A perspective on C. elegans neurodevelopment: from early visionaries to a booming neuroscience research

Abstract

The formation of the nervous system and its striking complexity is a remarkable feat of development. C. elegans served as a unique model to dissect the molecular events in neurodevelopment, from its early visionaries to the current booming neuroscience community. Soon after being introduced as a model, C. elegans was mapped at the level of genes, cells, and synapses, providing the first metazoan with a complete cell lineage, sequenced genome, and connectome. Here, I summarize mechanisms underlying C. elegans neurodevelopment, from the generation and diversification of neural components to their navigation and connectivity. I point out recent noteworthy findings in the fields of glia biology, sex dimorphism and plasticity in neurodevelopment, highlighting how current research connects back to the pioneering studies by Brenner, Sulston and colleagues. Multifaceted investigations in model organisms, connecting genes to cell function and behavior, expand our mechanistic understanding of neurodevelopment while allowing us to formulate emerging questions for future discoveries.

Keywords:

• Neurodevelopment
• C. elegans
• glia
• neurons
• pathfinding
• synapses
• plasticity
• dimorphism

Introduction: from pioneer feats to a booming neuroscience community

The nervous system, the set of cells involved in perceiving external or internal stimuli and responding with animal behavior, displays an alluring degree of sophistication. Exploring what shapes its complexity is an endeavor deep-rooted in the descriptions of Golgi, Cajal and their predecessors from the 1800s to today’s flourishing neuroscience community. Neurodevelopment follows similar patterns across organisms; cells commit to neural fates to generate neurons and glia, which migrate and extend processes to connect with target cells. It is shaped by cell death, fine-tuned by synaptic pruning, retains plasticity during development, and presents sex-dimorphism to support sex-specific behavior. Research in various models enables a sheer number of discoveries that connect genes to cells and behavior, revealing that neurodevelopment is driven by genetic pathways that are strikingly conserved. This fosters today’s misconception that the major principles of neurodevelopment have been addressed. Yet, many questions remain unanswered. How does patterning, cell compartmentalization, and communication coordinate to establish connectivity? What mechanisms remodel the nervous system or drive differences across sexes or related species? How is connectivity shaped by glia, the underappreciated non-neuronal cells? Multifaceted investigations in genetically tractable models allow for the continuous formulation of emerging questions and discoveries.

The non-parasitic nematode Caenorhabditis elegans, a well-established genetic model, has been instrumental to key breakthroughs in neurodevelopment for decades. Its defined nervous system consists of 5000 synapses, 302 neurons in a hermaphrodite, 387 neurons in a male, 50 sex-shared ectodermal glia, and 6 associated mesodermal glia-like cells and other hypodermal cells. C. elegans was honed for the analysis of the nervous system by visionaries Sydney Brenner and John Sulston, among others. Brenner’s and Sulston’s early practices gave rise to everyday rituals in all C. elegans labs. Importantly, their discoveries and the community they pioneered enable numerous mechanistic findings in neurodevelopment. Studies in C. elegans that dissect neural patterning, guidance and connectivity reveal underlying conserved genes, opening doorways to explore complex nervous systems.

This edition is a tribute to Brenner and Sulston as pioneers of a model and a booming community, using C. elegans as a showcase of nervous system biology. We compiled manuscripts that discuss the emerging themes in C. elegans neurodevelopment and reflect on established and open questions. Here, I delineate key neurodevelopment aspects, featuring the powers of C. elegans neuroscience research in light of Brenner’s and Sulston’s contributions (Figure 1).

Figure 1. Timeline of some milestones in research of C. elegans neurodevelopment. This timeline presents a variety of milestones in the research of neurodevelopment, in cellular, genetic, genomic and mechanistic aspects. As all milestones of a research field cannot be presented, here I highlight early works that solidified different research directions. For details and citations of the events mentioned, see in text.

The C. elegans nervous system: from cellular to anatomical and genome maps

In the pre-Brenner years, C. elegans was initially used in research from the 1900s by Maupas, Nigon, and Dougherty (Félix & Nigon, 2017). Later, Sydney Brenner chose C. elegans to study development and the nervous system. Brenner, Sulston, and their colleagues, contributed to the major steps that turn species into model organisms (Matthews & Vosshall, 2020). They proceeded from a vision of answering key questions to decrypting the animal’s genetic and cellular maps and inspiring a research community to dissect them.

Setting his mind on studying animal development, Brenner chose ‘… a multicellular organism which has a short life cycle, can be easily cultivated, and is small enough to be handled in large numbers, like a micro-organism. It should have relatively few cells, so that exhaustive studies of lineage and patterns can be made, and should be amenable to genetic analysis.’ (Brenner, 1963 MRC funding proposal). He further proposed ‘…to dissect the genetic specification of a nervous system in much the same way as was done for biosynthetic pathways in bacteria or for bacteriophage assembly … what was needed was an experimental organism which was suitable for genetical study and in which one could determine the complete structure of the nervous system’ (Brenner, 1974). Bravely following his lead, his trainees Sulston and White, addressed the extraordinary goal of determining the entire nervous system structure. Sulston devised methods to transfer and visualize animals with Nomarksi microscopy and then embarked on a remarkable venture, to map the C. elegans path from a single cell to an adult worm. This was seemingly impossible, since embryonic divisions are fast and poorly discernible away from the egg’s surface. Day after day for a year and a half, Sulston sat in a dark room for 12 h to track each division and daughter cell in developing embryos. Sulston confesses it was ‘… a challenge in the jigsaw-puzzling sense to get it all,’ (Check, 2002). ‘It was hugely exciting, looking at those cells dividing for the first time and knowing that I could see, I could find out. … then there was the group thing … the lineage was something that people really wanted.’ (Gitschier, 2006). Sulston’s determination and advances, narrated by Martin Chalfie in Sulston’s Obituary (Chalfie, 2018) provided a complete map of how a fertilized egg gives rise to a hermaphrodite (Sulston, Schierenberg, White, & Thomson, 1983; Sulston & Horvitz, 1977), the first road map to study metazoan development at the cellular level.

Meanwhile, John White invested his programming expertise into reconstructing the ultrastructure of the C. elegans nervous system. White recalled that ‘… the project was ridiculously ambitious, given the computer hardware available at the time (1970). Yet, with the courage of innocence we forged ahead’ (White, 2013). Together with the ‘remarkable electron microscopist’ Nichol Thomson and his ‘meticulous technician’ Eileen Southgate, they first reconstructed the animal’s ventral nerve cord and proceeded in tracing neurites of the central neuropil, also known as the nerve ring. Their decade-long labor culminated in the ultrastructural analysis of an entire nervous system, which serves as a reference for all studies of neural circuitry and connectivity (White, Southgate, Thomson, & Brenner, 1976, 1986). The tremendous amount of neurite trajectories and connections is incorporated into the WormAtlas, a C. elegans anatomy database generated by David Hall and colleagues, and complemented by recent nervous system reconstructions.

While generating C. elegans maps, cell identity was functionally linked to development using cell ablations. White developed laser ablation protocols, employed by Chalfie, Sulston, Bargmann, and others to correlate cell function to nervous system structure and animal behavior (Bargmann & Horvitz, 1991; Chalfie et al., 1985; J. E. Sulston & White, 1980). Meanwhile, Brenner and Sulston connected cellular maps to genetics by establishing landmark physical genetic maps of the C. elegans genome, a prerequisite for its sequencing (Brenner, 1974; Sulston & Brenner, 1974). Sulston also spearheaded the sequencing of the animal’s genome with Bob Waterston heading the sequencing effort at the Genome Sequencing Center (Washington University of St Louis). C. elegans was the first multicellular organism to have its genome sequenced (C. elegans sequencing consortium, 1998; Waterston & Sulston, 1995), giving the first complete genetic content required to build a nervous system (Bargmann, 1999).

Early studies of the genetics of the C. elegans nervous system

‘The relationship between genes and development is unknown’, Sulston and Horvitz wrote (Horvitz & Sulston, 1980). However, their work opened doors to numerous studies of C. elegans development that combined overturned this statement. Prior to sequencing the C. elegans genome, Brenner, Sulston and Horvitz systematically isolated and analyzed mutations affecting animal physiology and behavior to reveal developmental mechanisms. Brenner’s expertise in phage genetics proved insightful in exploiting the value of mutational analysis. This was facilitated by the clonal propagation of hermaphrodites and the freezing protocol developed by Sulston, for strain storage without the need for continuous propagation. This advance allows maintenance and sharing of thousands of isolates from the early mutants to all engineered strains and the creation of consortia such as the Caenorhabditis Genetics Center.

The systematic generation of mutants was a productive conceptual leap for connecting animal and cell physiology to genetic information. Sequencing the C. elegans genome and the subsequently established methodologies for genetic mapping (Wicks, Yeh, Gish, Waterston, & Plasterk, 2001) enabled the identification of all mutations that impair nervous system development. Luckily, albeit unanticipated by Brenner, the external application of double-stranded RNA in C. elegans suppresses gene expression. Needless to say, genome sequencing allows for the recent CRISPR/Cas9 genome editing and reverse genetics by RNA interference (Dickinson & Goldstein, 2016; Kamath et al., 2003). This array of unbiased and targeted gene manipulations allows for comprehensive research of neurodevelopment.

Core neurodevelopment events: birth and diversification, navigation and connectivity

Neurodevelopment proceeds through core processes of cell diversification, pathfinding, target selection, and connectivity (Figure 2). Investigations in C. elegans advance our molecular understanding of these events, pointing to principles that shape the prodigious complexity of nervous systems.

Figure 2. Core neurodevelopment processes that support the formation of the nervous system. The schematics summarizes selected events in nervous system formation, implicating cell autonomous factors, cell interactions and non cell-autonomous cues. Through development (from left to right), neuroblasts give rise to nervous system components, neurons and glia migrate, grow and diversify processes to then reach targets and generate synaptic connections. These connections are subject to maintenance and plasticity by mechanisms acting in neuronal or associated, non-neuronal cells, such as glial cells and hypodermal cells. Specifically, non-neuronal glia and hypodermal cells can generate postembryonic neurons, by division or transdifferentiation and in other instances they function for synapse maintenance or plasticity. For details and citations of the events and underlying mechanisms mentioned here, see in text.

Generation and diversification of nervous system components

An early step in neurodevelopment is the allotment of ectodermal neural precursors. In C. elegans master regulators that specify the ectoderm founders remained unknown (Maduro, 2010). Neural specification is driven by transcription factors and cell interactions (Lee et al., 2019; Stefanakis, Carrera, & Hobert, 2015). Early clues came from work by Sulston and colleagues on mutants of lineage iterations, creating supernumerary cells (Chalfie, Horvitz, & Sulston, 1981). It was demonstrated that upon commitment, the daughter cells of neuroblasts adopt distinct size and fate while in the absence of proneural transcription factors or specific kinases the daughter cells assume characteristics of their hypodermal sisters (Forrester, Dell, Perens, & Garriga, 1999; Frank, Baum, & Garriga, 2003; Singhvi & Garriga, 2009). Neural development is also shaped by cell death. Early work in C. elegans showed that of the 1090 somatic cells, 131 die before differentiating, most of which are in the ectodermal lineage (Sulston et al., 1983). These also led to the identification of dedicated apoptotic factors driving these deaths (Horvitz, Shaham, & Hengartner, 1994; Shaham, 1998). It is now known that asymmetric divisions of neuroblasts and the death of their daughter cells, which shape the nervous system, depend on a complex array of transcription factors, kinases, GTPases, and contractile forces driving cell asymmetry (Cordes, Frank, & Garriga, 2006; Hirose & Horvitz, 2013; Metzstein, Hengartner, Tsung, Ellis, & Horvitz, 1996; Mishra, Wei, & Conradt, 2018; Ou, Stuurman, D'Ambrosio, & Vale, 2010; Teuliere, Cordes, Singhvi, Talavera, & Garriga, 2014).

Nervous system sophistication relies on diversification of its components, starting from the fates of daughter cells after neuroblast division. Diversification depends on patterning along the body axis and determination of subtype fate. The earliest mutants of neuronal specification affected homeobox genes (Finney, Ruvkun, & Horvitz, 1988; Way & Chalfie, 1988; White, Southgate, & Thomson, 1992). Neurodevelopmental roles of transcription factors are being refined over the years. Distinct transcription factors specify left-right neuron asymmetry (Lesch & Bargmann, 2010; Sarin, Antonio, Tursun, & Hobert, 2009) or regulate terminal differentiation of motoneuron, interneuron, or sensory modalities (Hobert, 2016; Kim, Kim, & Sengupta, 2010; Masoudi et al., 2018; Poole, Bashllari, Cochella, Flowers, & Hobert, 2011; Satterlee et al., 2001; Sengupta, Chou, & Bargmann, 1996). Interestingly, neurons of the same neurotransmitter identity are subject to regulation by shared factors, the terminal selectors (Gendrel, Atlas, & Hobert, 2016; Kratsios, Stolfi, Levine, & Hobert, 2012; Pereira et al., 2015). Conversely, pan-neuronal identity is defined by redundant, parallel-acting cis-regulatory modules that direct expression to broad domains of the nervous system (Hobert, Carrera, & Stefanakis, 2010; Hobert & Kratsios, 2019). Each neuron subtype is now shown to present a unique combination of homeobox transcription factors (Reilly, Cros, Varol, Yemini, & Hobert, 2020). MicroRNAs are also shown to modulate fate; some can be subject to early priming in neuroblasts while others influence calcium signaling (Cochella & Hobert, 2012; Hsieh, Chang, & Chuang, 2012). Hence, new studies continue to uncover an increasing sophistication in mechanisms of neural differentiation.

Glia, non-neuronal ectodermal cells, also undergo diversification. This is regulated by transcription factors that segregate in non-neuronal daughter cells after progenitor division and act in glial cells (Labouesse, Hartwieg, & Horvitz, 1996) or by transcription factors that affect both glial and neuronal fate (Yoshimura, Murray, Lu, Waterston, & Shaham, 2008; Zhang, Noma, & Yan, 2020). Interestingly, the homeobox protein Prospero safeguards the postembryonic gene expression and function of glia (Wallace, Singhvi, Liang, Lu, & Shaham, 2016), suggesting that glia show maintenance and plasticity during the animal’s life.

Altogether, specification of the nervous system relies on inherited factors and intercellular signaling (Bertrand, Bisso, Poole, & Hobert, 2011). In this edition, Barrière and Bertrand (2020) review how the nervous system diversifies in nematode species, through a combination of transcription factors and cell interactions through morphogen signaling. Sulston’s cell lineaging and Brenner’s mutants were key to analyzing cell interactions (Sulston et al., 1983; Sulston & Horvitz, 1977) and their work in distant nematode species attempted to assign analogous neurons and features (Sulston, Dew, & Brenner, 1975). Barrière and Bertrand (2020) reflect on the evolvability of neural cell anatomy, cell fate, and wiring.

Nervous system morphogenesis: soma migrations and process pathfinding

Nervous system components migrate to adopt final positions in mature neuropils. Transcription factors and kinesin motors were early recognized to drive such migrations (Garriga, Guenther, & Horvitz, 1993; Wolf, Hung, Wightman, Way, & Garriga, 1998) while recent studies add the roles of actin, cytoskeletal scaffolds, and kinases (Levy-Strumpf & Culotti, 2007; Stringham & Schmidt, 2009; Tian et al., 2015; Withee, Galligan, Hawkins, & Garriga, 2004). Cell interactions were implicated early in migration (Garriga, Desai, & Horvitz, 1993); they involve cell adhesion (Solecki, 2012), guidance cues (Sundararajan & Lundquist, 2012), morphogens, and the planar cell polarity pathway for rosette formation (Shah et al., 2017). Recent research focuses on non-cell-autonomous factors of neuron migration, such as epidermal microRNAs (Pedersen et al., 2013), heparan sulfate proteoglycans, and extracellular matrix (Saied-Santiago et al., 2017; Tornberg et al., 2011). Glial migrations are shown to depend on nutrient supply (Zhang, Ackley, & Yan, 2020) but remain far less studied.

Concurrently to or after migration, neurons grow neurites to reach their partners. The specification of neurites to become axons or dendrites and dendrite development, in particular, were not subject of early research. However, later work uncovered that axon-dendrite sorting is polarized by ankyrin and kinesin, while guidance cues and neuronal asymmetry define the site of axon formation (Adler, Fetter, & Bargmann, 2006; Maniar et al., 2011). Glial-like mesodermal cells also specify certain axons through calcium signaling (Meng, Zhang, Jin, & Yan, 2016). Recent work reveals that the morphogenesis of dendrites and axons differ. Sensory dendrites form by retrograde extension upon extracellular attachment during neuronal migration (Heiman & Shaham, 2009). Some mechanosensory dendrites grow extensive arborization, driven by hypodermal cues, extracellular matrix, adhesion, and actin effectors (Dong, Liu, Howell, & Shen, 2013; Liu & Shen, 2012; Oren-Suissa, Hall, Treinin, Shemer, & Podbilewicz, 2010; Salzberg et al., 2013; W. Zou et al., 2018). Dendrites are also shaped by self-avoidance and axon-dendrite fasciculation (Chen, Hsu, Chang, & Pan, 2019; Smith, Watson, Vanhoven, Colón-Ramos, & Miller, 2012).

Axon formation driven by anterograde navigation was the subject of early scientific investigations. The earliest cues implicated in the process are the conserved Netrin, Robo, Semaphorin, Ephrin, and their receptors, now recognized as ‘canonical guidance proteins’ across organisms (Chédotal & Richards, 2010). Some of these were first identified in C. elegans. Brenner isolated mutants of the Netrin pathway and their roles in migration were characterized by Culotti, Wadsworth, Hall, and their colleagues (Culotti & Merz, 1998; Hedgecock, Culotti, & Hall, 1990; Keino-Masu et al., 1996; Wadsworth, Bhatt, & Hedgecock, 1996). Soon after, Robo, Semaphorin, and TGF-β were implicated in pathfinding by the Bargmann and Culotti labs (Colavita & Culotti, 1998; Ikegami, Zheng, Ong, & Culotti, 2004; Roy, Zheng, Warren, & Culotti, 2000; Zallen, Yi, & Bargmann, 1998) while the roles of the fibroblast growth factor and ephrins were discovered later (Boulin, Pocock, & Hobert, 2006; Bulow & Hobert, 2004; Grossman, Giurumescu, & Chisholm, 2013). A rich array of studies highlights the roles of guidance cues in attraction or repulsion and identified their effectors, many conserved across organisms (Huang, Cheng, Tessier-Lavigne, & Jin, 2002; Xu & Quinn, 2012; Zheng, Diaz-Cuadros, & Chalfie, 2016). Axon pathfinding is also affected by factors driving cell migration, such as the Wnt morphogen, heparan sulfate proteoglycans, and the planar cell polarity pathway (Bülow et al., 2008; Pan et al., 2006; Sanchez-Alvarez et al., 2011). It is now clear that guidance pathways are subject to significant crosstalk, with receptors and cues binding in more than one-to-one configurations (Fujisawa, Wrana, & Culotti, 2007; Rapti, Li, Shan, Lu, & Shaham, 2017; Yu, Hao, Lim, Tessier-Lavigne, & Bargmann, 2002). Downstream of guidance receptors, axons grow by dedicated growth cones with filopodia dependent on actin polymerization and GTPases (Demarco, Struckhoff, & Lundquist, 2012; Gujar, Sundararajan, Stricker, & Lundquist, 2018; Lebrand et al., 2004; Lundquist, Herman, Shaw, & Bargmann, 1998; Sundararajan & Lundquist, 2012). The precision of axon paths is controlled by additional adhesion proteins, transcription factors, microRNAs, and the environment (Baum, Guenther, Frank, Pham, & Garriga, 1999; Pocock & Hobert, 2008; Poinat et al., 2002; Schmitz, Kinge, & Hutter, 2007; Steimel et al., 2010; Troemel, Sagasti, & Bargmann, 1999; Y. Zou, Chiu, Domenger, Chuang, & Chang, 2012). Future investigations will need to better trace how all these factors cooperate in vivo.

Notwithstanding the wide-ranging study of nervous system pathfinding, certain aspects remain less clear. How do the above pathways coordinate in vivo, in diverse contexts, in neurons and glia? What are their primary effects? How do they instruct the early formation of the nervous system? How are major axon tracts formed in the embryo and how do neurons and glia communicate in this process? Pioneers were proposed to drive the formation of the C. elegans ventral nerve cord, in early electron microscopy studies (Durbin, 1987), and their functional importance was shown with ablations (Hutter, 2003). Pioneers of the brain-like nerve ring remained elusive. It is now shown that brain pioneers cooperate with glia to drive hierarchical brain assembly, using diverse signaling cues (Kennerdell, Fetter, & Bargmann, 2009; Rapti et al., 2017). This highlights the need to identify in the future the full array of neuron-glia interactions.

Nervous system connectivity: synapse formation and functional specification

Functional maturation and neural connectivity require protein localization in defined cell compartments. For example, sensory cilia consist of organelles with a defined proteome, including factors for extracellular vesicle release (Inglis, Blacque, & Leroux, 2009; Nechipurenko, Berciu, Sengupta, & Nicastro, 2017; Silva et al., 2017; Wang et al., 2014). Neurite polarity and compartmentalization are defined by finely-tuned anterograde and retrograde transport. Uncovering factors of axonal transport benefited early from Brenner’s genetic screens; dozens of his isolated Unc mutants affect kinesins and interactors for anterograde or retrograde transport (Brenner, 1974). In this edition, Vasudevan and Koushika (2020) review the molecular mechanisms of protein trafficking in C. elegans, highlighting how polarized transport shapes neurons’ forms, paths, and connectivity.

Upon navigation, neurites terminate their growth and form synapses to establish proper connectivity. Some Unc mutants from Brenner’s screens were shown to affect transcription factors that drive synaptic differentiation, sometimes in coordination with neurotransmitter signaling (Jin, 2005; Kratsios et al., 2015; Miller et al., 1992). Kinases, GTPases, and calcium mechanisms were also recognized early to regulate synaptogenesis (Crump, Zhen, Jin, & Bargmann, 2001; Rongo & Kaplan, 1999). Later studies dissected a rich array of synaptogenesis mechanisms including key signaling mechanisms. These include roles for gap junctions, insulins, and heparan sulfates (Grill et al., 2007; Hung et al., 2013; Lázaro-Peña, Díaz-Balzac, Bülow, & Emmons, 2018; Yeh et al., 2009) as well as adhesion and scaffolding complexes, some of which act hierarchically (Dai et al., 2006; Patel et al., 2006; Philbrook et al., 2018; Shen & Bargmann, 2003). On the postsynaptic end, dendrites are shown to differentiate functional spines, apposing presynaptic sites (Cuentas-Condori et al., 2019; Philbrook et al., 2018). Non-neuronal cells also influence synaptic connectivity; glia can affect postembryonic synapse localization (Colón-Ramos, Margeta, & Shen, 2007), and the epidermis acts to maintain peripheral synapses (Cherra, Goncharov, Boassa, Ellisman, & Jin, 2020; this edition). The full array of interactions driving synaptogenesis remains to be identified.

The establishment of functional connectivity culminates with neurotransmitter release and specialized localization and activity of neurotransmitter receptors. Some of Brenner’s Unc mutants affected conserved proteins of synaptic vesicle formation and release (Richmond, Davis, & Jorgensen, 1999; Weimer et al., 2003). Later studies identified roles of clathrin-mediated and clathrin-independent mechanisms in synaptic vesicle endocytosis (Gan & Watanabe, 2018). In addition to mutants affecting synaptic vesicles, some of Brenner’s mutants affected neurotransmitter receptors and their chaperones (Eimer et al., 2007). Precise connectivity depends on the localization of neurotransmitter receptors. In central synapses, receptor abundance depends on conserved cytoplasmic calcium- or clathrin-binding proteins, but the underlying cell interactions remain elusive (Burbea, Dreier, Dittman, Grunwald, & Kaplan, 2002; Hoerndli et al., 2015). At the neuromuscular junctions, synaptic localization of neurotransmitter receptors depends on extracellular domain interactions (Gally, Eimer, Richmond, & Bessereau, 2004; Gendrel, Rapti, Richmond, & Bessereau, 2009; Pinan-Lucarré et al., 2014). Precise nervous system activity also entails modulation of synaptic strength and gating of neurotransmitter receptors by auxiliary proteins (Boulin et al., 2012; Lei, Mellem, Brockie, Madsen, & Maricq, 2017; R. Wang et al., 2012). In addition to chemical synapses, the complex integration of neural connectivity is achieved by an array of electrical connections (Bhattacharya, Aghayeva, Berghoff, & Hobert, 2019; Schafer, 2018).

This morphogenetic and functional diversification supports a dynamic nervous system activity throughout development. Mechanisms of circuit integration that drive C. elegans behavior are reviewed elsewhere (Goodman & Sengupta, 2019; Whittaker & Sternberg, 2004).

Nervous system maturation, maintenance, and plasticity throughout animal life

After establishing its embryonic structure, the C. elegans nervous system shows plasticity and diversifies its components postembryonically. Although the pre-Brenner field considered that nematodes did not add somatic cells postembryonically, Sulston’s lineages showed otherwise. Sulston recognized that some dopaminergic neurons are generated by postembryonic divisions, adding to embryonic cells. He also noticed instances of postembryonic transdifferentiation; certain epithelial cells differentiate into neurons or male glia change their morphology and interactions (Sulston et al., 1983; Sulston & Horvitz, 1977; Walthall & Chalfie, 1988). These observations are corroborated by recent studies that dissect these transdifferentiation events. Epithelia-to-neuron (Y-to-PDA) transdifferentiation occurs through epigenetic mechanisms (Jarriault, Schwab, & Greenwald, 2008; Zuryn et al., 2014) while tail glia (PHso1) transdifferentiate in male-specific neurons by cell-intrinsic mechanisms (Molina-García et al., 2019). Recent research work also reveals an instance of glia-born neurons that was missed by the early cell lineaging. Specific sex-shared glia (AMso) divide postembryonically to generate male-specific neurons (MCM), which is essential for sexually dimorphic behavior (Sammut et al., 2015).

Recent investigations reveal that nervous system plasticity also occurs at the connectome level. After its establishment, connectivity is plastic in response to internal or external states. Sulston described early that embryonic and postembryonic lineages showed different motoneurons, suggesting that circuit wiring changes developmentally (Sulston, 1976; Sulston et al., 1983; Sulston & Horvitz, 1977). Indeed, specific motoneurons undergo a switch in neuronal polarity, presynaptic and postsynaptic regions. In this edition, Cuentas-Condori and Miller (2020), review the mechanisms of this synaptic remodeling: the underlying mechanisms of transcription regulation, downstream cascades of protein recycling, microtubule dynamics, cell death, and extracellular interactions. Intriguingly, some implicated factors were initially isolated in Brenner’s screens (Brenner, 1974). Cuentas-Condori and Miller (2020) highlight early and recent research work that shapes our understanding of synapse refinement.

The developmental plasticity of C. elegans connectivity is not limited to this specific switch. Recent ultrastructural studies by Zhen and colleagues describe global developmental changes in connectivity; they show that sensory and motor pathways gain new connections, while decision-making circuitry is maintained, and the brain becomes progressively more modular and feedforward (Witvliet et al., 2020). The nervous system also changes in response to environmental stimuli. During the harsh environment-induced dauer stage, specific sensory neurons remodel their axon arborization, which retracts when animals return to a favorable environment (Schroeder et al., 2013). The C. elegans electrical connectome is also dynamic, as gap junctions show striking changes in environment-induced diapause (Bhattacharya et al., 2019). Last but not least, neural responses also change with aging as reviewed elsewhere (Melentijevic et al., 2017; Stein & Murphy, 2012).

Besides its plasticity, the nervous system maintains its overall, embryonically-established structure throughout postembryonic life. This requires mechanisms that safeguard neuronal fate, through transcription factor autoregulation, as well as the position and axonal integrity through fibroblast growth factor (FGF) and immunoglobulin-domain signaling (Bénard, Blanchette, Recio, & Hobert, 2012; Bénard & Hobert, 2009; Bulow & Hobert, 2004). Maintenance of glia is less studied but depends on FGF receptor and solute carrier factors (Shao, Watanabe, Christensen, Jorgensen, & Colón-Ramos, 2013).

Along with the mechanisms for maintaining neural cell bodies and processes, additional mechanisms are in place to maintain connectivity. In this edition, Cherra et al. (2020), present how the epidermis regulates synapse density at neuromuscular junctions. An MPP5 factor controls the localization of an immunoglobulin-domain, adhesion protein that regulates CED-1-dependent phagocytosis (Cherra et al., 2020; Cherra & Jin, 2016). The CED-1 role in synapse engulfment comes a long way from its function in cell corpse engulfment, recognized by the Horvitz group (Zhou, Hartwieg, & Horvitz, 2001). Cherra and Jin (2016) suggest that the epidermis acts like glia for synapse elimination, at the C. elegans neuromuscular junctions that lack glia associations.

Glia in nervous system development; studying key roles of non-neuronal components

Glia are non-neuronal cells, abundant in complex nervous systems. They were long considered as connective tissue (‘glue’) providing trophic support to neurons and were neglected in quest of neuronal signaling. Glia are now implicated in nervous system development and function, coming closer to the center of attention. Glia communicate with neurons’ chemical and electrical connections, regulate nutrients, neurite morphogenesis, and connectivity. They are linked to neurological disorders including epilepsy, autism spectrum disorders, and Alzheimer’s (Allen & Lyons, 2018; Zuchero & Barres, 2015).

C. elegans glia associate with the sensory organs and axon-rich neuropils and were first described as ‘nervous system support cells’, in early studies by Sulston, Brenner, White, and Ward. (Shaham, 2015; Sulston et al., 1983; Sulston & Horvitz, 1977; Ward, Thomson, White, & Brenner, 1975; White et al., 1986). Twenty-five bilateral pairs of C. elegans ectoderm-derived glia come in different flavors. Twelve pairs of sheath glia wrap around axons or sensory endings (ADEsh, AMsh, CEPsh, ILsh, OLLsh, OLQsh PDEsh, PHsh). Some associate with synapses (CEPsh), while 13 pairs (socket glia) associate with sheath glia, generating pores for neurons to access the environment (ADEso, AMso, CEPso, ILo, OLLso, OLQso, PDEso, PHso). Early on, C. elegans glia were proposed to drive migration and to phagocytose dying cells (Sulston et al., 1983), yet they were understudied for decades.

Contrary to vertebrate glia, C. elegans glia appear dispensable for trophic support of mature neurons. Shai Shaham, who pioneered C. elegans glia research, recognized that the cell autonomy of many cell death events suggested glia may not be crucial for neuron survival in this animal (Shaham & Horvitz, 1996; Shaham, pers comm). This conjecture was verified by the Shaham group for a number of glia-neuron associations (Bacaj, Tevlin, Lu, & Shaham, 2008; Katz, Corson, Iwanir, Biron, & Shaham, 2018; Shaham, 2015), enabling a unique experimental setting to uncouple trophic support from glia-neuron functional interactions. Taking advantage of this knowledge, recent studies dissect glia functions and interactions, which are key in shaping the C. elegans nervous system.

C. elegans glia are now implicated in numerous aspects of neural development: in morphogenesis of sensory dendrites and microvilli, axon pathfinding and initiation of brain assembly, synapse positioning and regulation of neurotransmission, male-specific generation of neurons, mechanosensation, animal longevity, and sleep (Bacaj et al., 2008; Frakes et al., 2020; Johnson, Fernandez-Abascal, Wang, Wang, & Bianchi, 2020; Low et al., 2019; Perens & Shaham, 2005; Rapti et al., 2017; Sammut et al., 2015; Singhvi et al., 2016; Wallace et al., 2016; Yin et al., 2017; Yoshimura et al., 2008). C. elegans glia present heterogeneity in fate and function. Specific glia (CEPsh) communicate with several axons through distinct pathways (Rapti et al., 2017) while dorsal and ventral glia of the same subtype (CEPsh) employ partly different transcription factors to regulate their fate (Yoshimura et al., 2008). Other C. elegans glia (Amsh) are plastic and respond to external or internal conditions. They remodel their morphology together with their associated dendrites and show dynamic changes of gene expression, in different developmental stages or in response to temperature changes, starvation, or osmotic stress (Fung, Wexler, & Heiman, 2020; Lee, Procko, Lu, & Shaham, 2020; Procko, Lu, & Shaham, 2011). These glia (AMsh) also dynamically maintain their functional fate (Wallace et al., 2016). Whether heterogeneity and plasticity apply to all C. elegans glia remains to be defined. Neuron-glia interactions remain under intense investigation and were reviewed often (Shaham, 2015; Singhvi & Shaham, 2019). Many questions persist in the quest of glial fate, morphogenesis, circuit formation and function.

For most of the 20^th century, C. elegans glia remained obscure and vertebrate glia were studied as a by-product of recording neuronal connections. Today, different C. elegans glia are more or less studied. One of the reasons is that glia operate beyond the reach of tools designed to probe electrical signals. In a chicken or the egg problem, the lack of knowledge on glia hinders the identification of tools required to study them and the lack of tools hampers glial functional dissection. Luckily, recent research focuses on glia as equal protagonists of the nervous system. In this edition, the Heiman group contributes a resource manuscript presenting reagents to drive expression in different C. elegans glia (Fung et al., 2020). Using previous literature, transcriptomics and new analysis, they present drivers for glia expression, comparing their specificity and robustness in relation to the animal’s states. Such efforts, highlighting glia tools, facilitate the study of diverse glia to dissect aspects of their development and function. Given the numerous glial subtypes and functions, dissecting their biology will enable a more comprehensive view of nervous system complexity.

Sexual dimorphism in nervous system development

The remarkable intricacy of the nervous system defines the diversity of its components within and across sexes, to drive sex-specific behaviors allowing selective pressure against speciation. Pioneering work by Sulston, White, and colleagues provided maps of nervous system anatomy and connectome in the C. elegans hermaphrodite and spotlighted the lesser-studied male, describing a series of sexual variations in cell birth, elimination, or transformation (Sulston et al., 1983; Sulston & Horvitz, 1977). Research of the male biology was facilitated by mutations that cause sex transformation, as studied by Brenner and Hodgkin (Hodgkin, Horvitz, & Brenner, 1979). Sexual diversification of the nervous system relies on the birth or death of sex-specific neurons, and the sex-dimorphic plasticity of sex-shared components. Early investigation highlighted that sex-dimorphic cell birth gives rise to sex-specific neurons, like the hermaphrodite HSN that innervate vulval muscle or the male-specific, sensory and pheromone-secreting CEM, while their opposite-sex counterparts undergo programmed cell death (Silva et al., 2017; Sulston, Albertson, & Thomson, 1980, Sulston et al., 1983; Sulston & Horvitz, 1977; Wang et al., 2014; Ward et al., 1975). Recent studies highlight glia that contribute to nervous system dimorphism: head and tail glia (AMso, PHso1) divide or differentiate, respectively, to produce the male interneuron MCM and the ciliated neuron PHD, which are required for male-specific behaviors (Molina-García et al., 2019; Sammut et al., 2015). Sex-shared neurons can also undergo sex-specific synaptic pruning or axon branching (Bayer & Hobert, 2018; Hart & Hobert, 2018; Oren-Suissa, Bayer, & Hobert, 2016).

In this edition, Walsh, Boivin, and Barr (2020) review the nervous system of C. elegans males and the mechanisms driving sexually dimorphic development and plasticity. They present challenges and strategies to explore nervous system sex-dimorphism (Walsh et al., 2020). In addition, recent mapping of the male connectome by Emmons and colleagues will facilitate the dissection of sex-shared nervous system components that give rise to sex-specific behaviors and the plasticity of sex-related variations in neurodevelopment (Cook et al., 2019).

Looking back and ahead; reflections beyond C. elegans neural development

The early completion of the C. elegans cell lineage, connectome, and genome sequence by Brenner, Sulston, and subsequent C. elegans researchers give the impression that neuroscientists discovered the instructions to build a nervous system (C.I. Bargmann, 1999). Until today, our comprehension of how the nervous system develops grows by leaps and bounds, through the continuous work of a booming neuroscience community (Figure 1). The spirit of open science fosters these tremendous advances. Data communication and reagent sharing started with platforms such as the Worm Breeder’s Gazette, and International Worm Meetings. They culminate in consortia and databases such as the WormBook, WormAtlas, WormBase, CGC (Caenorhabditis Genetic Center), MMP (Million Mutation Project), modERN, modENCODE, and others. Sulston and his colleagues were early advocates of open science, an attribute highlighted when Sulston and Waterston led the public effort of the International Human Genome Consortium. Besides succeeding in this monumental achievement that altered the world, Sulston and Waterston insisted on making the data and reagents publicly available, years before publication. Sulston, Brenner, and their close colleagues and successors deserve our admiration, for pioneering scientific work in C. elegans and the first metazoan genome as well as for their scientific commitment that inspires C. elegans researchers to this day to make great strides toward understanding the intricacies of nervous system development and function.

Brenner and Sulston shared with Horvitz the 2002 Nobel Prize in Physiology or Medicine ‘for their discoveries concerning genetic regulation of organ development and programmed cell death.’ In their Nobel Prize interview they reflected on future research: ‘We are drowning in an ocean of data but we are starving for knowledge … you must have a theoretical framework to embed this … what we are going to need is human intelligence,’ highlights Brenner, and ‘we should use our creativity’ adds Sulston. ‘If we understand the worm, we understand life. Which of course we’re nowhere near.’, Sulston further contemplates (The Guardian, 2002, John Sulston interview: One man and his worm).

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No potential conflict of interest was reported by the author(s).

Additional information

Funding

G.R. is supported by the European Molecular Biology Laboratory.