Therapeutic application of decellularized porcine small intestinal submucosa scaffold in conjunctiva reconstruction.

The objective of this study was to investigate the biological feasibility and surgical applicability of decellularized porcine small intestinal submucosa (DSIS) in conjunctiva reconstruction. A total of 52 Balb/c mice were included in the study. We obtained the DSIS by decellularization, evaluated the physical and biological properties of DSIS in vitro, and further evaluated the effect of surgical transplantation of DSIS scaffold in vivo. The histopathology and ultrastructural analysis results showed that the scaffold retained the integrity of the fibrous morphology while removing cells. Biomechanical analysis showed that the elongation at break of the DSIS (239.00 ± 12.51%) were better than that of natural mouse conjunctiva (170.70 ± 9.41%, P < 0.05). Moreover, in vivo experiments confirmed the excellent biocompatibility of the decellularized scaffolds. In the DSIS group, partial epithelialization occurred at day-3 after operation, and the conjunctival injury healed at day-7, which was significantly faster than that in human amniotic membrane (AM) and sham surgery (SHAM) group (P < 0.05). The number and distribution of goblet cells of transplanted DSIS were significantly better than those of the AM and SHAM groups. Consequently, the DSIS scaffold shows excellent biological characteristics and surgical applicability in the mouse conjunctival defect model, and DSIS is expected to be an alternative scaffold for conjunctival reconstruction.

Extended depth-of-field wide-field fluorescence microscopy with a micro-mirror array lens system for versatile cellular examination.

We present a versatile extended depth-of-field (EDOF) wide-field fluorescence microscopy using a new, to the best of our knowledge, active device, micro-mirror array lens system (MALS) for calibration-free and orientation-insensitive EDOF imaging. The MALS changed the focal plane during image acquisition, and the system could be operated in any orientation. Two EDOF imaging modes of high-speed accumulation and low-speed surface sectioning were implemented. The performance was demonstrated in non-contact imaging of conjunctival goblet cells in live mice and depth-resolved cellular examination of ex-vivo human cancer specimens. MALS-based EDOF microscopy has potential for versatile cellular examination.

Developmental events in the mandibular salivary gland of Indian sheep (Ovis aries): Morphological and histochemical studies.

At 22nd day of fetal development, the primordial anlage of mandibular gland was first noticed as a solid epithelial bud from oral epithelium. The terminal buds were arranged in the form of clusters with undifferentiated epithelial cells and terminated in a bulb like structure in 28-day-old fetus. The lumenization and branching of the main cord was noticed at 35th day. The primary septa, which divide the glandular mass into lobes was observed from 53rd day onwards which resulted in the formation of distinct lobulation at 58th day. At 61st day, the capsule formation was initiated by the aggregation of mesenchymal tissue. The terminal tubules differentiated to form the secretory end pieces and the tubular portion leads to the formation of intercalated and striated ducts at 98th day. Predominantly mucous types of acinar cells were seen from 108th day onwards. The number of lobules increased with steep increase in parenchyma from 128th day onwards. Micrometrical studies revealed that the mean diameter of acinar cells and all ducts was increased with the advancement of age and the significant differences were observed between groups. Localization of acidic and neutral mucopolysaccharides was observed in mucous cells and goblet cells. RESEARCH HIGHLIGHTS: The primordial anlage of mandibular salivary gland was seen at 22nd day. Lobulation of gland was appeared at 53rd day of development, however; it was completed at 58th day. At 98th day, the terminal tubules differentiated to form the secretory end pieces. The parenchyma of the gland showed predominantly mucous type of cells from 108th day onwards. Myoepithelial cells were first appeared as flattened basal cells initially around the developing acinar cells at 132nd day. Localization of acidic as well as neutral mucopolysaccharides was observed in mucous cells and goblet cells. Fine lipid droplets were observed in intralobular as well as interlobular connective tissue, however; phospholipids were observed in the cell membrane of secretory cells and ducts.

Imaging assessment of conjunctival goblet cells in dry eye disease.

Dry eye disease (DED) is a widespread, multifactorial, and chronic disorder of the ocular surface with disruption of tear film homeostasis as its core trait. Conjunctival goblet cells (CGCs) are specialised secretory cells found in the conjunctival epithelium that participate in tear film formation by secreting mucin. Changes in both the structure and function of CGCs are hallmarks of DED, and imaging assessment of CGCs is important for the diagnosis, classification, and severity evaluation of DED. Existing imaging methods include conjunctival biopsy, conjunctival impression cytology and in vivo confocal microscopy, which can be used to assess the morphology, distribution, and density of the CGCs. Recently, moxifloxacin-based fluorescence microscopy has emerged as a novel technique that enables efficient, non-invasive and in vivo imaging of CGCs. This article presents a comprehensive overview of both the structure and function of CGCs and their alterations in the context of DED, as well as current methods of CGCs imaging assessment. Additionally, potential directions for the visual evaluation of CGCs are discussed.

Porcine Deltacoronavirus Infection Disrupts the Intestinal Mucosal Barrier and Inhibits Intestinal Stem Cell Differentiation to Goblet Cells via the Notch Signaling Pathway.

Goblet cells and their secreted mucus are important elements of the intestinal mucosal barrier, which allows host cells to resist invasion by intestinal pathogens. Porcine deltacoronavirus (PDCoV) is an emerging swine enteric virus that causes severe diarrhea in pigs and causes large economic losses to pork producers worldwide. To date, the molecular mechanisms by which PDCoV regulates the function and differentiation of goblet cells and disrupts the intestinal mucosal barrier remain to be determined. Here, we report that in newborn piglets, PDCoV infection disrupts the intestinal barrier: specifically, there is intestinal villus atrophy, crypt depth increases, and tight junctions are disrupted. There is also a significant reduction in the number of goblet cells and the expression of MUC-2. In vitro, using intestinal monolayer organoids, we found that PDCoV infection activates the Notch signaling pathway, resulting in upregulated expression of HES-1 and downregulated expression of ATOH-1 and thereby inhibiting the differentiation of intestinal stem cells into goblet cells. Our study shows that PDCoV infection activates the Notch signaling pathway to inhibit the differentiation of goblet cells and their mucus secretion, resulting in disruption of the intestinal mucosal barrier. IMPORTANCE The intestinal mucosal barrier, mainly secreted by the intestinal goblet cells, is a crucial first line of defense against pathogenic microorganisms. PDCoV regulates the function and differentiation of goblet cells, thereby disrupting the mucosal barrier; however, the mechanism by which PDCoV disrupts the barrier is not known. Here, we report that in vivo, PDCoV infection decreases villus length, increases crypt depth, and disrupts tight junctions. Moreover, PDCoV activates the Notch signaling pathway, inhibiting goblet cell differentiation and mucus secretion in vivo and in vitro. Thus, our results provide a novel insight into the mechanism underlying intestinal mucosal barrier dysfunction caused by coronavirus infection.

Epigenetic modifier balances Mapk and Wnt signalling in differentiation of goblet and Paneth cells.

Differentiation and lineage specification are controlled by cooperation of growth factor signalling. The involvement of epigenetic regulators in lineage specification remains largely elusive. Here, we show that the histone methyltransferase Mll1 prevents intestinal progenitor cells from differentiation, whereas it is also involved in secretory lineage specification of Paneth and goblet cells. Using conditional mutagenesis in mice and intestinal organoids, we demonstrate that loss of Mll1 renders intestinal progenitor cells permissive for Wnt-driven secretory differentiation. However, Mll1-deficient crypt cells fail to segregate Paneth and goblet cell fates. Mll1 deficiency causes Paneth cell-determined crypt progenitors to exhibit goblet cell features by unleashing Mapk signalling, resulting in increased numbers of mixed Paneth/goblet cells. We show that loss of Mll1 abolishes the pro-proliferative effect of Mapk signalling in intestinal progenitor cells and promotes Mapk-induced goblet cell differentiation. Our data uncover Mll1 and its downstream targets Gata4/6 as a regulatory hub of Wnt and Mapk signalling in the control of lineage specification of intestinal secretory Paneth and goblet cells.

An Improved Ocular Impression Cytology Method: Quantitative Cell Transfer to Microscope Slides Using a Novel Polymer.

PURPOSE: To develop a more efficient impression cytology (IC) method for the transfer of ocular surface cells onto glass microscope slides for cytochemical, immunocytochemical, and immunofluorescence studies. METHODS: Cells are lifted off the ocular surface with a mixed cellulose ester membrane and then firmly attached to a glass slide using a novel triblock copolymer comprised of collagen type I, polyethylenimine and poly-L-lysine (CPP), and crosslinking cells and glass slide by heating and cooling. The membrane is removed intact after softening it with a butanol/ethanol solution. Transfer of cells is complete in about 10-15 minutes and is ready for staining. The efficiency of our cell transfer method was compared to current methods based on poly-L-lysine and albumin paste. RESULTS: Our method ensured almost complete transfer of cells. In contrast, the transfer of rabbit conjunctiva cells onto poly-L-lysine-covered slides was 37.5 ± 6.3% lower, and onto albumin-paste covered slides 62.5 ± 5.6% lower (mean ± SD); the transfer of rabbit goblet cells was even less efficient. The new method was also more efficient for transfer of cells from human oral mucosa obtained by IC. Transferred cells were successfully stained with H&E, chemiluminescence, and immunofluorescence agents. Using our method, we stained ocular surface cells for S100A4 and ATF4, both of which play a role in the pathophysiology of dry eye disease. We obtained similar results with oral mucosal cells, suggesting the generalizability of our approach. We propose an explanation for the strong adhesion of cells to the glass slide, which is based on their interactions with the triblock copolymer. CONCLUSIONS: We developed a novel approach for the efficient and rapid transfer of cells obtained by IC onto glass microscope slides using a novel copolymer. Compared to available methods, our improved approach makes IC robust and simple, and should increase its diagnostic yield and clinical applicability.

NF-κB determines Paneth versus goblet cell fate decision in the small intestine.

Although the role of the transcription factor NF-κB in intestinal inflammation and tumor formation has been investigated extensively, a physiological function of NF-κB in sustaining intestinal epithelial homeostasis beyond inflammation has not been demonstrated. Using NF-κB reporter mice, we detected strong NF-κB activity in Paneth cells, in '+4/+5' secretory progenitors and in scattered Lgr5+ crypt base columnar stem cells of small intestinal (SI) crypts. To examine NF-κB functions in SI epithelial self-renewal, mice or SI crypt organoids ('mini-guts') with ubiquitously suppressed NF-κB activity were used. We show that NF-κB activity is dispensable for maintaining SI epithelial proliferation, but is essential for ex vivo organoid growth. Furthermore, we demonstrate a dramatic reduction of Paneth cells in the absence of NF-κB activity, concomitant with a significant increase in goblet cells and immature intermediate cells. This indicates that NF-κB is required for proper Paneth versus goblet cell differentiation and for SI epithelial homeostasis, which occurs via regulation of Wnt signaling and Sox9 expression downstream of NF-κB. The current study thus presents evidence for an important role for NF-κB in intestinal epithelial self-renewal.

Mucus threads from surface goblet cells clear particles from the airways.

BACKGROUND: The mucociliary clearance system driven by beating cilia protects the airways from inhaled microbes and particles. Large particles are cleared by mucus bundles made in submucosal glands by parallel linear polymers of the MUC5B mucins. However, the structural organization and function of the mucus generated in surface goblet cells are poorly understood. METHODS: The origin and characteristics of different mucus structures were studied on live tissue explants from newborn wild-type (WT), cystic fibrosis transmembrane conductance regulator (CFTR) deficient (CF) piglets and weaned pig airways using video microscopy, Airyscan imaging and electron microscopy. Bronchoscopy was performed in juvenile pigs in vivo. RESULTS: We have identified a distinct mucus formation secreted from the surface goblet cells with a diameter less than two micrometer. This type of mucus was named mucus threads. With time mucus threads gathered into larger mucus assemblies, efficiently collecting particles. The previously observed Alcian blue stained mucus bundles were around 10 times thicker than the threads. Together the mucus bundles, mucus assemblies and mucus threads cleared the pig trachea from particles. CONCLUSIONS: These results demonstrate that normal airway mucus is more complex and has a more variable structural organization and function than was previously understood. These observations emphasize the importance of studying young objects to understand the function of a non-compromised lung.

H1-Receptor Antagonist Olopatadine Inhibits MUC5AC Secretion by Conjunctival Goblet Cells.

The study examined the effect of H1-receptor antagonist olopatadine on the secretory function of cultured rat conjunctival goblet cells (CGC) assessed by enzyme-linked lectin assay employing UEA-I lectin. The level of mRNA for membrane-bound protein MUC16 in histaminestimulated CGC was assayed by reverse transcription PCR in the control and after preliminary application of olopatadine. The intracellular calcium concentration [Ca2+]i was measured by the calcium colorimetric method using GENMED kits. The effects of histamine and olopatadine on p-ERK level were assessed by Western blotting. Histamine up-regulated secretion of mucin MUC5AC and expression of membrane-bound protein MUC16 in CGC. In addition, it increased both [Ca2+]i and the level of phosphorylated ERK. These effects were diminished by preliminary application of olopatadine that probably acted via the ERK signaling pathway. Thus, olopatadine reduced [Ca2+]i and down-regulated ERK phosphorylation by binding to H1-receptors, thereby inhibiting secretion of mucin from histamine-stimulated CGC.

Characterisation of Gel-Forming Mucins Produced In Vivo and In Ex Vivo Conjunctival Explant Cultures.

One key element to the health of the ocular surface encompasses the presence of gel-forming mucins in the pre-ocular tear film. Conjunctival goblet cells are specialized epithelial cells that secrete mucins necessary for tear film stability and general homeostasis. Their dysfunction can be linked to a range of ocular surface inflammation disorders and chronic injuries. To obtain new perspectives and angles to tackle mucin deficiency, the need for an accurate evaluation of their presence and corresponding mucin secretion in ex vivo conjunctival cultures has become a requisite. In vitro, goblet cells show a significant decrease in the production and secretion of gel-forming mucins, accompanied by signs of dedifferentiation or transdifferentiation. Explant cultures on laminin-treated CLP-PEG hydrogels can, however, support the production of gel-forming mucins. Together, we challenge the current paradigm to evaluate the presence of cultured goblet cells solely based on their general mucin (MUC) content through imaging analyses, showing the need for additional techniques to assess the functionality of goblet cells. In addition, we broadened the gel-forming mucin profile of in vivo goblet cells with MUC5B and MUC6, while MUC2 and MUC6 is added to the profile of cultured goblet cells.

Mechanobiology of conjunctival epithelial cells exposed to wall shear stresses.

The human conjunctival epithelial cells (HCEC) line the inner sides of the eyelids and the anterior part of the sclera. They include goblet cells that secret mucus into the tear film that protects the ocular surface. The conjunctival epithelium is subjected to mechano-physical stimuli due to eyelid movement during blinking, during wiping and rubbing the eyes, and when exposed to wind and air currents. We cultured primary HCEC under air-liquid interface (ALI) conditions in custom-designed wells that can be disassembled for installation of the in vitro model in a flow chamber. We exposed the HCEC after ALI culture of 8-10 days to steady and oscillatory airflows. The in vitro model of HCEC was exposed to steady wall shear stresses (sWSS) of 0.5 and 1.0 dyne/cm2 for lengths of 30 and 60 min and to oscillatory wall shear stresses (oWSS) of 0.5 and 0.77 dyne/cm2 amplitudes for a length of 10 min. Cytoskeletal alterations and MUC5AC mucin secretion in response to WSS were investigated using immunohistochemically fluorescent staining and enzyme-linked lectin assay (ELLA), respectively. The results revealed that both exposure times and sWSS values increased the polymerization of F-actin filaments while mucin secretion decreased. However, after a recovery of 24 h in the incubator we observed a decrease of F-actin fibers and mucin secretion only for exposure of 30 min. The length of exposure was more influential on cytoskeletal alterations than the level of sWSS. The very small effect of sWSS on mucin secretion is most likely related to the much smaller amount of goblet cell than in other mucus-secreting tissue. The results for both oWSS amplitudes revealed similar trends regarding F-actin and mucin secretion. Immediately post-exposure we observed an increase in polymerization of F-actin filaments while mucin secretion decreased. However, after 24-h recovery we observed that both F-actin and mucin secretion returned to the same values as for unexposed cultures. The results of this study suggest that WSS should be considered while exploring the physiological characteristics of HCEC.

Acceleration of Small Intestine Development and Remodeling of the Microbiome Following Hyaluronan 35 kDa Treatment in Neonatal Mice.

The beneficial effects of human milk suppressing the development of intestinal pathologies such as necrotizing enterocolitis in preterm infants are widely known. Human milk (HM) is rich in a multitude of bioactive factors that play major roles in promoting postnatal maturation, differentiation, and the development of the microbiome. Previous studies showed that HM is rich in hyaluronan (HA) especially in colostrum and early milk. This study aims to determine the role of HA 35 KDa, a HM HA mimic, on intestinal proliferation, differentiation, and the development of the intestinal microbiome. We show that oral HA 35 KDa supplementation for 7 days in mouse pups leads to increased villus length and crypt depth, and increased goblet and Paneth cells, compared to controls. We also show that HA 35 KDa leads to an increased predominance of Clostridiales Ruminococcaceae, Lactobacillales Lactobacillaceae, and Clostridiales Lachnospiraceae. In seeking the mechanisms involved in the changes, bulk RNA seq was performed on samples from the terminal ileum and identified upregulation in several genes essential for cellular growth, proliferation, and survival. Taken together, this study shows that HA 35 KDa supplemented to mouse pups promotes intestinal epithelial cell proliferation, as well as the development of Paneth cells and goblet cell subsets. HA 35 KDa also impacted the intestinal microbiota; the implications of these responses need to be determined.

Yap/Taz inhibit goblet cell fate to maintain lung epithelial homeostasis.

Proper lung function relies on the precise balance of specialized epithelial cells that coordinate to maintain homeostasis. Herein, we describe essential roles for the transcriptional regulators YAP/TAZ in maintaining lung epithelial homeostasis, reporting that conditional deletion of Yap and Wwtr1/Taz in the lung epithelium of adult mice results in severe defects, including alveolar disorganization and the development of airway mucin hypersecretion. Through in vivo lineage tracing and in vitro molecular experiments, we reveal that reduced YAP/TAZ activity promotes intrinsic goblet transdifferentiation of secretory airway epithelial cells. Global gene expression and chromatin immunoprecipitation sequencing (ChIP-seq) analyses suggest that YAP/TAZ act cooperatively with TEA domain (TEAD) transcription factors and the NuRD complex to suppress the goblet cell fate program, directly repressing the SPDEF gene. Collectively, our study identifies YAP/TAZ as critical factors in lung epithelial homeostasis and offers molecular insight into the mechanisms promoting goblet cell differentiation, which is a hallmark of many lung diseases.

Immunomodulatory responses in plectasin-supplemented broilers under tropical environmental conditions.

The present study was aimed to determine the immunomodulatory effects of dietary supplementation of the antimicrobial peptide (AMP) plectasin on broiler chickens. The experiment involved 300-day-old Ross chicks reared in a conventional housing system and subjected to ambient temperature and relative humidity. The birds were randomly allocated to five treatment groups: the non-supplemented negative control group (T1), enramycin-supplemented group (T2), and groups supplemented with varying doses of plectasin at 150 ppm, 300 ppm, and 450 ppm (T3, T4, and T5, respectively) from day 1 to 35. The results indicated that plectasin supplementation increased jejunal and ileal goblet cell (GC) counts, serum interferon-gamma (IFN-γ) levels at neonatal age, and serum immunoglobulin Y (IgY) titer on days 7, 21, 28, and 35. These findings confirmed that plectasin induces positive immunomodulatory responses by specifically enhancing gut mucosal barriers, early innate immunity, and humoral immune response. Specifically, supplementation at 150 ppm may be considered as the optimal dose for inclusion in broiler chicken feeds.

An intercrypt subpopulation of goblet cells is essential for colonic mucus barrier function.

The intestinal mucus layer, an important element of epithelial protection, is produced by goblet cells. Intestinal goblet cells are assumed to be a homogeneous cell type. In this study, however, we delineated their specific gene and protein expression profiles and identified several distinct goblet cell populations that form two differentiation trajectories. One distinct subtype, the intercrypt goblet cells (icGCs), located at the colonic luminal surface, produced mucus with properties that differed from the mucus secreted by crypt-residing goblet cells. Mice with defective icGCs had increased sensitivity to chemically induced colitis and manifested spontaneous colitis with age. Furthermore, alterations in mucus and reduced numbers of icGCs were observed in patients with both active and remissive ulcerative colitis, which highlights the importance of icGCs in maintaining functional protection of the epithelium.

Optimization of goblet cell density quantification methods.

The purpose of this study was to develop a standardized, accurate and efficient method for estimating conjunctival goblet cell density (GCD) via optimizing sample storage conditions and quantification methods. Conjunctival impression cytology (CIC) membranes were collected from both eyes of 32 participants and were randomized to two storage durations (2-3 weeks, 6-7 weeks) and two storage container types (microcentrifuge tube, flat histology cassette). The CIC membranes were stained and subdivided into 25 areas (5 mm × 5 mm) for imaging and the GCs were counted under 200X magnification using three different methods: (1) full CIC membrane GC count of the 25 images with cell-counting software ("full"; reference method), (2) partial membrane GC count of 9 images with cell-counting software ("partial"), and (3) manual counting of the 25 images ("manual"). In all cases, GCD was determined by dividing the GC count by the counting area. The average time required for quantification was recorded to gauge efficiency. Results showed no significant difference in GC count between the two storage durations (p = 0.745) or storage container types (p = 0.552). The median (interquartile range (IQR)) time required to quantify a CIC membrane for the full, partial, and manual methods of GC counting, was 14.8(17.6), 4.6(5.2) and 5.0 (5.0) minutes, respectively. The agreement of GCD values between the full and manual methods (bias: 0.4, 95% LOA: [-4.6, 5.5]) was stronger than that comparing the full and partial methods (bias: 0.5, 95% LOA: [-18, 17]). All together, through systematic examination of key procedural variables, an optimized method for GCD quantification within 7 weeks of sample collection was outlined. Adaption of procedures described in this paper to facilitate accurate and efficient GCD quantification may serve as a valuable step in clinical trials investigating DED pathophysiology and/or novel DED treatment strategies.

Thyroid Hormones Regulate Goblet Cell Differentiation and Fgf19-Fgfr4 Signaling.

Hypothyroidism is a common pathological condition characterized by insufficient activity of the thyroid hormones (THs), thyroxine (T4), and 3,5,3'-triiodothyronine (T3), in the whole body or in specific tissues. Hypothyroidism is associated with inadequate development of the intestine as well as gastrointestinal diseases. We used a zebrafish model of hypothyroidism to identify and characterize TH-modulated genes and cellular pathways controlling intestine development. In the intestine of hypothyroid juveniles and adults, the number of mucus-secreting goblet cells was reduced, and this phenotype could be rescued by T3 treatment. Transcriptome profiling revealed dozens of differentially expressed genes in the intestine of hypothyroid adults compared to controls. Notably, the expression of genes encoding to Fgf19 and its receptor Fgfr4 was markedly increased in the intestine of hypothyroid adults, and treatment with T3 normalized it. Blocking fibroblast growth factor (FGF) signaling, using an inducible dominant-negative Fgfr transgenic line, rescued the number of goblet cells in hypothyroid adults. These results show that THs inhibit the Fgf19-Fgfr4 signaling pathway, which is associated with inhibition of goblet cell differentiation in hypothyroidism. Both the TH and Fgf19-Fgfr4 signaling pathways can be pharmaceutical targets for the treatment of TH-related gastrointestinal diseases.

DOCK4 stimulates MUC2 production through its effect on goblet cell differentiation.

The intestinal mucosa is in continuous contact with milliard of microorganisms, thus intestinal epithelial barrier is a critical component in the arsenal of defense mechanisms required to prevent infection and inflammation. Mucin 2 (MUC2), which is produced by the goblet cells, forms the skeleton of the intestinal mucus and protects the intestinal tract from self-digestion and numerous microorganisms. Dedicator of cytokinesis 4 (DOCK4) is a member of the DOCK-B subfamily of the DOCK family of guanine nucleotide exchange factors. It is reported that DOCK4 plays a critical role in the repair of the barrier function of the intestinal epithelium after chemical damage. In this study, the role of DOCK4 in the goblet cell differentiation and MUC2 production is explored. Disordered intestinal epithelium and shortage of goblet cells were observed in DOCK4 gene knockout mice. Furthermore, DOCK4 deletion contributed to the low expression of MUC2 and the goblet cell differentiation/maturation factors including growth factor independent 1 (Gfi1) and SAM pointed domain epithelial-specific transcription factor (Spdef) in mouse ileums and colons. Overexpression of DOCK4 caused a marked increase in Gfi1, Spdef, and MUC2, while siRNA knockdown of endogenous DOCK4 significantly decreased Gfi1, Spdef, and MUC2 in HT-29 cells. In addition, MUC2, DOCK4, and the goblet cell differentiation/maturation factors mRNA levels were decreased in colorectal cancer samples compared with normal colons. A significant positive correlation was found between MUC2 and DOCK4. In conclusion, DOCK4 may serve as a critical regulator of goblet cell differentiation and MUC2 production in the intestine.

Mitochondria Define Intestinal Stem Cell Differentiation Downstream of a FOXO/Notch Axis.

Differential WNT and Notch signaling regulates differentiation of Lgr5+ crypt-based columnar cells (CBCs) into intestinal cell lineages. Recently we showed that mitochondrial activity supports CBCs, while adjacent Paneth cells (PCs) show reduced mitochondrial activity. This implies that CBC differentiation into PCs involves a metabolic transition toward downregulation of mitochondrial dependency. Here we show that Forkhead box O (FoxO) transcription factors and Notch signaling interact in determining CBC fate. In agreement with the organoid data, Foxo1/3/4 deletion in mouse intestine induces secretory cell differentiation. Importantly, we show that FOXO and Notch signaling converge on regulation of mitochondrial fission, which in turn provokes stem cell differentiation into goblet cells and PCs. Finally, scRNA-seq-based reconstruction of CBC differentiation trajectories supports the role of FOXO, Notch, and mitochondria in secretory differentiation. Together, this points at a new signaling-metabolic axis in CBC differentiation and highlights the importance of mitochondria in determining stem cell fate.