Activated Notch1 alters differentiation of embryonic stem cells into mesodermal cell lineages at multiple stages of development

Signals of Notch transmembrane receptors function to regulate a wide variety of developmental cell fates. Here we investigate the role of Notch signaling in the development of mesodermal cell types by expressing a tamoxifen-inducible, activated form of Notch1 in embryonic stem cells (ESC). For differentiation of ESC into first mesodermal progenitor cells and then endothelial, mural, cardiac muscle and hematopoietic cells, the OP9 stroma co-culture system was used. Timed activation of Notch signaling by the addition of tamoxifen at various stages during differentiation of ESC into mesodermal cell lineages results in profound alterations in the generation of all of these cells. Differentiation of ESC into Flk1(+) mesodermal cells is inhibited by activated Notch. When Notch signaling is activated in mesodermal cells, generation of cardiac muscle, endothelial and hematopoietic cells is inhibited, favoring the generation of mural cells. Activation of Notch signaling in hematopoietic cells reduces colony formation and maintenance of hematopoiesis. These data suggest that Notch signaling plays a regulatory role in mesodermal development, cardiomyogenesis, the balanced generation of endothelial versus mural cells of blood vessels and hematopoietic development.     

Colony-forming activity of hematopoietic stem cells in tolerant mice as affected by antigens

The effect of sheep red blood cells (SRBC) and human red blood cells (HRBC) on the amount of CFUs in the bone marrow and spleen of (CBA X C57BL/6) FI SRBC-tolerant mice was studied. The increase in the number of bone marrow and spleen CFUs was demonstrated in SRBC-tolerant mice injected with HRBC. Using SRBC test injection the increase in CFUs amount was observed in the spleen, but not the bone marrow, where the amount of CFUs remained unchanged.     

Rapid expansion of intestinal secretory lineages following a massive small bowel resection in mice

Following massive small bowel resection (SBR) in mice, there are sustained increases in crypt depth and villus height, resulting in enhanced mucosal surface area. The early mechanisms responsible for resetting and sustaining this increase are presently not understood. We hypothesized that expansion of secretory lineages is an early and sustained component of the adaptive response. This was assessed in the ileum by quantitative morphometry at 12 h, 36 h, 7 days, and 28 days and by quantitative RT-PCR of marker mRNAs for proliferation and differentiated goblet, Paneth cell, and enterocyte genes at 12 h after 50% SBR or sham operation. As predicted, SBR elicited increases of both crypt and villus epithelial cells, which were sustained though the 28 days of the experiment. Significant increases in the overall number and percentage of both Paneth and goblet cells within intestinal epithelium occurred by 12 h and were sustained up to 28 days after SBR. The increases of goblet cells after SBR were initially observed within villi at 12 h, with marked increases occurring in crypts at 36 h and 7 days. Consistent with this finding, qRT-PCR demonstrated significant increases in the expression of mRNAs associated with proliferation (c-myc) and differentiated goblet cells (Tff3, Muc2) and Paneth cells (lysozyme), whereas mRNA associated with differentiated enterocytes (sucrase-isomaltase) remained unchanged. From these data, we speculate that early expansion of intestinal secretory lineages within the epithelium of the ileum occurs following SBR, possibly serving to amplify the signal responsible for initiating and sustaining intestinal adaptation.     

Diversification of haematopoietic stem cells to specific lineages

Diverse types of blood cell (lineages) are produced from rare haematopoietic stem cells that reside in the bone marrow. This process, known as haematopoiesis, provides a valuable model for examining how genetic programs are established and executed in vertebrates, and also how homeostasis of blood formation is altered in leukaemias. So, how does an apparently small group of critical lineage-restricted nuclear regulatory factors specify the diversity of haematopoietic cells? Recent findings not only indicate how this may be achieved but also show the extraordinary plasticity of tissue stem cells in vivo.     

Notch activity in neural cells triggered by a mutant allele with altered glycosylation

The receptor protein Notch is inactive in neural precursor cells despite neighboring cells expressing ligands. We investigated specification of the R8 neural photoreceptor cells that initiate differentiation of each Drosophila ommatidium. The ligand Delta was required in R8 cells themselves, consistent with a lateral inhibitor function for Delta. By contrast, Delta expressed in cells adjacent to R8 could not activate Notch in R8 cells. The split mutation of Notch was found to activate signaling in R8 precursor cells, blocking differentiation and leading to altered development and neural cell death. split did not affect other, inductive functions of Notch. The Ile578-->Thr578 substitution responsible for the split mutation introduced a new site for O-fucosylation on EGF repeat 14 of the Notch extracellular domain. The O-fucose monosaccharide did not require extension by Fringe to confer the phenotype. Our results suggest functional differences between Notch in neural and non-neural cells. R8 precursor cells are protected from lateral inhibition by Delta. The protection is affected by modifications of a particular EGF repeat in the Notch extracellular domain. These results suggest that the pattern of neurogenesis is determined by blocking Notch signaling, as well as by activating Notch signaling.     

Natural and antibody-dependent cell-mediated activity against Salmonella typhimurium by peripheral and intestinal lymphoid cells in mice

Cell-mediated immune responses were assessed employing a 2-hr in vitro cytotoxicity assay against S. typhimurium. It was observed that lymphocytes from GALT as well as from peripheral lymphoid organs possessed natural antibacterial activity, whereas macrophages were devoid of this spontaneous activity. The distribution of this newly described natural activity was PPL greater than MnL greater than IEL = SpL = PBL greater than PoL; this did not correlate with the organ distribution of NK activity against YAC-1 tumor cells, which was PBL greater than SpL = IEL greater than MnL = PoL = PPL. Moreover, the phenotype of the splenic effector cell of the natural activity against S. typhimurium showed some differences from that of NK activity. In fact, both these cells were asialo GM1+, Fc-receptor+, nonadherent, and nonphagocytic, but the former was Thy-1.2- and the latter Thy-1.2+. The effector cell of the natural antibacterial activity in the Peyer's patches had the same phenotype as the splenic one. It was then observed that the antibacterial activity could be augmented by the addition of immune antibodies against S. typhimurium. This was particularly evident employing IEL, SpL, and PBL as effector cells, whereas PPL and MnL did not show any antibody-dependent antibacterial activity. Furthermore, these last two populations could not mediate ADCC against CRBC. Employing selective methods to deplete cell populations, we observed that, at least at the splenic level, there is also a cell that differs in its phenotypic characteristics from that mediating natural antibacterial activity but that plays a role in the antibody-dependent reactions. In conclusion, these results suggest that natural and antibody-dependent antibacterial mechanisms might be important in defense against S. typhimurium, particularly at the gastrointestinal level, where many bacterial infections first take place and begin to interact with the host immune system.     

MicroRNA-34a induces transdifferentiation of glioma stem cells into vascular endothelial cells by targeting Notch pathway

The function of microRNA-34a (miR-34a) in transdifferentiation of glioma stem cells (GSCs) into vascular endothelial cells (VECs) was explored by focusing on Notch ligand Delta-like 1 (Dll1). MiR-34a mimics was transfected into CD133 + glioma cell U251. The angiogenesis feature of miR-34a transfected U251 cells was investigated and the expressions of CD31, CD34, Vwf, Notch 1, and Dll1 were quantified. Length of branching vessel-like structures in the miR-34a transfected U251 cells was significantly higher than control cells. The VEC feature of miR-34a overexpressed U251 cells was further confirmed by the expressions of CD31, CD34, and vWF. Transfection of miR-34a decreased the expression of Notch 1 and Dll1. Furthermore, the miR-34a overexpression-enhanced tube formation of GSCs was suppressed when the decreased expression of Dll1 was restored. The current study highlighted the potential of miR-34a as an inducer in GSCs’ transdifferentiation into VECs by targeting Dll1.     

Epididymal initial segment-specific Cre recombinase activity in Lcn8-Cre knock-in mice

Molecular Biology Reports - Sperm acquire the ability to fertilize ova through a complex process of epididymal maturation. To identify the functions of genes expressed in the proximal epididymis,...     

Differentiation of monkey embryonic stem cells into neural lineages

Embryonic stem (ES) cells are self-renewing, pluripotent, and capable of differentiating into all of the cell types found in the adult body. Therefore, they have the potential to replace degenerated or damaged cells, including those in the central nervous system. For ES cell-based therapy to become a clinical reality, translational research involving nonhuman primates is essential. Here, we report monkey ES cell differentiation into embryoid bodies (EBs), neural progenitor cells (NPCs), and committed neural phenotypes. The ES cells were aggregated in hanging drops to form EBs. The EBs were then plated onto adhesive surfaces in a serum-free medium to form NPCs and expanded in serum-free medium containing fibroblast growth factor (FGF)-2 before neural differentiation was induced. Cells were characterized at each step by immunocytochemistry for the presence of specific markers. The majority of cells in complex/cystic EBs expressed antigens (alpha-fetal protein, cardiac troponin I, and vimentin) representative of all three embryonic germ layers. Greater than 70% of the expanded cell populations expressed antigenic markers (nestin and musashi1) for NPCs. After removal of FGF-2, approximately 70% of the NPCs differentiated into neuronal phenotypes expressing either microtubule-associated protein-2C (MAP2C) or neuronal nuclear antigen (NeuN), and approximately 28% differentiated into glial cell types expressing glial fibrillary acidic protein. Small populations of MAP2C/NeuN-positive cells also expressed tyrosine hydroxylase (approximately 4%) or choline acetyltransferase (approximately 13%). These results suggest that monkey ES cells spontaneously differentiate into cells of all three germ layers, can be induced and maintained as NPCs, and can be further differentiated into committed neural lineages, including putative neurons and glial cells.     

Sending the right signal: Notch and stem cells

Background

Notch signaling plays a critical role in multiple developmental programs and not surprisingly, the Notch pathway has also been implicated in the regulation of many adult stem cells, such as those in the intestine, skin, lungs, hematopoietic system, and muscle.

Scope of review

In this review, we will first describe molecular mechanisms of Notch component modulation including recent advances in this field and introduce the fundamental principles of Notch signaling controlling cell fate decisions. We will then illustrate its important and varied functions in major stem cell model systems including: Drosophila and mammalian intestinal stem cells and mammalian skin, lung, hematopoietic and muscle stem cells.

Major conclusions

The Notch receptor and its ligands are controlled by endocytic processes that regulate activation, turnover, and recycling. Glycosylation of the Notch extracellular domain has important modulatory functions on interactions with ligands and on proper receptor activity. Notch can mediate cell fate decisions including proliferation, lineage commitment, and terminal differentiation in many adult stem cell types. Certain cell fate decisions can have precise requirements for levels of Notch signaling controlled through modulatory regulation.

General significance

We describe the current state of knowledge of how the Notch receptor is controlled through its interaction with ligands and how this is regulated by associated factors. The functional consequences of Notch receptor activation on cell fate decisions are discussed. We illustrate the importance of Notch's role in cell fate decisions in adult stem cells using examples from the intestine, skin, lung, blood, and muscle. This article is part of a Special Issue entitled Biochemistry of Stem Cells.     

Physiological properties of rod photoreceptor cells in green-sensitive cone pigment knock-in mice

Rod and cone photoreceptor cells that are responsible for scotopic and photopic vision, respectively, exhibit photoresponses different from each other and contain similar phototransduction proteins with distinctive molecular properties. To investigate the contribution of the different molecular properties of visual pigments to the responses of the photoreceptor cells, we have generated knock-in mice in which rod visual pigment (rhodopsin) was replaced with mouse green-sensitive cone visual pigment (mouse green). The mouse green was successfully transported to the rod outer segments, though the expression of mouse green in homozygous retina was approximately 11% of rhodopsin in wild-type retina. Single-cell recordings of wild-type and homozygous rods suggested that the flash sensitivity and the single-photon responses from mouse green were three to fourfold lower than those from rhodopsin after correction for the differences in cell volume and levels of several signal transduction proteins. Subsequent measurements using heterozygous rods expressing both mouse green and rhodopsin E122Q mutant, where these pigments in the same rod cells can be selectively irradiated due to their distinctive absorption maxima, clearly showed that the photoresponse of mouse green was threefold lower than that of rhodopsin. Noise analysis indicated that the rate of thermal activations of mouse green was 1.7 x 10(-7) s(-1), about 860-fold higher than that of rhodopsin. The increase in thermal activation of mouse green relative to that of rhodopsin results in only 4% reduction of rod photosensitivity for bright lights, but would instead be expected to severely affect the visual threshold under dim-light conditions. Therefore, the abilities of rhodopsin to generate a large single photon response and to retain high thermal stability in darkness are factors that have been necessary for the evolution of scotopic vision.     

Expansion of intestinal stem cells associated with long-term adaptation following ileocecal resection in mice

Sustained increases in mucosal surface area occur in remaining bowel following massive intestinal loss. The mechanisms responsible for expanding and perpetuating this response are not presently understood. We hypothesized that an increase in the number of intestinal stem cells (ISC) occurs following intestinal resection and is an important component of the adaptive response in mice. This was assessed in the jejunum of mice 2-3 days, 4-5 days, 6-7 days, 2 wk, 6 wk, and 16 wk following ileocecal resection (ICR) or sham operation. Changes in ISC following ICR compared with sham resulted in increased crypt fission and were assayed by 1) putative ISC population (SP) by flow cytometry, 2) Musashi-1 immunohistochemistry, and 3) bromodeoxyuridine (BrdU) label retention. Observed early increases in crypt depth and villus height were not sustained 16 wk following operation. In contrast, long-term increases in intestinal caliber and overall number of crypts per circumference appear to account for the enhanced mucosal surface area following ICR. Flow cytometry demonstrated that significant increases in SP cells occur within 2-3 days following resection. By 7 days, ICR resulted in marked increases in crypt fission and Musashi-1 immunohistochemistry staining. Separate label-retention studies confirmed a 20-fold increase in BrdU incorporation 6 wk following ICR, confirming an overall increase in the number of ISC. These studies support that expansion of ISC occurs following ICR, leading to an overall increase number of crypts through a process of fission and intestinal dilation. Understanding the mechanism expanding ISCs may provide important insight into management of intestinal failure.     

Distinct levels of Notch activity for commitment and terminal differentiation of stem cells in the adult fly intestine

Tight regulation of self-renewal and differentiation of adult stem cells ensures that tissues are properly maintained. In the Drosophila intestine, both commitment, i.e. exit from self-renewal, and terminal differentiation are controlled by Notch signaling. Here, we show that distinct requirements for Notch activity exist: commitment requires high Notch activity, whereas terminal differentiation can occur with lower Notch activity. We identified the gene GDP-mannose 4,6-dehydratase (Gmd), a modulator of Notch signaling, as being required for commitment but dispensable for terminal differentiation. Gmd loss resulted in aberrant, self-renewing stem cell divisions that generated extra ISC-like cells defective in Notch reporter activation, as well as wild-type-like cell divisions that produced properly terminally differentiated cells. Lowering Notch signaling using additional genetic means, we provided further evidence that commitment has a higher Notch signaling requirement than terminal differentiation. Our work suggests that a commitment requirement for high-level Notch activity safeguards the stem cells from loss through differentiation, revealing a novel role for the importance of Notch signaling levels in this system.     

Studies of intestinal stem cells using normal, chimeric, and transgenic mice.

The mouse intestinal epithelium represents a continuous developmental system. Its four principal differentiated cell types--enterocytes, goblet, enteroendocrine, and Paneth cells--are derived from a common multipotent stem cell located near the base of monoclonal crypts. Members of these four lineages undergo rapid and perpetual renewal along an anatomically well-defined pathway. The gut epithelium provides a unique mammalian model for studying the biological features of stem cells (e.g., their ability to undergo asymmetric division, their enormous proliferative potential, their capacity for functional anchorage in a niche), examining how stem cell hierarchies are established and maintained in renewing cell populations, analyzing the relationships between passage through the cell cycle and lineage allocation (commitment), and defining the mechanisms that give stem cells a "positional address" along the cephalocaudal axis, allowing them to generate regional differences in the differentiation programs of their derived lineages (axial pattern formation).