The PVD neuron has male-specific structure and mating function in Caenorhabditis elegans

Abstract

Neurons display unique shapes and establish intricate networks, which may differ between sexes. In complex organisms, studying sex differences in structure and function of individual neurons is difficult. The nematode Caenorhabditis elegans hermaphrodites and males present an exceptional model for studying neuronal morphogenesis in a simple, sexually dimorphic system. We focus on the polymodal sensory bilateral neuron pair PVD, which forms a complex but stereotypic dendritic tree composed of multiple subunits that resemble candelabra. PVD is well studied in hermaphrodites, but not in males. We show here that during larval development, male PVD extends a similar architecture to the hermaphrodite utilizing the sexually shared Menorin patterning mechanism. In early adulthood, however, male PVD develops a unique extension into the copulatory tail structure. Alongside established tail ray neurons RnA and RnB, we show PVD is a third, previously unrecognized, neuron within the tail rays. Unlike RnA and RnB, PVD extends anterogradely, branches and turns within the ray hypodermis, and is nonciliated. This PVD sexually dimorphic arborization is absent in mutant backgrounds which perturb the Menorin guidance complex. SAX-7/L1CAM, a hypodermal component of this complex, shows a male-specific expression pattern which precedes PVD extension, and its presence allows PVD to enter the tail rays. Further, our results reveal that genetically altered arborization or ablation of the PVD results in male mating behavioral defects, particularly as males turn around the hermaphrodite. These results uncover an adult-stage sexual dimorphism of dendritic branching and a function for PVD in male sexual behavior.

Keywords: Caenorhabditis elegans; mating behavior; neuronal arborization; neuronal morphogenesis; sexual dimorphism.

Fig. 1.

WT male PVD development shows postlarval sexual dimorphism. (A–C) Hermaphrodite PVD development in L4 through to adult. PVD branch orders are marked yellow in panel C. Anterior is Left, ventral down. ser2prom3::GFP PVD marker. (Scale bar, 50 µm.) (D–F) Male PVD development in L4 through to adult; white numbers and arrows note tail rays; PVD branch orders are marked in yellow in panel F. ser2prom3::GFP PVD marker. Asterisk notes PVD branching into the phasmids. (Scale bar, 50 µm.) (G) Analysis of PVD branches. t test, Holm–Šídák multiple comparison correction. Black bars show mean, error bars show ± SD. *P < 0.05, **P < 0.01, ***P < 0.001; ****P < 0.0001. (H) Ectopic branches around the cell body region in 1-d adult animals, normalized per 100 µm of the primary branch for the different branching orders; n = 16 (6 PVDL,10 PVDR) hermaphrodite and n = 12 (7 PVDL, 5 PVDR) males. t test, Holm–Šídák multiple comparison correction. Black bars show mean, error bars show ± SD (except male ectopic tertiaries and ectopic quaternaries, showing + SD). (I) Cartoon depiction of the male tail organ; lateral view contains a tracing of the neuron in panel F: projection of three-dimensional trace, branch orders annotated blue (1°), purple (2°), red (3°), cyan (4°), and yellow (5° and 6° order ectopics). (Scale bar, 50 µm.) PVD branch orders are marked in black following panel F. Ventral and dorsal cartoons are not drawn to scale.

Fig. 2.

PVD processes are not ciliated and enter the rays after, but not over, RnB ciliated ray neurons. (A–D) Examples of him-5(e1490) male tails expressing the RnB neuron marker pkd-2::GFP (expressed in all but R6B; cyan), and pF49H12.4::mCherry PVD marker (magenta). (A) Lateral view of an L4 WT. RnB dendrites enter the rays using a retrograde mechanism (23, 51, 55) before the PVD initiates its anterograde extension. Rays 4 and 5 marked for orientation purposes. (Scale bar, 25 µm.) (B) Ventral view of an adult WT (1 to 2 d, A1-2d). In the majority of rays and RnB axon paths (56), PVD (pF49H12.4::mCherry) and RnB (pkd-2::GFP) processes show zero overlap (75/144 RnB dendrites and 65/66 RnB axons), with partial-length, minimal overlap in the remaining cases. When quantifying tapered tip morphology in these animals, 3/67 ray PVD processes show thinner endings compared with 101/124 RnB dendrites. A ciliated tip example of R2B (Top) is highlighted by a white bracket in both the panel and its inset. Rays 4, 5, and 6 indicated for orientation. The orange arrowhead highlights a U-turn example, yellow arrowheads highlight intraray branching, and the white arrowhead points to a nonoverlapping PVD dendritic branch and R2B/R3B axon(s) (56). One ray 3 is missing [occasional in WT, (16)] with R3B localized to its base and PVD projecting alongside (asterisk, Top). (Scale bar, 25 µm; Inset Scale bar, 5 µm.) (C) Lateral view of an 1 to 2 d adult (A1-2d) WT him-5(e1490) animal expressing PVD (pF49H12.4::mCherry) and RnB (pkd-2::GFP) markers. Rays 4, 5, and 6 are indicated for orientation. The orange arrowhead highlights a U-turn example; the white arrowhead points to a nonoverlapping PVD dendritic branch and R3B dendrite, in both the panel and its inset. (Scale bar, 25 µm; Inset Scale bar, 10 µm.) (D) Lateral view of an adult mab-21(bx53); him-5(e1490) male expressing PVD (pF49H12.4::mCherry) and RnB (pkd-2::GFP) markers. Rays 4 and 6 are “fused,” with ray 6 translocating anteriorly to ray 5, to join ray 4 (dashed arrow). Note that, unlike the WT (56), this fusion causes ray 6 to express pkd-2::GFP [ray 6 pkd-2::GFP signal appears in 15/15 mab-21(bx53), 0/23 WT]. A single PVD branch enters the fused ray and performs a U-turn (orange arrowhead). (Scale bar, 25 µm.) (E) Approximate RnB cilium lengths (cyan) and distance from the distal end of PVD to the tip of RnB (black) in WT him-5(e1490) 1 to 2 d adults expressing pkd-2::GFP RnB marker (cyan cartoon) and pF49H12.4::mCherry PVD marker (magenta cartoon), measured based on fluorescent signal. R6B is not analyzed owing to absence of pkd-2::GFP signal. n = 116 tapering RnB measurements and 127 RnB-PVD dendrite pair distances. Red lines indicate medians. (F) Progression of intraray branching (yellow) and U-turn (orange) phenotypes of PVD within open rays (i.e., all but ray 6, Left), and the closed ray 6 (Right), during the first three days of adulthood, in WT him-5(e1490); ser2prom3::GFP males. A U-turn is defined by the distal tip executing a complete folding back and continuing medially toward the body, whereas a Branched process is any forked endpoint extending from a branch within the ray. A1d = one day adult; A2d = two day adult; A3d = three day adult. n = 62 A1d,186 A2d and 108 A3d open rays and n = 12 A1d, 37 A2d and 22 A3d ray 6 cases scored for the three age groups, respectively. Fisher’s exact test comparing A1d to A3d and straight to “wandering” branched, ****P < 0.0001. (G) PVD does not express the ciliated neuron reporter osm-6p::OSM-6::GFP. Tail region of an adult him-5(e1490) male expressing osm-6p::OSM-6::GFP, targeted to ciliated endings, and pF49H12.4::mCherry PVD marker. No colocalization was detected in n = 94 rays observed across 13 animals. Rays 2 and 6 indicated for orientation. (Scale bar, 25 µm.) Inset of dashed orange rectangle: examples of two cilia (brackets) and their transition zones (TZ). (Scale bar, 10 µm.) (H) Tail region of an adult him-5(e1490) male expressing osm-6p::GFP, expressed in ciliated neurons including PDE and PQR, and pF49H12.4::mCherry, expressed in PVD, AQR, and PQR. 0/15 adult males show colocalization of the two markers in the PVD soma (see also SI Appendix, Fig. S6). Rays 2 and 6 indicated for orientation. (Scale bar, 25 µm.) Inset of dashed orange rectangle: example of R2A and R2B converging in a ray. White arrows in the inset point to the two neurons. (Scale bar, 5 µm.)

Fig. 3.

Male PVD enters the tail rays outside the neuron channel and shares mutant phenotypes with hermaphrodite-characterized morphogenesis genes. (A) Cartoon depiction of a longitudinal section across an open male tail ray, noting hypodermis (hyp), structural cell (str), fan cuticle, ray neurons RnA (magenta), RnB (orange) and their cilia, and PVD (cyan). Based on (16). (B) Transmission electron microscopy image of a thin cross-section of a young adult WT ray 6, noting hypodermis (hyp), structural cell (str), fan cuticle and ray neurons RnA (magenta), RnB (orange), and PVD (cyan) as in panel A. (Scale bar, 1 µm.) (C) Top: ventral view of a two-day adult male, ser2prom3::GFP PVD marker. (Scale bar, 50 µm.) Bottom: cartoon tracing of respective PVDL (blue) and PVDR (green). Arrows denote PVD branching into the rays. (D) Male one-day adult lin-44(n1792); him-5(e1490) with ser2prom3::GFP PVD marker. Yellow arrows indicate a ray with PVD signal; white arrows indicate rays without PVD signal. (Scale bar, 50 µm.) (E) Male one-day adult mec-3(e1338); him-5(e1490) expressing ser2prom3::GFP PVD marker; the orange arrowhead marks the posterior-most branch. A green arrow points to a process of the PDE (31, 65), most visible here but also present in panel D and Fig. 1 D and E (also see Materials and Methods). (Scale bar, 50 µm.) (F) Male one-day adult sax-7(dz156) expressing pF49H12.4::GFP PVD marker; yellow arrow points to example of characteristic altered structure, and the orange arrowhead marks the posterior-most branch. (Scale bar, 50 µm.) (G) Male one-day adult mnr-1(dz175) expressing pF49H12.4::GFP PVD marker; yellow arrows point to examples of characteristic altered structure, and the orange arrowhead marks the posterior-most branch. (Scale bar, 50 µm.) (H) Male one-day adult dma-1(tm5159) expressing pF49H12.4::GFP PVD marker; the yellow arrow points to characteristic altered structure, and the orange arrowhead marks the posterior-most branch. (Scale bar, 50 µm.) (I) Male one-day adult lect-2(ok2617) expressing ser2prom3::GFP PVD marker; the yellow arrow points to characteristic altered structure, and the orange arrowhead marks the posterior-most branch. (Scale bar, 50 µm.) (J) mab-20(bx61ts); him-5(e1490) 1-2-day adult expressing ser2prom3::GFP PVD marker at a semirestrictive 22 °C temperature; the asterisk marks a characteristic “fusion” joining rays 3 and 4. Yellow arrows indicate rays with PVD signal, white arrows indicate rays without PVD signal. (Scale bar, 50 µm.) (K) PVD genetic ablation ser2prom3::deg-1(u662); him-5(e1490) two-day adult expressing ser2prom3::Kaede PVD marker showing signal remnant (arrow) next to the PVD soma, corresponding to PDE, with ventral truncated axon-like process. (Scale bar, 50 µm.) (L) Quantification of PVD entry into the tail rays in different mutant backgrounds and ages; all animals assayed express the integrated ser2prom3::GFP PVD marker and are him-5(e1490), with the exception of deg-3(u662) genetic ablation which utilizes a Kaede fluorescent marker, lect-2(ok2617) which has no him-5 background, and sax-7(dz156), mnr-1(dz175), and dma-1(tm5159) which express an integrated pF49H12.4::GFP PVD marker and are without him-5(e1490); YA: young adult; A1d, A1.5d, A2d, A3d, A4d: 1, 1.5, 2, 3, and 4-day adult, respectively. Day 1 adult = L4 + 1 d. n = 15, 28, 10, 21, 11, 13, 13, 10, 5, 10, 15, 12, 12, 13, respectively. For mab-20(bx61ts), one day from L4 at room temperature is comparable to L4 + 28 h at 20 °C and as such marked 1.5 d adult. One-way ANOVA, Šídák multiple comparison correction. Black bars show mean, error bars show ± SD. **P <0.01, ****P <0.0001.

Fig. 4.

SAX-7 in the hypodermis is required for PVD extension into the rays. (A) Male L4 tail with sax-7p::sax-7::GFP reporter (inverted). Signal observed in 43/43 nascent rays. (B) Matching DIC images to panel A. (C) Ventral view of a male adult tail with reporter (inverted). (D) Matching DIC images to panel C. Red arrows point to tail rays. (Scale bar, 50 µm.) (E) Adult lateral view of merged sax-7 reporter (magenta) with PVD marker (cyan), “inverted” pseudocolors are used for clarity. 113/113 one-day adult rays observed with SAX-7 signal, of which 79/113 expressed PVD signal. (F) One day adult sax-7(dz156); him-5(e1490) male expressing pF49H12.4::GFP PVD marker, nontransgenic (Non-Tg) control for animals carrying a dpy-7p::sax-7(s) short variant transgene (29, 30). The orange arrowhead marks the posterior-most branch, nontransgenic (Non-Tg) control. (Scale bar, 50 µm.) (G) One day adult sax-7(dz156); him-5(e1490) male expressing pF49H12.4::GFP PVD marker, transgenic (Tg) animal expressing dpy-7p::sax-7(s) short variant (29, 30), determined by coinjection marker (pharyngeal muscle myo-2p::GFP) presence. White arrows show PVD entry into rays 3 and 6. (Scale bar, 50 µm.) (H) Analysis of PVD entry into the male tail rays in animals carrying extrachromosomal dpy-7p::sax-7(s) short variant (29, 30) comparing transgenic (Tg) with nontransgenic (Non-Tg) animals based on coinjection marker expression (pharyngeal muscle myo-2p::GFP). Unpaired t test, *P < 0.05. A1d one-day adult animals are analyzed from two independent transgenic lines (n = 16, 10, 16, 14 respectively).

Fig. 5.

Mating behavior is impaired under PVD genetic ablation. (A) Cartoon of mating steps. Male presented in purple, hermaphrodite in green. (B–D) Hermaphrodite recognition, turning, and vulva recognition, respectively, in WT and ser2prom3::deg-3(u662) animals where PVD undergoes early degeneration. (B) n = 18 WT, 18 ser2prom3::deg-3(u662). (C) n = 16 WT, 18 ser2prom3::deg-3(u662). (D) n = 18 WT, 18 ser2prom3::deg-3(u662). Perfect turn is defined as a turn completed successfully without the male tail losing contact with the hermaphrodite. t test, black bars show mean, error bars show ± SD. *P < 0.05, **P < 0.01, ****P < 0.0001.