Quantitative analysis of protein synthesis in mouse embryos. II: Differentiation of endoderm, mesoderm, and ectoderm

Abstract

The changes in protein synthesis that occur during differentiation of the primitive germ layers were examined by high-resolution, two-dimensional gel electrophoresis of proteins synthesized in 6.5 and 7.5 days postcoitum (d.p.c.) mouse embryos. For 6.5 d.p.c. embryos, protein synthesis patterns were compared between whole extraembryonic and embryonic regions and between embryonic visceral endoderm and embryonic ectoderm. For 7.5 d.p.c. embryos, comparisons were made between extraembryonic and embryonic regions and between isolated embryonic endoderm, mesoderm, and ectoderm. Each of the isolated 7.5 d.p.c. germ layers was divided into anterior and posterior fragments in order to evaluate possible regional differences in gene expression along the anterior-posterior axis. Comparisons of protein synthesis patterns revealed the greatest difference between isolated endoderm and ectoderm, indicating that by as early as 6.5 d.p.c. patterns of gene expression differ significantly between these tissues. The greatest similarities were found between ectoderm and whole embryonic regions and between endoderm and whole extraembryonic regions, which most likely reflects the overall cellular compositions of the embryonic and extraembryonic regions. Based on their patterns of synthesis, four groups of proteins were identified that were preferentially synthesized in either endoderm or ectoderm. These provide useful markers for studying differentiation in these tissues. One other protein, migrating at the position expected for vimentin, was synthesized at an elevated rate in isolated mesoderm. We also observed differences in rates of synthesis of alpha-tubulin and tropomyosin-5 indicative of potential differences in cytoskeletal composition among the germ layers beyond those previously described. The difference in overall protein synthesis patterns between anterior and posterior regions was greatest in the embryonic endoderm, indicating that differentiation along the anterior-posterior axis may be initiated sooner or may proceed more rapidly in the endoderm than in the other germ layers. These data provide the first quantitative evaluation of the degree to which differentiation of the three primitive germ layers affects protein synthesis patterns and reveal potentially useful markers of endoderm and ectoderm differentiation.

Differential Protein Expression at the Stage of Neural Tube Closure in the Mouse Embryo

“…Moreover, use of silver staining gave a global display of the sample, whereas previous analyses of [ 35 S]methionine-labeled proteins was, by necessity, limited to those proteins synthesized during the labeling period (8,9). The approach taken in this study enabled detection of changes in the abundance of individual protein species and these observations correlated closely with the abundance of the corresponding proteins as determined by Western blot.…”

“…Previously, [ 35 S]methionine labeling was used to examine the pattern of protein synthesis in mouse embryos (7)(8)(9). In one study (7), 600 -800 protein species were resolved by two-dimensional electrophoresis but only one spot differed in intensity at E8, 9, and 10.…”

“…Among these, 11 spots differed in level in embryonic tissues at E7-9, although the identity of these proteins was not determined. A potential drawback of these studies (7)(8)(9) is that embryos were labeled in tissue culture medium that does not support normal physiological development, the optimal culture medium for organogenesis-stage embryos being rat serum (10). Labeling proteins under non-physiological conditions introduces the potential for spurious results that do not reflect in vivo development.…”

Modulation of protein synthesis in rabbit inner cell mass-derived cells by FGF-2

“…In 7.5-day embryos, comparison of protein syntheses revealed the difference among embryonic and extraembryonic regions, and between isolated embryonic endoderm, mesoderm and ectoderm. In summary, these data revealed both up-regulation and downregulation of protein synthesis with a predominantly increased rate of synthesis in the extraembryonic region and endoderm compared to embryonic region and ectoderm [15]. Changes in embryonic protein synthesis were also observed later in development, from day 7.5 to day 9.…”

“…Such databases were published for one-cell stage to blastocyst stage mouse embryos [3,14,16,19]. Others focus on the quantitative analysis of proteins synthesized by 6.5-7.5 day mouse embryos with respect to differentiation of ectoderm, endoderm and mesoderm [15], proteins specific to major organogenesis in the mouse from day 7.5 to day 9.0 of development [25] or the developing heart of a 9.5 day mouse embryo [26]. Concerning potential morphogens, of all the growth factors that are known to affect embryonic development, only the effects of insulin growth factor on protein synthesis in eightcell mouse embryos and blastocysts [20] and porcine embryonic discs [8], and the effect of TGFs on total protein synthesis in mouse blastocysts [1] were investigated.…”