Figure 6.

Nnat-mediated Ca²⁺ signaling increases p-Erk1/2 and cross talks with FGF/Erk pathway in neural induction. (A): The inhibitory effect of PD173074 (a FGF-R blocker) and PD184352 (a p-Erk1/2 blocker) on neural induction. Without the presence of blockers, Nnat-KD ESCs failed to initiate neural induction (fail to generate Sox1-eGFP⁺/Nes⁺ [red] NSCs), which was rescued by Tg treatment (Nnat-KD+Tg). In the presence of the blockers (+PD173074 or +PD184352), neural induction in control and Nnat-KD+Tg ESCs was abolished, whereas PD173074 partially and PD184352 completely inhibited neural induction in Nnatα-OE ESCs. (B): The rescue effects of FGF4 or FGF5 on Nnat-KD phenotype in the production of Sox1-eGFP⁺/Nes⁺ (red) NSCs and NeuN⁺ (red) neurons. (C): Quantification of NSC and neuron population generated from control, Nnat-KD, and Nnat-KD ESCs treated with FGF4 (Nnat-KD+FGF4) or FGF5 (Nnat-KD+FGF5) using fluorescence-activated cell sorting analysis. Data shown are the mean ± SD (n = 3). *, p < .05 and **, p < .01, significantly different from the control group; ^#, p < .05, significantly different from the Nnat-KD group, two-tailed Student's t test. (D): The FGF4 and FGF5 rescue of neural induction in Nnat-KD ESCs were abrogated by the presence of PD173074 or PD184352. (E): Increase in the phosphorylation of Erk1/2 after 5 minutes treated with various concentrations of Tg (top panel), the time points of the increment with 25 nM Tg treatment (middle panel), and the effect of PD173074 on Tg-induced p-Erk1/2 (bottom panel). Scale bar = 50 μm and all nuclei were stained with DAPI (blue). Abbreviations: DAPI, 4′,6′-diamidino-2-phenylindole; eGFP, enhanced green fluorescent protein; FGF4, fibroblast growth factor 4; FGF5, Fibroblast growth factor 5; GFP, green fluorescent protein; Nnatα-OE, neuronatin α-overexpression; Nnat-KD, neuronatin-knockdown; NSC, neural stem cell; Tg, thapsigargin; w/o, without.