Isolation of differentially expressed genes from wild‐type and Twist mutant mouse limb buds

Abstract

In the mouse, Twist is required for normal limb and craniofacial development. We show that the aristaless-like transcription factors, Alx3 and Alx4 are downregulated in the Twist(-/-) mutant and may be potential targets of Twist. By suppression subtractive hybridization we isolated 31 and 18 unique clones representing mRNAs that are putatively downregulated and upregulated respectively in Twist(-/-) forelimb buds. These included genes encoding cytoskeletal components, metabolic enzymes, hemoglobin molecules, membrane transport proteins, components of transcription and translation complexes, protein modification enzymes and proteins related to cell proliferation and apoptosis. Differential expression of selected clones was validated by whole mount in situ hybridization to E10.5 wild-type and Twist(-/-) embryos. We show that four novel clones are expressed in the Twist-expressing craniofacial tissues and paraxial mesoderm and downregulated in Twist(-/-) embryos, raising the possibility that they are, in addition to genes of the Alx family, downstream targets of Twist.

Differential configurations involving binding of USF transcription factors and Twist1 regulateAlx3promoter activity in mesenchymal and pancreatic cells

“…Thus the results of the present study provide a mechanistic explanation for the previous observations that the phenotypic features observed in Twist1-deficient embryos [35][36][37]39] can be partially recapitulated by loss-of-function mutations in aristalesslike genes [10,11,14,15]. In addition, they provide a mechanistic interpretation to the previously observed down-regulation of Alx3 expression associated with Twist1-deficiency in embryonic tissues [36][37][38].…”

“…In addition, during embryonic development in mice, Twist1 is expressed in the head mesenchyme, branchial arches and limb buds at a time that coincides with expression of aristaless-like genes [30]. Indeed, in Twist1-deficient embryos the expression of Alx3, Alx4 and Cart1 is down-regulated both in the forehead mesenchyme [36,37] and in the developing limb buds [38,39]. Thus the results of the present study provide a mechanistic explanation for the previous observations that the phenotypic features observed in Twist1-deficient embryos [35][36][37]39] can be partially recapitulated by loss-of-function mutations in aristalesslike genes [10,11,14,15].…”

Genomic screen for genes involved in mammalian craniofacial development

“…The subtractive approach has also been employed successfully to address the genetics of specific processes such as sex determination (Bowles et al, 2000), pituitary development (Douglas et al, 2001), and limb development (Loebel et al, 2002). In these cases, gene expression in the tissue of interest has been compared with that in the corresponding tissue in either a bipotential system (sex determination) or from a mouse mutant where a gene important in the development of that structure has been perturbed (limb development, pituitary development).…”

Normal and disease-related biological functions of Twist1 and underlying molecular mechanisms

“…Along with the altered expression of FGFR2, FGF4, FGF8 and FGF10, Twist1 deletion in the limb bud also reduced the overall activities of genes involved in SHH signaling in the limb bud mesenchyme, which include Shh, Gli1, Gli2, Gli3 and Ptch [65,66]. Twist1 also appears essential to Bmp4 expression in the apical ectoderm, as well as Alx3, Alx4, Pax1 and Pax3 activities in the mesenchyme [65,67].…”