Abstract
The expression of B lineage associated genes during early B cell differentiation stages is not firmly established. Using cell surface markers and multiparameter flow cytometry, bone marrow (BM) cells can be resolved into six fractions, representing sequential stages of development; i.e., pre-Pro-B, early Pro-B, late Pro-B/large Pre-B, small Pre-B, immature B, and mature B cells. Here we quantitate the levels of several B lineage associated genes in each of these fractions by RT-PCR, demonstrating different patterns of expression. We find that expression of terminal deoxynucleotidyl transferase (TdT), lambda 5, and VpreB is predominantly restricted to the Pro-B stages. Rag-1 and Rag-2 expression is also tightly regulated, and is found largely in the Pro-B through small Pre-B stages. Mb-1 is present from Pro-B throughout the pathway at high levels. Finally, Bcl-2 is expressed at high levels only at the pre-Pro-B and mature B stages, whereas it is low during all the intermediate stages. We also correlate this expression data with an analysis of the onset of Ig gene rearrangement as assessed by amplifying D-JH, VH-DJH, and VK-JK. Finally, we report differences in gene expression during B lymphopoiesis at two distinct ontogenic timings, in fetal liver and adult BM: both TdT and the precursor lymphocyte regulated myosin-like light chain are expressed at high levels in the Pro-B cell stage in bone marrow, but are absent from the corresponding fraction in fetal liver. In contrast, lambda 5, VpreB, Rag-1, and Rag-2 are expressed at comparable levels.
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- Alt F. W., Yancopoulos G. D., Blackwell T. K., Wood C., Thomas E., Boss M., Coffman R., Rosenberg N., Tonegawa S., Baltimore D. Ordered rearrangement of immunoglobulin heavy chain variable region segments. EMBO J. 1984 Jun;3(6):1209–1219. doi: 10.1002/j.1460-2075.1984.tb01955.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Alt F., Rosenberg N., Lewis S., Thomas E., Baltimore D. Organization and reorganization of immunoglobulin genes in A-MULV-transformed cells: rearrangement of heavy but not light chain genes. Cell. 1981 Dec;27(2 Pt 1):381–390. doi: 10.1016/0092-8674(81)90421-9. [DOI] [PubMed] [Google Scholar]
- Carlson L. M., Oettinger M. A., Schatz D. G., Masteller E. L., Hurley E. A., McCormack W. T., Baltimore D., Thompson C. B. Selective expression of RAG-2 in chicken B cells undergoing immunoglobulin gene conversion. Cell. 1991 Jan 11;64(1):201–208. doi: 10.1016/0092-8674(91)90221-j. [DOI] [PubMed] [Google Scholar]
- Carmack C. E., Shinton S. A., Hayakawa K., Hardy R. R. Rearrangement and selection of VH11 in the Ly-1 B cell lineage. J Exp Med. 1990 Jul 1;172(1):371–374. doi: 10.1084/jem.172.1.371. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Chirgwin J. M., Przybyla A. E., MacDonald R. J., Rutter W. J. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry. 1979 Nov 27;18(24):5294–5299. doi: 10.1021/bi00591a005. [DOI] [PubMed] [Google Scholar]
- Coffman R. L., Weissman I. L. B220: a B cell-specific member of th T200 glycoprotein family. Nature. 1981 Feb 19;289(5799):681–683. doi: 10.1038/289681a0. [DOI] [PubMed] [Google Scholar]
- Desiderio S. V., Yancopoulos G. D., Paskind M., Thomas E., Boss M. A., Landau N., Alt F. W., Baltimore D. Insertion of N regions into heavy-chain genes is correlated with expression of terminal deoxytransferase in B cells. Nature. 1984 Oct 25;311(5988):752–755. doi: 10.1038/311752a0. [DOI] [PubMed] [Google Scholar]
- Feeney A. J. Lack of N regions in fetal and neonatal mouse immunoglobulin V-D-J junctional sequences. J Exp Med. 1990 Nov 1;172(5):1377–1390. doi: 10.1084/jem.172.5.1377. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gu H., Förster I., Rajewsky K. Sequence homologies, N sequence insertion and JH gene utilization in VHDJH joining: implications for the joining mechanism and the ontogenetic timing of Ly1 B cell and B-CLL progenitor generation. EMBO J. 1990 Jul;9(7):2133–2140. doi: 10.1002/j.1460-2075.1990.tb07382.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gu H., Kitamura D., Rajewsky K. B cell development regulated by gene rearrangement: arrest of maturation by membrane-bound D mu protein and selection of DH element reading frames. Cell. 1991 Apr 5;65(1):47–54. doi: 10.1016/0092-8674(91)90406-o. [DOI] [PubMed] [Google Scholar]
- Gu H., Tarlinton D., Müller W., Rajewsky K., Förster I. Most peripheral B cells in mice are ligand selected. J Exp Med. 1991 Jun 1;173(6):1357–1371. doi: 10.1084/jem.173.6.1357. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hardy R. R., Carmack C. E., Shinton S. A., Kemp J. D., Hayakawa K. Resolution and characterization of pro-B and pre-pro-B cell stages in normal mouse bone marrow. J Exp Med. 1991 May 1;173(5):1213–1225. doi: 10.1084/jem.173.5.1213. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hardy R. R., Hayakawa K. A developmental switch in B lymphopoiesis. Proc Natl Acad Sci U S A. 1991 Dec 15;88(24):11550–11554. doi: 10.1073/pnas.88.24.11550. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hayakawa K., Carmack C. E., Shinton S. A., Hardy R. R. Selection of autoantibody specificities in the Ly-1 B subset. Ann N Y Acad Sci. 1992 May 4;651:346–353. doi: 10.1111/j.1749-6632.1992.tb24634.x. [DOI] [PubMed] [Google Scholar]
- Hayashi S., Kunisada T., Ogawa M., Sudo T., Kodama H., Suda T., Nishikawa S., Nishikawa S. Stepwise progression of B lineage differentiation supported by interleukin 7 and other stromal cell molecules. J Exp Med. 1990 May 1;171(5):1683–1695. doi: 10.1084/jem.171.5.1683. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hockenbery D. M., Zutter M., Hickey W., Nahm M., Korsmeyer S. J. BCL2 protein is topographically restricted in tissues characterized by apoptotic cell death. Proc Natl Acad Sci U S A. 1991 Aug 15;88(16):6961–6965. doi: 10.1073/pnas.88.16.6961. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hombach J., Leclercq L., Radbruch A., Rajewsky K., Reth M. A novel 34-kd protein co-isolated with the IgM molecule in surface IgM-expressing cells. EMBO J. 1988 Nov;7(11):3451–3456. doi: 10.1002/j.1460-2075.1988.tb03219.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Karasuyama H., Kudo A., Melchers F. The proteins encoded by the VpreB and lambda 5 pre-B cell-specific genes can associate with each other and with mu heavy chain. J Exp Med. 1990 Sep 1;172(3):969–972. doi: 10.1084/jem.172.3.969. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kitamura D., Rajewsky K. Targeted disruption of mu chain membrane exon causes loss of heavy-chain allelic exclusion. Nature. 1992 Mar 12;356(6365):154–156. doi: 10.1038/356154a0. [DOI] [PubMed] [Google Scholar]
- Kudo A., Thalmann P., Sakaguchi N., Davidson W. F., Pierce J. H., Kearney J. F., Reth M., Rolink A., Melchers F. The expression of the mouse VpreB/lambda 5 locus in transformed cell lines and tumors of the B lineage differentiation pathway. Int Immunol. 1992 Aug;4(8):831–840. doi: 10.1093/intimm/4.8.831. [DOI] [PubMed] [Google Scholar]
- Kurosawa Y., von Boehmer H., Haas W., Sakano H., Trauneker A., Tonegawa S. Identification of D segments of immunoglobulin heavy-chain genes and their rearrangement in T lymphocytes. Nature. 1981 Apr 16;290(5807):565–570. doi: 10.1038/290565a0. [DOI] [PubMed] [Google Scholar]
- Landau N. R., Schatz D. G., Rosa M., Baltimore D. Increased frequency of N-region insertion in a murine pre-B-cell line infected with a terminal deoxynucleotidyl transferase retroviral expression vector. Mol Cell Biol. 1987 Sep;7(9):3237–3243. doi: 10.1128/mcb.7.9.3237. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lawler A. M., Lin P. S., Gearhart P. J. Adult B-cell repertoire is biased toward two heavy-chain variable-region genes that rearrange frequently in fetal pre-B cells. Proc Natl Acad Sci U S A. 1987 Apr;84(8):2454–2458. doi: 10.1073/pnas.84.8.2454. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lin J., Justement L. B. The MB-1/B29 heterodimer couples the B cell antigen receptor to multiple src family protein tyrosine kinases. J Immunol. 1992 Sep 1;149(5):1548–1555. [PubMed] [Google Scholar]
- Lo K., Landau N. R., Smale S. T. LyF-1, a transcriptional regulator that interacts with a novel class of promoters for lymphocyte-specific genes. Mol Cell Biol. 1991 Oct;11(10):5229–5243. doi: 10.1128/mcb.11.10.5229. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ma A., Fisher P., Dildrop R., Oltz E., Rathbun G., Achacoso P., Stall A., Alt F. W. Surface IgM mediated regulation of RAG gene expression in E mu-N-myc B cell lines. EMBO J. 1992 Jul;11(7):2727–2734. doi: 10.1002/j.1460-2075.1992.tb05338.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Melchers F., Strasser A., Bauer S. R., Kudo A., Thalmann P., Rolink A. Cellular stages and molecular steps of murine B-cell development. Cold Spring Harb Symp Quant Biol. 1989;54(Pt 1):183–189. doi: 10.1101/sqb.1989.054.01.023. [DOI] [PubMed] [Google Scholar]
- Nemazee D., Buerki K. Clonal deletion of autoreactive B lymphocytes in bone marrow chimeras. Proc Natl Acad Sci U S A. 1989 Oct;86(20):8039–8043. doi: 10.1073/pnas.86.20.8039. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Oettinger M. A., Schatz D. G., Gorka C., Baltimore D. RAG-1 and RAG-2, adjacent genes that synergistically activate V(D)J recombination. Science. 1990 Jun 22;248(4962):1517–1523. doi: 10.1126/science.2360047. [DOI] [PubMed] [Google Scholar]
- Oltz E. M., Yancopoulos G. D., Morrow M. A., Rolink A., Lee G., Wong F., Kaplan K., Gillis S., Melchers F., Alt F. W. A novel regulatory myosin light chain gene distinguishes pre-B cell subsets and is IL-7 inducible. EMBO J. 1992 Jul;11(7):2759–2767. doi: 10.1002/j.1460-2075.1992.tb05341.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Park Y. H., Osmond D. G. Post-irradiation regeneration of early B-lymphocyte precursor cells in mouse bone marrow. Immunology. 1989 Mar;66(3):343–347. [PMC free article] [PubMed] [Google Scholar]
- Perlmutter R. M., Kearney J. F., Chang S. P., Hood L. E. Developmentally controlled expression of immunoglobulin VH genes. Science. 1985 Mar 29;227(4694):1597–1601. doi: 10.1126/science.3975629. [DOI] [PubMed] [Google Scholar]
- Reichman-Fried M., Bosma M. J., Hardy R. R. B-lineage cells in mu-transgenic scid mice proliferate in response to IL-7 but fail to show evidence of immunoglobulin light chain gene rearrangement. Int Immunol. 1993 Mar;5(3):303–310. doi: 10.1093/intimm/5.3.303. [DOI] [PubMed] [Google Scholar]
- Sauter H., Paige C. J. B cell progenitors have different growth requirements before and after immunoglobulin heavy chain commitment. J Exp Med. 1988 Oct 1;168(4):1511–1516. doi: 10.1084/jem.168.4.1511. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Schatz D. G., Oettinger M. A., Baltimore D. The V(D)J recombination activating gene, RAG-1. Cell. 1989 Dec 22;59(6):1035–1048. doi: 10.1016/0092-8674(89)90760-5. [DOI] [PubMed] [Google Scholar]
- Schlissel M. S., Baltimore D. Activation of immunoglobulin kappa gene rearrangement correlates with induction of germline kappa gene transcription. Cell. 1989 Sep 8;58(5):1001–1007. doi: 10.1016/0092-8674(89)90951-3. [DOI] [PubMed] [Google Scholar]
- Sentman C. L., Shutter J. R., Hockenbery D., Kanagawa O., Korsmeyer S. J. bcl-2 inhibits multiple forms of apoptosis but not negative selection in thymocytes. Cell. 1991 Nov 29;67(5):879–888. doi: 10.1016/0092-8674(91)90361-2. [DOI] [PubMed] [Google Scholar]
- Shapiro M. A., Weigert M. How immunoglobulin V kappa genes rearrange. J Immunol. 1987 Dec 1;139(11):3834–3839. [PubMed] [Google Scholar]
- Sheehan K. M., Brodeur P. H. Molecular cloning of the primary IgH repertoire: a quantitative analysis of VH gene usage in adult mice. EMBO J. 1989 Aug;8(8):2313–2320. doi: 10.1002/j.1460-2075.1989.tb08358.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Strasser A., Harris A. W., Cory S. bcl-2 transgene inhibits T cell death and perturbs thymic self-censorship. Cell. 1991 Nov 29;67(5):889–899. doi: 10.1016/0092-8674(91)90362-3. [DOI] [PubMed] [Google Scholar]
- Strasser A., Harris A. W., Vaux D. L., Webb E., Bath M. L., Adams J. M., Cory S. Abnormalities of the immune system induced by dysregulated bcl-2 expression in transgenic mice. Curr Top Microbiol Immunol. 1990;166:175–181. doi: 10.1007/978-3-642-75889-8_22. [DOI] [PubMed] [Google Scholar]
- Strasser A., Rolink A., Melchers F. One synchronous wave of B cell development in mouse fetal liver changes at day 16 of gestation from dependence to independence of a stromal cell environment. J Exp Med. 1989 Dec 1;170(6):1973–1986. doi: 10.1084/jem.170.6.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Strasser A., Whittingham S., Vaux D. L., Bath M. L., Adams J. M., Cory S., Harris A. W. Enforced BCL2 expression in B-lymphoid cells prolongs antibody responses and elicits autoimmune disease. Proc Natl Acad Sci U S A. 1991 Oct 1;88(19):8661–8665. doi: 10.1073/pnas.88.19.8661. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Toda M., Hirama T., Takeshita S., Yamagishi H. Excision products of immunoglobulin gene rearrangements. Immunol Lett. 1989 Jun 15;21(4):311–316. doi: 10.1016/0165-2478(89)90025-4. [DOI] [PubMed] [Google Scholar]
- Turka L. A., Schatz D. G., Oettinger M. A., Chun J. J., Gorka C., Lee K., McCormack W. T., Thompson C. B. Thymocyte expression of RAG-1 and RAG-2: termination by T cell receptor cross-linking. Science. 1991 Aug 16;253(5021):778–781. doi: 10.1126/science.1831564. [DOI] [PubMed] [Google Scholar]
- Vaux D. L., Cory S., Adams J. M. Bcl-2 gene promotes haemopoietic cell survival and cooperates with c-myc to immortalize pre-B cells. Nature. 1988 Sep 29;335(6189):440–442. doi: 10.1038/335440a0. [DOI] [PubMed] [Google Scholar]
- Wu G. E., Paige C. J. VH gene family utilization in colonies derived from B and pre-B cells detected by the RNA colony blot assay. EMBO J. 1986 Dec 20;5(13):3475–3481. doi: 10.1002/j.1460-2075.1986.tb04672.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Yancopoulos G. D., Desiderio S. V., Paskind M., Kearney J. F., Baltimore D., Alt F. W. Preferential utilization of the most JH-proximal VH gene segments in pre-B-cell lines. Nature. 1984 Oct 25;311(5988):727–733. doi: 10.1038/311727a0. [DOI] [PubMed] [Google Scholar]
- Yancopoulos G. D., Malynn B. A., Alt F. W. Developmentally regulated and strain-specific expression of murine VH gene families. J Exp Med. 1988 Jul 1;168(1):417–435. doi: 10.1084/jem.168.1.417. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Yaoita Y., Matsunami N., Choi C. Y., Sugiyama H., Kishimoto T., Honjo T. The D-JH complex is an intermediate to the complete immunoglobulin heavy-chain V-region gene. Nucleic Acids Res. 1983 Nov 11;11(21):7303–7316. doi: 10.1093/nar/11.21.7303. [DOI] [PMC free article] [PubMed] [Google Scholar]
