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Formation of the left/right body axis is a critical early step in embryogenesis. The heart loop is one of the first clearly recognizable morphological asymmetries, and the molecular pathway which dictates this laterality is now beginning to be understood. We report here that the left and right precardiac fields of chick differ in their sensitivity to retinoic acid (RA); while RA applied to the right precardiac field at gastrulation randomizes heart looping, left side treatment induces situs inversus only at high RA concentrations. We identified two extracellular matrix proteins, the heart-specific lectin-associated matrix protein-1 (hLAMP1) and the fibrillin-related protein recognized by the antibody JB3, which are distributed asymmetrically within the precardiac fields at the head process stage. In normal embryos, JB3 expression is enhanced within the right precardiac field, and hLAMP-1 is enriched within the left. RA treatment predictably altered the expression of these proteins in a manner consistent with subsequent heart laterality: RA treatments which randomize heart loop direction also equalized or reversed the left/right JB3 and hLAMP-1 distribution prior to heart tube fusion. The existence of asymmetrically expressed extracellular matrix proteins within precardiac regions suggests that interactions between cardiocytes and their environment may contribute to heart laterality determination and looping.

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It was demonstrated previously that a polyclonal antibody (ES1) raised against EDTA extractable proteins from embryonic chicken heart blocks cardiac endothelial-mesenchymal transformation in a culture bioassay and stains extracellular matrix at sites of embryonic inductive interactions, e.g., developing heart, limb buds, and neural crest forming region [Krug et al., 1987, Dev Biol 120:348-355; Mjaatvedt et al., 1991, Dev Biol 145:219-230). In the present study, by using an antiserum (ES3) to a similar immunogen, we affinity purified four major EDTA-soluble proteins. These proteins migrated as 27, 44, 63, and 70 kD molecules under reduced conditions and 27, 41, 52, and 59 kD under nonreduced conditions, respectively, on SDS-PAGE. Based on several criteria, the protein migrating at 70/59 kD (reduced/nonreduced) was indistinguishable from chicken transferrin (conalbumin): 1) amino acid sequencing showed that eight N-terminal residues were identical to those of chicken transferrin, 2) acid hydrolysates of both proteins had nearly identical compositions, 3) the protein co-migrated exactly with chicken transferrin under both reduced and nonreduced conditions, and 4) ES3 IgG recognized both the 70/59 kD protein and chicken transferrin by western blot analysis of nonreduced samples, but not with reduced samples. Immunohistochemistry of chicken embryonic heart with antibodies against transferrin demonstrated that anti-transferrin immunoreactivity is present in myocardium but absent in cardiac endothelium before the initiation of cardiac endothelial-mesenchymal formation. However, both cardiac endothelium and migrating mesenchymal cells became immunoreactive with anti-transferrin at the time transformation occurred. These findings suggest a possible involvement of transferrin in the inductive process of cardiac endothelial-mesenchymal transformation.

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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 ectoderrn/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 to 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 epithelialmesenchymal 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 path-0 1995 WILEY-LISS, INC. way in the cellular remodeling accompanying embryonic growth and differentiation.Q 1995 Wiley-Liss, Inc.

A high level of the post-translational modification, acetylation, is found on the N-terminal regions of the core histones H2A, H2B, H3, and H4 and is primarily located in the nucleosomes of active genes. An in vitro transcription system was applied, which utilizes T7 RNA polymerase and template DNAs that are either moderately or highly positively coiled, to determine whether acetylation alters the dynamics of histone displacement from these templates during transcription. To measure displacement, an excess of a competitor (negatively coiled DNA reconstituted with unlabeled H3-H4) was included during the transcription process. Acetylated but not unacetylated (3)H-labeled H3-H4 was found to displace with high frequency from the moderately positively coiled template. This displacement of acetylated H3-H4 was not observed when the template was highly positively coiled. Acetylated (3)H-labeled H2A-H2B also preferentially displaced to the competitor, but in this instance, transcription-induced stress on the highly positively coiled template was required. The histone chaperone, NAP1, was found to facilitate the displacement of both H3-H4 and H2A-H2B. Surprisingly, when acetylated H2A-H2B and acetylated H3-H4 were reconstituted together in the same nucleosomes, the displacement of acetylated H2A-H2B was much reduced during transcription. We conclude that acetylation alters nucleosome stability by enhancing displacement of H3-H4, while decreasing the displacement of H2A-H2B. These results are discussed with regard to potential in vivo conditions in which these observations may be relevant.