Expression of the α6A 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 α6β1 exists in two different splice variant forms of the α chain (α6A and α6B), the α6A 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 α6 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 α6B is constitutively expressed by ES cells. In contrast, α6A 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 β1 supplement) or inhibit (TGFβ1 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 TGFβ1 supplement does, however, lead to a relatively greater expression of α6A compared to α6B than the TGFβ1 plus 10 −9 M RA supplement after 6 days in culture, suggesting that α6A expression is favoured under conditions that stimulate cardiac muscle differentiation. The switch towards α6A expression in ES cell aggregates is paralleled by expression of the binding receptor for TGFβ (TβRII). Stable expression of a mutated (dominant negative) TβRII in ES cells, however, still resulted in (TGFβ-independent) upregulation of α6A, 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 α6A integrin subunit expression suggests that this model system could be a valuable tool in the study of the role of the α6Aβ1 integrin in cardiac muscle differentiation.