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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.
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