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1.
J Biol Chem ; 300(3): 105772, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38382674

RESUMO

Pre-mRNA splicing is a precise regulated process and is crucial for system development and homeostasis maintenance. Mutations in spliceosomal components have been found in various hematopoietic malignancies (HMs) and have been considered as oncogenic derivers of HMs. However, the role of spliceosomal components in normal and malignant hematopoiesis remains largely unknown. Pre-mRNA processing factor 31 (PRPF31) is a constitutive spliceosomal component, which mutations are associated with autosomal dominant retinitis pigmentosa. PRPF31 was found to be mutated in several HMs, but the function of PRPF31 in normal hematopoiesis has not been explored. In our previous study, we generated a prpf31 knockout (KO) zebrafish line and reported that Prpf31 regulates the survival and differentiation of retinal progenitor cells by modulating the alternative splicing of genes involved in mitosis and DNA repair. In this study, by using the prpf31 KO zebrafish line, we discovered that prpf31 KO zebrafish exhibited severe defects in hematopoietic stem and progenitor cell (HSPC) expansion and its sequentially differentiated lineages. Immunofluorescence results showed that Prpf31-deficient HSPCs underwent malformed mitosis and M phase arrest during HSPC expansion. Transcriptome analysis and experimental validations revealed that Prpf31 deficiency extensively perturbed the alternative splicing of mitosis-related genes. Collectively, our findings elucidate a previously undescribed role for Prpf31 in HSPC expansion, through regulating the alternative splicing of mitosis-related genes.


Assuntos
Fatores de Processamento de RNA , Proteínas de Peixe-Zebra , Peixe-Zebra , Animais , Desenvolvimento Embrionário , Mutação , Precursores de RNA/metabolismo , Fatores de Processamento de RNA/metabolismo , Células-Tronco/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimento , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/metabolismo
2.
Mol Cancer Res ; 22(4): 386-401, 2024 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-38294692

RESUMO

Calcium homeostasis is critical for cell proliferation, and emerging evidence shows that cancer cells exhibit altered calcium signals to fulfill their need for proliferation. However, it remains unclear whether there are oncogene-specific calcium homeostasis regulations that can expose novel therapeutic targets. Here, from RNAi screen, we report that adenosylhomocysteinase like protein 1 (AHCYL1), a suppressor of the endoplasmic reticulum (ER) calcium channel protein inositol trisphosphate receptor (IP3R), is selectively upregulated and critical for cell proliferation and tumor growth potential of human NRAS-mutated melanoma, but not for melanoma expressing BRAF V600E. Mechanistically, AHCYL1 deficiency results in decreased ER calcium levels, activates the unfolded protein response (UPR), and triggers downstream apoptosis. In addition, we show that AHCYL1 transcription is regulated by activating transcription factor 2 (ATF2) in NRAS-mutated melanoma. Our work provides evidence for oncogene-specific calcium regulations and suggests AHCYL1 as a novel therapeutic target for RAS mutant-expressing human cancers, including melanoma. IMPLICATIONS: Our findings suggest that targeting the AHCYL1-IP3R axis presents a novel therapeutic approach for NRAS-mutated melanomas, with potential applicability to all cancers harboring RAS mutations, such as KRAS-mutated human colorectal cancers.


Assuntos
Adenosil-Homocisteinase , Retículo Endoplasmático , Melanoma , Humanos , Adenosil-Homocisteinase/metabolismo , Cálcio , Linhagem Celular Tumoral , Retículo Endoplasmático/metabolismo , GTP Fosfo-Hidrolases/genética , Homeostase , Melanoma/metabolismo , Melanoma/patologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mutação , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo
3.
Nature ; 623(7989): 1034-1043, 2023 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-37993715

RESUMO

Diet-derived nutrients are inextricably linked to human physiology by providing energy and biosynthetic building blocks and by functioning as regulatory molecules. However, the mechanisms by which circulating nutrients in the human body influence specific physiological processes remain largely unknown. Here we use a blood nutrient compound library-based screening approach to demonstrate that dietary trans-vaccenic acid (TVA) directly promotes effector CD8+ T cell function and anti-tumour immunity in vivo. TVA is the predominant form of trans-fatty acids enriched in human milk, but the human body cannot produce TVA endogenously1. Circulating TVA in humans is mainly from ruminant-derived foods including beef, lamb and dairy products such as milk and butter2,3, but only around 19% or 12% of dietary TVA is converted to rumenic acid by humans or mice, respectively4,5. Mechanistically, TVA inactivates the cell-surface receptor GPR43, an immunomodulatory G protein-coupled receptor activated by its short-chain fatty acid ligands6-8. TVA thus antagonizes the short-chain fatty acid agonists of GPR43, leading to activation of the cAMP-PKA-CREB axis for enhanced CD8+ T cell function. These findings reveal that diet-derived TVA represents a mechanism for host-extrinsic reprogramming of CD8+ T cells as opposed to the intrahost gut microbiota-derived short-chain fatty acids. TVA thus has translational potential for the treatment of tumours.


Assuntos
Linfócitos T CD8-Positivos , Neoplasias , Ácidos Oleicos , Animais , Bovinos , Humanos , Camundongos , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , AMP Cíclico/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Laticínios , Ácidos Graxos Voláteis/farmacologia , Ácidos Graxos Voláteis/uso terapêutico , Leite/química , Neoplasias/dietoterapia , Neoplasias/imunologia , Ácidos Oleicos/farmacologia , Ácidos Oleicos/uso terapêutico , Carne Vermelha , Ovinos
4.
iScience ; 26(11): 108103, 2023 Nov 17.
Artigo em Inglês | MEDLINE | ID: mdl-37867960

RESUMO

DEAH-Box Helicase 38 (DHX38) is a pre-mRNA splicing factor and also a disease-causing gene of autosomal recessive retinitis pigmentosa (arRP). The role of DHX38 in the development and maintenance of the retina remains largely unknown. In this study, by using the dhx38 knockout zebrafish model, we demonstrated that Dhx38 deficiency causes severe differentiation defects and apoptosis of retinal progenitor cells (RPCs) through disrupted mitosis and increased DNA damage. Furthermore, we found a significant accumulation of R-loops in the dhx38-deficient RPCs and human cell lines. Finally, we found that DNA replication stress is the prerequisite for R-loop-induced DNA damage in the DHX38 knockdown cells. Taken together, our study demonstrates a necessary role of DHX38 in the development of retina and reveals a DHX38/R-loop/replication stress/DNA damage regulatory axis that is relatively independent of the known functions of DHX38 in mitosis control.

5.
Development ; 149(17)2022 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-35929537

RESUMO

Mutations that occur in RNA-splicing machinery may contribute to hematopoiesis-related diseases. How splicing factor mutations perturb hematopoiesis, especially in the differentiation of erythro-myeloid progenitors (EMPs), remains elusive. Dhx38 is a pre-mRNA splicing-related DEAH box RNA helicase, for which the physiological functions and splicing mechanisms during hematopoiesis currently remain unclear. Here, we report that Dhx38 exerts a broad effect on definitive EMPs as well as the differentiation and maintenance of hematopoietic stem and progenitor cells (HSPCs). In dhx38 knockout zebrafish, EMPs and HSPCs were found to be arrested in mitotic prometaphase, accompanied by a 'grape' karyotype, owing to the defects in chromosome alignment. Abnormal alternatively spliced genes related to chromosome segregation, the microtubule cytoskeleton, cell cycle kinases and DNA damage were present in the dhx38 mutants. Subsequently, EMPs and HSPCs in dhx38 mutants underwent P53-dependent apoptosis. This study provides novel insights into alternative splicing regulated by Dhx38, a process that plays a crucial role in the proliferation and differentiation of fetal EMPs and HSPCs.


Assuntos
Processamento Alternativo , Peixe-Zebra , Processamento Alternativo/genética , Animais , Hematopoese/genética , Células-Tronco Hematopoéticas , Células Progenitoras Mieloides , Peixe-Zebra/genética , Peixe-Zebra/metabolismo
6.
Cell Chem Biol ; 29(7): 1200-1208.e6, 2022 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-35429459

RESUMO

Environmental stresses, including hypoxia or detachment for anchorage independence, or attenuation of mitochondrial respiration through inhibition of electron transport chain induce reductive carboxylation in cells with an enhanced fraction of citrate arising through reductive metabolism of glutamine. This metabolic process contributes to redox homeostasis and sustains biosynthesis of lipids. Reductive carboxylation is often dependent on cytosolic isocitrate dehydrogenase 1 (IDH1). However, whether diverse cellular signals induce reductive carboxylation differentially or through a common signaling converging node remains unclear. We found that induction of reductive carboxylation commonly requires enhanced tyrosine phosphorylation and activation of IDH1, which, surprisingly, is achieved by attenuation of a cytosolic protein tyrosine phosphatase, Src homology region 2 domain-containing phosphatase-2 (SHP-2). Mechanistically, diverse signals induce reductive carboxylation by converging at upregulation of NADPH oxidase 2, leading to elevated cytosolic reactive oxygen species that consequently inhibit SHP-2. Together, our work elucidates the signaling basis underlying reductive carboxylation in cancer cells.


Assuntos
Isocitrato Desidrogenase , Neoplasias , Linhagem Celular Tumoral , Ciclo do Ácido Cítrico , Glutamina/metabolismo , Isocitrato Desidrogenase/metabolismo , Oxirredução , Fosforilação , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo
7.
PLoS Genet ; 18(3): e1009841, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-35245286

RESUMO

Neural retina leucine zipper (NRL) is an essential gene for the fate determination and differentiation of the precursor cells into rod photoreceptors in mammals. Mutations in NRL are associated with the autosomal recessive enhanced S-cone syndrome and autosomal dominant retinitis pigmentosa. However, the exact role of Nrl in regulating the development and maintenance of photoreceptors in the zebrafish (Danio rerio), a popular animal model used for retinal degeneration and regeneration studies, has not been fully determined. In this study, we generated an nrl knockout zebrafish model via the CRISPR-Cas9 technology and observed a surprising phenotype characterized by a reduced number, but not the total loss, of rods and over-growth of green cones. We discovered two waves of rod genesis, nrl-dependent and -independent at the embryonic and post-embryonic stages, respectively, in zebrafish by monitoring the rod development. Through bulk and single-cell RNA sequencing, we characterized the gene expression profiles of the whole retina and each retinal cell type from the wild type and nrl knockout zebrafish. The over-growth of green cones and mis-expression of green-cone-specific genes in rods in nrl mutants suggested that there are rod/green-cone bipotent precursors, whose fate choice between rod versus green-cone is controlled by nrl. Besides, we identified the mafba gene as a novel regulator of the nrl-independent rod development, based on the cell-type-specific expression patterns and the retinal phenotype of nrl/mafba double-knockout zebrafish. Gene collinearity analysis revealed the evolutionary origin of mafba and suggested that the function of mafba in rod development is specific to modern fishes. Furthermore, the altered photoreceptor composition and abnormal gene expression in nrl mutants caused progressive retinal degeneration and subsequent regeneration. Accordingly, this study revealed a novel function of the mafba gene in rod development and established a working model for the developmental and regulatory mechanisms regarding the rod and green-cone photoreceptors in zebrafish.


Assuntos
Degeneração Retiniana , Peixe-Zebra , Animais , Fatores de Transcrição de Zíper de Leucina Básica/genética , Proteínas do Olho/metabolismo , Mamíferos/metabolismo , Células Fotorreceptoras Retinianas Cones/metabolismo , Degeneração Retiniana/genética , Degeneração Retiniana/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismo
8.
Biochim Biophys Acta Mol Basis Dis ; 1867(5): 166096, 2021 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-33524530

RESUMO

Previous reports revealed that mutation of mitochondrial inner-membrane located protein SFXN1 led to pleiotropic hematological and skeletal defects in mice, associated with the presence of hypochromic erythroid cell, iron overload in mitochondrion of erythroblast and the development of sideroblastic anemia (SA). However, the potential role of sfxn1 during erythrocyte differentiation and the development of anemia, especially the pathological molecular mechanism still remains elusive. In this study, the correlation between sfxn1 and erythroid cell development is explored through zebrafish in vivo coupled with human hematopoietic cells assay ex vivo. Both knockdown and knockout of sfxn1 result in hypochromic anemia phenotype in zebrafish. Further analyses demonstrate that the development of anemia attributes to the biosynthetic deficiency of hemoglobin, which is caused by the biosynthetic disorder of heme that associates with one­carbon (1C) metabolism process of mitochondrial branch in erythrocyte. Sfxn1 is also involved in the differentiation and maturation of erythrocyte in inducible human umbilical cord blood stem cells. In addition, we found that functional disruption of sfxn1 causes hypochromic anemia that is distinct from SA. These findings reveal that sfxn1 is genetically conserved and essential for the maturation of erythrocyte via facilitating the production of hemoglobin, which may provide a possible guidance for the future clinical treatment of sfxn1 mutation associated hematological disorders.


Assuntos
Anemia/patologia , Embrião não Mamífero/patologia , Eritrócitos/patologia , Hemoglobinas/metabolismo , Mutação , Transportador 1 de Glucose-Sódio/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Anemia/metabolismo , Animais , Diferenciação Celular , Embrião não Mamífero/metabolismo , Eritrócitos/metabolismo , Eritropoese , Fenótipo , Transportador 1 de Glucose-Sódio/genética , Peixe-Zebra , Proteínas de Peixe-Zebra/genética
9.
Nucleic Acids Res ; 49(4): 2027-2043, 2021 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-33476374

RESUMO

Dysfunction of splicing factors often result in abnormal cell differentiation and apoptosis, especially in neural tissues. Mutations in pre-mRNAs processing factor 31 (PRPF31) cause autosomal dominant retinitis pigmentosa, a progressive retinal degeneration disease. The transcriptome-wide splicing events specifically regulated by PRPF31 and their biological roles in the development and maintenance of retina are still unclear. Here, we showed that the differentiation and viability of retinal progenitor cells (RPCs) are severely perturbed in prpf31 knockout zebrafish when compared with other tissues at an early embryonic stage. At the cellular level, significant mitotic arrest and DNA damage were observed. These defects could be rescued by the wild-type human PRPF31 rather than the disease-associated mutants. Further bioinformatic analysis and experimental verification uncovered that Prpf31 deletion predominantly causes the skipping of exons with a weak 5' splicing site. Moreover, genes necessary for DNA repair and mitotic progression are most enriched among the differentially spliced events, which may explain the cellular and tissular defects in prpf31 mutant retinas. This is the first time that Prpf31 is demonstrated to be essential for the survival and differentiation of RPCs during retinal neurogenesis by specifically modulating the alternative splicing of genes involved in DNA repair and mitosis.


Assuntos
Processamento Alternativo , Células-Tronco Neurais/metabolismo , Neurogênese/genética , Retina/embriologia , Proteínas de Peixe-Zebra/fisiologia , Animais , Apoptose , Sistemas CRISPR-Cas , Sobrevivência Celular , Dano ao DNA , Reparo do DNA , Éxons , Técnicas de Inativação de Genes , Pontos de Checagem da Fase M do Ciclo Celular , Células-Tronco Neurais/citologia , Neurônios Retinianos/citologia , Neurônios Retinianos/metabolismo , Fuso Acromático/ultraestrutura , Proteína Supressora de Tumor p53/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
10.
Entropy (Basel) ; 22(9)2020 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-33286799

RESUMO

Incomplete data are unavoidable for survival analysis as well as life testing, so more and more researchers are beginning to study censoring data. This paper discusses and considers the estimation of unknown parameters featured by the Kumaraswamy distribution on the condition of generalized progressive hybrid censoring scheme. Estimation of reliability is also considered in this paper. To begin with, the maximum likelihood estimators are derived. In addition, Bayesian estimators under not only symmetric but also asymmetric loss functions, like general entropy, squared error as well as linex loss function, are also offered. Since the Bayesian estimates fail to be of explicit computation, Lindley approximation, as well as the Tierney and Kadane method, is employed to obtain the Bayesian estimates. A simulation research is conducted for the comparison of the effectiveness of the proposed estimators. A real-life example is employed for illustration.

11.
FASEB J ; 34(9): 11997-12008, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32738093

RESUMO

Hematopoietic stem and progenitor cells (HSPCs) have the ability to self-renew and differentiate into various blood cells, thus playing an important role in maintenance of lifelong hematopoiesis. Brahma-related gene 1 (BRG1), which acts as the ATP subunit of mammalian SWI-SNF-related chromatin remodeling complexes, is involved in human acute myeloid leukemia and highly expresses in short-term HSPCs. But its role and regulatory mechanism for HSPC development have not yet been well established. Here, we generated a brg1 knockout zebrafish model using TALEN technology. We found that in brg1-/- embryo, the primitive hematopoiesis remained well, while definitive hematopoiesis formation was significantly impaired. The number of hemogenic endothelial cells was decreased, further affecting definitive hematopoiesis with reduced myeloid and lymphoid cells. During embryogenesis, the nitric oxide (NO) microenvironment in brg1-/- embryo was seriously damaged and the reduction of HSPCs could be partially rescued by a NO donor. Chromatin immunoprecipitation (ChIP) assays showed that BRG1 could bind to the promoter of KLF2 and trigger its transcriptional activity of NO synthase. Our findings show that Brg1 promotes klf2a expression in hemogenic endothelium and highlight a novel mechanism for HSPC formation and maintenance.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Embrião não Mamífero/embriologia , Hematopoese , Células-Tronco Hematopoéticas/metabolismo , Nicho de Células-Tronco , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Inativação de Genes , Células-Tronco Hematopoéticas/citologia , Fatores de Transcrição Kruppel-Like/biossíntese , Fatores de Transcrição Kruppel-Like/genética , Óxido Nítrico/genética , Óxido Nítrico/metabolismo , Elementos de Resposta , Transcrição Gênica , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/biossíntese , Proteínas de Peixe-Zebra/genética
12.
Biochim Biophys Acta Mol Basis Dis ; 1865(6): 1273-1283, 2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-30684641

RESUMO

Mutations in the photoreceptor cell-specific nuclear receptor gene Nr2e3 increased the number of S-cone photoreceptors in human and murine retinas and led to retinal degeneration that involved photoreceptor and non-photoreceptor cells. The mechanisms underlying these complex phenotypes remain unclear. In the hope of understanding the precise role of Nr2e3 in photoreceptor cell fate determination and differentiation, we generated a line of Nr2e3 knockout zebrafish using CRISPR technology. In these Nr2e3-null animals, rod precursors undergo terminal mitoses but fail to differentiate as rods. Rod-specific genes are not expressed and the outer segment (OS) fails to form. Formation and differentiation of cone photoreceptors is normal. Specifically, there is no increase in the number of UV-cone or S-cone photoreceptors. Laminated retinal structure is maintained. After normal development, L-/M-cones selectively degenerate, with progressive shortening of OS that starts at age 1 month. The amount of cone phototransduction proteins is concomitantly reduced, whereas UV- and S-cones have normal OS lengths even at age 10 months. In vitro studies show Nr2e3 synergizes with Crx and Nrl to enhance rhodopsin gene expression. Nr2e3 does not affect cone opsin expression. Our results extend the knowledge of Nr2e3's roles and have specific implications for the interpretation of the phenotypes observed in human and murine retinas. Furthermore, our model may offer new opportunities in finding treatments for enhanced S-cone syndrome (ESCS) and other retinal degenerative diseases.


Assuntos
Diferenciação Celular/genética , Mutação , Receptores Citoplasmáticos e Nucleares/genética , Células Fotorreceptoras Retinianas Cones/patologia , Degeneração Retiniana/genética , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Proteínas de Peixe-Zebra/genética , Animais , Sequência de Bases , Sistemas CRISPR-Cas , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Microscopia Eletrônica de Transmissão , Receptores Citoplasmáticos e Nucleares/metabolismo , Retina/embriologia , Retina/crescimento & desenvolvimento , Retina/ultraestrutura , Células Fotorreceptoras Retinianas Cones/metabolismo , Degeneração Retiniana/metabolismo , Degeneração Retiniana/patologia , Células Fotorreceptoras Retinianas Bastonetes/citologia , Peixe-Zebra , Proteínas de Peixe-Zebra/metabolismo
13.
Blood ; 133(8): 805-815, 2019 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-30482793

RESUMO

Hematopoietic stem and progenitor cells (HSPCs) originate from the hemogenic endothelium via the endothelial-to-hematopoietic transition, are self-renewing, and replenish all lineages of blood cells throughout life. BCAS2 (breast carcinoma amplified sequence 2) is a component of the spliceosome and is involved in multiple biological processes. However, its role in hematopoiesis remains unknown. We established a bcas2 knockout zebrafish model by using transcription activator-like effector nucleases. The bcas2 -/- zebrafish showed severe impairment of HSPCs and their derivatives during definitive hematopoiesis. We also observed significant signs of HSPC apoptosis in the caudal hematopoietic tissue of bcas2 -/- zebrafish, which may be rescued by suppression of p53. Furthermore, we show that the bcas2 deletion induces an abnormal alternative splicing of Mdm4 that predisposes cells to undergo p53-mediated apoptosis, which provides a mechanistic explanation of the deficiency observed in HSPCs. Our findings revealed a novel and vital role for BCAS2 during HSPC maintenance in zebrafish.


Assuntos
Embrião não Mamífero/embriologia , Desenvolvimento Embrionário , Células-Tronco Hematopoéticas/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Animais , Animais Geneticamente Modificados/embriologia , Animais Geneticamente Modificados/genética , Técnicas de Silenciamento de Genes , Proteínas de Neoplasias/genética , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética
14.
FASEB J ; 33(1): 696-710, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30044923

RESUMO

The proper development of atrioventricular (AV) valves is critical for heart morphogenesis and for the formation of the cardiac conduction system. Defects in AV valve development are the most common type of congenital heart defect. Cardiac troponin I ( ctnni), a structural and regulatory protein involved in cardiac muscle contraction, is a subunit of the troponin complex, but the functions and molecular mechanisms of ctnni during early heart development remain unclear. We created a knockout zebrafish model in which troponin I type 1b ( tnni1b) ( Tnni-HC, heart and craniofacial) was deleted using the clustered regularly interspaced short palindromic repeat/clustered regularly interspaced short palindromic repeat-associated protein system. In the homozygous mutant, the embryos showed severe pericardial edema, malformation of the heart tube, reduction of heart rate without contraction and with almost no blood flow, heart cavity congestion, and lack of an endocardial ring or valve leaflet, resulting in 88.8 ± 6.0% lethality at 7 d postfertilization. Deletion of tnni1b caused the abnormal expression of several markers involved in AV valve development, including bmp4, cspg2, has2, notch1b, spp1, and Alcam. Myocardial re-expression of tnni1b in mutants partially rescued the pericardial edema phenotype and AV canal (AVC) developmental defects. We further showed that tnni1b knockout in zebrafish and ctnni knockdown in rat h9c2 myocardial cells inhibited cardiac wnt signaling and that myocardial reactivation of wnt signaling partially rescued the abnormal expression of AVC markers caused by the tnni1b deletion. Taken together, our data suggest that tnni1b plays a vital role in zebrafish AV valve development by regulating the myocardial wnt signaling pathway.-Cai, C., Sang, C., Du, J., Jia, H., Tu, J., Wan, Q., Bao, B., Xie, S., Huang, Y., Li, A., Li, J., Yang, K., Wang, S., Lu, Q. Knockout of tnni1b in zebrafish causes defects in atrioventricular valve development via the inhibition of myocardial wnt signaling pathway.


Assuntos
Nó Atrioventricular/patologia , Embrião não Mamífero/patologia , Valvas Cardíacas/patologia , Miocárdio/patologia , Troponina I/antagonistas & inibidores , Via de Sinalização Wnt , Proteínas de Peixe-Zebra/antagonistas & inibidores , Peixe-Zebra/embriologia , Animais , Animais Geneticamente Modificados/embriologia , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/metabolismo , Nó Atrioventricular/metabolismo , Sistemas CRISPR-Cas , Células Cultivadas , Embrião não Mamífero/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Valvas Cardíacas/embriologia , Valvas Cardíacas/metabolismo , Miocárdio/metabolismo , Organogênese , Ratos , Troponina I/genética , Troponina I/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
15.
Hum Genet ; 137(10): 779-794, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30242501

RESUMO

Most cases of Usher syndrome type II (USH2) are due to mutations in the USH2A gene. There are no effective treatments or ideal animal models for this disease, and the pathological mechanisms of USH2 caused by USH2A mutations are still unknown. Here, we constructed a ush2a knockout (ush2a-/-) zebrafish model using TALEN technology to investigate the molecular pathology of USH2. An early onset auditory disorder and abnormal morphology of inner ear stereocilia were identified in the ush2a-/- zebrafish. Consequently, the disruption of Ush2a in zebrafish led to a hearing impairment, like that in mammals. Electroretinography (ERG) test indicated that deletion of Ush2a affected visual function at an early stage, and histological analysis revealed that the photoreceptors progressively degenerated. Rod degeneration occurred prior to cone degeneration in ush2a-/- zebrafish, which is consistent with the classical description of the progression of retinitis pigmentosa (RP). Destruction of the outer segments (OSs) of rods led to the down-regulation of phototransduction cascade proteins at late stage. The expression of Ush1b and Ush1c was up-regulated when Ush2a was null. We also found that disruption of fibronectin assembly at the retinal basement membrane weakened cell adhesion in ush2a-/- mutants. In summary, for the first time, we generated a ush2a knockout zebrafish line with auditory disorder and retinal degeneration which mimicked the symptoms of patients, and revealed that disruption of fibronectin assembly may be one of the factors underlying RP. This model may help us to better understand the pathogenic mechanism and find treatment for USH2 in the future.


Assuntos
Proteínas da Matriz Extracelular , Técnicas de Inativação de Genes , Síndromes de Usher , Proteínas de Peixe-Zebra , Peixe-Zebra , Animais , Modelos Animais de Doenças , Eletrorretinografia , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Humanos , Síndromes de Usher/genética , Síndromes de Usher/metabolismo , Síndromes de Usher/patologia , Síndromes de Usher/fisiopatologia , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
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