Entry to and exit from diapause arrest in Caenorhabditis elegans are both regulated by a steroid hormone pathway

Diapause arrest in animals such as Caenorhabditis elegans is tightly regulated so that animals make appropriate developmental decisions amidst environmental challenges. Fully understanding diapause requires mechanistic insight of both entry and exit from the arrested state. While a steroid hormone pathway regulates the entry decision into Caenorhabditis elegans dauer diapause, its role in the exit decision is less clear. A complication to understanding steroid hormonal regulation of dauer has been the peculiar fact that steroid hormone mutants such as daf-9 form partial dauers under normal growth conditions. Here, we corroborate previous findings that daf-9 mutants remain capable of forming full dauers under unfavorable growth conditions, and we establish that the daf-9 partial dauer state is likely a partially exited dauer that has initiated but cannot complete the dauer exit decision. We show that the steroid hormone pathway is both necessary for and promotes complete dauer exit, and that the spatiotemporal dynamics of steroid hormone regulation during dauer exit resembles that of dauer entry. Overall, dauer entry and dauer exit are distinct developmental decisions that are both controlled by steroid hormone signaling. Summary Statement In animals such as Caenorhabditis elegans, a steroid hormone pathway controls both the entry and exit decisions into and out of the developmentally arrested dauer state in response to environmental signaling.

To test possibility (1), in which daf-9 partial dauers require unfavorable conditions 187 to become full dauers, we grew daf-9(dh6) mutants under favorable conditions to first 188 form partial dauers and then transferred them to unfavorable conditions to determine if 189 they could form WT-dauers ( Fig. 3A-C). We found that, despite a 24-hour incubation 190 under unfavorable conditions, daf-9(dh6) partial dauers did not transition towards a full 191 dauer state. These daf-9(dh6) animals continued to move and pump at high rates in 192 comparison to daf-9(dh6) full dauers. Thus, we find it unlikely that the partial dauer state 193 obtained under favorable growth conditions represents a transition state that is en route 194 to becoming full dauer. 195 To assess possibility (2), in which daf-9 partial dauers are first full dauers that 196 then partially exit dauer, we grew daf-9(dh6) mutants under unfavorable conditions to 197 first form full dauers, and then we transferred them to favorable conditions to examine if 198 they became partial dauers. We found that 24 hours post-transfer, daf-9(dh6) larvae 199 actively pumped, moved significantly more, and had an enlarged pharynx compared to 200 before the transfer (Fig. 3D-G, and Video 2), thereby recapitulating the daf-9 partial 201 dauer state. These partially exited dauers slowly continued to grow radially and develop 202 a larger pharynx even past the 24-hour mark, although they never develop into healthy 203 reproductive adults (data not shown). We obtained similar results using daf-9 alleles 204 e1406 (another putative null mutation) and m540 (a weaker loss-of-function allele) 205 ( Fig. 3E, F). From both mutants, we obtained full dauers under unfavorable growth 206 conditions that became partial dauers following transfer to favorable conditions. The 207 major allele-specific difference was that daf-9(m540), which only has a mild Daf-c 208 phenotype, was sometimes able to form healthily exited dauers rather than partially 209 exited dauers upon returning to favorable growth conditions (see Fig. 3F, m540 210 pumping frequency). Together, these findings suggest that transfer to favorable 211 conditions causes full daf-9 dauers to initiate dauer exit and engage in concomitant 212 behavioral and morphological changes such as increased pumping, motility, and 213 pharyngeal expansion. Strong loss-of-function mutants such as daf-9(dh6) and 214 daf-9(e1406) are halted in this exit process, while weaker loss-of-function mutants like We also examined whether this partial dauer exit phenotype could be 217 recapitulated at the level of the nuclear hormone receptor DAF-12/FXR, which acts 218 genetically after DAF-9 to specify the dauer decision. Although most loss-of-function 219 alleles for daf-12 are Daf-d, there exists numerous Daf-c alleles that bear mutations 220 altering DAF-12's putative ligand binding domain (Antebi et al., 1998(Antebi et al., , 2000. These 221 Daf-c strains have also been described to form partial dauers under favorable growth 222 conditions, although at a lower penetrance than daf-9 putative null mutants (Antebi et al., 223 1998). We took such a Daf-c mutant, daf-12(rh273), and found that we were able to 224 induce full dauers under unfavorable growth conditions that could become partial 225 dauers upon transfer to favorable conditions (Fig. S1B, C). Therefore, daf-12(rh273) 226 mutants can phenocopy the partial dauer exit phenotype of daf-9 putative null mutants, 227 consistent with DAF-12/FXR mediating this phenotype. 228 A feature of dauer exit is its irreversibility: wild-type dauers that have been shifted 229 to favorable conditions commit to dauer exit within an hour since shifting them back onto 230 unfavorable conditions afterwards cannot maintain or restore the dauer state (Golden 231 and Riddle, 1984). We asked whether the daf-9 dauer-like state represented a 232 committed or irreversible state of partial dauer exit, or if a return to unfavorable 233 conditions could cause the animal to become a full dauer again. We grew daf-9(dh6) 234 mutants under unfavorable conditions to induce full dauers, transferred the resulting 235 dauers to favorable conditions to stimulate partial dauer formation, and then transferred 236 them back onto unfavorable conditions to see if they could become full dauers again 237 ( Fig. S1D-F). Transfer into unfavorable conditions neither dramatically altered pumping 238 rate nor movement speed compared to the mock transfer control, nor did it produce 239 larvae that were similar to full dauers, even 24 hours after a return to unfavorable 240 conditions. This observation suggests that partial dauers may be animals that have 241 committed to, but can only partially complete, dauer exit. 242 243 Evaluating if daf-9 partial dauers pass through a transient state of full dauer 244 Under the hypothesis that daf-9 partial dauers were once full dauers that then 245 partially exited, it should be possible to observe daf-9 mutants pass through a period of 246 being full dauers before they become partial dauers. We thus grew daf-9(dh6) mutants under favorable conditions and scored animals every two hours as being L2d, full dauer, 248 or partial dauer based on important metrics such as pharyngeal pumping, locomotion, 249 and morphology (Fig. 4A, B, and see Materials and Methods). Pharyngeal pumping 250 was the most prominent and earliest cue for scoring partial dauers. As a control, we 251 also grew daf-9(dh6) and wild-type animals under unfavorable conditions in parallel. To 252 maintain synchrony across the different growth conditions, we grew all animals at a high 253 temperature of 25.5°C but withheld pheromone from the daf-9(dh6) mutants grown 254 under favorable conditions. Although growth at 25.5°C favors dauer formation, it alone 255 cannot induce dauer formation in wild-type animals (Ailion and Thomas, 2000). At 44 256 hours post egg-lay, the vast majority of animals were L2d (Fig. 4A). By 49 hours, 257 around 25% of daf-9(dh6) mutants grown in the absence of pheromone could be scored 258 as full dauers, while by 52 hours, a surprising 75% of animals were found to be full 259 dauers. After 52 hours, the percentage of full dauers decreased such that by 69 hours, 260 the large majority of animals were partial dauers. In contrast, both the wild-type and daf-261 9(dh6) animals grown under high pheromone conditions showed a steady increase in 262 the proportion of dauers over time, and few if any partial dauers could be found at any 263 time point (Fig. 4A). These results show that a proportion of daf-9(dh6) mutants grown 264 in the absence of pheromone become full dauers for some period of time. To determine what fraction of daf-9(dh6) mutants pass through a transient dauer 266 state, we repeated the above experiment but with single animals. We grew daf-9(dh6) 267 mutants without pheromone at 25.5°C and, after 43 hours post egg-lay, we transferred 268 the resulting L2ds onto new plates without pheromone (one per plate), and we scored 269 individual animals over time (Fig. 4C). In concordance with our bulk tracking assay, we 270 observed full dauers between 45 and 50 hours that later became partial dauers. Of 12 271 tracked animals, we observed seven that went through a period of being full dauers. For 272 these animals, we observed an L2d molt in which the animal detached from and 273 sometimes rolled inside its cuticle (Singh and Sulston, 1978). Afterwards, the animal 274 would cease both movement and pharyngeal pumping before completing radial 275 constriction to become a full dauer. Within a few hours, these dauers slowly began 276 pumping and moving more (a sign of partial dauer exit), but radial expansion did not 277 occur until many hours later (data not shown). Some animals were never observed as having formed full dauers (Fig. 4C), which may be because their transition through full 279 dauers occurred in between time points. 280 We also performed the above single animal observation experiments under more 281 favorable conditions by lowering the temperature to 20°C. However, under these 282 conditions, we were unable to find any daf-9(dh6) larvae that went through a full dauer 283 state, despite making observations every hour (Fig. S2). Following the same L2d 284 molting process that typically precedes full dauer formation, the animal instead passed 285 through an intermediate state that involved both elements of being a dauer (a darkened 286 body) as well as partial dauer (pumping, motility), before becoming well-recognizable 287 partial dauers usually within one hour. These observations suggest that high 288 temperatures facilitate formation of full dauers in daf-9(dh6) mutant animals in the 289 absence of exogenously added pheromone. 290 291
We speculated that a potential reason for the partial dauer exit phenotype could 309 be that a small amount of reproduction-promoting steroid hormones continues to be 310 produced even in daf-9 putative null mutants, and that these steroid hormones might 311 trigger partial dauer exit. We reasoned that withholding cholesterol, a precursor for the 312 vast majority of DAF-12 steroid hormone ligands (Aguilaniu et al., 2016), would hinder 313 partial dauer exit formation and keep larvae in their full dauer state. It has been shown 314 that withholding cholesterol exacerbates the developmental defects of many daf-9 315 partial loss-of-function mutants by restricting precursor availability (Gerisch et al., 2001;316 Jeong et al., 2010). 317 We allowed daf-9(dh6) partial dauers to form using two distinct methods: (1) 318 constant growth on favorable conditions and (2) growth under unfavorable conditions 319 followed by a transfer to favorable conditions. We found that withholding cholesterol 320 from the NGM media did not hinder the formation of partial dauers using either of these 321 methods (Fig. 5C, D). While these results are inconsistent with the hypothesis that non-322 daf-9-dependent reproduction-promoting steroid hormones promote partial dauer exit, it 323 is also possible that there was sufficient cholesterol or sterol derivatives contained in the 324 medium and/or passed on by previous generations to induce a partial dauer exit state. 325 We attempted to further deprive cholesterol availability by growing daf-9(dh6) mutants 326 for two generations in the absence of cholesterol, but these second-generation animals 327 became very sick and could not grow past the L2 stage (data not shown). 328 329 daf-9 dependent steroid hormone biosynthesis is necessary for and promotes 330 dauer exit 331 Having confirmed that steroid hormone mutants retain the ability to form full 332 dauers, we proceeded to assess the role of the steroid hormone biosynthesis pathway 333 in dauer exit using daf-9(dh6) full dauers for our analyses. daf-9 encodes a cytochrome 334 P450 enzyme that catalyzes the formation of all known steroid hormones (  whether the XXX cells are dispensable for dauer exit, we bilaterally ablated the XXX 385 cells in dauers using a laser microbeam in dauer animals and transferred the ablated 386 animals to a recovery plate under favorable conditions to induce dauer exit. We found 387 that all XXX-ablated dauer larvae were able to exit dauer, similar to their mock ablated 388 counterparts (Fig. 7C). To validate this finding, we also genetically ablated the XXX 389 cells by expressing the human caspase gene ICE from the XXX-specific promoter 390 eak-3p using our cGal bipartite expression system for C. elegans (Wang et al., 2017). 391 We confirmed ablation of the XXX cells by noting the loss of GFP in XXX under 392 fluorescence microscopy in a strain expressing the UAS::ICE construct (Fig. S3). With 393 the enhanced throughout of the genetic ablation method compared to laser ablation, we 394 recovered animals onto different pheromone concentrations to tease out more subtle 395 changes in dauer exit phenotypes. Genetic ablation of the XXX cells did not 396 substantially prevent dauer larvae from exiting more when compared to control animals 397 expressing the UAS::ICE effector transgene without the XXX cell-specific driver 398 (Fig. 7D). In tandem with our laser ablation results, these findings suggest that the XXX 399 cells may not be essential for dauer exit. 400

Discussion 402
Partial dauers formed by steroid hormone mutants are likely partially exited 403 dauers 404 In this study, we evaluated how the steroid hormone pathway regulates both the 405 dauer entry and dauer exit developmental decisions by first addressing why daf-9 406 mutants form partial dauers. Our evidence favors the hypothesis that daf-9 partial 407 dauers, along with other partial dauers formed by mutations in steroid hormone 408 biosynthesis, represent a state in which dauers have commenced but cannot complete 409 dauer exit. We find that forming daf-9 full dauers under unfavorable conditions followed 410 by transfer to favorable conditions to induce dauer exit produces animals that resemble 411 the partial dauers that are formed when daf-9 mutants are grown constantly under 412 favorable conditions (Fig. 2, 3E-G). We also show that even when pheromone is 413 omitted from the growth medium, some daf-9(dh6) animals pass through a state of full 414 dauer before initiating dauer exit owing to the favorable environment (Fig. 4). But given 415 the lack of reproduction-promoting steroid hormones such as ∆ 7-DA, these larvae can 416 only partially exit from dauer, resulting in a partial dauer state that slowly grows to 417 unhealthy and sterile adulthood. 418 Under completely favorable conditions (i.e., no pheromone and low temperature), 419 daf-9(dh6) larvae could not be found in a full dauer state (Fig. S2). Following the L2d 420 molt, we were only able to find daf-9(dh6) mutants in a transient, indeterminate state 421 that looked like a hybrid between an L2d and a partial dauer in terms of morphology and 422 behavior. Within an hour, these animals then quickly went on to become familiar partial 423 dauers. While it is difficult to speculate why this intermediate state arises, one possibility 424 has to do with the fact that dauer formation affects multiple tissues, including the 425 epithelium, intestine, pharynx, and body muscles (Androwski et al., 2017). While this 426 process normally happens synchronously, in the case of daf-9(dh6) mutants grown 427 under favorable conditions, there could be asynchrony as different tissues start to 428 engage dauer exit programs before other tissues finish executing dauer entry programs. 429 For instance, as daf-9(dh6) mutants conclude the L2d molt, the nervous system could 430 immediately sense food and the lack of pheromone to trigger dauer exit programs that 431 promote pumping and locomotion. These dauer exit behaviors could start before 432 epidermal tissues finish the radial constriction process associated with dauer entry. The 433 result would be an animal that looks and behaves neither like a partially exited dauer 434 nor a full dauer, as we see in our experiments. When conditions are tuned to be slightly 435 less favorable (such as by increasing the temperature), the different tissues of daf-9 436 mutants can engage dauer entry in a more synchronous fashion, therefore allowing 437 observation of the full dauer state by eye. Another possibility to explain these results is 438 simply that daf-9 mutants completely skip or fail to enter the full dauer state under 439 completely favorable growth conditions because some tissues never fully execute their 440 dauer entry programs, but why that might happen remains unclear. Experiments that 441 can track how individual tissues engage dauer entry and exit programs will be helpful in 442 teasing these possibilities apart. 443 Our conclusions regarding the daf-9 partial dauer state as being one of partial 444 dauer exit aligns with those of a previous group concerning the partial dauer phenotype 445 of daf-12 Daf-c alleles such as daf-12(rh273), in which they suggest that daf-12 Daf-c 446 mutants initiate all the programs necessary for full dauer entry, but immediately detect 447 favorable conditions upon dauer entry and thus promptly attempt to exit (Antebi et al., 448 1998). In further support of these conclusions, we find that the Daf-c daf-12(rh273) 449 strain phenocopies daf-9(dh6) when it comes to partial dauer exit (Fig. S1B, C). 450

451
The daf-9 partial dauer state provides new insights into the dauer exit process 452 Recognizing that the daf-9 partial dauer state may be a partially exited dauer 453 raises many intriguing questions about dauer exit biology. It prompts a consideration of 454 what genetic, developmental, and physiological factors may be responsible for 455 triggering partial dauer exit. We considered that residual steroid hormone production, 456 either through non-daf-9-dependent biosynthesis or transgenerational rescue, suffices 457 to trigger partial dauer exit. The former is improbable because there are no 458 characterized biochemical pathways to form DAF-12 ligands that do not involve DAF-9 459 (Aguilaniu et al., 2016). The latter, while difficult to prove since we could not limit 460 cholesterol deprivation past one generation, seems unlikely. Growing daf-9(dh6) 461 mutants on media lacking cholesterol does not suppress partial dauer formation 462 (Fig. 5C, D). Moreover, a double mutant for both daf-9 and daf-36, which would 463 presumably have lower steroid hormone levels than daf-9 single mutants alone, 464 phenocopies daf-9 to produce partial dauers (Rottiers et al., 2006). 465 The partial dauer exit phenotype can be suppressed by mutations in important 466 components of the dauer pathway such as DAF-2/InsR and DAF-7/GDF11 (Fig. 5A, B). and steroid hormone pathway activity in full and partial dauers will help delineate such a 482 model. The daf-9 partial dauer exit state, therefore, provides an avenue by which other 483 regulatory components underlying dauer exit can be explored. 484 Could other described partial dauers, such as those produced by double mutant 485 strains carrying daf-16 (Ailion and Thomas, 2000;Vowels and Thomas, 1992), also be 486 characterized as partially exited dauers? The features of those partial dauers closely 487 match those of the daf-9 partial dauers that we and others have described including 488 pumping, an enlarged pharynx, and intermediate intestinal darkness (Gerisch et al., 489 2001;Vowels and Thomas, 1992). One distinction is that these double mutant partial 490 dauers were described to quickly and spontaneously exit to adulthood (Vowels and 491 Thomas, 1992), while daf-9(dh6) partial dauers cannot ever fully exit. The spontaneity of 492 complete exit in the double mutant partial dauers suggests that they may be in a 493 partially exited dauer state waiting for a threshold event to complete dauer exit. We 494 have not rigorously tested those strains using our partial dauer exit analyses, and it 495 remains to be determined whether these double mutant partial dauers have the capacity 496 to enter full dauers under unfavorable conditions as can steroid hormone mutants. 497 498 Steroid hormone biosynthesis governs dauer exit in a manner similar to the L1 to 499 L2 versus L2d dauer entry decision 500 We evaluated how the C. elegans steroid hormone pathway regulates dauer exit 501 in comparison to dauer entry. We established that steroid hormones are essential for full 502 dauer exit by showing that daf-9(dh6) dauers become competent for complete dauer via 503 supplementation of ∆ 7-DA at an EC50 in the low nanomolar range (Fig. 6B)  suggest that similar concentrations of ∆ 7-DA mediate both dauer entry and dauer exit in 508 daf-9(dh6) animals. We further show that hypodermal overexpression of daf-9 during 509 dauer promotes dauer exit (Fig. 6C), therefore demonstrating a parallel role for daf-9-510 dependent steroid hormones in regulating both dauer entry and dauer exit. 511 The dauer entry decision can be split into two distinct subdecisions, both of 512 which are regulated by the steroid hormone pathway: (1) the L1 to L2 versus L2d and (2) 513 the L2d to L3 versus dauer (Golden and Riddle, 1984). We asked whether the steroid 514 hormone pathway regulates the dauer exit decision in a manner that resembles the 515 former, the latter, or neither dauer entry subdecision. Our results collectively suggest 516 that the role of the steroid hormone pathway in dauer exit more closely mirrors that of 517 the L1 to L2 versus L2d decision for multiple reasons. 518 First, our daf-9::gfp translational fusion analysis shows that hypodermal 519 upregulation of daf-9::gfp begins around 10-14 hours following transfer of dauers onto 520 favorable conditions (Fig. 7A). This delay in hypodermal daf-9::gfp expression nearly 521 matches that of the L1 to L2 vs. L2d decision, in which it was shown that daf-9::gfp 522 fluorescence increased starting at 24 to 27 hours post hatch in animals grown under 523 favorable conditions, while larvae commit to reproductive adulthood much earlier at 524 around 14 to 16 hours post hatch (Schaedel et al., 2012). In stark contrast, during the L2d to dauer versus L3 decision, hypodermal DAF-9::GFP fluorescence closely aligned 526 with the time window in which L2d larvae committed to reproductive adulthood 527 (Schaedel et al., 2012). Importantly, given that dauer exit commitment occurs within one 528 to two hours following transfer onto favorable conditions (Golden and Riddle, 1984), the 529 fact that hypodermal DAF-9::GFP fluorescence does not appear until hours later 530 suggests that hypodermal upregulation of daf-9 may be a consequence of, rather than a 531 cause of, the commitment to exit dauer. 532 Secondly, our XXX ablation experiments argue against an essential role for the 533 XXX cells in dauer exit, as laser or genetic ablation of XXX cells do not prevent dauers 534 from exiting (Fig. 7C, D). Such observations are consistent with the nonessential role of 535 XXX cells in the L1 to L2 versus L2d decision, in which groups have reported that laser 536 ablation of XXX cells in L1 larvae has only a minor effect on dauer entry (Gerisch et al., 537 2001;Ohkura et al., 2003). In contrast, the XXX cells are required for reproductive 538 adulthood in the L2d to L3 versus dauer subdecision, as ablation of the XXX cells in L2d 539 larvae prevents development into the L3 stage even under favorable conditions 540 (Schaedel et al., 2012). Taken together, these results suggest higher similarity between 541 dauer exit and the L1 to L2 versus L2d dauer entry decision, at least as far as the daf-9-542 dependent steroid hormone pathway is concerned. 543 544 Dauer entry and dauer exit are asymmetric developmental decisions 545 Despite dauer entry and dauer exit sharing similar features, including their 546 sensory inputs and regulatory pathways, our work highlights that these two decisions 547 are inherently asymmetric. One basic distinction is that dauer entry comprises two 548 developmental subdecisions, while dauer exit consists of just one. Moreover, the two 549 dauer entry subdecisions involve a choice between two mutually exclusive 550 developmental paths that must be chosen within a certain timeframe. On the other hand, 551 dauer exit has a different decision architecture involving a continuous decision made 552 over an indeterminate timeframe (i.e., animals will exit dauer or they will stay as dauers). 553 The differences in these decision-making structures are highlighted by the daf-9 partial 554 dauer exit phenotype in that there does not seem to be an analogous "partial dauer 555 entry" phenotype for daf-9 mutants. This difference is because the dauer entry decision  DA to induce full dauer exit (Fig. 6B). Such conversation suggests that mechanistic 569 knowledge of how steroid hormones control dauer exit in C. elegans could yield 570 potential therapeutic insights to combat other parasitic species. 571 Diapause is evolutionarily conserved and phylogenetically widespread, and 572 hormonal control of both diapause entry and exit is especially well-studied in insects 573 (Denlinger et al., 2012). In Heliothis and Helicoverpa species of moth, diapause entry is 574 likely caused by insufficient levels of diapause hormone (DH) and prothoracicotropic 575 hormone (PTTH) (Xu and Denlinger, 2003 including their genotypes and origins, can be found in Table S1. Maintenance and 597 propagation of C. elegans, with the exception of daf-9 loss-of-function mutants, were 598 performed under typical growth conditions with Nematode Growth Medium (NGM) agar 599 seeded with OP50 E. coli cultures as described previously (Brenner, 1974). The daf-600 9(dh6), daf-9(e1406), and daf-9(m540) mutants were propagated in the presence of 100 601 nM ∆ 7-Dafachronic Acid (∆7-DA) to promote reproductive development. The daf-9(dh6) 602 strain was obtained by propagating non-array carrying animals from PS5511 (daf-9(dh6); 603  To induce full dauers in wild-type and daf-9 mutants, 10-20 young adults were placed 607 on 35 mm diameter Petri dishes containing 2 mL of NGM agar (without peptone) 608 supplemented with a quantity of crude pheromone extract (Schroeder and Flatt, 2014) 609 that normally induce 95-100% of dauers in wild-type animals -typically 10-30 µL per 2 610 mL of agar. Plates were seeded with 10 µL of 8% w/v E. coli OP50 that was heat-killed 611 at 95°C for 5 minutes. Adults were picked onto the plate and allowed to lay eggs at 612 room temperature (RT; 22-23°C) for 5-9 hours before being removed, during which time 613 they typically laid 200-300 eggs. The plates were then further seeded with an additional 614 20 µL of heat-killed OP50. Afterwards, the plates were wrapped with parafilm and 615 incubated at 25.5°C for 60-72 hours. 616 617

Dauer Exit Assay
Dauers were formed according to Dauer Entry Induction, above. In most cases, dauers 619 were selected for by an SDS wash (2%, 30 minutes, 25°C) to kill non-dauers before 620 being washed 3x in M9 solution (1 minute, room temperature, 1000 x g). Surviving 621 dauers were then plated onto dauer exit plates, which were identical to dauer entry 622 plates but contained a lower concentration of crude pheromone extract (typically 0.5-2 623 µL per 2 mL of agar) that had been determined to induce ~40-60% of wild-type dauers 624 to exit within 24 hours. In the case of the genetic ablation assay using the UAS::ICE 625 construct, the SDS wash step was omitted since dauers bearing the UAS::ICE construct 626 were SDS sensitive. Dauers were instead washed directly onto the dauer exit plate. 627 Following 24 hours after dauers were transferred onto dauer exit plates, dauer exit was 628 scored using the following criteria. Larvae were scored as having exited dauer if they 629 showed any pharyngeal pumping or if their body had thickened and lightened 630 considerably. Additional factors that favored scoring an animal as having exited dauer 631 included whether the larva showed foraging behavior (such as increased head turns) or 632 increased and consistent locomotory behavior, both of which are absent from dauers. 633 634

Microscopy and Image Analysis 635
Worms were immobilized on a 4-10% agarose pad (10% for dauer imaging, 4% for 636 others) in 1-2 µL of 10 mg/mL levamisole or 50 mM sodium azide. Imaging was 637 performed on a Zeiss AxioImager2 equipped with a Colibri 7 for LED fluorescence 638 illumination and an Axiocam 506 Mono camera (Carl Zeiss Inc., White Plains, NY). 639 Pharyngeal bulb width measurements were performed using Zen Blue 2.3 (Zeiss) 640 software by using the length tool to measure the widest section of the posterior 641 pharyngeal bulb. Images were processed using FIJI (ImageJ). visualized under fluorescence, and the laser was fired at ~5 Hz for a total 20-30 pulses, or until all discernable fluorescence was gone. Cellular damage could often be