Dynamic, Non-binary Specification of Sexual State in the C. elegans Nervous System

Abstract

Biological sex in animals is often considered a fixed, individual-level characteristic. However, not all sex-specific features are static: for example, C. elegans males (XO) can sometimes exhibit hermaphrodite (XX)-like feeding behavior [1, 2]. (C. elegans hermaphrodites are somatic females that transiently produce self-sperm.) Essentially all somatic sex differences in C. elegans are governed by the master regulator tra-1, whose activity is controlled by chromosomal sex and is necessary and sufficient to specify the hermaphrodite state [3]. One aspect of this state is high expression of the chemoreceptor odr-10. In hermaphrodites, high odr-10 expression promotes feeding, but in males, low odr-10 expression facilitates exploration [4]. However, males suppress this sex difference in two contexts: juvenile males exhibit high odr-10 expression and food deprivation activates odr-10 in adult males [4-6]. Remarkably, we find that both of these phenomena require tra-1. In juvenile (L3) males, tra-1 is expressed in numerous neurons; this expression diminishes as individuals mature into adulthood, a process that requires conserved regulators of sexual maturation. tra-1 remains expressed in a small number of neurons in adult males, where it likely has a permissive role in odr-10 activation. Thus, the neuronal functions of tra-1 are not limited to hermaphrodites; rather, tra-1 also acts in the male nervous system to transiently suppress a sexual dimorphism, developmentally and in response to nutritional stress. Our results show that the molecular and functional representation of sexual state in C. elegans is neither static nor homogeneous, challenging traditional notions about the nature of biological sex.

Keywords: biological sex; chemosensation; developmental timing; sex determination; sex difference; sexual differentiation; sexual dimorphism; sexual maturation.

Figure 1.. tra-1 is dynamically expressed in the C. elegans male nervous system and is regulated by the heterochronic genes lep-2 and let-7.

(A) The C. elegans somatic sex determination pathway. Red indicates genes typically active in hermaphrodites (XX); blue, males (XO). X and A indicate the X chromosome and the autosomes, respectively. Dots indicate signal element genes. (B, C) Merged and individual images of GFP::TRA-1 and pan-neural nRFP fluorescence in wild-type L3, L4, and young adult hermaphrodites (B) and males (C). In the merged images, GFP::TRA-1 is pseudocolored blue and nRFP is pseudocolored red. (D) Number of neurons with detectable GFP::TRA-1 fluorescence in L3, L4, and young adult males. (E) Number of ciliated sensory neurons with detectable GFP::TRA-1 fluorescence in L3, L4, and young adult males. (F) Number of neurons with detectable GFP::TRA-1 fluorescence in wild-type and lep-2 young adult males. (G) Number of neurons with detectable GFP::TRA-1 fluorescence in wild-type and lin-28 L3 males. (H) Number of neurons with detectable GFP::TRA-1 fluorescence in wild-type and let-7 young adult males. In each bar graph, numbers above each bar indicate numbers of animals scored. *0.01 < p < 0.05; **0.001 < p < 0.01; ***p < 0.001. Dotted gray brackets indicate p > 0.05. See also Figure S1.

Figure 2.. tra-1 functions in the L3 nervous system to promote odr-10 expression.

(A) L3 and adult hermaphrodites, as well as L3 males, express high levels of odr-10 (represented by green head neuron), while adult males express low levels (light green neuron). (B) ODR-10::GFP expression in L3, L4, and adult hermaphrodites, scored qualitatively on a four-point scale (see STAR Methods). (C) ODR-10::GFP expression in L3, L4, and young adult WT XO males and tra-1 XX pseudomales. (D) ODR-10::GFP expression in young adult WT XO males, lep-2 XO males, tra-1 XX pseudomales, and tra-1; lep-2 XX pseudomales. (E) ODR-10::GFP expression in L3, L4, and young adult WT XO males and fsIs15[Prab-3::fem-3] XO males. (F) Psrj-54::YFP expression in L3, L4, and young adult WT XO males and tra-1 XX pseudomales. (G) Merged images showing GFP::TRA-1 (blue) and Pgpa-14Δ6::mCherry (red) fluorescence in wild-type L3 hermaphrodites (upper) and males (lower). The position of the AWA neuron is indicated. (H) ODR-10::GFP expression in L3 XO males, XX pseudomales, and XX pseudomales bearing a Prab-3::tra-1a transgene. (I) ODR-10::GFP expression in L3 XO males, XX pseudomales, and XX pseudomales bearing a Posm-5::tra-1a transgene. In each bar graph, numbers above each bar indicate numbers of animals scored. *0.01 < p < 0.05; **0.001 < p < 0.01; ***p < 0.001. Dotted gray brackets indicate p > 0.05.

Figure 3.. tra-1 is required for the starvation-induced feminization of odr-10 expression in adult males.

(A) ODR-10::GFP expression in fed and starved (stv) WT XO males and tra-1 XX pseudomales. (B) ODR-10::GFP expression in fed and starved WT XO males and Prab-3::fem-3 hyper-masculinized XO males. (C) ODR-10::GFP expression in fed and starved WT XO males and Posm-5::fem-3 XO males. (D) ODR-10::GFP expression in fed and starved (stv) WT XO males and Pgpa-4Δ6::fem-3 (AWA::fem-3) XO males. (E) ODR-10::GFP expression in fed and starved (stv) WT XO males and Ptax-4::fem-3 (results from two independent lines are shown). In each bar graph, numbers above each bar indicate numbers of animals scored. *0.01 < p < 0.05; **0.001 < p < 0.01; ***p < 0.001. Dotted gray brackets indicate p > 0.05. See also Figure S2.

Figure 4.. tra-1 likely acts permissively in the activation of odr-10 expression in starved males.

(A) Number of neurons with detectable GFP::TRA-1 fluorescence in fed and starved (stv) WT XO adult males. (B) ODR-10::GFP expression in well-fed WT, daf-2, fsIs15[Prab-3::fem-3], and daf-2; fsIs15[Prab-3::fem-3] adult XO males. (C) ODR-10::GFP expression in well-fed WT, daf-7, fsIs15[Prab-3::fem-3], and daf-7; fsIs15[Prab-3::fem-3] adult XO males. (D) Pdaf-7::GFP fluorescence in the ASJ neuron in fed and starved WT and fsIs15[Prab-3::fem-3] males. (E) Pdaf-7::GFP fluorescence in the ASJ neuron in fed and starved tra-1 XX pseudomales. (F) Schematic diagram of dynamic, heterogeneous expression and function of tra-1 in the male nervous system. Red ovals indicate neurons expressing tra-1, blue ovals indicate neurons without detectable tra-1 expression. For simplicity, the number of neurons shown is reduced and is not meant to depict precise cell counts. In each bar graph, numbers above each bar indicate numbers of animals scored. *0.01 < p < 0.05; **0.001 < p < 0.01; ***p < 0.001. Dotted gray brackets indicate p > 0.05.