Robert K. Montgomery

Mouse telomerase reverse transcriptase (mTert) expression marks slowly cycling intestinal stem cells

The intestinal epithelium is maintained by a population of rapidly cycling (Lgr5+) intestinal stem cells (ISCs). It has been postulated, however, that slowly cycling ISCs must also be present in the intestine to protect the genome from accumulating deleterious mutations and to allow for a response to tissue injury. Here, we identify a subpopulation of slowly cycling ISCs marked by mouse telomerase reverse transcriptase (mTert) expression that can give rise to Lgr5+ cells. mTert-expressing cells distribute in a pattern along the crypt–villus axis similar to long-term label-retaining cells (LRCs) and are resistant to tissue injury. Lineage-tracing studies demonstrate that mTert+ cells give rise to all differentiated intestinal cell types, persist long term, and contribute to the regenerative response following injury. Consistent with other highly regenerative tissues, our results demonstrate that a slowly cycling stem cell population exists within the intestine.

Development of the human gastrointestinal tract: Twenty years of progress

A combination of approaches has begun to elucidate the mechanisms of gastrointestinal development. This review describes progress over the last 20 years in understanding human gastrointestinal development, including data from both human and experimental animal studies that address molecular mechanisms. Rapid progress is being made in the identification of genes regulating gastrointestinal development. Genes directing initial formation of the endoderm as well as organ-specific patterning are beginning to be identified. Signaling pathways regulating the overall right-left asymmetry of the gastrointestinal tract and epithelial-mesenchymal interactions are being clarified. In searching for extrinsic developmental regulators, numerous candidate trophic factors have been proposed, but compelling evidence remains elusive. A critical gene that initiates pancreas development has been identified, as well as a number of genes regulating liver, stomach, and intestinal development. Mutations in genes affecting neural crest cell migration have been shown to give rise to Hirschsprungs disease. Considerable progress has been achieved in understanding specific phenomena, such as the transcription factors regulating expression of sucrase-isomaltase and fatty acid-binding protein. The challenge for the future is to integrate these data into a more complete understanding of the physiology of gastrointestinal development.

Differentiation-Specific Histone Modifications Reveal Dynamic Chromatin Interactions and Partners for the Intestinal Transcription Factor CDX2

VIDEO ABSTRACT Cell differentiation requires remodeling of tissue-specific gene loci and activities of key transcriptional regulators, which are recognized for their dominant control over cellular programs. Using epigenomic methods, we characterized enhancer elements specifically modified in differentiating intestinal epithelial cells and found enrichment of transcription factor-binding motifs corresponding to CDX2, a critical regulator of the intestine. Directed investigation revealed surprising lability in CDX2 occupancy of the genome, with redistribution from hundreds of sites occupied only in proliferating cells to thousands of new sites in differentiated cells. Knockout mice confirmed distinct Cdx2 requirements in dividing and mature adult intestinal cells, including responsibility for the active enhancer configuration associated with maturity. Dynamic CDX2 occupancy corresponds with condition-specific gene expression and, importantly, to differential co-occupancy with other tissue-restricted transcription factors, such as GATA6 and HNF4A. These results reveal dynamic, context-specific functions and mechanisms of a prominent transcriptional regulator within a cell lineage.

Generation of mTert-GFP mice as a model to identify and study tissue progenitor cells

Stem cells hold great promise for regenerative medicine, but remain elusive in many tissues in part because universal markers of “stemness” have not been identified. The ribonucleoprotein complex telomerase catalyzes the extension of chromosome ends, and its expression is associated with failure of cells to undergo cellular senescence. Because such resistance to senescence is a common characteristic of many stem cells, we hypothesized that telomerase expression may provide a selective biomarker for stem cells in multiple tissues. In fact, telomerase expression has been demonstrated within hematopoietic stem cells. We therefore generated mouse telomerase reverse transcriptase (mTert)-GFP-transgenic mice and assayed the ability of mTert-driven GFP to mark tissue stem cells in testis, bone marrow (BM), and intestine. mTert-GFP mice were generated by using a two-step embryonic stem cell-based strategy, which enabled primary and secondary screening of stably transfected clones before blastocyst injection, greatly increasing the probability of obtaining mTert reporter mice with physiologically appropriate regulation of GFP expression. Analysis of adult mice showed that GFP is expressed in differentiating male germ cells, is enriched among BM-derived hematopoietic stem cells, and specifically marks long-term BrdU-retaining intestinal crypt cells. In addition, telomerase-expressing GFP+ BM cells showed long-term, serial, multilineage BM reconstitution, fulfilling the functional definition of hematopoietic stem cells. Together, these data provide direct evidence that mTert-GFP expression marks progenitor cells in blood and small intestine, validating these mice as a useful tool for the prospective identification, isolation, and functional characterization of progenitor/stem cells from multiple tissues.

Small intestinal stem cell markers

Stem cells hold great promise for regenerative medicine but remain elusive in many tissues, including the small intestine, where it is well accepted that the epithelium is maintained by intestinal stem cells located in the crypts. The lack of established markers to prospectively identify intestinal stem cells has necessitated the use of indirect analysis, e.g. long‐term label retention, which is based on the hypothesis that intestinal stem cells are slow‐cycling. Several intestinal stem cell markers have been proposed, including Musashi‐1, BMPR1α, phospho‐PTEN, DCAMKL1, Eph receptors and integrins, but their validity, using functional and/or lineage tracing assays, has yet to be confirmed. Recently, Lgr5 has been identified by lineage tracing as an intestinal stem cell marker. In this review we summarize what is known about the currently reported intestinal stem cell markers and provide a rationale for developing model systems whereby intestinal stem cells can be functionally validated.

Lactase gene expression during early development of rat small intestine

Expression of lactase messenger (m) RNA and protein in rat small intestine during fetal and postnatal development was analyzed using in situ hybridization and immunohistochemistry. Lactase mRNA was first identified at 18 days of development, and lactase protein was first detected at day 20. Lactase mRNA and protein were present along the entire villus. Lactase mRNA increased, reaching a maximum at day 20. Just before birth a decrease in lactase mRNA was observed. In newborn intestine, lactase mRNA was present only from the base of the villus up to the mid-villus region and was undetectable up to the villus tips. Lactase protein continued to be expressed along the entire villus. These data show that expression of lactase mRNA and protein do not parallel, indicating a posttranscriptional control in fetal development. Lactase gene transcription is initiated late in gestation concomitant with villus formation and is exclusively seen in villus epithelial cells. The restriction after birth of lactase mRNA expression to cells at the villus base suggests the occurrence of a previously unknown step in postnatal differentiation of the enterocyte.

Restriction of lactase gene expression along the proximal-to-distal axis of rat small intestine occurs during postnatal development

BACKGROUND/AIMS Developmental changes of lactase activity along the proximal-to-distal axis of the small intestine are poorly understood. A study of delineate lactase gene expression at the cellular level was undertaken. METHODS The topographical regulation of lactase was studied in conjunction with sucrase-isomaltase in proximal, middle, and distal segments of 0-, 7-, 14-, 16-, 18-, 21-, and 28-day-old and adult rats using in sity hybridization, immunohistochemistry, and ribonuclease protection assays. RESULTS From 0 to 16 days, lactase messenger RNA (mRNA) and protein were abundant along the total length of the small intestine. However, at weaning, lactase mRNA and protein were no longer detectable in the terminal ileum. After 28 days, zones of reduced lactase expression were found in the duodenum and terminal ileum. These zones demonstrated expression of lactase protein in scattered enterocytes along the villus (patchy expression). In contrast, sucrase-isomaltase was first detected at 16 days, with patchy expression along the total small intestine; at 21 days it was abundant. CONCLUSIONS Concordant changes in both lactase mRNA and protein detection during development suggest that the horizontal gradient of lactase enzyme expression is dependent on lactase mRNA abundance. Furthermore, zones of patchy lactase expression appear around weaning and flank the area of high lactase expression in the midintestine. Patchy expression is also found for sucrase-isomaltase before weaning.

Autonomous biochemical and morphological differentiation in fetal rat intestine transplanted at 17 and 20 days of gestation

Abstract The morphological and biochemical development of fetal rat intestine was examined for up to 5 weeks following transplantation to syngeneic hosts at 17 and 20 days of gestation. In transplants of both ages, normal villi bearing mature enterocytes developed. In addition, the disaccharidases lactase, maltase, and sucrase, as well as alkaline phosphatase, underwent normal patterns of development. Lactase activity, initially high, fell significantly, while maltase and sucrase activities increased significantly in the interval between 2 and 5 weeks following transplantation. During this same period, alkaline phosphatase developed the proximally located, high-activity form. The transplanted intestine also developed normal topographical distributions of enzyme activities. Measurement of corticosterone levels demonstrated that, except for a transient upsurge at the time of operation, hormone levels did not change significantly during the period of transplant maturation. These data indicate that the brush-border enzymes of the small intestine develop according to an intrinsic program which is already established as early as 17 days of gestation.

Messenger RNA sorting in enterocytes Co-localization with encoded proteins

This study describes the intracellular compartmentalization of three different mRNAs in the polarized rat fetal enterocyte. They encode proteins that are known to be localized within different regions of the epithelial cell namely (i) the apical, membrane‐bound glycoprotein, lactase‐phlorizin hydrolase (lactase), (ii) the mitochondrially localized enzyme, carbamoylphosphate synthetase (CPS), and (iii) the cytoplasmically localized enzyme, phosphoenolpyruvate carboxykinase (PEPCK). These mRNAs are found in close proximity to their respective protein products, i.e. the apical membrane, mitochondria and cytoplasm, respectively. The significance or these observations is twofold: (i) they indicate that mRNAs are sorted into specific domains of the cytosol of intestinal epithelial cells: and (ii) they imply the presence or two distinct pathways of mRNA targeting one that allows transport of mRNAs that are translated on ribosomes associated with the rough endoplasmic reticulum (lactase mRNA), and the other that allows sorting of mRNAs that are translated on free polysames (CPS and PEPCK mRNA).

Cortisone and thyroxine modulate intestinal lactase and sucrase mRNA levels and activities in the suckling rat

Glucocorticoids and thyroxine modulate postnatal intestinal sucrase and lactase activities. Whether changes in enzyme activity are accompanied by changes in enzyme mRNA levels were determined in day 6 rats given thyroxine, cortisone, or thyroxine plus cortisone and killed 3 days later. Cortisone induced precocious expression of jejunal sucrase activity which was enhanced when cortisone plus thyroxine was administered; sucrase mRNA changed in parallel. Jejunal lactase activity was unaffected by thyroxine and was increased after cortisone, but not after thyroxine plus cortisone. Jejunal lactase mRNA levels increased equally after cortisone or after cortisone plus thyroxine. Thus, cortisone induces coordinated increases in sucrase and lactase activities and in corresponding mRNA levels. Thyroxine only enhances cortisone induced sucrase expression and antagonizes cortisone by depressing lactase activity post-translationally.