Figure 9. Neuronal differentiation via forced Neurog1 expression is affected by FGF signaling.

A. Treatment groups. N7 cells were plated in defined medium (time = 0h) and exposed to a 12h pulse of 5 nM SU5402. One group (pre-induction) was treated with SU5402 at the time of plating, after which the medium was exchanged for fresh medium supplemented with 1 µg/ml doxycycline (+Dox) for the remainder of the culture period. The second group (simultaneous) was plated in defined medium for 12h followed by addition of SU5402 and Dox. At 24h, the medium was exchanged for fresh medium supplemented with Dox for the remainder of the culture period. The final group (post-induction) was plated in defined medium for 12h followed by addition of Dox. At 24h, a 12-hour pulse of SU5402 was administered, and, at 36h, the medium was exchanged for fresh medium supplemented with Dox for the remainder of the culture period.

B. Oct4/Sox3/TuJ1 cell counts. There was a decrease in the number of Sox3+ cells between the pre-induction and simultaneous treatment groups that was not significant (**, p = 0.058), while the decrease in the number of Sox3⁺ cells in the post-dox group was slight but significant compared with the other treatment groups. There was a striking increase in TuJ1 expression with delayed SU5402 treatment and a corresponding decrease in the number of Oct4+ cells, while the number of Sox3+ cells observed dropped only slightly with delayed treatment. Untreated controls demonstrated markedly increased neuronal differentiation, a concomitant reduction in neuronal precursor cells, with very few cells expressing Oct4. At least 1000 cells from 10 randomly selected fields in two replicate dishes were scored for Oct4, Sox3, and TuJ1, and analyzed by pairwise Student’s t-test.

C. Oct4/Sox3/TuJ1 immunohistochemistry. The cell counts described above were obtained from cell cultures that were fixed after 72h and co-stained with TuJ1 and Oct4 antibodies (left panels) or TuJ1 and Sox3 antibodies (right panels). Nuclei are labeled with Hoechst 33258. Blocking FGF signaling resulted in fewer neurons (TuJ1⁺) in a manner depending on the timing of treatment. Control cells receiving no SU5402 show robust neuronal differentiation.