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The expression and function of the Src family protein tyrosine kinase Fyn in Xenopus laevis embryos have been examined. In situ hybridization analysis demonstrated nervous system-specific expression of Fyn mRNA in tail-bud embryos. However, a class of primary sensory neurons; that is, Rohon-Beard (RB) neurons, which is positive for immunoglobulin superfamily cell adhesion molecules (CAM), neural cell adhesion molecule (N-CAM) and contactin, is devoid of Fyn expression. Injection of Fyn mRNA into one of the blastomeres at the 2-cell stage led to overexpression of Fyn in the injected half of the tail-bud embryos. Immunolabeling of the embryos with anti-HNK-1 antibody revealed that the peripheral axons of RB neurons were partially misguided and bound to each other to form abnormal subcutaneous fascicles. Similar abnormality was induced by injection of the Fyn overexpression vector. The incidence of abnormality appeared dose-dependent, being 68-92% of the injected embryos at 50-400 pg of mRNA. Co-injection of the contactin antisense vector depleted contactin mRNA accumulation without affecting Fyn overexpression and reduced the incidence of the abnormal RB-cell phenotype. However, the N-CAM antisense was ineffective in reducing this abnormality. These results suggest that Fyn can modify signals regulating axonal guidance or fasciculation in the developing X. laevis nervous system and that contactin may affect this action of Fyn.
Fig. 1. Expression of Fyn in developing embryos, as revealed by whole-mount in situ hybridization. (A–C) Lateral view of the embryos
at stages 20, 26 and 32, respectively, with the anterior to the left. Note in (A) that strong Fyn signals (arrowheads) are detectable in
the brain region of neural tube and that medium to weak signals (arrows) are detectable in all the length of remaining neural tube.
(D,E) Cross-sections of the stained embryo at the levels of (D) hindbrain and (E) spinal cord. (F) A section through the spinal cord
showing location of Rohon–Beard neurons (arrows) that are labeled using a contactin antisense probe. fb, forebrain; hb, hindbrain;
mb, mid-brain; n, notochord; sp., spinal cord. Bars, 100 μm.
Fig. 2. Translation of the injected mRNA in embryos. (A) Distribution
of -galactosidase (-gal) activity (blue) and HNK-1
immunoreactivities (brown) in the stage 32 embryo injected
unilaterally with a mixture of 200 pg Fyn and 100 pg nucleartargeted
-gal (nuc-gal) mRNA at the 2-cell stage. Dorsolateral
view of the embryo with the anterior to the left. (B) Western blot
analysis of the Fyn protein. A single 60 kDa band is clearly seen
in the adult brain (Br) and the embryos injected with 200 pg Fyn
mRNA (Fyn-injected). The levels of 60 kDa protein are much lower
in the H2O-injected embryos (control).
Fig. 3. Abnormal development
of RohonâBeard (RB) neurons
caused by Fyn overexpression
and recovery with the contactin
antisense. Parts of stage 32
embryos were immunolabeled
with an anti-HNK-1 antibody. (A)
The control embryo injected with
100 pg nuclear-targeted -gal
(nuc-gal) mRNA alone, showing
HNK-1-positive fascicles in the
spinal cord (arrows) and the subcutaneous
nerve plexus formed
by axons of RB neurons (arrowheads).
(B) Dorsal and (CâE)
lateral views and (F,G) a crosssection
of the embryos injected
with 200 pg Fyn mRNA. The
peripheral axons of RB neurons
bind to each other to form abnormal
bundles (arrowheads in BâE).
The medial longitudinal fascicles
in the hindbrain (arrow in F) and
the dorsal lateral fascicle in spinal
cord (arrow in G) are disintegrated.
(H) Lateral view of the
embryo injected with 200 pg of the
Fyn overexpression vector. An
arrow indicates abnormal fasciculation
of peripheral axons of RB
neurons. (I,J) Lateral view of the
embryos injected with 200 pg
each of Fyn mRNA and the contactin
antisense vector. The spinal
fascicles and the peripheral axons
of RB neurons seem normal in (I),
but the latter exhibit abnormal
bundle formation in (J) (arrowheads).
(AâE, HâJ) Bars, 100 μm;
(F,G) bars, 200 μm.
Fig. 4. (A) Effects of contactin
and neural cell adhesion molecule
(N-CAM) antisense vectors
on mRNA accumulation. Both
blastomeres of 2-cell stage
embryos were injected with (+)
or without (â) 200 pg Fyn mRNA
together with (+) or without (â)
200 pg of either pXeX-XF3.AS
or pXeX-XNCAM.AS. Total RNA
was isolated separately from
each embryo at stage 24
and contactin and N-CAM transcripts
were detected by reverse
transcriptionâpolymerase chain
reaction. Elongation factor 1
(EF1) was amplified as a loading
control and the reaction without
reverse transcriptase (âRT)
served as a genomic DNA
control. (B) Contactin antisense vector does not affect Fyn overexpression induced by mRNA microinjection. One blastomere of 2-cell
stage embryos was injected with indicated amounts of Fyn mRNA and pXeX-XF3.AS, and Fyn protein was detected in the pooled
embryos at stage 32 by western blotting.
Fig. 5. N-CAM antisense does not reduce Fyn-induced abnormality of RohonâBeard (RB) neurons. Lateral view of the stage 32 embryos
immunolabeled with an anti--tubulin antibody. (A) The control embryo showing subcutaneous extension of peripheral axons of
RB neurons (arrows) and ciliated epithelial cells (arrowheads). (B) The embryo injected with 200 pg of the N-CAM antisense vector,
showing abortive extension of subcutaneous axons (arrows). (C) The embryo injected unilaterally with 200 pg each of Fyn mRNA and
N-CAM antisense vector, showing abnormal fasciculation of subcutaneous axons (arrows). sp, Spinal cord. Bars, 100 μm.
