Expression of the alpha 6A integrin splice variant in developing mouse embryonic stem cell aggregates and correlation with cardiac muscle differentiation
S. Thorsteinsdottir et al., Expression of the alpha 6A integrin splice variant in developing mouse embryonic stem cell aggregates and correlation with cardiac muscle differentiation, DIFFERENTIA, 64(3), 1999, pp. 173-184
Mouse embryonic stem (ES) cells grown in aggregates give rise to several di
fferent cell types, including cardiac muscle. Given the lack of cardiac mus
cle cell lines, ES cells can be a useful tool in the study of cardiac muscl
e 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 car
diac 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 exp
ression appears later and overlaps in time with a period when the muscle ma
rker myosin light chain-2V (MLC-2V) is expressed, but no MyoD is present, w
hich indicates the presence of cardiac muscle cells in the aggregates. We f
urther show that GATA-4 is present at the same time. Culturing the aggregat
es under conditions that stimulate (transforming growth factor beta 1 suppl
ement) or inhibit (TGF beta 1 plus 10(-9) M retinoic acid supplement) cardi
ac muscle differentiation does not lead to any qualitative differences in t
he timing of expression of these genes, but quantitative changes cannot be
excluded. The TGF beta 1 supplement does, however, lead to a relatively gre
ater expression of alpha 6A compared to alpha 6B than the TGF beta 1 plus 1
0-9 M RA supplement after 6 days in culture, suggesting that alpha 6A expre
ssion is favoured under conditions that stimulate cardiac muscle differenti
ation. The switch towards alpha 6A expression in ES cell aggregates is para
lleled by expression of the binding receptor for TGF beta (T beta RII). Sta
ble expression of a mutated (dominant negative) T beta RII in ES cells, how
ever, still resulted in (TGF beta-independent) upregulation of alpha 6A, de
monstrating that these events were not causally related and that parallel o
r alternative regulatory pathways exist. The initial characterisation of di
fferentiating ES cell aggregates in terms of alpha 6A integrin subunit expr
ession suggests that this model system could be a valuable tool in the stud
y of the role of the alpha 6 beta 1 integrin in cardiac muscle differentiat
ion.