Muscle-directed mechanosensory feedback activates egg-laying circuit activity and behavior in C. elegans

Mechanosensory feedback of internal reproductive state drives decisions about when and where to reproduce.1 For instance, stretch in the Drosophila reproductive tract produced by artificial distention or from accumulated eggs regulates the attraction to acetic acid to ensure optimal oviposition.2 How such mechanosensory feedback modulates neural circuits to coordinate reproductive behaviors is incompletely understood. We previously identified a stretch-dependent homeostat that regulates egg laying in Caenorhabditis elegans. Sterilized animals lacking eggs show reduced Ca2+ transient activity in the presynaptic HSN command motoneurons that drive egg-laying behavior while animals forced to accumulate extra eggs show dramatically increased circuit activity that restores egg laying.3 Interestingly, genetic ablation or electrical silencing of the HSNs delays, but does not abolish, the onset of egg laying3–5 with animals recovering vulval muscle Ca2+ transient activity upon egg accumulation.6 Using an acute gonad microinjection technique to mimic changes in pressure and stretch resulting from germline activity and egg accumulation, we find that injection rapidly stimulates Ca2+ activity in both neurons and muscles of the egg-laying circuit. Injection-induced vulval muscle Ca2+ activity requires L-type Ca2+ channels but is independent of presynaptic input. Conversely, injection-induced neural activity is disrupted in mutants lacking the vulval muscles, suggesting ‘bottom-up’ feedback from muscles to neurons. Direct mechanical prodding activates the vulval muscles, suggesting they are the proximal targets of the stretch-dependent stimulus. Our results show that egg-laying behavior in C. elegans is regulated by a stretch-dependent homeostat that scales postsynaptic muscle responses with egg accumulation in the uterus.


Summary 8
Mechanosensory feedback of internal reproductive state drives decisions about 9 when and where to reproduce. 1 For instance, stretch in the Drosophila reproductive 10 tract produced by artificial distention or from accumulated eggs regulates the attraction 11 to acetic acid to ensure optimal oviposition. 2 How such mechanosensory feedback 12 modulates neural circuits to coordinate reproductive behaviors is incompletely 13 understood. We previously identified a stretch-dependent homeostat that regulates egg 14 laying in Caenorhabditis elegans. Sterilized animals lacking eggs show reduced Ca 2+ 15 transient activity in the presynaptic HSN command motoneurons that drive egg-laying 16 behavior while animals forced to accumulate extra eggs show dramatically increased 17 circuit activity that restores egg laying. 3 Interestingly, genetic ablation or electrical 18 silencing of the HSNs delays, but does not abolish, the onset of egg laying 3-5 with 19 animals recovering vulval muscle Ca 2+ transient activity upon egg accumulation. 6 Using 20 an acute gonad microinjection technique to mimic changes in pressure and stretch 21 resulting from germline activity and egg accumulation, we find that injection rapidly 22 stimulates Ca 2+ activity in both neurons and muscles of the egg-laying circuit. Injection-23 induced vulval muscle Ca 2+ activity requires L-type Ca 2+ channels but is independent of 24 presynaptic input. Conversely, injection-induced neural activity is disrupted in mutants 25 lacking the vulval muscles, suggesting 'bottom-up' feedback from muscles to neurons. 26 Direct mechanical prodding activates the vulval muscles, suggesting they are the 27 proximal targets of the stretch-dependent stimulus. Our results show that egg-laying 28 behavior in C. elegans is regulated by a stretch-dependent homeostat that scales 29 postsynaptic muscle responses with egg accumulation in the uterus.

Results and Discussion 31
While microinjection into the gonad of the C. elegans is a common technique for 32 generating transgenic strains, 7,8 we noticed injection often led to vulval opening and egg 33 release. While we feel this phenomenon is widely known among C. elegans 34 researchers, it is not well-described in the literature. To determine if microinjection 35 promotes egg laying, we inserted a needle into the syncytial gonad of wild-type animals 36 and performed a brief (3 s) injection delivering a median volume of ~250 pL of injection 37 buffer (see STAR methods) while monitoring egg release ( Figure 1A). Microinjection 38 drove egg release in nearly half (~46 ± 9%) of animals ( Figure 1B) but not in unc-39 54(e190) muscle-specific myosin mutants which cannot contract the vulval muscles 40 ( Figure 1B). 9 41 As muscle contraction requires Ca 2+ activity, 10-12 we performed ratiometric 42 GCaMP5 recordings of the vulval muscles. 6 Gonad injections triggered a strong and 43 rapid induction of vulval muscle Ca 2+ activity which peaked on average 5.6 s after the 44 onset of injection ( Figure 1C; Video S1) with subsequent injections showing weaker 45 induction of Ca 2+ (Figure S1A). Direct contact with the injected buffer did not seem to 46 be required for a robust muscle response, as Ca 2+ activity was induced immediately 47 of an immobilized C. elegans worm (top). A 3 s pulse is applied to inject a standard 277 microinjection buffer into the gonad syncytium. Unlaid eggs (cyan asterisks) are then 278 monitored for injection-induced egg release through the vulva (white bracket).

279
(B) Bar       Slopes were binned as 'slow' (highlighted in orange) or 'fast' depending on whether they 469 were below or above the median slope, respectively. (black), wild-type sterilized with FUDR (red), and fog-2(oz40) mutant animals (green

Lead contact 521
Further information and requests for resources and reagents should be directed to and 522 will be fulfilled by the lead contact, Kevin M. Collins (kevin.collins@miami.edu). 523

Materials availability 524
Plasmids and strains generated in this study are available from the lead contact by 525

request. 526
Data and code availability 527 • Microscopy, ratiometric traces, and summary data analyzed for statistical 528 significance reported in this paper will be shared by the lead contact upon 529 request. 530 • This paper does not report original code. 531 • Any additional information required to reanalyze the data reported in this paper is 532 available from the lead contact upon request. 533

Egg accumulation at onset of egg laying 714
To determine the extent of egg accumulation at onset of egg laying, both wild-type and 715 MIA26 egl-1(n986dm) mutant animals were staged following the L4 molt as previously 716 described. 3 Animals were observed for the first instance of egg laying every 30 minutes 717 (after a 4 h period for wild type and after a 16 h period for egl-1(dm) mutants). Once an 718 animal had laid its first egg, it was dissolved in a 20% bleach solution, and the eggs in 719 the uterus were counted as described. 65 The number of eggs in the uterus plus the number of eggs laid on the plate were considered the total number of eggs accumulated 721 at the onset of egg laying. 722

Microinjection protocol and flow rate estimate 723
To induce acute increases in pressure within C. elegans worms, we performed 724 microinjections essentially as described. 7 To create microinjection needles, filamented

Brightfield and fluorescence recordings 762
To record egg-laying behavior during injections, a Grasshopper 3 camera (FLIR) was 763 used to capture 2 x 2 binned, 1,024 x 1,024 8-bit JPEG image sequences. Vulval 764 muscle, HSN, VC, or uv1 Ca 2+ responses were recorded as described. 6,64 GCaMP5 and mCherry fluorescence was excited at 470 nm and 590 nm, respectively, for 10 msec 766 using a Colibri.2 LED illumination system. Recordings were collected at 20 fps and a 767 256x256 pixel resolution (4x4 binning) using a Hamamatsu Flash 4.0 V2 sCMOS 768 camera recording at 16-bit depth through a 20x Apochromatic objective (0.8NA). 769 GCaMP5 and mCherry channels were separated via a Gemini beam-splitter. Worms 770 were recorded for 30 seconds prior to injection to establish baseline levels of cell activity 771 and were completed within five minutes to reduce effects from worm desiccation after 772 prolonged exposure to halocarbon oil. Image sequences were imported into Volocity 773 (Version 6.5.1, Quorum Technologies Inc.) for GCaMP5/mCherry ratiometric analysis, 774 image segmentation, Ca 2+ quantitation of GCaMP5/mCherry ratio changes (∆R/R), and 775 Ca 2+ transient peak finding, as described previously. 6,64 776

Optimization of microinjection protocol 777
To ensure injections into control animals and the different mutants were comparable, 778 the correlation between flow rate and recorded calcium transient amplitudes was 779 analyzed. From this comparison, we found that flow rate had no significant effect on the 780 amplitude of the resulting vulval muscle Ca 2+ transients (p-value = 0.4860; Figure S1C) 781 and, as a result, the injection flow rate was allowed to vary. Additionally, the correlation 782 between flow rate and an egg-laying response was also analyzed. This relationship also 783 showed no significant correlation between flow rate and percent of injection-induced 784 egg laying (p-value = 0.1665; Figure S1E. Individual animals were injected twice to 785 determine if subsequent responses were comparable. We found that subsequent 786 injections resulted in lower Ca 2+ transient amplitudes and were but equally likely to 787 result in an egg-laying event ( Figure S1A) However, given the wide range of flow rates obtained among multiple injections, we limited our analyses of injection-induced Ca 2+ 789 responses to each animal's first injection. 790 To determine if the composition of the injection buffer influenced the Ca 2+ 791 response, worms were injected with deionized water or Milli-Q water. Injections with 792 both types of water did not differ in their ability to induce comparable levels of Ca 2+ 793 transient activity from the vulval muscles (p-value >0.05; Figure S1F). However, we 794 observed that when the injection buffer was made with Na + salts instead of K + salts, a 795 decrease in the average vulval muscle Ca 2+ response was observed (p-value = 0.0003; 796 Figure S1F) when compared to the standard K + salt containing buffer. To ensure that 797 the injection-induced response is not a consequence of a disruption of the Na + /K + 798 gradient, low flow rate injections were performed by decreasing injection pressure to 20 799 psi. In these low-flow experiments, we observed that injections were more likely to fail to 800 elicit a Ca 2+ response even when the 'optimal' K + buffer was used. Injections were 801 performed into either the anterior or posterior gonad, with no significant difference in 802 vulval muscle Ca 2+ transient amplitude or proportion of injection-induced egg laying 803 observed (Figure S1G-H). Attempts to determine if injections performed in other 804 compartments (e.g. the uterus) also drove vulval muscle Ca 2+ activity were unsuccessful 805 due to injection needles frequently getting clogged (data not shown). 806

Injections with bromophenol blue 807
To visualize injections, and to determine if Ca 2+ dynamics correlated with the rate of 808 liquid spread at lower flow rates (injection pressure 25 psi), a solution of 450 µM 809 bromophenol blue made in injection buffer was used as a fluorescent tracker (Figure  810 S2A). Bromophenol blue was excited at 590 nm alongside co-expressed mCherry and normal, a nonparametric equivalence test was performed (e.g., Mann-Whitney or 926 Kruskal-Wallis). All tests were corrected for multiple comparisons (Bonferroni for 927 ANOVA, Dunn for Kruskal-Wallis). Details of which test was performed for specific 928 strains can be found in figure legends. 929 Standard least squares regression for vulval muscle Ca 2+ transient amplitude 930 To determine which factor, as a consequence of injection, best predicted the vulval 931 muscle Ca 2+ response, a standard least squares regression analysis was performed 932