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The transformer gene in Ceratitis capitata provides a genetic basis for selecting and remembering the sexual fate

Dipartimento di Genetica, Biologia Generale e Molecolare, Università degli Studi di Napoli ‘Federico II’, Via Mezzocannone 8, 80134 Napoli, Italy

View larger version (58K): [in a new window]   Fig. 1. Analyses of Cctra transcripts. (A) Northern blot analysis on poly A+ RNA from embryos (E), larvae (L), and adult males (M) and females (F), using as probe the F1 cDNA clone. In males, two predominant transcripts 1.9 kb and 2 kb long are detected, while two different transcripts 1.6 kb and 3 kb long are present in females. In embryos, two transcripts are detected and in larvae all four transcripts are detected. (B) RT-PCR amplification of Cctra on adult males (M) and females (F) total mRNA samples. Three main products are present in the female lane (F), which are 0.7 kb, 1.3 kb and 2.1 kb long. In the male lane (M) four bands are detectable which are 1 kb, 1.1 kb, 1.3 kb and 2.1 kb long. Male (cM) and female (cF) RT-PCR negative controls (reactions without reverse transcriptase) are shown.

View larger version (15K): [in a new window]   Fig. 2. Genomic organization of the Ceratitis capitata tra gene. (A) The top line represents the genomic DNA encompassing the Cctra locus. The positions of exons in the Cctra mRNAs are shown above the line, with Ex1, Ex2 and Ex3 representing exons in common between the male and the female mRNAs, the blue boxes representing male-specific exons, the yellow box indicating a male-specific exon in the M1 mRNA, and the red box representing a male-specific exon included in the M2 mRNA. Numbered green ovals indicate TRA/TRA-2-binding sites (see B). Introns are represented by solid lines. Open boxes represent the ORF of the female-specific 1.6 kb long mRNA (Female F1) encoding the putative 429 amino acid TRA protein (see Fig. 3). Gray boxes indicate 5' and 3' untranslated regions. Arrows above the first line represent the positions of the oligonucleotides used in the RT-PCR experiments. The bar indicates the scale of the figure. (B) Sequence alignment of eight putative TRA/TRA-2 binding sites found in the Cctra genomic sequence (see A). Conserved positions between Ceratitis and Drosophila are indicated in bold.

View larger version (62K): [in a new window]   Fig. 3. Multiple sequence alignment of TRA proteins. Ceratitis capitata (Cc), D. melanogaster (Dm), D. erecta (De), D. simulans (Ds), D. virilis (Dv) and D. hydei (Dh). Asterisks indicate amino acid identity in all species. Intron/exon boundaries are indicated by vertical arrows. Amino acid residues occurring in the conserved regions are indicated by capital letters.

View larger version (71K): [in a new window]   Fig. 4. Phenotypic analysis of RNAi intersexes. (A) Wild-type female has long pigmented bristles on the femur pointing towards the coxa of the foreleg (arrow in E) and the ovopositor (A,I). (B) Wild-type male exhibits two spatulated bristles on the head (B), a row of non-pigmented bristles on the ventral part of the femur towards the coxa of the foreleg, short pigmented bristles grouped on the dorsal part of the femur (arrow in F) close to the coxa of the foreleg (F) and male genitalia (B,O). (C,D) Intersexes obtained by dsRNA injection into the anterior pole of the embryos exhibit male-specific spatulated bristles on the head (arrow in C), male-specific bristles (upper arrow in H) and female-specific bristles (arrow in G; lower arrow in H) mixed together on the femur of the foreleg (G,H) and female genitalia (C,D). Some intersexes show various degrees of abnormal gonadal development exhibiting bent (arrow in D), deformed (L-N) or completely absent (arrow in P) genitalia. Scale bar in D applies at A-D; scale bar in H applies to E-H; scale bar in I applies to I; scale bar in L applies to L; and scale bar in P applies to M-P.

View larger version (70K): [in a new window]   Fig. 5. Karyotypic analysis of RNAi treated individuals. (A) PCR with Y-specific oligonucleotides carried on medfly genomic DNA. From lane 1 to 10, PCR on single males developed from dsRNA-injected embryos; lanes 11 and 12, PCR on single wild-type females; lanes 13 and 14, PCR on single wild-type males. PCR on flies of mixed sexes and a negative control are shown, respectively, in lanes 15 and 16. The PCR amplification patterns in lanes 1,2,6 and 7 correspond to those of wild-type males, indicating that the analysed adults have an XY karyotype. By contrast, no bands are detected in lanes 3-5,8-10 indicating that these males lack a Y chromosome and therefore are XX sexually transformed males. (B) Positive PCR control with Cctra specific primers (Cctra164+ and Cctra481–) showing that medfly genomic DNA is present in all samples. Lane M (A,B) presents the molecular weight marker.

View larger version (40K): [in a new window]   Fig. 6. Analysis of Cctra and Ccdsx splicing patterns in adult individuals. (A) RT-PCR with Cctra specific primers Cctra164+ and Cctra900– on XY and XX males from dsRNA-injected embryos (lanes 1 and 2) and on wild-type males (lane 3) and females (lane 4). Lanes c1-c4 show RT-PCR negative controls. The dsRNA injection in XX embryos induces a permanent shift in the splicing pattern of Cctra that turns from a female to a male mode. (B) RT-PCR with Ccdsx-specific primers (Ccdsx1400+, Ccdsx1130– and Ccdsx2000–) on the same cDNA samples used in A. The 0.6 kb fragment corresponds to a region of Ccdsx female-specific transcript, while the 0.3 kb fragment represents a region of Ccdsx male-specific transcript. A consequence of the Cctra-specific RNAi is a persistent change in Ccdsx regulation that turns from a female-specific to a male-specific splicing mode. A molecular weight marker is also shown in lane M (A,B).

View larger version (16K): [in a new window]   Fig. 7. Model for sex determination in Ceratitis capitata. (A) In XX embryos, a maternal Cctra mRNA provides full-length CcTRA protein that initiates a positive feedback regulation. This protein drives a female-specific splicing of the zygotically transcribed Cctra pre-mRNA so that new CcTRA protein can be produced. The newly synthesized protein controls the maintenance of Cctra autoregulation and the female-specific splicing of Ccdsx pre-mRNA. Therefore a CcDSXF protein is produced that induces, at least in part, female development. (B) In XY embryos, Cctra autoregulation is impaired by the male determining M factor. The M factor could prevent the translation of the maternal Cctra transcript (1) or inhibit the function of the protein that is produced by this mRNA (2). It is also conceivable that the M could interact with the spliceosome or repress Cctra transcription initiation in the zygote (3). In any case, the result is always that a full-length CcTRA protein is not produced in XY embryos and, thus, the autoregulatory loop can not initiate. In absence of CcTRA protein, Ccdsx is expressed by default to produce the CcDSXM isoform, which induces, in turn, male development.