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INTRODUCTION: Triggering receptor expressed on myeloid cells 2 (TREM2) agonists are being clinically evaluated as disease-modifying therapeutics for Alzheimer's disease. Clinically translatable pharmacodynamic (PD) biomarkers are needed to confirm drug activity and select the appropriate therapeutic dose in clinical trials. METHODS: We conducted multi-omic analyses on paired non-human primate brain and cerebrospinal fluid (CSF), and stimulation of human induced pluripotent stem cell-derived microglia cultures after TREM2 agonist treatment, followed by validation of candidate fluid PD biomarkers using immunoassays. We immunostained microglia to characterize proliferation and clustering. RESULTS: We report CSF soluble TREM2 (sTREM2) and CSF chitinase-3-like protein 1 (CHI3L1/YKL-40) as PD biomarkers for the TREM2 agonist hPara.09. The respective reduction of sTREM2 and elevation of CHI3L1 in brain and CSF after TREM2 agonist treatment correlated with transient microglia proliferation and clustering. DISCUSSION: CSF CHI3L1 and sTREM2 reflect microglial TREM2 agonism and can be used as clinical PD biomarkers to monitor TREM2 activity in the brain. HIGHLIGHTS: CSF soluble triggering receptor expressed on myeloid cells 2 (sTREM2) reflects brain target engagement for a novel TREM2 agonist, hPara.09. CSF chitinase-3-like protein 1 reflects microglial TREM2 agonism. Both can be used as clinical fluid biomarkers to monitor TREM2 activity in brain.
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Biomarcadores , Encéfalo , Proteína 1 Similar a Quitinasa-3 , Glicoproteínas de Membrana , Microglía , Receptores Inmunológicos , Proteína 1 Similar a Quitinasa-3/líquido cefalorraquídeo , Receptores Inmunológicos/metabolismo , Animales , Humanos , Encéfalo/metabolismo , Biomarcadores/líquido cefalorraquídeo , Microglía/efectos de los fármacos , Microglía/metabolismo , Células Madre Pluripotentes Inducidas , Enfermedad de Alzheimer/líquido cefalorraquídeo , Enfermedad de Alzheimer/tratamiento farmacológico , MasculinoRESUMEN
Intelligent data acquisition (IDA) strategies, such as a real-time database search (RTS), have improved the depth of proteome coverage for experiments that utilize isobaric labels and gas phase purification techniques (i.e., SPS-MS3). In this work, we introduce inSeqAPI, an instrument application programing interface (iAPI) program that enables construction of novel data acquisition algorithms. First, we analyze biotinylated cysteine peptides from ABPP experiments to demonstrate that a real-time search method within inSeqAPI performs similarly to an equivalent vendor method. Then, we describe PairQuant, a method within inSeqAPI designed for the hyperplexing approach that utilizes protein-level isotopic labeling and peptide-level TMT labeling. PairQuant allows for TMT analysis of 36 conditions in a single sample and achieves â¼98% coverage of both peptide pair partners in a hyperplexed experiment as well as a 40% improvement in the number of quantified cysteine sites compared with non-RTS acquisition. We applied this method in the ABPP study of ligandable cysteine sites in the nucleus leading to an identification of additional druggable sites on protein- and DNA-interaction domains of transcription regulators and on nuclear ubiquitin ligases.
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Cisteína , Proteoma , Proteómica , Proteoma/análisis , Proteómica/métodos , Cisteína/química , Cisteína/metabolismo , Cisteína/análisis , Humanos , Reproducibilidad de los Resultados , Algoritmos , Péptidos/química , Péptidos/análisis , Marcaje Isotópico/métodos , Programas InformáticosRESUMEN
Recent advances in targeted covalent inhibitors have aroused significant interest for their potential in drug development for difficult therapeutic targets. Proteome-wide profiling of functional residues is an integral step of covalent drug discovery aimed at defining actionable sites and evaluating compound selectivity in cells. A classical workflow for this purpose is called IsoTOP-ABPP, which employs an activity-based probe and two isotopically labeled azide-TEV-biotin tags to mark, enrich, and quantify proteome from two samples. Here we report a novel isobaric 11plex-AzidoTMT reagent and a new workflow, named AT-MAPP, that significantly expands multiplexing power as compared to the original isoTOP-ABPP. We demonstrate its application in identifying cysteine on- and off-targets using a KRAS G12C covalent inhibitor ARS-1620. However, changes in some of these hits can be explained by modulation at the protein and post-translational levels. Thus, it would be crucial to interrogate site-level bona fide changes in concurrence to proteome-level changes for corroboration. In addition, we perform a multiplexed covalent fragment screening using four acrylamide-based compounds as a proof-of-concept. This study identifies a diverse set of liganded cysteine residues in a compound-dependent manner with an average hit rate of 0.07% in intact cell. Lastly, we screened 20 sulfonyl fluoride-based compounds to demonstrate that the AT-MAPP assay is flexible for noncysteine functional residues such as tyrosine and lysine. Overall, we envision that 11plex-AzidoTMT will be a useful addition to the current toolbox for activity-based protein profiling and covalent drug development.
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Cisteína , Proteoma , Proteoma/metabolismo , Cisteína/metabolismo , Proteómica , Procesamiento Proteico-Postraduccional , Descubrimiento de DrogasRESUMEN
The growing opportunities recognized for covalent drug inhibitors, like KRAS G12C inhibitors, are driving the need for mass spectrometry methods that can quickly and robustly measure therapeutic drug activity in vivo for drug discovery research and development. Effective front-end sample preparation is critical for proteins extracted from tumors but is generally labor intensive and impractical for large sample numbers typical in pharmacodynamic (PD) studies. Herein, we describe an automated and integrated sample preparation method for the measurement of activity levels of KRAS G12C drug inhibitor alkylation from complex tumor samples involving high throughput detergent removal and preconcentration followed by quantitation using mass spectrometry. We introduce a robust assay with an average intra-assay coefficient of variation (CV) of 4% and an interassay CV of 6% obtained from seven studies, enabling us to understand the relationship between KRAS G12C target occupancy and the therapeutic PD effect from mouse tumor samples. Further, the data demonstrated that the drug candidate GDC-6036, a KRAS G12C covalent inhibitor, shows dose-dependent target inhibition (KRAS G12C alkylation) and MAPK pathway inhibition, which correlate with high antitumor potency in the MIA PaCa-2 pancreatic xenograft model.
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Antineoplásicos , Proteínas Proto-Oncogénicas p21(ras) , Humanos , Animales , Ratones , Proteínas Proto-Oncogénicas p21(ras)/genética , Línea Celular Tumoral , Mutación , Antineoplásicos/farmacología , Modelos Animales de EnfermedadRESUMEN
The ability to quantitatively measure a small molecule's interactions with its protein target(s) is crucial for both mechanistic studies of signaling pathways and in drug discovery. However, current methods to achieve this have specific requirements that can limit their application or interpretation. Here we describe a complementary target-engagement method, HIPStA (Heat Shock Protein Inhibition Protein Stability Assay), a high-throughput method to assess small molecule binding to endogenous, unmodified target protein(s) in cells. The methodology relies on the change in protein turnover when chaperones, such as HSP90, are inhibited and the stabilization effect that drug-target binding has on this change. We use HIPStA to measure drug binding to three different classes of drug targets (receptor tyrosine kinases, nuclear hormone receptors, and cytoplasmic protein kinases), via quantitative fluorescence imaging. We further demonstrate its utility by pairing the method with quantitative mass spectrometry to identify previously unknown targets of a receptor tyrosine kinase inhibitor.
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Proteínas HSP90 de Choque Térmico/metabolismo , Ensayos Analíticos de Alto Rendimiento/métodos , Chaperonas Moleculares/metabolismo , Bibliotecas de Moléculas Pequeñas/metabolismo , Benzoquinonas/farmacología , Compuestos Bicíclicos Heterocíclicos con Puentes/metabolismo , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Línea Celular Tumoral , Receptor alfa de Estrógeno/metabolismo , Técnica del Anticuerpo Fluorescente , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Humanos , Hidroxibutiratos/metabolismo , Hidroxibutiratos/farmacología , Lactamas Macrocíclicas/farmacología , Espectrometría de Masas , Chaperonas Moleculares/antagonistas & inhibidores , Proteínas Nucleares/metabolismo , Estabilidad Proteica/efectos de los fármacos , Proteoma/análisis , Proteínas Proto-Oncogénicas c-raf/metabolismo , Receptor ErbB-2/metabolismoRESUMEN
Activity-based probes (ABPs) are widely used to monitor the activity of enzyme families in biological systems. Inferring enzyme activity from probe reactivity requires that the probe reacts with the enzyme at its active site; however, probe-labeling sites are rarely verified. Here we present an enhanced chemoproteomic approach to evaluate the activity and probe reactivity of deubiquitinase enzymes, using bioorthogonally tagged ABPs and a sequential on-bead digestion protocol to enhance the identification of probe-labeling sites. We confirm probe labeling of deubiquitinase catalytic Cys residues and reveal unexpected labeling of deubiquitinases on non-catalytic Cys residues and of non-deubiquitinase proteins. In doing so, we identify ZUFSP (ZUP1) as a previously unannotated deubiquitinase with high selectivity toward cleaving K63-linked chains. ZUFSP interacts with and modulates ubiquitination of the replication protein A (RPA) complex. Our reactive-site-centric chemoproteomics method is broadly applicable for identifying the reaction sites of covalent molecules, which may expand our understanding of enzymatic mechanisms.
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Enzimas Desubicuitinizantes/química , Procesamiento Proteico-Postraduccional , Proteómica/métodos , Proteína de Replicación A/metabolismo , Coloración y Etiquetado/métodos , Biocatálisis , Dominio Catalítico , Cisteína/química , Cisteína/metabolismo , Enzimas Desubicuitinizantes/clasificación , Enzimas Desubicuitinizantes/genética , Enzimas Desubicuitinizantes/metabolismo , Células HEK293 , Células HeLa , Humanos , Lisina/química , Lisina/metabolismo , Sondas Moleculares , Proteína de Replicación A/genética , Sumoilación , UbiquitinaciónRESUMEN
The ubiquitin system regulates essential cellular processes in eukaryotes. Ubiquitin is ligated to substrate proteins as monomers or chains and the topology of ubiquitin modifications regulates substrate interactions with specific proteins. Thus ubiquitination directs a variety of substrate fates including proteasomal degradation. Deubiquitinase enzymes cleave ubiquitin from substrates and are implicated in disease; for example, ubiquitin-specific protease-7 (USP7) regulates stability of the p53 tumour suppressor and other proteins critical for tumour cell survival. However, developing selective deubiquitinase inhibitors has been challenging and no co-crystal structures have been solved with small-molecule inhibitors. Here, using nuclear magnetic resonance-based screening and structure-based design, we describe the development of selective USP7 inhibitors GNE-6640 and GNE-6776. These compounds induce tumour cell death and enhance cytotoxicity with chemotherapeutic agents and targeted compounds, including PIM kinase inhibitors. Structural studies reveal that GNE-6640 and GNE-6776 non-covalently target USP7 12 Å distant from the catalytic cysteine. The compounds attenuate ubiquitin binding and thus inhibit USP7 deubiquitinase activity. GNE-6640 and GNE-6776 interact with acidic residues that mediate hydrogen-bond interactions with the ubiquitin Lys48 side chain, suggesting that USP7 preferentially interacts with and cleaves ubiquitin moieties that have free Lys48 side chains. We investigated this idea by engineering di-ubiquitin chains containing differential proximal and distal isotopic labels and measuring USP7 binding by nuclear magnetic resonance. This preferential binding protracted the depolymerization kinetics of Lys48-linked ubiquitin chains relative to Lys63-linked chains. In summary, engineering compounds that inhibit USP7 activity by attenuating ubiquitin binding suggests opportunities for developing other deubiquitinase inhibitors and may be a strategy more broadly applicable to inhibiting proteins that require ubiquitin binding for full functional activity.
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Aminopiridinas/química , Aminopiridinas/farmacología , Indazoles/química , Indazoles/farmacología , Fenoles/química , Fenoles/farmacología , Piridinas/química , Piridinas/farmacología , Peptidasa Específica de Ubiquitina 7/antagonistas & inhibidores , Ubiquitina/metabolismo , Animales , Unión Competitiva , Línea Celular Tumoral , Sinergismo Farmacológico , Femenino , Humanos , Ratones , Ratones SCID , Modelos Moleculares , Neoplasias/tratamiento farmacológico , Neoplasias/enzimología , Neoplasias/patología , Unión Proteica , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Proteínas Proto-Oncogénicas c-pim-1/antagonistas & inhibidores , Especificidad por Sustrato , Proteína p53 Supresora de Tumor/deficiencia , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Ubiquitina/química , Peptidasa Específica de Ubiquitina 7/química , Peptidasa Específica de Ubiquitina 7/deficiencia , Peptidasa Específica de Ubiquitina 7/metabolismoRESUMEN
Many diseases are associated with endoplasmic reticulum (ER) stress, which results from an accumulation of misfolded proteins. This triggers an adaptive response called the "unfolded protein response" (UPR), and prolonged exposure to ER stress leads to cell death. Caspases are reported to play a critical role in ER stress-induced cell death but the underlying mechanisms by which they exert their effect continue to remain elusive. To understand the role caspases play during ER stress, a systems level approach integrating analysis of the transcriptome, proteome, and proteolytic substrate profile was employed. This quantitative analysis revealed transcriptional profiles for most human genes, provided information on protein abundance for 4476 proteins, and identified 445 caspase substrates. Based on these data sets many caspase substrates were shown to be downregulated at the protein level during ER stress suggesting caspase activity inhibits their cellular function. Additionally, RNA sequencing revealed a role for caspases in regulation of ER stress-induced transcriptional pathways and gene set enrichment analysis showed expression of multiple gene targets of essential transcription factors to be upregulated during ER stress upon inhibition of caspases. Furthermore, these transcription factors were degraded in a caspase-dependent manner during ER stress. These results indicate that caspases play a dual role in regulating the cellular response to ER stress through both post-translational and transcriptional regulatory mechanisms. Moreover, this study provides unique insight into progression of the unfolded protein response into cell death, which may help identify therapeutic strategies to treat ER stress-related diseases.
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Caspasas/metabolismo , Estrés del Retículo Endoplásmico , Perfilación de la Expresión Génica/métodos , Proteómica/métodos , Respuesta de Proteína Desplegada , Apoptosis , Muerte Celular , Regulación hacia Abajo , Retículo Endoplásmico/metabolismo , Regulación de la Expresión Génica , Células HCT116 , Células HeLa , Humanos , Proteolisis , Análisis de Secuencia de ARN , Factores de Transcripción/genéticaRESUMEN
The KEAP1/Nrf2 pathway senses and responds to changes in intracellular oxidative stress. Mutations that result in constitutive activation of Nrf2 are present in several human tumors, especially non-small cell lung cancer. Therefore, compounds that inhibit Nrf2 activity might be beneficial in treating patients whose tumors show activation of this pathway. Recent reports suggest that the natural product brusatol can potently and selectively inhibit Nrf2 activity, resulting in cell cytotoxicity, and can be effectively combined with chemotherapeutic agents. Here, we analyzed the effects of brusatol on the cellular proteome in the KEAP1 mutant non-small cell lung cancer cell line A549. Brusatol was found to rapidly and potently decrease the expression of the majority of detected proteins, including Nrf2. The most dramatically decreased proteins are those that display a short half-life, like Nrf2. This effect was confirmed by restricting the analysis to newly synthesized proteins using a labeled methionine analogue. Moreover, brusatol increased the expression of multiple components of the ribosome, suggesting that it regulates the function of this macromolecular complex. Finally, we show that brusatol induces its potent cellular cytotoxicity effects on multiple cancer cell lines in a manner independent of KEAP1/Nrf2 activity and with a profile similar to the protein translation inhibitor silvestrol. In conclusion, our data show that the activity of brusatol is not restricted to Nrf2 but, rather, functions as a global protein synthesis inhibitor.
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Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Neoplasias Pulmonares/metabolismo , Espectrometría de Masas/métodos , Factor 2 Relacionado con NF-E2/metabolismo , Inhibidores de la Síntesis de la Proteína/farmacología , Cuassinas/farmacología , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Proteína 1 Asociada A ECH Tipo Kelch/genética , Neoplasias Pulmonares/tratamiento farmacológico , Mutación , Proteómica/métodosRESUMEN
Mutations in the transactive response DNA binding protein-43 (TARDBP/TDP-43) gene, which regulates transcription and splicing, causes a familial form of amyotrophic lateral sclerosis (ALS). Here, we characterize and report the first tardbp mutation in zebrafish, which introduces a premature stop codon (Y220X), eliminating expression of the Tardbp protein. Another TARDBP ortholog, tardbpl, in zebrafish is shown to encode a Tardbp-like protein which is truncated compared with Tardbp itself and lacks part of the C-terminal glycine-rich domain (GRD). Here, we show that tardbp mutation leads to the generation of a novel tardbpl splice form (tardbpl-FL) capable of making a full-length Tardbp protein (Tardbpl-FL), which compensates for the loss of Tardbp. This finding provides a novel in vivo model to study TDP-43-mediated splicing regulation. Additionally, we show that elimination of both zebrafish TARDBP orthologs results in a severe motor phenotype with shortened motor axons, locomotion defects and death at around 10 days post fertilization. The Tardbp/Tardbpl knockout model generated in this study provides an excellent in vivo system to study the role of the functional loss of Tardbp and its involvement in ALS pathogenesis.
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Esclerosis Amiotrófica Lateral/genética , Axones/metabolismo , Proteínas de Unión al ADN/genética , Neuronas Motoras/metabolismo , Empalme del ARN , Proteínas de Pez Cebra/genética , Pez Cebra/metabolismo , Esclerosis Amiotrófica Lateral/embriología , Esclerosis Amiotrófica Lateral/metabolismo , Animales , Proteínas de Unión al ADN/metabolismo , Modelos Animales de Enfermedad , Femenino , Técnicas de Inactivación de Genes , Humanos , Masculino , Mutación , Pez Cebra/embriología , Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
Somites are formed from the presomitic mesoderm (PSM) and give rise to the axial skeleton and skeletal muscles. The PSM is dynamic; somites are generated at the anterior end, while the posterior end is continually renewed with new cells entering from the tailbud progenitor region. Which genes control the conversion of tailbud progenitors into PSM and how is this process coordinated with cell movement? Using loss- and gain-of-function experiments and heat-shock transgenics we show in zebrafish that the transcription factor Mesogenin 1 (Msgn1), acting with Spadetail (Spt), has a central role. Msgn1 allows progression of the PSM differentiation program by switching off the progenitor maintenance genes ntl, wnt3a, wnt8 and fgf8 in the future PSM cells as they exit from the tailbud, and subsequently induces expression of PSM markers such as tbx24. msgn1 is itself positively regulated by Ntl/Wnt/Fgf, creating a negative-feedback loop that might be crucial to regulate homeostasis of the progenitor population until somitogenesis ends. Msgn1 drives not only the changes in gene expression in the nascent PSM cells but also the movements by which they stream out of the tailbud into the PSM. Loss of Msgn1 reduces the flux of cells out of the tailbud, producing smaller somites and an enlarged tailbud, and, by delaying exhaustion of the progenitor population, results in supernumerary tail somites. Through its combined effects on gene expression and cell movement, Msgn1 (with Spt) plays a key role both in genesis of the paraxial mesoderm and in maintenance of the progenitor population from which it derives.
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Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/fisiología , Diferenciación Celular/genética , Movimiento Celular/genética , Células Madre Embrionarias/fisiología , Mesodermo/embriología , Proteínas de Pez Cebra/fisiología , Animales , Animales Modificados Genéticamente , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Rastreo Celular , Desarrollo Embrionario/genética , Células Madre Embrionarias/metabolismo , Mesodermo/citología , Mesodermo/metabolismo , Somitos/embriología , Somitos/metabolismo , Proteínas de Dominio T Box/genética , Proteínas de Dominio T Box/metabolismo , Proteínas de Dominio T Box/fisiología , Cola (estructura animal)/embriología , Torso/embriología , Pez Cebra/embriología , Pez Cebra/genética , Pez Cebra/metabolismo , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
The pancreaticobiliary ductal system connects the liver and pancreas to the intestine. It is composed of the hepatopancreatic ductal (HPD) system as well as the intrahepatic biliary ducts and the intrapancreatic ducts. Despite its physiological importance, the development of the pancreaticobiliary ductal system remains poorly understood. The SRY-related transcription factor SOX9 is expressed in the mammalian pancreaticobiliary ductal system, but the perinatal lethality of Sox9 heterozygous mice makes loss-of-function analyses challenging. We turned to the zebrafish to assess the role of SOX9 in pancreaticobiliary ductal system development. We first show that zebrafish sox9b recapitulates the expression pattern of mouse Sox9 in the pancreaticobiliary ductal system and use a nonsense allele of sox9b, sox9b(fh313), to dissect its function in the morphogenesis of this structure. Strikingly, sox9b(fh313) homozygous mutants survive to adulthood and exhibit cholestasis associated with hepatic and pancreatic duct proliferation, cyst formation, and fibrosis. Analysis of sox9b(fh313) mutant embryos and larvae reveals that the HPD cells appear to mis-differentiate towards hepatic and/or pancreatic fates, resulting in a dysmorphic structure. The intrahepatic biliary cells are specified but fail to assemble into a functional network. Similarly, intrapancreatic duct formation is severely impaired in sox9b(fh313) mutants, while the embryonic endocrine and acinar compartments appear unaffected. The defects in the intrahepatic and intrapancreatic ducts of sox9b(fh313) mutants worsen during larval and juvenile stages, prompting the adult phenotype. We further show that Sox9b interacts with Notch signaling to regulate intrahepatic biliary network formation: sox9b expression is positively regulated by Notch signaling, while Sox9b function is required to maintain Notch signaling in the intrahepatic biliary cells. Together, these data reveal key roles for SOX9 in the morphogenesis of the pancreaticobiliary ductal system, and they cast human Sox9 as a candidate gene for pancreaticobiliary duct malformation-related pathologies.
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Conductos Biliares Intrahepáticos/crecimiento & desarrollo , Hígado/crecimiento & desarrollo , Páncreas/crecimiento & desarrollo , Factor de Transcripción SOX9/genética , Proteínas de Pez Cebra/genética , Pez Cebra , Animales , Conductos Biliares Intrahepáticos/embriología , Conductos Biliares Intrahepáticos/metabolismo , Codón sin Sentido , Regulación del Desarrollo de la Expresión Génica , Hígado/embriología , Hígado/metabolismo , Morfogénesis/genética , Páncreas/embriología , Páncreas/metabolismo , Receptores Notch/genética , Receptores Notch/metabolismo , Factor de Transcripción SOX9/metabolismo , Transducción de Señal , Pez Cebra/embriología , Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
Recent zebrafish studies have shown that the late appearing pancreatic endocrine cells are derived from pancreatic ducts but the regulatory factors involved are still largely unknown. Here, we show that the zebrafish sox9b gene is expressed in pancreatic ducts where it labels the pancreatic Notch-responsive cells previously shown to be progenitors. Inactivation of sox9b disturbs duct formation and impairs regeneration of beta cells from these ducts in larvae. sox9b expression in the midtrunk endoderm appears at the junction of the hepatic and ventral pancreatic buds and, by the end of embryogenesis, labels the hepatopancreatic ductal system as well as the intrapancreatic and intrahepatic ducts. Ductal morphogenesis and differentiation are specifically disrupted in sox9b mutants, with the dysmorphic hepatopancreatic ducts containing misdifferentiated hepatocyte-like and pancreatic-like cells. We also show that maintenance of sox9b expression in the extrapancreatic and intrapancreatic ducts requires FGF and Notch activity, respectively, both pathways known to prevent excessive endocrine differentiation in these ducts. Furthermore, beta cell recovery after specific ablation is severely compromised in sox9b mutant larvae. Our data position sox9b as a key player in the generation of secondary endocrine cells deriving from pancreatic ducts in zebrafish.
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Hepatopáncreas/embriología , Islotes Pancreáticos/fisiología , Factor de Transcripción SOX9/fisiología , Proteínas de Pez Cebra/fisiología , Pez Cebra/embriología , Animales , Factores de Crecimiento de Fibroblastos/fisiología , Hepatopáncreas/fisiología , Páncreas/citología , Páncreas/fisiología , Receptores Notch/fisiología , Regeneración , Transducción de Señal , Pez Cebra/fisiologíaRESUMEN
The intrahepatic biliary ducts transport bile produced by the hepatocytes out of the liver. Defects in biliary cell differentiation and biliary duct remodeling cause a variety of congenital diseases including Alagille Syndrome and polycystic liver disease. While the molecular pathways regulating biliary cell differentiation have received increasing attention (Lemaigre, 2010), less is known about the cellular behavior underlying biliary duct remodeling. Here, we have identified a novel gene, claudin 15-like b (cldn15lb), which exhibits a unique and dynamic expression pattern in the hepatocytes and biliary epithelial cells in zebrafish. Claudins are tight junction proteins that have been implicated in maintaining epithelial polarity, regulating paracellular transport, and providing barrier function. In zebrafish cldn15lb mutant livers, tight junctions are observed between hepatocytes, but these cells show polarization defects as well as canalicular malformations. Furthermore, cldn15lb mutants show abnormalities in biliary duct morphogenesis whereby biliary epithelial cells remain clustered together and form a disorganized network. Our data suggest that Cldn15lb plays an important role in the remodeling process during biliary duct morphogenesis. Thus, cldn15lb mutants provide a novel in vivo model to study the role of tight junction proteins in the remodeling of the biliary network and hereditary cholestasis.
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Conductos Biliares Intrahepáticos/crecimiento & desarrollo , Claudinas/metabolismo , Hepatocitos/metabolismo , Morfogénesis/fisiología , Uniones Estrechas/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/crecimiento & desarrollo , Animales , Conductos Biliares Intrahepáticos/citología , Conductos Biliares Intrahepáticos/metabolismo , Línea Celular , Polaridad Celular/fisiología , Claudinas/genética , Perros , Células Epiteliales/metabolismo , Técnica del Anticuerpo Fluorescente , Hibridación in Situ , Larva/crecimiento & desarrollo , Larva/metabolismo , Microscopía Electrónica de Transmisión , Mutación/genética , Pez Cebra/metabolismo , Proteínas de Pez Cebra/genéticaRESUMEN
Disc1 is a schizophrenia risk gene that engages multiple signaling pathways during neurogenesis and brain development. Using the zebrafish as a tool, we analyze the function of zebrafish Disc1 (zDisc1) at the earliest stages of brain and body development. We define a "tool" as a biological system that gives insight into mechanisms underlying a human disorder, although the system does not phenocopy the disorder. A zDisc1 peptide binds to GSK3ß, and zDisc1 directs early brain development and neurogenesis, by promoting ß-catenin-mediated Wnt signaling and inhibiting GSK3ß activity. zDisc1 loss-of-function embryos additionally display a convergence and extension phenotype, demonstrated by abnormal movement of dorsolateral cells during gastrulation, through changes in gene expression, and later through formation of abnormal, U-shaped muscle segments, and a truncated tail. These phenotypes are caused by alterations in the noncanonical Wnt pathway, via Daam and Rho signaling. The convergence and extension phenotype can be rescued by a dominant negative GSK3ß construct, suggesting that zDisc1 inhibits GSK3ß activity during noncanonical Wnt signaling. This is the first demonstration that Disc1 modulates the noncanonical Wnt pathway and suggests a previously unconsidered mechanism by which Disc1 may contribute to the etiology of neuropsychiatric disorders.
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Proteínas del Tejido Nervioso/metabolismo , Vía de Señalización Wnt , Proteínas de Pez Cebra/metabolismo , Pez Cebra/embriología , Pez Cebra/metabolismo , beta Catenina/metabolismo , Secuencia de Aminoácidos , Animales , Animales Modificados Genéticamente , Secuencia de Bases , Sitios de Unión , Encéfalo/embriología , Encéfalo/metabolismo , Secuencia Conservada , Cartilla de ADN/genética , Desarrollo Embrionario/genética , Desarrollo Embrionario/fisiología , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3 beta , Humanos , Mutagénesis , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Proteínas del Tejido Nervioso/genética , Neurogénesis/genética , Neurogénesis/fisiología , Oligodesoxirribonucleótidos Antisentido/genética , Homología de Secuencia de Aminoácido , Transducción de Señal , Especificidad de la Especie , Pez Cebra/genética , Proteínas de Pez Cebra/antagonistas & inhibidores , Proteínas de Pez Cebra/genéticaRESUMEN
Studies of the zebrafish epithalamus have provided recent insights into the development of left-right brain asymmetry, which is crucial to normal human brain function. The habenular nuclei of zebrafish are robustly asymmetric, with dense elaboration of neuropil only in the left lateral subnucleus. Because this feature is tightly correlated with asymmetric expression of K(+) channel tetramerization domain-containing proteins 12.1 and 12.2 (Kctd12.1/12.2), we screened for Kctd12.1-interacting proteins to identify molecular mechanisms leading to neuropil asymmetry, and uncovered a novel interaction between Kctd12.1 and Unc-51-like kinase 2 (Ulk2). We show here that knockdown of Ulk2 or overexpression of Kctd12 proteins reduces asymmetric neuropil elaboration. Conversely, overexpression of Ulk2 or mutation of kctd12 genes causes excess neuropil elaboration. We conclude that Ulk2 activity promotes neuropil elaboration while Kctd12 proteins limit Ulk2 activity asymmetrically. This work describes a regulatory mechanism for neuronal process extension that may be conserved in other developmental contexts in addition to the epithalamus.
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Lateralidad Funcional/fisiología , Habénula/citología , Inhibición Psicológica , Neurópilo/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , Alquilantes/farmacología , Animales , Animales Modificados Genéticamente , Proteínas de Transporte de Catión/genética , Proteínas de Transporte de Catión/metabolismo , Etilnitrosourea/farmacología , Lateralidad Funcional/efectos de los fármacos , Lateralidad Funcional/genética , Regulación del Desarrollo de la Expresión Génica/genética , Regulación del Desarrollo de la Expresión Génica/fisiología , Proteínas Fluorescentes Verdes/genética , Habénula/efectos de los fármacos , Habénula/crecimiento & desarrollo , Habénula/lesiones , Inmunoprecipitación , Larva , Mutación/genética , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Neurópilo/efectos de los fármacos , Proteínas Proto-Oncogénicas c-myc/metabolismo , ARN Mensajero/metabolismo , Tubulina (Proteína)/metabolismo , Técnicas del Sistema de Dos Híbridos , Pez Cebra , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
Myogenic regulatory factors of the myod family (MRFs) are transcription factors essential for mammalian skeletal myogenesis. Here we show that a mutation in the zebrafish myod gene delays and reduces early somitic and pectoral fin myogenesis, reduces miR-206 expression, and leads to a persistent reduction in somite size until at least the independent feeding stage. A mutation in myog, encoding a second MRF, has little obvious phenotype at early stages, but exacerbates the loss of somitic muscle caused by lack of Myod. Mutation of both myod and myf5 ablates all skeletal muscle. Haploinsufficiency of myod leads to reduced embryonic somite muscle bulk. Lack of Myod causes a severe reduction in cranial musculature, ablating most muscles including the protractor pectoralis, a putative cucullaris homologue. This phenotype is accompanied by a severe dysmorphology of the cartilaginous skeleton and failure of maturation of several cranial bones, including the opercle. As myod expression is restricted to myogenic cells, the data show that myogenesis is essential for proper skeletogenesis in the head.
Asunto(s)
Huesos/embriología , Regulación del Desarrollo de la Expresión Génica/fisiología , Haploinsuficiencia/genética , Desarrollo de Músculos/fisiología , Proteína MioD/genética , Cráneo/embriología , Pez Cebra/embriología , Animales , Cartílago/embriología , Haploinsuficiencia/fisiología , Inmunohistoquímica , Hibridación in Situ , Larva/fisiología , Músculo Esquelético/embriología , Mutación/genética , Proteína MioD/metabolismo , Extremidad Superior/embriología , Pez Cebra/genéticaRESUMEN
TILLING, for Targeting Induced Local Lesions in Genomes, is a reverse genetics strategy that identifies mutations in specific genes of interest in chemically mutagenized populations. First described in 2000 for mutation detection in Arabidopsis, TILLING is now used in a wide range of plants including soybean, rice, barley and maize as well as for animal model systems, including Arabidopsis, Drosophila, Caenorhabditis elegans, rat, medaka and zebrafish and for the discovery of naturally occurring polymorphisms in humans. This review summarizes current TILLING methodologies as they have been applied to the zebrafish, ongoing TILLING projects and resources in the zebrafish community, and the future of zebrafish TILLING.