Skip to main page content
U.S. flag
See More

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2005 Sep 14;25(37):8578-86.
doi: 10.1523/JNEUROSCI.1656-05.2005.

Absence of Fyn and Src causes a reeler-like phenotype

Gloria Kuo  1 , Lionel Arnaud, Priscilla Kronstad-O'Brien, Jonathan A Cooper
Affiliations

Absence of Fyn and Src causes a reeler-like phenotype

Gloria Kuo et al. J Neurosci. .

Abstract

Nonreceptor protein tyrosine kinases of the Src family regulate the survival, proliferation, differentiation, and motility of many cell types, but their roles in brain development are unclear. Biochemical and in vitro experiments implicate Src and Fyn in the Reelin-dependent tyrosine phosphorylation of Dab1, which controls the positioning of radially migrating neurons in many brain regions. However, genetic evidence that either Src or Fyn mediates Reelin-dependent migrations in vivo has been lacking. Here, we report that, although Src is dispensable and although the absence of Fyn causes an intermediate phenotype, the combined absence of Src and Fyn almost abolishes tyrosine phosphorylation of Dab1 and causes defects in the fetal cortex and cerebellum very similar to those of dab1 mutants of the same age. Neurogenesis is not detectably affected, but the layering of neurons in the cortex is inverted, and the formation of the Purkinje plate is impaired. This implies that Src and Fyn are needed for Reelin-dependent events during brain development.

PubMed Disclaimer

Figures

Figure 1.
Figure 1.
Expression levels and tyrosine phosphorylation of Dab1 in mutant embryonic brain and neuron cultures. a, b, Protein extracts were prepared from E16.5 embryo brains. fyn–/– littermate embryos with various src genotypes were obtained from a src+/–fyn–/– intercross. Wild-type embryo brains were from another litter. a, Protein samples were analyzed directly by immunoblotting for phosphotyrosine, neuron-specific βIII tubulin, Src-family kinases, and Dab1. The asterisks indicates proteins whose phosphorylation was reduced by src fyn mutation. Note the reduction in Src-family kinase expression and the increase in Dab1 protein levels as src and fyn gene dosages were reduced. b, Dab1 was immunoprecipitated from the protein extracts in a and immunoblotted for phosphotyrosine and Dab1. Relative phosphorylation levels were calculated after densitometry. c, Protein extracts from independent E16.5 embryo brains were analyzed by immunoprecipitation of Dab1, p190 Rho GAP, and FAK and immunoblotted with the indicated antibodies. Mutation of src and fyn caused decreases in tyrosine phosphorylation of Dab1, p190, and FAK, a smaller decrease in FAK autophosphorylation (pTyr397), and increases in Dab1 protein level, with no effect on protein levels of p190 or FAK.d, Number of experiments, mean, and SE of the stoichiometry of Dab1 tyrosine phosphorylation from different experiments. e, Neurons from littermate fyn–/– embryos of various src genotypes were cultured for 5 d and stimulated for 15 min with Reelin-containing or mock supernatants. Samples were analyzed by immunoblotting with antibodies to phosphotyrosine and Dab1. f, Stoichiometry of tyrosine phosphorylation of Dab1 after invitro stimulation with Reelin. Both basal and Reelin-stimulated tyrosine phosphorylation of Dab1 were greatly decreased in src–/–fyn–/– (s–/–f–/–) neurons. WB, Western blot; IP, immunoprecipitation.
Figure 2.
Figure 2.
Dab1 staining is increased in the src–/–fyn–/– neocortex. Coronal sections of E18.5 neocortices were double stained with antibodies against Reelin and Dab1. The separate channels are shown. All images were captured using the same exposure time, and levels were adjusted equally. a–d, Reelin was expressed equally by scattered neurons in the marginal zone across all genotypes. However, Dab1 staining was absent in dab–/– (b′) and elevated in fyn–/– (c′) and src–/–fyn–/– (d′) neocortices relative to wild-type (a′) neocortex. ps, Pial surface; MZ, marginal zone; II and V, layers II/III and V/VI of the cortical plate, respectively; SP, subplate; IZ, intermediate zone; v, ventricle.
Figure 3.
Figure 3.
The src–/–fyn–/– mutant shows defects in preplate splitting. a, b, CSPG antibody stained the subplate in wild-type E18.5 neocortex (a) and faintly stained the superplate and scattered subplate cells in dab1–/– (b). c, d, Subplate staining was dispersed in fyn–/– (c), and CSPG+ cells were dispersed through the cortical plate in src–/–fyn–/– (d). e, f, CSPG staining at E16.5 in wild-type (e) and src–/–fyn–/– (f) showed the same pattern seen at E18.5. g, h, Calretinin staining was detected in the subplate of wild type (g) but stained fibers and cells throughout the src–/–fyn–/– cortex (h). Scale bars: a (for a–f), g (for g, h), 100 μm. ps, Pial surface; sp, subplate; v, ventricle; CalR, calretinin.
Figure 4.
Figure 4.
Cortical lamination is inverted in the src–/–fyn–/– mutant. a–d, E18.5 cortices stained with antibodies to Tbr1 (red) and Brn1 (green). e–h, Staining with antibodies to Cux1 (red) and Brn1 (green). Tbr1 marks subplate and early-born cortical plate neurons, whereas Cux1 and Brn1 mark overlapping populations of late-born cortical plate neurons. Note approximate layer inversion in dab1–/– and src–/–fyn–/– relative to wild type. i–k, Quantification of phenotypes. The percentage of Tbr1+ (i), Brn1+ (j), and Cux1+ (k) in different layers from marginal zone (bin 1) to the top of the ventricular zone (bin 9) was calculated based on replicate sections from one (Cux1) or two (Tbr1 and Brn1) embryos of each genotype. Tbr1 and Brn1 staining of a total of five E18.5 and four E16.5 src–/–fyn–/– cortices showed that all had an inverted cortical plate. ps, Pial surface; v, ventricle; WT, wild type.
Figure 5.
Figure 5.
Purkinje cell migration defects in src–/–fyn–/– cerebellum. Coronal sections of E18.5 cerebellum were stained with antibody to calbindin and 4′,6′-diamidino-2-phenylindole dihydrochloride (DAPI) to show nuclei. Each figure is a montage of high-power images. a, Purkinje cells in the wild-type cerebellum had largely migrated out to form the Purkinje cell layer. b, In the dab1–/– cerebellum, Purkinje cells were located in clusters deep in the cerebellum. c, A partial phenotype was seen in the src–/–fyn–/– cerebellum, where a vestigial partial Purkinje cell layer had formed, but most Purkinje cells were in clusters deep in the cerebellum. In all cases, an external granule layer could be detected. mb, midbrain; pcl, Purkinje cell layer; Pj, ectopic Purkinje cells; egl, external granule layer; vz, ventricular zone; v4, fourth ventricle. The asterisks indicate fiber tracts.
See this image and copyright information in PMC

References

    1. Angevine JB, Sidman RL (1961) Autoradiographic study of cell migration during histogenesis of cerebral cortex in the mouse. Nature 25: 766–768. - PubMed
    1. Arnaud L, Ballif BA, Forster E, Cooper JA (2003a) Fyn tyrosine kinase is a critical regulator of disabled-1 during brain development. Curr Biol 13: 9–17. - PubMed
    1. Arnaud L, Ballif BA, Cooper JA (2003b) Regulation of protein tyrosine kinase signaling by substrate degradation during brain development. Mol Cell Biol 23: 9293–9302. - PMC - PubMed
    1. Arthur WT, Petch LA, Burridge K (2000) Integrin engagement suppresses RhoA activity via a c-src-dependent mechanism. Curr Biol 10: 719–722. - PubMed
    1. Beggs HE, Soriano P, Maness PF (1994) NCAM-dependent neurite outgrowth is inhibited in neurons from fyn-minus mice. J Cell Biol 127: 825–833. - PMC - PubMed

Publication types