Loss of beta1 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 beta1 integrin gene results in embryonal death in mice around the time of implantation. In vitro, differentiation of embryonic stem (ES) cells which lack beta1 integrin (beta1-/-) into the cardiogenic lineage is delayed and results in a disordered cellular specification (Fässler et al., J. Cell Sci. 109, 2989-2999, 1996). To analyze beta1 integrin function during myogenesis and neurogenesis we studied differentiation of beta1-/- ES cells via embryoid bodies into skeletal muscle and neuronal cells in vitro. beta1-/- cells showed delayed and reduced myogenic differentiation compared to wildtype and heterozygous (beta1+/-) ES cells. RT-PCR analysis demonstrated delayed expression of skeletal muscle-specific genes in the absence of beta1 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 beta1-/- ES cells appeared earlier than wildtype and heterozygous (beta1+/-) ES cells. This was shown by the accelerated expression of neuron-specific genes and an increased number of neuronal cells in beta1-/- embryoid bodies. However, neuronal outgrowth was retarded in the absence of beta1 integrin. No functional difference between wildtype and beta1-/- cells was found with respect to secretion of gamma-aminobutyric acid, the main neurotransmitter of ES cell-derived neuronal cells. The lineage-specific effects of loss of beta1 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 beta1-/- cells, we analyzed in wildtype and beta1-/- 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 beta1-/- cells by modifying mesodermal and neuronal pathways. The reduced expression of BMP-4 in beta1-/- cells may account for the accelerated neuronal differentiation in beta1-/- ES cells.

Rohwedel J ，Guan K ，Zuschratter W ，Jin S ，Ahnert-Hilger G ，Fürst D ，Fässler R ，Wobus AM ... -  《DEVELOPMENTAL BIOLOGY》

Defects in adhesion and migration, but not in proliferation and differentiation, of embryonic stem cells upon replacement of integrin subunit beta1A by beta1D.

:Beta1D is a skeletal muscle-specific splice variant of the beta1 integrin subunit, while beta1A integrin subunit has a wide tissue distribution. We have previously shown that replacement of beta1A by beta1D 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 beta1D instead of beta1A, and analyzed the ability of beta1D to support ES cell differentiation in vitro and in teratomas in vivo. Beta1D knockin (KI) ES cells grew at a similar rate but as more compact colonies than the beta1A-expressing cells. Increased cell cohesiveness, however, did not appear to involve changes in cadherin activity. Although in both beta1A and beta1D-KI ES cells only one beta1 allele is active; the expression of beta1 integrins in the beta1D-KI ES cells was reduced by 50%, compared with that in the beta1A-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 alpha- and beta-myosin heavy chains, contracting cardiomyocytes were detected in similar numbers and at the same time in embryoid bodies (EB) derived from beta1D-KI and from beta1A cells. Furthermore, replacement of beta1A by beta1D in ES cells did not affect neurite differentiation in embryoid bodies in the presence of retinoic acid suggesting that beta1D supports neurogenesis. However, the impaired migration of other cells from the EB, including endodermal cells, prevented the normal outgrowth of neurites in beta1D-KI EB. Finally, injection of beta1D-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 beta1D, in spite of its specific cytoplasmic domain, supports the differentiation of many cell types. This further suggests that the embryonic lethality in the beta1D-KI embryos was mainly due to the different ability of beta1 A and beta1D to mediate cell adhesion and migration.

Gimond C ，Baudoin C ，Sonnenberg A 《DIFFERENTIATION》

Loss of beta 1 integrin function results in upregulation of connexin expression in embryonic stem cell-derived cardiomyocytes.

Czyz J ，Guan K ，Zeng Q ，Wobus AM ... -  《INTERNATIONAL JOURNAL OF DEVELOPMENTAL BIOLOGY》

Bone morphogenetic protein regulation of forkhead/winged helix transcription factor Foxc2 (Mfh1) in a murine mesodermal cell line C1 and in skeletal precursor cells.

Nifuji A ，Miura N ，Kato N ，Kellermann O ，Noda M ... -  《JOURNAL OF BONE AND MINERAL RESEARCH》

Lithium influences differentiation and tissue-specific gene expression of mouse embryonic stem (ES) cells in vitro.

Schmidt MM ，Guan K ，Wobus AM 《INTERNATIONAL JOURNAL OF DEVELOPMENTAL BIOLOGY》