Primo, Pasquale (2021) .Functional evolutionary studies of the sex-determining pathway of Ceratitis capitata: MoY ectopic expression and CRISPR/Cas9 targeting of Cctra and Ccdsx genes lead to sex reversals. [Tesi di dottorato]

Preview

Text Primo_Pasquale_33.pdf Download (11MB) | Preview

Collection description

The Ceratitis capitata, also known as the Mediterranean fruit fly or Medfly, is one of the most dangerous agricultural pest insects considering its ability to parasite hundreds of fruits, nuts and crops. Ceratitis capitata and Drosophila melanogaster share a relatively recent common ancestry of about 120 million years ago. Based on this phylogenetic relationship, Ceratitis and Drosophila preserved segments of their sex respective determination pathways. In Drosophila, the sex determination pathway starts from a primary signal based on the number of X chromosomes. The presence of two chromosomes allows the functional and female-specific splicing of the master gene regulator of sex determination Sex-lethal (Sxl). Sxl is a splicing factor that drives the female-specific splicing of the downstream gene transformer. The transformer gene encodes for an SR-rich protein that regulates the female-specific splicing of the doublesex gene, a transcriptional factor involved in the transcription of the somatic female differentiation genes and the repression of the male-specific ones. Whilst, in the case of just one X chromosome, Sxl male-specific and tra non-sex-specific mRNAs contain truncated, non-functional, open reading frames, and doublesex produces by default a male-specific protein isoform promoting male somatic differentiation. In brief, the Drosophila sex determination pathway is based on the hierarchy Sxl>tra>dsx. Drosophila, also in Ceratitis female, a functional female-specific tra (Cctra), drives doublesex female-specific splicing. Differently from Drosophila, Cctra can autoregulate in XX embryos until adulthood,promoted by maternal mRNA, and maintained by a positive feedback loop its activation (Pane et al., 2002), with the help of the auxiliary factor Cctra-2 (Salvemini et al., 2009). In males bearing the Y chromosome, tra mRNAs contain truncated open reading frames leading to non-functional peptides, and doublesex produces by default a male-specific isoform. However, a different primary signal is present in this species, based on a male-specific Y-linked dominant signal, M, repressing Cctra in XY, which in the master gene regulator is a transformer (Cctra). Recently, we have identified the primary Y-linked dominant signal necessary for male differentiation: Maleness-on-the-Y (MoY) (Meccariello et al., 2019). MoY encodes for a 70aa long protein, showing no apparent relationship with other Tephritidae. I have shown that injections of recombinant MOY in medfly XX embryos have partial masculinisation activity at the phenotypic and molecular level. We have also shown that transient RNAi targeting MoY confirms that MoY has a crucial role in maleness determination and that XY females are surprisingly fertile. During my PhD thesis, I have extended these studies and showed that embryos injections of Bactrocera dorsalis and Bactrocera oleae MOY recombinant proteins in XX-only embryos led to a molecular Cctra male-specific splicing but no to a significative phenotype alteration. Considering the efficacy of the CRISPR/Cas9 approach by Cas9 protein complexes embryos injections in the Medfly (Meccariello et al., 2017; idem, 2019), I have applied this technique targeted the Cctransformer and, more recently, the Ccdoublesex genes. Interestingly, I have found that Cctra Cas9 targeting led to a complete female to male sex reversal even in the absence of changes in the DNA sequence due to an unexpected CRISPR/Cas9 interference (Primo et al., 2020). Furthermore, I have found that Ccdsx Cas9 targeting (common region present in both sex-specific isoforms) led either to masculinisation of XX individuals or feminisation of XY individuals rather than an expected intersexual phenotype. We have discovered that Ccdsx sex-specific isoforms seem to have regulatory feedbacks on the upstream Cctra gene, to either CcdsxM to stabilise male-specific Cctra splicing or CcdsxF to stabilise Cctra female-specific splicing. We asked if these novel dsx feedback tra are evolutionarily conserved in Drosophila melanogaster, and we have found preliminary evidence supporting this model. I have achieved the fully female-to-male reversion even without a high rate of indels introduction. In the end, I have targeted the Ccdoublesex gene showing that its disruption affects both doublesex splicing and transformer splicing, its upstream regulator.

Downloads

Actions (login required)

View Item