title: Expression of the alpha 6A integrin splice variant in developing mouse embryonic stem cell aggregates and correlation with cardiac muscle differentiation.
abstract: Mouse embryonic stem (ES) cells grown in aggregates give rise to several different cell types, including cardiac muscle. Given the lack of cardiac muscle cell lines, ES cells can be a useful tool in the study of cardiac muscle differentiation. The laminin-binding integrin alpha 6 beta 1 exists in two different splice variant forms of the alpha chain (alpha 6A and alpha 6B), the alpha 6A form having been implicated as possibly playing a role in cardiac muscle development, based on its distribution pattern [4, 53]. In this study we characterise the ES cell model system in terms of the expression of the two different alpha 6 splice variants. We correlate their expression with that of muscle markers and the transcription factor GATA-4, using the reverse transcription-polymerase chain reaction (RT-PCR). We confirm that alpha 6B is constitutively expressed by ES cells. In contrast, alpha 6A expression appears later and overlaps in time with a period when the muscle marker myosin light chain-2V (MLC-2V) is expressed, but no MyoD is present, which indicates the presence of cardiac muscle cells in the aggregates. We further show that GATA-4 is present at the same time. Culturing the aggregates under conditions that stimulate (transforming growth factor beta 1 supplement) or inhibit (TGF beta 1 plus 10(-9) M retinoic acid supplement) cardiac muscle differentiation does not lead to any qualitative differences in the timing of expression of these genes, but quantitative changes cannot be excluded. The TGF beta 1 supplement does, however, lead to a relatively greater expression of alpha 6A compared to alpha 6B than the TGF beta 1 plus 10(-9) M RA supplement after 6 days in culture, suggesting that alpha 6A expression is favoured under conditions that stimulate cardiac muscle differentiation. The switch towards alpha 6A expression in ES cell aggregates is paralleled by expression of the binding receptor for TGF beta (T beta RII). Stable expression of a mutated (dominant negative) T beta RII in ES cells, however, still resulted in (TGF beta-independent) upregulation of alpha 6A, demonstrating that these events were not causally related and that parallel or alternative regulatory pathways exist. The initial characterisation of differentiating ES cell aggregates in terms of alpha 6A integrin subunit expression suggests that this model system could be a valuable tool in the study of the role of the alpha 6A beta 1 integrin in cardiac muscle differentiation.
authors.affiliation: Department of Zoology, Faculty of Sciences, University of Lisbon, Portugal. solveig@fc.ul.pt
authors.forename: S
authors.initials: S
authors.lastname: Thorsteinsdóttir
authors.forename: B A
authors.initials: BA
authors.lastname: Roelen
authors.forename: M J
authors.initials: MJ
authors.lastname: Goumans
authors.forename: D
authors.initials: D
authors.lastname: Ward-van Oostwaard
authors.forename: A C
authors.initials: AC
authors.lastname: Gaspar
authors.forename: C L
authors.initials: CL
authors.lastname: Mummery
mesh_terms: D000818:Animals; D015703:Antigens, CD / Q000235:genetics* / Q000378:metabolism*; D015415:Biomarkers; D002454:Cell Differentiation / Q000187:drug effects; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000378:metabolism; D050991:GATA4 Transcription Factor; D018507:Gene Expression Regulation, Developmental; D006321:Heart / Q000196:embryology; D039503:Integrin alpha6; D051379:Mice; D009206:Myocardium / Q000166:cytology*; D017346:Protein Serine-Threonine Kinases; D012326:RNA Splicing; D000077294:Receptor, Transforming Growth Factor-beta Type II; D018125:Receptors, Transforming Growth Factor beta / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013234:Stem Cells / Q000187:drug effects / Q000502:physiology*; D014157:Transcription Factors / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000378:metabolism / Q000494:pharmacology; D014212:Tretinoin / Q000494:pharmacology; D015854:Up-Regulation
chemical_list: D015703:Antigens, CD; D015415:Biomarkers; D004268:DNA-Binding Proteins; D050991:GATA4 Transcription Factor; D039503:Integrin alpha6; D018125:Receptors, Transforming Growth Factor beta; D014157:Transcription Factors; D016212:Transforming Growth Factor beta; D014212:Tretinoin; D017346:Protein Serine-Threonine Kinases; D000077294:Receptor, Transforming Growth Factor-beta Type II