A mouse model of Alagille syndrome: Notch2 as a genetic modifier of Jag1 haploinsufficiency

Alagille syndrome is a human autosomal dominant developmental disorder characterized by liver, heart, eye, skeletal, craniofacial and kidney abnormalities. Alagille syndrome is caused by mutations in the Jagged 1 (JAG1) gene, which encodes a ligand for Notch family receptors. The majority of JAG1 mutations seen in Alagille syndrome patients are null alleles, suggesting JAG1 haploinsufficiency as a primary cause of this disorder. Mice homozygous for a Jag1 null mutation die during embryogenesis and Jag1/+ heterozygous mice exhibit eye defects but do not exhibit other phenotypes characteristic of Alagille syndrome patients ( Xue, Y., Gao, X., Lindsell, C. E., Norton, C. R., Chang, B., Hicks, C., Gendron-Maguire, M., Rand, E. B., Weinmaster, G. and Gridley, T. (1999) HUM: Mol. Genet. 8, 723-730). Here we report that mice doubly heterozygous for the Jag1 null allele and a Notch2 hypomorphic allele exhibit developmental abnormalities characteristic of Alagille syndrome. Double heterozygous mice exhibit jaundice, growth retardation, impaired differentiation of intrahepatic bile ducts and defects in heart, eye and kidney development. The defects in bile duct epithelial cell differentiation and morphogenesis in the double heterozygous mice are similar to defects in epithelial morphogenesis of Notch pathway mutants in Drosophila, suggesting that a role for the Notch signaling pathway in regulating epithelial morphogenesis has been conserved between insects and mammals. This work also demonstrates that the Notch2 and Jag1 mutations interact to create a more representative mouse model of Alagille syndrome and provides a possible explanation of the variable phenotypic expression observed in Alagille syndrome patients.     

NOTCH2 mutations cause Alagille syndrome, a heterogeneous disorder of the notch signaling pathway

Alagille syndrome (AGS) is caused by mutations in the gene for the Notch signaling pathway ligand Jagged1 (JAG1), which are found in 94% of patients. To identify the cause of disease in patients without JAG1 mutations, we screened 11 JAG1 mutation-negative probands with AGS for alterations in the gene for the Notch2 receptor (NOTCH2). We found NOTCH2 mutations segregating in two families and identified five affected individuals. Renal manifestations, a minor feature in AGS, were present in all the affected individuals. This demonstrates that AGS is a heterogeneous disorder and implicates NOTCH2 mutations in human disease.     

Notch signaling regulates bile duct morphogenesis in mice

Background

Alagille syndrome is a developmental disorder caused predominantly by mutations in the Jagged1 (JAG1) gene, which encodes a ligand for Notch family receptors. A characteristic feature of Alagille syndrome is intrahepatic bile duct paucity. We described previously that mice doubly heterozygous for Jag1 and Notch2 mutations are an excellent model for Alagille syndrome. However, our previous study did not establish whether bile duct paucity in Jag1/Notch2 double heterozygous mice resulted from impaired differentiation of bile duct precursor cells, or from defects in bile duct morphogenesis.

Methodology/Principal Findings

Here we characterize embryonic biliary tract formation in our previously described Jag1/Notch2 double heterozygous Alagille syndrome model, and describe another mouse model of bile duct paucity resulting from liver-specific deletion of the Notch2 gene.

Conclusions/Significance

Our data support a model in which bile duct paucity in Notch pathway loss of function mutant mice results from defects in bile duct morphogenesis rather than cell fate specification.     

Human Jagged 1 mutants cause liver defect in Alagille syndrome by overexpression of hepatocyte growth factor

Alagille syndrome (AGS, MIM 118450) is an autosomal dominant inherited disease. Paucity of interlobular bile ducts is one of the major abnormalities. To explore the molecular mechanism by which mutation in the human Jagged 1 gene (JAG1, MIM 601920) causes liver defects, we investigated the gene regulation of JAG1 to hepatocyte growth factor gene (HGF). By transfecting wild-type and mutant JAG1 into COS-7 cells in vitro, we found that HGF is a target gene of JAG1 downstream. Wild-type JAG1 is inhibitory for HGF expression and mutant JAG1s relieve the inhibition. Several domain disruptions in mutant JAG1 protein reveal a reduced inhibition to HGF expression at different levels. JAG1 mutations actually result in HGF overexpression. Furthermore, JAG1 controls HGF expression by a dosage-dependent regulation and Notch2 signaling seems to mediate JAG1 function. Given that HGF plays a critical role in differentiation of hepatic stem cells, overexpression of HGF acts on off-balanced cell fate determination in AGS patients. Hepatic stem cells may differentiate towards more hepatocytes but less biliary cells, thus causing the paucity of interlobular bile ducts in liver development of AGS. Our novel findings demonstrated that dosage-dependent regulation by mutations of JAG1 is a fundamental mechanism for liver abnormality in AGS.     

Conditional JAG1 mutation shows the developing heart is more sensitive than developing liver to JAG1 dosage

Mutations of Jagged 1 (JAG1), a ligand in the Notch signaling pathway, cause Alagille syndrome (AGS). AGS is an autosomal dominant, multisystem disorder with variable expressivity, characterized by bile duct paucity and resultant liver disease in combination with cardiac, ocular, skeletal, and facial findings. JAG1 mutations in AGS include gene deletions and protein truncating, splicing, and missense mutations, suggesting that haploinsufficiency is the mechanism of disease causation. With limited exceptions, there is no genotype-phenotype correlation. We have studied a JAG1 missense mutation (JAG1-G274D) that was previously identified in 13 individuals from an extended family with cardiac defects of the type seen in patients with AGS (e.g., peripheral pulmonic stenosis and tetralogy of Fallot) in the absence of liver dysfunction. Our data indicate that this mutation is "leaky." Two populations of proteins are produced from this allele. One population is abnormally glycosylated and is retained intracellularly rather than being transported to the cell surface. A second population is normally glycosylated and is transported to the cell surface, where it is able to signal to the Notch receptor. The JAG1-G274D protein is temperature sensitive, with more abnormally glycosylated (and nonfunctional) molecules produced at higher temperatures. Carriers of this mutation therefore have >50% but <100% of the normal concentration of JAG1 molecules on the cell surface. The cardiac-specific phenotype associated with this mutation suggests that the developing heart is more sensitive than the developing liver to decreased dosage of JAG1.     

Jagged1 functions downstream of Twist1 in the specification of the coronal suture and the formation of a boundary between osteogenic and non-osteogenic cells

The Notch pathway is crucial for a wide variety of developmental processes including the formation of tissue boundaries. That it may function in calvarial suture development and figure in the pathophysiology of craniosynostosis was suggested by the demonstration that heterozygous loss of function of JAGGED1 in humans can cause Alagille syndrome, which has craniosynostosis as a feature. We used conditional gene targeting to examine the role of Jagged1 in the development of the skull vault. We demonstrate that Jagged1 is expressed in a layer of mesoderm-derived sutural cells that lie along the osteogenic-non-osteogenic boundary. We show that inactivation of Jagged1 in the mesodermal compartment of the coronal suture, but not in the neural crest compartment, results in craniosynostosis. Mesodermal inactivation of Jagged1 also results in changes in the identity of sutural cells prior to overt osteogenic differentiation, as well as defects in the boundary between osteogenic and non-osteogenic compartments at the coronal suture. These changes, surprisingly, are associated with increased expression of Notch2 and the Notch effector, Hes1, in the sutural mesenchyme. They are also associated with an increase in nuclear β-catenin. In Twist1 mutants, Jagged1 expression in the suture is reduced substantially, suggesting an epistatic relationship between Twist1 and Jagged1. Consistent with such a relationship, Twist1-Jagged1 double heterozygotes exhibit a substantial increase in the severity of craniosynostosis over individual heterozygotes. Our results thus suggest that Jagged1 is an effector of Twist1 in coronal suture development.     

Spectrum and frequency of jagged1 (JAG1) mutations in Alagille syndrome patients and their families.

Alagille syndrome (AGS) is a dominantly inherited disorder characterized by liver disease in combination with heart, skeletal, ocular, facial, renal, and pancreatic abnormalities. We have recently demonstrated that Jagged1 (JAG1) is the AGS gene. JAG1 encodes a ligand in the Notch intercellular signaling pathway. AGS is the first developmental disorder to be associated with this pathway and the first human disorder caused by a Notch ligand. We have screened 54 AGS probands and family members to determine the frequency of mutations in JAG1. Three patients (6%) had deletions of the entire gene. Of the remaining 51 patients, 35 (69%) had mutations within JAG1, identified by SSCP analysis. Of the 35 identified intragenic mutations, all were unique, with the exceptions of a 5-bp deletion in exon 16, seen in two unrelated patients, and a C insertion at base 1618 in exon 9, also seen in two unrelated patients. The 35 intragenic mutations included 9 nonsense mutations (26%); 2 missense mutations (6%); 11 small deletions (31%), 8 small insertions (23%), and 1 complex rearrangement (3%), all leading to frameshifts; and 4 splice-site mutations (11%). The mutations are spread across the coding sequence of the gene within the evolutionarily conserved motifs of the JAG1 protein. There is no phenotypic difference between patients with deletions of the entire JAG1 gene and those with intragenic mutations, which suggests that one mechanism involved in AGS is haploinsufficiency. The two missense mutations occur at the same amino acid residue. The mechanism by which these missense mutations lead to the disease is not yet understood; however, they suggest that mechanisms other than haploinsufficiency may result in the AGS phenotype.     

Familial deafness,congenital heart defects,and posterior embryotoxon caused by cysteine substitution in the first epidermal-growth-factor-like domain of jagged 1

In the present study, we report a kindred with hearing loss, congenital heart defects, and posterior embryotoxon, segregating as autosomal dominant traits. Six of seven available affected patients manifested mild-to-severe combined hearing loss, predominantly affecting middle frequencies. Two patients were diagnosed with vestibular pathology. All patients had congenital heart defects, including tetralogy of Fallot, ventricular septal defect, or isolated peripheral pulmonic stenosis. No individual in this family met diagnostic criteria for any previously described clinical syndrome. A candidate-gene approach was undertaken and culminated in the identification of a novel Jagged 1 (JAG1) missense mutation (C234Y) in the first cysteine of the first epidermal-growth-factor-like repeat domain of the protein. JAG1 is a cell-surface ligand in the Notch signaling pathway. Mutations in JAG1 have been identified in patients with Alagille syndrome. Our findings revealed a unique phenotype with highly penetrant deafness, posterior embryotoxon, and congenital heart defects but with variable expressivity in a large kindred, which demonstrates that mutation in JAG1 can cause hearing loss.     

JAG1 Mutation Spectrum and Origin in Chinese Children with Clinical Features of Alagille Syndrome

Alagille syndrome is an autosomal dominant disorder that results from defects in the Notch signaling pathway, which is most frequently due to JAG1 mutations. This study investigated the rate, spectrum, and origin of JAG1 mutations in 91 Chinese children presenting with at least two clinical features of Alagille syndrome (cholestasis, heart murmur, skeletal abnormalities, ocular abnormalities, characteristic facial features, and renal abnormalities). Direct sequencing and/or multiplex-ligation-dependent probe amplification were performed in these patients, and segregation analysis was performed using samples available from the parents. JAG1 disease-causing mutations were detected in 70/91 (76.9%) patients, including 29/70 (41.4%) small deletions, 6/70 (8.6%) small insertions, 16/70 (22.9%) nonsense mutations, 8/70 (11.4%) splice-site mutations, 6/70 (9.4%) missense mutations, and 5/70 (7.1%) gross deletions. Of the mutations detected, 45/62 (72.6%) were novel, and almost all were unique, with the exception of c.439C>T, c.439+1G>A, c.703C>T, c.1382_1383delAC, c.2698C>T, and c.2990C>A, which were detected in two cases each; three cases exhibited entire gene deletions. A majority (69.2%) of the point and frameshift mutations could be detected by the sequencing of eleven exons (exons 3, 5, 6, 11, 14, 16, 18, 21, and 23–25). The mutation detection rate was 50.0% (10/20) in atypical cases that only presented with two or three clinical features of Alagille syndrome. Segregation analysis revealed that 81.1% (30/37) of these mutations were de novo. In conclusion, JAG1 mutations are present in the majority of Chinese pediatric patients with clinical features of Alagille syndrome, and the mutations concentrate on different exons from other reports. Genetic study is important for the diagnosis of atypical Alagille syndrome in Chinese patients.     

Alagille Syndrome Mimicking Biliary Atresia in Early Infancy

Alagille syndrome may mimic biliary atresia in early infancy. Since mutations in JAG1 typical for Alagille syndrome type 1 have also been found in biliary atresia, we aimed to identify JAG1 mutations in newborns with proven biliary atresia (n = 72). Five biliary atresia patients with cholestasis, one additional characteristic feature of Alagille syndrome and ambiguous liver histology were single heterozygotes for nonsense or frameshift mutations in JAG1. No mutations were found in the remaining 67 patients. All “biliary atresia” carriers of JAG1 null mutations developed typical Alagille syndrome at the age of three years. Our data do not support association of biliary atresia with JAG1 mutations, at least in Czech patients. Rapid testing for JAG1 mutations could prevent misdiagnosis of Alagille syndrome in early infancy and improve their outcome.     

Analysis of JAG1 gene variant in Chinese patients with Alagille syndrome

Alagille syndrome (AGS) is an autosomal dominant disorder characterized by bile duct paucity. It can be caused by variations in the JAG1 gene encoding a protein of Notch ligand and by variations in the NOTCH2 gene encoding a Notch receptor. In this study we identified 15 different JAG1 gene variations in 17 Chinese patients, nine of which were novel alterations including c.766G > T, c.819delC, c.826delT, c.3099_3100delCA, c.1323_1326delCTGG, c.1771_1775delGTGCGinsT, c.1868delG, c. 2791_2792insA and c.866delG. These alterations were located in the extracellular domain of JAG1, in particular in the DSL and EGF-like repeat domain. All the specific variations in five inheritance cases investigated were de novo. Furthermore, no sequence variation of NOTCH2 was detected in JAG1 alteration negative patients.     

Spectrum of <Emphasis Type="Italic">JAG1</Emphasis> gene mutations in Polish patients with Alagille syndrome

Alagille syndrome (ALGS) is an autosomal dominant disorder characterized by developmental abnormalities in several organs including the liver, heart, eyes, vertebrae, kidneys, and face. The majority (90-94 %) of ALGS cases are caused by mutations in the JAG1 (JAGGED1) gene, and in a small percent of patients (～1 %) mutations in the NOTCH2 gene have been described. Both genes are involved in the Notch signaling pathway. To date, over 440 different JAG1 gene mutations and ten NOTCH2 mutations have been identified in ALGS patients. The present study was conducted on a group of 35 Polish ALGS patients and revealed JAG1 gene mutations in 26 of them. Twenty-three different mutations were detected including 13 novel point mutations and six large deletions affecting the JAG1 gene. Review of all mutations identified to date in individuals from Poland allowed us to propose an effective diagnostic strategy based on the mutations identified in the reported patients of Polish descent. However, the distribution of mutations seen in this cohort was not substantively different than the mutation distribution in other reported populations.     

The Notch ligand JAG1 is required for sensory progenitor development in the mammalian inner ear

In mammals, six separate sensory regions in the inner ear are essential for hearing and balance function. Each sensory region is made up of hair cells, which are the sensory cells, and their associated supporting cells, both arising from a common progenitor. Little is known about the molecular mechanisms that govern the development of these sensory organs. Notch signaling plays a pivotal role in the differentiation of hair cells and supporting cells by mediating lateral inhibition via the ligands Delta-like 1 and Jagged (JAG) 2. However, another Notch ligand, JAG1, is expressed early in the sensory patches prior to cell differentiation, indicating that there may be an earlier role for Notch signaling in sensory development in the ear. Here, using conditional gene targeting, we show that the Jag1 gene is required for the normal development of all six sensory organs within the inner ear. Cristae are completely lacking in Jag1-conditional knockout (cko) mutant inner ears, whereas the cochlea and utricle show partial sensory development. The saccular macula is present but malformed. Using SOX2 and p27^kip1 as molecular markers of the prosensory domain, we show that JAG1 is initially expressed in all the prosensory regions of the ear, but becomes down-regulated in the nascent organ of Corti by embryonic day 14.5, when the cells exit the cell cycle and differentiate. We also show that both SOX2 and p27^kip1 are down-regulated in Jag1-cko inner ears. Taken together, these data demonstrate that JAG1 is expressed early in the prosensory domains of both the cochlear and vestibular regions, and is required to maintain the normal expression levels of both SOX2 and p27^kip1. These data demonstrate that JAG1-mediated Notch signaling is essential during early development for establishing the prosensory regions of the inner ear.     

Distinct Biological Roles for the Notch Ligands Jagged-1 and Jagged-2

Notch signaling is activated in a subset of non-small cell lung cancer cells because of overexpression of Notch3, but the role of Notch ligands has not been fully defined. On the basis of gene expression profiling of a panel of non-small cell lung cancer cell lines, we found that the predominant Notch ligands were JAG1, JAG2, DLL1, and DLL3. Given that Notch ligands reportedly have overlapping receptor binding specificities, we postulated that they have redundant biological roles. Arguing against this hypothesis, we found that JAG1 and JAG2 were differentially regulated; JAG1 expression was dependent upon epidermal growth factor receptor (EGFR) activation in HCC827 cells, which require EGFR for survival, whereas JAG2 expression was EGFR-independent in these cells. Furthermore, HCC827 cells underwent apoptosis following depletion of JAG1 but not JAG2, whereas co-culture experiments revealed that depletion of JAG2, but not JAG1, enhanced the ability of HCC827 cells to chemoattract THP-1 human monocytes. JAG2-depleted HCC827 cells expressed high levels of inflammation-related genes, including interleukin 1 (IL1) and a broad range of IL1-regulated cytokines, which was attenuated by inhibition of IL1 receptor (IL1R). Our findings suggest that JAG1 and JAG2 have distinct biological roles including a previously undiscovered role for JAG2 in regulating the expression of cytokines that can promote antitumor immunity.     

Maintenance of Bone Homeostasis by DLL1‐Mediated Notch Signaling

Adult bone mass is maintained through a balance of the activities of osteoblasts and osteoclasts. Although Notch signaling has been shown to maintain bone homeostasis by controlling the commitment, differentiation, and function of cells in both the osteoblast and osteoclast lineages, the precise mechanisms by which Notch performs such diverse and complex roles in bone physiology remain unclear. By using a transgenic approach that modified the expression of delta‐like 1 (DLL1) or Jagged1 (JAG1) in an osteoblast‐specific manner, we investigated the ligand‐specific effects of Notch signaling in bone homeostasis. This study demonstrated for the first time that the proper regulation of DLL1 expression, but not JAG1 expression, in osteoblasts is essential for the maintenance of bone remodeling. DLL1‐induced Notch signaling was responsible for the expansion of the bone‐forming cell pool by promoting the proliferation of committed but immature osteoblasts. However, DLL1‐Notch signaling inhibited further differentiation of the expanded osteoblasts to become fully matured functional osteoblasts, thereby substantially decreasing bone formation. Osteoblast‐specific expression of DLL1 did not alter the intrinsic differentiation ability of cells of the osteoclast lineage. However, maturational arrest of osteoblasts caused by the DLL1 transgene impaired the maturation and function of osteoclasts due to a failed osteoblast‐osteoclast coupling, resulting in severe suppression of bone metabolic turnover. Taken together, DLL1‐mediated Notch signaling is critical for proper bone remodeling as it regulates the differentiation and function of both osteoblasts and osteoclasts. Our study elucidates the importance of ligand‐specific activation of Notch signaling in the maintenance of bone homeostasis. J. Cell. Physiol. 232: 2569–2580, 2017. © 2016 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals Inc.     

Notch signaling in human development and disease

Mutations in Notch signaling pathway members cause developmental phenotypes that affect the liver, skeleton, heart, eye, face, kidney, and vasculature. Notch associated disorders include the autosomal dominant, multi-system, Alagille syndrome caused by mutations in both a ligand (Jagged1 (JAG1)) and receptor (NOTCH2) and autosomal recessive spondylocostal dysostosis, caused by mutations in a ligand (Delta-like-3 (DLL3)), as well as several other members of the Notch signaling pathway. Mutations in NOTCH2 have also recently been connected to Hajdu-Cheney syndrome, a dominant disorder causing focal bone destruction, osteoporosis, craniofacial morphology and renal cysts. Mutations in the NOTCH1 receptor are associated with several types of cardiac disease and mutations in NOTCH3 cause the dominant adult onset disorder CADASIL (cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy), a vascular disorder with onset in the 4th or 5th decades. Studies of these human disorders and their inheritance patterns and types of mutations reveal insights into the mechanisms of Notch signaling.