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. 2011 Feb 10:11:41.
doi: 10.1186/1471-2148-11-41.

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

Marco Salvemini  1 , Umberto Mauro, Fabrizio Lombardo, Andreina Milano, Vincenzo Zazzaro, Bruno Arcà, Lino C Polito, Giuseppe Saccone
Affiliations

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

Marco Salvemini et al. BMC Evol Biol. .

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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Figures

Figure 1
Figure 1
Aeadsx gene. (A) Genomic organization, splicing variants and protein isoforms of dsx in Aedes aegypti. Male-specific and female-specific exons/protein regions are marked in blue and red, respectively. Exons and introns are not shown to scale. Translational start and stop sites and the poly(A) addition sites are marked. Rectangular striped box within exon 2 represents a 63-bp intronic sequence alternatively removed in Aeadsx transcripts of both sexes (see Figure 5B.3 for further details). All transcripts shown in this picture retain the 63-bp intronic sequence, which encodes an in-frame non-conserved 21-aa sequence. Transcripts without the 63-bp are not shown in this picture and in subsequent paper figures. (B) RT-PCR amplification of Aeadsx sex-specific transcripts. Primers used in this amplification are indicated as short red arrows in Figure 1A.
Figure 2
Figure 2
Comparative genomic structure of the D. melanogaster, Ae. aegypti and An. gambiae dsx genes. Comparative genomic structure of the D. melanogaster, Ae. aegypti and An. gambiae dsx genes. Green boxes represent the OD1 and OD2 domain-encoding exons. Black boxes represent exons encoding protein regions conserved in mosquitoes but not in fruit flies. Alternative male-specific and female-specific exons are represented as blue boxes and pink boxes, respectively. Green dots represent canonical acceptor/donor splicing sites. Red dots represent weak acceptor/donor splicing sites. White and green rectangles represent, respectively, TRA/TRA-2 binding sites and Nasonia dsxRE. In Drosophila, the Dmdsx gene is located in a 45-kb region on chromosome 3R and is organized into six exons and five introns, with three common exons followed by a female-specific and two male-specific exons. DmdsxF translation initiates at the AUG within exon 2 and terminates within the female-specific exon 4, while in the case of DmdsxM, translation begins at the same AUG and terminates within the first male-specific exon 5.
Figure 3
Figure 3
Multiple sequence alignment of DSX homologues. Protein sequence alignment of DSX isoforms in Drosophila melanogaster, Anopheles gambiae and Aedes aegypti. The sequences are divided into a region that is common to males and females (A), a first female-specific region (B), a second female-specific region (C) and a male-specific region (D). The amino-terminal DNA binding (OD1) and oligomerization domains (OD2) are boxed in grey. The asterisk (*) indicates six amino acids whose replacements has been shown to abolish DNA-binding activity in D. melanogaster; (**) double asterisks indicate the three amino acids specific for the DSX DM domain. Intron positions are indicated by solid triangles. The amino acid stretch marked in rectangular box corresponds to the 63-bp sequence removed in some but not all Aeadsx transcripts. This event leads to the in-frame deletion of the indicated 21-amino acid tract (see Figure 5B.3 for further details). Also the conserved removed amino acid stretch of An. gambiae is marked in rectangular box. Bold letters indicate amino acid identity in the homologous proteins. Gaps were introduced in the alignments to maximize similarity. The comparison of protein sequences was performed using Clustal-W (1.82).
Figure 4
Figure 4
Phylogenetic and molecular evolutionary analyses. (A) A phylogenetic tree based on the combined dsx nucleotide sequences encoding OD1 and OD2 domains in six dipteran families and two lepidopteran species. The consensus of six equally parsimonious trees (tree length = 757 and parsimony-informative characters = 206) and the neighbor-joining tree (417 total characters) obtained using the combined nucleotide sequences are shown with bootstrap support above branches (shown only when greater than 50%). Taxonomic relationships are indicated in the right margin of the trees. The topology was rooted with the dsx corresponding sequences from the lepidopteran B. mori and Danaus plexippus. (B) Comparison of dipteran female-specific dsx coding sequences and localization of OD1 and OD2 domains. Pairwise synonymous (dS) and non-synonymous (dN) substitution rates and the mean pairwise ratio (dN/dS) values are placed above the corresponding coding sequence.
Figure 5
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.
Figure 6
Figure 6
Putative cis-acting elements of the Aeadsx gene. The distribution of the putative cis-acting elements in the sex-specifically spliced region of Aeadsx is reported. Black lowercase letters indicate intronic regions. Dark purple letters indicate the female-specific 5a and 5b exons. Translational stop codons are reported in red uppercase letters. The putative polyadenylation signal of exon 5a is underlined.
Figure 7
Figure 7
Model for sex determination in Aedes aegypti. In male embryos a male-determining factor (M) inhibits the function of an SR-F protein required for the female-specific splicing of exon 5b and activates the function of an SR-M protein required for the male-specific repression of exon 5a. It is also conceivable that the M could directly control the splicing of Aeadsx exon 5a. In any case, the result is the skipping of both female-specific exons and only a male-specific product of dsx gene is produced in male embryos and this product induces the male development. In female embryos the absence of the M leads to the default splicing of female-specific exons 5a and 5b and to the activation of the SR-F factor which in turn regulates the female-specific splicing of exon 5b. As a result two female-specific DSX proteins are produced that induce female development. Alternative male-specific and female-specific exons are represented as blue boxes and pink boxes, respectively. Green dot represent NvdsxRE and TRA-2-ISS elements. White dot represents TRA/TRA-2 binding sites.
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References

    1. Raymond SC, Shamu EC, Shen MM, Seifert JK, Hirsch B. et al.Evidence for evolutionary conservation of sex-determining genes. Nature. 1998;391(6668):691–695. doi: 10.1038/35618. - DOI - PubMed
    1. Zarkower D. Invertebrates may not be so different after all. Novartis Found Symp. 2002;244:115–126. discussion 126-135, 203-116, 253-117. full_text. - PubMed
    1. Sanchez L. Sex-determining mechanisms in insects. Int J Dev Biol. 2008;52(7):837–856. doi: 10.1387/ijdb.072396ls. - DOI - PubMed
    1. An W, Cho S, Ishii H, Wensink CP. Sex-specific and non-sex-specific oligomerization domains in both of the doublesex transcription factors from Drosophila melanogaster. Mol Cell Biol. 1996;16(6):3106–3111. - PMC - PubMed
    1. Baker SB, Wolfner FM. A molecular analysis of doublesex, a bifunctional gene that controls both male and female sexual differentiation in Drosophila melanogaster. Genes Dev. 1988;2(4):477–489. doi: 10.1101/gad.2.4.477. - DOI - PubMed

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