Apoptotic and anti-apoptotic factors in early human mandible development.

The regulators of apoptosis Bcl-2, Bax, caspase-3, p53, and Hsp70 were analyzed immunohistochemically in the developing human mandible of eight human conceptuses from weeks 5 to 10 of gestation. During this period, all proteins displayed an increased pattern of expression in the mandible ectomesenchyme and in newly formed bone, except for caspase-3, which showed decreased expression in the ectomesenchyme, but appeared first in the ossification zone at the 7th wk of development. Simultaneously, the oral epithelium showed weak (p53) to strong (hsp70) expression of all proteins investigated, while in Meckel's cartilage cells, bcl-2 was expressed weakly and hsp70 was expressed moderately. Cells on the surface of the forming bone were predominantly bax positive, and only occasionally bcl-2 positive. Only a few cells on the surface and inside the bony spicules co-expressed bax and bcl-2. Terminal deoxynucleotidyl transferase (TdT)-mediated biotin-dUTP nick-end labelling (TUNEL)-positive cells were found to be apoptotic osteoblasts. The expression of all proteins investigated changed dynamically during early mandible development and the subsequent differentiation of Meckel's cartilage and bone. While interactions between those factors might be associated with the survival of Meckel's cartilage, in the ossification zone they might participate in the control of cell numbers, mineralization, and bone remodelling. Among many other factors, precise orchestration of pro- and anti-apoptotic factors contributes to normal mandible development.

Involvement of FGF and BMP family proteins and VEGF in early human kidney development.

The spatial and temporal pattern of the appearance of the fibroblast growth factor proteins (FGF-8 and FGF-10), the bone morphogenetic proteins (BMP-2/4 subfamily and BMP-7) and the vascular endothelial growth factor protein (VEGF) was investigated in the human mesonephros and metanephros of the 5-9 week-old conceptuses. In the mesonephros, both FGF's and BMP's were found in all structures and their expression slightly decreased in the early fetal period. VEGF positivity appeared in all mesonephric structures, and increased in the fetal period coincidently with formation of the mesonephric blood vessel network. In the metanephros, FGF-8 first appeared only in the metanephric mesenchyme, but from the 7th week on, its reactivity increased and spread to other metanephric structures. FGF-10 positive cells appeared in all metanephric structures already in the 5th week, and slightly intensified with progression of development. Cell survival and nephrogenesis in the permanent kidney might be associated with the appearance of both growth factors. Both BMP-2/4 and BMP-7 displayed a similar pattern of reactivity in all metanephric structures, and their reactivity intensified with advancing development. Alterations in their pattern of appearance might lead to the formation of small and dysplastic kidneys. Already in the earliest developmental stages, VEGF protein appeared in all metanephric structures. At later stages, VEGF showed more intense reaction in the collecting system than in the differentiating nephrons and interstitium. Due to VEGF involvement in vasculogenesis and angiogenesis, abnormal VEGF appearance might lead to impaired formation of the blood vessel network in the human permanent kidney.

Developmental patterns of caspase-3, bax and bcl-2 proteins expression in the human spinal ganglia.

The distribution of the bcl-2, bax and caspase-3 proteins was investigated in the cells of developing human spinal ganglia. Paraffin sections of 10 human conceptuses between 5th and 9th gestational weeks were analysed morphologically, immunohistochemically and by TUNEL-method. Cells positive to caspase-3 had brown stained nuclei or nuclear fragmentations. At earliest stages, 6% of ganglion population were caspase-3 positive cells. Later on, a significant increase in number of caspase-3 positive cells appeared, particularly in the ventral part of ganglia (12%), and subsequently decreased to 6%. TUNEL-positive cells had the same distribution pattern as caspase-3 positive cells. Bax-positive cells followed the developmental pattern similar to caspase-3 cells, changing in range between 20% and 32%. There were 8% of bcl-2 positive cells at earliest stages. They increased significantly in dorsal part of the ganglion during the 7th week (28%), and than dropped to 15% by the end of the 8th week. These findings suggest a ventro-dorsal course of development in human spinal ganglia. Number of bcl-2, bax and caspase-3 positive cells changed in a temporally and spatially restricted manner, coincidently with ganglion differentiation. While apoptosis might control cell number, bcl-2 could act in suppression of apoptosis and enhancement of cell differentiation.

Expression of intermediate filaments, EGF and TGF-alpha in early human kidney development.

The spatial and temporal expression patterns of cytokeratins, vimentin, epithelial growth factor (EGF) and transforming growth factor alpha (TGF-alpha), were investigated in the 5-9-week old human mesonephros and metanephros. Vimentin was found in all mesonephric structures, while cytokeratins were seen only in the mesonephric tubules. EGF and TGF-alpha were detected early in all mesonephric structures, and immunoreactivity to both factors decreased in later stages. In the 5-6-week metanephros, vimentin immunoreactivity was found in all structures and later increased in the collecting system and interstitium. In the 5th week, cytokeratins 8 and 19 appeared in the ureteric bud and ampullae, and later showed increasing immunoreactivity in the collecting system and nephrons. The coexpression of intermediate filament proteins in metanephric development is a temporary feature and might be associated with mesenchymal to epithelial transformation of developing nephrons. In adult kidneys, such coexpression is associated with fibrosis or carcinomatous changes. At early stages, immunoreactivity to EGF and TGF-alpha was detected in all metanephric structures and from the 7th week onward, it decreased in differentiating nephrons. EGF and TGF-alpha patterns of appearance indicate their role in induction, proliferation and growth of metanephric structures. Disturbances in that pattern might cause reduction in kidney growth.

Role of mitotic, pro-apoptotic and anti-apoptotic factors in human kidney development.

The expression pattern of mitotic Ki-67 and anti-apoptotic bcl-2 proteins, as well as apoptotic caspase-3 and p53 proteins, were investigated in the human mesonephros and metanephros of 5-9 week-old human conceptuses. Apoptotic cells were additionally detected using the terminal deoxynucleotidyl transferase (TdT) nick-end labelling (TUNEL) method. Between the 5th and 7th developmental weeks Ki-67, caspase-3 and TUNEL-positive cells characterized all mesonephric structures, indicating importance of cell proliferation in the growth of the mesonephros and role of apoptosis in nephrogenesis. From the 7th week on, p53 and bcl-2 positive cells appeared in the mesonephros as well. Regressive changes in the mesonephros could be regulated by activation of p53, while bcl-2 could contribute to selective survival of some tubules giving rise to adult structures. In the early human metanephros (5-7 weeks), Ki-67 positive cells characterized all metanephric structures, indicating a role of cell proliferation in branching of the ureteric bud and in nephron formation. During the same period bcl-2, caspase-3 and TUNEL-positive cells were found only in the metanephric mesenchyme and nephrons. Bcl-2 protein probably protected nephrons from apoptosis, while caspase-3 protein controlled cell death in the mesenchyme. At later stages (7-9-weeks), appearance of p53-expressing cells could participate in further morphogenesis of the metanephric collecting system. The factors investigated had a spatially and temporally restricted pattern of appearance in developing kidneys. Changes in that pattern might lead to serious disturbances of kidney formation and function in early childhood.