Developmental analysis of tropomyosin gene expression in embryonic stem cells and mouse embryos.

Muthuchamy, Mariappan; Pajak, Laura; Howles, Philip N.; Doetschman, Thomas; Wieczorek, David F.

doi:10.1128/mcb.13.6.3311

Cited by 118 publications

(99 citation statements)

References 53 publications

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“…We observed that S␣TM, a highly-specific protein marker of striated muscle cells and a very early marker of stem-cell cardiogenesis (15), was strongly induced by Shz small molecules in P19CL6 cells. Staining with the CH1 S␣TM-specific mAb identified large clusters of cardiac progenitor cells, found exclusively in Shz small-molecule-treated cultures (Fig.…”

Section: Activation Of Sarcomeric ␣-Tropomyosin Expression By Shz Smallmentioning

confidence: 80%

Cardiogenic small molecules that enhance myocardial repair by stem cells

Sadek

Hannack

Choe

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

115

134

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The clinical success of stem cell therapy for myocardial repair hinges on a better understanding of cardiac fate mechanisms. We have identified small molecules involved in cardiac fate by screening a chemical library for activators of the signature gene Nkx2.5, using a luciferase knockin bacterial artificial chromosome (BAC) in mouse P19CL6 pluripotent stem cells. We describe a family of sulfonylhydrazone (Shz) small molecules that can trigger cardiac mRNA and protein expression in a variety of embryonic and adult stem/progenitor cells, including human mobilized peripheral blood mononuclear cells (M-PBMCs). Small-molecule-enhanced M-PBMCs engrafted into the rat heart in proximity to an experimental injury improved cardiac function better than control cells. Recovery of cardiac function correlated with persistence of viable human cells, expressing humanspecific cardiac mRNAs and proteins. Shz small molecules are promising starting points for drugs to promote myocardial repair/ regeneration by activating cardiac differentiation in M-PBMCs.cardiogenesis ͉ chemical biology ͉ high-throughput screen ͉ myocardial repair

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Section: Activation Of Sarcomeric ␣-Tropomyosin Expression By Shz Smallmentioning

confidence: 80%

Cardiogenic small molecules that enhance myocardial repair by stem cells

Sadek

Hannack

Choe

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

115

134

View full text Add to dashboard Cite

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“…Most recently, a developmental change of TM isoforms has been demonstrated using mouse embryonic stem cells and embryos; ␣-TM-SM mRNA is expressed in undifferentiated embryonic stem cells and in all stages of developing embryoid bodies, whereas the mRNA is detectable in postcoitum embryo (4.5 days) and increases during development, suggesting that expression of ␣-TM-SM occurs at very early developmental stages (54). However, these results are unclear with respect to localization of TM isoform mRNAs, because such study has been performed by RT-PCR using RNAs from whole embryoid bodies and embryos.…”

Section: Figmentioning

confidence: 99%

Coordinate Expression of α-Tropomyosin and Caldesmon Isoforms in Association with Phenotypic Modulation of Smooth Muscle Cells

Kashiwada¹,

Nishida²,

Hayashi³

et al. 1997

Journal of Biological Chemistry

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Isoform diversity of tropomyosin is generated from the limited genes by a combination of differential transcription and alternative splicing. In the case of the ␣-tropomyosin (␣-TM) gene, exon 2a rather than exon 2b is specifically spliced in ␣-TM-SM mRNA, which is one of the major tropomyosin isoforms in smooth muscle cells. Here we demonstrate that expressions of ␣-tropomyosin and caldesmon isoforms are coordinately regulated in association with phenotypic modulation of smooth muscle cells. Molecular cloning and Western and Northern blottings have revealed that in addition to the downregulation of ␤-TM-SM, ␣-TM-SM converted to ␣-TM-F1 and ␣-TM-F2 by a selectional change from exon 2a to exon 2b during dedifferentiation of smooth muscle cells in culture. Simultaneously, a change of caldesmon isoforms from high M r type to low M r type was also observed by alternative selection between exons 3b and 4 in the caldesmon gene during this process. In contrast, cultured smooth muscle cells maintaining a differentiated phenotype continued to express ␣-TM-SM, ␤-TM-SM, and high M r caldesmon. In situ hybridization revealed specific coexpression of ␣-TM-SM and high M r caldesmon in smooth muscle in developing embryos. These results suggest a common splicing mechanism for phenotype-dependent expression of tropomyosin and caldesmon isoforms in both visceral and vascular smooth muscle cells.It is important to elucidate the molecular mechanism of phenotypic modulation of smooth muscle cells (SMCs) 1 such as vasculogenesis, enterogenesis, atherosclerosis, hypertension, and leiomyogenic tumorigenesis. The SMCs are derived from mesodermal precursors, but the intracellular and extracellular factors determining the SMC lineage and its phenotype remain unclear. The search for molecular parameters indicating SMC phenotype is a first step in analyzing phenotypic modulation of SMCs. Several cytoskeletal and contractile proteins are such candidates. Among them, changes of actin (1, 2), caldesmon (CaD) (1, 3, 4), myosin heavy chain (5, 6), and vinculin/metavinculin (1, 7) isoforms are closely associated with phenotypic modulation of SMCs. Recent studies have focused on the gene regulation of such parameters (8 -14). In addition to these isoform changes, expression of ␣-smooth muscle actin (␣-SM actin) (15, 16), CaD (1, 3, 4), myosin heavy and light chains (5, 6, 17), meta-vinculin (1, 7), SM22, and calponin (18 -20) have been reported to be up-regulated during differentiation of SMCs, but down-regulated during dedifferentiation, suggesting the involvement of SMC phenotype-dependent transcriptional regulation in the SMC-specific parameter genes. In fact, the transcriptional machineries of ␣-SM actin (8, 9), CaD (12), myosin heavy chain (13), SM22 (21), and calponin (22) have been partially characterized.Tropomyosin (TM) is a predominant helical protein that binds to actin groves. Recent evidence suggests that Ca 2ϩ -dependent actin-myosin interaction in smooth and nonmuscle cells is controlled by myosin-and actin-linked dual regulation....

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“…In contrast to bladder and aorta, the other tissues were more sensitive to the levels of transgene expression and could only support exon skipping at lower levels of expression. Expression of exon 2 containing ␣TM isoforms has been detected previously in murine ES cells at stages of embryonic development preceding the formation of SM tissues (25). However, that analysis did not look at the contemporaneous expression of exon 3-containing isoforms, so it is not clear whether the detected exon 2-containing ␣TM was a major or minor isoform.…”

Section: Figmentioning

confidence: 71%

Regulated Tissue-specific Alternative Splicing of Enhanced Green Fluorescent Protein Transgenes Conferred by α-Tropomyosin Regulatory Elements in Transgenic Mice

Ellis

Smith

Kemp

2004

Journal of Biological Chemistry

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The mutually exclusive exons 2 and 3 of ␣-tropomyosin (␣TM) have been used as a model system for strictly regulated alternative splicing. Exon 2 inclusion is only observed at high levels in smooth muscle (SM) tissues, whereas striated muscle and non-muscle cells use predominantly exon 3. Experiments in cell culture have shown that exon 2 selection results from repression of exon 3 and that this repression is mediated by regulatory elements flanking exon 3. We have now tested the cell culture-derived model in transgenic mice. We show that by harnessing the intronic splicing regulatory elements, expression of an enhanced green fluorescent protein transgene with a constitutively active promoter can be restricted to SM cells. Splicing of both endogenous ␣TM and a series of transgenes carrying regulatory element mutations was analyzed by reverse transcriptase-PCR. These studies indicated that although SM-rich tissues are equipped to regulate splicing of high levels of endogenous or transgene ␣TM RNA, other non-SM tissues such as spleen, which express lower amounts of ␣TM, also splice significant proportions of exon 2, and this splicing pattern can be recapitulated by transgenes expressed at low levels. We confirm the importance in vivo of the negatively acting regulatory elements for regulated skipping of exon 3. Moreover, we provide evidence that some of the regulatory factors responsible for exon 3 skipping appear to be titratable, with loss of regulated splicing sometimes being associated with high transgene expression levels.

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Developmental analysis of tropomyosin gene expression in embryonic stem cells and mouse embryos.

Cited by 118 publications

References 53 publications

Cardiogenic small molecules that enhance myocardial repair by stem cells

Cardiogenic small molecules that enhance myocardial repair by stem cells

Coordinate Expression of α-Tropomyosin and Caldesmon Isoforms in Association with Phenotypic Modulation of Smooth Muscle Cells

Regulated Tissue-specific Alternative Splicing of Enhanced Green Fluorescent Protein Transgenes Conferred by α-Tropomyosin Regulatory Elements in Transgenic Mice

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