We investigated gene expression during polyembryogenesis of embryos of C. floridanum treated with JH in the two-cell stage. Additionally, we constructed a pipeline for functional gene annotation of C. floridanum and performed molecular network analysis.
Our previous study revealed that JH and methoprene accelerate polyembryogenesis; a phenomenon that was also observed when JH I or JH II were added to the culture medium . Methoprene had the strongest effect on promotion of polyembryogenesis, while farnesol, farnesyl acetate, and methyl caproate did not promote polyembryogenesis . Moreover, polyembryogenesis was not promoted by ecdysone (Supplementary Fig. 1). Hence, only JH or methoprene promote polyembryogenesis in C. floridanum.
The insect hormone JH is unique in its structure; it has a-, and b-unsaturated methyl ester groups and epoxy groups at both ends of the terpenoid backbone . The importance of JH in processes such as regulation of molting, pheromone biosynthesis, maturation of gonads, egg development, homeostasis, maintenance of population, and body color change has been reported . Thus, JH is a critical element of insect physiology.
Krüppel homolog 1 (Kr-h1) is a JH-responsive gene . Kr-h1 was not affected by JH in this study (Supplementary Fig. 2). Retinoid X receptor (RXR), a type of nuclear receptor that binds to 9-cis retinoic acid , may be able to bind to several chemicals that share the structure of retinoic acid . Reportedly, the JH analog methoprene and methoprene acid can bind to RXR , which also functions as a JH receptor in D. melanogaster . In this study, the expression of C. floridanum RXR-alpha-B tended to be increased following JH treatment (Supplementary Fig. 2). Retinoic acid is involved in embryonic development and cell differentiation through its interaction with RXR . This suggests that JH could act in a similar manner to retinoic acid to affect embryonic development and participate in the retinoic acid signaling pathway in our model of polyembryony.
Additionally, C. floridanum RefSeq datasets were constructed by analyzing male and female adult C. floridanum transcriptome. A reference genome sequence of C. floridanum from male has been published (https://www.ncbi.nlm.nih.gov/assembly/GCF_000648655.2/). Overall, 17,038 of the estimated proteins have been registered in the NCBI Genome database (https://www.ncbi.nlm.nih.gov/genome/12734?genome_assembly_id=358239).
Previously, we identified human homologs of Bombyx mori and D. melanogaster through systematic BLAST analysis. We found B. mori and D. melanogaster to contain 58 and 63 % of human homologs, respectively . In the present study, we found, C. floridanum exhibits 76% gene similarity with humans. The large number of human homologs that we observed in C. floridanum is therefore comparable with these model insects. In the present study, 88 C. floridanum genes that showed differential expression when comparing JH-treated and control groups corresponded to human genes, and we input these genes to gene enrichment and molecular network analyses.
Gene enrichment analysis revealed the expression of lipid metabolism-related (GO: 0006633) and platelet degranulation (GO: 0002576) related genes were upregulated in the JH-treated group. The characteristics of genes containing these GO terms have been shown to be involved with vesicle-associated V-soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) in cellular membrane adhesion . Additionally, genes encoding synaptosomal-associated protein (SNAP)25 and 29, (SNAPC) 3 and 4, SNARE-associated protein Snapin (SNAPIN), and syntaxin-binding protein (STXBP) were shown to be commonly expressed in both groups of the present study. Reportedly, these genes are involved in the membrane fusion of neurosecretory cells . Notably, STXBP interacts with these proteins to inactivate membrane fusion . Thus the decreased mRNA expression of STXBP that we observed following JH treatment may lead to activation of cellular membrane fusion. As the molura promotes fusion with the extraembryonic syncytium, it is possible that JH treatment could accelerate morula to polyembrogenesis.
The genes SNAP25 and SNAP29, SNAPC3 and SNAPC4, SNAPIN, and STXBP, which we observed to be expressed in C. floridanum morula embryos, also play a role for mediates membrane fusion during exocytosis in the neurosecretory cells of humans . Therefore, C. floridanum might employ similar molecular mechanisms for cellular membrane adhesion as human neurosecretory cells.
The morula comprises the outer extraembryonic syncytium and the inner embryonic cell; the extraembryonic syncytium separates by dividing the embryonic cell . Embryonic cells adhere to the extraembryonic syncytium via integrin in the morula. When integrin expression is decreased following exposure to JH, this adhesion may be loose, causing the extraembryonic syncytium to invaginate into the cell gap of embryonic cells. These cells might then divide, resulting in polyembryony. The actin filament crosslinking protein, FLNA, which is present in non-muscle cells  might be involved in the division of embryos interacting with SSH2 and ITGA4. Reportedly, SSH2 mediates actin dynamics , while ITGA4 belongs to the integrin family and plays a role in cell adhesion . These molecular interactions might contribute to the polyembryony.
Xanthine dehydrogenase/oxidase is the rate-limiting enzyme of purine metabolism, and a key component in uric acid synthesis. During egg development of B. mori the amount of uric acid gradually declines until blastokinesis, after which it increases until egg pigmentation . Remarkably, XHD knockdown has been shown to enhance cell mobility and invasion of HepG2 cells, although no effect on cell proliferation was observed . Furthermore, XDH converts retinoic acid to 9-cis retinal , and activity of the enzyme is essential in order for JH to induce bristle formation and cuticle production on the abdominal epidermis during pupal and adult development .
The chemical structures of JH III and retinoic acid are similar, and it is possible that JH III binds to XDH. Then, XDH-bound JH could enter the nucleus via XPO1, and released JH might bind to RXR to control subsequent transcription; this molecular correlation could be a novel mechanism triggered by JH. Accordingly, the following model could be proposed for the progression of forming molura to polymolura: To start, embryos are wrapped with extraembryonic syncytium (Fig. 6a). During the formation of polymolurae, the degradation of actin is avoided by reducing SSH2 expression followed by suppression of ITGA4 expression. As the adhesion of embryonic cells is loose (Fig. 6b), the syncytial membrane facilitates fractionation (Fig. 6c and d) and promotes polyembryony (Fig. 6e). Hence, XDH plays a key role in embryogenesis via JH, as well as in uric acid synthesis. Overall, the present study reveals novel molecular interactions involved in polyembryogenesis and demonstrates the connections that are required for the progression of cell separation in polyembryogenesis.