Insulin-like growth factor 1 (IGF-1)-induced twist expression is involved in the anti-apoptotic effects of the IGF-1 receptor.
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In this study we investigated the molecular mechanisms whereby insulin-like growth factor 1 (IGF-1) induced Twist gene expression and the role of Twist in the anti-apoptotic actions of the IGF-1 receptor. In NIH-3T3 fibroblasts overexpressing the human IGF-1 receptor (NWTb3), treatment with IGF-1 (10(-8) m) for 1 and 4 h increased the level of Twist mRNA as well as protein by 3-fold. In contrast, insulin at physiological concentrations did not stimulate Twist expression in NIH-3T3 fibroblasts overexpressing the human insulin receptor. The IGF-1 effect was specific for the IGF-1 receptor since, in cells overexpressing a dominant negative IGF-1 receptor, IGF-1 failed to increase Twist expression. Pre-incubation with the ERK1/2 inhibitor U0126 or expression of a dominant negative MEK-1 abolished the effect of IGF-1 on Twist mRNA expression in NWTb3 cells, suggesting that Twist induction by IGF-1 occurs via the mitogen-activated protein kinase signaling pathway. In vivo, IGF-1 injection increased the mRNA level of Twist in mouse skeletal muscle, the major site of Twist expression. Finally, using an antisense strategy, we demonstrated that a reduction of 40% in Twist expression decreased significantly the ability of IGF-1 to rescue NWTb3 cells from etoposide-induced apoptosis. Taken together, these results define Twist as an important factor involved in the anti-apoptotic actions of the IGF-1 receptor. (+info)
R-twist gene expression during rat palatogenesis.
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Palatal clefting is often associated with premature fusion of cranial sutures in human craniosynostosis syndromes, many of which are characterised by mutations affecting the fibroblast growth factor receptor (FGFR) gene family. In palatal fusion, epithelio-mesenchymal transition (EMT) contributes to the dispersion of the midline epithelial seam. EMT has also been observed in neoplastic epithelial cells in relation to the acquisition of malignant characteristics where morphological changes are accompanied by rapid switching in the expression of fgfr2 from the epithelial type (kgfr) to the mesenchymal type (bek). The twist gene codes for a basic helix-loop-helix transcription factor putatively involved in regulation of transcription of fgfr2. Mutations in the TWIST gene have been described as being responsible for the Saethre-Chotzen syndrome, an autosomal dominant craniosynostosis associated with cleft palate as well as other disturbances of the facial skeleton. In this study we have analysed the distribution of twist transcripts during rat palatogenesis in vivo from 14.5 to 17.5 days post coitum by in situ hybridisation with digoxygenin-labelled ssDNA probes. twist transcripts were found to be concentrated in mesenchymal cells beneath the epithelium at the tip of the palatal shelves immediately prior to, and during fusion as well as in a localised epithelial area at the tip of the shelves prior to fusion, thereby implicating twist gene expression in the process of palatogenesis. This pattern of expression illuminates the disturbances of maxillary growth that occur in human craniosynostotic syndromes. (+info)
Increased bone formation and decreased osteocalcin expression induced by reduced Twist dosage in Saethre-Chotzen syndrome.
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The Saethre-Chotzen syndrome is characterized by premature fusion of cranial sutures resulting from mutations in Twist, a basic helix-loop-helix (bHLH) transcription factor. We have identified Twist target genes using human mutant calvaria osteoblastic cells from a child with Saethre-Chotzen syndrome with a Twist mutation that introduces a stop codon upstream of the bHLH domain. We observed that Twist mRNA and protein levels were reduced in mutant cells and that the Twist mutation increased cell growth in mutant osteoblasts compared with control cells. The mutation also caused increased alkaline phosphatase and type I collagen expression independently of cell growth. During in vitro osteogenesis, Twist mutant cells showed increased ability to form alkaline phosphatase-positive bone-like nodular structures associated with increased type I collagen expression. Mutant cells also showed increased collagen synthesis and matrix production when cultured in aggregates, as well as an increased capacity to form a collagenous matrix in vivo when transplanted into nude mice. In contrast, Twist mutant osteoblasts displayed a cell-autonomous reduction of osteocalcin mRNA expression in basal conditions and during osteogenesis. The data show that genetic deletion of Twist causing reduced Twist dosage increases cell growth, collagen expression, and osteogenic capability, but inhibits osteocalcin gene expression. This provides one mechanism that may contribute to the premature cranial ossification induced by deletion of the bHLH Twist domain in Saethre-Chotzen syndrome. (+info)
Patterns of gene expression during Drosophila mesoderm development.
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The transcription factor Twist initiates Drosophila mesoderm development, resulting in the formation of heart, somatic muscle, and other cell types. Using a Drosophila embryo sorter, we isolated enough homozygous twist mutant embryos to perform DNA microarray experiments. Transcription profiles of twist loss-of-function embryos, embryos with ubiquitous twist expression, and wild-type embryos were compared at different developmental stages. The results implicate hundreds of genes, many with vertebrate homologs, in stage-specific processes in mesoderm development. One such gene, gleeful, related to the vertebrate Gli genes, is essential for somatic muscle development and sufficient to cause neural cells to express a muscle marker. (+info)
Gastrulation defective, a complement factor C2/B-like protease, interprets a ventral prepattern in Drosophila.
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gastrulation defective (gd) encodes a serine protease required for specification of dorsal-ventral cell fates during Drosophila embryogenesis. Using RNA microinjection, I show that wild-type gd RNA can restore ventrolateral pattern elements with correct polarity with respect to egg shape in embryos lacking gd function. While low RNA concentrations restore ventrolateral pattern elements, higher concentrations ventralize the embryo. Gastrulation defective concentration has a rate-limiting effect on the domain of high Dorsal concentration but little effect upon the slope of the gradient. In embryos from pipe-null females, much higher RNA concentrations generate an ectopic axis oriented with respect to the site of injection. The data suggest that the Dorsal gradient is not directly determined by asymmetric cues in the eggshell but arises de novo within the perivitelline space as a consequence of self-regulatory properties of the protease cascade. A homology to the mammalian complement factors C2 and B is also described. (+info)
Common regulation of growth arrest and differentiation of osteoblasts by helix-loop-helix factors.
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Cellular differentiation entails the coordination of cell cycle arrest and tissue-specific gene expression. We investigated the involvement of basic helix-loop-helix (bHLH) factors in differentiation of osteoblasts using the human osteoblastic cell line MG63. Serum starvation induced growth arrest at G1 phase, accompanied by expression of cyclin-dependent kinase inhibitor p21(WAF1/Cip1). Reporter assays with the p21 gene promoter demonstrated that the combination of E2A (E12 or E47) and coactivator CBP was responsible for p21 induction independent of p53. Twist inhibited E2A-CBP-dependent activation of the exogenous and endogenous p21 promoters. Ids similarly inhibited the exogenously transfected p21 promoter; however less antagonistic effect on the endogenous p21 promoter was observed. Twist was predominantly present in nuclei in MG63 cells growing in complete medium, while it localized mainly in the cytoplasm after serum starvation. The fibroblast growth factor receptor 3 gene (FGFR3), which generates signals leading to differentiation of osteoblasts, was found to be controlled by the same transcriptional regulation as the p21 gene. E2A and Twist influenced alkaline phosphatase expression, a consensus marker of osteoblast differentiation. Expression of E2A and FGFR3 was seen at the location of osteoblast differentiation in the calvaria of mouse embryos, implicating bHLH molecules in physiological osteoblast differentiation. These results demonstrate that a common regulatory system is involved in at least two distinct steps in osteoblastic differentiation. Our results also provide the molecular basis of Saethre-Chotzen syndrome, caused by mutations of the TWIST and FGFR3 genes. (+info)
Developmental expression of chick twist and its regulation during limb patterning.
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We have isolated a chick Twist gene (cTwist) and examined its expression pattern during development by whole mount in situ hybridization. In early embryos, cTwist transcripts are found in the developing somites, lateral plate mesoderm, limb mesenchyme, branchial arches and head mesenchyme. At later stages, cTwist expression is found in the sclerotome and dermatome, limb bud mesenchyme, interdigital regions, and distal mesenchyme of the maxillary and mandibular processes. In the developing feathers, cTwist is expressed in the mesenchyme of the buds and becomes restricted to the proximal region of the feather filaments. Additionally, we report that the expression of cTwistin the limb mesenchyme is regulated by the AER, FGFs, RA and SHH. The FGFs secreted by the AER seem to have a critical role in maintaining cTwist expression. SHH is also able to maintain cTwist expression but only in the presence of the AER. Overall, our results provide new evidence that reinforce the existence of an interplay between the cTwist and FGF signalling pathways. (+info)
Dimerization partners determine the activity of the Twist bHLH protein during Drosophila mesoderm development.
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The basic helix-loop-helix transcription factor Twist regulates a series of distinct cell fate decisions within the Drosophila mesodermal lineage. These twist functions are reflected in its dynamic pattern of expression, which is characterized by initial uniform expression during mesoderm induction, followed by modulated expression at high and low levels in each mesodermal segment, and finally restricted expression in adult muscle progenitors. We show two distinct partner-dependent functions for Twist that are crucial for cell fate choice. We find that Twist can form homodimers and heterodimers with the Drosophila E protein homologue, Daughterless, in vitro. Using tethered dimers to assess directly the function of these two particular dimers in vivo, we show that Twist homodimers specify mesoderm and the subsequent allocation of mesodermal cells to the somatic muscle fate. Misexpression of Twist-tethered homodimers in the ectoderm or mesoderm leads to ectopic somatic muscle formation overriding other developmental cell fates. In addition, expression of tethered Twist homodimers in embryos null for twist can rescue mesoderm induction as well as somatic muscle development. Loss of function analyses, misexpression and dosage experiments, and biochemical studies indicate that heterodimers of Twist and Daughterless repress genes required for somatic myogenesis. We propose that these two opposing roles explain how modulated Twist levels promote the allocation of cells to the somatic muscle fate during the subdivision of the mesoderm. Moreover, this work provides a paradigm for understanding how the same protein controls a sequence of events within a single lineage. (+info)