1 Conditional *nmy-1* and *nmy-2* alleles establish that non-muscle myosins are required for late *C. elegans* embryonic elongation

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6 Kelly Molnar¹,², Shashi Kumar Suman¹,³, Jeanne Eichelbrenner¹,⁴, Camille N. Plancke¹,⁵,

7 François B. Robin¹, Michel Labouesse¹*,

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9 ¹Laboratoire de Biologie du Développement - UMR7622, Institut de Biologie Paris Seine, Sorbonne

10 Université, 7-9 quai Saint Bernard 75005 Paris, France.

11 ²Present Address: Laboratoire Matière et Systèmes Complexes, Université Paris Cité – CNRS

12 UMR7057, 10 Rue Alice Domon et Léonie Duquet - 75013 Paris, France

13 ³Present Address: Development and stem cell program, IGBMC, UMR7104, U964, Université de

14 Strasbourg, 1 rue Laurent Fries, BP10142, 67400 Illkirch, France

15 ⁴Present Address: CIML - UMR7280, 163 avenue de Luminy, 13288 Marseille Cedex 09, France

16 ⁵Present address: Institut Pierre-Gilles de Gennes for Microfluidics, 6 rue Jean Calvin 75005 Paris,

17 France

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19 * Author for correspondence: michel.labouesse@sorbonne-universite.fr

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26 The elongation of *C. elegans* embryos allows examination of mechanical interactions between
27 adjacent tissues. Muscle contractions during late elongation induce the remodelling of
28 epidermal circumferential actin filaments through mechanotransduction. Force inputs from
29 the muscles deform circumferential epidermal actin filament, which causes them to be
30 severed, eventually reformed and shortened. This squeezing force drives embryonic
31 elongation. We investigated the possible role of the non-muscle myosins NMY-1 and NMY-2
32 in this process using *nmy-1* and *nmy-2* thermosensitive alleles. Our findings show these
33 myosins act redundantly in late elongation, since double *nmy-2(ts)*; *nmy-1(ts)* mutants
34 immediately stop elongation when raised to 25°C. Their inactivation does not reduce muscle
35 activity, as measured from epidermis deformation, suggesting that they are directly involved
36 in the multi-step process of epidermal remodeling. Furthermore, NMY-1 and NMY-2
37 inactivation is reversible when embryos are kept at the non-permissive temperature for a few
38 hours. However, after longer exposure to 25°C double mutant embryos fail to resume
39 elongation, presumable because NMY-1 was seen to form protein aggregates. We propose
40 that the two C. elegans non-muscle myosin II act during actin remodeling either to bring
41 severed ends or hold them.

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45 **Keywords:** *C. elegans*, morphogenesis, non-muscle myosin, mechanotransduction,
46 aggregates

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54 **INTRODUCTION**

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56 Morphogenesis refers to the process by which organisms develop their 3D-shape, which 57 generally involves cell shape changes (Lecuit, 2005). Multicellular organisms must coordinate 58 the morphogenesis of multiple tissues in time and space (GILMOUR et al. 2017; GOODWIN AND 59 NELSON 2021), which they achieve in part through mechanical inputs to ensure the smooth 60 development of adjacent tissues (AIGOUY et al. 2010; ZHANG et al. 2011; COLLINET et al. 61 2015; LYE et al. 2015). A classical paradigm is that proteins associated with a transmembrane 62 receptor, such as integrin or E-cadherin, undergoes a conformational change that favours the 63 binding of additional proteins, influencing protein trafficking, planar polarity, junction 64 remodelling, or cytoskeleton dynamics, (DEL RIO et al. 2009; AIGOUY et al. 2010; LE DUC et 65 al. 2010; YONEMURA et al. 2010; LEVAYER et al. 2011; ZHANG et al. 2011; LARDENNOIS et al. 66 2019).

67 Recent results have emphasized permanent viscoplastic changes induced by mechanical 68 inputs and the key role of the actomyosin cortex (BONAKDAR et al. 2016; DOUBROVINSKI et 69 al. 2017; KHALILGHARIBI et al. 2019; LARDENNOIS et al. 2019; STADDON et al. 2019; 70 MOLNAR AND LABOUESSE 2021). Nevertheless, although several proteins relaying mechanical 71 stress have been identified, our understanding of mechanotransductive pathways is 72 incomplete (MOORE et al. 2010; HU et al. 2017; YAP et al. 2018; NIETHAMMER 2021), in 73 particular when repeated mechanical inputs are involved(MARTIN et al. 2009; SOLON et al. 74 2009; AIGOUY et al. 2010; RAUZI et al. 2010; ZHANG et al. 2011; MAITRE et al. 2015; 75 LARDENNOIS et al. 2019).

76 *C. elegans* represents a powerful system to analyze mechanical inputs. During embryonic 77 morphogenesis, an ellipsoidal cell aggregate elongates into a vermiform shape in two distinct 78 stages that rely on epidermal cell shape change (VUONG-BRENDER et al. 2016). The driving
forces for elongation come from the epidermal actomyosin cortex and from the muscles for
the early and late stages, respectively (WILLIAMS AND WATERSTON 1994; PIEKNY et al. 2003;
GALLY et al. 2009; ZHANG et al. 2011). The early stage involves lateral cells which have a
greater concentration of non-muscle myosin and a disordered actin network (PIEKNY et al.
2003; GALLY et al. 2009), (VUONG-BRENDER et al. 2017). Stiffness anisotropy originating
from circumferential actin bundles in dorso-ventral cells is also important (VUONG-BRENDER
et al. 2017).

In the late stage, muscles, which are tightly linked to the dorso-ventral epidermis via
hemidesmosomes, deform epidermal cells when they contract (ZHANG AND LABOUESSE 2010;
ZHANG et al. 2011). Muscle activity transiently bends dorso-ventral actin bundles beyond a
critical angle (Fig. 1A), which is thought to induce their severing and further shortening
(LARDENNOIS et al. 2019), the p21-activated kinase PAK-1, alpha-spectrin SPC-1 and
atypical formin FHOD-1 restabilize them (Fig. 1A) (LARDENNOIS et al. 2019). In the absence
of PAK-1 and SPC-1, or FHOD-1 and SPC-1, embryos elongate up to the 1.5-fold stage and
then regress to their initial lima bean shape (roughly 1.2-fold stage) due to the loss of actin
filament integrity (LARDENNOIS et al. 2019). Muscles thus progressively promote actin
filament shortening and embryo elongation.

The model described above assumes that the ends of severed actin filaments do not get much
further apart before the PAK-1/SPC-1/FHOD-1 complex stabilizes them. This might not be
the case since hydrostatic pressure exerts a radial force that might pull severed actin ends
apart. Since muscles are arranged orthogonally to actin bundles, they cannot contribute to
bringing actin ends closer. Although spectrins are actin-binding proteins that could fulfil the
ascribed function of holding actin ends (LENNE et al. 2000; LAW et al. 2003; CHOI AND WEIS
2011), we wondered whether additional proteins might act upstream of PAK-1/SPC-1/FHOD-
1 (Fig. 1).
We focused on non-muscle myosins, because they are obvious actin-dependent motor proteins. Non-muscle myosins are hetero-hexamers consisting of two myosin heavy chains, two regulatory light chains (MLC-4 in *C. elegans*), which must be phosphorylated to enable motor activity, and two essential light chains (MLC-5 in *C. elegans*) (VICENTE-MANZANARES *et al.* 2009). *C. elegans* has two non-muscle heavy chains, NMY-1 and NMY-2. NMY-1 is expressed at the time of elongation, is partially required for elongation and is essential for fertility (PIEKNY *et al.* 2003; KOVACEVIC *et al.* 2013). NMY-2 is essential to establish early embryonic polarity and for cytokinesis (GUO AND KEMPHUES 1996; SHELTON *et al.* 1999; MUNRO *et al.* 2004; LIU *et al.* 2010). Both paralogues appear to act redundantly in embryonic morphogenesis, although the precise stage at which they are acting together has not been determined (PIEKNY *et al.* 2003).

To define more precisely how the two heavy chains act, we used temperature sensitive mutants to inactivate NMY-1/-2 at different stages. Our data establish that NMY-1 and NMY-2 are jointly required once muscles become active. We discuss their role compared to spectrins.

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120 MATERIALS AND METHODS

121 Strains and genetic methods

122 The *C. elegans* control strain N2 and other strains were maintained under standard conditions
123 (Brenner 1974), and were propagated at 20°C unless noted otherwise. A complete list of
124 strains and associated genotypes used in this study are included in Table S1.

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126 Generation of a *nmy-1* thermosensitive allele

127 We introduced a mutation in *nmy-1* by CRISPR (Clustered Regularly Interspaced Short
128 Palindromic Repeats) at the position corresponding to the allele *nmy-2(ne3409ts)* changing
129 NMY-2 Leucine-981 to Proline, which is conserved among non-muscle myosins (see Fig.
130 S1). The mutation in NMY-1 was generated using an oligonucleotide repair template
131 (GAAACCGTCCGTGATCTCGAGGAGCAACTCGAGCAAGAtGAACAAGCTAGACAG
132 AAACTGCTTccGGATAAGACGAATGTTGACCAGAGACTTCGAAACCTGGAAGAGC
133 G) carrying a mutation to create a non-functional PAM (protospacer adjacent motif) site
134 (AGG to ATG), and another to introduce the desired mutation (TTG to CCG). We co-injected
135 the plasmid encoding Cas9 (CRISPR-associated endonuclease 9) and the sgRNA (single-
136 molecule guide RNA) for *nmy-1* (5'GAGGAGCAACTCGAGCAAG) at 50 ng/µl, the *nmy-1*
137 oligonucleotide repair template at 20 ng/µl, along with the plasmids pRF4 and pBSK2 each at
138 100 ng/µl in the strain ML2540 (10.7554/eLife.23866) carrying a CRISPR-generated NMY-
139 1::GFP fusion, so as to track the putative mutant protein. We identified among the 88 F1
140 Roller animals those carrying the mutation using the PCR-primers
141 5'TCAAGCTCACCGCTTTAATTATGAAC and 5'CCCATTTTCTCGGCCAAGTGATCT.
142 One positive hit was named *nmy-1(mc90ts)*, which had two mutations verified by sequencing
143 homozygous *nmy-1(mc90ts)* animals, corresponding to the desired L>P change and to the
144 PAM mutation, such that the protein sequence 959-QEEQARQKLLL became 959-
145 QDEQARQKLLP. Homozygous *nmy-1*(mc90ts) animals were sterile at 25°C but not at 15°C
146 (see results).

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148 RNA interference (RNAi)

149 RNAi experiments were performed by feeding on *HT115 Escherichia coli* bacteria strains
150 generating double-stranded RNA (dsRNA) from the Ahringer-MRC feeding RNA
151 interference (RNAi) library (Kamath et al., 2003) using L4 larvae at 20°C. RNAi feeding was
152 performed for 24 hrs using standard procedures, with 100 µg ml⁻¹ ampicillin/1 mM IPTG
153 (Sigma). Empty L4440 RNAi vector served as a control.

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155 Hatch count, arrest count protocols and microscopy

156 Strains carrying a thermosensitive mutation were maintained at 15°C on agar plates. Embryos
157 at different developmental stages on the plates were removed with an eyelash tool, washed in
158 M9, and put on a 5% agarose pad with M9 on a slide sealed in paraffin oil. The slide was then
159 imaged using a Roper Scientific spinning disk system with an immersion oil 40x objective
160 (Zeiss Axio Observer Z1 microscope, Yokogawa CSUX1-A1 spinning disk confocal head,
161 Photometrics Evolve 512 EMCCD camera, Metamorph software). Images were initially taken
162 at 20°C until the incubation chamber attached to the microscope increased to 25°C in less than
163 5 minutes, and imaging was continued overnight (8 hrs). A count was then performed to
164 determine how many embryos of a particular initial developmental stage could make it to
165 hatching, and at what stage they had arrested, if so. Temperature downshifts were done by
166 bringing the microscopy slide on a pre-equilibrated metal block set in a 15°C incubator.

167 Spinning disk fluorescence imaging was performed with a 63× or 100× oil-immersion
168 objective, NA=1.4. Images of embryos were acquired using time-lapse mode with a 110 ms
169 exposure at intervals depending on the experiment. To image MLC-5::GFP, NMY-2::mKate
and NMY-1::GFP (Fig. 1B-E), we randomly picked embryos and staged them based on their length. Laser power and exposure times were kept constant throughout the experiments for specific strains and their controls. Images of Fig. 1F were acquired with a LSM 980 Airyscan2 Zeiss confocal system equipped with 488 nm and 561 nm excitation laser lines and an oil immersion objective with NA=1.4 simultaneously acquiring the green and red channels in confocal mode. The images were deconvolved with the Huygens software.

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## Image processing and quantification

All images were treated by first subtracting the background with a rolling ball radius of 50 pixel. Stacks were projected using maximum intensity. In Fig. 4, the average displacement was obtained by tracking stable points of high intensity in the epidermis and calculating the displacement using the following equation:

$$d = \sqrt{(x_{i+1} - x_i)^2 + (y_{i+1} - y_i)^2}.$$

Fig. 4 images were three-plane projections from stacks taken with an inter-plane distance of 0.8 µm to capture the epidermis. The distribution of the signal was obtained by measuring the mean intensity and standard deviation of a square of 45X33 pixels inside the H₁ cell of the lateral epidermis, distribution = σ/|I|. Images with an even distribution of signal (meaning no bright spots) have a low standard deviation σ, and therefore a low distribution. In the case of bright spots against a black background, the value of σ increases, and therefore so will the distribution.

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## 190 RESULTS and DISCUSSION

## 192 The concentration of non-muscle myosins increases until the 2-fold stage

To understand when non-muscle myosins are required during elongation, we examined the fluorescence of three CRISPR knockin strains marking the essential light chain MLC-5, NMY-2 and NMY-1. We observed that the fluorescence of MLC-5::GFP increased between the lima bean and 2-fold stages; it was followed by a slight plateau in the lateral cells, and a steady value in the dorso-ventral (DV) cells (Fig. 1B-C). Using an NMY-2::mKate; NMY-1::GFP double knockin strain, we observed that only NMY-2 declined after the beginning of the lima bean stage, whereas NMY-1 increased after that stage (Fig. 1D-E). At higher magnification, we observed that NMY-2 was faint with no clear pattern in the lateral cells but formed aligned puncta in DV cells, whereas NMY-1 was enriched in the lateral epidermis cells as aligned puncta and colocalized in DV epidermal cells with NMY-2 puncta (Fig. 1F). Hence, the myosin population is dynamic.

## 205 Inhibition of NMY-1 and NMY-2 impairs late elongation

To test the functions of the two non-muscle myosin heavy chains, we used conditional alleles. A temperature sensitive *nmy-2* allele had previously been described, *nmy-2(ne3409ts)*, which changes a conserved Leucine residue among heavy chains into a Proline, NMY-2(L981P) (*LIU et al.* 2010). We engineered an NMY-1(L970P) change by CRISPR at the position homologous to that of *nmy-2(ne3409)*; the strategy also involved a change of E960 into D (see Methods and Fig. S1). The resulting allele, named *nmy-1(mc90ts)*, induced a thermosensitive sterile phenotype but very little lethality (Table 1; Fig. 2A, 2B-line4). We assume that sterility was due to the L970P mutation rather than to the conservative E960D change, because the *nmy-2(ne3409ts)* at the homologous position is conditional, but cannot
exclude additive effects of both changes. At the non-permissive temperature, the allele *nmy-1*(mc90ts) behaved like the previously known *nmy-1* missense allele *sb113*, which induced <10% lethality and partial sterility, but was not as severe as the presumptive null allele *sb115*, which induced over 50% embryonic and larval lethality (PIEKNY *et al.* 2003). Since the mutant protein NMY-2(ne3409ts) unfolds and becomes very rapidly inactive when animals are shifted to 25°C (LIU *et al.* 2010), we expected the mutant protein NMY-1(mc90ts) to behave similarly.

Using these two conditional alleles, we investigated at which stage they are required during elongation by performing temperature shift experiments from 20°C to 25°C (Fig. 2A). Homozygous *nmy-2(ne3409ts)* embryos displayed embryonic lethality when upshifted at an early elongation stage, but could elongate if shifted at or beyond the 2-fold stage (Fig. 2B-line8). By contrast, double *nmy-2(ne3409ts)*; *nmy-1(mc90ts)* mutants displayed 100% embryonic lethality irrespective of the time at which they were shifted to 25°C (Fig. 2B-line10), yet could generally hatch if maintained at 20°C (Fig. 2B-line9).

We further examined at which elongation stage non-muscle myosin mutants arrested. When *nmy-2(ne3409ts)* embryos were shifted to 25°C at the pre-comma stage, about 40% remained pre-comma and 40% made it to the comma stage (Fig. 2D). When shifted at the comma stage, 50% *nmy-2(ne3409ts)* progressed to the 2-fold stage. When double *nmy-2(ne3409ts)*; *nmy-1(mc90ts)* mutants were shifted to 25°C, they immediately stopped elongation at the stage at which the temperature had been raised (Fig. 2C-D). Importantly, they did not regress to an earlier body morphology (Fig. 2C), as we had previously observed in *spc-1 pak-1* or *fhod-1*; *spc-1* double mutants (LARDENNOIS *et al.* 2019) Hence, NMY-2 is required during elongation, prior to muscle activity. Moreover, we conclude that NMY-1 and NMY-2 are continuously required for elongation at all stages, that they act partially redundantly, but that they are not required to maintain body shape. Finally, we suggest that both non-myosin mutant proteins
240 become very rapidly inactive at 25°C. The arrest phenotype observed for *nmy-2(ne3409ts)*;
241 *nmy-1(mc90ts)* mutants after an early upshift resembles that of strong *let-502*, *mlc-4* or *mlc-5*
242 deficient embryos (WISSMANN *et al.* 1997; GALLY *et al.* 2009), except that *let-502 sb118ts)*
243 elongate normally if upshifted to 25.5°C at the 2-fold stage (DIOGON *et al.* 2007). As
244 previously suggested (GALLY *et al.* 2009), this difference may reflect that the kinases PAK-1
245 or MRCK-1 act in parallel to LET-502/Rho-kinase to phosphorylate MLC-4/RMLC beyond
246 the 2-fold stage.

247 Our results extend an earlier study suggesting that the two non-muscle myosins NMY-1 and
248 NMY-2 are redundantly required during elongation (PIEKNY *et al.* 2003). Those previous
249 experiments involved RNA interference against *nmy-2* in the background of the non-
250 conditional and putative null alleles *nmy-1 sb113*) or *nmy-1 sb115*). As RNAi against *nmy-2*
251 had to be mild to allow cytokinesis, it did not enable the authors to define when NMY-1 and
252 NMY-2 act. Our results establish that neither NMY-1 nor NMY-2 alone is required, but that
253 they are constantly and redundantly required during late embryonic elongation, which
254 depends on muscle input. Moreover, we conclude that NMY-2 has a more critical role during
255 early elongation since all single *nmy-2* mutants, but only a few single *nmy-1* mutants, arrested
256 when shifted to 25°C during early elongation. Consistent with these results, NMY-1, NMY-2,
257 and the myosin essential light chain MLC-5 are expressed in the epidermis throughout
258 elongation.

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260 Elongation of *nmy-2* (*ne3409ts*); *nmy-1婚*(*mc90ts*) resumes after arrest

261 The double *nmy-2(ne3409ts)*; *nmy-1(mc90ts)* mutant always arrested during elongation,
262 regardless of their stage at the time of upshift. We thus wondered whether elongation could
263 resume after an arrest. To test this possibility, we shifted *nmy-2(ne3409ts)*; *nmy-1(mc90ts)*
264 embryos at 25°C for 45 min, then back to 15°C for 5 hrs before imaging. As described in Fig.
265 3A, 60% of 2-fold *nmy-2(ne3409ts)*; *nmy-1(mc90ts)* embryos hatched, as compared to 0% at
266 25°C. This resumption suggests that the temporary absence of NMY-1 and NMY-2 did not
267 permanently damage any of the molecular- components involved. Finally, we investigated for
268 how long could elongation be paused and restarted. By increasing the duration of the pause,
269 we found that the curve was sigmoidal, with a mid-height value of about 2,5 hours, and that it
270 levelled off around 5 hours (Fig. 3B). For the embryos that developed and hatched after the
271 reversal of their arrest, no phenotype was observed.

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273 *nmy-2 (ne3409ts); nmy-1(mc90ts) has normal muscle activity at 25°C*

274 We considered two explanations for *nmy-2(ne3409ts)*; *nmy-1(mc90ts)* embryos stopping
275 elongation when shifted to 25°C at the 2-fold stage. One was that these non-muscle myosins
276 are required in muscles, perhaps to enable strong contractions or to better transmit the force to
277 the epidermis. Another possibility was that they act in the epidermis.

278 We examined whether *nmy-2(ne3409ts)*; *nmy-1(mc90ts)* embryos twitch normally, relying on
279 the GFP fluorophore marking *nmy-1(mc90ts)*. Specifically, we used irregularities in the
280 NMY-1::GFP pattern as landmarks to monitor the twitching of control *nmy-1::gfp* embryos,
281 of embryos in which muscles had been made inactive by RNAi against *unc-112* which is
282 essential to assemble myofilaments (ROGALSKI *et al.* 2000), and of *nmy-2(ne3409ts)*; *nmy-
283 1(mc90ts)* double mutants.

284 Analysis of NMY-1::GFP videograms showed that embryonic movements can be
285 decomposed into two fundamental movements: rotation which corresponds to 90° changes in
286 the body position within the eggshell (YANG 2017), and twitching which corresponds to
287 lateral displacement of epidermal landmarks in-between body rotations (Fig. 4A, lower
288 panels). We found that both control and double non-muscle myosin mutants at 25°C could
rotate on average every 12 seconds (Fig. 4A-B upper panels; Fig. 4D), and contract locally over 0.73 ± 0.06 µm/sec, as compared to the control *nmy-1::gfp*, 0.81 ± 0.06 µm/sec when placed at 25°C (Fig. 4E). By contrast the muscle deficient strain exhibited no rotations and contracted 0.21 ± 0.01 µm/sec (Fig. 4C-E). We conclude that the double mutant is not lacking the mechanical input from the muscles needed to drive elongation, and that the forces are properly transmitted to the epidermis. Although *NMY-1/-2* could have ancillary roles in muscle contractions, different types of myosin heavy chains (e.g., UNC-54) are required to generate force in striated muscles.

The results above suggest that both non-muscle myosins are required in the epidermis. Which function could non-muscle myosins perform in the epidermis? One possibility could be that non-muscle myosins keep pulling on actin filaments of opposite polarity in the lateral epidermis as they do until the 2-fold stage. This scenario seems unlikely since actin filaments become organized beyond the 2-fold stage (GILLARD *et al.* 2019), implying that pulling should be less efficient. Alternatively, non-muscle myosins might keep actin ends together, prior to FHOD-1 activity and in parallel to spectrin, after the repeated muscle contractions induce their severing (LARDENNOIS *et al.* 2019). Non-muscle myosins have two well-characterized activities, actin binding and actin pulling. We did not directly test in which cells *NMY-1* and *NMY-2* act. However, we note that when wild-type MLC-4 is expressed under a dorso-ventral epidermal promoter in *mlc-4* null mutant embryos, the embryos elongate up to the 2.5-fold but do not reach full elongation (GALLY *et al.* 2009), indicating that MLC-4 activity, and hence *NMY-1/2* activity, is most likely also required in dorso-ventral cells. In part because the precise organization and polarity of actin filaments within dorso-ventral actin bundles are unknown (COSTA *et al.* 1997), we cannot currently determine whether non-muscle myosins are only required to maintain actin ends together or if they pull on severed actin filaments to bring them closer before FHOD-1 activity. If actin filaments all have the same
314 polarity, it would tend to exclude a pulling function, since pulling of actin filaments by non-
315 muscle myosin relies on filaments of opposite polarity.

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317 **Inactivated NMY-1 forms aggregates**

318 We noticed that the NMY-1(mc90ts>::GFP protein aggregated in the epidermis at the non-
319 permissive temperature (Fig. 5A). By quantifying the distribution of these aggregates in the
320 lateral cells (see Methods), we measured a value of 0.5 ± 0.1 at 25°C for control NMY-
321 1::GFP embryos, the most uniform distribution of all the strains. At 20°C, the NMY-
322 1(mc90ts) displayed some degree of aggregation compared to control NMY-1::GFP (see Fig.
323 5A). At 25°C, we observed a marked aggregation for the single mutant *nmy-1 mc90ts*) (1.4 ±
324 0.1) as well as for the double mutant *nmy-2(ne3409ts); nmy-1 mc90ts*) (1.5 ± 0.1) (Fig. 5A-
325 B). Interestingly, oxygen depletion, which has a marked effect on the regulatory light chain
326 MLC-4 distribution (GALLY *et al.* 2009), did not modify that of wild-type NMY-1::GFP. It
327 implies that the aggregation of NMY-1(mc90ts) does not result from ATP-depletion,
328 consistent with the fact that the *mc90* mutation is located in the coiled coil region of NMY-1
329 (Fig. S1). Left at 25°C, these aggregates formed small clusters, which began to fuse for
330 roughly 1.5 hours, until a sudden transition occurred within 3 minutes. At that point, the
331 aggregates disappeared in a highly coordinated fashion. Note that the images used for the
332 quantification of the distribution of the aggregates were taken on average one hour after the
333 temperature shift had occurred, and thus correspond to roughly the half-point in the time-
334 evolution of the particles. Our findings are consistent with the literature, indicating that non-
335 functional non-muscle myosins in both *C. elegans* oocytes, and human thrombocytes tend to
336 aggregate (ALTHAUS AND GREINACHER 2009; SUN *et al.* 2020).

337

338 **Conclusion**
339 Altogether, our data demonstrate that either NMY-1 or NMY-2 can support elongation but
340 that if both are absent elongation stops immediately during late elongation. Importantly, late
341 elongation arrest could not be attributed to less muscle movement, and thus to abnormal
342 mechanotransduction in the epidermis. Elongation arrest could be reversed by returning the
343 embryos to < 20°C, indicating that the absence of both non-muscle myosin heavy chains did
344 not permanently affect actin integrity, nor any other epidermal structure. Our data are
345 compatible with the possibility that this myosin pair acts to reduce the length of the epidermal
346 actin filaments by holding or pulling the severed ends together.
347

347

348 **Data Availability Statement**

349 Strains and plasmids are available upon request. The authors affirm that all data necessary for
350 confirming the conclusions of the article are present within the article, figures, and tables.
351

351

352 **Acknowledgements**

353 The authors thank Martine Ben Amar for discussions, and greatly acknowledge France
354 Lam from the IBPS Imaging Facility for running the Zeiss Airyscan confocal microscope and
355 for performing deconvolution of the images. The IBPS Imaging facility is supported by
356 Region-Île-de-France, Sorbonne-University and CNRS. This work was supported by an
357 Agence Nationale de la Recherche grant to Michel Labouesse (project number ANR-18-
358 CE13-0008-01).
359

359

360 **Conflict of Interest**

361 The authors declare no competing or conflict of interests.
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**Figure 1.** The two non-muscle myosins are present in the epidermis throughout morphogenesis

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(A) Cross-section through an embryo illustrating the main anatomical features of the embryo: the dorsal and ventral epidermis (pink, red), the lateral epidermis (yellow) and the muscles (orange); other tissues not represented for clarity. Muscles are attached to the apical extracellular matrix (not illustrated) surrounding the embryo through hemidesmosomes in the epidermis (blue dots). Actin filaments (green) run circumferentially as bundles. The current model holds that in a typical contraction cycle, muscles will locally displace the actin filaments (black arrows in 2^nd^ drawing), which will trigger their severing (3^rd^ drawing), before they eventually get stabilized by a complex of actin-binding proteins (dark green stars). At the end of the cycle, when the underlying muscles relax, the embryo has become thinner and longer (4^th^ drawing). It is unclear whether the actin-binding complex can hold actin ends to counter the hydrostatic pressure (blue arrows in 3^rd^ drawing) if they drift apart. (B)

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Fluorescence micrographs showing the distribution of the essential myosin light chain MLC-5 (marked by a GFP knockin) at three different stages: lima bean, 2-fold, 3-fold, N=41. Note that MLC-5 is enriched in the lateral cells (yellow arrow), and 2F stands for two-fold. (C)

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Quantification of MLC-5::GFP fluorescence over time in the lateral and dorso-ventral epidermis. (D) Fluorescence micrographs showing the distribution of the two large non-muscle myosin chains NMY-1 (marked by a GFP knockin) and NMY-2 (marked by a mKate knockin) at different stages; timing starts approximately 350 minutes after the 2-cell stage, N=20 (black line in E; see video1). (E) Quantification of NMY-1::GFP and NMY-2::mKate fluorescence starting after the time of epidermal differentiation by measuring the average intensity in an ROI spanning the lateral and DV cells. (F) Deconvoluted confocal micrographs of NMY-1::GFP and NMY-2::mKate in the lateral (yellow box) and dorso-ventral

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390
391 (pink box) epidermis of a 3-fold embryo. Higher magnification of the area marked by a dotted rectangle are shown on the left for lateral cells (three consecutive focal planes; 1.75x magnification), or in the top right corner for the dorso-ventral epidermis (4.5x magnification) revealing short circumferential alignments. Scale bars, 10 µm (B, D, F), except 4 µm for the enlargements.
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396

397 **Figure 2. NMY-1 and NMY-2 are required during the morphogenetic phase driven by muscles**
398

399 (A) Outline of the temperature shift experiments: embryos were maintained at 20°C and
400 shifted to 25°C at different stages (prior to comma, comma, or two-fold). (B) Percentage of
401 hatching when embryos were left at 20°C or shifted to 25°C at the stage indicated in the top
402 row. (C) DIC images of *nmy-2(ne3409ts)*; *nmy-1(mc90ts)* left at 15°C (top row) or shifted to
403 25°C (bottom row). Note that both lima bean and 1.8-fold embryos essentially did not
404 elongate any further after the shift to 25°C at t0, nor did they retract; scale bar 10 µm. (D)
405 Percentage and arrest stage of single *nmy-2(ne3409ts)*, *nmy-1(mc90ts)* and double mutants
406 shifted to 25°C at the stage indicated on the X axis. Note that for the double mutant, all 2-fold
407 embryos arrested at the 2-fold stage. The number of embryos was at least 100 for each
408 genotype.
409

410

410 **Figure 3. Resumption of elongation.**

411 (A) Double *nmy-2(ne3409ts)*; *nmy-1(mc90ts)* mutants were shifted to 25°C at the stage
412 indicated at the bottom and left overnight at 25°C (left three columns, taken from Fig. 2D) or
413 left at 25°C for 1 hour, causing arrest, then shifted for 5 hrs at 15°C (right three columns).
414 Note that elongation could resume in many cases, N=80. (B) Quantification of the percentage
415 of *nmy-2(ne3409ts)*; *nmy-1(mc90ts)* embryos that could resume elongation after being left at
416 25°C for various duration and then shifted back to 15°C (N>500, embryos at least 1.5-fold).

417 The polynomial fit probability is 0.9903.

418

419 **Figure 4. Depletion of NMY-1 and NMY-2 does not affect muscle contractions**

420 (A) Spinning disk micrographs from Video2 illustrating a rotation (top two images; see
421 yellow arrow) or local contractions (bottom four images) in *nmy-1[mc82(nmy-1::gfp)]*

422 knockin embryos grown at 25°C; timing refers to the video. Note in the bottom four images
423 the local magnitude of contraction between two brighter NMY-1::GFP points (yellow

424 brackets with their size above). (B) Spinning disk micrographs from Video3 illustrating a

425 rotation (top two images; see yellow arrow) or local contractions in double *nmy-2(ne3409ts)*;
426 *nmy-1(mc90ts)*::gfp shifted to 25°C when they reached the 2-fold stage; timing refers to the

427 video. Note the local contractions (yellow brackets with their size above). (C) Spinning disk
428 micrographs from Video4 the local contractions in a *nmy-1[mc82(nmy-1::gfp)]* embryo

429 treated by RNAi against the gene *unc-112* and raised at 25°C; timing refers to the video. Note
430 the local contractions (yellow brackets with their size above). (D) Quantification of the time
431 elapsed between embryo rotations, defined as at least 90° about the centre line of the embryo,
432 as observed in videos similar to those shown in (A-C); *unc-112(RNAi)* failed to rotate, error
433 bars due to the rotation event occurring over several seconds, 10%. (E) Quantification of
434 bright NMY-1::GFP particles lateral movements consecutive to muscle twitching measured
435 from videos such as those in (A-C). The number of embryos was at least 18 for each genotype
436 in all quantifications, and the error bars resulted from particle tracking which was done by
437 hand, 7%.

438

439 **Figure 5. NMY-1(mc90ts>::GFP spots aggregate at the non-permissive temperature**
440 (A) Spinning disk micrographs illustrating NMY-1::GFP particles in different genotypes
441 when embryos were raised at 20°C, shifted to 25°C after reaching the 2-fold stage, or grown
442 at 25°C in anaerobic conditions. Enlargements (1.75x) of the boxed areas are shown in the
443 bottom right corner. (B) Spinning disk micrographs illustrating the further aggregation of
444 NMY-1(mc90ts)::GFP particles, until their rapid dissolution; images taken from Video6. (C)
445 Quantification of the distribution of NMY-1::GFP particles brightness measured in the H1
446 lateral cell and expressed as standard deviation of the signal divided by the mean. The number
447 of embryos was at least 25 for each genotype, error bars 8%.

448

449

450

451 Supplementary figures

452 Supplementary figure 1. Sequence alignment of non-muscle heavy chains and positions
453 of the *nmy-1* and *nmy-2* conditional alleles

454 Upper bar, schematized structure of a non-muscle heavy chain, indicating the position of the
455 residue L981 which is mutated to a proline in *nmy-2(ne3409ts)*. Below, alignment of seven
456 heavy chains starting in the IQ region. The residue altered in *nmy-2(ne3409ts)* (red arrow) is
457 conserved and was also mutated in *nmy-1(mc90ts)* (green circle). Red asterisk, position of the
458 secondary mutation introduced to mutate the PAM site (959-QEEQARQKLLL to 959-
459 QDEQARQKLLL).

460

461 Table 1. Progeny counts for temperature sensitive strains.

462 For each strain, 10 plates, each with one adult, were left for 24 hrs at 20°C. The same was
463 done at 25°C. The top table represents the sum total of the progeny, whereas the bottom table
464 is the average number of progeny per adult. Of note is the low progeny for *nmy-1(mc90ts)* and
465
466 *nmy-2(ne3409ts);nmy-1(mc90ts)* even at 20°C, and the total sterility of the latter strain at
467 25°C.

468

469

470 **Supplementary videos**

471 **Video1:** Temporal distributions of a strain carrying NMY-2::mKate and NMY-1::GFP knockins (strain FBR241). The acquisition rate was 1 image every 15 minutes, and the playback speed is 6 frames per second.

474

475 **Video2:** Two-fold embryo of *nmy-1(mc82[nmy-1::gfp]* control strain. Acquisition rate, 1 frame per second; playback speed, 6 frames per second. Still images of Fig. 3A were taken from this video, with the timing indicated on each image corresponding to the real timing on this videogram.

479

480 **Video3:** Double *nmy-2(ne3409ts); nmy-1(mc90ts) :: gfp* mutant embryo shifted to 25°C after
481 2-fold stage. Acquisition rate, 1 frame per second; playback speed, 6 frames per second. Still
482 images of Fig. 3B were taken from this video, with the timing indicated on each image
483 corresponding to the real timing on this videogram.

484

485 **Video4:** A *unc-112(RNAi); nmy-1(mc82[nmy-1::gfp]* embryo beyond the 2-fold stage, which
486 had been raised at 25°C. Acquisition rate, 1 frame per second; playback speed, 6 frames per
487 second. Still images of Fig. 3D come from this video, with the timing indicated on each image
488 corresponding to the real timing on this videogram.

489
490 **Video5:** A two-fold *nmy-1(mc90ts)*::gfp embryo raised at 25°C. Protein aggregates, embryo rotations and twitching, can be observed. Acquisition rate, 1 frame per second; playback speed, 6 frames, per second.

493
494 **Video6:** A 1.5-fold *nmy-2(ne3409ts)*; *nmy-1(mc90ts)*::gfp embryo which had been shifted to 25°C. Note the gradual fusion of the NMY-1::GFP aggregates in the epidermis over time.

496 Acquisition rate, 1 frame per 1 minute; playback speed, 6 frames per second.

497
498
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<figure><img src="image_1.png" /></figure>

<figure><img src="image_2.png" /></figure>

<figure><img src="image_3.png" /></figure>

<figure><img src="image_4.png" /><figcaption>Figure 1</figcaption></figure>
<figure><img src="image_5.png" /><figcaption>Figure 2</figcaption></figure>

<table><caption>B</caption><thead><tr><th>Line</th><th>Genotype</th><th>Temp (Celsius)</th><th>N</th><th>pre comma (% elong.)</th><th>comma (% elong.)</th><th>Two-fold (%elong.)</th></tr></thead><tbody><tr><td>1</td><td>wild type</td><td>20</td><td>130</td><td>100</td><td>95</td><td>100</td></tr><tr><td>2</td><td>wild type</td><td>25</td><td>105</td><td>100</td><td>100</td><td>100</td></tr><tr><td>3</td><td>nmy-1(mc90ts):gfp</td><td>20</td><td>98</td><td>96</td><td>100</td><td>100</td></tr><tr><td>4</td><td>nmy-1(mc90ts):gfp</td><td>25</td><td>130</td><td>89</td><td>96</td><td>100</td></tr><tr><td>5</td><td>nmy-1::gfp</td><td>20</td><td>115</td><td>100</td><td>100</td><td>100</td></tr><tr><td>6</td><td>nmy-1::gfp</td><td>25</td><td>91</td><td>100</td><td>100</td><td>100</td></tr><tr><td>7</td><td>nmy-2(ne3409ts)</td><td>20</td><td>150</td><td>89</td><td>100</td><td>100</td></tr><tr><td>8</td><td>nmy-2(ne3409ts)</td><td>25</td><td>124</td><td>11</td><td>46</td><td>100</td></tr><tr><td>9</td><td>nmy-2(ne3409ts); nmy-1(mc90ts):gfp</td><td>20</td><td>155</td><td>70</td><td>85</td><td>100</td></tr><tr><td>10</td><td>nmy-2(ne3409ts); nmy-1(mc90ts):gfp</td><td>25</td><td>130</td><td>0</td><td>0</td><td>0</td></tr></tbody></table>

<figure><img src="image_6.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_7.png" /></figure>
A

<figure><img src="image_8.png" /></figure>

B

<figure><img src="image_9.png" /><figcaption>Figure 3</figcaption></figure>
<figure><img src="image_10.png" /><figcaption>Figure 4</figcaption></figure>
<figure><img src="image_11.png" /></figure>

sigma divided by mean in H1

<caption>**Figure 5**</caption>
<table><caption>Table 1</caption><thead><tr><th colspan="3">total number of eggs layed by 10 mothers overnight</th></tr><tr><th>Genotype</th><th>20°C</th><th>25°C</th></tr></thead><tbody><tr><td>wild-type</td><td>556</td><td>651</td></tr><tr><td>nmy-1(mc90ts)</td><td>26</td><td>5</td></tr><tr><td>nmy-2(ne3409ts)</td><td>463</td><td>261</td></tr><tr><td>nmy-2(ne3409); nmy-1(mc90ts)</td><td>8</td><td>0</td></tr></tbody></table><table><thead><tr><th colspan="3">total number of eggs layed overnight per mother *</th></tr><tr><th>Genotype</th><th>20°C</th><th>25°C</th></tr></thead><tbody><tr><td>wild-type</td><td>55.6</td><td>65.1</td></tr><tr><td>nmy-1(mc90ts)</td><td>2.9</td><td>0.5</td></tr><tr><td>nmy-2(ne3409ts)</td><td>46.3</td><td>26.1</td></tr><tr><td>nmy-2(ne3409); nmy-1(mc90ts)</td><td>0.8</td><td>0</td></tr></tbody></table>

<footnote>* each entry corresponds to the entry in the upper part divided by 10</footnote>