The Journal of Neuroscience, July 7, 2004, ():
Fibroblast Growth Factor Receptor 1 Is Required for the Proliferation of Hippocampal Progenitor Cells and for Hippocampal Growth in Mouse
J. Neurosci. Ohkubo et al.
24: 6057
Supplemental data
Supplemental Figures
Files in this Data Supplement:
- Supplemental Fig. 1
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Supplemental Figure 1. Characterization of Yu302, an antiserum to the intracellular FGFR1 domain. A and C are Western blots of the indicated cell lysates probed with Yu302 (A) or preimmune serum (C) (1:1000 dilution). B and D are immunoprecipitates using either Yu302 (B) or preimmune serum (D) probed with the same antisera. Lanes are as follows: rFGFR1, recombinant human FGFR1 from Sigma; 3T3-R1, 3T3 cells permanently transfected with human Fgfr1; 3T3-R2, 3T3 cells permanently transfected with human Fgfr2; K-R3, keratinocytes permanently transfected with human Fgfr3. Arrowheads indicate different post-translational variants of FGFR1 likely attributable to different degrees of phosphorylation. The light bands present in the 3T3-R2 lanes are [lt]100 kDa, lower than the expected molecular weight for FGFR2 and may therefore be unspecific. The light 120 kDa band present in K-R3 may be attributable to slight cross-reactivity of the antiserum with FGFR3. Cell lines are gifts from J. Schlessinger and C. Basilico.
- Supplemental Fig. 2
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Supplemental Figure 2. Fgfr1 recombination. A, Schematic diagrams of wild-type (Fgfr1; top), floxed Fgfr1 (Fgfr1flox; middle), and recombined allele (Fgfr1?flox; bottom). In the Fgfr1flox allele, the transmembrane region and the majority of the intracellular kinase domain of the Fgfr1 gene are flanked by loxP elements. Red boxes, loxP sites; yellow, transmembrane domain (TM); blue, kinase domain. B, The truncated Fgfr1?flox allele and the intact Fgfr1flox allele were detected by genomic PCR in various tissue to assess the specificity and efficiency of Fgfr1 recombination. Top, The Fgfr1?flox recombined allele was amplified with primers in intron 7 and exon 16 (A, black and blue arrowheads) and was detected only in the cerebral cortex and hippocampus but not in other body regions. Middle, The nonrecombined intact allele (Fgfr1flox) was amplified with primers in intron 7 (A, black and white arrowheads). In hGFAP-Cre/+; Fgfr1flox/flox mice, a faint band was detected in the cortex but not in the hippocampus. As expected, this nonrecombined allele is fully amplified in the tail and in all tissues of hGFAP-Cre/- mice. Bottom, A 116 bp control fragment present in both the floxed and recombined alleles (A, red and blue arrowheads) was used as a PCR control to normalize the genomic DNA template. C, The transcription of the intact and truncated Fgfr1 alleles was assessed by semiquantitative RT-PCR using primers corresponding to the Ig domain 1 in the extracellular region or to the floxed region (A, indicated by arrows). Top, In E16.5 hGFAP-Cre/+; Fgfr1flox/flox mice, the extracellular domain mRNA product was virtually undetectable in the hippocampus and decreased in the cerebral cortex compared with the Cre/- controls. Middle, In hGFAP-Cre/+; Fgfr1flox/flox mice, the transmembrane domain mRNA product was virtually undetectable from the hippocampus and decreased in the cerebral cortex compared with the Cre/- controls. Glyceraldehyde 3-phosphate dehydrogenase is used as positive control for RT-PCR sensitivity (bottom panel). E, The FGFR1 protein (~140 kDa; black arrow) was undetectable by Western blotting in hGFAP-Cre/+; Fgfr1flox/flox, but it was detected in Cre-negative brain tissue. Lane rFGFR1 is an extract of human 293T cells transfected with hFgfr1 gene as a positive control (Sigma; F2897). The bottom bands are nonspecific. The blot was also probed with anti-Actin antibody (Roche Diagnostics) as a positive control for equal loading on the gel. Cre-, Fgfr1flox/flox; Cre+, hGFAP-Cre/+; Fgfr1flox/flox.
- Supplemental Fig. 3
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Supplemental Figure 3. Regional patterns of Cre recombinase activity directed by the hGFAP-Cre transgene as revealed by Rosa26R reporter mice. Tissue from hGFAP-Cre; ROSA26R double transgenic mice was assayed for lacZ activity or ?-gal expression at the indicated ages. A, Coronal section of E13.5 brain showing Cre activity in cortical neuroepithelium (Ctx) and dorsal archicortex (hippocampal primodium; Hip) but not in ventral archicortex, thalamus (Th), and ganglionic eminences (Ge). B, Higher magnification of blue dashed square in A, showing that the Cre activity is present in the ventricular zone (VZ). C, D, Coronal (C) and sagittal (D) sections from E16.5 brains showing that recombination is evident in cells of the hippocampus (Hip), dorsal cortex (Ctx), and trigeminal ganglion (TG) but not in thalamus (Th). E, F, Coronal sections from 6-week-old mice. Dense LacZ staining is present in the neocortex, which is less strong in lower cortical layers; only sparse staining is present in striatum (St), probably because of axonal projections from cortex. In the hippocampus, cells of the CA field and DG are strongly stained. G, H, Double immunostaining using anti-?-gal antibodies in the adult hippocampus. Parvalbumin-positive cells (G, green; white arrows) are not positive for ?-gal (red). Some GFAP-positive cells are positive for ?-gal (H, white arrowheads). Am, Amygdala; OB, olfactory bulb. Scale bars: A, B, 500 ?m; C, 1100 ?m; D, 1250 ?m; E, F, 2500 ?m; G, H, 12.3 ?m.
