Subthreshold serotonin signals combined by the G proteins Gαq and Gαs activate the C. elegans egg-laying muscles

Individual neuron or muscle cells express many G protein coupled receptors (GPCRs) for neurotransmitters and neuropeptides. It remains unclear how these cells integrate multiple GPCR signals that all must act through the same few G proteins. We investigated how two serotonin GPCRs, Gαq-coupled SER-1 and Gαs-coupled SER-7, function together on the C. elegans egg-laying muscles to promote contraction and thus cause eggs to be laid. Using receptor null mutations and cell-specific knockdowns, we found that serotonin signaling through either SER-1/Gαq or SER-7/Gαs alone does not induce egg laying, but these subthreshold signals can combine to promote egg laying. However, using designer receptors or optogenetics to artificially induce high levels of either Gαq signaling or Gαs signaling in the muscles was sufficient to induce egg laying. Conversely, knocking down both Gαq and Gαs in the egg-laying muscle cells induced egg-laying defects stronger than those of a ser-7 ser-1 double knockout. These results suggest that, in the egg-laying muscles, multiple GPCRs for serotonin and other signals each produce weak effects that individually do not result in strong behavioral outcomes. However, they can combine to produce sufficient levels of Gαq and Gαs signaling to promote muscle activity and egg laying.


Introduction 29
Individual neuron or muscle cells can express many different G protein coupled receptors 30 (GPCRs), which in turn act through just three main types of heterotrimeric G proteins: Gs, Gq/11, 31 and Gi/o (Kaur et (Marder and Bucher, 2007). In this study, we focus on the C. elegans egg-laying circuit, where we 47 proteins. Even so, we note that the strong egg-laying defect that resulted from Gαq/Gαs double 233 knockdown in the egg-laying muscles (Figures 5) is stronger than the egg-laying defects observed 234 in animals with complete knockouts of both ser-1 and ser-7, or in animals with a tph-1 knockout 235 that completely eliminates endogenous serotonin ( Figure 1E, G, and H and Figure 1-figure  236 supplement 2A and 2C). Therefore, serotonin appears not to be the only signal that generates Gαq 237 and Gαs activity in the egg-laying muscles to stimulate egg laying. These data indicate that there 238 must be other GPCRs on the egg-laying muscles, in addition to SER-1 and SER-7, that signal 239 through Gαq and Gαs to stimulate egg laying. Therefore, normal levels of egg-laying activity result 240 from Gαq and Gαs acting in the egg-laying muscles to combine signals from SER-1, SER-7, and 241 additional GPCRs. 242 243 Overexpressed SER-1 is sufficient to allow serotonin to induce egg laying in the absence of 244

SER-7 245
Results presented above show that knocking out or knocking down either the SER-1 or 246 SER-7 serotonin receptors result in severe defects in the ability of serotonin to induce egg laying. 247 In some cases, the defects observed were as strong as those caused by knocking out both SER-1 248 and SER-7 at the same time or as strong as those seen when completely eliminating serotonin with 249 a tph-1 null mutation ( Figure 1E). These results raise the question of whether serotonin absolutely 250 requires both SER-1/Gαq and SER-7/Gαs signaling to induce egg laying, or whether these two 251 signaling pathways might rather combine to induce egg laying in a more nuanced fashion. Thus, 252 we designed several different experiments to determine if increasing the strength of just one of the 253 two pathways could induce egg laying in the absence of the other pathway. 254 13 The first method was to simply overexpress one serotonin GPCR by increasing the copy 255 number of the GPCR gene. Previous genetic studies have shown that overexpression can increase 256 the normal functions of a GPCR in a manner that is suppressed by knocking out the endogenous 257 ligand for that GPCR (Ringstad and Horvtiz, 2008;Harris et al., 2010;Brewer et al., 2019;258 Fernandez et al., 2020), suggesting that the overexpressed GPCR is activated by its endogenous 259 ligand to signal at a higher level than would the endogenous levels of the GPCR. Indeed, 260 overexpressing SER-1 in C. elegans was shown to increase egg laying in a manner completely 261 dependent on endogenous serotonin (Fernandez et al., 2020). 262 To overexpress serotonin receptors, we used chromosomally-integrated transgenes that 263 carry multiple copies of the complete ser-1 or ser-7 genes, including their own promoters, resulting 264 in overexpression of these genes in the same cells that normally express them (Fernandez et al., 265 2020). We tested the ability of exogenous serotonin to induce egg laying in animals overexpressing 266 one serotonin receptor while also carrying a deletion mutation for the other serotonin receptor. Our 267 results are graphed in Figure 6, and the design and logic of this experiment are schematized in 268 Figure 6-figure supplement 1. We found that animals overexpressing ser-7 in a ser-1-null 269 background were not able to lay eggs in response to exogenous serotonin, similar to animals that 270 simply lacked ser-1. However, animals overexpressing ser-1 in a ser-7-null background did lay 271 eggs in response to exogenous serotonin, unlike animals that simply lacked ser-7. 272 These results show that while SER-1/Gαq signaling is normally not sufficient to allow 273 serotonin to induce egg laying in the absence of SER-7/Gαs signaling, artificially increasing SER-274 1 expression levels overcomes this limitation. It is difficult to interpret the negative result from the 275 converse experiment, in which overexpressed SER-7 failed to induce egg laying in the absence of 276 SER-1. It could be that our SER-7 overexpression experiment may have not increased SER-7/Gαs 277 14 signaling to a high enough level to induce egg laying in the absence of SER-1/Gαs signaling or it 278 could mean that SER-7 alone is incapable of driving egg-laying. Thus, we devised additional 279 methods, described below, to artificially increase Gαq and/ (Prömel et al., 2016). We acutely activated Gαq-signaling specifically 286 in the egg-laying muscles by transgenically expressing this designer Gαq-coupled receptor using 287 the egg-laying muscle specific promoter and treating the worms with CNO. This induced egg 288 laying ( Figure 7A and B). In contrast, worms carrying a control transgene were unable to lay eggs 289 in response to CNO ( Figure 7A). 290 Next, we determined if Gαs-signaling in the egg-laying muscles was sufficient to drive egg 291 laying. To date there is no designer Gαs-coupled receptor that is functional in C. elegans (Prömel 292 et al., 2016) and we were unsuccessful in further attempts to design such a receptor (data not 293 shown). Gαs signals by activating adenylyl cyclase, which in turn generates cAMP. A 294 photoactivatable adenylyl cyclase (PAC) has been successfully used in the cholinergic neurons 295 and body wall muscles of C. elegans to evoke changes in locomotion (Steuer Costa et al., 2017;296 Henss et al., 2022). We generated transgenic animals that express PAC in their egg-laying muscles 297 and found that blue light activation of PAC was able to induce egg laying in these worms, whereas 298 control worms carrying an empty vector transgene were unable to lay eggs in response to blue light 299 ( Figure 7C and D, and Video 1). These results demonstrate that activation of either the Gαq or Gαs 300 15 pathways in the egg-laying muscles is sufficient to induce egg laying, and that these G protein 301 signals can originate from sources other than a serotonin receptor. 302

303
The combination of two subthreshold signals from different Gαq-coupled receptors in the 304 egg-laying muscles is sufficient to drive egg laying 305 The results above demonstrate that artificially-induced Gαq or Gαs signaling in the egg-306 laying muscles can be sufficient to induce egg laying. However, we also found that neither 307 endogenous SER-1/Gαq signaling alone nor endogenous SER-7/Gαs signaling alone in these same 308 egg-laying muscles is sufficient to drive egg laying; instead, both these endogenous signaling 309 pathways must be active at the same time to induce egg laying. To reconcile these findings, we 310 hypothesized that the endogenous levels of SER-1/Gαq and SER-7/Gαs signaling are both 311 "subthreshold," i.e., occur at low levels that are not sufficient to properly activate egg laying on 312 their own, and together sum to reach the threshold necessary to activate egg laying. 313 To test this hypothesis, we generated an artificial subthreshold G protein signal that was 314 unable to activate egg laying on its own and determined if it was capable of activating egg laying 315 when combined with another subthreshold G protein signal. The designer Gαq-coupled receptor 316 offered the potential to tune the levels of Gαq signaling it induces: we titrated the concentration of 317 its CNO ligand to find a concentration (2 mM) that was just below the threshold required to activate 318 egg laying on its own ( Figure 8A). We then expressed the designer Gαq-coupled receptor in the 319 egg-laying muscles of worms lacking the SER-7 receptor, so that serotonin could only signal to 320 induce egg laying via SER-1/Gαq ( Figure 8C). Exposure to either exogenous serotonin or to 2 mM 321 CNO was unable to induce egg laying, as expected. However, when both exogenous serotonin and 322 16 2 mM CNO were applied to the worm at the same time, these two subthreshold Gαq-coupled 323 signals combined to activate egg laying ( Figures 8B and C). 324 325 Discussion 326 The principal finding of this study is that the SER-1 and SER-7 serotonin receptors, as well as 327 additional Gαq and/or Gαs coupled receptors, all signal together in the C. elegans egg-laying 328 muscles to help induce their coordinated contraction and thus the laying of eggs. While signaling 329 from endogenous levels of just one of these receptors alone is not strong enough to induce egg 330 laying, together the signals from multiple types of GPCRs on the same cells combine to reach a 331 threshold that can activate egg laying ( Figure 9). This study is perhaps the most detailed to date of 332 how cells within an intact organism integrate signaling by multiple GPCRs to generate a concerted 333 response to the complex mixture of chemical signals impinging upon them. Such signal integration 334 is a challenge faced by virtually all cells within multicellular organisms, and the findings from our 335 study of how this is accomplished in the C. elegans egg-laying muscles likely generalize to similar 336 situations faced by other cells. 337 338

Multiple GPCRs signal through Gαq and Gαs to activate excitable cells 339
We found that knocking down both Gαq and Gαs in the egg-laying muscles resulted in a 340 dramatic defect in egg laying, while loss of the SER-1 and SER-7 serotonin receptors that activate 341 these G proteins, either from the entire animal or from the egg-laying muscles alone, only had a 342 modest effect. Therefore, serotonin appears to combine with other endogenous signals to generate 343 sufficient Gαq and Gαs signaling in the egg-laying muscles to induce egg laying. Treating animals 344 with a high concentration of exogenous serotonin is sufficient to induce egg laying, and, even in 345 this artificial situation, both the SER-1 and SER-7 receptors must operate in parallel on the egg-346 laying muscles to mediate this effect, as loss of either receptor from the egg-laying muscles results 347 in almost complete loss of the ability of exogenous serotonin to induce egg laying. We were able 348 to generate artificial circumstances in which activation of a single type of GPCR on the egg-laying 349 muscles (either overexpressed SER-1 or the designer CNO receptor) could induce egg laying. 350 Additionally, activation of the signaling pathway downstream of SER-7 with a photoactivatable 351 adenylyl cyclase was sufficient to induce egg laying. Nonetheless, our results show that the normal 352 situation in wild-type animals is that egg laying is induced by the combined signaling from 353 multiple Gαq-and Gαs-coupled receptors. 354 What other signals besides serotonin might be acting on the egg-laying muscles to promote 355 egg laying? The neuropeptide NLP-3 is co-released with serotonin onto the egg-laying muscles to 356 promote egg laying (Brewer et al., 2019), and it is possible that the NLP-3 receptor, which has not 357 yet been identified, is one of the additional GPCRs expressed on these muscles. If so, the NLP-3 358 receptor would combine its effects with those of the SER-1 and SER-7 serotonin receptors to 359 induce egg laying. NLP-3 signaling on its own, like signaling from the serotonin receptors, 360 produces modest effects, with dramatic defects in egg laying seen only when both NLP-3 and 361 serotonin signaling are lost simultaneously. A systematic analysis of the expression of all C. 362 elegans neurotransmitter GPCRs on the egg-laying muscles (Fernandez et al., 2020) found that, 363 besides SER-1 and SER-7, three additional Gαq-and Gαs-coupled GPCRs are expressed on these 364 cells: the dopamine receptor DOP-4, the tyramine receptor TYRA-3, and the metabotropic 365 acetylcholine receptor GAR-3. Just as for SER-1 and SER-7, knockouts for any one of these 366 receptors have, at most, modest effects on the accumulation of unlaid eggs (Fernandez et al., 2020), 367 18 consistent with the hypothesis that the G proteins Gαq and Gαs integrate signals from a variety of 368 GPCRs on the egg-laying muscles to maintain proper egg laying. 369 Our finding that multiple GPCRs combine signaling in the egg-laying muscles of C. 370 elegans to induce strong behavioral effects appears to be a general feature of GPCR signaling in 371 excitable cells within multicellular organisms. Heterotrimeric G protein signaling in C. elegans 372 neurons and muscles has been studied intensively for decades (reviewed by Koelle, 2018). These receptors which, like ryanodine receptors, are Ca 2+ channels that release Ca 2+ from internal stores 438 to promote muscle contraction. In this mechanism, Gαq directly activates the enzyme 439 phospholipase C (Smrcka et al., 1991;Taylor et al., 1991), which generates the second messenger 440 IP3 that directly binds and activates the IP3 receptor. Gαs signaling, as noted above, activates the 441 protein kinase PKA, which can phosphorylate and activate IP3 receptors (Taylor, 2017). 442 The mechanism by which Gαq  promoter region and each exon-rich gene region that was targeted by RNAi. During the 516 knockdown of G proteins, mCherry was expressed in the egg-laying muscles to demonstrate that 517 the G protein knockdown did not interfere with muscle development ( Figure 5-supplement 1). 518 There was no statistical difference between the number of eggs retained in animals in expression 519 (data not shown). 520 521

Calcium imaging 522
Animals were staged as late-L4 larvae and recorded 24 hours later. Freely-behaving animals were 523 mounted between a glass coverslip and a ~1 cm 2 chunk from an NGM plate containing OP50 food 524 for imaging as previously described (Collins and  serotonin creatine sulfate monohydrate (Sigma, H7752-5G) were poured and seeded with OP50 545 one day prior to assay. Animals were staged as late L4 larvae for assay 24 hours later. At time 0 546 of the assay, 5-10 worms were placed on the serotonin plates, spaced in a manner that it was 547 unlikely they would be able to crawl near each other prior to being paralyzed by the serotonin. Animals were staged as L4 larva 30 hours prior to assay. Quantitation of unlaid eggs was 578 performed as described in Chase and Koelle (2004). 579 580

Statistical analysis 581
Error bars shown in all graphs represent 95% confidence intervals. All statistical analysis was 582 analyzed using GraphPad Prism version 9.3.1 software. Calcium imaging transients in the vm1 583 and vm2 muscles (Figure 3) were analyzed using a contingency analysis and Fisher's exact test 584 with two-sided P-values and Bonferroni correction method for multiple comparisons. Egg-laying 585 assays involving CNO-induced activation of the DREADD Gαq receptor were analyzed in Figure  586 7 using the unpaired t-test with a two-tailed P value with the assumption that both populations 587 had the same standard deviation and in Figure 8