Research ArticleStem Cells

RhoA inhibits neural differentiation in murine stem cells through multiple mechanisms

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Science Signaling  26 Jul 2016:
Vol. 9, Issue 438, pp. ra76
DOI: 10.1126/scisignal.aaf0791

Halting neural differentiation with RhoA

Bone morphogenetic proteins (BMPs) are important for maintaining the pluripotency and self-renewal of embryonic stem cells (ESCs). Spontaneous differentiation of ESCs into neurons involves the production of Noggin, a secreted protein that inhibits BMP signaling. The GTPase RhoA maintains ESC pluripotency by preventing neural differentiation. Yang et al. found that the RhoA-specific guanine nucleotide exchange factor Syx prevented neural differentiation of murine ESCs (mESCs) by limiting both the abundance of Noggin and its secretion. Thus, regulation of secretion, as well as production of differentiation-promoting factors, provides multiple layers of control over stem cell fate.


Spontaneous neural differentiation of embryonic stem cells is induced by Noggin-mediated inhibition of bone morphogenetic protein 4 (BMP4) signaling. RhoA is a guanosine triphosphatase (GTPase) that regulates cytoskeletal dynamics and gene expression, both of which control stem cell fate. We found that disruption of Syx, a gene encoding a RhoA-specific guanine nucleotide exchange factor, accelerated retinoic acid–induced neural differentiation in murine embryonic stem cells aggregated into embryoid bodies. Cells from Syx+/+ and Syx−/− embryoid bodies had different abundances of proteins implicated in stem cell pluripotency. The differentiation-promoting proteins Noggin and RARγ (a retinoic acid receptor) were more abundant in cells of Syx−/− embryoid bodies, whereas the differentiation-suppressing proteins SIRT1 (a protein deacetylase) and the phosphorylated form of SMAD1 (the active form of this transcription factor) were more abundant in cells of Syx+/+ embryoid bodies. These differences were blocked by the overexpression of constitutively active RhoA, indicating that the abundance of these proteins was maintained, at least in part, by RhoA activity. The peripheral stress fibers in cells from Syx−/− embryoid bodies were thinner than those in Syx+/+ cells. Furthermore, less Noggin and fewer vesicles containing Rab3d, a GTPase that mediates Noggin trafficking, were detected in cells from Syx−/− embryoid bodies, which could result from increased Noggin exocytosis. These results suggested that, in addition to inhibiting Noggin transcription, RhoA activity in wild-type murine embryonic stem cells also prevented neural differentiation by limiting Noggin secretion.

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