0301-472X/05 $–see

doi: 10.1016/j.exp

Experimental Hematology 33 (2005) 1055–1061

Regulation of hematopoiesis by retinoid signaling

Todd Evans

Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, Bronx, NY, USA

The discovery that retinoic acid efficiently stimulates the terminal differentiation ofgranulocytic leukemia cells had a major impact on clinical hematology, but has also inspiredresearch into the normal function of the retinoid signaling pathway during hematopoiesis.New animal models and loss-of-function approaches have successfully revealed requirementsfor the pathway at defined embryonic stages that are relevant for distinct hematopoietic cellpopulations. For example, novel insight has been gained regarding the function of retinoids inyolk sac hematovascular development, fetal erythropoiesis, T-cell homing, and hematopoieticstem and progenitor cell biology. The lessons learned so far indicate that future developmentof sophisticated animal models will be needed to fully understand the intricacy and specificityof this complex signaling pathway, but that this effort will be productive and continue toinform both basic and clinical research on many fronts. � 2005 International Society forExperimental Hematology. Published by Elsevier Inc.

Retinoic acid (RA) is a key signaling molecule thatregulates the patterning and development of the body planand many organ systems during embryogenesis [1,2]. RAcontinues to be required for homeostasis in the adult, whichis reflected by the adverse effects of a human diet deficientin vitamin A, the essential precursor from which bioactiveretinoid ligands are generated. It was early epidemiologicalstudies, followed-up by nutrition-based animal models [3],which first implicated RA as an important regulator ofsteady-state hematopoiesis. Indeed, hematopoietic defectscaused by a deficiency of vitamin A are still a seriousproblem in the developing world, while in the developedworld, with diets that generally are sufficient for vitamin A,RA signaling might be thought of as less relevant to clinicalhematology.

However, the retinoid signaling pathway was broughtfront and center to the attention of hematologists uponrecognition that RA stimulates the differentiation ofmyeloid leukemia cells [4,5], and the eventual discoverythat the t(15:17) translocation, a cytogenetic hallmark ofacute promyelocytic leukemia (APL), leads to expressionof an oncoprotein consisting of a retinoic acid receptor(RARa) fused to a previously unknown protein called PML[6,7]. Pharmacological doses of RA are now used clinicallyto activate the RA-APL fusion protein, leading to cell-cyclearrest and terminal differentiation of the leukemic cells.

Offprint requests to: Todd Evans, Ph.D., Department of Developmental

and Molecular Biology, Albert Einstein College of Medicine, 1300 Morris

Park Avenue, Bronx, NY 10461; E-mail: tevans@aecom.yu.edu

front matter. Copyright � 2005 International Society for

hem.2005.06.007

Leukemia studies also stimulated investigation into thetarget genes for RARs in hematopoietic cells, and the roleof retinoids during normal myeloid development. As will bedescribed here, evidence has accumulated suggesting thatRA plays at least a modulator function for myelopoiesis.Meanwhile, unsuspected functions for RA signaling havemore recently emerged for defined stages of hematopoiesis,namely in yolk sac (primitive) hematovascular develop-ment, fetal liver erythropoiesis, T-cell homing, andhematopoietic stem cell biology. This review focuses onthe normal role of RA signaling during these developmentalstages of hematopoiesis. It seems likely that the activity ofthe APL oncoprotein, which has over the years stimulatedso much interest in RA signaling, is at least partly due tothe RA fusion partner moiety and/or alterations in thereciprocal translocation loci, and for a historical perspectivethe reader is referred to reviews focused on retinoids andleukemia [4,8–12].

RA signaling pathwayThe pathway is complex, with levels of regulation rangingfrom metabolism and presentation of the ligand, expressionof receptor isoforms, and cross-interaction with other keysignaling pathways. Retinoids must first be metabolizedbecause they are neither synthesized de novo nor obtaineddirectly from the diet. Bioactive retinoid ligands, princi-pally all-trans RA and 9-cis RA, are derived from vitaminA, which is a family of retinol and retinyl esters convertedfrom beta-carotene, which itself is typically obtained in the

Experimental Hematology. Published by Elsevier Inc.

1056 T. Evans/Experimental Hematology 33 (2005) 1055–1061

diet from fruits and vegetables [1]. The concentration ofRA in a cell is regulated at several levels [13]. Retinol is notfreely diffusible, but is associated with retinol bindingprotein (RBP), which needs to interact with a cell-surfacereceptor (RPBr) for transfer across the plasma membrane.Once in the cell, retinol associates with high-affinitycellular retinol binding proteins (CRBPs) and is convertedfirst to retinal by alcohol dehydrogenases, and then to RAby retinal dehydrogenase. RA is complexed with cellularRA binding proteins (CRABPs), which may influence theclearance or nuclear entry of RA. The details of how RBP,RPBR, CRBPs, and CRABPs control retinoid signaling arebeyond further discussion here, but it is pertinent to notethat expression levels and distinct isoforms of theseproteins provide considerable regulatory potential. Withrespect to hematopoiesis, for example, in distinct stem andprogenitor cell niches, this represents largely unchartedterritory for investigation.

Once in the nucleus, RA binds to receptor complexesthat are also DNA-binding transcription factors and directlyaffect the expression of target genes [14]. There are twosubclasses of nuclear receptors, the retinoid (RAR) andrexinoid (RXR) receptors, and each subclass includes threeseparate genes (a, B, g), and in some cases distinct splice-derived isoforms [15]. RAR-RXR heterodimers are themajor functional complex that binds to retinoic acidresponse elements (RAREs) in promoters or other regula-tory regions. In contrast, RXR binds specifically 9-cis-RAand can heterodimerize with various nuclear receptors inaddition to RAR. There has been much progress over thepast several years in understanding how receptor complexescontrol gene expression [16,17]. Broadly speaking, theunliganded receptor complex is fully capable of binding toan RARE, and in doing so recruits corepressors (e.g., N-CoR and SMRT), which in turn recruit chromatinmodification complexes and cause locus silencing. Ligandbinding releases corepressors and facilitates exchange withcoactivator and alternative chromatin-modifying complexesthat mediate transcriptional activation. The complexity andredundancy of receptor isoforms, and the dual role ofreceptors as unliganded repressors and liganded activators,creates challenges for defining the function of the pathwayby disrupting individual components.

Experimental approachesfor studying retinoid functionOne of the most perplexing characteristics of the RAsignaling pathway is that similar phenotypes are seen fromeither loss of signaling or hyperactivation of the pathway.For example, excess RA causes many of the sameembryonic developmental defects seen with vitamin Adeficiency (VAD), which is why women are cautioned tomonitor intake of vitamin A during pregnancy. The RApathway invokes both positive and negative feedback, e.g.,

by activating expression of its own receptors, or RA-degrading enzymes such as CYP26, respectively. Althoughthere is substantial literature describing the effects of exo-genous retinoids on hematopoietic cells, loss-of-functionprovides a more interpretable experimental format forunderstanding the normal role of retinoids and, for thisreason, several distinct approaches have been developed.

Knockouts and mutantsOne of the most direct approaches to test the function of theRA pathway has been to analyze the phenotype of micewith targeted disruptions of the RAR and RXR genes. Eachof the genes (and several specific isoforms) has beenknocked out alone or in combination [18]. InterpretingRXR knockouts can be complicated because these receptorsare also used in other signaling pathways. None of theindividual RAR genes are essential for hematopoiesis, butcombinatorial knockouts have phenotypes as will bediscussed here. The RALDH2 knockout is also a usefulmodel because this gene is one of the family membersrequired for generation of functional RA ligand [19]. In thezebrafish system, a mutation in the RALDH2 gene isresponsible for the neckless phenotype [20], and this mutantprovides an excellent model for probing the developmentalconsequences of defective RA signaling.

Inhibitors and agonistsSpecific small molecule receptor antagonists are availablethat effectively block RA signaling and these can be addedor removed at specific stages of development [21]. Thisapproach obviates issues of functional redundancy forreceptors or RALDH genes [22,23], and is particularlyuseful for embryos that can be cultured (chick, frog,zebrafish), or for mammalian cell culture experiments.Antagonists and agonists that are specific to RARa havealso been developed [24].

VAD modelsA complete VAD effectively and specifically eliminates theretinoid signaling pathway because the ligand (in contrastto the receptors) is nonredundant. The rationale behinda VAD model is that the embryo must metabolize RA frommaternal stores of vitamin A. The mother is healthy as longas her diet is supplemented with RA, which is, however, notpassed on to the embryo. Both rodent and avian modelshave been used effectively. The VAD quail survives untilaround day 4, and it has been particularly useful forstudying the role of RA in the formation of nervous andcardiovascular systems [25,26]. Furthermore, the VADquail is completely rescued if RA is added in ovo or tocultured embryos prior to the 5 somite stage, which provesthat retinoid signaling is not essential until this definedstage of development.

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Regulation of yolk sac hematovascular developmentThe most obvious phenotype of the VAD quail embryo, andthe cause of lethality, is lack of a connection between theyolk sac vasculature and the presumptive inflow tract of theprimordial heart tube [26]. A more recent analysis foundthat primitive erythropoiesis is also disrupted in the bloodislands of VAD embryos [27]. The defect is clearlydocumented by a decrease in staining for hemoglobin, butthe total number of erythroid cells is also quantitativelyreduced severalfold, and the blood that does form containsa high percentage of morphologically abnormal cells. RNAlevels for the transcription factor GATA-2 are decreasedthroughout the caudal region of VAD embryos and thiscorrelates with the hematopoietic defect. There is a well-established epistatic relationship between RA and the bonemorphogentic protein (BMP) pathway relevant to cellsurvival in the developing vertebrate limb [28]. GATA-2 isa known downstream target of the BMP signaling pathway,and so it was considered whether RA might regulatehematopoiesis indirectly through the BMP pathway. In-deed, addition of BMP4 rescued erythropoiesis in culturedVAD quail explants, while the BMP antagonist nogginblocked erythropoiesis when added to cultured normalembryo explants. The VAD embryos have high levels ofabnormal apoptosis, which is consistent with the lack ofa normal cell survival signal, directly or indirectlycontrolled by BMP signaling.

Addition of exogenous RA to cultured Xenopus embryoscauses a phenotype grossly equivalent to VAD, in terms ofblocking embryonic globin expression [29]. In this case, themajor alteration noted was on GATA-1 expression ratherthan GATA-2, which might suggest a block to erythroid celldifferentiation. However, retinoids also regulate HOX geneexpression along the A/P axis, and so the failure inerythropoiesis caused by excess retinoids can be interpretedas a secondary consequence of alterations in mesodermpatterning. It may be that RA has an early function inestablishing hematopoietic mesoderm and a later functionmore specific to primitive erythropoiesis, or even forregulating HOX genes that are implicated more directly inhematopoiesis [30–32]. It will be informative to evaluatehematopoiesis in frog or fish models using VAD or inhibitorapproaches. The inhibitors are particularly well suited forthis question, because they can be added or removed at anystage of early development.

Meanwhile, analysis of yolk sac development in theRALDH2 knockout mouse revealed that RA functions atthe interface of a complex signaling network that is criticalfor development of the embryonic vasculature. The mutantembryos develop intrinsic defects in the vasculature (priorto hematopoietic or cardiac dysfunction) that appear to berelated to altered cell-cycle control. RA normally inducesexpression of the cyclin-dependent kinases p21Cip1 andp27Kip1 [33], which suppresses endothelial cell growth. Inthe RALDH2 mutant embryo, there is abnormal endothelial

cell proliferation due to altered cell-cycle progression, andin addition a defect in vascular plexus remodeling [33]. RAis normally generated via RALDH2 in the visceralendoderm and activates RARa1 and RARa2, both ofwhich are expressed in endothelial cells. Two distinct setsof signals act downstream of RA, based on analysis of themutant embryos and the ability of exogenous factors torescue vasculogenesis [34]. First, RA-dependent expressionof transforming growth factor-b1 and fibronectin is requiredfor survival of visceral endoderm and normal expression ofintegrin a5, which signals to suppress endothelial cellproliferation and induce maturation. Second, RA is requiredindirectly for expression of factors from visceral endoderm(vascular endothelial growth factor-A, Indian hedgehog,and basic fibroblast growth factor) that subsequentlypromote vascular remodeling and formation of a normalyolk sac plexus. It is possible that defects in primitivehematopoiesis noted in the VAD quail are secondary tovascular abnormalities. While the vascular defects of theRALDH2 mutant embryo occur prior to any hemodynamicabnormality, altered signaling from defective blood islandsmay also contribute to the vascular phenotype, given theclose developmental association of yolk sac hematopoieticand endothelial cells.

Regulation of erythropoietinand definitive hematopoiesisThe erythropoietin (EPO) gene is regulated by a 3#enhancer that serves as a paradigm for understandingHIF1a-mediated induction by hypoxia [35]. Adjacent to theHIF1a-binding site is a consensus RARE, shown inhepatocytes to bind to the orphan receptor HNF4 (and notRAR). While it had been noted previously that the gene canbe induced by exogenous RA [36], it was unclear whetherthis was physiologically significant, because erythropoieticphenotypes are not described for the RAR knockouts.However, a careful study of the RXRa knockout mousedemonstrated a stage-specific function for retinoid signal-ing during the establishment of fetal liver erythropoiesis[37]. This was not an obvious phenotype because themutant embryos die around embryonic day 15 (E15) fromcardiac failure, and when analyzed at this stage the fetalliver appears normal. However, during a transient stagearound E12.5 there is a marked delay in fetal liverdevelopment and definitive erythropoiesis, which correlateswith decreased levels of EPO expression. Unlike the VADquail embryos, primitive erythropoiesis is apparentlynormal enough in the RXRa knockout embryo yolk sacto compensate for the transient fetal liver defect, whicheventually recovers by E15. The analysis of RXRa andEPO trans-heterozygotes embryos confirmed a geneticinteraction between the two genes.

Therefore, during establishment of fetal liver erythro-poiesis, the EPO enhancer is dependent on functional RAR/

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RXR receptors that bind to the RARE in the 3# enhancer.As development proceeds, the RAR complex is replaced bythe orphan receptor HNF4, which binds to the same cis-element, but now facilitates a productive interaction withHIF1 bound to the adjacent site, placing the EPO geneunder oxygen responsiveness. According to this mecha-nism, EPO transitions from an RA-regulated gene toa hypoxic-regulated gene in the fetal liver around day11.5. A similar erythropoietic defect for the RAR mutantsis not reported, but this might not have been analyzed ascarefully or may require studying a conditional tripleknockout, since each of the RAR genes are expressed at thisstage in the fetal liver [38].

Regulation of myeloid progenitorsThere has perhaps been the most interest in the role ofretinoids for granulocyte differentiation, in light of thehistory of using retinoids as a therapy for myeloidleukemia. There is, however, at this point, little evidencethat RA signaling is required in vivo for normal steady-stategranulocyte development. It is true that exogenous RAdrives terminal differentiation of granulocytes from com-mitted myeloid progenitors, particularly at the expense oferythroid fate [39–41]. But granulocyte fate is not the onlypathway induced by RA. In granulocyte macrophagecolony-stimulating factor (GM-CSF)-dependent cultures,reduction of the serum retinol results in a decrease indevelopment of myeloid dendritic cells (DC), which arerescued (at the expense of granulocyte development) whenRA is added back to the cultures [42]. In these experiments,RA influenced the expression on DC of MHC class II andcostimulatory molecules, which is a particularly interestingobservation considering the influence of RAwith respect toDC-mediated T-cell development (discussed later). Thus, atleast during in vitro culture conditions, RA signaling canmodulate myeloid development and this is dependent on thecontext of other cytokine-signaling pathways.

To better understand this context, several groups haveinvestigated how RARs interact with cell-cycle controlproteins and components of cytokine-signaling pathways.Both RARa and RARg genes are expressed in thegranulocyte lineage, so the knockout phenotypes of thesegenes should be revealing. However, the double knockout isembryonic lethal and, to date, the phenotype of conditionaldouble-knockout bone marrow has not been reported. Atleast with respect to granulocyte differentiation, thephenotype of RARa/RARg double knockout E14.5 fetalliver cells [43] and RARa1/RARg double-knockout E18bone marrow cells [44] is normal compared to wild-typecells. However, behavior in vitro of the mutant progenitorsis not normal. RARa/RARg null fetal liver cells differen-tiate with enhanced kinetics in response to granulocytecolony-stimulating factor (G-CSF) and stem cell factorcompared to wild-type. Surprisingly, the RARa1/RARg

mutant bone marrow cells show precisely the oppositebehavior, and are impaired for colony differentiation. Theseresults suggest that the RARa2 isoform has specificcapabilities for blocking progenitor cell differentiation,and again supports the general concept that RA signalingcan modulate granulocyte differentiation, but may notnormally do so under steady-state conditions.

Mostly driven by research related to APL, RA targetgenes and interacting signaling pathways have beeninvestigated in myeloid cells. CCAAT/enhancer bindingprotein (C/EBP) transcription factors are major regulatorsof myeloid cell development and relevant targets of RA-induced granulocytic differentiation in APL cells [45]. C/EBPa is required for myelopoiesis [46], while C/EBP3 isrequired specifically for granulocyte differentiation [47].The promoter for C/EBP3 contains a functional RARE,which is a target of repression for the RARa-PML fusionprotein [48]. RA-mediated induction of C/EBP3 results insubsequent activation of the myc antagonist Mad andp27Kip1 [49,50], both of which are required for granulocyteprogenitor cell-cycle arrest. Isoform-specific agonists wereused to show that this arrest is specific to RARa, suggestingthat different RARs act through discrete target gene sets[24]. Expression of p21Cip1 is also directly inducible by RA[51], although the p21 knockout does not appear to bedefective in granulopoiesis.

Not surprisingly, the net effect of RA signaling duringhematopoiesis is influenced by the cytokine environment.For example, G-CSF and GM-CSF enhance RARa expres-sion and granulopoiesis in the FDCP-mixA4 system, whileEPO represses RARa expression during erythroid induction[52]. Similarly, GM-CSF or interleukin-3 (IL-3) enhancesRAR activity in themultipotent EMLcell line [53]. However,in this case there is no change in either the expression levelsof the RA receptors or corepressor/coactivators. Rather, thecytokine response is mediated by STAT5, downstream ofstimulation by Jak2 [54]. RARE derived from the RARb2promoter contains an overlapping STAT/RARE cis elementthat binds activated STAT5, and STAT5 and RARs caninteract physically in an IL-3–dependent fashion. Thus,retinoid and JAK-STAT signaling pathways influence eachother and progenitor biology is presumably dependent onspecificity of RAR and STAT isoforms. While it is not yetknown how these observations relate to normal bone marrowmyelopoiesis, it seems likely that cytokine pathwaysinteracting with RA signaling might at least partially explainthe differential sensitivity ofAPLpatients to retinoid therapy.

Regulation of lymphopoiesisPast studies using the VAD rat model implicated RAsignaling in the regulation of lymphopoiesis. For example,serum IgM and IgG levels are consistently lower in VADrats injected with b-lactoglobulin compared to controls, andthe function of T cells from VAD animals are impaired

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[55], being characterized as displaying excessive Th1 andimpaired Th2 response [56]. Exogenous RA causes thecorresponding opposite effect in vitro on isolated T cells[57]. There is a particularly strong literature documentingimpaired immune responses in the gut of VAD animals[58]. The physiological basis for this immune failure hasperhaps been revealed finally through studies on T-cellhoming [59]. Naı̈ve T cells do not normally migrate intononlymphoid organs until they become activated byantigen, and for the gut this requires expression of a specificchemokine receptor CCR9 and the a4b7 integrin. Expres-sion of these homing receptors is dependent on antigenpresentation by gut DC, and ensures that effector/memoryT cells home to the gut instead, for example, to the skin.Exposure of T cells to RA induces expression of the gut-homing receptors and represses expression of skin-homingreceptors. Intestinal DC express RALDH1 and, therefore,can metabolize vitamin A. In vitro studies had shownpreviously that gut-derived DCs are able to instruct or‘‘imprint’’ gut-homing behavior [60]. Therefore, it seemslikely that by interacting with DC in the gut, T cells areexposed to RA and imprinted to home specifically to thegut, and this model is supported by RA inhibitors and VAD,which cause a decrease in the numbers of a4b71 memory/activated T cells. While many of the details regarding whenand how RA is presented to the T cells are yet to be workedout, this remarkable study provides an example for how RAcan influence cell fate in the hematopoietic system withhigh specificity, and provides a paradigm for future studiesrelevant to homing of other hematopoietic stem andprogenitor cell types.

Regulation of hematopoieticstem and progenitor cellsAs discussed here already, much of the in vitro dataindicates that RA has a propensity to push granulocytedifferentiation from a myeloid progenitor cell. However,there is also evidence that RA can modulate proliferation orsurvival of earlier precursors. This may still favorgranulocyte differentiation at the expense of erythroid orother fates, as shown, for example, by studying descendentsof single CD341 fetal liver cells in culture [61]. On theother hand, RA induces cell-cycle arrest of early human ormouse progenitor cells, and this results in an inhibition ofmyeloid colony formation [62,63]. Mice treated with RARantagonists show an increased number of granulocytes thatcorrelates with enhanced numbers of progenitors [64], alsosuggesting that in vivo the RA pathway normally limitsgranulocyte expansion from an early progenitor. It is notdifficult to imagine that RA could have opposite effects oncell number or fate, depending on the precise developmen-tal stage that the progenitor is induced.

Of considerable interest in this regard is whether RAregulates development of the hematopoietic stem cell. RA

can enhance both short- and long-term repopulating activityof lin2/c-kit1/Sca11 cells when these are grown insuspension culture or tested by serial transplantation assays[65,66]. CD341 cells removed from appropriate growthfactor conditions will undergo apoptosis, and this ispartially prevented by exposure to RA, without alteringcolony-forming capability [67]. It might be possible toexpand stem cell activity by treating purified cells withappropriate doses of RA ex vivo prior to transplantation. Insupport of this concept, RAR antagonists reduce stem cellactivity [65]. It is interesting to note that these stem cellstudies indicate that RA has a positive effect on maintainingstem cell activity, which is the opposite of how RA acts topromote differentiation of myeloid cells. Yet, in apparentcontrast, RA enhances expression of CD38 inCD341CD382 cultured progenitor cells, while RA antag-onists delayed differentiation and increased the numbers ofCD341CD382 cells [68]. Results on stem cells are likelysensitive to the specific subset tested and the cultureconditions, and it will ultimately require the appropriateconditional loss-of-function experiments in defined trans-plantation assays in order to fully evaluate the physiologicalrole of RA in hematopoietic stem cell biology.

Future prospectiveUnraveling the specificity of RA signaling with respect tohematopoiesis is an ongoing challenge that will requiredevelopment of new animal models. Conditional andcombinatorial targeting of RARs and perhaps RALDHs isneeded to overcome issues of early embryonic lethality andfunctional redundancy. At the molecular level, it will beimportant to define the target genes of RA signaling andhow these differ among distinct stem, progenitor, anddifferentiated cell types. Some progress is being made atdefining RA-responsive genes by microarray experiments,confirming, for example, GATA-2 as an RA-activated targetgene [69]. This approach can next be applied to subsets ofRA-responsive hematopoietic cells. HOX genes are rele-vant targets of the RA-signaling pathway in limb, vertebral,and neural development, and it would be surprising if thiswere not also the case also for hematopoiesis, where theimportance of HOX gene regulation is well established[70,71]. In addition, the transcription factors that interactwith downstream components of RA signaling need to bedefined. Interestingly, GATA-2 is an example of an RAtarget gene that also can interact directly with RARa. Bybinding to GATA-2, RARa confers RA-responsiveness toGATA-2, enhances GATA-2 transactivation activity, and(for reasons that are not yet clear) inhibits the ability ofGATA-2 to stimulate embryonic stem cell-derived hemato-poietic colony formation [72]. Physical interactions ofRARs with other hematopoietic transcriptional regulatoryproteins will likely reveal other examples of functionalcross-talk and relevant genetic networks.

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At the cellular level, there is much to be discoveredabout how RA production, turnover, and presentation toreceptor complexes are regulated in specific niches, andhow thresholds of RA concentrations are interpreted byresponding cells. RA activates both BMP and sonichedgehog pathways in various embryonic contexts, andthese may then indirectly mediate RA affects, or evensynergize or modulate RAR-dependent pathways. Theintriguing ability of RA to educate T cells suggests thatthe retinoid pathway may also have cellular functions inregulating stem and progenitor cell homing. While it hasnot been a topic of this review, the ability of RA to stimulateleukemic cell differentiation is, of course, a topic of majorclinical significance, and further progress in understandingthe role of retinoids in normal hematopoietic cell biology iscertain to impact future adaptation of cancer therapies.

AcknowledgmentsThe author is supported by grants from the National Institutes ofHealth (HL64282 and HL56182) and the Irma T. Hirschl Trust. Heis particularly grateful to Dr. Maija Zile (Michigan StateUniversity) for providing an enjoyable education and collabora-tion with the VAD quail.

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