Transcription factor Zeb2 regulates commitment toplasmacytoid dendritic cell and monocyte fateXiaodi Wua, Carlos G. Briseñoa, Gary E. Grajales-Reyesa, Malay Haldara, Arifumi Iwataa, Nicole M. Kretzera,Wumesh KCa, Roxane Tussiwanda, Yujiro Higashib, Theresa L. Murphya, and Kenneth M. Murphya,c,1

aDepartment of Pathology and Immunology, School of Medicine, Washington University, St. Louis, MO 63110; bDepartment of Perinatology, Institute forDevelopmental Research, Aichi Human Service Center, Kasugai, Aichi 480-0392, Japan; and cHoward Hughes Medical Institute, School of Medicine,Washington University, St. Louis, MO 63110

Contributed by Kenneth M. Murphy, November 14, 2016 (sent for review July 20, 2016; reviewed by Christophe Benoist and Richard A. Flavell)

Dendritic cells (DCs) and monocytes develop from a series of bone-marrow–resident progenitors in which lineage potential is regu-lated by distinct transcription factors. Zeb2 is an E-box–bindingprotein associated with epithelial–mesenchymal transition and iswidely expressed among hematopoietic lineages. Previously, weobserved that Zeb2 expression is differentially regulated in pro-genitors committed to classical DC (cDC) subsets in vivo. Usingsystems for inducible gene deletion, we uncover a requirementfor Zeb2 in the development of Ly-6Chi monocytes but not neutro-phils, and we show a corresponding requirement for Zeb2 in ex-pression of the M-CSF receptor in the bone marrow. In addition,we confirm a requirement for Zeb2 in development of plasmacy-toid DCs but find that Zeb2 is not required for cDC2 development.Instead, Zeb2 may act to repress cDC1 progenitor specification inthe context of inflammatory signals.

monocyte | plasmacytoid dendritic cell | transcription factor

Dendritic cells (DCs) comprise several related lineages thatinitiate and regulate immune responses (1). Classical DCs

(cDCs) present antigens to prime naive T cells and produce cyto-kines to activate T cells and innate lymphoid cells. They can becategorized into two distinct lineages, termed cDC1 and cDC2 (2),that rely on different transcription factors for their development andfunction. The cDC1 subset includes lymphoid-resident CD8α+cDCs and tissue-resident CD103+ cDCs that function in cross-presentation of viral antigen and defense against intracellular patho-gens. The cDC2 subset includes heterogeneous populations ofCD172a (Sirp-α)+ cDCs that promote TH17-type responses to bac-teria and fungi and TH2-type responses to parasites. Plasmacytoid DCs(pDCs) are a lineage distinct from cDCs identified by surface ex-pression of CD45R (B220), Siglec-H, and CD317 (Bst2). They do notfunction directly in T-cell priming (3) but are specialized for produc-tion of large quantities of type I IFN in response to infection (4–6).DCs arise from a series of progenitors with progressively restricted

potential (1). Within lineage (Lin)−Kit+Sca-1−IL-7Rα− bone marrow(BM) cells, CD16/32 (FcγRII/III)loCD34+ common myeloid pro-genitors give rise to all myeloid lineages through FcγRII/IIIhiCD34+

granulocyte–macrophage progenitors (GMPs) and FcγRII/IIIloCD34−

megakaryocyte–erythrocyte progenitors. Macrophage–DC pro-genitors (MDPs) differ from GMPs by decreased expression of Kitand increased expression of the chemokine receptor CX3CR1.MDPs express the receptors M-CSFR and Flt3 and give riseto Kit+M-CSFR+Flt3−Ly-6C+

–committed monocyte progenitors(7) and to KitintM-CSFR+Flt3+ common DC progenitors (CDPs).From CDPs, pDCs develop via KitintM-CSFR−IL-7Rα−Flt3+progenitors (8). Committed progenitors of cDCs also developfrom CDPs, and progenitors committed to either the cDC1 or thecDC2 lineage have been identified in the BM and blood (9, 10).Several transcription factors are required for development of DCs

(11). cDC1 development requires Irf8, Nfil3, Id2, and Batf3, whereaspDC development requires Irf8 and Tcf4 (E2-2). cDC2 developmentwas thought to require Irf4; however, recent analysis has shown thatcDC2s develop in the absence of Irf4 but lack CD4 expression and

have impaired migration from tissues (12, 13). Notch2 is required forcDC2s in the spleen and mesenteric lymph node (LN) to acquireexpression of CD4 and ESAM and produce IL-23 in response topathogens (14–16). Klf4 expression in cDC2s is required to induceprotective TH2 responses to Schistosoma mansoni infection (17).A recent study has argued that the transcription factor Zeb2

(Sip1, Zfhx1b) regulates commitment to the cDC2 lineage by re-pression of Id2 (18). Zeb2 interacts with Smad proteins and containsN- and C-terminal zinc finger domains flanking a Smad-bindingdomain, homeodomain, and a C-terminal–binding protein in-teraction domain (19). Zeb2 represses E-cadherin and other com-ponents of cell junctions during epithelial–mesenchymal transition(20, 21), and germline deletion of Zeb2 leads to embryonic lethalityin mice (22, 23). Heterozygous Zeb2 defects in humans are associ-ated with Hirschprung’s disease and Mowat–Wilson syndrome, andZeb2 expression is dysregulated in several human cancers (19). Inthe nervous system, Zeb2 controls myelination by modulating theactivity of Smads activated by bone morphogenetic proteins, mem-bers of the TGF-β superfamily (24). In oligodendrocyte precursors,where Zeb2 expression is low in abundance, activated Smads bindthe coactivator histone acetyltransferase p300 and activate the ex-pression of negative regulatory genes such as Id2 and Hes1; bycontrast, in differentiating oligodendrocytes, expression of Olig1 andOlig2 induces Zeb2, which binds Smad–p300 complexes and re-presses expression of Id2 and Hes1 (24). Within the hematopoieticsystem, Zeb2 cooperates with Tbx21 (T-bet) to promote terminalmaturation of natural killer (NK) cells and CD8+ T cells (25–27),and its inactivation results in broadly dysregulated hematopoiesiswith prominent neutrophilia and loss of B cells and monocytes (28).

Significance

Distinct transcription factors regulate the development of im-mune cell lineages, and changes in their expression can alter thebalance of cell types responding to infection. Recent studieshave identified Zeb2 as a transcription factor important for thefinal maturation of natural killer cells and effector CD8+ T cells.In this study, we show that Zeb2 is required for the developmentof two myeloid cell types, the monocyte and the plasmacytoiddendritic cell, and clarify that this factor is not required for thedevelopment of classical dendritic cells.

Author contributions: X.W., W.K., T.L.M., and K.M.M. designed research; X.W., C.G.B.,G.E.G.-R., M.H., A.I., N.M.K., R.T., and T.L.M. performed research; Y.H. contributed newreagents/analytic tools; X.W., C.G.B., A.I., R.T., T.L.M., and K.M.M. analyzed data; andX.W. and K.M.M. wrote the paper.

Reviewers: C.B., Harvard Medical School; and R.A.F., Yale School of Medicine, HowardHughes Medical Institute.

The authors declare no conflict of interest.

Freely available online through the PNAS open access option.

Data deposition: Gene expression microarray data have been deposited in the GeneExpression Omnibus (accession nos. GSE87882, GSE87883, and GSE87884).1To whom correspondence should be addressed. Email: kmurphy@wustl.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1611408114/-/DCSupplemental.

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Previously, we and others have observed that Zeb2 is down-regulated upon specification of the CDP to the cDC1 lineage (9,29). Id2 is induced by TGF-β and is required for development ofcDC1s but is not required for development of cDC2s (30, 31).Furthermore, the balance between Id2 and E2-2 influences cDC1and pDC development (32–34), and exogenous TGF-β applied toBM progenitors accelerates differentiation to cDCs rather thanpDCs (35). Modest decreases in pDC and cDC2 frequency havebeen observed in mice with conditional deletion of Zeb2 inCD11c+ cells, leading to the interpretation that Zeb2 regulatescommitment of pDC and cDC2 lineages by controlling Id2 ex-pression (18). However, expression of CD11c occurs coordinatelywith lineage specification or, in the case of the committed cDC1progenitor, actually occurs after specification (9). Thus, condi-tional deletion of Zeb2 in CD11c+ cells may not fully eliminate theactions of that transcription factor during lineage specification. Toaddress these issues, we used several systems to control the timingof Zeb2 deletion during DC development, and we find that, incontrast to Scott et al. (18), deletion in early progenitors regulatesspecification to the pDC lineage but not to the cDC2 lineage. Thisfinding is consistent with reports that Id2 is required for the de-velopment of cDC1s but not cDC2s (30, 36). Finally, we foundthat loss of Zeb2 impaired both the expression of M-CSFR andthe development of Ly-6Chi monocytes, implicating Zeb2 activityin the diversification of multiple myeloid lineages.

ResultsWe generated mice in which Zeb2 is conditionally deleted in cellsexpressing Cre recombinase driven by the Itgax promoter (CD11c-Cre) (14). Compared with Zeb2-sufficient (Zeb2fl/fl) mice, CD11c-Cre–driven Zeb2-deficient [Zeb2fl/fl;CD11c-Cre(tg)] mice showedsubstantially decreased pDC frequency in the spleen and skin-draining LN but not in the BM (Fig. 1A). Within the cDC com-partment, Zeb2-deficient mice showed an increased ratio of spleniccDC1s to cDC2s (identified by expression of CD24 and Sirp-α,respectively) and an increased ratio of equivalent cell types inother organs (Fig. 1 B and C). Neither spleen nor BM cellularitywas significantly different between the two groups (Fig. S1). Usingan inducible model of Zeb2 deficiency driven by the type I IFN-inducible Mx1-Cre, pDCs were ablated in vivo 7–9 d after twotreatments with poly(I:C) (Fig. 1D), and this ablation persisted forat least 1 y (Fig. 1E). By contrast, the ratio of CD24+:Sirp-α+ cDCswas perturbed only slightly in that system (Fig. 1F). Neither spleennor BM cellularity was significantly different between the twogroups in the second week after poly(I:C) treatment (Fig. S1),although splenomegaly was grossly evident in Zeb2-deficient mice1 y after treatment. In summary, robust deletion of Zeb2 usingMx1-Cre showed a complete dependence for this factor in pDCdevelopment and not in cDC development.We overexpressed Zeb2 by retroviral transduction in Kithi BM

progenitors cultured in the presence of Flt3L (Fig. 2A). Trans-duction efficiency was consistently lower for Zeb2-expressingretrovirus compared with control retrovirus, yet the transducedfraction showed markedly increased frequency of pDCs with nodetectable change in the ratio of CD24:Sirp-α cDCs (Fig. 2A).To test whether the requirement for Zeb2 was intrinsic to de-veloping pDCs, we mixed CD45.1+CD45.2+ Zeb2+/− BM cells withCD45.1+CD45.2− wild-type (WT) BM cells and cultured themin vitro in the presence of the cytokine Flt3L. In this setting, weobserved that Zeb2 haploinsufficiency produced a partial defect inthe development of pDCs, whereas we observed no defect in Sirp-α+cDC development from Zeb2-haploinsufficient BM (Fig. 2B). Weused another model of conditional Zeb2 deletion driven by the4-hydroxytamoxifen (4-OHT)–inducible Gt(ROSA)26Sor-Cre-ERT2 (R26-iCre) to study the effect of complete Zeb2 deficiencyin vitro. As expected, 4-OHT treatment eliminated pDC devel-opment in Flt3L-treated cultures of Zeb2fl/fl;R26iCre/iCre BM but didnot perturb pDC development in cultures of WT BM (Fig. 2C).

Next, we analyzed the development of CD19−Ly-6G−KithiFlt3+

progenitors in the presence of Flt3L and 4-OHT, mixing CD45.2+

Zeb2fl/fl;R26iCre/iCre cells with congenically marked CD45.1+ Zeb2-sufficient cells (B6.SJL) (Fig. 2D). We observed that Zeb2-defi-cient progenitors were unable to support substantial pDC devel-opment, but CD24+ cDCs and Sirp-α+ cDCs developed from bothZeb2-deficient and Zeb2-sufficient progenitors (Fig. 2D). Thus,

Fig. 1. Zeb2 is required for pDC development in vivo. (A) Samples preparedfrom the spleen, skin-draining LN (SLN), and BM, harvested from mice of theindicated genotypes, are compared for frequency of pDCs. Shown are repre-sentative two-color histograms comparing splenic populations (Top) and a plotdisplaying frequency of pDCs as a proportion of all singlet lymphocytes (Bot-tom). Numbers at the Top indicate percentage of cells within the indicatedgate; dots at the Bottom each represent a distinct biological replicate and arerepresentative of multiple independent experiments. (B and C) Samples pre-pared in A are compared for frequency of cDCs as a proportion of all singletlymphocytes (B) and for frequency of CD24+ cDCs and Sirp-α+ cDCs as a pro-portion of cDCs (C). Shown is a table (B) and representative two-color histo-grams (C). Numbers in C indicate the percentage of cells within the indicatedgate. (D) Samples prepared from the indicated organs, harvested frommice ofthe indicated genotypes 7–9 d after administration of poly(I:C), are comparedfor frequency of pDCs. Shown is a plot displaying frequency of pDCs as aproportion of all singlet lymphocytes. Dots each represent a distinct biologicalreplicate and are representative of multiple independent experiments.(E) Samples prepared from the indicated organs, harvested from mice of theindicated genotypes 1 y after administration of poly(I:C), are compared forfrequency of pDCs. Shown are representative two-color histograms (n = 3 miceper group pooled over two independent, consecutive experiments). Numbersindicate the percentage of cells within the indicated gate. (F) Samples pre-pared in D are compared for frequency of cDCs as in C. res., resident.

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the selective requirement for Zeb2 in pDC but not cDC devel-opment was cell-intrinsic to progenitors.We also examined the effect of complete Zeb2 deficiency on DC

development in vitro using the type I IFN-inducibleMx1-Cre systembecause type I IFN itself can induce pDC death (37) or promotepDC development (38, 39). We cultured CD19−Ly-6G−KithiFlt3+

progenitors in vitro with Flt3L and in the absence or presence ofIFN-α. In this setting, Zeb2-sufficient progenitors skewed awayfrom CD24+ cDC development and toward pDC development(Fig. 3A). However, IFN-α treatment of Zeb2fl/fl;Mx1-Cre(tg)progenitors diverted progenitors toward CD24+ cDC developmentwith nearly complete loss of pDCs and substantial loss of Sirp-α+

cDCs (Fig. 3A). Mixed cultures of Zeb2-sufficient and Zeb2fl/fl;Mx1-Cre(tg) progenitors showed that this action of Zeb2 was cell-intrinsic because Zeb2-sufficient cells contributed exclusively topDC development and to the majority of Sirp-α+ cDC develop-ment, whereas Zeb2-deficient progenitors were the predominant

source of CD24+ cDCs (Fig. 3B). Thus, loss of Zeb2 in DC pro-genitors treated with IFN-α not only abrogated development ofpDCs but diverted progenitors to the CD24+ cDC lineage at theexpense of Sirp-α+ cDC development.We examined targets of Zeb2 regulated during DC develop-

ment by gene expression microarray analysis of WT and CD11c-Cre–driven Zeb2-deficient splenic Sirp-α+ cDCs (Fig. 4). Fewgenes showed more than a threefold change between groups.Among those were Itgad, which encodes CD11d and is part ofthe CD11c-Cre transgenic integration (14), and Ms4a1, whichencodes CD20 (Fig. S2A). Expression of Lyz2 (encoding lyso-zyme) was decreased more than twofold and that of Csf1r(encoding M-CSFR) was halved in Zeb2-deficient Sirp-α+ cDCscompared with WT (Fig. 4A and Fig. S2A). Of transcription-factor–encoding genes, Ifi203 and Id2 were each increased inexpression less than twofold in Zeb2-deficient Sirp-α+ cDCscompared with WT (Fig. 4B). By reverse transcription quanti-tative PCR (RT-qPCR), we confirmed that Id2 expression wasincreased in CD11c-Cre–driven Zeb2-deficient Sirp-α+ cDCscompared with WT Sirp-α+ cDCs (Fig. 4C).Because Ifi204 encodes a protein known to antagonize the

function of Id2 by cytoplasmic translocation (40, 41), we overex-pressed Ifi204 in BM cultured in the presence of Flt3L and analyzedits effect on DC development. However, expression of Ifi204 in-duced no substantial change in the frequency of pDCs or cDCs (Fig.S3A). Because Zeb2 promotes transcription of Smad7 in the centralnervous system (24), we also overexpressed Smad7 in BM culturedin the presence of Flt3L. Again, however, we observed no sub-stantial change in the relative frequency of DC subsets (Fig. S3B).Using the type I IFN-inducibleMx1-Cre system, we also observed

long-term changes in the development of other myeloid lineagesafter deletion of Zeb2. Poly(I:C) treatment induced transient loss ofLy-6Chi blood monocytes in both Zeb2fl/fl;Mx1-Cre(tg) mice andZeb2fl/fl control mice; however, monocytes were restored to pre-treatment frequencies within 3 d in control mice but remaineddepleted in Zeb2fl/fl;Mx1-Cre(tg) mice (Fig. 5A). Similarly, mono-cytes were depleted in the BM and spleen of Zeb2fl/fl;Mx1-Cre(tg)mice compared with Zeb2fl/fl control mice 1 wk after poly(I:C)treatment (Fig. S4). By contrast, neutrophil frequency was not de-creased in Zeb2-deficient mice (Fig. S4). We found that depletionof monocytes persisted for at least 1 y after poly(I:C)-induced Zeb2deletion (Fig. 5B). Consistent with these observations, the fre-quency of M-CSFR+ cells in the BM was substantially decreasedafter Mx1-Cre–driven Zeb2-deletion (Fig. 5C). By contrast, thefrequency of GMPs (Lin−Sca1−Kit+FcγRII/III+) was not decreased1 wk after Mx1-Cre–driven Zeb2 deletion compared with Zeb2-sufficient control mice (Fig. 5C). Using a Zeb2-GFP fusionprotein reporter mouse (42), we found that Zeb2 was expressed inall Lin−M-CSFR+ cells in the BM, supporting the notion that Zeb2may be required for M-CSFR expression (Fig. S5).To determine if commitment to the monocyte lineage was im-

paired by loss of Zeb2, we used gene expression microarrays tocompare neutrophils and Ly-6Chi monocytes from Zeb2fl/fl controlmice or poly(I:C)-treated Zeb2fl/fl;Mx1-Cre(tg) mice (Fig. 6A).Although Zeb2-deficient and Zeb2-sufficient neutrophils werenearly indistinguishable from each other, residual Ly-6Chi mono-cytes developing in poly(I:C)-treated Zeb2fl/fl;Mx1-Cre(tg) miceshowed increased expression of numerous genes compared withZeb2-sufficient Ly-6Chi monocytes (Fig. 6A). In particular, Zeb2-deficient Ly-6Chi monocytes showed increased expression of ∼50genes that as a group are characteristic of WT neutrophils (Fig.6B). For example, Ltf (encoding lactotransferrin), Mmp9 (encod-ing matrix metallopeptidase 9), and Camp (encoding cathelicidinantimicrobial peptide) were more abundantly expressed in Zeb2-deficient Ly-6Chi monocytes and in WT neutrophils than in WTLy-6Chi monocytes. Zeb2-deficient Ly-6Chi monocytes alsoshowed increased expression of several genes characteristic of theGMP (43) and not highly expressed in neutrophils (Fig. 6 B–D).

Fig. 2. Plasmacytoid DCs require Zeb2 in a dose-dependent and cell-intrinsicmanner. (A) Single-cell suspensions of Kithi BM progenitors isolated from WTmice were cultured in the presence of Flt3L and then retrovirally transduced1 d later to overexpress Thy1.1 alone (Empty RV) or Zeb2 and Thy1.1 (Zeb2 RV).Shown are two-color histograms comparing pDC and cDC frequencies amongtransduced (Thy1.1+) cells or 7 d after transduction. Data are representative ofat least two independent experiments. (B) Single-cell suspensions of whole BMisolated from WT or Zeb2+/− mice were mixed with congenically marked(CD45.1+ CD45.2−) single-cell suspensions of whole BM isolated fromWT B6.SJLmice and cultured in the presence of Flt3L. Shown is the proportion of cellsexpressing CD45.2 (blue) or not expressing CD45.2 (orange) among progenywithin the indicated subsets. (C) Single-cell suspensions of whole BM isolatedfrom mice of the indicated genotypes were cultured in the presence of Flt3L(FL) and either 100 nM 4-OHT dissolved in ethanol or ethanol alone (vehicle).Shown are representative two-color histograms comparing a proportion ofSiglec-H+ pDCs among progeny after 9 d of culture (n > 2 biological replicatesper group over at least two independent experiments). (D) BM Kithi Flt3+

progenitors isolated from R26iCre/+ mice or Zeb2fl/fl;R26iCre/iCre mice were mixedwith congenically marked (CD45.1+) BM Kithi Flt3+ progenitors isolated fromWT B6.SJL mice and cultured in the presence of Flt3L and 4-OHT. Shown aretwo-color histograms comparing the proportion of cells within the indicatedsubsets expressing CD45.1 or CD45.2.

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For example, Mpo (encoding myeloperoxidase), Elane (encodingneutrophil-expressed elastase; the transcript is not highly expressedin neutrophils), and Prtn3 (encoding proteinase 3) were moreabundantly expressed in Zeb2-deficient Ly-6Chi monocytes com-pared with WT Ly-6Chi monocytes and neutrophils (Fig. 6C), andthey are also more abundantly expressed in GMPs than in Ly-6Chi

monocytes or neutrophils (Fig. 6D).The transcription-factor–encoding genes Id2 and Myc are each

increased in expression in Zeb2-deficient Ly-6Chi monocytescompared with Zeb2-sufficient Ly-6Chi monocytes (Fig. 6E).Other transcription factor–encoding genes such as Cebpe andZeb1 also showed increased expression in Zeb2-deficient Ly-6Chi

monocytes compared with Zeb2-sufficient counterparts and arealso more highly expressed in neutrophils than in Zeb2-sufficientLy-6Chi monocytes. Several transcription-factor–encoding geneswere expressed less abundantly in Zeb2-deficient Ly-6Chi mono-cytes than in Zeb2-sufficient Ly-6Chi monocytes, including Tcf4,Prdm1 (also known as Blimp1), Klf4 [a target of Irf8 essential formonocyte development (44–46)], Irf4, Fosb, and Atf3 (Fig. 6E).In summary, deletion of Zeb2 resulted in loss of M-CSFR

expression in the BM and in the long-term ablation of Ly-6Chi

monocytes. Residual Ly-6Chi monocytes that develop in the ab-sence of Zeb2 showed increased expression of Id2 along withexpression of genes more characteristic of neutrophils or GMPs.

DiscussionHere, we describe an action of Zeb2 in promoting M-CSFRexpression and repressing expression of neutrophil genes in favorof monocyte development. After inducing deletion of Zeb2, weobserved severe loss of M-CSFR expression on progenitor cellsin the BM and a long-term impairment of monocyte develop-ment in the peripheral blood. Residual Zeb2-deficient Ly-6Chi

monocytes showed increased expression of Id2 compared withWT Ly-6Chi monocytes, and they expressed genes characteristicof WT neutrophils and GMPs. These findings agree with pre-vious reports that Id2 represses PU.1-mediated induction ofCsf1r (47) and that M-CSFR–deficient (op/op) mice have se-verely decreased blood monocyte frequency (48).In agreement with a recent study (18), we confirm a re-

quirement for Zeb2 in the development of pDCs. We observedthat deletion of Zeb2 abrogated pDC development in vitro andin vivo. We also found that expression of Id2 was increased incDC2s lacking Zeb2 in agreement with previous studies (18, 24).Because Id2 overexpression inhibits pDC development (49), andbecause the balance between Id2 and E2-2 regulates the ratio of

cDC1s to pDCs (32–34), our evidence supports a model in whichZeb2 regulates the choice between pDC and cDC1 fate throughrepression of Id2 (Fig. S6).Previously, the similarly incomplete abrogation of pDC and

cDC2 development resulting from CD11c-Cre–driven Zeb2 de-letion suggested a role for this transcription factor in commitmentto both lineages (18). Using inducible systems to delete Zeb2 inearly progenitors, we observed that Zeb2 was not essential forcDC2 lineage specification and survival. Instead, we found only atwofold decrease in cDC2 frequency in Zeb2-deficient micethat showed abrogation of pDC development. Furthermore,Zeb2-deficient cDC2s closely resembled their WT counterpartsin global gene expression. Moreover, because cDC2s appear topersist normally in Id2-deficient mice (30, 36), defects in their

""

Fig. 3. Type I IFN diverts Zeb2-deficient progenitorsto the CD24+ cDC lineage. (A) BM Kithi Flt3+ progen-itors isolated from mice of the indicated genotypeswere cultured in the presence of Flt3L (FL) and IFN-α.Shown are representative two-color histograms com-paring progeny of those isolated cells as analyzedafter 7–7.5 d of culture (n ≥ 3 biological replicates pergroup over two independent experiments). (B) BMKithi Flt3+ progenitors isolated as in A were mixedwith congenically marked (CD45.1+) BM Kithi Flt3+

progenitors isolated from WT B6.SJL mice and cul-tured as in A. Shown are representative two-colorhistograms comparing their progeny as analyzed after7 d of culture (Top) and one-color histograms in-dicating the proportion of pDCs (magenta), CD24+

cDCs (cyan), and Sirp-α+ cDCs (orange) lacking ex-pression of CD45.1 (n ≥ 3 biological replicates pergroup over two independent experiments). In allpanels, numbers indicate the percentage of cellswithin the indicated gate.

Fig. 4. Zeb2-deficient Sirp-α+ cDCs express more abundant Id2 mRNA.(A) Scatterplot comparing changes in gene expression between Zeb2-deficientSirp-α+ cDCs and WT Sirp-α+ cDCs (y axis) to changes in gene expression be-tween WT CD24+ cDCs and WT Sirp-α+ cDCs (x axis). Both axes are logarithmic,and probe sets with less than 1.9-fold changes in expression along either di-mension are omitted for clarity. (B) Heat map showing relative expression in theindicated populations as determined by gene expression microarray analysis,filtered for probe sets assigned to genes encoding nuclear protein products withthe greatest increase in expression in Zeb2-deficient Sirp-α+ cDCs compared withWT Sirp-α+ cDCs. In A and B, probe sets in gray have scant expression (linearnormalized value < 64) in Zeb2-deficient Sirp-α+ cDCs (n ≥ 3 biological replicatesper group pooled over two independent experiments). (C) Plot showing rela-tive expression of Id2 mRNA normalized to Hprt mRNA in Sirp-α+ cDCs isolatedfrom mice of the indicated genotypes as determined by RT-qPCR; each dotrepresents a biological replicate averaged from technical replicates.

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development would not be expected to arise from changes inexpression of Id2. As such, our results are more consistent withthe interpretation that Zeb2 control of Id2 expression regulatesspecification between pDC and cDC1 fates.Addition of type I IFN substantially increased the pDC output

of WT Kithi progenitors in vitro. The cytokine itself can eitherinduce pDC death (37) or promote pDC development (38, 39),and our observation raises the possibility that a developmentalfeed-forward loop could promote pDC development in thecontext of viral infection. Such a mechanism would potentiatethe well-studied molecular feed-forward loop that promotes typeI IFN production (50, 51). In contrast, treatment of Zeb2fl/fl;Mx1-Cre(tg) Kithi progenitors with type I IFN diverted nearly allprogenitors to the cDC1 fate. This observation is not explainedby selective death of pDCs and cDC2s because we observed thatZeb2-deficient progenitors outcompete WT progenitors in gen-erating cDC1s in mixed cultures. Thus, pDC development re-quires cell-intrinsic Zeb2 expression and is not compensated bytype I IFN signaling. Instead, Zeb2 acts to repress specificationof progenitors to the cDC1 fate under these conditions.Taken together, our findings suggest that Zeb2 activity may

engage similar mechanisms to suppress alternative fates inmultiple developing lineages. As lymphoid progenitors not pre-sented here express and require Zeb2 (28), those cells and theirprogeny represent further avenues to explore the mechanisms by

which this transcription factor regulates the development ofimmune lineages.

Materials and MethodsMice carrying the conditional Zeb2fl [B6;129(Cg)-Zfhx1btm1.1Yhi] allele (22)were derived from biological material provided by the RIKEN BioResourceCenter through the National BioResource Project of the Ministry of Edu-cation, Culture, Sports, Science and Technology, Japan. Other mouse strainswere obtained from The Jackson Laboratory as described in SI Materialsand Methods. Mice were bred and maintained in a specific-pathogen–free

Fig. 5. Zeb2 is required for monocyte development in vivo. (A) Blood fromZeb2fl/fl and Zeb2fl/fl;Mx1-Cre(tg) mice is compared for neutrophil and monocytefrequency before and after administration of poly(I:C). Shown are two-colorhistograms analyzed on the indicated days. Numbers indicate the percentage ofcells within the indicated gate. (B) Blood from poly(I:C)-treated mice of theindicated genotypes are compared using two gating schemes for monocytefrequency 1 y after administration of poly(I:C). Shown are representative two-color histograms (n = 3 mice per group pooled over two independent, con-secutive experiments). Numbers indicate percentage of cells within the in-dicated gate. (C) BM from poly(I:C)-treated mice are compared for M-CSFRexpression and frequency of GMPs (identified as Lin− Sca-1−Kit+ FcγRII/III+) 7–9 dafter treatment. Shown are representative two-color histograms (Top) and aplot displaying frequency of cells identified by the indicated surface markers asa proportion of all singlet lymphocytes (Bottom). Numbers at Top indicatepercentage of cells within the indicated gate; dots at Bottom each represent adistinct biological replicate and are pooled from independent experiments.

Fig. 6. Zeb2-deficient Ly-6Chi monocytes express neutrophil-associatedgenes. (A) Volcano plots showing changes in gene expression between Zeb2-deficient and Zeb2-sufficient Ly-6Chi monocytes (mo.) or between Zeb2-deficient and Zeb2-sufficient neutrophils, all isolated from poly(I:C)-treatedmice. (B) Scatterplot comparing changes in gene expression between Zeb2-deficient Ly-6Chi monocytes and Zeb2-sufficient Ly-6Chi monocytes (y axis) tochanges in gene expression between Zeb2-sufficient neutrophils and Zeb2-sufficient Ly-6Chi monocytes (x axis). Compared with their expression inZeb2-sufficient monocytes, some probe sets are more abundantly expressedin Zeb2-deficient monocytes but not in Zeb2-sufficient neutrophils (solidgreen), more abundantly expressed in Zeb2-sufficient neutrophils but not inZeb2-deficient monocytes (solid red), or more abundantly expressed both inZeb2-deficient monocytes and in Zeb2-sufficient neutrophils (dashed yel-low). Numbers in each outlined region indicate absolute probe set count.(C) Heat map showing relative expression of 22 probe sets outlined in B(solid green) among the indicated populations. (D) Relative expression ofnine probe sets clustered in C among the indicated populations in the BM(data from ImmGen). (E) Sparklines showing relative expression of tran-scription-factor–encoding genes more (Top) or less (Bottom) abundantlyexpressed in Zeb2-deficient Ly-6Chi monocytes than in Zeb2-sufficient Ly-6Chi

monocytes. In all panels, results shown are from at least four biologicalreplicates in each group pooled over two independent experiments.

Wu et al. PNAS | December 20, 2016 | vol. 113 | no. 51 | 14779

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animal facility according to institutional guidelines and under protocolsapproved by the Animal Studies Committee of Washington University inSt. Louis.

Flow cytometry samples were stained in magnetic-activated cell-sorting(MACS) buffer at 4 °C and, unless staining for FcγRII/III, in the presence of FcBlock (2.4G2, BD Biosciences). Antibodies and other reagents were pur-chased as described in SI Materials and Methods. Cells were analyzed using aFACSCanto II, LSR II, LSR Fortessa, FACSAria II, or FACSAria Fusion flowcytometer (BD), and data were analyzed using FlowJo software (FlowJo). Allgating strategies incorporated size and doublet discrimination based onforward and side scatter parameters.

Induction of gene deletion, cell preparation, cell culture, microarray analysis,and RT-qPCR were performed as described in SI Materials and Methods.

ACKNOWLEDGMENTS. We thank J. Michael White and the technical staffof the Transgenic Knockout Micro-Injection Core in the Department ofPathology and Immunology at Washington University School of Medicinefor mouse rederivation and the Genome Technology Access Center in theDepartment of Genetics at Washington University School of Medicine. TheGenome Technology Access Center is partially supported by National Can-cer Institute Cancer Center Support Grant P30 CA91842 to the SitemanCancer Center and by Institute of Clinical and Translational Sciences/Clinical & Translational Science Award Grant UL1 TR000448 from the Na-tional Center for Research Resources, a component of the NIH, and by NIHRoadmap for Medical Research. This work was supported by the HowardHughes Medical Institute (K.M.M.), NIH Grants 1K08AI106953 (to M.H.)and 1F31CA189491-01 (to G.E.G.-R.), American Heart Association Grant12PRE12050419 (to W.K.), and the Burroughs Wellcome Fund CareerAward for Medical Scientists (to M.H.).

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