Loss of β₁ integrin function results in a retardation of myogenic, but an acceleration of neuronal, differentiation of embryonic stem cells in vitro

Integrin cell surface receptors play an important role for cell adhesion, migration, and differentiation during embryonic development by mediating cell-cell and cell-matrix interactions. Less is known about the function of integrins during commitment and lineage determination of early embryogenesis. Homozygous inactivation of the β₁ integrin gene results in embryonal death in mice around the time of implantation. In vitro, differentiation of embryonic stem (ES) cells which lack β₁ integrin (β₁(- /-) into the cardiogenic lineage is delayed and results in a disordered cellular specification (Fassler et al., J. Cell Sci. 109, 9989-2999, 1996). To analyze β₁ integrin function during myogenesis and neurogenesis we studied differentiation of β₁(-/-) ES cells via embryoid bodies into skeletal muscle and neuronal cells in vitro. β₁(-/-) cells showed delayed and reduced myogenic differentiation compared to wildtype and heterozygous (β₁(+/-)) ES cells. RT-PCR analysis demonstrated delayed expression of skeletal muscle-specific genes in the absence of β₁ integrin. Immunofluorescence studies with antibodies against the sarcomeric proteins myosin heavy chain, titin, nebulin, and slow C-protein showed that myotubes formed, but their number was reduced and the assembly of sarcomeric structures was retarded. In contrast, neuronal cells differentiating from β₁(-/-) ES cells appeared earlier than wildtype and heterozygous (β₁(+/- )) ES cells. This was shown by the accelerated expression of neuron-specific genes and an increased number of neuronal cells in β₁(-/-) embryoid bodies. However, neuronal outgrowth was retarded in the absence of β₁ integrin. No functional difference between wildtype and β₁(-/-) cells was found with respect to secretion of γ-aminobutyric acid, the main neurotransmitter of ES cell-derived neuronal cells. The lineage-specific effects of loss of β₁ integrin function, that is the inhibition of mesodermal and acceleration of neuroectodermal differentiation, were supported by differential expression of genes encoding lineage-specific transcription factors (Brachyury, Pax-6, Mash1) and signaling molecules (BMP-4 and Wnt-1). Because of the reduced and delayed expression of the BMP-4 encoding gene in β₁(-/-) cells, we analyzed in wildtype and β₁(-/-) cells the regulatory role of exogenously added BMP- 4 on the expression of the mesodermal and neuronal marker genes, Brachyury and wnt-1, respectively. The data suggest that BMP-4 plays a regulatory role during differentiation of wildtype and β₁(-/-) cells by modifying mesodermal and neuronal pathways. The reduced expression of BMP-4 in β₁(- /-) cells may account for the accelerated neuronal differentiation in β₁(- /-) ES cells.