Oxidative stress is considered causal of aging and pathological cell death, however, very little is known about its function in the natural processes that support the formation of an organism. It is generally thought that cells must continuously protect themselves from the possible damage caused by reactive oxygen species (ROS) (passive ROS function). However, presently, ROS are recognized as physiologically relevant molecules that mediate cell responses to a variety of stimuli, and the activities of several molecules, some developmentally relevant, are directly or indirectly regulated by oxidative stress (active ROS function). Here we review recent data that are suggestive of specific ROS functions during development of animals, particularly mammals.

Programmed cell death (PCD) is considered one of the most important cellular processes in the morphogenesis of organs and tissues during animal development. Although the embryonic limb has been established as a classic model for the study of PCD, detailed studies on this process' contribution to morphogenesis are still lacking. In the present work, using modern computer-aided techniques, we estimated the contribution of PCD to mouse limb morphogenesis. For the detection of apoptotic cell death, we stained whole embryonic limbs with acridine orange or, in some instances, used the TUNEL technique, and visualized the tissues by confocal laser scanning microscopy. We found that cell death patterns are dynamic during limb development, and occur in gradients oriented with the main limb axes, anteroposterior, dorsoventral and distoproximal. Interdigital apoptosis in the autopod was initially detected at the most distal region, and then more proximally as development proceeded. Interestingly, we found that digit separation is more pronounced on the dorsal side, contrary to what is expected from the apoptotic cell distribution, which shows more abundant cell death in the ventral region. Using 2-D and 3-D models, we found that most digit individualization occurs rather 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, reducing interdigital growth, which results in protrusion of digits. We determined the expression pattern of fgf-8 during the period of digit individualization, 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 interdigits, fgf-8 expression became restricted to the tip of the growing digits. Therefore, FGF-8 could be one of the factors responsible for differential digit-interdigit growth, and might also act as a survival factor on interdigital tissue. We also found that the expression patterns of rar-␤, bmp-2, bmp-4, bmp-7, msx-1, and msx-2 genes, proposed to be involved in the activation of interdigital cell death, did not overlap with, or were not highly expressed in the major zones of cell death in the developing limb.

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During mammalian development, the first cell lineage diversification event occurs in the blastocyst, when the trophectoderm (TE) and the inner cell mass (ICM) become established. Part of the TE (polar) remains in contact with the ICM and differs from the mural TE (mTE) which is separated from the ICM by a cavity known as the blastocoele. The presence of filopodia connecting ICM cells with the distant mural TE cells through the blastocoelic fluid was investigated in this work. We describe two types of actin-based cell projections found in freshly dissected and in vitro cultured expanding blastocysts: abundant short filopodia projecting into the blastocoelic cavity that present a continuous undulating behavior; and long, thin traversing filopodia connecting the mural TE with the ICM. Videomicroscopy analyses revealed the presence of vesicle-like structures moving along traversing filopodia and dynamic cytoskeletal rearrangements. These observations, together with immunolocalization of the FGFR2 and the ErbB3 receptors to these cell extensions, suggest that they display signal transduction activity. We propose that traversing filopodia are employed by mitotic mTE cells to receive the required signals for cell division after they become distant to the ICM.