LOSS OF BETA(1) 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 adhesi on, migration, and differentiation during embryonic development by med iating cell-cell and cela-matrix interactions. Less is known about the function of integrins during commitment and lineage determination of early embryogenesis. Homozygous inactivation of the beta(1) integrin g ene results in embryonal death in mice around the time of implantation . In vitro, differentiation of embryonic stem (ES) cells which lack be ta(1) integrin(beta(1)(-/-)) into the cardiogenic Lineage is delayed a nd results in a disordered cellular specification (Fassler et al., J. Cell Sci. 109, 2989-2999, 1996). To analyze beta(1) integrin function during myogenesis and neurogenesis we studied differentiation of beta( 1)(-/-) ES cells via embryoid bodies into skeletal muscle and neuronal cells in vitro. beta(1)(-/-) cells showed delayed and reduced myogeni c differentiation compared to wildtype and heterozygous (beta(1)(+/-)) ES cells. RT-PCR analysis demonstrated delayed expression of skeletal muscle-specific genes in the absence of beta(1) integrin. Immunofluor escence 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 st ructures was retarded. In contrast, neuronal cells differentiating fro m beta(1)(-/-) ES cells appeared earlier than wildtype and heterozygou s (beta(1)(+/-)) ES cells. This was shown by the accelerated expressio n of neuron-specific genes and an increased number of neuronal cells i n beta(1)(-/-) embryoid bodies. However, neuronal outgrowth was retard ed in the absence of beta(1) integrin. No functional difference betwee n wildtype and beta(1)(-/-) cells was found with respect to secretion of gamma-aminobutyric acid, the main neurotransmitter of ES cell-deriv ed neuronal cells. The lineage-specific effects of loss of beta(1) int egrin function, that is the inhibition of mesodermal and acceleration of neuroectodermal differentiation, were supported by differential exp ression of genes encoding lineage-specific transcription factors (Brac hyury, Pax-6,;Mash1) and signaling molecules (BMP-4 and Wnt-1). Becaus e of the reduced and delayed expression of the BMP-4 encoding gene in beta(1)(-/-) cells, we analyzed in wildtype and beta(1)(-/-) cells the regulatory role of exogenously added BMP-4 on the expression of the m esodermal and neuronal marker genes, Brachyury and wnt-1, respectively . The data suggest that BMP-4 plays a regulatory role during different iation of wildtype and beta(1)(-/-) cells by modifying mesodermal and neuronal pathways. The reduced expression of BMP-4 in beta(1)(-/-) cel ls may account for the accelerated neuronal differentiation in beta(1) (-/-) ES cells. (C) 1998 Academic Press.