ANKRD26 is a new regulator of type I cytokine receptor signaling in normal and pathological hematopoiesis.

Sustained ANKRD26 expression associated with germline ANKRD26 mutations causes thrombocytopenia 2 (THC2), an inherited platelet disorder associated with a predisposition to leukemia. Some patients also present with erythrocytosis and/or leukocytosis. Using multiple human-relevant in vitro models (cell lines, primary patients' cells and patient-derived induced pluripotent stem cells) we demonstrate for the first time that ANKRD26 is expressed during the early steps of erythroid, megakaryocyte and granulocyte differentiation, and is necessary for progenitor cell proliferation. As differentiation progresses, ANKRD26 expression is progressively silenced, to complete the cellular maturation of the three myeloid lineages. In primary cells, abnormal ANKRD26 expression in committed progenitors directly affects the proliferation/differentiation balance for the three cell types. We show that ANKRD26 interacts with and crucially modulates the activity of MPL, EPOR and G-CSFR, three homodimeric type I cytokine receptors that regulate blood cell production. Higher than normal levels of ANKRD26 prevent the receptor internalization that leads to increased signaling and cytokine hypersensitivity. These findings afford evidence how ANKRD26 overexpression or the absence of its silencing during differentiation is responsible for myeloid blood cell abnormalities in patients with THC2.

Stepwise GATA1 and SMC3 mutations alter megakaryocyte differentiation in a Down syndrome leukemia model.

Acute megakaryoblastic leukemia of Down syndrome (DS-AMKL) is a model of clonal evolution from a preleukemic transient myeloproliferative disorder requiring both a trisomy 21 (T21) and a GATA1s mutation to a leukemia driven by additional driver mutations. We modeled the megakaryocyte differentiation defect through stepwise gene editing of GATA1s, SMC3+/-, and MPLW515K, providing 20 different T21 or disomy 21 (D21) induced pluripotent stem cell (iPSC) clones. GATA1s profoundly reshaped iPSC-derived hematopoietic architecture with gradual myeloid-to-megakaryocyte shift and megakaryocyte differentiation alteration upon addition of SMC3 and MPL mutations. Transcriptional, chromatin accessibility, and GATA1-binding data showed alteration of essential megakaryocyte differentiation genes, including NFE2 downregulation that was associated with loss of GATA1s binding and functionally involved in megakaryocyte differentiation blockage. T21 enhanced the proliferative phenotype, reproducing the cellular and molecular abnormalities of DS-AMKL. Our study provides an array of human cell-based models revealing individual contributions of different mutations to DS-AMKL differentiation blockage, a major determinant of leukemic progression.

Lyl-1 regulates primitive macrophages and microglia development.

During ontogeny, macrophage populations emerge in the Yolk Sac (YS) via two distinct progenitor waves, prior to hematopoietic stem cell development. Macrophage progenitors from the primitive/"early EMP" and transient-definitive/"late EMP" waves both contribute to various resident primitive macrophage populations in the developing embryonic organs. Identifying factors that modulates early stages of macrophage progenitor development may lead to a better understanding of defective function of specific resident macrophage subsets. Here we show that YS primitive macrophage progenitors express Lyl-1, a bHLH transcription factor related to SCL/Tal-1. Transcriptomic analysis of YS macrophage progenitors indicate that primitive macrophage progenitors present at embryonic day 9 are clearly distinct from those present at later stages. Disruption of Lyl-1 basic helix-loop-helix domain leads initially to an increased emergence of primitive macrophage progenitors, and later to their defective differentiation. These defects are associated with a disrupted expression of gene sets related to embryonic patterning and neurodevelopment. Lyl-1-deficiency also induce a reduced production of mature macrophages/microglia in the early brain, as well as a transient reduction of the microglia pool at midgestation and in the newborn. We thus identify Lyl-1 as a critical regulator of primitive macrophages and microglia development, which disruption may impair resident-macrophage function during organogenesis.

Dual role of EZH2 in megakaryocyte differentiation.

EZH2, the enzymatic component of PRC2, has been identified as a key factor in hematopoiesis. EZH2 loss-of-function mutations have been found in myeloproliferative neoplasms, particularly in myelofibrosis, but the precise function of EZH2 in megakaryopoiesis is not fully delineated. Here, we show that EZH2 inhibition by small molecules and short hairpin RNA induces megakaryocyte (MK) commitment by accelerating lineage marker acquisition without change in proliferation. Later in differentiation, EZH2 inhibition blocks proliferation and polyploidization and decreases proplatelet formation. EZH2 inhibitors similarly reduce MK polyploidization and proplatelet formation in vitro and platelet levels in vivo in a JAK2V617F background. In transcriptome profiling, the defect in proplatelet formation was associated with an aberrant actin cytoskeleton regulation pathway, whereas polyploidization was associated with an inhibition of expression of genes involved in DNA replication and repair and an upregulation of cyclin-dependent kinase inhibitors, particularly CDKN1A and CDKN2D. The knockdown of CDKN1A and to a lesser extent CDKN2D could partially rescue the percentage of polyploid MKs. Moreover, H3K27me3 and EZH2 chromatin immunoprecipitation assays revealed that CDKN1A is a direct EZH2 target and CDKN2D expression is not directly regulated by EZH2, suggesting that EZH2 controls MK polyploidization directly through CDKN1A and indirectly through CDKN2D.

Role of Rho-GTPases in megakaryopoiesis.

Megakaryocytes (MKs) are the bone marrow (BM) cells that generate blood platelets by a process that requires: i) polyploidization responsible for the increased MK size and ii) cytoplasmic organization leading to extension of long pseudopods, called proplatelets, through the endothelial barrier to allow platelet release into blood. Low level of localized RHOA activation prevents actomyosin accumulation at the cleavage furrow and participates in MK polyploidization. In the platelet production, RHOA and CDC42 play opposite, but complementary roles. RHOA inhibits both proplatelet formation and MK exit from BM, whereas CDC42 drives the development of the demarcation membranes and MK migration in BM. Moreover, the RhoA or Cdc42 MK specific knock-out in mice and the genetic alterations in their down-stream effectors in human induce a thrombocytopenia demonstrating their key roles in platelet production. A better knowledge of Rho-GTPase signalling is thus necessary to develop therapies for diseases associated with platelet production defects.Abbreviations: AKT: Protein Kinase BARHGEF2: Rho/Rac Guanine Nucleotide Exchange Factor 2ARP2/3: Actin related protein 2/3BM: Bone marrowCDC42: Cell division control protein 42 homologCFU-MK: Colony-forming-unit megakaryocyteCIP4: Cdc42-interacting protein 4mDIA: DiaphanousDIAPH1; Protein diaphanous homolog 1ECT2: Epithelial Cell Transforming Sequence 2FLNA: Filamin AGAP: GTPase-activating proteins or GTPase-accelerating proteinsGDI: GDP Dissociation InhibitorGEF: Guanine nucleotide exchange factorHDAC: Histone deacetylaseLIMK: LIM KinaseMAL: Megakaryoblastic leukaemiaMARCKS: Myristoylated alanine-rich C-kinase substrateMKL: Megakaryoblastic leukaemiaMLC: Myosin light chainMRTF: Myocardin Related Transcription FactorOTT: One-Twenty Two ProteinPACSIN2: Protein Kinase C And Casein Kinase Substrate In Neurons 2PAK: P21-Activated KinasePDK: Pyruvate Dehydrogenase kinasePI3K: Phosphoinositide 3-kinasePKC: Protein kinase CPTPRJ: Protein tyrosine phosphatase receptor type JRAC: Ras-related C3 botulinum toxin substrate 1RBM15: RNA Binding Motif Protein 15RHO: Ras homologousROCK: Rho-associated protein kinaseSCAR: Suppressor of cAMP receptorSRF: Serum response factorSRC: SarcTAZ: Transcriptional coactivator with PDZ motifTUBB1: Tubulin ß1VEGF: Vascular endothelial growth factorWAS: Wiskott Aldrich syndromeWASP: Wiskott Aldrich syndrome proteinWAVE: WASP-family verprolin-homologous proteinWIP: WASP-interacting proteinYAP: Yes-associated protein.

Vitamin E but Not GSH Decreases Reactive Oxygen Species Accumulation and Enhances Sperm Production during In Vitro Maturation of Frozen-Thawed Prepubertal Mouse Testicular Tissue.

Freezing-thawing procedures and in vitro culture conditions are considered as a source of stress associated with increased reactive oxygen species (ROS) generation, leading to a damaged cell aerobic metabolism and consequently to oxidative stress. In the present study, we sought to investigate whether vitamin E (Vit E) or reduced glutathione (GSH) enhances sperm production by decreasing ROS accumulation during in vitro maturation of prepubertal mice testes. Testes of prepubertal mice were cryopreserved using a freezing medium supplemented or not supplemented with Vit E and were cultured after thawing. In presence of Rol alone in culture medium, frozen-thawed (F-T) testicular tissues exhibited a higher ROS accumulation than fresh tissue during in vitro culture. However, Vit E supplementation in freezing, thawing, and culture media significantly decreased cytoplasmic ROS accumulation in F-T testicular tissue during in vitro maturation when compared with F-T testicular tissue cultured in the presence of Rol alone, whereas GSH supplementation in culture medium significantly increased ROS accumulation associated with cytolysis and tissue disintegration. Vit E but not GSH promoted a better in vitro sperm production and was a suitable ROS scavenger and effective molecule to improve the yield of in vitro spermatogenesis from F-T prepubertal mice testes. The prevention of oxidative stress in the cytoplasmic compartment should be regarded as a potential strategy for improving testicular tissue viability and functionality during the freeze-thaw procedure and in vitro maturation.

Stallion spermatozoa: putative target of estrogens; presence of the estrogen receptors ESR1, ESR2 and identification of the estrogen-membrane receptor GPER.

Among mammals, the stallion produces the largest amount of testicular estrogens. These steroid hormones are produced mainly by Leydig and Sertoli cells in the testis and also in the epididymis. Their role in horse testicular physiology and their ability to act on spermatozoa are still unknown. In order to determine if spermatozoa are targets for estrogens, the presence of estrogen receptors in mature ejaculated spermatozoa has been investigated. The presence of a single isoform of ESR1 (66kDa) and ESR2 (61kDa) was found by Western-blot analysis in samples from seven stallions. Confocal analysis mainly showed a flagellar localization for both receptors. Immuno-TEM experiments revealed that they are mostly located near the membranes, which are classically associated with rapid, non-genomic, effects. Moreover, we evidenced the expression of the seven transmembrane estradiol binding receptor GPER in colt testis. The protein was also localized at the connecting piece in mature spermatozoa. In conclusion, our results suggest that horse spermatozoa are a target for estrogens, which could act on several receptors either during the epididymal transit and/or in the female genital tract.

[Cryopreservation of testicular tissue in children]. / Cryopréservation du tissu testiculaire chez l'enfant.

The toxicity of cancer therapies can affect all organs and tissues. Some treatments damage spermatogonial stem cells (SSCs), with a risk of infertility. Storage and reimplantation of frozen testicular tissue is a recent approach tofertilitypreservationfor young boys. However, thawed frozen prepubertal testicular tissue must undergo a maturation process to restore sperm production. This process, currently being studied in animal models, can be achieved by in vivo transplantation of SSCs into seminiferous tubules or by testicular grafting, possibly following in vitro maturation.