Differential Effects of Four Canonical Notch-Activating Ligands on c-Kit+ Cardiac Progenitor Cells.

Notch signaling, an important signaling pathway in cardiac development, has been shown to mediate the reparative functions of c-kit+ progenitor cells (CPCs). However, it is unclear how each of the four canonical Notch-activating ligands affects intracellular processes in c-kit+ cells when used as an external stimulus. Neonatal c-kit+ CPCs were stimulated using four different chimeric Notch-activating ligands tethered to Dynabeads, and the resulting changes were assessed using TaqMan gene expression arrays, with subsequent analysis by principal component analysis (PCA). Additionally, functional outcomes were measured using an endothelial cell tube formation assay and MSC migration assay to assess the paracrine capacity to stimulate new vessel formation and recruit other reparative cell types to the site of injury. Gene expression data showed that stimulation with Jagged-1 is associated with the greatest pro-angiogenic gene response, including the expression of VEGF and basement membrane proteins, while the other canonical ligands, Jagged-2, Dll-1, and Dll-4, are more associated with regulatory and epigenetic changes. The functional assay showed differential responses to the four ligands in terms of angiogenesis, while none of the ligands produced a robust change in migration. These data demonstrate how the four Notch-activating ligands differentially regulate CPC gene expression and function.

Three novel missense variants in two families with JAG2-associated limb-girdle muscular dystrophy.

Limb-girdle muscular dystrophy recessive 27 is associated with biallelic variants in JAG2, encoding the JAG2 notch ligand. Twenty-four affected individuals from multiple families have been described in two reports. We present two Australian families with three novel JAG2 missense variants: (c.1021G>T, p.(Gly341Cys)) homozygous in two siblings of Pakistani origin, and compound heterozygous variants (c.703T>C, p.(Trp235Arg); c.2350C>T, p.(Arg784Cys)) in a proband of European ancestry. Patients presented with childhood-onset limb-girdle-like myopathy with difficulty or inability walking. MRI revealed widespread torso and limb muscle involvement. Muscle pathology showed myopathic changes with fatty infiltration. Muscle RNA sequencing revealed significant downregulation of myogenesis genes PAX7, MYF5, and MEGF10 similar to previous JAG2-related muscular dystrophy cases or Jag2-knockdown cells. In absence of functional assays to characterise JAG2 variants, clinical, MRI and transcriptomic profiling collectively may help discern JAG2-related muscular dystrophy, diagnosis of which is essential for patients and families given the severity of disease and reoccurrence risk.

Jag1/2 maintain esophageal homeostasis and suppress foregut tumorigenesis by restricting the basal progenitor cell pool.

Basal progenitor cells are crucial for maintaining foregut (the esophagus and forestomach) homeostasis. When their function is dysregulated, it can promote inflammation and tumorigenesis. However, the mechanisms underlying these processes remain largely unclear. Here, we employ genetic mouse models to reveal that Jag1/2 regulate esophageal homeostasis and foregut tumorigenesis by modulating the function of basal progenitor cells. Deletion of Jag1/2 in mice disrupts esophageal and forestomach epithelial homeostasis. Mechanistically, Jag1/2 deficiency impairs activation of Notch signaling, leading to reduced squamous epithelial differentiation and expansion of basal progenitor cells. Moreover, Jag1/2 deficiency exacerbates the deoxycholic acid (DCA)-induced squamous epithelial injury and accelerates the initiation of squamous cell carcinoma (SCC) in the forestomach. Importantly, expression levels of JAG1/2 are lower in the early stages of human esophageal squamous cell carcinoma (ESCC) carcinogenesis. Collectively, our study demonstrates that Jag1/2 are important for maintaining esophageal and forestomach homeostasis and the onset of foregut SCC.

Jagged2 targeting in lung cancer activates anti-tumor immunity via Notch-induced functional reprogramming of tumor-associated macrophages.

Signaling through Notch receptors intrinsically regulates tumor cell development and growth. Here, we studied the role of the Notch ligand Jagged2 on immune evasion in non-small cell lung cancer (NSCLC). Higher expression of JAG2 in NSCLC negatively correlated with survival. In NSCLC pre-clinical models, deletion of Jag2, but not Jag1, in cancer cells attenuated tumor growth and activated protective anti-tumor T cell responses. Jag2-/- lung tumors exhibited higher frequencies of macrophages that expressed immunostimulatory mediators and triggered T cell-dependent anti-tumor immunity. Mechanistically, Jag2 ablation promoted Nr4a-mediated induction of Notch ligands DLL1/4 on cancer cells. DLL1/4-initiated Notch1/2 signaling in macrophages induced the expression of transcription factor IRF4 and macrophage immunostimulatory functionality. IRF4 expression was required for the anti-tumor effects of Jag2 deletion in lung tumors. Antibody targeting of Jagged2 inhibited tumor growth and activated IRF4-driven macrophage-mediated anti-tumor immunity. Thus, Jagged2 orchestrates immunosuppressive systems in NSCLC that can be overcome to incite macrophage-mediated anti-tumor immunity.

miR-2392 functions as tumour suppressor and inhibits malignant progression of hepatocellular carcinoma via directly targeting JAG2.

BACKGROUND & AIMS: Dysregulation of microRNA (miRNA) expression in various cancers and their vital roles in malignant progression of cancers are well investigated. Our previous studies have analysed miRNAs that promote malignant progression in hepatocellular carcinoma (HCC); this study aims to systematically elucidate the mechanism of metastasis suppressor miRNAs in HCC. METHODS: High-throughput RNA sequencing was used to identify anti-metastatic miRNAs. The relative expression levels of miRNAs were confirmed by qRT-PCR. The biological functions of miRNAs were detected in vitro and in vivo. Circulating tumour cells (CTCs) were enriched from blood samples of HCC patients and cultured by three-dimensional (3D) system. Kaplan-Meier and Cox regression were used to analyse the value of potential target mRNAs on overall survival. RESULTS: miR-2392 was significantly down-regulated in HCC. Overexpression of miR-2392 suppressed proliferation, clonogenicity, mobility, spheroid formation and maintenance of cancer stem cells (CSC)-like characteristics in HCC cells. CTCs from HCC patients with lower serum miR-2392 level had stronger cell spheroid formation ability. A negative correlation between the content of miR-2392 in serum and the number of CTC spheroids had been found. We identified Jagged2 (JAG2) as a direct target of miR-2392. miR-2392 inhibited the expression of JAG2 by targeting 3'-UTR of JAG2. Down-regulation of JAG2 inhibited the overexpression effects of miR-2392 in vitro and in vivo. JAG2 is highly expressed in HCC and is closely related to poor prognosis and survival of patients. CONCLUSIONS: miR-2392 may play a role as a tumour suppressor to guide the individualized precise treatment of HCC.

Delta/Jagged-mediated Notch signaling induces the differentiation of agr2-positive epidermal mucous cells in zebrafish embryos.

Teleosts live in aquatic habitats, where they encounter ionic and acid-base fluctuations as well as infectious pathogens. To protect from these external challenges, the teleost epidermis is composed of living cells, including keratinocytes and ionocytes that maintain body fluid ionic homeostasis, and mucous cells that secret mucus. While ionocyte progenitors are known to be specified by Delta-Notch-mediated lateral inhibition during late gastrulation and early segmentation, it remains unclear how epidermal mucous cells (EMCs) are differentiated and maintained. Here, we show that Delta/Jagged-mediated activation of Notch signaling induces the differentiation of agr2-positive (agr2+) EMCs in zebrafish embryos during segmentation. We demonstrated that agr2+ EMCs contain cytoplasmic secretory granules and express muc5.1 and muc5.2. Reductions in agr2+ EMC number were observed in mib mutants and notch3 MOs-injected notch1a mutants, while increases in agr2+ cell number were detected in notch1a- and X-Su(H)/ANK-overexpressing embryos. Treatment with γ-secretase inhibitors further revealed that Notch signaling is required during bud to 15 hpf for the differentiation of agr2+ EMCs. Increased agr2+ EMC numbers were also observed in jag1a-, jag1b-, jag2a- and dlc-overexpressing, but not jag2b-overexpressing embryos. Meanwhile, reductions in agr2+ EMC numbers were detected in jag1a morphants, jag1b mutants, jag2a mutants and dlc morphants, but not jag2b mutants. Reduced numbers of pvalb8-positive epidermal cells were also observed in mib or jag2a mutants and jag1a or jag1b morphants, while increased pvalb8-positive epidermal cell numbers were detected in notch1a-overexpressing, but not dlc-overexpressing embryos. BrdU labeling further revealed that the agr2+ EMC population is maintained by proliferation. Cell lineage experiments showed that agr2+ EMCs are derived from the same ectodermal precursors as keratinocytes or ionocytes. Together, our results indicate that specification of agr2+ EMCs in zebrafish embryos is induced by DeltaC/Jagged-dependent activation of Notch1a/3 signaling, and the cell population is maintained by proliferation.

A form of muscular dystrophy associated with pathogenic variants in JAG2.

JAG2 encodes the Notch ligand Jagged2. The conserved Notch signaling pathway contributes to the development and homeostasis of multiple tissues, including skeletal muscle. We studied an international cohort of 23 individuals with genetically unsolved muscular dystrophy from 13 unrelated families. Whole-exome sequencing identified rare homozygous or compound heterozygous JAG2 variants in all 13 families. The identified bi-allelic variants include 10 missense variants that disrupt highly conserved amino acids, a nonsense variant, two frameshift variants, an in-frame deletion, and a microdeletion encompassing JAG2. Onset of muscle weakness occurred from infancy to young adulthood. Serum creatine kinase (CK) levels were normal or mildly elevated. Muscle histology was primarily dystrophic. MRI of the lower extremities revealed a distinct, slightly asymmetric pattern of muscle involvement with cores of preserved and affected muscles in quadriceps and tibialis anterior, in some cases resembling patterns seen in POGLUT1-associated muscular dystrophy. Transcriptome analysis of muscle tissue from two participants suggested misregulation of genes involved in myogenesis, including PAX7. In complementary studies, Jag2 downregulation in murine myoblasts led to downregulation of multiple components of the Notch pathway, including Megf10. Investigations in Drosophila suggested an interaction between Serrate and Drpr, the fly orthologs of JAG1/JAG2 and MEGF10, respectively. In silico analysis predicted that many Jagged2 missense variants are associated with structural changes and protein misfolding. In summary, we describe a muscular dystrophy associated with pathogenic variants in JAG2 and evidence suggests a disease mechanism related to Notch pathway dysfunction.

Cholera toxin induces food allergy through Th2 cell differentiation which is unaffected by Jagged2.

AIMS: Cholera toxin is often used to induce food allergies. However, its exact mode of action and effect remain ambiguous. In this study, we established a BALB/c mouse cholera toxin/ovalbumin-induced food allergy model to determine the molecular basis and signaling mechanisms of the immune regulation of cholera toxin during food allergy. MATERIALS AND METHODS: The adjuvant activity of cholera toxin was analyzed by establishing mouse allergy model, and the allergic reaction of each group of mice was evaluated. The effect of cholera toxin on Th1/Th2 cell differentiation was analyzed to further explore the role of cholera toxin in allergen immune response. We stimulated bone marrow-derived dendritic cells (BMDCs) with cholera toxin in vitro to investigate the effect of cholera toxin on Notch ligand expression. BMDCs and naive CD4+T cells were co-cultured in vitro, and their cytokine levels were examined to investigate whether cholera toxin regulates Th cell differentiation via the Jagged2 Notch signaling pathway. KEY FINDINGS: The results showed that in the presence of allergens, cholera toxin promotes Th2 cell differentiation and enhances the body's immune response. Cholera toxin induces expression of the Notch ligand Jagged2, but Jagged2 Notch signaling pathway is not required to promote BMDCs-mediated differentiation of Th2 cells. SIGNIFICANCE: This study initially revealed the mechanism by which cholera toxin plays an adjuvant role in food allergy, and provides reference for future related research.

[Association between the JAG2 gene polymorphism and the occurrence of nonsyndromic cleft lip with or without cleft palate in northwest Chinese population].

OBJECTIVE: To assess the association of JAG2 gene single nucleotide polymorphisms with the occurrence of nonsyndromic cleft lip with or without cleft palate (NSCLP) among northwest Chinese population. METHODS: A case-control study was carried out on 301 NSCLP patients and 304 healthy controls. An iMLDR(TM) genotyping technique was used to detect three single nucleotide polymorphisms (SNPs) [rs741859 (T/C), rs11621316 (A/G) and rs1057744(C/T)] of the JAG2 gene. Allelic and genotypic frequencies and haplotypic distribution among the two groups were compared. RESULTS: A significant difference was found in the frequency of C and T alleles for rs741859 between the two groups. The CT genotype of rs741859 could significantly reduce the risk for NSCLP to 65% (P< 0.05) and the risk for cleft lip with or without cleft palate (CL/P) to 62% (P< 0.05). rs11621316 and rs1057744 are in the same linkage disequilibrium (LD) region with a high degree of linkage (γ 2> 0.8), whose distribution difference between the two groups was not statistically significant (P> 0.05). CONCLUSION: The CT genotype of the JAG2 gene rs741859 may confer a protective effect for NSCLP among northwest Chinese population.

Multiple myeloma exploits Jagged1 and Jagged2 to promote intrinsic and bone marrow-dependent drug resistance.

Multiple myeloma is still incurable due to an intrinsic aggressiveness or, more frequently, to the interactions of malignant plasma cells with the bone marrow (BM) microenvironment. Myeloma cells educate BM cells to support neoplastic cell growth, survival, acquisition of drug resistance resulting in disease relapse. Myeloma microenvironment is characterized by Notch signaling hyperactivation due to the increased expression of Notch1 and 2 and the ligands Jagged1 and 2 in tumor cells. Notch activation influences myeloma cell biology and promotes the reprogramming of BM stromal cells. In this work we demonstrate, in vitro, ex vivo and by using a zebrafish multiple myeloma model, that Jagged inhibition causes a decrease in both myeloma-intrinsic and stromal cell-induced resistance to currently used drugs, i.e. bortezomib, lenalidomide and melphalan. The molecular mechanism of drug resistance involves the chemokine system CXCR4/SDF1α. Myeloma cell-derived Jagged ligands trigger Notch activity in BM stromal cells. These, in turn, secrete higher levels of SDF1α in the BM microenvironment increasing CXCR4 activation in myeloma cells, which is further potentiated by the concomitant increased expression of this receptor induced by Notch activation. Consistently with the augmented pharmacological resistance, SDF1α boosts the expression of BCL2, Survivin and ABCC1. These results indicate that a Jagged-tailored approach may contribute to disrupting the pharmacological resistance due to intrinsic myeloma cell features or to the pathological interplay with BM stromal cells and, conceivably, improve patients' response to standard-of-care therapies.

Haematopoietic stem cells in perisinusoidal niches are protected from ageing.

With ageing, intrinsic haematopoietic stem cell (HSC) activity decreases, resulting in impaired tissue homeostasis, reduced engraftment following transplantation and increased susceptibility to diseases. However, whether ageing also affects the HSC niche, and thereby impairs its capacity to support HSC function, is still widely debated. Here, by using in-vivo long-term label-retention assays we demonstrate that aged label-retaining HSCs, which are, in old mice, the most quiescent HSC subpopulation with the highest regenerative capacity and cellular polarity, reside predominantly in perisinusoidal niches. Furthermore, we demonstrate that sinusoidal niches are uniquely preserved in shape, morphology and number on ageing. Finally, we show that myeloablative chemotherapy can selectively disrupt aged sinusoidal niches in the long term, which is linked to the lack of recovery of endothelial Jag2 at sinusoids. Overall, our data characterize the functional alterations of the aged HSC niche and unveil that perisinusoidal niches are uniquely preserved and thereby protect HSCs from ageing.

Crosstalk between BMP and Notch Induces Sox2 in Cerebral Endothelial Cells.

Bone morphogenetic protein (BMP) and Notch signaling are critical for endothelial cell (EC) differentiation in vascular development. Recent studies have shown that excess BMP activity induces Notch signaling in cerebral ECs resulting in arteriovenous malformation (AVMs). However, it is unclear how the crosstalk between BMP and Notch signaling affects cerebral EC differentiation at the gene regulatory level. Here, we report that BMP6 activates the activin receptor-like kinase (ALK) 3, a BMP type 1 receptor, to induce Notch1 receptor and Jagged1 and Jagged2 ligands. We show that increased expression of the Notch components alters the transcriptional regulatory complex in the SRY-Box 2 (Sox2) promoter region so as to induce its expression in cerebral ECs. Together, our results identify Sox2 as a direct target of BMP and Notch signaling and provide information on how altered BMP and Notch signaling affects the endothelial transcriptional landscape.

Determinant roles of dendritic cell-expressed Notch Delta-like and Jagged ligands on anti-tumor T cell immunity.

BACKGROUND: Notch intercellular communication instructs tissue-specific T-cell development and function. In this study, we explored the roles of dendritic cell (DC)-expressed Notch ligands in the regulation of T-cell effector function. METHODS: We generated mice with CD11c lineage-specific deletion of Notch Delta-like ligand (Dll)1 and Jagged (Jag)2. Using these genetically-ablated mice and engineered pharmacological Notch ligand constructs, the roles of various Delta-like and Jagged ligands in the regulation of T-cell-mediated immunity were investigated. We assessed tumor growth, mouse survival, cytokine production, immunophenotyping of myeloid and lymphoid populations infiltrating the tumors, expression of checkpoint molecules and T-cell function in the experimental settings of murine lung and pancreatic tumors and cardiac allograft rejection. Correlative studies were also performed for the expression of NOTCH ligands, NOTCH receptors and PD-1 on various subsets of myeloid and lymphoid cells in tumor-infiltrating immune cells analyzed from primary human lung cancers. RESULTS: Mice with CD11c lineage-specific deletion of Notch ligand gene Dll1, but not Jag2, exhibited accelerated growth of lung and pancreatic tumors concomitant with decreased antigen-specific CD8+T-cell functions and effector-memory (Tem) differentiation. Increased IL-4 but decreased IFN-γ production and elevated populations of T-regulatory and myeloid-derived suppressor cells were observed in Dll1-ablated mice. Multivalent clustered DLL1-triggered Notch signaling overcame DC Dll1 deficiency and improved anti-tumor T-cell responses, whereas the pharmacological interference by monomeric soluble DLL1 construct suppressed the rejection of mouse tumors and cardiac allograft. Moreover, monomeric soluble JAG1 treatment reduced T-regulatory cells and improved anti-tumor immune responses by decreasing the expression of PD-1 on CD8+Tem cells. A significant correlation was observed between DC-expressed Jagged and Delta-like ligands with Tem-expressed PD-1 and Notch receptors, respectively, in human lung tumor-infiltrates. CONCLUSION: Our data show the importance of specific expression of Notch ligands on DCs in the regulation of T-cell effector function. Thus, strategies incorporating selectively engineered Notch ligands could provide a novel approach of therapeutics for modulating immunity in various immunosuppressive conditions including cancer.

Long noncoding RNA ENST00000455974 plays an oncogenic role through up-regulating JAG2 in human DNA mismatch repair-proficient colon cancer.

DNA mismatch repair-proficient colon cancer is the most common type of colon cancer, but its initiation and progression are still unknown. Our previous study has revealed that a long noncoding RNA (lncRNA) ENST00000455974 was significantly associated with TNM stage and distant metastasis in patients with DNA mismatch repair-proficient (pMMR) colon cancer (CC). Here, firstly, we observed that ENST00000455974 was gradual increased across colon normal-adenoma-carcinoma-metastasis sequence by quantitative real-time PCR. Secondly, ENST00000455974 showed a better sensitivity and specificity than CEA and CA19-9 in the diagnosis of pMMR CC by drawing the receiver operating characteristic (ROC) curve. Thirdly, a higher level of ENST00000455974 was associated with a poorer patient survival. Furthermore, Knockdown of ENST00000455974 led to reduced proliferation and migration of colon cancer cells. Mechanistically, ENST00000455974 was mainly located in the nucleus of colon cancer cells and it promoted the growth and metastasis of pMMR CC cells through up-regulating JAG2.

Jagged2 promotes cancer stem cell properties of triple negative breast cancer cells and paclitaxel resistance via regulating microRNA-200.

OBJECTIVE: To investigate the role of Jagged2 in triple negative breast cancer (TNBC) and its underlying mechanism. PATIENTS AND METHODS: Breast cancer tissues of patients diagnosed with TNBC in Fujian Medical University Affiliated MinDong Hospital from January 2015 to September 2017 were selected. TNBC patients were divided into the paclitaxel-resistant group (n=34) and non-resistance group (n=11). Jagged2 expression in paclitaxel-resistant group and non-resistance group before and after treatment was detected by quantitative Real-time-polymerase chain reaction (qRT-PCR), respectively. After Jagged2 knockdown in paclitaxel-resistant MDA-MB-231 cells (MDA-MB-231/TXR), expression of CD44+CD24-ESA+ subset was detected by flow cytometry. MicroRNA-200 expression was detected after Jagged2 knockdown in MDA-MB-231/TXR cells. RESULTS: Jagged2 was highly expressed in paclitaxel-resistant TNBC tissues and cells. Jagged2 expression was found to be associated with cancer stem cell (CSC) properties of TNBC cells. Knockdown of Jagged2 inhibited CSC properties and paclitaxel resistance, whereas upregulated microRNA-200 expression. The inhibited CSC properties and paclitaxel resistance induced by Jagged2 knockdown were reversed by microRNA-200 knockdown. CONCLUSIONS: Jagged2 maintains CSC properties of TNBC cells and paclitaxel resistance via regulating microRNA-200.

Integrated genomic characterization of oral carcinomas in post-hematopoietic stem cell transplantation survivors.

OBJECTIVES: Secondary oral squamous cell carcinoma (OSCC) is a late complication in allogeneic hematopoietic stem cell transplantation (HSCT) patients, but little is known about long-term outcomes and prognostication. Additionally, molecular alterations and immunologic insights unique to this disease remain largely unexplored. METHODS: We present a cohort of 31 patients with post-HSCT OSCC and reported on clinicopathologic predictors of survival. Whole-exome sequencing was performed on 6 (19%) matched pairs of peripheral blood (post-conditioning, pre-HSCT) and tumor samples. The entire cohort had archival tumor available for immunoprofiling with PD-1/L1 immunohistochemistry. RESULTS: Five-year overall survival (OS) was 57% (95% CI: 46.1-69.8) with a median disease-free survival (DFS) of 13.3 months. Advanced initial staging, a buccal or oral tongue subsite, chronic oral graft-versus-host disease (GVHD) and smoking all negatively impacted survival. High tumor mutational burden (TMB) (median 11.3 vs. 5.0) and unique mutational signatures were noted between unrelated and related donor groups - with a strong correlation between infiltrating PD-1+ lymphocytes and TMB (R = 0.98, p < 0.01). Some differences were observed when comparing commonly mutated genes among our cohort and TCGA, with a predominance of TP53 events. CONCLUSION: Survival outcomes appear similar in HSCT survivors with OSCC compared with non-HSCT OSCC populations. We identified somatic alterations in genes with therapeutic potential unique to this subpopulation of oral cancers.

Endothelial jagged-2 sustains hematopoietic stem and progenitor reconstitution after myelosuppression.

Angiocrine factors, such as Notch ligands, supplied by the specialized endothelial cells (ECs) within the bone marrow and splenic vascular niche play an essential role in modulating the physiology of adult hematopoietic stem and progenitor cells (HSPCs). However, the relative contribution of various Notch ligands, specifically jagged-2, to the homeostasis of HSPCs is unknown. Here, we show that under steady state, jagged-2 is differentially expressed in tissue-specific vascular beds, but its expression is induced in hematopoietic vascular niches after myelosuppressive injury. We used mice with EC-specific deletion of the gene encoding jagged-2 (Jag2) to demonstrate that while EC-derived jagged-2 was dispensable for maintaining the capacity of HSPCs to repopulate under steady-state conditions, by activating Notch2 it did contribute to the recovery of HSPCs in response to myelosuppressive conditions. Engraftment and/or expansion of HSPCs was dependent on the expression of endothelial-derived jagged-2 following myeloablation. Additionally, jagged-2 expressed in bone marrow ECs regulated HSPC cell cycle and quiescence during regeneration. Endothelial-deployed jagged-2 triggered Notch2/Hey1, while tempering Notch2/Hes1 signaling in HSPCs. Collectively, these data demonstrate that EC-derived jagged-2 activates Notch2 signaling in HSPCs to promote hematopoietic recovery and has potential as a therapeutic target to accelerate balanced hematopoietic reconstitution after myelosuppression.

Aberrant Global and Jagged-Mediated Notch Signaling Disrupts Segregation Between wt1-Expressing and Steroidogenic Tissues in Zebrafish.

Although the zebrafish interrenal tissue has been used as a model for steroidogenesis and genesis of the adrenal gland, its specification and morphogenesis remains largely unclear. In the present study, we explored how the Wilms tumor 1 (WT1)-expressing cells are segregated from the SF-1-expressing steroidogenic cells in the zebrafish model. The interrenal tissue precursors expressing ff1b, the equivalent of mammalian SF-1, were derived from wt1-expressing pronephric primordia in the zebrafish embryo. Through histochemistry and in situ hybridization, we demonstrated that the size of functionally differentiated interrenal tissue was substantially increased on global inhibition of the Notch signaling pathway and was accompanied by a disrupted segregation between the wt1- and ff1b-expressing cells. As the Notch pathway was conditionally activated during interrenal specification, differentiation, but not ff1b expression, of interrenal tissue was drastically compromised. In embryos deficient for Notch ligands jagged 1b and 2b, transgenic reporter activity of wt1b promoter was detected within the steroidogenic interrenal tissue. In conclusion, our results indicate that Jagged-Notch signaling is required (1) for segregation between wt1-expressing cells and differentiated steroidogenic tissue; and (2) to modulate the extent of functional differentiation in the steroidogenic interrenal tissue.

Endoderm Jagged induces liver and pancreas duct lineage in zebrafish.

Liver duct paucity is characteristic of children born with Alagille Syndrome (ALGS), a disease associated with JAGGED1 mutations. Here, we report that zebrafish embryos with compound homozygous mutations in two Notch ligand genes, jagged1b (jag1b) and jagged2b (jag2b) exhibit a complete loss of canonical Notch activity and duct cells within the liver and exocrine pancreas, whereas hepatocyte and acinar pancreas development is not affected. Further, animal chimera studies demonstrate that wild-type endoderm cells within the liver and pancreas can rescue Notch activity and duct lineage specification in adjacent cells lacking jag1b and jag2b expression. We conclude that these two Notch ligands are directly and solely responsible for all duct lineage specification in these organs in zebrafish. Our study uncovers genes required for lineage specification of the intrahepatopancreatic duct cells, challenges the role of duct cells as progenitors, and suggests a genetic mechanism for ALGS ductal paucity.The hepatopancreatic duct cells connect liver hepatocytes and pancreatic acinar cells to the intestine, but the mechanism for their lineage specification is unclear. Here, the authors reveal that Notch ligands Jagged1b and Jagged2b induce duct cell lineage in the liver and pancreas of the zebrafish.

Notch signaling in T cells is essential for allergic airway inflammation, but expression of the Notch ligands Jagged 1 and Jagged 2 on dendritic cells is dispensable.

BACKGROUND: Allergic asthma is characterized by a TH2 response induced by dendritic cells (DCs) that present inhaled allergen. Although the mechanisms by which they instruct TH2 differentiation are still poorly understood, expression of the Notch ligand Jagged on DCs has been implicated in this process. OBJECTIVE: We sought to establish whether Notch signaling induced by DCs is critical for house dust mite (HDM)-driven allergic airway inflammation (AAI) in vivo. METHODS: The induction of Notch ligand expression on DC subsets by HDM was quantified by using quantitative real-time PCR. We used an HDM-driven asthma mouse model to compare the capacity of Jagged 1 and Jagged 2 single- and double-deficient DCs to induce AAI. In addition, we studied AAI in mice with a T cell-specific deletion of recombination signal-binding protein for immunoglobulin Jκ region (RBPJκ), a downstream effector of Notch signaling. RESULTS: HDM exposure promoted expression of Jagged 1, but not Jagged 2, on DCs. In agreement with published findings, in vitro-differentiated and HDM-pulsed Jagged 1 and Jagged 2 double-deficient DCs lacked the capacity to induce AAI. However, after in vivo intranasal sensitization and challenge with HDM, DC-specific Jagged 1 or Jagged 2 single- or double-deficient mice had eosinophilic airway inflammation and a TH2 cell activation phenotype that was not different from that in control littermates. In contrast, RBPJκ-deficient mice did not experience AAI and airway hyperreactivity. CONCLUSION: Our results show that the Notch signaling pathway in T cells is crucial for the induction of TH2-mediated AAI in an HDM-driven asthma model but that expression of Jagged 1 or Jagged 2 on DCs is not required.