β-gal and BrdU co-staining scenarios. Wnt-reporter mouse intestines were injected with BrdU 2 days prior to analyses to assess the proliferative status of the β-gal positive crypt-based cells. (A) Approximately 7.5% of crypts contained a cell that was dual-labeled for β-gal and BrdU, reflecting cells that have been retained within the crypt (label-retaining cells) and that were Wnt-activated. (B) Approximately 27.3% of crypts contained a single BrdU-positive cell, possibly representing a "stem cell" that is not designated by the Wnt signaling pathway. This would be in line with the recently identified Bmi-1 positive stem cell. (C) 6.1% of crypts contained a single positive β-gal cell. This cell likely represents a cell that is activated by the Wnt signal after the effective BrdU labeling half-life in the animal. Finally, (D) a small percentage of crypts, 1.6%, contained a β-gal-positive cell and a BrdU-positive cell distinct from one another, likely representing a combination of the described scenarios. These scenarios are schematized in cartoon form beneath the corresponding fluorescent image that describes our perception of what each scenario may represent. In classical stem cell hierarchy, the lowest circle represents a progenitor cell residing near the base of the crypt and upper circles represent the progeny. Solid green circles represent BrdU-positive cells, solid red circles represent an activated Wnt cell, open red circles represent a cell that may have been Wnt-activated prior to BrdU labeling. These many different scenarios reflect the complex nature of the role of Wnt signaling on the stem cell hierarchy within the intestinal crypt.