The intestinal epithelium is the most rapidly self-renewing tissue in adult mammals. We have recently demonstrated the presence of about six cycling Lgr5(+) stem cells at the bottoms of small-intestinal crypts. Here we describe the establishment of long-term culture conditions under which single crypts undergo multiple crypt fission events, while simultanously generating villus-like epithelial domains in which all differentiated cell types are present. Single sorted Lgr5(+) stem cells can also initiate these cryptvillus organoids. Tracing experiments indicate that the Lgr5(+) stem-cell hierarchy is maintained in organoids. We conclude that intestinal cryptvillus units are self-organizing structures, which can be built from a single stem cell in the absence of a non-epithelial cellular niche.

Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche.

The canonical Wnt cascade has emerged as a critical regulator of stem cells. In many tissues, activation of Wnt signalling has also been associated with cancer. This has raised the possibility that the tightly regulated self-renewal mediated by Wnt signalling in stem and progenitor cells is subverted in cancer cells to allow malignant proliferation. Insights gained from understanding how the Wnt pathway is integrally involved in both stem cell and cancer cell maintenance and growth in the intestinal, epidermal and haematopoietic systems may serve as a paradigm for understanding the dual nature of self-renewal signals.

Wnt signalling in stem cells and cancer

Stem cell fate is influenced by specialized microenvironments that remain poorly defined in mammals. To explore the possibility that haematopoietic stem cells derive regulatory information from bone, accounting for the localization of haematopoiesis in bone marrow, we assessed mice that were genetically altered to produce osteoblast-specific, activated PTH/PTHrP receptors (PPRs). Here we show that PPR-stimulated osteoblastic cells that are increased in number produce high levels of the Notch ligand jagged 1 and support an increase in the number of haematopoietic stem cells with evidence of Notch1 activation in vivo. Furthermore, ligand-dependent activation of PPR with parathyroid hormone (PTH) increased the number of osteoblasts in stromal cultures, and augmented ex vivo primitive haematopoietic cell growth that was abrogated by gamma-secretase inhibition of Notch activation. An increase in the number of stem cells was observed in wild-type animals after PTH injection, and survival after bone marrow transplantation was markedly improved. Therefore, osteoblastic cells are a regulatory component of the haematopoietic stem cell niche in vivo that influences stem cell function through Notch activation. Niche constituent cells or signalling pathways provide pharmacological targets with therapeutic potential for stem-cell-based therapies.

/pdf/osteoblastic-cells-regulate-the-haematopoietic-stem-cell-17prhh2a28.pdf

Osteoblastic cells regulate the haematopoietic stem cell niche

The Canonical Notch Signaling Pathway: Unfolding the Activation Mechanism

https://www.cell.com/cell/pdf/S0092-8674(05)00540-4.pdf

SLAM Family Receptors Distinguish Hematopoietic Stem and Progenitor Cells and Reveal Endothelial Niches for Stem Cells

The Notch genes encode single-pass transmembrane receptors that transduce the extracellular signals responsible for cell fate determination during several steps of metazoan development. The mechanism by which extracellular signals affect gene transcription and ultimately cell fate decisions is beginning to emerge for the Notch signalling pathway. One paradigm is that ligand binding to Notch triggers a Presenilin1-dependent proteolytic release of the Notch intracellular domain from the membrane, resulting in low amounts of Notch intracellular domain which form a nuclear complex with CBF1/Su(H)/Lag1 to activate transcription of downstream targets. Not all observations clearly support this processing model, and the most rigorous test of it is to block processing in vivo and then determine the ability of unprocessed Notch to signal. Here we report that the phenotypes associated with a single point mutation at the intramembranous processing site of Notch1, Val1,744-->Gly, resemble the null Notch1 phenotype. Our results show that efficient intramembranous processing of Notch1 is indispensable for embryonic viability and proper early embryonic development in vivo.

Embryonic lethality in mice homozygous for a processing-deficient allele of Notch1

The Human Homolog of Rat Jagged1Expressed by Marrow Stroma Inhibits Differentiation of 32D Cells through Interaction with Notch1

Notch as a Mediator of Cell Fate Determination in Hematopoiesis: Evidence and Speculation

Hematopoiesis can be considered a developmental process in which pluripotent stem cells give rise to committed progeny that undergo proliferation and differentiation, resulting in the continuous production of appropriate numbers of mature blood cells throughout the lifetime of a vertebrate organism (for reviews, see references 33 and 38). Considerable progress has been made in understanding the regulation of hematopoiesis, including the effects of and interactions among cytokines and the interactions of progenitors with stromal elements (for reviews, see references 23 and 30). Despite these advances, many aspects of hematopoiesis remain obscure, including the mechanisms by which multipotent progenitors choose to differentiate along one of multiple pathways or to self-renew and remain multipotent. In other developmental systems, cell fate decisions by multipotent progenitors are mediated by members of the Notch family (for reviews, see references 2, 9, and 46). We have previously demonstrated the expression of Notch genes in hematopoietic progenitors (27) and the functional activity of Notch1 in 32D myeloid progenitors (20, 26) and have proposed that members of this receptor family play a similar role in the determination of hematopoietic cell fates.

The general function of Notch as a mediator of cell fate decisions has been highly conserved throughout evolution (for a review, see reference 1), and this is reflected in the conservation of its molecular structure (Figure ​(Figure1).1). The extracellular domain of Notch, which contains 33 to 36 tandem epidermal growth factor repeats and three lin-12/Notch repeats (LNR), functions as a receptor in cell-cell interactions. There is a single transmembrane domain. The intracellular domain contains six cdc10/SWI6/ankyrin repeats, putative nuclear localization signals (NLS), and a C-terminal OPA/PEST region. The cdc10 region is the most highly conserved portion of the molecule and is crucial for intracellular signal transduction (6, 10, 15, 21, 32, 34, 36, 40). Activation of the Notch molecule by ligand (DSL proteins, such as Delta, Serrate, and Lag-2) binding to the extracellular domain inhibits differentiation along a specific cell fate pathway in response to inductive signals (5, 20, 22). Thus, among a group of cells having equivalent cell fate potentials, a limited number of cells will adopt the specific cell fate while others (those expressing higher levels of Notch) will remain multipotent and competent to subsequently adopt an alternate cell fate (for reviews, see references 1 and 13). 



FIG. 1

Diagram of the full-length Notch molecule and the activated intracellular Notch construct. Both Notch1 and Notch2 consist of an extracellular domain containing 36 epidermal growth factor (EGF) repeats and 3 LNR; there is a single transmembrane domain ...



Evaluation of the phenotypic effects of mutant Notch molecules in several different systems has helped elucidate the functions of different parts of the Notch molecule (21, 34, 40). These studies have demonstrated that truncated Notch molecules lacking most or all of the extracellular domain behave as constitutively activated forms of Notch (10, 21, 40). Thus, expression of only the intracellular domain (or a portion of the intracellular domain) results in effects comparable to those observed or expected from unregulated or continuous Notch activation through ligand binding. We have previously demonstrated that expression of a truncated intracellular form of mNotch1 (as illustrated in Fig. ​Fig.1)1) in 32D myeloid cells inhibits granulocyte colony-stimulating factor (G-CSF)-induced granulocytic differentiation and permits the expansion of undifferentiated progenitors, effects consistent with Notch activity in other systems (26). More recently, we have demonstrated that activation of full-length mNotch1 by the Notch ligand, Jagged1, results in the same functional effects on 32D differentiation (20). These findings suggest that signaling through the Notch pathway may function in hematopoiesis to regulate cell fate decisions and to maintain progenitor populations.

In contrast to Drosophila, in which a single Notch molecule mediates a variety of cell fate decisions during the development of different tissues (2, 4, 7, 31, 37), mammals express at least four distinct Notch genes (11, 18, 41, 44, 45). These individual Notch molecules have both overlapping and distinct patterns of expression, but differences in function, if any, have not been characterized. In Caenorhabditis elegans, two Notch homologs, lin-12 and glp-1, are expressed in different types of progenitors and mediate the cell fate determination of vulval or germ line cells, respectively (3, 39, 46); however, these two Notch homologs appear to be functionally interchangeable (8, 36). We have found that Notch1, Notch2, and Notch3 are all expressed in hematopoietic progenitors, including the mouse myeloid progenitor cell lines 32D and FDCP mixA4 (reference 26 and unpublished observations). In preliminary studies to compare Notch1 and Notch2 function in hematopoietic cells, we found that the activated intracellular form of Notch1, but not Notch2, inhibited G-CSF-induced differentiation of 32D myeloid cells (26). This observation suggested that Notch1 and Notch2 might have distinct functions in hematopoietic differentiation.

In the studies presented here, we show that activated forms of Notch1 and Notch2, when constitutively expressed in 32D myeloid progenitors, have effects that depend on the specific cytokine inductive signal. Activated Notch1 specifically inhibits differentiation in response to G-CSF, whereas Notch2 inhibits differentiation only in response to granulocyte-macrophage colony-stimulating factor (GM-CSF). In addition, we provide evidence that a previously uncharacterized region, termed the Notch cytokine response (NCR) region, modulates these specific effects. We also show that the NCR region is associated with different posttranslational modifications and subcellular localizations of Notch1 and Notch2 molecules, supporting the conclusion that structural differences between the Notch1 and Notch2 NCR regions contribute to functional specificity in this system. We propose a model through which the NCR region could modulate the activity of Notch1 and Notch2 in response to different cytokines.

Laurie A. Milner

Papers

Osteoblastic cells regulate the haematopoietic stem cell niche

Embryonic lethality in mice homozygous for a processing-deficient allele of Notch1

The Human Homolog of Rat Jagged1Expressed by Marrow Stroma Inhibits Differentiation of 32D Cells through Interaction with Notch1

Notch as a Mediator of Cell Fate Determination in Hematopoiesis: Evidence and Speculation

Notch1 and Notch2 Inhibit Myeloid Differentiation in Response to Different Cytokines