Regulation of Stem Cell Proliferation and Cell Fate Specification by Wingless/Wnt Signaling Gradients Enriched at Adult Intestinal Compartment Boundaries

Intestinal stem cell (ISC) self-renewal and proliferation are directed by Wnt/β-catenin signaling in mammals, whereas aberrant Wnt pathway activation in ISCs triggers the development of human colorectal carcinoma. Herein, we have utilized the Drosophila midgut, a powerful model for ISC regulation, to elucidate the mechanisms by which Wingless (Wg)/Wnt regulates intestinal homeostasis and development. We provide evidence that the Wg signaling pathway, activation of which peaks at each of the major compartment boundaries of the adult intestine, has essential functions. Wg pathway activation in the intestinal epithelium is required not only to specify cell fate near compartment boundaries during development, but also to control ISC proliferation within compartments during homeostasis. Further, in contrast with the previous focus on Wg pathway activation within ISCs, we demonstrate that the primary mechanism by which Wg signaling regulates ISC proliferation during homeostasis is non-autonomous. Activation of the Wg pathway in absorptive enterocytes is required to suppress JAK-STAT signaling in neighboring ISCs, and thereby their proliferation. We conclude that Wg signaling gradients have essential roles during homeostasis and development of the adult intestine, non-autonomously controlling stem cell proliferation inside compartments, and autonomously specifying cell fate near compartment boundaries.     

EGFR, Wingless and JAK/STAT signaling cooperatively maintain Drosophila intestinal stem cells

Tissue-specific adult stem cells are commonly associated with local niche for their maintenance and function. In the adult Drosophila midgut, the surrounding visceral muscle maintains intestinal stem cells (ISCs) by stimulating Wingless (Wg) and JAK/STAT pathway activities, whereas cytokine production in mature enterocytes also induces ISC division and epithelial regeneration, especially in response to stress. Here we show that EGFR/Ras/ERK signaling is another important participant in promoting ISC maintenance and division in healthy intestine. The EGFR ligand Vein is specifically expressed in muscle cells and is important for ISC maintenance and proliferation. Two additional EGFR ligands, Spitz and Keren, function redundantly as possible autocrine signals to promote ISC maintenance and proliferation. Notably, over-activated EGFR signaling could partially replace Wg or JAK/STAT signaling for ISC maintenance and division, and vice versa. Moreover, although disrupting any single one of the three signaling pathways shows mild and progressive ISC loss over time, simultaneous disruption of them all leads to rapid and complete ISC elimination. Taken together, our data suggest that Drosophila midgut ISCs are maintained cooperatively by multiple signaling pathway activities and reinforce the notion that visceral muscle is a critical component of the ISC niche.     

Casein kinase 1‐epsilon or 1‐delta required for Wnt‐mediated intestinal stem cell maintenance

The intestinal epithelium holds an immense regenerative capacity mobilized by intestinal stem cells (ISCs), much of it supported by Wnt pathway activation. Several unique regulatory mechanisms ensuring optimal levels of Wnt signaling have been recognized in ISCs. Here, we identify another Wnt signaling amplifier, CKIε, which is specifically upregulated in ISCs and is essential for ISC maintenance, especially in the absence of its close isoform CKIδ. Co‐ablation of CKIδ/ε in the mouse gut epithelium results in rapid ISC elimination, with subsequent growth arrest, crypt–villous shrinking, and rapid mouse death. Unexpectedly, Wnt activation is preserved in all CKIδ/ε‐deficient enterocyte populations, with the exception of Lgr5⁺ ISCs, which exhibit Dvl2‐dependent Wnt signaling attenuation. CKIδ/ε‐depleted gut organoids cease proliferating and die rapidly, yet survive and resume self‐renewal upon reconstitution of Dvl2 expression. Our study underscores a unique regulation mode of the Wnt pathway in ISCs, possibly providing new means of stem cell enrichment for regenerative medicine.     

The role of intestinal stem cell within gut homeostasis: Focusing on its interplay with gut microbiota and the regulating pathways

Intestinal stem cells (ISCs) play an important role in maintaining intestinal homeostasis via promoting a healthy gut barrier. Within the stem cell niche, gut microbiota linking the crosstalk of dietary influence and host response has been identified as a key regulator of ISCs. Emerging insights from recent research reveal that ISC and gut microbiota interplay regulates epithelial self-renewal. This article reviews the recent knowledge on the key role of ISC in their local environment (stem cell niche) associating with gut microbiota and their metabolites as well as the signaling pathways. The current progress of intestinal organoid culture is further summarized. Subsequently, the key challenges and future directions are discussed.     

Mathematical modelling and experiments for the proliferation and differentiation of Drosophila intestinal stem cells II

The Drosophila posterior midgut epithelium mainly consists of intestinal stem cells (ISCs); semi-differentiated cells, i.e. enteroblasts (EBs); and two types of fully differentiated cells, i.e. enteroendocrine cells (EEs) and enterocytes (ECs), which are controlled by signalling pathways. In [M. Kuwamura, K. Maeda, and T. Adachi-Yamada, Mathematical modeling and experiments for the proliferation and differentiation of Drosophila intestinal stem cells I, J. Biol. Dyn. 4 (2009), pp. 248-257], on the basis of the functions of the Wnt and Notch signalling pathways, we studied the regulatory mechanism for the proliferation and differentiation of ISCs under the assumption that the Wnt proteins are supplied from outside the cellular system of ISCs. In this paper, we experimentally show that the Wnt proteins are specifically expressed in ISCs, EBs, and EEs, and theoretically show that the cellular system of ISCs can be self-maintained under the assumption that the Wnt proteins are produced in the cellular system of ISCs. These results provide a useful basis for determining whether an environmental niche is required for maintaining the cellular system of tissue stem cells.     

Mathematical modelling and experiments for the proliferation and differentiation of Drosophila intestinal stem cells II

The Drosophila posterior midgut epithelium mainly consists of intestinal stem cells (ISCs); semi-differentiated cells, i.e. enteroblasts (EBs); and two types of fully differentiated cells, i.e. enteroendocrine cells (EEs) and enterocytes (ECs), which are controlled by signalling pathways. In [M. Kuwamura, K. Maeda, and T. Adachi-Yamada, Mathematical modeling and experiments for the proliferation and differentiation of Drosophila intestinal stem cells I, J. Biol. Dyn. 4 (2009), pp. 248–257], on the basis of the functions of the Wnt and Notch signalling pathways, we studied the regulatory mechanism for the proliferation and differentiation of ISCs under the assumption that the Wnt proteins are supplied from outside the cellular system of ISCs. In this paper, we experimentally show that the Wnt proteins are specifically expressed in ISCs, EBs, and EEs, and theoretically show that the cellular system of ISCs can be self-maintained under the assumption that the Wnt proteins are produced in the cellular system of ISCs. These results provide a useful basis for determining whether an environmental niche is required for maintaining the cellular system of tissue stem cells.     

The intestinal stem cell signature identifies colorectal cancer stem cells and predicts disease relapse

A frequent complication in colorectal cancer (CRC) is regeneration of the tumor after therapy. Here, we report that a gene signature specific for adult intestinal stem cells (ISCs) predicts disease relapse in CRC patients. ISCs are marked by high expression of the EphB2 receptor, which becomes gradually silenced as cells differentiate. Using EphB2 and the ISC marker Lgr5, we have FACS-purified and profiled mouse ISCs, crypt proliferative progenitors, and late transient amplifying cells to define a gene program specific for normal ISCs. Furthermore, we discovered that ISC-specific genes identify a stem-like cell population positioned at the bottom of tumor structures reminiscent of crypts. EphB2 sorted ISC-like tumor cells display robust tumor-initiating capacity in immunodeficient mice as well as long-term self-renewal potential. Taken together, our data suggest that the ISC program defines a cancer stem cell niche within colorectal tumors and plays a central role in CRC relapse.     

Rap-GEF signaling controls stem cell anchoring to their niche through regulating DE-cadherin-mediated cell adhesion in the Drosophila testis

Stem cells will undergo self-renewal to produce new stem cells if they are maintained in their niches. The regulatory mechanisms that recruit and maintain stem cells in their niches are not well understood. In Drosophila testes, a group of 12 nondividing somatic cells, called the hub, identifies the stem cell niche by producing the growth factor Unpaired (Upd). Here, we show that Rap-GEF/Rap signaling controls stem cell anchoring to the niche through regulating DE-cadherin-mediated cell adhesion. Loss of function of a Drosophila Rap-GEF (Gef26) results in loss of both germline and somatic stem cells. The Gef26 mutation specifically impairs adherens junctions at the hub-stem cell interface, which results in the stem cells "drifting away" from the niche and losing stem cell identity. Thus, the Rap signaling/E-cadherin pathway may represent one mechanism that regulates polarized niche formation and stem cell anchoring.     

Dual role of BMP signaling in the regulation of Drosophila intestinal stem cell self-renewal

Many adult organs including Drosophila adult midguts rely on resident stem cells to replenish damaged cells during tissue homeostasis and regeneration. Previous studies have shown that, upon injury, intestinal stem cells (ISCs) in the midguts can increase proliferation and lineage differentiation to meet the demand for tissue repair. Our recent study has demonstrated that, in response to certain injury, midguts can expand ISC population size as an additional regenerative mechanism. We found that injury elicited by bleomycin feeding or bacterial infection increased the production of two BMP ligands (Dpp and Gbb) in enterocytes (ECs), leading to elevated BMP signaling in progenitor cells that drove an expansion of ISCs by promoting their symmetric self-renewing division. Interestingly, we also found that BMP signaling in ECs inhibits the production of Dpp and Gbb, and that this negative feedback mechanism is required to reset ISC pool size to the homeostatic state. Our findings suggest that BMP signaling exerts two opposing influences on stem cell activity depending on where it acts: BMP signaling in progenitor cells promotes ISC self-renewal while BMP signaling in ECs restricts ISC self-renewal by preventing excessive production of BMP ligands. Our results further suggest that transient expansion of ISC population in conjunction with increasing ISC proliferation provides a more effective strategy for tissue regeneration.     

Intestinal stem cells and stem cell-based therapy for intestinal diseases

Currently, many gastrointestinal diseases are a major reason for the increased mortality rate of children and adults every year. Additionally, these patients may cope with the high cost of the parenteral nutrition (PN), which aids in the long-term survival of the patients. Other treatment options include surgical lengthening, which is not sufficient in many cases, and intestinal transplantation. However, intestinal transplantation is still accompanied by many challenges, including immune rejection and donor availability, which may limit the transplant’s success. The development of more safe and promising alternative treatments for intestinal diseases is still ongoing. Stem cell-based therapy (SCT) and tissue engineering (TE) appear to be the next promising choices for the regeneration of the damaged intestine. However, suitable stem cell source is required for the SCT and TE process. Thus, in this review we discuss how intestinal stem cells (ISCs) are a promising cell source for small intestine diseases. We will also discuss the different markers were used to identify ISCs. Moreover, we discuss the dominant Wnt signaling pathway in the ISC niche and its involvement in some intestinal diseases. Additionally, we discuss ISC culture and expansion, which are critical to providing enough cells for SCT and TE. Finally, we conclude and recommend that ISC isolation, culture and expansion should be considered when SCT is a treatment option for intestinal disorders. Therefore, we believe that ISCs should be considered a cell source for SCT for many gastrointestinal diseases and should be highlighted in future clinical applications.     

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.     

Steep differences in wingless signaling trigger Myc-independent competitive cell interactions

Wnt signaling is a key regulator of development that?is often associated with cancer. Wingless,?a Drosophila Wnt homolog, has been reported to be a survival factor in wing imaginal discs. However, we found that prospective wing cells survive in the absence of Wingless as long as they are not surrounded by Wingless-responding cells. Moreover, local autonomous overactivation of Wg signaling (as a result of a mutation in APC or axin) leads to the elimination of surrounding normal cells. Therefore, relative differences in Wingless signaling lead to competitive cell interactions. This process does not involve Myc, a well-established cell competition factor. It does, however, require Notum, a conserved secreted feedback inhibitor of Wnt signaling. We suggest that Notum could amplify local differences in Wingless signaling, thus serving as an early trigger of Wg signaling-dependent competition.     

EGFR/Ras/MAPK signaling mediates adult midgut epithelial homeostasis and regeneration in Drosophila

Many tissues in higher animals undergo dynamic homeostatic growth, wherein damaged or aged cells are replaced by the progeny of resident stem cells. To maintain homeostasis, stem cells must respond to tissue needs. Here we show that in response to damage or stress in the intestinal (midgut) epithelium of adult Drosophila, multiple EGFR ligands and rhomboids (intramembrane proteases that activate some EGFR ligands) are induced, leading to the activation of EGFR signaling in intestinal stem cells (ISCs). Activation of EGFR signaling promotes ISC division and midgut epithelium regeneration, thereby maintaining tissue homeostasis. ISCs defective in EGFR signaling cannot grow or divide, are poorly maintained, and cannot support midgut epithelium regeneration after enteric infection by the bacterium Pseudomonas entomophila. Furthermore, ISC proliferation induced by Jak/Stat signaling is dependent upon EGFR signaling. Thus the EGFR/Ras/MAPK signaling pathway plays central, essential roles in ISC maintenance and the feedback system that mediates intestinal homeostasis.     

小肠干细胞的命运调控

小肠上皮具有快速更新的能力,是研究成体干细胞的理想系统.小肠上皮由绒毛和隐窝两部分组成,而位于小肠隐窝底部的小肠干细胞是其持续更新的源泉.近年来,以Lgr5为代表的小肠干细胞标记物的发现、Lgr5+小肠干细胞的分离培养和多种转基因小鼠模型的出现,极大地促进了对小肠干细胞自我更新和分化调控的研究,使得人们可以更加深入地认识小肠干细胞命运决定的分子机制.本文简要综述了近年来人们对Wnt,BMP,Notch和EGF等信号如何在小肠干细胞命运调控中发挥作用的认识.     

Regulation of hematopoiesis and the hematopoietic stem cell niche by Wnt signaling pathways

Hematopoietic stem cells （HSCs） are a rare population of cells that are responsible for life-long generation of blood cells of all lineages. In order to maintain their numbers, HSCs must establish a balance between the opposing cell fates of self-renewal （in which the ability to function as HSCs is retained） and initiation of hematopoietic differentiation. Multiple signaling pathways have been implicated in the regulation of HSC cell fate. One such set of pathways are those activated by the Wnt family of ligands. Wnt signaling pathways play a crucial role during embryogenesis and deregulation of these pathways has been implicated in the formation of solid tumors. Wnt signaling also plays a role in the regulation of stem cells from multiple tissues, such as embryonic, epidermal, and intestinal stem cells. However, the function of Wnt signaling in HSC biology is still controversial. In this review, we will discuss the basic characteristics of the adult HSC and its regulatory microenvironment, the ＂niche＂, focusing on the regulation of the HSC and its niche by the Wnt signaling pathways.