PMC

Differences in regulation of lag-2 in continuous development and dauer life history. (A) lag-2 regulation during continuous development (). (Left) LIN-1 represses lag-2 expression in VPCs in which Ras signaling is low via a cis-acting sequence called VPCrep. (Right) Deletion of VPCrep or loss of lin-1/Elk1 activity permits transcription of lag-2 in all VPCs (). (B and C) Effect of dauer life history on repression of lag-2 in VPCs. Percentage of larvae in which each VPC expresses the indicated reporter out of the total larvae examined (n). *P < 0.01 compared with L2d-Dauer molt (Fisher’s exact test); ^†P < 0.01 compared with P6.p at L2d-Dauer molt in lin-1(+) (Fisher's exact test).

Effect of dauer formation mutants on down-regulation of lag-2 transcription. (A) Schematic representation of the major signaling pathways that regulate the dauer formation decision (). Boxes indicate active components under the indicated conditions. (B) Percentage of larvae expressing lag-2p::yfp in P6.p at 25 °C (n = 13–37). (C) Percentage of larvae expressing lag-2p::yfp in P6.p at 25 °C (“+” indicates exogenous pheromone was added to create an unfavorable environment condition as in A, Right). (Left) n = 13–34. (Right) Laser ablation (abl) of the gonad (n = 8) compared with mock-ablated (mock) larvae (n = 7). (D) Percentage of larvae expressing lag-2p::yfp in P6.p at 25 °C (“+” indicates exogenous pheromone was present; n = 23–44). *P < 0.01 (Fisher’s exact test). NS, not significant.

Constitutive activity of LIN-12/Notch or LIN-45/Raf appears to be blocked in dauer larvae. (A and B) Percentage of larvae in which each VPC expresses a 1° fate reporter (lag-2p::yfp, red) or a 2° fate reporter (lst-5p::yfp, blue) out of the total larvae examined (n). daf-7(e1372) is present in larvae scored during dauer life history. Similar results were seen in dauer larvae isolated from starved and crowded plates in daf-7(+) strains. All strains were grown at 25 °C. (A) lin-31p::LIN-45[AAED], transgene expressing activated LIN-45/RAF in all VPCs. *P < 0.01 compared with Dauer (row 5) (Fisher’s exact test). (B) lin-31p::LIN-12(intra∆P), transgene expressing activated LIN-12 in all VPCs. *P < 0.01 compared with L2d-D molt (row 4) (Fisher’s exact test).

Cell-fate plasticity in dauer larvae. (A) VPC specification in wild-type hermaphrodites. An EGF-like signal (red) from the anchor cell (AC) activates Ras signaling in P6.p, causing it to adopt 1° fate and produce ligands, including LAG-2 and APX-1, which activate LIN-12/Notch in P5.p and P7.p (). (B) Comparison of cell fate in P5.p, P6.p, and P7.p (ovals) during continuous development (Upper) and dauer life history (Lower). Vertical lines represent molts. Red, 1° fate; blue, 2° fate; purple, multipotential; white, unknown before this work. (C–H) Expression of VPC specification markers in different conditions. Reddish bars indicate 1° fate markers, bluish bars indicate 2° fate markers, and thickened black lines indicate no expression. Expression of VPC identity markers in dauer life history is shown in Fig. S1. (C) *P < 0.01, compared with wild-type L2 (G) (n = 23–42; Fisher's exact test). (D) *P < 0.01, compared with Dauer (n = 16–46; Fisher's exact test). (E and F) Representative images of lag-2p::yfp expression before (E) and during (F) dauer in lin-28(0) larvae. P6.p has generated four descendants in both larvae shown. lag-2p::yfp is also expressed in the gonad (labeled “G”) in continuous and dauer life histories. (G) *P < 0.01, compared with wild-type L2 (n = 22–39; Fisher's exact test). (H) *P < 0.01, compared with Dauer (n = 20–43; Fisher's exact test).

daf-16 promotes cell-fate plasticity in dauer larvae. (A) Percentage of dauer larvae expressing lag-2p::yfp in P6.p at 25 °C. daf-7(e1372) is present in all strains (“+” indicates exogenous pheromone was present; n = 17–71). (B) Percentage of the indicated VPCs that have divided one or more rounds in dauer larvae (n = 30). These divisions occur during dauer because they are never observed at the L2d-Dauer molt (0/29). (C) “Whole worm”: daf-7(e1372) lag-2p::yfp larvae were subject to RNAi for either lacZ (control) or daf-16 (n = 43–63). RNAi works efficiently in all nonneuronal tissues because these larvae are wild-type for rde-1 (, , ). “VPC only”: daf-7 lag-2p::yfp; rde-1(0); lin-31p::RDE-1 larvae were subject to RNAi for either lacZ (control) or daf-16 (n = 22). These larvae are unable to carry out RNAi in any tissue except the VPCs where lin-31p drives expression of rde-1 (indicated by “VPC only”) (, , ). (D) A total of 33/33 daf-7 postdauer L4 larvae displayed normal vulval morphology including the characteristic “Christmas-tree” structure (bracketed in image). In addition, normal vulval morphology was observed in 13/13 daf-16 larvae grown continuously. By contrast, 13/15 postdauer daf-16(0); daf-7 larvae displayed overtly abnormal morphology, such as the lack of Christmas tree. *P < 0.01 (Fisher’s exact test). NS, not significant.