Defects in adhesion and migration, but not in proliferation and differentiation, of embryonic stem cells upon replacement of integrin subunit β1A by β1D

Abstract

β1D is a skeletal muscle-specific splice variant of the β1 integrin subunit, while β1A integrin subunit has a wide tissue distribution. We have previously shown that replacement of β1A by β1D by homologous recombination (knockin) in all mouse tissues was embryonic lethal. Through two successive rounds of homologous recombination, we have now produced embryonic stem (ES) cells expressing β1D instead of β1A, and analyzed the ability of β1D to support ES cell differentiation in vitro and in teratomas in vivo. β1D knockin (KI) ES cells grew at a similar rate but as more compact colonies than the β1A-expressing cells. Increased cell cohesiveness, however, did not appear to involve changes in cadherin activity. Although in both β1A and β1D-KI ES cells only one β1 allele is active, the expression of β1 integrins in the β1D-KI ES cells was reduced by 50 %, compared with that in the β1A-expressing cells; this correlated with impaired adhesive and migratory capacities. It appeared that during in vitro cardiac differentiation, in spite of a slight delay in the induction of two cardiac-specific transcripts, the α- and β-myosin heavy chains, contracting cardiomyocytes were detected in similar numbers and at the same time in embryoid bodies (EB) derived from β1D-KI and from β1A cells. Furthermore, replacement of β1A by β1D in ES cells did not affect neurite differentiation in embryoid bodies in the presence of retinoic acid suggesting that β1D supports neurogenesis. However, the impaired migration of other cells from the EB, including endodermal cells, prevented the normal outgrowth of neurites in β1D-KI EB. Finally, injection of β1D-KI ES cells in the flank of syngeneic mice gave rise to fully developed teratomas containing simple and pluristratified epithelia, muscle, cartilage, blood vessels, and tissues from the neural lineage. These results show that the muscle-specific splice variant β1D, in spite of its specific cytoplasmic domain, supports the differentiation of many cell types. This further suggests that the embryonic lethality in the β1D-KI embryos was mainly due to the different ability of β1A and β1D to mediate cell adhesion and migration.