P9.p fusion with hyp7 during the mid-to-late L2. In all panels showing GFP fluorescence, an unfused Pn.p cell expresses AJM-1–GFP (observed as a green line at the ventral side of the cell, toward the bottom of the figure). The junction of adjacent unfused Pn.p cells is marked by a bright dot (arrowhead). (A–C) Early L2. Unfused P(3–6).p (A) and P(9–11).p (B, C) with AJM-1–GFP expression. (D, E) Mid-L2. Unfused P9-11.p cells retained AJM-1–GFP expression. (F–H) Mid-late L2. AJM-1–GFP expression was observed in P10.p and P11.p (G, H) but absent in P(5–6).p (F) and P9.p. Left lateral views. Scale bar in A, 20 μm for A–H.

Wnt and EGF signaling cooperate during 1°HCG specification. (A) P11.p lineages in Wnt mutants. Our data suggests that of the five Wnt-like genes in C. elegans, only mutations in lin-44 caused defects in 1°HCG specification. However, the P11.p proliferation defect of lin-44(lf) mutants was mild and P11.p always adopted a non-3° fate. When Wnt activity was further reduced in lin-44(n1792); HS::CAM-1 animals (heat-shocked for 2 hours), P11.p adopted a 3° fate in 2 of 32 animals. n, number of animals in which cell lineages were observed; 3°, cell did not divide or divided once (red); non-3°, cell generated 3–8 descendants (3–6 (yellow); wild-type 7 (light blue); 8 (green), more than 2 (gray)). (B) P11.p lineages in EGF or LIN-17/Frizzled Wnt Receptor mutants. P11.p in 25% of lin-17(n671lf) mutants adopts the 3° fate (wild-type males as compared to lin-17(n671) males, *p=0.0471, Fisher’s Exact Test. Decreased EGF signaling by lin-3 RNAi enhanced the 1° lineage defect of lin-17(lf) mutants and caused P11.p to adopt the 3° fate instead of a non-3° fate more frequently (lin-17(n671); lin-3 RNAi males as compared to lin-17(n671) males, **p=0.0095, Fisher’s Exact Test); while increased EGF signaling by a let-60(gf) mutation prevented 3° fate transformation of P11.p in a lin-17(lf) background, causing P11.p to adopt an abnormal non-3° fate instead of a 3° fate (lin-17(n671); let-60(n1046) males as compared to lin-17(n671) males, *p=0.0471, Fisher’s Exact Test). Color scheme as in (A). (C) P10.p lineages in Wnt signaling mutants. P10.p in animals with lower levels of Wnt or that carried a lin-17/Frizzled null allele often adopted the 3° fate (wild-type males as compared to lin-44(n1792); egl-20(hu120) males, ***p<0.0001, Fisher’s Exact Test; HS::CAM-1 males heat-shocked for 45 minutes as compared to lin-44(n1792); HS::CAM-1 males heat-shocked for 45 minutes, **p=0.0010, Fisher’s Exact Test; HS::CAM-1 males heat-shocked for 45 minutes as compared to lin-44(n1792); HS::CAM-1 males heat-shocked for 2 hours, **p=0.0003, Fisher’s Exact Test; wild-type males as compared to lin-17(n671) males, ***p<0.0001, Fisher’s Exact Test; wild-type males as compared to lin-17(n671) males, ***p<0.0001, Fisher’s Exact Test. In addition, in lin-17(lf) males in which P10.p divided, P10.p generated an abnormal non-3° fate with 3-to-8 descendants. Color scheme as in (A), however, for non-3°, cell generated 3–7 descendants (yellow) and wild-type 9 descendants (dark blue).

lin-17::GFP and BAR-1–GFP expression in the HCG. (A–B) Wild-type transcriptional lin-17::GFP expression (A1–2) Early L3. lin-17::GFP in P10.p was barely detectable but stronger in P11.p. No expression was detected in P9.p. (B1–2) Mid-L3. P11.p descendants had brighter lin-17::GFP expression than P10.p descendants. Pn.px refers to Pn.pa and Pn.pp. (C–F) Wild-type dynamic BAR-1–GFP expression in P11.p. (C1–2) L1. Faint BAR-1–GFP expression observed in P12 daughters but not in the undivided P11. (D1–2) Late L1. Faint BAR-1–GFP expression observed in P11.p. (E1–2) Mid-L2. Bright cytoplasmic punctate GFP granules (small arrowheads) and faint nuclear GFP expression in P11.p. (F1–2) Mid-L3. BAR-1–GFP expression in P11.p became predominantly nuclear. (G1–2) Early L3 lin-17(lf) mutant. No BAR-1–GFP was observed in P11.p. Panels (D2), (F2) and (G2) were exposed for longer than images in the other panels. In fluorescence images, cells are outlined based on corresponding Nomarski images. P11.p (large arrow), P12.pp corpse (large arrowhead), other cells (small arrows). Left lateral views. Scale bar in A1, 20 μm for A–H.

Development of the male hook sensillum competence group (HCG). (A) Cell division patterns of P(9–11).p, adapted from Sulston et al. (1980). so, socket cell; sh, sheath cell. Three-letter names refer to specific neurons. (B) HCG divisions during the L3 stage. Left lateral views. } indicates sister cells, L indicates left plane, R indicates right plane. (C) Mid-L2. Distances from P9.p to P10.p and from P10.p to P11.p are similar. (D) Early L3. P10.p and P11.p migrate posteriorly. (E) Late L4. P10.papp, the hook structure cell, formed an invagination (arrowhead) just anterior to the anus. HOA and HOB are hook neurons generated by the 2°P10.p lineage. (F) Adult sclerotic hook structure (arrowhead). (G, H) eat-4::GFP expression in PVV, a P11.p (1°) descendant. (I) osm-6::GFP in HOA and HOB. (J) ceh-26::GFP in HOB. For B–J: Left lateral views (anterior left, ventral down). Cell nucleus (arrows). Scale bar in I, 20 μm for C–J. (K) Arrangement of nuclei in the adult, adapted from Sulston et al., 1980. Ventral view.

The lin-15(e1763) mutation causes a partial 2°-fate transformation of P9.p in males with wild-type P12 specification. (A–C) The P10.p-derived wild-type hook invagination (h-in) was accompanied by a pair of hook neurons HOA and HOB, expressing osm-6::GFP (n=23). However, the P9.p-derived ectopic hook invagination was not accompanied by a pair of neurons expressing osm-6::GFP. Left lateral views. Scale bar in C, 20 μm for A–C.

Abnormal HCG lineages in lin-17(lf) males. (A) End of L3 lethargus in wild type, cell divisions of P10.p and P11.p were complete. (B) An early L4 lin-17(n671) male, just after the L3 molt, in which P11.p and P10.p adopted an uninduced 3° fate. P10.px refers to P10.pa and P10.pp. (C, D) A hookless n671 adult with five eat-4::GFP-positive neurons (1°). (E) A L4 lin-17(n671); lin-12(gf)/lin-12(null) male in which P(9–11).p had proliferated in response to the activated LIN-12 pathway but the alignment of cells was abnormal, indicating a failure to differentiate correctly due to the lack of LIN-17 function. Left lateral views. Scale bar in A, 20 μm for A–E.

Comparison of VPC and HCG patterning networks in C. elegans and Pristionchus pacificus. In the C. elegans hermaphrodite, the EGF signal is produced by the anchor cell and induces the 1°VPC fate. The Wnt pathway is required for VPC competence and has a minor role in induction. In the C. elegans male, the EGF and Wnt pathways participate in HCG specification. However, the relative contributions of these two pathways in hook development are likely different from their contributions in vulval development, as Wnt signaling plays a relatively major role in this process. In response to a high level of Wnt and EGF signal(s), the LIN-17 and LET-23 receptors, respectively, on the cell surface of P11.p activate downstream pathways to specify the 1° fate, which produces ligands (DSL) for LIN-12/Notch. In P10.p, activated LIN-12/Notch signaling by the adjacent 1°P11.p cell acts with a weak Wnt and/or EGF signal to promote the 2°HCG fate. P9.p receives little (if any) signal, and therefore usually fuses with hyp7, adopting a 3° fate. In P. pacificus, different Wnt ligands act to induce as well as inhibit vulval development. A lateral signal from P6.p induces P5.p and P7.p. to adopt the 2° fates. It is not known if this is mediated by LIN-12/Notch.