Saito R et al. (2001), Overexpression of Fyn tyrosine kinase causes ab...

XB-ART-8837

Dev Growth Differ 2001 Jun 01;433:229-38. doi: 10.1046/j.1440-169x.2001.00568.x.

Show Gene links Show Anatomy links

Overexpression of Fyn tyrosine kinase causes abnormal development of primary sensory neurons in Xenopus laevis embryos.

Saito R , Fujita N , Nagata S .

???displayArticle.abstract???
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.

???displayArticle.pubmedLink??? 11422288
???displayArticle.link??? Dev Growth Differ

Species referenced: Xenopus laevis
Genes referenced: b3gat1l fyn ncam1 uqcc6

???attribute.lit??? ???displayArticles.show???

	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 Rohon–Beard (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 (C–E) 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 B–E). 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). (A–E, H–J) 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 (–) 200 pg Fyn mRNA together with (+) or without (–) 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 transcription–polymerase chain reaction. Elongation factor 1 (EF1) was amplified as a loading control and the reaction without reverse transcriptase (–RT) 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 Rohon–Beard (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.