RESUMEN
The morphological effects of mutation and disease are often critical to our understanding of normal and abnormal function. The power and popularity of zebrafish as a forward and reverse genetic vertebrate model system, combined with its small size, have made it an ideal model in which to study the genetics of histologically scorable phenotypes. The presence of multiple tissue types in this organism's small larvae also makes it a potentially important model for toxicological analysis. Studying histological phenotypes is greatly enhanced by high-throughput methods of histology. Here, we describe details of high-throughput histology of the zebrafish using larval arrays, along with recent advances in mold design and discussion of work in progress that will lead to easier ways for people in the field to more rapidly score phenotypes in arrays. These detailed descriptions, together with the troubleshooting guide, should enable any laboratory with ties to a histology facility to perform high-throughput histology of zebrafish.
Asunto(s)
Técnicas Histológicas , Análisis de Matrices Tisulares/métodos , Pez Cebra/anatomía & histología , Animales , Técnicas Histológicas/instrumentación , Larva , Microtomía/instrumentación , Adhesión en Parafina/instrumentación , Fenotipo , Programas Informáticos , Fijación del Tejido , Pez Cebra/crecimiento & desarrolloRESUMEN
Lighter variations of pigmentation in humans are associated with diminished number, size, and density of melanosomes, the pigmented organelles of melanocytes. Here we show that zebrafish golden mutants share these melanosomal changes and that golden encodes a putative cation exchanger slc24a5 (nckx5) that localizes to an intracellular membrane, likely the melanosome or its precursor. The human ortholog is highly similar in sequence and functional in zebrafish. The evolutionarily conserved ancestral allele of a human coding polymorphism predominates in African and East Asian populations. In contrast, the variant allele is nearly fixed in European populations, is associated with a substantial reduction in regional heterozygosity, and correlates with lighter skin pigmentation in admixed populations, suggesting a key role for the SLC24A5 gene in human pigmentation.
Asunto(s)
Antiportadores/genética , Pigmentación de la Piel/genética , Proteínas de Pez Cebra/genética , Pez Cebra/genética , Negro o Afroamericano/genética , Alanina/genética , Alelos , Secuencia de Aminoácidos , Animales , Antiportadores/química , Antiportadores/fisiología , Pueblo Asiatico/genética , Evolución Biológica , Población Negra/genética , Calcio/metabolismo , Frecuencia de los Genes , Genes , Variación Genética , Haplotipos , Heterocigoto , Humanos , Transporte Iónico , Melaninas/análisis , Melanosomas/química , Melanosomas/ultraestructura , Ratones , Datos de Secuencia Molecular , Herencia Multifactorial , Mutación , Epitelio Pigmentado Ocular/química , Epitelio Pigmentado Ocular/ultraestructura , Polimorfismo de Nucleótido Simple , Selección Genética , Treonina/genética , Población Blanca/genética , Pez Cebra/embriología , Pez Cebra/metabolismo , Proteínas de Pez Cebra/química , Proteínas de Pez Cebra/fisiologíaRESUMEN
During early zebrafish (Danio rerio) development zygotic transcription does not begin until the mid-blastula transition (MBT) 3 h after fertilization. MBT demarcates transition from synchronous short cell cycles of S and M phases exclusively to full cycles encompassing G1 and G2 phases. Transcriptional profiling and RT-PCR analyses during these phases enabled us to determine that this shift corresponds to decreased transcript levels of S/M phase cell cycle control genes (e.g. ccna2, ccnb1, ccnb2 and ccne) and increased transcript levels of ccnd1, encoding cyclin D1, and orthologs of p21 (p21-like) and retinoblastoma (Rb-like 1). To investigate the regulation of this process further, the translation of ccnd1 mRNA, a G1/S checkpoint control element, was impaired by microinjection of ccnd1-specific morpholino phosphorodiamidate (MO) 20mer or hydroxyprolyl-phosphono peptide nucleic acid (HypNA-pPNA) 16mer antisense oligonucleotides. The resulting downregulation of cyclin D1 protein resulted in microophthalmia and microcephaly, but not lethality. The phenotypes were not seen with 3-mismatch MO 20mers or 1-mismatch HypNA-pPNA 16mers, and were rescued by an exogenous ccnd1 mRNA construct with five mismatches. Collectively, these results indicate that transcription of key molecular determinants of asynchronous cell cycle control in zebrafish embryos commences at MBT and that the reduction of cyclin D1 expression compromises zebrafish eye and head development.
