A search among genes that are strongly upregulated in response to Wnt signaling revealed one, called Lgr5, that is expressed in gut stem cells specifically. The technique described in Figure 22–5 can be used to create a genetic mark in a random subset of Lgr5-expressing cells—a mark that is inherited by the progeny of each cell. These Lgr5 cells divide with a cycle time of about 24 hours, and within a few days marked clones are seen extending from the crypt bases up along the sides of the villi. After as long as 60 days or more, many of these clones still persist, retaining one or more members at the crypt base and extending all the way up to the tips of the villi (Figure 22–6). Moreover, each single clone typically contains all the major differentiated gut cell types—absorptive, goblet, Paneth, and enteroendocrine—in their normal proportions. The Lgr5-expressing cells, therefore, are true stem cells that are multipotent—that is, able to generate a diverse set of differentiated cell types.
1 day 5 days 60 days Figure 22–6 Lgr5-expressing stem cells and their progeny in the small intestine.
the method shown in Figure 22–5 was used here to mark single intestinal stem cells and trace the fates of their progeny. the Lgr5 gene encodes a member of the family of G-protein-linked transmembrane receptors, and it is expressed specifically in stem cells near the crypt base. because the Lgr5 promoter was used to drive expression of CreERT2, treatment with a low dose of tamoxifen resulted in occasional stem cells expressing LacZ. these cells and all of their progeny could subsequently be detected with a blue histochemical stain. all of the blue cells in these images derive from a single Lgr5expressing stem cell. after 60 days, the blue progeny of this cell are seen to extend all the way up a villus. these progeny 100 µm can be shown to include all types of differentiated cells, as well as persistent the two daughters of a Stem Cell do not always have to Lgr5-expressing cells at the crypt base. become different this proves that Lgr5-expressing cells are multipotent stem cells. (From n. barker et If the number of stem cells in a crypt is to remain stable, each stem-cell division al., Nature 449:1003–1007, 2007. with must on average generate one daughter that remains a stem cell and one that permission from macmillan publishers ltd.) becomes committed to differentiation. In principle, this could be achieved in at least two ways (Figure 22–7).
One mechanism—the simplest at first sight—would be through asymmetric division: processes internal to the dividing stem cell could distribute regulatory factors asymmetrically to its two daughters, as occurs in Drosophila neuroblast divisions (see Figure 21–36). The factors inherited by one daughter would cause it to remain a stem cell, while those inherited by the other would drive it toward differentiation. This strategy would guarantee that the original stem cell would give rise to precisely one stem cell in every subsequent cell generation.
An alternative strategy would be based on a choice that each daughter makes independently of its sister: in normal circumstances, each would have a 50% probability of remaining as a stem cell and a 50% probability of commitment to differentiation. Sometimes the two daughters of a stem cell would thus have opposite fates, sometimes the same. The choice that each cell makes might either be stochastic, like the flip of a coin, or governed by the environment in which the cell finds itself. A strategy of independent choices is more flexible than that of strict asymmetric division. In particular, environmental factors can control the balance of probabilities, adjusting them in favor of the stem-cell option where more stem cells are needed, as they often are, either for growth or for damage repair.
Clonal analysis gives a way to distinguish between the two strategies, since they give quite different predictions as to the expected number of clones of different sizes produced from individual stem cells (see Figure 22–7). For the gut, the findings seem clear: the independent-choice theory fits the observations, and the asymmetric-division theory does not.
paneth Cells Create the Stem-Cell niche