the crypt/villus hierarchy
By the process of proliferation, the murine intestinal epithelium renews itself approximately every 5 days. Intestinal epithelial cell proliferation is confined to the crypt compartment and is initiated by asymmetric stem cell division which results in the production of an identical stem cell (retaining the original DNA strand as a protective mechanism against replication errors) and a differentiated progenitor cell (a committed transit-amplifying cell). The transit-amplifying cell cycles very quickly compared to the original stem cell. As the transit-amplifying cell undergoes several divisions, it migrates upwards along the crypt-villus axis in the small intestine and along the crypt axis in the colon, before undergoing differentiation (23). The first few of about 6 cell generations in the small intestine and 8 cell generations in the colon cells retain some stemness and are capable of replacing the ultimate stem cells when they die. These cells are referred to as clonogenic cells and constitute a reserve force to supply additional stem cells to preserve crypt survival. The clonogenic cell population is about 30-40 cells per crypt while about 124 dividing transit committed cells completely lose their stemness (24). As these committed precursor cells migrate upwards, they gradually differentiate when they reach the crypt-villus junction in the small intestine and the upper part of the large intestinal crypt. They differentiate predominantly into absorptive enterocytes and secretory lineages, particularly goblet cells and enteroendocrine cells (4, 12, 24-26). Paneth cells represent the fourth cell lineage in the small intestine and result from the downward migration of committed precursor cells. Paneth cells reside in the crypt base for around 20 days (27). The upwardly migrating epithelial cells are
33 organised in coherent lines from the upper third of the crypt until they reach the villus tip in the small intestine and inter-crypt table in the colon where they are eventually removed by apoptosis (26).
Regulation of proliferation and differentiation is very important for maintaining homeostasis within intestinal epithelia. This equilibrium is dynamic and depends upon tissue conditions. For instance, tissue injury triggers more proliferation through rapid division of precursor cells and an increase in clonogenic cell number (28). Two vital signalling pathways that appear to control intestinal proliferation and differentiation are the Wnt/β-catenin and Notch pathways.
1.3.1 Wnt/β-catenin signalling pathway
Wnt/β-catenin(canonical Wnt) signals influence cell proliferation by regulating progression through the cell cycle. Active Wnt/β-catenin signalling at the bottom of crypts maintains transit amplifying progenitors in a proliferative state. Inactivating Wnt signalling by over expression of the inhibitor DΚΚ1 results in loss of crypt proliferation (29, 30). Conversely, a constitutively active Wnt/β-catenin/TCF pathway keeps intestinal epithelial cells in a proliferative state and suppresses differentiation (31). A cytoplasmic protein β-catenin is a key element of the canonical Wnt signalling pathway. It translocates into the nucleus and stimulates the subsequent transcriptional regulation of Wnt target genes such as the c-myc
oncogene. Therefore, nuclear accumulation of β-catenin is considered to be a hallmark of an activated canonical Wnt signalling. When the trigger of Wnt disappears, β-catenin is removed from the nucleus and is degraded by the tumour
34 suppressor gene product Adenomatous Polyposis Coli (APC). APC therefore acts as a suppressor of Wnt/β-catenin signalling (26).
The role of Wnt signalling in influencing cell proliferation is probably mediated by suppressing the expression of p21CIP1/WAF1. Additionally, it has been shown that Wnt-dependent down regulation of p21CIP1/WAF1 is mediated by c-Myc. Therefore, it has been suggested that Wnt signalling regulates cell cycle progression through up- regulation of c-Myc, which subsequently leads to down-regulation of p21CIP1/WAF1 (31, 32).
1.3.2 Notch signalling pathway
Whereas both Wnt and Notch signals are important for maintaining a proliferative state in the lower crypt, many studies have suggested that the key determinant of cell fate and differentiation in the intestinal epithelium is the Notch signalling pathway. This pathway is stimulated by the interaction between Notch receptors and their ligands. This leads to cleavage of an intracellular domain (ICD) from the transmembrane domain of the receptor and the subsequent translocation of ICD into the nucleus, where it binds to the DNA binding protein RBP-J. Subsequently this complex activates the transcription of Notch target genes such as Hes1 (33, 34). These Notch signalling pathway elements, ligands, receptors and the target gene
Hes1, are expressed in the proliferative cell compartment of the intestinal crypt (35). Inhibiting Notch signalling through intestinal-specific deletion of RBP-J resulted in increased expression of Math1 (which is usually repressed by active Notch signalling), and the replacement of the transit amplifying (proliferative) compartment by goblet cells. This was associated with repression of all epithelial
35 cell division while Wnt signalling stayed active (36, 37). Interrupting the Notch signalling pathway in Zebrafish by mutating DeltaD (which is a Notch ligand) and
mind bomb (in which all Delta-Notch signalling is blocked) also resulted in an increase in goblet cell number in the gut epithelium (35, 38). Murine intestinal epithelium deficient in Hes1 displayed an increase in the number of secretory cell types (goblet cells, entero-endocrine cells and Paneth cells) and a decrease in enterocytes. This suggests that Hes1 functions as a negative regulator of secretory cell type differentiation (39). Additionally, Hes1 inhibits Math1 which is a positive regulator of secretory cell lineage differentiation. Math1 deletion in mice caused a reduction in intestinal epithelial goblet cells, entero-endocrine cells and Paneth cells without affecting enterocytes (40). Therefore, the commitment choice of progenitor cells to differentiate into either absorptive or secretory cell types depends to a certain extent on the balance between Hes1 and Math1 expression (Figure 1.3). However, further downstream regulation of Math1-specified secretory cell lineages is necessary for the differentiation of individual secretory cell lineages. This downstream choice involves the zinc-finger transcriptional repressor Gfi1 which is shown to be lost in Math1-null embryonic intestines. Deleting Gfi1 in mice was shown to cause loss of Paneth cells and a reduction in the number of goblet cells and an increase in the number of enteroendocrine cells. This suggests that Gfi1 is essential for the differentiation of Paneth and goblet cells and that it is a negative regulator of the differentiation of enteroendocrine cells (41). Deleting neurogenin 3 (Ngn3) in mice resulted in complete loss of enteroendocrine cells, while the other three cell types (enterocytes, goblet cells and Paneth cells) developed normally. This suggests that Ngn3 is specifically essential for the enteroendocrine cell fate decision
36 Figure 1.3: Schematic diagram showing the contribution of several regulators to fate decision from intestinal epithelial stem cells.
in intestinal multipotent stem cells (42). Homozygous Kruppel-like factor 4 (Klf4)- null mice die within 15 hours of birth. The colon of these Klf4-null mice showed a 90% decrease in goblet cell numbers. Therfore Klf4 is likely to be a goblet cell- specific differentiation factor (43). Sox9 has been shown to be crucial for the differentiation of Paneth cells (44) (Figure 1.3).
37