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Developmental separation of V(D)J recombinase expression and initiation of IgH recombination in B lineage progenitors in vivo.

Borghesi L, Gerstein RM - J. Exp. Med. (2004)

Bottom Line: To distinguish between these possibilities, we developed a transgenic V(D)J recombination substrate that is not governed by the same chromatin remodeling constraints as endogenous immunoglobulin heavy chain (IgH) loci and examined the requirements for V(D)J recombination to initiate in early B lineage progenitors.We find that single B lineage precursors express an active V(D)J recombinase in vivo before the stage when IgH rearrangements are frequently detectable.Our results indicate that the onset of recombinase activity and the initiation of IgH recombination are developmentally distinct events in the B lineage.

View Article: PubMed Central - PubMed

Affiliation: University of Massachusetts Medical School, Molecular Genetics and Microbiology, 55 Lake Ave. North, Worcester 01655, USA.

ABSTRACT
In B lineage progenitors, V(D)J recombination occurs only during distinct stages of development and is restricted to immunoglobulin loci. This process is thought to be controlled by both regulated expression of the V(D)J recombinase and by limited accessibility of target loci to the recombinase complex. However, it is unknown whether these two processes occur concomitantly in developing B lineage progenitors or whether these events are temporally distinct and, therefore, potentially independently regulated. To distinguish between these possibilities, we developed a transgenic V(D)J recombination substrate that is not governed by the same chromatin remodeling constraints as endogenous immunoglobulin heavy chain (IgH) loci and examined the requirements for V(D)J recombination to initiate in early B lineage progenitors. We find that single B lineage precursors express an active V(D)J recombinase in vivo before the stage when IgH rearrangements are frequently detectable. Our results indicate that the onset of recombinase activity and the initiation of IgH recombination are developmentally distinct events in the B lineage.

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Simultaneous analysis of rag2 transcription and recombinase activity in B lineage progenitors. (A) Bone marrow obtained from the F1 progeny of RAG2 GFP × H2-SVEX (SB110) animals (fourth column) was stained with antibodies to identify B lineage progenitors. The B220+CD43+DX5−Ly6C−IgM− subset of bone marrow was then gated to examine CD19−CD24− pre-pro-B cells (first row), CD19−CD24+ early pro-B (second row), and CD19+CD24+ late pro-B (third row) cells. Gated samples were simultaneously examined for GFP expression, an indicator of rag2 transcription, and VEX expression, an indicator of V(D)J recombinase activity. Because the GFP expression in RAG2 GFP NG animals is so bright (12), a small percentage (<3%) of the GFPBRIGHTVEX− cells fall into the GFP+VEX+ quadrant due to limitations of compensation. Therefore, bone marrow from single transgenic RAG2 GFP NG mice (second column), single transgenic H2-SVEX transgenic mice (third column), and nontransgenic B6 control mice is provided for comparison. The percentage of cells in each region is given. (B) VEX and GFP expression was similarly examined in Ly6C−DX5− fraction A2(AA4.1+B220+CD24−CD4−) bone marrow cells. The data are representative of two to five independent experiments.
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fig2: Simultaneous analysis of rag2 transcription and recombinase activity in B lineage progenitors. (A) Bone marrow obtained from the F1 progeny of RAG2 GFP × H2-SVEX (SB110) animals (fourth column) was stained with antibodies to identify B lineage progenitors. The B220+CD43+DX5−Ly6C−IgM− subset of bone marrow was then gated to examine CD19−CD24− pre-pro-B cells (first row), CD19−CD24+ early pro-B (second row), and CD19+CD24+ late pro-B (third row) cells. Gated samples were simultaneously examined for GFP expression, an indicator of rag2 transcription, and VEX expression, an indicator of V(D)J recombinase activity. Because the GFP expression in RAG2 GFP NG animals is so bright (12), a small percentage (<3%) of the GFPBRIGHTVEX− cells fall into the GFP+VEX+ quadrant due to limitations of compensation. Therefore, bone marrow from single transgenic RAG2 GFP NG mice (second column), single transgenic H2-SVEX transgenic mice (third column), and nontransgenic B6 control mice is provided for comparison. The percentage of cells in each region is given. (B) VEX and GFP expression was similarly examined in Ly6C−DX5− fraction A2(AA4.1+B220+CD24−CD4−) bone marrow cells. The data are representative of two to five independent experiments.

Mentions: For these experiments, we analyzed VEX expression in progenitor B subsets derived from the progeny of crosses between H2-SVEX and NG transgenic RAG2 reporter mice (12). This approach enables us to examine both RAG2 gene transcription (GFP) and V(D)J recombinase activity (VEX) in single lymphocyte progenitors. VEX and GFP have distinct excitation requirements, and thus can be readily resolved (23). Fig. 2 A depicts GFP versus VEX (within B220+CD43+DX5−Ly6C−IgM− cells) for pre-pro-B (CD19−CD24−/LO), early pro-B (CD19−CD24+), and late pro-B (CD19+CD24+) cells.


Developmental separation of V(D)J recombinase expression and initiation of IgH recombination in B lineage progenitors in vivo.

Borghesi L, Gerstein RM - J. Exp. Med. (2004)

Simultaneous analysis of rag2 transcription and recombinase activity in B lineage progenitors. (A) Bone marrow obtained from the F1 progeny of RAG2 GFP × H2-SVEX (SB110) animals (fourth column) was stained with antibodies to identify B lineage progenitors. The B220+CD43+DX5−Ly6C−IgM− subset of bone marrow was then gated to examine CD19−CD24− pre-pro-B cells (first row), CD19−CD24+ early pro-B (second row), and CD19+CD24+ late pro-B (third row) cells. Gated samples were simultaneously examined for GFP expression, an indicator of rag2 transcription, and VEX expression, an indicator of V(D)J recombinase activity. Because the GFP expression in RAG2 GFP NG animals is so bright (12), a small percentage (<3%) of the GFPBRIGHTVEX− cells fall into the GFP+VEX+ quadrant due to limitations of compensation. Therefore, bone marrow from single transgenic RAG2 GFP NG mice (second column), single transgenic H2-SVEX transgenic mice (third column), and nontransgenic B6 control mice is provided for comparison. The percentage of cells in each region is given. (B) VEX and GFP expression was similarly examined in Ly6C−DX5− fraction A2(AA4.1+B220+CD24−CD4−) bone marrow cells. The data are representative of two to five independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2211822&req=5

fig2: Simultaneous analysis of rag2 transcription and recombinase activity in B lineage progenitors. (A) Bone marrow obtained from the F1 progeny of RAG2 GFP × H2-SVEX (SB110) animals (fourth column) was stained with antibodies to identify B lineage progenitors. The B220+CD43+DX5−Ly6C−IgM− subset of bone marrow was then gated to examine CD19−CD24− pre-pro-B cells (first row), CD19−CD24+ early pro-B (second row), and CD19+CD24+ late pro-B (third row) cells. Gated samples were simultaneously examined for GFP expression, an indicator of rag2 transcription, and VEX expression, an indicator of V(D)J recombinase activity. Because the GFP expression in RAG2 GFP NG animals is so bright (12), a small percentage (<3%) of the GFPBRIGHTVEX− cells fall into the GFP+VEX+ quadrant due to limitations of compensation. Therefore, bone marrow from single transgenic RAG2 GFP NG mice (second column), single transgenic H2-SVEX transgenic mice (third column), and nontransgenic B6 control mice is provided for comparison. The percentage of cells in each region is given. (B) VEX and GFP expression was similarly examined in Ly6C−DX5− fraction A2(AA4.1+B220+CD24−CD4−) bone marrow cells. The data are representative of two to five independent experiments.
Mentions: For these experiments, we analyzed VEX expression in progenitor B subsets derived from the progeny of crosses between H2-SVEX and NG transgenic RAG2 reporter mice (12). This approach enables us to examine both RAG2 gene transcription (GFP) and V(D)J recombinase activity (VEX) in single lymphocyte progenitors. VEX and GFP have distinct excitation requirements, and thus can be readily resolved (23). Fig. 2 A depicts GFP versus VEX (within B220+CD43+DX5−Ly6C−IgM− cells) for pre-pro-B (CD19−CD24−/LO), early pro-B (CD19−CD24+), and late pro-B (CD19+CD24+) cells.

Bottom Line: To distinguish between these possibilities, we developed a transgenic V(D)J recombination substrate that is not governed by the same chromatin remodeling constraints as endogenous immunoglobulin heavy chain (IgH) loci and examined the requirements for V(D)J recombination to initiate in early B lineage progenitors.We find that single B lineage precursors express an active V(D)J recombinase in vivo before the stage when IgH rearrangements are frequently detectable.Our results indicate that the onset of recombinase activity and the initiation of IgH recombination are developmentally distinct events in the B lineage.

View Article: PubMed Central - PubMed

Affiliation: University of Massachusetts Medical School, Molecular Genetics and Microbiology, 55 Lake Ave. North, Worcester 01655, USA.

ABSTRACT
In B lineage progenitors, V(D)J recombination occurs only during distinct stages of development and is restricted to immunoglobulin loci. This process is thought to be controlled by both regulated expression of the V(D)J recombinase and by limited accessibility of target loci to the recombinase complex. However, it is unknown whether these two processes occur concomitantly in developing B lineage progenitors or whether these events are temporally distinct and, therefore, potentially independently regulated. To distinguish between these possibilities, we developed a transgenic V(D)J recombination substrate that is not governed by the same chromatin remodeling constraints as endogenous immunoglobulin heavy chain (IgH) loci and examined the requirements for V(D)J recombination to initiate in early B lineage progenitors. We find that single B lineage precursors express an active V(D)J recombinase in vivo before the stage when IgH rearrangements are frequently detectable. Our results indicate that the onset of recombinase activity and the initiation of IgH recombination are developmentally distinct events in the B lineage.

Show MeSH