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

Programmed cell death (PCD) is considered one of the most important cellula r processes in the morphogenesis of organs and tissues during animal develo pment. Although the embryonic limb has been established as a classic model for the study of PCD, detailed studies on this process' contribution to mor phogenesis are still lacking, In the present work, using modern computer-ai ded techniques, we estimated the contribution of PCD to mouse limb morphoge nesis. For the detection of apoptotic cell death, we stained whole embryoni c limbs with acridine orange or, in some instances, used the TUNEL techniqu e, and visualized the tissues by confocal laser scanning microscopy. We fou nd that cell death patterns are dynamic during limb development, and occur in gradients oriented with the main limb axes, anteroposterior, dorsoventra l and distoproximal. Interdigital apoptosis in the autopod was initially de tected at the most distal region, and then more proximally as development p roceeded. Interestingly, we found that digit separation is more pronounced on the dorsal side, contrary to what is expected from the apoptotic cell di stribution, which shows more abundant cell death in the ventral region. Usi ng 2-D and 3-D models, we found that most digit individualization occurs ra ther by digit growth than by interdigital cell death. Therefore, digits do not mainly individualize by degeneration of preformed interdigital tissue, but probably by a dynamic balance between proliferation and cell death, red ucing interdigital growth, which results in protrusion of digits. We determ ined the expression pattern of fgf-8 during the period of digit individuali zation, as the product of this gene could participate in defining the limb growth pattern, Initially, fgf-8 expression was coincident with the apical ectodermal ridge, but when cell death was first detected in the inter-digit s, fgf-8 expression became restricted to the tip of the growing digits. The refore, FGF-8 could be one of the factors responsible for differential digi t-interdigit growth, and might also act as a survival factor on interdigita l tissue. We also found that the expression patterns of rar-beta, bmp-2, bm p-4, bmp-7, msx-1, and msx-2 genes, proposed to be involved in the activati on of inter-digital cell death, did not overlap with, or were not highly ex pressed in the major zones of cell death in the developing limb. (C) 2001 W iley-Liss, Inc.