Zebrafish B Cell Development without a Pre–B Cell Stage, Revealed by CD79 Fluorescence Reporter Transgenes

Abstract

CD79a and CD79b proteins associate with immunoglobulin receptors as integral signaling components of the B cell antigen receptor complex. In order to study B cell development in zebrafish, we isolated orthologs of these genes and performed in situ hybridization, finding that their expression co-localized with immunoglobulin heavy chain (IgH)-μ in the kidney, which is the site of B cell development. CD79 transgenic lines were made by linking the promoter and upstream regulatory segments of CD79a and CD79b to EGFP to identify B cells, as demonstrated by PCR analysis of IgH-μ expression in sorted cells. We crossed these CD79-GFP lines to a recombination activating gene 2 (Rag2):mCherry transgenic line to identify B cell development stages in kidney marrow. Initiation of CD79:GFP expression in Rag2:mCherry+ cells and the timing of immunoglobulin heavy and light chain expression revealed simultaneous expression of both IgH-μ and IgL-κ chains, without progressing through the stage of IgH-μ chain alone. Rag2:mCherry+ cells without CD79:GFP showed the highest Rag1 and Rag2 mRNAs compared to CD79a and CD79b:GFP+ B cells which showed strongly reduced Rag mRNAs. Thus, B cell development in zebrafish does not go through a Raghi CD79+IgH-μ+ pre-B cell stage, different from mammals. After the generation of CD79:GFP+B cells, decreased CD79 expression occurred upon differentiation to Ig secretion, as detected by alteration from membrane to secreted IgH-μ exon usage, similar to in mammals. This confirmed a conserved role for CD79 in B cell development and differentiation, without the requirement of a pre-B cell stage in zebrafish.

Simultaneous B and T cell acute lymphoblastic leukemias in zebrafish driven by transgenic MYC: implications for oncogenesis and lymphopoiesis

“…3A(e), 5, 7C,E). A recent report of zebrafish B cell development despite low rag1 supports our observation (34). Apart from rag1 , pre-B ALL expressed other classic B-lymphoblast genes like rag2 , surrogate light chain components, pax5, cd79a/b, and others (Figs.…”

“…Our results also demonstrate multiple features of abnormal B and T lymphopoiesis in hMYC fish. Of interest, pre-B ALL GEPs closely matched the gene expression pattern of a recently-described ighz + B cell population dubbed “fraction 2” (34), suggesting hMYC is oncogenically active in this cell population. Supporting this, every pre-B ALL we identified was ighz + (Fig.…”

Molecularly distinct models of zebrafishMyc-induced B cell leukemia

“…By contrast, hMYC / ighz + B-ALLs were enriched for intracellular signaling, protein binding, and germinal center B cell maturation pathways. In support of our findings, Liu et al recently reported the identification of molecularly and biological distinct ighz + and ighm + B cell lineages using rag2:mCherry; cd79b:GFP transgenic zebrafish 7 . In the context of normal B cell development, ighz + B cells were mCherry hi /GFP lo while ighm + B cells were mCherry hi /GFP hi .…”

Themacrophage-expressed gene(mpeg)1identifies a subpopulation of B cells in the adult zebrafish

“…This We next sought to determine whether the two subsets of B-cells existed in peripheral tissues. Using the same mpeg1.1:mCherry;ighm:GFP double transgenic fish, we analyzed the spleen, gut and skin, known to host IgM-expressing B lymphocytes 26,33 . While the proportion of IgM + cells greatly varied among tissues, the majority of IgM + cells showed co-expression with mpeg1.1 in all samples examined Figure 5B).…”