Fig. 6

LFP induction requires FGFR activation. a Expression pattern of shh mRNA on transverse section of E4 brachial spinal cord showing shh expression restricted to MFP cells at this stage. b-d Time course of shh expression in spinal cord explants dissected at E4–4.5 and cultivated for 9 h (b), 1 day (c) or 2 days (d). e-f Immunodetection of Nkx2.2 on transverse sections of explants cultivated for 1 day (e) or 2 days (f). Note that dorsal extension of shh expression overlaps the Nkx2.2-positive domain to form the LFP at day 1 (compare c and e) while dorsal extension of the Nkx2.2 domain to form the p* domain is detected only at day 2 (f). g-h Time course of shh expression through spinal cord explants cultivated in presence of SU5402 for 1 day (g) or 2 days (h). Note that dorsal extension of shh expression to form the LFP fails to occur in presence of the FGFR inhibitor. i-j Expression of shh in spinal cord explants cultivated for 2 days and treated for a time period of 24 h by SU5402, either during the first (i) or the second day in culture (j). Note that LFP fails to form even when the treatment is interrupted after the first day in culture (i), whereas it forms readily when the inhibitor is applied only during the second day of culture (j). k-n Transverse section of control explants (k, m) or explants treated with cyclopamine for 2 days (l, n) and immunolabeled for Olig2 and Nkx2.2 (k, l) or processed for shh mRNA detection (m, n). Note that cyclopamine treatment inhibits Olig2 expression and prevents dorsal extension of the Nkx2.2 domain (brackets, l), whereas the treatment has no effect on dorsal extension of shh mRNA expression (n). o Relative location of shh mRNA-expressing cells (purple in o, o') and dnFGFR electroporated cells (green) on transverse sections of spinal cord explant cultivated for 2 days. Note inhibition of shh expression in the ventral-most dnFGFR-overexpressing cells (bracket in o’). p-q: Double immunodetection of Olig2 (red) and Nkx2.2 (blue) in explants electroporated with the control vector (green in p) or with the dnFGFR vector (green in q). Note that limiting overexpression of dnFGFR to the Nkx2.2-positive domain (q) is sufficient to prevent generation of Olig2/Nkx2.2-coexpressing OPCs. r Quantification of Olig2/Nkx2.2-positive cells in the mantle zone (MZ) of non-electroporated sides of explants and sides of explants electroporated with the control (n = 4) or dnFGFR (n = 5) vectors. Results are presented as mean number of cells ± sem (**p ≤ 0.01). hiv = hour in vitro, div = day in vitro. Scale bars = 50 μm