DEV033548 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Representative ImagePro Plus image of an E13.5 wild-type embryo eye following H&E staining. (A) Showing how the posterior region of the developing primary vitreous is traced in green to define the area of interest (AOI). (B) Showing the same field after selecting all the space within the AOI that is covered by a cell (excluding erythrocytes). The percentage cellularity is calculated by dividing the number of pixels highlighted in B by the total number of pixels in the AOI (A).

• Supplemental Figure S2 -

  Fig. S2. Representative photomicrographs of H&E-stained sections of eyes from Tgfb2^+/- and Tgfb2^+/+ mice at P0. Note the cornea thinning (arrowhead), retinal folding (*) and hyperplastic vitreous (arrow) observed in Tgfb2^-/- eyes (bottom). Eyes from heterozygous (top) and wild-type animals were normal.

• Supplemental Figure S3 -

  Fig. S3. Tgfβ2 blocks cell proliferation in the mouse primary vitreous. Representative photomicrographs of cryostat sections through the vitreous of E13.5 mouse embryos of the indicated genotypes, stained for DAPI (blue) and Ki67 (green), as a marker of proliferation. Quantitative analyses showed increased numbers of vitreous cells are Ki67-positive in Tgfb2^−/− embryos.

• Supplemental Figure S4 -

  Fig. S4. Tgfβ2 promotes p19^Arf expression in MEFs but does not influence the expression of other Cdk inhibitors. (A) Representative western blot for the indicated proteins in lysates from wild-type (WT) MEFs treated with Tgfβ2 for the indicated times; from Arf-deficient 10T1/2 cells transduced with Arf (A) or Gfp (G) encoding retrovirus; and from NIH3T3 cells. Note that the small Cdk4 repression at 72 hours, which has been established to be mediated by p53 (Ewen et al., 1995), also occurred in Arf^-/- MEFs (data not shown) and is likely inconsequential. Ectopically-expressed p19^Arf (arrow) distinguished it from the cross-reactive band (*). (B) Representative western blot showing no apparent induction of p19^Arf at 24 hours. Note that the cross-reactive band (A, *) is cropped from B. (C) Representative photomicrograph showing normal phenotype of a H&E-stained p21^-/- mouse eye.

  Reference

  Ewen, M. E., Oliver, C. J., Sluss, H. K., Miller, S. J. and Peeper, D. S. (1995). p53-dependent repression of cdk4 translation in TGFb-induced G1 cell-cycle arrest. Genes Dev. 9, 204-217.

• Supplemental Figure S5 -

  Fig. S5. Generation of Arf^lacZ/+ mouse ES cells and knock-in mice. (A) Schematic diagram of targeting plasmid in which Arf exon 1β is replaced by a lacZ-Neo cassette. Also included is the diphtheria toxin gene for selection against cells with non-homologous integration of plasmid. (B) Southern blot of AflII-digested genomic DNA from a representative 129/Sv ES cell clone shows expected ∼12 kb band (arrow). For comparison, Southern blot of similarly digested Arf^-/- cells (Kamijo et al., 1997; Kamijo et al., 1999) is shown. (C) Representative PCR genotyping of Arf ^acZ/lacZ, Arf^+/lacZ and wild-type mice.

  Reference

  Kamijo, T., Bodner, S., van de Kamp, E., Randle, D. H. and Sherr, C. J. (1999). Tumor spectrum in ARF-deficient mice. Cancer Res. 59, 2217-2222.

• Supplemental Figure S6 -

  Fig. S6. X-gal staining in Arf^lacZ/+ and Arf^lacZ/lacZ mice parallels the known expression pattern of Arf. (See Zindy et al., 2003; Martin et al., 2004). (A) Representative photomicrographs of testes from wild type (a, left, and b) and Arf^lacZ/lacZ (a, right, and c) shown as whole mounts (a) and cryostat sections (b,c). (B) X-gal staining identified Arf-expressing, perivascular cells enveloping the hyaloid vessels in a newborn Arf^lacZ/+ mouse eye shown at low (left) and high (right) magnification. No X-gal-stained cells are evident in the wild-type postnatal eye (Thornton et al., 2005). (C) X-gal stain of MEFs at the indicated number of passes shows increased β-galactosidase in Arf^lacZ/lacZ MEFs. Notably, tumor predisposition in Arf^lacZ/lacZ mice and developmental eye disease also parallels previously published Arf-deficient models (Kamijo et al., 1999; Zindy et al., 2003; McKeller et al., 2002; Martin et al., 2004)

  Reference

  Thornton, J. D., Silva, R. L. A., Martin, A. C. and Skapek, S. X. (2005). The Arf tumor suppressor regulates Platelet-derived growth factor receptor β signaling: A new view through the eyes of Arf -/- mice. Cell Cycle 4, 1316-1319.

• Supplemental Figure S7 -

  Fig. S7. Tgfβ2 increases Arf mRNA in MEFs but does not alter p19^Arf stability. (A) Quantitative RT-PCR in wild-type MEFs shows Arf expression following exposure to Tgfβ1 or Tgfβ2 (each at 5 ng/ml) for the indicated number of hours. Arf expression was normalized to Gapdh expression. Differences between Tgfβ and vehicle were significant at 72 hours (P<0.001). The capacity for the lacZ reporter to detect induction at 48 hours probably relates to relatively higher sensitivity to detect small changes in transcription (see Fig. 2D). (B) Western blotting for ectopically expressed p19^Arf in Arf-null MEFs shows decreased protein following cycloheximide (CHX) exposure, with no apparent stabilization by 24 hours of Tgfβ2 pretreatment (Pre-Tx). Top and bottom represent two different experiments. V, vehicle; T, Tgfβ2. Similar findings are evident with endogenous p19^Arf in wild-type MEFs.

• Supplemental Figure S8 -

  Fig S8. Arf expression is detectable in perivascular cells flanking the embryonic umbilical artery. (A) Representative photograph of ethidium bromide-stained gel following electrophoresis of RT-PCR amplification of Arf or CD31 from P4 aorta (Ao) or right umbilical artery (UA). Because Arf-expressing cells are perivascular, the endothelial-specific CD31 serves as a loading control. Serial dilutions of cDNA (RT+) indicate amplification is in linear range. Note that no product is amplified when reverse transcriptase is excluded from reaction (RT−). (B) Representative photomicrographs of sections through the right umbilical artery in E14.5 embryos of the indicated genotypes following dual immunofluorescence staining for β-galactosidase (green) and Pdgfrβ (red). No β-galactosidase expression is detected in sections from wild-type mice. Original magnification: 600×.

• Supplemental Figure S9 -

  Fig. S9. Representative photomicrographs of H&E-stained eyes taken from ~13-week-old phenotypically normal Arf^+/- mice and from Arf^-/- mice. No obvious differences in the cornea were evident at that age, or in the early postnatal period.