Divergence of cortical force-generating mechanisms underlies differences in spindle behavior between C. elegans and C. inopinata

Shun Oomura; Koji Kyoda; Shuichi Onami; Nami Haruta; Asako Sugimoto

doi:10.1371/journal.pone.0352057

. 2026 Oct 27;21(10):e0352057. doi: 10.1371/journal.pone.0352057

Divergence of cortical force-generating mechanisms underlies differences in spindle behavior between C. elegans and C. inopinata

Shun Oomura ¹, Koji Kyoda ², Shuichi Onami ², Nami Haruta ¹, Asako Sugimoto ^1,^*

Editor: Bob Goldstein³

PMCID: PMC13712525 PMID: 42945194

Abstract

Microtubule-dependent pronuclear migration and mitotic spindle positioning are fundamental processes during the first embryonic division in many animals. In the one-cell embryo of Caenorhabditis elegans, these events are regulated by well-characterized pulling forces acting on astral microtubules, including cortical forces mediated by the Gα-GPR-LIN-5 dynein complex. Although the overall framework of these dynamics is conserved, recent studies have revealed substantial interspecies variation in their regulation. Here, we investigated nuclei and mitotic spindle behaviors in one-cell embryos of Caenorhabditis inopinata, the closest known relative of C. elegans, using live-cell imaging and functional perturbation. We found that C. inopinata embryos exhibit altered pronuclear migration, reduced anaphase spindle oscillations, and slower centrosome diffusion during telophase compared with C. elegans. These differences suggest weaker cortical pulling forces. Functional analyses using RNA interference showed that GPR retains its essential role in force generation, whereas the contribution of the microtubule depolymerizing kinesin KLP-7 is reduced in C. inopinata. Our results point to evolutionary changes in microtubule-regulated spindle dynamics, and provide insight into how conserved cellular processes can diversify through subtle changes in their underlying mechanisms.

Introduction

Precise regulation of microtubules is essential for the spatiotemporal control of intracellular dynamics. These structures play a central role in key events during early embryogenesis, including pronuclear migration and mitotic spindle positioning.

In the one-cell-stage embryo of Caenorhabditis elegans, nuclear and mitotic spindle dynamics are regulated by a combination of cytoplasmic and cortical forces acting on astral microtubules. Upon fertilization, centrosomes near the male pronucleus nucleate microtubules that guide the female pronucleus toward the posterior, resulting in the formation of a nucleus-centrosome complex (NCC) [1]. This complex then migrates to the cell center and rotates along the anterior-posterior axis. The spindle is positioned by two distinct types of forces: (1) cytoplasmic, length-dependent forces involving cytoplasmic dynein along microtubules [2], and (2) cortical pulling forces generated by interactions between astral microtubules and the Gα-GPR-LIN-5 dynein complex at the cell cortex [3]. Posterior enrichment of this complex causes net posterior displacement of the spindle and generates transverse oscillations during anaphase [3–8].

Although the molecular components of this force-generating machinery are broadly conserved, studies across Rhabditida nematodes have revealed substantial variation in spindle dynamics, suggesting evolutionary flexibility in the regulation of these forces [9–11]. Understanding the basis of this variation provides an opportunity to investigate how microtubule regulatory mechanisms have diversified across species.

Caenorhabditis inopinata, the closest known relative of C. elegans, provides a useful model for such comparative studies. Despite their close phylogenetic relationship, having diverged ~10.5 million years ago, these two species differ in adult morphology, reproductive systems, and ecological adaptations [12]. These differences suggest possible divergence in intracellular dynamics, including microtubule-dependent events. However, intracellular dynamics during C. inopinata development remains poorly characterized.

In this study, we conducted a comparative analysis of the dynamics of pronuclear migration and mitotic spindles in one-cell-stage embryos of C. inopinata and C. elegans using differential interference contrast (DIC) and fluorescence live imaging. We identified differences in pronuclear migration and mitotic spindle behavior, suggesting that cortical pulling forces are weaker in C. inopinata. RNAi-based assays further suggested that reduced activity of the microtubule-depolymerizing kinesin KLP-7 may contribute to this weakened pulling force. These findings demonstrate that even subtle changes in microtubule regulatory pathways can lead to divergent intracellular behaviors between closely related species.

Materials and methods

Strains and culture conditions

C. elegans strains were cultured as described by Brenner (1974) [13]. Worms were cultured at 20 °C on nematode growth medium (NGM) plates seeded with Escherichia coli OP50. The following strains were used: N2 (wild-type), SA1161 (tjSi193[eft-3p::gfp::his-58::tbb-2 3’UTR, rps-27p::NeoR::unc-54 3’UTR]) [14], and SA250 (tjIs54[pie-1p::gfp::tbb-2, pie-1p::2xmCherry::tbg-1, unc-119+]; tjIs57[pie-1p::mCherry::his-48, unc-119+]) [15]. The wild-type strain of C. briggsae (HK104) was cultured under the same conditions.

C. inopinata strains were cultured at 27 °C on modified NGM plates containing 2.5% agar and half the standard concentration of MgSO₄ (0.5 mM), seeded with Escherichia coli HT115 (DE3) [14]. For routine culture, gravid females were transferred onto these plates to lay eggs, and their progeny reached adult stage after approximately 4 days. The following strains were used: NKZ35 (wild-type), SA1438 (tjIs372[eft-3p::gfp::his-58::tbb-2 3’UTR, rps-0p::HygR::unc-54 3’UTR]) [14], and SA1648 (tjIs375[pie-1p::gfp::tbb-2::tbb-2 3’UTR, rps-0p::HygR::unc-54 3’UTR]) [14].

Microscopy

For live-cell imaging, gravid C. elegans hermaphrodites or gravid C. inopinata females were dissected in M9 buffer to release embryos, which were then mounted on 2% agarose pads. DIC images were acquired every 0.5 s using an ORCA-R2 digital CCD camera (Hamamatsu Photonics) mounted on an Axioplan 2 imaging microscope (Zeiss) equipped with a C-Apochromat 63x/1.2 W Corr M27 water-immersion objective lens (Zeiss).

Fluorescent live images of GFP::histone and GFP::β-tubulin were acquired using a CSU-X1 spinning disc confocal system (Yokogawa Electric Corporation). GFP::histone images were acquired every 30 s with 23 z-slices at 1 μm intervals using an iXonEM+ EMCCD camera (Andor technology) mounted on an Axioplan 2 imaging microscope (Zeiss) equipped with a C-Apochromat 63x/1.2 W Corr M27 water-immersion objective lens (Zeiss). GFP::β-tubulin images were acquired every 30 s for non-RNAi-treated C. elegans embryos or 60 s for RNAi-treated C. elegans and C. inopinata embryos with 10 z-slices at 1 μm intervals using an ORCA-flash4.0 V3 digital CMOS camera (Hamamatsu Photonics) mounted on an IX71 microscope (Olympus) equipped with a UPlanSApo 60 × /1.30 silicone oil-immersion objective lens (Olympus). MetaMorph software (Molecular Devices) was used to control the microscopes.

All live imaging experiments were performed at 25°C unless otherwise noted. During imaging, the room temperature was maintained using the air conditioning system, and the temperature around the sample was measured immediately before imaging.

Tracking of pronuclei, chromosomes, centrosomes, and spindle movements

ImageJ/Fiji software (NIH; https://imagej.net/Fiji) was used for image processing and analysis. Fluorescent images were processed by z-projection using averaged intensity values across z-slices.

Pronuclei, centrosomes, and chromosomes were manually tracked, and their positions were recorded relative to embryo length (anterior-posterior axis) and width (dorsal-ventral axis). Spindle position was defined as the midpoint between the two centrosomes, spindle angle as the angle of the line connecting the centrosomes, and spindle length as the distance between the centrosomes.

Quantification of mitotic spindle oscillation

Centrosome positions were tracked at 1-s intervals in DIC images from nuclear envelope break down (NEBD) to the completion of cytokinesis. During spindle oscillation, rocking peaks were defined as the time points at which a continuous increase (or decrease) in spindle angle lasting at least 4 s switched to a continuous decrease (or increase) lasting at least 4 s. For the initial rocking, the continuous angular displacement up to the first peak was measured. The maximum angular displacement was defined as the largest change in spindle angle between consecutive rocking peaks. Oscillation frequency was determined as the dominant frequency component between 0.02 and 0.10 Hz by Fast Fourier transform (FFT) with a Hamming window function.

Statistical analysis

Quantification method, sample sizes, and statistical analyses are described in the main text and figure legends. Data were analyzed using GraphPad Prism 9. Statistical significance was assessed using one-way ANOVA followed by Tukey’s multiple comparisons test.

Ortholog search

Orthologs of C. elegans genes in the C. inopinata genome were identified using BLAST+ search [16–18] with amino acid sequences from the C. inopinata v7.10 gene set [12]. The amino acid sequence of the longest isoform of each C. elegans gene was used as the query, and the C. inopinata gene with the lowest E-value was selected as the ortholog. C. inopinata orthologs are referred to with the prefix “Cin”.

RNA interference

For C. elegans, RNAi was performed using the soaking method [19]. Target sequences of gpr-1 (1063 bp), let-99 (1024 bp), and klp-7 (1025 bp) were amplified from cDNA. dsRNA was synthesized using T7 RiboMAX Express Large Scale RNA Production System (Promega) and purified by phenol-chloroform extraction. Twenty L4 hermaphrodites were soaked in RNAi solution containing 2 µg/µl dsRNA at 24.5 °C for 24 h, recovered, and then incubated on NGM plates at 24.5 °C for an additional 24 h before imaging. To assess embryonic lethality, five adult hermaphrodites were allowed to lay eggs at 24.5 °C for 2 h, and the unhatched rate was measured 24 h later.

For C. inopinata, RNAi was performed using the feeding method [12,20], because the recovery rate was very low for the soaking method. Target sequences of Cin-gpr-1 (1017 bp), Cin-let-99 (1040 bp), and Cin-klp-7 (1040 bp) were amplified from genomic DNA and cloned into the T444T feeding RNAi vector [21]. E. coli HT115(DE3) transformed with each RNAi plasmid was seeded onto RNAi plates (NGM supplemented with 1 mM IPTG, 50 μg/mL ampicillin, and 10 μg/mL tetracycline) and incubated at room temperature for one day. Twenty untreated gravid females (P0) were transferred onto the RNAi plates to lay eggs. After 96–120 h at 27 °C, F1 males and females reached the adult stage and were allowed to mate each other. For live imaging, F2 embryos were dissected from RNAi-treated F1 gravid females. To assess embryonic lethality, 20 RNAi-treated F1 gravid females were allowed to lay eggs (F2) at 27 °C for 8 h, and the unhatched rate was measured 24 h later.

Results

The asymmetry of the first embryonic division in C. inopinata is similar to that in C. elegans

We first compared egg size, the duration of the first cell division, and division asymmetry between one-cell-stage embryos of C. elegans (N2) and C. inopinata (NKZ35) using differential interference contrast (DIC) microscopy (Fig 1). Although C. inopinata adults are more than twice as long as C. elegans adults [12], the egg length of C. inopinata (58.8 ± 3.6 µm, mean ± SD, n = 11) was only 5.7% longer than that of C. elegans (55.7 ± 2.9 µm, n = 10, p < 0.05), and egg width did not differ significantly between the two species (C. elegans: 32.5 ± 1.3 µm, n = 10; C. inopinata: 32.2 ± 0.8 µm, n = 11) (Fig 1B). Because more than 80% of embryos hatched at 10–28°C for C. elegans and 16–32°C for C. inopinata (S1 Fig and S1 Table), all observations were conducted at 25°C, a permissive temperature for both species. Under these conditions, the duration of the first cell division, measured from nuclear envelope breakdown (NEBD) to the completion of cytokinesis, was 1.6-fold longer in C. inopinata (7.7 ± 0.4 min, n = 9) than in C. elegans (4.7 ± 0.4 min, n = 10, p < 0.0005) (Fig 1C). The position of the first cleavage furrow, expressed as the relative position (%) = distance from the anterior end of the embryo/ embryo length, was 56.0 ± 0.9% (n = 11) in C. inopinata and 56.7 ± 1.4% (n = 10) in C. elegans, indicating no significant difference in the asymmetry of the first embryonic division between the two species (Fig 1D).

Fig 1 — (A) DIC live imaging of wild-type embryos and fluorescent live imaging of embryos expressing GFP::histone or GFP::β-tubulin. Embryos are oriented with the anterior side to the left. Time is shown relative to the pronuclear meeting (s). All images were acquired at 25°C. Scale bars, 10 µm. (B) Egg length and width measured from DIC images of *C. elegans* (n = 10) and *C. inopinata* (n = 11) embryos. (C) Duration of the first cell division, defined as the time from NEBD to the completion of cytokinesis, based on DIC live imaging of *C. elegans* (n = 10) and *C. inopinata* (n = 9) embryos. ****P < 0.001; unpaired t-test. (D) Cleavage furrow position measured from DIC images of *C. elegans* (n = 10) and *C. inopinata* (n = 9) embryos. ns, no significant; unpaired t-test.

Pronuclear dynamics differ between C. elegans and C. inopinata embryos

Next, we compared pronuclear dynamics between one-cell-stage embryos of C. elegans and C. inopinata using the fluorescent live-cell imaging of GFP::histone (Figs 1A and 2A). In C. elegans, the female and male pronuclei formed at the anterior and posterior poles, respectively (Fig 2A and S2 Table). In C. inopinata, the male pronucleus formed at the posterior pole, as in C. elegans, whereas the female pronucleus formed at various positions; in more than half of the embryos, it formed on the posterior side (6/10) (Fig 2A and S2 Table). The position of the female pronucleus in one-cell-stage embryos reflects the position of the nucleus in mature oocytes [22]. In C. elegans, the oocyte nucleus migrates from the center to the distal cortex during oocyte maturation (Fig 2C and 2D and S2 Table). In contrast, in C. inopinata, the oocyte nucleus did not migrate distally, even in oocytes adjacent to the spermatheca (Fig 2C and 2D). This difference may explain the various positions of female pronuclear formation. In both species, the female and male pronuclei move toward each other at the posterior side. However, the male pronucleus exhibited less anterior movement in C. inopinata than in C. elegans (Fig 2A and S2 Table). Consequently, the pronuclei met at a more posterior in C. inopinata (80.1 ± 3.8%, n = 10) than in C. elegans (66.2 ± 3.4%, n = 10, p < 0.0005) (Fig 2B).

Fig 2 — (A) Time course of pronuclear movement. Pronuclear positions were tracked every 30 s using fluorescent live imaging of embryos expressing GFP::histone: *C. elegans* (n = 10), *C. inopinata* (n = 10). The horizontal axis indicates the relative position (%) along the embryo length, and the transverse axis indicates the relative position (%) along the embryo width. Symbols indicate the following positions: circle, female pronuclear formation; square, male pronuclear formation; arrowhead, pronuclear meeting. Each colored line represents an individual embryo. The number near the female pronuclear formation indicates the time in seconds until the pronuclear meeting. (B) Position of pronuclear meeting: *C. elegans* (n = 10), *C. inopinata* (n = 10). ****P < 0.001; unpaired t-test. (C) DIC images of the proximal gonad in adult *C. elegans* hermaphrodites and *C. inopinata* females. −1, −2, and −3 indicate proximal oocytes adjacent to the spermatheca. Scale bars, 25 µm. (D) Distance between the oocyte nucleus and the distal cortex in proximal oocytes: *C. elegans* (n = 10), *C. inopinata* (n = 11). Oocytes at the corresponding positions were compared between *C. elegans* and *C. inopinata*. **P < 0.01; ****P < 0.0001; unpaired t-test.

NCC and mitotic spindle movements differ between C. elegans and C. inopinata embryos

In C. elegans, after pronuclear meeting, the nucleus-centrosome complex (NCC) migrates toward the center of the embryo, a process known as centration, and rotates 90 degrees to align the centrosomes along the anterior-posterior (A/P) axis [23]. The NCC then undergoes nuclear envelope breakdown (NEBD), and the mitotic spindle forms along the A/P axis. The spindle is displaced slightly posteriorly by the end of anaphase. To compare NCC and spindle movements between C. elegans and C. inopinata embryos, we performed fluorescent live-cell imaging of GFP::β-tubulin, as a microtubule marker (Figs 1A and 3A). During centration, the NCC in C. inopinata migrated anteriorly by 39.9 ± 6.3% of the embryo length (n = 7), approximately twice the distance observed in C. elegans (20.3 ± 1.9%, n = 9), and reached the anterior side of the embryo (Fig 3A and S3 Table). Consequently, the spindle formed more anteriorly in C. inopinata (42.1 ± 5.2%, n = 11) than in C. elegans (52.1 ± 1.5%, n = 10, p < 0.0005), a phenotype referred to here as “overcentration” (Fig 3B). In C. elegans, the NCC rotated to align the centrosomes along the A/P axis (initial spindle angle; 9.1 ± 6.2 degrees, n = 10). In contrast, the NCC in C. inopinata exhibited limited rotation, resulting in a larger initial spindle angle (54.3 ± 25.4 degrees, n = 10, p < 0.0005) (Fig 3B). The initial spindle length did not differ significantly between the species (C. elegans: 10.0 ± 0.7 µm, n = 10; C. inopinata: 10.5 ± 1.0 µm, n = 11) (Fig 3B). By the end of metaphase, however, the spindle became longer in C. inopinata (17.1 ± 1.3 µm, n = 10) than in C. elegans (12.1 ± 0.8 µm, n = 10, p < 0.0005) (Fig 3C and S3 Table). At this stage, the spindle in C. inopinata was positioned slightly posterior to the center of the embryo (58.9 ± 2.3%, n = 11), similar to its position in C. elegans (57.1 ± 1.8%, n = 10), and was aligned along the A/P axis (Figs 1A and 3D and S3 Table).

Fig 3 — (A) Time course of spindle positions from pronuclear meeting to the completion of cytokinesis. Spindle positions were tracked using fluorescent live imaging of GFP::β-tubulin every 30 s in *C. elegans* (n = 9) and 60 s in *C. inopinata* (n = 7). Spindle positions are shown as relative positions (%) along the embryo length. Time is indicated relative to anaphase onset (t = 0 s). (B) Spindle position, angle, and length at spindle formation. The measurement model is shown in the upper left panel. *C. elegans* (n = 10), *C. inopinata* (n = 11). ****P < 0.0001; ns, not significant; unpaired t-test. (C) Spindle length at the end of metaphase. *C. elegans* (n = 10), *C. inopinata* (n = 11). ****P < 0.0001; unpaired t-test. (D) Spindle position at the completion of cytokinesis. *C. elegans* (n = 10), *C. inopinata* (n = 11). ns, not significant; unpaired t-test.

Mitotic spindle oscillation is smaller in C. inopinata than in C. elegans and C. briggsae

During anaphase, cortical microtubule pulling forces induce transverse spindle oscillations in C. elegans [4,8]. To compare spindle oscillation among species, we tracked centrosomes using DIC live imaging in C. inopinata, C. elegans, and C. briggsae, the latter of which is known to show smaller oscillations than C. elegans [11,24] (Fig 4A and S4 Table). The number of rocking cycles in C. inopinata (3.4 ± 1.5, n = 9) was similar to that in C. briggsae (3.7 ± 0.8, n = 9) but significantly lower than in C. elegans (5.8 ± 1.4, n = 10, p < 0.005) (Fig 4B and S4 Table). The maximum angular displacement in C. inopinata (13.1 ± 5.1°, n = 9) was similar to that in C. briggsae (14.7 ± 7.1°, n = 9) but significantly smaller than that in C. elegans (26.8 ± 11.7°, n = 10, p < 0.005) (Fig. 4B and S4 Table). The oscillation frequency in C. inopinata (38.7 ± 6.0 mHz, n = 9) was significantly lower than those in both C. elegans (56.8 ± 7.1 mHz, n = 10, p < 0.0005) and C. briggsae (62.2 ± 4.3 mHz, n = 9, p < 0.0005) (Fig 4B and S4 Table). Taken together, the spindle oscillation in C. inopinata is smaller than in C. elegans, possibly reflecting differences in the interaction between astral microtubules and the cell cortex between the two species.

Fig 4 — (A) Time course of spindle angle. The upper panel shows the period from NEBD to the completion of cytokinesis. The lower panel focuses on the oscillation period. Time is indicated relative to the completion of oscillation (t = 0 s). The spindle angle was calculated every second using DIC live imaging. *C. elegans* (n = 10), *C. inopinata* (n = 9), *C. briggsae* (n = 9). (B) Comparison of the number of rocking cycles, maximum angular displacement (θ°), and oscillation frequency (Hz). Frequency was determined by fast Fourier transform. *C. elegans* (n = 10), *C. inopinata* (n = 9), *C. briggsae* (n = 9). *P < 0.05; **P < 0.01; ****P < 0.0001; ns, not significant; Tukey’s multiple comparisons test.

Centrosome disassembly during telophase occurs more slowly in C. inopinata than in C. elegans

We next compared centrosome disassembly during telophase, which is thought to be associated with the strength of cortical microtubule pulling forces [25,26], using fluorescent live-cell imaging of GFP::β-tubulin. In C. elegans, the posterior bias of cortical pulling forces led to the rapid expansion and fragmentation of the posterior centrosome, followed by the anterior centrosome (n = 10) (Fig 5). In C. inopinata, the posterior centrosome also disassembled before the anterior centrosome, but both centrosome signals gradually decreased without marked expansion or fragmentation (n = 11) (Fig 5). These results suggest that cortical pulling forces in C. inopinata are biased posteriorly, as in C. elegans, but are weaker overall.

Fig 5 — Centrosomes were recorded using fluorescent live imaging of embryos expressing GFP::β-tubulin every 30 s in *C. elegans* and 60 s in *C. inopinata*. Time is indicated relative to anaphase onset (s). Scale bars, 5 µm.

Comparative analysis of genes involved in the regulation of cortical microtubule pulling forces

The smaller mitotic spindle oscillation during anaphase and the slower posterior centrosome dispersal were consistent with weaker cortical microtubule pulling forces in C. inopinata than in C. elegans. In C. elegans, the Gα-GPR-LIN-5 complex functions as a cortical pulling force generator, and its posterior enrichment is regulated by multiple protein interactions [3–5]. In addition, spindle oscillation is affected by microtubule dynamics [27–30]. Genes involved in these processes were generally conserved between C. elegans and C. inopinata (Table 1). We therefore hypothesized that the contribution of each gene might differ between the species. To test this hypothesis, we selected three genes, gpr-1/2, let-99, and klp-7, and compared their reduction-of-function phenotypes by RNAi. In C. elegans, gpr-1/2 encode two functionally redundant GPR proteins [3], whereas C. inopinata has a single ortholog. LET-99 inhibits both GPR and LIN-5 localization [3,5]. KLP-7 is a kinesin-13 microtubule depolymerization factor involved in regulating astral microtubule levels [29,30].

Table 1. Genes involved in the control of cortical microtubule pulling forces in C. elegans.

C. elegans				C. inopinata
Protein	Gene	Protein length	Protein motif or domain: Function	Ortholog	Gene ID	Protein length	Protein identity
GOA-1	goa-1	354 aa	G protein α subunit: Gα-GPR-LIN-5 component	Cin-goa-1	Sp34_10095600	354 aa	352/354 (99%)
GPA-16	gpa-16	357 aa	G protein α subunit: Gα-GPR-LIN-5 component	Cin-gpa-16	Sp34_10230800	357 aa	343/357 (96%)
GPR-1	gpr-1	525 aa	GoLoco motif protein: Gα-GPR-LIN-5 component	Cin-gpr-1	Sp34_30269300	529 aa	338/526 (64%)
GPR-2	gpr-2	525 aa	GoLoco motif protein: Gα-GPR-LIN-5 component	Cin-gpr-1	Sp34_30269300	529 aa	335/526 (64%)
LIN-5	lin-5	821 aa	Dynein binding protein: Gα-GPR-LIN-5 component	Cin-lin-5	Sp34_10075000	819 aa	392/829 (47%)
LET-99	let-99	698 aa	DEPdomain protein: GPR-1/2 and LIN-5 localization inhibitor	Cin-let-99	Sp34_30193200	699 aa	445/699 (64%)
CSNK-1	csnk-1	407 aa	Casein kinase: GPR-1/2 and LIN-5 localization regulator	Cin-csnk-1	Sp34_10184910	443 aa	382/389 (98%)
PPK-1	ppk-1	611 aa	PIP2 synthetase: GPR-1/2 and LIN-5 localization regulator	Cin-ppk-1	Sp34_10244700	611 aa	543/600 (91%)
KLP-7	klp-7	747 aa	Kinesin 13 family: Microtubule depolymerization factor	Cin-klp-7	Sp34_30111700	693 aa	573/696 (82%)

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Gene ID was based on C. inopinata v7.10 gene set. Protein identities were determined using BLAST+ searches.

RNAi knockdown of Cin-gpr-1, Cin-let-99, and Cin-klp-7 resulted in high embryonic lethality in C. inopinata, similar to the phenotypes observed for their orthologs in C. elegans (Fig 6A), indicating that RNAi was sufficiently effective to disrupt normal development. In C. elegans, gpr-1/2(RNAi) embryos exhibited a decreased number of rocking cycles during the spindle oscillation (Fig 6B and 6C and S5 Table), whereas let-99(RNAi) embryos exhibited earlier onset and increased amplitude of spindle oscillations (Fig 6B and 6D and S5 Table). Similar phenotypes were observed in Cin-gpr-1(RNAi) and Cin-let-99(RNAi) embryos (Fig 6B–6D and S5 Table), suggesting that the roles of Cin-gpr-1 and Cin-let-99 are conserved in C. inopinata. In contrast, while klp-7(RNAi) embryos of C. elegans exhibited spindle misalignment along the A/P axis and most failed to complete cell division (6/8 embryos), Cin-klp-7(RNAi) embryos of C. inopinata completed asymmetric cell division despite a decreased number of rocking cycles (Fig 6B, 6C and 6E and S5 Table). These results suggest that although Cin-klp-7 is essential for embryogenesis in C. inopinata, its contribution to the first asymmetric division is limited.

Fig 6 — (A) Embryonic lethality observed in each RNAi experiment. For *C. elegans* soaking RNAi, each value represents the mean of four independent measurements: control, 0% (0/47, 0/43, 0/50, 0/37); *gpr-1/2(RNAi)*, 100% (48/48, 26/26, 48/48, 32/32); *let-99(RNAi)*, 100% (29/29, 38/38, 33/33, 45/45); *klp-7(RNAi)*, 51.5% (22/46, 31/48, 22/52, 9/17). For *C. inopinata* feeding RNAi, each value represents the mean of two independent measurements: control, 9.5% (4/97, 19/127); *Cin-gpr-1(RNAi)*, 90.1% (25/28, 30/33); *Cin-let-99(RNAi)*, 63.9% (75/125, 84/124); *Cin-klp-7(RNAi)*, 74.7% (72/103, 70/88). (B) Time course of spindle angle in each RNAi experiment. Upper and lower panels show *C. elegans* and *C. inopinata*, respectively, from NEBD to completion of cytokinesis (t = 0). The spindle angle was measured every second using DIC live imaging. (C) Number of rocking cycles. (D) Angular displacement during initial rocking. (E) Centrosome orientation at cytokinesis completion. Sample sizes for (B–E) are as follows: *C. elegans*: control, n = 16; *gpr-1/2(RNAi)*, n = 13; *let-99(RNAi)*, n = 9; *klp-7(RNAi)*, n = 8. *C. inopinata*: control, n = 10; *Cin-gpr-1(RNAi)*, n = 8; *Cin-let-99(RNAi)*, n = 6; *Cin-klp-7(RNAi)*, n = 6. *P < 0.05, **P < 0.01, ***P < 0.001; ns, not significant; unpaired t-test or Tukey’s multiple comparisons test.

Discussion

In this study, we identified differences in microtubule-dependent pronuclear migration and mitotic spindle dynamics in one-cell-stage embryos of C. elegans and C. inopinata. In C. inopinata, the female pronucleus formed at various positions and met the male pronucleus at a more posterior position than in C. elegans. After pronuclear meeting, the NCC migrated toward the anterior side of the embryo and showed limited rotation, resulting in spindle formation at a more anterior position than in C. elegans. During cell division, spindle oscillations during anaphase were smaller, and centrosome dispersal during telophase occurred more slowly in C. inopinata than in C. elegans. These observations are consistent with the possibility that cortical microtubule pulling forces are weaker in C. inopinata than in C. elegans. RNAi-based assays further suggested that reduced activity of the microtubule-depolymerizing kinesin KLP-7 in C. inopinata may contribute to this difference in microtubule pulling force.

In C. elegans, spindle oscillation is caused by cortical microtubule pulling forces generated by the Gα-GPR-LIN-5 complex. Our RNAi experiments indicated that, in C. inopinata, this complex is also involved in spindle positioning, but its force-generating activity may be reduced. This reduction may result from a lower force-generating capacity of the Gα-GPR-LIN-5 complex in C. inopinata, or from fewer centrosomal microtubules interacting with the complex. We also found that the microtubule depolymerization factor KLP-7 contributes less to positioning of the first mitotic spindle in C. inopinata. This suggests that the regulation of centrosomal microtubules differs between the two species, which may affect the interaction between centrosomal microtubules and cortical force generators.

Technical limitations in C. inopinata prevented direct comparison of RNAi efficiency, which may have influenced to the differences in pronuclear and spindle dynamics between C. elegans and C. inopinata. Nevertheless, RNAi targeting Cin-gpr-1, Cin-let-99, or Cin-klp-7 induced high embryonic lethality in C. inopinata, comparable to that observed in C. elegans, indicating that the RNAi treatments produced biologically significant knockdown of gene function. Therefore, while variations in RNAi efficiency may have played a role, they are unlikely to account for all of our findings.

In addition to differences in spindle dynamics, we observed NCC overcentration in C. inopinata. In C. elegans, anterior movement of the NCC is mainly driven by microtubule length-dependent pulling forces [2]. The overcentration of the NCC in C. inopinata may reflect a transient imbalance between anterior-directed, length-dependent pulling forces and cortical pulling forces anchoring the NCC near the center of the embryo. Specifically, the anterior-directed forces may initially predominate, causing the NCC to move beyond the center. Further RNAi experiments targeting factors involved in length-dependent pulling forces, such as cytoplasmic dyneins and kinesins, will help clarify the molecular basis of these differences.

Comparative analysis among 34 Rhabditid species related to C. elegans has shown diverse cellular behaviors during early cell division. However, two features, 90-degree NCC rotation (31/34 species) and NEBD at the center of the embryo (30/34), are well conserved [10]. Notably, these features were rarely observed in C. inopinata. Among Caenorhabditis nematodes, C. inopinata exhibited one of the most anterior spindle formation positions and one of the smallest spindle oscillations [11]. Thus, the cellular behaviors of C. inopinata during the first cell division have diverged substantially from those of closely related species.

In C. inopinata, transposable elements are desilenced and expanded [12,31,32], which is believed to contribute to faster phenotype evolution than other related species. For example, optimal growth temperature of C. inopinata is 25–29°C, which is significantly higher than that of C. elegans (15–24°C) [12]. Because microtubule dynamics are temperature-sensitive, adaptation to higher temperatures and the regulatory mechanisms of microtubule-dependent processes may have co-evolved. Consistent with this idea, closely related frog species inhabiting different temperature environments show distinct microtubule growth rates and catastrophe frequencies [33].

In summary, our comparison of C. elegans and C. inopinata demonstrates that modulation of microtubule regulatory mechanisms can affect nuclear and spindle dynamics during early embryogenesis. Further comparative analyses among closely related species will contribute to the understanding of the evolution of cellular dynamics.

Supporting information

S1 Fig. Hatching rates of C. elegans and C. inopinata over a broad temperature range.

At each temperature, gravid C. elegans hermaphrodites or C. inopinata females were allowed to lay eggs on NGM plates for 2–6 h until at least 10 eggs were laid. The adults were then removed, and the total number of eggs was counted. After 2 days of incubation at the indicated temperature, unhatched eggs were counted to calculate the hatching rate. Data are presented as mean with SD. Temperatures at which no surviving worms were observed after 2 days are indicated by hatched boxes. https://nc.rdx.tohoku.ac.jp/s/H7izQb3tBZwcG7t.

(TIF)

pone.0352057.s001.tif^{(62.9KB, tif)}

S1 Table. Fig 1 raw data.

https://nc.rdx.tohoku.ac.jp/s/kRsBKxQLKTH6DRf.

(XLSX)

pone.0352057.s002.xlsx^{(24KB, xlsx)}

S2 Table. Fig 2 raw data.

https://nc.rdx.tohoku.ac.jp/s/kppyWQ8DJygdydk.

(XLSX)

pone.0352057.s003.xlsx^{(36.5KB, xlsx)}

S3 Table. Fig 3 raw data.

https://nc.rdx.tohoku.ac.jp/s/ZZQ2aMsRoZ53atH.

(XLSX)

pone.0352057.s004.xlsx^{(20.8KB, xlsx)}

S4 Table. Fig 4 raw data.

https://nc.rdx.tohoku.ac.jp/s/BBqwacj74PsaeBd.

(XLSX)

pone.0352057.s005.xlsx^{(134.7KB, xlsx)}

S5 Table. Fig 6 raw data.

https://nc.rdx.tohoku.ac.jp/s/2gcAT42E4f3XwQX.

(XLSX)

pone.0352057.s006.xlsx^{(433KB, xlsx)}

Acknowledgments

We thank members of the Sugimoto Lab for helpful discussion. Some strains were provided by the Caenorhabditis Genetics Center.

Data Availability

The minimal data set is available in the Tohoku University Repository (TOUR) at https://hdl.handle.net/10097/0002008489.

Funding Statement

This work was supported by Japan Science and Technology Agency (JST; https://www.jst.go.jp/), Core Research for Evolutionary Science and Technology (CREST; https://www.jst.go.jp/kisoken/crest/en/) Grant Number JPMJCR18S7 and Japan Society for the Promotion of Science (JSPS; https://www.jsps.go.jp/) KAKENHI Grant Number JP23K27152 to A.S., and JSPS KAKENHI Grant Number JP23KJ0148 and MEXT’s University Fellowship Founding Project for Innovation Creation in Science and Technology (https://pgd.tohoku.ac.jp/rpc/fellowship.html) to S.O. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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PLoS One. doi: 10.1371/journal.pone.0352057.r001

Decision Letter 0

Bob Goldstein

28 Jun 2026

C. elegans and C. inopinata

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Reviewer #1: Oomura et al document differerences in pronuclear positioning and migration, and mitotic spindle morphology in C. inopinata relative to C. elegans oocytes and one-cell zygotes, with C. inopinata being the one known nematode species that is most closely related to C. elegans. As these differences in cell division dynamics likely reflect differences in microtubule dynamics, the authors compare the impact of knockdowns known to influence these events in C. elegans. They find that RNAi knockdown of genes encoding proteins that influence cortical and cytoplasmic pulling forces on microtubules have substantial impacts on these dynamics in both species, while RNAi knockdown of the microtubule depolymerase KLP-7 only affects these processes in C. elegans and not in inopinata. The authors conclude that relatively minor changes in microtubule regulation can account for species differences in cell division dynamics. These are interesting results, and the comparison of dynamics in the two species is especially compelling. This manuscript would be suitable for publication in PLOS One if the authors can first address the following significant concern.

The conclusion that differences in the contribution of different microtubule regulators can account for the differences in pronuclear and spindle dynamics rests entirely on the use of RNAi to reduce gene function. As RNAi is known to differ in effectiveness for knocking down gene function for different genes in C. elegans, the authors cannot exclude the possibilities that (i) knocking down the force generators would have potentially even more severe impacts on C. inopinata, and more significantly (ii) knockdown of KLP-7 in C. inopinata might be weak relative to the knockdown in C. elegans, and hence account for the lack of an effect in C. inopinata. While it would be interesting and potentially much more conclusive to compare the impact of klp-7 null alleles in both species, the authors need to at least acknowledge this caveat concerning the variable effectiveness of RNAi and their conclusions as the different relative contributions of KLP-7 and the other microtubule regulators in the two species.

Reviewer #2: In this manuscript, Oomura et al. examine pronuclear migration and positioning of the mitotic spindle in the one-cell embryo of the closest known relative of Caenorhabditis elegans, Caenorhabditis inopinata. They find that the first mitotic division takes longer in C. inopinata, and that several events during pronuclear migration, centration and mitotic spindle assembly are different. Despite these differences, the end result – asymmetric cell division along the embryo anterior-posterior axis – is strikingly similar to C. elegans. They also compare the effect of depleting three spindle assembly or positioning factors (gpr-1, let-99 and klp-7) on embryonic lethality and spindle dynamics in C. inopinata and C. elegans. Overall, this manuscript provides an initial characterization of the first embryonic division in C. inopinata and lays the groundwork for more mechanistic comparisons of spindle assembly dynamics between these two sister species. While this work is largely well-done, there are a few places where additional experimentation would strengthen the authors’ conclusions or where the conclusions should be revised in view of current limitations.

Major points

Pronuclear and spindle dynamics in both species were assessed at 25�C, which near the upper limit of what C. elegans tolerates and may be near the lower end of what C. inopinata normally experiences. While the rationale for comparing the two species at the same temperature is reasonable, the report would be strengthened by evidence that the observations made for C. inopinata at 25°C are similar to what is seen under culture conditions closer to the preferred temperature for this species.

The RNAi approaches for C. elegans and C. inopinata are quite different. For C. elegans, L4 larvae were soaked and self-fertilized embryos were analyzed from the resulting adults. For C. inopinata, gravid females were fed RNAi bacteria, and embryos were analyzed from the mated progeny of these females. No comparison of RNAi efficiency, other than embryonic lethality, is provided. Without assessing the degree of depletion, it is difficult to compare the severity of the resulting phenotypes between species. This is particularly important for Cin-klp-7, which shows a weaker phenotype than might be expected. The authors should show that this is not simply due to incomplete depletion. If depletion efficiency cannot be measured directly, the authors should, at minimum, acknowledge this limitation in the Results and Discussion sections.

The data presented in Fig. 6 should be quantified similarly to what was done in Fig. 4. While the Cin-gpr-1 RNAi phenotype is clear, the effect of Cin-let-99 depletion is more difficult to assess. This is particularly true for the increased amplitude of spindle oscillations reported by the authors. This difference is not obvious from the data as shown and should be quantified to support the authors’ interpretations.

In C. inopinata, the NCC forms more posteriorly, then moves more anteriorly, before moving back to its final position at ~60% of A-P axis. Posterior formation of the NCC is attributed to the position of the maternal pronucleus/oocyte nucleus, as well as reduced movement of the male pronucleus. What do the authors think underlies the subsequent over-centration (i.e. increased movement of the NCC to the anterior) in C. inopinata?

Minor points

Lines 189, 217 – 219, 291 – 292, and possibly elsewhere: Add references to original papers for C. elegans observations, e.g. PMID: 8625834 DOI: 10.1242/dev.122.5.1467 should be referenced for oocyte nuclear positioning in C. elegans.

Line 62: typo/grammar, remove “the” before “intracellular dynamics”

Lines 82-85: Since C. inopinata is a relatively new model, it would be helpful to add technical details regarding culture conditions here, rather than directing readers only to previous work (i.e. ref 14). Both modifications to NGM plates and any differences in propagation due to gonochorism should be added.

Line 102 – 103: The Methods state that all imaging experiments were performed at 25C. The authors should indicate how this temperature control was achieved.

Line 147: Were antibiotics used in RNAi plates as is typical for RNAi by feeding for C. elegans? If so indicate which antibiotic was used and at what final concentration. If not, it would be helpful to state this and briefly explain the rationale, especially for researchers who may wish to perform RNAi in C. inopinata.

Lines 147 – 151: For readers unfamiliar with C. inopinata, please clarify the RNAi workflow. Were the “gravid adults” used for embryo dissection and imaging mated females, whose parents, both female and male, had been RNAi treated since hatching? Similarly, for embryonic lethality tests, were the females and males used the progeny of the original RNAi-treated “gravid females”?

Line 165: Add to main text that the duration of the first cell cycle was measured as the time between NEBD and the completion of cytokinesis. This is currently only specified in the figure legend.

Line 182 and throughout: Replace “pronucleus” with “pronuclear”, when the word is used as an adjective, as in “pronuclear dynamics”. This terminology/grammar is inconsistent throughout the manuscript.

Lines 190 – 191 and Fig. 2C: Did the authors measure oocyte volume/size? Given the similar size of the embryos, I was surprised that the oocytes and zygote shown in Fig. 2C appear substantially larger in C. inopinata than in C. elegans.

Line 278: The phrase “both centrosomes diffused slowly” implies diffusion of intact centrosomes. Consider revising to indicate slower, progressive centrosome disassembly, possibly by diffusion of centrosome components into the cytoplasm.

Line 285: Add a description to the figure legend for Fig. 5 as to what “1.5 x enhanced” refers to.

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PLoS One. 2026 Oct 27;21(10):e0352057. doi: 10.1371/journal.pone.0352057.r002

Author response to Decision Letter 1

17 Sep 2026

Reviewer 1

Reviewer comment

Response

We agree with the reviewer that variability in RNAi efficiency is an important consideration, particularly when comparing gene function between C. elegans and C. inopinata. Because of the technical difficulty of obtaining sufficient quantities of specific stages of early C. inopinata embryos for quantitative analyses such as qPCR, we were unable to directly measure and compare the extent of gene depletion between the two species. Moreover, because genome-editing methods have not yet been established in C. inopinata, it is currently not feasible to generate and analyze klp-7 null mutants in this species. We therefore cannot exclude the possibility that differences in RNAi efficiency contributed to the observed differences in pronuclear and spindle dynamics, including the relatively mild effect of klp-7(RNAi) in C. inopinata.

Nevertheless, RNAi targeting gpr-1/2, let-99, or klp-7 caused high embryonic lethality in C. inopinata, at levels comparable to those observed in C. elegans. These results indicate that the RNAi treatments produced biologically substantial reductions in gene function, although they do not establish that the extent of depletion was equivalent between the two species. Thus, while differences in RNAi efficiency may have contributed to the observed phenotypic differences, they are unlikely to account fully for all of our findings.

Following the reviewer’s suggestion, we have revised the Results and Discussion sections (Page 14, line 357; Pages 16–17, lines 418–431) to explicitly acknowledge the technical limitations of RNAi analysis in C. inopinata and to qualify our conclusion regarding the differential contributions of KLP-7 and other microtubule regulators in the two species.

Reviewer #2:

Major points 1

Response

We thank the reviewer for raising this important point regarding the temperature conditions used for the interspecies comparison. Prior to conducting this experiment, we measured the hatching rates of C. elegans and C. inopinata over a broad temperature range. More than 80% of C. elegans embryos hatched at temperatures between 10°C and 28°C, whereas more than 80% of C. inopinata embryos hatched between 16°C and 32°C. These results indicate that 25°C lies well within the permissive range for embryonic development in both species and is not close to the lower limit for C. inopinata.

Although these hatching data do not directly demonstrate that all aspects of pronuclear and spindle dynamics are identical across the permissive temperature range, they support the conclusion that the dynamics observed at 25°C were examined under physiologically permissive conditions for C. inopinata. Moreover, the use of the same temperature for both species allowed us to compare their embryonic dynamics under equivalent experimental conditions.

To clarify this rationale, we have added the temperature-dependent hatching data to the Supporting Information and described the permissive temperature ranges in the Results section (Page 8, lines 183–184).

Major point 2

Response

We agree that differences in the RNAi protocols and efficiencies between the two species are an important limitation, particularly when interpreting the relatively mild Cin-klp-7(RNAi) phenotype. As this concern overlaps with that raised by Reviewer #1, we refer the reviewer to our detailed response above. Briefly, although high embryonic lethality indicates that RNAi substantially reduced gene function, we were unable to directly compare depletion efficiency between the two species and therefore cannot exclude the possibility that incomplete or variable knockdown contributed to the observed phenotypic differences. We have revised the Results and Discussion sections accordingly (Page 14, line 357; Pages 16–17, lines 418–431).

Major point 3

Response

Following the reviewer’s suggestion, we have quantified the data presented in Fig. 6 and added new panels (Fig. 6C–6E) in the revised manuscript. These newly added panels present the number of rocking cycles, the angular displacement during initial rocking, and the centrosome orientation upon completion of cytokinesis, respectively.

These quantitative data demonstrate that gpr-1/2 RNAi reduced the number of rocking cycles in both species, and let-99 RNAi increased the rocking amplitude in both species. Notably, while klp-7 RNAi reduced the number of rocking cycles in both species, it induced spindle misalignment at the completion of cytokinesis exclusively in C. elegans, but not in C. inopinata.

We have added these new data and descriptions into the Results section (Page 14-15, lines 358–368). In addition, to reflect these quantitative analyses, we have revised the Materials and Methods section under "Quantification of mitotic spindle oscillation" (Page 6, lines 125–131).

Major point 4

Response

We thank the reviewer for this insightful question. We speculate that the excessive anterior displacement of the NCC in C. inopinata reflects a transient imbalance between anterior-directed, microtubule length-dependent pulling forces and cortical pulling forces that normally position and stabilize the NCC near the center of the embryo. Specifically, the anterior-directed forces may initially predominate, causing the NCC to move beyond the center, after which cortical pulling forces reposition it at approximately 60% of the A–P axis. We have added this possible explanation to the Discussion section (Page 17, lines 435–438).

Minor point 1

Response

Following the reviewer's suggestion, we have added references to original papers for C. elegans observations throughout the manuscript.

For the oocyte nuclear positioning (Page 9, line 217):

・ Goldstein, B., & Hird, S. N. Specification of the anteroposterior axis in Caenorhabditis elegans. Development 1 May 1996; 122 (5): 1467–1474. doi: https://doi.org/10.1242/dev.122.5.1467

For the NCC and mitotic spindle dynamics (Page 10, Line 338):

・ Albertson, D. G. Formation of the first cleavage spindle in nematode embryos. Developmental Biology, Volume 101, Issue 1, 1984, Pages 61-72, ISSN 0012-1606, doi: https://doi.org/10.1016/0012-1606(84)90117-9.

For the description of the Gα-GPR-LIN-5 complex (Page 13, Line 337):

・ Park, D. H., & Rose, L. S. (2008). Dynamic localization of LIN-5 and GPR-1/2 to cortical force generation domains during spindle positioning. Developmental biology, 315(1), 42-54.

・ Grill, S. W., Gönczy, P., Stelzer, E. H., & Hyman, A. A. (2001). Polarity controls forces governing asymmetric spindle positioning in the Caenorhabditis elegans embryo. Nature, 409(6820), 630-633.

・ Tsou, M. F. B., Hayashi, A., DeBella, L. R., McGrath, G., & Rose, L. S. (2002). LET-99 determines spindle position and is asymmetrically enriched in response to PAR polarity cues in C. elegans embryos.

Minor point 2

Line 62: typo/grammar, remove “the” before “intracellular dynamics”

Response

Following the reviewer's suggestion, we have revised the manuscript accordingly (Page 3, Lines 61). We have also corrected similar grammatical errors elsewhere in the manuscript (Page 3, Lines 62).

Minor point 3

Response

Following the reviewer's suggestion, we have added technical details regarding the culture conditions of C. inopinata to the Materials and Methods section (Page 4, Lines 84–89).

Minor point 4

Line 102 – 103: The Methods state that all imaging experiments were performed at 25℃. The authors should indicate how this temperature control was achieved.

Response

Following the reviewer's suggestion, we have added the temperature control method to the Materials and Methods section (Page 5, lines 110–112).

Minor point 5

Response

Yes, ampicillin (final concentration, 50 μg/mL) and tetracycline (final concentration, 10 μg/mL) were used in the RNAi plates for C. inopinata. Following the reviewer's suggestion, we have added this information to the Materials and Methods section (Page 7, Lines 160).

Minor point 6

Response

Following the reviewer’s suggestion, we have clarified the RNAi workflow in the Materials and Methods section (Page 7, lines 161–165). Briefly, untreated adult females (P0) were transferred to RNAi plates and allowed to lay eggs. The F1 males and females were continuously exposed to feeding RNAi from hatching and allowed to mate with each other. The resulting mated F1 females carrying embryos were referred to as “gravid adults.” Their embryos (F2) were used for both live imaging and embryonic lethality assays.

Minor point 7

Line 165: Add to main text that the duration of the first cell cycle was measured as the time between NEBD and the completion of cytokinesis. This is currently only specified in the figure legend.

Response to minor point 7

Following the reviewer’s suggestion, we have revised the main text to define the duration of the first cell division as the interval from nuclear envelope breakdown (NEBD) to the completion of cytokinesis (Page 8, lines 185–186), consistent with the figure legend.

Minor point 8

Response to minor point 8

Following the reviewer's suggestion, we have replaced “pronucleus” with “pronuclear” throughout the manuscript when used adjectivally.

Minor point 9

Response

Although we did not directly measure oocyte volume, C. inopinata eggs are significantly larger than those of C. elegans, with an approximately 5.7% greater egg length (Fig. 1B). This difference may reflect a corresponding difference in oocyte size.

We also found that the scale bar in the legend for Fig. 2C was incorrectly labeled as 100 µm rather than 25 µm, which likely contributed to the apparent size discrepancy. We have corrected this error in the revised manuscript (Page 9, line 236).

Minor point 10

Response

Following the reviewer’s suggestion, we have revised the Results section by replacing “both centrosomes diffused slowly” with “the centrosome signals gradually decreased,” which more accurately describes the progressive loss of centrosomal fluorescence observed in our imaging (Page 12, line 320).

Minor point 11

Line 285: Add a description to the figure legend for Fig. 5 as to what “1.5 x enhanced” refers to.

Response

We thank the reviewer for pointing this out. The label “1.5 × enhanced” referred to an adjustment of the display range in ImageJ, from 90–700 for C. elegans to 90–500 for C. inopinata, which increased the apparent brightness by approximately 1.5-fold. However, because Fig. 5 shows different transgenes in the two species and is not intended for quantitative comparison of fluorescence intensity, we agreed that this label could be misleading. We have therefore removed “1.5 × enhanced” from Fig. 5 and its legend.

Comments to the Author

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #2: No

Response

In response to the reviewer’s comment, we have provided all underlying numerical data required to replicate the findings of this study as "Supporting Information" in the public repository "Tohoku University Research Data Lake IZUMI." The corresponding URL has been incorporated into the revised manuscript (Page 23-24, lines 582–604).

Attachment

Submitted filename: Response to Reviewers_Oomura.pdf

pone.0352057.s007.pdf^{(241.9KB, pdf)}

PLoS One. doi: 10.1371/journal.pone.0352057.r003

Decision Letter 1

Bob Goldstein

24 Sep 2026

Divergence of cortical force-generating mechanisms underlies differences in spindle behavior between C. elegans and C. inopinata

PONE-D-26-27765R1

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PLoS One. doi: 10.1371/journal.pone.0352057.r004

Acceptance letter

Bob Goldstein

PONE-D-26-27765R1

PLOS One

Dear Dr. Sugimoto,

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Fig. Hatching rates of C. elegans and C. inopinata over a broad temperature range.

(TIF)

pone.0352057.s001.tif^{(62.9KB, tif)}

S1 Table. Fig 1 raw data.

https://nc.rdx.tohoku.ac.jp/s/kRsBKxQLKTH6DRf.

(XLSX)

pone.0352057.s002.xlsx^{(24KB, xlsx)}

S2 Table. Fig 2 raw data.

https://nc.rdx.tohoku.ac.jp/s/kppyWQ8DJygdydk.

(XLSX)

pone.0352057.s003.xlsx^{(36.5KB, xlsx)}

S3 Table. Fig 3 raw data.

https://nc.rdx.tohoku.ac.jp/s/ZZQ2aMsRoZ53atH.

(XLSX)

pone.0352057.s004.xlsx^{(20.8KB, xlsx)}

S4 Table. Fig 4 raw data.

https://nc.rdx.tohoku.ac.jp/s/BBqwacj74PsaeBd.

(XLSX)

pone.0352057.s005.xlsx^{(134.7KB, xlsx)}

S5 Table. Fig 6 raw data.

https://nc.rdx.tohoku.ac.jp/s/2gcAT42E4f3XwQX.

(XLSX)

pone.0352057.s006.xlsx^{(433KB, xlsx)}

Attachment

Submitted filename: Response to Reviewers_Oomura.pdf

pone.0352057.s007.pdf^{(241.9KB, pdf)}

Data Availability Statement

The minimal data set is available in the Tohoku University Repository (TOUR) at https://hdl.handle.net/10097/0002008489.

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PERMALINK

Divergence of cortical force-generating mechanisms underlies differences in spindle behavior between C. elegans and C. inopinata

Shun Oomura

Koji Kyoda

Shuichi Onami

Nami Haruta

Asako Sugimoto

Roles

Abstract

Introduction

Materials and methods

Strains and culture conditions

Microscopy

Tracking of pronuclei, chromosomes, centrosomes, and spindle movements

Quantification of mitotic spindle oscillation

Statistical analysis

Ortholog search

RNA interference

Results

The asymmetry of the first embryonic division in C. inopinata is similar to that in C. elegans

Fig 1. The first asymmetric cell division in C. elegans and C. inopinata embryos.

Pronuclear dynamics differ between C. elegans and C. inopinata embryos

Fig 2. Pronuclear dynamics in C. elegans and C. inopinata embryos.

NCC and mitotic spindle movements differ between C. elegans and C. inopinata embryos

Fig 3. NCC and spindle dynamics in C. elegans and C. inopinata embryos.

Mitotic spindle oscillation is smaller in C. inopinata than in C. elegans and C. briggsae

Fig 4. Comparison of mitotic spindle oscillation parameters in C. elegans, C. inopinata, and C. briggsae embryos.

Centrosome disassembly during telophase occurs more slowly in C. inopinata than in C. elegans

Fig 5. Centrosome disassembly during telophase in C. elegans and C. inopinata embryos.

Comparative analysis of genes involved in the regulation of cortical microtubule pulling forces

Table 1. Genes involved in the control of cortical microtubule pulling forces in C. elegans.

Fig 6. Reduction-of-function phenotypes of gpr-1/2, let-99, and klp-7, and their orthologs in C. elegans and C. inopinata embryos.

Discussion

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Bob Goldstein

Roles

Author response to Decision Letter 1

Decision Letter 1

Bob Goldstein

Roles

Acceptance letter

Bob Goldstein

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

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