The Journal of Neuroscience, March 31, 2004, ():
Dual Control of Neurogenesis by PC3 through Cell Cycle Inhibition and Induction of Math1
J. Neurosci. Canzoniere et al.
24: 3355
Supplementary Figures
Supplementary Figures
Files in this Data Supplement:
- Supplementary Fig. 1
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Figure 1. Analysis of cyclin D2, D3, and N-myc expression in Tg ?ACT-tTA/TRE-PC3 cerebella at P1. A, Analysis by Western blot of cyclin D1, D2, and D3 levels in lysates of cerebella from activated ([minus]doxy) or control (+doxy) transgenic mice. A representative experiment is shown. B, ISH analysis of cyclin D2 and cyclin D3 mRNAs on sagittal cerebellar sections in proximity of the midline. An evident decrease of cyclin D1 expression in the activated transgene is observed. Scale bars, 240 ?m. C, Sagittal sections of cerebella from Tgs activated in the absence of doxycycline ([minus]doxy) or control (+doxy) were stained by an antibody against N-myc and revealed by TRITC-conjugated secondary antibodies. Representative enlarged views of cerebellar lobes in the anterodorsal region, in proximity of the midline, are shown. No evident difference of N-myc immunostaining in the EGL is detectable between active and control Tg. Scale bars, 160 ?m.
- Supplementary Fig. 2
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Figure 2. Specificity of Math1 induction by PC3. No detectable induction of Math1 mRNA, as observed by semiquantitative RT-PCR analysis, follows the infection of primary cultures of rat cerebellar granule cells in vitro with recombinant adenoviruses, encoding the cyclin-dependent kinase inhibitors p21 (Adeno-p21; A) or p27 (Adeno-p27; B). Cerebellar granule cells, obtained from P8 rats, were infected after 5 d in culture and harvested 24 hr later. The amplified PCR products were visualized by Southern blotting, followed by hybridization to a [lsqb]32P[rsqb]-labeled probe (see Materials and Methods). RT-PCR analysis of Math1 induction was performed in parallel in granule cell cultures infected with Adeno-PC3, as positive control (shown in B). As in Figure 9A, Rev.T.ase(+/[minus]) indicates amplifications with or without reverse transcriptase, to control for genomic DNA contamination. One representative experiment of three is shown.
- Supplementary Fig. 3
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Figure 3. Detection by ISH of endogenous Math1 and PC3 mRNAs in cerebellum, by digoxigenin-labeled riboprobes. Each couple of images at the same developmental stage is obtained from adjacent sagittal sections. A, B, Cerebellar primordium at E14. The arrow indicates the rhombic lip. C--L, Full view of the cerebellum at different stages. In the rhombic lip and in the developing cerebellum, expression of Math1 and PC3 mRNAs overlap, the only difference being that Math1 mRNA expression in the EGL is more restricted to the outer part. I, J, At P10, the expression of both Math1 and PC3 mRNA becomes restricted to the rostral and central lobes in the region of the vermis (indicated by arrowheads; the white asterisk indicates a false signal attributable to folding of the section). K, L, At P30, both PC3 and Math1 mRNAs are barely detectable above background. Riboprobes were transcribed by T7 or T3 RNA polymerase and comprised the full ORF of mouse Math1 or PC3 (Tis21) cDNAs. No signal was given by sense riboprobes. Scale bars: A--J, 160 ?m; K, L, 130 ?m. Me, Mesencephalon; rh, rhombic lip; cb, cerebellar primordium; ch., choroidal plexus.
