Reporter Insertions Reveal a Strictly B Lymphoid-Specific Expression Pattern of Pax5 in Support of Its Identity Function This information is current as of September 25, 2021. Martin Fuxa and Meinrad Busslinger J Immunol 2007; 178:3031-3037; ; doi: 10.4049/jimmunol.178.5.3031 http://www.jimmunol.org/content/178/5/3031 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2007 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Reporter Gene Insertions Reveal a Strictly B Lymphoid-Specific Expression Pattern of Pax5 in Support of Its B Cell Identity Function1

Martin Fuxa2 and Meinrad Busslinger3

The Pax5 is essential for B cell commitment and development. Although the detailed Pax5 expression pattern within the hemopoietic system is still largely unknown, we previously reported that Pax5 is monoallelically transcribed in pro-B and mature B cells. In this study, we have investigated the expression of Pax5 at single-cell resolution by inserting a GFP or human .Cd2 indicator gene under the translational control of an internal ribosomal entry sequence into the 3؅ untranslated region of Pax5 ihCd2/ihCd2 iGFP/iGFP These insertions were noninvasive, as B cell development was normal in Pax5 and Pax5 mice. Transhetero- Downloaded from zygous Pax5ihCd2/iGFP mice coexpressed GFP and human CD2 at similar levels from pro-B to mature B cells, thus demonstrating biallelic expression of Pax5 at all stages of B cell development. No reporter could be detected in plasma cells and non-B cells of the hemopoietic system. Moreover, the vast majority of common lymphoid progenitors and pre-pro-B cells in the bone marrow of Pax5iGFP/iGFP mice did not yet express GFP, indicating that Pax5 expression is fully switched on only during the transition from uncommitted pre-pro-B cells to committed pro-B cells. Hence, the transcriptional initiation and B cell-specific expression of Pax5 is entirely consistent with its B cell lineage commitment function. The Journal of Immunology, 2007, 178: http://www.jimmunol.org/ 3031–3037.

emopoietic stem cells give rise to B lymphocytes by first of wild-type mice (4, 5). The restoration of Pax5 expression sup- differentiating to lymphoid-primed multipotent progen- presses the multilineage potential of Pax5Ϫ/Ϫ pro-B cells, while H itors (MPP)4 (1), which subsequently develop to com- simultaneously promoting their development to mature B cells (9). mon lymphoid progenitors (CLP) with their characteristic B, T, Conversely, the loss of Pax5 expression by conditional gene in- and NK cell potential (2). The CLP enters the B lymphoid lineage activation converts committed pro-B cells with a restricted B lym- by differentiating via the pre-pro-B cell stage to early pro-B cells, phoid potential into lymphoid progenitors with a broad develop- by guest on September 25, 2021 which undergo B cell lineage commitment followed by develop- mental potential (13). Together, these data identified Pax5 as the ment along the B cell pathway (3–5). critical B cell commitment factor. Pax5 expression is continuously The entry of lymphoid progenitors into the B cell lineage and required to maintain B cell lineage commitment even at later the subsequent developmental progression to terminally differen- stages of B cell development, as conditional Pax5 deletion leads to tiated plasma cells is controlled by a multitude of transcriptional loss of the identity and function of mature B cells (14, 15). At the regulators (3, 6). Pax5 (BSAP) is one of the earliest expressed molecular level, Pax5 fulfills its commitment function by repress- transcription factors that is essential for B cell development, as its ing B cell lineage-inappropriate and activating B cell-spe- mutation arrests B lymphopoiesis at an early progenitor B cell Ϫ/Ϫ cific genes, which results in the shutdown of inappropriate signal- stage in the bone marrow (7, 8). Pax5 pro-B cells possess an ing systems and the simultaneous promotion of B lymphoid signal extensive self-renewal and broad developmental potential (9–11), transduction (15–17). correspond to lymphoid progenitors with a latent myeloid potential The expression of Pax5 was initially studied in cultured cell (12) and, in this regard, resemble the uncommitted pre-pro-B cells lines representing different stages of B cell development, indicat- ing that Pax5 is expressed from pro-B to mature B cells, but not in Research Institute of Molecular Pathology, Biocenter, Vienna, plasmacytoma and other hemopoietic cell lines (18, 19). Given the Received for publication October 10, 2006. Accepted for publication December essential role of Pax5 in B cell lineage commitment, the question 1, 2006. arises when and how Pax5 transcription is initiated at the onset of The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance B cell development. RT-PCR analyses of Pax5 expression in with 18 U.S.C. Section 1734 solely to indicate this fact. FACS-sorted progenitors have generated conflicting results. In two 1 This work was supported by Boehringer Ingelheim and the Austrian Industrial Re- studies, the Pax5 gene is shown to be expressed in pre-pro-B cells search Promotion Fund. already at ϳ40% (5) or close to 100% (20) of the level observed 2 Current address: Department of Immunology and Molecular Pathology, University in committed pro-B cells. Other reports identified even the earlier College London, 46 Cleveland Street, London W1T 4JF, U.K. CLP cell as the developmental stage where Pax5 transcription is 3 Address correspondence and reprint requests to Dr. Meinrad Busslinger, Research Institute of Molecular Pathology, Vienna Biocenter, Dr. Bohr-Gasse 7, A-1030 efficiently initiated (21, 22). At face value, these results are in- Vienna, Austria. E-mail address: [email protected] compatible with the B cell lineage commitment function of Pax5, 4 Abbreviations used in this paper: MPP, multipotent progenitor; CLP, common lym- as both the CLP and pre-pro-B cells are uncommitted progenitors phoid progenitor; IRES, internal ribosomal entry site; DN, double negative; LSK, despite their apparent Pax5 expression (2, 4, 5, 20). To study how LinϪSca1highc-Kithigh progenitor. Pax5 transcription is initiated in early B cell development, we Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00 previously took advantage of the fact that expression of the two www.jimmunol.org 3032 MONITORING Pax5 EXPRESSION IN VIVO

FIGURE 1. Generation of two Pax5 alleles with different reporter gene insertions. A, Structure of the tar- geted Pax5 alleles. The indicated ex- pression cassette was inserted 149 bp downstream of the translation stop codon in exon 10 of Pax5. The cas- sette contained a puromycin (puro) resistance gene, flanked by two loxP sites (arrowheads), upstream of an IRES and an intracellularly truncated (t) human (h) Cd2 gene or GFP gene. Downloaded from Correct targeting was verified by Southern blot analysis of BamHI-di- gested DNA using the indicated 1-kb PCR probe. The BamHI (B) fragment indicative of each allele is shown together with its length (in kilobase). http://www.jimmunol.org/ The two promoters of Pax5 are shown. DT, diphtheria toxoid A; HSV-tk, herpes simplex virus thymi- dine kinase; pA, polyadenylation se- quence. B, Southern blot analysis of BamHI-digested DNA isolated from the indicated mice. C, Normal B cell numbers in homozygous knock-in mice. Absolute numbers of CD19ϩ ϩ B220 B cells are shown for the bone by guest on September 25, 2021 marrow and spleen of 6-wk-old mice of the indicated genotypes (n ϭ 3 each).

Pax5 alleles can be individually monitored in B lymphocytes of into the 3Ј untranslated region of the endogenous Pax5 gene. B cell heterozygous Pax5lacZ/ϩ mice carrying a lacZ reporter gene inser- development was normal in Pax5ihCd2/ihCd2 and Pax5iGFP/iGFP tion in the mutant Pax5 allele (7, 23). Flow cytometric analyses mice, indicating that the two noninvasive reporter gene insertions revealed that Pax5 is predominantly expressed from only one allele can be used to monitor the expression of individual Pax5 alleles. in pro-B and mature B cells, whereas it switches to a biallelic Transheterozygous Pax5ihCd2/iGFP mice coexpressed GFP and hu- transcription mode in pre-B and immature B cells (23, 24). As man CD2 at similar levels from committed pro-B cells in the bone we could confirm these results in genetically unmanipulated B marrow to mature B cells in the spleen, thus demonstrating bial- lymphocytes by single-cell RT-PCR and RNA-FISH analyses, lelic expression of Pax5 at all stages of B cell development. No we proposed that Pax5 is initiated in a stochastic manner from reporter gene expression could, however, be detected in plasma only one allele at the onset of B cell development (23, 24). Our cells and non-B cells of the hemopoietic system. Pax5 is therefore hypothesis of stage-specific monoallelic expression of Pax5 exclusively expressed within the B lymphoid lineage and is re- was subsequently challenged by Rhoades et al. (25), who pro- pressed during terminal differentiation. Importantly, vided evidence for biallelic Pax5 expression in unfractionated the vast majority of CLP and pre-pro-B cells in Pax5iGFP/iGFP (immature and mature) B cells of the spleen by single-cell RT- mice did not express GFP, whereas a minor fraction of these un- PCR analysis. committed progenitors started to up-regulate GFP expression. To study the regulation of Pax5 at single-cell resolution in vivo, These data demonstrate that the onset and B cell specificity of we inserted a GFP or human Cd2 indicator gene under the trans- Pax5 expression are entirely consistent with the B cell lineage lational control of an internal ribosomal entry site (IRES) element commitment function of Pax5. The Journal of Immunology 3033

FIGURE 2. Similar expression of both Pax5 al- leles during B cell development. A, B cell-specific expression of Pax5. Bone marrow cells of 6-wk-old Pax5ihCd2/iGFP mice were analyzed by flow cytom- etry for expression of the pan-B cell marker CD19, and the expression of human CD2 and GFP is dis- played for CD19Ϫ non-B cells and CD19ϩ B cells. B, Pax5 expression at different B cell developmental stages in the bone marrow. Bone marrow cells of 3- and 6-wk-old mice were stained and gated as indicated. GFP and human CD2 expression is shown for pro-B (CD19ϩc-Kitϩ), pre-B (CD19ϩ Downloaded from CD25ϩIgMϪ), immature B (B220intIgMϩ), and recirculating B (B220highIgDhigh) cells. C, Com- parison of the GFP expression levels detected in the gated cell populations of the indicated B cell developmental stages. http://www.jimmunol.org/

Materials and Methods CD16/CD32 Fc block solution (BD Pharmingen). Single-cell suspensions by guest on September 25, 2021 Generation of Pax5 knock-in mice were stained with the respective Abs and analyzed on a Canto flow cy- tometer (BD Biosciences). A wide forward and side light scatter gate was The Pax5ihCd2 and Pax5iGFP targeting vectors were assembled in a pSP64 used for analysis of the different hemopoietic tissues. Bone marrow cells plasmid containing a polylinker with appropriate loxP and restriction sites. and thymocytes were stained with the following PE-coupled lineage A 1.6-kb XhoI-SalI PCR fragment and a 4.5-kb BamHI fragment of the Pax5 marker Abs for identifying MPPs (LSK), CLPs, and double-negative gene were inserted as homology arms together with a floxed puromycin re- (DN)1/2 cells (anti-B220, CD4, CD8␣, CD11b/Mac1, CD11c, CD19, Gr1, sistance gene (positive selection) and an IRES linked to either an intracellu- Ly6C, Pan-NK, TCR-␤, and Ter119) and pre-pro-B cells (same Ab mixture larly truncated human Cd2 gene (hCd2t) or to a codon-improved (enhanced) but lacking anti-B220 and CD4). Subsequently, the cells were washed GFP gene. The HSV-tk and DT-A genes (negative selection) were inserted once, incubated with anti-PE beads (Miltenyi Biotec), and lineage-depleted Ϫ upstream of the short homology arm. FspI-linearized DNA (15 ␮g) was elec- by MACS sorting (Miltenyi Biotec). The progenitor-enriched Lin cells troporated into HM-1 ES cells (1 ϫ 107) followed by selection with 2.5 ␮g/ml were then stained with the corresponding Abs for identifying the MPPs Ϫ Ϫ Ϫ puromycin and 2 ␮M gancyclovir. PCR-positive clones were verified by (LSK) as Lin Sca1highc-KithighIL-7R␣ cells, the CLP as Lin Sca1lowc- ϩ Ϫ Southern blot analysis of BamHI-digested DNA before injection into C57BL/6 KitlowIL-7R␣ cells, and the pre-pro-B cells as Lin c-KitlowB220low cells. Ϫ Ϫ blastocysts and the generation of Pax5ϩ/puro-ihCd2 and Pax5ϩ/puro-iGFP mice, DN1 and DN2 thymocytes were identified as Lin c-KithighCD44highCD25 Ϫ ϩ respectively. The floxed puromycin gene was deleted in the germline by and Lin c-KithighCD44highCD25 cells, respectively. Plasma cells were de- ϩ Ϫ Ϫ Ϫ Ϫ Ϫ crossing Pax5ϩ/puro-ihCd2 and Pax5ϩ/puro-iGFP mice with the Mox2-cre line fined as CD138highCD28 Lin (CD4 CD8␣ F4/80 CD21 ) cells in the (26) to generate Pax5ϩ/ihCd2 and Pax5ϩ/iGFP mice, respectively. The bone marrow and spleen as described (15). For isolating B-1a cells, 6-wk-old Pax5ihCd2 and Pax5iGFP alleles were backcrossed for seven generations or mice were i.p. injected with 10 ml of prewarmed RPMI 1640 medium. After more into the C57BL/6 genetic background. The Pax5ihCd2 and Pax5iGFP massaging the filled peritoneum, the B-1a cell suspension was aspirated, alleles were genotyped by PCR amplification of a 327- and 448-bp DNA washed, and stained with the corresponding Abs. fragment, respectively. These fragments were amplified from the human Cd2 and GFP gene with the forward primers 5Ј-GAGACAAGAGCCCA Immunization with sheep RBC CAGAGTA-3Ј and 5Ј-CCGACCACTACCAGCAGAACA-3Ј, respec- Sheep RBC were washed in PBS and resuspended at 109 cells/ml followed tively, and the common reverse primer 5Ј-ACAGAGGAGCAAGACGGT by i.p. injection of 150 ␮l into an adult mouse. ATT-3Ј in Pax5 exon 10. Results Abs and flow cytometry Generation of two Pax5 alleles with distinct reporter gene The following biotin-, PE-, CyChrome-, allophycocyanin-, PE Cy7-, or insertions allophycocyanin Cy7-coupled Abs were used for flow cytometry: anti- B220 (RA3-6B2), CD4 (GK1.5), CD5 (53-7.3), CD8␣ (53-6.7), CD11b/ The expression of Pax5 was so far determined in cultured B cell lines Mac1 (M1/70), CD11c (HL3), CD19 (1D3), CD21 (7G6), CD23 (B3B4), and sorted lymphocyte fractions with methods that allowed quantifi- CD44 (1M7), CD117/c-Kit (ACK4), F4/80, Gr1 (RB6-8C5), IgD (1.19), cation of Pax5 expression only at the cell population level (5, 18–20). IgM (M41.42), Ly6C (6C3), Pan-NK (DX5), Sca1/Ly6A (D7), TCR-␤ (H57-597), and Ter119 Abs from BD Pharmingen and anti-CD25/IL-2R␣ To be able to measure allele-specific regulation of Pax5 at single-cell (PC61), CD127/IL-7R␣ (A7R34), and human CD2 (RPA-2.10) Abs from resolution, we generated two Pax5 knock-in alleles expressing each a eBioscience. Unspecific Ab binding was suppressed by preincubation with distinct reporter gene from the Pax5 promoters. To this end, we used 3034 MONITORING Pax5 EXPRESSION IN VIVO

FIGURE 3. Pax5 expression in B cells of peripheral lymphoid organs. A, Pax5 expression in splenic B cells. Splenocytes of 6-wk-old Pax5ihCd2/iGFP mice were stained and gated as indicated to reveal human CD2 and GFP expression in immature (IgMhighIgDlow) and ma- ture (IgMlowIgDhigh) B cells as well as in transitional 1 (T1; B220ϩCD21ϪCD23Ϫ), transitional 2 (T2; B220ϩ Downloaded from CD21highCD23high), marginal zone (MZ; B220ϩCD21high CD23low/Ϫ) and follicular (FO; B220ϩCD21intCD23high) B cells. B, Pax5 expression in peritoneal IgMϩCD5ϩ B-1a cells of Pax5ihCd2/iGFP mice. C, Comparison of the GFP expression levels between the indicated B cell subtypes. http://www.jimmunol.org/ by guest on September 25, 2021

homologous recombination in ES cells to insert either a GFP or in- not in non-B cells, which demonstrates exclusive expression of tracellularly truncated human Cd2 indicator gene under the transla- Pax5 within the B cell lineage of the hemopoietic system. We next tional control of an IRES element into the 3Ј untranslated region (exon investigated Pax5 expression during B lymphopoiesis by monitor- 10) of Pax5 (Fig. 1A). Blastocyst injection of correctly targeted ES ing GFP and human CD2 expression at different B cell develop- cells resulted in Pax5ϩ/puro-ihCd2 and Pax5ϩ/puro-iGFP mice, mental stages (Fig. 2B). Both Pax5 alleles were coexpressed in which were crossed with the Mox2-cre line (26) to achieve pro-B (CD19ϩc-Kitϩ), pre-B (CD19ϩCD25ϩIgMϪ), immature B germline deletion of the puromycin resistance gene (Fig. 1B). (B220lowIgMϩ), and recirculating B (B220ϩIgDhigh) cells of the Heterozygous Pax5ϩ/ihCd2 and Pax5ϩ/GFP mice were inter- bone marrow. Flow cytometric analysis of Pax5ihCd2/iGFP spleno- crossed to obtain homozygous Pax5ihCd2/ihCd2 and Pax5iGFP/iGFP cytes also revealed coexpression of GFP and human CD2 in im- mice (Fig. 1B), which were born at Mendelian frequency and mature (IgMhighIgDlow) and mature (IgMlowIgDhigh) B cells as showed no apparent abnormalities. Importantly, flow cytometric well as in transitional 1 (B220ϩCD21ϪCD23Ϫ), transitional 2 (B220ϩ analysis revealed similar numbers of B220ϩCD19ϩ B cells in the CD21highCD23high), marginal zone (B220ϩCD21highCD23low/Ϫ), bone marrow and spleen of Pax5ihCd2/ihCd2 and Pax5iGFP/iGFP and follicular (B220ϩCD21intCD23high) B cells (Fig. 3A). Likewise, mice compared with wild-type control mice (Fig. 1C). We conclude all B-1a cells (IgMϩCD5ϩMac1ϩ) in the peritoneum of therefore that both reporter gene insertions are noninvasive, as they do Pax5ihCd2/iGFP mice coexpressed the human CD2 and GFP in- not interfere with normal expression and function of Pax5. dicator (Fig. 3B). Direct comparison of GFP expression revealed similar levels of Pax5 expression at all stages of B Similar expression of both Pax5 alleles throughout B cell lymphopoiesis except for marginal zone B cells, which ex- development pressed Pax5 at a ϳ2-fold higher level (Figs. 2C and 3C). To- To monitor the expression of each Pax5 allele, we analyzed the gether these data unequivocally demonstrate that Pax5 is ex- bone marrow of transheterozygous Pax5ihCd2/iGFP mice, which ex- pressed within the B lymphoid lineage at similar levels and in press human CD2 from one and GFP from the other Pax5 allele. a biallelic manner from the pro-B to the mature B cell stage. We used the pan-B cell marker CD19 to separate bone marrow cells into non-B cells (CD19Ϫ) and B lymphocytes (CD19ϩ)by Loss of Pax5 expression during terminal plasma cell flow cytometric analysis and then displayed human CD2 and GFP differentiation expression for both cell populations. As shown in Fig. 2A, the GFP We next studied Pax5 expression in late B lymphopoiesis by an- and human CD2 proteins were coexpressed in B lymphocytes, but alyzing germinal center B cells, which undergo Ig class switch The Journal of Immunology 3035 Downloaded from http://www.jimmunol.org/

FIGURE 4. Loss of Pax5 expression during terminal plasma cell dif- ferentiation. A, Pax5 expression in germinal center (GC) B cells. Pax5iGFP/iGFP (green) and wild-type (dashed line) mice at the age of 3 mo were immunized with sheep RBC and analyzed 11 days later by flow cytometry for GFP expression in germinal center B cells (B220ϩPNAϩ)of the spleen. B, Loss of Pax5 expression in plasma cells, which were iden- tified as CD138highCD28ϩLinϪ (CD4ϪCD8␣ϪF4/80ϪCD21Ϫ) cells in the bone marrow and spleen (15). GFP expression is shown for plasma cells iGFP/iGFP of Pax5 (green) and wild-type (dashed) mice 18 days after by guest on September 25, 2021 immunization.

recombination and somatic hypermutation before differentiation into memory B or plasma cells (27). Eleven days after immuniza- tion with sheep RBC, the germinal center B cells (B220ϩ CD19ϩPNAhigh) of homozygous Pax5iGFP/iGFP mice expressed similar GFP levels (Fig. 4A) as the mature B cells in the spleen (Fig. 3A). The terminal differentiation of germinal center B cells FIGURE 5. Initiation of Pax5 expression in early hemopoietic progen- generates short-lived plasma cells in the spleen as well as long- itors. A, Pax5 expression pattern in bone marrow progenitors. Bone marrow iGFP/iGFP lived plasma cells that home to the bone marrow (27). We have cells of 4- to 5-wk-old Pax5 and wild-type control mice were recently described the unambiguous identification of plasma cells depleted of lineage (Lin)-positive cells before flow cytometric analysis high ϩ Ϫ Ϫ Ϫ Ϫ Ϫ using the indicated Abs (for details, see Materials and Methods). GFP as CD138 CD28 Lin (CD4 CD8␣ F4/80 CD21 ) cells by Ϫ Ϫ expression is displayed for the MPPs (Lin c-KithighSca1highIL-7R␣ ) (or flow cytometric analysis (15). The long-lived plasma cells in the Ϫ low low ␣ϩ Ϫ iGFP/iGFP LSK), CLPs (Lin c-Kit Sca1 IL-7R ), pre-pro-B cells (Lin c- bone marrow of immunized Pax5 mice failed to express KitϩB220ϩCD19Ϫ) and pro-B cells (c-KitϩCD19ϩ)ofPax5iGFP/iGFP GFP, whereas the short-lived plasma cells in the spleen retained a (green) and wild-type (dashed line) mice. The percentages of GFPlow and very low GFP level, possibly due to the long half-life of the GFP GFPhigh cells within each progenitor cell population are shown. B, Absence (Fig. 4B). The loss of Pax5 expression is also reflected by of Pax5 expression in early thymic progenitors. The earliest CD4ϪCD8Ϫ the down-regulation of CD19 on plasma cells (28), consistent with DN thymocytes were identified by flow cytometry as LinϪc-Kithigh Ϫ the fact that Pax5 is absolutely required for transcription of its CD44high cells, which were further subdivided into CD25 DN1 and ϩ target gene Cd19 (16). In summary, these results indicate that Pax5 CD25 DN2 pro-T cells. GFP expression is shown (right) was absent in iGFP/iGFP expression is lost during the development of germinal center B DN1 and DN2 cells of Pax5 mice (green) like in wild-type control mice (dashed line). cells to terminally differentiated plasma cells.

Initiation of Pax5 expression in lymphoid progenitors cells from the bone marrow of homozygous Pax5iGFP/iGFP and Turning our attention to early lymphopoiesis, we were particularly wild-type control mice followed by staining with appropriate Abs interested to see whether the initiation of Pax5 expression would to identify the different progenitor cell populations (Fig. 5A). No be consistent with the previously described B cell lineage com- GFP expression could be detected in early MPPs (LSK; LinϪIL- mitment function of Pax5 (9, 10). To determine Pax5 expression in 7R␣Ϫc-KithighSca1high), which also include the hemopoietic stem early hemopoietic progenitors, we first eliminated lineage-positive cells of Pax5iGFP/iGFP mice (Fig. 5A). The majority (93.5%) of 3036 MONITORING Pax5 EXPRESSION IN VIVO

CLPs (LinϪIL-7R␣ϩc-KitlowSca1lowB220ϪCD19Ϫ) and most gene in one of the two Il4 alleles (39, 40). All three methods (83.7%) of the pre-pro-B cells (LinϪc-KitϩB220ϩCD19Ϫ) did not produced concordant results for the Il4 gene, thus unequivocally yet express GFP. A small percentage (3.5%) of CLPs and a slightly demonstrating that this gene is subject to stochastic allele-specific larger fraction (7.8%) of pre-pro-B cells expressed GFP at a low regulation. In contrast, FISH and single-cell RT-PCR analyses re- level, indicating that these cells started to up-regulate Pax5 vealed tight monoallelic regulation of the Il2 gene in mature CD4ϩ expression (Fig. 5A). Similar GFP expression levels as found in T cells (41), whereas the majority of CD4ϩ T cells expressed both committed pro-B cells were observed only in 3% and 8.5% of Il2 alleles in mice containing an Il2GFP knock-in allele (42). These CLPs and pre-pro-B cells, respectively (Fig. 5A). In marked discrepancies question whether negative results obtained by FISH contrast, all pro-B cells (c-KitϩB220ϩCD19ϩ) homogeneously and single-cell RT-PCR analyses always reflect the failure of an expressed GFP both in Pax5iGFP/iGFP (Fig. 5A) and transhet- allele to be expressed or rather result from the limitation of these erozygous Pax5ihCd2/iGFP mice (Fig. 2B). CLPs and pre-pro-B methods in reliably detecting the expression of both alleles, as cells (also referred to as CLP2 or EPLM) are known to have a discussed by Rhoades et al. (25). broad lymphoid and latent myeloid developmental potential in Based on single-cell RT-PCR and RNA-FISH analyses of ge- contrast to the B cell lineage-committed CD19ϩ pro-B cells (2, 4, netically unmodified B cells as well as FACS analysis of 5, 20). As GFP expression of the tagged Pax5 allele is fully Pax5lacZ/ϩ B lymphocytes, we previously concluded that the Pax5 switched on only in committed pro-B cells, we conclude that the gene is monoallelically expressed in pro-B and mature B cells initiation of Pax5 expression is entirely consistent with the B cell during early and late B cell development (23, 24). In an attempt to lineage commitment function of Pax5. verify this hypothesis with a more reliable method for detecting

In addition to the lymphoid progenitors in bone marrow, the allele-specific gene transcription, we tagged the two Pax5 alleles Downloaded from earliest CD4ϪCD8Ϫ DN progenitors (DN1) in the thymus are also with IRES-hCd2 or IRES-GFP insertions. Analysis of transhetero- uncommitted and retain some B cell developmental potential (29, zygous Pax5ihCd2/iGFP mice revealed that Pax5 is biallelically ex- 30). No GFP expression could, however, be observed in DN1 pressed throughout B cell development. Our new data thus confirm (LinϪc-KithighCD44highCD25Ϫ) or DN2 (LinϪc-KithighCD44high and significantly extend the single-cell RT-PCR results of Rhoades CD25ϩ) prothymocytes of Pax5iGFP/iGFP mice (Fig. 5B). More- et al. (25), who demonstrated biallelic Pax5 expression only in

over, a subset of plasmacytoid dendritic cells was shown to exhibit unfractioned splenic B cells. Hence, the negative data of our pub- http://www.jimmunol.org/ low-level expression of lymphoid genes including Pax5 (31). How- lished single-cell RT-PCR and RNA-FISH analyses have misled ever, plasmacytoid dendritic cells (CD19ϪB220ϩLy6CϩCD11cϩ)in us despite careful control experiments (23, 24). We analyzed Pax5 the bone marrow of Pax5iGFP/iGFP mice failed to express detectable expression by RT-PCR only in those B cells displaying biallelic levels of GFP, indicating that these cells do not transcribe significant expression of the equally rare Cd19 mRNA, and the transcription amounts of Pax5 mRNA (data not shown). Hence, the absence of factor gene Ikaros revealed biallelic expression under the same Pax5 expression in uncommitted progenitors and hemopoietic cell RNA-FISH conditions used for the Pax5 gene (23, 24). Our recent types other than B lymphocytes is fully compatible with the known identification of a potent B cell-specific enhancer in intron 5 of role of Pax5 in controlling the B cell identity. Pax5 provides a likely explanation why the lacZ gene is infre- quently transcribed from the endogenous Pax5 promoters in pro-B by guest on September 25, 2021 Discussion and mature B cells of heterozygous Pax5lacZ/ϩ mice (23). This The transcription factor Pax5 is essential for B cell commitment Pax5 enhancer may efficiently interact and thus specifically service and subsequent development to the mature B cell stage (9, 10, 14). the promoter of the neomycin resistance gene in intron 2 of the In this study we have analyzed the hemopoietic expression of Pax5 targeted Pax5 allele (7), as this promoter is located more closely to by monitoring the transcription of individual Pax5 alleles, which the enhancer than the endogenous Pax5 promoters, thus resulting was achieved by noninvasive insertion of different reporter genes in a transgenic artifact. Interestingly, five of the nine mammalian into the two Pax5 alleles. The Pax5 gene was shown to be exclu- are associated with mouse developmental mutant and sively expressed within the B lymphoid lineage of the hemopoietic human disease syndromes, as heterozygous loss-of-function mu- system from the committed pro-B to the mature B cell stage. Both tations in these genes lead to haploinsufficient phenotypes (43). Pax5 alleles were similarly expressed at all stages of B cell de- The Pax6 gene, which is associated with aniridia in humans and velopment, demonstrating that Pax5 is biallelically transcribed in Small eye (Sey) in mice, was shown to be biallelically expressed contrast to our previous report suggesting stage-specific monoal- during lens placode development, thus ruling out the possibility lelic expression of Pax5 (23). that monoallelic Pax6 expression in this tissue causes the haplo- Genes coding for receptors, which convey clonal specificity to a insufficient eye phenotype of Sey mice (44). cell, are known to be subject to allele-specific regulation in addi- The B cell-specific expression of Pax5 from the pro-B to the tion to imprinted and X-chromosomal genes. This class of mono- mature B cell stage is entirely consistent with the B cell identity allelically expressed gene codes for Ag receptors (32), NK cell function of Pax5, as conditional Pax5 inactivation in pro-B or receptors (33), odorant receptors (34), and cadherin-related neu- mature B cells results in loss of the B cell phenotype and ronal receptors (35). Allele-specific regulation has also been de- retrodifferentiation to uncommitted lymphoid progenitors (13, scribed for certain cytokine genes. For instance, the closely linked 14) (C. Cobaleda and M. Busslinger, unpublished data). More- Il4 and Il13 genes are monoallelically transcribed in CD4ϩ Th2 over, the loss of Pax5 expression during plasma cell differen- cells, which was demonstrated by three independent methods. tiation is in agreement with the fact that Pax5-repressed, B cell First, single nucleotide polymorphisms were used to discriminate, lineage-inappropriate genes are reactivated and Pax5-activated, by RT-PCR analysis, the allelic origin of Il4 and Il13 mRNA in B cell-specific genes are down-regulated in plasma cells (15) ϩ ϫ CD4 T cell clones generated from BALB/c CAST/Ei F1 hybrid (A. Schebesta and M. Busslinger, unpublished data). Finally, mice (36, 37). Second, nascent transcripts at one or both alleles of the initiation of Pax5 expression at the onset of B cell devel- the same cytokine genes were visualized by RNA-FISH in nuclei opment fully supports a critical role for Pax5 in B cell lineage of Th2 cells (38). Finally, FACS analysis was used to monitor the commitment. 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