Ubiquitin-protein conjugates selectively distribute during early chicken embryogenesis

The major mechanism for proteolysis in eucaryotes involves an ATP-dependent pathway for which the covalent attachment of ubiquitin targets proteins for degradation. The involvement of ubiquitin conjugation in early embryonic vertebrate development was investigated by examining the amounts and localization of ubiquitin conjugates at different stages of development in the chicken using an affinity-purified antibody specific for conjugated ubiquitin. Solid phase immunochemical assays measuring whole embryo pools of free and conjugated ubiquitin demonstrated a progressive increase in conjugate pools to stage 18, followed by a decline to stage 24. In contrast, levels of free polypeptide showed a dramatic increase after stage 5, indicating a change in the dynamics of the two pools during development. Immunohistochemistry revealed that the distribution of ubiquitin adducts between stages 3 and 22 was pronounced in regions undergoing extensive cellular remodeling. Ubiquitin conjugates were detected in the primitive streak where cells ingress during gastrulation. The presence of these degradative intermediates in both neuroectodermal cells of the neural folds and subsequent neural crest cells migrating from the dorsum of the neural tube is consistent with an involvement in key morphogenetic events. The localization of ubiquitin conjugates at other selected tissue interfaces including limb bud ectoderm/mesoderm, and cardiac atrioventricular myocardium/endothelium suggests an active role for ubiquitin-mediated protein modification in similar developmental interactions. Conjugates were distributed first between somites, then in myotomes with a pattern spatially identical that of the ubiquitin conjugating enzyme, E214K, the major cognate isozyme for isopeptide ligase (E3)-dependent degradation. The potential involvement of ubiquitin conjugation at sites of epithelial-mesenchymal associations was further analyzed in culture using atrioventricular canal (AV) endothelium. Immunoreactivity was abundant in cells immediately prior to and during their transformation into mesenchyme. Collectively, the specific temporal and spatial changes in ubiquitin conjugates during early vertebrate development suggest a regulatory role for this degradative pathway in the cellular remodeling accompanying embryonic growth and differentiation.