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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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MZB cell defect in the MMTV-Cre;Mib1f/f mice. (A) The splenocytes from the indicated mice were analyzed by flow cytometry for the expression of CD21 and CD23 gated on B220+ cells. MZB and FOB cells were defined as CD21hiCD23lo/− and CD21intCD23hi, respectively. Percentages indicated are mean ± SD from 10 independent experiments. (B) The splenocytes from the MMTV-Cre;Mib1+/f (left) and MMTV-Cre;Mib1f/f (right) mice were stained for CD1d and CD9 gated on B220+ cells. Note the defective CD1dhiCD9hi MZB cells in the mutant mice. Percentages indicated are mean ± SD from 10 independent experiments. (C) FOB cells from the MMTV-Cre;Mib1+/f and MMTV-Cre;Mib1f/f mice were analyzed by flow cytometry for the expression of CD21. A representative of four independent experiments is shown. (D) The splenocytes were stained with antibodies against CD21, CD23, and IgM and were analyzed by flow cytometry for the expression of CD21 and IgM, gated on the CD23− splenocytes for T1 B cells, and gated on the CD23+ splenocytes for T2 B cells. Numbers indicate mean ± SD from five independent experiments. (E) The splenocytes from the indicated mice were analyzed by flow cytometry for the expression of CD23 and IgM gated on B220+AA4.1+ cells. The transitional T2 B cells were defined as CD23+IgMhi. Percentages indicated are mean ± SD from three independent experiments.
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fig4: MZB cell defect in the MMTV-Cre;Mib1f/f mice. (A) The splenocytes from the indicated mice were analyzed by flow cytometry for the expression of CD21 and CD23 gated on B220+ cells. MZB and FOB cells were defined as CD21hiCD23lo/− and CD21intCD23hi, respectively. Percentages indicated are mean ± SD from 10 independent experiments. (B) The splenocytes from the MMTV-Cre;Mib1+/f (left) and MMTV-Cre;Mib1f/f (right) mice were stained for CD1d and CD9 gated on B220+ cells. Note the defective CD1dhiCD9hi MZB cells in the mutant mice. Percentages indicated are mean ± SD from 10 independent experiments. (C) FOB cells from the MMTV-Cre;Mib1+/f and MMTV-Cre;Mib1f/f mice were analyzed by flow cytometry for the expression of CD21. A representative of four independent experiments is shown. (D) The splenocytes were stained with antibodies against CD21, CD23, and IgM and were analyzed by flow cytometry for the expression of CD21 and IgM, gated on the CD23− splenocytes for T1 B cells, and gated on the CD23+ splenocytes for T2 B cells. Numbers indicate mean ± SD from five independent experiments. (E) The splenocytes from the indicated mice were analyzed by flow cytometry for the expression of CD23 and IgM gated on B220+AA4.1+ cells. The transitional T2 B cells were defined as CD23+IgMhi. Percentages indicated are mean ± SD from three independent experiments.

Mentions: Several recent reports demonstrated that Notch2 is critical for the generation of MZB cells, which is mediated through a specific interaction with Dll1 (14–16). To determine which E3 ubiquitin ligase is required for MZB cell development by regulating Dll1, we examined B cell differentiation in the MMTV-Cre;Mib1f/f, Mib2−/−, Neur1−/−, and Neur2−/− mice. When the splenocytes from the mice were analyzed, the fraction of B220+CD21+CD23lo/− MZB cells was markedly reduced only in the MMTV-Cre;Mib1f/f mice, with a concomitant increase in the fraction of B220+CD21+CD23+ FOB cells (Fig. 4 A). The impaired MZB cell development in the MMTV-Cre;Mib1f/f mice was further confirmed with other markers, CD1d and CD9, which are expressed at high levels in MZB cells (Fig. 4 B) (42, 43). Notch signaling reportedly induces the expression of CD21 (44, 45), and the inactivation of Notch2 in the splenocytes down-regulates the expression of CD21 (14). Consistent with these findings, the FOB cells in the spleens from the MMTV-Cre;Mib1f/f mice also showed reduced expression of CD21 (Fig. 4 C). Consistent with the results from the MMTV-Cre;Mib1f/f mice, the defect in MZB cell development was also found in the spleen of the Mx1-Cre;Mib1f/f mice at 12 wk after the last pIpC injections (Fig. S2, A–C, available at http://www.jem.org/cgi/content/full/jem.20081344/DC1). Surprisingly, MZB cell development was not disturbed in the other mutant mice, the Mib2−/−, Neur1−/−, Neur2−/−, and even Neur1−/−;Neur2−/− mice (Fig. 4 A). Therefore, our results demonstrate that Mib1 is essential for MZB cell specification, whereas the other three E3 ligases are dispensable.


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)

MZB cell defect in the MMTV-Cre;Mib1f/f mice. (A) The splenocytes from the indicated mice were analyzed by flow cytometry for the expression of CD21 and CD23 gated on B220+ cells. MZB and FOB cells were defined as CD21hiCD23lo/− and CD21intCD23hi, respectively. Percentages indicated are mean ± SD from 10 independent experiments. (B) The splenocytes from the MMTV-Cre;Mib1+/f (left) and MMTV-Cre;Mib1f/f (right) mice were stained for CD1d and CD9 gated on B220+ cells. Note the defective CD1dhiCD9hi MZB cells in the mutant mice. Percentages indicated are mean ± SD from 10 independent experiments. (C) FOB cells from the MMTV-Cre;Mib1+/f and MMTV-Cre;Mib1f/f mice were analyzed by flow cytometry for the expression of CD21. A representative of four independent experiments is shown. (D) The splenocytes were stained with antibodies against CD21, CD23, and IgM and were analyzed by flow cytometry for the expression of CD21 and IgM, gated on the CD23− splenocytes for T1 B cells, and gated on the CD23+ splenocytes for T2 B cells. Numbers indicate mean ± SD from five independent experiments. (E) The splenocytes from the indicated mice were analyzed by flow cytometry for the expression of CD23 and IgM gated on B220+AA4.1+ cells. The transitional T2 B cells were defined as CD23+IgMhi. Percentages indicated are mean ± SD from three independent experiments.
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fig4: MZB cell defect in the MMTV-Cre;Mib1f/f mice. (A) The splenocytes from the indicated mice were analyzed by flow cytometry for the expression of CD21 and CD23 gated on B220+ cells. MZB and FOB cells were defined as CD21hiCD23lo/− and CD21intCD23hi, respectively. Percentages indicated are mean ± SD from 10 independent experiments. (B) The splenocytes from the MMTV-Cre;Mib1+/f (left) and MMTV-Cre;Mib1f/f (right) mice were stained for CD1d and CD9 gated on B220+ cells. Note the defective CD1dhiCD9hi MZB cells in the mutant mice. Percentages indicated are mean ± SD from 10 independent experiments. (C) FOB cells from the MMTV-Cre;Mib1+/f and MMTV-Cre;Mib1f/f mice were analyzed by flow cytometry for the expression of CD21. A representative of four independent experiments is shown. (D) The splenocytes were stained with antibodies against CD21, CD23, and IgM and were analyzed by flow cytometry for the expression of CD21 and IgM, gated on the CD23− splenocytes for T1 B cells, and gated on the CD23+ splenocytes for T2 B cells. Numbers indicate mean ± SD from five independent experiments. (E) The splenocytes from the indicated mice were analyzed by flow cytometry for the expression of CD23 and IgM gated on B220+AA4.1+ cells. The transitional T2 B cells were defined as CD23+IgMhi. Percentages indicated are mean ± SD from three independent experiments.
Mentions: Several recent reports demonstrated that Notch2 is critical for the generation of MZB cells, which is mediated through a specific interaction with Dll1 (14–16). To determine which E3 ubiquitin ligase is required for MZB cell development by regulating Dll1, we examined B cell differentiation in the MMTV-Cre;Mib1f/f, Mib2−/−, Neur1−/−, and Neur2−/− mice. When the splenocytes from the mice were analyzed, the fraction of B220+CD21+CD23lo/− MZB cells was markedly reduced only in the MMTV-Cre;Mib1f/f mice, with a concomitant increase in the fraction of B220+CD21+CD23+ FOB cells (Fig. 4 A). The impaired MZB cell development in the MMTV-Cre;Mib1f/f mice was further confirmed with other markers, CD1d and CD9, which are expressed at high levels in MZB cells (Fig. 4 B) (42, 43). Notch signaling reportedly induces the expression of CD21 (44, 45), and the inactivation of Notch2 in the splenocytes down-regulates the expression of CD21 (14). Consistent with these findings, the FOB cells in the spleens from the MMTV-Cre;Mib1f/f mice also showed reduced expression of CD21 (Fig. 4 C). Consistent with the results from the MMTV-Cre;Mib1f/f mice, the defect in MZB cell development was also found in the spleen of the Mx1-Cre;Mib1f/f mice at 12 wk after the last pIpC injections (Fig. S2, A–C, available at http://www.jem.org/cgi/content/full/jem.20081344/DC1). Surprisingly, MZB cell development was not disturbed in the other mutant mice, the Mib2−/−, Neur1−/−, Neur2−/−, and even Neur1−/−;Neur2−/− mice (Fig. 4 A). Therefore, our results demonstrate that Mib1 is essential for MZB cell specification, whereas the other three E3 ligases are dispensable.

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