Abstract
Sexually dimorphic behaviours require underlying differences in the nervous system between males and females. The extent to which nervous systems are sexually dimorphic and the cellular and molecular mechanisms that regulate these differences are only beginning to be understood. We reveal here a novel mechanism to generate male-specific neurons in Caenorhabditis elegans, through the direct transdifferentiation of sex-shared glial cells. This glia-to-neuron cell fate switch occurs during male sexual maturation under the cell-autonomous control of the sex-determination pathway. We show that the neurons generated are cholinergic, peptidergic and ciliated putative proprioceptors which integrate into male-specific circuits for copulation. These neurons ensure coordinated backward movement along the mate’s body during mating. One step of the mating sequence regulated by these neurons is an alternative readjustment movement performed when intromission becomes difficult to achieve. Our findings reveal programmed transdifferentiation as a developmental mechanism underlying flexibility in innate behaviour.
Introduction
The coordinated execution of innate, stereotyped sexual behaviours, such as courtship and mating, requires sexually dimorphic sensory-motor circuits that are genetically specified during development (reviewed in 1–3). Studies in the nematode Caenorhabditis elegans, in which the development and function of neural circuits can be interrogated with single cell resolution, have revealed two general developmental mechanisms underlying sexual dimorphism in the nervous system. The first involves the acquisition of sexually dimorphic features in sex-shared neurons during sexual maturation, which include changes in terminal gene expression, such as odorant receptors, neurotransmitters and synaptic regulators 4-9,10,11. The second mechanism involves the generation of sex-specific neurons 12–14. Sex-specific neurons are primarily involved in controlling distinct aspects of reproductive behaviours, such as egg-laying in the hermaphrodite and mating in the male (reviewed in 15). Generation of sex-specific neurons requires sex-specific cell death 16 or neurogenesis events resulting from sex differences in the cell division patterns and neurodevelopmental programmes of post-embryonic cell lineages (reviewed in 3). Here we identify a third, novel way to generate sexual dimorphism in the nervous system.
In one of his seminal papers, John Sulston described a sexual dimorphism in the phasmid sensilla of adult animals 13. The phasmid sensillum is one of the seven classes of sense organs that are common to both sexes in C. elegans. These sense organs are organised in sensilla which are concentrated in the head and the tail 17-19,20. Each sensillum is composed of the dendrites of one or more sensory neurons enveloped by a channel, usually composed of a single sheath glial cell and a single socket glial cell. These sensilla can be viewed as part of an epithelium, continuous with the skin, and are shaped by mechanisms shared with other epithelia 21. Socket glial cells are highly polarised and adhere to the hypodermis at the distal end of their process where they form a small, ring-like hollow pore in the cuticle through which the sensory dendrites can access the outside world. The bilateral phasmid sensilla (Fig.1), situated in the tail, are unusual in that they are each composed of two socket glial cells (PHso1 and PHso2). John Sulston observed that in juvenile animals (L2 stage) of both sexes, PHso1 forms the primary pore (13; Fig.1A). At adulthood, the hermaphrodite retains a similar structure (Fig.1B). In males, however, it is PHso2 that forms the main pore and PHso1 was described as having retracted from the hypodermis and to protrude into the phasmid sheath (Fig.1C). It was also described to contain basal bodies, a structural component of cilia, in the region enveloped by the phasmid sheath. As sensory neurons are the only ciliated cells in C. elegans 22, this is suggestive of neuronal fate, yet Sulston observed no other neuronal characteristics 13. Because we have previously shown that in the amphid sensillum (a similar organ located in the head), the amphid socket glial cell (AMso) acts as a male-specific neural progenitor that, during sexual maturation, divides to self-renew and generate the MCM neurons 14, we sought to investigate the PHso1 cells in more detail.
We find that during sexual maturation (L4 stage), the sex-shared PHso1 glial cells acquire sexually dimorphic function by undergoing a direct glia-to-neuron transdifferentiation that results in the production of male-specific neurons. This plasticity is regulated cell-intrinsically by the sex-determination pathway. These previously unnoticed neurons, which we term PHDs, are putative proprioceptors that regulate male locomotion during specific steps of mating. One of these steps is a novel readjustment movement performed when intromission becomes difficult to achieve. Our results reveal sex-specific direct transdifferentiation as a novel mechanism for generating sex-specific neurons and also show the importance of proprioceptive feedback during the complex steps of mating for successful reproduction.
Results
The sex-shared PHso1 cells undergo glia-to-neuron transdifferentiation in males
Using a lin-48/OVO1 reporter transgene to identify and visualise the PHso1 cell bodies both before and during sexual maturation 23,24, we observed no distinguishable differences between the hermaphrodite and male PHso1 cells at the L3 stage (Fig. 2A). The PHso1 cells display a polarised morphology and a visible socket structure in both sexes. In hermaphrodites, this morphology is maintained during the transition to adulthood and PHso1 cells elongate as the animal grows. In males, in contrast, PHso1 displays morphological changes that can be observed during the L4 stage, the last larval stage before adulthood (Fig. 2A). We find that in early L4 PHso1 retracts its socket process and extends a short dendrite-like posterior projection into the PHsh cell and a long axon-like anterior process projecting towards the pre-anal ganglion region. A time-series of carefully staged animals reveals that axon-like outgrowth is complete by the time of tail-tip retraction (Fig. 2B). These observations corroborate those of John Sulston and identify the stage of sexual maturation as the time during which the PHso1 cells undergo radical remodelling in males. This remodelling involves quite a remarkable change in morphology from a socket-glial morphology to a neuron-like morphology. Importantly, we do not observe any cell division of the PHso1 cells during this process. We only ever observe one bilateral pair of cells expressing lin-48 and only two bilateral pairs of cells (PHso1 and PHso2) expressing the glial subtype marker grl-2 during the change in morphology (Fig. 2 and Fig. 3). Together, these observations indicate that PHso1 cells undergo direct remodelling during which they may directly acquire neuronal fate.
To determine whether the PHso1 cells acquire neuronal characteristics at the level of gene expression in addition to morphology, we analysed and compared the expression of glial and neuronal markers in the PHso1 and PHso2 cells of males and hermaphrodites. We find that both cells express the panglial microRNA mir-228 25 and the AMso/PHso glial subtype marker grl-2 (a hedgehog-like and Ground-like gene protein 26) at equivalent levels in adult hermaphrodites (Fig. 4A). In males, in contrast, mir-228 and grl-2 expression is noticeably dimmer in PHso1 than in PHso2 by the mid-to-late L4 stage and completely absent by day two of adulthood (Fig. 4A, B and C). Importantly, expression of these genes is equal in brightness in both cells at the late L3 and early L4 stages (Fig. 4B and C). By the L4 moult, PHso1 begins to express the pan-neuronal marker rab-3 (a synaptic vesicle associated Ras GTPase 27) in males but not in hermaphrodites, and this expression persists throughout adulthood (Fig. 4B and C). In addition to rab-3 expression we also observe expression of other neuronal markers such as: the vesicle acetylcholine transporter unc-17 28,29 (Fig. 4D); the phogrin orthologue for dense-core vesicle secretion ida-1 30 (Fig. 4E); the intraflagellar transport component required for proper sensory cilium structure osm-6 31 (Fig. 4F); and the IG domain containing protein oig-8 32 (Fig. 6A). None of these neuronal markers are observed in PHso2 or in the hermaphrodite PHso1. The switch in gene expression in PHso1 is therefore initiated concomitantly with morphological changes during sexual maturation and in a male-specific manner. Together, these data demonstrate that the male PHso1 glial cells transdifferentiate into a novel class of ciliated neurons which we have termed phasmid D neurons (PHDs).
Biological sex regulates PHso1-to-PHD transdifferentiation cell-autonomously
We next addressed whether the PHso1-to-PHD cell fate switch is regulated by the genetic sex of the cell rather than the sex of the rest of the animal, in a manner similar to that of several other sexual dimorphisms in C. elegans 5-7,10,14,33-36. To uncouple the sex of PHso1 from the rest of the animal, we drove expression of fem-3 in PHso1 (and PHso2) under the grl-2 promoter. fem-3 inhibits the expression of tra-1, a downstream target of the sex-determination pathway that activates hermaphrodite development and inhibits male development 37 (and reviewed in 38). Therefore, cell-specific expression of a fem-3 transgene will masculinise a cell in an otherwise hermaphrodite background. We find that fem-3 expression specifically in the PHso1 transforms it into a PHD-like neuron, resulting in the upregulation of ida-1 and rab-3 expression and the acquisition of neuronal morphology (Fig. 5). This indicates that the competence of PHso1 to transdifferentiate into PHD is cell-intrinsic and based on genetic sex.
PHDs are sensory neurons of male-specific copulation circuits
To further establish the neuronal characteristics of PHDs, we examined their synapses and ultrastructure, and mapped their full wiring diagram. We identified a ~1kb promoter region that specifically drives the expression of oig-8 in the PHD neurons. Expression of a rab-3 translational fusion under the control of this oig-8 promoter reveals that PHDs form synapses in the pre-anal ganglion, where the synaptic-vesicle associated mCherry-tagged RAB-3 protein can be observed (Fig. 6A). At the ultrastructural level, we observed both synaptic and dense core vesicles in PHD, proximal to the cell body (Fig. 6B). Ultrastructural analysis of the PHD dendrite in seven different animals independently confirms John Sulston’s original observation on the presence of cilia, including the basal body and axonemes. The basal body of PHD lies just dorsal and anterior to the phasmid sheath channel containing the PHA and PHB cilia, with 2, 1 or zero short cilia extending within the phasmid sheath very close to PHA and PHB cilia, but within a separate sheath channel. Interestingly, the PHD cilia can lie in variable positions relative to PHA and PHB cilia, medial, lateral, dorsal or ventral. In addition, we observe that the dendrite is more elaborate than previously described and has a number of unciliated finger-like villi within the phasmid sheath cell, proximal to the basal body (Fig. 6C and Fig. S1). Through reconstruction of serial electron micrographs, we identified all the synaptic partners of PHD (Table S1). PHD neurons project from the dorsolateral lumbar ganglia, anteriorly and then ventrally along the posterior lumbar commissure and into the pre-anal ganglion. In the pre-anal ganglion, these establish chemical synapses and gap junctions with sensory neurons and interneurons, most of which are male-specific (Fig. 6D). Previously, the axonal process of PHD was attributed to the R8B ray neuron 4. A re-examination of the lumbar commissure allowed us to disambiguate PHD’s axon. PHDs receive synaptic input from male-specific ray sensory neurons involved in the initiation of the mating sequence in response to mate contact 39,40,41. The main PHD output is to the male-specific EF interneurons (35.4% of chemical synapses), both directly and through their second major post-synaptic target, the sex-shared PVN interneuron. The EF interneurons are GABAergic 8 and synapse onto the AVB pre-motor interneurons which drive forward locomotion. Other PHD outputs include the PVV (male-specific) and PDB (sex-shared but highly dimorphic) interneurons, which synapse onto body-wall muscle, and the cholinergic male-specific interneurons PVY and PVX whose output is to the AVA pre-motor interneurons, which drive backward locomotion. PVN, PVY and PVX form disynaptic feed-forward triplet motifs that connect PHD strongly to the locomotion circuit interneurons AVB and AVA. The pattern of connectivity of PHD suggests a possible role in male mating behaviour.
PHDs are putative proprioceptive neurons
We next sought to establish the function of the PHD neurons. We began by asking what sensory stimuli might activate them. To monitor neuronal activity, we co-expressed GCaMP6f and RFP in the PHDs and performed ratiometric measurements of fluorescence. We imaged calcium transients in restrained animals glued to a slide without anaesthetic. To our surprise, without applying any exogenous stimulation, we observed intermittent calcium transients in the PHDs of wildtype animals, every 30 seconds on average (Fig. 6E, F and Movie 1). We observed calcium transients in 73% of the neurons imaged and no peaks could be identified in the remaining traces (n=19). Since in restrained animals there are small tail and spicule movements due to the defecation cycle and sporadic muscle contractions, one explanation could be that PHDs may be proprioceptive. We reasoned that if PHD activity resulted from internal tissue deformation caused by muscle contractions, inhibition of muscle activity should eliminate or reduce calcium transients. To inhibit muscle contractions, we generated transgenic worms in which muscles can be silenced in an inducible manner through the expression of the Drosophila histamine-gated chloride channel HisCl1 42, under the myo-3 promoter. To increase the efficiency of muscle silencing we introduced this transgene into an unc-51(e369) mutant background that renders animals lethargic. unc-51 encodes a serine/threonine kinase required for axon guidance and is expressed in motor neurons and body wall muscle 43. Histamine-treated myo-3::HisCl1;unc-51(e369) animals were highly immobile and the frequency of calcium transients in the PHDs was strongly reduced (Fig. 6E, F and Movies 2 and S1). Transients were completely eliminated in half of the neurons (Fig. 6F). The reduction in transient frequency was specific to the silencing of the muscles because in histamine-treated unc-51(e369) and histamine non-treated myo-3::HisCl1;unc-51(e369) control animals, frequency was similar to that of wildtype animals (Fig. 6E, F and Movie S2).
Although PHDs are sensory neurons, they do receive some small synaptic input from other neurons (Fig. 6D) and therefore, the activity observed in restrained animals in response to muscle contractions could arise either directly through PHD sensory input or indirectly through presynaptic neurons. To test this, we imaged PHD activity in mutant males with impaired synaptic neurotransmission, unc-13(e51) 44 and dense core vesicle secretion, unc-31(e169) 45. In these mutants, PHD calcium transients persisted at similar frequencies to those in wildtype animals (Fig. 6E and F). This suggests that PHD activity does not require chemical input from the network. However, we cannot completely rule out that residual neurotransmission in the hypomorphic unc-13(e51) mutants may be sufficient to trigger wildtype levels of PHD activity in response to muscle contractions. Together, these results suggest that PHDs may respond directly to internal cues arising from muscle contractions, and that they may be proprioceptive neurons. PHDs may sense internal tissue deformations through their elaborate ciliated dendrites, which are deeply encased in the phasmid sheath and not exposed to the outside environment.
A novel readjustment step during male-mating
The connectivity of PHD to male-specific neurons in the tail suggests that they may play a role in male reproductive behaviours controlled by tail circuits. In C. elegans, these include food-leaving behaviour, an exploratory strategy to search for mates 46,47,48, and mating behaviour. Mating consists in a temporal sequence of discrete behavioural steps: response (to mate contact); scanning (backward locomotion while scanning the mate’s body during vulva search); turning (at the end of the mate’s body to continue vulva search); location of vulva (stop at vulva); spicule insertion (intromission); and sperm transfer (reviewed in 3; Fig. 7). The coordinated execution of these mating step relies on sensory cues from the mate 40 and presumably, proprioceptive inputs within the male’s copulation circuit as well 13 (and reviewed in 49). This sensory information guides the male to either initiate the next step of the sequence or to reattempt the current, unsuccessful step through readjustment of movement and/or posture (reviewed in 3).
During our behavioural analysis of wildtype males, we identified a novel readjustment movement that has not been previously described which we have termed the ‘Molina manoeuvre’. This movement occurs when the male has been trying to insert its spicules in the mate’s vulva for a period of time without success and subsequently loses vulva aposition. Unsuccessful spicule insertion attempts occurred in 65% of males, resulting in either a long displacement from the vulva, which led to the re-initiation of the scanning sequence, or in a small displacement (less than two tail-tip length) from the vulva (98% of vulva losses), as previously described 50. We find that these small displacements from the vulva lead to either local shifts to relocate the vulva (68% of total vulva losses) or in a Molina manoeuvre (32% of vulva losses) (Fig. 7). The Molina manoeuvre consists in the initiation of forward locomotion along the mate’s body away from the vulva to the end or middle of the mate’s body, at which point the male tail acquires a deep arched posture, followed by a return to the vulva with backward locomotion along the same route (Movie 3). Males perform this movement towards either end (head or tail) of the mate as a smooth, continuous sequence. Although we used paralysed unc-51 mutant hermaphrodites to aid our mating analysis, males also perform this readjustment movement with moving wildtype mates (Movie S3).
The PHD neurons are required for coordinated backward locomotion and effective intromission during mating
To test the hypothesis that PHDs regulate reproductive behaviours, we performed behavioural tests of intact and PHD-ablated males and functional imaging of PHD activity in freely behaving males during mating. We found no defects in food-leaving behaviour, response to mate contact, turning, location of vulva, or spicule insertion (Fig. 7A, B, D, E and H). However, these experiments revealed a role for PHDs in initiation and/or maintenance of backward locomotion during scanning and during the Molina manoeuvre. PHD-ablated males often switched their direction of locomotion during scanning, performing fewer continuous backward scans during vulva search compared to intact males (Fig. 7C). Consistent with this, we observed higher activity (i.e. Ca2+ levels) in PHD during backward locomotion than during forward locomotion while scanning (Fig. 7C). PHD activity also peaked just after the switch to backward locomotion during Molina manoeuvres, when the tail tip displays an acute bent posture, and PHD-ablated males performed defective, discontinuous manoeuvres, often stopping at the transition from forward to backward locomotion to return to the vulva (Fig. 7F and Movie 4). Importantly, we did not find significant differences between intact and PHD-ablated males in number of events that trigger the initiation of manoeuvres (Fig. 7G), indicating that PHDs are specifically required for coordinated locomotion during the manoeuvre itself, at the transition point to backward locomotion.Together, these data demonstrate that without intact PHD neurons, backward movement along the mating partner becomes slightly erratic, often interrupted by a switch in direction (during scanning), or its initiation is being delayed by a stop (during Molina manoeuvres). The qualitative difference in locomotion defects between these two steps of mating may result from differences in the state of the neural network before and after spicule insertion attempts 50–52.
In addition to the afore mentioned changes in neuronal activity, the highest level of PHD activation was observed during intromission, the penultimate step of the mating sequence, which precedes sperm transfer and involves full insertion of the spicules into the mate’s vulva while sustaining backward locomotion. PHD activity increased two-fold upon spicule insertion and remained high for several seconds while the spicules were inserted (Fig. 7H). PHDs are required for the efficiency of these two last steps of mating since PHD-ablated males that were observed to complete the mating sequence, including intromission and ejaculation, during a single mating with one individual mate, produced fewer cross-progeny than intact males in the same conditions (Fig. 7H). Intact PHDs may increase the efficiency of sperm transfer by controlling the male’s posture during intromission. While the cues and network interactions that activate PHDs during mating are presently not known, the high levels of PHD activity upon spicule insertion are consistent with a putative proprioceptive role for these neurons.
Discussion
The data presented here extend John Sulston’s original observations and demonstrate that male PHso1 cells undergo a direct glia-to-neuron transdifferentiation to produce a novel class of bilateral cholinergic, peptidergic and ciliated sensory neurons, the phasmid D neurons. This updates the anatomy of the C. elegans male to be comprised of 387 neurons (93 of which are male-specific) and 90 glia. This is the second example of neurons arising from glia in C. elegans, confirming that glia can act as neural progenitors across metazoan taxa. In both cases we have demonstrated that fully differentiated, functional glia can retain neural progenitor properties during normal development. The complete glia-to-neuron cell fate switches we observe strongly suggest the production of neurons from glia is a process of natural transdifferentiation. In the first case, this is an indirect transdifferentiation as it occurs via an asymmetric cell division, leading to self-renewal of the glial cell and the production of a neuron 14. In the work described here it is a direct transdifferentiation and as such is the second direct transdifferentiation to be described in C. elegans. The first involves the transdifferentiation of the rectal epithelial cell Y into the motor neuron PDA, which occurs in hermaphrodites at earlier larval stages, before sexual maturation 53. It will be interesting to determine whether the mechanisms that regulate the PHso1-to-PHD cell fate switch are similar or distinct from those that have been described for Y-to-PDA 54–56. Intriguingly, direct conversion of glial-like neural stem cells has been observed in the adult zebrafish brain 57, suggesting that this may be a conserved mechanism for postembryonic generation of neurons.
Based on our manipulations and imaging of PHD activity in restrained and freely mating animals, we propose that PHDs may be proprioceptive neurons which become activated by tail tip deformations and engage the circuits for backward locomotion. The high levels of PHD activity during intromission and upon tail tip bending during the Molina manoeuvre, steps which require inhibition of forward locomotion and sustained backward movement, are consistent with such model. PHDs may ensure sustained backward locomotion through excitation of their postsynaptic targets PVY, PVX and EF interneurons, all of which have been shown to promote backward movement through AVA and AVB during scanning and location of vulva 58,59. As wildtype hermaphrodites are highly uncooperative during mating and actively move away 60, any erratic and uncoordinated movement by the male is likely to result in the loss of its mate.
Sensory input from the mating partner is essential for successful mating and accordingly, many sensory neurons in copulation circuits are dedicated to this purpose (reviewed in 61 and 3). However, coordinated motor control in all animals requires also proprioception, sensory feedback from internal tissues that inform the individual about its posture and strength exerted during movement 62,63. Several putative proprioceptive neurons have been identified in C. elegans (reviewed in 64). Within the male copulation circuit, only the spicule neuron SPC has been attributed a putative proprioceptive role, based on the attachment of its dendrite to the base of the spicules 13. The SPC neuron is required for full spicule protraction during intromission 39,65.The presence of proprioceptive neurons in copulation circuits may be a broadly employed mechanism to regulate behavioural transitions during mating. In this light, it would be interesting to determine whether the mechanosensory neurons found in mating circuits of several organisms 66–68 may play a role in self-sensory feedback.
In summary our results provide new insight into the developmental mechanisms underlying the neural substrates of sexually dimorphic behaviour. The glia-to-neuron transdifferentiation that results in PHD represents an extreme form of sexual dimorphism acquired by differentiated sex-shared glial cells during sexual maturation. The PHD neurons, perhaps through proprioception, enable the smooth and coordinated readjustment of the male’s movement along its mate when spicule insertion becomes difficult to attain. This readjustment movement represents an alternative step within the stereotyped mating sequence. These findings reveal the extent to which both cell fate and innate behaviour can be plastic yet developmentally wired.
Materials and Methods
Strains
For a list of strains generated and/or used in this study see Table S2
DNA constructs and transgenic strains
The oig-8::gfp reporter was built by amplifying a 964 bp promoter fragment upstream of the oig-8 ATG adding the restriction sites of SphI and XmaI. This PCR product was digested and ligated into SphI/XmaI-digested pPD95.75 vector. To generate oig-8p::mCherry::rab-3, the same oig-8 promoter fragment was ligated into SphI/XmaI-digested pkd-2p::mCherry::rab-3 (a gift from M. Lázaro-Peña). The oig-8::GCaMP6f::SL2::RFP construct was created by PCR fusion 69. A ~ 1 kb promoter region of the oig-8 locus was amplified with primers: (A) gggagtgacctatgcaaacc and (B) CGACGTGATGAGTCGACCATtgttttacctgaaatctttt, which has a tail overlapping with the GCaMP sequence for the fusion PCR. The myo-3::HisCl1::sl2::mCherry construct was created as PCR fusion. A 2.2 kb promoter region of the myo-3 locus was amplified with primers: (A) cgtgccatagttttacattcc and (B) gctagttgggctttgcatGCttctagatggatctagtggtc, which has a tail overlapping with the HisCl1 sequence for the fusion PCR. The lin-48::tdTomato construct was a kind gift from Mike Boxem and contains a 6.8kb promoter fragment upstream of the coding sequence of tdTomato and the unc-54 3’ UTR. It was injected into him-5(e1490) animals and spontaneously integrated generating drpIs3 (see Table S2).
The grl-2::gfp reporter was generated by integrating sEx12852 26.
Cell ablations
PHD was ablated with a laser microbeam as previously described 70. L4 males were staged and placed in a seeded plate the night before. Ablations were carried out at 1 day adulthood and PHD was identified by oig-8::gfp or unc-17::gfp reporter expression. Mock-ablated animals underwent the same treatment as ablated males except for laser trigger. Animals were left to recover overnight to perform behavioural assays the next day. After behavioural assays, animals were checked for lack of GFP expression in PHD to confirm correct cell ablation. The few animals in which PHD had not been efficiently ablated were discarded from the data.
Cell-type specific sex transformations
We used two previously described strains each containing an array that drives fem-3 expression from a grl-2 promoter fragment 14. In the case of oleEx24 the presence/absence of the array in PHso1 and PHso2 was monitored by the mCherry expression from the array itself and the expression of an ida-1::gfp reporter (inIs179), in the background of the strain was used to monitor neuronal fate in PHso1/PHD. In the case of oleEx18, we had difficulty visualising the mCherry from the array in PHD and crossed a lin-48::tdTomato (drpIs3) into the strain to visualise PHso1/PHD. The presence/absence of the array in whole animals was assessed using the array co-injection marker elt-2::gfp. Neuronal fate was monitored using a rab-3::yfp reporter. See Table S2 for full strain details.
Behavioural assays
All behavioural assays were scored blind to the manipulation. Males carried either an oig-8::gfp or an unc-17::gfp transgene to identify the PHD neurons for ablation.
Food leaving
Animals were tested at 3 days of adulthood (the day after they had been tested for mating). Assays were performed and scored as previously described 47.
Mating
Assays were performed and scored as previously described 14. Males were tested at 2 days of adulthood with 1 day-old unc-51(e369) hermaphrodites picked the night before as L4s. Each male was tested once for all steps of mating. Those males that were not successful at inserting their spicules with the first hermaphrodite were tested again with a maximum of three hermaphrodites to control for hermaphrodite-specific difficulty in penetration 39. Assays were replicated at least three times on different days and with different sets of males. Videos of matings were recorded and scored blind by two independent observers. Displacements from the vulva were scored as movements of one or two tail-tip distance away from the vulva.
Fertility assays
each individual male was monitored for all mating steps during a single mating until it ejaculated. After disengagement from the mate, the hermaphrodite was picked and placed in a fresh plate to lay progeny. The adult hermaphrodite was transferred to a fresh plate each day during three consecutive days. After three days from the eggs being laid, L4 larvae and adult progeny were counted as Unc self-progeny of Wt cross-progeny.
Microcopy and imaging
Worms were anesthetized using 50mM sodium azide and on mounted on 5% agarose pads on glasss slides. Images were acquired on a Zeiss AxioImager using a Zeiss Colibri LED fluorescent light source and custom TimeToLive multichannel recording software (Caenotec). Representative images are shown following maximum intensity projections of 2-10 1µm z-stack slices and was perfumed in ImageJ.
Ca2+ imaging
Imaging was performed in an upright Zeiss Axio Imager 2 microscope with a 470nm LED and a GYR LED (CoolLED) with a dual-band excitation filter F59-019 and dichroic F58-019 (Chroma) in the microscope turret. Emission filters ET515/30M and ET641/75 and dichroic T565lprx-UF2 were placed in the cube of a Cairn OptoSplit II attached between the microscope and an ORCA-Flash 4 camera (Hamamatsu). Acquisition was performed at 20 fps.
Imaging during mating was performed with a 20× long working distance objective (LD Plan-NEOFLUAR numerical aperture 0.4), placing the male on an agar pad with food and 20 hermaphrodites. The ~ 50 mm per side agar pad was cut out from a regular, seeded NGM plate and placed on a glass slide. The hermaphrodites were placed in a ~ 100 mm2 centre region. A fresh pad was used every two recordings.
Imaging in restrained animals was performed for 2.5 to 3 minutes with a 63× objective (LD C-apochromat numerical aperture 1.15). Animals were glued with Wormglu along the body to a 5% agarose pad on a glass slide and covered with M9 or 20 mM histamine (Sigma, H7125) and a coverslip.
Ca2+ imaging analysis
A moving region of interest in both channels was identified and mean fluorescent ratios (GFP/RFP) were calculated with custom-made Matlab scripts 71, kindly shared by Zoltan Soltesz and Mario de Bono. For recordings in restrained animals, bleach correction was applied to those traces in which an exponential decay curve fitted with an R square > 0.6. Ratios for each recording were smoothened using a 8 frame rolling average. For ΔR/R0 values, R0 for each recording period was calculated as the mean of the lowest 10th percentile of ratio values. Traces that were locked to behavioural transitions had their ΔR/R0 values added or subtracted such that all events had the same value at t=0. Peaks were identified manually by an observer blind to the genotype and treatment. Peaks were called as signals above 0.2 ΔR/Rmax (where Rmax was calculated as the highest 5th percentile of ratio values), above 2σ from local basal and a minimum duration of 5 seconds.
Electron microscopy and serial reconstruction
The samples were fixed by chemical fixation or high-pressure freezing and freeze substitution as previously described 72. Several archival print series from wildtype male tails in the MRC/LMB collection were compared to wildtype adult males prepared in the Hall lab, showing the same features overall. Ultrathin sections were cut using a RMC Powertome XL, collected onto grids, and imaged using a Philips CM10 TEM. The PHD cell bodies were identified in the EM sections based on position and morphology. This was followed by serial tracing of the projections to establish their morphology and connectivity. The method of quantitative reconstruction using our custom software is described in detail in 4,73. The connectivity of the PHD neurons was determined from the legacy N2Y EM series 13. Circuit diagrams were generated using Cytoscape 74. PHD neuronal maps and connectivity tools are available at www.wormwiring.org.
Author contributions
L.M.G. performed, analysed and interpreted the behavioural experiments; B.K. identified neuronal characteristics in PHD and generated the oig-8 reporter constructs; S.J.C. with S.W.E reconstructed the connectivity; R.B. performed the sex-transformation experiments and with M.S. and J.M.O. performed the characterization of the PHso1-to-PHD molecular transdifferentiation; S.P.R.G scored grl-2 expression and histone condensation in larvae; D.J.E. provided technical assistance; D.H.H. provided and analysed the electron micrographs; A.B. conceived, performed and interpreted the behavioural and Ca2+ imaging experiments and co-wrote the manuscript; R.J.P. conceived and performed the analysis of the PHso1-to-PHD transdifferentiation and co-wrote the manuscript.
Movie Legends
Movies 1 and 2: Imaging of neuronal activity in PHD neurons with GCaMP6f (left channel) and RFP (right channel) in restrained animals. Animals are expressing a oig-8::GCaMP6f::sl2::rfp transgene. Movies play at 100 fps (recorded at 20 fps).
wildtype male
unc-51(e359) male expressing a histamine-inducible silencing transgene in muscle (myo-3::HisCl1::mCherry) and treated with 20 mM histamine.
Movies 3 and 4: Males performing Molina manoeuvres during mating. Movies are played at 40 fps.
wildtype male performing a Molina manoeuvre during mating with a paralysed unc-51(e359) hermaphrodite.
PHD-ablated male performing a defective, discontinuous Molina manoeuvre.
Acknowledgements
We thank Mario de Bono and Zoltan Soltesz for kindly sharing custom-made software for ratiometric analysis of Ca2+ imaging in moving animals; Rene Garcia for worm cartoons; Ken Nguyen for help with EM; John G. White and Jonathan Hodgkin for their help in transferring archival TEM data from the MRC/LMB to the Hall Lab at the Albert Einstein College of Medicine for long-term curation and study; Shai Shaham for sharing the mir-228::gfp strain prior to publication; Mike Boxem, María Lázaro-Peña and Cori Bargmann for additional strains and reagents; Sheila Poole for edits on the manuscript; Baris Kuru for aid with behavioural experiments. Additional strains were obtained from the CGC, which is supported NIH grant P40 OD010440. Christopher Brittin was influential in designing and creating www.wormwiring.org. This work was supported by a Newton Fellowship from the Royal Society to L.M.G., a Wellcome Trust PhD studentship to R.B., Marie Curie CIG grant 618779 and Wellcome Trust Enhancement Funding (095722/Z/11/A) to R.J.P., NIH R01 GM066897 grant and the G. Harold & Leila Y. Mathers Charitable Foundation to S.W.E, NIH OD 010943 to D.H.H., NIH T32GM007491 and F32 MH115438 01 to S.J.C.; R.J.P. was supported by a Wellcome Trust Research Career Development Fellow (095722/Z/11/Z) and is currently a Wellcome Senior Fellow in Basic Biomedical Science (207483/Z/17/Z). A.B and R.J.P are members of COST Action BM1408.
Footnotes
This paper is dedicated to the memory of John Sulston