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Fig. 2. Neogenin mRNA is expressed in the developing Xenopus forebrain. (A) The predicted structure of Xenopus Neogenin is shown schematically. Xenopus Neogenin contains an open reading frame of 4326 bp and encodes a protein of 1442 amino acids. It has four immunoglobulin-like (Ig) domains, six fibronectin type III (FnIII) domains, a transmembrane region (TM) and a cytoplasmic tail containing three highly conserved domains (P1�P3). The positions of three alternatively spliced exons are indicated (splice 1, bp 1273�1332; splice 2, bp 2534�2581; splice 3, bp 3653�3680). The inclusion of splice 3 (28 bp) imparts a frameshift and premature stop codon, resulting in a truncated protein. Xenopus Neogenin possesses an SQTG amino acid motif in its cytoplasmic tail, instead of the usual conserved C(C/T)TD motif for caspase-3 cleavage, suggesting that Neogenin is unlikely to be involved in caspase-3-mediated cell death in Xenopus. The region used for synthesis of antisense probe is identified. (B) Lateral view of Neogenin mRNA expression on a whole-mount stage 30�31 Xenopus embryo, dorsal to the top and rostral to the left. Neogenin expression is predominant in the head (asterisk) and spinal cord (arrowhead) and is also present in the gut and somites. (C) Lateral view of Neogenin mRNA expression in isolated CNS tissue from a stage 30�31 Xenopus embryo. The forebrain (fb), midbrain (mb), hindbrain (hb), forebrain�midbrain boundary (FMB) and midbrain�hindbrain boundary (MHB) are indicated. Neogenin expression (indicated by the dashed line) is strong in the dorsal forebrain and extends into the ventral midbrain. Expression is also strong in the ventral hindbrain. Neogenin is absent from the hypothalamus (h), epiphysis (e), dorsal midbrain, midbrain�hindbrain boundary and dorsal hindbrain. (D) Dorsorostral view of an isolated stage 30�31 Xenopus brain, dorsal to the top (indicated by the epiphysis (e)), showing strong expression of Neogenin extending from both sides of the midline to the lateral margins of the dorsal forebrain. The estimated position of the nPT relative to these zones of Neogenin expression is indicated (hatched circles), based on double-labeling experiments (see panel F). (E�F) Isolated CNS from stage 30�31 Xenopus embryos, double-labeled for Neogenin mRNA expression (blue) and the NOC-2+ subpopulation of forebrain axons (brown), revealing their spatial relationship. (E) Magnification of a region of the forebrain in lateral view, equivalent to that indicated by a box in panel C, double-labeled for Neogenin (blue) and NOC-2 (brown stain, which appears deep purple in this double label). Neogenin is expressed in the nucleus of the presumptive telencephalon (nPT, indicated by the dashed circle) and in the neuroepithelium underlying the supraoptic tract (SOT) and the longitudinally oriented tract of the post-optic commissure (TPOC). The anterior commissure (AC) is indicated. (F) Dorsorostral view of a double-labeled brain, showing that Neogenin is expressed by the nPT (arrows). The AC is indicated at the rostral margin of the forebrain, and the epiphysis (e) is indicated at the dorsal surface of the brain. (G) Schematic representation of Neogenin mRNA expression in relation to the axon scaffold in the Xenopus forebrain. Scale bar in panel B = 500 μm, panels C�F = 60 μm. |
