Notch-regulated Ankyrin Repeat Research Articles

Notch-regulated Ankyrin Repeat Protein Is Overexpressed in Different Subtypes of Breast Cancer

Introduction: Breast cancer (BC) is a highly heterogeneous disease that has been classified into several subtypes at the molecular level, each with a distinct outcome. Recently, Notch-regulated ankyrin-repeat protein (NRARP), a gene expressed followed by Notch signal activation, has attracted interest due to its aberrant expression in different types of cancer. Accordingly, this study evaluated the expression levels of NRARP in different subtypes of BC. Methods: The MCF-7, SKBR3, MDA-MB-468, and MDA-MB-231 human BC cell lines, which represent the luminal, human epidermal growth factor receptor 2 (HER2) overexpression, basal A, and basal B subtypes, respectively, as well as MCF-10A as a normal breast epithelial cell for comparison, were selected and grown in the appropriate medium. The relative expression of NRARP in BC cell lines was then determined using the quantitative real-time polymerase chain reaction (qRT-PCR). Results: The results of the qRT-PCR demonstrated that the expression level of NRARP in all BC cell lines was significantly higher than that of the normal breast epithelial cells (P<0.05). However, the significance was more noteworthy in luminal, HER2-overexpressing, and basal A (7.72, 5.81, and 4.6 folds, respectively). Conclusion: NRARP is a potential target gene for further interventional studies due to its abnormal expression in different subtypes of BC.

Downregulation of NRARP exerts anti-tumor activities in the breast tumor cells depending on Wnt/β-catenin-mediated signals: The role of miR-130a-3p.

The Notch-regulated ankyrin repeat protein (NRARP) functions as a molecular link between Notch and Wnt signaling pathways. Although it has recently been identified to be overexpressed in breast cancer (BC), the molecular mechanisms that regulate NRARP remain unknown. Since microRNAs (miRNAs) regulate gene expression post-transcriptionally, miRNA dysregulation could explain the abnormal gene expression. Here, we identified miR-130a-3p as an NRARP regulator and evaluated its effects on the behavior of BC cells. Quantitative real-time PCR was performed to assess the transcriptional levels of miR-130a-3p and NRARP in BC cells. Next, miR-130a-3p was transiently transfected into BC cells to assess its influence on NRARP expression. Owing to the positive regulatory effects of NRARP on the Wnt/β-catenin signaling pathway, we also analyzed the expression levels of five Wnt/β-catenin pathway genes and one downstream target gene in BC cells. We then assessed anti-tumor activities of miR-130a-3p in BC cells using the MTT proliferation assay, the soft agar colony formation assay for anchorage-independent growth (AIG), as well as scratch and transwell assays for cell migration. The results showed that miR-130a-3p was downregulated in BC cells, whereas NRARP was upregulated. Overexpression of miR-130a-3p inhibited the expression of NRARP and some Wnt/β-catenin signaling pathway genes, as well as exerted anti-tumor effects as evidenced by decreased cell proliferation, AIG, and migration of BC cells. In conclusion, the tumor-suppressive function of miR-130a-3p in BC may be mediated by inhibiting NRARP and Wnt/β-catenin signaling pathway. As a result, miR-130a-3p could be introduced as a therapeutic target for miRNA therapy in BC.

First-in-human study of LY3039478, an oral Notch signaling inhibitor in advanced or metastatic cancer

BackgroundDeregulated Notch signaling due to mutation or overexpression of ligands and/or receptors is implicated in various human malignancies. γ-Secretase inhibitors inhibit Notch signaling by preventing cleavage of transmembrane domain of Notch protein. LY3039478 is a novel, potent Notch inhibitor decreases Notch signaling and its downstream biologic effects. In this first-in-human study, we report the safety, pharmacokinetic (PK) profile, pharmacodynamic effects, and antitumor activity of LY3039478 in patients with advanced or metastatic cancer. MethodsThis phase I, open-label, multicenter, nonrandomized, and dose-escalation phase study determined and confirmed the recommended phase II dose of LY3039478 (oral dose: 2.5–100mg, thrice weekly (TIW) on a 28-day cycle). The primary objectives are to determine (part A) and confirm (part B) a recommended phase II dose that may be safely administered to patients with advanced or metastatic cancer, and secondary objectives include evaluation of safety, tolerability, PK parameters, and preliminary antitumor activity of LY3039478. ResultsA total of 110 patients were treated with LY3039478 monotherapy between 31 October 2012 and 15 July 2016. Dose-limiting toxicities were thrombocytopenia, colitis, and nausea. Most adverse events were gastrointestinal. The recommended phase II dose was 50mg TIW, because of its better tolerability compared with 75mg. The PKs of LY3039478 appeared dose proportional. Pharmacodynamic data indicate an ∼80% inhibition of plasma Aβ, and >50% inhibition of Notch-regulated genes hairy and enhancer of split-1, cyclin D1, and Notch-regulated ankyrin repeat at 45–100-mg dose. Clinical activity (tumor necrosis, metabolic response, or tumor shrinkage) was observed in patients with breast cancer, leiomyosarcoma, and adenoid cystic carcinoma. ConclusionPotent inhibition of Notch signaling by LY3039478 was well tolerated at doses associated with target engagement, and demonstrated evidence of clinical activity in heavily pretreated patients. Further investigation with LY3039478 as monotherapy and in combination with targeted agent or chemotherapy is ongoing. Clinicaltrials.gov IDNCT01695005.

Abstract 2787: Identifying predictive markers of endocrine response in high-grade serous ovarian cancer using RNA sequencing

Abstract Epithelial ovarian cancer is a complex disease and patients vary considerably in response to treatment. A subset of patients responds well to endocrine therapy, however hormone receptor positivity does not predict response and there are no biomarkers in clinical use to help select patients that would benefit from endocrine agents. We aimed to identify markers of endocrine response in high-grade serous ovarian cancer (HGSOC) by analysis of genes that are differentially expressed in tumors from patients that are endocrine responders compared with non-responders, combined with analysis of estrogen (E2) regulated genes that differ between E2 sensitive and E2 insensitive HGSOC cell lines. Methods: Among women recruited to the Australian Ovarian Cancer Study, we identified 10 HGSOC patients treated with endocrine therapy (5 responders and 5 non-responders, based on GCIG CA125 response criteria) and performed RNAseq on cryopreserved tumor tissue. We also utilized paired E2 receptor (ER) positive HGSOC cell lines, derived from the same patient, with differential sensitivity to the growth effect of E2, PEO1 (E2 insensitive) and PEO4 (E2 sensitive). RNA was extracted following treatment with 0.1 nM E2 (or vehicle) for 24 hrs and gene expression changes in response to E2 were determined using RNAseq. Expression of selected ER responsive genes was validated using PCR array. Differential gene expression was determined using EdgeR. Pathway and Gene Ontology enrichment analysis of differentially expressed genes were performed using Metacore. Results: Between endocrine responders and non-responders, 27 genes were significantly differentially expressed (False Discovery Rate (FDR)<0.05, absolute log2 Fold-Change (FC)≥1). In the paired HGSOC cell lines, significantly more genes were regulated by E2 in E2-sensitive PEO4, compared with E2-insensitive PEO1 (896 genes compared to 56 genes, respectively, FDR<0.05, absolute log2FC≥1). In response to E2, up-regulation of NRP1 in PEO1, up-regulation of LTBP1, MYC, PDZK1 and down-regulation of S100A6 and CYP1A1 in PEO4 were validated by PCR array profiling of ER-related genes. Genes regulated by E2 in cell lines and also differentially expressed between endocrine responders and non-responders included anoctamin 1 (ANO1) and NOTCH-Regulated Ankyrin Repeat Protein (NRARP). Both were up-regulated in endocrine responders and also up-regulated by E2 in PEO4. Conclusion: The use of RNAseq in both tumors and cell line models was able to identify plausible sets of genes with prior evidence of association with endocrine sensitivity. In breast cancer, ANO1 has been associated with good prognosis following tamoxifen treatment, while NOTCH signaling, implicated in endocrine resistance, is negatively regulated by NRARP. Further validation is required to test the utility of these genes as predictive biomarkers that may aid in identifying HGSOC patients likely to respond to endocrine treatment. Citation Format: Cristina Mapagu, Sian Fereday, Australian Ovarian Cancer Study Group, David D. Bowtell, Paul R. Harnett, Anna deFazio. Identifying predictive markers of endocrine response in high-grade serous ovarian cancer using RNA sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2787. doi:10.1158/1538-7445.AM2017-2787

Overexpression of NOTCH-regulated Ankyrin Repeat Protein is associated with papillary thyroid carcinoma progression

Papillary thyroid cancer (PTC) is one of the endocrine cancers with high clinical and genetic heterogeneity. NOTCH signaling and its downstream NOTCH-Regulated Ankyrin Repeat Protein (NRARP) have been implicated in oncogenesis of many cancers, but the roles in PTCs are less studied. In this study, we show that NRARP is frequently over-expressed in thyroid carcinoma. The over-activation of NRARP is highly and positively correlated with NOTCH genes. Moreover, we find that the expression of NRARP is highly associated with several epithelial mesenchymal transition (EMT) markers and contributes to poor survival outcomes. Therefore, these results indicate that NRARP is an important clinical biomarker in thyroid carcinoma and it promotes EMT induction as well as the progression of PTCs via NOTCH signaling activation.

Abstract CT048: Population pharmacokinetics and pharmacodynamics for an oral Notch inhibitor, LY3039478, in the first-in-man study

Abstract Background: Deregulation of Notch signalling is involved in multiple human cancers. LY3039478 is a potent Notch inhibitor that prevents release of the Notch Intracellular Domain (NICD) by inhibiting proteolytic activity of the gamma (γ) -secretase complex and thereby decreasing Notch signalling and its downstream biologic effects. Since γ-secretase also possesses proteolytic activity to cleave amyloid precursor proteins, LY3039478 is expected to inhibit production of plasma Amyloid-beta (Aβ), which can be used as a biomarker to determine pharmacodynamic (PD) effects of LY3039478 in patients. Additionally, expression of Notch regulated genes [Hairy and enhancer of split-1 (Hes1), Cyclin D1 (CCND1), and Notch-Regulated Ankyrin Repeat (NRARP)] in skin can be used to monitor additional PD effects. The evaluation of pharmacokinetic (PK) - PD (biomarker) relationships is explored in this phase I does escalation of LY3039478 in patients (pts) with advanced cancer. Methods: In the dose escalation part of the first human dose study of LY3039478, patients received oral doses of 2.5 up to 100 mg thrice weekly (TIW) via 25 or 50 mg capsules. Plasma samples were collected up to 24 to 30 hours after the 1st and 10th dose, and urine samples up to 8 to10 hours after the 1st dose. Skin samples were collected prior to and after approximately 6 to 8 hours after the 1st dose. PK and PD parameter estimations were performed using non-linear mixed effects models in NONMEM Version 7.3 using a sequential approach. Results: A population PK/PD model was developed using preliminary data collected from 55 pts with age range 30 to 75 years and weight between 43 to 103 kg. A PK model with 3 transit absorption compartments, central and peripheral plasma compartments, plus a urine compartment best described the LY3039478 disposition. LY3039478 was rapidly absorbed with peak concentrations occurring within 1 to 2 hours. Renal clearance contributed to ∼20% of the apparent plasma clearance. Simple maximal effects (Emax) models were used to describe the PK/PD relationships for maximal plasma A-beta inhibition and inhibition of gene expression in relation to LY3039478 exposure. The results showed that 80% of maximal effects of plasma Aβ inhibition occurred at approximately 3300 μg*h/L (90% prediction intervals (PI) 1200 - 13000 μg*h/L). For gene expression of HES1, NRARP and CCND1, 80% of maximal effects occurred at average exposures of 1600 μg*h/L (90% PI 1000-3000 μg*h/L), 1300 μg*h/L (90% PI 1000- 1800 μg*h/L) and 1000 μg*h/L (90% PI 600 - 2000 μg*h/L), respectively. These exposures correspond to a dose range of 15 to 50 mg of LY3039478. Conclusion: The plasma Aβ and gene expression data showed that 80% of maximal biomarker effects were obtained at approximately 15 to 50 mg LY3039478, supporting the use of the 50 mg TIW LY3039478 dosing regimen currently used in the dose-expansion phase of the first in man study. Citation Format: Eunice Yuen, Bharvin Patel, Claire Smith, Maria Posada, Robert Bell, Ute Ohnmacht, Christophe Massard, Jordi Rodon, Karim Benhadji. Population pharmacokinetics and pharmacodynamics for an oral Notch inhibitor, LY3039478, in the first-in-man study. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr CT048.

Downregulation of Notch-regulated Ankyrin Repeat Protein Exerts Antitumor Activities against Growth of Thyroid Cancer.

Background:The Notch-regulated ankyrin repeat protein (NRARP) is recently found to promote proliferation of breast cancer cells. The role of NRARP in carcinogenesis deserves extensive investigations. This study attempted to investigate the expression of NRARP in thyroid cancer tissues and assess the influence of NRARP on cell proliferation, apoptosis, cell cycle, and invasion in thyroid cancer.Methods:Thirty-four cases with thyroid cancer were collected from the Department of General Surgery, Xinhua Hospital, Shanghai Jiao Tong University School of Medicine between 2011 and 2012. Immunohistochemistry was used to detect the level of NRARP in cancer tissues. Lentivirus carrying NRARP-shRNA (Lenti-NRARP-shRNA) was applied to down-regulate NRARP expression. Cell viability was tested after treatment with Lenti-NRARP-shRNA using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Apoptosis and cell cycle distribution were determined by flow cytometry. Cell invasion was tested using Transwell invasion assay. In addition, expressions of several cell cycle-associated and apoptosis-associated proteins were examined using Western blotting after transfection. Student's t-test, one-way analysis of variance (ANOVA), or Kaplan–Meier were used to analyze the differences between two group or three groups.Results:NRARP was highly expressed in thyroid cancer tissues. Lenti-NRARP-shRNA showed significantly inhibitory activities against cell growth at a multiplicity of infection of 10 or higher (P < 0.05). Lenti-NRARP-shRNA-induced G1 arrest (BHT101: 72.57% ± 5.32%; 8305C: 75.45% ± 5.26%) by promoting p21 expression, induced apoptosis by promoting bax expression and suppressing bcl-2 expression, and inhibited cell invasion by suppressing matrix metalloproteinase-9 expression.Conclusion:Downregulation of NRARP expression exerts significant antitumor activities against cell growth and invasion of thyroid cancer, that suggests a potential role of NRARP in thyroid cancer targeted therapy.

Notch1 is required in newly postmitotic cells to inhibit the rod photoreceptor fate

Several models of cell fate determination can be invoked to explain how single retinal progenitor cells (RPCs) produce different cell types in a terminal division. To gain insight into this process, the effects of the removal of a cell fate regulator, Notch1, were studied in newly postmitotic cells using a conditional allele of Notch1 (N1-CKO) in mice. Almost all newly postmitotic N1-CKO cells became rod photoreceptors, whereas wild-type (WT) cells achieved a variety of fates. Single cell profiling of wild-type and N1-CKO retinal cells transitioning from progenitor to differentiated states revealed differential expression of inhibitor of DNA binding factors Id1 and Id3, as well as Notch-regulated ankyrin repeat protein (Nrarp). Misexpression of Id1 and Id3 was found to be sufficient to drive production of Müller glial cells and/or RPCs. Moreover, Id1 and Id3 were shown to partially rescue the production of bipolar and Müller glial cells in the absence of Notch1 in mitotic and newly postmitotic cells. Misexpression of Nrarp, a downstream target gene and inhibitor of the Notch signaling pathway, resulted in the overproduction of rod photoreceptors at the expense of Müller glial cells. These data demonstrate that cell fate decisions can be made in newly postmitotic retinal cells, and reveal some of the regulators downstream of Notch1 that influence the choice of rod and non-rod fates. Taken together, our results begin to address how different signals downstream from a common pathway lead to different fate outcomes.

Transcriptomic Analysis of the Developing and Adult Mouse Cochlear Sensory Epithelia

The adult mammalian cochlea lacks regenerative ability and the irreversible degeneration of cochlear sensory hair cells leads to permanent hearing loss. Previous data show that early postnatal cochlea harbors stem/progenitor-like cells and shows a limited regenerative/repair capacity. These properties are progressively lost later during the postnatal development. Little is known about the genes and pathways that are potentially involved in this difference of the regenerative/repair potentialities between early postnatal and adult mammalian cochlear sensory epithelia (CSE). The goal of our study is to investigate the transcriptomic profiles of these two stages. We used Mouse Genome 430 2.0 microarray to perform an extensive analysis of the genes expressed in mouse postnatal day-3 (P3) and adult CSE. Statistical analysis of microarray data was performed using SAM (Significance Analysis of Microarrays) software. We identified 5644 statistically significant differentially expressed transcripts with a fold change (FC) >2 and a False Discovery Rate (FDR) ≤0.05. The P3 CSE signature included 3,102 transcripts, among which were known genes in the cochlea, but also new transcripts such as, Hmga2 (high mobility group AT-hook 2) and Nrarp (Notch-regulated ankyrin repeat protein). The adult CSE overexpressed 2,542 transcripts including new transcripts, such as Prl (Prolactin) and Ar (Androgen receptor), that previously were not known to be expressed in the adult cochlea. Our comparative study revealed important genes and pathways differentially expressed between the developing and adult CSE. The identification of new candidate genes would be useful as potential markers of the maintenance or the loss of stem cells and regenerative/repair ability during mammalian cochlear development.

Notch1 counteracts WNT/β-catenin signaling through chromatin modification in colorectal cancer

Crosstalk between the Notch and wingless-type MMTV integration site (WNT) signaling pathways has been investigated for many developmental processes. However, this negative correlation between Notch and WNT/β-catenin signaling activity has been studied primarily in normal developmental and physiological processes in which negative feedback loops for both signaling pathways are intact. We found that Notch1 signaling retained the capability of suppressing the expression of WNT target genes in colorectal cancers even when β-catenin destruction by the adenomatous polyposis coli (APC) complex was disabled. Activation of Notch1 converted high-grade adenoma into low-grade adenoma in an Apcmin mouse colon cancer model and suppressed the expression of WNT target genes in human colorectal cancer cells through epigenetic modification recruiting histone methyltransferase SET domain bifurcated 1 (SETDB1). Extensive microarray analysis of human colorectal cancers also showed a negative correlation between the Notch1 target gene, Notch-regulated ankyrin repeat protein 1 (NRARP), and WNT target genes. Notch is known to be a strong promoter of tumor initiation, but here we uncovered an unexpected suppressive role of Notch1 on WNT/β-catenin target genes involved in colorectal cancer.

The Notch‐regulated ankyrin repeat protein is required for proper anterior–posterior somite patterning in mice

The Notch-regulated ankyrin repeat protein (Nrarp) is a component of a negative feedback system that attenuates Notch pathway-mediated signaling. In vertebrates, the timing and spacing of formation of the mesodermal somites are controlled by a molecular oscillator termed the segmentation clock. Somites are also patterned along the rostral-caudal axis of the embryo. Here, we demonstrate that Nrarp-deficient embryos and mice exhibit genetic background-dependent defects of the axial skeleton. While progression of the segmentation clock occurred in Nrarp-deficient embryos, they exhibited altered rostrocaudal patterning of the somites. In Nrarp mutant embryos, the posterior somite compartment was expanded. These studies confirm an anticipated, but previously undocumented role for the Nrarp gene in vertebrate somite patterning and provide an example of the strong influence that genetic background plays on the phenotypes exhibited by mutant mice. genesis 50:366–374, 2012. © 2011 Wiley Periodicals, Inc.

The period of the somite segmentation clock is sensitive to Notch activity

The number of vertebrae is defined strictly for a given species and depends on the number of somites, which are the earliest metameric structures that form in development. Somites are formed by sequential segmentation. The periodicity of somite segmentation is orchestrated by the synchronous oscillation of gene expression in the presomitic mesoderm (PSM), termed the "somite segmentation clock," in which Notch signaling plays a crucial role. Here we show that the clock period is sensitive to Notch activity, which is fine-tuned by its feedback regulator, Notch-regulated ankyrin repeat protein (Nrarp), and that Nrarp is essential for forming the proper number and morphology of axial skeleton components. Null-mutant mice for Nrarp have fewer vertebrae and have defective morphologies. Notch activity is enhanced in the PSM of the Nrarp(-/-) embryo, where the ~2-h segmentation period is extended by 5 min, thereby forming fewer somites and their resultant vertebrae. Reduced Notch activity partially rescues the Nrarp(-/-) phenotype in the number of somites, but not in morphology. Therefore we propose that the period of the somite segmentation clock is sensitive to Notch activity and that Nrarp plays essential roles in the morphology of vertebrae and ribs.

The Dll4/Notch pathway controls postangiogenic blood vessel remodeling and regression by modulating vasoconstriction and blood flow

AbstractBlood vessel remodeling is crucial to the formation of the definitive vasculature, but little is known about the mechanisms controlling this process. We show that Delta-like ligand 4 (Dll4)/Notch pathway regulates vessel regression in normal pathologic conditions. Genetic and pharmacologic inhibition of Dll4/Notch prevented retinal capillary regression in the oxygen-induced retinopathy (OIR) model and during normal development. Deletion of the Notch-regulated ankyrin repeat protein, a negative regulator of the Notch pathway, produced an opposite phenotype. Inhibition of Dll4/Notch reduced vessel occlusion, maintaining blood flow that is essential for survival of microvessels. Dll4/Notch inhibition up-regulated the expression of vasodilators adrenomedullin and suppressed the expression of vasoconstrictor angiotensinogen. Angiotensin II induced rapid nonperfusion and regression of developing retinal capillaries, whereas Ace1 and AT1 inhibitors and adrenomedullin attenuated vasoobliteration in OIR, indicating that both pathways are involved in modulating vessel remodeling. In contrast, inhibition of vascular endothelial growth factor-A (VEGF-A) did not result in a pervasive loss of retinal capillaries, demonstrating that reduced expression of VEGF-A is not the proximate cause of capillary regression in OIR. Modulation of VEGF-A and DII4/Notch signaling produced distinct changes in blood vessel morphology and gene expression, indicating that these pathways can have largely independent functions in vascular remodeling.

Notch-regulated ankyrin repeat protein（NRARP）のヒト乳がん細胞株における機能解析

Notch-regulated ankyrin repeat protein（NRARP）のヒト乳がん細胞株における機能解析

Nrarp Coordinates Endothelial Notch and Wnt Signaling to Control Vessel Density in Angiogenesis

When and where to make or break new blood vessel connections is the key to understanding guided vascular patterning. VEGF-A stimulation and Dll4/Notch signaling cooperatively control the number of new connections by regulating endothelial tip cell formation. Here, we show that the Notch-regulated ankyrin repeat protein (Nrarp) acts as a molecular link between Notch- and Lef1-dependent Wnt signaling in endothelial cells to control stability of new vessel connections in mouse and zebrafish. Dll4/Notch-induced expression of Nrarp limits Notch signaling and promotes Wnt/Ctnnb1 signaling in endothelial stalk cells through interactions with Lef1. BATgal-reporter expression confirms Wnt signaling activity in endothelial stalk cells. Ex vivo, combined Wnt3a and Dll4 stimulation of endothelial cells enhances Wnt-reporter activity, which is abrogated by loss of Nrarp. In vivo, loss of Nrarp, Lef1, or endothelial Ctnnb1 causes vessel regression. We suggest that the balance between Notch and Wnt signaling determines whether to make or break new vessel connections.

Nrarp functions to modulate neural-crest-cell differentiation by regulating LEF1 protein stability

Nrarp (Notch-regulated ankyrin repeat protein) is a small protein that has two ankyrin repeats. Although Nrarp is known to be an inhibitory component of the Notch signalling pathway that operates in different developmental processes, the in vivo roles of Nrarp have not been fully characterized. Here, we show that Nrarp is a positive regulator in the Wnt signalling pathway. In zebrafish, knockdown of Nrarp-a expression by an antisense morpholino oligonucleotide (MO) results in altered Wnt-signalling-dependent neural-crest-cell development. Nrarp stabilizes LEF1 protein, a pivotal transcription factor in the Wnt signalling cascade, by blocking LEF1 ubiquitination. In accordance with this, the knockdown phenotype of lef1 is similar to that of nrarp-a, at least in part, in its effect on the development of multiple tissues in zebrafish. Furthermore, activation of LEF1 does not affect Notch activity or vice versa. These findings reveal that Nrarp independently regulates canonical Wnt and Notch signalling by modulating LEF1 and Notch protein turnover, respectively.

Developmentally regulated expression of two members of the Nrarp family in zebrafish

Delta-Notch signaling is essential for somitogenesis in vertebrate embryos. In a search for genes that control somite formation in zebrafish we have identified two paralogues encoding proteins related to Nrarp (Notch regulated ankyrin repeat protein). Zebrafish nrarp-a and- b encode small proteins with two ankyrin repeat domains. Here, we report the expression patterns of both genes in normal and mutant embryos.