Fig 1.
(A) The C. elegans cell lineages of the three blast cells (V5, V6 and T) that make the rays (GPs) and phasmids. Three cells make each ray: one becomes the “structural cell” (st) and the other two become A- and B-type neurons (inset). After the anlagen for the GPs originate in the lateral hypodermis in L3, the ray cells cluster in early L4. Rays then differentiate, leaving the ray structural cells at the lateral surface (mid-L4) at about the same time as tail tip morphogenesis occurs [13]. After male tail morphogenesis is complete, the adult has finger-like rays in the same anteroposterior order as the structural cells in the mid-L4 stage [11]. The rays were initially named 1-9, counting anterior to posterior in adult males. Names for homologous GPs are v1-v7, ad and pd [14]. Ray 5 (homologous to ad) is derived from a lineage posterior to that of ray 6. That is, R6 (= V6.ppppa) and R5 (= V6.ppppp) are labeled out of numerical order, with R6 being anterior instead of posterior to R5 in the cell lineage and relative positions of Rn cells in the lateral epidermis. Originally, Sulston & Horvitz [15] had identified V6.ppppa as contributing to ray 6 and V6.ppppp to ray 5 in C. elegans. In their paper more fully characterizing C. elegans male development, Sulston et al. [16] labeled the Rn cells in anteroposterior order (V6.ppppa as R5 and V6.ppppp as R6), but this was subsequently reversed (Fig 2 of [17]) to be consistent with the relative positions of the resulting rays in adult C. elegans males. For consistency, we follow the most recent (out-of-order) labeling of these Rn cells and their descendants for all of the species characterized here. (B) The Panagrellus redivivus male cell lineages of the blast cells V6 and T. This species has three T cells: T1 on the left side and T2 + T3 on the right side. T1 has the lineage depicted here; T2 + T3 contribute to a similar lineage. V5 does not produce a GP; the most anterior lineage from V6 makes seam cells instead of a GP. Thus, adult males have 7 GPs and the homologs of v1 and v2 are absent in this species. (C) Schematic of the archetypical arrangement of GP cell groups and epidermal Rn.p cells early and late during GP development. The GP cell groups are color coded as in Figs 7–11). The archetype allows assignment of ray homologies across species [11,14]. (D) Two hypotheses for how phasmids in the anterior position could develop (see text for further details).
Fig 2.
Male tail character evolution on the phylogeny of Rhabditina.
The phylogeny is based on molecular characters [31], with taxa designated as in [34]. See text for more information about the phylogeny and character tracing.
Table 1.
Cell ablation experiments1.
Fig 3.
Results of ablations of T lineage cells in Pelodera strongyloides DF5022.
(A) DIC image (top) and schematic drawing (bottom) of the tail of an early L1 larva before the first division of T. Some nuclei are outlined. Numbers in the nuclei refer to the hyp cells in the tail tip. (B) DIC image (top) and schematic drawing (bottom) of the tail of an L1 larva after the first division of T. Colored arrows indicate which cells were ablated, scale bars in A and B = 10 µm. (C) Drawing of the tail of an adult male in ventral view. A blue X indicates the GPs missing upon ablation of T.a, a green X indicates the phasmid that is missing upon ablation of T.p. (D, E, F) Examples of the result upon ablation of T or its progeny. DIC images of three focal planes taken in ventral view are shown for each animal. Scale bars = 20 µm. (D) Male #44 (see Table 1): after ablation of the right T cell, the phasmid and the three posterior (T) GPs are missing on the right side (asterisk); the left phasmid and T GPs are present and pd is out of focus. (E) Male #46: after ablation of the left T.a; the left three T GPs are missing and phasmids are present on both sides. (F) Male #13: after ablation of the T.p; the left phasmid is missing and all T GPs are present. Raw image data are provided in S1–S5 Figs.
Fig 4.
Results of ablations of cells in the T lineage in Cruznema tripartitum SB202.
(A) DIC image (top) and schematic drawing (bottom) of the tail of an early L1 larva before the first division of T. Some nuclei are outlined. Numbers refer to the hyp cells in the tail tip. (B) DIC image (top) and schematic drawing (bottom) of the tail of an L1 larva after the first division of T. (C) DIC image and schematic drawing of DIC image (top) and schematic drawing (bottom) of the tail of an L1 larva after the second division of T. Colored arrows indicate which cells were ablated, scale bars in A, B, C = 10 µm. (D) Drawings of the tail of an adult male in ventral (left) and lateral (right) view. A blue X indicates the GPs missing upon ablation of T.a and a green X indicates the phasmid that is missing upon ablation of T.p; scale bar 20 µm. (E, F, G) Examples of the result upon ablation of T or its progeny. DIC images of three focal planes are shown for each animal. Scale bars = 20 µm. (E) Male #17 (ventral view): after ablation of the right T cell, the phasmid and two of the four posterior GPs are missing on the right side (asterisk). (F) Male #31 (ventral view): after ablation of the left T.p, the left phasmid is missing (asterisk). (G) Male #25 (left side view): after ablation of the left T.ap, two of the four posterior GPs are missing on the left side (asterisk). Raw image data are provided in S6–S11 Figs.
Fig 5.
Results of ablations of cells in the T lineage in Diplogasteroides nasuensis SB335.
(A) DIC images (top) and schematic drawing (bottom) of the tail of an early L2 larva right after hatching. The top DIC image is focused on the apical surface and shows the location of the phasmid opening. The identity of the T-lineage cells as marked in the schematic was derived from the ablation results. Colored arrows indicate which cells were ablated, scale bar = 10 µm. (B) Schematic drawing of the tail of an adult male in ventral view. Red Xs indicate GPs that are missing when V5.p is ablated, a blue X marks GPs that were missing when the cell identified as T.ap is ablated, and a green X marks the phasmid that is missing when T.pa is ablated. (C) Male #36 (tail in ventral view): after ablation of the left V6.p cell, five GPs are missing. The remaining rays are v1 near the cloaca and three in the posterior (v6, v7 and pd). (D) Male #18 (posterior end of the tail in ventral view): after ablation of the left T.ap, three GPs (v6, v7, pd) are missing but the left phasmid is present. Raw image data are provided in S12–S14 Figs.
Fig 6.
Results of ablations of cells in the T lineage in Poikilolaimus oxycercus EUK103.
(A) DIC image (top) and schematic drawing (bottom) of the tail of an L1 larva after the first division of T. (B) Male after ablation of the left T.a: the left posterior GPs are missing (asterisk). (C) Schematic drawing of a P. oxycercus male in ventral view. Scale bar in A = 10 µm, in B = 20 µm. Raw image data are provided in S15, S16 Figs.
Fig 7.
MH27 antibody staining of adherens junctions in thetail of male P. oxycercus (strain EUK106) larvae.
Reconstructions of the adherens junction patterns (top) and part of the z-projected photomicrographs it is based on (bottom) for (A, B) two early L3 males, (C) a stage 1 male and (D) a stage 3 male (stages as in [11]). (E) Schematic drawing of an adult male. In (D), the photomicrographs are from two different z-plane merges separated by a line. Cells and GP cell groups are labeled based on our hypotheses for the homologies of these structures, and cells from the same lineage are marked with the same color. P. oxycercus has only 8 GP cell groups. In (B–D), a large undivided cell (brown) that we think is R2 separates the most anterior GP group (v1) from the next (v3). Raw image data are provided in S17–S20 Figs.
Fig 8.
MH27 antibody staining of adherens junctions in the tail of male H. subulatum SB303 larvae.
Reconstructions of the adherens junction patterns of (A, B) males early during GP development, (C) a stage 1 male and (D) a stage 3 male (stages as in [11]). (E) Schematic drawing of an adult male. In (C and D), part of the z-projected photographs is shown that are the basis for the reconstruction. The most anterior GP cell group (V1) was outside of the captured images in (B–D). In this species, the phasmid opening is positioned anterior of GP groups v5–7 throughout GP development. Raw image data are provided in S21–S24 Figs.
Fig 9.
MH27 antibody staining of adherens junctions in the tail of male D. nasuensis SB335 larvae.
Reconstructions of the adherens junction patterns (top) and part of the z-projected photomicrographs on which it is based (bottom) for (A, B) two L3 males, (C) a stage 1 male, and (D) a stage 3 male. (E) Schematic drawing of an adult male, left side. Raw image data are provided in S25–S28 Figs.
Fig 10.
MH27 antibody staining of adherens junctions in the tail of male B. saprophaga SB261 larvae.
Reconstructions of the adherens junction patterns (top) and part of the z-projected photomicrographs on which it is based (bottom) for (A) an early male, (B) a stage 1 male and (C) a stage 3 male (stages as in [11]). (D) Schematic drawing of an adult male. In C, the box shows a separate set of merged z-slices. In early males, the phasmid opening is located anterior of all GP groups. By stage 3, it is seen anterior of the three most posterior ray groups v5, 6 and 7. This is where it is found in adult males and (D) a stage 3 male. (E) Schematic drawing of an adult male. The phasmid opening is positioned anterior of the three posterior GP precursor cells before GP development begins. Raw image data are provided in S29–S31 Figs.
Fig 11.
Reconstruction of the AJ pattern of Pelodera strongyloides and C. elegans males early during GP development.
(A) P. strongyloides after the first division of most Rn cells [11]. (B, C) Drawing after the ajm-1::GFP images from WormAtlas [39] for C. elegans males before (A) and after (B) the first division of the Rn cells.
Fig 12.
Photomicrograph of a P. oxycercus male with two ectopic GPs on the left body side.
Raw image data are provided in S32 Fig.