Probes for INS

The uptake of glucose is mediated mainly by the sodium-glucose cotransporter, SGLT1. Previous studies using quantitative PCR showed that SGLT1 mRNA was induced in the yolk sac and in the small intestine prior to hatch. However, PCR analysis did not allow for the localization of cells expressing SGLT1 mRNA. The objective of this study was to use in situ hybridization to identify cells in the yolk sac and small intestine that expressed SGLT1 mRNA during the transition from late embryogenesis to early post-hatch. Expression of SGLT1 mRNA in yolk sac epithelial cells was low from embryonic d 11 to 17, peaked at embryonic d 19, and declined at day of hatch. In the small intestine, cells expressing SGLT1 mRNA were present not only along the intestinal villi but also in the crypts. There was greater expression of SGLT1 mRNA in the intestinal epithelial cells that line the villus than in the olfactomedin 4-expressing stem cells located in the crypts. The latter result suggests that stem cells have the ability to import glucose. Expression of SGLT1 mRNA in the intestine increased from embryonic d 19 to day of hatch and then maintained a high level of expression from d 1 to d 7 post-hatch. For both the yolk sac and small intestine, the temporal pattern of SGLT1 mRNA expression detected by in situ hybridization was consistent with the pattern revealed by PCR.

The chicken yolk sac (YS) and small intestine are essential for nutrient absorption during the pre-hatch and post-hatch periods, respectively. Absorptive enterocytes and secretory cells line the intestinal villi and originate from stem cells located in the intestinal crypts. Similarly, in the YS, there are absorptive and secretory cells that presumably originate from a stem cell population. Leucine-rich repeat containing G protein-coupled receptor 5 (Lgr5) and olfactomedin 4 (Olfm4) are 2 widely used markers for intestinal stem cells. The objective of this study was to map the distribution of putative stem cells expressing LGR5 and OLFM4 mRNA in the chicken small intestine from the late embryonic period to early post hatch and the YS during embryogenesis. At embryonic d 11, 13, 15, 17, and 19, the YS was collected (n = 3), and small intestine was collected at embryonic d 19, d of hatch (doh), and d 1, 4, and 7 post hatch (n = 3). Cells expressing OLFM4 and LGR5 mRNA were identified by in situ hybridization. In the YS, cells expressing only LGR5 and not OLFM4 mRNA were localized to the vascular endothelial cells lining the blood vessels. In the small intestine, cells in the intestinal crypt expressed both LGR5 and OLFM4 mRNA. Staining for OLFM4 mRNA was more intense than LGR5 mRNA, demonstrating that Olfm4 is a more robust marker for stem cells than Lgr5. At embryonic d 19 and doh, cells staining for OLFM4 mRNA were already present in the rudimentary crypts, with the greatest staining in the duodenal crypts. The intensity of OLFM4 mRNA staining increased from doh to d 7 post hatch. Dual label staining at doh for the peptide transporter PepT1 and Olfm4 revealed a population of cells above the crypts that did not express Olfm4 or PepT1 mRNA. These cells are likely progenitor transit amplifying cells. Thus, avians and mammals share similarity in the ontogeny of stem cells in the intestinal crypts.

In the adult mouse spinal cord, the ependymal cell population that surrounds the central canal is thought to be a promising source of quiescent stem cells to treat spinal cord injury. Relatively little is known about the cellular origin of ependymal cells during spinal cord development, or the molecular mechanisms that regulate ependymal cells during adult homeostasis. Using genetic lineage tracing based on the Wnt target gene Axin2, we have characterized Wnt-responsive cells during spinal cord development. Our results revealed that Wnt-responsive progenitor cells are restricted to the dorsal midline throughout spinal cord development, which gives rise to dorsal ependymal cells in a spatially restricted pattern. This is contrary to previous reports that suggested an exclusively ventral origin of ependymal cells, suggesting that ependymal cells may retain positional identities in relation to their neural progenitors. Our results further demonstrated that in the postnatal and adult spinal cord, all ependymal cells express the Wnt/β-catenin signaling target gene Axin2, as well as Wnt ligands. Genetic elimination of β-catenin or inhibition of Wnt secretion in Axin2-expressing ependymal cells in vivo both resulted in impaired proliferation, indicating that Wnt/β-catenin signaling promotes ependymal cell proliferation. These results demonstrate the continued importance of Wnt/β-catenin signaling for both ependymal cell formation and regulation. By uncovering the molecular signals underlying the formation and regulation of spinal cord ependymal cells, our findings thus enable further targeting and manipulation of this promising source of quiescent stem cells for therapeutic interventions.

Wnt signaling plays an important role in uterine organogenesis and oncogenesis. Our mRNA expression data documents the expression of various Wnt pathway members during the key stages of uterine epithelial gland development. Our data illustrates the expression of Wnt signaling inhibitors (Axin2, Sfrp2, Sfrp4, Dkk1 and Dkk3) in mice uteri at postnatal day 6 (PND 6) and day 15 (PND 15). They also describe the expression pattern of the Wnt ligands (Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt5b, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, Wnt10a and Wnt10b) in mice uteri with or without progesterone treatment. Detailed interpretation and discussion of these data is presented in the research article entitled “Differential Wnt signaling activity limits epithelial gland development to the anti-mesometrial side of the mouse uterus” [1].

The epithelial lining of the Fallopian tube is vital for fertility, providing nutrition to gametes, and facilitating their transport. It is composed of two major cell types: secretory cells and ciliated cells. Interestingly, human ovarian cancer precursor lesions are primarily consisting of secretory cells. It is unclear why secretory cells are the dominant cell type in these lesions. Additionally, the underlying mechanisms governing Fallopian tube epithelial homoeostasis are currently unknown. In the present study, we showed that across the different developmental stages of mouse oviduct, secretory cells are the most frequently dividing cells of the oviductal epithelium. In vivo genetic cell lineage tracing showed that secretory cells not only self-renew, but also give rise to ciliated cells. Analysis of a Wnt reporter mouse model and different Wnt target genes showed that the Wnt signaling pathway is involved in oviductal epithelial homoeostasis. By developing two triple transgenic mouse models, we showed that Wnt/β-catenin signaling is essential for self-renewal as well as differentiation of secretory cells. In summary, our results provide mechanistic insight into oviductal epithelial homoeostasis.

The canonical Wnt/β-catenin signalling pathway governs diverse developmental, homeostatic and pathological processes. Palmitoylated Wnt ligands engage cell-surface frizzled (FZD) receptors and LRP5 and LRP6 co-receptors, enabling β-catenin nuclear translocation and TCF/LEF-dependent gene transactivation. Mutations in Wnt downstream signalling components have revealed diverse functions thought to be carried out by Wnt ligands themselves. However, redundancy between the 19 mammalian Wnt proteins and 10 FZD receptors and Wnt hydrophobicity have made it difficult to attribute these functions directly to Wnt ligands. For example, individual mutations in Wnt ligands have not revealed homeostatic phenotypes in the intestinal epithelium-an archetypal canonical, Wnt pathway-dependent, rapidly self-renewing tissue, the regeneration of which is fueled by proliferative crypt Lgr5+ intestinal stem cells (ISCs). R-spondin ligands (RSPO1-RSPO4) engage distinct LGR4-LGR6, RNF43 and ZNRF3 receptor classes, markedly potentiate canonical Wnt/β-catenin signalling, and induce intestinal organoid growth in vitro and Lgr5+ ISCs in vivo. However, the interchangeability, functional cooperation and relative contributions of Wnt versus RSPO ligands to in vivo canonical Wnt signalling and ISC biology remain unknown. Here we identify the functional roles of Wnt and RSPO ligands in the intestinal crypt stem-cell niche. We show that the default fate of Lgr5+ ISCs is to differentiate, unless both RSPO and Wnt ligands are present. However, gain-of-function studies using RSPO ligands and a new non-lipidated Wnt analogue reveal that these ligands have qualitatively distinct, non-interchangeable roles in ISCs. Wnt proteins are unable to induce Lgr5+ ISC self-renewal, but instead confer a basal competency by maintaining RSPO receptor expression that enables RSPO ligands to actively drive and specify the extent of stem-cell expansion. This functionally non-equivalent yet cooperative interaction between Wnt and RSPO ligands establishes a molecular precedent for regulation of mammalian stem cells by distinct priming and self-renewal factors, with broad implications for precise control of tissue regeneration.

Hair growth disorders often carry a major psychological burden. Therefore, more effective human hair growth-modulatory agents urgently need to be developed. Here, we used the hypertrichosis-inducing immunosuppressant, Cyclosporine A (CsA), as a lead compound to identify new hair growth-promoting molecular targets. Through microarray analysis we identified the Wnt inhibitor, secreted frizzled related protein 1 (SFRP1), as being down-regulated in the dermal papilla (DP) of CsA-treated human scalp hair follicles (HFs) ex vivo. Therefore, we further investigated the function of SFRP1 using a pharmacological approach and found that SFRP1 regulates intrafollicular canonical Wnt/β-catenin activity through inhibition of Wnt ligands in the human hair bulb. Conversely, inhibiting SFRP1 activity through the SFRP1 antagonist, WAY-316606, enhanced hair shaft production, hair shaft keratin expression, and inhibited spontaneous HF regression (catagen) ex vivo. Collectively, these data (a) identify Wnt signalling as a novel, non-immune-inhibitory CsA target; (b) introduce SFRP1 as a physiologically important regulator of canonical β-catenin activity in a human (mini-)organ; and (c) demonstrate WAY-316606 to be a promising new promoter of human hair growth. Since inhibiting SFRP1 only facilitates Wnt signalling through ligands that are already present, this 'ligand-limited' therapeutic strategy for promoting human hair growth may circumvent potential oncological risks associated with chronic Wnt over-activation.

In vitro differentiation of human intestinal organoids (HIOs) from pluripotent stem cells is an unparalleled system for creating complex, multicellular three-dimensional structures capable of giving rise to tissue analogous to native human tissue. Current methods for generating HIOs rely on growth in an undefined tumour-derived extracellular matrix (ECM), which severely limits the use of organoid technologies for regenerative and translational medicine. Here, we developed a fully defined, synthetic hydrogel based on a four-armed, maleimide-terminated poly(ethylene glycol) macromer that supports robust and highly reproducible in vitro growth and expansion of HIOs, such that three-dimensional structures are never embedded in tumour-derived ECM. We also demonstrate that the hydrogel serves as an injection vehicle that can be delivered into injured intestinal mucosa resulting in HIO engraftment and improved colonic wound repair. Together, these studies show proof-of-concept that HIOs may be used therapeutically to treat intestinal injury.

Tissues that undergo rapid cellular turnover, such as the mammalian haematopoietic system or the intestinal epithelium, are dependent on stem and progenitor cells that proliferate to provide differentiated cells to maintain organismal health. Stem and progenitor cells, in turn, are thought to rely on signals and growth factors provided by local niche cells to support their function and self-renewal. Several cell types have been hypothesized to provide the signals required for the proliferation and differentiation of the intestinal stem cells in intestinal crypts1-6. Here we identify subepithelial telocytes as an important source of Wnt proteins, without which intestinal stem cells cannot proliferate and support epithelial renewal. Telocytes are large but rare mesenchymal cells that are marked by expression of FOXL1 and form a subepithelial plexus that extends from the stomach to the colon. While supporting the entire epithelium, FOXL1+ telocytes compartmentalize the production of Wnt ligands and inhibitors to enable localized pathway activation. Conditional genetic ablation of porcupine (Porcn), which is required for functional maturation of all Wnt proteins, in mouse FOXL1+ telocytes causes rapid cessation of Wnt signalling to intestinal crypts, followed by loss of proliferation of stem and transit amplifying cells and impaired epithelial renewal. Thus, FOXL1+ telocytes are an important source of niche signals to intestinal stem cells.

In mice, implantation always occurs towards the antimesometrial side of the uterus, while the placenta develops at the mesometrial side. What determines this particular orientation of the implanting blastocyst remains unclear. Uterine glands are critical for implantation and pregnancy. In this study, we showed that uterine gland development and active Wnt signalling activity is limited to the antimesometrial side of the uterus. Dkk2, a known antagonist of Wnt signalling, is only present at the mesometrial side of the uterus. Imaging of whole uterus, thick uterine sections (100-1000μm), and individual glands revealed that uterine glands are simple tubes with branches that are directly connected to the luminal epithelium and are only present towards the antimesometrial side of the uterus. By developing a unique mouse model targeting the uterine epithelium, we demonstrated that Wnt/β-catenin signaling is essential for prepubertal gland formation and normal implantation, but dispensable for postpartum gland development and regeneration. Our results for the first time have provided a probable explanation for the antimesometrial bias for implantation.

The constant regeneration of stomach epithelium is driven by long-lived stem cells, but the mechanism that regulates their turnover is not well understood. We have recently found that the gastric pathogen Helicobacter pylori can activate gastric stem cells and increase epithelial turnover, while Wnt signalling is known to be important for stem cell identity and epithelial regeneration in several tissues. Here we find that antral Wnt signalling, marked by the classic Wnt target gene Axin2, is limited to the base and lower isthmus of gastric glands, where the stem cells reside. Axin2 is expressed by Lgr5+ cells, as well as adjacent, highly proliferative Lgr5- cells that are able to repopulate entire glands, including the base, upon depletion of the Lgr5+ population. Expression of both Axin2 and Lgr5 requires stroma-derived R-spondin 3 produced by gastric myofibroblasts proximal to the stem cell compartment. Exogenous R-spondin administration expands and accelerates proliferation of Axin2+/Lgr5- but not Lgr5+ cells. Consistent with these observations, H. pylori infection increases stromal R-spondin 3 expression and expands the Axin2+ cell pool to cause hyperproliferation and gland hyperplasia. The ability of stromal niche cells to control and adapt epithelial stem cell dynamics constitutes a sophisticated mechanism that orchestrates epithelial regeneration and maintenance of tissue integrity.