A Role for Notch Signaling in Stromal Survival and Differentiation during Prostate Development

Notch1 signaling is involved in epithelial growth and differentiation of prostate epithelia, and we have examined the role that notch signaling plays in the stroma of the developing prostate. We initially observed expression of δ-like 1 (Dlk1) and Notch2 in gene profiling studies of prostatic mesenchyme, and anticipated that they might be expressed in a key subset of inductive mesenchyme. Using quantitative RT-PCR, Northern blotting, and whole mount in situ hybridization, we confirmed that both Dlk1 and Notch2 mRNAs showed a restricted expression pattern within subsets of the stroma during prostate development. Localization of Dlk1 and Notch2 proteins mirrored the transcript expression, and showed both distinct and overlapping expression patterns within the stroma. Dlk1 and Notch2 were coexpressed in condensed inductive mesenchyme of the ventral mesenchymal pad (VMP), and were partially colocalized in the smooth muscle (SM) layer of the urethral stroma. In addition, Dlk1 was not expressed in SM adjacent to the VMP in female urethra. The function of notch signaling was examined using organ cultures of prostate rudiments and a small molecule inhibitor of notch receptor activity. Inhibition of notch signaling led to a loss of stromal tissue in both prostate and female VMP cultures, suggesting that this pathway was required for stromal survival. Inhibition of notch signaling also led to changes in both epithelial and stromal differentiation, which was evident in altered distributions of SM α-actin and p63 in prostates grown in vitro. The effects of notch signaling upon the stroma were only evident in the presence of testosterone, in contrast to effects upon epithelial differentiation. Studies on the expression of delta-like 1 homolog (Dlk1) and Notch 2 in prostatic mesenchyme shows that inhibition of notch signaling leads to defects in mesenchymal differentiation.

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POS0042 NOTCH 1 INHIBITION INCREASES OSTEOCLAST PROGENITOR ACTIVITY IN THE MOUSE MODEL OF RHEUMATOID ARTHRITIS

Annals of the Rheumatic Diseases ◽

10.1136/annrheumdis-2021-eular.2601 ◽

2021 ◽

Vol 80 (Suppl 1) ◽

pp. 226.3-226

Author(s):

M. Filipović ◽

A. Šućur ◽

D. Flegar ◽

Z. Jajić ◽

M. Ikić Matijašević ◽

...

Keyword(s):

Flow Cytometry ◽

Notch Signaling ◽

Neutralizing Antibodies ◽

Osteoclast Differentiation ◽

Notch Receptor ◽

Notch 1 ◽

Collagen Induced Arthritis ◽

Notch Receptors

Background:Osteoclasts mediate periarticular and systemic bone loss in rheumatoid arthritis (RA). Osteoclast progenitor cells (OCPs) derived from the myeloid lineage are susceptible to regulation through Notch signaling. Murine bone marrow and splenic OCPs, identified as CD45+Ly6G-CD3-B220-NK1.1-CD11blo/+CD115+CCR2+ cells, are specifically increased in arthritis. We previously identified an increased frequency of OCPs expressing Notch receptors in arthritic mice.Objectives:Several studies suggested that Notch signaling modulation affects the course of experimental arthritis. We aimed to determine the effects of Notch receptor signaling inhibition on OCP activity and arthritis severity in murine collagen-induced arthritis (CIA).Methods:Male C57/Bl6 and DBA mice were immunized with chicken type II collagen and treated with i.p. injections of anti-Notch 1 neutralizing antibodies (1mg/kg). Notch receptor 1 through 4 expression on OCPs was analyzed by flow cytometry in periarticular bone marrow (PBM) and spleen (SPL). Gene expression of Notch receptors, ligands and transcription targets as well as osteoclast differentiation genes RANK, cFos and cFms was determined by qPCR from tissues and sorted OCPs. FACS sorted OCPs were stimulated by osteoclastogenic factors (M-CSF and RANKL), in control, IgG, Jagged (Jag)1 or Delta-like (DLL)1 coated wells, with or without anti-Notch 1 antibodies. Research was approved by the Ethics Committee.Results:We confirmed the expression of Notch receptors on OCPs by flow cytometry with Notch 1 and 2 being most abundantly expressed (around 25% and 40% positive OCPs in PBM and 35% and 20% in SPL respectively), with a significant increase of Notch 2 expression in arthritis. Seeding OCPs on DLL1 coated wells significantly increased while seeding on Jag1 coated wells significantly decreased osteoclastogenesis as reflected on the number of TRAP+ osteoclasts and expression of osteoclast differentiation genes. The addition of anti-Notch 1 antibodies to ligand-stimulated OCPs resulted in an increased number of TRAP+ osteoclasts, partially reversing Jag1 inhibition. In vivo treatment with anti-Notch 1 antibodies did not affect total OCP frequency, but increased expression of Notch 4 both in PBM and SPL as seen by flow cytometry and qPCR. Additionally, anti-Notch 1 treatment stimulated Notch transcription factors HES and HEY. Both PBM and SPL cultured OCPs from anti-Notch 1 treated mice produced a higher number of large TRAP+ osteoclasts, doubling the area covered with osteoclasts in the latter compared to untreated mice. Increased osteoclastogenesis in vitro was further confirmed by an increased expression of osteoclast differentiation genes in the treated group.Conclusion:Our results confirm that Notch signaling may represent an important therapeutic target for the regulation of osteoclast activity in arthritis. Both in vitro and in vivo anti-Notch 1 neutralizing antibodies enhanced osteoclastogenesis in CIA model, implying an inhibitory role of Notch 1 signaling in osteoclast differentiation. As Notch 2 expression is increased on OCPs of arthritic mice, we next plan to determine the effects of Notch 2 neutralization on osteoclast activity and arthritis severity.References:[1]Ikić Matijašević M, Flegar D, Kovačić N, Katavić V, Kelava T, Šućur A, et al. Increased chemotaxis and activity of circulatory myeloid progenitor cells may contribute to enhanced osteoclastogenesis and bone loss in the C57BL/6 mouse model of collagen-induced arthritis. Clin Exp Immunol. 2016;186(3):321–35.[2]Šućur A, Filipović M, Flegar D, Kelava T, Šisl D, Lukač N, et al. Notch receptors and ligands in inflammatory arthritis – a systematic review. Immunology Letters 2020 Vol. 223, p. 106–14.Acknowledgements:The work has been supported by Croatian Science Foundation projects IP-2018-01-2414, UIP-2017-05-1965 and DOK-2018-09-4276.Disclosure of Interests:None declared.

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Estrogen Signaling Drives Ciliogenesis in Human Endometrial Organoids

Endocrinology ◽

10.1210/en.2019-00314 ◽

2019 ◽

Vol 160 (10) ◽

pp. 2282-2297 ◽

Cited By ~ 11

Author(s):

Sandra Haider ◽

Magdalena Gamperl ◽

Thomas R Burkard ◽

Victoria Kunihs ◽

Ulrich Kaindl ◽

...

Keyword(s):

Notch Signaling ◽

Cell Fate ◽

Expression Patterns ◽

Estrogen Signaling ◽

Ciliated Cells ◽

Cell Fate Decisions ◽

Primary Driver ◽

Endometrial Epithelium ◽

Cyclic Changes

Abstract The human endometrium is the inner lining of the uterus consisting of stromal and epithelial (secretory and ciliated) cells. It undergoes a hormonally regulated monthly cycle of growth, differentiation, and desquamation. However, how these cyclic changes control the balance between secretory and ciliated cells remains unclear. Here, we established endometrial organoids to investigate the estrogen (E2)-driven control of cell fate decisions in human endometrial epithelium. We demonstrate that they preserve the structure, expression patterns, secretory properties, and E2 responsiveness of their tissue of origin. Next, we show that the induction of ciliated cells is orchestrated by the coordinated action of E2 and NOTCH signaling. Although E2 is the primary driver, inhibition of NOTCH signaling provides a permissive environment. However, inhibition of NOTCH alone is not sufficient to trigger ciliogenesis. Overall, we provide insights into endometrial biology and propose endometrial organoids as a robust and powerful model for studying ciliogenesis in vitro.

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Notch-Dependent Control of Blood Lineage Development is Modified by Fucosylation

Blood ◽

10.1182/blood.v112.11.2448.2448 ◽

2008 ◽

Vol 112 (11) ◽

pp. 2448-2448

Author(s):

Lan Zhou ◽

Quanjian Yan ◽

David Yao ◽

Lebing W Li ◽

Stanton L. Gerson ◽

...

Keyword(s):

Ligand Binding ◽

Notch Signaling ◽

Es Cells ◽

Notch Receptor ◽

Wild Type ◽

Notch Ligand ◽

Hematopoietic Reconstitution ◽

Myeloid Development

Abstract Notch receptors are conserved cell surface molecules essential for hematopoietic cell fate determination. Activated Notch enhances self-renewal of hematopoietic stem cells and promotes T lymphopoiesis. O-linked fucose moieties attached to the EGF domains of Notch receptors and its modification by Fringe can strongly modulate Notch signaling. Our recently published results indicate that Notch-dependent signaling controls myelopoiesis both in vitro and in vivo, and identify a requirement for Notch fucosylation in the expression of Notch ligand binding activity and Notch signaling efficiency in hematopoietic progenitor cells. In the current study, we tested the hypothesis that fucosylation controlled Notch signaling regulates hematopoietic lineage homeostasis. Genetically-modified mouse embryonic stem (ES) cells deficient in Notch1 receptor (NOTCH1−/−) or pofut1 (POFUT1−/−) that controls O-fucose modification of Notch receptor EGF repeats are studied in an in vitro co-culture assay with Notch ligand-expressing OP9 cells. Activation of Notch in wild type ES cells promotes T lymphopoiesis, while exposure of NOTCH1−/− or POFUT1−/− ES cells to Notch ligand failed to generate T lymphocytes but sustained granulocytic production. When probed with recombinant Notch ligand Dll1 or Dll4, hematopoietic cells derived from wild type ES line displayed robust Notch ligand binding, but cells from NOTCH1−/− or POFUT1−/− ES lines showed completely absent or reduced Notch ligand interaction, respectively. In comparison, ES cells deficient in pofut2 (POFUT2−/−) that controls O-fucose modification on thrombospondin repeats (TSR) displayed a wild type lineage development phenotype and normal Notch ligand binding ability. When examined for their in vivo hematopoietic reconstitution, blood cells derived from NOTCH1−/− or POFUT1−/− ES lines, but not POFUT2−/− ES line, showed enhanced granulocytic but suppressed T and B lymphoid lineage development. These results are consistent with our bone marrow transplantation findings that hematopoietic reconstitution by fucosylation-deficient marrow progenitor cells exhibited increased granulocytopoiesis while wild type or fucosylation-intact marrow cells have normal lineage distribution. Our observations indicate that Notch signaling maintains blood lineage homeostasis by promoting lymphoid lineage development and suppressing overt myeloid development. O-fucose modification of EGF repeats on Notch receptor is essential for this Notch-dependent control of blood lineage homeostasis as deficiency of fucose on Notch receptor results in enhanced myeloid development.

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Notch pathway activation targets AML-initiating cell homeostasis and differentiation

Journal of Experimental Medicine ◽

10.1084/jem.20121484 ◽

2013 ◽

Vol 210 (2) ◽

pp. 301-319 ◽

Cited By ~ 94

Author(s):

Camille Lobry ◽

Panagiotis Ntziachristos ◽

Delphine Ndiaye-Lobry ◽

Philmo Oh ◽

Luisa Cimmino ◽

...

Keyword(s):

Tumor Suppressor ◽

Notch Signaling ◽

Myeloproliferative Neoplasms ◽

Notch Pathway ◽

Notch Signaling Pathway ◽

Notch Receptor ◽

Pathway Activation ◽

Function Models

Notch signaling pathway activation is known to contribute to the pathogenesis of a spectrum of human malignancies, including T cell leukemia. However, recent studies have implicated the Notch pathway as a tumor suppressor in myeloproliferative neoplasms and several solid tumors. Here we report a novel tumor suppressor role for Notch signaling in acute myeloid leukemia (AML) and demonstrate that Notch pathway activation could represent a therapeutic strategy in this disease. We show that Notch signaling is silenced in human AML samples, as well as in AML-initiating cells in an animal model of the disease. In vivo activation of Notch signaling using genetic Notch gain of function models or in vitro using synthetic Notch ligand induces rapid cell cycle arrest, differentiation, and apoptosis of AML-initiating cells. Moreover, we demonstrate that Notch inactivation cooperates in vivo with loss of the myeloid tumor suppressor Tet2 to induce AML-like disease. These data demonstrate a novel tumor suppressor role for Notch signaling in AML and elucidate the potential therapeutic use of Notch receptor agonists in the treatment of this devastating leukemia.

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PPARγ Regulates the Anti-Inflammatory Effects of Carbon Monoxide on Macrophages: A Gene Profiling Study.

Blood ◽

10.1182/blood.v104.11.3445.3445 ◽

2004 ◽

Vol 104 (11) ◽

pp. 3445-3445

Author(s):

Martin Bilban ◽

Sherrie L. Otterbein ◽

Emeka Ifedigbo ◽

Keiji Enjyoji ◽

Anny Usheva ◽

...

Keyword(s):

Carbon Monoxide ◽

Inflammatory Response ◽

Acute Phase Proteins ◽

Expression Patterns ◽

Endotoxic Shock ◽

Gene Profiling ◽

In Vivo Experiments ◽

Anti Inflammatory

Abstract Carbon monoxide (CO) at low concentrations has generated recent interest due to its ability to modulate the inflammatory response associated with chronic graft rejection, vascular injury and septic shock. Both in vivo and in vitro CO can inhibit the expression of pro-inflammatory genes such as TNFα in macrophages while simultaneously increasing the expression of the anti-inflammatory cytokine IL-10. The mechanisms by which this occurs are still unclear. To better understand the mechanisms underlying the effects of CO, we employed the Affymetrix GeneChip technology to evaluate the time-dependent expression patterns of >12,000 genes in macrophages stimulated with bacterial endotoxin (LPS) in the presence or absence of a low concentration of CO previously demonstrated to evoke an anti-inflammatory response. We were particularly interested whether CO would, by itself, modulate in a specific manner the expression of proteins that might explain the anti-inflammatory effects observed following subsequent administration of endotoxin. RAW 264.7 murine macrophages were grown to 75% confluency and then exposed to CO (250 ppm) for 3 hr prior to administration of LPS (10 ng/ml). At 0, 15, 30, 60, 120 and 240 min thereafter, total RNA was isolated by standard methods and the RNA was then labeled and hybridized to U74Av2 GeneChips. Of >12,000 genes assessed, 116 of 270 that were LPS-responsive were affected by CO treatment. CO inhibited the majority of LPS-induced pro-inflammatory cytokines and acute phase proteins including expression of early growth response-1 (Egr-1), a transcription factor that serves as a central intermediary regulating many genes. Egr-1 was nearly completely inhibited by CO as was Egr-1-dependent expression of tissue factor (TF) and PAI-1. Treatment of cells with CO alone led to a rapid early increase in PPARγ, the expression of which was essential for the anti-inflammatory effects of CO. Inhibition of PPARγ using the selective chemical inhibitor GW9662 reversed the CO inhibitory effects on LPS-induced Egr-1 and TF expression. Correlative in vivo experiments in mice showed that CO pre-treatment blocked endotoxin-induced Egr-1 expression and decreased markers of lung inflammmation the effects of which were also lost with inhibition of PPARγ. Our analyses of gene expression patterns has led to the first molecular understanding of how treatment with CO, in this case by inducing PPARγ, blocks the pro-inflammatory response. These experiments provide novel insights into the mechanisms and pathophysiology of endotoxic shock and identify cellular targets by which CO mediates these cytoprotective effects.

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Notch: From fly wings to human hematological tumors

Archive of oncology ◽

10.2298/aoo0904072m ◽

2009 ◽

Vol 17 (3-4) ◽

pp. 72-77

Author(s):

Leonardo Mirandola ◽

Sara Larocca ◽

Katia Rea ◽

Giovanni Palma ◽

Paola Comi ◽

...

Keyword(s):

Notch Signaling ◽

Biological Effects ◽

Lymphoblastic Leukemia ◽

Fruit Fly ◽

Tumor Burden ◽

Notch Receptor ◽

Stem Cell Maintenance ◽

Associated Proteins

Notch history begins in 1919 with Thomas Hunt Morgan studies on fruit fly mutants. From then, this gene aroused lively interest in the scientific community since it is involved in a wide variety of processes, including morphogenesis, tissue homeostasis, and stem cell maintenance. Deregulation of Notch signaling characterizes several human tumors. Hematopoietic system is affected by mutations of Notch receptors, Notch ligands, and proteins controlling their stability. Approximately 60% T acute lymphoblastic leukemia (T-ALL) patients carry activating Notch1 mutations prompting blasts growth. In addition, multiple myeloma is characterized by Notch signaling hyper-activation due to an abnormal expression of the Jagged2 ligand; this affects not only myeloma cells, but also their interaction with bone marrow microenvironment, influencing tumor burden and bone disease. These findings make Notch a rational target of a therapeutic approach. Inhibitors of the Notch activating enzyme, ?-Secretase, have been successfully used in vitro and in vivo and are currently under clinical trials for T-ALL and breast cancer. Yet a wide use of these inhibitors is prevented by frequently occurring drug resistance. To elucidate the mechanism underlying this phenomenon, a number of pathways have been identified mediating Notch biological effects: AKT and c-Myc are frequently deregulated in leukemic patients and account for resistance to ?-Secretase inhibitors by acting downstream Notch receptor. Therefore, the interaction of Notch with other cancer-associated proteins should be clarified to predict the biological outcome of a Notch targeted therapy and possibly, to exploit combined treatments against the key deregulated elements in Notch-associated cancers.

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SEL-10 Is an Inhibitor of Notch Signaling That Targets Notch for Ubiquitin-Mediated Protein Degradation

Molecular and Cellular Biology ◽

10.1128/mcb.21.21.7403-7415.2001 ◽

2001 ◽

Vol 21 (21) ◽

pp. 7403-7415 ◽

Cited By ~ 226

Author(s):

Guangyu Wu ◽

Svetlana Lyapina ◽

Indranil Das ◽

Jinhe Li ◽

Mark Gurney ◽

...

Keyword(s):

Notch Signaling ◽

Protein Degradation ◽

Notch Receptor ◽

Dominant Negative ◽

Intracellular Domain ◽

Notch Receptors ◽

Intracellular Domains ◽

Wd40 Repeats ◽

In Cells

ABSTRACT Notch receptors and their ligands play important roles in both normal animal development and pathogenesis. We show here that the F-box/WD40 repeat protein SEL-10 negatively regulates Notch receptor activity by targeting the intracellular domain of Notch receptors for ubiquitin-mediated protein degradation. Blocking of endogenous SEL-10 activity was done by expression of a dominant-negative form containing only the WD40 repeats. In the case of Notch1, this block leads to an increase in Notch signaling stimulated by either an activated form of the Notch1 receptor or Jagged1-induced signaling through Notch1. Expression of dominant-negative SEL-10 leads to stabilization of the intracellular domain of Notch1. The Notch4 intracellular domain bound to SEL-10, but its activity was not increased as a result of dominant-negative SEL-10 expression. SEL-10 bound Notch4 via the WD40 repeats and bound preferentially to a phosphorylated form of Notch4 in cells. We mapped the region of Notch4 essential for SEL-10 binding to the C-terminal region downstream of the ankyrin repeats. When this C-terminal fragment of Notch4 was expressed in cells, it was highly labile but could be stabilized by the expression of dominant-negative SEL-10. Ubiquitination of Notch1 and Notch4 intracellular domains in vitro was dependent on SEL-10. Although SEL-10 interacts with the intracellular domains of both Notch1 and Notch4, these proteins respond differently to interference with SEL-10 function. Thus, SEL-10 functions to promote the ubiquitination of Notch proteins; however, the fates of these proteins may differ.

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The Notch signaling pathway promotes basophil responses during helminth-induced type 2 inflammation

Journal of Experimental Medicine ◽

10.1084/jem.20180131 ◽

2019 ◽

Vol 216 (6) ◽

pp. 1268-1279 ◽

Cited By ~ 5

Author(s):

Lauren M. Webb ◽

Oyebola O. Oyesola ◽

Simon P. Früh ◽

Elena Kamynina ◽

Katherine M. Still ◽

...

Keyword(s):

Gene Expression ◽

Notch Signaling ◽

Gene Expression Regulation ◽

Notch Receptor ◽

Receptor Expression ◽

Helminth Parasites ◽

Trichuris Muris ◽

Type 2 Inflammation

Type 2 inflammation drives the clearance of gastrointestinal helminth parasites, which infect over two billion people worldwide. Basophils are innate immune cells that support host-protective type 2 inflammation during murine infection with the helminth Trichuris muris. However, the mechanisms required for basophil function and gene expression regulation in this context remain unclear. We show that during T. muris infection, basophils localized to the intestine and up-regulated Notch receptor expression, rendering them sensitive to Notch signals that rapidly regulate gene expression programs. In vitro, Notch inhibition limited basophil cytokine production in response to cytokine stimulation. Basophil-intrinsic Notch signaling was required for T. muris–elicited changes in genome-wide basophil transcriptional programs. Mice lacking basophil-intrinsic functional Notch signaling had impaired worm clearance, decreased intestinal type 2 inflammation, altered basophil localization in the intestine, and decreased CD4+ T helper 2 cell responses following infection. These findings demonstrate that Notch is required for basophil gene expression and effector function associated with helminth expulsion during type 2 inflammation.

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Ezh1 Inhibits Commitment to Hemogenic Fate and HSPC Formation during Vertebrate Development

Blood ◽

10.1182/blood-2019-131464 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3710-3710

Author(s):

Rebecca Soto ◽

Edroaldo Lummertz da rocha ◽

Linda T Vo ◽

Mariam Hachimi ◽

Jenna M Frame ◽

...

Keyword(s):

Notch Signaling ◽

Notch Pathway ◽

Notch Receptor ◽

Gene Set Enrichment Analysis ◽

Patient Specific ◽

Sequencing Data ◽

Zebrafish Model ◽

Hematopoietic Stem

Understanding how hematopoietic stem cells (HSCs) are specified from mesodermal precursors is essential to the goal of generating patient-specific HSCs capable of multi-potent long-term function. HSCs are born from hemogenic endothelium in select arterial niches during embryonic development through a transdifferentiation process turned endothelial-to-hematopoietic transistion (EHT). Despite increasing efforts to recapitulate this process in vitro, current differentiation protocols largely fail to produce long-lived multi-lineage progenitors from human induced pluripotent stem cell (iPSC) sources. Recently, an in vitro loss-of-function screen in human hematopoietic progenitors identified the Polycomb group protein, Enhancer of Zeste Homolog 1 (EZH1), as a regulator of definitive hematopoietic commitment, as assayed by acquisition of lymphoid competence. To determine the mechanism by which Ezh1 regulates HSPC fate in vivo we employed functional knockdown and epistasis investigations using the zebrafish model. Morpholino-mediated knockdown of ezh1 promoted expression of the conserved HSC markers runx1 and c-myb in the ventral wall of the dorsal aorta (VDA) at 36 hours post fertilization (hpf), as assessed by whole mount in situ hybridization (WISH); additionally, expression of the lymphoid marker rag1 was found to be enhanced at 120 hpf, as assayed by WISH and fluorescent activated cell sorting (FACS), in line with our in vitro observations. An impact on HSPCs was confirmed and quantified by qPCR for runx1 (**p < 0.01) and FACS using the CD41:GFP reporter line (**p < 0.01), indicating significantly increased HSPC number. Importantly, this enhancement in HSPC production had no effect on gross vascular morphology of the niche as determined by confocal microscopy for flk1. Assessment of arterial versus venous fate indicated that while the latter was unchanged in morphant embryos, expression of the arterial markers, epbrinb2a, dll4, dlc and tbx20, was strongly reduced by WISH and qPCR (**p < 0.01, *p < 0.05, **p < 0.01, and **** p < 0.001, respectively). In contrast, markers of hemogenic commitment, gata2b, and scl/flk1, were significantly increased, suggesting that loss of ezh1 enhanced hematopoietic potential at the expense of maintaining arterial fate. Profiling of single-cell RNA-sequencing data obtained from sorted populations of E10.5 mouse embryos revealed EZH1 to be more highly expressed in cells undergoing the endothelial-to-hematopoietic transition, consistent with a role of EZH1 in regulating arterial verses hematopoietic fate. Gene set enrichment analysis (GSEA) from our prior in vitro studies revealed the Notch pathway to be significantly altered following EZH1 knockdown. As Notch signaling has been implicated in both arterial specification and HSC emergence, we next examined the potential role of Notch signaling in ezh1 knockdown-mediated HSPC expansion. Consistent with a hypothesized interaction, differential regulation of Notch ligands and receptors was observed in ezh1 morphants compared to wild-type siblings; specifically, expression of arterial ligands, dll4 and dlc were decreased, while hematopoietic ligands and receptors, jag1a and notch1a were enhanced. Notably, the effect on Notch signaling was specific to ezh1 knockdown, as ezh2 loss shows a distinct pattern and temporal impact, reducing HSC production rather than enhancing it, consistent with recent reports. The strong conservation of ezh1-mediated regulation of HSC number, and our identification of its mechanistic role at the level of Notch receptor/ligand interactions, position zebrafish as a platform to identify chemical mediators that can be used to regulate ezh1 function during in vitro differentiation to unlock multi-lineage HSC commitment of human iPSC for therapeutic application. Disclosures Daley: Epizyme, Inc: Other: Equity & Consulting Fees; 28/7 Therapeutics: Other: Equity & Consulting Fees.

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Expression of Hes-1 (Notch-1 Effector) during “In Vitro” Normal Erythroid Differentiation.

Blood ◽

10.1182/blood.v106.11.4287.4287 ◽

2005 ◽

Vol 106 (11) ◽

pp. 4287-4287

Author(s):

Maria D. Cappellini ◽

Ilaria V. Libani ◽

Elisabetta Calzavara ◽

Luisa Ronzoni ◽

Raffaella Chiaramonte ◽

...

Keyword(s):

Stem Cells ◽

Notch Signaling ◽

Cell Fate ◽

Notch Pathway ◽

Erythroid Differentiation ◽

Notch Receptor ◽

Erythroid Progenitors ◽

Notch 1 ◽

Multipotent Stem Cells

Abstract Hematopoiesis involves highly regulated proliferation and differentiation during which a small number of multipotent stem cells give rise to differentiated progenies. In several developmental systems stem cells fate is influenced by soluble molecules acting via cell-cell interaction, including those mediate by the Notch receptor family. Members of Notch family transmembrane receptors are found on primitive hematopoietic precursors, suggesting a role for Notch signaling in mammalian blood cells development. Notch signaling regulates cell fate controlling asymmetric cell division during stem/progenitor cell differentiation. A previous study on K562 cell line showed that Notch signaling inhibits erythroid/megakaryocytic development by suppressing GATA-1 activity. Furthermore there are evidences that Notch is expressed in early murine erythroid precursors. Probably Notch signaling in these uncommittted precursors may lead to enhanced survival, preserving multilineage potential. The role of Notch pathway during human adult erytropiesis has not been described. The aim of this study is to investigate the modulation of Notch activity during “in vitro” human erythropiesis. Human CD34+ from perpheral blood of normal adult subjects were differentiate “in vitro” for two weeks by the addiction of IL-3, SCF and Epo. This method of colture reproduces all stages of adult erythropoiesis. We analized the modulation of the expression of Notch-1, of its effector Hes-1 and of several erythoid specific genes, at different stages of differentiation using real time PCR. Our analysis shows that Hes-1 expression, which indicates the activation of Notch-1 pathway, is very high in the early steps of differentiation (BFU-E, CFU-E) while in the late stages rapidly decreases to undetectable levels. The Notch-1 gene expression doesn’t seem to be modulated way, but we didn’t invstigate the protein levels yet. These data suggest that Notch pathway is involved in the early stages of erythroid differentiation where it may enhance erythroid progenitors survival up to CFU-E, as hypothisized in mouse model, preventing them from apoptotic stimuli and promoting their proliferation. Involvement of Notch-1 signaling in preventing erythroid progenitors from apoptosis during erythroid differentiation could be important in some erythropoietic disorders such as b-Thalassemia syndromes or diserythropoietic anemias.

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