Discovery of two GLP-1/Notch target genes that account for the role of GLP-1/Notch signaling in stem cell maintenance

A stem cell’s immediate microenvironment creates an essential “niche” to maintain stem cell self-renewal. Many niches and their intercellular signaling pathways are known, but for the most part, the key downstream targets of niche signaling remain elusive. Here, we report the discovery of two GLP-1/Notch target genes, lst-1 (lateral signaling target) and sygl-1 (synthetic Glp), that function redundantly to maintain germ-line stem cells (GSCs) in the nematode Caenorhabditis elegans. Whereas lst-1 and sygl-1 single mutants appear normal, lst-1 sygl-1 double mutants are phenotypically indistinguishable from glp-1/Notch mutants. Multiple lines of evidence demonstrate that GLP-1/Notch signaling activates lst-1 and sygl-1 expression in GSCs within the niche. Therefore, these two genes fully account for the role of GLP-1/Notch signaling in GSC maintenance. Importantly, lst-1 and sygl-1 are not required for GLP-1/Notch signaling per se. We conclude that lst-1 and sygl-1 forge a critical link between Notch signaling and GSC maintenance.

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Faculty Opinions recommendation of Discovery of two GLP-1/Notch target genes that account for the role of GLP-1/Notch signaling in stem cell maintenance.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.718287283.793493719 ◽

2014 ◽

Author(s):

Morris Maduro

Keyword(s):

Stem Cell ◽

Notch Signaling ◽

Target Genes ◽

Stem Cell Maintenance ◽

Cell Maintenance

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Faculty Opinions recommendation of Discovery of two GLP-1/Notch target genes that account for the role of GLP-1/Notch signaling in stem cell maintenance.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.718287283.793492360 ◽

2014 ◽

Author(s):

Robert K Herman

Keyword(s):

Stem Cell ◽

Notch Signaling ◽

Target Genes ◽

Stem Cell Maintenance ◽

Cell Maintenance

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Progression from a stem cell–like state to early differentiation in the C. elegans germ line

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.0912704107 ◽

2010 ◽

Vol 107 (5) ◽

pp. 2048-2053 ◽

Cited By ~ 67

Author(s):

Olivier Cinquin ◽

Sarah L. Crittenden ◽

Dyan E. Morgan ◽

Judith Kimble

Keyword(s):

Stem Cell ◽

Germ Cells ◽

Cell Biology ◽

Germ Line ◽

Stem Cell Biology ◽

Self Renewal ◽

Early Differentiation ◽

C Elegans ◽

Stem Cell Maintenance ◽

System A

Controls of stem cell maintenance and early differentiation are known in several systems. However, the progression from stem cell self-renewal to overt signs of early differentiation is a poorly understood but important problem in stem cell biology. The Caenorhabditis elegans germ line provides a genetically defined model for studying that progression. In this system, a single-celled mesenchymal niche, the distal tip cell (DTC), employs GLP-1/Notch signaling and an RNA regulatory network to balance self-renewal and early differentiation within the “mitotic region,” which continuously self-renews while generating new gametes. Here, we investigate germ cells in the mitotic region for their capacity to differentiate and their state of maturation. Two distinct pools emerge. The “distal pool” is maintained by the DTC in an essentially uniform and immature or “stem cell–like” state; the “proximal pool,” by contrast, contains cells that are maturing toward early differentiation and are likely transit-amplifying cells. A rough estimate of pool sizes is 30–70 germ cells in the distal immature pool and ≈150 in the proximal transit-amplifying pool. We present a simple model for how the network underlying the switch between self-renewal and early differentiation may be acting in these two pools. According to our model, the self-renewal mode of the network maintains the distal pool in an immature state, whereas the transition between self-renewal and early differentiation modes of the network underlies the graded maturation of germ cells in the proximal pool. We discuss implications of this model for controls of stem cells more broadly.

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The F-Box Protein NIPA Limits Hematopoietic Stem Cell Survival and Transplantation Efficiency

Blood ◽

10.1182/blood.v122.21.1175.1175 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1175-1175

Author(s):

Stefanie Kreutmair ◽

Anna Lena Illert ◽

Rouzanna Istvanffy ◽

Christina Eckl ◽

Christian Peschel ◽

...

Keyword(s):

Cell Cycle ◽

Stem Cell ◽

Spleen Cells ◽

Self Renewal ◽

Hematopoietic Stem ◽

Mitotic Entry ◽

Stem Cell Maintenance ◽

F Box Protein

Abstract Hematopoietic stem cells (HSCs) are characterized by their ability to self-renewal and multilineage differentiation. Since mostly HSCs exist in a quiescent state re-entry into cell cycle is essential for their regeneration and differentiation. We previously characterized NIPA (Nuclear Interaction Partner of ALK) as a F-Box protein that defines an oscillating ubiquitin E3 ligase and contributes to the timing of mitotic entry. To examine the function of NIPA in vivo, we generated Nipa deficient animals, which are viable but sterile due to a defect in testis stem cell maintenance. To further characterize the role of NIPA in stem cell maintenance and self-renewal we investigated hematopoiesis in Nipa deficient animals. FACS analyses of spleen cells and bone marrow (BM) showed differences in Leucocyte subpopulations. Measuring the CD4 and CD8 positivity within all Thy1.2+ cells, the balance in NIPA-/- T-lymphocytes is destabilised in favour of CD4 positive cells. Besides CD43/CD19 positive as well as CD43/B220 positive cells within all leukocytes are increased in NIPA deficient spleen cells. Analysing more primitive cells, FACS data of bone marrow showed significantly decreased numbers of Lin-Sca1+cKit+ (LSK) cells in NIPA-/- mice (age > 20 month), where LSKs were reduced to 40% of wildtype (wt) littermates (p=0,0171). Additionally, in such older NIPA-/- mice, only half the number of multipotent myeloid progenitors were detected in comparison to wt mice. To examine efficient response of stem cells to myeloid depression, mice were treated with 5-FU four days before BM harvest. We found that in NIPA-/- mice, both the number of myeloid progenitors as well as the number of LSKs were severely reduced compared to those in wt levels after 5-FU treatment (p<0.001). Interestingly, the reduction of progenitors and LSK cells was not dependent on age of the NIPA ko mice, suggesting a role for NIPA in stem cell activation or regeneration. This statement was studied in vitro by methylcellulose assays with 10 000 BM cells seeded in methylcellulose with cytokines and replated for three times after 10 days. Nipa deficient hematopoietic progenitors showed a reduced ability to proliferate and differentiate into colonies compared to their controls with an increasing difference after each replating (p(third replating) < 0.0001). Dynamic cell cycle analysis of seeded BM cells with BRDU and PI uncovered delayed cell cycle progress and mitotic entry in NIPA-/- BM cells in contrast to wt BM cells. Using competitive BM transplantation assay we investigated the role of NIPA for hematopoietic reconstitution in vivo. These experiments showed that NIPA-/- BM cells were severely deficient in hematopoietic recovery as recipient mice of NIPA-/- BM cells showed only half the amount of donor-derived peripheral blood cells in contrast to recipient mice of wt BM cells after 4, 11, 17 and over 23 weeks after transplantation. Furthermore NIPA-/- cells contributed only 7% in BM of transplanted mice 6 month after transplantation compared to 33% in recipients transplanted with wt BM cells (p<0.005). To further explore this defect in hematopoietic repopulation capacity and apply to more primitive progenitors serial transplantation assays were conducted with LSK cells transplanted together with support BM cells. Taken together our results demonstrate a critical role of NIPA in regulating the primitive hematopoietic compartment as a regulator of self-renewal, cycle capacity and HSC expansion. Disclosures: No relevant conflicts of interest to declare.

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Reduction of Derlin activity suppresses Notch-dependent tumours in the C. elegans germ line

PLoS Genetics ◽

10.1371/journal.pgen.1009687 ◽

2021 ◽

Vol 17 (9) ◽

pp. e1009687

Author(s):

Ramya Singh ◽

Ryan B. Smit ◽

Xin Wang ◽

Chris Wang ◽

Hilary Racher ◽

...

Keyword(s):

Stem Cell ◽

Er Stress ◽

Notch Signaling ◽

Germ Line ◽

Notch Signaling Pathway ◽

Notch Receptor ◽

Loss Of Function ◽

Gain Of Function ◽

Self Renewal ◽

Proliferation Suppression

Regulating the balance between self-renewal (proliferation) and differentiation is key to the long-term functioning of all stem cell pools. In the Caenorhabditis elegans germline, the primary signal controlling this balance is the conserved Notch signaling pathway. Gain-of-function mutations in the GLP-1/Notch receptor cause increased stem cell self-renewal, resulting in a tumour of proliferating germline stem cells. Notch gain-of-function mutations activate the receptor, even in the presence of little or no ligand, and have been associated with many human diseases, including cancers. We demonstrate that reduction in CUP-2 and DER-2 function, which are Derlin family proteins that function in endoplasmic reticulum-associated degradation (ERAD), suppresses the C. elegans germline over-proliferation phenotype associated with glp-1(gain-of-function) mutations. We further demonstrate that their reduction does not suppress other mutations that cause over-proliferation, suggesting that over-proliferation suppression due to loss of Derlin activity is specific to glp-1/Notch (gain-of-function) mutations. Reduction of CUP-2 Derlin activity reduces the expression of a read-out of GLP-1/Notch signaling, suggesting that the suppression of over-proliferation in Derlin loss-of-function mutants is due to a reduction in the activity of the mutated GLP-1/Notch(GF) receptor. Over-proliferation suppression in cup-2 mutants is only seen when the Unfolded Protein Response (UPR) is functioning properly, suggesting that the suppression, and reduction in GLP-1/Notch signaling levels, observed in Derlin mutants may be the result of activation of the UPR. Chemically inducing ER stress also suppress glp-1(gf) over-proliferation but not other mutations that cause over-proliferation. Therefore, ER stress and activation of the UPR may help correct for increased GLP-1/Notch signaling levels, and associated over-proliferation, in the C. elegans germline.

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Ing4 Regulates Hematopoiesis through Suppression of NF-Kb

Blood ◽

10.1182/blood-2018-99-120033 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 642-642

Author(s):

Zanshe Thompson ◽

Vera Binder ◽

Michelle Ammerman ◽

Ellen Durand ◽

Leonard I. Zon ◽

...

Keyword(s):

Gene Expression ◽

Stem Cell ◽

Progenitor Cells ◽

Progenitor Cell ◽

Target Gene ◽

Target Genes ◽

Zebrafish Embryos ◽

Self Renewal ◽

Hematopoietic Stem

Abstract Hematopoiesis is tightly regulated by a network of transcription factors and complexes that are required for the maintenance and development of HSCs. In a screen for epigenetic regulators of hematopoiesis in zebrafish, we identified a requirement of the tumor suppressor protein, Ing4, in hematopoietic stem and progenitor cell (HSPC) specification. Though the Ing4 mechanism of action remains poorly characterized, it has been shown to promote stem-like cell characteristics in malignant cells. This activity is, in part, due to the inhibitory role of Ing4 in the NF-kB signaling pathway. In the absence of Ing4, there is a significant increase in NF-kB target gene expression. As in the zebrafish, we have identified a requirement for Ing4 in murine hematopoiesis, where Ing4 deficiency impairs hematopoietic stem cell (HSC) function, but enhances multipotent progenitor cell (MPP) regenerative capacity. Given the role of Ing4 in both normal hematopoiesis and cancer, this gene likely has a critical role in regulation of stem cell self-renewal and maintenance. To define the role of Ing4 in zebrafish HSPCs, we designed an anti-sense morpholino oligo against Ing4 and injected into zebrafish embryos at the single cell stage. Embryos were screened using in situ hybridizations for c-myb and runx1 expression, which are highly expressed in the aorta, gonad, mesonephros (AGM) region in the developing zebrafish embryo. We found that Ing4-deficient zebrafish embryos lose >90% of runx1+/c-myb+ cells in the AGM, demonstrating a lack of HSPC specification. Analysis of ephrinB2 expression showed normal specification of the aorta in Ing4 morphant embryos, signifying that the step of HSPC specification is affected in the absence of Ing4. Overexpression of human Ing4 in zebrafish embryos resulted in increased HSPC marker staining suggesting that normal expression levels of Ing4 are required for HSC specification. As Ing4 is an epigenetic regulator that binds specific gene loci, we examined the chromatin occupancy of Ing4 in human peripheral blood CD34+ progenitor cells. Using ChIP-seq for Ing4 in CD34+ cells, we show that Ing4 binds to many regulators of blood development including MYB, LMO2, RUNX1, and IKAROS, and several NF-kB target genes. In other tissues, Ing4 negatively regulates NF-kB, so accordingly, loss of Ing4 results in an overabundance of NF-kB signaling. To address NF-kB target gene expression in Ing4-deficient zebrafish embryos, we performed qPCR analysis at 36hpf. These assays showed an increase in the expression of a subset of NF-kB target genes (IKBKE, IL-19, IL-1b, IL-20R). Simultaneous knockdown of both Ing4 and RelA, through combined morpholino injections against both factors, resulted in the rescue of HSC marker expression in the aorta. These results suggest that NF-kB inhibition could remediate the loss of Ing4. A mouse model for Ing4 deficiency was generated to further evaluate the role of Ing4 in differentiated immune cells. These mice are developmentally normal but are hypersensitive to stimulation with LPS. Interestingly, we found that Ing4-/- mice showed skewed hematopoiesis resulting in an increase in the number of short term-HSCs (ST-HSCs) (11.4% vs 31.7%) and a dramatic decrease in multipotent progenitor cells (MPPs) (47.9% vs 19.3%) along with concurrent modest increase in the population of long-term HSCs (LT-HSCs) (2.4% vs 5.5%). Additionally, there were alterations in stress hematopoiesis following hematopoietic stem cell transplant. Sorted LT-HSCs fail to engraft, suggesting an evolutionarily conserved requirement for Ing4 in HSCs. Surprisingly, competitive transplantation assay with Ing4-defecient MPPs versus wild-type showed dramatic increase in peripheral blood multilineage chimerism up to 9 months post-transplantation (19% vs. 59%). This lends to the hypothesis that Ing4 deficient MPPs gain self-renewal capabilities. Based on these exciting findings, we hypothesize that Ing4 normally functions as a critical suppressor for genes required for self-renewal and developmental potency in MPPs. Overall, our findings suggest that Ing4 plays a crucial role in the regulation of hematopoiesis and provides key tools for further identification and characterization of Ing4 pathways and functions. Disclosures No relevant conflicts of interest to declare.

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Ing4 Suppresses Quiescence and Inflammation in Hematopoietic Stem Cells

Blood ◽

10.1182/blood-2020-143388 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 16-16

Author(s):

Zanshe Thompson ◽

Melanie Rodriguez ◽

Georgina Anderson ◽

Seth Gabriel ◽

Vera Binder ◽

...

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Peripheral Blood ◽

Target Genes ◽

Publicly Traded ◽

Private Company ◽

Self Renewal ◽

Hematopoietic Stem ◽

Marker Expression

Hematopoietic stem and progenitor cell (HSPC) development and maintenance is regulated through a complex regulatory network. In a screen of epigenetic regulators of hematopoiesis in zebrafish, we identified a requirement for the tumor suppressor protein, Inhibitor of growth 4 (Ing4) in HSPC specification. Ing4 acts to regulate transcription through interactions with transcription factors, including HIF, NF-kB, and p53. It is often mis-expressed in many human cancers and has been shown to promote stem cell-like characteristics in malignant cells, in part, due to the inhibitory role of Ing4 in the NF-kB signaling pathway. The transcription factor NF-kB is a regulator of inflammatory response and serves an important role in embryonic HSPC emergence, survival, differentiation and proliferation. The Ing4 protein binds to the p65/RelA subunit of NF-kB, inhibiting DNA binding and suppressing NF-kB cytokines and inflammatory pathways. In the absence of Ing4 there is an overexpression of NF-kB target genes that have inhibitory effects on hematopoietic programming. Given the regulatory role of Ing4 in both hematopoiesis and cancer, it is likely critical to the regulation of stem cell self-renewal, maintenance and specialization. To better define the role of Ing4 on hematopoiesis we use two Ing4 loss-of-function models: zebrafish and mouse. For the zebrafish model of Ing4 deficiency, Ing4-deficient zebrafish embryos lose >90% of runx1+/c-myb+ cells in the aorta, gonad, mesonephros (AGM) region of the developing zebrafish embryo, demonstrating a lack of HSPC specification. 36 hours post fertilization (hpf) Ing4 morphants display increased expression of NF-kB target genes when Ing4 is absent. Genetic epistasis experiments performed to block translation of RelA, IL-1b, and additional NF-kB target gene mRNA revealed recovered HSC marker expression in the aorta. To discover small molecule inhibitors that would mimic these effects, we conducted an in vivo chemical screen of NF-kB pathway inhibitors assessing their ability to rescue HSC specification in Ing4 morphant zebrafish. Ing4 morphants treated with NF-kB inhibitors had reduced NF-kB cytokine expression, as well as a dose-dependent rescue of HSC marker expression in the aorta. These results suggest that NF-kB inhibition could remediate the effects of Ing4 loss on hematopoiesis. To more thoroughly profile the effects of Ing4 loss on HSC specification and the bone marrow niche, an Ing4-/-mouse model was used. These mice are developmentally normal but are hypersensitive to stimulation with LPS due to increased inflammatory signaling. Peripheral blood analysis reveals an increase in Mac-1 cells in the Ing4-/- mouse. Ing4-/- bone marrow progenitors are skewed toward granulocyte-myocyte progenitor cells (GMPs) lending to the shift in cell populations present in the peripheral blood. Ing4 loss further disrupts the mouse hematopoietic program resulting in a dramatic increase in the number of short term-HSCs (ST-HSC) (WT: 11.4%, Null: 31.7%), a modest increase in long term-HSCs (LT-HSC) (WT: 2.4%, Null: 5.52%), and a dramatic decrease in multipotent progenitors (MPPs) (WT: 47.9%, Null: 19.3%). We also found significant alterations in stress hematopoiesis following competitive HSC transplant where sorted Ing4-/- LT-HSCs failed to engraft. Following myeloablative insult, we found no significant change in Ing4-/- LT-HSC (-1.18%) when compared with ST-HSC (-14.43%) indicating reduced sensitivity to 5-FU ablation in the Ing4-/- LT-HSC group. Cell cycle analysis identified 92.9% of Ing4-/- LT-HSCs are in G0 compared to 76.2% of wildtype LT-HSCs. ST-HSCs were also more quiescent with 27% of Ing4-/- ST-HSCs in G0 compared to 11.1% of wildtype ST-HSCs. Previously published work reports hyper proliferative HSCs that exhibit loss of quiescence as a result of proinflammatory NF-kB signaling. We believe that the interaction between Ing4 and the HIF-1a pathway may play a role in the observed phenotype of Ing4-/- LT-HSCs resulting in increased quiescence and disruption of the balance between self-renewal and differentiation critical to reconstitution of the hematopoietic compartment. Overall, our findings suggest that the regulatory effects of Ing4 play a crucial role in hematopoiesis and provides key tools for further identification and characterization of Ing4 pathways and functions. Disclosures Zon: CAMP4 Therapeutics: Current equity holder in private company, Other: Founder; Fate Therapeutics: Current equity holder in publicly-traded company, Other: Founder; Scholar Rock: Current equity holder in publicly-traded company, Other: Founder; Amagma Therapeutics: Current equity holder in private company, Other: Founder; Cellarity: Consultancy; Celularity: Consultancy.

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Reduction of Derlin activity suppresses Notch-dependent tumours in the C. elegans germ line

10.1101/2021.07.02.450841 ◽

2021 ◽

Author(s):

Ramya Singh ◽

Ryan Smit ◽

Xin Wang ◽

Chris Wang ◽

Hilary Racher ◽

...

Keyword(s):

Stem Cell ◽

Er Stress ◽

Notch Signaling ◽

Germ Line ◽

Notch Receptor ◽

Loss Of Function ◽

Gain Of Function ◽

Self Renewal ◽

C Elegans ◽

Proliferation Suppression

Regulating the balance between self-renewal (proliferation) and differentiation is key to the long-term functioning of all stem cell pools. In the Caenorhabditis elegans germline, the primary signal controlling this balance is the conserved Notch signaling pathway. Gain-of-function mutations in the GLP-1/Notch receptor cause increased stem cell self-renewal, resulting in a tumor of proliferating germline stem cells. Notch gain-of-function mutations activate the receptor, even in the presence of little or no ligand, and have been associated with many human diseases, including cancers. We demonstrate that reduction in CUP-2 and DER-2 function, which are Derlin family proteins that function in endoplasmic-reticulum-associated degradation (ERAD), suppresses the C. elegans germline over-proliferation phenotype associated with glp-1(gain-of-function) mutations. We further demonstrate that their reduction does not suppress other mutations that cause over-proliferation, suggesting that over-proliferation suppression due to loss of Derlin activity is specific to glp-1/Notch (gain-of-function) mutations. Reduction of CUP-2 Derlin activity reduces the expression of a read-out of GLP-1/Notch signaling, suggesting that the suppression of over-proliferation in Derlin loss-of-function mutants is due to a reduction in the activity of the mutated GLP-1/Notch(GF) receptor. Over-proliferation suppression in cup-2 mutants is only seen when the Unfolded Protein Response (UPR) is functioning properly, suggesting that the suppression, and reduction in GLP-1/Notch signaling levels, observed in Derlin mutants may be the result of activation of the UPR. Chemically inducing ER stress also suppress glp-1(gf) over-proliferation but not other mutations that cause over-proliferation. Therefore, ER stress and activation of the UPR may help correct for increased GLP-1/Notch signaling levels, and associated over-proliferation, in the C. elegans germline.

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