Fujita N et al. (2000), An essential role of the neuronal cell adhesion...

XB-ART-11094

Dev Biol 2000 May 15;2212:308-20. doi: 10.1006/dbio.2000.9692.

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An essential role of the neuronal cell adhesion molecule contactin in development of the Xenopus primary sensory system.

Fujita N , Saito R , Watanabe K , Nagata S .

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Contactin is a glycosylphosphatidylinositol-anchored immunoglobulin-like neuronal cell adhesion molecule that has been implicated in cellular interaction during development of the vertebrate central nervous system. Here we report evidence for an essential role of contactin in development of the Xenopus nervous system. Contactin mRNA is detectable by in situ hybridization in subsets of neurons in the brain, primary sensory neurons in the spinal cord, and cells along the trigeminal nerves of tailbud embryos. Contactin immunoreactivities preferentially distribute on axon tracts of the brain, the spinal cord, and the trigeminal sensory nerves. Most prominently, cell bodies and peripheral and spinal axons of primary sensory neurons, Rohon-Beard (RB) cells, are strongly contactin positive. Injection of the contactin overexpression vector into one blastomere of two-cell stage embryos leads to misdirected elongation of the peripheral axons of RB neurons in the injected half. Overexpression of antisense transcript causes depletion of contactin mRNA accumulation and abnormal development of RB neurons. In 52.3% of the injected embryos, RB neurons decrease in number and their peripheral axons in dorsal lateral tracts are defasciculated. These results demonstrate that contactin plays an essential role in development of the Xenopus primary sensory system.

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Species referenced: Xenopus laevis
Genes referenced: b3gat1l cntn1 eef1a2 ncam1 tec uqcc6
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	FIG. 1. Expression of contactin during Xenopus embryonic development. (A) RT–PCR analysis of contactin mRNA. Total RNA was isolated from the embryos at each stage indicated and contactin transcript was amplified using specific primers. EF1a was amplified as a loading control; (2R) Reaction using RNA from stage 32 embryos without reverse transcriptase. (B) Western blot analysis of contactin protein. A sample equivalent to a single embryo extract was analyzed at each stage. (Br) Adult brain extract.
	FIG. 2. Patterns of contactin mRNA expression in tailbud embryos, revealed by whole-mount in situ hybridization. (A and B) Lateral views of the embryos at stage 24 (A) and stage 32 (B), with the anterior to the left. Weak contactin signals (arrowheads) are detectable in the anterior spinal cord, the hindbrain, and the trigeminal nerves in (A). Intensity of the overall contactin signal increases and the areas of expression expand by stage 32 (B). (C and D) Cross sections of the whole-mount hybridized stage 32 embryo at the levels of the midbrain (C) and spinal cord (D). Contactin mRNA is detected in the trigeminal nerve (small arrowheads), the fasciculus longitudinalis medialis, and the ventral fascicles (large arrowheads) in the midbrain section (C). It is also detectable in RB neurons (arrowheads), lateral fascicles (lf), and ventral fascicles (vf) in the spinal cord section (D). hb, hindbrain; nc, notochord; sp, spinal cord; tgn, trigeminal nerve. Scale bars, 100 mm.
	FIG. 3. Distribution of contactin protein in stage 37/8 embryos, visualized by whole-mount immunocytochemistry. (A) A lateral view of the anterior 2/3 of the stained embryo, showing nervous tissue-specific distribution of contactin immunoreactivity. (B) An enlarged dorsal view of the brain portion of the embryo shown in (A). Axon tracts in the brain and primary olfactory region (arrowhead) are contactin positive. (C, D, and G) Cross sections of the stained embryo at the levels of midbrain (C), hindbrain (D), and posterior spinal cord (G). Contactin is expressed in the postoptic commissure and the optic chiasm of the lateral diencephalon (arrowheads in C) and the fasciculus longitudinalis medialis in the ventral marginal region (arrowheads in D). (E and F) Enlarged lateral (E) and dorsal (F) views of a part of the embryos, showing contactin-positive dorsal and ventral spinal fascicles. An arrow and an arrowhead in (E) represent contactin-positive cell bodies of a RB neuron and an EM neuron, respectively. (G) A pair of RB neurons (small arrows), an EM neuron with peripheral axon (large arrow), and dorsal fascicles (arrowheads) are strongly contactin positive. ac, anterior commissure; dr, dorsal lateral tract; df, dorsal fascicle; flm, fasciculus longitudinalis medialis; hb, hindbrain; mb, midbrain; nc, notochord; ov, otic vesicle; pc, posterior commissure; poc, postoptic commissure; sp, spinal cord; tec, optic tectum; tgn, trigeminal nerve; vf, ventral fascicle. Scale bars, 100 mm (A–F); 200 mm (G).
	FIG. 4. Expression of b-galactosidase and contactin in embryos co-injected with the overexpression vectors. (A) Dorsal view of an embryo unilaterally injected with pXeX-cbgal and pXeX-XF3 vectors and stained for expression of b-galactosidase (blue) and contactin (brown). The predominant expression occurs in the left half of the embryo. The white line represents the dorsal midline. (B) Lateral view of the embryo, showing mosaic patterns of b-galactosidase (blue) and contactin (brown) expression. The mosaic patterns largely overlap, but there are also patches expressing either b-galactosidase (green arrowheads) or contactin (red arrowheads) alone. Scale bar, 250 mm.
	FIG. 5. Injection of the antisense vector suppresses accumulation of the contactin transcripts. The contactin antisense vector pXeXXF3. AS was injected into both blastomeres of two-cell stage embryos and a total RNA fraction was prepared from each embryo at stage 21 (AS injected). Control embryos (control) were injected with sterile water. Transcripts of contactin, contactin antisense (contactin AS), NCAM, and EF1a were amplified for each embryo by RT–PCR. The number of each lane represents an individual embryo of the same injection group, and the 2R lane indicates the reaction-omitted reverse transcriptase.
	FIG. 6. Injection of the antisense vector results in abnormal development of RB neurons. (A) Dorsal view of a control stage 37 embryo injected with H2O, showing HNK-1-positive longitudinal spinal tracts, dorsal lateral tracts (dr), and cell bodies of RB cells. (B and C) Dorsal view of representative antisense-injected embryos, showing defasciculation or absence of HNK-1-positive dorsal lateral tracts (arrows) in the injected half. (D) Dorsal view of a antisense-injected stage 37 embryo, showing fewer RB cells in the injected (lower) half than those (arrowheads) in the opposite uninjected half. (E1–10) Serial cross sections of an antisense-injected stage 37 embryo, showing distribution of HNK-1 (black) and contactin (brown) immunoreactivities in the spinal cord. The number of RB and EM cells (arrowheads) is fewer in the injected (right) half than in the uninjected (left) half. Scale bars, 100 mm (A–C); 50 mm (D and E).
	FIG. 7. Contactin overexpression leads to misdirected axonal extension by RB neurons. (A) Lateral view of the uninjected side of the stage 37 embryo unilaterally injected with contactin overexpression vector at the two-cell stage. The micrograph is focused on the intramyotomal fascicles (arrowheads) and further ventral extension of peripheral axons. (B) Lateral view of the same embryo, showing misdirected extension of the peripheral axons (arrowheads) in the injected side. Subcutaneous extension of RB cell axons seems normal. Scale bar, 100 mm.
	cntn1 (contactin 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 32, lateral view, anterior left, dorsal up.