JAK/STAT signaling coordinates stem cell proliferation and multilineage differentiation in the Drosophila intestinal stem cell lineage

Adult stem cells are the most primitive cells of a lineage and are distinguished by the properties of self-renewal and multipotency. Coordinated control of stem cell proliferation and multilineage differentiation is essential to ensure a steady output of differentiated daughter cells necessary to maintain tissue homeostasis. However, little is known about the signals that coordinate stem cell proliferation and daughter cell differentiation. Here we investigate the role of the conserved JAK/STAT signaling pathway in the Drosophila intestinal stem cell (ISC) lineage. We show first, that JAK/STAT signaling is normally active in both ISCs and their newly formed daughters, but not in terminally differentiated enteroendocrine (ee) cells or enterocyte (EC) cells. Second, analysis of ISC lineages shows that JAK/STAT signaling is necessary but not sufficient for daughter cell differentiation, indicating that competence to undergo multilineage differentiation depends upon JAK/STAT. Finally, our analysis reveals JAK/STAT signaling to be a potent regulator of ISC proliferation, but not ISC self-renewal. On the basis of these findings, we suggest a model in which JAK/STAT signaling coordinates the processes of stem cell proliferation with the competence of daughter cells to undergo multilineage differentiation, ensuring a robust cellular output in the lineage.     

Live and let die in the intestinal epithelium

The intestinal epithelium is a relatively simple developmental system and a prime example of tissue renewal from a source of multipotent stem cells. Throughout adulthood, intestinal epithelial proliferation, cell-fate specification and differentiation are coupled to migration in discrete units known as crypts of Lieberkühn. Physically guided by Eph receptors and their ligands, the ephrins, stem cell progeny transit through the proliferation/differentiation switch, and Notch diversifies their subsequent fates. Wnt signalling appears to control most of these events.     

On the utility of a compartmental population kinetics model of intestinal epithelial stem cell proliferation and differentiation

Background

The small intestinal epithelium is a dynamic system with specialized cell types. The various cell populations of this tissue are continually renewed and replenished from stem cells that reside in the small intestinal crypt. The cell types and their locations in the crypt and villus are well known, but the details of the kinetics of stem cell division, and precursor cell proliferation and differentiation into mature enterocytes and secretory cells are still being studied. These proliferation and differentiation events have been extensively modeled with a variety of computational approaches in the past.

Methods

A compartmental population kinetics model, incorporating experimentally measured proliferation rates for various intestinal epithelial cell types, is implemented for a previously reported scheme for the intestinal cell dynamics. A sensitivity analysis is performed to determine the effect that varying the model parameters has upon the model outputs, the steady-state cell populations.

Results

The model is unable to reproduce the experimentally known timescale of renewal of the intestinal epithelium if literature values for the proliferation rates of stem cells and transit amplifying cells are employed. Unphysically large rates of proliferation result when these parameters are allowed to vary to reproduce this timescale and the steady-state populations of terminally differentiated intestinal epithelial cells. Sensitivity analysis reveals that the strongest contributor to the steady-state populations is the transit amplifying cell proliferation rate when literature values are used, but that the differentiation rate of transit amplifying cells to secretory progenitor cells dominates when all parameters are allowed to vary.

Conclusions

A compartmental population kinetics model of proliferation and differentiation of cells of the intestinal epithelium can provide a simplifying means of understanding a complicated multistep process. However, when literature values for proliferation rates of the crypt based columnar and transit amplifying cell populations are employed in the model, it cannot reproduce the experimentally known timescale of intestinal epithelial renewal. Nevertheless, it remains a valuable conceptual tool, and its sensitivity analysis provides important clues for which events in the process are the most important in controlling the steady-state populations of specialized intestinal epithelial cells.

     

Wnt control of stem cells and differentiation in the intestinal epithelium

The intestinal epithelium represents a very attractive experimental model for the study of integrated key cellular processes such as proliferation and differentiation. The tissue is subjected to a rapid and perpetual self-renewal along the crypt-villus axis. Renewal requires division of multipotent stem cells, still to be morphologically identified and isolated, followed by transit amplification, and differentiation of daughter cells into specialized absorptive and secretory cells. Our understanding of the crucial role played by the Wnt/beta-catenin signaling pathway in controlling the fine balance between cell proliferation and differentiation in the gut has been significantly enhanced in recent years. Mutations in some of its components irreversibly lead to carcinogenesis in humans and in mice. Here, we discuss recent advances related to the Wnt/beta-catenin signaling pathway in regulating intestinal stem cells, homeostasis, and cancer. We emphasize how Wnt signaling is able to maintain a stem cell/progenitor phenotype in normal intestinal crypts, and to impose a very similar phenotype onto colorectal adenomas.     

Stem cell self-renewal in intestinal crypt

As a rapidly cycling tissue capable of fast repair and regeneration, the intestinal epithelium has emerged as a favored model system to explore the principles of adult stem cell biology. However, until recently, the identity and characteristics of the stem cell population in both the small intestine and colon has remained the subject of debate. Recent studies based on targeted lineage tracing strategies, combined with the development of an organotypic culture system, have identified the crypt base columnar cell as the intestinal stem cell, and have unveiled the strategy by which the balance between proliferation and differentiation is maintained. These results show that intestinal stem cells operate in a dynamic environment in which frequent and stochastic stem cell loss is compensated by the proliferation of neighboring stem cells. We review the basis of these experimental findings and the insights they offer into the mechanisms of homeostatic stem cell regulation.     

STORM: a general model to determine the number and adaptive changes of epithelial stem cells in teleost, murine and human intestinal tracts

Intestinal stem cells play a pivotal role in the epithelial tissue renewal, homeostasis and cancer development. The lack of a general marker for intestinal stem cells across species has hampered analysis of stem cell number in different species and their adaptive changes upon intestinal lesions or during development of cancer. Here a two-dimensional model, named STORM, has been developed to address this issue. By optimizing epithelium renewal dynamics, the model examines the epithelial stem cell number by taking experimental input information regarding epithelium proliferation and differentiation. As the results suggest, there are 2.0-4.1 epithelial stem cells on each pocket section of zebrafish intestine, 2.0-4.1 stem cells on each crypt section of murine small intestine and 1.8-3.5 stem cells on each crypt section of human duodenum. The model is able to provide quick results for stem cell number and its adaptive changes, which is not easy to measure through experiments. Its general applicability to different species makes it a valuable tool for analysis of intestinal stem cells under various pathological conditions.     

The balance of two opposing factors Mad and Myc regulates cell fate during tissue remodeling

Cell proliferation and differentiation are two distinct yet coupled processes in development in diverse organisms. Understanding the molecular mechanisms that regulate this process is a central theme in developmental biology. The intestinal epithelium is a highly complex tissue that relies on the coordination of cell proliferation within the crypts and apoptosis mainly at the tip of the villi, preservation of epithelial function through differentiation, and homeostatic cell migration along the crypt-villus axis. Small populations of adult stem cells are responsible for the self-renewal of the epithelium throughout life. Surprisingly, much less is known about the mechanisms governing the remodeling of the intestine from the embryonic to adult form. Furthermore, it remains unknown how thyroid hormone (T3) affects stem cell development during this postembryonic process, which is around birth in mammals when T3 level increase rapidly in the plasma. Tissue remodeling during amphibian metamorphosis is very similar to the maturation of the mammalian organs around birth in mammals and is regulated by T3. In particular, many unique features of Xenopus intestinal remodeling during metamorphosis has enabled us and others to elucidate how adult stem cells are formed during postembryonic development in vertebrates. In this review, we will focus on recent findings on the role of Mad1/c-Myc in cell death and proliferation during intestinal metamorphosis and discuss how a Mad1–c-Myc balance controls intestinal epithelial cell fate during this T3-dependent process.     

Another notch in stem cell biology: Drosophila intestinal stem cells and the specification of cell fates

Previous work has suggested that many stem cells can be found in microanatomic niches, where adjacent somatic cells of the niche control the differentiation and proliferation states of their resident stem cells. Recently published work examining intestinal stem cells (ISCs) in the adult Drosophila midgut suggests a new paradigm where some stem cells actively control the cell fate decisions of their daughters. Here, we review recent literature((1)) demonstrating that, in the absence of a detectable stem cell niche, multipotent Drosophila ISCs modulate the Notch signaling pathway in their adjacent daughter cells in order to specify the differentiated lineages of their descendants. These observations made in Drosophila are challenging and advancing our understanding of stem cell biology.     

Increased centrosome amplification in aged stem cells of the Drosophila midgut

Age-related changes in long-lived tissue-resident stem cells may be tightly linked to aging and age-related diseases such as cancer. Centrosomes play key roles in cell proliferation, differentiation and migration. Supernumerary centrosomes are known to be an early event in tumorigenesis and senescence. However, the age-related changes of centrosome duplication in tissue-resident stem cells in vivo remain unknown. Here, using anti-γ-tubulin and anti-PH3, we analyzed mitotic intestinal stem cells with supernumerary centrosomes in the adult Drosophila midgut, which may be a versatile model system for stem cell biology. The results showed increased centrosome amplification in intestinal stem cells of aged and oxidatively stressed Drosophila midguts. Increased centrosome amplification was detected by overexpression of PVR, EGFR, and AKT in intestinal stem cells/enteroblasts, known to mimic age-related changes including hyperproliferation of intestinal stem cells and hyperplasia in the midgut. Our data show the first direct evidence for the age-related increase of centrosome amplification in intestinal stem cells and suggest that the Drosophila midgut is an excellent model for studying molecular mechanisms underlying centrosome amplification in aging adult stem cells in vivo.     

Quantitative rather than qualitative differences in gene expression predominate in intestinal cell maturation along distinct cell lineages

Several cell types are present in the intestinal epithelium that likely arise from a common precursor, the stem cell, and each mature cell type expresses a unique set of genes that characterizes its functional phenotype. Although the process of differentiation is intimately linked to the cessation of proliferation, the mechanisms that dictate intestinal cell fate determination are not well characterized. To investigate the reprogramming of gene expression during the cell lineage allocation/differentiation process, we took advantage of a unique system of two clonal derivatives of HT29 cells, Cl16E and Cl19A cells, which spontaneously differentiate as mucus producing goblet and chloride-secreting cells, respectively, as a function of time. By profiling gene expression, we found that these two cell lines show remarkably similar kinetics of change in gene expression and common clusters of coordinately regulated genes. This demonstrates that lineage-specific differentiation of intestinal epithelial cells is characterized overall by the sequential recruitment of functionally similar gene sets independent of the final phenotype of the mature cells.     

Rac1 drives intestinal stem cell proliferation and regeneration

Adult stem cells are responsible for maintaining the balance between cell proliferation and differentiation within self-renewing tissues. The molecular and cellular mechanisms mediating such balance are poorly understood. The production of reactive oxygen species (ROS) has emerged as an important mediator of stem cell homeostasis in various systems. Our recent work demonstrates that Rac1-dependent ROS production mediates intestinal stem cell (ISC) proliferation in mouse models of colorectal cancer (CRC). Here, we use the adult Drosophila midgut and the mouse small intestine to directly address the role of Rac1 in ISC proliferation and tissue regeneration in response to damage. Our results demonstrate that Rac1 is necessary and sufficient to drive ISC proliferation and regeneration in an ROS-dependent manner. Our data point to an evolutionarily conserved role of Rac1 in intestinal homeostasis and highlight the value of combining work in the mammalian and Drosophila intestine as paradigms to study stem cell biology.     

Heparan sulfate maintains adult midgut homeostasis in Drosophila

Tissue homeostasis is controlled by the differentiated progeny of residential progenitors (stem cells). Adult stem cells constantly adjust their proliferation/differentiation rates to respond to tissue damage and stresses. However, how differentiated cells maintain tissue homeostasis remains unclear. Here, we find that heparan sulfate (HS), a class of glycosaminoglycan (GAG) chains, protects differentiated cells from loss to maintain intestinal homeostasis. HS depletion in enterocytes (ECs) leads to intestinal homeostasis disruption, with accumulation of intestinal stem cell (ISC)‐like cells and mis‐differentiated progeny. HS‐deficient ECs are prone to cell death/stress and induced cytokine and epidermal growth factor (EGF) expression, which, in turn, promote ISC proliferation and differentiation. Interestingly, HS depletion in ECs results in the inactivation of decapentaplegic (Dpp) signaling. Moreover, ectopic Dpp signaling completely rescued the defects caused by HS depletion. Together, our data demonstrate that HS is required for Dpp signal activation in ECs, thereby protecting ECs from ablation to maintain midgut homeostasis. Our data shed light into the regulatory mechanisms of how differentiated cells contribute to tissue homeostasis maintenance.     

hnRNP I Inhibits Notch Signaling and Regulates Intestinal Epithelial Homeostasis in the Zebrafish

Regulated intestinal stem cell proliferation and differentiation are required for normal intestinal homeostasis and repair after injury. The Notch signaling pathway plays fundamental roles in the intestinal epithelium. Despite the fact that Notch signaling maintains intestinal stem cells in a proliferative state and promotes absorptive cell differentiation in most species, it remains largely unclear how Notch signaling itself is precisely controlled during intestinal homeostasis. We characterized the intestinal phenotypes of brom bones, a zebrafish mutant carrying a nonsense mutation in hnRNP I. We found that the brom bones mutant displays a number of intestinal defects, including compromised secretory goblet cell differentiation, hyperproliferation, and enhanced apoptosis. These phenotypes are accompanied by a markedly elevated Notch signaling activity in the intestinal epithelium. When overexpressed, hnRNP I destabilizes the Notch intracellular domain (NICD) and inhibits Notch signaling. This activity of hnRNP I is conserved from zebrafish to human. In addition, our biochemistry experiments demonstrate that the effect of hnRNP I on NICD turnover requires the C-terminal portion of the RAM domain of NICD. Our results demonstrate that hnRNP I is an evolutionarily conserved Notch inhibitor and plays an essential role in intestinal homeostasis.     

Distinct Human Stem Cell Populations in Small and Large Intestine

The intestine is composed of an epithelial layer containing rapidly proliferating cells that mature into two regions, the small and the large intestine. Although previous studies have identified stem cells as the cell-of-origin for intestinal epithelial cells, no studies have directly compared stem cells derived from these anatomically distinct regions. Here, we examine intrinsic differences between primary epithelial cells isolated from human fetal small and large intestine, after in vitro expansion, using the Wnt agonist R-spondin 2. We utilized flow cytometry, fluorescence-activated cell sorting, gene expression analysis and a three-dimensional in vitro differentiation assay to characterize their stem cell properties. We identified stem cell markers that separate subpopulations of colony-forming cells in the small and large intestine and revealed important differences in differentiation, proliferation and disease pathways using gene expression analysis. Single cells from small and large intestine cultures formed organoids that reflect the distinct cellular hierarchy found in vivo and respond differently to identical exogenous cues. Our characterization identified numerous differences between small and large intestine epithelial stem cells suggesting possible connections to intestinal disease.     

Use of transgenic mice to characterize the multipotent intestinal stem cell and to analyze regulation of gene expression in various epithelial cell lineages as a function of their position along the cephalocaudal and crypt-to-villus (or crypt-to-surface epithelial cuff) axes of the gut

The processes of cell proliferation, lineage allocation and differentiation occur continuously and rapidly along the crypt-to-villus axis of the small intestine and the crypt-to-surface epithelial cuff axis of the colon. The four principal epithelial cell lineages in the gut are derived from a multipotent stem cell. Current evidence suggests that each small intestinal and colonic crypt contains a single active stem cell. The biological properties of these stem cells can be inferred from the properties of their amplified, spatially constrained, descendants. Recent studies in transgenic mice have provided insights about how axial pattern formation is maintained in this perpetually renewing epithelium.     

Generation-dependent control mechanisms in cell proliferation and differentiation—the power of two

There is evidence that the proliferation of cells is controlled by the number of divisions after leaving a multi-potent (stem) cell. A detailed study of the growth of tissue in the small intestinal tract, more precisely the growth of crypts and villi, suggests that not only the proliferation but also the differentiation of cells obey the same biological law. We postulate a theory of a cellular internal control mechanism: the cell-generation control of differentiation and proliferation. This basic mechanism, together with external influences, determines the kinetic behaviour of the crypt-villus system.     

Intestinal stem cells in the adult Drosophila midgut

Drosophila has long been an excellent model organism for studying stem cell biology. Notably, studies of Drosophila's germline stem cells have been instrumental in developing the stem cell niche concept. The recent discovery of somatic stem cells in adult Drosophila, particularly the intestinal stem cells (ISCs) of the midgut, has established Drosophila as an exciting model to study stem cell-mediated adult tissue homeostasis and regeneration. Here, we review the major signaling pathways that regulate the self-renewal, proliferation and differentiation of Drosophila ISCs, discussing how this regulation maintains midgut homeostasis and mediates regeneration of the intestinal epithelium after injury.