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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.
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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.
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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).
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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.
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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.
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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).
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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.
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cntn1 (contactin 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 32, lateral view, anterior left, dorsal up.
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