Introduction
Electroporation, a technique that creates pores in cell membranes through voltage stimuli, is widely used to introduce macromolecules, such as DNA and RNA, into host cells1, 2–3. The in vivo electroporation method has been used for tissue-specific gene functional analyses in various organisms, including vertebrates4, 5, 6, 7–8 as well as insects such as the silkworm, Bombyx mori9 the butterfly Papilio polytes10 and the dragonfly Nannophya pygmaea11. Ando and Fujiwara9 established a protocol to maintain stable expression of exogenous DNA in insect somatic cells. Key advantages of this method include the short time from the start of the experiment to the acquisition of results, the availability of both gain- and loss-of-function analyses, and the generation of genetic mosaic clones of cells within a single organism9.
The Japanese rhinoceros beetle, Trypoxylus dichotomus (Coleoptera: Scarabaeidae: Dynastini), a large insect species reaching up to 90 mm in body length (Fig. 1), has been the focus of studies in various areas including evolutionary developmental biology, behavioral ecology, and materials science12, 13, 14, 15, 16–17. Molecular studies on horn morphogenesis in this species have utilized a loss-of-function method, systemic larval RNA interference (RNAi)18, 19, 20, 21, 22, 23, 24–25; however, gain-of-function and tissue-specific analyses remain underdeveloped, limiting the elucidation of the molecular mechanisms involved. Additionally, generating germline transgenic lines and performing genome-editing in T. dichotomus are impractical because it takes an extremely long time to obtain results in T. dichotomus due to the species’ long generation time of 10 to 12 months14. Therefore, we aimed to establish a tissue-specific gain-of-function analysis method applicable to T. dichotomus larvae based on the protocol by Ando and Fujiwara9.
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Fig. 1
Comparison of male and female adult sizes (A), and first, early second, late second, and early third instar larval sizes (B) in the Japanese rhinoceros beetle, Trypoxylus dichotomus. Scale bars: 5 mm.
In this study, we first identified a suitable promoter, T. dichotomus actin A3 promoter, which is active in postembryonic tissues of T. dichotomus. We then determined the developmental stages applicable for electroporation because the body size of T. dichotomus is far larger than those of other insects in which in vivo electroporation was successful. We could efficiently introduce plasmid DNA between the first and early second instar, when larval weight is below 1.57 g. This study represents the first success of a region-specific gain-of-function analysis method for T. dichotomus. Finally, we found T. dichotomus actin A3 promoter is also active at post embryonic stages in other insects, including the harlequin ladybug Harmonia axyridis and the silkworm B. mori, suggesting that this promoter can be a versatile promoter across Coleoptera and Lepidoptera.
Results and discussion
The cytoplasmic actin A3 promoter is active at postembryonic stages in Trypoxylus dichotomus
To establish an in vivo electroporation method in T. dichotomus tissues, we first cloned the promoter of actin A3 gene, which is presumably ubiquitously expressed in postembryonic tissues. The actin A3 gene encodes a cytoplasmic actin protein conserved in eukaryotes26. The promoter of Drosophila melanogaster Actin 5 C, the ortholog of actin A3, is ubiquitously expressed in various tissues27,28 and widely used for heterologous expression of the genes of interest in various insect cell lines including flies, mosquitos, and lepidopteran cells29. Therefore, we first tested whether the T. dichotomus ortholog of this promoter could be applied to overexpression in in vivo tissue in T. dichotomus. Briefly, we identified the T. dichotomus actinA3 ortholog (hereinafter referred to as Tdic-actA3) by performing BLAST search using the beetle genome database30 (Fig. S1). We determined the transcription start site using the 5´ rapid amplification of cDNA ends (5´ RACE) method. We cloned the 1113 bp region including 321 bp upstream region from the transcription start site, the whole 5´ UTR, and the 1st intronic region (hereinafter referred to as Tdic-actA3 promoter) (Fig. S2). As a reporter gene, we inserted the Enhanced Green Fluorescent Protein (EGFP) gene fused with a nuclear translocation signal downstream of the Tdic-actA3 promoter (pBac[Tdic-actA3-nls-EGFP_Dmel-hsp70-DsRed2]). In this vector, we also inserted DsRed2 expression gene cassette driven by the D. melanogaster hsp70 promoter that is active in various insects such as the silkworm B. mori31, the red flour beetle Tribolium castaneum32 and the butterflies Bicyclus anynana33 and Papilio xuthus9 in order to assess its promoter activity simultaneously.
To examine the promoter activity of the Tdic-actA3 promoter in the larval tissues of T. dichotomus, we targeted the prothoracic region of the first instar larvae where the body width is minimal (approximately 3 mm). We injected the reporter plasmid into the hemolymph beneath the thoracic epidermis and performed electroporation targeting the thoracic epidermis (poring pulse: 300 V, 5 msec × 2; transferring pulse: 20 V, 90 msec × 10). In this condition, EGFP-positive cells were detected in the targeted tissues 1 day after electroporation (Fig. 2A). Thus, we concluded that the Tdic-actA3 promoter has enough transcription activity to express the downstream gene in the larval tissues of T. dichotomus. On the other hand, DsRed2 expression driven by the Dmel-hsp70 promoter was below the detection limit of fluorescence stereomicroscopy, suggesting that the activity of Tdic-actA3 promoter is considerably higher than that of Dmel-hsp70 in T. dichotomus.
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Fig. 2
Search for possible stage of introduction of exogenous plasmid DNA by the electroporation method. Expression of exogenous Enhanced green fluorescent protein in the first (A), early second (B), late second (C), and early third instar (D) larvae of the Japanese rhinoceros beetle, Trypoxylus dichotomus. The expression was detected on the right side of the thorax. L and R indicate the direction from which the larvae were observed (L: left side; R: right side). Yellow arrowheads indicate the injection sites. Scale bars: 500 μm.
The optimal developmental stages for electroporation in T. dichotomus
T. dichotomus larvae are larger than other insect larvae in which electroporation has been successfully applied (Fig. 1B). This large size serves as a barrier to the success of electroporation because its high electrical resistance lowers the electric current necessary to form pores in the cell membranes. To identify the postembryonic stage applicable for exogenous DNA introduction by electroporation, we investigated the efficiency of exogenous DNA introduction at three larval stages, the first, the second, and the early third (last) instar. Moreover, to examine how the efficiency of exogenous DNA introduction varies in each instar, we used larval body weight as a growth index at each instar according to Johns et al.14. EGFP-positive cells were detected in each stage. However, the DNA vector introduction rate varied among the three developmental stages as described below.
In the first instar (< 0.57 g), 82% (23/28) of the electroporated larvae exhibited EGFP expression (Figs. 2A and 3A). Statistical analysis revealed no significant effect of body weight on the presence or absence of EGFP expression (Model selection based on Akaike’s Information Criterion (AIC) and Generalized Linear Model (GLM) analysis, Fig. 3A, Tables 1A and 2A). This result indicates that in first instar larvae, a high proportion of individuals can incorporate exogenous plasmid DNA at any growth stage with electroporation.
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Fig. 3
Relationship between larval weight and the result of observations conducted one day after electroporation in the first (A), second (B), and early third (C) instar larvae of the Japanese rhinoceros beetle, Trypoxylus dichotomus. Larvae with at least one cell positive for Enhanced green fluorescent protein observed one day post-electroporation are categorized as “positive”; otherwise, they are categorized as “negative.” Each filled circle represents a single larva.
Table 1. Results of model selection based on the Akaike information criterion for generalized linear models of factors that affected the result of observation one day after electroporation in first (A), second (B), and early third (C) instar larvae of Japanese rhinoceros beetle, Trypoxylus dichotomus.
Model | AIC | ΔAIC | |
---|---|---|---|
(A) First instar | Null | 28.276 | – |
Weight | 28.287 | 0.011 | |
(B) Second instar | Weight | 21.303 | – |
Null | 35.542 | 14.239 | |
(C) Early third instar | Null | 9.6064 | – |
Weight | 11.594 | 1.9876 |
Table 2. Results from generalized linear model analysis for the result of observation one day after electroporation in first (A), second (B), and early third (C) instar larvae of the Japanese rhinoceros beetle, Trypoxylus dichotomus.
Explanatory variable | Estimate of coefficient | S.E. | z-value | p-value | |
---|---|---|---|---|---|
(A) First instar | Intercept | 1.5261 | 0.4934 | 3.093 | 0.00198 |
(B) Second instar | Intercept | 5.300 | 2.381 | 2.226 | 0.0260 |
Weight | − 3.370 | 1.359 | − 2.480 | 0.0131 | |
(C) Early third instar | Intercept | − 2.773 | 1.031 | − 2.69 | 0.00715 |
In the second instar (0.49–3.56 g), 35% (9/26) of the electroporated larvae expressed EGFP, and the number of EGFP-positive cells per larva was lower compared to the first instar (Figs. 2A–C and 3B). Our statistical analysis (AIC-GLM analysis) indicated that body weight significantly affected the presence or absence of EGFP expression (Fig. 3B, Tables 1B and 2B). This indicated that the percentage of individuals introduced with exogenous plasmid DNA is higher in early second instar larvae (< 1.57 g) and lower in late 2nd instar larvae (> 1.57 g).
In the early third instar (> 2.07 g), only 6% (1/17) of the electroporated larvae expressed EGFP (Figs. 2D and 3C). Our statistical analysis revealed no significant effect of body weight on the presence or absence of EGFP expression (Fig. 3C, Tables 1C and 2C), suggesting that the electroporation condition in this developmental stage is suboptimal.
In summary, we concluded that the gene delivery under the current electroporation conditions is practically applicable to first and early second instar larvae weighing less than 1.57 g. The low efficiency of DNA vector incorporation in the targeted tissues of late second and early third instar larvae, as assessed by the number of EGFP-positive cells, implies that low electrical resistance in the early larval stages due to smaller body size and/or thinner cuticle is associated with a high efficiency of gene delivery using electroporation.
Long-term stable somatic gene expression in T. dichotomus using the piggyBac transgenic system
We also tested somatic transgenesis system9 to achieve long-term stable gene expression during development. We designed the reporter vector pBac[Tdic-actA3-nls-EGFP_Dmel-hsp70-DsRed2] so that the gene cassette is flanked with piggyBac transposase recognition sequences, Inverted Terminal Repeats (ITRs), at both ends. Thus, we additionally injected a helper DNA vector pBac[phsp-pBac]34 to supply piggyBac transposase driven by the D. melanogaster hsp70 promoter and to facilitate stable integration of the gene cassette into the host genome. In this experiment, we performed electroporation using the first instar larvae below 0.15 g to maximize gene delivery efficiency. When we injected only the reporter plasmid before electroporation, EGFP expression disappeared within 30 days (Fig. 4A, n = 3). In contrast, co-injection of the reporter and the helper plasmid pBac[phsp-pBac] before electroporation resulted in stable EGFP expression maintained for at least 30 days (Fig. 4B, n = 9). These results suggest that the donor DNA vector can be stably incorporated into the host genome of targeted tissues in T. dichotomus larvae and maintained for a long period, as in the silkworms9. In addition, the Tdic-actA3 promoter maintains its transcription activity from the first instar to the early third instar (30 days after hatching), suggesting that Tdic-actA3 promoter works throughout the whole larval stages. Future studies using inverse PCR-based deep-sequencing analysis will facilitate a better understanding of the specificity, integrity and stability of piggyBac insertions into the somatic T. dichotomus genome.
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Fig. 4
Long-term stabilization of exogenous gene expression in somatic cells of T. dichotomus larvae. Extrachromosomal plasmid expression of Enhanced green fluorescent protein (EGFP) in the first instar larvae of the Japanese rhinoceros beetle, Trypoxylus dichotomus. The thorax of the larvae was electroporated with donor pBac[Tdic-actA3-nls-EGFP_Dmel-hsp70-DsRed2] and piggyBac helper plasmids, or with the donor plasmid alone. The expression was detected on the right side of the thorax. The number of days after electroporation is indicated in the upper right corner of each panel. “L” and “R” denote the direction from which the larvae were observed. Arrowheads indicate EGFP-positive cells. Yellow arrowheads indicate the injection sites. Scale bars: 500 μm.
Tdic-actA3 promoter is a versatile promoter applicable to various coleopteran and lepidopteran insects
Finally, we examined whether the Tdic-actA3 promoter is also active in other insect tissues. We set up electroporation experiments using pBac[Tdic-actA3-nls-EGFP_Dmel-hsp70-DsRed2] in another coleopteran insect, the harlequin ladybug H. axyridis, and a distantly related lepidopteran, the silkworm B. mori. In H. axyridis, we performed electroporation targeting the abdominal epidermis of the third (penultimate) instar larvae, and EGFP expression was observed in 5 out of 9 larvae within 1 day (Fig. 5A). In B. mori, we targeted the abdominal epidermis of the second and third instar larvae. The EGFP expression was observed in 1 of the 4 second instar larvae and both of the 2 third instar larvae tested within 1 day (Fig. 5B). These results suggest that the Tdic-actA3 promoter is a versatile promoter with high transcription activity not only in T. dichotomus but also in a broad range of coleopteran and lepidopteran insect species.
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Fig. 5
Usefulness of Tdic-actA3 promoter in Harmonia axyridis and Bombyx mori. Electroporation-mediated overexpression of the exogenous Enhanced green fluorescent protein in third instar larvae of the ladybird beetle H. axyridis (A) and in second instar larvae of the silkworm B. mori (B). The anterior is oriented to the left in both images. The ventral side of H. axyridis and the dorsal side of B. mori are shown. Scale bars: 500 μm.
Conclusions
In this study, we established an in vivo electroporation system for Trypoxylus dichotomus larval tissues. Our electroporation experiments revealed that a Tdic-actA3 promoter is highly active in postembryonic stages in T. dichotomus. Tdic-actA3 promoter exhibited activity not only in T. dichotomus but in the harlequin ladybug Harmonia axyridis and the silkworm Bombyx mori, suggesting its potential for heterologous gene expression across a broad range of insect species. Moreover, we also established the electroporation-mediated somatic transgenesis method using the piggyBac transgenic system. piggyBac transposase stably integrates exogenous gene cassette to the host somatic genome, allowing for long-term stable gene expression throughout development. The protocol for T. dichotomus described in the present study was optimized to introduce exogenous DNA into the thoracic larval epidermis. Fine-tuning of the electroporation parameters will extend targeted tissues variety including appendages and neurons as reported previously9. Our method extends the toolkit for genetic research in T. dichotomus, facilitating gain-of-function and tissue-specific genetic analyses. We believe that this approach will contribute to a more profound understanding of the molecular mechanisms underlying morphogenesis in the Japanese rhinoceros beetle, T. dichotomus.
Materials and methods
Insects
Trypoxylus dichotomus
The third instar larvae of T. dichotomus were purchased from Loiinne (Gunma, Japan) and Kishita (Shizuoka, Japan). Each larva was individually reared on humus (Dorcas Owners Shop, Japan) in plastic containers under natural daylength and room temperature conditions. After hatching, adults were fed with insect jelly (Loiinne, Japan) and reared on conifer mats (Dorcas Owners Shop, Japan) under the long day condition (LD = 16 h:8 h) at 25 °C. Adults provided from Dr. Kojima were also reared under the same conditions. Females were mated with males at least 7 days after hatching and placed in plastic cages containing humus; after additional 7 days, the eggs laid by the females in humus were collected and incubated under the natural day length and room temperature conditions until use in experiments.
Harmonia axyridis
H. axyridis was reared in the laboratory under the natural day length at room temperature and fed with either artificial diet or the pea aphid Acyrthosiphon pisum according to Niimi et al. (2005)35 until use in the experiment.
Bombyx mori
Silkworm (Daizo) eggs were incubated under long day condition (LD = 16 h:8 h, 25 °C). After hatching, the larvae were reared on artificial diet (NOSAN, Japan) under the same photoperiod (LD= 16 h:8 h) and temperature (25 °C) until use in the experiment.
Determination of the transcription start site of actin A3 in T. dichotomus
The sequence of actin A3 gene was identified through BLAST search using the predicted gene information of T. dichotomus genome30. Total RNA was first extracted from head and thoracic horn primordia of T. dichotomus with the TRI reagent (Molecular Research Center, USA) according to the manufacturer’s protocol. First-stranded cDNA synthesis was performed using the SMARTer™ PCR cDNA Amplification Kit (Takara Bio USA, USA) with the primers listed in Table S1, following the manufacturer’s instructions. To determine the transcription start site of actin A3 in T. dichotomus, we applied the 5´ rapid amplification of cDNA ends (RACE) method. The obtained 5´ end sequence of the actin A3 cDNA and the predicted open reading frame of actin A3 gene information of T. dichotomus30 were mapped to the genome of T. dichotomus (TdicSN1.0)30 to determine the exon-intron structure (Fig. S2).
DNA vector construction
To construct the DNA vector pBac[Tdic-actA3-nls-EGFP_Dmel-hsp70-DsRed2], pBac[3xP3-ECFPafm]36 was first digested with the restriction enzyme BglII (New England Biolabs, USA) and used as the plasmid backbone. Subsequently, the region from the translation start site to 321 bp upstream of the Tdic-actA3 transcription start site (1113 bp in total) was considered to be the promoter region of the actin A3 gene of T. dichotomus (Accession number LC849809) (Fig. S2). This putative promoter region was cloned using T. dichotomus genomic DNA as a template and Q5 High-Fidelity DNA Polymerase (New England Biolabs, USA). Furthermore, the nls-EGFP-hsp70-polyA-SV40-polyA-DsRed and Dmel-hsp70 promoter were cloned from pBac[TetO-EGFP, 3xP3-DsRed]37 and pBac[hsp70-tTA, 3xP3-ECFP]37respectively, using Q5 High Fidelity DNA Polymerase. The cloned DNA fragments and backbone vector were then assembled using NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs, USA) according to the manufacturer’s protocol. The primers used are summarized in Table S1.
Electroporation
We injected 2 µl of DNA solution (containing 5 µg of donor plasmid and 5 µg of helper plasmid) and 4 µl of DNA solution (containing 10 µg of donor plasmid and 10 µg of helper plasmid) into the right side of the first thoracic segment (T1 segment) of larvae below and above 0.15 g, respectively, of T. dichotomus (Fig. S3A). After a larva was held by a hand, the injection was performed using a microinjector FemtoJet (Eppendorf, Germany) and a split-tip glass needle (NATSUME OPTICAL CORP, Japan). After injection, a platinum electrode (NEPAGENE, Japan) with a diameter of 3 mm was placed on the T1 segment of the larvae (Fig. S3B). A conductive gel including electrolyte, LOGIQLEAN (GE HealthCare Japan, Japan), was applied between the platinum electrode and the T1 segment, and electric pulses were delivered using the electroporator, NEPA21 Super Electroporator (NEPAGENE, Japan) (poring pulse, two 5-msec pulses of 300 V at 50-msec intervals; transfer pulse, 10 90-msec pulses of 20 V at 100-msec intervals).
In the same manner as in T. dichotomus, 0.5 µl of DNA solution (containing 1.25 µg of donor plasmid and 1.25 µg of helper plasmid) was injected into the abdomen of third instar larvae of H. axyridis. Following CO2 anesthesia and injection, a platinum electrode (NEPAGENE, Japan) with a diameter of 3 mm was placed, and the NEPA21 Super Electroporator (NEPAGENE, Japan) was used for applying electricity (poring pulse, two 5-msec pulses of 100 V at 50-msec intervals; transfer pulse, 10 90-msec pulses of 20 V at 100-msec intervals).
In B. mori, electroporation was conducted following the protocol by Ando and Fujiwara9. In brief, B. mori larvae were anesthetized at 4 °C, and 0.5 µl of DNA solution (containing 0.5 µg of donor plasmid and 0.5 µg of helper plasmid) was injected beneath the epidermis using a microinjector FemtoJet (Eppendorf, Germany) and a split-tipped glass needle (NATSUME OPTICAL CORP, Japan). Voltage pulses were applied immediately after the injection (third instar larvae, five 280-msec pulses of 45 V in 5 sec; second instar larvae, five 280-msec pulses of 20 V in 5 sec).
EGFP fluorescence was observed under a fluorescence stereomicroscope THUNDER (Leica, Japan) or a customized fluorescence stereomicroscope (Fluorescence Stereomicroscope: Nikon, SMZ18; Camera: Nikon, DS-Fi3; Software: Nikon, NIS-Elements for Desktop PC, version 5.21.00, (https://www.microscope.healthcare.nikon.com/products/software/nis-elements); Fluorescence Filter: Nikon, GFP-L (EX 460–500, DM 505, BA 510) (Fig. S3C).
Statistical analysis
A generalized linear model with binomial distribution and logit link was applied to evaluate the effect of body weight on the presence or absence of EGFP expression in R version 4.1.2 (R Core Team 2021, https://www.R-project.org/).
Molecular phylogenetic analysis
We obtained the amino acid sequences of actin A3 and actin related protein 1 of Drosophila melanogaster (NP_001014725 and NP_524331), B. mori (XP_037872546 and NP_001040336), T. castaneum (XP_008199969 and XP_015839668) from NCBI, respectively. The sequence of actin related protein 1 was identified by BLAST search from the predicted gene information of T. dichotomus genome30. We also obtained the amino acid sequence of actin related protein 3 of D. melanogaster (NP_001261559) from NCBI as the outgroup. We then aligned the amino acid sequences obtained above using MAFFT version 7 with the L-INS-i option. Subsequently, we conducted a phylogenetic analysis using raxmlGUI 2.0. For this analysis, we employed the maximum-likelihood method with the LG + FO + I model and set bootstrap replicates to 1000, designating the amino acid sequence of actin related protein 3 of D. melanogaster as the outgroup.
Acknowledgements
We thank Tatsuro Konagaya for providing us eggs of B. mori, and Wataru Kojima for providing us adults of T. dichotomus. We thank the Model Organisms Facility (Model Plant)/Data Integration and Analysis Facility/Emerging Model Organisms Facility/Trans-Omics Facility of NIBB Trans-Scale Biology Center for technical assistance. This work was supported by NIBB Collaborative research projects for integrative genomics (23NIBB402) and MEXT KAKENHI Grant Numbers 16H01452, 18H04766, 20H04933, 20H05944 (to T. N.) and JSPS KAKENHI Grant Numbers 19K16181, 21K15135 (to S. M.), and 17K19275 (to T. A.).
Author contributions
KS, SM, and TN designed this study. SM, SS, TN, and KY constructed the donor plasmid. KS, SM, and TA conducted the electroporation experiments in T. dichotomus, H. axyridis, and B. mori, respectively. KS, SM, and TN wrote the original draft of the manuscript. All authors commented on and approved the final version of the manuscript.
Data availability
The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.
Declarations
Competing interests
The authors declare no competing interests.
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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Abstract
The Japanese rhinoceros beetle, Trypoxylus dichotomus, possesses large horns on its head and thorax, features whose biological significance has been explored across various fields, including evolutionary developmental biology, behavioral ecology, and materials science. To investigate the molecular basis of these characteristics, systemic larval RNA interference (RNAi) has been employed as a primary loss-of-function genetic tool. However, gain-of-function analyses and region-specific gene function assessments remain underdeveloped, thereby limiting the comprehensive understanding of the molecular mechanisms involved. To address this limitation, we developed an in vivo electroporation technique to introduce exogenous DNA vectors directly into the somatic tissues of T. dichotomus larvae to express the genes of interest. Additionally, we utilized the piggyBac transposon system to insert the exogenous DNA vectors into the host genome for stable gene expression. Our findings indicate that the T. dichotomus actin A3 gene promoter exhibits sufficient transcriptional activity in the early postembryonic stage of T. dichotomus via electroporation. Furthermore, we observed that this promoter functions effectively across a diverse range of insect species, including the harlequin ladybug, Harmonia axyridis and the silkworm, Bombyx mori, suggesting the broad applicability of the T. dichotomus actin A3 promoter in various insects.
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1 National Institute for Basic Biology, Division of Evolutionary Developmental Biology, Okazaki, Japan (GRID:grid.419396.0) (ISNI:0000 0004 0618 8593)
2 National Institute for Basic Biology, Division of Evolutionary Developmental Biology, Okazaki, Japan (GRID:grid.419396.0) (ISNI:0000 0004 0618 8593); The Graduate University for Advanced Studies, SOKENDAI, Basic Biology Program, Okazaki, Japan (GRID:grid.275033.0) (ISNI:0000 0004 1763 208X)
3 National Institute for Basic Biology, Division of Evolutionary Developmental Biology, Okazaki, Japan (GRID:grid.419396.0) (ISNI:0000 0004 0618 8593); Kyoto University, The Hakubi Center for Advanced Research, Kyoto, Japan (GRID:grid.258799.8) (ISNI:0000 0004 0372 2033)
4 National Institute for Basic Biology, Division of Evolutionary Developmental Biology, Okazaki, Japan (GRID:grid.419396.0) (ISNI:0000 0004 0618 8593); Nagoya University, Graduate School of Bioagricultural Sciences, Chikusa, Nagoya, Japan (GRID:grid.27476.30) (ISNI:0000 0001 0943 978X)
5 The Graduate University for Advanced Studies, SOKENDAI, Basic Biology Program, Okazaki, Japan (GRID:grid.275033.0) (ISNI:0000 0004 1763 208X); National Institute for Basic Biology, Laboratory of Evolutionary Genomics, Okazaki, Japan (GRID:grid.419396.0) (ISNI:0000 0004 0618 8593); National Institute for Basic Biology, Trans-Omics Facility, Okazaki, Japan (GRID:grid.419396.0) (ISNI:0000 0004 0618 8593)
6 National Institute for Basic Biology, Division of Evolutionary Developmental Biology, Okazaki, Japan (GRID:grid.419396.0) (ISNI:0000 0004 0618 8593); The Graduate University for Advanced Studies, SOKENDAI, Basic Biology Program, Okazaki, Japan (GRID:grid.275033.0) (ISNI:0000 0004 1763 208X); Nagoya University, Graduate School of Bioagricultural Sciences, Chikusa, Nagoya, Japan (GRID:grid.27476.30) (ISNI:0000 0001 0943 978X)