A new interpretation of the necrotic changes occurring in the developing limb bud paddle of mouse embryos based upon...

Degenerative changes occurring in the apical ectodermal ridge (a.e.r.) and undifferentiated distal mesoderm of developing limb buds were studied macro- and microscopically in day-11 to day-13 mouse embryos displaying the normal (+/+), oligosyndactylous (Os/+), polydactylous (Xpl/+) and hybrid (Os/+/Xpl/+) phenotypes. Isolated limb buds were submitted either to supravital staining with Nile blue sulfate or to lectin binding staining in serial paraffin sections, taking advantage of strong binding affinites of macrophage cells for peanut agglutinin after neuraminidase treatment and for ricinus communis agglutinin. Necrotic changes detected in three definite areas of the distal mesoderm of normal limb buds exhibit characteristic spatial temporal relationships with earlier cytolytic changes affecting the pre- and postaxial parts of the a.e.r. Two of them, known as the primary preaxial site (fpp) and the anterior marginal necrotic zone (AMNZ) appeared deeply modified in mutant embryos as compared to the posterior marginal necrotic zone (PMNZ) which remained unaffected. Macrophage cells loaded with cell debris appear in advance and in excessive number in the fpp of Os/+ limb buds. Conversely, they were found absent or locally reduced in number in the fpp and AMNZ of Xpl/+ limb buds which otherwise develop in the same area a preaxial protrusion covered with a healthy portion of the a.e.r. Hybrid Os/+/Xpl/+ limb buds expressing both mutant genes develop a smaller and macrophage-free preaxial protrusion which coexists with residual and locally excessive necrotic changes in its immediate surrounding and is covered with a normally necrotic portion of the a.e.r. Microscopic observations collected in the limb buds of all phenotypes, though more frequently in Os/+ limb buds, strongly suggest that in all three necrotic sites examined, macrophage cells of vascular origin somehow contribute to the clearance of ectodermal necrotic debris and eventually return in the blood stream through the marginal vein and its affluents.