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.
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.
Sumoy
L
L
Department of BioStructure and Function, School of Dental Medicine, University of Connecticut Health Center, Farmington 06030, USA.
Wang
C K
CK
Lichtler
A C
AC
Pierro
L J
LJ
Kosher
R A
RA
Upholt
W B
WB
D000818:Animals; D001483:Base Sequence; D002645:Chickens / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics*; D004475:Ectoderm / Q000378:metabolism*; D004742:Enhancer Elements, Genetic; D005091:Exons / Q000235:genetics; D005121:Extremities / Q000196:embryology*; D018507:Gene Expression Regulation, Developmental; D015723:Gene Library; D017930:Genes, Reporter; D018398:Homeodomain Proteins / Q000096:biosynthesis / Q000235:genetics*; D017403:In Situ Hybridization; D051379:Mice; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D016415:Sequence Alignment; D012689:Sequence Homology, Nucleic Acid; D001616:beta-Galactosidase / Q000096:biosynthesis / Q000235:genetics
D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; C084119:MSX2 protein; D011993:Recombinant Fusion Proteins; D001616:beta-Galactosidase