RESUMEN
Vertebrate Hox clusters are comprised of multiple Hox genes that control morphology and developmental timing along multiple body axes. Although results of genetic analyses using Hox-knockout mice have been accumulating, genetic studies in other vertebrates have not been sufficient for functional comparisons of vertebrate Hox genes. In this study, we isolated all of the seven hox cluster loss-of-function alleles in zebrafish using the CRISPR-Cas9 system. Comprehensive analysis of the embryonic phenotype and X-ray micro-computed tomography scan analysis of adult fish revealed several species-specific functional contributions of homologous Hox clusters along the appendicular axis, whereas important shared general principles were also confirmed, as exemplified by serial anterior vertebral transformations along the main body axis, observed in fish for the first time. Our results provide insights into discrete sub/neofunctionalization of vertebrate Hox clusters after quadruplication of the ancient Hox cluster. This set of seven complete hox cluster loss-of-function alleles provide a formidable resource for future developmental genetic analysis of the Hox patterning system in zebrafish.
Asunto(s)
Genes Homeobox/genética , Familia de Multigenes , Pez Cebra/genética , Pez Cebra/fisiología , Animales , Sistemas CRISPR-Cas , Desarrollo Embrionario/genética , Evolución Molecular , Femenino , Duplicación de Gen , Regulación del Desarrollo de la Expresión Génica , Masculino , Mutación , Esqueleto/anatomía & histología , Esqueleto/crecimiento & desarrollo , Especificidad de la Especie , Microtomografía por Rayos X , Pez Cebra/embriologíaRESUMEN
The presumptive somite boundary in the presomitic mesoderm (PSM) is defined by the anterior border of the expression domain of Tbx6 protein. During somite segmentation, the expression domain of Tbx6 is regressed by Ripply-meditated degradation of Tbx6 protein. Although the expression of zebrafish tbx6 remains restricted to the PSM, the transcriptional regulation of tbx6 remains poorly understood. Here, we show that the expression of zebrafish tbx6 is maintained by transcriptional autoregulation. We find that a proximal-located cis-regulatory module, TR1, which contains two putative T-box sites, is required for somite segmentation in the intermediate body and for proper expression of segmentation genes. Embryos with deletion of TR1 exhibit significant reduction of tbx6 expression at the 12-somite stage, although its expression is initially observed. Additionally, Tbx6 is associated with TR1 and activates its own expression in the anterior PSM. Furthermore, the anterior expansion of tbx6 expression in ripply gene mutants is suppressed in a TR1-dependent manner. The results suggest that the autoregulatory loop of zebrafish tbx6 facilitates immediate removal of Tbx6 protein through termination of its own transcription at the anterior PSM.
Asunto(s)
Tipificación del Cuerpo/genética , Homeostasis/genética , Somitos/embriología , Proteínas de Dominio T Box/metabolismo , Transcripción Genética , Proteínas de Pez Cebra/metabolismo , Pez Cebra/embriología , Pez Cebra/genética , Animales , Sitios de Unión/genética , Embrión no Mamífero/metabolismo , Elementos de Facilitación Genéticos/genética , Eliminación de Gen , Regulación del Desarrollo de la Expresión Génica , Genes Reporteros , Homocigoto , Dominios Proteicos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Somitos/metabolismo , Proteínas de Dominio T Box/química , Proteínas de Dominio T Box/genética , Proteínas de Pez Cebra/química , Proteínas de Pez Cebra/genéticaRESUMEN
In the vertebrate body, a metameric structure is present along the anterior-posterior axis. Zebrafish tbx6-/- larvae, in which somite boundaries do not form during embryogenesis, were shown to exhibit abnormal skeletal morphology such as rib, neural arch and hemal arch. In this study, we investigated the role of somite patterning in the formation of anterior vertebrae and ribs in more detail. Using three-dimensional computed tomography scans, we found that anterior vertebrae including the Weberian apparatus were severely affected in tbx6-/- larvae. In addition, pleural ribs of tbx6 mutants exhibited severe defects in the initial ossification, extension of ossification, and formation of parapophyses. Two-colour staining revealed that bifurcation of ribs was caused by fusion or branching of ribs in tbx6-/- . The parapophyses in tbx6-/- juvenile fish showed irregular positioning to centra and abnormal attachment to ribs. Furthermore, we found that the ossification of the distal portion of ribs proceeded along myotome boundaries even in irregularly positioned myotome boundaries. These results provide evidence of the contribution of somite patterning to the formation of the Weberian apparatus and rib in zebrafish.
Asunto(s)
Tipificación del Cuerpo/genética , Costillas/embriología , Somitos/enzimología , Pez Cebra/embriología , Animales , Animales Modificados Genéticamente , Regulación del Desarrollo de la Expresión Génica , Costillas/diagnóstico por imagen , Somitos/diagnóstico por imagen , Proteínas de Dominio T Box/genética , Tomografía Computarizada por Rayos X , Pez Cebra/genética , Proteínas de Pez Cebra/genéticaRESUMEN
In the zebrafish segmentation clock, hairy/enhancer of split-related genes her1, her7, and hes6 encodes components of core oscillators. Since the expression of cyclic genes proceeds rapidly in the presomitic mesoderm (PSM), these hairy-related mRNAs are subject to strict post-transcriptional regulation. In this study, we demonstrate that inhibition of the CCR4-NOT deadenylase complex lengthens poly(A) tails of hairy-related mRNAs and increases the amount of these mRNAs, which is accompanied by defective somite segmentation. In transgenic embryos, we show that EGFP mRNAs with 3'UTRs of hairy-related genes exhibit turnover similar to endogenous mRNAs. Our results suggest that turnover rates of her1, her7, and hes6 mRNAs are differently regulated by the CCR4-NOT deadenylase complex possibly through their 3'UTRs in the zebrafish PSM.