Chicken Rbfox3 isoforms and their expression in chick neural tube. (A) Diagrams of two alternatively spliced isoforms of Rbfox3. The dashed line indicates the deletion of 31 aa. Numbers represent aa. Two critical elements, RNP1 and RNP2, of the RRM for RNA binding are indicated. (B) Developmentally regulated expression of splice variants of Rbfox3 mRNAs. mRNAs isolated from the embryos at the indicated embryonic days were analyzed by RT-PCR using primers P1 and P2 indicated in A. An ethidium bromide stained agarose gel is shown. M, size marker. (C) Developmentally regulated expression of Rbfox3 proteins. Embryonic extracts harvested at the indicated embryonic days were analyzed by immunoblotting for the indicated proteins. The top and bottom (indicated by an arrowhead) bands of Rbfox3 (top) presumably represent Rbfox3-full and Rbfox3-d31, respectively. (D–G) Expression of Rbfox3 protein in the developing chick neural tube. The transverse sections of the neural tube at the indicated embryonic days were stained with anti-Rbfox3 (red) and DAPI (cyan). Bars, 50 µm. (H–L) Restricted expression of Rbfox3 in late postmitotic neurons. Transverse sections of the E4 neural tube were stained for proteins indicated at the top. Brackets in I, J, and L indicate the areas where double-negative cells exist. Arrowheads in K indicate double-positive cells. Bars, 20 µm. (M) A schematic diagram to illustrate the time of the onset of expression and the localization of Rbfox3 and the marker proteins in the ventral neural tube at E4. A vertical solid line and a dashed line indicate the ventricular surface and the time point of cell cycle exit, respectively. The gradient gray scales represent the relative expression levels.