Identification of a Spatially Specific Enhancer Element in the Chicken Msx-2 Gene That Regulates Its Expression in the Apical Ectodermal Ridge of the Developing Limb Buds of Transgenic Mice

Abstract

Msx-2 is a member of the Msx family of homeobox-containing genes expressed in a variety of embryonic tissues involved in epithelial-mesenchymal interactions and pattern formation. In the developing chick limb bud, Msx-2 is expressed in the apical ectodermal ridge, which plays a crucial role in directing the growth and patterning of limb mesoderm. In addition, Msx-2 is expressed in the anterior nonskeletal-forming mesoderm of the limb bud, in the posterior necrotic zone, and in the interdigital mesenchyme. Studies of the altered expression patterns of Msx-2 in amelic and polydactylous mutant chick limbs have suggested that the apical ectodermal ridge and mesodermal domains of Msx-2 expression are independently regulated and that there might be separate cis-regulatory elements in the Msx-2 gene controlling its spatially distinct domains of expression. To test this hypothesis, we have isolated the chicken Msx-2 gene and have tested the ability of various regions of the gene to target expression of LacZ reporter gene to specific regions of the limbs of transgenic mice. A variety of these constructs are consistently expressed only in the apical ectodermal ridge and the ectoderm of the genital tubercle and are not expressed in the mesoderm of the limb bud or in other regions of the embryo where the endogenous Msx-2 gene is expressed. These results suggest the presence of spatially specific cis-regulatory elements in the Msx-2 gene. We identified a 348-bp region in the 5' flanking region of the Msx-2 gene which can act as an apical ectodermal ridge enhancer element when placed in reverse orientation in front of the reporter gene with transcription initiation directed by the minimal hsp68 promoter.

Application of Ultrasound Stimulation in Bone Tissue Engineering

“…Increased osteoblast cells and higher mineralized nodule formation explain the enhancement of proliferation by LIPUS (12,22,23). In addition, LIPUS affects on differentiation of osteoblast cells, which is shown by increased ALPase, and transcriptional factors, Runx2 (3,22,(24)(25)(26)(27)(28). Ultrasound stimulates PEG2 and COX-2 in osteoblast cells, and the expression of signals accelerates the bone regeneration in tissue engineering (1,11,(29)(30)(31).…”

“…Dlx5 expression is related with osteoblast differentiation, and occurs in the final stages of osteoblast differentiation in vitro (26). Msx2 is predominantly expressed by proliferating osteoblasts and preosteoblasts, but its expression decreases according to terminal osteoblast differentiation (26)(27)(28).…”

Daily low-intensity pulsed ultrasound-mediated osteogenic differentiation in rat osteoblasts

“…The homeobox proteins Dlx5 and Msx2 appear to regulate the development of mineralized tissues, including bone, cartilage, and dentin [26][27][28][29][30]. Dlx5 expression correlates with osteoblast differentiation and is at a maximum in the final stages of osteoblast differentiation in vitro when the extracellular matrix mineralizes, suggesting that Dlx5 may be involved in the differentiation of osteogenic cells [31].…”

Differential tissue growth and patterns of cell death in mouse limb autopod morphogenesis

“…FGFs have been previously shown to regulate msx-1 and msx-2 expression (Sumoy et al, 1995;Wang and Sassoon, 1995), and on the basis of experimental evidence, it has been proposed that these genes participate in controlling cell proliferation and regeneration of digit tips in the developing mouse limb (Reginelli et al, 1995). However, the expression pattern in the limb and other embryo regions has suggested that these genes also participate in the process of cell death and that they are regulated by BMPs.…”