RESUMEN
The genetic basis of morphological variation provides a major topic in evolutionary developmental biology. Fish of the genus Danio display colour patterns ranging from horizontal stripes, to vertical bars or spots. Stripe formation in zebrafish, Danio rerio, is a self-organizing process based on cell-contact mediated interactions between three types of chromatophores with a leading role of iridophores. Here we investigate genes known to regulate chromatophore interactions in zebrafish that might have evolved to produce a pattern of vertical bars in its sibling species, Danio aesculapii. Mutant D. aesculapii indicate a lower complexity in chromatophore interactions and a minor role of iridophores in patterning. Reciprocal hemizygosity tests identify the potassium channel gene obelix/Kcnj13 as evolved between the two species. Complementation tests suggest evolutionary change through divergence in Kcnj13 function in two additional Danio species. Thus, our results point towards repeated and independent evolution of this gene during colour pattern diversification.
Asunto(s)
Color , Pigmentación/genética , Canales de Potasio de Rectificación Interna/genética , Proteínas de Pez Cebra/genética , Pez Cebra/genética , Animales , Cromatóforos/metabolismo , Evolución Molecular , Hibridación Genética , Fenotipo , Especificidad de la Especie , Pez Cebra/clasificaciónRESUMEN
Color patterns are prominent features of many animals and are of high evolutionary relevance. In basal vertebrates, color patterns are composed of specialized pigment cells that arrange in multilayered mosaics in the skin. Zebrafish (Danio rerio), the preeminent model system for vertebrate color pattern formation, allows genetic screens as powerful approaches to identify novel functions in a complex biological system. Adult zebrafish display a series of blue and golden horizontal stripes, composed of black melanophores, silvery or blue iridophores, and yellow xanthophores. This stereotyped pattern is generated by self-organization involving direct cell contacts between all three types of pigment cells mediated by integral membrane proteins [1-5]. Here, we show that neuropeptide signaling impairs the striped pattern in a global manner. Mutations in the genes coding either for galanin receptor 1A (npm/galr1A) or for its ligand galanin (galn) result in fewer stripes, a pale appearance, and the mixing of cell types, thus resembling mutants with thyroid hypertrophy [6]. Zebrafish chimeras obtained by transplantations of npm/galr1A mutant blastula cells indicate that mutant pigment cells of all three types can contribute to a normal striped pattern in the appropriate host. However, loss of galr1A expression in a specific region of the brain is sufficient to cause the mutant phenotype in an otherwise wild-type fish. Increased thyroid hormone levels in mutant fish suggest that galanin signaling through Galr1A in the pituitary is an upstream regulator of the thyroid hormone pathway, which in turn promotes precise interactions of pigment cells during color pattern formation.
Asunto(s)
Tipificación del Cuerpo/fisiología , Galanina/genética , Receptor de Galanina Tipo 1/genética , Transducción de Señal , Proteínas de Pez Cebra/genética , Pez Cebra/genética , Animales , Encéfalo/metabolismo , Color , Embrión no Mamífero/metabolismo , Desarrollo Embrionario , Femenino , Galanina/metabolismo , Morfogénesis , Mutación , Pigmentación/genética , Receptor de Galanina Tipo 1/metabolismo , Pez Cebra/embriología , Pez Cebra/metabolismo , Proteínas de Pez Cebra/metabolismoRESUMEN
The development of the pigmentation pattern in zebrafish is a tightly regulated process that depends on both the self-organizing properties of pigment cells and extrinsic cues from other tissues. Many of the known mutations that alter the pattern act cell-autonomously in pigment cells, and our knowledge about external regulators is limited. Here, we describe novel zebrafish mau mutants, which encompass several dominant missense mutations in Aquaporin 3a (Aqp3a) that lead to broken stripes and short fins. A loss-of-function aqp3a allele, generated by CRISPR-Cas9, has no phenotypic consequences, demonstrating that Aqp3a is dispensable for normal development. Strikingly, the pigment cells from dominant mau mutants are capable of forming a wild-type pattern when developing in a wild-type environment, but the surrounding tissues in the mutants influence pigment cell behaviour and interfere with the patterning process. The mutated amino acid residues in the dominant alleles line the pore surface of Aqp3a and influence pore permeability. These results demonstrate an important effect of the tissue environment on pigment cell behaviour and, thereby, on pattern formation.
Asunto(s)
Acuaporina 3/genética , Mutación/genética , Pigmentación , Proteínas de Pez Cebra/genética , Pez Cebra/metabolismo , Secuencia de Aminoácidos , Aletas de Animales/anatomía & histología , Aletas de Animales/citología , Animales , Acuaporina 3/química , Acuaporina 3/metabolismo , Cromatóforos/metabolismo , Genes Dominantes , Proteínas Fluorescentes Verdes/metabolismo , Mutación Missense/genética , Permeabilidad , Fenotipo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Pez Cebra/química , Proteínas de Pez Cebra/metabolismoRESUMEN
α2ß1 integrin is one of the most important collagen-binding receptors, and it has been implicated in numerous thrombotic and immune diseases. α2ß1 integrin is a potent tumour suppressor, and its downregulation is associated with increased metastasis and poor prognosis in breast cancer. Currently, very little is known about the mechanism that regulates the cell-surface expression and trafficking of α2ß1 integrin. Here, using a quantitative fluorescence-microscopy-based RNAi assay, we investigated the impact of 386 cytoskeleton-associated or -regulatory genes on α2 integrin endocytosis and found that 122 of these affected the intracellular accumulation of α2 integrin. Of these, 83 were found to be putative regulators of α2 integrin trafficking and/or expression, with no observed effect on the internalization of epidermal growth factor (EGF) or transferrin. Further interrogation and validation of the siRNA screen revealed a role for KIF15, a microtubule-based molecular motor, as a significant inhibitor of the endocytic trafficking of α2 integrin. Our data suggest a novel role for KIF15 in mediating plasma membrane localization of the alternative clathrin adaptor Dab2, thus impinging on pathways that regulate α2 integrin internalization.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Neoplasias de la Mama/genética , Membrana Celular/metabolismo , Integrina alfa2beta1/metabolismo , Cinesinas/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Proteínas Reguladoras de la Apoptosis , Colágeno/metabolismo , Citoesqueleto/genética , Endocitosis/genética , Femenino , Pruebas Genéticas/métodos , Células HeLa , Humanos , Integrina alfa2beta1/genética , Cinesinas/genética , Microscopía Fluorescente , Metástasis de la Neoplasia , Unión Proteica/genética , Transporte de Proteínas/genética , Interferencia de ARN , ARN Interferente Pequeño/genéticaRESUMEN
Endocytosis is one of the most essential cellular processes, which enables cells to internalise diverse -material. It is crucial for regulation of receptor activity and signalling, cell polarisation, attachment and motility, and a great number of other cellular functions. A number of diverse endocytosis pathways are described by now; however, their specificity for different cellular cargoes is poorly resolved. Only few of endocytosis regulators are well-characterised and even less are attributed to the specific cargo. That is very true for the integrin endocytosis pathway, which is a key process in cell migration, adhesion, and signalling. The recent advent of quantitative fluorescent microscopy and cell arrays opened an exciting possibility to systematically characterise molecules playing a role in this crucially important process. Here, we describe a fluorescent screening microscopy-based assay to identify regulators of integrin α2 internalisation. The experimental procedure is the best suited for a highly parallel screening format, such as cell arrays, albeit can be used in single experiments. We provide protocols for sample preparation, fabrication of cell arrays and quantification of integrin α2 internalisation. The approach can be modified to quantify endocytosis of other cargo, and can be used under the conditions of knock-down and knock-in as well as for chemical screening.
Asunto(s)
Comunicación Celular/genética , Endocitosis/genética , Integrina alfa2/metabolismo , Microscopía Fluorescente/métodos , Análisis por Matrices de Proteínas/métodos , Interferencia de ARN , Comunicación Celular/fisiología , Endocitosis/fisiologíaRESUMEN
In Ustilago maydis the pheromone signal is transmitted via a mitogen-activated protein kinase (MAP kinase) module to the transcription factor Prf1. Prf1 activates transcription of a and b mating type genes by binding to pheromone response elements (PREs) located in regulatory regions of these genes. Here we show that the CCAAT-box binding protein Hap2 from U. maydis regulates prf1 expression. Hap2 was initially identified as a potential interaction partner of the MAP kinase Kpp6 in yeast two-hybrid screens and was subsequently also shown to interact with the MAPK Kpp2. Deletion of hap2 in haploid cells abolished mating, resulting from a defect in pheromone-induced gene expression. Crosses of haploid hap2 deletion strains were completely impaired in pathogenicity. Constitutive expression of prf1 complemented the pheromone response defect in Δhap2 strains. Chromatin immunoprecipitation assays indicated that Hap2 binds directly to CCAAT motifs in the prf1 promoter. Point mutations in two putative MAPK phosphorylation sites in Hap2 attenuated the pheromone response. In a solopathogenic strain hap2 deletion affected filamentation and the mutants showed reduced pathogenicity symptoms. These data suggest that Hap2 is a novel regulator of prf1 with additional functions after cell fusion.