TRANSFORMING GROWTH-FACTOR-BETA IN THE EARLY MOUSE EMBRYO - IMPLICATIONS FOR THE REGULATION OF MUSCLE FORMATION AND IMPLANTATION

In a search for functions of transforming growth factor-beta during ea rly embryonic development we used two different experimental approache s. In the first we made use of embryonic stem (ES) cells. ES cells in culture differentiate to derivatives of all three germ layers and mimi c some aspects of organogenesis when grown as aggregates in suspension to form embryoid bodies. Differentiation procedes further when the em bryoid bodies attach to suitable substrates. Muscle and neuronal cells are among the most readily identified cell types then formed. We exam ined the effect of all-trans retinoic acid (RA) and members of the tra nsforming growth factor-beta family (TGF-beta1, TGF-beta2) under these conditions in an assay where single aggregates formed in hanging micr odrops in medium supplemented with serum depleted of lipophilic substa nces which would include retinoids. Endoderm-like cells formed under a ll conditions tested. RA at concentrations of 10(-8) M and 10(-7) M in duced the formation of neurons but in the absence of RA or at concentr ations up to 10(-9) M, neurons were not observed. Instead, beating mus cle formed in about one-third of the plated aggregates; this was great ly reduced when RA concentrations increased above 10(-9) M. Immunofluo rescent staining for muscle specific myosin showed that two muscle cel l types could be distinguished: elongated, non-contractile myoblasts a nd mononucleate flat cells. The mononucleate flat cells appeared to co rrespond with rhythmically contracting muscle. The number of non-contr actile myoblasts increased 3-fold over controls in the presence of 10( -9) M RA. TGF-betas increased the number of contractile and non-contra ctile muscle cells by a factor 3 to 7 over controls, depending on the TGF-beta isoform added and the muscle cell type formed. TGF-beta2 also invariably increased the rate at which contracting muscle cells were first observed in replated aggregates. The stimulatory effect of TGF-b etas on the formation of mononucleate flat cells was completely abroga ted by RA at 10(-9) M while the number of myoblasts under similar cond itions was unchanged. These data suggest that a complex interplay betw een retinoids and TGF-beta isoforms may be involved in regulation of d ifferentiation in early myogenesis. In the second approach, neutralizi ng polyclonal rabbit antibodies specific for TGF-beta2 were injected i nto the cavity of mouse blastocysts 3.5 days post coitum (pc). After 1 day in culture, embryos were transferred to pseudopregnant females. T he number of decidua, embryos and resorptions were counted at day 8.5- 9.5 pc. Control antibody injected embryos implanted with high efficien cy (87%) compared with anti-TGF-beta2 injected embryos which implanted with an efficiency of only 43%. If empty decidua (resorptions) were i ncluded, the overall recovery was 71% and 32% for control and experime ntal embryos, respectively. Embryos that were recovered showed no over t macroscopic abnormalities. These results together imply functions fo r TGF-betas in implantation as well as in later development of the emb ryo.