UNLABELLED: The development of functional B lymphocytes during chicken embryogenesis relies on a series of tightly regulated processes. Precursor B cells migrate from the spleen via the blood to the bursa of Fabricius, where they colonize the bursal follicles to undergo further maturation and differentiation. To better understand the molecular mechanisms underlying early B cell migration in the chicken embryo, transcriptome analysis of B cells isolated from the spleen, blood, and bursa at embryonic days (ED) 12, ED14, and ED16 was performed. These findings suggest that sphingosine-1-phosphate (S1P) and its receptors regulate B cell presence in the bloodstream, while CCR7 and CXCR4 guide B cells to the bursa. Additionally, integrins and cell adhesion molecules, such as PECAM1, appear to facilitate transendothelial migration into the bursal mesenchyme. This study highlights a coordinated interplay between chemokines, integrins and cell adhesion molecules involved in B cell recruitment and colonization of the bursa microenvironment. These findings enhance our understanding of early B cell migration and shed light on the mechanisms governing B cell trafficking during chicken embryonic development. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12864-025-11749-w.

Center for Infection Prevention Technical University of Munich Freising Germany

Division of Animal Physiology and Immunology TUM School of Life Sciences Technical University of Munich Freising Germany

Institute of Molecular Genetics Czech Academy of Sciences Prague Czech Republic

Reproductive Biotechnology TUM School of Life Sciences Technical University of Munich Freising Germany

Zobrazit více v PubMed

Houssaint E, Mansikka A, Vainio O. Early separation of B and T lymphocyte precursors in chick embryo. J Exp Med. 1991;174(2):397–406. PubMed DOI PMC

Dieterlen-Lievre F. On the origin of Haemopoietic stem cells in the avian embryo: an experimental approach. J Embryol Exp Morphol. 1975;33(3):607–19. PubMed

Le Douarin NM, Houssaint E, Jotereau FV, Belo M. Origin of Hemopoietic stem cells in embryonic bursa of Fabricius and bone marrow studied through interspecific chimeras. Proc Natl Acad Sci U S A. 1975;72(7):2701–5. PubMed DOI PMC

Benatar T, Iacampo S, Tkalec L, Ratcliffe MJ. Expression of Immunoglobulin genes in the avian embryo bone marrow revealed by retroviral transformation. Eur J Immunol. 1991;21(10):2529–36. PubMed DOI

Pink JR, Vainio O, Rijnbeek AM. Clones of B lymphocytes in individual follicles of the bursa of Fabricius. Eur J Immunol. 1985;15(1):83–7. PubMed DOI

Cooper MD, Peterson RD, Good RA. Delineation of the thymic and bursal lymphoid systems in the chicken. Nature. 1965;205:143–6. PubMed DOI

Ratcliffe MJ. Antibodies, Immunoglobulin genes and the bursa of Fabricius in chicken B cell development. Dev Comp Immunol. 2006;30(1–2):101–18. PubMed DOI

Weill JC, Reynaud CA. The chicken B cell compartment. Science. 1987;238(4830):1094–8. PubMed DOI

McCormack WT, Tjoelker LW, Thompson CB. Immunoglobulin gene diversification by gene conversion. Prog Nucleic Acid Res Mol Biol. 1993;45:27–45. PubMed DOI

Reynaud CA, Bertocci B, Dahan A, Weill JC. Formation of the chicken B-cell repertoire: ontogenesis, regulation of Ig gene rearrangement, and diversification by gene conversion. Adv Immunol. 1994;57:353–78. PubMed DOI

Lassila O. Emigration of B cells from chicken bursa of Fabricius. Eur J Immunol. 1989;19(5):955–8. PubMed DOI

Reynaud CA, Imhof BA, Anquez V, Weill JC. Emergence of committed B lymphoid progenitors in the developing chicken embryo. EMBO J. 1992;11(12):4349–58. PubMed DOI PMC

Pickel JM, McCormack WT, Chen CH, Cooper MD, Thompson CB. Differential regulation of V(D)J recombination during development of avian B and T cells. Int Immunol. 1993;5(8):919–27. PubMed DOI

Sayegh CE, Ratcliffe MJ. Perinatal deletion of B cells expressing surface Ig molecules that lack V(D)J-encoded determinants in the bursa of Fabricius is not due to intrafollicular competition. J Immunol. 2000;164(10):5041–8. PubMed DOI

Schusser B, Collarini EJ, Yi H, Izquierdo SM, Fesler J, Pedersen D, et al. Immunoglobulin knockout chickens via efficient homologous recombination in primordial germ cells. Proc Natl Acad Sci U S A. 2013;110(50):20170–5. PubMed DOI PMC

Nagy N, Busalt F, Halasy V, Kohn M, Schmieder S, Fejszak N, et al. In and out of the Bursa-The role of CXCR4 in chicken B cell development. Front Immunol. 2020;11:1468. PubMed DOI PMC

Laparidou M, Schlickenrieder A, Thoma T, Lengyel K, Schusser B. Blocking of the CXCR4-CXCL12 interaction inhibits the migration of chicken B cells into the Bursa of Fabricius. Front Immunol. 2019;10:3057. PubMed DOI PMC

Sayegh CE, Demaries SL, Iacampo S, Ratcliffe MJ. Development of B cells expressing surface Immunoglobulin molecules that lack V(D)J-encoded determinants in the avian embryo bursa of Fabricius. Proc Natl Acad Sci U S A. 1999;96(19):10806–11. PubMed DOI PMC

Fonfria J, Moreno J, Gomez del Moral M, Alonso L, Zapata AG. The diffusely-infiltrated lymphoid tissue of the bursa of Fabricius of Sturnus unicolor. Histological organization and functional significance. Histol Histopathol. 1994;9(2):333–8. PubMed

Springer TA. Traffic signals for lymphocyte recirculation and leukocyte emigration: the multistep paradigm. Cell. 1994;76(2):301–14. PubMed DOI

Fabbri M, Bianchi E, Fumagalli L, Pardi R. Regulation of lymphocyte traffic by adhesion molecules. Inflamm Res. 1999;48(5):239–46. PubMed DOI

Sinha RK, Mage RG. Developing neonatal rabbit appendix, a primary lymphoid organ, is seeded by immature blood-borne B cells: evidence for roles for CD62L/PNAd, CCR7/CCL21, alpha4beta1 and LFA-1. Dev Comp Immunol. 2004;28(7–8):829–41. PubMed DOI

Masteller EL, Lee KP, Carlson LM, Thompson CB. Expression of Sialyl Lewis(x) and Lewis(x) defines distinct stages of chicken B cell maturation. J Immunol. 1995;155(12):5550–6. PubMed DOI

Lasky LA. Selectins: interpreters of cell-specific carbohydrate information during inflammation. Science. 1992;258(5084):964–9. PubMed DOI

Palojoki E, Jalkanen S, Toivanen P. Sialyl LewisX carbohydrate is expressed differentially during avian lymphoid cell development. Eur J Immunol. 1995;25(9):2544–50. PubMed DOI

Sinha RK, Alexander C, Mage RG. Regulated expression of peripheral node addressin-positive high endothelial venules controls seeding of B lymphocytes into developing neonatal rabbit appendix. Vet Immunol Immunopathol. 2006;110(1–2):97–108. PubMed DOI

Chatzikonstantinou S, Poulidou V, Arnaoutoglou M, Kazis D, Heliopoulos I, Grigoriadis N et al. Signaling through the S1P-S1PR Axis in the gut, the immune and the central nervous system in multiple sclerosis: implication for pathogenesis and treatment. Cells. 2021;10(11). PubMed PMC

Sic H, Kraus H, Madl J, Flittner KA, von Munchow AL, Pieper K, et al. Sphingosine-1-phosphate receptors control B-cell migration through signaling components associated with primary immunodeficiencies, chronic lymphocytic leukemia, and multiple sclerosis. J Allergy Clin Immunol. 2014;134(2):420–8. PubMed DOI

Juarez JG, Harun N, Thien M, Welschinger R, Baraz R, Pena AD, et al. Sphingosine-1-phosphate facilitates trafficking of hematopoietic stem cells and their mobilization by CXCR4 antagonists in mice. Blood. 2012;119(3):707–16. PubMed DOI

Crousillac S, Colonna J, McMains E, Dewey JS, Gleason E. Sphingosine-1-phosphate elicits receptor-dependent calcium signaling in retinal Amacrine cells. J Neurophysiol. 2009;102(6):3295–309. PubMed DOI PMC

Bradaric MJ, Barua A, Penumatsa K, Yi Y, Edassery SL, Sharma S, et al. Sphingosine-1 phosphate receptor (S1p1), a critical receptor controlling human lymphocyte trafficking, is expressed in Hen and human ovaries and ovarian tumors. J Ovarian Res. 2011;4(1):4. PubMed DOI PMC

Wilson SM, Chambers AF. Experimental metastasis assays in the chick embryo. Curr Protoc Cell Biol. 2004;Chap. 19:Unit 19 6. PubMed

Cossarizza A, Chang HD, Radbruch A, Akdis M, Andra I, Annunziato F, et al. Guidelines for the use of flow cytometry and cell sorting in immunological studies. Eur J Immunol. 2017;47(10):1584–797. PubMed DOI PMC

Andrews S. FastQC 2010 [Available from: https://www.bioinformatics.babraham.ac.uk/projects/fastqc/

Ewels P, Magnusson M, Lundin S, Kaller M. MultiQC: summarize analysis results for multiple tools and samples in a single report. Bioinformatics. 2016;32(19):3047–8. PubMed DOI PMC

Chen S, Zhou Y, Chen Y, Gu J. Fastp: an ultra-fast all-in-one FASTQ preprocessor. Bioinformatics. 2018;34(17):i884–90. PubMed DOI PMC

Kim D, Paggi JM, Park C, Bennett C, Salzberg SL. Graph-based genome alignment and genotyping with HISAT2 and HISAT-genotype. Nat Biotechnol. 2019;37(8):907–15. PubMed DOI PMC

Liao Y, Smyth GK, Shi W. FeatureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics. 2014;30(7):923–30. PubMed DOI

Love MI, Huber W, Anders S. Moderated Estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014;15(12):550. PubMed DOI PMC

Galaxy C. The galaxy platform for accessible, reproducible and collaborative biomedical analyses: 2022 update. Nucleic Acids Res. 2022;50(W1):W345–51. PubMed DOI PMC

Kucukural A, Yukselen O, Ozata DM, Moore MJ, Garber M. DEBrowser: interactive differential expression analysis and visualization tool for count data. BMC Genomics. 2019;20(1):6. PubMed DOI PMC

Heberle H, Meirelles GV, da Silva FR, Telles GP, Minghim R. InteractiVenn: a web-based tool for the analysis of sets through Venn diagrams. BMC Bioinformatics. 2015;16(1):169. PubMed DOI PMC

Institute B. Morpheus [RRID: SCR_017386:[Available from: https://software.broadinstitute.org/morpheus

Thomas PD, Ebert D, Muruganujan A, Mushayahama T, Albou LP, Mi H. PANTHER: making genome-scale phylogenetics accessible to all. Protein Sci. 2022;31(1):8–22. PubMed DOI PMC

Shannon P, Markiel A, Ozier O, Baliga NS, Wang JT, Ramage D, et al. Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res. 2003;13(11):2498–504. PubMed DOI PMC

Huang da W, Sherman BT, Lempicki RA. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat Protoc. 2009;4(1):44–57. PubMed DOI

Blake JA, Richardson JE, Davisson MT, Eppig JT. The mouse genome database (MGD). A comprehensive public resource of genetic, phenotypic and genomic data. The mouse genome informatics group. Nucleic Acids Res. 1997;25(1):85–91. PubMed DOI PMC

Kanehisa M, Furumichi M, Sato Y, Kawashima M, Ishiguro-Watanabe M. KEGG for taxonomy-based analysis of pathways and genomes. Nucleic Acids Res. 2023;51(D1):D587–92. PubMed DOI PMC

Szocs E, Balic A, Soos A, Halasy V, Nagy N. Characterization and ontogeny of a novel lymphoid follicle inducer cell during development of the bursa of Fabricius. Front Immunol. 2024;15:1449117. PubMed DOI PMC

Kothlow S, Morgenroth I, Graef Y, Schneider K, Riehl I, Staeheli P, et al. Unique and conserved functions of B cell-activating factor of the TNF family (BAFF) in the chicken. Int Immunol. 2007;19(2):203–15. PubMed DOI

Kothlow S, Morgenroth I, Tregaskes CA, Kaspers B, Young JR. CD40 ligand supports the long-term maintenance and differentiation of chicken B cells in culture. Dev Comp Immunol. 2008;32(9):1015–26. PubMed DOI

Forster R, Schubel A, Breitfeld D, Kremmer E, Renner-Muller I, Wolf E, et al. CCR7 coordinates the primary immune response by Establishing functional microenvironments in secondary lymphoid organs. Cell. 1999;99(1):23–33. PubMed DOI

Okada T, Ngo VN, Ekland EH, Forster R, Lipp M, Littman DR, et al. Chemokine requirements for B cell entry to lymph nodes and Peyer’s patches. J Exp Med. 2002;196(1):65–75. PubMed DOI PMC

McHeik S, Van Eeckhout N, De Poorter C, Gales C, Parmentier M, Springael JY. Coexpression of CCR7 and CXCR4 during B cell development controls CXCR4 responsiveness and bone marrow homing. Front Immunol. 2019;10:2970. PubMed DOI PMC

Huttenlocher A, Horwitz AR. Integrins in cell migration. Cold Spring Harb Perspect Biol. 2011;3(9):a005074. PubMed DOI PMC

Muller WA, Weigl SA, Deng X, Phillips DM. PECAM-1 is required for transendothelial migration of leukocytes. J Exp Med. 1993;178(2):449–60. PubMed DOI PMC

Martin-Padura I, Lostaglio S, Schneemann M, Williams L, Romano M, Fruscella P, et al. Junctional adhesion molecule, a novel member of the Immunoglobulin superfamily that distributes at intercellular junctions and modulates monocyte transmigration. J Cell Biol. 1998;142(1):117–27. PubMed DOI PMC

Donate C, Ody C, McKee T, Ruault-Jungblut S, Fischer N, Ropraz P, et al. Homing of human B cells to lymphoid organs and B-cell lymphoma engraftment are controlled by cell adhesion molecule JAM-C. Cancer Res. 2013;73(2):640–51. PubMed DOI

Kabashima K, Haynes NM, Xu Y, Nutt SL, Allende ML, Proia RL, et al. Plasma cell S1P1 expression determines secondary lymphoid organ retention versus bone marrow tropism. J Exp Med. 2006;203(12):2683–90. PubMed DOI PMC

Allende ML, Tuymetova G, Lee BG, Bonifacino E, Wu YP, Proia RL. S1P1 receptor directs the release of immature B cells from bone marrow into blood. J Exp Med. 2010;207(5):1113–24. PubMed DOI PMC

Ko KH, Lee IK, Kim G, Gu MJ, Kim HY, Park BC, et al. Changes in bursal B cells in chicken during embryonic development and early life after hatching. Sci Rep. 2018;8(1):16905. PubMed DOI PMC