Reporter Gene Insertions Reveal a Strictly B Lymphoid-Specific Expression Pattern of Pax5 in Support of Its B Cell 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 References This article cites 44 articles, 16 of which you can access for free at: http://www.jimmunol.org/content/178/5/3031.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 25, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts 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 transcription factor 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 gene expression 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 genes 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, Vienna Biocenter, Vienna, Austria 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).