August 1, 2013;
MRAS GTPase is a novel stemness marker that impacts mouse embryonic stem cell plasticity and Xenopus embryonic cell fate.
Pluripotent mouse embryonic stem cells (mESCs), maintained in the presence of the leukemia inhibitory factor (LIF
) cytokine, provide a powerful model with which to study pluripotency and differentiation programs. Extensive microarray studies on cultured cells have led to the identification of three LIF
signatures. Here we focus on muscle
ras oncogene homolog (MRAS
), which is a small GTPase of the Ras family encoded within the Pluri gene cluster. To characterise the effects of Mras
on cell pluripotency and differentiation, we used gain- and loss-of-function strategies in mESCs and in the Xenopus laevis embryo
, in which Mras
gene structure and protein sequence are conserved. We show that persistent knockdown of Mras
in mESCs reduces expression of specific master genes and that MRAS
plays a crucial role in the downregulation of OCT4
and NANOG protein levels upon differentiation. In Xenopus, we demonstrate the potential of Mras
to modulate cell fate at early steps of development and during neurogenesis. Overexpression of Mras
cells to retain responsiveness to fibroblast
growth factor (FGF) and activin. Collectively, these results highlight novel conserved and pleiotropic effects of MRAS
in stem cells and early steps of development.
[+] show captions
Fig. 6. Mras expression is biphasic during Xenopus laevis development. RT-PCR analysis of Mras expression. (A) Egg and embryonic stages (st). (B) Stage 9 or 10.5 dissected embryos. AP, animal part; VP, ventral part; DM, dorsal mesoderm; VM, ventral mesoderm; EN, endoderm. An1, Vg1, Chordin (Chd), Wnt8 and Sox17 are used as controls. Total embryo (E) was analysed in parallel. (C) Adult tissues. Sk.m, skeletal muscle. Ornithine decarboxylase (Odc) and Ribosome protein like 8 (Rpl8) were used as controls, and a reaction was performed in the absence of reverse transcriptase (-RT).
Fig. 7. Mras is expressed in Rohon-Beard cells and trigeminal neurons in the Xenopus embryo. (A-H) In situ hybridisation with Mras antisense probe (A,B,E,F) or combined with immunostaining with the muscle-specific antibody 12/101 (C,D,G,H) on stage 24 (A-D) or stage 33 (E-H) embryos. Lateral (A,C,E,G) and dorsal (B,F) views. The plane of the transverse sections shown in D and H are indicated in C and G, respectively. Arrowheads indicate Mras expression in Rohon-Beard cells. Ep, epiphysis; No, notochord; Nt, neural tube; RB, Rohon-Beard cells; S, somites; Tn, trigeminal neurons.
Fig. 8. Inhibition of Mras blocks neuronal differentiation. MO Mras, MO mismatch or MO standard were injected into one blastomere of 2-cell stage Xenopus embryos and morphants were analysed by in situ hybridisation for N-tubulin, Elrc, Runx1, Pak3 and Islet1 expression. In rescue experiments, 500 pg Mras mRNA was co-injected with MO Mras (MO Mras + Mras). The injected side is on the right. Motor (m) inter- (i) and sensory (s) primary neurons and the trigeminal placode (tg) are shown (arrowhead). Arrows indicate change in expression induced by MO Mras. Dorsal views, anterior to bottom.
Fig. 9. Mras acts at a late step of neurogenesis and is required for neuronal differentiation. MO Mras, MO standard, or 500 pg mRNA encoding a constitutively active form (G22V) or a dominant-negative form (S27N) of MRAS was injected into one blastomere of 2-cell stage Xenopus embryos. Embryos were analysed by in situ hybridisation for Sox2, Neurogenin or N-tubulin expression. The injected side is on the right. Arrows indicate change in expression. Dorsal views, anterior to bottom.
Fig. 10. Mras can maintain the competence of Xenopus gastrula embryo cells for FGF and activin induction but is not required for mesoderm induction. RT-PCR analysis of gene expression in Xenopus animal caps cultured until the control embryo (lane E) reached stage 12.5. (A) Activin, BMP or FGF2 treatment followed by Mras and Xbra expression analysis. (B) Animal caps from Mras morphants (MO) or control embryos treated with 50 or 100 ng/ml FGF2 (FGF50 or FGF100) and analysed for Xbra expression. (C,D) Animal caps dissected at stage 8 (Early) or stage 11 (Late) from embryos injected with Mras or Brg1 mRNAs or uninjected embryo (Control) were treated with FGF2 (F) or activin (A) or left untreated (-) before expression analysis of Xbra, Cdx4, Pnp, Esr5, Xnr, Chordin (Chd) or Sox17 (Sox). Odc was used as control, and a reaction was performed in the absence of reverse transcriptase (-RT).
Fig. S5. Inhibition of Mras mRNA translation by morpholinos. Mras mRNA was translated in vitro in the presence of [35S]methionine and 40 ng
(+) or 400 ng (++) morpholinos directed against Mras (Mras MO) or 400 ng of a standard control (Std) or mismatch (Mis) morpholino. Translation
products were analysed by SDS-PAGE followed by autoradiography. Lane c contains a blank translation (no mRNA). MRAS is 24 kDa.
Fig. S6. Mras stimulates neuronal differentiation of pluripotent animal cap cells. Two-cell stage Xenopus embryos were injected with 500 pg
Mras mRNA with (+) or without (�) 50 pg noggin mRNA and animal caps were dissected at stage 8 (Early) or stage 11 (Late) and analysed by RTPCR
when the control embryo (E) had reached stage 23 for N-tubulin (N-tub) and N-cam expression. Odc was used as loading control.