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1.
PLoS Genet ; 12(2): e1005821, 2016 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-26918822

RESUMEN

Heterotaxy, a birth defect involving left-right patterning defects, and primary ciliary dyskinesia (PCD), a sinopulmonary disease with dyskinetic/immotile cilia in the airway are seemingly disparate diseases. However, they have an overlapping genetic etiology involving mutations in cilia genes, a reflection of the common requirement for motile cilia in left-right patterning and airway clearance. While PCD is a monogenic recessive disorder, heterotaxy has a more complex, largely non-monogenic etiology. In this study, we show mutations in the novel dynein gene DNAH6 can cause heterotaxy and ciliary dysfunction similar to PCD. We provide the first evidence that trans-heterozygous interactions between DNAH6 and other PCD genes potentially can cause heterotaxy. DNAH6 was initially identified as a candidate heterotaxy/PCD gene by filtering exome-sequencing data from 25 heterotaxy patients stratified by whether they have airway motile cilia defects. dnah6 morpholino knockdown in zebrafish disrupted motile cilia in Kupffer's vesicle required for left-right patterning and caused heterotaxy with abnormal cardiac/gut looping. Similarly DNAH6 shRNA knockdown disrupted motile cilia in human and mouse respiratory epithelia. Notably a heterotaxy patient harboring heterozygous DNAH6 mutation was identified to also carry a rare heterozygous PCD-causing DNAI1 mutation, suggesting a DNAH6/DNAI1 trans-heterozygous interaction. Furthermore, sequencing of 149 additional heterotaxy patients showed 5 of 6 patients with heterozygous DNAH6 mutations also had heterozygous mutations in DNAH5 or other PCD genes. We functionally assayed for DNAH6/DNAH5 and DNAH6/DNAI1 trans-heterozygous interactions using subthreshold double-morpholino knockdown in zebrafish and showed this caused heterotaxy. Similarly, subthreshold siRNA knockdown of Dnah6 in heterozygous Dnah5 or Dnai1 mutant mouse respiratory epithelia disrupted motile cilia function. Together, these findings support an oligogenic disease model with broad relevance for further interrogating the genetic etiology of human ciliopathies.


Asunto(s)
Síndrome de Heterotaxia/genética , Síndrome de Kartagener/genética , Animales , Dineínas Axonemales/genética , Dineínas Axonemales/metabolismo , Tipificación del Cuerpo , Cilios/fisiología , Embrión no Mamífero , Técnicas de Silenciamiento del Gen , Heterocigoto , Humanos , Macrófagos del Hígado/patología , Ratones Noqueados , Mutación , ARN Interferente Pequeño/genética , Pez Cebra/embriología , Pez Cebra/genética , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismo
2.
Birth Defects Res C Embryo Today ; 93(3): 281-7, 2011 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-21932436

RESUMEN

Zebrafish have become an invaluable vertebrate animal model to interrogate small molecule libraries for modulators of complex biological pathways and phenotypes. We have recently described the implementation of a quantitative, high-content imaging assay in multi-well plates to analyze the effects of small molecules on Fibroblast Growth Factor (FGF) signaling in vivo. Here we have evaluated the capability of the assay to identify compounds that hyperactivate FGF signaling from a test cassette of agents with known biological activities. Using a transgenic zebrafish reporter line for FGF activity, we screened 1040 compounds from an annotated library of known bioactive agents, including FDA-approved drugs. The assay identified two molecules, 8-hydroxyquinoline sulfate and pyrithione zinc, that enhanced FGF signaling in specific areas of the brain. Subsequent studies revealed that both compounds specifically expanded FGF target gene expression. Furthermore, treatment of early stage embryos with either compound resulted in dorsalized phenotypes characteristic of hyperactivation of FGF signaling in early development. Documented activities for both agents included activation of extracellular signal-related kinase (ERK), consistent with FGF hyperactivation. To conclude, we demonstrate the power of automated quantitative high-content imaging to identify small molecule modulators of FGF.


Asunto(s)
Animales Modificados Genéticamente , Embrión no Mamífero/efectos de los fármacos , Factores de Crecimiento de Fibroblastos/metabolismo , Ensayos Analíticos de Alto Rendimiento , Preparaciones Farmacéuticas/metabolismo , Transducción de Señal/efectos de los fármacos , Pez Cebra/genética , Pez Cebra/metabolismo , Animales , Pez Cebra/embriología
3.
J Leukoc Biol ; 86(3): 713-25, 2009 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-19454651

RESUMEN

Foxp3+ T regulatory cells are required to prevent autoimmune disease, but also prevent clearance of some chronic infections. While natural T regulatory cells are produced in the thymus, TGF-beta1 signaling combined with T-cell receptor signaling induces the expression of Foxp3 in CD4+ T cells in the periphery. We found that ICAM-1-/- mice have fewer T regulatory cells in the periphery than WT controls, due to a role for ICAM-1 in induction of Foxp3 expression in response to TGF-beta1. Further investigation revealed a functional deficiency in the TGF-beta1-induced translocation of phosphorylated Smad3 from the cytoplasmic compartment to the nucleus in ICAM-1-deficient mice. This impairment in the TGF-beta1 signaling pathway is most likely responsible for the decrease in T regulatory cell induction in the absence of ICAM-1. We hypothesized that in the presence of an inflammatory response, reduced production of inducible T regulatory cells would be evident in ICAM-1-/- mice. Indeed, following Mycobacterium tuberculosis infection, ICAM-1-/- mice had a pronounced reduction in T regulatory cells in the lungs compared with control mice. Consequently, the effector T-cell response and inflammation were greater in the lungs of ICAM-1-/- mice, resulting in morbidity due to overwhelming pathology.


Asunto(s)
Molécula 1 de Adhesión Intercelular/inmunología , Mycobacterium tuberculosis/inmunología , Transducción de Señal/inmunología , Linfocitos T Reguladores/inmunología , Factor de Crecimiento Transformador beta/inmunología , Animales , Inflamación , Molécula 1 de Adhesión Intercelular/metabolismo , Interferón gamma/inmunología , Interferón gamma/metabolismo , Ratones , Ratones Noqueados , Mycobacterium tuberculosis/metabolismo , Linfocitos T Reguladores/metabolismo , Factor de Crecimiento Transformador beta/metabolismo
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