Functional Dissection of Protein Kinases in Sexual Development and Female Receptivity of Drosophila

Abstract

Protein phosphorylation is crucial for a variety of biological functions, but how it is involved in sexual development and behavior is rarely known. In this study, we performed a screen of RNA interference targeting 177 protein kinases in Drosophila and identified 13 kinases involved in sexual development in one or both sexes. We further identified that PKA and CASK promote female sexual behavior while not affecting female differentiation. Knocking down PKA or CASK in about five pairs of pC1 neurons in the central brain affects the fine projection but not cell number of these pC1 neurons and reduces virgin female receptivity. We also found that PKA and CASK signaling is required acutely during adulthood to promote female sexual behavior. These results reveal candidate kinases required for sexual development and behaviors and provide insights into how kinases would regulate neuronal development and physiology to fine tune the robustness of sexual behaviors.

Keywords: Akt; CASK; Drosophila; PKA; female receptivity; protein kinase; sexual dimorphism.

FIGURE 1

Specific kinases are required for sexual development and female receptivity. (A) Statistical results of screening for developmental defects of sex traits. The number in the colored graph represents the number of different RNAi lines targeting protein kinases. (B) Protein kinases were divided into two categories, one crucial for somatic sexual development, and the other not required for female differentiation, which were further assayed for virgin female receptivity. (C) A summary of protein kinases that are required for somatic sexual development. (D,E) Knockdown of Akt in dsx-expressing cells led to developmental defects in external genitalia, abdominal cuticular pigmentation (scale bar, 0.2 mm) and sex comb (scale bar, 0.1 mm), as indicated by arrowheads. (F) Statistical results of screening for unreceptive females. The number in the colored graph represents the number of different RNAi lines targeting protein kinases. (G) Knockdown of 17 protein kinases in dsx-expressing cells severely reduced female receptivity. The number at the top of the bar indicates the number of tested flies. The genotype of the control group was dsx^GAL4/+; and other genotypes were abbreviations of specific kinase RNAi driven by dsx^GAL4 .

FIGURE 2

Identification of protein kinases that are required in dsx^brain neurons for virgin female receptivity. (A) Expression pattern of dsx^brain in female CNS. Scale bar, 100 μm. (B) Compared to the control, female receptivity was significantly decreased with specific kinases knocked down in dsx^brain neurons. The number at the top of the bar indicates the number of tested flies. The control genotype (dsx^brain/+) is Otd-Flp/+; dsx^GAL4,tub > GAL80>/+, and other genotypes were abbreviations of specific kinase RNAi driven by dsx^brain . (C) Copulation rates were significantly decreased in virgin females with PKA, CASK or msn knocked down in dsx^brain neurons compared with control females. *p < 0.05 and ***p < 0.001 at 10 min time point, Chi-square test. The number in parentheses indicates the number of tested virgin females paired with wild-type males.

FIGURE 3

PKA and CASK signaling in pC1 neurons is essential for virgin female receptivity. (A) Expression pattern of pC1-SS2 in female CNS. Scale bar, 100 μm. (B) Compared to control females, copulation rates were significantly decreased in virgin females with PKA or CASK knocked down in pC1 neurons, whereas copulation rate was not significantly different in msn knockdown females. ns, not significant; ***p < 0.001 at 10 min time point, Chi-square test. (C) Knocking down PKA or CASK in 4–6 days old virgin females for 2 days significantly reduced female receptivity. ***p < 0.001 at 10 min time point, Chi-square test. (D) Diagram of the PKA and CASK signaling pathway. (E) Knockdown of other positive regulators, rather than the negative regulator (Dnc), of PKA and CASK signaling pathway significantly decreased virgin female receptivity. ns, not significant; ***p < 0.001 at 10 min time point. **p < 0.01 at 30 min time point, Chi-square test. The number in parentheses indicates the number of tested virgin females paired with wild-type males.

FIGURE 4

PKA and CASK regulate fine neuronal projection of pC1 neurons. (A–C) Morphology of pC1 neurons of different genotypes. Compared with the control (A), pC1 neurons with PKA knockdown (B) or CASK knockdown (C) exhibited unchanged number of pC1 cell bodies (dashed circles), loss of the vertical projection (arrows in the left panels, red rectangles in the right panels), and increase of the nerve endings in SMP region (arrowheads in the left panels, red circles in the right panels). Scale bars, 50 μm. (D) pC1 cell numbers per hemisphere were not affected by knocking down PKA or CASK. n = 18, 18 and 16, respectively from left to right. ns, not significant, Mann-Whitney U test. Error bars indicate SEM. (E) PKA or CASK knockdown resulted in significant projection defects of pC1 neurons. Type A: regular morphology of pC1 neurons; type B: loss of the vertical projection and increase of the nerve endings in SMP region; type C: weak/unilateral vertical projection and increase of the nerve endings in SMP region. n = 9, 9 and 8, respectively from left to right.

FIGURE 5

A summary of the functions of protein kinases in sexual development and behavior. (A) Protein kinases such as Akt and Prpk are crucial in Dsx-expressing cells for sexual differentiation. (B) PKA and CASK regulate both fine neuronal projection of pC1 neurons and their physiology to promote virgin female receptivity.