Expression analysis of the family of 14-3-3 proteins in zebrafish development.

14-3-3 proteins comprise a family of dimeric multi-functional proteins present in all eukaryotes, that are important in a whelm of ubiquitous biological processes. We have analyzed the genomic structure of all 14-3-3s from zebrafish comprising 11 genes and have analyzed their phylogeny. The gene family was cloned and its expression pattern in zebrafish embryogenesis was analyzed by whole mount in situ hybridization and microarray analysis with gene specific probes. We demonstrate that maternal mRNA of 14-3-3s is expressed evenly at the first cell division. At later stage all genes are expressed in a patterned way with, in most cases, intricate patterns in the developing brain. Our result shows distinct expression patterns of various genes. Microarray results show that differences in expression levels of highly similar 14-3-3 genes also occur in the adult stage.

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

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-beta, 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.

Reactive oxygen species participate in the control of mouse embryonic cell death.

Programmed cell death or apoptosis is an essential process during the morphogenesis of a large number of structures. Evidence obtained over the past few years indicates that, in some cases, the generation of reactive oxygen species (ROS) is an important event during the course of apoptosis. Using an in vitro culture system in which digit individualization of developing limbs normally occurs, we assayed the effect of different antioxidants on the cell death that takes place at interdigits. The addition of phenol, dimethyl sulfoxide, or 2',7'-dichlorodihydrofluorescein diacetate (DCDHF-DA) to murine developing limbs in culture prevented digit individualization as well as the typical interdigital cell death. Two ROS-sensitive dyes, 3-(4,5-dimethylthiazol)-2,5-diphenyl tetrazolium bromide and DCDHF-DA, stained interdigits and the so-called "necrotic zones," implying that they contain cells under oxidative stress. Very few interdigital cells were doubly stained with the ROS probes and two cell death indicators (i.e., acridine orange and propidium iodide), suggesting that they detect a different stage during the course of apoptosis. Furthermore, we found cells stained for ROS that did not express a specific macrophage marker and in a few cases were seen surrounded by a macrophage. Surprisingly, many regions of the midgestation mouse embryo that are undergoing cell death correlated with those that have a markedly higher level of ROS. Our data suggest that the generation of oxidative stress is a common requirement for cell death that occurs during mouse embryonic development.