Worm sex and the art of pruning

Sexually reproducing animals generally come in two sexes that can differ dramatically in anatomy and behavior to facilitate mating and reproduction. Interestingly, individuals of each sex are often born with the instincts and ability to mate with the opposite sex. These sex differences in behavior are due to sex differences in the nervous system. How do the neural circuits that control sexually dimorphic behaviors differ, and what mechanisms underlie the generation of those differences?

The nematode worm, C. elegans, is an excellent organism to study these questions. These small worms have a quick generation time and their biology is relatively well understood. On top of that, C. elegans worms occur in two sexes: a male and a hermaphrodite. Hermaphrodite C. elegans are worms with both male and female tissue that can produce sperm, self-fertilize and lay eggs.

Male and hermaphrodite worms differ dramatically in regards to behavior. Males have a strong mating drive beginning in adulthood after they undergo sexual maturation during late larval life. Males will seek hermaphrodite worms by detecting hermaphrodite-specific pheromones. When he encounters the hermaphrodite, he initiates mating through a series of steps. The first starting with a search for the hermaphrodite’s vulva (i.e., the female external genitalia) by running his tail and feeling along the length of the hermaphrodite’s body. Eventually when he encounters the vicinity of the vulva, he detects a hermaphrodite-derived signal that makes him stop. After finding the vulva more precisely, he inserts a pair of spicules into the vulva and transfers sperm into the hermaphrodite’s reproductive organ.

Hermaphrodites, on the other hand, do not initiate mating with the male. Instead, they carry out the process of fertilization and the laying of eggs.

How does the nervous system of worms differ between males and hermaphrodites, and what developmental and molecular mechanisms underlie the establishment of sexually dimorphic neural circuits and behavior?

A work published in the journal Nature in 2016 by Dr. Meital Oren-Suissa and her colleagues at Columbia University provided a unique glimpse into how genes can guide the development of sexually dimorphic neural circuitry and behavior in C. elegans. This study demonstrated a surprising twist to how these neural circuits develop.

The researchers focused on a specific pair of neurons known as PHB that is present in both male and hermaphrodite worms. PHB is a sensory neuron that is associated with a sensory organ called the phasmid located at the worm’s tail. Although PHB and the phasmid organ are shared between the sexes, they contribute to very different behaviors in adult male and hermaphrodite worms.

In a previous study from another research group, the PHB neuron was found to regulate the worm’s repulsion to chemical repellants. However, in that case the researchers only studied the hermaphroditic worms. When Dr. Oren-Suissa started her studies, she analyzed the function of the PHB neuron in males. Surprisingly, the PHB neuron in males did not function in chemorepulsion, but instead was important during mating. When the PHB neurons were functionally destroyed in males, the male attempted to mate with the hermaphrodite as it normally would but was unable to effectively locate and stop at the hermaphrodite vulva.

How can the PHB sensory control such different behaviors in males and hermaphrodite worms? Dr. Oren-Suissa hypothesized that the PHB neuron was wired into different types of neurons and circuits. In both males and hermaphrodites, PHB connects to sex-specific interneurons that essentially act as relay centers. These relay centers then trigger and regulate sex-specific behaviors. The corresponding relay center that PHB connects to in the male is made in part of an interneuron called AVG. Hermaphrodites also have an AVG interneuron, but interestingly, PHB is not connected to AVG; instead, PHB wires into another interneuron that is also present in both sexes called AVA. Through the unique neuronal connections between the two sexes, PHB can regulate two different behaviors.

These findings then prompted the researchers to ask the million-dollar question: What’s the mechanism that causes PHB to wire into two different sex-shared neurons? Dr. Oren-Suissa and her colleagues envisioned two possibilities by which the PHB neuron could connect to AVG in males but AVA in hermaphrodites. The first they called the “pruning mechanism,” and the second, a “prepatterning mechanism.”

In the pruning mechanism, the PHB neurons would be initially connected to the AVG and AVA neurons in both sexes. When the neural circuits for reproductive behavior are established right before adulthood, the PHB neuron would get rewired such that it maintains connectivity with AVA in hermaphrodites, while, in males AVG connectivity is retained. In other words, synaptic connections between PHB and AVG and AVA would get sex-specifically pruned.

In the prepatterning mechanism, the PHB, AVG and AVA neurons would be present throughout the worm’s pre-adult life, but synaptic connections between them would emerge late when the circuits for reproductive behaviors are built during the final larval stage before adulthood.

To figure out which of these models were true, Dr. Oren-Suissa investigated the connectivity between PHB and AVG and AVA in juvenile worms. Remarkably, juvenile male and hermaphrodite worms have the same exact connections: PHB is connected to both AVG and AVA. As the worm undergoes sexual maturation during late larval life, the connectivity between PHB and its downstream target neurons gets sex-specifically pruned. In males, PHB’s connection to AVA is lost, whereas in hermaphrodites, PHB’s connection to AVG is removed. Furthermore, in juvenile hermaphrodites and males, the PHB neuron regulates chemorepulsion as it does in adult hermaphrodites. These findings told the researchers that a sex-specific neural circuit motif in the adult worm arises from the reconfiguration of a sex-non-specific default circuit that exists in juvenile worms (See Figure below).

Dr. Oren-Suissa and her colleagues then questioned what specific molecular mechanisms may guide this pruning process. To answer this question the researchers took a deeper look into the inner workings of these neurons. They first focused on identifying whether the sexual identity of these neurons was essential for pruning to take place in a sex-specific manner. By expressing transcription factors that regulate sexual development in the PHB, AVG and AVA neurons, Dr. Oren-Suissa found that feminization of the PHB neurons in a male caused the connection between PHB and AVG to get pruned, whereas synapses between PHB and AVA were maintained. Conversely, when the PHB neurons were masculinized in hermaphrodites, PHB’s connectivity was reversed. The masculinization and feminization of the worms resulted in cutting away the previous neuronal connections and replacing them with the opposite sexes.

Next, Dr. Oren-Suissa set out to identify genes that regulate the pruning process. They focused on a group of evolutionarily conserved transcription factors that regulate sexual development in many different animals. The lab then carefully narrowed it down to two key transcription factors called dmd-5 and dmd-11. These two unassuming transcription factors are expressed in males and are required to prevent pruning of the connection between PHB and AVG neurons in the males. However, in hermaphrodites dmd-5 and dmd-11 are not expressed and pruning proceeds.

This work has given us a remarkable view into how neural circuits for sexually dimorphic behaviors are created during development. What we’ve seen is that dimorphic circuits can arise by reconfiguring circuits that initially develop in both sexes. This contrasts with the notion that sex-specific circuits arise anew in each sex. It will be interesting to see if this mode of circuit development occurs in other animals.