S. Holtzer

Myogenic and chondrogenic lineages in developing limb buds grown in vitro

Embryonic chick leg bud mesoderms from stages 17 through 25 were cultured from 1 to 3 weeks as explants or as monolayers (1×104 to 2×106 cells per 35 mm dish). Differentiation of muscle and cartilage was exmained in living and fixed cultures. In media known to permit either type of terminal differentiation, first myotubes and later chondrified clusters of cells appeared in all explant cultures. However, stage 21 was the earliest these cell types were obtained in low and high density monolayers. Myogenic clones were derived from stage 21–22 cells; “mixed clones,” with muscle and cartilage, did not occur. Only a small percentage of cells in any one cultured population terminally differentiated. The small percentage of cells whose progeny terminally differentiated increased with culture of older stages. These results are consistent with mesoderm development by means of distinct and multicompartmented lineages, the cells of which are asynchronously distributed in vivo and in vitro. Alternatives for myogenic and chondrogenic lineages are discussed.

Unanticipated temporal and spatial effects of sarcomeric α‐actinin peptides expressed in PtK2 cells

To understand the multiple roles of alpha-actinin in the assembly of (1) Z bands in muscle, and (2) a variety of cytoskeletal structures in non-muscle cells, 4 sarcomeric alpha-actinin derived cDNAs tagged with a MYC epitope were constructed. The constructs were: (1) full-length (FL/MYC); (2) minus EF-hands (-EF/MYC); (3) actin-binding site (+A/MYC); and (4) minus actin-binding site (-A/MYC). These four cDNAs were individually transfected into PtK2 cells. The exogenous sarcomeric alpha-actinin (s-alpha-actinin/MYC) was followed with labeled anti-MYC, the endogenous non-sarcomeric alpha-actinin (non-s-alpha-actinin) with labeled anti-non-s-alpha-actinin. The salient findings were: (1) the selective intracellular localizations of each expressed MYC-tagged peptide differed one from the other; (2) their respective localizations in the 10-24-h post-transfection (p.t.) period differed from their localizations in the 48-72-h p.t. period; (3) each MYC-positive peptide was cytotoxic, but each in a distinctive way; and (4) while the selective targeting of FL/MYC to dense bodies, adhesion plaques, adherens junctions, and ruffled membranes was consistent with binding studies in cell-free systems, the incorporation of the mutated peptides, particularly +A/MYC and -A/MYC was not. Changes in localization over time and the distinctive cytopathologies probably reflect domain-specific targeting. They also suggest unexpected cooperative involvement of multiple domains of alpha-actinin with specific receptors in distal cytoskeletal structures. To date, such qualitative in vivo interactions have not been described either in in vitro binding studies, or in short-term experiments involving localization and/or fate of microinjected labeled molecules into living cells.