The RED domain of Paired is specifically required for Drosophila accessory gland maturation

Abstract

The evolutionarily conserved paired domain consists of the N-terminal PAI and the C-terminal RED domains, each containing a helix-turn-helix motif capable of binding DNA. Despite its conserved sequence, the physiological functions of the RED domain remain elusive. Here, we constructed a prd transgene expressing a truncated Paired (Prd) protein without the RED domain, and examined its rescue ability in prd mutants. We found that the RED domain is specifically required for the expression of Acp26Aa and sex peptide in male accessory glands, and the induction of female post-mating response. Our data thus identified an important physiological function for the evolutionarily conserved RED domain.

Keywords: Drosophila; Paired; RED domain; accessory gland; post-mating response.

Figure 1.

The RED domain is dispensable for the embryonic functions of Prd. (a) Schematic of the coding structure of prd-Prd and prd-PrdΔPBC, and their ability to rescue Prd functions in Drosophila development. The rescue ability is scored by + if the transgene is sufficient for rescue or by − if no rescue is obtained. (b–d) The cuticle of a prd^+/− (b), or a prd^−/− (c) or a prd^−/−; prd-PrdΔPBC/+ (d) embryo is shown under dark-field illumination with anterior up. prd-PrdΔPBC is able to rescue the cuticle phenotype in prd⁻ (d). Expression patterns of Gsb (e–g), Wg (h–j) and En (k–m) in prd^+/− (e,h,k), prd^−/−embryo carrying no (f,i,l) or one copy of prd-PrdΔPBC (g,j,m) are shown during the extended germ band stage of embryogenesis. Embryos are oriented with their anterior to the left and dorsal side up. Expression of Gsb, Wg and En in prd mutants (f,i,l) is fully rescued by prd-PrdΔPBC (g,j,m).

Figure 2.

The RED domain is dispensable for the viability and male fertility functions of Prd. (a) prd-PrdΔPBC is able to rescue prd mutants to adulthood at a similar rate as prd-Prd or the endogenous prd. Viability is scored by the actual number over the expected number of (i) prd^+/−, (ii) prd^−/−+prd-PrdΔPBC or (iii) prd^−/− + prd-Prd from corresponding crosses. There is no significant difference in the viability among prd^+/− (92/102 expected), prd^−/−; prd-Prd/+ (72/64 expected) and prd^−/−; prd-PrdΔPBC/+ (31/71 expected) flies. (b) prd-PrdΔPBC rescues the fertility of prd mutant males to a similar extent as prd-Prd, but slightly lower than that of endogenous prd. Number of flies examined (fertile/sterile): prd^+/− (n = 24; 24/0), prd^−/−; prd-Prd/+ (n = 44; 35/9) and prd^−/−; prd-PrdΔPBC/+ (n = 22; 15/7). (c,d) prd mutant males rescued by prd-PrdΔPBC have a reduced fecundity when compared with those rescued by prd-Prd or the heterozygous controls. The number of progenies produced per vial per day (c) and the total number of progenies for 10 days (d) of prd^+/− (n = 27), prd^−/−; prd-Prd/+ (n = 24) and prd^−/−; prd-PrdΔPBC/+ (n = 17) are shown as mean ± s.e.m. For statistical analysis: *p < 0.05; **p < 0.01; ***p < 0.001; n.s., not significant. (e–h) Light micrographs showing AG from a 3-day-old prd^+/− (e), prd^−/−; prd-Res/+ (f), prd^−/−; prd-Prd/+ (g) or prd^−/−; prd-PrdΔPBC/+ (h) male. The loss of AG phenotype in prd mutant males (f) was rescued by prd-Prd (g) or prd-PrdΔPBC (h).

Figure 3.

The RED domain is imperative for female post-mating response (PMR). (a,b) Females mated with prd^−/−; prd-PrdΔPBC/+ males exhibit a virgin-like behaviour with a high receptivity (a) and low rejection (b) to second mating, whereas those that copulated with the heterozygous controls or prd^−/−; prd-Prd/+ males exhibit an opposite behaviour with a low receptivity (a) and high rejection (b). (a) The percentage of virgin females (17/20) mated to naive w¹¹¹⁸ males within 1 h or the percentage of non-virgin females previously mated to prd^+/−(2/20), prd^−/−; prd-PrdΔPBC/+ (19/20) or prd^−/−; prd-Prd/+ (3/20) males successfully re-mating within 1 h. (b) The percentage of virgin females (2/20) not mated within 2 h or the percentage of non-virgin females previously mated to prd^+/−(16/20), prd^−/−; prd-PrdΔPBC/+ (1/20) or prd^−/−; prd-Prd/+ (14/20) males not re-mated within 2 h. For statistical analysis: *p < 0.05; **p < 0.01; n.s., not significant. (c) Egg-laying of virgin females or females mated to (i) prd^+/−, (ii) prd^−/−; prd-Prd/+ or (iii) prd^−/−; prd-PrdΔPBC/+ males (n = 20, respectively). Females mated with heterozygous controls or prd^−/−; prd-Prd/+ show dramatic increase in egg-laying that lasts for a few days with a gradual reduction. However, females mated with prd^−/−; prd-PrdΔPBC/+ males fail to trigger the increased oviposition and lay few eggs per day, as do virgin females. The statistical analysis of egg laying using one-way ANOVA followed by Bonferroni's multiple comparison test is shown on the right. Significant differences are indicated as *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 4.

The RED domain is required for the expression of Acp26Aa and SP in the AG. The expression of Acp26Aa (a–c) and SP (d–f) in AG from a 3-day-old prd^+/− (a or d), prd^−/−; prd-PrdΔPBC/+ (b or e) or prd^−/−; prd-Prd/+ (c or f) male. The expression of Acp26Aa (b) or SP (e) in prd mutant males rescued by prd-PrdΔPBC was undetectable when compared with controls (a,d) or prd mutants rescued by prd-Prd (c,f). (g) qRT-PCR assay showing the transcription level of prd, acp26A and SP in AG of indicated genotypes (n = 10 per genotype). Significant differences are indicated as *p < 0.05; *** p < 0.001, n.s., not significant.