Defining a transcriptional fingerprint of murine splenic B-cell development

Abstract

B-cell development occurs in a stepwise fashion that can be followed by the expression of B cell-specific surface markers. In this study, we wished to identify proteins that could contribute to the changes in expression of such markers. By using RNA from freshly isolated B220 þ cells, we hoped to reduce the effect of artifacts that occur during the isolation and amplification steps necessary to use flow cytometry analysis-sorted subsets in microarray experiments. Analyses comparing expression patterns from B220 þ 2-week bone marrow (pro-B, pre-B, immature B cells), 2-week spleen (predominantly transitional cells) and 8-week spleen (mainly mature B cells) yielded hundreds of genes. We also examined the B cell-activating factor (BAFF)-dependent effects on immature splenic B cells by comparing expression patterns in the spleen between 2-week A/J vs 2-week A/WySnJ mice, which lack functional BAFF receptor signaling. Genes that showed the expression differences between spleen and bone marrow samples were then analyzed through quantitative PCR on B-cell subsets isolated using two different sorting protocols. A comparison of the results from our study with the results from other analyses showed not only some overlap of preferentially expressed genes but also an expansion of other genes potentially involved in B-cell development.

Zfp318 Regulates IgD Expression by Abrogating Transcription Termination within the Ighm/Ighd Locus

“…Our previous screen of maturing and mature B cells for the expression of Zfp318 utilized gene grid array analysis of pooled age- and tissue-specific B cells(6). To expand and specify the time frame of Zfp318 expression during B cell development, we FACS sorted and isolated Pro, Pre and Newly Formed (NF) B cells from the bone marrow, and T1, T2, MZ and FO B cells from the spleen (Supplemental Fig.…”

“…A number of studies have sought to define the key transcriptional regulators and kinetics that control this pathway. In one such analysis, we examined the expression of transcriptional regulators during B cell differentiation comparing B220+ B cells obtained from the marrow of 2 week (wk) old animals (thus lacking mature recirculating B cells), spleens from 2 wk old animals (enriched for transitional 1 and transitional 2 B cells)(T1, T2, respectively) and from B220+ B cells from mature but naïve spleens (enriched for FO and MZ B cells) (6). That gene expression screen identified a number of candidate transcriptional regulatory proteins whose expression increased with B cell maturation.…”

Zinc-finger protein ZFP318 is essential for expression of IgD, the alternatively splicedIghproduct made by mature B lymphocytes

“…Microarray comparisons of gene expression in B-cell subsets have identified Zfp318 as a member of a set of mRNAs that increases during maturation of immature B cells into mature follicular B cells (22)(23)(24). By analyzing flow-sorted B-cell subsets, we confirmed expression of Zfp318 mRNA closely parallels IgD heavy chain (Ighd) mRNA during B-cell development ( Two alternatively spliced isoforms of Zfp318 mRNA are annotated in the genome and described in the literature (18) (Fig.…”

CD21 signaling via C3 regulates Purkinje cell protein 4 expression

“…Additionally, past analysis of maturing B cells demonstrated increasing Pcp4 expression with differentiation (Debnath et al, 2008). Pcp4 expression differences were confirmed by RT-PCR for both BALB/c and C57BL/6 WT and CD21/35−/− naïve (non-immunized) spleen samples (Fig.…”