Parker B Antin

PMID: 8917097;Abstract:

We report the cloning of a bromodeoxyuridine (BrdU)-sensitive transcript of 918 bp from an immortalized quail heart cell line containing an open reading frame (ORF) of 215 amino acids (aa) (≃ 23 kDa). Analysis of the secondary structure predicts two amphipathic α-helices with oppositely oriented amphipathic surfaces at the C-terminus of the protein. Each of the helices contains an LEA (late embryogenesis abundant) consensus sequence (A/TAEKAK/RETKD) which has been previously described only in a group of plant seed-specific proteins. Temporal and spatial distribution patterns of the transcript during chick embryo development were examined by whole-mount in situ hybridization and Northern blot analysis. At H and H (Hamburger and Hamilton, 1951) stages 11-14, the message was expressed strongly in blood islands in the area opaca. At day 5, strong signals were found in the liver primordia, mesonephrons, and nephric duct. Frozen sections of whole mount-stained embryonic liver demonstrated that the message was restricted to developing blood cells. The expression pattern of this transcript suggests that its protein product may be involved in hematopoiesis during avian development.

PMID: 9216998;Abstract:

An in vitro assay has been developed to investigate tissue interactions regulating myocardial cell specification in birds. Explants from the posterior region of stage XI-XIV blastulas were found to form heart muscle at high frequency with a timing that corresponded to onset of cardiac myocyte differentiation in vivo. Isolation and recombination experiments demonstrated that a signal from the hypoblast was required to induce cardiac myogenesis in the epiblast, and regional differences in epiblast responsiveness and hypoblast inductiveness restrict appearance of cardiac myocytes to the posterior region. Explantation studies provided evidence that myocardial cell specification is underway by stage 3, indicating that the hypoblast-derived signal occurs shortly before specification is detected. Recombinations were also performed to compare cardiac-inducing capacities of pregastrula hypoblast and stage 5 anterior lateral endoderm. The hypoblast possessed broad capacity to induce heart muscle cells in pregastrula and mid-gastrula epiblast, and modest ability to induce cardiac myogenesis in stage 4 posterior primitive streak. Stage 5 anterior lateral endoderm, in contrast, showed no ability to induce heart development in epiblast cells but was a potent inducer of cardiac myogenesis in cells from stage 4 posterior primitive streak. These findings suggest that the hypoblast-derived signal likely acts upstream of proposed heart-inducing signals provided by anterior lateral endoderm. Experiments were also performed to investigate whether activin, or an activin-like molecule, is involved in regulating cardiac myogenesis. Follistatin blocked cardiac myogenesis in stage XI-XIV posterior region explants and activin induced cardiac myogenesis in a dose-dependent fashion in posterior epiblast. These findings indicate that activin, or an activin-like molecule, is required for and is sufficient to stimulate cardiac myogenesis in posterior region pregastrula epiblast. Three models are presented to explain these results.

PMID: 1985014;Abstract:

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 α-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. © 1991 Academic Press, Inc.

PMID: 1985013;Abstract:

Myogenic cell lines have proven extremely valuable for studying myogenesis in vitro. Although a number of mammalian muscle cell lines have been isolated, attempts to produce cell lines from other classes of animals have met with only limited success. We report here the isolation and characterization of seven avian myogenic cell lines (QM1-4 and QM6-8), derived from the quail fibrosarcoma cell line QT6. A differentiation incompetent QM cell derivative was also isolated (QM5DI). The major features of QM cell differentiation in vitro closely resemble those of their mammalian counterparts. Mononucleated QM cells replicate in medium containing high concentrations of serum components. Upon switching to medium containing low serum components, cells withdraw from the cell cycle and fuse to form elongated multinucleated myotubes. Cultures typically obtain fusion indices of 43-49%. Northern blot and immunoblot analyses demonstrate that each differentiated QM cell line expresses a wide variety of genes encoding muscle specific proteins: desmin, cardiac troponin T, skeletal troponin T, cardiac troponin C, skeletal troponin I, α-tropomyosin, muscle creatine kinase, myosin light chain 2, and a ventricular isoform of myosin heavy chain. While all QM lines analyzed to date express at least some myosin light chain 2, only one line, QM7, expresses this gene at high levels. Surprisingly, none of the QM lines reported here express any known form of α-actin. The absence of sarcomeric actin expression may explain the absence of myofibrils in QM myotubes. These novel features of muscle gene expression in QM cells may prove useful for studying the role of specific muscle proteins during myogenesis. More importantly, however, the isolation of QM cell lines indicates that it may be feasible to isolate other avian myogenic cells lines with general utility for the study of muscle development. © 1991 Academic Press, Inc.