Multifaceted involvements of Paneth cells in various diseases within intestine and systemically.

Serving as the guardians of small intestine, Paneth cells (PCs) play an important role in intestinal homeostasis maintenance. Although PCs uniquely exist in intestine under homeostasis, the dysfunction of PCs is involved in various diseases not only in intestine but also in extraintestinal organs, suggesting the systemic importance of PCs. The mechanisms under the participation of PCs in these diseases are multiple as well. The involvements of PCs are mostly characterized by limiting intestinal bacterial translocation in necrotizing enterocolitis, liver disease, acute pancreatitis and graft-vs-host disease. Risk genes in PCs render intestine susceptible to Crohn's disease. In intestinal infection, different pathogens induce varied responses in PCs, and toll-like receptor ligands on bacterial surface trigger the degranulation of PCs. The increased level of bile acid dramatically impairs PCs in obesity. PCs can inhibit virus entry and promote intestinal regeneration to alleviate COVID-19. On the contrary, abundant IL-17A in PCs aggravates multi-organ injury in ischemia/reperfusion. The pro-angiogenic effect of PCs aggravates the severity of portal hypertension. Therapeutic strategies targeting PCs mainly include PC protection, PC-derived inflammatory cytokine elimination, and substituting AMP treatment. In this review, we discuss the influence and importance of Paneth cells in both intestinal and extraintestinal diseases as reported so far, as well as the potential therapeutic strategies targeting PCs.

Gut microbiota promotes stem cell differentiation through macrophage and mesenchymal niches in early postnatal development.

Intestinal stem cell maturation and development coincide with gut microbiota exposure after birth. Here, we investigated how early life microbial exposure, and disruption of this process, impacts the intestinal stem cell niche and development. Single-cell transcriptional analysis revealed impaired stem cell differentiation into Paneth cells and macrophage specification upon antibiotic treatment in early life. Mouse genetic and organoid co-culture experiments demonstrated that a CD206+ subset of intestinal macrophages secreted Wnt ligands, which maintained the mesenchymal niche cells important for Paneth cell differentiation. Antibiotics and reduced numbers of Paneth cells are associated with the deadly infant disease, necrotizing enterocolitis (NEC). We showed that colonization with Lactobacillus or transfer of CD206+ macrophages promoted Paneth cell differentiation and reduced NEC severity. Together, our work defines the gut microbiota-mediated regulation of stem cell niches during early postnatal development.

Paneth cell maturation is related to epigenetic modification during neonatal-weaning transition.

Paneth cells are antimicrobial peptide-secreting epithelial cells located at the bottom of the intestinal crypts of Lieberkühn. The crypts begin to form around postnatal day 7 (P7) mice, and Paneth cells usually appear within the first 2 weeks. Paneth cell dysfunction has been reported to correlate with Crohn's disease-like inflammation, showing narrow crypts or loss of crypt architecture in mice. The morphology of dysfunctional Paneth cells is similar to that of Paneth/goblet intermediate cells. However, it remains unclear whether the formation of the crypt is related to the maturation of Paneth cells. In this study, we investigated the histological changes including epigenetic modification in the mouse ileum postnatally and assessed the effect of the methyltransferase inhibitor on epithelium development using an organoid culture. The morphological and functional maturation of Paneth cells occurred in the first 2 weeks and was accompanied by histone H3 lysine 27 (H3K27) trimethylation, although significant differences in DNA methylation or other histone H3 trimethylation were not observed. Inhibition of H3K27 trimethylation in mouse ileal organoids suppressed crypt formation and Paneth cell maturation, until around P10. Overall, our findings show that post-transcriptional modification of histones, particularly H3K27 trimethylation, leads to the structural and functional maturation of Paneth cells during postnatal development.

Screening for modulators of the cellular composition of gut epithelia via organoid models of intestinal stem cell differentiation.

The cellular composition of barrier epithelia is essential to organismal homoeostasis. In particular, within the small intestine, adult stem cells establish tissue cellularity, and may provide a means to control the abundance and quality of specialized epithelial cells. Yet, methods for the identification of biological targets regulating epithelial composition and function, and of small molecules modulating them, are lacking. Here we show that druggable biological targets and small-molecule regulators of intestinal stem cell differentiation can be identified via multiplexed phenotypic screening using thousands of miniaturized organoid models of intestinal stem cell differentiation into Paneth cells, and validated via longitudinal single-cell RNA-sequencing. We found that inhibitors of the nuclear exporter Exportin 1 modulate the fate of intestinal stem cells, independently of known differentiation cues, significantly increasing the abundance of Paneth cells in the organoids and in wild-type mice. Physiological organoid models of the differentiation of intestinal stem cells could find broader utility for the screening of biological targets and small molecules that can modulate the composition and function of other barrier epithelia.

Regulation of Paneth Cell Function by RNA-Binding Proteins and Noncoding RNAs.

Paneth cells are specialized intestinal epithelial cells that are located at the base of small intestinal crypts and play a vital role in preserving the gut epithelium homeostasis. Paneth cells act as a safeguard from bacterial translocation across the epithelium and constitute the niche for intestinal stem cells in the small intestine by providing multiple niche signals. Recently, Paneth cells have become the focal point of investigations defining the mechanisms underlying the epithelium-microbiome interactions and pathogenesis of chronic gut mucosal inflammation and bacterial infection. Function of Paneth cells is tightly regulated by numerous factors at different levels, while Paneth cell defects have been widely documented in various gut mucosal diseases in humans. The post-transcription events, specific change in mRNA stability and translation by RNA-binding proteins (RBPs) and noncoding RNAs (ncRNAs) are implicated in many aspects of gut mucosal physiology by modulating Paneth cell function. Deregulation of RBPs and ncRNAs and subsequent Paneth cell defects are identified as crucial elements of gut mucosal pathologies. Here, we overview the posttranscriptional regulation of Paneth cells by RBPs and ncRNAs, with a particular focus on the increasing evidence of RBP HuR and long ncRNA H19 in this process. We also discuss the involvement of Paneth cell dysfunction in altered susceptibility of the intestinal epithelium to chronic inflammation and bacterial infection following disrupted expression of HuR and H19.

Secretory Sorcery: Paneth Cell Control of Intestinal Repair and Homeostasis.

Paneth cells are professional secretory cells that classically play a role in the innate immune system by secreting antimicrobial factors into the lumen to control enteric bacteria. In this role, Paneth cells are able to sense cues from luminal bacteria and respond by changing production of these factors to protect the epithelial barrier. Paneth cells rely on autophagy to regulate their secretory capability and capacity. Disruption of this pathway through mutation of genes, such as Atg16L1, results in decreased Paneth cell function, dysregulated enteric microbiota, decreased barrier integrity, and increased risk of diseases such as Crohn's disease in humans. Upon differentiation Paneth cells migrate downward and intercalate among active intestinal stem cells at the base of small intestinal crypts. This localization puts them in a unique position to interact with active intestinal stem cells, and recent work shows that Paneth cells play a critical role in influencing the intestinal stem cell niche. This review discusses the numerous ways Paneth cells can influence intestinal stem cells and their niche. We also highlight the ways in which Paneth cells can alter cells and other organ systems.

Cell fate coordinates mechano-osmotic forces in intestinal crypt formation.

Intestinal organoids derived from single cells undergo complex crypt-villus patterning and morphogenesis. However, the nature and coordination of the underlying forces remains poorly characterized. Here, using light-sheet microscopy and large-scale imaging quantification, we demonstrate that crypt formation coincides with a stark reduction in lumen volume. We develop a 3D biophysical model to computationally screen different mechanical scenarios of crypt morphogenesis. Combining this with live-imaging data and multiple mechanical perturbations, we show that actomyosin-driven crypt apical contraction and villus basal tension work synergistically with lumen volume reduction to drive crypt morphogenesis, and demonstrate the existence of a critical point in differential tensions above which crypt morphology becomes robust to volume changes. Finally, we identified a sodium/glucose cotransporter that is specific to differentiated enterocytes that modulates lumen volume reduction through cell swelling in the villus region. Together, our study uncovers the cellular basis of how cell fate modulates osmotic and actomyosin forces to coordinate robust morphogenesis.

Simultaneous real-time analysis of Paneth cell and intestinal stem cell response to interferon-γ by a novel stem cell niche tracking method.

Paneth cells and Lgr5+ intestinal stem cells (Lgr5+ ISCs) constitute the stem cell niche and maintain small intestinal epithelial integrity by recognizing various niche factors derived from subepithelial cells and external antigens. Although it has been known that interferon-γ (IFN-γ), a Th1 cytokine, is associated with intestinal epithelial disruption during inflammation as a niche factor, dynamics of Paneth cells and Lgr5+ ISCs in response to IFN-γ remain to be understood. Here we show that CAG-tdTomato;Lgr5-EGFP (CT-LE) mice generated in this study enable to identify Paneth cells and Lgr5+ ISCs separately by fluorescence signals. Lgr5+ ISCs underwent cell death a little earlier than Paneth cells in response to IFN-γ by simultaneous tracking using CT-LE mice. In addition, the timing of cell death in most Paneth cells overlapped with Lgr5+ ISCs, suggesting that Paneth cell depletion is induced directly by IFN-γ. Taken together, we established a novel simultaneous stem cell niche tracking method and clarified the involvement of both Paneth cells and Lgr5+ ISCs in stem cell niche damage induced by IFN-γ, further contribute to understanding the mechanism for maintaining intestinal homeostasis by stem cell niche.

Regulation of the Paneth cell niche by exogenous L-arginine couples the intestinal stem cell function.

Although previous studies show that exogenous nutrients regulate the stem cell function, little is known about the effects of L-arginine on intestinal stem cells (ISCs). In this study, we utilize mice, small intestinal (SI) organoids, and ISC-Paneth cell co-cultured models to clarify the role of L-arginine in ISC function. We find that exogenous L-arginine is essential for ISCs proliferation and intestinal epithelial renewal. Our data show that Paneth cells, a critical component of the ISCs niche, augment the ISCs function in response to L-arginine. Moreover, enhanced the expression of Wnt3a in Paneth cells, which is a ligand of the Wnt/ß-catenin signaling pathway, mediates the effects of L-arginine on ISCs function. Pre-treatment with L-arginine enhances the ISCs pool and protects the gut in response to injury provoked by murine tumor necrosis factor α (TNF-α) and 5-Fluorouracil (5-FU). Our findings establish that the regulation of Wnt3a in the Paneth cell niche by exogenous L-arginine couples ISCs function and favours a model in which the ISCs niche couples the nutrient levels to ISCs function.

An Update Review on the Paneth Cell as Key to Ileal Crohn's Disease.

The Paneth cells reside in the small intestine at the bottom of the crypts of Lieberkühn, intermingled with stem cells, and provide a niche for their neighbors by secreting growth and Wnt-factors as well as different antimicrobial peptides including defensins, lysozyme and others. The most abundant are the human Paneth cell α-defensin 5 and 6 that keep the crypt sterile and control the local microbiome. In ileal Crohn's disease various mechanisms including established genetic risk factors contribute to defects in the production and ordered secretion of these peptides. In addition, life-style risk factors for Crohn's disease like tobacco smoking also impact on Paneth cell function. Taken together, current evidence suggest that defective Paneth cells may play the key role in initiating inflammation in ileal, and maybe ileocecal, Crohn's disease by allowing bacterial attachment and invasion.

The Paneth Cell: The Curator and Defender of the Immature Small Intestine.

Paneth cells were first described in the late 19th century by Gustav Schwalbe and Josef Paneth as columnar epithelial cells possessing prominent eosinophilic granules in their cytoplasm. Decades later there is continued interest in Paneth cells as they play an integral role in maintaining intestinal homeostasis and modulating the physiology of the small intestine and its associated microbial flora. Paneth cells are highly specialized secretory epithelial cells located in the small intestinal crypts of Lieberkühn. The dense granules produced by Paneth cells contain an abundance of antimicrobial peptides and immunomodulating proteins that function to regulate the composition of the intestinal flora. This in turn plays a significant role in secondary regulation of the host microvasculature, the normal injury and repair mechanisms of the intestinal epithelial layer, and the levels of intestinal inflammation. These critical functions may have even more importance in the immature intestine of premature infants. While Paneth cells begin to develop in the middle of human gestation, they do not become immune competent or reach their adult density until closer to term gestation. This leaves preterm infants deficient in normal Paneth cell biology during the greatest window of susceptibility to develop intestinal pathology such as necrotizing enterocolitis (NEC). As 10% of infants worldwide are currently born prematurely, there is a significant population of infants contending with an inadequate cohort of Paneth cells. Infants who have developed NEC have decreased Paneth cell numbers compared to age-matched controls, and ablation of murine Paneth cells results in a NEC-like phenotype suggesting again that Paneth cell function is critical to homeostasis to the immature intestine. This review will provide an up to date and comprehensive look at Paneth cell ontogeny, the impact Paneth cells have on the host-microbial axis in the immature intestine, and the repercussions of Paneth cell dysfunction or loss on injury and repair mechanisms in the immature gut.

Paneth Cell Dysfunction Mediates Alcohol-related Steatohepatitis Through Promoting Bacterial Translocation in Mice: Role of Zinc Deficiency.

BACKGROUND AND AIMS: Microbial dysbiosis is associated with alcohol-related hepatitis (AH), with the mechanisms yet to be elucidated. The present study aimed to determine the effects of alcohol and zinc deficiency on Paneth cell (PC) antimicrobial peptides, α-defensins, and to define the link between PC dysfunction and AH. APPROACH AND RESULTS: Translocation of pathogen-associated molecular patterns (PAMPs) was determined in patients with severe AH and in a mouse model of alcoholic steatohepatitis. Microbial composition and PC function were examined in mice. The link between α-defensin dysfunction and AH was investigated in α-defensin-deficient mice. Synthetic human α-defensin 5 (HD5) was orally given to alcohol-fed mice to test the therapeutic potential. The role of zinc deficiency in α-defensin was evaluated in acute and chronic mouse models of zinc deprivation. Hepatic inflammation was associated with PAMP translocation and lipocalin-2 (LCN2) and chemokine (C-X-C motif) ligand 1 (CXCL1) elevation in patients with AH. Antibiotic treatment, lipopolysaccharide injection to mice, and in vitro experiments showed that PAMPs, but not alcohol, directly induced LCN2 and CXCL1. Chronic alcohol feeding caused systemic dysbiosis and PC α-defensin reduction in mice. Knockout of functional α-defensins synergistically affected alcohol-perturbed bacterial composition and the gut barrier and exaggerated PAMP translocation and liver damage. Administration of HD5 effectively altered cecal microbial composition, especially increased Akkermansia muciniphila, and reversed the alcohol-induced deleterious effects. Zinc-regulated PC homeostasis and α-defensins function at multiple levels, and dietary zinc deficiency exaggerated the deleterious effect of alcohol on PC bactericidal activity. CONCLUSIONS: Taken together, the study suggests that alcohol-induced PC α-defensin dysfunction is mediated by zinc deficiency and involved in the pathogenesis of AH. HD5 administration may represent a promising therapeutic approach for treating AH.

Generating an Artificial Intestine for the Treatment of Short Bowel Syndrome.

Intestinal failure is defined as the inability to maintain fluid, nutrition, energy, and micronutrient balance that leads to the inability to gain or maintain weight, resulting in malnutrition and dehydration. Causes of intestinal failure include short bowel syndrome (ie, the physical loss of intestinal surface area and severe intestinal dysmotility). For patients with intestinal failure who fail to achieve enteral autonomy through intestinal rehabilitation programs, the current treatment options are expensive and associated with severe complications. Therefore, the need persists for next-generation therapies, including cell-based therapy, to increase intestinal regeneration, and development of the tissue-engineered small intestine.

Functional Changes of Paneth Cells in the Intestinal Epithelium of Mice with Obstructive Jaundice and After Internal and External Biliary Drainage.

OBJECTIVE: To investigate the functional changes of Paneth cells in the intestinal epithelium of mice with obstructive jaundice (OJ) and after internal biliary drainage (ID) and external biliary drainage (ED). METHODS: The experiment was divided into two stages. First stage: Mice were randomly assigned to two groups: (I) sham operation (SH); (II) OJ. The mice were sacrificed before the operation and on the 1st, 3rd, 5th and 7th day after the operation to collect specimens. Second stage: Mice were randomly assigned to four groups: (I) SH; (II) OJ; (III) OJ and ED; and (IV) OJ and ID. They were reoperated on day 5 for biliary drainage procedure. The specimens were collected on day 10. RESULTS: The expressions of lysozyme and cryptdin-4 increased first and then decreased over time in group OJ, and the number of Paneth cells decreased gradually with the extension of OJ time（p<0.05. After the secondary operation on the mice to relieve OJ, the number of Paneth cells and expressions of lysozyme and cryptdin-4 in group ID increased more significantly than those in group ED（p<0.05）. CONCLUSION: OJ could cause intestinal Paneth cells to dysfunction in mice. ID was more significant than ED in restoring the function of Paneth cells. It might be one of the mechanisms that make ID superior to ED.

Enteroids expressing a disease-associated mutant of EpCAM are a model for congenital tufting enteropathy.

Congenital tufting enteropathy (CTE) is an autosomal recessive disease characterized by severe intestinal failure in infancy and mutations in the epithelial cell adhesion molecule (EPCAM) gene. Previous studies of CTE in mice expressing mutant EpCAM show neonatal lethality. Hence, to study the cellular, molecular, and physiological alterations that result from EpCAM mutation, a tamoxifen-inducible mutant EpCAM enteroid model has been generated. The presence of mutant EpCAM in the model was confirmed at both mRNA and protein levels. Immunofluorescence microscopy demonstrated the reduced expression of mutant EpCAM. Mutant enteroids had reduced budding potential as well as significantly decreased mRNA expression for epithelial lineage markers (Mucin 2, lysozyme, sucrase-isomaltase), proliferation marker Ki67, and secretory pathway transcription factors (Atoh1, Hnf1b). Significantly decreased numbers of Paneth and goblet cells were confirmed by staining. These findings were correlated with intestinal tissue from CTE patients and the mutant mice model that had significantly fewer Paneth and goblet cells than in healthy counterparts. FITC-dextran studies demonstrated significantly impaired barrier function in monolayers derived from mutant enteroids compared with control monolayers. In conclusion, we have established an ex vivo CTE model. The role of EpCAM in the budding potential, differentiation, and barrier function of enteroids is noted. Our study establishes new facets of EpCAM biology that will aid in understanding the pathophysiology of CTE and role of EpCAM in health and disease.NEW & NOTEWORTHY Here, we develop a novel ex vivo enteroid model for congenital tufting enteropathy (CTE) based on epithelial cell adhesion molecule (EPCAM) gene mutations found in patients. With this model we demonstrate the role of EpCAM in maintaining the functional homeostasis of the intestinal epithelium, including differentiation, proliferation, and barrier integrity. This study further establishes a new direction in EpCAM biology that will help in understanding the detailed pathophysiology of CTE and role of EpCAM.

Mist1 Expression Is Required for Paneth Cell Maturation.

BACKGROUND: Paneth cells are professional secretory cells found within the small intestinal crypt epithelium. Although their role as part of the innate immune complex providing antimicrobial secretory products is well-known, the mechanisms that control secretory capacity are not well-understood. MIST1 is a scaling factor that is thought to control secretory capacity of exocrine cells. METHODS: Mist1+/+ and Mist1-/- mice were used to evaluate the function of MIST1 in small intestinal Paneth cells. We used histologic and immunofluorescence staining to evaluate small intestinal tissue for proliferation and lineage allocation. Total RNA was isolated to evaluate gene expression. Enteroid culture was used to evaluate the impact of the absence of MIST1 expression on intestinal stem cell function. RESULTS: Absence of MIST1 resulted in increased numbers of Paneth cells exhibiting an intermediate cell phenotype but otherwise did not alter overall epithelial cell lineage allocation. Muc2 and lysozyme staining confirmed the presence of intermediate cells at the crypt base of Mist1-/- mice. These changes were not associated with changes in mRNA expression of transcription factors associated with lineage allocation, and they were not abrogated by inhibition of Notch signaling. However, the absence of MIST1 expression was associated with alterations in Paneth cell morphology including decreased granule size and distended rough endoplasmic reticulum. Absence of MIST1 was associated with increased budding of enteroid cultures; however, there was no evidence of increased intestinal stem cell numbers in vivo. CONCLUSIONS: MIST1 plays an important role in organization of the Paneth cell secretory apparatus and managing endoplasmic reticulum stress. This role occurs downstream of Paneth cell lineage allocation.

Dithizone-induced Paneth cell disruption significantly decreases intestinal perfusion in the murine small intestine.

PURPOSE: Necrotizing enterocolitis is associated with decreased intestinal perfusion and ischemia. Paneth cells, specialized epithelial cells, have been shown to regulate the intestinal vasculature and disruption of these cells has been associated with NEC. We hypothesized that Paneth cell disruption in immature mice intestine would decrease the perfusion of the intestinal microvasculature. METHODS: Paneth cells were disrupted in P14-16 mice using chemical (dithizone) and transgenic (diphtheria toxin) methodology. Six hours after Paneth cell disruption, Dylight 488 was injected directly into the left ventricle and allowed to perfuse for 5 minutes prior to intestinal harvesting. Tissue samples were evaluated with confocal fluorescence microscopy to quantify intestinal perfusion and samples were quantified by real time RT-PCR for gene expression. RESULTS: Dithizone treatment significantly decreased intestinal perfusion compared to controls (p < 0.01). However, diphtheria toxin treatment demonstrated no significant difference in perfusion (p > 0.21). Intestines from all treatment groups had similar PECAM staining, but intestines treated with dithizone had significantly decreased nNOS and iNOS gene expression compared to controls (p < 0.007). CONCLUSIONS: Paneth cell disruption significantly decreases the perfusion of the small intestinal microvasculature in a dithizone-specific manner. Dithizone has no effect on the amount of microvasculature, but does impact genes critical to nitric oxide signaling likely contributing to mesenteric vasoconstriction.

The mTORC1 signaling modulated by intracellular C3 activation in Paneth cells promotes intestinal epithelial regeneration during acute injury.

Complement activation is associated with regional inflammation during acute gastrointestinal injury (AGI). This study is designed to explore how intracellular C3 activation in Paneth cells (PCs) affects regeneration of intestinal epithelium during AGI. AGI was induced in wildtype C57BL/6 mice, with sham operation employed as control. Exogenous C3 (1 mg, I.P.) was applied at 6 h post-surgery. Intestinal crypts harvested from ileum were cultured with presence or absence of C3 (20 µg/ml), with small interfering RNA against BST1 and complement activation inhibitor selectively applied in vitro. The intestinal integrity, percentage of PCs and intestinal stem cells (ISCs) were evaluated. Importantly, cADPR, C3 fragments, and S6-related proteins were detected in PCs to inspect the mammalian target of rapamycin complex 1 (mTORC1) signaling. AGI caused breakdown of intestinal mucosa integrity and regional inflammation. Exogenous C3 by itself failed to promote the growth of intestinal epithelium, but distinctly enhanced the activity of PCs via intracellular activation, which subsequently supported the expansion of ISCs inside of intestinal crypts. Inhibition of C3 activation was associated with decreased expressions of S6, S6K1 and cADPR, with blocking BST1 found to depress cADPR only. Collectively, these data confirmed intracellular activation of C3 in PCs enhanced expansion of ISCs in response to acute injury. The mTORC1 signaling pathway in PCs contributed to this crosstalk during exogenous C3 treatment.

Intracellular activation of complement C3 in Paneth cells improves repair of intestinal epithelia during acute injury.

AIM: To explore whether Paneth cells (PCs) and complement system collaborate in the repair of enteric epithelia during acute gastrointestinal injury (AGI). METHODS: Wild-type C57BL/6 mice were employed to induce AGI by performing colon ascendens stent surgery, with sham-operated as control. Exogenous C3 treatment was applied at 6-h postsurgery. After 48 h, overall survival, intestinal damage severity, and C3 intracellular activation were assessed in both epithelial cells and PCs. RESULTS: AGI caused a high mortality, while C3 therapy significantly attenuated epithelial damages and improved survival. Besides, exogenous C3 in vitro enhanced the proliferation and activity of PCs. Importantly, intracellular C3 activation was observed inside of PCs under C3 co-stimulation in vitro. CONCLUSION: C3 immunotherapy might play a valuable role in turnover of gut epithelia through intracellular activation in PCs.

Spatial Patterning from an Integrated Wnt/ß-catenin and Notch/Delta Gene Circuit.

Classically, the Wnt/ß-catenin and Notch /Delta signaling pathways were thought to operate through separate mechanisms, performing distinct roles in tissue patterning. However, it has been shown that b-catenin activates transcription of Hesl, a signaling intermediate in the Notch /Delta pathway that controls its lateral inhibition mechanism. To investigate this non-canonical crosstalk mechanism, a new gene circuit, integrating the two pathways, is proposed and simulated in two-cell and multi-cell environments. This model also captures both Paneth cell- mediated and mesenchymal Wnt production. The simulations verify that the gene circuit is temporally bistable and capable of forming a pattern on a multi-cell grid. Last, the model exhibits a bifurcation based on the steady state concentration of Wnt and the relative amount of control b-catenin has over the Hesl promoter, providing a possible mechanism to explain why a homogeneous population of transit amplifying cells is observed directly above the more diverse stem niche.