Wnt signaling is involved in self-renewal and maintenance of hematopoietic stem cells (HSCs); however, the particular role of noncanonical Wnt signaling in regulating HSCs in vivo is largely unknown. ...Here, we show Flamingo (Fmi) and Frizzled (Fz) 8, members of noncanonical Wnt signaling, both express in and functionally maintain quiescent long-term HSCs. Fmi regulates Fz8 distribution at the interface between HSCs and N-cadherin+ osteoblasts (N-cad+OBs that enrich osteoprogenitors) in the niche. We further found that N-cad+OBs predominantly express noncanonical Wnt ligands and inhibitors of canonical Wnt signaling under homeostasis. Under stress, noncanonical Wnt signaling is attenuated and canonical Wnt signaling is enhanced in activation of HSCs. Mechanistically, noncanonical Wnt signaling mediated by Fz8 suppresses the Ca2+-NFAT- IFNγ pathway, directly or indirectly through the CDC42-CK1α complex and also antagonizes canonical Wnt signaling in HSCs. Taken together, our findings demonstrate that noncanonical Wnt signaling maintains quiescent long-term HSCs through Fmi and Fz8 interaction in the niche.
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► Flamingo regulates Frizzled8 between HSC and N-cadherin+ osteoblast (N-cad+OB) ► N-cad+OBs and HSCs maintain noncanonical Wnt signaling in homeostasis ► Flamingo and Frizzled8 functionally maintain quiescent long-term HSCs ► Noncanonical Wnt signaling suppresses NFAT pathway and canonical Wnt signaling
Niche-derived noncanonical Wnt signals promote the maintenance of quiescent long-term hematopoietic stem cells, unlike canonical Wnt signaling, which promotes stem cell activation.
The mammalian imprinted Dlk1-Gtl2 locus produces multiple non-coding RNAs (ncRNAs) from the maternally inherited allele, including the largest miRNA cluster in the mammalian genome. This locus has ...characterized functions in some types of stem cell, but its role in hematopoietic stem cells (HSCs) is unknown. Here, we show that the Dlk1-Gtl2 locus plays a critical role in preserving long-term repopulating HSCs (LT-HSCs). Through transcriptome profiling in 17 hematopoietic cell types, we found that ncRNAs expressed from the Dlk1-Gtl2 locus are predominantly enriched in fetal liver HSCs and the adult LT-HSC population and sustain long-term HSC functionality. Mechanistically, the miRNA mega-cluster within the Dlk1-Gtl2 locus suppresses the entire PI3K-mTOR pathway. This regulation in turn inhibits mitochondrial biogenesis and metabolic activity and protects LT-HSCs from excessive reactive oxygen species (ROS) production. Our data therefore show that the imprinted Dlk1-Gtl2 locus preserves LT-HSC function by restricting mitochondrial metabolism.
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•Transcriptome profiling reveals Gtl2-derived ncRNA enrichment in LT-HSCs•Loss of Dlk1-Gtl2 imprinting leads to functional defects in fetal liver HSCs•miRNAs of the Dlk1-Gtl2 locus suppress components of the entire PI3K-mTOR pathway•PI3K-mTOR inhibition restricts mitochondrial metabolism to preserve LT-HSC function
Qian and colleagues show that ncRNAs expressed from the imprinted locus Dlk1-Gtl2 maintain fetal liver and adult LT-HSCs through multiplexed inhibition of PI3K-mTOR signaling that in turn keeps mitochondrial biogenesis and metabolic activity in check.
Although it is established that some general transcription factors are inactivated at mitosis, many details of mitotic transcription inhibition (MTI) and its underlying mechanisms are largely ...unknown. We have identified mitotic transcriptional activation (MTA) as a key regulatory step to control transcription in mitosis for genes with transcriptionally engaged RNA polymerase II (Pol II) to activate and transcribe until the end of the gene to clear Pol II from mitotic chromatin, followed by global impairment of transcription reinitiation through MTI. Global nascent RNA sequencing and RNA fluorescence in situ hybridization demonstrate the existence of transcriptionally engaged Pol II in early mitosis. Both genetic and chemical inhibition of P-TEFb in mitosis lead to delays in the progression of cell division. Together, our study reveals a mechanism for MTA and MTI whereby transcriptionally engaged Pol II can progress into productive elongation and finish transcription to allow proper cellular division.
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•Mitotic transcription inhibition occurs in early mitosis•P-TEFb is required for mitotic transcriptional activation and release of paused Pol II•Nascent RNA-seq and RNA FISH reveal active transcription at the onset of mitosis•Inhibition of mitotic transcriptional activation delays cell-cycle progression
How transcription is shut down as cells begin to condense chromosomes during mitosis is poorly understood. Liang et al. report the requirement of mitotic transcriptional activation by P-TEFb to release paused Pol II as a prerequisite for this process, and ultimately for proper cell-cycle progression.
The epigenetic regulation of imprinted genes by monoallelic DNA methylation of either maternal or paternal alleles is critical for embryonic growth and development. Imprinted genes were recently ...shown to be expressed in mammalian adult stem cells to support self-renewal of neural and lung stem cells; however, a role for imprinting per se in adult stem cells remains elusive. Here we show upregulation of growth-restricting imprinted genes, including in the H19-Igf2 locus, in long-term haematopoietic stem cells and their downregulation upon haematopoietic stem cell activation and proliferation. A differentially methylated region upstream of H19 (H19-DMR), serving as the imprinting control region, determines the reciprocal expression of H19 from the maternal allele and Igf2 from the paternal allele. In addition, H19 serves as a source of miR-675, which restricts Igf1r expression. We demonstrate that conditional deletion of the maternal but not the paternal H19-DMR reduces adult haematopoietic stem cell quiescence, a state required for long-term maintenance of haematopoietic stem cells, and compromises haematopoietic stem cell function. Maternal-specific H19-DMR deletion results in activation of the Igf2-Igfr1 pathway, as shown by the translocation of phosphorylated FoxO3 (an inactive form) from nucleus to cytoplasm and the release of FoxO3-mediated cell cycle arrest, thus leading to increased activation, proliferation and eventual exhaustion of haematopoietic stem cells. Mechanistically, maternal-specific H19-DMR deletion leads to Igf2 upregulation and increased translation of Igf1r, which is normally suppressed by H19-derived miR-675. Similarly, genetic inactivation of Igf1r partly rescues the H19-DMR deletion phenotype. Our work establishes a new role for this unique form of epigenetic control at the H19-Igf2 locus in maintaining adult stem cells.
Chromosomal translocations of the mixed-lineage leukemia (MLL) gene with various partner genes result in aggressive leukemia with dismal outcomes. Despite similar expression at the mRNA level from ...the wild-type and chimeric MLL alleles, the chimeric protein is more stable. We report that UBE2O functions in regulating the stability of wild-type MLL in response to interleukin-1 signaling. Targeting wild-type MLL degradation impedes MLL leukemia cell proliferation, and it downregulates a specific group of target genes of the MLL chimeras and their oncogenic cofactor, the super elongation complex. Pharmacologically inhibiting this pathway substantially delays progression, and it improves survival of murine leukemia through stabilizing wild-type MLL protein, which displaces the MLL chimera from some of its target genes and, therefore, relieves the cellular oncogenic addiction to MLL chimeras. Stabilization of MLL provides us with a paradigm in the development of therapies for aggressive MLL leukemia and perhaps for other cancers caused by translocations.
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•UBE2O acts downstream of the interleukin-1 pathway to regulate MLL/COMPASS stability•Stabilizing wild-type MLL protein inhibits MLL leukemia cell proliferation•UBE2O and IRAK inhibition alters a common set of MLL chimera target genes•Targeting the IL-1 pathway is a potential therapeutic strategy for MLL leukemia
Stabilizing wild-type MLL proteins is a potential therapeutic approach for leukemia resulting from MLL translocations.
Loss of sensory hair cells leads to deafness and balance deficiencies. In contrast to mammalian hair cells, zebrafish ear and lateral line hair cells regenerate from poorly characterized support ...cells. Equally ill-defined is the gene regulatory network underlying the progression of support cells to differentiated hair cells. scRNA-Seq of lateral line organs uncovered five different support cell types, including quiescent and activated stem cells. Ordering of support cells along a developmental trajectory identified self-renewing cells and genes required for hair cell differentiation. scRNA-Seq analyses of
mutants, in which hair cell regeneration is increased, demonstrates that Fgf and Notch signaling inhibit proliferation of support cells in parallel by inhibiting Wnt signaling. Our scRNA-Seq analyses set the foundation for mechanistic studies of sensory organ regeneration and is crucial for identifying factors to trigger hair cell production in mammals. The data is searchable and publicly accessible via a web-based interface.
Background & Aims Identification of intestinal stem cells (ISCs) has relied heavily on the use of transgenic reporters in mice, but this approach is limited by mosaic expression patterns and ...difficult to directly apply to human tissues. We sought to identify reliable surface markers of ISCs and establish a robust functional assay to characterize ISCs from mouse and human tissues. Methods We used immunohistochemistry, real-time reverse-transcription polymerase chain reaction, and fluorescence-activated cell sorting (FACS) to analyze intestinal epithelial cells isolated from mouse and human intestinal tissues. We compared different combinations of surface markers among ISCs isolated based on expression of Lgr5–green fluorescent protein. We developed a culture protocol to facilitate the identification of functional ISCs from mice and then tested the assay with human intestinal crypts and putative ISCs. Results CD44+ CD24lo CD166+ cells, isolated by FACS from mouse small intestine and colon, expressed high levels of stem cell–associated genes. Transit-amplifying cells and progenitor cells were then excluded based on expression of GRP78 or c-Kit. CD44+ CD24lo CD166+ GRP78lo/– putative stem cells from mouse small intestine included Lgr5-GFPhi and Lgr5-GFPmed/lo cells. Incubation of these cells with the GSK inhibitor CHIR99021 and the E-cadherin stabilizer Thiazovivin resulted in colony formation by 25% to 30% of single-sorted ISCs. Conclusions We developed a culture protocol to identify putative ISCs from mouse and human tissues based on cell surface markers. CD44+ CD24lo CD166+ , GRP78lo/– , and c-Kit− facilitated identification of putative stem cells from the mouse small intestine and colon, respectively. CD44+ CD24−/lo CD166+ also identified putative human ISCs. These findings will facilitate functional studies of mouse and human ISCs.
The eukaryotic cell cycle, driven by both transcriptional and posttranslational mechanisms, is the central molecular oscillator underlying tissue growth throughout animals. Although genome-wide ...studies have investigated cell-cycle-associated transcription in unicellular systems, global patterns of periodic transcription in multicellular tissues remain largely unexplored. Here we define the cell-cycle-associated transcriptome of the developing Drosophila wing epithelium and compare it with that of cultured Drosophila S2 cells, revealing a core set of periodic genes and a surprising degree of context specificity in periodic transcription. We further employ RNAi-mediated phenotypic profiling to define functional requirements for more than 300 periodic genes, with a focus on those required for cell proliferation in vivo. Finally, we investigate uncharacterized genes required for interkinetic nuclear migration. Combined, these findings provide a global perspective on cell-cycle control in vivo, and they highlight a critical need to understand the context-specific regulation of cell proliferation.
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•The cell-cycle-associated transcriptome of Drosophila is highly context dependent•RNAi screen validates more than 100 periodic genes implicated in wing growth•Genes controlling cell-cycle phasing, mitosis, and other processes are reported•A putative long ncRNA, CR32027, is required for IKNM but not mitotic progression
Liang et al. report the physiological diversity of cell-cycle-associated transcriptomes in Drosophila melanogaster. Systematic RNAi knockdowns further implicate >100 periodic genes in growth, cell-cycle phasing, mitosis, and interkinetic nuclear migration (IKNM) in the wing disc. A putative long ncRNA, CR32027, is specifically required for IKNM but not mitotic progression.
Previous studies have shown that fibroblast growth factor (FGF) signaling promotes hematopoietic stem and progenitor cell (HSPC) expansion in vitro. However, it is unknown whether FGF promotes HSPC ...expansion in vivo. Here we examined FGF receptor 1 (FGFR1) expression and investigated its in vivo function in HSPCs. Conditional knockout (CKO) of Fgfr1 did not affect phenotypical number of HSPCs and homeostatic hematopoiesis, but led to a reduced engraftment only in the secondary transplantation. When treated with 5-fluorouracil (5FU), the Fgfr1 CKO mice showed defects in both proliferation and subsequent mobilization of HSPCs. We identified megakaryocytes (Mks) as a major resource for FGF production, and further discovered a novel mechanism by which Mks underwent FGF-FGFR signaling dependent expansion to accelerate rapid FGF production under stress. Within HSPCs, we observed an up-regulation of nuclear factor κB and CXCR4, a receptor for the chemoattractant SDF-1, in response to bone marrow damage only in control but not in Fgfr1 CKO model, accounting for the corresponding defects in proliferation and migration of HSPCs. This study provides the first in vivo evidence that FGF signaling facilitates postinjury recovery of the mouse hematopoietic system by promoting proliferation and facilitating mobilization of HSPCs.
Embryonic cells that migrate long distances must critically balance cell division in order to maintain stream dynamics and population of peripheral targets. Yet details of individual cell division ...events and how cell cycle is related to phases of migration remain unclear. Here, we examined these questions using the chick cranial neural crest (NC). In vivo time-lapse imaging revealed that a typical migrating NC cell division event lasted ~1 hour and included four stereotypical steps. Cell tracking showed that dividing NC cells maintained position relative to non-dividing neighbors. NC cell division orientation and the time and distance to first division after neural tube exit were stochastic. To address how cell cycle is related to phases of migration, we used FACs analysis to identify significant spatiotemporal differences in NC cell cycle profiles. Two-photon photoconversion of single and small numbers of mKikGR-labeled NC cells confirmed that lead NC cells exhibited a nearly fourfold faster doubling time after populating the branchial arches. By contrast, Ki-67 staining showed that one out of every five later emerging NC cells exited the cell cycle after reaching proximal head targets. The relatively quiescent mitotic activity during NC cell migration to the branchial arches was altered when premigratory cells were reduced in number by tissue ablation. Together, our results provide the first comprehensive details of the pattern and dynamics of cell division events during cranial NC cell migration.
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