Probes for INS

Adult stem cells play an essential role in adult organ physiology and tissue repair and regeneration. While much has been learnt about the property and function of various adult stem cells, the mechanisms of their development remain poorly understood in mammals. Earlier studies suggest that the formation of adult mouse intestinal stem cells takes place during the first few weeks after birth, the postembryonic period when plasma thyroid hormone (T3) levels are high. Furthermore, deficiency in T3 signaling leads to defects in adult mouse intestine, including reduced cell proliferation in the intestinal crypts, where stem cells reside. Our earlier studies have shown that protein arginine methyltransferase 1 (PRMT1), a T3 receptor coactivator, is highly expressed during intestinal maturation in mouse.We have analyzed the expression of PRMT1 by immunohistochemistry and studied the effect of tissue-specific knockout of PRMT1 in the intestinal epithelium.We show that PRMT1 is expressed highly in the proliferating transit amplifying cells and crypt base stem cells. By using a conditional knockout mouse line, we have demonstrated that the expression of PRMT1 in the intestinal epithelium is critical for the development of the adult mouse intestine. Specific removal of PRMT1 in the intestinal epithelium results in, surprisingly, more elongated adult intestinal crypts with increased cell proliferation. In addition, epithelial cell migration along the crypt-villus axis and cell death on the villus are also increased. Furthermore, there are increased Goblet cells and reduced Paneth cells in the crypt while the number of crypt base stem cells remains unchanged.Our finding that PRMT1 knockout increases cell proliferation is surprising considering the role of PRMT1 in T3-signaling and the importance of T3 for intestinal development, and suggests that PRMT1 likely regulates pathways in addition to T3-signaling to affect intestinal development and/or homeostasis, thus affecting cell proliferating and epithelial turn over in the adult.

Acute intestinal mucositis is a common off-target effect of chemotherapy, leading to co-morbidities such as vomiting, diarrhea, sepsis, and death. We previously demonstrated that the presence of enteric bacteria modulates the extent of jejunal epithelial damage induced by doxorubicin (DXR) in mice. Despite conventional thinking of the crypt as a sterile environment, recent evidence suggests that bacterial signaling influences aISC function. In this study, we labeled aISCs using transgenic Lgr5-driven fluorescence or with immunostaining for OLFM4. We examined the effect of DXR in both germ free (GF) mice and mice depleted of microbiota using an established antimicrobial treatment protocol (AMBx). We found differences in DXR-induced loss of aISCs between GF mice and mice treated with AMBx. aISCs were decreased after DXR in GF mice, whereas AMBx mice retained aISC expression after DXR. Neither group of mice exhibited an inflammatory response to DXR, suggesting the difference in aISC retention was not due to differences in local tissue inflammation. Therefore, we suspected that there was a protective microbial signal present in the AMBx mice that was not present in the GF mice. 16S rRNA sequencing of jejunal luminal contents demonstrated that AMBx altered the fecal and jejunal microbiota. In the jejunal contents, AMBx mice had increased abundance of Ureaplasma and Burkholderia. These results suggest pro-survival signaling from microbiota in AMBx-treated mice to the aISCs, and that this signaling maintains aISCs in the face of chemotherapeutic injury. Manipulation of the enteric microbiota presents a therapeutic target for reducing the severity of chemotherapy-associated mucositis.

Mammary gland homeostasis is maintained by adult tissue stem-progenitor cells residing within the luminal and basal epithelia. Dysregulation of mammary stem cells is a key mechanism for cancer development. However, stem cell characterization is challenging because reporter models using cell-specific promoters do not fully recapitulate the mammary stem cell populations. We previously found that a 270-basepair Runx1 enhancer element, named eR1, marked stem cells in the blood and stomach. Here, we identified eR1 activity in a rare subpopulation of the ERα-negative luminal epithelium in mouse mammary glands. Lineage-tracing using an eR1-CreERT2 mouse model revealed that eR1+ luminal cells generated the entire luminal lineage and milk-secreting alveoli - eR1 therefore specifically marks lineage-restricted luminal stem cells. eR1-targeted-conditional knockout of Runx1 led to the expansion of luminal epithelial cells, accompanied by elevated ERα expression. Our findings demonstrate a definitive role for Runx1 in the regulation of the eR1-positive luminal stem cell proliferation during mammary homeostasis. Our findings identify a mechanistic link for Runx1 in stem cell proliferation and its dysregulation in breast cancer. Runx1 inactivation is therefore likely to be an early hit in the cell-of-origin of ERα+ luminal type breast cancer.

Infection with the protozoan parasite Eimeria can cause the economically devastating disease coccidiosis, which is characterized by gross tissue damage and inflammation resulting in blunted villi and altered intestinal homeostasis. Male broiler chickens at 21 d of age were given a single challenge with Eimeria acervulina. Temporal changes in intestinal morphology and gene expression were investigated at 0, 3, 5, 7, 10, and 14 d post-infection (dpi). There were increased crypt depths for chickens infected with E. acervulina starting at 3 dpi and continuing to 14 dpi. At 5 and 7 dpi, infected chickens had decreased Mucin2 (Muc2), and Avian beta defensin (AvBD) 6 mRNA at 5 and 7 dpi and decreased AvBD10 mRNA at 7 dpi compared to uninfected chickens. Liver-enriched antimicrobial peptide 2 (LEAP2) mRNA was decreased at 3, 5, 7, and 14 dpi compared to uninfected chickens. After 7 dpi, there was increased Collagen 3a1 and Notch 1 mRNA compared to uninfected chickens. Marker of proliferation Ki67 mRNA was increased in infected chickens from 3 to 10 dpi. In addition, the presence of E. acervulina was visualized by in situ hybridization (ISH) with an E. acervulina sporozoite surface antigen (Ea-SAG) probe. In E. acervulina infected chickens, Ea-SAG mRNA was only detectable on 5 and 7 dpi by both ISH and qPCR. To further investigate the site of E. acervulina infection, Ea-SAG and Muc2 probes were examined on serial sections. The Muc2 ISH signal was decreased in regions where the Ea-SAG ISH signal was present, suggesting that the decrease in Muc2 by qPCR may be caused by the loss of Muc2 in the localized regions where the E. acervulina had invaded the tissue. Eimeria acervulina appears to manipulate host cells by decreasing their defensive capabilities and thereby allows the infection to propagate freely. Following infection, the intestinal cells upregulate genes that may support regeneration of damaged intestinal tissue.

Symbionts play an indispensable role in gut homeostasis, but underlying mechanisms remain elusive. To clarify the role of lactic-acid-producing bacteria (LAB) on intestinal stem-cell (ISC)-mediated epithelial development, we fed mice with LAB-type symbionts such as Bifidobacterium and Lactobacillus spp. Here we show that administration of LAB-type symbionts significantly increased expansion of ISCs, Paneth cells, and goblet cells. Lactate stimulated ISC proliferation through Wnt/β-catenin signals of Paneth cells and intestinal stromal cells. Moreover, Lactobacillus plantarum strains lacking lactate dehydrogenase activity, which are deficient in lactate production, elicited less ISC proliferation. Pre-treatment with LAB-type symbionts or lactate protected mice in response to gut injury provoked by combined treatments with radiation and a chemotherapy drug. Impaired ISC-mediated epithelial development was found in mice deficient of the lactate G-protein-coupled receptor, Gpr81. Our results demonstrate that LAB-type symbiont-derived lactate plays a pivotal role in promoting ISC-mediated epithelial development in a Gpr81-dependent manner.

RUNX transcription factors play pivotal roles in embryonic development and neoplasia. We previously identified the single missense mutation R122C in RUNX3 from human gastric cancer. However, how RUNX3R122C mutation disrupts stem cell homeostasis and promotes gastric carcinogenesis remained unclear.To understand the oncogenic nature of this mutation in vivo, we generated the RUNX3R122C knock-in mice. Stomach tissues were harvested, followed by histological and immunofluorescence staining, organoid culture, flow cytometry to isolate gastric corpus isthmus and non-isthmus epithelial cells, and RNA extraction for transcriptomic analysis.The corpus tissue of RUNX3R122C/R122C homozygous mice exhibited a precancerous phenotype such as spasmolytic polypeptide-expressing metaplasia (SPEM). We observed mucous neck cell hyperplasia, massive reduction of pit, parietal, and chief cell populations, as well as a dramatic increase in the number of rapidly proliferating isthmus stem/progenitor cells in the corpus of RUNX3R122C/R122C mice. Transcriptomic analyses of the isolated epithelial cells showed that the cell cycle-related MYC target gene signature was enriched in the corpus epithelial cells of RUNX3R122C/R122C mice compared with the wild-type corpus. Mechanistically, RUNX3R122C mutant protein disrupted the regulation of the restriction point where cells decide to enter either proliferative or quiescent state, thereby driving stem cell expansion and limiting the ability of cells to terminally differentiate.RUNX3R122C missense mutation is associated with the continuous cycling of isthmus stem/progenitor cells, maturation arrest and development of a precancerous state. This work highlights the importance of RUNX3 in prevention of metaplasia and gastric cancer.