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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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Structure and expression of BgTai and their isoforms in Blattella germanica.(A) Organization of BgTai protein in different domains; in addition to the bHLH region and the two PAS domains, it contains seven LxxLL motifs indicates as black bars, five regions rich in Glutamine, indicated with “Q”, and two Insertions/Deletions towards the 3′ region one (IN-1) of 276 bp and the other (IN-2) of 74 bp; also indicated is the region were the dsTai-core used in RNAi studies was designed. (B) Scheme representing the C-terminal region of the four isoforms of BgTai. (C) Expression of BgTai mRNA in whole body of female nymphs in penultimate (N5) and last (N6) instar; from top to down it is showed the expression of the ensemble of isoforms (amplified with primers designed to regions common to all them), and that of each isoform A, B, C and D (amplified with respective specific primers). (D) Effect of JH III treatment (20 µg) on BgTai expression, on the ensemble of isoforms, and specifically on each one; JH was topically applied in freshly emerged N6, and BgMet mRNA levels were measured 6 and 48 h later. (E) Expression of each BgTai isoform in different tissues of females in N6D0: corpora allata (CA), epidermis of the thoracic discs (EP), ovaries (O), muscle (M), brain (B), fat body (FB), and in testicles (TST) from males of the same age. Each point in C and D represents 4 biological replicates and results are expressed as the mean ± SEM, whereas those in E represent a pool of 5 specimens; data in C and E are expressed as copies of BgTai mRNA per 1000 copies of BgActin-5c mRNA; data in D 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].
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pgen-1004769-g001: Structure and expression of BgTai and their isoforms in Blattella germanica.(A) Organization of BgTai protein in different domains; in addition to the bHLH region and the two PAS domains, it contains seven LxxLL motifs indicates as black bars, five regions rich in Glutamine, indicated with “Q”, and two Insertions/Deletions towards the 3′ region one (IN-1) of 276 bp and the other (IN-2) of 74 bp; also indicated is the region were the dsTai-core used in RNAi studies was designed. (B) Scheme representing the C-terminal region of the four isoforms of BgTai. (C) Expression of BgTai mRNA in whole body of female nymphs in penultimate (N5) and last (N6) instar; from top to down it is showed the expression of the ensemble of isoforms (amplified with primers designed to regions common to all them), and that of each isoform A, B, C and D (amplified with respective specific primers). (D) Effect of JH III treatment (20 µg) on BgTai expression, on the ensemble of isoforms, and specifically on each one; JH was topically applied in freshly emerged N6, and BgMet mRNA levels were measured 6 and 48 h later. (E) Expression of each BgTai isoform in different tissues of females in N6D0: corpora allata (CA), epidermis of the thoracic discs (EP), ovaries (O), muscle (M), brain (B), fat body (FB), and in testicles (TST) from males of the same age. Each point in C and D represents 4 biological replicates and results are expressed as the mean ± SEM, whereas those in E represent a pool of 5 specimens; data in C and E are expressed as copies of BgTai mRNA per 1000 copies of BgActin-5c mRNA; data in D 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].

Mentions: BLAST search of Tai in transcriptomes of B. germanica followed by PCR amplifications gave an ORF sequence of 4914 bp whose conceptual translation rendered a 1638 amino acid protein with sequence similarity to insect Tai/FISC/SRC proteins and that we called BgTai. The sequence contained the bHLH, PAS A and PAS B domains, as well as seven LxxLL motifs and several glutamine-rich regions characteristics of Tai/FISC/SRC proteins (Figure 1A), and top BLAST scores were obtained from Tai/FISC/SRC orthologs of other insects. The phylogenetic analysis of vertebrate SRC and insect Tai/FISC/SRC shows that insect proteins cluster a part, as a sister group of the vertebrate SRCs that is formed by three separated nodes corresponding to SRC-1, SRC-2 and SRC-3 subgroups (Figure S1). Therefore, it seems appropriate to use a single name, Taiman, for all insect representatives of the SRC superfamily of proteins. BgTai protein shows the highest amino acid identity with the Tai ortholog of the hemimetabolan species Pediculus humanus, both in the overall protein sequence and in the bHLH domain (57 and 82% identity, respectively); identity values decreased for D. melanogaster (25 and 49%) and A. aegypti (32 and 51%) (Figure S2).


A role for Taiman in insect metamorphosis.

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

Structure and expression of BgTai and their isoforms in Blattella germanica.(A) Organization of BgTai protein in different domains; in addition to the bHLH region and the two PAS domains, it contains seven LxxLL motifs indicates as black bars, five regions rich in Glutamine, indicated with “Q”, and two Insertions/Deletions towards the 3′ region one (IN-1) of 276 bp and the other (IN-2) of 74 bp; also indicated is the region were the dsTai-core used in RNAi studies was designed. (B) Scheme representing the C-terminal region of the four isoforms of BgTai. (C) Expression of BgTai mRNA in whole body of female nymphs in penultimate (N5) and last (N6) instar; from top to down it is showed the expression of the ensemble of isoforms (amplified with primers designed to regions common to all them), and that of each isoform A, B, C and D (amplified with respective specific primers). (D) Effect of JH III treatment (20 µg) on BgTai expression, on the ensemble of isoforms, and specifically on each one; JH was topically applied in freshly emerged N6, and BgMet mRNA levels were measured 6 and 48 h later. (E) Expression of each BgTai isoform in different tissues of females in N6D0: corpora allata (CA), epidermis of the thoracic discs (EP), ovaries (O), muscle (M), brain (B), fat body (FB), and in testicles (TST) from males of the same age. Each point in C and D represents 4 biological replicates and results are expressed as the mean ± SEM, whereas those in E represent a pool of 5 specimens; data in C and E are expressed as copies of BgTai mRNA per 1000 copies of BgActin-5c mRNA; data in D 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].
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4214675&req=5

pgen-1004769-g001: Structure and expression of BgTai and their isoforms in Blattella germanica.(A) Organization of BgTai protein in different domains; in addition to the bHLH region and the two PAS domains, it contains seven LxxLL motifs indicates as black bars, five regions rich in Glutamine, indicated with “Q”, and two Insertions/Deletions towards the 3′ region one (IN-1) of 276 bp and the other (IN-2) of 74 bp; also indicated is the region were the dsTai-core used in RNAi studies was designed. (B) Scheme representing the C-terminal region of the four isoforms of BgTai. (C) Expression of BgTai mRNA in whole body of female nymphs in penultimate (N5) and last (N6) instar; from top to down it is showed the expression of the ensemble of isoforms (amplified with primers designed to regions common to all them), and that of each isoform A, B, C and D (amplified with respective specific primers). (D) Effect of JH III treatment (20 µg) on BgTai expression, on the ensemble of isoforms, and specifically on each one; JH was topically applied in freshly emerged N6, and BgMet mRNA levels were measured 6 and 48 h later. (E) Expression of each BgTai isoform in different tissues of females in N6D0: corpora allata (CA), epidermis of the thoracic discs (EP), ovaries (O), muscle (M), brain (B), fat body (FB), and in testicles (TST) from males of the same age. Each point in C and D represents 4 biological replicates and results are expressed as the mean ± SEM, whereas those in E represent a pool of 5 specimens; data in C and E are expressed as copies of BgTai mRNA per 1000 copies of BgActin-5c mRNA; data in D 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].
Mentions: BLAST search of Tai in transcriptomes of B. germanica followed by PCR amplifications gave an ORF sequence of 4914 bp whose conceptual translation rendered a 1638 amino acid protein with sequence similarity to insect Tai/FISC/SRC proteins and that we called BgTai. The sequence contained the bHLH, PAS A and PAS B domains, as well as seven LxxLL motifs and several glutamine-rich regions characteristics of Tai/FISC/SRC proteins (Figure 1A), and top BLAST scores were obtained from Tai/FISC/SRC orthologs of other insects. The phylogenetic analysis of vertebrate SRC and insect Tai/FISC/SRC shows that insect proteins cluster a part, as a sister group of the vertebrate SRCs that is formed by three separated nodes corresponding to SRC-1, SRC-2 and SRC-3 subgroups (Figure S1). Therefore, it seems appropriate to use a single name, Taiman, for all insect representatives of the SRC superfamily of proteins. BgTai protein shows the highest amino acid identity with the Tai ortholog of the hemimetabolan species Pediculus humanus, both in the overall protein sequence and in the bHLH domain (57 and 82% identity, respectively); identity values decreased for D. melanogaster (25 and 49%) and A. aegypti (32 and 51%) (Figure S2).

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