Les plaquettes sanguines ont comme rôle principal d’arrêter les saignements. Elles sont produites dans la moelle osseuse par des mégacaryocytes (MK) qui proviennent de la différenciation des cellules souches hématopoïétiques (CSH). L’objectif de ma thèse a été d’identifier les éléments cellulaires du microenvironnement contrôlant la mégacaryopoïèse. Mon travail a permis d’identifier une population particulière de progéniteurs hépatiques du foie foetal capable de promouvoir in vitro les étapes précoces de la mégacaryopoïèse à partir de CSH humaines et murines (Brouard et al., 2017). Le rôle des cellules endothéliales (EC) dans les étapes tardives de maturation a été étudié après purification à partir de moelle humaine dans des expériences de co-culture avec des MK prédifférenciés. Mes résultats montrent que ces EC ont la propriété unique, par comparaison avec des EC d’autres tissus, de promouvoir la maturation des MK. Une analyse transcriptionnelle différentielle a permis d’identifier des effecteurs possibles ouvrant des pistes pour mieux comprendre les mécanismes de la mégacaryopoïèse et pour améliorer la production des plaquettes en culture. / The main role of platelets is to stop bleeding. They are produced in the bone marrow by megakaryocytes (MK) that are produced by the differentiation of hematopoietic stem cells (HSC). The objective of my thesis was to identify the cellular elements of the microenvironment controlling megakaryopoiesis. My work has identified a particular population of hepatic progenitors from the fetal liver capable of promoting in vitro the early stages of megakaryopoiesis from human and murine HSC (Brouard et al., 2017). The role of endothelial cells (EC), purified from human bone marrow, in late maturation stages was studied in co-culture experiments with predifferentiated MK. My results show that these EC have the unique property in comparison with EC from other tissues, of promoting the maturation of MK. A differential transcriptional analysis identified possible effectors that could lead to a better understanding of the mechanisms of megakaryopoiesis and improve platelet production in culture.

La niche des cellules souches hématopoïétiques représente un compartiment complexe et radiosensible. Sa protection est nécessaire pour la restauration de l'hématopoïèse faisant suite à la myélosuppression due à l'exposition aux rayonnements ionisants. Nous avons dans un premier temps étudié l'effet des RI sur les progéniteurs endothéliaux et mésenchymateux de la niche par une étude de radiosensiblilité et une étude d'évaluation de la mort cellulaire. Nous avons proposé par la suite une stratégie innovante de thérapie génique basée sur la sécrétion locale et à court terme du morphogène Sonic hedgehog visant à favoriser la réparation de niche vasculaire et de stimuler les cellules souches hématopoïétiques et les cellules progénitrices résiduelles. Nous avons étudié la réponse hématopoïétique des singes irradiés à 8-Gy gamma après une seule injection intra-osseuse de cellules souches mésenchymateuses xénogéniques, multipotentes et d'origine adipocytaire transfectées avec un plasmide pIRES2-eGFP codant la protéine Shh. La durée de thrombocytopénie et celle de neutropénie ont été significativement réduites chez les animaux greffés et les clonogènes sont normalisés à partir du 42e jour. Les aires sous la courbe des numération des plaquettes et des neutrophiles entre 0 et 30 jours ont été significativement plus élevée chez les animaux traités que chez les témoins. La greffe d'explants de MatrigelTM colonisés ou non avec des ASC chez des souris immunodéprimées a démontré une activité pro-angiogénique notable des ASC transfectées avec le plasmide Shh . Le suivi à long terme (180 à 300 jours) a confirmé une reconstitution durable dans les quatre singes greffés. Globalement cette étude suggère que la greffe de cellules souches multipotentes Shh-peut représenter une nouvelle stratégie pour la prise en charge des dommages radio-induits de la niche. / The hematopoietic stem cell niche represents a complex radiosensitive compartment whose protection is required for recovery from radiation-induced myelosuppression. We initially studied RI effects on endothelial and mesenchymal progenitors by an evaluating radiosensitivity and cell death. Then, we have proposed a new gene therapy strategy based on local and short term secretion of Sonic hedgehog morphogene to favour vascular niche repair and to stimulate residual hematopoietic stem and progenitor cells. We investigated the hematopoietic response of 8-Gy gamma irradiated monkeys to a single intra-osseous injection of xenogeneic multipotent mesenchymal stem cells transduced with a Shh pIRES2 plasmid. Thrombocytopenia and neutropenia duration were significantly reduced in grafted animals and clonogenics normalized from day 42. Areas under the curve of PLTs and ANCs between day 0 and day 30 were significantly higher in treated animals than in controls. Grafting MatrigelTM colonized or not with ASC in immunocompromized mice demonstrated a notable pro-angiogenic activity for Shh-ASC. Long term follow up (180-300 days) confirmed a durable recovery in the four grafted monkeys. Globally this study suggests that grafting Shh-multipotent stem cells may represent a new strategy to cure radiation-induced niche damage.

<p>During development of the mammalian embryo, spatial and temporal expression of fibroblast growth factors (FGFs) and their cognate receptors are vital in the regulation of a number of patterning processes. Inappropriate or decreased expression leads to severe malformations and even embryonic death. The objectives of this thesis have been to evaluate the usefulness of differentiating embryonic stem (ES) cells as a model to study FGF and FGF receptors in endothelial and hematopoietic cell function in vitro and in vivo, and the effect of an activating mutation in the platelet-derived growth factor receptor-β (PDGFR-β) on endothelial cells and vessel formation.</p><p>Aggregates of differentiating ES cells, denoted embryoid bodies, faithfully recapitulate many developmental processes. Embryoid bodies cultured in fetal calf serum spontaneously develop cardiomyocytes and endothelial cells. The endothelial cells organize into lumen-containing vessels carrying erythroblasts. Administration of FGF or vascular endothelial growth factor (VEGF)-A promotes development of specific vascular phenotypes. About 20% of endothelial cells in embryoid bodies and teratomas express FGFR-1, and these FGFR-1-expressing endothelial cells are mitogenically active in the absence of exogenous stimuli and respond to VEGF-A to the same extent as endothelial cells lacking FGFR-1 expression. FGFR-1 deficiency leads to arrest in hematopoietic differentiation, whereas endothelial cell development is enhanced. As a consequence, teratomas derived from ES cells lacking FGFR-1 expression display vessels composed of a double layer of endothelial cells. The hyperactivity of endothelial cells derived from FGFR-1-deficient ES cells is suggested to be due to hyperactivity of VEGF receptor-2, as well as to loss of negative regulators of angiogenesis, such as interleukin-4.</p><p>Mutation of platelet-derived factor receptor-β (PDGFR-β) to replace D849 in the activating loop in the kinase domain with V leads to ligand-independent kinase activity, increased basal signal transduction, and enhanced expression of VEGF-A as well as VEGFR-2. As a result, endothelial cell sprouts covered with pericyte-like cells are formed in a VEGF-A/VEGFR-2 dependent manner in ES cells expressing the mutated PDGFR-β.</p><p>In conclusion, embryoid bodies represent a high-quality model for the study of growth factor-regulated vascular development and sprouting angiogenesis.</p>

<p>The Mad/Max/Myc transcription factor network takes part in the control of vital cellular functions such as growth, proliferation, differentiation and apoptosis. Dimerization with the protein Max is necessary for the Myc-family of oncoproteins and their antagonists, the Mad-family proteins, to regulate target genes and carry out their intended functions. Myc functions as a positive regulator of proliferation, antagonized by the growth inhibitory Mad-proteins that potentially functions as tumor supprerssors. Deregulated Myc expression is found in a variety of tumors and signals negatively regulating Myc expression and/or activity could therefore be of potential use in treating tumors with deregulated Myc.</p><p>Our aim was to therefore to investigate possible negative effects on Myc expression and activity by growth inhibitory cytokines and by the Myc antagonists, the Mad-family proteins.Two different cellular model systems of neuronal and hematopoietic origin have been utilized for these studies.</p><p>Our results show that Mad1 is upregulated during induced neuronal differentiation of SH-SY5Y cells. Further, the growth inhibitory cytokine interferon-g (IFN-g) was shown to cooperate with retinoic acid (RA) and the phorbol ester TPA in inducing growth arrest and differentiation in N-<i>myc</i> amplified neuroblastoma cell lines. In contrast to treatment with either agent alone, the combined treatment of TPA+IFN-g and RA+IFN-g led to upregulation of Mad1 and to downregulation of N-Myc, respectively, thus correlating with the enhanced growth inhibition and differentiation observed after combination treatment. Ectopic expression of an inducible Mad1 in monoblastic U-937 cells led to growth inhibition but did not lead to differentiation or enhancement of differentiation induced by RA, vitamin D3 or TPA. In v-Myc transformed U-937 cells Mad1 expression reestablished the TPA-induced G1 cell cycle arrest, but did not restore differentiation, blocked by v-Myc. The growth inhibitory cytokine TGF-b was found to induce Mad1 expression and Mad1:Max complex formation in v-Myc transformed U-937 cells correlating with reduced Myc activity and G1 arrest. </p><p>In conclusion, our results show that the Myc-antagonist Mad1 is upregulated by growth inhibitory cytokines and/or differentiation signals in neuronal and hematopoietic cells and that enforced Mad1 expression in hematopoietic cells results in growth inhibition and increased sensitivity to anti-proliferative cytokines. Mad1 and cytokine-induced signals therefore seem to cooperate in counteracting Myc activity.</p>

The Mad/Max/Myc transcription factor network takes part in the control of vital cellular functions such as growth, proliferation, differentiation and apoptosis. Dimerization with the protein Max is necessary for the Myc-family of oncoproteins and their antagonists, the Mad-family proteins, to regulate target genes and carry out their intended functions. Myc functions as a positive regulator of proliferation, antagonized by the growth inhibitory Mad-proteins that potentially functions as tumor supprerssors. Deregulated Myc expression is found in a variety of tumors and signals negatively regulating Myc expression and/or activity could therefore be of potential use in treating tumors with deregulated Myc. Our aim was to therefore to investigate possible negative effects on Myc expression and activity by growth inhibitory cytokines and by the Myc antagonists, the Mad-family proteins.Two different cellular model systems of neuronal and hematopoietic origin have been utilized for these studies. Our results show that Mad1 is upregulated during induced neuronal differentiation of SH-SY5Y cells. Further, the growth inhibitory cytokine interferon-g (IFN-g) was shown to cooperate with retinoic acid (RA) and the phorbol ester TPA in inducing growth arrest and differentiation in N-myc amplified neuroblastoma cell lines. In contrast to treatment with either agent alone, the combined treatment of TPA+IFN-g and RA+IFN-g led to upregulation of Mad1 and to downregulation of N-Myc, respectively, thus correlating with the enhanced growth inhibition and differentiation observed after combination treatment. Ectopic expression of an inducible Mad1 in monoblastic U-937 cells led to growth inhibition but did not lead to differentiation or enhancement of differentiation induced by RA, vitamin D3 or TPA. In v-Myc transformed U-937 cells Mad1 expression reestablished the TPA-induced G1 cell cycle arrest, but did not restore differentiation, blocked by v-Myc. The growth inhibitory cytokine TGF-b was found to induce Mad1 expression and Mad1:Max complex formation in v-Myc transformed U-937 cells correlating with reduced Myc activity and G1 arrest. In conclusion, our results show that the Myc-antagonist Mad1 is upregulated by growth inhibitory cytokines and/or differentiation signals in neuronal and hematopoietic cells and that enforced Mad1 expression in hematopoietic cells results in growth inhibition and increased sensitivity to anti-proliferative cytokines. Mad1 and cytokine-induced signals therefore seem to cooperate in counteracting Myc activity.

During development of the mammalian embryo, spatial and temporal expression of fibroblast growth factors (FGFs) and their cognate receptors are vital in the regulation of a number of patterning processes. Inappropriate or decreased expression leads to severe malformations and even embryonic death. The objectives of this thesis have been to evaluate the usefulness of differentiating embryonic stem (ES) cells as a model to study FGF and FGF receptors in endothelial and hematopoietic cell function in vitro and in vivo, and the effect of an activating mutation in the platelet-derived growth factor receptor-β (PDGFR-β) on endothelial cells and vessel formation. Aggregates of differentiating ES cells, denoted embryoid bodies, faithfully recapitulate many developmental processes. Embryoid bodies cultured in fetal calf serum spontaneously develop cardiomyocytes and endothelial cells. The endothelial cells organize into lumen-containing vessels carrying erythroblasts. Administration of FGF or vascular endothelial growth factor (VEGF)-A promotes development of specific vascular phenotypes. About 20% of endothelial cells in embryoid bodies and teratomas express FGFR-1, and these FGFR-1-expressing endothelial cells are mitogenically active in the absence of exogenous stimuli and respond to VEGF-A to the same extent as endothelial cells lacking FGFR-1 expression. FGFR-1 deficiency leads to arrest in hematopoietic differentiation, whereas endothelial cell development is enhanced. As a consequence, teratomas derived from ES cells lacking FGFR-1 expression display vessels composed of a double layer of endothelial cells. The hyperactivity of endothelial cells derived from FGFR-1-deficient ES cells is suggested to be due to hyperactivity of VEGF receptor-2, as well as to loss of negative regulators of angiogenesis, such as interleukin-4. Mutation of platelet-derived factor receptor-β (PDGFR-β) to replace D849 in the activating loop in the kinase domain with V leads to ligand-independent kinase activity, increased basal signal transduction, and enhanced expression of VEGF-A as well as VEGFR-2. As a result, endothelial cell sprouts covered with pericyte-like cells are formed in a VEGF-A/VEGFR-2 dependent manner in ES cells expressing the mutated PDGFR-β. In conclusion, embryoid bodies represent a high-quality model for the study of growth factor-regulated vascular development and sprouting angiogenesis.

Se realizó el estudio histológico del timo de lubina, con el fin de conocer la arquitectura del microambiente tímico y las relaciones estructurales de sus diferentes componentes. Las células linfoides sufren procesos de muerte celular programada por apoptosis, no descrita previamente en timo de peces; indicando la existencia de procesos de selección negativa durante su diferenciación intratímica. Existen células interdigitadas, no descritas previamente en peces. Hay áreas mielopoyéticas, con actividad eritropoyética y granulopoyética heterófila. La fragilidad de la barrera epitelial, que separa la cavidad branquial del parénquima tímico, puede verse incrementada por la presencia de macrófagos intraepiteliales y macrófagos con inmunoglobulinas de superficie. El transito de células sanguíneas a través de la capa de células epitelio-reticulares limitantes y de capilares sanguíneos indican la carencia de barrera hematotímica. Estos datos sugieren la existencia de procesos antígen o dependiente y la persistencia de funciones propias de órgano linfoide secundario en timo de lubina. / The histological study of the sea bass thymus was performed to know the architecture of the thymic microenvironment and the structural relationships of its different components. The lymphoid cells suffer processes of programmed cell death by apoptosis, not described previously in fish thymus, indicating the existence of negative selection processes during its intrathymic differentiation. There are interdigitating cells, which has not described before in fish. Exist myelopoiesis areas, with erythropoietic and heterophilic granulopoietic activity. The fragility of the epithelial barrier, that separates the branchial cavity from the thymic parenchyma, is increased by the presence of intraepithelial macrophages and macrophages with surface immunoglobulins. The traffic of blood cells across the layer of limiting reticular epithelial cells and blood capillaries indicate the lack of hemato-thymic barrier. This information suggests the existence of antigen dependent process and the p ersistence of own functions of secondary lymphoid organ in thymus of sea bass.

During the last 40 years, Drosophila melanogaster has become an invaluable tool in understanding innate immunity. The innate immune system of Drosophila consists of a humoral and a cellular component. While many details are known about the humoral immune system, our knowledge about the cellular immune system is comparatively small. Blood cells or hemocytes constitute the cellular immune system. Three blood types have been described for Drosophila larvae. Plasmatocytes are phagocytes with a plethora of functions. Crystal cells mediate melanization and contribute to wound healing. Plasmatocytes and crystal cells constitute the blood cell repertoire of a healthy larva, whereas lamellocytes are induced in a demand-adapted manner after infection with parasitoid wasp eggs. They are involved in the melanotic encapsulation response against parasites and form melanotic nodules that are also referred to as tumors. In my thesis, I focused on unraveling the mechanisms of how the immune system orchestrates the cellular immune response. In particular, I was interested in the hematopoiesis of lamellocytes. In Article I, we were able to show that ectopic expression of key components of a number of signaling pathways in blood cells induced the development of lamellocytes, led to a proliferative response of plasmatocytes, or to a combination of lamellocyte activation and plasmatocyte proliferation. In Article II, I combined newly developed fluorescent enhancer-reporter constructs specific for plasmatocytes and lamellocytes and developed a “dual reporter system” that was used in live microscopy of fly larvae. In addition, we established flow cytometry as a tool to count total blood cell numbers and to distinguish between different blood cell types. The “dual reporter system” enabled us to differentiate between six blood cell types and established proliferation as a central feature of the cellular immune response. The combination flow cytometry and live imaging increased our understanding of the tempo-spatial events leading to the cellular immune reaction. In Article III, I developed a genetic modifier screen to find genes involved in the hematopoiesis of lamellocytes. I took advantage of the gain-of-function phenotype of the Tl10b mutation characterized by an activated cellular immune system, which induced the formation blood cell tumors. We screened the right arm of chromosome 3 for enhancers and suppressors of this mutation and uncovered ird1. Finally in Article IV, we showed that the activity of the Toll signaling pathway in the fat body, the homolog of the liver, is necessary to activate the cellular immune system and induce lamellocyte hematopoiesis.

Τα μυελοδυσπλαστικά σύνδρομα αποτελούν ετερογενή ομάδα νοσημάτων, με δυσμενή πρόγνωση και δυσκολία θεραπευτικής προσέγγισης. Η κατανόηση των παθογενετικών μηχανισμών που διέπουν την παθολογική αιμοποίηση που παρατηρείται στα σύνδρομα αυτά in vitro και in vivo, μπορεί να βοηθήσει στην αποτελεσματικότερη αντιμετώπισή τους. Σκοπός αυτής της διατριβής ήταν η μελέτη της αιμοποίησης των ΜΔΣ με τη μέθοδο των καλλιεργειών αιμοποιητικών κυττάρων μακράς διάρκειας και η επίδραση διαφόρων παραγόντων στην in vitro αιμοποίηση, με στόχο την αναγωγή των ευρημάτων και στην in vivo διαδικασία. Οι καλλιέργειες βραχείας διάρκειας ανέδειξαν την αδυναμία των προγονικών κυττάρων των ασθενών να δημιουργήσουν φυσιολογικές αποικίες. Η προσθήκη μίγματος αυξητικών παραγόντων στο καλλιεργητικό υλικό είχε θετική επίδραση στην πλειοψηφία των περιπτώσεων. Με μακράς διάρκειας καλλιέργειες συγκρίθηκαν τα αποτελέσματα των ΜΔΣ με αυτά φυσιολογικών μαρτύρων και των υποκατηγοριών ΜΔΣ μεταξύ τους. Αξιολογήθηκαν η έκταση του στρώματος, η διάρκεια ζωής, η εβδομαδιαία και η συνολική κυτταρική απόδοση. Σε όλες τις περιπτώσεις η ανάπτυξη υπολειπόταν ποιοτικά και ποσοτικά στους ασθενείς, σε σχέση με τους μάρτυρες. Η προσθήκη παραγόντων στο καλλιεργητικό υλικό αποσκοπούσε στη βελτίωση των παραμέτρων που προαναφέρθηκαν. Η IFN-α, η βιταμίνη D3, η Ara-c και ο συνδυασμός της με IFN-α δεν βελτίωσαν τα αποτελέσματα. Η προσθήκη IL-3 είχε ευοδωτική επίδραση κυρίως στις κυτταρικές αποδόσεις. Η IL-6 είχε επίσης ευοδωτική δράση, κυρίως στον σχηματισμό στρώματος. Ο συνδυασμός IL-3+IL-6 απέβη ο πιο σημαντικός τροποποιητής της συμπεριφοράς των καλλιεργειών των ασθενών με ΜΔΣ ευοδώνοντας όλες τις παραμέτρους και προκάλεσε διαφορές πολύ σημαντικές σε σχέση με την control καλλιέργεια. Αναδείχθησαν λοιπόν ευρήματα συνέργειας των δύο ιντερλευκινών, σε όλες τις άλλες παραμέτρους αξιολόγησης των καλλιεργειών μακράς διάρκειας. Με βάση τον τρόπο ανάπτυξης και την συμπεριφορά των κυττάρων των ασθενών με ΜΔΣ στην προσθήκη των κυτταροκινών, διακρίθηκαν δύο μοντέλα in vitro ανάπτυξης της καλλιεργειών: Το δυσπλαστικό και το λευχαιμικό. Συμπερασματικά οι μακράς διάρκειας καλλιέργειες στους ασθενείς με ΜΔΣ αποτελούν χρήσιμη προγνωστική μέθοδο και μπορούν να διακρίνουν τους ασθενείς που θα εξελιχθούν ταχέως, από εκείνους που θα έχουν χρονιότερη και ηπιότερη πορεία. Συνδυασμός κυτταροκινών και άλλων παραγόντων μπορεί να βελτιώσει την προβληματική-παθολογική in vitro αιμοποίηση των προγονικών κυττάρων των ασθενών με ΜΔΣ. / Myelodysplastic syndromes comprise a heterogeneous group of hematopoietic stem-cell disorders, with dismal prognosis and difficulty in their therapeutic approach. The revealing of the underlying pathogenetic mechanisms, implicated in the impaired hematopoiesis of these syndromes, is crucial for the development a more comprehensive and effective treatment approaches. The aim of this thesis was the study of hematopoiesis of MDS, by using long term cultures of hemopoietic cells and the investigation of the influence of various exogenous modulating factors-drugs in vitro, in an effort to obtain results, which could direct their use in vivo. Short term cultures revealed the disability of the progenitor cells of patients to form normal colonies. The addition of a mixture of growth factors in the conditioned medium had a positive influence in the majority of cases. By using long term cultures we compared the results obtained from patients with MDS, with those from normal controls, and between the different MDS subgroups For this comparison we used: the extent of the area of the stroma-layer formed, the longevity of the culture, the weekly cell production and the total cell yield of each culture. In all cases the development of cultures derived from patients was inferior to those of controls. The addition of modulating factors to the culture medium was aimed to improve the above parameters. IFN-α, vitamin D3, Ara-c and the combination of IFN-α and Ara-c did not improve any of the culture’s parameter. The addition of IL-3 had a clearly favorable effect mainly to the weekly and the total cell yield. Interleukin-6 had similarly a favourable effect, particularly promi-nent in the stroma-cell formation. The combination of IL-3 plus IL-6 was proved as the most important favourable modulator of the MDS cultures. It improved all culture parameters and produced statistically significant differences in comparison to the control cultures. According to the developmental model obtained by the long-term culture the dysplastic and the leukemic pattern of in vitro growth could be distinguished. In conclusion, long term cultures of hematopoietic cells of MDS patients represent a useful prognostic tool and can distinguish patients who will more rapidly evolve to leukemia from those who will have a more prolonged and stable clinical course. The use of a combination of cytokines and might have a favourable effect on in vitro hematopoiesis of the progenitor cells of MDS patients.

Hematopoiesis is an intricately regulated homeostatic process that maintains all of the differentiated blood cell lineages. N-ethyl-N-nitrosurea (ENU) is a powerful mutagen that induces point mutations randomly in the genome. ENU was used in a dominant forward genetic screen to identify novel mutations in regulators of hematopoiesis and to create new mouse models of hematopoietic disease. The objectives of this thesis were to characterize two mutants that originated from the dominant screen (7192 and 7238) and to develop a pharmacologically sensitized screen that would detect a unique set of mutations undetectable in the dominant screen. The 7192 mutant from the ENU dominant screen presented with elevated microcytic red blood cells (RBC) and increased polychromasia. The causative mutation was identified as a nonsense mutation in Ank1 (Q895X) that coded for a truncated ANK1 protein. Ank17192 is a novel mouse model of hereditary spherocytosis (HS), a human disease that results from increased RBC fragility. We have demonstrated that Ank17192/+ mice model a mild HS and Ank17192/7192 mice model severe HS. The 7238 mutant from the dominant ENU screen was macrothrombocytic and carried a missense mutation in Myh9 (Q1443L). The Myh97238/7238 mice are viable and have a more severe phenotype of macrothrombocytopenia. Myh97238 is the first mouse model for Myh9 related disorders that accurately models the genetic origins and the systemic manifestations of the disorder. A pharmacologically sensitized screen using chemotherapeutic drugs was designed to induce stress hematopoiesis to detect mutations that alter cell cycle of hematopoietic progenitors or stress hematopoiesis. Analysis of both peripheral blood and progenitor recovery kinetics, determined that 5-fluorouracil (5FU) and phenylhydrazine were good candidates for a pharmacologically sensitized screen. 5FU was successfully incorporated into an ENU dominant screen, and 13 platelet recovery outliers were detected. From these outliers, three mutant lines were successfully established.