), besides their action in the fission machinery, regulate mitochondrial fusion through direct interaction with the fusion proteins Mfn1 and Mfn2, suggesting that MIEFs participate in not only fission but also fusion. Elevated levels of MIEFs enhance mitochondrial fusion in an Mfn1/2- and OPA1-dependent but Drp1-independent manner. Moreover, mitochondrial localization and self-association of MIEFs are crucial for their fusion-promoting ability. In addition, we show that MIEF1/2 can competitively decrease the interaction of hFis1 with Mfn1 and Mfn2, alleviating hFis1-induced mitochondrial fragmentation and contributing to mitochondrial fusion. CONCLUSIONS: Our study suggests that MIEFs serve as a central hub that interacts with and regulates both the fission and fusion machineries, which uncovers a novel mechanism for balancing these opposing forces of mitochondrial dynamics in mammals.

Roles of Notch Signaling in the Tumor Microenvironment.

The Notch signaling pathway is an architecturally simple signaling mechanism, well known for its role in cell fate regulation during organ development and in tissue homeostasis. In keeping with its importance for normal development, dysregulation of Notch signaling is increasingly associated with different types of tumors, and proteins in the Notch signaling pathway can act as oncogenes or tumor suppressors, depending on the cellular context and tumor type. In addition to a role as a driver of tumor initiation and progression in the tumor cells carrying oncogenic mutations, it is an emerging realization that Notch signaling also plays a role in non-mutated cells in the tumor microenvironment. In this review, we discuss how aberrant Notch signaling can affect three types of cells in the tumor stroma-cancer-associated fibroblasts, immune cells and vascular cells-and how this influences their interactions with the tumor cells. Insights into the roles of Notch in cells of the tumor environment and the impact on tumor-stroma interactions will lead to a deeper understanding of Notch signaling in cancer and inspire new strategies for Notch-based tumor therapy.

Emerging links between cerebrovascular and neurodegenerative diseases-a special role for pericytes.

Neurodegenerative and cerebrovascular diseases cause considerable human suffering, and therapy options for these two disease categories are limited or non-existing. It is an emerging notion that neurodegenerative and cerebrovascular diseases are linked in several ways, and in this review, we discuss the current status regarding vascular dysregulation in neurodegenerative disease, and conversely, how cerebrovascular diseases are associated with central nervous system (CNS) degeneration and dysfunction. The emerging links between neurodegenerative and cerebrovascular diseases are reviewed with a particular focus on pericytes-important cells that ensheath the endothelium in the microvasculature and which are pivotal for blood-brain barrier function and cerebral blood flow. Finally, we address how novel molecular and cellular insights into pericytes and other vascular cell types may open new avenues for diagnosis and therapy development for these important diseases.

Notch activation in the mouse mammary luminal lineage leads to ductal hyperplasia and altered partitioning of luminal cell subtypes.

Hyperactivated Notch signalling has been implicated in breast cancer, but how elevated levels of Notch signalling contribute to mammary dysplasia and tumorigenesis is not fully understood. In this study, we express an activated form of Notch1 in the mouse mammary luminal lineage and analyse the consequences for tumour formation and the transcriptomic landscape in the luminal lineage. Simultaneous conditional activation of a Notch1 intracellular domain (Notch1 ICD) and EGFP in the luminal lineage was achieved by removal of a stop cassette by CRE-recombinase expression from the whey acidic protein (WAP) promoter. Mice in which Notch1 ICD was activated in the luminal lineage (WAP-CRE;R26-N1ICD mice) exhibit ductal hyperplasia after lactation with an increase in branching frequency and in the number of side-branch ends in the ductal tree. A subset of the mice developed mammary tumours and the majority of the tumour cells expressed EGFP (as a proxy for Notch1 ICD), indicating that the tumours originate from the Notch1 ICD-expressing cells. Single-cell transcriptome analysis of the EGFP-positive mammary cells identified six subtypes of luminal cells. The same six subtypes were found in control mice (WAP-CRE;R26-tdTomato mice expressing the tdTomato reporter from WAP-CRE-mediated activation), but the proportion of cells in the various subtypes differed between the WAP-CRE;R26-N1ICD and control WAP-CRE;R26-tdTomato mice. In conclusion, we show that Notch1 ICD expression in the luminal lineage produces a ductal hyperplasia and branching phenotype accompanied by altered luminal cell subtype partitioning.

The phosphorylation status of Ser-637 in dynamin-related protein 1 (Drp1) does not determine Drp1 recruitment to mitochondria.

Recruitment of the GTPase dynamin-related protein 1 (Drp1) to mitochondria is a central step required for mitochondrial fission. Reversible Drp1 phosphorylation has been implicated in the regulation of this process, but whether Drp1 phosphorylation at Ser-637 determines its subcellular localization and fission activity remains to be fully elucidated. Here, using HEK 293T cells and immunofluorescence, immunoblotting, RNAi, subcellular fractionation, co-immunoprecipitation assays, and CRISPR/Cas9 genome editing, we show that Drp1 phosphorylated at Ser-637 (Drp1pS637) resides both in the cytosol and on mitochondria. We found that the receptors mitochondrial fission factor (Mff) and mitochondrial elongation factor 1/2 (MIEF1/2) interact with and recruit Drp1pS637 to mitochondria and that elevated Mff or MIEF levels promote Drp1pS637 accumulation on mitochondria. We also noted that protein kinase A (PKA), which mediates phosphorylation of Drp1 on Ser-637, is partially present on mitochondria and interacts with both MIEFs and Mff. PKA knockdown did not affect the Drp1-Mff interaction, but slightly enhanced the interaction between Drp1 and MIEFs. In Drp1-deficient HEK 293T cells, both phosphomimetic Drp1-S637D and phospho-deficient Drp1-S637A variants, like wild-type Drp1, located to the cytosol and to mitochondria and rescued a Drp1 deficiency-induced mitochondrial hyperfusion phenotype. However, Drp1-S637D was less efficient than Drp1-WT and Drp1-S637A in inducing mitochondrial fission. In conclusion, the Ser-637 phosphorylation status in Drp1 is not a determinant that controls Drp1 recruitment to mitochondria.

Beta-amyloid deposition around hepatic bile ducts is a novel pathobiological and diagnostic feature of biliary atresia.

BACKGROUND AND AIMS: Biliary atresia (BA) is a poorly understood and devastating obstructive bile duct disease of newborns. It is often diagnosed late, is incurable and frequently requires liver transplantation. In this study, we aimed to investigate the underlying pathogenesis and molecular signatures associated with BA. METHODS: We combined organoid and transcriptomic analysis to gain new insights into BA pathobiology using patient samples and a mouse model of BA. RESULTS: Liver organoids derived from patients with BA and a rhesus rotavirus A-infected mouse model of BA, exhibited aberrant morphology and disturbed apical-basal organization. Transcriptomic analysis of BA organoids revealed a shift from cholangiocyte to hepatocyte transcriptional signatures and altered beta-amyloid-related gene expression. Beta-amyloid accumulation was observed around the bile ducts in BA livers and exposure to beta-amyloid induced the aberrant morphology in control organoids. CONCLUSION: The novel observation that beta-amyloid accumulates around bile ducts in the livers of patients with BA has important pathobiological implications, as well as diagnostic potential. LAY SUMMARY: Biliary atresia is a poorly understood and devastating obstructive bile duct disease of newborns. It is often diagnosed late, is incurable and frequently requires liver transplantation. Using human and mouse 'liver mini-organs in the dish', we unexpectedly identified beta-amyloid deposition - the main pathological feature of Alzheimer's disease and cerebral amyloid angiopathy - around bile ducts in livers from patients with biliary atresia. This finding reveals a novel pathogenic mechanism that could have important diagnostic and therapeutic implications.

Canonical Notch signaling is dispensable for adult steady-state and stress myelo-erythropoiesis.

Although an essential role for canonical Notch signaling in generation of hematopoietic stem cells in the embryo and in thymic T-cell development is well established, its role in adult bone marrow (BM) myelopoiesis remains unclear. Some studies, analyzing myeloid progenitors in adult mice with inhibited Notch signaling, implicated distinct roles of canonical Notch signaling in regulation of progenitors for the megakaryocyte, erythroid, and granulocyte-macrophage cell lineages. However, these studies might also have targeted other pathways. Therefore, we specifically deleted, in adult BM, the transcription factor recombination signal-binding protein J κ (Rbpj), through which canonical signaling from all Notch receptors converges. Notably, detailed progenitor staging established that canonical Notch signaling is fully dispensable for all investigated stages of megakaryocyte, erythroid, and myeloid progenitors in steady state unperturbed hematopoiesis, after competitive BM transplantation, and in stress-induced erythropoiesis. Moreover, expression of key regulators of these hematopoietic lineages and Notch target genes were unaffected by Rbpj deficiency in BM progenitor cells.

Mouse Model of Alagille Syndrome and Mechanisms of Jagged1 Missense Mutations.

BACKGROUND & AIMS: Alagille syndrome is a genetic disorder characterized by cholestasis, ocular abnormalities, characteristic facial features, heart defects, and vertebral malformations. Most cases are associated with mutations in JAGGED1 (JAG1), which encodes a Notch ligand, although it is not clear how these contribute to disease development. We aimed to develop a mouse model of Alagille syndrome to elucidate these mechanisms. METHODS: Mice with a missense mutation (H268Q) in Jag1 (Jag1+/Ndr mice) were outbred to a C3H/C57bl6 background to generate a mouse model for Alagille syndrome (Jag1Ndr/Ndr mice). Liver tissues were collected at different timepoints during development, analyzed by histology, and liver organoids were cultured and analyzed. We performed transcriptome analysis of Jag1Ndr/Ndr livers and livers from patients with Alagille syndrome, cross-referenced to the Human Protein Atlas, to identify commonly dysregulated pathways and biliary markers. We used species-specific transcriptome separation and ligand-receptor interaction assays to measure Notch signaling and the ability of JAG1Ndr to bind or activate Notch receptors. We studied signaling of JAG1 and JAG1Ndr via NOTCH 1, NOTCH2, and NOTCH3 and resulting gene expression patterns in parental and NOTCH1-expressing C2C12 cell lines. RESULTS: Jag1Ndr/Ndr mice had many features of Alagille syndrome, including eye, heart, and liver defects. Bile duct differentiation, morphogenesis, and function were dysregulated in newborn Jag1Ndr/Ndr mice, with aberrations in cholangiocyte polarity, but these defects improved in adult mice. Jag1Ndr/Ndr liver organoids collapsed in culture, indicating structural instability. Whole-transcriptome sequence analyses of liver tissues from mice and patients with Alagille syndrome identified dysregulated genes encoding proteins enriched at the apical side of cholangiocytes, including CFTR and SLC5A1, as well as reduced expression of IGF1. Exposure of Notch-expressing cells to JAG1Ndr, compared with JAG1, led to hypomorphic Notch signaling, based on transcriptome analysis. JAG1-expressing cells, but not JAG1Ndr-expressing cells, bound soluble Notch1 extracellular domain, quantified by flow cytometry. However, JAG1 and JAG1Ndr cells each bound NOTCH2, and signaling from NOTCH2 signaling was reduced but not completely inhibited, in response to JAG1Ndr compared with JAG1. CONCLUSIONS: In mice, expression of a missense mutant of Jag1 (Jag1Ndr) disrupts bile duct development and recapitulates Alagille syndrome phenotypes in heart, eye, and craniofacial dysmorphology. JAG1Ndr does not bind NOTCH1, but binds NOTCH2, and elicits hypomorphic signaling. This mouse model can be used to study other features of Alagille syndrome and organ development.