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The developmentally regulated expression of Twisted gastrulation reveals a role for bone morphogenetic proteins in the control of T cell development.

Graf D, Nethisinghe S, Palmer DB, Fisher AG, Merkenschlager M - J. Exp. Med. (2002)

Bottom Line: The evolutionarily conserved, secreted protein Twisted gastrulation (Tsg) modulates morphogenetic effects of decapentaplegic (dpp) and its orthologs, the bone morphogenetic proteins 2 and 4 (BMP2/4), in early Drosophila and vertebrate embryos.We have uncovered a role for Tsg at a much later stage of mammalian development, during T cell differentiation in the thymus.BMP4 is expressed by thymic stroma and inhibits the proliferation of CD4(-)CD8(-) double-negative (DN) thymocytes and their differentiation to the CD4(+)CD8(+) double-positive (DP) stage in vitro.

View Article: PubMed Central - PubMed

Affiliation: Lymphocyte Development Group, Medical Research Council Clinical Sciences Centre, Imperial College of Medicine, London W12 0NN, United Kingdom.

ABSTRACT
The evolutionarily conserved, secreted protein Twisted gastrulation (Tsg) modulates morphogenetic effects of decapentaplegic (dpp) and its orthologs, the bone morphogenetic proteins 2 and 4 (BMP2/4), in early Drosophila and vertebrate embryos. We have uncovered a role for Tsg at a much later stage of mammalian development, during T cell differentiation in the thymus. BMP4 is expressed by thymic stroma and inhibits the proliferation of CD4(-)CD8(-) double-negative (DN) thymocytes and their differentiation to the CD4(+)CD8(+) double-positive (DP) stage in vitro. Tsg is expressed by thymocytes and up-regulated after T cell receptor signaling at two developmental checkpoints, the transition from the DN to the DP and from the DP to the CD4(+) or CD8(+) single-positive stage. Tsg can synergize with the BMP inhibitor chordin to block the BMP4-mediated inhibition of thymocyte proliferation and differentiation. These data suggest that the developmentally regulated expression of Tsg may allow thymocytes to temporarily withdraw from inhibitory BMP signals.

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BMP4 inhibits the cell cycle activity and the developmental progression of DN thymocytes to the DP stage. (a) E15.5 thymic lobes were cultured in hanging drops for 24–36 h with BMP4 (100 ng/ml, 6 nM approximately), BMP4 (100 ng/ml) plus neutralizing anti-BMP4 (10 μg/ml) or chordin (2 μg/ml, 20 nM approximately). Thymocytes were stained for CD4 and CD8, fixed, permeabilized, and DNA content was visualized by 7AAD. The fraction of cells with a DNA content >G1 is shown relative to control cultures (16 ± 7% >G1). (b) Reduced cell cycle activity and impaired developmental progression in BMP4-treated E15.5 thymic organ cultures (36 h, methods as in a). (c) BMP4 (100 ng/ml) inhibited the DN to DP transition in E15.5 thymic organ cultures by 52 ± 21% (from 50 ± 10% DP to 25 ± 13% at 36 h, top row) and in Rag1o/o thymic organ cultures treated for 72 h with anti-CD3ε (1 μg/ml) by 46 ± 22% (from 57 ± 4% DP to 32 ± 12%, row two). BMP4 reduced the generation of DP cells in proteolytically dissociated E15.5 thymus suspensions by 52 ± 17% (from 59 ± 13% DP to 28 ± 9%, third row), in mechanically prepared E15.5 thymocyte suspensions by 72 ± 8% (from 44 ± 19% DP to 13 ± 6%, row four), in DN cells prepared by CD8 depletion of E17 thymocytes by 80 ± 13% (from 33 ± 16% to 8 ± 8%, row five), in DN/CD8 transitional thymocytes prepared by CD4 depletion of E17 thymocytes by 36 ± 10% (from 66 ± 23% DP to 44 ± 20%, row six), in highly purified DN by 72% (from 39% DP to 11%) and 73% (from 40% DP to 10%) in two experiments (row seven), and in highly purified CD8 transitional thymocytes by 26% (from 91% DP to 67%) and 29%, (from 83% DP to 59%) in two experiments (row eight, all suspension culture experiments read out at 18 h). Recombinant BMPR-IA/Fc (bottom row, 1–3 μg/ml) increased the generation of DP cells in E15.5 thymic lobes cultured for 18 h by 67 ± 28% (from 23 to 38%, n = 4). (d) E15.5 thymus suspensions were cultured with 300 ng/ml of BMP4 or BMP7 and analyzed as in Fig. 3, b and c. BMP4 reduced the generation of DP cells by 73 ± 7% (from 50 ± 27% DP to 14 ± 9%) but BMP7 did not (1 ± 5%).
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fig3: BMP4 inhibits the cell cycle activity and the developmental progression of DN thymocytes to the DP stage. (a) E15.5 thymic lobes were cultured in hanging drops for 24–36 h with BMP4 (100 ng/ml, 6 nM approximately), BMP4 (100 ng/ml) plus neutralizing anti-BMP4 (10 μg/ml) or chordin (2 μg/ml, 20 nM approximately). Thymocytes were stained for CD4 and CD8, fixed, permeabilized, and DNA content was visualized by 7AAD. The fraction of cells with a DNA content >G1 is shown relative to control cultures (16 ± 7% >G1). (b) Reduced cell cycle activity and impaired developmental progression in BMP4-treated E15.5 thymic organ cultures (36 h, methods as in a). (c) BMP4 (100 ng/ml) inhibited the DN to DP transition in E15.5 thymic organ cultures by 52 ± 21% (from 50 ± 10% DP to 25 ± 13% at 36 h, top row) and in Rag1o/o thymic organ cultures treated for 72 h with anti-CD3ε (1 μg/ml) by 46 ± 22% (from 57 ± 4% DP to 32 ± 12%, row two). BMP4 reduced the generation of DP cells in proteolytically dissociated E15.5 thymus suspensions by 52 ± 17% (from 59 ± 13% DP to 28 ± 9%, third row), in mechanically prepared E15.5 thymocyte suspensions by 72 ± 8% (from 44 ± 19% DP to 13 ± 6%, row four), in DN cells prepared by CD8 depletion of E17 thymocytes by 80 ± 13% (from 33 ± 16% to 8 ± 8%, row five), in DN/CD8 transitional thymocytes prepared by CD4 depletion of E17 thymocytes by 36 ± 10% (from 66 ± 23% DP to 44 ± 20%, row six), in highly purified DN by 72% (from 39% DP to 11%) and 73% (from 40% DP to 10%) in two experiments (row seven), and in highly purified CD8 transitional thymocytes by 26% (from 91% DP to 67%) and 29%, (from 83% DP to 59%) in two experiments (row eight, all suspension culture experiments read out at 18 h). Recombinant BMPR-IA/Fc (bottom row, 1–3 μg/ml) increased the generation of DP cells in E15.5 thymic lobes cultured for 18 h by 67 ± 28% (from 23 to 38%, n = 4). (d) E15.5 thymus suspensions were cultured with 300 ng/ml of BMP4 or BMP7 and analyzed as in Fig. 3, b and c. BMP4 reduced the generation of DP cells by 73 ± 7% (from 50 ± 27% DP to 14 ± 9%) but BMP7 did not (1 ± 5%).

Mentions: To assess the functional effects of BMPs we established thymic organ cultures at embryonic day 15.5 (E15.5), a time in ontogeny when all thymocytes are DN, and analyzed the cell cycle distribution as well as the phenotype of thymocytes that developed in these cultures. Addition of BMP4 at 100 ng/ml (∼6 nM) reduced the percentage of thymocytes in S and G2/M phase of the cell cycle by 42 ± 9% (Fig. 3 , a and b). This inhibition was largely abrogated by a neutralizing antibody to BMP4 (Fig. 3 a). Conversely, treatment of thymic organ cultures with recombinant chordin 2 μg/ml (∼20 nM) increased the percentage of thymocytes in S and G2/M by 59 ± 30% (Fig. 3 a). As chordin is an extracellular inhibitor specifically of BMPs but not of TGFβ or activin (21), the increased proliferation of thymocytes in intact thymic lobes treated with chordin suggests that thymocyte cell cycle activity is subject to BMP-mediated inhibition in situ.


The developmentally regulated expression of Twisted gastrulation reveals a role for bone morphogenetic proteins in the control of T cell development.

Graf D, Nethisinghe S, Palmer DB, Fisher AG, Merkenschlager M - J. Exp. Med. (2002)

BMP4 inhibits the cell cycle activity and the developmental progression of DN thymocytes to the DP stage. (a) E15.5 thymic lobes were cultured in hanging drops for 24–36 h with BMP4 (100 ng/ml, 6 nM approximately), BMP4 (100 ng/ml) plus neutralizing anti-BMP4 (10 μg/ml) or chordin (2 μg/ml, 20 nM approximately). Thymocytes were stained for CD4 and CD8, fixed, permeabilized, and DNA content was visualized by 7AAD. The fraction of cells with a DNA content >G1 is shown relative to control cultures (16 ± 7% >G1). (b) Reduced cell cycle activity and impaired developmental progression in BMP4-treated E15.5 thymic organ cultures (36 h, methods as in a). (c) BMP4 (100 ng/ml) inhibited the DN to DP transition in E15.5 thymic organ cultures by 52 ± 21% (from 50 ± 10% DP to 25 ± 13% at 36 h, top row) and in Rag1o/o thymic organ cultures treated for 72 h with anti-CD3ε (1 μg/ml) by 46 ± 22% (from 57 ± 4% DP to 32 ± 12%, row two). BMP4 reduced the generation of DP cells in proteolytically dissociated E15.5 thymus suspensions by 52 ± 17% (from 59 ± 13% DP to 28 ± 9%, third row), in mechanically prepared E15.5 thymocyte suspensions by 72 ± 8% (from 44 ± 19% DP to 13 ± 6%, row four), in DN cells prepared by CD8 depletion of E17 thymocytes by 80 ± 13% (from 33 ± 16% to 8 ± 8%, row five), in DN/CD8 transitional thymocytes prepared by CD4 depletion of E17 thymocytes by 36 ± 10% (from 66 ± 23% DP to 44 ± 20%, row six), in highly purified DN by 72% (from 39% DP to 11%) and 73% (from 40% DP to 10%) in two experiments (row seven), and in highly purified CD8 transitional thymocytes by 26% (from 91% DP to 67%) and 29%, (from 83% DP to 59%) in two experiments (row eight, all suspension culture experiments read out at 18 h). Recombinant BMPR-IA/Fc (bottom row, 1–3 μg/ml) increased the generation of DP cells in E15.5 thymic lobes cultured for 18 h by 67 ± 28% (from 23 to 38%, n = 4). (d) E15.5 thymus suspensions were cultured with 300 ng/ml of BMP4 or BMP7 and analyzed as in Fig. 3, b and c. BMP4 reduced the generation of DP cells by 73 ± 7% (from 50 ± 27% DP to 14 ± 9%) but BMP7 did not (1 ± 5%).
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fig3: BMP4 inhibits the cell cycle activity and the developmental progression of DN thymocytes to the DP stage. (a) E15.5 thymic lobes were cultured in hanging drops for 24–36 h with BMP4 (100 ng/ml, 6 nM approximately), BMP4 (100 ng/ml) plus neutralizing anti-BMP4 (10 μg/ml) or chordin (2 μg/ml, 20 nM approximately). Thymocytes were stained for CD4 and CD8, fixed, permeabilized, and DNA content was visualized by 7AAD. The fraction of cells with a DNA content >G1 is shown relative to control cultures (16 ± 7% >G1). (b) Reduced cell cycle activity and impaired developmental progression in BMP4-treated E15.5 thymic organ cultures (36 h, methods as in a). (c) BMP4 (100 ng/ml) inhibited the DN to DP transition in E15.5 thymic organ cultures by 52 ± 21% (from 50 ± 10% DP to 25 ± 13% at 36 h, top row) and in Rag1o/o thymic organ cultures treated for 72 h with anti-CD3ε (1 μg/ml) by 46 ± 22% (from 57 ± 4% DP to 32 ± 12%, row two). BMP4 reduced the generation of DP cells in proteolytically dissociated E15.5 thymus suspensions by 52 ± 17% (from 59 ± 13% DP to 28 ± 9%, third row), in mechanically prepared E15.5 thymocyte suspensions by 72 ± 8% (from 44 ± 19% DP to 13 ± 6%, row four), in DN cells prepared by CD8 depletion of E17 thymocytes by 80 ± 13% (from 33 ± 16% to 8 ± 8%, row five), in DN/CD8 transitional thymocytes prepared by CD4 depletion of E17 thymocytes by 36 ± 10% (from 66 ± 23% DP to 44 ± 20%, row six), in highly purified DN by 72% (from 39% DP to 11%) and 73% (from 40% DP to 10%) in two experiments (row seven), and in highly purified CD8 transitional thymocytes by 26% (from 91% DP to 67%) and 29%, (from 83% DP to 59%) in two experiments (row eight, all suspension culture experiments read out at 18 h). Recombinant BMPR-IA/Fc (bottom row, 1–3 μg/ml) increased the generation of DP cells in E15.5 thymic lobes cultured for 18 h by 67 ± 28% (from 23 to 38%, n = 4). (d) E15.5 thymus suspensions were cultured with 300 ng/ml of BMP4 or BMP7 and analyzed as in Fig. 3, b and c. BMP4 reduced the generation of DP cells by 73 ± 7% (from 50 ± 27% DP to 14 ± 9%) but BMP7 did not (1 ± 5%).
Mentions: To assess the functional effects of BMPs we established thymic organ cultures at embryonic day 15.5 (E15.5), a time in ontogeny when all thymocytes are DN, and analyzed the cell cycle distribution as well as the phenotype of thymocytes that developed in these cultures. Addition of BMP4 at 100 ng/ml (∼6 nM) reduced the percentage of thymocytes in S and G2/M phase of the cell cycle by 42 ± 9% (Fig. 3 , a and b). This inhibition was largely abrogated by a neutralizing antibody to BMP4 (Fig. 3 a). Conversely, treatment of thymic organ cultures with recombinant chordin 2 μg/ml (∼20 nM) increased the percentage of thymocytes in S and G2/M by 59 ± 30% (Fig. 3 a). As chordin is an extracellular inhibitor specifically of BMPs but not of TGFβ or activin (21), the increased proliferation of thymocytes in intact thymic lobes treated with chordin suggests that thymocyte cell cycle activity is subject to BMP-mediated inhibition in situ.

Bottom Line: The evolutionarily conserved, secreted protein Twisted gastrulation (Tsg) modulates morphogenetic effects of decapentaplegic (dpp) and its orthologs, the bone morphogenetic proteins 2 and 4 (BMP2/4), in early Drosophila and vertebrate embryos.We have uncovered a role for Tsg at a much later stage of mammalian development, during T cell differentiation in the thymus.BMP4 is expressed by thymic stroma and inhibits the proliferation of CD4(-)CD8(-) double-negative (DN) thymocytes and their differentiation to the CD4(+)CD8(+) double-positive (DP) stage in vitro.

View Article: PubMed Central - PubMed

Affiliation: Lymphocyte Development Group, Medical Research Council Clinical Sciences Centre, Imperial College of Medicine, London W12 0NN, United Kingdom.

ABSTRACT
The evolutionarily conserved, secreted protein Twisted gastrulation (Tsg) modulates morphogenetic effects of decapentaplegic (dpp) and its orthologs, the bone morphogenetic proteins 2 and 4 (BMP2/4), in early Drosophila and vertebrate embryos. We have uncovered a role for Tsg at a much later stage of mammalian development, during T cell differentiation in the thymus. BMP4 is expressed by thymic stroma and inhibits the proliferation of CD4(-)CD8(-) double-negative (DN) thymocytes and their differentiation to the CD4(+)CD8(+) double-positive (DP) stage in vitro. Tsg is expressed by thymocytes and up-regulated after T cell receptor signaling at two developmental checkpoints, the transition from the DN to the DP and from the DP to the CD4(+) or CD8(+) single-positive stage. Tsg can synergize with the BMP inhibitor chordin to block the BMP4-mediated inhibition of thymocyte proliferation and differentiation. These data suggest that the developmentally regulated expression of Tsg may allow thymocytes to temporarily withdraw from inhibitory BMP signals.

Show MeSH
Related in: MedlinePlus