RESUMEN
XTcf-3 functions as a transcriptional regulator in the canonical Wnt signaling cascade and can repress or activate downstream target genes. Expression of XTcf-3 is differentially regulated in time and place during development (Molenaar et al. [1998] Mech Dev. 75:151-154), but little is known about the mechanisms that control transcriptional activation and repression. A 15-kb genomic fragment of Tcf-3 sequences from Xenopus tropicalis was cloned, including the 5' untranslated region; exons 1, 2, and 3; and intron sequences. We used 5' deletion constructs for transgenesis and episomal luciferase assays in Xenopus to examine temporal and spatial regulation of the promoter during early development. A -3054/+34-bp Tcf-3 upstream region was identified that drives a green fluorescent protein (GFP) reporter transgene in a pattern similar to endogenous expression of XtTcf-3 from gastrula to tail bud stages. At stage 12, expression of the reporter is restricted to the middle and posterior neurectoderm. At stage 22, expression is strongest in the neural plate, the eye anlagen and branchial arches. At stage 35/36, expression is found in the head mesenchyme, the branchial arches, the heart, the mesencephalon, eyes, otic vesicles, notochord, somites and the lateral plate mesoderm. Part of the cis-acting elements driving this GFP reporter transgene expression map between -372 and -95 bp of the transcription start site. Furthermore, two TCF/LEF sites are necessary for full activity of the promoter during gastrula stages in episomal luciferase assays.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Regiones Promotoras Genéticas , Transactivadores , Factores de Transcripción/metabolismo , Xenopus/embriología , Regiones no Traducidas 5' , Animales , Animales Modificados Genéticamente , Mapeo Cromosómico , Clonación Molecular , Análisis Mutacional de ADN , Embrión no Mamífero , Desarrollo Embrionario , Exones , Gástrula , Eliminación de Gen , Genes Reporteros , Genoma , Proteínas Fluorescentes Verdes/metabolismo , Hibridación in Situ , Intrones , Luciferasas/metabolismo , Oocitos/metabolismo , Factores de Tiempo , Distribución Tisular/genética , Factores de Transcripción/genética , Transgenes , Xenopus/genética , Xenopus/metabolismoRESUMEN
Xenopus laevis type XVIII collagen occurs in three variants, 22 + 1285 amino acid residues (signal peptide + mature protein), 23 + 1581 residues and 23 + 1886 residues in length, differing in their N-terminal non-collagenous domains. The region showing highest homology to mammalian counterparts is the C-terminal endostatin domain. All three variants are expressed, at different levels, during early and late stages of development, as demonstrated by reverse transcription-polymerase chain reaction. Whole-mount in situ hybridization shows that the short variant is expressed at high levels in the developing eye, the central nervous system, the otic vesicle, the head mesenchyme, the branchial arches and the pronephros, and at the boundaries between somites. The middle variant is expressed in the head mesenchyme, the branchial arches, the peripheral nervous system, the pronephros and the pronephric duct, and at the somite boundaries. The longest variant is weakly expressed in the head mesenchyme and branchial arches.