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Mind bomb 1 in the lymphopoietic niches is essential for T and marginal zone B cell development.

Song R, Kim YW, Koo BK, Jeong HW, Yoon MJ, Yoon KJ, Jun DJ, Im SK, Shin J, Kong MP, Kim KT, Yoon K, Kong YY - J. Exp. Med. (2008)

Bottom Line: In addition, the endocytosis of Dll1 was impaired in the Mib1- microenvironment.Moreover, the block in T cell development and the failure of Dll1 endocytosis were also observed in coculture system by Mib1 knockdown.Our study reveals that Mib1 is the essential E3 ligase in T and MZB cell development, through the regulation of Notch ligands in the thymic and splenic microenvironments.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Science, Pohang University of Science and Technology, Pohang, Kyungbuk 790-784, South Korea.

ABSTRACT
Notch signaling regulates lineage decisions at multiple stages of lymphocyte development, and Notch activation requires the endocytosis of Notch ligands in the signal-sending cells. Four E3 ubiquitin ligases, Mind bomb (Mib) 1, Mib2, Neuralized (Neur) 1, and Neur2, regulate the Notch ligands to activate Notch signaling, but their roles in lymphocyte development have not been defined. We show that Mib1 regulates T and marginal zone B (MZB) cell development in the lymphopoietic niches. Inactivation of the Mib1 gene, but not the other E3 ligases, Mib2, Neur1, and Neur2, abrogated T and MZB cell development. Reciprocal bone marrow (BM) transplantation experiments revealed that Mib1 in the thymic and splenic niches is essential for T and MZB cell development. Interestingly, when BM cells from transgenic Notch reporter mice were transplanted into Mib1- mice, the Notch signaling was abolished in the double-negative thymocytes. In addition, the endocytosis of Dll1 was impaired in the Mib1- microenvironment. Moreover, the block in T cell development and the failure of Dll1 endocytosis were also observed in coculture system by Mib1 knockdown. Our study reveals that Mib1 is the essential E3 ligase in T and MZB cell development, through the regulation of Notch ligands in the thymic and splenic microenvironments.

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Regulation of T cell development by Mib1 in the thymic microenvironment. (A) The lethally irradiated CD45.1 mice were injected intravenously with BM cells from the 12–15-wk-old MMTV-Cre;Mib1+/f and MMTV-Cre;Mib1f/f mice. At 12 wk after transplantation, the thymocytes were analyzed by flow cytometry for the expression of CD4 and CD8 gated on the CD45.2+ populations (top), CD19 and B220 gated on the CD4−CD8− DN thymocytes (middle), and CD24 and c-kit gated on CD4−CD8−CD25−CD44+ cells (bottom). Numbers indicate mean ± SD from four independent experiments. (B) The lethally irradiated MMTV-Cre;Mib1+/f and MMTV-Cre;Mib1f/f mice were transplanted with CD45.1 BM cells. At 5–6 wk after reconstitution with CD45.1 BM cells, the thymocytes were stained for CD4 and CD8 (top) and B220 gated on the CD4−CD8− DN thymocytes (bottom). Numbers indicate mean ± SD from three independent experiments. (C) Genomic DNA was prepared from the CD45− cells sorted from thymi. Quantitative real-time PCR was performed to analyze the Mib1 deletion using deleted allele-specific primers. Expression of nondeleted allele served as a control for relative quantification. Data are mean ± SD from triplicate experiments. *, P < 0.01.
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fig5: Regulation of T cell development by Mib1 in the thymic microenvironment. (A) The lethally irradiated CD45.1 mice were injected intravenously with BM cells from the 12–15-wk-old MMTV-Cre;Mib1+/f and MMTV-Cre;Mib1f/f mice. At 12 wk after transplantation, the thymocytes were analyzed by flow cytometry for the expression of CD4 and CD8 gated on the CD45.2+ populations (top), CD19 and B220 gated on the CD4−CD8− DN thymocytes (middle), and CD24 and c-kit gated on CD4−CD8−CD25−CD44+ cells (bottom). Numbers indicate mean ± SD from four independent experiments. (B) The lethally irradiated MMTV-Cre;Mib1+/f and MMTV-Cre;Mib1f/f mice were transplanted with CD45.1 BM cells. At 5–6 wk after reconstitution with CD45.1 BM cells, the thymocytes were stained for CD4 and CD8 (top) and B220 gated on the CD4−CD8− DN thymocytes (bottom). Numbers indicate mean ± SD from three independent experiments. (C) Genomic DNA was prepared from the CD45− cells sorted from thymi. Quantitative real-time PCR was performed to analyze the Mib1 deletion using deleted allele-specific primers. Expression of nondeleted allele served as a control for relative quantification. Data are mean ± SD from triplicate experiments. *, P < 0.01.

Mentions: The MMTV-Cre and Mx1-Cre transgenic lines delete the floxed genomic sequences in both hematopoietic and nonhematopoietic cells (36, 50), and the Mib1 mRNA is expressed in both the CD45+ hematopoietic and CD45− nonhematopoietic cells from the thymus (Fig. 1 A). Therefore, the impaired T cell development in the MMTV-Cre;Mib1f/f and Mx1-Cre;Mib1f/f mice could be caused by either an autonomous defect in hematopoietic cells or a nonhematopoietic defect in other components residing in the thymus, such as stromal cells. To distinguish between these possibilities, lethally irradiated CD45.1 WT mice were reconstituted with BM cells from either the MMTV-Cre;Mib1+/f or MMTV-Cre;Mib1f/f mice. At 12 wk after transplantation, all of the CD45.1 recipient mice displayed normal thymocyte development, including B cell and ETP generation (Fig. 5 A), indicating that the defective thymocyte development in the MMTV-Cre;Mib1f/f and Mx1-Cre;Mib1f/f mice is not caused by the inactivation of Mib1 in hematopoietic cells.


Mind bomb 1 in the lymphopoietic niches is essential for T and marginal zone B cell development.

Song R, Kim YW, Koo BK, Jeong HW, Yoon MJ, Yoon KJ, Jun DJ, Im SK, Shin J, Kong MP, Kim KT, Yoon K, Kong YY - J. Exp. Med. (2008)

Regulation of T cell development by Mib1 in the thymic microenvironment. (A) The lethally irradiated CD45.1 mice were injected intravenously with BM cells from the 12–15-wk-old MMTV-Cre;Mib1+/f and MMTV-Cre;Mib1f/f mice. At 12 wk after transplantation, the thymocytes were analyzed by flow cytometry for the expression of CD4 and CD8 gated on the CD45.2+ populations (top), CD19 and B220 gated on the CD4−CD8− DN thymocytes (middle), and CD24 and c-kit gated on CD4−CD8−CD25−CD44+ cells (bottom). Numbers indicate mean ± SD from four independent experiments. (B) The lethally irradiated MMTV-Cre;Mib1+/f and MMTV-Cre;Mib1f/f mice were transplanted with CD45.1 BM cells. At 5–6 wk after reconstitution with CD45.1 BM cells, the thymocytes were stained for CD4 and CD8 (top) and B220 gated on the CD4−CD8− DN thymocytes (bottom). Numbers indicate mean ± SD from three independent experiments. (C) Genomic DNA was prepared from the CD45− cells sorted from thymi. Quantitative real-time PCR was performed to analyze the Mib1 deletion using deleted allele-specific primers. Expression of nondeleted allele served as a control for relative quantification. Data are mean ± SD from triplicate experiments. *, P < 0.01.
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fig5: Regulation of T cell development by Mib1 in the thymic microenvironment. (A) The lethally irradiated CD45.1 mice were injected intravenously with BM cells from the 12–15-wk-old MMTV-Cre;Mib1+/f and MMTV-Cre;Mib1f/f mice. At 12 wk after transplantation, the thymocytes were analyzed by flow cytometry for the expression of CD4 and CD8 gated on the CD45.2+ populations (top), CD19 and B220 gated on the CD4−CD8− DN thymocytes (middle), and CD24 and c-kit gated on CD4−CD8−CD25−CD44+ cells (bottom). Numbers indicate mean ± SD from four independent experiments. (B) The lethally irradiated MMTV-Cre;Mib1+/f and MMTV-Cre;Mib1f/f mice were transplanted with CD45.1 BM cells. At 5–6 wk after reconstitution with CD45.1 BM cells, the thymocytes were stained for CD4 and CD8 (top) and B220 gated on the CD4−CD8− DN thymocytes (bottom). Numbers indicate mean ± SD from three independent experiments. (C) Genomic DNA was prepared from the CD45− cells sorted from thymi. Quantitative real-time PCR was performed to analyze the Mib1 deletion using deleted allele-specific primers. Expression of nondeleted allele served as a control for relative quantification. Data are mean ± SD from triplicate experiments. *, P < 0.01.
Mentions: The MMTV-Cre and Mx1-Cre transgenic lines delete the floxed genomic sequences in both hematopoietic and nonhematopoietic cells (36, 50), and the Mib1 mRNA is expressed in both the CD45+ hematopoietic and CD45− nonhematopoietic cells from the thymus (Fig. 1 A). Therefore, the impaired T cell development in the MMTV-Cre;Mib1f/f and Mx1-Cre;Mib1f/f mice could be caused by either an autonomous defect in hematopoietic cells or a nonhematopoietic defect in other components residing in the thymus, such as stromal cells. To distinguish between these possibilities, lethally irradiated CD45.1 WT mice were reconstituted with BM cells from either the MMTV-Cre;Mib1+/f or MMTV-Cre;Mib1f/f mice. At 12 wk after transplantation, all of the CD45.1 recipient mice displayed normal thymocyte development, including B cell and ETP generation (Fig. 5 A), indicating that the defective thymocyte development in the MMTV-Cre;Mib1f/f and Mx1-Cre;Mib1f/f mice is not caused by the inactivation of Mib1 in hematopoietic cells.

Bottom Line: In addition, the endocytosis of Dll1 was impaired in the Mib1- microenvironment.Moreover, the block in T cell development and the failure of Dll1 endocytosis were also observed in coculture system by Mib1 knockdown.Our study reveals that Mib1 is the essential E3 ligase in T and MZB cell development, through the regulation of Notch ligands in the thymic and splenic microenvironments.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Science, Pohang University of Science and Technology, Pohang, Kyungbuk 790-784, South Korea.

ABSTRACT
Notch signaling regulates lineage decisions at multiple stages of lymphocyte development, and Notch activation requires the endocytosis of Notch ligands in the signal-sending cells. Four E3 ubiquitin ligases, Mind bomb (Mib) 1, Mib2, Neuralized (Neur) 1, and Neur2, regulate the Notch ligands to activate Notch signaling, but their roles in lymphocyte development have not been defined. We show that Mib1 regulates T and marginal zone B (MZB) cell development in the lymphopoietic niches. Inactivation of the Mib1 gene, but not the other E3 ligases, Mib2, Neur1, and Neur2, abrogated T and MZB cell development. Reciprocal bone marrow (BM) transplantation experiments revealed that Mib1 in the thymic and splenic niches is essential for T and MZB cell development. Interestingly, when BM cells from transgenic Notch reporter mice were transplanted into Mib1- mice, the Notch signaling was abolished in the double-negative thymocytes. In addition, the endocytosis of Dll1 was impaired in the Mib1- microenvironment. Moreover, the block in T cell development and the failure of Dll1 endocytosis were also observed in coculture system by Mib1 knockdown. Our study reveals that Mib1 is the essential E3 ligase in T and MZB cell development, through the regulation of Notch ligands in the thymic and splenic microenvironments.

Show MeSH
Related in: MedlinePlus