Ectopic Expression ofMsx-2in Posterior Limb Bud Mesoderm Impairs Limb Morphogenesis While InducingBMP-4Expression, Inhibiting Cell Proliferation, and Promoting Apoptosis

Abstract

During early stages of chick limb development, the homeobox-containing geneMsx-2is expressed in the mesoderm at the anterior margin of the limb bud and in a discrete group of mesodermal cells at the midproximal posterior margin. These domains ofMsx-2expression roughly demarcate the anterior and posterior boundaries of the progress zone, the highly proliferating posterior mesodermal cells underneath the apical ectodermal ridge (AER) that give rise to the skeletal elements of the limb and associated structures. Later in development as the AER loses its activity,Msx-2expression expands into the distal mesoderm and subsequently into the interdigital mesenchyme which demarcates the developing digits. The domains ofMsx-2expression exhibit considerably less proliferation than the cells of the progress zone and also encompass several regions of programmed cell death including the anterior and posterior necrotic zones and interdigital mesenchyme. We have thus suggested thatMsx-2may be in a regulatory network that delimits the progress zone by suppressing the morphogenesis of the regions of the limb mesoderm in which it is highly expressed. In the present study we show that ectopic expression ofMsx-2via a retroviral expression vector in the posterior mesoderm of the progress zone from the time of initial formation of the limb bud severely impairs limb morphogenesis.Msx-2-infected limbs are typically very 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 ectopicMsx-2expression the morphogenesis of large portions of the posterior mesoderm has been suppressed. We further show thatMsx-2impairs limb morphogenesis by reducing cell proliferation and promoting apoptosis in the regions of the posterior mesoderm in which it is ectopically expressed. The domains of ectopicMsx-2expression in the posterior mesoderm also exhibit ectopic expression ofBMP-4, a secreted signaling molecule that is coexpressed withMsx-2during normal limb development in the anterior limb mesoderm, the posterior necrotic zone, and interdigital mesenchyme. This indicates thatMsx-2regulatesBMP-4expression and that the suppressive effects ofMsx-2on limb morphogenesis might be mediated in part by BMP-4. These studies indicate that during normal limb developmentMsx-2is a key component of a regulatory network that delimits the boundaries of the progress zone by suppressing the morphogenesis of the regions of the limb mesoderm in which it is highly expressed, thus restricting the outgrowth and formation of skeletal elements and associated structures to the progress zone. We also report that rather large numbers of apoptotic cells as well as proliferating 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 are continuously undergoing programmed cell death at the same time that new AER cells are being generated by cell proliferation. Thus, a balance between cell proliferation and programmed cell death may play a very important role in maintaining the activity of the AER.