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A role for Taiman in insect metamorphosis.

Lozano J, Kayukawa T, Shinoda T, Belles X - PLoS Genet. (2014)

Bottom Line: Concomitant depletion of all four Tai isoforms in B. germanica resulted in 100% mortality, but when only the insertion 1 (IN-1) isoforms were depleted, mortality was significantly reduced and about half of the specimens experienced precocious adult development.Reporter assays indicated that both T. castaneum Tai isoforms, one that contains the IN-1 and another that does not (DEL-1) activated a JH response element (kJHRE) in Krüppel homolog 1 in conjunction with Met and JH.The results indicate that Tai is involved in the molecular mechanisms that repress metamorphosis, at least in B. germanica, and highlight the importance of distinguishing Tai isoforms when studying the functions of this transcription factor in development and other processes.

View Article: PubMed Central - PubMed

Affiliation: Institut de Biologia Evolutiva (CSIC-UPF), Barcelona, Spain.

ABSTRACT
Recent studies in vitro have reported that the Methoprene-tolerant (Met) and Taiman (Tai) complex is the functional receptor of juvenile hormone (JH). Experiments in vivo of Met depletion have confirmed this factor's role in JH signal transduction, however, there is no equivalent data regarding Tai because its depletion in larval or nymphal stages of the beetle Tribolium castaneum and the bug Pyrrhocoris apterus results in 100% mortality. We have discovered that the cockroach Blattella germanica possesses four Tai isoforms resulting from the combination of two indels in the C-terminal region of the sequence. The presence of one equivalent indel-1 in Tai sequences in T. castaneum and other species suggests that Tai isoforms may be common in insects. Concomitant depletion of all four Tai isoforms in B. germanica resulted in 100% mortality, but when only the insertion 1 (IN-1) isoforms were depleted, mortality was significantly reduced and about half of the specimens experienced precocious adult development. This shows that Tai isoforms containing IN-1 are involved in transducing the JH signal that represses metamorphosis. Reporter assays indicated that both T. castaneum Tai isoforms, one that contains the IN-1 and another that does not (DEL-1) activated a JH response element (kJHRE) in Krüppel homolog 1 in conjunction with Met and JH. The results indicate that Tai is involved in the molecular mechanisms that repress metamorphosis, at least in B. germanica, and highlight the importance of distinguishing Tai isoforms when studying the functions of this transcription factor in development and other processes.

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Functional study of IN-2-containing isoforms of BgTai and reporter assays to assess the contribution of IN-1 of TbTai to transduce the JH signal.(A) Effects, at transcript level, of dsTai-in-2 treatment in N4; N4 females received two 3-µg doses of dsTai-in-2, one on N4D0 and the other on N4D3, and transcript levels (of Tai-A, Tai-B, Tai-C, Tai-D, Met, Kr-h1 and BR-C) were measured on N5D6; controls received an equivalent treatment with dsMock. (B) Effects of the dsTai-in-2 treatment on the expression of EcR, RXR, E75A and ILP-1 measured also on N5D6. (C) Effects, at transcript level, of dsTai-in-1 plus dsTai-2 treatment in N4; specimens received an injection of dsTai-in-1 (3 µg) plus dsTai-in-2 (3 µg) in N4D0, and the treatment was repeated in N4D3; controls received an equivalent treatment with dsMock. (D) Effects of the dsTai-in-1 plus dsTai-in-2 treatment on the expression of EcR, RXR, E75A and ILP-1 measured also on N5D6. (E–G) Dorsal and ventral view of specimens resulting from dsTai-in-1 plus dsTai-in-2 treatment; normal N5 obtained from this or from dsMock treatments (E); nymphoids with adult features obtained (instead of N6) from dsTai-in-1 plus dsTai-in-2 treatments (F); normal N6 obtained from dsMock treatments (G). Each point of quantitative data in histograms A to D represents 4 biological replicates and results are expressed as the mean ± SEM; data are normalized against the dsMock-treated samples (reference value = 1), and the asterisk indicates statistically significant differences with respect to controls (p<0.05), according to the REST software tool [38]. Scale bars in E–G = 3 mm. (H) Reporter assays to study the JH-dependent interaction of TcMet and TcTai DEL-1 or TcTai IN-1 with kJHRE in Drosophila S2 cells. The cells were transfected with a kJHRE-reporter vector (−477 to +1883, pGL4.14), a reference reporter plasmid carrying Renilla luciferase (pIZT-Rluc) and a plasmid expressing the full ORF of TcMet and/or TcTai IN-1 or TcTai DEL-1. Cells were treated with 1 µM of JH III for 24 h. Reporter activity was measured using a Dual-Luciferase Reporter Assay System. Each bar indicates the mean ± SEM (n = 6). The asterisks indicate statistically significant differences between measurements (***p<0.001; **p<0.01) using a two-tailed t-test (n = 6).
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pgen-1004769-g003: Functional study of IN-2-containing isoforms of BgTai and reporter assays to assess the contribution of IN-1 of TbTai to transduce the JH signal.(A) Effects, at transcript level, of dsTai-in-2 treatment in N4; N4 females received two 3-µg doses of dsTai-in-2, one on N4D0 and the other on N4D3, and transcript levels (of Tai-A, Tai-B, Tai-C, Tai-D, Met, Kr-h1 and BR-C) were measured on N5D6; controls received an equivalent treatment with dsMock. (B) Effects of the dsTai-in-2 treatment on the expression of EcR, RXR, E75A and ILP-1 measured also on N5D6. (C) Effects, at transcript level, of dsTai-in-1 plus dsTai-2 treatment in N4; specimens received an injection of dsTai-in-1 (3 µg) plus dsTai-in-2 (3 µg) in N4D0, and the treatment was repeated in N4D3; controls received an equivalent treatment with dsMock. (D) Effects of the dsTai-in-1 plus dsTai-in-2 treatment on the expression of EcR, RXR, E75A and ILP-1 measured also on N5D6. (E–G) Dorsal and ventral view of specimens resulting from dsTai-in-1 plus dsTai-in-2 treatment; normal N5 obtained from this or from dsMock treatments (E); nymphoids with adult features obtained (instead of N6) from dsTai-in-1 plus dsTai-in-2 treatments (F); normal N6 obtained from dsMock treatments (G). Each point of quantitative data in histograms A to D represents 4 biological replicates and results are expressed as the mean ± SEM; data are normalized against the dsMock-treated samples (reference value = 1), and the asterisk indicates statistically significant differences with respect to controls (p<0.05), according to the REST software tool [38]. Scale bars in E–G = 3 mm. (H) Reporter assays to study the JH-dependent interaction of TcMet and TcTai DEL-1 or TcTai IN-1 with kJHRE in Drosophila S2 cells. The cells were transfected with a kJHRE-reporter vector (−477 to +1883, pGL4.14), a reference reporter plasmid carrying Renilla luciferase (pIZT-Rluc) and a plasmid expressing the full ORF of TcMet and/or TcTai IN-1 or TcTai DEL-1. Cells were treated with 1 µM of JH III for 24 h. Reporter activity was measured using a Dual-Luciferase Reporter Assay System. Each bar indicates the mean ± SEM (n = 6). The asterisks indicate statistically significant differences between measurements (***p<0.001; **p<0.01) using a two-tailed t-test (n = 6).

Mentions: As a next step, we attempted the depletion of the isoforms containing the insertion 2, using a dsRNA encompassing the 74 nucleotides of this insertion (dsTai-in-2). This dsRNA should specifically deplete the isoforms Tai-A and Tai-C (Figure 1B). We injected two 3-µg doses of dsTai-in-2 in N4 females, one in N4D0 and the other in N4D3, whereas controls received an equivalent treatment with dsMock. On N5D6, transcript measurements indicated that dsTai-in-2 targeted the isoforms Tai-A and Tai-C, as expected, which resulted in a rather modest depletion (approximately 55 and 60% for Tai-A and Tai-C, respectively), whereas Tai-B and Tai-D transcripts were unaffected (Figure 3A). These treatments with dsTai-in-2 did not reduce the mRNA levels of Met, Kr-h1 and BR-C (Figure 3A). In the same samples, we also measured mRNA levels of the ecdysone pathway genes (EcR, RXR and E75A), as well as those of ILP-1, and also in these genes, dsTai-in-2-treatment did not reduce transcript levels; only Kr-h1 showed somewhat lower average levels, although differences with respect to controls were not statistically significant (Figure 3B).


A role for Taiman in insect metamorphosis.

Lozano J, Kayukawa T, Shinoda T, Belles X - PLoS Genet. (2014)

Functional study of IN-2-containing isoforms of BgTai and reporter assays to assess the contribution of IN-1 of TbTai to transduce the JH signal.(A) Effects, at transcript level, of dsTai-in-2 treatment in N4; N4 females received two 3-µg doses of dsTai-in-2, one on N4D0 and the other on N4D3, and transcript levels (of Tai-A, Tai-B, Tai-C, Tai-D, Met, Kr-h1 and BR-C) were measured on N5D6; controls received an equivalent treatment with dsMock. (B) Effects of the dsTai-in-2 treatment on the expression of EcR, RXR, E75A and ILP-1 measured also on N5D6. (C) Effects, at transcript level, of dsTai-in-1 plus dsTai-2 treatment in N4; specimens received an injection of dsTai-in-1 (3 µg) plus dsTai-in-2 (3 µg) in N4D0, and the treatment was repeated in N4D3; controls received an equivalent treatment with dsMock. (D) Effects of the dsTai-in-1 plus dsTai-in-2 treatment on the expression of EcR, RXR, E75A and ILP-1 measured also on N5D6. (E–G) Dorsal and ventral view of specimens resulting from dsTai-in-1 plus dsTai-in-2 treatment; normal N5 obtained from this or from dsMock treatments (E); nymphoids with adult features obtained (instead of N6) from dsTai-in-1 plus dsTai-in-2 treatments (F); normal N6 obtained from dsMock treatments (G). Each point of quantitative data in histograms A to D represents 4 biological replicates and results are expressed as the mean ± SEM; data are normalized against the dsMock-treated samples (reference value = 1), and the asterisk indicates statistically significant differences with respect to controls (p<0.05), according to the REST software tool [38]. Scale bars in E–G = 3 mm. (H) Reporter assays to study the JH-dependent interaction of TcMet and TcTai DEL-1 or TcTai IN-1 with kJHRE in Drosophila S2 cells. The cells were transfected with a kJHRE-reporter vector (−477 to +1883, pGL4.14), a reference reporter plasmid carrying Renilla luciferase (pIZT-Rluc) and a plasmid expressing the full ORF of TcMet and/or TcTai IN-1 or TcTai DEL-1. Cells were treated with 1 µM of JH III for 24 h. Reporter activity was measured using a Dual-Luciferase Reporter Assay System. Each bar indicates the mean ± SEM (n = 6). The asterisks indicate statistically significant differences between measurements (***p<0.001; **p<0.01) using a two-tailed t-test (n = 6).
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pgen-1004769-g003: Functional study of IN-2-containing isoforms of BgTai and reporter assays to assess the contribution of IN-1 of TbTai to transduce the JH signal.(A) Effects, at transcript level, of dsTai-in-2 treatment in N4; N4 females received two 3-µg doses of dsTai-in-2, one on N4D0 and the other on N4D3, and transcript levels (of Tai-A, Tai-B, Tai-C, Tai-D, Met, Kr-h1 and BR-C) were measured on N5D6; controls received an equivalent treatment with dsMock. (B) Effects of the dsTai-in-2 treatment on the expression of EcR, RXR, E75A and ILP-1 measured also on N5D6. (C) Effects, at transcript level, of dsTai-in-1 plus dsTai-2 treatment in N4; specimens received an injection of dsTai-in-1 (3 µg) plus dsTai-in-2 (3 µg) in N4D0, and the treatment was repeated in N4D3; controls received an equivalent treatment with dsMock. (D) Effects of the dsTai-in-1 plus dsTai-in-2 treatment on the expression of EcR, RXR, E75A and ILP-1 measured also on N5D6. (E–G) Dorsal and ventral view of specimens resulting from dsTai-in-1 plus dsTai-in-2 treatment; normal N5 obtained from this or from dsMock treatments (E); nymphoids with adult features obtained (instead of N6) from dsTai-in-1 plus dsTai-in-2 treatments (F); normal N6 obtained from dsMock treatments (G). Each point of quantitative data in histograms A to D represents 4 biological replicates and results are expressed as the mean ± SEM; data are normalized against the dsMock-treated samples (reference value = 1), and the asterisk indicates statistically significant differences with respect to controls (p<0.05), according to the REST software tool [38]. Scale bars in E–G = 3 mm. (H) Reporter assays to study the JH-dependent interaction of TcMet and TcTai DEL-1 or TcTai IN-1 with kJHRE in Drosophila S2 cells. The cells were transfected with a kJHRE-reporter vector (−477 to +1883, pGL4.14), a reference reporter plasmid carrying Renilla luciferase (pIZT-Rluc) and a plasmid expressing the full ORF of TcMet and/or TcTai IN-1 or TcTai DEL-1. Cells were treated with 1 µM of JH III for 24 h. Reporter activity was measured using a Dual-Luciferase Reporter Assay System. Each bar indicates the mean ± SEM (n = 6). The asterisks indicate statistically significant differences between measurements (***p<0.001; **p<0.01) using a two-tailed t-test (n = 6).
Mentions: As a next step, we attempted the depletion of the isoforms containing the insertion 2, using a dsRNA encompassing the 74 nucleotides of this insertion (dsTai-in-2). This dsRNA should specifically deplete the isoforms Tai-A and Tai-C (Figure 1B). We injected two 3-µg doses of dsTai-in-2 in N4 females, one in N4D0 and the other in N4D3, whereas controls received an equivalent treatment with dsMock. On N5D6, transcript measurements indicated that dsTai-in-2 targeted the isoforms Tai-A and Tai-C, as expected, which resulted in a rather modest depletion (approximately 55 and 60% for Tai-A and Tai-C, respectively), whereas Tai-B and Tai-D transcripts were unaffected (Figure 3A). These treatments with dsTai-in-2 did not reduce the mRNA levels of Met, Kr-h1 and BR-C (Figure 3A). In the same samples, we also measured mRNA levels of the ecdysone pathway genes (EcR, RXR and E75A), as well as those of ILP-1, and also in these genes, dsTai-in-2-treatment did not reduce transcript levels; only Kr-h1 showed somewhat lower average levels, although differences with respect to controls were not statistically significant (Figure 3B).

Bottom Line: Concomitant depletion of all four Tai isoforms in B. germanica resulted in 100% mortality, but when only the insertion 1 (IN-1) isoforms were depleted, mortality was significantly reduced and about half of the specimens experienced precocious adult development.Reporter assays indicated that both T. castaneum Tai isoforms, one that contains the IN-1 and another that does not (DEL-1) activated a JH response element (kJHRE) in Krüppel homolog 1 in conjunction with Met and JH.The results indicate that Tai is involved in the molecular mechanisms that repress metamorphosis, at least in B. germanica, and highlight the importance of distinguishing Tai isoforms when studying the functions of this transcription factor in development and other processes.

View Article: PubMed Central - PubMed

Affiliation: Institut de Biologia Evolutiva (CSIC-UPF), Barcelona, Spain.

ABSTRACT
Recent studies in vitro have reported that the Methoprene-tolerant (Met) and Taiman (Tai) complex is the functional receptor of juvenile hormone (JH). Experiments in vivo of Met depletion have confirmed this factor's role in JH signal transduction, however, there is no equivalent data regarding Tai because its depletion in larval or nymphal stages of the beetle Tribolium castaneum and the bug Pyrrhocoris apterus results in 100% mortality. We have discovered that the cockroach Blattella germanica possesses four Tai isoforms resulting from the combination of two indels in the C-terminal region of the sequence. The presence of one equivalent indel-1 in Tai sequences in T. castaneum and other species suggests that Tai isoforms may be common in insects. Concomitant depletion of all four Tai isoforms in B. germanica resulted in 100% mortality, but when only the insertion 1 (IN-1) isoforms were depleted, mortality was significantly reduced and about half of the specimens experienced precocious adult development. This shows that Tai isoforms containing IN-1 are involved in transducing the JH signal that represses metamorphosis. Reporter assays indicated that both T. castaneum Tai isoforms, one that contains the IN-1 and another that does not (DEL-1) activated a JH response element (kJHRE) in Krüppel homolog 1 in conjunction with Met and JH. The results indicate that Tai is involved in the molecular mechanisms that repress metamorphosis, at least in B. germanica, and highlight the importance of distinguishing Tai isoforms when studying the functions of this transcription factor in development and other processes.

Show MeSH
Related in: MedlinePlus