The Journal of Neuroscience, September 14, 2005, ():
Absence of Fyn and Src Causes a Reeler-Like Phenotype
J. Neurosci. Kuo et al.
25: 8578
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Supplemental figures
Files in this Data Supplement:
- Supplemental Fig. 1
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Figure S1. Gross morphology of src-/-fyn-/- mutant cortex.
Cresyl violet staining at E18.5 illustrates the poorly-defined marginal zone and lack of columnar organization in the double src-/-fyn-/- mutant and dab1-/- cortex, compared with wildtype.
Supplementary method: Cryostat sections were thawed and soaked for 5 min each in 1 part chloroform: 1 part absolute ethanol, absolute ethanol, 70% ethanol, dH2O, and cresyl violet solution (0.5 % cresyl violet in 20 % ethanol, 1.65 % glacial acetic acid), then rinsed in 95% ethanol, then two 5 min soaks in absolute ethanol, and two soaks in histoclear. Slides were then dried and coverslipped with cytoseal.
- Supplemental Fig. 2
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Figure S2. Normal development of src-/- cortex.
E18.5 src-/- cortex stained with (a) Cresyl violet, (b) Tbr1 (red) and Brn1 (green) and (c) Cux1 (red) and Brn1 (green). The marginal zone is cell-free and early born and late born populations of neurons are distinctly and correctly located. Abbreviations: ps, pial surface; MZ, marginal zone; SP, subplate; IZ, intermediate zone; v, ventricle.
- Supplemental Fig. 3
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Figure S3. Cell proliferation is unaffected in the double mutant.
(a-c) E16.5 cortices were stained with antibodies to phosphohistone H3 (red) and DAPI (blue). The phosphohistone channel alone is shown alone (a'-c'). Most proliferating cells were located at the edge of the lateral ventricle for all genotypes (68+5% for src-/-fyn-/-, 77+1% for fyn-/- and 74+3% for wildtype; differences not significant by student t test). Total numbers of proliferating cells per section were also similar (52+9 for src-/-fyn-/-, 44+6 for fyn-/- and 48+5 for wildtype; differences not significant by student t test). Abbreviations: ps, pial surface; v, ventricle.
Supplementary method: Sections were subjected to a 5 min post-fix in cold methanol prior to blocking, and no antigen retrieval step was performed. The primary antibody was anti-phosphohistone H3 (Upstate Biotechnology) at 1:500 dilution.
- Supplemental Fig. 4
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Figure S4. The marginal zones of both fyn-/- and src-/-fyn-/- are crowded.
The cell-sparse marginal zone can be clearly distinguished in DAPI stained E18.5 wildtype cortex (a) and is absent in the dab1-/- cortex (b). Both the fyn-/- (c) and src-/-fyn-/- (d) cortices have a reduced and hypercellular marginal zone.
- Supplemental Fig. 5
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Figure S5. E18.5 cerebellum.
Coronalsections of wildtype (a) and src-/-fyn-/- (b) E18.5 cerebellum were stained with antibody to Dab1 and DAPI to show nuclei. Note ectopic Dab1+ Purkinje cells in (b). Dab1 images were not acquired at equal exposure, so the increased expression of Dab1 in double mutant cerebellum is not evident from these images. Abbreviations: mb, midbrain; pcl, Purkinje cell layer; Pj, Purkinje cells; egl, external granule layer; vz, ventricular zone; v4, fourth ventricle; *, fiber tracts.
