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See online version for legend and references.1198 Cell 152, February 28, 2013 ©2013 Elsevier Inc. DOI http://dx.doi.org/10.1016/j.cell.2013.02.030

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1198.e1 Cell 152, February 28, 2013 ©2013 Elsevier Inc. DOI http://dx.doi.org/10.1016/j.cell.2013.02.030

SnapShot: The Intestinal CryptHans Clevers1 and Eduard Batlle2,3

1Hubrecht Institute, KNAW and University Medical Centre Utrecht, Uppsalalaan 8, 3584CT Utrecht, the Netherlands2Institute for Research in Biomedicine (IRB Barcelona), 08028 Barcelona, Spain3Institució Catalana de Recerca i Estudis Avançats (ICREA), 08010 Barcelona, Spain

Organization of the Small Intestine and the ColonThe inner surface of the intestinal tube is lined by a simple epithelium, which displays distinct morphologies along the proximo-distal axis. The proximal region, the small intestine (duodenum, jejunum, and ileum), is arranged in invaginations (crypts) intercalated with finger-like protrusions (villus) that represent units specialized in the absorption of micronu-trients. The colon consists only of crypts and is largely dedicated to the compaction of stool. The identity of each of these segments is, in part, specified through the expression of the homeotic transcription factor Cdx2, which represses the expression of genes characteristic of the stomach, and the zinc finger Gata4, which confers proximal identity to the duodenum and ileum. Despite their distinctive morphologies, epithelial cell types of both the small intestine and colon are organized following a bottom-to-top axis into three compartments: the stem cell compartment that is located at the crypt base, the transient-amplifying (TA) compartment that occupies the middle portion of the crypts, and the differentiation zone, which expands from the top third of the crypt and the surface epithelium to the tip of the villus.

Renewal of the Epithelial Layer and Lineage SpecificationSix differentiated cell types populate the intestinal epithelium. The most abundant cells are the enterocytes (i.e., adsorptive cells) and goblet cells (i.e., mucosecreting cells). Other rare secretory cell types are scattered throughout the epithelium; they are enteroendocrine cells, which produce a diverse array of hormones, and the tuft cells, which are believed to secrete prostanoids. Microfold cells (M cells) are specialized epithelial cells situated over Peyer’s patches (PP) that transport antigens into intraepithelial pockets accessed by antigen-presenting cells. Finally, Paneth cells reside at the crypt base and perform a dual role; they secrete antimicrobial substances but also nurture the intestinal stem cell (ISC) population.

All differentiated cell types in the intestinal epithelium are short lived. Enterocytes, goblet cells, and the other secretory lineages are born in the crypts and follow an upward migratory flow that carries them to the tip of the villi in ~1 week. At this location, they are extruded into the lumen. Paneth cells are the exception and are retained at the base of the crypts, where they live for 6–8 weeks. Renewal of the epithelial layer is sustained throughout life by a small number of ISCs (n = 10–15 per crypt), which are located at the bottom-most positions intermingled with Paneth cells. ISCs proliferate with a rate of approximately one division per day. Their progeny is amplified through a series of very rapid divisions (about one division every 12 hr). While these TA cells migrate upward, they become progressively committed toward one of six lineages that are present in the intestine.

The activity of the bHLH transcription factor Math1 commits precursor cells to a secretory phenotype. Math1 also promotes elevated levels of delta-like ligands (Dll1/Dll4) in secretory precursors. In contrast, expression of Math1 is repressed in enterocytes by the Notch downstream effector Hes1. As a result of lateral inhibition, differential Notch activity in adjacent precursors operates as a binary switch to specify absorptive versus secretory cell types. Following commitment, a complex cascade of transcription factors drives the differentiation of the secretory precursor into distinct mature cell types (goblet, enteroendocrine, tuft, or Paneth cells). Differentiation of M cells requires the transcrip-tion factor Spi-B, the expression of which is switched on downstream of RANK signaling. RANK-L, the ligand for RANK receptor, is secreted by stromal cells present at Peyer’s patches.

Quiescent versus Proliferative ISCsIn homeostasis, Lgr5+ ISCs generate all cell types present in the epithelium (Barker et al., 2007; Sato et al., 2009). An independent class of quiescent ISCs marked by Bmi1, Lrig1, Tert, and Hopx has been proposed to serve as “reserve” stem cells. A recent study provides a simple view of the connection between these two stem cell types (Buczacki et al., 2013). The study shows that a small subset of Lgr5+ cells can enter a quiescent state. These label-retaining cells (LRCs) localize around crypt position +3. Lgr5+ LRCs express most of the markers previously postulated for the quiescent ISC population, but also several Paneth-cell-specific genes. These LRCs are not ISCs but, rather, transient precursors of Paneth cells, as they are short lived under homeostatic conditions (2–3 weeks) and they all differentiate toward mature Paneth cells. However, upon loss of the ISC pool, they can revert to become cycling Lrg5+ ISCs. Dedifferentiation of Dll1+-committed secretory precursors upon irradiation followed by regeneration of the ISC pool has also been demonstrated (van Es et al., 2012).

Neutral Competition in the ISC NicheAn intestinal crypt contains about 14 equal ISCs that all divide each day. Their dynamics are consistent with a model in which the resident ISCs double their numbers each day and stochastically adopt either stem or TA fates. Thus, ISCs divide symmetrically while competing for a niche of limited size. As a consequence, their turnover follows a pattern of neutral drift dynamics and crypts tend toward clonality within a period of 1–6 months. Therefore, ISCs persist for life as a population, yet only the lineage of one particular ISC is present in each crypt at any given time (Snippert et al., 2010).

Signaling in ISCsISCs are specified by high levels of WNT signaling in the crypts. R-spondin binds to LGR4/LGR5 receptors and potentiates WNT signals in ISCs (de Lau et al., 2011). On the contrary, the activity of Znfr3/Rnf43 negatively controls WNT signals in the ISC pool by ubiquitinating Frizzled receptors (Koo et al., 2012). Paneth cells secrete the WNT3 ligand constitutively, but an additional Wnt source also exists in the surrounding stroma. Notch signaling mainly acts by inhibiting the secretory fate in ISCs (Pellegrinet et al., 2011). Notch ligands (Dll1 and Dll4) are expressed by surrounding secretory cells, including the Paneth cells. Mitogenic stimuli regulate the size of the proliferative compartment. Lrig1, a marker for stem and early TA cells, acts as a negative regulator of receptor tyrosine kinase (RTK) activity (Wong et al., 2012). BMP signaling inhibits the stem cell fate in the intestine (Haramis et al., 2004) probably by antagonizing WNT signaling. BMPs are mainly expressed by stromal cells that surround the epithelium, whereas ISCs are protected from their action by the presence of local inhibitors, including Gremlin.

RefeRences

Barker, N., van Es, J.H., Kuipers, J., Kujala, P., van den Born, M., Cozijnsen, M., Haegebarth, A., Korving, J., Begthel, H., Peters, P.J., et al. (2007). Identification of stem cells in small intestine and colon by marker gene Lgr5. Nature 449, 1003–1007.

Buczacki, S.J.A., Zecchini, H.I., Nicholson, A.M., Russell, R., Vermeulen, L., Kemp, R., and Winton, D.J. (2013). The intestinal label-retaining cell expresses Lgr5 and is a committed secretory precursor. Nature. Published online February 27, 2013. http://dx.doi.org/10.1038/nature11965.

de Lau, W., Barker, N., Low, T.Y., Koo, B.K., Li, V.S., Teunissen, H., Kujala, P., Haegebarth, A., Peters, P.J., van de Wetering, M., et al. (2011). Lgr5 homologues associate with Wnt receptors and mediate R-spondin signalling. Nature 476, 293–297.

Haramis, A.P., Begthel, H., van den Born, M., van Es, J., Jonkheer, S., Offerhaus, G.J., and Clevers, H. (2004). De novo crypt formation and juvenile polyposis on BMP inhibition in mouse intestine. Science 303, 1684–1686.

Koo, B.K., Spit, M., Jordens, I., Low, T.Y., Stange, D.E., van de Wetering, M., van Es, J.H., Mohammed, S., Heck, A.J., Maurice, M.M., and Clevers, H. (2012). Tumour suppressor RNF43 is a stem-cell E3 ligase that induces endocytosis of Wnt receptors. Nature 488, 665–669.

SnapShot: The Intestinal CryptHans Clevers1 and Eduard Batlle2,3

1Hubrecht Institute, KNAW and University Medical Centre Utrecht, Uppsalalaan 8, 3584CT Utrecht, the Netherlands2Institute for Research in Biomedicine (IRB Barcelona), 08028 Barcelona, Spain3Institució Catalana de Recerca i Estudis Avançats (ICREA), 08010 Barcelona, Spain

Pellegrinet, L., Rodilla, V., Liu, Z., Chen, S., Koch, U., Espinosa, L., Kaestner, K.H., Kopan, R., Lewis, J., and Radtke, F. (2011). Dll1- and dll4-mediated notch signaling are required for homeostasis of intestinal stem cells. Gastroenterology 140, 1230–1240.e1–e7.

Sato, T., Vries, R.G., Snippert, H.J., van de Wetering, M., Barker, N., Stange, D.E., van Es, J.H., Abo, A., Kujala, P., Peters, P.J., and Clevers, H. (2009). Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature 459, 262–265.

Snippert, H.J., van der Flier, L.G., Sato, T., van Es, J.H., van den Born, M., Kroon-Veenboer, C., Barker, N., Klein, A.M., van Rheenen, J., Simons, B.D., and Clevers, H. (2010). Intestinal crypt homeostasis results from neutral competition between symmetrically dividing Lgr5 stem cells. Cell 143, 134–144.

van Es, J.H., Sato, T., van de Wetering, M., Lyubimova, A., Nee, A.N., Gregorieff, A., Sasaki, N., Zeinstra, L., van den Born, M., Korving, J., et al. (2012). Dll1+ secretory progenitor cells revert to stem cells upon crypt damage. Nat. Cell Biol. 14, 1099–1104.

Wong, V.W., Stange, D.E., Page, M.E., Buczacki, S., Wabik, A., Itami, S., van de Wetering, M., Poulsom, R., Wright, N.A., Trotter, M.W., et al. (2012). Lrig1 controls intestinal stem-cell homeostasis by negative regulation of ErbB signalling. Nat. Cell Biol. 14, 401–408.

1198.e2 Cell 152, February 28, 2013 ©2013 Elsevier Inc. DOI http://dx.doi.org/10.1016/j.cell.2013.02.030