RESUMEN
Gastrointestinal motility disorders include a spectrum of mild to severe clinical phenotypes that are caused by smooth muscle dysfunction. We investigated the genetic etiology of severe esophageal, gastric, and colonic dysmotility in two unrelated families with autosomal dominant disease presentation. Using exome sequencing, we identified a 2 base pair insertion at the end of the myosin heavy chain 11 (MYH11) gene in all affected members of Family 1 [NM_001040113:c.5819_5820insCA(p.Gln1941Asnfs*91)] and a 1 base pair deletion at the same genetic locus in Proband 2 [NM_001040113:c.5819del(p.Pro1940Hisfs*91)]. Both variants are predicted to result in a similarly elongated protein product. Heterozygous dominant negative MYH11 pathogenic variants have been associated with thoracic aortic aneurysm and dissection while biallelic null alleles have been associated with megacystis microcolon intestinal hypoperistalsis syndrome. This report highlights heterozygous protein-elongating MYH11 variants affecting the SM2 isoforms of MYH11 as a cause for severe gastrointestinal dysmotility, and we hypothesize that the mechanistic pathogenesis of this disease, dominant hypercontractile loss-of-function, is distinct from those implicated in other diseases involving MYH11 dysfunction.
Asunto(s)
Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Músculo Liso/metabolismo , Músculo Liso/fisiopatología , Mutación , Cadenas Pesadas de Miosina/genética , Fenotipo , Adulto , Niño , Análisis Mutacional de ADN , Electromiografía , Endoscopía del Sistema Digestivo , Trastornos de la Motilidad Esofágica/diagnóstico , Trastornos de la Motilidad Esofágica/genética , Femenino , Gastroparesia/diagnóstico , Gastroparesia/genética , Estudios de Asociación Genética/métodos , Estudio de Asociación del Genoma Completo , Humanos , Lactante , Enfermedades Intestinales/diagnóstico , Enfermedades Intestinales/genética , Masculino , Persona de Mediana Edad , Linaje , Polimorfismo de Nucleótido Simple , Radiografía , Síndrome , Adulto JovenRESUMEN
Chronic kidney disease (CKD) is a complex gene-environmental disease affecting close to 10% of the US population. Genome-wide association studies (GWASs) have identified sequence variants, localized to non-coding genomic regions, associated with kidney function. Despite these robust observations, the mechanism by which variants lead to CKD remains a critical unanswered question. Expression quantitative trait loci (eQTL) analysis is a method to identify genetic variation associated with gene expression changes in specific tissue types. We hypothesized that an integrative analysis combining CKD GWAS and kidney eQTL results can identify candidate genes for CKD. We performed eQTL analysis by correlating genotype with RNA-seq-based gene expression levels in 96 human kidney samples. Applying stringent statistical criteria, we detected 1,886 genes whose expression differs with the sequence variants. Using direct overlap and Bayesian methods, we identified new potential target genes for CKD. With respect to one of the target genes, lysosomal beta A mannosidase (MANBA), we observed that genetic variants associated with MANBA expression in the kidney showed statistically significant colocalization with variants identified in CKD GWASs, indicating that MANBA is a potential target gene for CKD. The expression of MANBA was significantly lower in kidneys of subjects with risk alleles. Suppressing manba expression in zebrafish resulted in renal tubule defects and pericardial edema, phenotypes typically induced by kidney dysfunction. Our analysis shows that gene-expression changes driven by genetic variation in the kidney can highlight potential new target genes for CKD development.
Asunto(s)
Regulación de la Expresión Génica , Variación Genética , Enfermedades Renales/genética , Animales , Secuencia de Bases , Técnicas de Silenciamiento del Gen , Redes Reguladoras de Genes , Estudio de Asociación del Genoma Completo , Humanos , Riñón/metabolismo , Riñón/patología , Enfermedades Renales/patología , Desequilibrio de Ligamiento/genética , Polimorfismo de Nucleótido Simple/genética , Sitios de Carácter Cuantitativo/genética , Carácter Cuantitativo Heredable , Reproducibilidad de los Resultados , Pez Cebra/genética , beta-Manosidasa/genéticaRESUMEN
Intratumoral hypoxia correlates with metastasis and poor survival in patients with sarcoma. Using an impedance sensing assay and a zebrafish intravital microinjection model, we demonstrated here that the hypoxia-inducible collagen-modifying enzyme lysyl hydroxylase PLOD2 and its substrate collagen type VI (COLVI) weaken the lung endothelial barrier and promote transendothelial migration. Mechanistically, hypoxia-induced PLOD2 in sarcoma cells modified COLVI, which was then secreted into the vasculature. Upon reaching the apical surface of lung endothelial cells, modified COLVI from tumor cells activated integrin ß1 (ITGß1). Furthermore, activated ITGß1 colocalized with Kindlin2, initiating their interaction with F-actin and prompting its polymerization. Polymerized F-actin disrupted endothelial adherens junctions and induced barrier dysfunction. Consistently, modified and secreted COLVI was required for the late stages of lung metastasis in vivo. Analysis of patient gene expression and survival data from The Cancer Genome Atlas (TCGA) revealed an association between the expression of both PLOD2 and COLVI and patient survival. Furthermore, high levels of COLVI were detected in surgically resected sarcoma metastases from patient lungs and in the blood of tumor-bearing mice. Together, these data identify a mechanism of sarcoma lung metastasis, revealing opportunities for therapeutic intervention. SIGNIFICANCE: Collagen type VI modified by hypoxia-induced PLOD2 is secreted by sarcoma cells and binds to integrin ß1 on endothelial cells to induce barrier dysfunction, which promotes sarcoma vascular dissemination and metastasis.
Asunto(s)
Neoplasias Pulmonares , Sarcoma , Humanos , Animales , Ratones , Colágeno Tipo VI/genética , Colágeno Tipo VI/metabolismo , Células Endoteliales/metabolismo , Pez Cebra/metabolismo , Actinas , Integrina beta1 , Hipoxia , Sarcoma/metabolismo , Pulmón/patologíaRESUMEN
The model organism zebrafish (Danio rerio) is particularly amenable to studies deciphering regulatory genetic networks in vertebrate development, biology, and pharmacology. Unraveling the functional dynamics of such networks requires precise quantitation of protein expression during organismal growth, which is incrementally challenging with progressive complexity of the systems. In an approach toward such quantitative studies of dynamic network behavior, we applied mass spectrometric methodology and rigorous statistical analysis to create comprehensive, high quality profiles of proteins expressed at two stages of zebrafish development. Proteins of embryos 72 and 120 h postfertilization (hpf) were isolated and analyzed both by two-dimensional (2D) LC followed by ESI-MS/MS and by 2D PAGE followed by MALDI-TOF/TOF protein identification. We detected 1384 proteins from 327,906 peptide sequence identifications at 72 and 120 hpf with false identification rates of less than 1% using 2D LC-ESI-MS/MS. These included only approximately 30% of proteins that were identified by 2D PAGE-MALDI-TOF/TOF. Roughly 10% of all detected proteins were derived from hypothetical or predicted gene models or were entirely unannotated. Comparison of proteins expression by 2D DIGE revealed that proteins involved in energy production and transcription/translation were relatively more abundant at 72 hpf consistent with faster synthesis of cellular proteins during organismal growth at this time compared with 120 hpf. The data are accessible in a database that links protein identifications to existing resources including the Zebrafish Information Network database. This new resource should facilitate the selection of candidate proteins for targeted quantitation and refine systematic genetic network analysis in vertebrate development and biology.
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Proteoma/análisis , Proteómica , Proteínas de Pez Cebra/análisis , Pez Cebra/embriología , Animales , Cromatografía Liquida , Bases de Datos de Proteínas , Electroforesis en Gel Bidimensional , Desarrollo Embrionario , Proteoma/genética , Proteoma/metabolismo , Espectrometría de Masa por Ionización de Electrospray , Pez Cebra/genética , Pez Cebra/metabolismo , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
The field of neuropharmacology has not yet achieved a full understanding of how the brain transitions between states of consciousness and drug-induced unconsciousness, or anesthesia. Many small molecules are used to alter human consciousness, but the repertoire of underlying molecular targets, and thereby the genes, are incompletely understood. Here we describe a robust larval zebrafish model of anesthetic action, from sedation to general anesthesia. We use loss of movement under three different conditions, spontaneous movement, electrical stimulation or a tap, as a surrogate for sedation and general anesthesia, respectively. Using these behavioral patterns, we find that larval zebrafish respond to inhalational and IV anesthetics at concentrations similar to mammals. Additionally, known sedative drugs cause loss of spontaneous larval movement but not to the tap response. This robust, highly tractable vertebrate model can be used in the detection of genes and neural substrates involved in the transition from consciousness to unconsciousness.
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Anestésicos/farmacología , Conducta Animal/efectos de los fármacos , Encéfalo/fisiología , Estado de Conciencia/efectos de los fármacos , Inconsciencia/fisiopatología , Animales , Encéfalo/efectos de los fármacos , Modelos Animales , Inconsciencia/psicología , Pez CebraRESUMEN
High-grade sarcomas are metastatic and pose a serious threat to patient survival. Undifferentiated pleomorphic sarcoma (UPS) is a particularly dangerous and relatively common sarcoma subtype diagnosed in adults. UPS contains large quantities of extracellular matrix (ECM) including hyaluronic acid (HA), which is linked to metastatic potential. Consistent with these observations, expression of the HA receptor, hyaluronan-mediated motility receptor (HMMR/RHAMM), is tightly controlled in normal tissues and upregulated in UPS. Moreover, HMMR expression correlates with poor clinical outcome in these patients. Deregulation of the tumor-suppressive Hippo pathway is also linked to poor outcome in these patients. YAP1, the transcriptional regulator and central effector of Hippo pathway, is aberrantly stabilized in UPS and was recently shown to control RHAMM expression in breast cancer cells. Interestingly, both YAP1 and RHAMM are linked to TGFß signaling. Therefore, we investigated crosstalk between YAP1 and TGFß resulting in enhanced RHAMM-mediated cell migration and invasion. We observed that HMMR expression is under the control of both YAP1 and TGFß and can be effectively targeted with small-molecule approaches that inhibit these pathways. Furthermore, we found that RHAMM expression promotes tumor cell proliferation and migration/invasion. To test these observations in a robust and quantifiable in vivo system, we developed a zebrafish xenograft assay of metastasis, which is complimentary to our murine studies. Importantly, pharmacologic inhibition of the TGFß-YAP1-RHAMM axis prevents vascular migration of tumor cells to distant sites. IMPLICATIONS: These studies reveal key metastatic signaling mechanisms and highlight potential approaches to prevent metastatic dissemination in UPS.YAP1 and TGFß cooperatively enhance proliferation and migration/invasion of UPS and fibrosarcomas.
Asunto(s)
Proteínas de la Matriz Extracelular/metabolismo , Receptores de Hialuranos/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Sarcoma/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Animales Modificados Genéticamente , Línea Celular Tumoral , Fibrosarcoma , Células HCT116 , Células HEK293 , Vía de Señalización Hippo , Humanos , Ratones , Ratones Desnudos , Metástasis de la Neoplasia , Sarcoma/patología , Factores de Transcripción/metabolismo , Proteínas Señalizadoras YAP , Pez CebraRESUMEN
OBJECTIVE: Prostaglandin E synthases (PGESs) are being explored as antiinflammatory drug targets as alternatives to cyclooxygenase (COX)-2. Located downstream of the cyclooxygenases, PGESs catalyze PGE(2) formation, and deletion of microsomal (m)-PGES-1 abrogates inflammation. We sought to characterize the developmental expression of COX and PGES in zebrafish. METHODS AND RESULTS: We cloned zebrafish cytosolic (c) and m-PGES orthologs and mapped them to syntenic regions of chromosomes 23 and 5. cPGES was widely expressed during development and was coordinately regulated with zCOX-1 in the inner ear, the pronephros, and intestine. COX-2 and mPGES-1 exhibited restricted expression, dominantly in the vasculature of the aortic arch. However, the enzymes were anatomically segregated within the vessel wall. Experiments with antisense morpholinos and with nonsteroidal antiinflammatory drugs suggest that these genes may not be critical for development. CONCLUSIONS: mPGES-1 is developmentally coregulated with COX-2 in vasculature. Given the high fecundidity and translucency of the zebrafish, this model may afford a high throughput system for characterization of novel PGES inhibitors. Microsomal prostaglandin E synthase (mPGES)-1, located downstream of COX-2, may represent a novel antiinflammatory drug target. Zebrafish cytosolic (c) PGES-1 and COX-1 were coordinately expressed; mPGES-1 and COX-2 were expressed particularly in the vasculature. Zebrafish may afford a high throughput system for detection of novel PGES inhibitors.
Asunto(s)
Oxidorreductasas Intramoleculares/genética , Prostaglandinas E/biosíntesis , Proteínas de Pez Cebra/genética , Pez Cebra/metabolismo , Animales , Vasos Sanguíneos/embriología , Vasos Sanguíneos/enzimología , Vasos Sanguíneos/crecimiento & desarrollo , Mapeo Cromosómico , Ciclooxigenasa 1 , Ciclooxigenasa 2 , Embrión no Mamífero , Regulación del Desarrollo de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Oxidorreductasas Intramoleculares/biosíntesis , Isoenzimas/biosíntesis , Isoenzimas/genética , Riñón/embriología , Riñón/enzimología , Riñón/crecimiento & desarrollo , Larva , Microsomas/enzimología , Datos de Secuencia Molecular , Especificidad de Órganos , Filogenia , Prostaglandina-E Sintasas , Prostaglandina-Endoperóxido Sintasas/biosíntesis , Prostaglandina-Endoperóxido Sintasas/genética , Canales Semicirculares/embriología , Canales Semicirculares/enzimología , Canales Semicirculares/crecimiento & desarrollo , Pez Cebra/crecimiento & desarrollo , Proteínas de Pez Cebra/biosíntesisRESUMEN
Cerebral cavernous malformation is a common human vascular disease that arises due to loss-of-function mutations in genes encoding three intracellular adaptor proteins, cerebral cavernous malformations 1 protein (CCM1), CCM2, and CCM3. CCM1, CCM2, and CCM3 interact biochemically in a pathway required in endothelial cells during cardiovascular development in mice and zebrafish. The downstream effectors by which this signaling pathway regulates endothelial function have not yet been identified. Here we have shown in zebrafish that expression of mutant ccm3 proteins (ccm3Delta) known to cause cerebral cavernous malformation in humans confers cardiovascular phenotypes identical to those associated with loss of ccm1 and ccm2. CCM3Delta proteins interacted with CCM1 and CCM2, but not with other proteins known to bind wild-type CCM3, serine/threonine protein kinase MST4 (MST4), sterile 20-like serine/threonine kinase 24 (STK24), and STK25, all of which have poorly defined biological functions. Cardiovascular phenotypes characteristic of CCM deficiency arose due to stk deficiency and combined low-level deficiency of stks and ccm3 in zebrafish embryos. In cultured human endothelial cells, CCM3 and STK25 regulated barrier function in a manner similar to CCM2, and STKs negatively regulated Rho by directly activating moesin. These studies identify STKs as essential downstream effectors of CCM signaling in development and disease that may regulate both endothelial and epithelial cell junctions.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/fisiología , Sistema Cardiovascular/embriología , Hemangioma Cavernoso del Sistema Nervioso Central/etiología , Péptidos y Proteínas de Señalización Intracelular/fisiología , Proteínas de la Membrana/fisiología , Proteínas Serina-Treonina Quinasas/fisiología , Proteínas Proto-Oncogénicas/fisiología , Transducción de Señal/fisiología , Proteínas de Pez Cebra/fisiología , Secuencia de Aminoácidos , Animales , Proteínas Reguladoras de la Apoptosis/química , Proteínas Reguladoras de la Apoptosis/genética , Secuencia Conservada , Humanos , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Proteínas de Microfilamentos/metabolismo , Proteínas Asociadas a Microtúbulos/fisiología , Datos de Secuencia Molecular , Proteínas Musculares , Fosforilación , Proteínas Proto-Oncogénicas/química , Proteínas Proto-Oncogénicas/genética , Alineación de Secuencia , Pez Cebra , Proteínas de Pez Cebra/química , Proteínas de Pez Cebra/genéticaRESUMEN
PURPOSE: Pre-mRNA processing factor 3 (PRPF3) is a spliceosomal component essential for pre-mRNA processing. Mutations in PRPF3 have been implicated in retinitis pigmentosa (RP) 18 through an unknown mechanism. The authors created and characterized Prpf3 knockout mice and zebrafish to determine whether RP18 is a result of haploinsufficiency. METHODS: Mice were produced from a Prpf3 gene trap cell line, and parameters of retinal function, structure, and RNA splicing were analyzed. The retinas of prpf3 insertional mutant zebrafish were also analyzed histologically. RESULTS: Homozygous Prpf3 knockout mice do not survive to 14 days postfertilization (dpf), implying that this allele is required for early embryonic development. Homozygous Prpf3 knockout zebrafish die by 4dpf, well beyond the mid-blastula transition at which transcription activates. Zebrafish knockout embryos reveal abnormally high levels of cell death in the developing eye. Heterozygous Prpf3 knockout mice have less than the expected 50% reduction in Prpf3 at the mRNA and protein levels, implying compensatory expression from the wild-type allele. The heterozygous mice develop normally, with no changes in retinal function, no evidence for photoreceptor degeneration at up to 23 months of age, and no decrease in pre-mRNA splicing of transcripts mutated in other forms of RP in the retina. Similarly, heterozygous prpf3 knockout zebrafish develop normally and show no retinal degeneration up to 12 months of age. CONCLUSIONS: These models suggest that RP18 is not a result of haploinsufficiency but instead arises from a toxic gain of function caused by missense mutations in PRPF3.
Asunto(s)
Células Madre Embrionarias/fisiología , Precursores del ARN/metabolismo , Células Fotorreceptoras Retinianas Conos/patología , Células Fotorreceptoras Retinianas Bastones/patología , Ribonucleoproteína Nuclear Pequeña U4-U6/metabolismo , Animales , Línea Celular , Quimera , Cartilla de ADN , Electrorretinografía , Ratones , Factores de Empalme de ARN , Ribonucleoproteína Nuclear Pequeña U4-U6/deficiencia , Pez CebraRESUMEN
Study of the cyclooxygenases (COXs) has been limited by the role of COX-2 in murine reproduction and renal organogenesis. We sought to characterize COX expression and function in zebrafish (z). Full-length cDNAs of zCOX-1 and zCOX-2 were cloned and assigned to conserved regions of chromosomes 5 and 2, respectively. The deduced proteins are 67% homologous with their human orthologs. Prostaglandin (PG) E(2) is the predominant zCOX product detected by mass spectrometry. Pharmacological inhibitors demonstrate selectivity when directed against heterologously expressed zCOX isoforms. Zebrafish thrombocyte aggregation ex vivo and hemostasis in vivo are sensitive to inhibition of zCOX-1, but not zCOX-2. Both zCOXs were widely expressed during development, and knockdown of zCOX-1 causes growth arrest during early embryogenesis. zCOX-1 is widely evident in the embryonic vasculature, whereas zCOX-2 exhibits a more restricted pattern of expression. Both zCOX isoforms are genetically and functionally homologous to their mammalian orthologs. The zebrafish affords a tractable model system for the study of COX biology and development.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Isoenzimas/genética , Prostaglandina-Endoperóxido Sintasas/genética , Secuencia de Aminoácidos , Animales , Ciclooxigenasa 1 , Ciclooxigenasa 2 , Dinoprostona/metabolismo , Humanos , Isoenzimas/química , Proteínas de la Membrana , Datos de Secuencia Molecular , Prostaglandina-Endoperóxido Sintasas/química , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Pez CebraRESUMEN
Mammalian studies have implicated important roles for the basic helix-loop-helix transcription factor Ptf1a-p48 in the development of both exocrine and endocrine pancreas. We have cloned the Ptf1a-p48 ortholog in Danio rerio. Early zebrafish ptf1a expression is observed in developing hindbrain and in endodermal pancreatic precursors. Analysis of ptf1a and insulin expression reveals a population of exocrine precursors that, throughout early development, are temporally and spatially segregated from endocrine elements. Morpholino-mediated knockdown of ptf1a confirms early divergence of these endocrine and exocrine lineages. Ptf1a morphants lack differentiated exocrine pancreas, but maintain normal differentiation and organization of the principal islet. In addition to the exocrine phenotype, ptf1a knockdown also reduces the prevalence of a small population of anterior endocrine cells normally found outside the principal islet. Together, these findings suggest the presence of distinct ptf1a-dependent and ptf1a-independent precursor populations in developing zebrafish pancreas.
Asunto(s)
Diferenciación Celular/fisiología , Regulación del Desarrollo de la Expresión Génica , Páncreas/embriología , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Pez Cebra/embriología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Linaje de la Célula/fisiología , Cartilla de ADN , ADN Complementario/genética , Técnica del Anticuerpo Fluorescente , Secuencias Hélice-Asa-Hélice/genética , Hibridación in Situ , Insulina/metabolismo , Datos de Secuencia Molecular , Morfogénesis , Páncreas/citología , Páncreas/metabolismo , Alineación de Secuencia , Análisis de Secuencia de ADN , Transactivadores/metabolismo , Pez Cebra/metabolismoRESUMEN
Mammalian studies have implicated important roles for the basic helix-loop-helix transcription factor Ptf1a-p48 in the development of both exocrine and endocrine pancreas. We have cloned the Ptf1a-p48 ortholog in Danio rerio. Early zebrafish ptf1a expression is observed in developing hindbrain and in endodermal pancreatic precursors. Analysis of ptf1a and insulin expression reveals a population of exocrine precursors that, throughout early development, are temporally and spatially segregated from endocrine elements. Morpholino-mediated knockdown of ptf1a confirms early divergence of these endocrine and exocrine lineages. Ptf1a morphants lack differentiated exocrine pancreas, but maintain normal differentiation and organization of the principal islet. In addition to the exocrine phenotype, ptf1a knockdown also reduces the prevalence of a small population of anterior endocrine cells normally found outside the principal islet. Together, these findings suggest the presence of distinct ptf1a-dependent and ptf1a-independent precursor populations in developing zebrafish pancreas.
Asunto(s)
Diferenciación Celular/fisiología , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio , Páncreas/embriología , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Pez Cebra/embriología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Linaje de la Célula/fisiología , Cartilla de ADN , ADN Complementario/genética , Técnica del Anticuerpo Fluorescente , Secuencias Hélice-Asa-Hélice/genética , Técnicas Histológicas , Hibridación in Situ , Insulina/metabolismo , Datos de Secuencia Molecular , Morfogénesis , Páncreas/citología , Páncreas/metabolismo , Alineación de Secuencia , Análisis de Secuencia de ADN , Transactivadores/metabolismo , Pez Cebra/metabolismoRESUMEN
Notch signaling regulates cell fate decisions in a variety of adult and embryonic tissues, and represents a characteristic feature of exocrine pancreatic cancer. In developing mouse pancreas, targeted inactivation of Notch pathway components has defined a role for Notch in regulating early endocrine differentiation, but has been less informative with respect to a possible role for Notch in regulating subsequent exocrine differentiation events. Here, we show that activated Notch and Notch target genes actively repress completion of an acinar cell differentiation program in developing mouse and zebrafish pancreas. In developing mouse pancreas, the Notch target gene Hes1 is co-expressed with Ptf1-P48 in exocrine precursor cells, but not in differentiated amylase-positive acinar cells. Using lentiviral delivery systems to induce ectopic Notch pathway activation in explant cultures of E10.5 mouse dorsal pancreatic buds, we found that both Hes1 and Notch1-IC repress acinar cell differentiation, but not Ptf1-P48 expression, in a cell-autonomous manner. Ectopic Notch activation also delays acinar cell differentiation in developing zebrafish pancreas. Further evidence of a role for endogenous Notch in regulating exocrine pancreatic differentiation was provided by examination of zebrafish embryos with homozygous mindbomb mutations, in which Notch signaling is disrupted. mindbomb-deficient embryos display accelerated differentiation of exocrine pancreas relative to wild-type clutchmate controls. A similar phenotype was induced by expression of a dominant-negative Suppressor of Hairless [Su(H)] construct, confirming that Notch actively represses acinar cell differentiation during zebrafish pancreatic development. Using transient transfection assays involving a Ptf1-responsive reporter gene, we further demonstrate that Notch and Notch/Su(H) target genes directly inhibit Ptf1 activity, independent of changes in expression of Ptf1 component proteins. These results define a normal inhibitory role for Notch in the regulation of exocrine pancreatic differentiation.