C. elegans possess a powerful chemosensory system for perceiving chemicals in the environment, including food, noxious elements, volatile compounds, gases, and mating signals (Bargmann et al., 1993; Troemel et al., 1997; Chang et al., 2006; White et al., 2007; Ryan et al., 2014). Chemosensation is important for a wide range of C. elegans behavior including chemotaxis, avoidance, and motility. Chemosensory neurons control behavior by signaling to downstream inter- or motor- neurons, and other tissues. In addition to controlling behavior, chemosensory neurons can regulate animal physiology and development by releasing secreted TGF-β-family neuropeptides. For example, the ASI chemosensory neurons can secrete DAF-7, a TGF-beta related peptide, to control dauer entry (Ren et al., 1996; Schackwitz et al., 1996).

Mechanosensory neurons sense external forces and internal tension generated by movement and convert them into electrical signals, through mechanotransduction (Goodman, 2006; Goldmann, 2014). Mechanotransduction is mediated by several mechano-electrical transduction ion channels, including proteins from the TRP and Degenerin/epithelial Na⁺ (DEG/ENaC) channel families. MEC-4 and MEC-10 are members of the (DEG/ENaC) channel family that are required for responding to gentle touch (Chalfie and Sulston, 1981; Huang and Chalfie, 1994; O’Hagan et al., 2005; Chatzigeorgiou et al., 2010). In C. elegans, several mechanosensory neurons are responsible for sensing touch, including the anterior AVM and ALMs, and posterior PVM and PLMs (Chalfie et al., 1985). Distinct neurons are involved, and consequently different behaviors are executed, depending on the severity or the location of the mechanical stimulus.

Several TFs control terminal differentiation of mechanosensory neurons and touch response behaviors. MEC-3, a LIM-homeobox TF, is expressed in several mechanosensory neurons, including AVM, ALMs, PVM, PLMs, FLPs, and PVDs and is required for touch response behavior (Way and Chalfie, 1989). The UNC-86 and LIN-32 TFs are the major developmental regulators of mec-3-expressing neurons. Both of these factors are required to develop the precursor lineage of sensory touch neurons, and likely regulate mec-3 expression indirectly (Way and Chalfie, 1989). MEC-3 and UNC-86 form a heterodimer that regulates mec-3 expression and two other genes required for mechanotransduction, mec-4 and mec-7 (Figure 3A; Way and Chalfie, 1989; Duggan et al., 1998).

The ASH sensory neurons are involved in several behaviors, including light touch response and odor avoidance (Kaplan and Horvitz, 1993; Troemel et al., 1995). The Prop1-like homeobox domain TF UNC-42 is required for mechanosensation and locomotion by regulating the terminal differentiation of several neurons, including the ASH neurons (Baran et al., 1999). While UNC-42 is not essential for determining ASH sensory identity, it partially controls the terminal differentiation of ASH by regulating expression of the putative chemosensory receptors SRA-6 and SRB-6 (Figure 3A). UNC-42 is also expressed in the AVA and AVD command interneurons, which are important for controlling rapid locomotion and the body touch response (Brockie et al., 2001; Wightman et al., 2005). When UNC-42 function is abolished, this leads to body touch defects (White et al., 1986; Baran et al., 1999).

Several other TFs can also control mechanosensory neuron development. LIN-32, EGL-5, and VAB-15 are involved in generating touch sensory neurons such as the PLM and AVM/PVM, and are therefore required for touch sensitivity (Chalfie and Au, 1989; Du and Chalfie, 2001). Other transcriptional regulators such as EGL-44, EGL-46, and SEM-4 repress touch receptor identity. For example, removing either egl-44 or egl-46 function results in neurons emerging with touch receptor identity that are absent in the wild-type animals (Mitani et al., 1993). In addition to controlling ets-5 expression in the BAG neurons, VAB-3 also controls other glutamatergic sensory neuron identities. This includes the potential mechanosensory neurons OLL (Perkins et al., 1986), where VAB-3 regulates eat-4, ser-2, ace-1, and grd-8 expression (Serrano-Saiz et al., 2013). However, specific behavioral defects in vab-3 mutants have not been reported, possibly due to their severe morphological defects (Figure 3A).

The AVA, AVD, and AVE are command interneurons that mediate backward locomotion (White et al., 1986; Schafer, 2015). The AVA and AVE interneuron identities are regulated by FAX-1 and UNC-42 in complementary and overlapping pathways. FAX-1, a nuclear hormone receptor, induces expression of the NMDA-type glutamate receptor subunits NMR-1 and NMR-2 (Wightman et al., 2005). UNC-42 controls expression of the AMPA-type glutamate receptor subunits glr-1, glr-5, and glr-4. UNC-42 regulates glr-1 expression in the AVA, AVE, and AVD interneurons to enable the nose touch response (Baran et al., 1999; Brockie et al., 2001). UNC-42 also induces glr-5 expression in the AVA, AVE, and AVD interneurons, and induces glr-4 expression solely in the AVA interneurons (Baran et al., 1999; Brockie et al., 2001; Wightman et al., 2005). UNC-42, but not FAX-1, also regulates axon guidance in the AVA, AVD, and AVE neurons (Figure 4A; Brockie et al., 2001; Wightman et al., 2005). Therefore, UNC-42 and FAX-1 control different pathways that define how the AVA, AVD, and AVE command interneurons can respond to different contextual inputs from upstream neurons.

The AIY interneurons receive and integrate information from several amphid sensory neurons, including AWA, AWB, AFD, and ASE, and are involved in locomotion, thermotaxis and chemotaxis (Mori and Ohshima, 1995; Tsalik and Hobert, 2003; Gray et al., 2005; Kuhara et al., 2011; Kocabas et al., 2012). The AIY and AIZ interneurons mediate thermosensory behavior by connecting the AFD thermosensory neurons to command motor neurons. Three homeodomain TFs, TTX-3, CEH-10, and CEH-23 control AIY neuron terminal differentiation. Among these, TTX-3 is required for thermosensory behaviors (Hobert et al., 1997). These TFs regulate each other linearly, such that CEH-10 regulates ttx-3 and TTX-3 regulates ceh-23. Additionally, TTX-3 and CEH-10 autoregulate their expression (Hobert et al., 1997; Forrester et al., 1998; Altun-Gultekin et al., 2001). TTX-3 also regulates expression of surface receptors and channels including ser-2, kal-1, unc-17, hen-1, and the serpentine receptor sra-11, which is also induced by CEH-23 (Figure 4A; Altun-Gultekin et al., 2001). Loss of TTX-3 function also causes defects in AIY neuron axon outgrowth and pathfinding (Hobert et al., 1997). Thus, TTX-3 controls thermosensory behaviors by establishing and maintaining AIY interneuron function.

The AIZ interneurons are involved in the odorant sensing pathway and receive signals from the AWA and AWC chemosensory neurons (Bargmann et al., 1993). The POU/homeobox TF UNC-86 controls AIZ development and is required for odor-attraction and odor-adaptation behavior. UNC-86 induces AIZ generation during embryogenesis and is required to maintain AIZ function throughout life. LIN-11 also acts in the AIZ interneurons to regulate odor sensing behavior and is required for AIZ axonal morphology but is not involved in AIZ fate determination (Figure 4A; Hobert et al., 1998; Sze and Ruvkun, 2003).

The ALA interneuron and the RIS GABAergic interneuron regulate a sleep-like behavior known as lethargus quiescence (Van Buskirk and Sternberg, 2007; Turek et al., 2013). Two homeodomain TFs, CEH-14, and CEH-17, collaborate to control ALA fate determination and are therefore required for lethargus quiescence (Van Buskirk and Sternberg, 2010). CEH-14 and CEH-17 induce the expression of let-23, a receptor tyrosine-protein kinase and plc-3 (phospholipase C) in the ALA neuron. LET-23 is activated by the epidermal growth factor-like LIN-3, which initiates a signaling pathway that inhibits pharyngeal pumping and locomotion (Van Buskirk and Sternberg, 2007). CEH-10 is also partially involved in lethargus quiescence, by regulating expression of plc-3, and the neuropeptide flp-7 (Van Buskirk and Sternberg, 2010). CEH-10, CEH-14, and CEH-17 also regulate ALA axon outgrowth, which is dispensable for lethargus quiescence (Van Buskirk and Sternberg, 2007). Finally, the RIS interneuron requires the APTF-1 TF, which induces locomotion quiescence through neuropeptide signaling (Figure 4A; Turek et al., 2013).

Motor neurons generate behavior-specific movements through neurotransmitter and neuropeptide release. Motor neuron outputs can be sex-specific, such that males generate mating-specific movements (Liu et al., 2007; Sherlekar et al., 2013; Mowrey et al., 2014; Choi et al., 2015), and can control rhythmic behaviors, such as egg-laying via HSN (hermaphrodite-specific neuron)-regulated vulval muscle contraction in hermaphrodites (Waggoner et al., 1998). In addition to correct fate determination, synaptic patterns are also essential for correct motor neuron diversity and function (Zhou and Walthall, 1998). Several TFs (detailed below) are critical for locomotion and are required for motor neuron fate determination or synapse formation.

TFs involved in motor neuron fate determination include LIM-4, an LHX6 ortholog, which determines SMB motor neuron fate. LIM-4 regulates flp-12, unc-17, and cho-1 expression, which are required for SMB function (Kim et al., 2015). The TF UNC-3 controls fate determination and synaptogenesis of the SAB motor neurons, which potentially controls head and neck movements in L1 larvae (Kerk et al., 2017). UNC-3 regulates SAB fate by activating ddr-2 and unc-129 expression, and mediates synaptogenesis by controlling madd-4, a secreted protein that organizes synapse formation by controlling AChR clustering on the muscle (Kratsios et al., 2015). Lack of unc-3 leads to defects in synapse formation between the SABs and head muscles (Kratsios et al., 2015). The VA and the VB motor neurons control backward and forward movements, respectively (Chalfie et al., 1985). The TFs UNC-3 and PAG-3 are expressed in the VA and VB motor neurons and are required for coordinated movements in C. elegans (Jia et al., 1996; Cameron et al., 2002). UNC-3 and PAG-3 collaborate to determine VA and VB neuron fate by suppressing VC fate in these neurons (Figure 4B; Jia et al., 1996; Cameron et al., 2002; Prasad et al., 2008).

Specific behavioral movements are achieved by forming neuron-specific synaptic connections between different motor neurons and command interneurons. Failure to establish these connectivity patterns causes defective locomotion. Synaptic patterns in the VA and VB neurons are controlled by the TFs UNC-4, UNC-37, and CEH-12 (Von Stetina et al., 2007). UNC-4 and UNC-37 are expressed in VA neurons and suppress CEH-12, a homeobox TF that regulates VB specific genes (Miller and Niemeyer, 1995; Pflugrad et al., 1997). CEH-12 is only expressed in the VB motor neurons and is likely required for generating the VB synaptic pattern (Von Stetina et al., 2007). Loss of UNC-4 leads to CEH-12 expression and induces the VB synaptic pattern in the VA neurons (Von Stetina et al., 2007). UNC-4 and UNC-37 are required for normal locomotion; however, CEH-12 is not, suggesting CEH-12 plays a subtler role in regulating VB neuron traits. Ventral and dorsal motor neurons (VD and DD motor neurons) are also involved in locomotion. The distinct synaptic patterns of these neurons are mediated by UNC-55, a nuclear hormone receptor. UNC-55 is required for synapse formation between the VD motor neurons and the DA and DB motor neurons. Loss of unc-55 also leads to the VDs acquiring a DD synaptic pattern and locomotory defects (Figure 4B; Zhou and Walthall, 1998).

As mentioned above, motor neurons can control rhythmic behaviors. An example of this are the HSNs, which control egg-laying through G protein-coupled receptor-mediated regulation of vulval muscle contraction (Dong et al., 2000; Ringstad and Horvitz, 2008; Collins et al., 2016). When HSN function is lost, egg-laying is defective, and eggs accumulate in the uterus (Trent et al., 1983). TFs from six different families control HSN neuron terminal differentiation and function. These TFs are: UNC-86 (POU domain), HLH-3 (bHLH domain), EGL-18 (GATA factor), AST-1 (Ets domain), SEM-4 and EGL-46 (zinc finger). Together, these TFs induce and maintain HSN-expressed genes, including tph-1 and cat-1, with UNC-86 acting as the master regulator for most of the other TFs (Lloret-Fernández et al., 2018). All of these TFs are expressed in the HSNs throughout life, except for HLH-3 which is not expressed after the L4 stage (Lloret-Fernández et al., 2018). The intricate regulatory mechanisms in the HSN neurons highlight the complexity of neuronal control of behavior (Figure 4B).

C. elegans locomotion is influenced by feeding and physiological status. C. elegans grown on a bacterial lawn typically exhibit three behavioral states: dwelling (feeding in a restricted area), quiescence (a sleep-like non-feeding state), or roaming (exploring the environment). Worms generally spend most of the time dwelling, however, animals shifted to an environment lacking food start to roam after 30 min to explore for nutrients (Bargmann, 2006). Serotonin released from the NSM and HSN motor neurons induces dwelling, and PDF neuropeptide signaling from the AIY, RIM, and RIA interneurons induce prolonged roaming (Flavell et al., 2013). Food seeking behavior is also regulated by several other neurons including the ADF, ASE, ASI, AWC, and BAG (Gray et al., 2004; Wakabayashi et al., 2004; Juozaityte et al., 2017; Rhoades et al., 2019). ETS-5, which controls the BAG neurons gas-sensing ability, is also involved in regulating foraging behavior and fat metabolism. ETS-5 controls foraging behavior by regulating the expression of neuropeptides, including flp-13 and flp-19, in the BAG and ASG neurons (Juozaityte et al., 2017). ETS-5 controls fat storage levels, which feeds back to control roaming and quiescence behaviors (Juozaityte et al., 2017). The AWC neurons also control foraging behavior by sensing volatile attractants from food. CEH-36, which is involved in AWC development, is also required for foraging. The lipid-TORC1 signaling pathway, including monomethyl branched-chain fatty acids from the intestine, induces CEH-36 expression during starvation, which promotes foraging (Figure 5A; Kniazeva et al., 2015).

Learning from and remembering experiences is critical for launching effective behavioral responses. C. elegans possess different learning processes, classified as: non-associative learning, associative learning, and imprinting. Learning from different environmental conditions, such as changes in temperature, gases, and odorants, establishes short- and long- term memories (Ardiel and Rankin, 2010). Various neurons mediate these processes, including the AWC, ASH, and AFD sensory neurons (Hawk et al., 2018; Eliezer et al., 2019) and the AIB, AIY, AVA, AVD, RIA, and RIM interneurons (Stetak et al., 2009; Jin et al., 2016).

Long-term memory formation is mediated by the CREB TF CRH-1, which has seven isoforms expressed across several tissues (Amano and Maruyama, 2011; Dahiya et al., 2019). The crh-1e isoform is expressed in RIM interneurons and is necessary for long-term memory formation of isoamyl alcohol exposure (Dahiya et al., 2019). CRH-1 is also involved in short-term memory formation against pathogens—where for a short time after conditioning to a pathogen, C. elegans tend to move toward that pathogen. After conditioning to Staphylococcus aureus, CRH-1 expression increases, and is required for chemotaxis toward S. aureus (Prithika et al., 2017). In another paradigm, animals that experience high or low salt concentrations during starvation learn to associate those salt concentrations with starvation, and therefore avoid them. This taste avoidance learning behavior is mediated by DAF-16, a FOXO TF that is the major target of insulin-like signaling in the ASER neuron. DAF-16 regulates neuropeptide production in ASER by controlling the expression of the neuropeptide processing enzyme EGL-21 (Figure 5B; Nagashima et al., 2019).

Distal tissues can act on the nervous system to control aversive learning behavior. The INS-11 insulin-like neuropeptide is an excellent example of this: where the presence of pathogenic bacteria increases ins-11 expression in intestinal cells through the HLH-30 TF and p38 MAPK pathway (Lee and Mylonakis, 2017). INS-11 secreted from the intestine regulates ins-6 (another insulin-like neuropeptide) and tph-1 (involved in serotonin biosynthesis) in the ASI and ADF neurons, respectively, which can adjust aversive behavior to the bacteria (Lee and Mylonakis, 2017). C. elegans can prepare for a pathogenic bacterial attack, solely by detecting bacterial odor. This process is controlled by release of serotonin from serotonergic neurons, which causes HSF-1 localization in nuclear bodies where it controls the expression of chaperone genes such as hsp-70 to prepare the animal against pathogens (Figure 5C; Ooi and Prahlad, 2017).

Sexual status impacts how the nervous system and behavioral programs develop. The sex determination pathway in C. elegans has three main players: X chromosome/autosome ratio (X:A), the master regulator XOL-1 and the Gli-type zinc finger TF TRA-1 (Wolff and Zarkower, 2008). In XX animals the X:A ratio is high, which supresses xol-1, leading to hermaphrodite sexual differentiation. Low XOL-1 levels triggers dosage compensation complex formation, which controls the level of X chromosome gene expression, represses expression of the secreted protein HER-1, and activates the sex-determining transmembrane factor TRA-2 (Figure 6A; Miller et al., 1988; Carmi and Meyer, 1999). In XO animals, the X:A ratio is low which leads to high XOL-1 levels. High XOL-1 triggers male sexual differentiation and inhibits assembly of the dosage compensation complex (Miller et al., 1988; Carmi and Meyer, 1999). Downstream of XOL-1, TRA-1 acts as the final step in the sex determination pathway. TRA-1 is active in XX animals and is required for the hermaphrodite phenotype, whereas TRA-1 repression in XO animals, by the FEM-1, FEM-2, and FEM-3 factors, is necessary for male phenotype formation (Figure 6A; Hodgkin and Brenner, 1977; Berkseth et al., 2013).

A useful feature of C. elegans sex determination is that it acts cell autonomously. Therefore, ectopically expressing FEM-3 to masculinize or TRA-2 to feminize a specific neuron can resolve how sexuality affects that neurons function. Mating behavior is a prominent example of sexual dimorphism. Mating success requires males to perceive pheromones (ascarosides) released from hermaphrodites, and to be attracted to them. In contrast, hermaphrodites are weakly repelled by pheromones (Ludewig and Schroeder, 2018). Males sense pheromones by CEM (male-specific) and ADF (sex-shared) neurons (Srinivasan et al., 2008; Fagan et al., 2018). Masculinizing or feminizing the ADF neurons can change mating behavior. Males with feminized ADF neurons are repelled by ascarosides, whereas hermaphrodites with masculinized ADF neurons are attracted to ascarosides (Fagan et al., 2018). The TF MAB-3 drives the masculine characteristics of ADF neurons in males and is required for attraction toward ascarosides. MAB-3 is expressed only in male ADFs and masculinizing the ADFs in hermaphrodites results in MAB-3 expression (Figure 6B; Fagan et al., 2018).

Another sexually dimorphic behavior is olfaction, where each sex shows different preferences to certain compounds. For instance, the sexuality of the AWA and AWC neurons can affect their preference to chemicals such as diacetyl and pyrazine (Lee and Portman, 2007). Masculinizing hermaphrodite animals, either by mutating tra-1 or by driving fem-3 using a pan-neuronal promoter, reverses olfactory preference. However, the genes and mechanisms that control olfaction preference in AWA and AWC downstream of the sex determination pathway remain to be determined (Figure 6B).

The PHC neurons are present in both sexes, but have sex-specific connectivity, morphology and physiology that are involved in male mating behavior. PHCs are highly connected with other neurons in males, whereas they are much less connected in hermaphrodites. The PHCs extend axons anteriorly and dendrites posteriorly. In immature animals, both sexes show similar morphology, but during maturation the PHC dendrites in males retract, whereas, in hermaphrodites they project to the tip of the tail (Serrano-Saiz et al., 2017). Also, during maturation the PHC axons in males grow anteriorly beyond the pre-anal ganglion, but in hermaphrodites they remain in an immature state. After maturation, male PHCs show a sex-specific expression pattern for some genes, including the neuropeptide flp-11, which is only expressed in L4 males (Serrano-Saiz et al., 2017). Male PHCs also express higher levels of the glutamate transporter eat-4 compared to hermaphrodites (Serrano-Saiz et al., 2017). All of these sex-specific characteristics in male PHCs are controlled cell-autonomously through the sex determination pathway and the Doublesex DNA domain TF DMD-3. DMD-3, which is controlled by TRA-1, is expressed only in male PHCs and is necessary and sufficient to induce male-specific PHC features (Figure 6C; Serrano-Saiz et al., 2017).

C. elegans hermaphrodites and males possess sex-specific connectomes that control their behavior. The sex-specific connectome results from synaptic pruning during the later stages of development. Both sexes in the larval stages have a similar connectome, but during sexual maturation synapses not required for that sex are pruned (Oren-Suissa et al., 2016). Sex specific connectomes are regulated by the sex determination pathway, and cell-autonomous feminization or masculinization leads to the opposite synaptic pattern forming. Sex-shared neurons with sex-specific connectivity are related to sexually dimorphic behavior. For example, differences in the connections between the PHB and AVG sensory neurons control forward locomotion and chemo-repulsive behaviors in hermaphrodites, and mating behavior in males (Hilliard et al., 2002; Oren-Suissa et al., 2016). This process is mediated by the DMD-5 and DMD-11 TFs, which are only expressed in male AVG neurons, and are required for mating behavior in males (Oren-Suissa et al., 2016). DMD-5 and DMD-11 do not act in synapse formation but supress the pruning process in the neuron (Figure 6D; Oren-Suissa et al., 2016). In addition to sex-specific TFs, other TFs also regulate pruning, such as MBR-1/Mblk-1. MBR-1 expression is controlled by UNC-86 and is necessary for pruning synaptic connections between the AIM neurons. However, MBR-1’s role in behavior remains to be studied (Kage et al., 2005).

Experience during development can also influence synaptic pruning. For example, males starved during the L1 stage develop the hermaphrodite synaptic pattern between PHA and AVG and PHB and AVA, a process controlled by octopamine and serotonin signaling from the ADF neurons (Bayer and Hobert, 2018). These changes in synaptic pruning alter behaviors, such as enhanced chemosensory avoidance and mating (Bayer and Hobert, 2018).