Transgene expression in the QM myogenic cell line.

We have isolated an avian muscle cell line (QM) which has the essential features of established mammalian muscle cell lines. The experiments reported here were undertaken to determine the suitability of QM cells for the introduction and analysis of cloned transgenes. The promoter of the cardiac troponin T (cTNT) gene has been previously shown to contain sequence elements which govern muscle-specific expression of the chloramphenicol acetyltransferase (CAT) gene in transiently transfected primary cell cultures. We show here that QM cells stably harboring cTNT promoter-CAT fusion genes up-regulate CAT expression in concert with myogenic differentiation, and that as few as 110 upstream nucleotides are sufficient for such differentiation-dependent regulation. In addition, both transient and stable transfection experiments demonstrate that differentiated QM cells possess trans-acting factors necessary for the expression of the skeletal alpha-actin promoter, despite the absence of mRNA or protein product from the endogenous sarcomeric actin genes in these cells. Finally, to follow the developmental potential of QM cells in vivo, we created a clone, QM2ADH, which constitutively expresses the histochemical marker transgene encoding Drosophila alcohol dehydrogenase. When surgically inserted into the limb buds of developing chick embryos, QM2ADH cells are incorporated into endogenous developing muscles, indicating that QM cells are capable of recognizing and responding to host cues governing muscle morphogenesis. Thus, QM cells are versatile as recipients of transgenes for the in vitro and in vivo analysis of molecular events in muscle development.

Dynamic activity of the filopodia of sea urchin embryonic cells and their role in directed migration of the primary mesenchyme in vitro.

Primary mesenchyme cells used in this study were isolated from Lytechinus pictus mesenchyme blastulae by their ability to preferentially adhere to the surface of a tissue culture dish in the presence of serum. Once isolated, primary mesenchyme cells were found to form thin, elongated, active filopodia which closely resemble the filopodia seen in vivo. The filopodia formed in vitro can move as stiffened bristles, bend gradually or very sharply, or be slowly withdrawn. The integrity of the filopodia is not affected by nocodazole but is totally disrupted by cytochalasin D. Filopodia exhibit several apparent functions in vitro: as organelles involved in contacting the external environment, as anchoring appendages that hold the cell bodies in place, and as intercellular connectives that can join cell bodies. The filopodia of primary mesenchyme cells appear to have similar roles within the embryo. The function of the filopodia has been explored by watching the behavior of isolated primary mesenchyme cells in close proximity to deposits of extracellular material (ECM) prepared from mesenchyme blastulae. When the filopodium from a mesenchyme cell makes contact with the nearby ECM, a response is initiated which causes the cell body to move in a directed manner toward the ECM deposit. The use of this type of response as a model system for the study of the migration of primary mesenchyme cells within the embryo is considered.

In vitro fusion and separation of sea urchin primary mesenchyme cells.

Time-lapse videomicroscopy of cultured primary mesenchyme cells from mesenchyme blastulae of the sea urchin Lytechinus pictus demonstrates the dramatic ability of these cells to undergo cell fusion and cell separation. Although this plasticity of cell associations is presumed to play a role in the formation of the syncytial cables that secrete the larval skeleton, the surfaces of these cells must be specialized for fusion and cell separation.

3H-amino acid uptake and incorporation in sea urchin gastrulae and exogastrulae: an autoradiographic study.

Autoradiographic analysis of gastrulae of the sea urchin, Lytechinus pictus, after brief incubation periods with 3H-amino acids has indicated that the cells of the ectoderm incorporate significantly greater levels of isotope than the cells of the interior. Further analysis based primarily on incorporation patterns of exogastrulae indicate that the greatly lowered incorporation by the cells of the archenteron reflects the inability of small molecular weight precursors to reach the invaginated cells. The maintenance of high levels of 3H-amino acid incorporation by ectodermal cells of exogastrulae indicates their relative independence with respect to macromolecular metabolism from the cells of the archenteron.

An effect of accumulated matrix on sulfation among cells in a cartilage colony: an autoradiographic study.

In this report an autoradiographic approach is used to compare synthetic activities of cells within differentiated cartilage colonies. While amino acid incorporation is umiform throughout the colony, H-3-uridine is incorporated more actively by cells having little matrix, cells which are typically in the peripheral regions of a colony. On the other hand S-35-O4 is incorporated most actively by cells in the colony centers. This difference in sulfation appears to occur independently of the mitotic state of the cells, since it is apparent in both growing and near-stationary cultures. Instead, there is a correlation between the accumulation of extracellular matrix and more active levels of sulfation. In support of the idea that matrix creates a microenvironment more favorable to chondrogenesis is the observation that a brief treatment with hyaluronidase, which removes about 60% of the S-35-O4 from prelabeled cultures, depresses isolation of labeled glycosaminoglycans. The possible role of extracellular matrices in altering the expression of differentiated functions by creating a more favorable microenvironment is considered.