Ann M. Wunsch

Distribution of connective tissue proteins during development and neovascularization of the epicardium

OBJECTIVE The epicardium is the site of initial cardiac neovascularization and formation of the coronary circulatory system. Recent evidence indicates that vascular progenitor cells are influenced by the connective tissue proteins of their extracellular environment, yet little is known about the composition or function of the embryonic epicardial extracellular matrix (ECM). This study examines the distribution of ECM proteins during the migration, growth and maturation of epicardial cells and also during the development of the coronary vascular network. METHODS Immunofluorescence microscopy was used to determine the distributions of vitronectin, fibronectin and a newly described fibrillin-like protein, the JB3 antigen, in the embryonic chicken heart. Immunoblot analysis was performed to compare the relative electrophoretic mobilities of the JB3 antigen and fibrillin-1. RESULTS The data show that vitronectin and fibronectin are present at sites of initial migration of the epicardial cells. The expression of vitronectin (and also fibronectin) becomes more pronounced as the epicardium thickens, undergoes remodeling and differentiates. The JB3 antigen is prominently expressed in the coronary arteries, allowing visualization of their connection to the systemic circulation and to the heart muscle, as well as vessel wall formation and organization. Immunoblot analysis suggests that the JB3 antibody recognizes a fibrillin-like polypeptide that is distinct from fibrillin-1. CONCLUSIONS The observed distributions of vitronectin and fibronectin are consistent with roles in migration of epicardial cells, in remodeling of the epicardium and as substratum components during blood vessel formation. The observed distribution of the JB3 antigen indicates a structural/organizational role in coronary arterial wall assembly and suggests that the JB3 antibody be considered an early marker for maturing coronary arteries.

Modulation of histone H3 variant synthesis during the myoblast-myotube transition of chicken myogenesis ☆

We have previously reported that nucleosomal histones are synthesized by cultured, postmitotic myotube cells at 9-29% of the rate in their dividing myoblast precursors (A. M. Wunsch, A. L. Haas, and J. Lough, 1987, Dev. Biol. 119, 85-93). In that study, histones were separated by two-dimensional polyacrylamide gels containing 8 M urea in the first-dimension to optimally separate variants of the H2A class. To separate and compare synthesis of variants in the H2B and H3 classes during myogenesis, 5.75 M urea has been used in the first dimension. Although no changes in the H2B variant pattern were discerned, a dramatic change in H3 variant synthesis was detected, in which a predominance of H3.2 synthesis in dividing myoblasts was almost completely replaced by a lower level of H3.3 synthesis after myotube formation. With increasing differentiation, H3.2 synthesis became undetectable, while H3.3 synthesis continued. Control experiments indicated that these results were not mediated by replicating cells in the myotube cultures, the effects of cytosine arabinoside, or contaminating non-histone proteins. These results suggest that histone H3.2 is replaced by histone H3.3 in nucleosomes during skeletal muscle maturation.

Synthesis and ubiquitination of histones during myogenesis

One and two-dimensional polyacrylamide gel electrophoresis have revealed that cultures of postmitotic (G0) chicken skeletal myotube cells synthesize significant but reduced quantities of histone proteins as compared to their proliferating myoblast precursors. In addition, modulation of variant synthesis within the histone H2A and H3 classes may accompany myotube formation. That the histone bands contain no nonhistone contaminants was shown by exclusion of [3H]tryptophan. It is unlikely that these results reflect synthesis of histone by contaminating replicating cells, since a single treatment with cytosine arabinoside at the time of fusion effectively removed unfused cells while suppressing synthesis of DNA in the myotube cultures. The relatively sparse incorporation of label by major variants of the H2A class in dividing myoblasts was shown to be caused by heterogeneity due to phosphorylation and extensive ubiquitination, which decline at the time of myotube formation. As determined by quantitative Western-blotting, dividing myoblasts and myotubes contain an average of 1.0 and 0.4 molecules of ubiquitinated H2A (uH2A), respectively, per 10 nucleosomes.

Histone variant patterns during vertebrate embryogenesis and limb development.

Two-dimensional gel electrophoresis was used to examine the relative content of core histone variants during early chicken embryogenesis and at selected stages of hindlimb development. Nuclei from stage 19 limb buds displayed a pattern similar to whole embryos at stage 1, at which time all of the known avian histone variants, including the minor isoprotein H3.3, were detected. Variant ratios did not change during limb development, up to stage 29. However, the portion of H2A variants migrating as ubiquitinated conjugates increased more than twofold during limb development, advancing from 4.5% of the total H2A proteins at stage 19 to 12% at stage 29.

Normal transitions in synthesis of replacement histones H2A.Z and H3.3 during differentiation of dystrophic myotube cells. A brief note.

We previously reported that differentiating G0 myotube cells cultured from normal chicken embryos exhibit a histone synthesis pattern that is highlighted by transitions in the expression of the minor replacement variants H3.3 and perhaps H2A.Z (Wunsch and Lough, Dev. Biol. 119 (1987) 94-99). Because these proteins may be synthesized to maintain chromatin structure during the differentiation and maturation of the skeletal muscle fiber, it was of interest to determine whether they are made at normal levels during the differentiation of dystrophic muscle. To this end, the synthesis of histone proteins in cultured myoblasts and myotubes from normal and dystrophic avian embryos has been characterized by two-dimensional polyacrylamide gel electrophoresis and fluorography. Proliferating myoblasts (day 1) as well as two stages of differentiating myotubes (days 3, 4) exhibited histone synthesis patterns that were indistinguishable when comparing normal and dystrophic cells. It is noteworthy that this study also revealed that, in both cell types, the change in H2A.Z synthesis during the myoblast/myotube transition was remarkable, increasing from approximately 20% of the non-ubiquitinated H2As in myoblasts to 80% in myotubes. Also, gel staining patterns and immunoblotting detected no differences in the degree of histone ubiquitination between normal and dystrophic cells. These findings indicate that, up to this point in dystrophic differentiation, neither the synthesis nor ubiquitination of histones are perturbed.