The intestinal epithelium is a dynamic self-renewing tissue that consists of multiple cell lineages that are actively and constantly renewed throughout the life of the organism. The crypts of Lieberkühn are the proliferative compartment of the intestine with stem cells residing near the base of colonic crypts. The stem cells give rise to rapidly proliferating transit-amplifying cells that differentiate into three main types of cells: colonocytes (enterocytes in the small intestine), goblet, and enteroendocrine cells. Paneth cells constitute the fourth major cell type that reside adjacent to stem cells at the base of small intestinal crypts. These major cell types are classified either as absorptive (colonocytes or enterocytes) or secretory (goblet, Paneth, and enteroendocrine cells), reflecting distinct genetic programs underlying differentiation of each class.
Notch Signaling is Essential for Making the Lineage Fate Decisions for Developing Epithelial Cells to Become Either an Absorptive or a Secretory Cell
Notch proteins (Notch1, 2, 3, and 4) are type I transmembrane receptors which require cleavage for their activation. When bound by ligands (Delta-like 1, 2, 3 and Jagged 1 and 2 proteins) on adjacent cells, the Notch receptors undergo cleavage at the extracellular and intracellular faces of the plasma membrane to release the Notch intracellular domain (NICD) [1
]. Cleavage of Notch occurs first on the extracellular side by the disintegrin and metalloproteinases ADAM10/ADAM17, which allows subsequent cleavage at the intracellular side by the γ-secretase complex to release NICD. NICD subsequently translocates to the nucleus, where it displaces transcriptional repressors and recruits transcriptional activators to convert the CSL (RBPJ
) complex from transcriptional repression to activation (). In the adult intestine, Notch1 and Notch2 are expressed in the crypt epithelium, and ligands Dll1, Dll4, and Jag1 are expressed in scattered cells within the crypts [2
]. Studies of transgenic animals expressing NICD in the intestinal epithelium found altered intestinal secretory cell production [3
]. Conversely, genetic models that selectively delete Notch1 and Notch2 or its DNA-binding partner, CSL (Rbpj
), in the intestinal epithelium of mice, produce extra intestinal secretory cells [5•
]. Similar effects were observed in transgenic mice in which Notch activity was modified by other genetic manipulations. Loss of Notch activity and conversion of progenitors to secretory cells was observed in animals with defective glycosylation of Notch, caused either by mutation of the fucosyl transferase POFUT1 or by mutation of the enzyme responsible for fucose synthesis (FX), or by mutation of Mindbomb1, a protein essential for endocytosis of Notch ligands and efficient Notch activation [8
]. Importantly, conversion of progenitors to secretory cells was also observed when animals are treated with small-molecule inhibitors of γ-secretase (GSIs), which prevent ligand-induced cleavage of Notch and production of NICD () [11
]. Altered cellular differentiation was also shown in zebrafish and fruit flies with abnormal Notch signaling [14
]. Thus, Notch activity has been shown to promote differentiation of intestinal absorptive cells and to block differentiation of intestinal secretory cells.
Fig. 1 Schematic representation of the role of NOTCH and ATOH1 in intestinal differentiation and carcinogenesis. a Notch signaling is activated upon engagement of the Notch receptor by its ligand (delta/serrate/lag-2 ligands; DSL) on an adjacent cell. Upon ligand (more ...) Notch Selects Hes1 Versus Atoh1 in the Intestinal Epithelium Hes1
(hairy and enhancer of split 1, a basic helix-loop-helix transcriptional repressor), a key target of Notch transcriptional activity, is important for differentiation of the intestinal absorptive cells. Hes1
-null mice develop too many goblet, Paneth, and enteroendocrine cells, at the expense of absorptive enterocytes [16
]. Atoh1 (
atonal homolog 1, also called Math1 or HATH1, a basic helix-loop-helix transcriptional activator) is transcriptionally repressed by Hes1
, and plays a reciprocal role to positively promote secretory lineage differentiation (). Atoh1
-null mice fail to form any secretory cell types and mice die shortly after birth [18
]. Intestine-specific Atoh1
knockout mice, which used the mosaic Fabp-cre
strain to produce a crypt-by-crypt mosaic of Atoh1
-null and adjacent wild-type tissue, demonstrated that Atoh1
is essential for intestinal secretory cell commitment in the adult intestine, with epithelial progenitors undergoing a secretory-to-absorptive fate switch [19
]. Conversely, transgenic Atoh1 overexpression converts progenitor cells into the secretory fate [20
]. Together, these studies show that Atoh1
and Notch play opposing roles in promoting secretory versus absorptive intestinal epithelial cell types. Thus, the relative activity of Notch versus Atoh1 determines whether an intestinal progenitor adopts an absorptive (Notch active) or secretory (Atoh1 active) fate ().
Mechanisms by which Notch and ATOH1 transcriptional activity are reciprocally regulated remain an area of investigation. Notch receptors are active in epithelial progenitor cells including the intestinal stem cells, which require Notch activity for their maintenance [6
]. Delta-like ligands Dll1 and Dll4 are expressed by goblet and Paneth cells, likely under the control of ATOH1 [22
], whereas the expression pattern of Jagged ligands Jag1 and Jag2 is less clear, but is likely regulated by the Wnt/β-catenin pathway [2
]. Thus, in the intestinal crypt, the level of Notch ligand exposure of intestinal stem and progenitor cells is likely to be critical for determining stem cell renewal or differentiation into absorptive or secretory lineage cells. However, in the absence of ATOH1, Notch activity is dispensable for stem cell renewal, proliferation, and absorptive cell differentiation [23•
], suggesting that the key role for Notch is to regulate expression of ATOH1 and consequently cell fate.
The Wnt/β-catenin pathway is also involved in regulating ATOH1 expression. GSK3-βcan phosphorylate ATOH1 and target it for ubiquitination and degradation by the proteasome () [28
]. This targeted degradation appears reciprocal to the involvement of GSK3-βin the Wnt/β-catenin pathway. When the Wnt pathway is inactive, β-catenin is phosphorylated by GSK3-βand targeted for degradation by the Axin/APC complex (). In this context, ATOH1 escapes GSK3-βphosphorylation and is spared from degradation [29•
]. Upon Wnt/β-catenin pathway stimulation, the APC/Axin complex is disrupted and β-catenin escapes phosphorylation and degradation; in this context, GSK3-βphosphorylates and targets ATOH1 for degradation (). Several genes have been identified as transcriptional targets of both active Notch and β-catenin, including Hes1 (), which in turn transcriptionally represses ATOH1 [26•
]. Together, the Notch and Wnt pathways use several mechanisms to regulate ATOH1 expression to control cell fate determination within the intestinal epithelium. A more complete understanding of how crosstalk between the Notch and Wnt pathways impacts intestinal homeostasis as well as tumorigenesis requires further investigation.