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Genomic organization and splicing evolution of the doublesex gene, a Drosophila regulator of sexual differentiation, in the dengue and yellow fever mosquito Aedes aegypti.

Salvemini M, Mauro U, Lombardo F, Milano A, Zazzaro V, Arcà B, Polito LC, Saccone G - BMC Evol. Biol. (2011)

Bottom Line: The sex-specific regulation is based on a combination of exon skipping, 5' alternative splice site choice and, most likely, alternative polyadenylation.Interestingly, when the Aeadsx gene is compared to the Anopheles dsx ortholog, there are differences in the in silico predicted default and regulated sex-specific splicing events, which suggests that the upstream regulators either are different or act in a slightly different manner.Furthermore, this study is a premise for the future development of transgenic sexing strains in mosquitoes useful for sterile insect technique (SIT) programs.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biological Sciences, Section of Genetics and Molecular Biology, University of Naples Federico II, Italy.

ABSTRACT

Background: In the model system Drosophila melanogaster, doublesex (dsx) is the double-switch gene at the bottom of the somatic sex determination cascade that determines the differentiation of sexually dimorphic traits. Homologues of dsx are functionally conserved in various dipteran species, including the malaria vector Anopheles gambiae. They show a striking conservation of sex-specific regulation, based on alternative splicing, and of the encoded sex-specific proteins, which are transcriptional regulators of downstream terminal genes that influence sexual differentiation of cells, tissues and organs.

Results: In this work, we report on the molecular characterization of the dsx homologue in the dengue and yellow fever vector Aedes aegypti (Aeadsx). Aeadsx produces sex-specific transcripts by alternative splicing, which encode isoforms with a high degree of identity to Anopheles gambiae and Drosophila melanogaster homologues. Interestingly, Aeadsx produces an additional novel female-specific splicing variant. Genomic comparative analyses between the Aedes and Anopheles dsx genes revealed a partial conservation of the exon organization and extensive divergence in the intron lengths. An expression analysis showed that Aeadsx transcripts were present from early stages of development and that sex-specific regulation starts at least from late larval stages. The analysis of the female-specific untranslated region (UTR) led to the identification of putative regulatory cis-elements potentially involved in the sex-specific splicing regulation. The Aedes dsx sex-specific splicing regulation seems to be more complex with the respect of other dipteran species, suggesting slightly novel evolutionary trajectories for its regulation and hence for the recruitment of upstream splicing regulators.

Conclusions: This study led to uncover the molecular evolution of Aedes aegypti dsx splicing regulation with the respect of the more closely related Culicidae Anopheles gambiae orthologue. In Aedes aegypti, the dsx gene is sex-specifically regulated and encodes two female-specific and one male-specific isoforms, all sharing a doublesex/mab-3 (DM) domain-containing N-terminus and different C-termini. The sex-specific regulation is based on a combination of exon skipping, 5' alternative splice site choice and, most likely, alternative polyadenylation. Interestingly, when the Aeadsx gene is compared to the Anopheles dsx ortholog, there are differences in the in silico predicted default and regulated sex-specific splicing events, which suggests that the upstream regulators either are different or act in a slightly different manner. Furthermore, this study is a premise for the future development of transgenic sexing strains in mosquitoes useful for sterile insect technique (SIT) programs.

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Developmental expression analyses of the Aeadsx gene. The analyses were performed on the following samples: E1 = 0-1.5 h embryos; E2 = 1.5-2 h embryos; E3 = 2-5 h embryos; E4 = 8-12 h embryos; E: 0-36 h embryos; O = dissected ovaries; FC = female carcasses depleted of ovaries; L12= early larvae; L34= late larvae; P = pupae; M = adult males; F = adult female. Except for M, F, O and FC all samples are composed of mixed sexes. Negative controls are not shown. (A) Amplification of Ae. aegypti rp49 transcripts with the Aearp49+/Aearp49- primer pair. The Aedes aegypti ribosomal gene rp49 is constitutively expressed throughout development. (B) Aeadsx developmental expression pattern. (B.1 and B.4) The dsx3/dsx5 primer combination amplified at adult stages a 0.5-kb male-specific cDNA fragment and two female-specific cDNA fragments (1.0 kb and 1.5 kb). These three bands were detected in pupae and late larvae, while the 1.5-kb band was absent in embryos and mid-larvae but present in ovaries and female carcasses. (B.2 and B.5) The dsx3-dsx4 primer combination amplified at adult stages a female-specific cDNA fragment. A cDNA product of identical size was amplified at all developmental stages, including embryos, suggesting an early Aeadsx female-specific regulation. (B.3 and B.6) The dsx1/dsx2 primer combination amplified in all samples two slightly different cDNA fragments (0.37 kb and 0.31 kb), corresponding to the alternatively spliced isoforms of exon 2 either containing (0.37 kb) or not containing (0.31 kb) the 63-bp intronic sequence. In contrast to the data reported in Figure B.1-3, the RT-PCR results in Figure B.4, B.5 and B.6 lack of a positive semiquantitative control and the apparent changes in expression levels of Aeadsx isoforms during embryonic stages have to be further investigated. (C) A northern blot analysis was performed on total RNA (20 μg) extracted from male and female Ae. aegypti adults. The genomic position of the utilized probe is indicated in Figure 5B. The observed molecular size of Aeadsx transcripts confirms that isolated Aeadsx cDNA clones were not full-length at the 3' and 5' ends.
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Figure 5: Developmental expression analyses of the Aeadsx gene. The analyses were performed on the following samples: E1 = 0-1.5 h embryos; E2 = 1.5-2 h embryos; E3 = 2-5 h embryos; E4 = 8-12 h embryos; E: 0-36 h embryos; O = dissected ovaries; FC = female carcasses depleted of ovaries; L12= early larvae; L34= late larvae; P = pupae; M = adult males; F = adult female. Except for M, F, O and FC all samples are composed of mixed sexes. Negative controls are not shown. (A) Amplification of Ae. aegypti rp49 transcripts with the Aearp49+/Aearp49- primer pair. The Aedes aegypti ribosomal gene rp49 is constitutively expressed throughout development. (B) Aeadsx developmental expression pattern. (B.1 and B.4) The dsx3/dsx5 primer combination amplified at adult stages a 0.5-kb male-specific cDNA fragment and two female-specific cDNA fragments (1.0 kb and 1.5 kb). These three bands were detected in pupae and late larvae, while the 1.5-kb band was absent in embryos and mid-larvae but present in ovaries and female carcasses. (B.2 and B.5) The dsx3-dsx4 primer combination amplified at adult stages a female-specific cDNA fragment. A cDNA product of identical size was amplified at all developmental stages, including embryos, suggesting an early Aeadsx female-specific regulation. (B.3 and B.6) The dsx1/dsx2 primer combination amplified in all samples two slightly different cDNA fragments (0.37 kb and 0.31 kb), corresponding to the alternatively spliced isoforms of exon 2 either containing (0.37 kb) or not containing (0.31 kb) the 63-bp intronic sequence. In contrast to the data reported in Figure B.1-3, the RT-PCR results in Figure B.4, B.5 and B.6 lack of a positive semiquantitative control and the apparent changes in expression levels of Aeadsx isoforms during embryonic stages have to be further investigated. (C) A northern blot analysis was performed on total RNA (20 μg) extracted from male and female Ae. aegypti adults. The genomic position of the utilized probe is indicated in Figure 5B. The observed molecular size of Aeadsx transcripts confirms that isolated Aeadsx cDNA clones were not full-length at the 3' and 5' ends.

Mentions: To analyze the developmental expression pattern of the Ae. aegypti dsx gene, an RT-PCR analysis was performed on total RNA extracted from different stages of development using primer pairs spanning the Aeadsx sex-specifically regulated region. The recently isolated Aedes aegypti (Aearp49) homologue of the Drosophila rp49 constitutively expressed gene was tested as the positive control for the RT-PCR analysis in non-saturating conditions [38-42]. Aearp49 was constitutively expressed from the embryonic stage of Aedes until adulthood, as in the fruitfly (Figure 5A).


Genomic organization and splicing evolution of the doublesex gene, a Drosophila regulator of sexual differentiation, in the dengue and yellow fever mosquito Aedes aegypti.

Salvemini M, Mauro U, Lombardo F, Milano A, Zazzaro V, Arcà B, Polito LC, Saccone G - BMC Evol. Biol. (2011)

Developmental expression analyses of the Aeadsx gene. The analyses were performed on the following samples: E1 = 0-1.5 h embryos; E2 = 1.5-2 h embryos; E3 = 2-5 h embryos; E4 = 8-12 h embryos; E: 0-36 h embryos; O = dissected ovaries; FC = female carcasses depleted of ovaries; L12= early larvae; L34= late larvae; P = pupae; M = adult males; F = adult female. Except for M, F, O and FC all samples are composed of mixed sexes. Negative controls are not shown. (A) Amplification of Ae. aegypti rp49 transcripts with the Aearp49+/Aearp49- primer pair. The Aedes aegypti ribosomal gene rp49 is constitutively expressed throughout development. (B) Aeadsx developmental expression pattern. (B.1 and B.4) The dsx3/dsx5 primer combination amplified at adult stages a 0.5-kb male-specific cDNA fragment and two female-specific cDNA fragments (1.0 kb and 1.5 kb). These three bands were detected in pupae and late larvae, while the 1.5-kb band was absent in embryos and mid-larvae but present in ovaries and female carcasses. (B.2 and B.5) The dsx3-dsx4 primer combination amplified at adult stages a female-specific cDNA fragment. A cDNA product of identical size was amplified at all developmental stages, including embryos, suggesting an early Aeadsx female-specific regulation. (B.3 and B.6) The dsx1/dsx2 primer combination amplified in all samples two slightly different cDNA fragments (0.37 kb and 0.31 kb), corresponding to the alternatively spliced isoforms of exon 2 either containing (0.37 kb) or not containing (0.31 kb) the 63-bp intronic sequence. In contrast to the data reported in Figure B.1-3, the RT-PCR results in Figure B.4, B.5 and B.6 lack of a positive semiquantitative control and the apparent changes in expression levels of Aeadsx isoforms during embryonic stages have to be further investigated. (C) A northern blot analysis was performed on total RNA (20 μg) extracted from male and female Ae. aegypti adults. The genomic position of the utilized probe is indicated in Figure 5B. The observed molecular size of Aeadsx transcripts confirms that isolated Aeadsx cDNA clones were not full-length at the 3' and 5' ends.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
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Figure 5: Developmental expression analyses of the Aeadsx gene. The analyses were performed on the following samples: E1 = 0-1.5 h embryos; E2 = 1.5-2 h embryos; E3 = 2-5 h embryos; E4 = 8-12 h embryos; E: 0-36 h embryos; O = dissected ovaries; FC = female carcasses depleted of ovaries; L12= early larvae; L34= late larvae; P = pupae; M = adult males; F = adult female. Except for M, F, O and FC all samples are composed of mixed sexes. Negative controls are not shown. (A) Amplification of Ae. aegypti rp49 transcripts with the Aearp49+/Aearp49- primer pair. The Aedes aegypti ribosomal gene rp49 is constitutively expressed throughout development. (B) Aeadsx developmental expression pattern. (B.1 and B.4) The dsx3/dsx5 primer combination amplified at adult stages a 0.5-kb male-specific cDNA fragment and two female-specific cDNA fragments (1.0 kb and 1.5 kb). These three bands were detected in pupae and late larvae, while the 1.5-kb band was absent in embryos and mid-larvae but present in ovaries and female carcasses. (B.2 and B.5) The dsx3-dsx4 primer combination amplified at adult stages a female-specific cDNA fragment. A cDNA product of identical size was amplified at all developmental stages, including embryos, suggesting an early Aeadsx female-specific regulation. (B.3 and B.6) The dsx1/dsx2 primer combination amplified in all samples two slightly different cDNA fragments (0.37 kb and 0.31 kb), corresponding to the alternatively spliced isoforms of exon 2 either containing (0.37 kb) or not containing (0.31 kb) the 63-bp intronic sequence. In contrast to the data reported in Figure B.1-3, the RT-PCR results in Figure B.4, B.5 and B.6 lack of a positive semiquantitative control and the apparent changes in expression levels of Aeadsx isoforms during embryonic stages have to be further investigated. (C) A northern blot analysis was performed on total RNA (20 μg) extracted from male and female Ae. aegypti adults. The genomic position of the utilized probe is indicated in Figure 5B. The observed molecular size of Aeadsx transcripts confirms that isolated Aeadsx cDNA clones were not full-length at the 3' and 5' ends.
Mentions: To analyze the developmental expression pattern of the Ae. aegypti dsx gene, an RT-PCR analysis was performed on total RNA extracted from different stages of development using primer pairs spanning the Aeadsx sex-specifically regulated region. The recently isolated Aedes aegypti (Aearp49) homologue of the Drosophila rp49 constitutively expressed gene was tested as the positive control for the RT-PCR analysis in non-saturating conditions [38-42]. Aearp49 was constitutively expressed from the embryonic stage of Aedes until adulthood, as in the fruitfly (Figure 5A).

Bottom Line: The sex-specific regulation is based on a combination of exon skipping, 5' alternative splice site choice and, most likely, alternative polyadenylation.Interestingly, when the Aeadsx gene is compared to the Anopheles dsx ortholog, there are differences in the in silico predicted default and regulated sex-specific splicing events, which suggests that the upstream regulators either are different or act in a slightly different manner.Furthermore, this study is a premise for the future development of transgenic sexing strains in mosquitoes useful for sterile insect technique (SIT) programs.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biological Sciences, Section of Genetics and Molecular Biology, University of Naples Federico II, Italy.

ABSTRACT

Background: In the model system Drosophila melanogaster, doublesex (dsx) is the double-switch gene at the bottom of the somatic sex determination cascade that determines the differentiation of sexually dimorphic traits. Homologues of dsx are functionally conserved in various dipteran species, including the malaria vector Anopheles gambiae. They show a striking conservation of sex-specific regulation, based on alternative splicing, and of the encoded sex-specific proteins, which are transcriptional regulators of downstream terminal genes that influence sexual differentiation of cells, tissues and organs.

Results: In this work, we report on the molecular characterization of the dsx homologue in the dengue and yellow fever vector Aedes aegypti (Aeadsx). Aeadsx produces sex-specific transcripts by alternative splicing, which encode isoforms with a high degree of identity to Anopheles gambiae and Drosophila melanogaster homologues. Interestingly, Aeadsx produces an additional novel female-specific splicing variant. Genomic comparative analyses between the Aedes and Anopheles dsx genes revealed a partial conservation of the exon organization and extensive divergence in the intron lengths. An expression analysis showed that Aeadsx transcripts were present from early stages of development and that sex-specific regulation starts at least from late larval stages. The analysis of the female-specific untranslated region (UTR) led to the identification of putative regulatory cis-elements potentially involved in the sex-specific splicing regulation. The Aedes dsx sex-specific splicing regulation seems to be more complex with the respect of other dipteran species, suggesting slightly novel evolutionary trajectories for its regulation and hence for the recruitment of upstream splicing regulators.

Conclusions: This study led to uncover the molecular evolution of Aedes aegypti dsx splicing regulation with the respect of the more closely related Culicidae Anopheles gambiae orthologue. In Aedes aegypti, the dsx gene is sex-specifically regulated and encodes two female-specific and one male-specific isoforms, all sharing a doublesex/mab-3 (DM) domain-containing N-terminus and different C-termini. The sex-specific regulation is based on a combination of exon skipping, 5' alternative splice site choice and, most likely, alternative polyadenylation. Interestingly, when the Aeadsx gene is compared to the Anopheles dsx ortholog, there are differences in the in silico predicted default and regulated sex-specific splicing events, which suggests that the upstream regulators either are different or act in a slightly different manner. Furthermore, this study is a premise for the future development of transgenic sexing strains in mosquitoes useful for sterile insect technique (SIT) programs.

Show MeSH
Related in: MedlinePlus