The many faces of Notch signaling in skin‐derived cells

Since the cloning of the Drosophila gene in the 1980s, decades of research have sought to dissect the intricacies of the mammalian Notch signaling cascade. The intrigue of this pathway undoubtedly lies in its ability to influence diverse cellular processes, including differentiation, cell fate, homeostasis, survival, proliferation and angiogenesis. Based on its evolutionary conservation and its fundamental role in development, it is not surprising that deregulation of the Notch signaling pathway can result in neoplastic growth. While originally of particular interest to immunologists based on its chief role in influencing T‐cell fate decisions and tumor oncogenesis in T‐cell acute lymphoblastic leukemia, pigment cell biologists have recently taken notice of the Notch cascade based on studies suggesting the importance of this pathway in regulating melanocyte stem cell survival and melanoma progression. We will review the Notch signaling literature as it relates to skin homeostasis, melanocytic stem cells and melanoma tumorigenesis.     

Notch in the pathway: the roles of Notch signaling in neural crest development

Here, we review recent studies that suggest that Notch signaling has two roles during neural crest development: first in establishing the neural crest domain within the ectoderm via lateral induction and subsequently in diversifying the fates of cells that arise from the neural crest via lateral inhibition. The first of these roles, specification of neural crest via lateral induction, has been explored primarily in the cranial neural folds from which the cranial neural crest arises. Evidence for such a role has thus far only been obtained from chick and frog; results from these two species differ, but share the feature that Notch signaling regulates genes that are expressed by cranial neural crest through effects on expression of Bmp family members. The second of these roles, diversification of neural crest progeny via lateral inhibition, has been identified thus far only in trunk neural crest. Evidence from several species suggests that Notch-mediated lateral inhibition functions in multiple episodes in this context, in each case inhibiting neurogenesis. In the 'standard' mode of lateral inhibition, Notch promotes proliferation and in the 'instructive' mode, it promotes specific secondary fates, including cell death or glial differentiation. We raise the possibility that a single molecular mechanism, inhibition of so-called proneural bHLH genes, underlies both modes of lateral inhibition mediated by Notch signaling.     

Notch信号通路调控猕猴胚胎干细胞的胆管样细胞分化(英文)

猕猴胚胎干细胞(rhesus monkey embryonic stem(rES))与人胚胎干细胞有相似的生物学特性,因此是理想的临床前研究的替代模型。Notch信号通路在胆管及胆管上皮细胞的形成中有重要的作用,然而,有关Notch信号通路在ES细胞的胆向分化中的作用了解甚少。该实验以rES为模型,对Notch信号通路对ES细胞的胆向分化过程中的作用进行了较为系统的研究。rES在细胞因子ActivinA诱导作用下产生约80%的限定性内胚层细胞。以Matrigel作为细胞外基质,在含BMP4和FGF1的无血清培养体系中继续诱导5～7d,rES细胞来源的限定性内胚层细胞分化产生约胆管样细胞。分化的细胞表达胆管细胞的特异性蛋白((CK7、CK18、CK19、CK20和OV-6)及基因(GSTPi、IB4和HNF1β)。在胆管样细胞的分化过程中检测到了Notch1和Notch2基因及下游信号分子hes1和hes5的表达。用Notch抑制剂L-685458处理分化过程中的细胞可导致Notch1和Notch2基因及下游信号分子hes1和hes5的表达下降,同时CK19阳性的胆管样细胞分化比率也从90%下降至约20%。这一...     

Multiple roles of Notch signaling in cochlear development

Notch signaling inhibits hair cell differentiation, based on studies on mice deficient in Notch signaling-related genes and its downstream genes. However, the precise mechanisms of this inhibition are unknown because it is difficult to control the timing and duration of the suppression of Notch signaling. Here, we developed a novel in vitro culture and analysis method for mouse fetal cochleae and examined the roles of Notch signaling by its reversible inhibition through the use of Notch signaling inhibitors of gamma-secretase and TNF-alpha-converting enzyme. Notch inhibition with Notch signaling inhibitor treatment increases the number of cochlear hair cells, as observed in gene deletion experiments. We elucidated that this increase is regulated by the dichotomy between hair cells and supporting cells from common progenitors. We also propose other roles of Notch signaling in cochlear development. First, Notch signaling arrests the cell cycle of the cochlear epithelium containing putative hair cells and supporting cell progenitors because Notch inhibition with inhibitor treatment increases the number of 5-bromo-2'-deoxyuridine (BrdU)-positive cells that can differentiate into hair cells or supporting cells. Second, Notch signaling is required for the induction of Prox1-positive supporting cells. Third, Notch signaling is required for the maintenance of supporting cells.     

Stable propagation of human embryonic and induced pluripotent stem cells on decellularized human substrates

Human pluripotent stem cells (hPSCs) that include human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) have gained enormous interest as potential sources for regenerative biomedical therapies and model systems for studying early development. Traditionally, mouse embryonic fibroblasts have been used as a supportive feeder layer for the sustained propagation of hPSCs. However, the use of nonhuman‐derived feeders presents concerns about the possibility of xenogenic contamination, labor intensiveness, and variability in experimental results in hPSC cultures. Toward addressing some of these concerns, we report the propagation of three different hPSCs on feeder‐free extracellular matrix (ECM)‐based substrates derived from human fibroblasts. hPSCs propagated in this setting were indistinguishable by multiple criteria, including colony morphology, expression of pluripotency protein markers, trilineage in vitro differentiation, and gene expression patterns, from hPSCs cultured directly on a fibroblast feeder layer. Further, hPSCs maintained a normal karyotype when analyzed after 15 passages in this setting. Development of this ECM‐based culture system is a significant advance in hPSC propagation methods as it could serve as a critical component in the development of humanized propagation systems for the production of stable hPSCs and its derivatives for research and therapeutic applications. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Identification of c-Kit receptor as a regulator of adult neural stem cells in the mammalian eye: interactions with Notch signaling

Neural stem cells are present in specific regions of the adult central nervous system (CNS). Recent evidence suggests that the ciliary epithelium (CE), a CNS derivative, in the adult mammalian eye, harbors a quiescent population of neural stem cells. Here, we report the identification of c-Kit signaling as one of the regulators of adult CE neural stem cells in vitro. c-Kit receptors are expressed in proliferating adult CE neural stem cells and colocalized with neural progenitor markers. Perturbation of c-Kit signaling influences the self-renewal and differentiation of CE neural stem cells, thus demonstrating the role of c-Kit signaling in the maintenance of these cells. In addition, we observed an influence of c-Kit-mediated signaling on the expression of Notch1, another critical regulator of neural stem cells. Our observations suggest that, given the importance of preservation of a stem cell pool for generating different cell types at different times, multiple signaling pathways act in concert for the maintenance of neural stem cells.     

Specific lectin biomarkers for isolation of human pluripotent stem cells identified through array-based glycomic analysis

Rapid and dependable methods for isolating human pluripotent stem cell (hPSC) populations are urgently needed for quality control in basic research and in cell-based therapy applications. Using lectin arrays, we analyzed glycoproteins extracted from 26 hPSC samples and 22 differentiated cell samples, and identified a small group of lectins with distinctive binding signatures that were sufficient to distinguish hPSCs from a variety of non-pluripotent cell types. These specific biomarkers were shared by all the 12 human embryonic stem cell and the 14 human induced pluripotent stem cell samples examined, regardless of the laboratory of origin, the culture conditions, the somatic cell type reprogrammed, or the reprogramming method used. We demonstrated a practical application of specific lectin binding by detecting hPSCs within a differentiated cell population with lectin-mediated staining followed by fluorescence microscopy and flow cytometry, and by enriching and purging viable hPSCs from mixed cell populations using lectin-mediated cell separation. Global gene expression analysis showed pluripotency-associated differential expression of specific fucosyltransferases and sialyltransferases, which may underlie these differences in protein glycosylation and lectin binding. Taken together, our results show that protein glycosylation differs considerably between pluripotent and non-pluripotent cells, and demonstrate that lectins may be used as biomarkers to monitor pluripotency in stem cell populations and for removal of viable hPSCs from mixed cell populations.     

The NOTCH signaling pathway in normal and malignant blood cell production

The NOTCH pathway is an evolutionarily conserved signalling network, which is fundamental in regulating developmental processes in invertebrates and vertebrates (Gazave et al. in BMC Evol Biol 9:249, 2009). It regulates self-renewal (Butler et al. in Cell Stem Cell 6:251–264, 2010), differentiation (Auderset et al. in Curr Top Microbiol Immunol 360:115–134, 2012), proliferation (VanDussen et al. in Development 139:488–497, 2012) and apoptosis (Cao et al. in APMIS 120:441–450, 2012) of diverse cell types at various stages of their development. NOTCH signalling governs cell-cell interactions and the outcome of such responses is highly context specific. This makes it impossible to generalize about NOTCH functions as it stimulates survival and differentiation of certain cell types, whereas inhibiting these processes in others (Meier-Stiegen et al. in PLoS One 5:e11481, 2010). NOTCH was first identified in 1914 in Drosophila and was named after the indentations (notches) present in the wings of the mutant flies (Bigas et al. in Int J Dev Biol 54:1175–1188, 2010). Homologs of NOTCH in vertebrates were initially identified in Xenopus (Coffman et al. in Science 249:1438–1441, 1990) and in humans NOTCH was first identified in T-Acute Lymphoblastic Leukaemia (T-ALL) (Ellisen et al. in Cell 66:649–61, 1991). NOTCH signalling is integral in neurogenesis (Mead and Yutzey in Dev Dyn 241:376–389, 2012), myogenesis (Schuster-Gossler et al. in Proc Natl Acad Sci U S A 104:537–542, 2007), haematopoiesis (Bigas et al. in Int J Dev Biol 54:1175–1188, 2010), oogenesis (Xu and Gridley in Genet Res Int 2012:648207, 2012), differentiation of intestinal cells (Okamoto et al. in Am J Physiol Gastrointest Liver Physiol 296:G23–35, 2009) and pancreatic cells (Apelqvist et al. in Nature 400:877–881, 1999). The current review will focus on NOTCH signalling in normal and malignant blood cell production or haematopoiesis.     

Chimeric contribution of human extended pluripotent stem cells to monkey embryos ex vivo

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Multiple functions of Notch signaling in self-renewing organs and cancer

In recent years a substantial body of evidence has accumulated to support the notion that signaling pathways known to be important during embryonic development play important roles in regulating self-renewing tissues. Moreover, the same pathways are often deregulated during tumorigenesis due to mutations of key elements of these pathways. The Notch signaling cascade meets all of the above-mentioned criteria. We discuss here the pleiotropic roles of the Notch signaling pathway in three different self-renewing organs (intestine, hematopoietic system and skin) and how its deregulation is involved in tumorigenesis.     

Notch signaling in intestinal homeostasis across species: the cases of Drosophila, Zebrafish and the mouse

Notch signaling has been recently shown to have a fundamental role in stem cell maintenance and control of proper homeostasis in the intestine of different species. Here, we briefly review the current literature on Notch signals in the intestine of Drosophila, Zebrafish and the mouse, and try to highlight conserved and divergent Notch functions across species. Notch signals show a remarkably conserved role in skewing cell fate choices in intestinal lineages throughout evolution. Genetic analysis demonstrates that loss of Notch signaling invariably leads to increased numbers of secretory cells and loss of enterocytes, while gain of Notch function will completely block secretory cell differentiation. Finally, we discuss the potential contribution of Notch signaling to the initiation of colorectal cancer by controlling the maintenance of the undifferentiated state of intestinal neoplastic cells and speculate on the therapeutic consequences of affecting cancer stem cells.     

The Notch signaling pathway in the cnidarian Hydra

Many of the major pathways that govern early development in higher animals have been identified in cnidarians, including the Wnt, TGFbeta and tyrosine kinase signaling pathways. We show here that Notch signaling is also conserved in these early metazoans. We describe the Hydra Notch receptor (HvNotch) and provide evidence for the conservation of the Notch signaling mode via regulated intramembrane proteolysis. We observed that nuclear translocation of the Notch intracellular domain (NID) was inhibited by the synthetic gamma-secretase inhibitor DAPT. Moreover, DAPT treatment of hydra polyps caused distinct differentiation defects in their interstitial stem cell lineage. Nerve cell differentiation proceeded normally but post-mitotic nematocyte differentiation was dramatically reduced. Early female germ cell differentiation was inhibited before exit from mitosis. From these results we conclude that gamma-secretase activity and presumably Notch signaling are required to control differentiation events in the interstitial cell lineage of Hydra.     

Deciphering the hierarchy of angiohematopoietic progenitors from human pluripotent stem cells

Identification of sequential progenitors leading to blood formation from pluripotent stem cells (PSCs) will be essential for understanding the molecular mechanisms of hematopoietic lineage specification and for development of technologies for in vitro production of hematopoietic stem cells (HSCs). It is well established that during development, blood and endothelial cells in the extraembryonic and embryonic compartments are formed in parallel from precursors with angiogenic and hematopoietic potentials. However, the identity and hierarchy of these precursors in human PSC (hPSC) cultures remain obscure. Using developmental stage-specific mesodermal and endothelial markers and functional assays, we recently identified discrete populations of angiohematopoietic progenitors from hPSCs, including mesodermal precursors and hemogenic endothelial cells with primitive and definitive hematopoietic potentials. In addition, we discovered a novel population of multipotent hematopoietic progenitors with an erythroid phenotype, which retain angiogenic potential. Here we introduce our recent findings and discuss their implication for defining putative HSC precursor and factors required for activation of self-renewal potential in hematopoietic cells emerging from endothelium.     

Notch signaling in blood vessels: from morphogenesis to homeostasis

Notch signaling is an evolutionarily conserved intercellular signaling pathway that plays numerous crucial roles in vascular development and physiology. Compelling evidence indicates that Notch signaling is vital for vascular morphogenesis including arterial and venous differentiation and endothelial tip and stalk cell specification during sprouting angiogenesis and also vessel maturation featured by mural cell differentiation and recruitment. Notch signaling is also required for vascular homeostasis in adults by keeping quiescent phalanx cells from re-entering cell cycle and by modulating the behavior of endothelial progenitor cells. We will summarize recent advances of Notch pathway in vascular biology with special emphasis on the underlying molecular mechanisms.     

The impact of trisomy 21 on early human hematopoiesis

Mitogen-activated protein kinases (MAPKs) are components of signaling cascades regulated by environmental stimuli. In addition to participating in the stress response, the MAPKs c-Jun N-terminal Kinases JNK1 and JNK2 regulate the proliferation of normal and neoplastic cells. JNKs contribute to these processes largely by phosphorylating c-Jun and thus contributing to the activation of the AP-1 complex. We here report that JNKs control entry into mitosis. We have observed that JNK activity and phosphorylation of c-Jun become elevated during the G2/M transition of the cell cycle in immortalized fibroblasts and ovarian granulosa cells. Pharmacological inhibition of JNK causes a profound cell cycle arrest at the G2/M transition in both cell types. This effect is specific as it occurs with two distinct small molecule compounds. Inactivation of JNK prior to mitosis prevents expression of Aurora B and phosphorylation of Histone-H3 at Ser 10. Silencing of JNK1 and 2 causes a similar effect, whereas overexpression of JNK1 and 2 causes the opposite effect. Inhibition of JNK delays activation of cdc-2 and prevents downregulation of Cyclin B1. We conclude that JNK signaling promotes entry into mitosis by promoting expression of Aurora B and thereby phosphorylation of Histone-H3.