Supporting Information

Files in this Data Supplement:

Fig. 5. Analysis of adult p63–/– chimeras. (A and B) Forelimbs of a partially rescued p63–/– chimera are characterized by split paw (A) and digit fusion (B). (C–F) X-gal staining of various tissues from a p63–/– chimera. Both p63+/+ b-gal-positive and p63–/– b-gal-negative cells contribute to organs that do not require p63 for development, including liver (D), stomach glands (E), and intestinal epithelium (F). In contrast, the epidermis consists of only p63+/+ b-gal-positive cells (C). (Magnification: ´100.)

Fig. 6. DNA analysis of b-gal-negative hepatocytes microdissected from putative p63–/– adult chimeras confirms that these mice derive from p63–/– blastocysts. Real-time PCR was performed with primers specific for the WT or p63–/– allele. The amount of WT allele relative to p63–/– allele (log WT:p63–/–) is shown on y axis. Bars 1–3 correspond to p63+/+, p63–/–, and p63+/– controls, respectively. Bars 5, 7, 9, 11, 13, and 15 correspond to b-gal-negative hepatocytes isolated from each of the six putative p63–/– chimeras. Bars 4, 6, 8, 10, 12, and 14 represent b-gal-positive hepatocytes isolated from the same mice. As expected, the WT allele is more abundant than the p63–/– allele in the purified b-gal (p63+/+) cells. In contrast, the p63–/– allele is more represented than the WT allele in the population of b-gal-negative cells. These results demonstrate that b-gal-negative hepatocytes from all of the six putative p63–/– chimeras are p63–/–. The fact that the p63–/– allele is detectable in the b-gal positive (p63+/+) cell samples and the WT allele is present in the b-gal negative (p63–/–) cells indicates that the microdissected samples contain variable amounts of contaminating cells. Values are the mean ± SD from independent microdissection experiments done at least twice.

Fig. 7. The levels of chimerism in the umbrella and basal/intermediate cell compartments of control chimeras is significantly different. C and D represent higher magnification (´400) of the marked regions in A and B (´40). (A and C) The basal/intermediate cell compartment is almost entirely b-gal negative, whereas »20% of umbrella cells are b-gal positive. (B and D) The basal/intermediate cells are entirely b-gal positive, whereas »80% of the umbrella cells are b-gal negative.