ECTOPIC EXPRESSION OF MSX-2 IN POSTERIOR LIMB BUD MESODERM IMPAIRS LIMB MORPHOGENESIS WHILE INDUCING BIMP-4 EXPRESSION, INHIBITING CELL-PROLIFERATION, AND PROMOTING APOPTOSIS

During early stages of chick limb development, the homeobox-containing gene Msx-2 is expressed in the mesoderm at the anterior margin of the limb bud and in a discrete group of mesodermal cells at the midproxim al posterior margin. These domains of Msx-2 expression roughly demarca te the anterior and posterior boundaries of the progress zone, the hig hly proliferating posterior mesodermal cells underneath the apical ect odermal ridge (AER) that give rise to the skeletal elements of the lim b and associated structures. Later in development as the AER loses its activity, Msx-2 expression expands into the distal mesoderm and subse quently into the interdigital mesenchyme which demarcates the developi ng digits. The domains of Msx-2 expression exhibit considerably less p roliferation than the cells of the progress zone and also encompass se veral regions of programmed cell death including the anterior and post erior necrotic zones and interdigital mesenchyme. We have thus suggest ed that Msx-2 may be in a regulatory network that delimits the progres s zone by suppressing the morphogenesis of the regions of the limb mes oderm in which it is highly expressed. In the present study we show th at ectopic expression of Msx-2 via a retroviral expression vector in t he posterior mesoderm of the progress zone from the time of initial fo rmation of the limb bud severely impairs Limb morphogenesis. Msx-2-inf ected limbs are typically Fiery narrow along the anteroposterior axis, are occasionally truncated, and exhibit alterations in the pattern of formation of skeletal elements, indicating that as a consequence of e ctopic Msx-2 expression the morphogenesis of large portions of the pos terior mesoderm has been suppressed We further show that Msx-2 impairs limb morphogenesis by reducing cell proliferation and promoting apopt osis in the regions of the posterior mesoderm in which it is ectopical ly expressed. The domains of ectopic Msx-2 expression in the posterior mesoderm also exhibit ectopic expression of BMP-4, a secreted signali ng molecule that is coexpressed with Msx-2 during normal limb developm ent in the anterior limb mesoderm, the posterior necrotic zone, and in terdigital mesenchyme. This Indicates that Msx-2 regulates BMP-4 expre ssion and that the suppressive effects of Msx-2 on limb morphogenesis might be mediated in part by BMP-4. These studies indicate that during normal limb development Msx-2 is a key component of a regulatory netw ork that delimits the boundaries of the progress zone by suppressing t he morphogenesis of the regions of the limb mesoderm in which it is hi ghly expressed, thus restricting the outgrowth and formation of skelet al elements rand associated structures to the progress zone. We also r eport that rather large numbers of apoptotic cells as well as prolifer ating cells are present throughout the AER during all stages of normal limb development we have examined, indicating that many of the cells of the AER ape continuously undergoing programmed cell death at the sa me time that new AER cells are being generated by cell proliferation. Thus, a balance between cell proliferation and programmed cell death m ay play a very important role in maintaining the activity of the AER. (C) 1998 Academic Press.