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First published online 11 February 2004
doi: 10.1242/dev.01027

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Cooperation between sonic hedgehog and fibroblast growth factor/MAPK signalling pathways in neocortical precursors

Nicoletta Kessaris^1,*, Françoise Jamen^1,*, Lee L. Rubin² and William D. Richardson^1,

¹ Wolfson Institute for Biomedical Research and Department of Biology, University College London, Gower Street, London WC1E 6BT, UK
² Curis, 61 Moulton Street, Cambridge, MA 02138, USA

View larger version (42K): [in a new window]   Fig. 1. FGF2 induces OLPs through FGFR1. (A) Dissociated cells from mouse E13.5 neocortex (boxed region) were cultured in defined medium in the presence or absence of different FGFs for 3 days in vitro (DIV). (B-D) The cultures were fixed and immunolabelled with polyclonal anti-NG2. Control cultures lacked NG2 immunoreactivity (B). Numerous NG2-positive cells developed in 10 ng/ml FGF2 (C) and 50 ng/ml FGF2 (D). (E) The numbers of OLPs induced by FGF8, 9 or 10 were at least 100x lower than with FGF2, suggesting that the inducing activity is mediated via FGFR1. NG2-positive cells were counted in more than 10 randomly selected fields on each of two coverslips (x63 microscope objective). At least two independent experiments gave similar results, one of which is illustrated. (F) Dorsal spinal cord explants from chick Hamilton-Hamburger stage12 (E2) developed O4-positive OLPs when cultured in the presence of FGF2, even in the presence of cyclopamine (FGF2 + cyc). The number of O4-positive explants and the total number of explants are shown above each bar.

View larger version (83K): [in a new window]   Fig. 2. Induction of OLPs by the Hedgehog agonist SHHAg1.2.Mouse E13.5 neocortical neuroepithelial cells were cultured in the presence or absence of SHHAg for 4 DIV. The cultures were fixed and immunolabelled with polyclonal anti-NG2. (A) Control cultures lacked NG2 immunoreactivity. (B) Numerous NG2-positive cells developed in the presence of 100 nM SHHAg1.2. (C) The dose-response curve shows induction of NG2-positive cells at a half-maximal concentration of SHHAg1.2 of 25 nM.

View larger version (67K): [in a new window]   Fig. 3. Rapid induction of OLIG2 by SHHAg or FGF2. Neocortical precursors from E13.5 mice were cultured in the presence or absence of FGF2 or SHHAg and assayed for OLIG2 immunoreactivity at different times. OLIG2-positive nuclei appeared within the first 20 hours in both FGF2-treated (C,F) and SHHAg-treated (B,E) cultures. The experiment was quantified (G) by counting OLIG2-positive cells in more than 10 randomly selected fields on each of two separate coverslips (x63 microscope objective) and are displayed as mean±s.d.

View larger version (17K): [in a new window]   Fig. 4. OLP induction by SHH requires FGFR. (A) E13.5 mouse neocortical cells were cultured for 2 DIV in the presence or absence of FGF2 or SHHAg, with or without the FGFR inhibitor PD173074 or the Hedgehog inhibitor cyclopamine. The cultures were assayed for OLIG2 immunoreactivity at DIV2 or NG2 immunoreactivity at DIV4. Induction of both OLIG2-positive and NG2-positive cells by SHH was inhibited by PD173074. The inducing activity of FGF2 was unaffected by cyclopamine. (B) Cortical cells from mouse E13.5 embryos were cultured in the presence or absence of SHHAg or FGF2, PD173074, a combination of FGFR1IIIc and FGFR1ßIIIc extracellular domains (sFGFR1) or cyclopamine. The inducing effect of FGF2 was inhibited by both PD173074 and sFGFR1 but not by cyclopamine. The effect of SHH was inhibited by PD173074 but not by sFGFR1, suggesting that SHHAg activity requires ligand-independent activation of FGFR. (C) Dose-response curve showing inhibition of OLP induction by SHHAg in the presence of increasing concentrations of PD173074 at DIV4. Half-maximal inhibition occurs at 25 nM PD173074, as described for inhibition of FGFR1 itself (Dimitroff et al., 1999).

View larger version (44K): [in a new window]   Fig. 5. OLP induction by SHH depends on activation of the MAPK pathway by FGFR1. (A) Neocortical neuroepithelial cells cultured for 24 hours in the presence of the MEK1/2 inhibitor U0126 and either SHHAg or FGF2 fail to develop Olig2-positive cells. The inhibitor of PI 3-kinase, LY294002, has no effect on the inducing activities of either SHHAg or FGF2. (B) To assess whether FGF2 and/or SHH activate the MAPK pathway we cultured E13.5 cortical cells in the absence (a-f) or presence of either SHHAg (g-j) or FGF2 (k-n), together with PD173074 (c,i) or cyclopamine (e,m) for 1 hour prior to immunolabelling with an anti-phospho-ERK1/2 antibody and Hoechst dye (b,d,f,h,j,l,n). FGF2 by itself caused strong activation of MAPK. SHH failed to activate MAPK above endogenous levels (compare a, g) and all MAPK activity was abolished by PD173074 (c,i). (C) Protein lysates from cortical cultures incubated with FGF2 or SHHAg and PD173074 or cyclopamine for 1 hour or 18 hours were separated by PAGE, and analysed for the presence of phosphorylated ERK1/2 (p42/p44) by western blot. SHHAg failed to activate MAPK above control levels, and PD173074 abolished all MAPK activity even in the presence of SHH.

View larger version (24K): [in a new window]   Fig. 6. Cell-autonomous activation of the MAPK pathway is required for OLIG2 induction by SHH. (A) Cultured neuroepithelial cells from E13.5 mouse embryos were infected with a control retrovirus (pBird) encoding GFP alone (a,b,c,d) or a retrovirus encoding GFP and a constitutively active form of RAS (pBird-RasV12) (e,f,g,h). Cells were then cultured for 48 hours in the presence of SHHAg. At DIV3, the cultures were assayed for OLIG2 immunoreactivity (c,g) and labelled with Hoechst dye (a,e). In control virus-infected cultures (a,b,c,d) there were no OLIG2-positive cells. However, in activated RAS virus-infected cultures, SHHAg induced OLIG2 expression in a subset of GFP-positive cells (e,f,g,h). (B) The number of OLIG2- and GFP-positive cells was counted and is presented as the percentage of all GFP-positive cells. In RAS virus-infected cultures in the presence of SHHAg, 17.0±1.3% of GFP-positive cells expressed OLIG2. No OLIG2-positive cells were induced by either in the absence of active MAPK (added U0126), or in the absence of SHHAg (added cyclopamine). In all cultures PD173074 was added to block MAPK activation via FGFR.

View larger version (10K): [in a new window]   Fig. 7. The progression of cells from being OLIG2-positive to NG2-positive requires both MAPK and PI 3-kinase activation. Neocortical cells were initially cultured in the presence or absence of SHHAg or FGF2 for 48 hours until OLIG2-positive cells appeared. The inducers were then removed and the medium replaced with defined medium containing U0126 or LY294002. The appearance of NG2-positive cells in the cultures was inhibited by both drugs.

View larger version (35K): [in a new window]   Fig. 8. Induction of OLPs in ventral spinal cord or ventral forebrain cultures is independent of FGFR-TK activity. (A) Ventral spinal cord explants from Hamilton and Hamburger stage 12 (E2) chicks developed O4-positive OLPs when cultured without exogenously added growth factors. Their development was strongly inhibited by cyclopamine but only weakly by PD173074. FGF2 in the absence of complementary Shh activity (i.e. in the presence of cyclopamine) was unable to induce O4-positive OLPs. (B-E) Dissociated cells from mouse E10.5 ventral forebrain (boxed region) were cultured in the presence of the cyclopamine or PD173074 for 10 days, then the cultures were fixed and immunolabelled with anti-NG2. Control cultures developed numerous NG2-positive cells (C). Their production was inhibited by cyclopamine (D) but not by PD173074 (E). (F) The experiment was quantified by counting NG2-positive cells in more than 10 randomly selected fields on each of two separate coverslips (x63 objective) and the results displayed as mean±s.d. Similar data were obtained in at least two independent experiments.