Impaired neuronal differentiation after Rbfox3-full knockdown. (A–J) Immunostaining of E4 neural tubes for Rbfox3 and neuronal markers. The right side of the neural tube in each panel has been transfected with siRNA indicated at the top and the GFP construct, as demonstrated by GFP in A and B. Sections were stained for proteins indicated on the left. Isl1⁺ cells indicated by open arrowheads in I are D2 interneurons. Bars, 50 µm. (K) Quantification of marker⁺ cells. Each bar represents the marker⁺ cell number in the transfected side divided by those in the contra-lateral untransfected side, except for Rbfox3 (mean ± SD, 5 embryos, 3 sections/embryo). In the case of Rbfox3, the fluorescence intensity is used instead of marker⁺ cell number. Isl1⁺ cells in the D2 domain are not included for quantification. *, P < 0.0001 compared with siCont (t test). (L) Diagram of the ventral neural tube specifying four motor subdomains. The red dashed lines indicate the borders of the subdomains. The mid-lines are halfway between the borders of the motor domains. TM, transfected medial motor domain; TL, transfected lateral motor domain; UM, untransfected medial motor domain; UL, untransfected lateral motor domain; MD, motor domain; pMN, motor neuron progenitor domain. (M–P) Differential effects of Rbfox3 knockdown on expression of differentiation markers and cellular distribution in different regions of the motor domain. The right side of the neural tube has been transfected with siRbfox3. Brackets in M and O indicate the areas with higher cell density compared with the untransfected side. Bars, 50 µm. (Q–T) Quantification of cell numbers with and without expression of motor neuron markers. Detailed statistical values are provided in Fig. S2 T. *, P < 0.006; **, P < 0.003; ***, P < 0.03 compared with corresponding untransfected subdomain. Black and white asterisks are for total and marker⁺ cell counts, respectively.