The intestine is a complex organ that has evolved to maximize the luminal surface area for the purpose of absorbing nutrients. The epithelial lining of the intestine must maintain itself in the face of a constant challenges from environmental factors such as microbes and food. Maintaining this epithelial lining, highly organized structure and cellular diversity is a highly proliferative population of intestinal stem cells. Intestinal stem cells are found in the crypt of Lieberkühn, which is the functional unit forming the stem cell niche. Aberrant regulation or mutations within the stem cells can lead to gastrointestinal pathologies. The niche within the crypt is complex, made up of cell-cell interactions, extracellular matrix and signaling pathways; chief among these is the WNT signaling pathway. The intestine has become a well-studied paradigmatic model system for understanding WNT signaling and its role in stem cell dynamics. The spatial restriction of WNT ligands by other pathway modulators, such as RSPO proteins, functions to balance the long term maintenance of intestinal stem cells while encouraging the rapid production of new progeny required for continual renewal of the epithelium in order to conduct its absorptive and protective roles. This chapter will specifically focus on the role of WNT signaling in the maintenance of intestinal stem cells, and will discuss the outcomes when this system is perturbed.
KeywordsIntestine, Gastrointestinal, Crypt base columnar, β-Catenin, Wnt, Stem cells
The gastrointestinal (GI) tract must maintain a barrier to food antigens and the microbial communities within the gut, while continually producing an array of specialized cell types to carry out digestive, absorptive, and regulatory functions unique to each regional segment. To fulfil this diversity of roles, the intestinal epithelium has evolved to maximize epithelial surface area through the formation of villi, which are finger-like projections that protrude into the intestinal lumen. The villi are covered in epithelial cells that take part in nutrient absorption and innate defense. Given the high burden placed on the intestinal epithelium, it is replaced rapidly, with the entire epithelial surface turning over approximately every 3–5 days. To accommodate this rapid turnover, the intestine possesses a specialized compartment at the base of the villi called the crypt of Lieberkühn, which houses highly proliferative stem cells at its base and transit-amplifying progenitor cells closer to the lumen. Cells within the intestine are born and die in a very ordered manner, when the intestinal stem cell (ISC) divides, one of the daughters moves luminally toward the villus and becomes a rapidly proliferating transit-amplifying cell, followed by terminal differentiation near the crypt opening. Subsequently, differentiated cells continue to move toward the villus tip like a conveyer belt until they undergo programmed detachment and cell death as they are shed into the lumen ( Fig. 7.1 ). The architecture of intestinal crypts sequesters and protects stem cells from gut microbe-derived metabolites that otherwise might inhibit stem cell proliferation. It has been speculated that crypts may have coevolved with microbial communities in our intestine as a way to physically protect stem cells from microbial metabolites that may be detrimental to ISC function.
Somatic stem cells, such as those found in the intestine, are required to both maintain normal tissue homeostasis and to repair tissue following injury or damage. In order for any cell to be considered a stem cell, two key requirements must be met: the stem cell must self-renew throughout life; and, it must give rise to daughter cells which can give rise to differentiated, specialized cell types. Stem cells and their progeny are frequently described as following a hierarchy in which stem cells sit at the top, where they undergo self-renewal, giving rise to highly proliferative transit-amplifying progenitor cells, each of which can then differentiate into one of multiple cell lineages. However, as in most biological systems, the intestine does not perfectly conform to the stereotype, and a remarkable amount of plasticity has been demonstrated within the crypt. Normal epithelial homeostasis in the intestine is maintained by a population of highly proliferative cells called the “crypt base columnar” (CBC) stem cells and the hierarchical model of stem cell dynamics is largely satisfied in this context. However, following a physiological stress, cells that have left the crypt and become committed progenitors to the secretory or absorptive lineages can revert back, once again adhering to the definition of a stem cell. In addition to this reversion, populations of “reserve” stem cells have been identified that are resistant to different types of damage, such as radiation injury.
The intestine has been a powerful model system to study stem cell dynamics, due to its organized tissue architecture; however, it has also been an intense focus of study due to a significant number of diseases that affect this system, such as inflammatory bowel diseases and intestinal cancers. Through these studies, as described throughout this chapter, WNT signaling has emerged as a key effector in both normal ISC homeostasis, and in disease. This chapter provides a brief historical perspective on our understanding of WNT signaling, a broad introduction to the WNT signaling network, and then discusses how WNT signaling plays a role in the intestine, during embryonic development, in the normal adult intestine, and in disease.
A Historical Perspective of WNT Signaling in the Intestine
The first WNT homolog, Wingless ( wg ), was described in Drosophila melanogaster in 1976, and was shown to be critical for wing development. Nüsslein-Volhard and Wieschaus subsequently identified wg in a forward genetic screen as a gene required for normal segmentation and polarity of the embryo. Around the same time, Nusse and Varmus found that a mouse mammary tumor virus induced tumors by integrating into the chromosome and activating the expression of an unknown gene that was named Int1 (integration 1). Int1 was then demonstrated to be a spontaneous loss-of-function mutat allele in the mouse that was described in 1967 which resulted in mice that lacked part of the cerebellum. Int1 and wg were subsequently identified as homologues, and gene-targeting experiments in mice confirmed that genetic deletion of Int1 led to aberrant cerebellar development. A portmanteau of the words wg and Int1 lead to coining the gene as “ Wnt ”.
Following the discovery of the initial WNT proteins, many more WNT pathway components were identified in forward genetic screens carried out in Drosophila , and mutations were identified that caused similar polarity and patterning defects to that of wg mutations. These components included armadillo (β-catenin), disheveled, porcupine, and zeste white 3 ( GSK3β ).
A connection between WNT signaling and the intestine was made with the discovery of a mouse with a dominant mutation that predisposed the animal to multiple intestinal neoplasia (MIN), which was subsequently demonstrated to be a mutation in the adenomatous polyposis coli ( Apc ) gene, and which makes up part of the destruction complex (see the next section). Null mutations in Apc lead to stabilization of β-catenin (encoded by the Ctnnb1 gene, we will use β-catenin throughout for both gene and protein), its subsequent nuclear localization and constitutive transcriptional activation. Following these discoveries, it was demonstrated that β-catenin bound with TCF/LEF transcription factors to modulate gene expression, that TCF4 was critical for mediating β-catenin-dependent transcription in the intestine, and that stabilizing β-catenin mutations led to colon cancer. Much of this early work helped shape the current understanding of the importance of WNT signaling in the ISC .
Historically, two types of ISCs were identified: CBCs cells and label-retaining (or “+ 4”) cells. These studies used techniques such as tracking of radiolabeled particles that were phagocytosed or incorporation of radioactive 3H-thymidine incorporation into DNA (label-retaining) in order to mark and follow these cells over time . It was previously thought that the + 4 cell was a largely quiescent stem cell that sat atop of the stem cell hierarchy and rarely divided, but ultimately would give rise to CBCs, which were more proliferative . More recently, however, it has been accepted that the + 4 cell is likely not a specialized reserve population of stem cells, rather, the evidence demonstrates that this cell is a secretory progenitor cell that has the capability of reverting into a stem-like state upon injury to the CBCs ( Fig. 7.1 ).
CBC cells have been defined using molecular markers and transgenic lineage tracing techniques (see Section 7.5 ), and are the proliferative engines that drive cellular production in the crypts, dividing daily with frequent turnover. The + 4 cells have been redefined as “reserve ISCs” (rISC) and appear to have a phenotype that is similar to an enteroendocrine progenitor cell, dividing infrequently under homeostatic conditions but being called upon to produce new CBCs in response to injury or other stimuli. Evidence also suggests that there is a large amount of plasticity among the cells in the crypts, particularly in the context of injury, and in addition to the rISCs, both secretory and absorptive progenitor cells can revert and give rise to new CBCs following injury.
The Nuts and Bolts of WNT Signaling
WNT signaling is mediated by secreted WNT ligands binding to receptors on the cell surface. This binding starts a complex cascade of events downstream of the ligand-receptor interaction, typically referred to as a “signal transduction pathway”. The term “pathway” invokes the notion of a linear path from one point to another, and it is worth noting that this concept far oversimplifies the complex events that actually occur downstream of the ligand-receptor interactions. Conceptually, it is more accurate to envision that there is a broad network of events that take place downstream of the ligand-receptor interaction, and that this network of events will be different in different cellular contexts; for example, the cascade of events in the ISC may be far different from those that occur in a different cell or tissue, and may be distinct from other cells within the crypt. However, the network of events that takes place downstream of the ligand-receptor interactions are incompletely characterized, and so for the purpose of this chapter, an overview of the basic elements of the WNT signaling “pathway” is provided. For additional reading, many excellent reviews cover this topic at length.
The WNT Signaling Module
WNT signaling takes place through a module made up of receptors, transducers, and effectors ( Fig. 7.2 ). WNT ligands bind to the Frizzled (FZD) family of G-coupled protein receptors along with a member of the LRP family of coreceptors. WNT-ligand binding to the FZD-LRP receptor complex initiates intracellular events that transduce the ligand-receptor interaction. The signaling strength of the ligand-receptor interaction is mediated, in part, by the ability of the receptors to accumulate at the cell surface. Accumulation of the FZD-LRP receptor complex is controlled by membrane bound E3-ubiquitin ligases ZNRF3 and RNF43, which act to stimulate ubiquitination, internalization, and degradation of the FZD-LRP complex. A second ligand-receptor interaction, which involves the R-spondin(RSPO) family of secreted ligands and LGR receptors, act to potentiate WNT signaling by binding to ZNRF3/RNF43. The LGR-RSPO-ZNRF3/RNF43 complex is then ubiquitinated in an E3 ligase-dependent manner, internalized, and targeted for degradation. This prevents the ubiquitination and internalization of the FZD-LRP receptor complex, allowing receptors to accumulate on the cell surface, thereby amplifying the number of WNT ligand-receptor complexes that form in order to stimulate downstream signaling ( Fig. 7.2 ).
One of the main transducers of WNT signaling in the ISC is β-catenin, a protein that is tightly regulated in both the inactive and active states. In the absence of WNT ligand (inactive state), β-catenin is primarily found at adherens junctions where it plays an important role in epithelial cell:cell adhesion . Free β-catenin that is not incorporated into adherens junctions is sequestered in the cytoplasm and actively targeted for degradation by a protein machine referred to as the destruction complex, which phosphorylates β-catenin near its amino terminus thus targeting it for ubiquitin- dependent proteasomal degradation. The destruction complex includes glycogen synthase kinase 3β GSK3β, CK1, AXIN and APC tumor suppressor proteins, where AXIN and APC function as scaffolds and interact with β-catenin directly. CK1 and GSK3β are Ser/Thr kinases that phosphorylate β-catenin, which is subsequently recognized by β-TrCP, a component of an E3 ubiquitin ligase complex. As a consequence, once ubiquitinated, β-catenin is degraded rapidly by the 26S proteasome. Following WNT ligand binding and dimerization of the FZD-LRP receptors, β-catenin accumulates in the cytoplasm and translocates into the nucleus, where it acts as a transcription factor; however, the mechanism by which β-catenin is stabilized is not entirely clear, and several models for this regulation exist. One study suggested that active WNT signaling inhibits β-catenin ubiquitination within the destruction complex, saturating the destruction complex with phosphorylated β-catenin and thus allowing newly synthesized β-catenin to accumulate in the cytoplasm and translocate to the nucleus. A second model proposed that phosphorylation of AXIN by GSK3 β kept AXIN active (“open”) in order to bind β-catenin. Upon ligand-receptor interactions, AXIN formed a complex with LRP, and the AXIN-LRP interaction subsequently promoted AXIN dephosphorylation and inactivated (“closed”) the AXIN complex through an intramolecular interaction. Inactivation of AXIN diminished its association with β-catenin and inhibited β-catenin phosphorylation. Yet another layer of complexity is added to the regulation of the destruction complex as AXIN itself forms a multimeric complex that is stabilized by APC, thus increasing the ability of the destruction complex to process β-catenin.
Once WNT signaling is active, β-catenin accumulates in the cytoplasm and is translocated to the nucleus where it interacts with the TCF/LEF family transcription factors, including TCF1, TCF3, TCF4 (also known as TCF7L2), and LEF1 in mammals, which guide β-catenin to specific DNA loci (reviewed in Refs. In the absence of β-catenin, TCF/LEF proteins bind to target DNA sequences and interact with Groucho/TLE corepressors to repress transcription of nearby target genes. On entering the nucleus, β-catenin displaces Groucho/TLE recruits additional coactivators to form a TCF-β-catenin complex that can initiate transcription. Genome-wide analysis of DNA binding by β-catenin in the murine intestinal epithelium and in colorectal cancer (CRC) cells demonstrated that the majority of β-catenin bound loci colocalized with TCF4 sites. A second class of β-catenin bound DNA colocalized with a minimal amount of TCF4; however, the latter drove only minimal transcription. When TCF/LEF and β-catenin are bound to DNA, transcriptional corepressors are displaced, and transcriptional coactivators are recruited. There is a large number of coactivators potentially recruited to the DNA; however, one of the major events that takes place is the recruitment of histone acetyltransferases (HATs) including CREB-binding protein (CBP) and its close relative p300, which act to acetylate histones. Increased histone methylation of lysine 4 (H3-K4me3) at various target promoters has also been documented, and both histone modifications are associated with increased transcription of target gene mRNA.
Development of the Intestinal Stem Cell
Intestine development is a complex process that includes regional patterning and morphogenetic events that give rise to region-specific functions and structures in the intestine. These early developmental processes rely on epithelial-mesenchymal cross talk to establish the cell-intrinsic regional identity of the epithelium. This signaling also controls an epithelial transition from a simple, relatively smooth, epithelial tube surrounded by mesenchyme to the formation of the stereotypical villus units that have evolved to provide an increased absorptive surface area; it is during this process of villus emergence that the future crypt domain is also established.
Evidence in adult mice has demonstrated that the ISCs are intrinsically programmed with their location/region specific function and gene expression patterns. This is supported by classical embryological studies, which have shown that the mesenchyme has potent inductive properties during early development and is instructive for the regional identity of the endoderm ; however, once the different regions of the intestine are specified (i.e., duodenum jejunum and ileum), the endodermal compartment then retains this positional information. Thus, it is not entirely surprising that when crypts from the duodenum, jejunum, or ileum of the adult were isolated and grown in culture for extended periods of time, they retained their region-specific gene expression programs. How this regional identity is established, is still not entirely clear. Recent evidence has suggested that gradients of growth factor signaling along the developing gastrointestinal tract may be responsible for specifying regional intestinal identity, with increasing levels of WNT and FGF signaling inducing progressively more posterior fates ; however, it is likely that anterior-posterior patterning is far more complicated than currently appreciated. Similarly, how the regional pattern is maintained is also not clear, but ex vivo culture of specific diverse regions of the intestine retain their identity, suggesting that the environment is not required to actively maintain regional identity. It is possible that epigenetic mechanisms play a role in this regional maintenance.
Formation of the proliferative compartment in the intestine begins concurrently with villus emergence during embryonic development. At this period of time, the flat epithelial tube transitions from a pseudostratified epithelium to a columnar epithelium during villus emergence, and proliferation becomes restricted to the domains between villi called the intervillus domains, which will ultimately deepen and give rise to bona fide crypts in the adult. Recent attention has been placed on understanding the transition from an immature progenitor cell state in the fetal intestine to the adult ISC. Through this work, it has become clear that there are significant differences between the fetal intestinal progenitor cell and the mature, postnatal, ISC, though the significance of these differences is yet to be fully appreciated.
Several studies have demonstrated that the ISC marker, LGR5, is expressed in intestinal progenitor cells as early as embryonic day (E) 8.5 during embryonic development in mice, whereas intervillus domains are not present until around E15.5, and bona fide crypts do not appear until postnatal week 2 in the mouse.
Interestingly, while Lgr5 is known to be a sensitive WNT-signaling target gene in adult ISCs, it appears that WNT-signaling is dispensable for regulation of Lgr5 in the early fetal intestine, before intervillus domains are present, at a time when Lgr5 gene expression is robust. Indeed, genetic studies have demonstrated that blocking WNT signaling prior to E15.5 has little effect on proliferation, whereas loss of WNT signaling following villus formation at E15.5 is catastrophic. Interestingly, while Lgr4 and Lgr5 play redundant roles to potentiate WNT signaling in the adult (see Section 7.5.2 ), loss of Lgr5 has only modest effects on cytodifferntiation when genetically deleted, and Lgr4 appears to be the dominant receptor in the developing intestine. Consistent with the notion that stem/progenitor cells are significantly different in the developing and adult intestine, studies have used transcriptional profiling (RNA sequencing) to show that the transcriptome is vastly different when comparing Lgr5 + cells in the fetal and adult intestine. Mechanistically, it appears that the transcription factor ID2 is expressed in the early fetal intestine and acts to repress WNT-signaling target genes, and genetic deletion of Id2 leads to precocious activation of target genes.
In addition to regional patterning and emergence of the stem cell domain (intervillus domain, crypt), it is also clear that there are major changes along the developmental continuum leading to maturation of intestinal epithelial cells into functional cell types. In this context, maturation is defined as the ability of the epithelium to express genes required for digesting nutrients in postnatal life. In mice, maturation is partly controlled by a transcription factor, BLIMP1 (PRDM1), which functions to repress genes expressed in the postnatal intestine, and thus Blimp1 is progressively downregulated as development progresses. In the adult intestine, a similar maturation must take place as ISC daughters transition from a proliferative state toward a differentiated cell type responsible for a specific cellular function, where the cell acquires the ability to perform specific cellular tasks through the expression of specific genes and proteins (i.e., an enterocyte must express digestive enzymes). In this context, it has been demonstrated that the stem cells retain their ability to make different cell fate choices by maintaining an accessible chromatin state. This allows access of cell-specific transcription factors to the necessary chromatin in order to drive appropriate transcriptional programs. In addition, the intestine-specific transcription factor CDX2 plays an important role in the transition from a stem cell state to a differentiated state by shifting the sites where it is bound to chromatin. That is, CDX2 is selectively bound to a smaller number of genomic loci in the stem cell state, but during differentiation into mature functional cell types, CDX2 binding expands to include more genomic loci where genes reside whose expression is required for cell-specific function.
Through studies of the developing immature and adult intestine (see also Section 7.5 ), we have begun to appreciate that there are major changes throughout an organism’s lifespan, and that intestinal stem/progenitor cells are regulated by multiple mechanisms across that time. It has recently been demonstrated that the fetal and adult gastric epithelium also changes across developmental time, but that during injury and repair of the adult stomach, a “fetal program” is reactivated. Thus, it is likely that insights into intestine development will inform our understanding of how regeneration and injury-repair takes, and changes associated with aging take place in the adult.
WNT/β-Catenin Signaling in the Adult ISC
Many different inter- and intracellular signals are integrated to maintain a stable pool of ISCs while simultaneously producing a rapidly proliferating progenitor population that can continuously reconstitute the epithelium lining the surface of the intestines. These include paracrine growth factor signaling pathways such as WNT, NOTCH, HIPPO/YAP, EGF, and other Receptor Tyorisine Kinases; however, ISC are also able to adjust their physiology based on nutrient states, the constituency of adjacent cells types, and epithelial integrity/injury. This section will focus on the role of WNT/β-catenin signaling activity, and its role in the maintenance of ISCs.
WNT/β-Catenin Signaling is the Major Driver of ISC Activity
WNT and RSPO Protein Activity in the ISC Niche
WNT ligands are cysteine-rich proteins that undergo extensive posttranslational modifications, including glycosylation and acylation, which affect protein secretion and stability. In humans, there are 19 WNT ligands, and more than 10 receptors/coreceptors from multiple protein families, this diversity often makes it difficult to discern which cells are sending and/or receiving WNT ligands within a complex environment such as the ISC niche. Indeed, within the intestine, at least 7 WNT ligands are expressed, in either the epithelial or mesenchymal compartments, with Wnt2B, Wnt4 , and Wnt5A being expressed in the mesenchyme and Wnt3, Wnt6, Wnt9b, and Wnt11 being expressed in the epithelial compartment. Studies had previously shown that Paneth cells are an important niche cell in the crypt, supporting the ISCs by secreting WNT ligands which are crucially required for their maintenance. Subsequent reports also showed that diverse cell types can secrete WNT proteins within the niche, such as stromal cells, and that these alternative sources of WNT ligand can act in a functionally redundant manner to support ISCs. For example, when Paneth cells are removed through genetic approaches, or epithelial WNT ligand secretion fails to develop or is blocked in the adult intestine with conditional mutations, the mesenchyme/stroma can provide an alternative source of WNT ligands. However, the precise mesenchymal/stromal cell types that secrete WNT proteins to support ISCs in the absence of Paneth cells are still not entirely clear. WNT ligand secretion from smooth muscle and from intestinal subepithelial myofiborblasts (ISEMFs) was shown to be dispensable, while a population of Foxl1 expressing mesenchymal cells was identified and shown to express specific Wnt mRNAs. When Foxl1- positive cells were genetically ablated, the ISCs failed to undergo self-renewal, and the cell population within the crypts ultimately collapsed. However, experiments have not conclusively demonstrated that WNT proteins secreted by Foxl1- positive cells are specifically able to support ISC function, and experiments blocking WNT secretion from this specific cell type will be needed to determine if this is the case.
The RSPO family of proteins acts to potentiate WNT signaling in the intestinal crypt (de Lau et al. ; see Section 7.3.1 ), and recent work has shown that RSPO3 caused increased proliferation when overexpressed, ultimately expanding the ISC compartment and leading to tumorigensis, consistent with known correlations between RSPO mutations and colon cancer in humans. Gain- and loss-of-function studies have recently shown that RSPO proteins, but not WNT proteins, act to limit the size of the ISC compartment. When WNT proteins were overexpressed systemically, there was little effect on the stem cells or crypt morphology. On the other hand, when RSPO proteins were overexpressed systemically, the crypt compartment expanded and housed more ISCs. This effect was amplified when both WNT and RSPO proteins were overexpressed, in which case, the proteins acted synergistically to further expand crypt size and stem cell number. Importantly, WNT proteins are an absolute requirement for ISC maintenance, since RSPO protein overexpression coupled with WNT protein inhibition leads to a loss of ISCs. These experiments collectively demonstrated that RSPO proteins act to limit the size of the crypt and the number of stem cells, and suggests that there are likely cells near the crypt that provide a local source of RSPO proteins that control crypt size. At this time; however, the specific cellular source of RSPO proteins necessary to support the ISCs within the crypt has not been identified.
Canonical WNT/β-Catenin Signaling in the ISC
WNT and RSPO proteins function at the cell surface to initiate the WNT signaling cascade (see Section 7.3.1 ), leading to the stabilization of WNT/β-catenin, which translocates to the nucleus and associates with TCF/LEF transcription factors to control transcription . β-catenin is the main transducer of the canonical WNT signaling cascade, and nuclear β-catenin levels are the highest in cells at the base of the crypt, including both Paneth cells and ISCs. This establishes an interesting paradigm where high levels of WNT/β-catenin signaling are required for terminal differentiation of Paneth cell themselves an important producer of WNT ligand, while high WNT levels are also required in the ISC niche to maintain the ISC population. Indeed, conditionally deleting the gene encoding for β-catenin, Ctnnb1 , causes ISCs to lose their stem cell identity and differentiate.
In the nucleus, β-catenin interacts with the TCF family of transcription factors. One of the first associations between β-catenin and TCF proteins in the intestine was identified in knockout mice that lacked Tcf4 , where it was demonstrated that loss of Tcf4 led to a halt in epithelial proliferation and a collapse of the intestine shortly after birth. Tcf4 was subsequently demonstrated to have an essential role in maintaining the ISCs of adult mice and zebrafish and was shown to be the dominant mediator of WNT/β-catenin-induced transcriptional activity.
Major Transcriptional Targets of WNT/β-Catenin/TCF That Regulate ISC Function
Myc and Ccnd1/2
β-catenin/TCF4 complexes are known to drive transcription of several target genes that are critical for normal ISC regulation. Myc (also known as c-Myc ), is a well-described β-catenin target gene in other tissues, and has been shown to be expressed in the crypt compartment and in the ISCs. Myc is a proto-oncogene, and forced expression of Myc recapitulates some, but not all, of the phenotype seen in mice with increased β-catenin-activated transcriptional activity caused by inactivating Apc mutations including increased proliferation and dysplasia. Myc is also an important downstream mediator of β-catenin transcription in the context of Apc mutations, and genetic deletion of Myc from the epithelium has been shown to rescue many of the cellular changes that occur following loss of Apc, including a rescue of perturbed differentiation, migration, proliferation and apoptosis. Rescue of Apc mutant epithelium occurred despite the high levels of nuclear β-catenin that were present, suggesting that MYC is an important mediator of β-catenin-dependent transcription in tissue with Apc mutations. Consistent with these data, acute genetic deletion of Myc from the intestinal epithelium led to a loss of mutant crypts and cells. In this case, mutant cells had reduced proliferation and were rapidly replaced with wild-type cells that had escaped genetic recombination. Paradoxically, MYC does not appear to be required for normal intestinal homeostasis. When Myc is genetically deleted in the intestinal epithelium shortly after birth, mice appear to adapt to the loss. Following an initial lag in growth and proliferation in the epithelium of juvenile mice, the epithelium recovered and maintained Myc deficient cells in the crypt.
Much like MYC, CYCLIND1 ( Ccnd1 ) is a cell cycle regulator, thought to be a direct β-catenin target gene. While CCND1 is required for WNT/β-catenin signaling mediated tumor growth, loss of Apc, and activation of WNT/β-catenin signaling did not lead to an increase in Ccnd1 gene expression until 20 days after genetic deletion. On the other hand, CyclinD2 ( Ccnd2 ) is upregulated immediately following the genetic deletion of Apc , and genetically removing Ccnd2 in Apc mutant mice led to a reduction of proliferation and inhibition of tumor formation and growth. These studies collectively suggested that Ccnd2, not Ccnd1 , may be immediate β-catenin target gene.
Following the discovery of LGR5 as a marker of ISCs, and the generation of a genetically modified mouse that allowed these cells to be identified and isolated using Lgr5 -driven green fluorescent protein (GFP), an “Lgr5+ stem cell signature” was identified using transcriptional and proteomic profiling. Among this gene signature was the transcription factor, ASCL2, which had also been identified as a WNT/β-catenin target gene in CRC cell lines, and which was shown to be expressed in the mouse intestinal crypt as well as human and mouse intestinal cancers. Consistent with this correlative data, overexpression of Ascl2 in the mouse intestine led to crypt hyperplasia, an expansion in size of the crypt domain and the formation of ectopic crypts, whereas genetic deletion of Ascl2 led to a loss of the LGR5+ ISCs. ASCL2 was subsequently shown to cooperate in the nucleus with both TCF4 and β-catenin, binding to DNA at several of the same target gene loci. In addition, ASCL2 was shown to bind DNA at its own genomic locus, thus forming an autoregulatory loop, where levels WNT/β-catenin signaling dictate Ascl2 gene expression, and once a threshold of Ascl2 is reached, it becomes autoactivating. In this way, ASCL2 acts as a bistable switch in the ISC, and helps to translate the WNT/β-catenin signaling gradient that exists along the crypt-villus axis in the intestinal epithelium so that only cells with high WNT/β-catenin signaling levels will lead to expression of ASCL2 and a subsequent “On” state. Conversely, when WNT/β-catenin signaling is low, sufficient ASCL2 is not induced and the autoregulatory state remains “Off”.
SOX9 is expressed throughout the crypt base, and was shown to be a direct target of β-catenin/TCF4. Disruption of WNT/β-catenin signaling by genetically deleting Tcf4 in the adult intestine led to a progressive loss of Sox9 expression, and removal of Ctnnb1 or other WNT signaling components in the embryonic intestinal epithelium also led to loss of Sox9 expression. Interestingly, genetically removing Sox9 led to increased proliferation and a loss of Paneth cell differentiation, suggesting that it plays two major roles in the intestine: first, acting as a negative feedback to dampen WNT/β-catenin- driven proliferation in the crypt, and second, acting to promote Paneth cell differentiation.
Within the crypt, different cell populations express distinct levels of SOX9 protein. The LGR5+ CBC ISCs express the lowest levels of SOX9, while the Paneth cells, and a Chromogranin A (CHGA)-positive population of cells express higher levels of the protein. The SOX9/CHGA-positive population of cells in the crypt is thought to be an enteroendocrine progenitor cell, which retains the capacity to serve as an alternative pool of ISCs throughout life and in response to injury. This pool of cells, which also expresses Bmi1 and Prox1 , is thought to act as a major source of stem cells following injury, such as radiation, which kills LGR5+ CBCs. SOX9 is critical to maintain the alternative ISC enteroendocrine progenitor pool, as genetic deletion of Sox9 leads to a loss of this population and the inability to undergo repair following radiation injury.
Lgr5 was first identified as a putative transcriptional target of WNT/β-catenin/TCF in the intestine in a screen of intestinal cancer cell lines in which WNT signaling was activated or blocked. Lgr5 was subsequently shown to be exclusively localized to the CBC ISCs in the crypt-base of the small intestine and colon, and the Lgr5 gene locus was used to generate a genetically modified mouse in which the Lgr5 + cells expressed GFP and an inducible form of Cre recombinase, allowing for genetic lineage tracing. This genetically modified mouse has been used to formally demonstrate that Lgr5+ cells are bona fide stem cells, undergoing self-renewal and giving rise to all lineages of the small and large intestinal epithelium. These mice have also allowed for the isolation, purification, and culture of these cells, facilitating a boon in our understanding of the biology of the ISC. Functionally, Lgr5 is dispensable for ISC self-renewal, and the functionally redundant Lgr4 has been shown to compensate in the event the Lgr5 is genetically deleted in the ISC. When both Lgr4 and Lgr5 are removed, the CBC ISC population is lost. Interestingly, research on the Lgr5+ ISC has also led to an appreciation that there is a remarkable amount of cellular plasticity within the adult crypt, since genetic, or radiation-mediated ablation of ISCs has revealed that these cells are dispensable for intestinal function, and that there are alternative/reserve stem cell pools that can compensate for loss of CBCs.