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Mouse embryonic stem cells with aberrant transforming growth factor beta signalling exhibit impaired differentiation
M J Goumans1, D Ward-van Oostwaard, F Wianny
1Hubrecht Laboratory, Netherlands Institute for Developmental Biology, Utrecht, The Netherlands.
Abstract:
Embryonic stem (ES) cells are resistant to transforming growth factor beta (TGF beta). We have shown previously that they lack type-II binding receptors (T beta RII) and in this respect resemble the inner cell mass and ectoderm cells of mouse embryos 4.5-7.5 days post coitum (dpc); they do however express type-I (alk-5) signalling receptors. Here we show that in contrast to several tumour cell lines, stable transfection of wtT beta RII is not sufficient for ES cells to become biologically sensitive to TGF beta. We analysed the expression of several down-stream molecules known to be involved in TGF beta signalling (Smads) and TGF beta-mediated cell cycle regulation (cyclins D) during the differentiation of control and wtT beta RII-expressing ES cells and showed that upregulation of these molecules correlated with (i) an increase in plasminogen activator inhibitor-1 (PAI-1) synthesis and (ii) growth inhibition, following addition of TGF beta 1. These TGF beta responses were reduced in an ES cell line expressing a dominant negative (truncated) T beta RII (delta T beta RII). The differentiation pattern of control and wtT beta RII-expressing ES cells was indistinguishable in monolayer culture and as embryoid bodies, but in delta T beta RII ES cells, the capacity to form mesodermal derivatives in monolayer cultures in response to the addition of retinoic acid (RA) and removal of leukemia inhibitory factor (LIF) was lost, and only endoderm-like cells formed. The T beta RII and delta T beta RII ES cells were, however, both distinguishable from control ES cells when allowed to differentiate in chimaeric embryos following aggregation with morula-stage hosts. Conceptuses containing mutant cells, recovered from pseudopregnant females at the equivalent of 9.5 dpc, exhibited highly defective yolk sac development; most strikingly, no blood vessels were present and in addition the yolk sacs with derivatives of ES cells containing wtT beta RII were blistered and lacked haematopoietic cells. The implications for understanding TGF beta signalling in early mouse development are discussed.
Insights
Embryonic stem cells resist transforming growth factor beta (TGF-β) due to lacking specific receptors. Introducing these receptors doesn't restore TGF-β sensitivity, impacting early mouse development and yolk sac formation.
Area of Science:
- Developmental Biology
- Stem Cell Biology
- Molecular Signaling
Background:
- Embryonic stem (ES) cells are resistant to transforming growth factor beta (TGF-β).
- ES cells lack TGF-β type-II binding receptors (TβRII), similar to early mouse embryonic cells.
- ES cells express TGF-β type-I (alk-5) signaling receptors.
Purpose of the Study:
- To investigate the biological sensitivity of ES cells to TGF-β upon stable transfection of wild-type TβRII (wtTβRII).
- To analyze the role of TβRII in TGF-β-mediated signaling pathways and differentiation.
- To understand the implications of TβRII expression on early mouse development, particularly yolk sac formation.
Main Methods:
- Stable transfection of ES cells with wtTβRII and dominant-negative TβRII (ΔTβRII).
- Analysis of downstream TGF-β signaling molecules (Smads, cyclins D, PAI-1) and cell cycle regulation.
- Differentiation assays in monolayer culture, as embryoid bodies, and in chimeric embryos.
Main Results:
- Stable transfection of wtTβRII did not confer TGF-β sensitivity to ES cells.
- TGF-β responses (Smad and cyclin D upregulation, PAI-1 synthesis, growth inhibition) were reduced in ΔTβRII cells.
- ΔTβRII ES cells lost the capacity to form mesodermal derivatives and showed defective yolk sac development in chimeric embryos, lacking blood vessels and hematopoietic cells.
Conclusions:
- TβRII expression alone is insufficient to restore TGF-β biological sensitivity in ES cells.
- TβRII plays a crucial role in TGF-β-mediated differentiation and development, particularly in mesodermal and yolk sac formation.
- Defects in TβRII signaling significantly impair early mouse embryonic development, highlighting its importance in vascularization and hematopoiesis.