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. 2002 Jul 23;99(15):10014-9.
doi: 10.1073/pnas.152327399. Epub 2002 Jul 15.

Molecular portraits of B cell lineage commitment

Markus Müschen  1 , Sanggyu Lee, Guolin Zhou, Niklas Feldhahn, Varun Singh Barath, Jianjun Chen, Cordula Moers, Martin Krönke, Janet D Rowley, San Ming Wang
Affiliations

Molecular portraits of B cell lineage commitment

Markus Müschen et al. Proc Natl Acad Sci U S A. .

Abstract

In an attempt to characterize early B cell development including the commitment of progenitor cells to the B cell lineage, we generated and compared genomewide gene expression profiles of human hematopoietic stem cells (HSCs) and pre-B cells (PBCs) by using serial analysis of gene expression. From more than 100,000 serial analysis of gene expression tags collected from human CD34(+) HSCs and CD10(+) CD19(+) PBCs, 42,399 unique transcripts were identified in HSCs but only 16,786 in PBCs, suggesting that more than 60% of transcripts expressed in HSCs were silenced during or after commitment to the B cell lineage. On the other hand, mRNAs of pre-B cell receptor (pre-BCR)-associated genes are virtually missing in HSCs but account for more than 10% of the transcriptome of PBCs, which also show increased expression of apoptosis-related genes. Both concentration of the transcriptional repertoire on pre-BCR-related genes together with marked up-regulation of apoptosis mediators in PBC might reflect selection for the expression of a functional pre-BCR within the bone marrow. Besides known regulator genes of early B cell development such as PAX5, E2A, and EBF, the most abundantly expressed genes in PBCs include ATM, PDGFRA, SIAH1, PIM2, C/EBPB, WNT16, and TCL1, the role of which has not been established yet in early B cell development.

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Figures

Figure 1
Figure 1
Phenotype of purified human PBCs. The FACS plots show enrichment of CD10+ CD19+ (CD3− CD15− CD20− CD138−) PBCs from mononuclear bone marrow cells before (A) and after (B) magnetic cell sorting enrichment. Using cDNA from the purified cells together with cDNAs from CD34+ HSC and memory B cells, the phenotype of purified PBCs was confirmed by semiquantitative RT-PCR.
Figure 2
Figure 2
Assessment of quantitative accuracy of SAGE data. To corroborate differential gene expression between HSCs and PBCs as assessed by SAGE, semiquantitative RT-PCR was performed for 45 selected genes. The RT-PCR was normalized for 18, 24, and 30 cycles by using a cDNA fragment of the COX6B gene (41 SAGE tags in the HSC and PBC libraries) as a standard. Shown is a comparison of the RT-PCR products (Left) to SAGE tag counts (Center), and the gene names are indicated (Right). For three of 45 genes, no or no specific amplification product was obtained (not shown).
Figure 3
Figure 3
Functional clustering of SAGE data in HSCs and PBCs. Shown is the graphic representation of SAGE data from HSC and PBC SAGE libraries on a color scale from black (no or low expression) to red (high expression levels). The data are arranged within functional clusters for costimulatory molecules and positive regulators of pre-BCR signaling (A), inhibitory molecules and negative regulators of pre-BCR signaling (B), lineage-specific molecules (C), and constituents of pre-BCR and genes related to V(D)J gene recombination (D) and are sorted based on the ratio of SAGE tag counts in PBCs and HSCs.
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