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Fig. 2.
Complementary patterns of NOS expression and BrdU incorporation in the developing Xenopus brain. Whole-mount brains from stage 43 (a–c, g) and 47 (d–f, h) tadpoles are shown labeled for BrdU (a, d, g, h) after 2 hr of survival and by in situ hybridization for XNOS (b, c, e–h). Dorsal views of brains (a, b, d, e) from different animals are aligned to show the relative position of BrdU- and XNOS-positive cells in brains of comparable stages. XNOS-positive cells are located in the telencephalon, midbrain, and hindbrain, neighboring but not overlapping sites of BrdU incorporation. Sagittal views of whole-mount brains (c, f) show the dorsoventral distribution of XNOS-positive cells. Two clusters of XNOS-positive cells are located in the caudal midbrain. One cluster lies in the caudal optic tectum on the roof of the midbrain near the tectal proliferative zone, and a second cluster is in the ventral midbrain. Whole-mount preparations, viewed sagitally (g, h), and horizontal sections (i–k) of the optic tectum were double-labeled for BrdU and XNOS expression by in situ hybridization. The dorsal cluster of XNOS-positive cells in the optic tectum (g–k, black arrows) lies adjacent to BrdU-positive cells in the proliferative zone (g–k, white arrows) at stages 43 and 47. The optic tectum is located on the dorsal aspect of the caudal midbrain, as shown in c and f.telen, Telencephalon; mb, midbrain;hb, hindbrain; R, rostral;C, caudal; D, dorsal; V, ventral. In i–k, rostral is at the top. Scale bar, 50 μm. |
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Fig. 1.
Structure and expression of XNOS.a, Structure of XNOS cDNA. cDNA (6.5 kb) was cloned from a stage 42 Xenopus cDNA library. It corresponds to a protein of 1419 aa, which has all of the regions of neuronal NOS isoforms conserved between mammals and Drosophila.FAD, Flavin-adenine dinucleotide; FMN, flavin mononucleotide. b, Expression of XNOS. Western blots of protein extracts were probed using a rat nNOS- and XNOS-specific polyclonal antibody. Each lane was loaded with 25 μg of protein isolated from 293 cells transfected with rat nNOS cDNA (first lane), 293 cells transfected with XNOS cDNA (second lane), and adultXenopus frog brain (third lane). The blot was treated with R20 polyclonal antibody raised against a 20 aa peptide, which is identical in both rat nNOS (positions 1400–1419) andXenopus XNOS (positions 1390–1409). c, Enzymatic activity of recombinant XNOS. 293 cells were transfected with XNOS cDNA, and the NOS activity in the cell extract was measured using the arginine-to-citrulline conversion assay in the presence or absence of various cofactors and inhibitors. d, In situ hybridization of XNOS RNA probe in whole-mount stage 47 tadpole brain and eyes. ret, Retina;telen, telencephalon; tectum, optic tectum; mb, midbrain; hb, hindbrain;R, rostral; C, caudal. e, Whole-mount NADPH-diaphorase staining of the tadpole brain at stage 46. The region depicted corresponds to the central left portion of d. Light stainingillustrates NADPH-diaphorase-positive cell bodies in the caudal optic tectum. Note that their position is similar to that of XNOS-positive cells in d. The cluster at thebottom corresponds to the XNOS-positive cells of the hindbrain in d. The dotted line marks the optic tectum; M, midline. Scale bars: d, 100 μm; e, 25 μm. |
