Fucose-deficient hematopoietic stem cells have decreased self-renewal and aberrant marrow niche...

T R A N S P L A N T A T I O N A N D C E L L U L A R E N G I N E E R I N G

Fucose-deficient hematopoietic stem cells have decreasedself-renewal and aberrant marrow niche occupancy_2745 2660..2669

Jay Myers, Yuanshuai Huang, Lebing Wei, Quanjian Yan, Alex Huang, and Lan Zhou

BACKGROUND: Modification of Notch receptors byO-linked fucose and its further elongation by the Fringefamily of glycosyltransferase has been shown to beimportant for Notch signaling activation. Our recentstudies disclose a myeloproliferative phenotype,hematopoietic stem cell (HSC) dysfunction, and abnor-mal Notch signaling in mice deficient in FX, which isrequired for fucosylation of a number of proteins includ-ing Notch. The purpose of this study was to assess theself-renewal and stem cell niche features of fucose-deficient HSCs.STUDY DESIGN AND METHODS: Homeostasis andmaintenance of HSCs derived from FX-/- mice werestudied by serial bone marrow transplantation, homingassay, and cell cycle analysis. Two-photon intravitalmicroscopy was performed to visualize and comparethe in vivo marrow niche occupancy by fucose-deficientand wild-type (WT) HSCs.RESULTS: Marrow progenitors from FX-/- mice hadmild homing defects that could be partially prevented byexogenous fucose supplementation. Fucose-deficientHSCs from FX-/- mice displayed decreased self-renewalcapability compared with the WT controls. This isaccompanied with their increased cell cycling activityand suppressed Notch ligand binding. When tracked invivo by two-photon intravital imaging, the fucose-deficient HSCs were found localized farther from theendosteum of the calvarium marrow than the WTHSCs.CONCLUSIONS: The current reported aberrant nicheoccupancy by HSCs from FX-/- mice, in the context of afaulty blood lineage homeostasis and HSC dysfunctionin mice expressing Notch receptors deficient inO-fucosylation, suggests that fucosylation-modifiedNotch receptor may represent a novel extrinsic regula-tor for HSC engraftment and HSC niche maintenance.

The selective adhesion of hematopoietic stemcells (HSCs) and hematopoietic progenitor cells(HPCs) to the extracellular matrix and thestromal cells plays an important role in the regu-

lation of stem cell self-renewal, lineage commitment, anddifferentiation.1 HSCs reside in the endosteal niche alongthe bone surface where the osteoblasts adhesively interactwith HSCs.2,3 A variety of intrinsic factors, includingc-Myc, p21, p27, and p53, are involved in the regulation ofHSC self-renewal.4-7 In addition, extrinsic cues, includingNotch ligand, cdc42, Ang-1/Tie2, retinoid acid, BMP, andCXCR4/CXCL12, may coordinate to regulate the ability ofHSCs to lodge and engraft at the endosteal niche.2,3,8-10

The Notch gene family is important for cell fate deter-mination in many processes including mammalianhematopoiesis.11 Notch receptors are decorated by variousposttranslation modifications including O-fucosylation,with fucose moieties attached to the conserved serine orthreonine residues of EGF-like repeats present on theNotch receptor extracellular domain. The O-fucose resi-dues can then be further modified by the Fringe family ofglycosyltransferases. These fucose-dependent modifica-tions modulate Notch signaling through ligand–receptorinteractions.12,13 Studies have shown that Notch activationpromotes HSC expansion in vitro and favors lymphoidover myeloid lineage outcome in vivo.14-17 In addition,

ABBREVIATIONS: 7-AAD = 7-aminoactinomycin D; APC =allophycocyanin; BrdU = bromodeoxyuridine; GFP = green

fluorescent protein; HPC(s) = hematopoietic progenitor cell(s);

HSC(s) = hematopoietic stem cell(s); NTg = Notch reporter

transgenic; WT = wild type.

From the Department of Pediatrics and the Department of

Pathology, Case Western Reserve University, Cleveland, Ohio.

Address reprint requests to: Lan Zhou, Department of

Pathology, Case Western Reserve University, Cleveland, OH

44106; e-mail: [email protected].

Received for publication February 4, 2010; revision

received April 27, 2010, and accepted April 29, 2010.

doi: 10.1111/j.1537-2995.2010.02745.x

TRANSFUSION 2010;50:2660-2669.

2660 TRANSFUSION Volume 50, December 2010

Notch activation by osteoblasts expressing the Notchligand, Jagged1, in the marrow niche promotes self-renewal of primitive hematopoietic cells.3 However, loss-of-function studies using the conditional knockout ofNotch1, Notch1 combined with Notch ligand Jagged1, orexpression of a dominant negative regulator of canonicalNotch transcriptional activation, did not reveal an essen-tial role of Notch1 signaling on HSC self-renewal.18-20 Itremains possible that other Notch/Notch ligand pairs maybe important for the maintenance of the HSC pool. Also, itis unclear whether aspects of Notch pathways upstream ofNotch activation, for example, its association with ligand-expressing osteoblasts, which dictates the physical asso-ciation of HSC with marrow microenvironment, may becritical for HSC biology.

We previously reported a chronic myeloproliferativephenotype and abnormal Notch signaling in FX-/- mice.13

Deletion of the FX locus eliminates constitutive GDP-fucose synthesis, but leaves intact a salvage pathway forGDP-fucose synthesis that uses exogenous fucose.21

Therefore, fucosylation of glycans and proteins are condi-tionally dependent on exogenous fucose via the salvagepathway in FX-/- mice. We disclosed that chronic myelo-proliferation found in FX-/- mice is a result of loss of con-trolled suppression of myelopoiesis exerted by Notch. Inthe context of a wild-type (WT) fucosylation phenotype,O-fucosylation of Notch, expressed on HSCs and HPCs, isrequired for effective interaction of these cells with Notchligand and the transduction of Notch signal activation.This effective interaction is required for Notch exertedsuppression of overt myeloid development. By contrast,fucosylation-deficient myeloid progenitors have lost theWT Notch ligand-binding phenotype, do not transcribeNotch target genes, and display uncontrolled myeloid dif-ferentiation. More recently, we found that HSCs deficientin cellular fucosylation have decreased lymphoid butincreased myeloid developmental potentials. These fea-tures were accompanied with their suppressed in vitrobinding ability with recombinant Notch ligand. Further,these cells displayed a 13.7-fold reduction of HSC fre-quency by limiting dilution transplant analysis.22 In thisstudy, we studied the mechanism underlying the dysfunc-tion of HSCs expressing fucosylation-deficient Notch byexamining their in vivo self-renewal capability. To explorewhether an altered self-renewal capability of fucose-deficient HSC is associated with their aberrant stem cellniche features, we performed two-photon intravitalmicroscopy to visualize and compare the in vivo marrowniche occupancy by fucose-deficient and WT HSCs.

MATERIALS AND METHODS

MiceThe animal research described in this article wasapproved by Case Western Reserve University Institutional

Animal Care and Use Committee. Mice used include 8- to24-week-old WT and FX-/- mice maintained and preparedas described.21 The Notch reporter transgenic (NTg)mouse was purchased from The Jackson Laboratory (BarHarbor, ME; Stock 005854). The NTg mouse carries a hem-izygous allele of Notch-signaling CBF-1 response elementand a minimal SV40 promoter driven enhanced greenfluorescent protein (GFP). This mouse has been used todefine active Notch signaling in HSCs in vivo.23 FX-/--NTgmouse was generated by crossing the NTg with the FX-/-

mouse and maintained as described.13

Flow cytometry analysis and cell sortingFlow cytometric analyses and cell sorting were performedas described.13 Briefly, marrow mononuclear cells wereincubated with biotin-conjugated rat anti-mouse anti-body cocktails (CD3, CD4, CD8, Gr-1, CD11b, B220,Ter119), followed by lineage depletion with goat anti-ratimmunoglobulin G (IgG) magnetic beads (MiltenyiBiotec, Auburn, CA). Lineage-depleted cells were furtherstained with streptavidin-allophycocyanin (APC)-Cy7,fluorescein isothiocyanate (FITC)–anti-Sca-1, and APC–anti-c-kit and sorted using a cell sorter (FACSAria, BD Bio-sciences, San Jose, CA). For cell cycle G1/G0 analysis in thestem cell compartment, marrow nucleated cells wereincubated with DNA dye Ho (4 mg/L) and RNA dye PY(1 mg/mL) at 37°C for 45 minutes, respectively, and thenlabeled with FITC–anti-lineage antibodies, APC–anti-c-kit, and PE–Cy7–anti-Sca-1. For S-G2/M cell cycle analy-sis, mice were injected intraperitoneally with one dose ofbromodeoxyuridine (BrdU) and fed with water containingBrdU for 3 days. The percentage of LSKs in S-G2/M phaseof the cell cycle was analyzed by anti-BrdU and7-aminoactinomycin D (7-AAD). Flow cytometry was per-formed on flow cytometers (FACSAria and BD LSR II, BDBiosciences).

Recombinant mouse Notch ligand (Dll4)binding assayRecombinant Notch ligand composed of the extracellulardomains of Dll4 fused with human IgG Fc were con-structed as described.24 Recombinant Dll4 were preparedfrom HEK 293 T cells transfected with hIgG-Dll4 or thevector and quantified by enzyme-linked immunosorbentassay. Binding assay was performed by incubating LSKcells with recombinant Dll4 in Hanks’ balanced salt solu-tions supplemented with Ca2+ and analyzed by flow usingPE–anti-human IgG Fc.

Bone marrow transplantation and homing assayPrimary and secondary bone marrow transplantation(BMT) was performed as described.13 Briefly, 2 ¥ 106

NOTCH AND HEMATOPOIETIC STEM CELL NICHE

Volume 50, December 2010 TRANSFUSION 2661

donor marrow cells were injected into lethally irradiated(950 Rad) female recipient mice. Recipient mice weremonitored daily for survival for more than 30 days. Themice were killed at 2 to 4 months, and marrow cells wereprepared from those mice and injected into new femalerecipients. To perform homing assays, marrow cells fromdonor animals (WT or FX-/- mice; Ly5.2) were prepared as2 ¥ 106 cells/200 mL volume in phosphate-buffered salineand were injected into lethally irradiated WT recipientmice. An aliquot is also plated in methylcellulose cul-tures (M3434, Stem Cell Technologies, Vancouver, BritishColumbia, Canada) to quantify committed progenitorcells of various lineages. Sixteen hours later, single-cellsuspensions were prepared from the marrow of therecipients and cultured in duplicate to assess donorcolony-forming unit (CFU) recovery in the recipientanimals. The number of homed CFUs per femur was cor-rected to represent the whole BM (multiplied by 16.9).25

The number of donor CFUs recovered after 16 hoursin BM was expressed as a percentage of total CFUsinfused.

Multiphoton intravital imaging studiesIntravital imaging of adoptively transplanted hematopoi-etic progenitors was performed as described.26 Briefly, iso-lated Lineage-c-kit+ (Lin-c-Kit+) cells (5 ¥ 105-50 ¥ 105)were injected into the tail vein of lethally irradiated recipi-ent mouse. At indicated times after intravenous (IV) trans-fer, mice were anesthetized and a small incision was madein the scalp so as to expose the underlying dorsal skullsurface. Donor cell homing to the skull marrow wasimaged using a photon microscope tuned to 860 nm(SP5/AOBS/2, Leica Microsystems & Coherent Inc.,Lawrenceville, GA) while mice are under inhaled anesthe-sia (1%-2% isoflurane) on a warmed microscope stage(37°C). To highlight the marrow vasculature, TRITC-dextran (Sigma, St Louis, MO) was injected into recipientmice 5 minutes before imaging experiments. Simulta-neous visualization of bone endosteum, vasculature, andHSC was achieved by second harmonic generationmicroscopy, dextran dye, and cells with GFP signals,respectively. Fluorescent images from optical sections ofindividual xy-planes were collected through predeter-mined, fixed optical z-slices. This data set was then ana-lyzed using imaging software (Imaris, BitPlane, Inc., StPaul, MN), which allows simultaneous tracking of objectpositions in three dimensions over time with statisticalcalculations.

Statistical analysisData are presented as means � SD, unless otherwisestated. Significance was assessed by t test.

RESULTS

Mild marrow homing defects of FX-/- cells can bepartially prevented by exogenous fucoseThe deletion of the FX locus abolishes the expression of allfucosylated glycans, including the counterreceptors forthe selectins, which are involved in the rolling and homingof HSCs to the marrow.27 To assess the contribution ofdefective homing to the stem cell defects we observed inthe FX-/- mice, we compared the CFUs of marrow cellsrecovered from recipient mice 16 hours after transplanta-tion of donor marrow cells derived from WT or FX-/- mice.As shown in Fig. 1A, the percentage of recovered HPCsthat homed to the BM was 12.2 � 0.9% in the WT controlgroup. As expected, the fucose-deficient FX-/- HPCs haddecreased homing by 52% when compared to the controlgroup. However, the homing ability of fucose-deficientFX-/- HPCs could be partially prevented with HPCs thatwere derived from FX-/- mice that had been reared onfucose-supplemented chow. Although the progenitors ofmyeloid lineage were relatively increased in FX-/- micemaintained on standard chow, the proportion of differentlineages of progenitors recovered was not changed com-pared to the cells infused (Fig. 1B). Therefore, to ensureequal homing of donor cells to the recipient marrow, thetransplantation experiments described thereafter wereperformed using cells from WT or FX-/- mice maintainedon fucose-supplemented chow, and the recipients wereprovided with fucose-supplemented drinking water for 9to 12 days after receiving IV injection of donor cells andthen maintained on standard chow.13

FX-/- HSCs have decreased self-renewal, are lessquiescent, and have increased cell cycling activityThe frequency of LSK cells (Lin-Sca-1+c-kit+) under steadystate is mildly decreased in FX-/- mice compared to WTcontrols or FX-/- mice with fucose supplementation (0.13%in WT, 0.10% and 0.12% in FX-/- mice without or withfucose supplementation, respectively). However, a reduc-tion of HSC frequency in FX-/- mice by competitive trans-plant after myeloablation suggests that fucose-deficientHSCs may have decreased engraftment or decreased self-renewal.22 Serial BMT was performed to test this hypoth-esis. We found that in the primary transplant setting, thepool of donor-derived marrow LSK cells was modestlydecreased in the WT recipient when the marrow cells arederived from FX-/- mice. LSKs were further decreased inthe secondary recipients receiving marrow cells derivedfrom the FX-/- donor. Interestingly, although the LSK fre-quency was not changed in the FX-/- recipients receivingWT donor cells in both primary and secondary transplantsettings, LSK pools were severely diminished when bothmarrow cells and the stroma are of the fucose-deficientgenotype, indicating a marrow stromal dependent

MYERS ET AL.


mechanism that maintains HSC pool by interacting withHSCs in these settings. Because stem cell quiescence iscritical for protection from myelotoxic injury and preser-vation of the stem cell pool, we decided to examine the cellcycling status of the HSCs in FX-/- mice. Using the RNA dyepyronin Y as a measure of quiescence among the LSK andHoechst 33342 low-staining marrow cells,28 we found thatFX-/- marrow progenitors were less quiescent by display-ing less cells in G0 phase (Fig. 2C). Further, we found thatmore FX-/- marrow LSKs were engaged in active cell divi-sion by using BrdU incorporation and 7-AAD staining(Fig. 2D). These results indicate that decreased self-renewal of HSCs expressing Notch receptors deficient inO-fucosylation could be accounted for by enhanced HSCcycling and depletion of the long-term HSC pool.

FX-/- HSCs display decreased Notch ligand bindingand aberrant endosteal niche occupancyProper endosteal niche occupancy has been shown to beessential for HSC self-renewal and marrow engraftment.In addition, there is evidence that perivascular space maydelineate another important niche for HSC engraftmentor expansion, as the major cell type, osteoblasts, in theendosteum, was also found in the perivascular regions.26

To understand the cellular mechanism by which the dif-ferential cellular positioning of HSCs may be regulated byNotch and its modification by O-fucose, we first examinedthe binding of Notch-expressing HSCs with Notch ligand.3

We then performed transplant experiments followed bytwo-photon microscopy to visualize the niche positioningof transplanted HSCs and/or HPCs that are either fucose-

replete or fucose-depleted in the calvarium marrow.29 Thecalvarium marrow has been shown to have HSC frequen-cies comparable to long bones26 and is easily accessible forintravital imaging by two-photon microscopy. Marrowcells were isolated from either FX-/--NTg mice that hadbeen fed standard chow (FX-/--NTg, - fucose) or WT-NTgmice. The NTg mouse carries a hemizygous allele ofNotch-signaling CBF-1 response element and a minimalSV40 promoter-driven enhanced GFP. GFP expressionindicates Notch activation, which is a hallmark of primi-tive HSCs, whereas HSCs undergoing proliferation anddifferentiation display downregulated Notch activation.23

By flow cytometric analysis, we found that GFP expressionwas decreased by approximately 50% in fucose-depletedLSK cells when compared to WT LSK cells. In addition,these GFP+ cells showed decreased binding ability torecombinant Notch ligands in vitro, such as Dll4 (Fig. 3A)and Jagged1 (data not shown).

Five million marrow Lin-c-kit+ cells from eitherWT-NTg mice or FX-/--NTg mice maintained on standardchow (no fucose supplementation) were transplanted intoWT recipient mice. High-resolution two-photon micros-copy images were obtained 16 hours later. Consistent withour previous finding that fucose-deficient progenitorshave decreased homing capability (Fig. 1), we observedless engrafted cells that are derived from FX-/--NTg donorscompared to that from the WT-NTg donors in the recipientcalvarium. Approximately 35% of GFP+ cells (green) fromWT-NTg mice were found lining along the blood vessels.In comparison, only approximately 20% of GFP+ cellsfrom FX-/--NTg donor (no fucose) were bound to the vas-cular segments (Fig. 3B), while more cells were found

Fig. 1. Mild homing defects associated with FX-/- marrow progenitors (BMNC) are corrected by exogenous fucose. Lethally irradi-

ated WT recipients were injected with marrow cells derived from WT mice, mice raised on standard chow (FX-/-, - fucose), or mice

raised on fucose-supplemented chow (FX-/-, + fucose). (A) CFUs were determined from the recipient bone marrow 16 hours after

injection and expressed as percentage recovery of infused CFU-Cs (n = 5 per group). (B) Proportions of different lineages of CFUs

were determined from the recovered marrow progenitors, including burst-forming unit–erythroid (BFU-E, ), myeloid lineage

colonies of granulocyte/macrophage/granulocyte macrophage (CFU-G/M/GM, ), and multipotential progenitors (CFU-

granulocyte, erythroid, macrophage, megakaryocyte [CFU-GEMM, ]).



scattered in the central marrow cavity (data not shown).No statistical difference was identified when the mean dis-tance of GFP+ cells from WT-NTg mice to the vessels wascompared to that from GFP+ cells from FX-/--NTg mice.Whether FX-/- and WT HSCs show any difference in theirphysical relationship to the osteoblasts that are embeddedin the vasculature remains to be determined.26

We then compared the endosteal niche localization ofHSCs that are either fucose-replete or fucose-depleted.Visualization of bone endosteum and HSC was achieved bysecond harmonic generation (SHG; blue) microscopy and

GFP signals (green) from HSCs, respectively.26 XYZ stackimages were collected and processed using Imaris software(Fig. 4A). The shortest three-dimensional distancebetween GFP+ cells and the endosteal surface was deter-mined (Fig. 4B). The mean distance of the fucose-depletedcells to the endosteum was 12.51 mm, which is greater thanthe 8.56 mm observed in fucose-replete cells. These find-ings suggest that the niche positioning of marrow progeni-tors from FX-/- mice is affected by the absence of fucose onthese cells, which correlates with their decreased Notchligand binding and Notch activation.

Fig. 2. FX-/- HSCs have decreased self-renewal in serial transplantation, are less quiescent, and have increased cell cycling activity.

Flow cytometry analysis of LSK cells in the primary recipients 4 months after transplantation (A) and secondary recipients 3

months after transplantation (B). n = 5 for each group, *p < 0.05, **p < 0.01 compared with primary (A) or secondary (B) transplant

WT recipients receiving WT donor cells. (C) Representative distribution of G0 versus G1 in the LSKs defining an increased cycling

fraction in FX-/- mice. Mouse marrow cells were stained with lineage antibodies, APC–anti-c-kit, PE–anti-Sca-1, pyronin Y (RNA

dye), and Hoechst 33342 (DNA dye). LSK cells were gated by means of a stringent variable. Cells residing in G0 appear at the bottom

of the G0/G1 peak, and G1 cells are the upper part as indicated. (D) Mice were injected intraperitoneally with one dose of BrdU and

fed with water containing BrdU for 3 days. The percentage of LSKs in S-G2/M phase of the cell cycle was analyzed by anti-BrdU and

7-AAD. Data shown in C and D are one representative of three similar experiments.

MYERS ET AL.


DISCUSSION

HSCs ensure sustained production of all blood cellsthrough complex processes regulated by intricate signal-ing pathways and interactions with the marrow microen-vironment and/or stem cell niche. HSCs expressingmembers of the Notch family of receptors are located inmarrow where they interact with surrounding stromalcells expressing Notch ligands.3,30-32 Such direct cell–cell

interaction via Notch and Notch ligand coupling mayprovide an important means for HSCs to position them-selves in the stem cell niche where they are exposed tocytokines and signals released from the niche-supportingcells. This hypothetical role of Notch has been suggestedby recent studies performed by Lo Celso and colleagues26

using a combination of confocal microscopy and two-photon imaging system, which showed that quiescentHSCs are significantly closer to osteoblasts lining the

Fig. 3. FX-/- HSCs have decreased Notch ligand binding but similar physical relationship to the vasculature compared to WT HSCs

after homing in mouse calvarium bone marrow. (A) Marrow LSK cells from WT-NTg or FX-/--NTg mice without fucose supplementa-

tion were examined for GFP expression. GFP+ cells were gated and analyzed for their binding with vector control or recombinant

Notch ligand Dll4 using PE–anti-hIgG Fc by flow cytometric analysis. Data shown are one representative of five similar experiments

as indicated by the mean fluorescent intensity (MFI) after binding of LSKs with vector or Dll4. (B) Shown are two representative

images of each taken by two-photon intravital microscopy of engrafted GFP+ marrow progenitors isolated from either WT-NTg (left

two panels) or FX-/--NTg (- fucose; right two panels) mouse donors, respectively. Calvarium blood vessels were highlighted by

TRITC-dextran.



endosteal surface than cells undergoing rapid prolifera-tion. Further, HSCs are found localized closer to theendosteal surface in a transgenic mouse model in whichincreased numbers of osteoblasts have been shown todrive stem cell expansion. Significantly, Calvi and col-

leagues3 showed osteoblasts that exert this regulatory roleexpress increased level of the Notch ligand, Jagged1.These findings suggest that HSC endosteal niche loca-lization may be facilitated by Notch and Notch ligandcoupling.

(µm)

A

0.00

5.00

10.00

15.00

20.00

25.00

Dis

tan

ce t

o E

nd

ost

eum

p < 0.05 8.56µm(n =15)

12.51µm(n =13)

B

FX-/--NTG FX-/--NTG + fucose -fucose

Fig. 4. Marrow HSCs without fucose are localized more distant to the endosteum in the calvarium. (A) A representative image of

three similar experiments showing HSCs (GFP+, green) localized in the marrow after transplantation. The shortest three-

dimensional distance (mm) between HSCs and the endosteum (blue) was determined and marked with white lines and numbers.

(B) GFP+ cells from fucose-replete mice are closer to the endosteum than GFP+ cells from fucose-depleted mice.

MYERS ET AL.


This notion is now supported by our observation thatHSCs with Notch activation are found localized closer tothe endosteal surface. Since the Notch/Notch ligand cou-pling can be modulated by Notch receptor O-fucose modi-fication,13,33 and the endosteal niche is essential for HSCquiescence maintenance, we speculate that HSC nicheoccupancy can be influenced by the status of Notch recep-tor glycosylation that may dictate the strength of interac-tion between Notch expressed on HSC and Notch ligandexpressed on osteoblasts. If this is the case, then HSCsexpressing nonfucosylated Notch molecules may exhibitaberrant niche occupancy resulting in more active celldivision and therefore are prone to differentiation alongthe myeloid lineage.13 Indeed, we found that fucose-deficient HSCs are positioned farther from the endostealsurface than fucose-replete HSCs. The altered physicalrelationship of fucose-deficient HSCs with the marrowendosteum correlates with their decreased quiescence,increased cell cycling, suppressed ligand binding abilityand suppressed Notch activation. Our observations areconsistent with the finding by Duncan and coworkers,23

who found that HSCs undergoing rapid proliferation anddifferentiation display down regulated Notch activation.Because the observed phenotype in the FX-/- mousemodel may not be specific for Notch, future studies will betargeted to confirm these results by using genetically engi-neered animal models targeting Notch-specific fucosemodification or by agents that directly target the Notchand/or Notch ligand binding.

Notch signaling is essential for T-cell fate specifica-tion and differentiation in other hematopoietic lineages.Whether canonical Notch intracellular signaling activa-tion is required for HSC self-renewal is controversial.34

The negative finding of an essential role of Notch canoni-cal signaling in HSC self-renewal suggests that aspects ofNotch pathways that are independent of the trans-criptional control of Notch signaling mediated byMastermind-like 1 may be critical for HSC functionalcontrol.20 To assess the contribution of Notch intracellu-lar signaling to HSC self-renewal that is independent ofglycosylation-modified Notch and ligand coupling, weexamined the consequence of restoring Notch1 intracel-lular activation on cell fate of marrow progenitors thatare fucose-deficient. Interestingly, forced expression ofNotch1 appears to be able to reverse the aberrant lineagecommitment in the marrow progenitors associatedwith fucose deficiency, but it also decreases the LSKfrequency.22 This latter finding could be caused bythe secondary leukemogenic effect of nonphysiologicdoes of Notch activation in promoting aberrant T-celldevelopment in the marrow HSC population.35 It remainsto be determined whether signaling from other Notchfamily members alone, or in concert with Notch1,may be required for HSC maintenance in the HSCcompartment.

In summary, our observations indicate that Notchand Notch ligand interaction may represent an importantmechanism being part of a complex network mediated bya group of adhesion and signaling molecules in a special-ized microenvironment that supports the stem cell niche.1

Therefore, similar to what has been described in Rb-/-

mice,36 we believe that the defective HSC phenotype andthe myeloproliferation are interrelated in FX-/- mice and islikely reflective of the role of fucosylation-modified Notchas a novel extrinsic regulator of HSC self-renewal and fatedetermination. Although evidence presented in this studyas well as our previous reports strongly support thatabnormal Notch ligand binding and Notch signaling couldaccount for the observed aberrant hematopoietic pheno-types and HSC defects in FX-/- mice, it remains possibleand unknown yet whether fucose deficiency could impacthematopoiesis mediated by other fucosylated proteins.O-fucose is present on EGF repeats of urinary plasmino-gen activator and some of the clotting factors, as well asthe thrombospondin type 1 repeats in many secreted andtransmembrane proteins.37-40 Recently, it was shown thatO-fucosylation sites on thrombospondin type 1 repeat ofADAMTS like-1/punctin-1 and ADAMTS13 are function-ally significant for secretion of these proteins.41,42 In addi-tion, other forms of fucosylated cell surface glycans andmatrix proteins may be implicated in a variety of biologicprocesses including cell growth and cell–cell adhesion.43 Itremains possible that lack of fucose on fucosylatedglycans other than Notch may directly or indirectly causeHSC defects mediated through altered intercellular signal-ing. Therefore, a clear link between Notch fucosylationand hematopoietic and stem cell defect awaits furtheranalysis of fucose deficiency specific for Notch pathway.Nevertheless, we hope that the findings from this studymay provide a rationale for future research in understand-ing the importance of Notch fucosylation, and fucosyla-tion of other relevant biologic molecules, for HSC nichecompetency and HSC functional control and may alsoadvance efforts in the future to use such knowledge instem cell therapy.

ACKNOWLEDGMENTS

We thank Dr John Lowe for providing FX-/- mouse line. This study

was supported in part by grants from the National Blood Foun-

dation Research Grant and NIH to Lan Zhou, by grants from the

St. Baldrick’s Foundation, the Dana Foundation, and the Gabri-

elle’s Angel’s Foundation to JM and AH. AH is a designated St.

Baldrick’s Scholar.

CONFLICT OF INTEREST

The authors certify that they have no conflict of interest of finan-

cial involvement with this manuscript.



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Fucose-deficient hematopoietic stem cells have decreased self-renewal and aberrant marrow niche...

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