Changes in bursal B cells in chicken during embryonic development and early life after hatching

Abstract

The bursa of Fabricius, the primary lymphoid organ for B cell development found only in birds, offers novel approaches to study B cell differentiation at various developmental stages. Here, we explored the changes and mechanism involved in the developmental stages of bursal B cells. The bursal B cells rapidly increased in the late embryonic stage and around hatching, which coincided with changes in specific cell surface markers. Moreover, the cells in the bursa were divided by size into small (low forward- and side-scatter) or large (high forward- and side-scatter) via flow cytometry. It is intriguing that the proportion of small and large B cells was reversed during this period. Because little is known about this phenomenon, we hypothesized that size-based B cell population could be used as an indicator to distinguish their status and stage during B cell development in chicken. The results demonstrated that large B cells are actively proliferating cells than small B cells. Additionally, large B cells showed higher mRNA expression of both proliferation- and differentiation-associated genes compared to small B cells. Taken together, these data show that large bursal B cells are the main source of proliferation and differentiation during B cell development in chickens.

Figure 1

Bursal B cells rapidly increased at the late embryonic stage in chickens. Bursal cells were taken from chicks aged E13 to 2–6 days old and stained with anti-Bu-1 antibody, which were regarded as bursal B cells. (a) Changes in bursal B cell population at E15, E17, E18, and hatching (H). Numbers in each area indicate the percentage of non-B or B cells in the bursa. (b) The percentage and absolute number of bursal B cells during embryonic and post-hatching stages. Each data point represents the mean value of four to ten chickens. The frequency of B cells in comparison to non-B cells from the spleen (c) and bone marrow (d) at the indicated stage. More than 5 samples per embryonic stage were analyzed, and all results represent at least two independent experiments. Each data point represents the mean of four to ten chickens with SD. **P < 0.01, ***P < 0.001.

Figure 2

The phenotype of bursal B cells rapidly changes at around hatching. Single cells produced from the bursa were stained with anti-Bu-1 antibody together with anti-IgY, -IgM or -MHC class II antibody. The percentage and absolute number of (a) IgY⁺ and (b) IgM⁺ bursal B cells before and after hatching was examined using flow cytometry. (c,d) Surface expression of MHC class II on bursal B cells was classified as a negative (−), low (+) and high (++). (c) Expression of MHC class II in bursal B cells at indicated stage. The number in each area indicates the percentage of cells. (d) The percentage and absolute number of MHC class II expression in bursal B cells during the embryonic and post-hatching stages. (e,f) B cells were distinguished by cell size as small (low forward-scatter) or large (high forward-scatter) using flow cytometry. (e) Differential display of small or large bursal B cells at indicated stage. Numbers in each area show the percentage of small or large cells. (f) The percentage and absolute number of small or large bursal B cells during the embryonic and post-hatching stages. Each data point represents the mean of four to ten chickens with SD.

Figure 3

Large B cells are proliferating cells with distinct surface marker expression. Single cells were produced from the bursa of (a,b) E17 or (c–f) 2-week-old chicks. Cell cycle and DNA content were assessed using propidium iodide (PI) and 7-amino-actinomycin D (7-AAD) intracellular staining, respectively. (a) The percentage of G0/G1, S, and G2/M phase and (b) 7-AAD expression in small and large B cells as mean fluorescence intensity (MFI) are shown. The cell surface expression of (c) MHC class II, (d) IgY, (e) IgM, and (f) IgA in small and large B cells from 2-week-old chicks. **P < 0.01, ***P < 0.001.

Figure 4

Large B cells show enhanced expression of genes for both proliferation and differentiation when compared to those of small B cells. Single cells were produced from the bursa at E17 and small and large B cells were sorted using a cell sorter. (a–h) The mRNA levels of genes associated with proliferation and colonization of B cells in small and large B cells were evaluated using qRT-PCR as follows: (a) BAFF, (b) BAFF receptor, (c) IL-7, (d) IL-7 receptor, (e) CD40, (f) CD40 ligand, (g) CXCL12, and (h) CXCR4. (i–s) qRT-PCR was used to determine the mRNA levels of genes associated with B cell differentiation in small and large B cells as follows: (i) RAG1, (j) RAG2, (k) FOXO1, (l) FOXO3, (m) PAX5, (n) BLNK, (o) Ikaros, (p) Helios, (q) Aiolos, (r) EBF, and (s) E2A. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 5

Large B cells are the main source of proliferation and differentiation in the embryonic stage. To explore whether the proportion of small and large B cells affects the status of B cells during the development, we chose two stages where the ratio of small and large B cells are dramatically reversed. Cells in the large B cell dominant stage (E18) and a small B cell dominant stage (E20) were sorted using Bu-1 positive selection, and then total RNA was extracted. (a–h) The mRNA expression of B cell proliferation- and colonization-associated genes in small and large B cells was evaluated using qRT-PCR as follows: (a) BAFF, (b) BAFF receptor, (c) IL-7, (d) IL-7 receptor, (e) CD40, (f) CD40 ligand, (g) CXCL12, and (h) CXCR4. (i–s) The mRNA levels of B cell differentiation-associated genes in small and large B cells were examined using qRT-PCR as follows: (i) RAG1, (j) RAG2, (k) FOXO1, (l) FOXO3, (m) PAX5, (n) BLNK, (o) Ikaros, (p) Helios, (q) Aiolos, (r) EBF, and (s) E2A. *P < 0.05, **P < 0.01, ***P < 0.001.