Biomedical subjects

David J Goldhamer

Publications and source records attributed to David J Goldhamer.

5 recordsLinked to original sources

PubMed2005

MyoD-cre transgenic mice: a model for conditional mutagenesis and lineage tracing of skeletal muscle.

The Cre-loxP recombination system has been used to great advantage in vivo for conditional gene targeting, lineage tracing, and other applications. To express cre in skeletal myoblasts and muscle fibers, we utilized the well-characterized transcriptional regulatory regions of the muscle determination gene, MyoD. Transgenic mouse lines were produced (F3/-2.5cre) in which the cre gene is driven by the MyoD promoter and core enhancer, which directs the early activation of MyoD. Specificity of cre expression and efficiency of recombination was determined by monitoring reporter gene expression after crossing to the Cre-dependent reporter lines, R26R and Z/AP. Efficient labeling of embryonic and fetal myoblasts and muscle fibers was observed, with timing that was similar (branchial arches and limb buds) or slightly delayed (myotomes) relative to the endogenous MyoD gene. In satellite cell cultures, a strict concordance between MyoD protein and reporter gene expression was observed, demonstrating the muscle specificity and efficiency of Cre-mediated recombination. Nascent muscle fibers were labeled following injury of adult muscle, indicating recombination in satellite cells or their daughters in vivo.

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PubMed2004

The core enhancer is essential for proper timing of MyoD activation in limb buds and branchial arches.

Transgenic analyses have defined two transcriptional enhancers that regulate MyoD expression in mammals, the core enhancer and distal regulatory region; these enhancers exhibit complementary activities and together are sufficient to recapitulate MyoD expression in developing and mature skeletal muscle. The core enhancer is activated in presumptive muscle cells and determined myoblasts, suggesting an important role in initiating MyoD expression. Here, targeted mutagenesis in the mouse is used to identify necessary and redundant core enhancer functions. The core enhancer is essential for the timely initiation of MyoD expression in limb buds and branchial arches, as enhancer deletion delayed MyoD activation by 1 to 2 days in these muscle lineages. Functionally, this delay in MyoD transcription delayed the onset of muscle differentiation, as assayed by expression of the gene encoding for the early differentiation marker, Myogenin. In addition to these lineage-specific defects, a generalized, modest reduction in MyoD expression was observed in all muscle lineages and at all embryonic stages examined. Interestingly, however, a specific defect was not observed in the nascent myocytes at the medial and lateral aspects of the myotome, suggesting the existence of at least one other enhancer with this specificity. The core enhancer was also dispensable for Myf-5- and Pax-3-dependent regulation of MyoD transcription. These data demonstrate a differential requirement for core enhancer activity in muscle lineages derived from migratory precursors and suggest redundancy in cis regulatory mechanisms controlling myotomal MyoD expression.

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PubMed2003

Skeletal muscle stem cells.

Satellite cells are myogenic stem cells responsible for the post-natal growth, repair and maintenance of skeletal muscle. This review focuses on the basic biology of the satellite cell with emphasis on its role in muscle repair and parallels between embryonic myogenesis and muscle regeneration. Recent advances have altered the long-standing view of the satellite cell as a committed myogenic stem cell derived directly from the fetal myoblast. The experimental basis for this evolving perspective will be highlighted as will the relationship between the satellite cell and other newly discovered muscle stem cell populations. Finally, advances and prospects for cell-based therapies for muscular dystrophies will be addressed.

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PubMed2002

Essential and redundant functions of the MyoD distal regulatory region revealed by targeted mutagenesis.

Transgenic analyses have defined two MyoD enhancers in mammals, the core enhancer and distal regulatory region (DRR); these enhancers exhibit complementary activities and together are sufficient to recapitulate MyoD expression in developing and mature skeletal muscle. DRR activity is restricted to differentiated muscle and persists postnatally, suggesting an important role in maintaining MyoD expression in myocytes and muscle fibers. Here, we use targeted mutagenesis in the mouse to define essential functions of the DRR in its normal chromosomal context. Surprisingly, deletion of the DRR resulted in reduced MyoD expression in all myogenic lineages at E10.5, at least 1 day prior to detection of DRR activity in limb buds and branchial arches of transgenic mice. At later embryonic and fetal stages, however, no defect in MyoD expression was observed, indicating that the DRR is dispensable for regulating MyoD during muscle differentiation. Expression analyses in wild-type and Myf-5 mutant embryos also indicate that the DRR is not an obligate target for Myf-5- and Pax-3-dependent regulation. In contrast to embryonic and fetal stages, deletion of the DRR resulted in a pronounced reduction in MyoD mRNA levels in adults, showing a functional requirement for DRR activity in mature muscle. These data reveal essential and redundant functions of the DRR and underscore the importance of loss-of-function enhancer analyses for understanding cis transcriptional circuitry.

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PubMed2002

Composition and function of AP-1 transcription complexes during muscle cell differentiation.

The role of activating protein-1 (AP-1) in muscle cells is currently equivocal. While some studies propose that AP-1 is inhibitory for myogenesis, others implicate a positive role in this process. We tested whether this variation may be due to different properties of the AP-1 subunit composition in differentiating cells. Using Western analysis we show that c-Jun, Fra-2, and JunD are expressed throughout the time course of differentiation. Phosphatase assays indicate that JunD and Fra-2 are phosphorylated in muscle cells and that at least two isoforms of each are expressed in muscle cells. Electrophoretic mobility shift assays combined with antibody supershifts indicate the appearance of Fra-2 as a major component of the AP-1 DNA binding complex in differentiating cells. In this context it appears that Fra-2 heterodimerizes with c-Jun and JunD. Studying the c-jun enhancer in reporter gene assays we observed that the muscle transcription factors MEF2A and MyoD can contribute to robust transcriptional activation of the c-jun enhancer. In differentiating muscle cells mutation of the MEF2 site reduces transactivation of the c-jun enhancer and MEF2A is the predominant MEF2 isoform binding to this cis element. Transcriptional activation of an AP-1 site containing reporter gene (TRE-Luc) is enhanced under differentiation conditions compared with growth conditions in C2C12 muscle cells. Further studies indicate that Fra-2 containing AP-1 complexes can transactivate the MyoD enhancer/promoter. Thus, an AP-1 complex containing Fra-2 and c-Jun or JunD is consistent with muscle differentiation, indicating that AP-1 function during myogenesis is dependent on its subunit composition.

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