Multiple doublesex-Related Genes Specify Critical Cell Fates in a C. elegans Male Neural Circuit

In most animal species, males and females exhibit differences in behavior and morphology that relate to their respective roles in reproduction. DM (Doublesex/MAB-3) domain transcription factors are phylogenetically conserved regulators of sexual development. They are thought to establish sexual traits by sex-specifically modifying the activity of general developmental programs. However, there are few examples where the details of these interactions are known, particularly in the nervous system.In this study, we show that two C. elegans DM domain genes, dmd-3 and mab-23, regulate sensory and muscle cell development in a male neural circuit required for mating. Using genetic approaches, we show that in the circuit sensory neurons, dmd-3 and mab-23 establish the correct pattern of dopaminergic (DA) and cholinergic (ACh) fate. We find that the ETS-domain transcription factor gene ast-1, a non-sex-specific, phylogenetically conserved activator of dopamine biosynthesis gene transcription, is broadly expressed in the circuit sensory neuron population. However, dmd-3 and mab-23 repress its activity in most cells, promoting ACh fate instead. A subset of neurons, preferentially exposed to a TGF-beta ligand, escape this repression because signal transduction pathway activity in these cells blocks dmd-3/mab-23 function, allowing DA fate to be established. Through optogenetic and pharmacological approaches, we show that the sensory and muscle cell characteristics controlled by dmd-3 and mab-23 are crucial for circuit function.In the C. elegans male, DM domain genes dmd-3 and mab-23 regulate expression of cell sub-type characteristics that are critical for mating success. In particular, these factors limit the number of DA neurons in the male nervous system by sex-specifically regulating a phylogenetically conserved dopamine biosynthesis gene transcription factor. Homologous interactions between vertebrate counterparts could regulate sex differences in neuron sub-type populations in the brain

The <i>C. elegans</i> Male Exercises Directional Control during Mating through Cholinergic Regulation of Sex-Shared Command Interneurons

<div><p>Background</p><p>Mating behaviors in simple invertebrate model organisms represent tractable paradigms for understanding the neural bases of sex-specific behaviors, decision-making and sensorimotor integration. However, there are few examples where such neural circuits have been defined at high resolution or interrogated.</p><p>Methodology/Principal Findings</p><p>Here we exploit the simplicity of the nematode <i>Caenorhabditis elegans</i> to define the neural circuits underlying the male’s decision to initiate mating in response to contact with a mate. Mate contact is sensed by male-specific sensilla of the tail, the rays, which subsequently induce and guide a contact-based search of the hermaphrodite’s surface for the vulva (the vulva search). Atypically, search locomotion has a backward directional bias so its implementation requires overcoming an intrinsic bias for forward movement, set by activity of the sex-shared locomotory system. Using optogenetics, cell-specific ablation- and mutant behavioral analyses, we show that the male makes this shift by manipulating the activity of command cells within this sex-shared locomotory system. The rays control the command interneurons through the male-specific, decision-making interneuron PVY and its auxiliary cell PVX. Unlike many sex-shared pathways, PVY/PVX regulate the command cells via cholinergic, rather than glutamatergic transmission, a feature that likely contributes to response specificity and coordinates directional movement with other cholinergic-dependent motor behaviors of the mating sequence. PVY/PVX preferentially activate the backward, and not forward, command cells because of a bias in synaptic inputs and the distribution of key cholinergic receptors (encoded by the genes <i>acr-18, acr-16</i> and <i>unc-29</i>) in favor of the backward command cells.</p><p>Conclusion/Significance</p><p>Our interrogation of male neural circuits reveals that a sex-specific response to the opposite sex is conferred by a male-specific pathway that renders subordinate, sex-shared motor programs responsive to mate cues. Circuit modifications of these types may make prominent contributions to natural variations in behavior that ultimately bring about speciation.</p></div

A circuit model for mate contact-induced backward locomotion in the male.

<p>A model for how mate contact alters the activity of the sex-shared locomotory system to induce directional change. Shown are the activity states of circuit components in the absence <i>(A)</i> or the presence of mate contact <i>(B),</i> with the corresponding male behavior depicted below. Cell type and sex-specificity is indicated by the symbol and color, respectively: sex-shared cells (pink), male-specific cells (blue), sensory neurons (triangles), interneurons (hexagons), motor neurons (circles). Color intensity indicates a cell activity state (intense color = high; weak color = low). The arrows indicate the positive action of an activated cell on its postsynaptic target. <b><i>A.</i></b> When not engaged in mating, the male moves with a forward directional bias due to high levels of activity in the forward pathway of the sex-shared locomotory system: forward command interneurons AVB and their motor neuron targets (FWD mns). This activity bias is conferred by the default state of this system <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060597#pone.0060597-Kawano1" target="_blank">[21]</a>, in the absence of specific cues, or by external or internal sensory cues that drive the male to explore his environment. <b><i>B.</i></b> Ray neuron stimulation by mate contact activates PVY and PVX, which release acetylcholine (ACh), preferentially stimulating the backward (AVA), and not forward, command interneurons. This is due to a bias in synaptic inputs and in cholinergic receptor expression (ACR-18, ACR-16 and UNC-29) in favor of the backward command cells. AVA activation in turn stimulates the sex-shared backward motor neurons (BK mns). The rays may also promote backward movement through an as yet uncharacterized PVY/PVX-independent pathway (represented as interneuron “X”).</p

Male movement during mating depends on functionally redundant cholinergic receptors.

<p><b><i>A–C.</i></b> The impact of cholinergic receptor mutations on three aspects of vulva search locomotion. Also see legend for Fig. 2. The X-axis indicates the genetic background examined. All strains carrying <i>unc-29</i> mutations also have the <i>rgIs1</i> array which rescues <i>unc-29</i> function in body wall muscles. <i>WT</i> (wild type) are <i>him-5</i> males. n: <i>WT</i> = 48, <i>acr-18</i> = 12, <i>unc-29; acr-16</i> = 14, <i>unc-29; acr-16 acr-18</i> = 12. Significance, *p<0.05; **p<0.005.</p

Male movement during mating depends on sex-shared and male-specific interneurons.

<p><b><i>A.</i></b> Schematic of an adult male (lateral view, left side) showing the anatomical location of cells ablated in experiments shown in <i>B–D</i> and in Fig. 3. Sex-shared cells (pink); male-specific cells (blue). The backward command interneurons (only the left AVA neuron is shown) have cell bodies in the head and send a process along the ventral nerve cord (VNC) where they synapse with motor neurons required for locomotion (the distribution of cell bodies for only a single motor neuron class is shown). PVY and PVX (located in the male pre-anal ganglion) receive inputs from the ray sensory neurons and have outputs onto the command interneuron processes. <b><i>B–D.</i></b> The impact of cell-specific ablations on three aspects of vulva search behavior related to locomotory control. <b><i>B</i></b><b>.</b> Contact response efficiency. <b><i>C.</i></b> Scanning speed. <b><i>D.</i></b> The number of times tail contact was lost per mating. The X-axis indicates the cells ablated in each treatment. A box plot representation of the data is shown, with median and mean values indicated by the line and the black dot within the box, respectively. Comparisons to the control (mock-ablated) were made using a ranksum test. The number of males assayed for each treatment, n: Control = 63; -PVY-PVX = 15; -PVY = 19; -PVX = 8; -AVA = 22. Significance, *p<0.05; **p<0.005.</p

Male mating behavior is characterized by distinct patterns of locomotion.

<p>A cartoon depicting the key changes observed in male movement and body posture that are triggered by mate contact. <b><i>A.</i></b> In the absence of mate contact, male locomotion resembles that of the hermaphrodite: the male moves with a forward locomotion bias and the sinusoidal body wave driving movement propagates along the full length of the body. <b><i>B.</i></b> Contact with a mate via the male tail induces contact response: the male presses his tail against the hermaphrodite surface and commences backward movement. <b><i>C.</i></b> Backward locomotion continues until the vulva is sensed, whereupon the male pauses and prods for the vulva slit opening with his copulatory spicules. The male sensory rays (shown in the inset for <i>B</i>), which sense hermaphrodite contact, are essential for the induction and maintenance of tail apposition and for directional control on the hermaphrodite surface.</p

Optogenetic manipulation of PVY and PVX activity affects male movement.

<p><b><i>A, B.</i></b> PVY and PVX artificial stimulation, using ChR2, induces backward locomotion that is male-specific and depends on sex-shared locomotory system cells. The graphs show the impact of ChR2 activation on <i>Pnlp-14::ChR2-YFP</i> transgenic animal locomotion. Except for H (hermaphrodites) in <i>(A)</i>, all animals tested were transgenic males (in <i>(A),</i> male treatments are designated “M”). Except for the “-ATR M” control treatment in <i>(A)</i>, all animals were cultured and assayed in the presence of OP50 <i>E.coli</i> food supplemented with ATR. The X-axis indicates the food supplementation conditions (+ATR or –ATR), animal sex or which cells were ablated. The controls in <i>(B)</i> correspond to mock-ablated animals. The Y-axis shows the distance traveled (in µm) in response to the flash, with the negative values indicating backward (BK) movement and the positive values indicating forward (FWD) movement (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060597#s4" target="_blank">Materials and Methods</a>). Statistical comparisons to the relevant controls were made using a ranksum test for differences in the median. Tabled below each graph is the percentage of animals in each treatment that backed, paused or continued to move forward (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060597#s4" target="_blank">Materials and Methods</a> for the µm range of each category). <i>n</i> is the number of worms assayed in each treatment. <b><i>C</i></b><b>.</b> Artificial hyperpolarization of PVY and PVX blocks backing in the context of mating. Shown is the pausing frequency of <i>Pnlp-14::NpHR-EYFP</i> males in response to yellow light flashes. The X-axis shows the food supplementation and mating (Mating or Not Mating) conditions used. The Y-axis indicates the percentage of light flashes (out of 5) that induced pausing. <i>n</i>: -ATR Mating, 26; +ATR Mating, 28; -ATR Not Mating, 10; +ATR Not Mating, 10. Comparisons between –ATR and +ATR treatments were made using a ranksum test. Significance, *p<0.05; **p<0.005.</p