Distinct ATOH1 and Neurog3 requirements define tuft cells as a new secretory cell type in the intestinal epithelium

Tuft cells represent a fourth type of intestinal secretory cell that constitutes the primary source of endogenous intestinal opioids and are the only epithelial cell that constitutively express cyclooxygenases

Presumed Symbolic Use of Diurnal Raptors by Neanderthals

In Africa and western Eurasia, occurrences of burials and utilized ocher fragments during the late Middle and early Late Pleistocene are often considered evidence for the emergence of symbolically-mediated behavior. Perhaps less controversial for the study of human cognitive evolution are finds of marine shell beads and complex designs on organic and mineral artifacts in early modern human (EMH) assemblages conservatively dated to ≈100–60 kilo-years (ka) ago. Here we show that, in France, Neanderthals used skeletal parts of large diurnal raptors presumably for symbolic purposes at Combe-Grenal in a layer dated to marine isotope stage (MIS) 5b (≈90 ka) and at Les Fieux in stratigraphic units dated to the early/middle phase of MIS 3 (60–40 ka). The presence of similar objects in other Middle Paleolithic contexts in France and Italy suggest that raptors were used as means of symbolic expression by Neanderthals in these regions

A semi-automated intestinal organoid screening method demonstrates epigenetic control of epithelial maturation

The intestinal epithelium maintains an important barrier throughout life. It consists of several epithelial cell lineages that are derived from LGR5+ intestinal stem cells. Although epigenetic regulation of embryonic stem cell differentiation is well established, its role in adult stem cell systems such as the intestinal epithelium is still undefined. Yet, targeting of epigenetic regulatory enzymes may be relevant for new therapeutics, for example in cancer treatment. Here, we combine a newly established organoid toolbox with an epigenetic probe library to identify epigenetic regulators of intestinal epithelial biology. We discover several probes that alter intestinal epithelial biology including those targeting HDACs, EP300/CREBBP, LSD1, and type I PRMTs. We conclude that epigenetic modifiers are primarily involved in mediating maturation of the epithelium rather than dictating specific cell lineage differentiation. Furthermore, we show that inhibiting type I PRMTs, which leads to epithelial maturation, blocks the growth of adenoma but not normal organoid cultures. Thus, epigenetic probes are a powerful tool in defining biological processes and demonstrate therapeutic potential

A semi-automated organoid screening method demonstrates epigenetic control of intestinal epithelial differentiation

Intestinal organoids are an excellent model to study epithelial biology. Yet, the selection of analytical tools to accurately quantify heterogeneous organoid cultures remains limited. Here, we developed a semi-automated organoid screening method, which we applied to a library of highly specific chemical probes to identify epigenetic regulators of intestinal epithelial biology. The role of epigenetic modifiers in adult stem cell systems, such as the intestinal epithelium, is still undefined. Based on this resource dataset, we identified several targets that affected epithelial cell differentiation, including HDACs, EP300/CREBBP, LSD1, and type I PRMTs, which were verified by complementary methods. For example, we show that inhibiting type I PRMTs, which leads enhanced epithelial differentiation, blocks the growth of adenoma but not normal organoid cultures. Thus, epigenetic probes are powerful tools to study intestinal epithelial biology and may have therapeutic potential

Intestinal epithelial stem cells do not protect their genome by asymmetric chromosome segregation

The idea that stem cells of adult tissues with high turnover are protected from DNA replication-induced mutations by maintaining the same 'immortal' template DNA strands together through successive divisions has been tested in several tissues. In the epithelium of the small intestine, the provided evidence was based on the assumption that stem cells are located above Paneth cells. The results of genetic lineage-tracing experiments point instead to crypt base columnar cells intercalated between Paneth cells as bona fide stem cells. Here we show that these cells segregate most, if not all, of their chromosomes randomly, both in the intact and in the regenerating epithelium. Therefore, the 'immortal' template DNA strand hypothesis does not apply to intestinal epithelial stem cells, which must rely on other strategies to avoid accumulating mutations

Krüppel-like Factor 4 Regulates Intestinal Epithelial Cell Morphology and Polarity

Krüppel-like factor 4 (KLF4) is a zinc finger transcription factor that plays a vital role in regulating cell lineage differentiation during development and maintaining epithelial homeostasis in the intestine. In normal intestine, KLF4 is predominantly expressed in the differentiated epithelial cells. It has been identified as a tumor suppressor in colorectal cancer. KLF4 knockout mice demonstrated a decrease in number of goblet cells in the colon, and conditional ablation of KLF4 from the intestinal epithelium led to altered epithelial homeostasis. However, the role of KLF4 in differentiated intestinal cells and colon cancer cells, as well as the mechanism by which it regulates homeostasis and represses tumorigenesis in the intestine is not well understood. In our study, KLF4 was partially depleted in the differentiated intestinal epithelial cells by a tamoxifen-inducible Cre recombinase. We found a significant increase in the number of goblet cells in the KLF4-deleted small intestine, suggesting that KLF4 is not only required for goblet cell differentiation, but also required for maintaining goblet cell numbers through its function in inhibiting cell proliferation. The number and position of Paneth cells also changed. This is consistent with the KLF4 knockout study using villin-Cre [1]. Through immunohistochemistry (IHC) staining and statistical analysis, we found that a stem cell and/or tuft cell marker, DCAMKL1, and a proliferation marker, Ki67, are affected by KLF4 depletion, while an enteroendocrine cell marker, neurotensin (NT), was not affected. In addition, we found KLF4 depletion altered the morphology and polarity of the intestinal epithelial cells. Using a three-dimensional (3D) intestinal epithelial cyst formation assay, we found that KLF4 is essential for cell polarity and crypt-cyst formation in human colon cancer cells. These findings suggest that, as a tumor suppressor in colorectal cancer, KLF4 affects intestinal epithelial cell morphology by regulating proliferation, differentiation and polarity of the cells

Dietary Pectin Increases Intestinal Crypt Stem Cell Survival following Radiation Injury

This research was performed as a project of the Intestinal Stem Cell Consortium, a collaborative research project funded by the National Institute of Diabetes and Digestive and Kidney Diseases (NIH U01 DK-085508 to CWH), and a grant from Oklahoma Center for the Advancement of Science and Technology to CWH.Gastrointestinal (GI) mucosal damage is a devastating adverse effect of radiation therapy. We have recently reported that expression of Dclk1, a Tuft cell and tumor stem cell (TSC) marker, 24h after high dose total-body gamma-IR (TBI) can be used as a surrogate marker for crypt survival. Dietary pectin has been demonstrated to possess chemopreventive properties, whereas its radioprotective property has not been studied. The aim of this study was to determine the effects of dietary pectin on ionizing radiation (IR)-induced intestinal stem cell (ISC) deletion, crypt and overall survival following lethal TBI. C57BL/6 mice received a 6% pectin diet and 0.5% pectin drinking water (pre-IR mice received pectin one week before TBI until death; post-IR mice received pectin after TBI until death). Animals were exposed to TBI (14 Gy) and euthanized at 24 and 84h post-IR to assess ISC deletion and crypt survival respectively. Animals were also subjected to overall survival studies following TBI. In pre-IR treatment group, we observed a three-fold increase in ISC/crypt survival, a two-fold increase in Dclk1+ stem cells, increased overall survival (median 10d vs. 7d), and increased expression of Dclk1, Msi1, Lgr5, Bmi1, and Notch1 (in small intestine) post-TBI in pectin treated mice compared to controls. We also observed increased survival of mice treated with pectin (post-IR) compared to controls. Dietary pectin is a radioprotective agent; prevents IR-induced deletion of potential reserve ISCs; facilitates crypt regeneration; and ultimately promotes overall survival. Given the anti-cancer activity of pectin, our data support a potential role for dietary pectin as an agent that can be administered to patients receiving radiation therapy to protect against radiation-induces mucositis.Yeshttp://www.plosone.org/static/editorial#pee

Isolation of Oct4-Expressing Extraembryonic Endoderm Precursor Cell Lines

BACKGROUND:The extraembryonic endoderm (ExEn) defines the yolk sac, a set of membranes that provide essential support for mammalian embryos. Recent findings suggest that the committed ExEn precursor is present already in the embryonic Inner Cell Mass (ICM) as a group of cells that intermingles with the closely related epiblast precursor. All ICM cells contain Oct4, a key transcription factor that is first expressed at the morula stage. In vitro, the epiblast precursor is most closely represented by the well-characterized embryonic stem (ES) cell lines that maintain the expression of Oct4, but analogous ExEn precursor cell lines are not known and it is unclear if they would express Oct4. METHODOLOGY/PRINCIPAL FINDINGS:Here we report the isolation and characterization of permanently proliferating Oct4-expressing rat cell lines ("XEN-P cell lines"), which closely resemble the ExEn precursor. We isolated the XEN-P cell lines from blastocysts and characterized them by plating and gene expression assays as well as by injection into embryos. Like ES cells, the XEN-P cells express Oct4 and SSEA1 at high levels and their growth is stimulated by leukemia inhibitory factor, but instead of the epiblast determinant Nanog, they express the ExEn determinants Gata6 and Gata4. Further, they lack markers characteristic of the more differentiated primitive/visceral and parietal ExEn stages, but exclusively differentiate into these stages in vitro and contribute to them in vivo. CONCLUSIONS/SIGNIFICANCE:Our findings (i) suggest strongly that the ExEn precursor is a self-renewable entity, (ii) indicate that active Oct4 gene expression (transcription plus translation) is part of its molecular identity, and (iii) provide an in vitro model of early ExEn differentiation

Modelling the Spatio-Temporal Cell Dynamics Reveals Novel Insights on Cell Differentiation and Proliferation in the Small Intestinal Crypt

We developed a slow structural relaxation model to describe cellular dynamics in the crypt of the mouse small intestine. Cells are arranged in a three dimensional spiral the size of which dynamically changes according to cell production demands of adjacent villi. Cell differentiation and proliferation is regulated through Wnt and Notch signals, the strength of which depends on the local cell composition. The highest level of Wnt activity is associated with maintaining equipotent stem cells (SC), Paneth cells and common goblet-Paneth cell progenitors (CGPCPs) intermingling at the crypt bottom. Low levels of Wnt signalling area are associated with stem cells giving rise to secretory cells (CGPCPs, enteroendocrine or Tuft cells) and proliferative absorptive progenitors. Deciding between these two fates, secretory and stem/absorptive cells, depends on Notch signalling. Our model predicts that Notch signalling inhibits secretory fate if more than 50% of cells they are in contact with belong to the secretory lineage. CGPCPs under high Wnt signalling will differentiate into Paneth cells while those migrating out from the crypt bottom differentiate into goblet cells. We have assumed that mature Paneth cells migrating upwards undergo anoikis. Structural relaxation explains the localisation of Paneth cells to the crypt bottom in the absence of active forces. The predicted crypt generation time from one SC is 4–5 days with 10–12 days needed to reach a structural steady state. Our predictions are consistent with experimental observations made under altered Wnt and Notch signalling. Mutations affecting stem cells located at the crypt floor have a 50% chance of being propagated throughout the crypt while mutations in cells above are rarely propagated. The predicted recovery time of an injured crypt losing half of its cells is approximately 2 days

The neuropeptide NMU amplifies ILC2-driven allergic lung inflammation

Type 2 innate lymphoid cells (ILC2s) both contribute to mucosal homeostasis and initiate pathologic inflammation in allergic asthma. However, the signals that direct ILC2s to promote homeostasis versus inflammation are unclear. To identify such molecular cues, we profiled mouse lung-resident ILCs using single-cell RNA sequencing at steady state and after in vivo stimulation with the alarmin cytokines IL-25 and IL-33. ILC2s were transcriptionally heterogeneous after activation, with subpopulations distinguished by expression of proliferative, homeostatic and effector genes. The neuropeptide receptor Nmur1 was preferentially expressed by ILC2s at steady state and after IL-25 stimulation. Neuromedin U (NMU), the ligand of NMUR1, activated ILC2s in vitro, and in vivo co-administration of NMU with IL-25 strongly amplified allergic inflammation. Loss of NMU-NMUR1 signalling reduced ILC2 frequency and effector function, and altered transcriptional programs following allergen challenge in vivo. Thus, NMUR1 signalling promotes inflammatory ILC2 responses, highlighting the importance of neuro-immune crosstalk in allergic inflammation at mucosal surfaces