Figure 3. Stages of colon crypt development and budding rates for mice of different ages.

(A) 3D confocal image of colonic crypts at the four stages of crypt development (defined as stages 0 to 3) as proposed in this study. An asymmetrical crypt production concept is proposed (budding), contrary to the symmetrical concept (fission). Schematic representations of the two concepts: red representing the development of a new bud; green sections depict the continuation of the original crypt; black dotted line represents the developing front of the crypt(s). Stages are defined based on the position of the new crypt base and the point of division of the crypts as described below the representative confocal image of each stage. Stage 0 is the only non-budding phase; Stage 1 represents the onset of crypt bud formation; Stage 2, in the midst of budding and Stage 3, late completion stage of budding. Stage 3 crypts transits into independent stage 0 crypts upon detachment from each other. (B) 2D images from image stacks showing the advantage provided by confocal 3D imaging of isolated crypts in capturing previously undetected buds of colonic crypts that are in stage 1 (white arrows). Onset of budding is difficult to detect using micro-dissection [17,32,33] or serial tissue sectioning [34]. Asymmetrical budding is also shown to occur in stage 1. P: Parental Crypt, D1: Daughter crypt 1, D2: Daughter crypt 2.