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First published online July 27, 2007
doi: 10.1242/10.1242/dev.02880

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FGF stimulation of the Erk1/2 signalling cascade triggers transition of pluripotent embryonic stem cells from self-renewal to lineage commitment

¹ Centre Development in Stem Cell Biology, Institute for Stem Cell Research, School of Biological Sciences, University of Edinburgh, Edinburgh, UK.
² Institut de Recherche en Immunologie et en Cancérologie, and Department of Pharmacology, Université de Montréal, Montréal, Québec, Canada.
³ Wellcome Trust Centre for Stem Cell Research, and Department of Biochemistry, University of Cambridge, Cambridge, UK.

View larger version (58K): [in this window] [in a new window]   Fig. 1. Fgf4-/- ES cells are deficient in neural induction. (A-E) Immunostaining (left; phase-contrast, right) for Oct4 and nestin (A,D) and Oct4 and TuJ1 (B,C,E) in wild-type (A,B), Fgf4+/- (C) and Fgf4-/- mouse ES cells on day 6 (A,D) and day 10 (B,C,E) of the monolayer neural differentiation protocol. (F) RT-PCR for neural markers Sox1 and nestin in Fgf4+/- and Fgf4-/- ES cells in self-renewing conditions on day 2 of the neural induction protocol. ß-actin was used as a loading control. (G) Quantitative RT-PCR for nestin on day 5 of the neural differentiation assay. TBP, TATA-binding protein. (H,I) Immunostaining for Oct4 and nestin (H) and Oct4 and TuJ1 (I) of Fgf4-/- ES cells cultured in FGF4 (5 ng/ml) for the first 24 hours of a 6 day (H) and 10 day (I) neural monolayer assay. Scale bar: 100 µm. (J) Cell counts of Oct4-positive, nestin-positive, and double-negative cells for wild-type, Fgf4-/- ES cells, and Fgf4-/- ES cells treated with FGF4 after 6 days culture in N2B27 (n=3 for each sample).

View larger version (76K): [in this window] [in a new window]   Fig. 2. Fgf4-/- and wild-type ES cells require FGFR signalling for multilineage commitment. (A-E) Immunostaining (below; phase-contrast, above) of Oct4 and TuJ1 in wild-type (E14Tg2a) mouse ES cells on day 7 of monolayer culture in N2B27 alone (A) or N2B27 with Bmp4 (B) and on Fgf4-/- ES cells in N2B27 alone (C), with BMP4 (D), or with BMP4 and FGF4 (E). Note that the green fluorescence in C and D is not specific and both immunopositivity and neuronal morphology are required to identify cells as neurons (Svendsen et al., 2001). (F,G) Immunostaining (right; phase-contrast, left) for Oct4 in a colony of E14Tg2a ES cells in the absence of LIF (F) or absence of LIF and presence of PD173074 (G). (H,I) Immunostaining for Oct4 and Sox2 in E14Tg2a ES cells cultured in BMP4 (H) or BMP4 and PD173074 (I). Scale bar: 100 µm. (J) RT-PCR for Id1 and Id3 after a 45-minute BMP4 stimulation. ß-actin was used as a loading control. (K,L) FACS analysis for Pdgfr expression of wild-type ES cells after 5 days on collagen IV plates in the absence (K) or presence (L) of the FGFR inhibitor PD173074. (M) RT-PCR analysis of wild-type cells after 4 days on collagen IV plates in the presence or absence of PD173074.

View larger version (59K): [in this window] [in a new window]   Fig. 3. Erk1/2 activity in ES cells is FGF dependent. (A-D) Immunostaining (left; phase-contrast, right) for phospho-Erk1/2 (pErk1/2) in mouse ES cells cultured in serum plus LIF for 14 hours in vehicle control (DMSO, A,C), inhibitor of MEK activation PD184352 (B), or FGFR inhibitor SU5402 (D). Each pair of images was taken at the same exposure time. Scale bar: 100 µm for A,B; 50 µm for C,D. (E) Immunoblot of Fgf4-/- and Fgf4+/- ES cell lysates cultured in serum-free N2B27 medium with or without LIF for 24 hours.

View larger version (41K): [in this window] [in a new window]   Fig. 4. Erk2-/- ES cells are severely deficient in neural and mesodermal commitment. (A) Immunoblotting for phospho-Erk1/2 (pErk1/2) and total Erk1/2 from serum-starved Erk2+/- and Erk2-/- mouse ES cells that were stimulated with foetal bovine serum (FBS), LIF, or unstimulated (-). (B-D) Immunostaining (left; phase-contrast, right) for nestin in Erk2+/- cells (A), and two independent Erk2-/- ES cell clones (B,C) fixed on day 6 of neural monolayer culture. Scale bar: 50 µm. (E) RT-PCR for Oct4, Fgf5, Rex1 and ß-actin (as loading control) on RNA isolated from two Erk2-/- ES cell lines (B1, B3) that were maintained in N2B27 medium without LIF. Parallel cultures were exposed to LIF for 2 days (+LIF) before isolation of RNA. (F) RT-PCR analysis of wild-type and two Erk2-/- (B1, B3) ES cell lines on day 4 of mesoderm monolayer differentiation. (G) FACS analysis for Pdgfr expression of wild-type and Erk2-/- ES cells on day 5 of mesoderm monolayer differentiation.