NPC1 defect results in abnormal platelet formation and function

NPC1 defect results in abnormal platelet formationand function: studies in Niemann–Pick diseasetype C1 patients and zebrafish

Sophie Louwette1, Luc Regal2, Christine Wittevrongel1, Chantal Thys1, Gwenny

Vandeweeghde1, Elisa Decuyper1, Peter Leemans3, Rita De Vos3, Chris Van Geet1,2,

Jaak Jaeken2 and Kathleen Freson1,∗

1Center for Molecular and Vascular Biology, 2Departement of Pediatrics and 3Department of Pathology, University of

Leuven, Leuven, Belgium

Received June 27, 2012; Revised and Accepted September 18, 2012

Niemann–Pick type C is a lysosomal storage disease associated with mutations in NPC1 or NPC2, resultingin an accumulation of cholesterol in the endosomal–lysosomal system. Niemann–Pick type C has a clinicalspectrum that ranges from a neonatal rapidly fatal disorder to an adult-onset chronic neurodegenerative dis-ease combined with remarkably, in some cases, hematological defects such as thrombocytopenia, anemiaand petechial rash. A role of NPC1 in hematopoiesis was never shown. Here, we describe platelet functionabnormalities in three unrelated patients with a proven genetic and biochemical NPC1 defect. Their plateletshave reduced aggregations, P-selectin expression and ATP secretions that are compatible with the observedabnormal alpha and reduced dense granules as studied by electron microscopy and CD63 staining afterplatelet spreading. Their blood counts were normal. NPC1 expression was shown in platelets and megakar-yocytes (MKs). In vitro differentiated MKs from NPC1 patients exhibit hyperproliferation of immature MKswith different CD631 granules and abnormal cellular accumulation of cholesterol as shown by filipin stain-ings. The role of NPC1 in megakaryopoiesis was further studied using zebrafish with GFP-labeled thrombo-cytes or DsRed-labeled erythrocytes. NPC1 depletion in zebrafish resulted in increased cell death in the brainand abnormal cellular accumulation of filipin. NPC1-depleted embryos presented with thrombocytopenia andmild anemia as studied by flow cytometry and real-time QPCR for specific blood cell markers. In conclusion,this is the first report, showing a role of NPC1 in platelet function and formation but further studies areneeded to define how cholesterol storage interferes with these processes.

INTRODUCTION

Niemann–Pick diseases are autosomal recessive defects insphingomyelin/cholesterol metabolism and belong to thelarger family of lysosomal storage diseases. NP diseasestypes A and B are primary defects in acid sphingomyelinaseactivity, while NP diseases type C (C1 and C2) have defectiveintracellular processing of low-density lipoprotein-derivedcholesterol, resulting in an accumulation of unesterified chol-esterol in the endosomal–lysosomal system (1,2). NP type Chas a prevalence of �1 in 100 000 live births in western coun-tries (1,2). Ninety-five percent of the patients have mutations

in the NPC1 gene (MIM257220), while the remaining 5%have mutations in the NPC2 gene (MIM607625) (1–3).Both genes encode for endosomal–lysomal proteins withNPC1 being a transmembrane protein and NPC2 a solubleprotein (4). The exact functions of the NPC1 and NPC2 pro-teins are still unclear. NPC is currently considered a cellularcholesterol trafficking defect but in the brain, the prominentlystored lipids are gangliosides.

The clinical spectrum of NPC ranges from a neonatalrapidly fatal disorder to an adult-onset chronic neurodegenera-tive disease (5,6). The neurological involvement defines thedisease severity in most patients but is typically preceded by

∗To whom correspondence should be addressed at: Center for Molecular and Vascular Biology, KU Leuven, Campus Gasthuisberg, O&N1, Herestraat49, Box 911, 3000 Leuven, Belgium. Email: [email protected]

# The Author 2012. Published by Oxford University Press. All rights reserved.For Permissions, please email: [email protected]

Human Molecular Genetics, 2013, Vol. 22, No. 1 61–73doi:10.1093/hmg/dds401Advance Access published on September 24, 2012

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systemic signs as cholestatic jaundice in the neonatal period orisolated spleno- or hepatosplenomegaly in infancy or child-hood. Loss-of-function mutations in the NPC1 gene lead toa failure of the calcium-mediated fusion of endosomes withlysosomes, resulting in the accumulation of cholesterol andother lipids in late endosomes and lysosomes (7). The initiallaboratory diagnosis requires skin fibroblasts to demonstrateaccumulation of unesterified cholesterol in lysosomes afterstaining with filipin (8). Pronounced abnormalities areobserved in �80% of the cases, while mild to moderate altera-tions in the remainder. Genotyping confirms the diagnosis andis essential for prenatal diagnosis. Symptomatic managementof patients is crucial and a first product, Miglustat, has beengranted marketing authorization in Europe and several othercountries for specific treatment of the neurological manifesta-tions (9).

Interestingly, in 1971, a report described for the first timecoagulation and platelet changes in a NP disease patient(10). Thrombocytopenia, anemia and petechial rash werealso described immediately after birth in patients with fetalonset NPC (5). NPC1 mutant mice in a C57BL/6J backgroundpresented with red blood cell abnormalities and abundantghost erythrocytes in addition to abnormalities in whitecells, such as cytoplasmic granulation and neutrophil hyper-segmentation, that included lymphopenia and atypias (11). Re-markably, these blood cell abnormalities were not presentwhen this NPC1 mutant was present in the original BALB/cbackground. It was also suggested that NPC2 plays a role inhematopoiesis as this protein, in the presence of thrombopoie-tin (TPO), increased human multipotential myeloid progenitorcells (CFU-GEMM) and decreased granulocyte–macrophageprogenitors (CFU-GM) (12). A hematopoietic defect inNPC2 deficient patients has never been described probablybecause other molecules in the TPO system compensate forthe loss of NPC2. Very recently, thrombocytopenia was alsoreported in most patients with NP disease type B, whileanemia and leucopenia were less common in these patients(13). To our knowledge, there are no detailed studies on therole of NPC1 in megakaryopoiesis and/or platelet function inNPC1-deficient patients or under normal physiological condi-tions.

Platelets are easy to isolate in a non-active state and areideal cells to evaluate signal transduction, secretion and adhe-sion in hemostatic disorders, but we have shown that thesekinds of functional platelet studies are also useful to character-ize the unknown role of a protein that is involved in a well-known disorder that includes only a mild clinical or even sub-clinical platelet defect (14,15). For example, we have foundthat Duchenne muscular dystrophy patients with dystrophinmutations have an impaired collagen platelet activation anda disorganized cytoskeletal platelet membrane organizationthat accounts for their increased blood loss during spinalsurgery without generating a spontaneous platelet-relatedbleeding phenotype in these patients (16). We have thereforeperformed different functional platelet tests in three unrelatedNPC1 patients, including one with a prolonged Ivy bleedingtime. The morphology and functional activity of their plateletswere studied in detail. NPC1 expression studies were per-formed in megakaryocyte (MK) cell lines and primary cells.The effect of NPC1 was also studied during the in vitro

differentiation of peripheral blood cell-derived hematopoieticstem cells (HSCs) from patients versus control into MKsand early erythroblasts. Evidence for a role of NPC1 in plateletand erythrocyte formation was further addressed in transgeniczebrafish models after NPC1 depletion.

RESULTS

Clinical description of three unrelated NPC1 patients

Patient 1 is a girl who presented at the age of 10 days withcholestasis, which improved spontaneously and disappearedaround at the age of 6 months, and with hepatosplenomegaly.Her length and weight followed the third percentile. Head cir-cumference followed between the 10th and 25th percentiles.Developmental delay was apparent from the age of 18months. At the age of 2 years, ptosis and internal strabismuswere noticed. From the age of 26 months, there was neuro-logical regression, with the loss of motor and cognitive mile-stones. Tube feeding was started. Developmental age was 10months at the age of 31 months. There was progressive ap-pearance of pyramidal dysfunction with spasticity, as well asanterior horn dysfunction with muscular atrophy and tonguefasciculations. EMG showed an axonal polyneuropathy andcerebral MRI showed progressive cortical atrophy and peri-ventricular white matter changes. She died at the age of 3.5years after further neurological and general deterioration.Studies in cultured skin fibroblasts showed strong reductionin exogenous cholesterol from lipoprotein, confirming thediagnosis of Niemann–Pick type C (Table 1) (17). Compoundheterozygosity for two mutations in NPC1, S425X (paternal)and p.231Vfs (maternal), was found.

Patient 2 is a girl who presented on day 14 with cholestasis,which improved spontaneously, and hepatosplenomegaly.Electron microscopy of a liver biopsy at the age of 15months showed lysosomal myelin-like inclusions in the hepa-tocytes and Kupffer cells. Mild developmental delay was ap-parent from the age of 18 months. From the age of 47months, neurological regression, starting with vertical supra-nuclear gaze palsy, was noted. Cerebral MRI at that timewas normal. In the following months, progressive supranucleargaze palsy, ataxia, spasticity, kataplexia and mental deterior-ation became apparent, and therapy with Miglustat wasstarted at the age of 5.5 years. The SARA score for ataxiawas 18 at that time, and has remained stable until now (age7.5 years). Studies in cultured skin fibroblasts that quantifiedthe exogenous lipoprotein-derived cholesterol confirmed thediagnosis of Niemann–Pick type C (Table 1). Compound het-erozygosity for the frequent I1061T mutation and a N169Kmutation in NPC1 was found.

Patient 3 is a boy who presented in the neonatal period withcholestasis, which improved spontaneously, hepatosplenome-galy and bilateral inguinal hernia. Growth and developmenthave been normal (current age is 6 years). Electron micros-copy of a liver biopsy at the age of 3 months showed lyso-somal myelin-like inclusions in the hepatocytes and Kupffercells. Biochemical studies in cultured skin fibroblasts con-firmed the diagnosis of Niemann–Pick type C (Table 1).Homozygosity for the frequent I1061T mutation in NPC1was found. Both parents are carriers.

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Table 1. Patients’ characteristics: NPC1 mutations, hematological counts and functional platelet studies

Normal values Hematological counts Functional platelet testsWBC RBC MCV PLT MPV Aggregation

epinephrine,1.25 mM

Aggregationcollagen,2 mg/ml(0.5 mg/ml)

AggregationADP, 10 mM

ATP secretioncollagen,2 mg/ml

ATP secretionADP, 10 mM

Electron microscopy

Patients Age(years);gender

NPC1 mutationsa

Cholesterol3H-oleate (16)a

2950+1200 pmol/mg

5.5–15.5 × 109/l

3.9–5.3 × 1012/l

75–87 fL

150–450 × 109/l

9–13fL

Amplitude,71.8+4.3%(n ¼ 40)

Amplitude,78.9+8.6%(n ¼ 40)

Amplitude,75.1+10.8%(n ¼ 40)

2.0+0.8 mM

(n ¼ 40)1.3+0.7 mM

(n ¼ 40)

Patient 1 Died3.5; F

S425X V231fs75 pmol/mg

10.112.5

4.64.82

7477

159122

10.010.1

20b 65 (52) 60 0.5 0.5 Lysosomal alphagranules, somewith reducedcontent, reduceddense granules

Patient 2 8; F I1061T N169K,10 pmol/mg

8.49.8

5.25.2

7366.5

177271

10.010.5

24b 56b (28) 55 0.28b 0.22 Normal alphagranules, reduceddense granules

Patient 3 7; M I1061T I1061T30 pmol/mg

5.88.0

4.64.3

7877

141187

8.67.8

34b 58b (39) 43b 0.24b 0b Lysosomal alphagranules, somewith reducedcontent, somegiant polygonalalpha granules,reduced densegranules

WBC, white blood cells; RBC, red blood cells; MCV, mean corpuscular volume; PLT, platelets; MPV, mean platelet volume.aPerformed by Dr M.T. Vanier.bz-Score+2.

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Morphological and functional platelet studies in NPC1patients

Interestingly, in addition to her other phenotypic featuresdescribed above, patient 1 also presented with a prolongedIvy bleeding time of 10 min. Her standard coagulation para-meters were normal but functional platelet testing at multipleoccasions showed reduced aggregation and secretion values.The three patients have in common a reduced aggregation re-sponse to low concentrations of Horm collagen and an absentsecondary response to epinephrine (aggregation amplitudesare reported in Table 1). Aggregation to high doses of ADPand collagen were normal. These results are typically seenin platelets with secretion defects (18). Indeed, ATP secretionwith collagen and especially ADP was reduced for thepatients. Flow cytometry showed decreased expression levelsof CD62P (P-selectin that is stored in platelet alpha granules)for patients (P2; 2760 and P3; 2563) versus control platelets(C1; 3696 and C2; 3548) after full activation with convulxin.No expression differences by flow cytometry were seen for themain platelet receptors GPIIIa (CD61), GPIIb (CD41) andGPIb/IX (CD42a/CD42b). Electron microscopy showed areduced number of dense granules in the patients’ plateletsand, patients 1 and 3, also have some giant and lysosomal-likealpha granules (Table 1 and Fig. 1A). Some platelets almosthave no granules in these patients. Staining of platelets spreadon fibrinogen for the lysosomal marker CD63 showed a morecentralized and fused localization of the platelets granules inthe NPC1 patients P2 and P3 compared with control platelets(Fig. 1B and C). CD63 is a typical marker for platelet denseand some type of alpha granules (19). Hematological countsshowed platelet numbers and mean platelet volume (MPV) atthe lower normal limits (Table 1). Patients had normal countsfor all types of white blood cells, reticulocytes and erythrocytes,but the mean corpuscular volume (MCV) was always low.Immunoblot analysis of NPC1 expression in platelets wasreduced in the patients (Fig. 1D). The platelet granule secretiondefect in our NPC1 patients only presented with a subclinicalplatelet defect as these patients did not yet present with anyspontaneous or trauma-related bleeding problems yet.

NPC1 expression in human (erythro)megakaryocytic cellsand platelets

Platelets with ultrastructural abnormalities that lead to granuleand secretion defects in combination with lower plateletcounts are often a result of a defective megakaryopoiesis.Therefore, we studied the expression of NPC1 in human MKcell lines (MEG01 and CHRF), in human erythromyeloblas-toid cells (K562) and in in vitro differentiated MKs fromCD34+ human HSCs versus normal platelets using immuno-blot analysis (Fig. 2A). NPC1 expression was showed for allcell lines and primary MKs though with a different intensityof the higher (glycosylated form) and lower molecularweights for NPC1 (190 and 170 kDa) (20).

HSC differentiation studies in NPC1 patients

Since it was not possible to obtain bone marrow from thesepatients to study the role of NPC1 in megakaryopoiesis and

platelet production, we isolated HSCs from the peripheralblood of patients P2 and P3 to compare their in vitro differen-tiation into MKs and erythroblasts (E) with a control subject.The differentiation experiments were performed in duplicatestarting each from different amounts of HSCs (Fig. 2B). Thepatients had increased colony numbers of differentiated MKsat days 6 and 10 (Fig. 2B). There were no differences incolony numbers between patients and control during erythro-blast differentiation at day 6, while the counts at a laterstage were not possible because the cells were overgrown.The MK colonies at day 10 were also larger and containedmore single cells for the patients compared with the control(Fig. 2C). However, the MK cultures for the control typicallyshowed proplatelet-forming MKs that were not observed in thecultures from P2 and P3. A hyperproliferation of only imma-ture MKs but almost no mature proplatelet-forming MKs istypically seen in patients and mice with a genetic defect thatcauses thrombocytopenia with granule defects such asGATA1 deficiency (21,22). To further confirm that all col-onies are MKs, a staining with an anti-GPIIIa antibody wasperformed on these colonies. These staining showed again ahyperproliferation of small immature MKs for P2 and P3,while the control condition clearly showed proplatelet-formingMKs (Fig. 3A). The duplicate MK cultures were stained withCD63 that typically reacts with the dense and some alphagranules in mature MKs but also with the granule precursor,the multivesicular bodies (MVBs) in immature MKs (19).Figure 3B showed a different kind of granular pattern thatcould be due to the dense/alpha granule staining in controlMKs versus MVBs staining in MKs from P2 and P3.Finally, filipin staining to reveal the cholesterol distributionin MKs was performed and showed some typical accumulationin MKs derived from the patients but in the control condition(Fig. 3C). We decided to confirm the effect of NPC1 on MKdifferentiation further using our available transgenic zebrafishmodels with fluorescently labeled blood cells (21,23) via mor-pholino (MO)-induced NPC1 depletion.

NPC1 is highly expressed in the developing brain, eyesand yolk syncytial layer of zebrafish embryos

To identify zebrafish NPC1, the human NPC1 amino acid se-quence was used in a TBLASTP search and a 1278-amino acidprotein was identified with 68% identity to the human NPC1protein that corresponds to a gene with accession numberNM_001243875.1. This sequence was later found to be iden-tical to the NPC1 gene used by others to characterize the roleof NPC1 in early embryonic zebrafish development (24). RT–PCR analysis confirmed NPC1 expression during differentstages of zebrafish embryonic development (Fig. 4A). Whole-mount in situ hybridization (WISH) was used to determine thespatiotemporal expression pattern and NPC1 was detected at24 hpf in developing brain and eyes but with the highestexpression in the yolk syncytial layer (YSL) (Fig. 4B).

NPC1 depletion results in increased cell death in the brainand abnormal cellular accumulation of cholesterol

Schwend et al. (24) found that NPC1 depletion did not disruptearly cell fate or survival, but early morphogenetic movements


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were delayed and increased cell death was detected. We usedanother NPC1 MO but showed that our ATG blocking MOcompletely inhibits NPC1 protein expression even at lowdose of MO (200 mM) (Fig. 4C). In addition, we also detected

embryos at 24 hfp with malformed heads and dysmorphicbrain and eyes with increased levels of apoptosis in theseregions, in a MO dose-dependent manner (Fig. 4D). Filipinstaining clearly showed an accumulation of free sterols at

Figure 1. Platelets from NPC1 patients. (A) Electron microscopy of platelets from NPC1 patients shows platelets with a paucity of granules (arrows), absentdense granules, lysosomal-like (black arrowheads) and giant (white arrowhead) alpha granules. (B) CD63 immunostaining in spread platelets shows a granularpattern, but granules are more centralized and fused for the NPC1 patients (P2 and P3) compared with control platelets (C1 and C2). (C) Quantification of theoverall coverage and the mean particle size for the CD63 staining in platelets from controls and patients. Bars represent the mean and standard deviations.∗P-value of 0.0006 by Student’s t-test. (D) Immunoblot analysis showing NPC1 expression in platelets from three control subjects and three NPC1 patients.Beta tubulin is the loading control.

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the plasma membrane of all cells (Fig. 4E), hereby confirmingthat NPC1 depletion in zebrafish also results in a defectiveintracellular processing of cholesterol.

NPC1 depletion leads to thrombocytopenia and mildanemia

To study the role of NPC1 in megakaryopoiesis, CD41 trans-genic zebrafish with GFP-labeled thrombocytes were used.Injected CD41 embryos were analyzed by life-screening forGFPhigh&low-labeled thrombocytes in the caudal hematopoietictissue (CHT) region at 72 hpf (Fig. 5A). According to the se-verity of the phenotype, embryos were divided into three sub-classes: a severe phenotype with no thrombocytes, a mildphenotype with only few (n , 10) thrombocytes and anormal phenotype with many non-circulating thrombocytesin the CHT region and some circulating thrombocytes. Evenat a low dose of MO (200 mM), 82% of the screenedembryos showed an almost complete absence of thrombocytesat 3 days post-fertilization (dpf). At a higher dose of MO (400and 800 mM), more than 90% of the embryos had severethrombocytopenia and all these embryos died at 5 dpf(Fig. 5A). In addition, immunoblot analysis showed reducedGFP levels in pooled samples (n ¼ 30 embryos/condition)from NPC1-depleted embryos for 200 and 400 mM NPC1

MO compared with Crl MO-injected embryos (Fig. 5B).Further quantification of thrombocytes was determined byflow cytometry, measuring a total of 900 versus 183GFPhigh&low-labeled thrombocytes in Crl MO versus NPC1MO-injected embryos, respectively (Fig. 5C). Especially, theGFPhigh-labeled thrombocytes were absent in the NPC1 condi-tion. Interestingly, FSC plots also showed differences inthrombocyte size distribution. Since our NPC1 patients notonly had lower normal limits of platelet count and MPV butalso relatively low values for MCV (Table 1) and becauseanemia was described in NPC1-deficient mice in a C57BL/6J background (11), we also evaluated the NPC1 depletionusing GATA1 transgenic fish for which erythrocytes areDSred labeled. Flow cytometric analysis of Crl MO versusNPC1 MO (200 mM) injected embryos showed a total of5767 versus 3022 DSred-labeled erythrocytes (Fig. 5C).Again, the FSC plot of the DSred-positive erythrocytes wasslightly different between the two groups.

Real-time RT–PCR confirmed a 68% reduction in relativeexpression of GATA1 after NPC1 depletion compared withcontrols (Fig. 5D). Relative expression of CD41 was also mea-sured by real-time PCR, but CD41 levels appeared to be toolow for detection after NPC1 depletion. It is assumed thatthe GFPhigh labeled (CD41 detection by RT–PCR) representthe mature thrombocytes, while the GFPlow population

Figure 2. NPC1 expression in MKs and erythroblasts. (A) Immunoblot analysis for NPC1 in K562, MEG01, CHRF, platelets (PLT) and differentiated MKs fromcontrol CD34+ HSCs (days 2, 6 and 10). Equal amounts of 100 mg total lysate were loaded for each condition. Membranes were stripped for Evi1 blotting. (B)Total amount of colony-forming cells (CFC) on days 6 and 10 from CD34+ HSCs from control (white bars) or patients P2 and P3 (gray bars). Bars with filled ordotted lines present the colonies starting from 0.5 and 1 × 104 HSC for MKs or 0.5 and 1 × 103 HSC for erythroblasts (E), respectively. Each condition was donein duplicate. Bars represent the mean. (B) Pictures of the CFU-MK colonies for control and patients at day 10. The arrows indicate the proplatelet-forming MKs.Magnification ×20.


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includes thrombocyte precursors and stem cells (25). Wedetected a strongly reduced but not absent level of GFP byimmunoblot analysis (Fig. 5B) and mainly absent populationof GFPhigh-labeled thrombocytes by flow cytrometry afterNPC1 deletion. This would mean that the remaining populationof GFPlow-labeled thrombocyte precursors and stem cells areonly partially affected by NPC1 depletion and represent thepopulation that is still detected by GFP immunoblot analysis.While the GFPhigh-labeled thrombocytes were not detectedafter NPC1 depletion (CD41 RT–PCR negative), GFPlow-labeled precursors and stem cells are also reduced (by 74%)but not absent as screened by RT–PCR for Scl (Fig. 5D).From these results, we concluded that the development of thecomplete myeloid lineage is affected by NPC1 depletion.

DISCUSSION

We have studied a patient (P1) with a prolonged Ivy bleedingtime of 10 min and showed abnormal platelet morphology andfunctional aggregation and secretion tests before she was

diagnosed with the NPC1 disease. Literature evidence for arole of NPC1 in hemostasis and/or hematopoiesis is verylimited. Although some evidence was present for a regulatoryrole of NPC1 in platelet function (6,10) and platelet and redblood cell formation (6,11), this definitely required morestudies. We therefore studied platelet count, function andmorphology in three unrelated Niemann–Pick patients witha proven genetic and biochemical NPC1 defect. It was clearform these studies that the NPC1 defect inhibits normal plate-let secretion and subsequent stimulation as shown by an absentresponse in the epinephrine-induced aggregation, typicallypresent in patients with dense granule platelet abnormalities(18). Abnormalities in granule number and/or contentusually originate from a defective granulopoiesis in the MKas for example found for GATA1 deficiency (21,22) or theGray platelet syndrome (26–28). Since it was not possibleto study megakaryopoiesis using bone marrow from NPC1patients, we studied in vitro MK and erythroblast differenti-ation experiments starting from peripheral blood-derivedHSCs from patients and an NPC1-depleted zebrafish model.

Figure 3. Immunostainings of differentiated MKs at day 10. (A) GPIIIa immunostaining in MKs from control (C) and patients (P2 and P3). Only the controlcondition presents with the typical proplatelet-forming MKs at day 10 (upper right panels). Scale bars represent 10 mm. (B) CD63 immunostaining in MKs fromcontrol (C) and patients (P2 and P3). The size and distribution of the granules is different between control and patients. Scale bars represent 10 mm. (C) Filipinimmunostaining in MKs from control (C) and patients (P2 and P3) that only showed accumulation in the patients’ cells (white arrows). Scale bars represent10 mm.


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During the HSC differentiation experiments, we could notdetect an early effect on the erythropoiesis, but NPC1 defi-ciency has an effect on the differentiation of MKs. Patientsshow a hyperproliferation of early MKs that do no’t seem toform the terminally differentiated proplatelet-forming MKs.We and others have described a similar megakaryopoiesisdefect in thrombocytopenia patients with GATA1 defects

and GATA1 knockout mice (21,22) that also present witha defective granulopoiesis and associated platelet granulesecretion defect. Therefore, these results together with theelectron microscopy data are strongly suggestive of a plate-let formation defect that causes a secondary functionaldefect (mainly at the levels of the granules) in NPC1disease.

Figure 4. NPC1 expression in zebrafish and phenotype analysis of NPC1-depleted embryos. (A) RT–PCR showing NPC1 expression during different develop-mental stages of zebrafish embryos (30 embryos pooled for each stage). (B) WISH (30 embryos analyzed for each condition; WISH experiment performed induplicate) in zebrafish shows NPC1 expression in the head, brain and eye at 24 hpf, using a zebrafish-specific antisense probe (left upper panel; arrow) whencompared with the sense probe, used as a negative control (right upper panel). In the lower panels, a magnification of the tail region is shown to indicate theNPC1 expression in the YSL, with the specific zebrafish antisense probe (left lower panel; arrowhead) but not using a sense probe (right lower panel). (C) Immu-noblot analysis for NPC1 in control (Crl) and NPC1 MO injected (200 mM) embryos (50 mg loaded from 30 deyolked, pooled and lysed embryos per condition).(D) Head phenotype analysis after NPC1 depletion using NPC1 MO at a concentration of 800, 400 and 200 mM and Crl MO, used as a negative control. A total of100 embryos (pooled from three independent experiments) were live screened and divided according to the severity of the phenotype as indicated. Severe pheno-type comprises embryos with severely dysmorphic brain and eyes and a high level of apoptosis, clearly visible as a large amount of grey cells in this region. Mildphenotype comprises embryos with brain ventricles that are still visible but showing serious malformation with apoptosis in the head and eyes. (E) Multiphotonimages of filipin staining in control and NPC1-depleted embryos. Filipin staining in control embryos (left panel) shows equal distribution of cholesterol at theplasma membrane, whereas in NPC1-depleted embryos (right panel) an accumulation of free sterols at the plasma membrane as well as in the cells is visible(experiment performed in duplicate for 30 embryos).


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We used CD41 transgenic zebrafish with GFP-labeledthrombocytes to show that the thrombocytopenia, obviouslyobserved in some NPC1 patients (5), is a direct consequence

of the genetic NPC1 defect and not caused by an increaseddestruction of platelets by their enlarged spleen and/or liver.MOs are only active during the first 4–7 days after injection,

Figure 5. Severe thrombocytopenia and anemia in NPC1-depleted embryos. (A) Thrombocyte phenotype after NPC1 depletion using NPC1 MO at concentra-tions of 800, 400 and 200 mM versus a Crl MO as negative control. A total of 100 embryos (pooled from three independent experiments) were live screened anddivided according to the severity of the phenotype: severe (green), mild (red) or normal (blue) phenotypes. Stereomicroscope images of the CHT region at 72 hpfto visualize GFP-labeled thrombocytes (×20 original magnification) to represent each phenotype are also shown. (B) Immunoblot analysis of GFP expression forCD41 transgenic embryos (50 mg loaded from 30 deyolked, pooled and lysed embryos per condition, experiment was performed in duplicate with similar find-ings). NPC1 MO was injected at the indicated concentrations (mM). (C) Flow cytometic analysis of transgenic CD41+ (upper panels) or GATA1 (lower panels)embryos injected with 200 mM of NPC1 MO or Crl MO. At 72 hpf, 30 embryos for each condition were pooled and the number of GFP-positive cells was deter-mined for a total of 500 000 total events (upper panels) or the number of DSred-positive cells was determined for a total of 100 000 total events (lower panels).These experiments were each performed in duplicate with similar findings. (D) Real-time RT–PCR using Syber Green I fluorescence and specific zebrafishprimer sets for Scl, GATA1 and CD41. All relative expression levels were normalized to the endogenous reference control elfa. Graph shows the relative ex-pression level of each marker for NPC1-depleted embryos (200 mM MO) compared with Crl MO-injected embryos. Complete experiment was performed intriplicate.


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which is synchronic with the time period of thrombocyte for-mation (usually clearly visible in the CHT region at 3 dpf),while the liver in zebrafish is not yet functional within thistime frame (29). The exact stage of zebrafish spleen formationis not known, but since it is part of the digestive track, fullyfunctional only at 4 dpf (30), it is unlikely that a platelet de-struction would be the origin of the thrombocytopenia,already observed at 3 dpf. This would indicate that NPC1has a major regulatory role in thrombocyte formation. Wedid observe severe thrombocytopenia in NPC1-depletedembryos, while the thrombocytopenia is typically mild infetal onset NPC1 patients (5). Platelet counts were even justwithin normal limits in our NPC1 patients. This differencecan be explained by the fact that a complete knockout in zeb-rafish is different from having autosomal recessive NPC1mutations. In addition, we of course studied fetal and notadult hematopoiesis in zebrafish. HSCs in zebrafish migrateto and seed the CHT at 48 hpf, which acts as a transient hem-atopoietic site that gives rise to erythroid, myeloid and throm-boid cells (31). The CHT is equivalent to mouse fetal liver orplacenta. HSCs from the aorta-gonad-mesonephros region col-onize kidney only around 48 hpf and this kidney marrow isfunctionally similar to mammalian bone marrow in that itgives rise to all blood lineages for the larval and adult zebra-fish. NPC1 could therefore have a different role in fetal versusadult hematopoiesis.

Since NPC1 depletion in zebrafish has a role in fetal throm-bopoiesis, it is expected that especially the lipid mobilizationduring early stages of development is important for inducing athrombocytopenia phenotype. During the first week of larvaldevelopment, the yolk contains the main supply of lipidsand other nutrients needed for larval development. At thesurface of the yolk, a syncytium, named the YSL, demarcatesthe extra-embryonic yolk and plays a crucial role in embryonicpatterning and morphogenesis, besides its function in thetransfer of nutrients to the embryo (32). We and others (24)showed that zebrafish NPC1 is highly expressed in this import-ant lipid transfer region. Interestingly, this YSL region is invery close proximity to the CHT region for fetal hematopoieis,but the link between defective lipid transfer and thrombocyteformation deserves further detailed studies. It is only knownthat disturbed cholesterol levels affect bone marrow cellmobilization (33), which indeed could result in changes ofblood lineage development. Interestingly, the NPC1 depletionin zebrafish was not thrombocyte specific as we also found ab-normalities in GATA1 and Scl expressing erythrocytes andstem cells, respectively. But also in some fetal onset NPC1patients, anemia and white blood cell abnormalities weredescribed (5). Our patients had normal erythrocyte and whiteblood cell counts but have borderline low MCV and at someoccasions, lower hemoglobin levels.

Several publications recently raised the possibility that notcholesterol but disturbed sphingosine storage is the primarycause of the NPC1 disease. Sphingosine storage in the acidiccompartment would lead to calcium depletion in these orga-nelles, which then also results in cholesterol, sphingomyelinand glycospingolipid storage (34). They also claim that thiscalcium imbalance is a new target for therapeutic intervention,since treatment of NPC1-deficient mice with curcumin, aSERCA antagonist, increased cytosolic calcium, thereby

normalizing sphingolipid and cholesterol levels. We treatedNPC1-depleted zebrafish with 5 and 7 mM curcumin in orderto try to rescue the thrombocytopenic phenotype, but we didnot observe any correction (data not shown). However, thepharmacokinetics of curcumin in zebrafish were recentlyshown to be different compared from those of rats and evento cause developmental defects including shorter body axiswith hooked tails, spinal column curving, edema in pericardialsac and retarded yolk sac resorption (35). The questionwhether disturbed cholesterol levels or imbalanced calciumwith sphingosine storage plays a dominant role in theNPC1-depleted zebrafish phenotypes remains open.

In conclusion, this study shows morphological and function-al platelet abnormalities in three unrelated NPC1 patients,more specifically, platelets with granulopoiesis defects typic-ally arising during megakaryopoiesis. In addition, NPC1 de-pletion in zebrafish further supports an important role forNPC1 in thrombocyte formation. We show that NPC1 deple-tion does not only affect megakaryopoiesis but also leads toanemia and a general defect in stem cell development;however, further studies are needed to link these phenotypesto a defective lipid storage metabolism.

MATERIALS AND METHODS

Patient study

Informed consent was obtained from all participants and/ortheir legal representatives. The Ethics Committee of the Uni-versity of Leuven approved the study.

Hematological counts and functional platelet studies

EDTA anticoagulated blood was analyzed on an automatedcell counter (Cell-Dyn 1300; Abbott Laboratories, AbottPark, IL, USA) to determine blood cell counts. Platelet-richplasma (PRP) was prepared by centrifugation (15 min at150g) of whole blood anticoagulated with 3.8% trisodiumcitrate (9:1). The PRP was used for functional plateletstudies and electron microscopy, as described previously(18). In short, aggregation studies were carried out byadding Horm collagen, epinephrine or ADP at the indicatedconcentrations. ATP secretion tests were performed afterstimulation of platelets with Horm collagen 2 mg/ml andADP 10 mM. Flow cytometry to measure expression ofCD61, CD41, CD42a, CD42b and CD62P was performed asdescribed previously (36).

Platelet spreading and staining

Platelet spreading and staining was performed as described(37). Platelets were incubated with a 1:50 anti-CD63 (BDBiosciences PharMingen, San Diego, CA, USA) of anti-GPIIIaantibody overnight at 48C. After five washing steps with PBS,cells were incubated with a specific secondary antibodydiluted 1:200 (Alexa Fluor; Life technologies) together withphalloidin rhodamine (Sigma, Poole, UK) for F-actin stainingduring 45 min at 378C. Analysis was done using a Zeiss Axio-vert 100M confocal microscope (Carl Zeiss Inc., Gottingen,Germany). Fluorescent intensities were quantified using the


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Java image processing program ImageJ 1.34g (NIH Imagesoftware).

Cell cultures and differentiation of CD341 HSCs

Human megakaryocytic cell lines MEG01 and CHRF andhuman erythromyeloblastoid cells K562 were grown asdescribed previously (38,39). Human CD34+ cells were iso-lated from peripheral blood by magnetic cell sorting (MiltenyiBiotec, Auburn, CA, USA) and were cultured in the StemSpanSFEM medium (Stem Cell Technologies, Vancouver,Canada), supplemented with 20 ng/ml TPO, 10 ng/ml SCF,10 ng/ml IL-6 and 10 ng/ml FLT-3 (Peprotech, London, UK)as described previously (38).

MKs and erythroblasts colony assays and stainings

Human CD34+ cells were isolated from peripheral blood bymagnetic cell sorting (Miltenyi Biotec, Bergisch Gladbach,Germany). Human CD34+ cells (0.5 and 1 × 104 and 0.5and 1 × 103; respectively) were cultured in duplicate inMegacult-C 04973 and Methocult 04964, according to themanufacturer’s instructions (Stem Cell Technologies). Thetotal number of MK and erythroblasts colonies were counted6 and 10 days later using a light microscope (Leica DMRBE, Wetzlar, Germany) in cultures performed in duplicatefor each start condition of HSCs. Slides were processed forstained as described by the manufacturer (Stem Cells). Stain-ings of MK cultures were done with the monoclonal mouseanti-GPIIIa antibody and 50 mg/ml filipin (Sigma ChemicalCo., St Louis, MO, USA) or with anti-CD63 and photographedat 40×/0.5 magnification with a Zeiss Axiovert microscopeand captured with Zeiss Axiovision.

Immunoblot analysis

Protein lysates were obtained from human cell lines (K562,MEG01 and CHRF), in vitro differentiated human MKs,human platelets and control MO versus NPC1 MO-injectedzebrafish embryos [20 deyolked embryos per condition asdescribed; (18)]. Protein fractions were resolved by SDS/PAGE and blots were incubated with a rabbit polyclonalanti-NPC1 antibody, anti-tubulin, anti-EVI1 (all from SantaCruz Biotechnology, Inc., Heidelberg, Germany) or a goatpolyclonal anti-GFP antibody (Rockland Immunochemicals,Inc., Gilbertsville, PA, USA). Membranes were next incubatedwith HRP-conjugated secondary antibody and staining wasperformed with the ECL detection reagent (GE Healthcare).

MO injection in zebrafish

Tg(cd41:EGF) (25) and Tg(gata1:dsred) (40) zebrafishembryos were injected at the 1-cell stage with a start codonMO ATG MO (5′-TGTGGTTTCTCCCCAGCAGAAGCAT-3′) at the indicated concentrations (200–800 mM). Off-targeteffects were assessed by including a standard control MO orbuffer-injected embryos. MOs were designed by Gene-ToolsLLC (Philomath, OR, USA). Embryos were life-screened at1, 2 and 3 dpf using a Zeiss Lumar V12 and images were cap-tured with a Zeiss Axiocam MRc camera using AxioVision

software (Carl Zeiss, Jena, Germany). All animal protocolswere approved by the Ethical Committee of the KatholiekeUniversiteit Leuven. Tg(cd41:EGF) zebrafish were a giftfrom Dr L. Zon (Hematology Division, Brigham andWomen’s Hospital’s, Boston, MA, USA) and Tg(gata1:dsred)zebrafish were a gift of Dr C. Verfaillie (Stem Cell Institute,KULeuven).

WISH of zebrafish

Total RNA was extracted from zebrafish embryos at variousstages of development to amplify a RT–PCR fragment ofthe zebrafish (NM_001243875.1) NPC1 orthologue. ThisNPC1 fragment was cloned into pGEM-T easy (Promega)for making sense and antisense probes by linearization ofthe plasmid and subjection to in vitro transcription using themMessage mMachine High Yield Capped RNA kit(Ambion). Primer sequences are available in the Supplemen-tary Material, Table. Developmental staging of injected zebra-fish was determined by somite number and embryos werefixed in 4% paraformaldehyde. Assays for RNA expressionusing WISH were performed as described previously (41).

Filipin staining in zebrafish embryos

For filipin staining of free sterols, embryos were fixed in 4%paraformaldehyde for 30 min, washed twice in PBS, stainedwith 50 mg/ml filipin in PBS solution for 30 min and washedagain twice with PBS. Images were captured on a ZeissLSM510 confocal laser scanning microscope.

Flow cytometric analysis of zebrafish thrombocytesand erythrocytes

Control versus NPC1 MO-injected Tg(cd41:EGFP) andTg(gata1:dsred) embryos were dechorionated 72 hfp and pro-cessed as described previously (42). We used the Cell Divasoftware for two-color immunofluorescence acquisition on aFACSCanto II flow cytometer (BD Biosciences) and for dataanalysis to determine the percentage of, respectively,GFP-positive thrombocytes and dsred-positive erythrocytes.

SUPPLEMENTARY MATERIAL

Supplementary Material is available at HMG online.

ACKNOWLEDGEMENTS

Biochemical and genetic analyses were performed by Dr M.T.Vainer (Laboratoire Gillet-Merieux; Groupe HospitalierLyon-Est; Lyon-Bron, France).

Conflict of Interest statement. None declared.

FUNDING

This work was supported by the ‘Excellentie financieringKULeuven’ (EF/05/013), by research grants G.0490.10N andG.0743.09 from the Fund for Scientific Research, Flanders


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http://hmg.oxfordjournals.org/lookup/suppl/doi:10.1093/hmg/dds401/-/DC1



(FWO-Vlaanderen, Belgium), GOA/2009/13 from the Re-search Council of the University of Leuven (OnderzoeksraadKULeuven, Belgium). C.V.G. is holder of a clinical-fundamental research mandate of the Fund for ScientificResearch-Flanders (F.W.O.-Vlaanderen, Belgium and of theBayer and Norbert Heimburger (CSL Behring) Chairs. L.R.received a clinical doctoral grant from F.W.O.-Vlaanderen.

REFERENCES

1. Vanier, M.T. and Millat, G. (2003) Niemann-Pick disease type C. Clin.Genet., 64, 269–281.

2. Vanier, M.T. (2010) Niemann-Pick disease type C. Orphanet J. Rare Dis.,5, 16.

3. Karten, B., Peake, K.B. and Vance, J.E. (2009) Mechanisms andconsequences of impaired lipid trafficking in Niemann-Pick typeC1-deficient mammalian cells. Biochim. Biophys. Acta, 1791, 659–670.

4. Ikonen, E. (2008) Cellular cholesterol trafficking andcompartmentalization. Nat. Rev. Mol. Cell. Biol., 9, 125–138.

5. Spiegel, R., Raas-Rothschild, A., Reish, O., Regev, M., Meiner, V.,Bargal, R., Sury, V., Meir, K., Nadjari, M., Hermann, G. et al. (2009) Theclinical spectrum of fetal Niemann-Pick type C. Am. J. Med. Genet. A,149A, 446–450.

6. Sturley, S.L., Patterson, M.C., Balch, W. and Liscum, L. (2004) Thepathophysiology and mechanisms of NP-C disease. Biochim. Biophys.Acta, 1685, 83–87.

7. Rosenbaum, A.I. and Maxfield, F.R. (2011) Niemann-Pick type C disease:molecular mechanisms and potential therapeutic approaches.J. Neurochem., 116, 789–795.

8. Vanier, M.T., Rodriguez-Lafrasse, C., Rousson, R., Duthel, S., Harzer, K.,Pentchev, P.G., Revol, A. and Louisot, P. (1991) Type C Niemann-Pickdisease: biochemical aspects and phenotypic heterogeneity. Dev.Neurosci., 13, 307–314.

9. Perez-Poyato, M.S. and Pineda, M. (2011) New agents and approaches totreatment in Niemann-Pick type C disease. Curr. Pharm. Biotechnol., 12,897–901.

10. Del Principe, D., Giardini, O., Ballati, G. and Torroni, A. (1971)Coagulation inhibiting factor and platelet changes in a patient withNiemann-Pick disease. Haematologica, 56, 36–48.

11. Parra, J., Klein, A.D., Castro, J., Morales, M.G., Mosqueira, M., Valencia,I., Cortes, V., Rigotti, A. and Zanlungo, S. (2011) Npc1 deficiency in theC57BL/6J genetic background enhances Niemann-Pick disease type Cspleen pathology. Biochem. Biophys. Res. Commun., 413, 400–406.

12. Heo, K., Jariwala, U., Woo, J., Zhan, Y., Burke, K.A., Zhu, L., Anderson,W.F. and Zhao, Y. (2006) Involvement of Niemann-Pick type C2 proteinin hematopoiesis regulation. Stem Cells, 24, 1549–1555.

13. Hollak, C.E., de Sonnaville, E.S., Cassiman, D., Linthorst, G.E., Groener,J.E., Morava, E., Wevers, R.A., Mannens, M., Aerts, J.M., Meersseman,W. et al. (2012) Acid sphingomyelinase (Asm) deficiency patients in TheNetherlands and Belgium: disease spectrum and natural course inattenuated patients. Mol. Genet. Metab., Epub ahead of print.

14. Freson, K., Labarque, V., Thys, C., Wittevrongel, C. and Van Geet, C.(2007) What’s new in using platelet research? To unravel thrombopathiesand other human disorders. Eur. J. Pediatr., 166, 1203–1210.

15. Van Geet, C., Izzi, B., Labarque, V. and Freson, K. (2009) Human plateletpathology related to defects in the G-protein signaling cascade. J. Thromb.Haemost., (Suppl. 1), 282–286.

16. Labarque, V., Freson, K., Thys, C., Wittevrongel, C., Hoylaerts, M.F., DeVos, R., Goemans, N. and Van Geet, C. (2008) Increased Gs signalling inplatelets and impaired collagen activation, due to a defect in thedystrophin gene, result in increased blood loss during spinal surgery.Hum. Mol. Genet., 17, 357–366.

17. Vanier, M.T., Rodriguez-Lafrasse, C., Rousson, R., Gazzah, N., Juge,M.C., Pentchev, P.G., Revol, A. and Louisot, P. (1991) Type CNiemann-Pick disease: spectrum of phenotypic variation in disruption ofintracellular LDL-derived cholesterol processing. Biochim. Biophys. Acta,1096, 328–337.

18. Di Michele, M., Thys, C., Waelkens, E., Overbergh, L., D’Hertog, W.,Mathieu, C., De Vos, R., Peerlinck, K., Van Geet, C. and Freson, K.

(2011) An integrated proteomics and genomics analysis to unravel aheterogeneous platelet secretion defect. J. Proteomics, 74, 902–913.

19. Polasek, M.D. (2005) Platelet secretory granules or secretory lysosomes?Platelets, 16, 500–501.

20. Millat, G., Marcais, C., Tomasetto, C., Chikh, K., Fensom, A.H., Harzer,K., Wenger, D.A., Ohno, K. and Vanier, M.T. (2001) Niemann-Pick C1disease: correlations between NPC1 mutations, levels of NPC1 protein,and phenotypes emphasize the functional significance of the putativesterol-sensing domain and of the cysteine-rich luminal loop. Am. J. Hum.Genet., 68, 1373–1385.

21. de Waele, L., Freson, K., Louwette, S., Thys, C., Wittevrongel, C., deVos, R., Debeer, A. and Van Geet, C. (2010) Severe gastrointestinalbleeding and thrombocytopenia in a child with an anti-GATA1autoantibody. Pediatr. Res., 67, 314–319.

22. Vyas, P., Ault, K., Jackson, C.W., Orkin, S.H. and Shivdasani, R.A.(1999) Consequences of GATA-1 deficiency in megakaryocytes andplatelets. Blood, 93, 2867–2875.

23. Louwette, S., Labarque, V., Wittevrongel, C., Thys, C., Metz, J., Gijsbers,R., Debyser, Z., Arnout, J., Van Geet, C. and Freson, K. (2012) Regulator ofG-protein signaling 18 controls megakaryopoiesis and the cilia-mediatedvertebrate mechanosensory system. FASEB J., 26, 2125–2136.

24. Schwend, T., Loucks, E.J., Snyder, D. and Ahlgren, S.C. (2011)Requirement of Npc1 and availability of cholesterol for early embryoniccell movements in zebrafish. J. Lipid Res., 52, 1328–1344.

25. Lin, H.F., Traver, D., Zhu, H., Dooley, K., Paw, B.H., Zon, L.I. andHandin, R.I. (2005) Analysis of thrombocyte development in CD41-GFPtransgenic zebrafish. Blood, 106, 3803–3810.

26. Albers, C.A., Cvejic, A., Favier, R., Bouwmans, E.E., Alessi, M.C.,Bertone, P., Jordan, G., Kettleborough, R.N., Kiddle, G., Kostadima, M.et al. (2011) Exome sequencing identifies NBEAL2 as the causative genefor gray platelet syndrome. Nat. Genet., 43, 735–737.

27. Gunay-Aygun, M., Falik-Zaccai, T.C., Vilboux, T., Zivony-Elboum, Y.,Gumruk, F., Cetin, M., Khayat, M., Boerkoel, C.F., Kfir, N., Huang, Y.et al. (2011) NBEAL2 is mutated in gray platelet syndrome and is requiredfor biogenesis of platelet alpha-granules. Nat. Genet., 43, 732–734.

28. Kahr, W.H., Hinckley, J., Li, L., Schwertz, H., Christensen, H., Rowley,J.W., Pluthero, F.G., Urban, D., Fabbro, S., Nixon, B. et al. (2011)Mutations in NBEAL2, encoding a BEACH protein, cause gray plateletsyndrome. Nat. Genet., 43, 738–740.

29. Chu, J. and Sadler, K.C. (2009) New school in liver development: lessonsfrom zebrafish. Hepatology, 50, 1656–1663.

30. Pack, M., Solnica-Krezel, L., Malicki, J., Neuhauss, S.C., Schier, A.F.,Stemple, D.L., Driever, W. and Fishman, M.C. (1996) Mutations affectingdevelopment of zebrafish digestive organs. Development, 123, 321–328.

31. Jing, L. and Zon, L.I. (2011) Zebrafish as a model for normal andmalignant hematopoiesis. Dis. Model. Mech., 4, 433–438.

32. Carvalho, L. and Heisenberg, C.P. (2010) The yolk syncytial layer in earlyzebrafish development. Trends Cell Biol., 20, 586–592.

33. Gomes, A.L., Carvalho, T., Serpa, J., Torre, C. and Dias, S. (2010)Hypercholesterolemia promotes bone marrow cell mobilization byperturbing the SDF-1:CXCR4 axis. Blood, 115, 3886–3894.

34. Lloyd-Evans, E., Morgan, A.J., He, X., Smith, D.A., Elliot-Smith, E.,Sillence, D.J., Churchill, G.C., Schuchman, E.H., Galione, A. and Platt,F.M. (2008) Niemann-Pick disease type C1 is a sphingosine storagedisease that causes deregulation of lysosomal calcium. Nat. Med., 14,1247–1255.

35. Wu, J.Y., Lin, C.Y., Lin, T.W., Ken, C.F. and Wen, Y.D. (2007)Curcumin affects development of zebrafish embryo. Biol. Pharm. Bull.,30, 1336–1339.

36. Hermans, C., Wittevrongel, C., Thys, C., Smethurst, P.A., Van Geet, C.and Freson, K. (2009) A compound heterozygous mutation inglycoprotein VI in a patient with a bleeding disorder. J. Thromb.Haemost., 7, 1356–1363.

37. Goubau, C., Jaeken, J., Levtchenko, E.N., Thys, C., Di Michele, M.,Martens, G.A., Gerlo, E., De Vos, R., Buyse, G.M., Goemans, N., VanGeet, C. and Freson, K. (2012) Homozygosity for aquaporin 7 G264V inthree unrelated children with hyperglyceroluria and a mild plateletsecretion defect. Genet Med., Epub ahead of print.

38. Di Michele, M., Peeters, K., Loyen, S., Thys, C., Waelkens, E.,Overbergh, L., Hoylaerts, M., Van Geet, C. and Freson, K. (2012)Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) impairs theregulation of apoptosis in megakaryocytes by activating NF-kappaB: aproteomic study. Mol. Cell Proteomics, 11, M111 007625.


Dow


ic.oup.com/hm


ber 2021

39. Serbanovic-Canic, J., Cvejic, A., Soranzo, N., Stemple, D.L., Ouwehand,W.H. and Freson, K. (2011) Silencing of RhoA nucleotide exchangefactor, ARHGEF3, reveals its unexpected role in iron uptake. Blood, 118,4967–4976.

40. Yaqoob, N., Holotta, M., Prem, C., Kopp, R. and Schwerte, T. (2009)Ontogenetic development of erythropoiesis can be studied non-invasivelyin GATA-1:DsRed transgenic zebrafish. Comp. Biochem. Physiol. A Mol.Integr. Physiol., 154, 270–278.

41. Schulte-Merker, S., Ho, R.K., Herrmann, B.G. and Nusslein-Volhard, C.(1992) The protein product of the zebrafish homologue of the mouse Tgene is expressed in nuclei of the germ ring and the notochord of the earlyembryo. Development, 116, 1021–1032.

42. Delvaeye, M., De Vriese, A., Zwerts, F., Betz, I., Moons, M., Autiero, M.and Conway, E.M. (2009) Role of the 2 zebrafish survivin genes invasculo-angiogenesis, neurogenesis, cardiogenesis and hematopoiesis.BMC Dev. Biol., 9, 25.


Dow


ic.oup.com/hm


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NPC1 defect results in abnormal platelet formation and function