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Two distinct steps during thymocyte maturation from CD4-CD8- to CD4+CD8+ distinguished in the early growth response (Egr)-1 transgenic mice with a recombinase-activating gene-deficient background.

Miyazaki T - J. Exp. Med. (1997)

Bottom Line: The early growth response (Egr)-1 is a zinc finger-containing transcription factor belonging to the immediate-early genes.In transgenic mice overexpressing Egr-1 in a recombinase-activating gene-deficient background, thymocytes bypassed the block at the CD25+CD44- DN stage and matured to the immature CD8 single-positive (ISP) cell stage, but not further to the CD4/CD8 double-positive (DP) cell stage.The first step, from the CD25+CD44- DN to the ISP stage, can be entirely promoted by overexpression of Egr-1.

View Article: PubMed Central - PubMed

Affiliation: Basel Institute for Immunology, Postfach CH-4005, Basel, Switzerland. miyazaki@bii.ch

ABSTRACT
The early growth response (Egr)-1 is a zinc finger-containing transcription factor belonging to the immediate-early genes. Its expression in CD4/CD8 double negative (DN) immature thymocytes suggests that Egr-1 expression may be involved in early thymocyte development. In transgenic mice overexpressing Egr-1 in a recombinase-activating gene-deficient background, thymocytes bypassed the block at the CD25+CD44- DN stage and matured to the immature CD8 single-positive (ISP) cell stage, but not further to the CD4/CD8 double-positive (DP) cell stage. When these mice were irradiated, thymocytes did develop to the DP stage, suggesting transcriptional induction of additional genes by irradiation that are required to promote thymocyte development from the ISP to the DP stage. These results provide genetic evidence for two distinct steps during early thymocyte development from the CD25+CD44- DN to the DP stage. The first step, from the CD25+CD44- DN to the ISP stage, can be entirely promoted by overexpression of Egr-1.

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Development of ISP cells induced by Egr-1 expression in  RAG-2−/− mice. Thymocyte suspensions from 6–8-wk-old transgenic  mice or negative littermates, both with a RAG-2−/− background, were  stained and analyzed by fluorocytometry. (a) CD4/CD8 profiles. Numbers indicate relative percentages of positive cells within a quadrant. The  average of the total thymocyte numbers of 10 of each type of mice are indicated above the profiles. (b) CD25 expression of total thymocytes from  transgenic or negative littermates (top) or CD8+ cells from transgenic  mice. (c) HSA histograms. (d) Side scatter (SSC)/forward scatter (FSC)  profiles indicating cell size. Only living cells were gated. (e) CD25/CD44  profiles of DN cells. Thymocytes were stained with anti-CD25–Tricolor,  anti-CD44–FITC, anti-CD4–PE, and anti-CD8–PE. PE-negative cells  (CD4/CD8 DN cells) were gated and analyzed for CD25/CD44 expression. Numbers indicate relative percentages of positive cells within a  quadrant.
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Figure 2: Development of ISP cells induced by Egr-1 expression in RAG-2−/− mice. Thymocyte suspensions from 6–8-wk-old transgenic mice or negative littermates, both with a RAG-2−/− background, were stained and analyzed by fluorocytometry. (a) CD4/CD8 profiles. Numbers indicate relative percentages of positive cells within a quadrant. The average of the total thymocyte numbers of 10 of each type of mice are indicated above the profiles. (b) CD25 expression of total thymocytes from transgenic or negative littermates (top) or CD8+ cells from transgenic mice. (c) HSA histograms. (d) Side scatter (SSC)/forward scatter (FSC) profiles indicating cell size. Only living cells were gated. (e) CD25/CD44 profiles of DN cells. Thymocytes were stained with anti-CD25–Tricolor, anti-CD44–FITC, anti-CD4–PE, and anti-CD8–PE. PE-negative cells (CD4/CD8 DN cells) were gated and analyzed for CD25/CD44 expression. Numbers indicate relative percentages of positive cells within a quadrant.

Mentions: The thymi of Egr-1 transgenic mice with a RAG-2−/− background (Egr-1 Tg-RAG−) were significantly larger than those of RAG-2−/− negative littermates (NL-RAG−). The absolute number of thymocytes was 3.5 (± 0.5) × 106 in Egr-1 Tg-RAG− (n = 10), compared to 1.2 (± 0.3) × 106 in NL-RAG− (n = 10). Typical staining profiles of thymocytes are presented in Fig. 2 a. The most impressive aberration in the transgenic thymi was the presence of a large population (30%) of CD4−CD8+ cells. These CD8+ cells are CD25-negative (Fig. 2 b) and display lower heat-stable antigen (HSA) levels than those on DN cells, but higher levels than those on DP cells (Fig. 2 c), indicating that they are ISP transitional stage thymocytes (15–18). Consistently, size profiles of total thymocytes show that smaller cells appeared (Fig. 2 d) and a significant population (18%) of CD25−CD44− DN cells was detected in Egr-1 Tg-RAG− thymi (Fig. 2 e). Thus, overexpression of Egr-1 promoted thymocyte development from the DN to the ISP stage, overcoming the β block in the RAG-2−/− background. In accordance with this, small numbers of ISP cells appeared to express CD4 also, falling into the CD4/ CD8-DP quadrant (Fig. 2 a). These cells form, however, a very small (2%), indistinct population, possibly representing the intermediate stage cells between the ISP and the DP. Neither mature single-positive thymocytes nor peripheral T cells were detected in either Egr-1 Tg-RAG− or NL-RAG− mice. ISP cells isolated from Egr-1 Tg-RAG− could not develop to the DP stage spontaneously in in vitro culture for 12–24 h (data presented in following section as Fig. 3 d).


Two distinct steps during thymocyte maturation from CD4-CD8- to CD4+CD8+ distinguished in the early growth response (Egr)-1 transgenic mice with a recombinase-activating gene-deficient background.

Miyazaki T - J. Exp. Med. (1997)

Development of ISP cells induced by Egr-1 expression in  RAG-2−/− mice. Thymocyte suspensions from 6–8-wk-old transgenic  mice or negative littermates, both with a RAG-2−/− background, were  stained and analyzed by fluorocytometry. (a) CD4/CD8 profiles. Numbers indicate relative percentages of positive cells within a quadrant. The  average of the total thymocyte numbers of 10 of each type of mice are indicated above the profiles. (b) CD25 expression of total thymocytes from  transgenic or negative littermates (top) or CD8+ cells from transgenic  mice. (c) HSA histograms. (d) Side scatter (SSC)/forward scatter (FSC)  profiles indicating cell size. Only living cells were gated. (e) CD25/CD44  profiles of DN cells. Thymocytes were stained with anti-CD25–Tricolor,  anti-CD44–FITC, anti-CD4–PE, and anti-CD8–PE. PE-negative cells  (CD4/CD8 DN cells) were gated and analyzed for CD25/CD44 expression. Numbers indicate relative percentages of positive cells within a  quadrant.
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Figure 2: Development of ISP cells induced by Egr-1 expression in RAG-2−/− mice. Thymocyte suspensions from 6–8-wk-old transgenic mice or negative littermates, both with a RAG-2−/− background, were stained and analyzed by fluorocytometry. (a) CD4/CD8 profiles. Numbers indicate relative percentages of positive cells within a quadrant. The average of the total thymocyte numbers of 10 of each type of mice are indicated above the profiles. (b) CD25 expression of total thymocytes from transgenic or negative littermates (top) or CD8+ cells from transgenic mice. (c) HSA histograms. (d) Side scatter (SSC)/forward scatter (FSC) profiles indicating cell size. Only living cells were gated. (e) CD25/CD44 profiles of DN cells. Thymocytes were stained with anti-CD25–Tricolor, anti-CD44–FITC, anti-CD4–PE, and anti-CD8–PE. PE-negative cells (CD4/CD8 DN cells) were gated and analyzed for CD25/CD44 expression. Numbers indicate relative percentages of positive cells within a quadrant.
Mentions: The thymi of Egr-1 transgenic mice with a RAG-2−/− background (Egr-1 Tg-RAG−) were significantly larger than those of RAG-2−/− negative littermates (NL-RAG−). The absolute number of thymocytes was 3.5 (± 0.5) × 106 in Egr-1 Tg-RAG− (n = 10), compared to 1.2 (± 0.3) × 106 in NL-RAG− (n = 10). Typical staining profiles of thymocytes are presented in Fig. 2 a. The most impressive aberration in the transgenic thymi was the presence of a large population (30%) of CD4−CD8+ cells. These CD8+ cells are CD25-negative (Fig. 2 b) and display lower heat-stable antigen (HSA) levels than those on DN cells, but higher levels than those on DP cells (Fig. 2 c), indicating that they are ISP transitional stage thymocytes (15–18). Consistently, size profiles of total thymocytes show that smaller cells appeared (Fig. 2 d) and a significant population (18%) of CD25−CD44− DN cells was detected in Egr-1 Tg-RAG− thymi (Fig. 2 e). Thus, overexpression of Egr-1 promoted thymocyte development from the DN to the ISP stage, overcoming the β block in the RAG-2−/− background. In accordance with this, small numbers of ISP cells appeared to express CD4 also, falling into the CD4/ CD8-DP quadrant (Fig. 2 a). These cells form, however, a very small (2%), indistinct population, possibly representing the intermediate stage cells between the ISP and the DP. Neither mature single-positive thymocytes nor peripheral T cells were detected in either Egr-1 Tg-RAG− or NL-RAG− mice. ISP cells isolated from Egr-1 Tg-RAG− could not develop to the DP stage spontaneously in in vitro culture for 12–24 h (data presented in following section as Fig. 3 d).

Bottom Line: The early growth response (Egr)-1 is a zinc finger-containing transcription factor belonging to the immediate-early genes.In transgenic mice overexpressing Egr-1 in a recombinase-activating gene-deficient background, thymocytes bypassed the block at the CD25+CD44- DN stage and matured to the immature CD8 single-positive (ISP) cell stage, but not further to the CD4/CD8 double-positive (DP) cell stage.The first step, from the CD25+CD44- DN to the ISP stage, can be entirely promoted by overexpression of Egr-1.

View Article: PubMed Central - PubMed

Affiliation: Basel Institute for Immunology, Postfach CH-4005, Basel, Switzerland. miyazaki@bii.ch

ABSTRACT
The early growth response (Egr)-1 is a zinc finger-containing transcription factor belonging to the immediate-early genes. Its expression in CD4/CD8 double negative (DN) immature thymocytes suggests that Egr-1 expression may be involved in early thymocyte development. In transgenic mice overexpressing Egr-1 in a recombinase-activating gene-deficient background, thymocytes bypassed the block at the CD25+CD44- DN stage and matured to the immature CD8 single-positive (ISP) cell stage, but not further to the CD4/CD8 double-positive (DP) cell stage. When these mice were irradiated, thymocytes did develop to the DP stage, suggesting transcriptional induction of additional genes by irradiation that are required to promote thymocyte development from the ISP to the DP stage. These results provide genetic evidence for two distinct steps during early thymocyte development from the CD25+CD44- DN to the DP stage. The first step, from the CD25+CD44- DN to the ISP stage, can be entirely promoted by overexpression of Egr-1.

Show MeSH