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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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Notch signaling defect in the thymi of Mib1 conditional KO mice. (A) The lethally irradiated MMTV-Cre;Mib1+/f (black) and MMTV-Cre;Mib1f/f (red) mice (7–9 wk old) were injected i.v. with BM cells from the TNR mice (51). At 4 wk after transplantation, the thymocytes were analyzed by flow cytometry for GFP expression gated on the CD4−CD8− DN thymocytes. Percentages indicated are mean ± SD from three independent experiments. (B–D) The lethally irradiated MMTV-Cre;Mib1+/f and MMTV-Cre;Mib1f/f mice were transplanted with CD45.1 BM cells. 6 wk after transplantation, the thymi were fixed and cryosections were immunostained with anti-Dll1 (B and C, red), anti-Hrs (B, green), anti-cytokeratin (C and D, green), and anti-Mib1 (D, red) antibodies with HOECHST (blue). Note that Dll1 was colocalized with Hrs (B, arrowheads) in the MMTV-Cre;Mib1+/f thymus, whereas it accumulated in the cytokeratin-positive cortical epithelial cells of the MMTV-Cre;Mib1f/f thymus (B and C, asterisks). The arrowheads in C and D show the expression of Dll1 and Mib1, respectively. A representative of three independent experiments is shown. Bars, 5 μm.
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fig7: Notch signaling defect in the thymi of Mib1 conditional KO mice. (A) The lethally irradiated MMTV-Cre;Mib1+/f (black) and MMTV-Cre;Mib1f/f (red) mice (7–9 wk old) were injected i.v. with BM cells from the TNR mice (51). At 4 wk after transplantation, the thymocytes were analyzed by flow cytometry for GFP expression gated on the CD4−CD8− DN thymocytes. Percentages indicated are mean ± SD from three independent experiments. (B–D) The lethally irradiated MMTV-Cre;Mib1+/f and MMTV-Cre;Mib1f/f mice were transplanted with CD45.1 BM cells. 6 wk after transplantation, the thymi were fixed and cryosections were immunostained with anti-Dll1 (B and C, red), anti-Hrs (B, green), anti-cytokeratin (C and D, green), and anti-Mib1 (D, red) antibodies with HOECHST (blue). Note that Dll1 was colocalized with Hrs (B, arrowheads) in the MMTV-Cre;Mib1+/f thymus, whereas it accumulated in the cytokeratin-positive cortical epithelial cells of the MMTV-Cre;Mib1f/f thymus (B and C, asterisks). The arrowheads in C and D show the expression of Dll1 and Mib1, respectively. A representative of three independent experiments is shown. Bars, 5 μm.

Mentions: The thymic stroma provides a unique microenvironment for T cell differentiation, and it expresses multiple Notch ligands (30, 32, 33). Several studies suggested that Dll1 and Dll4 in the thymic stroma might be the critical Notch ligands to activate Notch1 in T cell progenitors (16, 32). Because Mib1 regulates multiple Notch ligands, including Dll1 and Dll4, and T lineage commitment is blocked in the MMTV-Cre;Mib1f/f and Mx1-Cre;Mib1f/f mice, we speculated that the inactivation of Mib1 in the thymic microenvironment would prevent the activation of Notch signaling in T cell progenitors. To test this possibility, the lethally irradiated MMTV-Cre;Mib1+/f and MMTV-Cre;Mib1f/f mice were reconstituted with BM cells from the transgenic Notch reporter (TNR) mice, which express EGFP in cells upon Notch/CBF1 activation (51). We readily observed EGFP expression in the DN thymocytes of the MMTV-Cre;Mib1+/f mice at 4 wk after TNR BM transplantation. In contrast, EGFP expression was almost absent in those of the MMTV-Cre;Mib1f/f mice (Fig. 7 A), indicating that the Mib1- thymic microenvironment cannot activate Notch signaling in T cell progenitors.


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)

Notch signaling defect in the thymi of Mib1 conditional KO mice. (A) The lethally irradiated MMTV-Cre;Mib1+/f (black) and MMTV-Cre;Mib1f/f (red) mice (7–9 wk old) were injected i.v. with BM cells from the TNR mice (51). At 4 wk after transplantation, the thymocytes were analyzed by flow cytometry for GFP expression gated on the CD4−CD8− DN thymocytes. Percentages indicated are mean ± SD from three independent experiments. (B–D) The lethally irradiated MMTV-Cre;Mib1+/f and MMTV-Cre;Mib1f/f mice were transplanted with CD45.1 BM cells. 6 wk after transplantation, the thymi were fixed and cryosections were immunostained with anti-Dll1 (B and C, red), anti-Hrs (B, green), anti-cytokeratin (C and D, green), and anti-Mib1 (D, red) antibodies with HOECHST (blue). Note that Dll1 was colocalized with Hrs (B, arrowheads) in the MMTV-Cre;Mib1+/f thymus, whereas it accumulated in the cytokeratin-positive cortical epithelial cells of the MMTV-Cre;Mib1f/f thymus (B and C, asterisks). The arrowheads in C and D show the expression of Dll1 and Mib1, respectively. A representative of three independent experiments is shown. Bars, 5 μm.
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fig7: Notch signaling defect in the thymi of Mib1 conditional KO mice. (A) The lethally irradiated MMTV-Cre;Mib1+/f (black) and MMTV-Cre;Mib1f/f (red) mice (7–9 wk old) were injected i.v. with BM cells from the TNR mice (51). At 4 wk after transplantation, the thymocytes were analyzed by flow cytometry for GFP expression gated on the CD4−CD8− DN thymocytes. Percentages indicated are mean ± SD from three independent experiments. (B–D) The lethally irradiated MMTV-Cre;Mib1+/f and MMTV-Cre;Mib1f/f mice were transplanted with CD45.1 BM cells. 6 wk after transplantation, the thymi were fixed and cryosections were immunostained with anti-Dll1 (B and C, red), anti-Hrs (B, green), anti-cytokeratin (C and D, green), and anti-Mib1 (D, red) antibodies with HOECHST (blue). Note that Dll1 was colocalized with Hrs (B, arrowheads) in the MMTV-Cre;Mib1+/f thymus, whereas it accumulated in the cytokeratin-positive cortical epithelial cells of the MMTV-Cre;Mib1f/f thymus (B and C, asterisks). The arrowheads in C and D show the expression of Dll1 and Mib1, respectively. A representative of three independent experiments is shown. Bars, 5 μm.
Mentions: The thymic stroma provides a unique microenvironment for T cell differentiation, and it expresses multiple Notch ligands (30, 32, 33). Several studies suggested that Dll1 and Dll4 in the thymic stroma might be the critical Notch ligands to activate Notch1 in T cell progenitors (16, 32). Because Mib1 regulates multiple Notch ligands, including Dll1 and Dll4, and T lineage commitment is blocked in the MMTV-Cre;Mib1f/f and Mx1-Cre;Mib1f/f mice, we speculated that the inactivation of Mib1 in the thymic microenvironment would prevent the activation of Notch signaling in T cell progenitors. To test this possibility, the lethally irradiated MMTV-Cre;Mib1+/f and MMTV-Cre;Mib1f/f mice were reconstituted with BM cells from the transgenic Notch reporter (TNR) mice, which express EGFP in cells upon Notch/CBF1 activation (51). We readily observed EGFP expression in the DN thymocytes of the MMTV-Cre;Mib1+/f mice at 4 wk after TNR BM transplantation. In contrast, EGFP expression was almost absent in those of the MMTV-Cre;Mib1f/f mice (Fig. 7 A), indicating that the Mib1- thymic microenvironment cannot activate Notch signaling in T cell progenitors.

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