The transcriptional coactivator Maml1 is required for Notch2-mediated marginal zone B-cell development

doi:10.1182/blood-2007-06-097030Prepublished online August 15, 2007;2007 110: 3618-3623   

 Lizi Wu, Ivan Maillard, Makoto Nakamura, Warren S. Pear and James D. Griffin marginal zone B-cell developmentThe transcriptional coactivator Maml1 is required for Notch2-mediated

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HEMATOPOIESIS

The transcriptional coactivator Maml1 is required for Notch2-mediated marginalzone B-cell developmentLizi Wu,1,2 Ivan Maillard,3,4 Makoto Nakamura,1 Warren S. Pear,4-6 and James D. Griffin1

1Department of Medical Oncology, Dana-Farber Cancer Institute, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA; 2Department ofMolecular Genetics and Microbiology, Shands Cancer Center, University of Florida, Gainesville; 3Division of Hematology-Oncology, 4Abramson Family CancerResearch Institute, 5Department of Pathology and Laboratory Medicine, and 6Institute of Medicine and Engineering, University of Pennsylvania School ofMedicine, Philadelphia

Signaling mediated by various Notch re-ceptors and their ligands regulates di-verse biological processes, including lym-phoid cell fate decisions. Notch1 isrequired during T-cell development, whileNotch2 and the Notch ligand Delta-like1control marginal zone B (MZB) cell devel-opment. We previously determined thatMastermind-like (MAML) transcriptionalcoactivators are required for Notch-induced transcription by forming ternarynuclear complexes with Notch and the

transcription factor CSL. The 3 MAMLfamily members (MAML1-MAML3) are col-lectively essential for Notch activity invivo, but whether individual MAMLs con-tribute to the specificity of Notch func-tions is unknown. Here, we addressedthis question by studying lymphopoiesisin the absence of the Maml1 gene. SinceMaml1�/� mice suffered perinatal lethal-ity, hematopoietic chimeras were generatedwith Maml1�/�, Maml1�/�, or wild-type fetalliver progenitors. Maml1 deficiency mini-

mally affected T-cell development, but wasrequired for the development of MZB cells,similar to the phenotype of Notch2 defi-ciency. Moreover, the number of MZB cellscorrelated with Maml1 gene dosage. Sinceall 3 Maml genes were expressed in MZBcells and their precursors, these resultssuggest that Maml1 is specifically requiredfor Notch2 signaling in MZB cells. (Blood.2007;110:3618-3623)

© 2007 by The American Society of Hematology

Introduction

The evolutionarily conserved Notch signaling pathway regu-lates multiple cell fate decisions during hematopoietic andlymphoid development (reviewed in Maillard et al,1 Radtke etal,2 and Tanigaki and Honjo3). Notch is essential for theemergence of definitive hematopoietic stem cells4,5 and atmultiple steps of T-cell development.6-15 Together with othermolecular pathways, Notch signaling also plays an essential rolein the generation of marginal zone B (MZB) cells in thespleen.16–19 MZB cells are a distinct subset of peripheral B cellsthat reside in the splenic marginal zone and are capable ofmounting rapid antibody responses, providing vital early protec-tion against bloodborne infections, particularly encapsulatedbacteria (reviewed in Martin and Kearney18).

Mammalian cells express 4 Notch receptors (Notch1-Notch4) and 5 ligands from the Jagged (Jagged1-Jagged2) orDelta-like families (Dll1, Dll3, and Dll4). Signaling mediatedby specific combinations of Notch receptors and ligands haveunique roles in lymphoid lineage commitment. For instance,loss of the Notch1 receptor perturbs T-cell development, yet theB-cell compartment seems unaffected.15 Conversely, the tar-geted inactivation of the Notch2 receptor or Dll1 ligand resultsin a loss of MZB cells, but T-cell development appearsnormal.16,17 These effects are consistent with phenotypes causedby inactivation of canonical Notch signaling in these compart-ments, because loss of the transcription factor CSL blocks bothT-cell development and MZB cell generation.13,19 Overall, thesestudies indicate that specific Notch ligands and receptors control

distinct cell fate decisions in the lymphoid system. However,such specificity is not completely explained by differentialexpression patterns or preferential molecular interactions ofNotch receptors or ligands, suggesting that other genes cancontribute to specifying Notch receptor functions.

Our group and others have previously identified a family ofMastermind-like (MAML) coactivators as essential regulators ofNotch-induced transcriptional events.20-25 This MAML familycurrently consists of 3 members, MAML1-MAML3. When acti-vated by ligand binding, the Notch receptor is cleaved, and itsintracellular domain (ICN) translocates to the nucleus.26 ICN theninteracts with the transcription factor CSL and recruits the MAMLcoactivator proteins to form an ICN/MAML/CSL transcriptionalcomplex, which regulates the transcription of specific targetgenes.27,28 Structurally, MAML proteins contain a basic domainthat is responsible for ICN binding, and a domain required for thetranscriptional activation of Notch target genes. Biochemically, allMAML proteins appear to form stable DNA-binding complexeswith all 4 ICNs and the transcription factor CSL. Functionally, the3 MAML proteins have overlapping and differential activitiesbased on reporter assays in cultured cells.23 MAML1 and MAML2are similarly able to coactivate all 4 Notch receptors, whereasMAML3 seems only to effectively coactivate Notch4. Importantly,interference with endogenous MAML function through expressionof a dominant-negative MAML1 mutant (DNMAML1) in bonemarrow cells caused early inhibition of T-cell development andMZB cell generation, indicating that Maml genes are collectively

Submitted June 21, 2007; accepted August 11, 2007. Prepublished online asBlood First Edition paper, August 15, 2007; DOI 10.1182/blood-2007-06-097030.

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essential for physiologic Notch functions.8,9,29 However, the pre-cise roles of individual MAML coactivators in vivo, and whetherthey contribute to the diverse biological effects of Notch signaling,have not been addressed.

Because individual Notch receptors have specific effects, thehematolymphoid system is an interesting context to evaluate theindividual contribution of MAML1-MAML3 to Notch-mediatedtranscriptional activation in vivo. To this end, we examined theeffect of Maml1 deficiency on lymphopoiesis using Maml1-deficient mice.30 Since Maml1 deficiency induces perinatal lethal-ity, we generated hematopoietic chimeras from Maml1�/�,Maml1�/�, or wild-type fetal liver progenitors, in order to compre-hensively assess all adult hematopoietic lineages in the absence ofthe Maml1 gene. The targeted deletion of Maml1 resulted in aspecific B-cell phenotype with an absence of the MZB cell lineage.Moreover, the number of MZB cells correlated with Maml1 genedosage. Conversely, T-cell development was largely unaffected,with only a modestly but significantly increased number of ��T cells. Taken together, these results identified a unique signalingcascade in which the ligand Dll1 activates the Notch2 receptor,resulting in a putative Notch2/MAML1/CSL transcriptional com-plex that is essential for MZB cell development. Furthermore, thesefindings demonstrate that individual MAML coactivators providemolecular specificity and are dose limiting in the regulation ofNotch signaling in vivo.

Materials and methods

Mice

Maml1�/� mice were generated as described.30 Briefly, Maml1 exon 1 wasreplaced with a neo cassette through homologous recombination in J129embryonic stem (ES) cells. After germ-line transmission, the null Maml1allele was maintained through heterozygous breeding due to the neonatallethality of Maml1�/� mice. Genotyping was performed by polymerasechain reaction (PCR), as described.30 C57BL/6.Ly5.2 mice (B6-SJL,CD45.1�) were purchased from Taconic (Germantown, NY). Experimentswere performed according to protocols approved by the InstitutionalAnimal Care and Use Committees (IACUC) of the Dana-Farber CancerInstitute and the University of Pennsylvania.

Fetal liver hematopoietic chimeras

Timed pregnant mice were generated through intercrossing of Maml1�/�

mice. Mice were killed at embryonic day (E) 15.5. A cell suspension wasprepared from individual fetal livers. After purification on Ficoll, cells werewashed and kept on ice until genotyping had been completed. Recipientfemale B6-SJL (CD45.1�) mice were irradiated (9 Gy [900 rad]) andreconstituted through tail-vein injection with 1 to 2 � 106 fetal liver cells(CD45.2�). Recipient mice were analyzed 12 weeks after reconstitution.

Antibodies

Antibodies were from Pharmingen (San Diego, CA), or eBioscience(San Diego, CA). The following antibodies were used: anti-CD4,anti-CD8�, anti-CD3, anti–T-cell receptor (TCR) �, anti-TCR�, anti–c-Kit, anti-CD25, anti-B220, anti-CD19, anti-sIgM, anti-CD43, anti-AA4.1, anti-CD21/35, anti-CD23, anti-CD11b, anti-Gr1, anti-NK1.1,anti-CD11c, anti-CD45.1, anti-Sca1, anti-IL-7R�, and anti-F1t3. Biotin-ylated antibodies were revealed with streptavidin–Pacific Blue (Molecu-lar Probes, Eugene, OR) or PE–Texas Red (Caltag, Burlingame, CA).Lineage� cells in the thymus were defined with anti-CD11b, anti-Gr1,anti-B220, anti-CD19, anti-CD11c, anti-CD8�, anti-CD3�, anti-TCR�,anti-TCR�, and anti-NK1.1.

Flow cytometry and cell sorting

Cells were stained on ice after blocking with rat/mouse IgG (Sigma, StLouis, MO). Cells were sorted on FACSAria (Becton Dickinson, San Jose,CA) or MoFlo (Cytomation, Ft Collins, CO). Analysis was performed onLSR II (Becton Dickinson). Doublets were excluded with forward scatter(FSC)–W and side scatter (SSC)–W characteristics. DAPI was used toexclude dead cells. Data were analyzed in FlowJo (Tree Star, San Carlos,CA).

Quantitative RT-PCR

RNA from sorted cell populations was isolated using the RNEasy Micro Kit(QIAGEN, Valencia, CA). cDNA was prepared with the Superscript II kit(Invitrogen, Carlsbad, CA). Real-time PCR was performed with SYBRGreenPCR Master Mix (Applied Biosystems, Foster City, CA) and analyzed onan ABI Prism 7900HT (Applied Biosystems). A standard curve wasgenerated from 10-fold dilutions of a positive control with a known amountof target DNA, so that the abundance of Maml1, Maml2, and Maml3transcripts could be compared. The following primers were used: Maml1,5-ACAAGTCCCAAGGGTGTCAG-3 and 5-TCACACAGCTGTTC-CCAGAC-3; Maml2, 5-TTTCCCCTCAGGATCAGATG-3 and5-AGAGGAGCCACCCGAATACT-3; Maml3, 5-CTGGTGAACTCG-GCTCTCTC-3 and 5-GACGGTTCCCATGACACTCT-3; and Hprt,5-CTCCTCAGACCGCTTTTTGC-3 and 5-TAACCTGGTTCATCA-TCGCTAATC-3.

Results

The 3 Maml genes are differentially expressed during lymphoiddevelopment

The 3 members of the Maml gene family (Maml1-Maml3) have anonoverlapping pattern of expression in nonhematopoietic tis-sues.23 To assess the effects of individual Maml genes duringNotch-mediated cell fate decisions in the hematolymphoid system,we first examined the relative expression levels of Maml1-Maml3during hematopoietic and lymphoid development, focusing ondevelopmental steps with known involvement of Notch signaling.We purified various cell populations including hematopoieticprecursors as well as cells at different stages of B and T lineagedevelopment from the bone marrow (BM), spleen, and thymus. Theexpression of Maml1, Maml2, and Maml3 genes was assessed byquantitative reverse transcription (RT)–PCR in such a way that therelative expression level of Maml family members could bedirectly compared.

As shown in Figure 1, the 3 Maml genes had different patternsof expression. Transcripts from all 3 Maml genes (Maml1-Maml3)were present in multipotent hematopoietic progenitors with aLin�Sca-1�c-kit� (LSK) phenotype, containing hematopoieticstem cells, as well as in early progenitors of the T-cell and B-celllineages in the thymus and bone marrow (ETP, early T lineageprogenitors; CLP, common lymphoid progenitors; Figure 1A,B).Maml1 expression was generally stable during T-cell developmentin the thymus (Figure 1A) and B-cell development in the BM(Figure 1B), as well as in splenic B-cell subsets (Figure 1C). Incontrast, Maml2 was expressed at 3- to 4-fold higher levels thanMaml1 in LSK progenitors, before decreasing during T- and B-celldevelopment (Figure 1A,B). Maml2 expression appeared to in-crease again in all splenic B-cell subsets, including MZB precur-sors (MZP B) and MZB cells, which undergo Notch2-mediatedsignaling. Finally, Maml3 transcripts were detected in LSK progeni-tors and during early lymphoid development, but decreasedsignificantly to very low levels at late stages of T- and B-celldevelopment.

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These results indicated that Maml1-Maml3 were differentiallyexpressed in hematopoietic and lymphoid progenitor compart-ments, but with more than 1 Maml family member expressed at allstages of development. We therefore asked if this pattern ofexpression was associated with functional redundancy of Maml1-Maml3, or if individual Maml family members could contribute tothe specificity of Notch functions in vivo.

Maml1 haploinsufficiency leads to a reduction of MZB cells

To assess specifically the role of Maml1 in Notch-mediated cell fatedecisions, we generated a null Maml1 allele through homologousrecombination in ES cells. We previously described Maml1-deficient mice, which fail to thrive, exhibit a severe musculardystrophy, and die within 10 days of birth.30

In contrast to Maml1� mice, Maml1�/� littermates wereviable and fertile and had no overt phenotypic abnormality.Therefore, we first assessed hematopoietic and lymphoid devel-opment in adult Maml1�/� mice compared with control litter-mates (Figure 2). The cellularity of the BM, thymus, and spleenwere similar in Maml1�/� mice and their Maml1�/� littermates(data not shown). T-cell development in the thymus was notaffected by the loss of 1 Maml1 allele, as shown by the normalgeneration of �� and �� T lineage cells in Maml1�/� thymi(Figure 2A; data not shown). Given the importance of Notch1 atmultiple steps of T-cell development, this indicated that Notch1functions were well preserved in Maml1 heterozygous mice. Wenext assessed splenic MZB cells through their characteristicB220�CD21hiCD23lo/neg immunophenotype (Figure 2B). Weobserved a statistically significant decrease in the percentage ofCD21hiCD23lo/neg among B220� cells in the spleen of Maml1�/�

mice compared with control littermates (Figure 2C). On aver-age, the number of MZB cells was approximately 2-fold lowerin the Maml1 heterozygous spleens. Such reduction of the MZBcell number in Maml1 heterozygous mice are correlated with thereduced level of Maml1 expression at the mRNA and proteinlevels as compared with the wild-type littermates, based on theassessment of mouse embryonic fibroblasts prepared frommouse embryos from our previous study.30

The significant reduction of MZB cell numbers in the Maml1heterozygous background suggested that Maml1 haploinsuffi-ciency could disrupt Notch2-mediated MZB cell development.Interestingly, MZB cell development is influenced by the genedosage of other individual components of the Notch pathway, sinceboth the Notch ligand gene Dll1 and the Notch receptor geneNotch2 are essential and haploinsufficient for MZB celldevelopment.16,17

Maml1 deletion results in a total loss of MZB cells in fetal liverhematopoietic chimeras

To study hematopoiesis in the complete absence of Maml1, it wasnecessary to bypass the early postnatal lethality of Maml1�/� mice.To accomplish this, we generated fetal liver hematopoietic chime-ras through fetal liver cell reconstitution of irradiated recipients.

Figure 1. Maml genes are differentially expressed in hematopoietic andlymphoid progenitor subsets. Quantitative RT-PCR was performed on cDNAisolated from multipotent BM progenitors, thymocyte subsets, BM B lineage progeni-tors, and splenic B-cell subsets from C57BL/6 mice fractioned by flow cytometry.Maml1-Maml3 transcript levels were normalized using Hprt expression. The Maml1/Hprt ratio in LSK progenitors was normalized to 1. The measurements wereperformed in triplicates. Results are shown as means plus or minus SD. (A) Relativeexpression of Maml1-Maml3 in multipotent BM progenitors (LSK subset) andthymocyte subsets. LSK indicates Lin�Sca-1�c-kit�; ETP, early T lineage progenitors(Lin�c-Kit�CD25�); DN2, CD4�CD8� double-negative 2 (Lin�c-Kit�CD25�); DN3,double-negative 3 (Lin�c-Kit�CD25�); and DP, CD4�CD8� double-positive thymo-cytes. (B) Maml expression in BM B-cell progenitor subsets. CLP indicates commonlymphoid progenitors (Lin�Sca-1loc-kitloIL-7R��Flt3�); ProB, pro-B cells(B220�CD43�AA4.1�CD19�sIgM�); PreB, pre-B cells (B220�CD43�AA4.1�sIgM�);and ImmB, immature B cells (B220�CD43�AA4.1�sIgM�). (C) Maml expression insplenic B-cell subsets. Tr1-Tr3 indicates successive stages of immature transitionalB cells (Tr1, B220�AA4.1�sIgMhiCD23�; Tr2, B220�AA4.1�sIgMhiCD23�; Tr3,B220�AA4.1�sIgMintCD23�); FoB, follicular B cells (most abundant mature B-celltype in the spleen; B220�AA4�sIgMintCD23�); MZP B cells, marginal zone B cellprecursors (B220�AA4.1�sIgMhiCD21hiCD23�); MZB, marginal zone B cells(B220�AA4.1�sIgMhiCD21hiCD23lo).

Figure 2. Normal T-cell development and decreased numbers ofMZB cells in Maml1�/� mice. (A) Flow cytometric analysis showed asimilar percentage of CD4�CD8� double-positive and CD4� or CD8�

single-positive cells in the thymus of Maml1�/� mice compared withwild-type littermates. Numbers indicate the percentage of cells in eachquadrant. (B) Flow cytometric analysis of splenic B-cell subsetsshowing a reduced percentage of B220�CD21hiCD23lo MZB cells inMaml1�/� compared with wild-type spleens. Numbers indicate thepercentage of cells in the rectangular box identifying MZB cells.(C) Data from multiple mice were compiled, indicating significantMaml1 dosage-dependent decrease in MZB cells. Each trianglerepresents a data point for a single mouse (Maml1�/�, Œ, n 5;Maml1�/�, ‚, n 5).

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Specifically, fetal liver cells were isolated from E15.5 wild-type,heterozygous, and Maml1�/� embryos and transplanted into le-thally irradiated wild-type recipient mice (B6-SJL, CD45.1�). Therecipient mice expressed a different CD45 allele from the donorcells, thus allowing us to specifically track donor-derived cells(CD45.2�). The donor-derived lymphoid cells were analyzed at12 weeks after transplantation by flow cytometry.

We found that BM B-cell development was preserved inrecipients of Maml1�/� progenitors (data not shown). The samewas true for immature transitional B cells in the spleen of theserecipients, indicating that B-cell production was normal in theabsence of Maml1. In contrast, there was a virtual absence of bothdonor-derived MZB precursors (MZP B, B220�CD21hiCD23�)and MZB cells in Maml1�/� fetal liver cell chimeras (Figure 3).Moreover, the number of MZB cells was reduced to approximately50% in Maml1�/� fetal liver chimeras compared with recipients ofwild-type progenitors, consistent with observations in Maml1�/�

adult mice (Figure 2). These data indicate that Maml1 is absolutelyrequired for MZB cell development and that in vivo Maml1 levelsare limiting downstream of Notch2-mediated signaling.

Modest abnormalities of T-cell development in the absence ofMaml1

We then studied T-cell development in recipients of Maml1�/� fetalliver progenitors. Because no significant impairment in T-celldevelopment had been detected in adult Maml1�/� mice comparedwith wild-type mice (Figure 2), we used recipients of Maml1�/�

progenitors as controls. The percentages of CD4�CD8� double-negative, CD4�CD8� double-positive (DP), and CD4� or CD8�

single-positive cells were similar in the thymi of Maml1�/� andMaml1�/� recipients (Figure 4A). When Lin� thymocytes wereexamined, we found no significant difference in the relative orabsolute numbers of early T lineage progenitors (c-Kit�CD25�)and DN2 (c-Kit�CD25�), DN3 (c-Kit�CD25�), or DN4(c-Kit�CD25�) thymocytes (Figure 4B). There was also no differ-ence in the absolute number of CD4�CD8� DP thymocytes,

although a nonsignificant trend for a modestly decreased number ofMaml1�/� DP thymocytes was apparent (Figure 4C). Thesefindings indicated that �� T-cell development was largely pre-served in the absence of Maml1 and suggested that Notch1signaling can function efficiently through the coactivators Maml2and/or Maml3.

Next, we examined �� T-cell development in the absence ofMaml1 (Figure 5). We found a modest but significant increase inthe percentage of TCR�� cells among CD3� thymocytes inrecipients of Maml1�/� cells compared with Maml1�/� controls(Figure 5A). This translated into a significant increase in theabsolute number of donor-derived �� T cells in the thymus (Figure5B) and spleen (data not shown) of Maml1�/� recipients.

Figure 3. Characterization of splenic B-cell subsets in fetal liver hematopoieticchimeras reveals an absolute dosage-dependent requirement for Maml1 inMZB development. (A) Representative contour plots showing that MZB precursors(MZP B) and MZB populations were absent in Maml1�/� fetal liver chimera, whilereduced to about half of normal in Maml1�/� fetal liver chimera compared with thewild-type controls. Host-derived CD45.1� cells were excluded from the analysis.(B) Compilation of data collected from all the fetal liver chimeras that were analyzed(Maml1�/�, n 9; Maml1�/�, n 21; Maml1�/�, n 9). MZB cells were essentiallyabsent in Maml1�/� chimeras and decreased in numbers in Maml1�/� heterozygouschimeras. Differences were highly statistically significant (P � .01; Student t test).

Figure 4. Preserved development of �� lineage T cells in the absence of Maml1.(A) Flow cytometric analysis showed a similar percentage of CD4�CD8� double-positive and CD4� or CD8� single-positive thymocytes in recipients of Maml1�/�

compared with Maml1�/� progenitors. Numbers indicate the percentage of cells ineach quadrant. Host-derived CD45.1� cells were excluded from the analysis.(B) Normal distribution of Lin� thymocyte progenitor subsets, as defined using c-Kitand CD25 expression, in recipients of Maml1�/� compared with Maml1�/� progeni-tors. Numbers indicate the percentage of cells in each quadrant. Host-derivedCD45.1� cells were excluded from the analysis. (C) Absolute number of donor-derived double-positive thymocytes in all the fetal liver chimeras that were analyzed(Maml1�/�, ‚, n 21; Maml1�/�, Œ, n 9). The trend for decreased numbers ofdouble-positive thymocytes in Maml1�/� compared with Maml1�/� progenitors wasnot statistically significant (P .14; Student t test).

Figure 5. Increased numbers of �� lineage T cells in the absence of Maml1.(A) Flow cytometric analysis showed an increased percentage of TCR�� cells amongCD3� thymocytes in recipients of Maml1�/� compared with Maml1�/� progenitors(representative examples). Numbers indicate the percentage of cells in each box.Host-derived CD45.1� cells were excluded from the analysis. (B) Absolute number ofdonor-derived CD3� �� T cells in the thymus of all the fetal liver chimeras that wereanalyzed (Maml1�/�, ‚, n 21; Maml1�/�, Œ, n 9). The increased number of ��T cells in Maml1�/� compared with Maml1�/� recipients was statistically significant(P � .05; Student t test).

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Discussion

Maml1 is 1 of 3 members of the MAML family of transcriptionalcoactivators, which are essential for mediating Notch signalingvia coactivation of target gene transcription. Importantly, how-ever, the Notch molecules display unique and important func-tions. This functional specificity is best exemplified by thegenetic mouse models in which Notch1 deficiency results inimpaired T-cell development, while Notch2 deficiency causes adramatic absence of MZB cells.15,17 Strikingly, it remains acritical question how various mammalian Notch receptors andligands exert their unique functional activities. In this study, wediscovered an unexpected, in vivo role for Maml1 in mediatingthe signaling of a specific Notch receptor, Notch2. Using theknockout mice we established recently, we observed that thetargeted deletion of 1 member of the MAML transcriptionalcoactivators, the Maml1 gene, led to the absence of MZB cells,similar to the phenotype of Notch2 or Dll1 ligand deficiency.Therefore, this work strongly implicates individual Maml coac-tivators in regulating molecular specificity of Notch receptorfunctions in vivo. Thus, our present study provides evidence forat least 1 mechanism by which this occurs: specifically, byunique transcriptional coactivators of the Notch pathway, theMAML family.

Our studies show only modest abnormalities in T-celldevelopment from Maml1�/� progenitors, indicating that Maml1is largely dispensable to mediate Notch1 function in the thymus,although it has mild effects on �� T-cell development. Previousstudies have provided different conclusions as to the role ofNotch signaling during �� T-cell development. Inactivation ofNotch signaling through Lck-Cre–mediated inactivation ofNotch1 or Lck-Cre–mediated expression of the Notch inhibitorDNMAML1 have not revealed significant effects on �� T-celldevelopment.8,14 In contrast, inactivation of the CSL/RBP-J genethrough the same Lck-Cre transgene was reported to cause anincrease in the number of thymic and splenic �� T cellsassociated with increased export of these cells into the periph-ery.12 In addition, recent data using the OP9-DL1 culture systemto drive T-cell development in vitro have revealed that �� T-celldevelopment is less sensitive to Notch inhibition than �� T-celldevelopment, due to a cooperative effect of Notch and pre-TCRsignals in the �� lineage.6,7 Our observations in the absence ofMaml1 reveal an increase in the ��/�� T-cell ratio, but also atrue increase in the absolute numbers of �� T cells in the thymusand in peripheral lymphoid organs. These findings are mostreminiscent of the effects observed after conditional deletion ofCSL/RBP-J,12 and cannot be completely explained by cooperat-ivity between Notch and pre-TCR signaling, since disruption ofthis effect might give rise to an increased ��/�� ratio but not toan increase in the absolute number of �� T cells. Therefore, theeffect of Notch signaling on �� T-cell development is morecomplex in vivo than it is in culture systems.

With respect to the B-cell phenotype, Maml1-deficient micedisplayed identical MZB cell phenotype as those mouse modelsthat lack Notch ligand Dll1, the receptor Notch2, or thetranscription factor CSL.16,17,19 Our data revealed that thetargeted deletion of Maml1 in hematopoietic cells led to thespecific loss of MZB cells, indicating an absolute requirementfor Maml1 in MZB cell development. Moreover, the Maml1haploinsufficiency leads to a reduction in MZB cells. Thoughthe possible functional compensation by other Maml members

and the effect of the Maml1 deficiency on Notch receptorfunctions in the Maml1 knockout mice remain to be furthercharacterized, we recently found no significant differences inthe expression levels of Maml2, Maml3, Notch2, or CSL in theMaml1-deficient embryonic fibroblasts (L.W., July 2007, unpub-lished data). Therefore, combined with previous studies show-ing that there is an absence of MZB cells in the absence of theNotch ligand Dll1, the receptor Notch2, or the transcriptionfactor CSL, our data indicate that a unique signaling cascadecontrols MZB cell development: (1) the Notch ligand Dll1activates the Notch2 receptor, resulting in ICN2 translocation tothe nucleus; and (2) this complex interacts with the CSLtranscription factor and recruits MAML1 as a coactivator todrive the transcription of genes involved in MZB cell formation.Thus, the Notch2/MAML1/CSL complex is essential for MZBdevelopment, and there is a functional haploinsufficiency foreach of its components.

Currently, the underlying basis for the specific interaction andfunction of the Notch2/MAML1/CSL complex in the nucleusremains unclear, and warrant further investigations because suchstudies will provide mechanistic explanations for specific modula-tion of various Notch receptor signaling in different cellularcontexts. Because both Maml1 and Maml2 transcripts were presentin splenic B cells (Figure 1), the specific requirement for Maml1could not be explained by the absence of other Maml familymembers in cells exposed to Notch2 signals. Instead, the datasuggested that MAML1 provided specific molecular features to theNotch2 transcriptional activation complex that could not besubstituted by Maml2. In addition, the preservation of nearlynormal T-cell development in the absence of Maml1 suggested thatNotch1 functions are either mediated by Maml2 and/or Maml3, orthat both Maml1 and other Mamls can interact well with Notch1 invivo. Another possibility to consider is that Maml1 might associatewith other, distinct cellular factors that are required for theexpression of genes necessary for MZB cell development. Finally,it is possible (although unlikely) that only a small subset of cells inthe pre-MZB population represents true MZB precursors, and thatthese cells might express only MAML1.

In summary, our study provides in vivo evidence for thefunctional specificity of Maml family members in a well-definedNotch function and indicates that Maml expression levels can belimiting in vivo. It should be noted that our work suggests theexistence of Notch2/MAML1/CSL complex based on the strongmouse genetic data from our and other groups, and currently we donot have direct biochemical data supporting that such uniquecomplex regulating MZB cell development due to the inherent lowlevel of endogenous Notch activity and the lack of specific Notchantibodies. Moreover, the specific interaction of Dll1/Notch2/MAML1 could not be clearly predicted from in vitro biochemicaldata showing that overexpressed MAML proteins appear to formstable complexes with all 4 ICNs in immunoprecipitation assays.23

Therefore, more sensitive assays will be required to test if a higherbinding affinity between MAML1 and Notch2 can be detected atphysiologic levels in vivo. In addition, detailed structural informa-tion is now available regarding the trimolecular complex betweenCSL, Notch1, and the N-terminal Notch-binding domain ofMAML1.27 Such emerging structural insights will provide a basisto investigate whether structural differences underlie the in vivospecificity of various transcriptional complexes involving combina-tions of individual Notch and MAML family members. In thefuture,

3622 WU et al BLOOD, 15 NOVEMBER 2007 � VOLUME 110, NUMBER 10

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this information might lead to novel strategies to inhibit some butnot all Notch functions in cancer and other diseases where Notchsignaling plays a pathogenic role.

Acknowledgments

This work was supported by grants from the National Institutes ofHealth (RO1CA097148 to L.W., RO1AI047833 to W.S.P., andRO1CA036167 to J.D.G.). I.M. was supported by a grant from theDamon Runyon Cancer Research Foundation (DRG-102–05).Additional support was provided by a Specialized Center ofResearch (SCOR) grant from the Leukemia and LymphomaSociety (to W.S.P.).

Authorship

Contribution: L.W. and I.M. participated in the design of the study,performed research, and wrote the manuscript. M.N. performedpart of the research. W.S.P. and J.D.G. were responsible for studyconception, design, and oversight as well as help with manuscriptpreparation. All authors read and approved the final manuscript.L.W. and I.M. contributed equally to this work.

Conflict-of-interest disclosure: The authors declare no compet-ing financial interests.

Correspondence: Lizi Wu, Cancer and Genetics ResearchComplex, Rm 362, Department of Molecular Genetics and Micro-biology, Shands Cancer Center University of Florida, 1376 MowryRd, Gainesville, FL 32610-3633; e-mail: [email protected].

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