regeneration factors expressed on myeloid expression in macrophage-like cells is required for tail regeneration in Xenopus laevis tadpoles.

Xenopus laevis tadpoles can regenerate whole tails after amputation. We have previously reported that interleukin 11 (il11) is required for tail regeneration. In this study, we have screened for genes that support tail regeneration under Il11 signaling in a certain cell type and have identified the previously uncharacterized genes Xetrov90002578m.L and Xetrov90002579m.S [referred to hereafter as regeneration factors expressed on myeloid.L (rfem.L) and rfem.S]. Knockdown (KD) of rfem.L and rfem.S causes defects of tail regeneration, indicating that rfem.L and/or rfem.S are required for tail regeneration. Single-cell RNA sequencing (scRNA-seq) revealed that rfem.L and rfem.S are expressed in a subset of leukocytes with a macrophage-like gene expression profile. KD of colony-stimulating factor 1 (csf1), which is essential for macrophage differentiation and survival, reduced rfem.L and rfem.S expression levels and the number of rfem.L- and rfem.S-expressing cells in the regeneration bud. Furthermore, forced expression of rfem.L under control of the mpeg1 promoter, which drives rfem.L in macrophage-like cells, rescues rfem.L and rfem.S KD-induced tail regeneration defects. Our findings suggest that rfem.L or rfem.S expression in macrophage-like cells is required for tail regeneration.

Cellular responses in the FGF10-mediated improvement of hindlimb regenerative capacity in Xenopus laevis revealed by single-cell transcriptomics.

Xenopus laevis tadpoles possess regenerative capacity in their hindlimb buds at early developmental stages (stages ~52-54); they can regenerate complete hindlimbs with digits after limb bud amputation. However, they gradually lose their regenerative capacity as metamorphosis proceeds. Tadpoles in late developmental stages regenerate fewer digits (stage ~56), or only form cartilaginous spike without digits or joints (stage ~58 or later) after amputation. Previous studies have shown that administration of fibroblast growth factor 10 (FGF10) in late-stage (stage 56) tadpole hindlimb buds after amputation can improve their regenerative capacity, which means that the cells responding to FGF10 signaling play an important role in limb bud regeneration. In this study, we performed single-cell RNA sequencing (scRNA-seq) of hindlimb buds that were amputated and administered FGF10 by implanting FGF10-soaked beads at a late stage (stage 56), and explored cell clusters exhibiting a differential gene expression pattern compared with that in controls treated with phosphate-buffered saline. The scRNA-seq data showed expansion of fgf8-expressing cells in the cluster of the apical epidermal cap of FGF10-treated hindlimb buds, which was reported previously, indicating that the administration of FGF10 was successful. On analysis, in addition to the epidermal cluster, a subset of myeloid cells and a newly identified cluster of steap4-expressing cells showed remarkable differences in their gene expression profiles between the FGF10- or phosphate-buffered saline-treatment conditions, suggesting a possible role of these clusters in improving the regenerative capacity of hindlimbs via FGF10 administration.

Effective enrichment of stem cells in regenerating Xenopus laevis tadpole tails using the side population method.

Xenopus laevis tadpoles have a strong regenerative ability and can regenerate their whole tails after tail amputation. Lineage-restricted tissue stem cells are thought to provide sources for the regenerating tissues by producing undifferentiated progenitor cells in response to tail amputation. However, elucidating the behavioral dynamics of tissue stem cells during tail regeneration is difficult because of their rarity, and there are few established methods of isolating these cells in amphibians. Here, to detect and analyze rare tissue stem cells, we attempted to enrich tissue stem cells from tail regeneration buds. High Hoechst dye efflux capacity is thought to be a common characteristic of several types of mammalian tissue stem cells; these stem cells, designated as the "side population (SP)," may be enriched by flow cytometry (SP method). To evaluate the effectiveness of stem cell enrichment using the SP method in regenerating X. laevis tadpole tails, we performed single-cell RNA sequencing (scRNA-seq) of SP cells from regeneration buds and analyzed the frequency of satellite cells, which are muscle stem/progenitor cells expressing pax7. The pax7-expressing cells were enriched in the SP compared with whole normal tails and regeneration buds. Furthermore, hes1-expressing cells, which are assumed to be neural stem/progenitor cells, were also enriched in the SP. Our findings suggest that the SP method is efficient for successfully enriching tissue stem cells in regenerating X. laevis tadpole tails, indicating that the combination of the SP method and scRNA-seq is useful for studying tissue stem cells that contribute to tail regeneration.

Low-temperature incubation improves both knock-in and knock-down efficiencies by the CRISPR/Cas9 system in Xenopus laevis as revealed by quantitative analysis.

The recent development of the CRISPR/Cas9-mediated gene editing technique has provided various gene knock-down and knock-in methods for Xenopus laevis. Gene-edited F0 individuals created by these methods, however, are mosaics with both mutated/knocked-in and unedited wild-type cells, and therefore precise determination and higher efficiency of knock-down and knock-in methods are desirable, especially for analyses of F0 individuals. To clarify the ratio of cells that are gene-edited by CRISPR/Cas9 methods to the whole cells in F0 individuals, we subjected Inference of CRISPR Edits analysis for knock-down experiments and flow cytometry for knock-in experiments to the F0 individuals. With these quantitative methods, we showed that low-temperature incubation of X. laevis embryos after microinjection improved the mutation rate in the individuals. Moreover, we applied low-temperature incubation when using a knock-in method with long single-strand DNA and found improved knock-in efficiency. Our results provide a simple and useful way to evaluate and improve the efficiency of gene editing in X. laevis.

An essential role of the arginine vasotocin system in mate-guarding behaviors in triadic relationships of medaka fish (Oryzias latipes).

To increase individual male fitness, males of various species remain near a (potential) mating partner and repel their rivals (mate-guarding). Mate-guarding is assumed to be mediated by two different types of motivation: sexual motivation toward the opposite sex and competitive motivation toward the same sex. The genetic/molecular mechanisms underlying how mate presence affects male competitive motivation in a triadic relationship has remained largely unknown. Here we showed that male medaka fish prominently exhibit mate-guarding behavior. The presence of a female robustly triggers male-male competition for the female in a triadic relationship (2 males and 1 female). The male-male competition resulted in one male occupying a dominant position near the female while interfering with the other male's approach of the female. Paternity testing revealed that the dominant male had a significantly higher mating success rate than the other male in a triadic relationship. We next generated medaka mutants of arginine-vasotocin (avt) and its receptors (V1a1, V1a2) and revealed that two genes, avt and V1a2, are required for normal mate-guarding behavior. In addition, behavioral analysis of courtship behaviors in a dyadic relationship and aggressive behaviors within a male group revealed that avt mutant males displayed decreased sexual motivation but showed normal aggression. In contrast, heterozygote V1a2 mutant males displayed decreased aggression, but normal mate-guarding and courtship behavior. Thus, impaired mate-guarding in avt and V1a2 homozygote mutants may be due to the loss of sexual motivation toward the opposite sex, and not to the loss of competitive motivation toward rival males. The different behavioral phenotypes between avt, V1a2 heterozygote, and V1a2 homozygote mutants suggest that there are redundant systems to activate V1a2 and that endogenous ligands activating the receptor may differ according to the social context.

Mblk-1 Transcription Factor Family: Its Roles in Various Animals and Regulation by NOL4 Splice Variants in Mammals.

Transcription factors play critical roles in regulation of neural development and functions. A transcription factor Mblk-1 was previously reported from a screen for factors possibly important for the higher brain functions of the honeybee. This review first summarizes how Mblk-1 was identified, and then provides an overview of the studies of Mblk-1 and their homologs. Mblk-1 family proteins are found broadly in animals and are shown to affect transcription activities. Studies have revealed that the mammalian homologs can interact with several cofactors and together regulate transcription. Interestingly, a recent study using the mouse homologs, Mlr1 and Mlr2, showed that one of their cofactor proteins, NOL4, have several splice variants with different effects on the transactivation activities of Mlr proteins. These findings suggest that there is an additional layer of the regulation of Mblk-1 family proteins by cofactor splice variants and provide novel insights into our current understanding of the roles of the conserved transcription factor family.

Phyhd1, an XPhyH-like homologue, is induced in mouse T cells upon T cell stimulation.

We previously identified XPhyH-like as a gene whose expression is enhanced in Xenopus blood cells during the refractory period, in which Xenopus tadpoles transiently lose their tail regenerative ability. Although we hypothesized that some autoreactive immune cells attack tail blastemal cells during the refractory period and XPhyH-like expressing immune cells were involved in the process, the nature of cells expressing XPhyH-like remain unknown, partly due to the lack of leukocyte markers available in Xenopus. In the present study, we used mice to analyze the expression pattern of XPhyH-like homologues. When we used quantitative reverse transcription-polymerase chain reaction (RT--PCR) to analyze the expression of mouse Phyhd1, an XPhyH-like orthologue, and Phyh, a Phyhd1 paralogue, both Phyhd1 and Phyh showed similar tissue-specific expression patterns. The expression pattern in leukocytes, however, differed between Phyhd1 and Phyh; Phyhd1 was considerably expressed in T cells and B cells. Moreover, the expression of Phyhd1 in T cells was up-regulated for approximately 3- to 7-times by T cell stimulation 3-4 days after the stimulation, unlike Phyh. Our findings suggest that Phyhd1 and Phyh have distinct roles in mouse leukocytes and Phyhd1 is related to T cell differentiation and/or function of effector T cells.

Acute phase response in amputated tail stumps and neural tissue-preferential expression in tail bud embryos of the Xenopus neuronal pentraxin I gene.

Regeneration of lost organs involves complex processes, including host defense from infection and rebuilding of lost tissues. We previously reported that Xenopus neuronal pentraxin I (xNP1) is expressed preferentially in regenerating Xenopus laevis tadpole tails. To evaluate xNP1 function in tail regeneration, and also in tail development, we analyzed xNP1 expression in tailbud embryos and regenerating/healing tails following tail amputation in the 'regeneration' period, as well as in the 'refractory' period, when tadpoles lose their tail regenerative ability. Within 10 h after tail amputation, xNP1 was induced at the amputation site regardless of the tail regenerative ability, suggesting that xNP1 functions in acute phase responses. xNP1 was widely expressed in regenerating tails, but not in the tail buds of tailbud embryos, suggesting its possible role in the immune response/healing after an injury. xNP1 expression was also observed in neural tissues/primordia in tailbud embryos and in the spinal cord in regenerating/healing tails in both periods, implying its possible roles in neural development or function. Moreover, during the first 48 h after amputation, xNP1 expression was sustained at the spinal cord of tails in the 'regeneration' period tadpoles, but not in the 'refractory' period tadpoles, suggesting that xNP1 expression at the spinal cord correlates with regeneration. Our findings suggest that xNP1 is involved in both acute phase responses and neural development/functions, which is unique compared to mammalian pentraxins whose family members are specialized in either acute phase responses or neural functions.

Ontogeny and Sexual Differences in Swimming Proximity to Conspecifics in Response to Visual Cues in Medaka Fish.

Adult medaka fish (Oryzias latipes) exhibit complex social behaviors that depend mainly on visual cues from conspecifics. The ontogeny of visually-mediated social behaviors from larval/juvenile to adult medaka fish, however, is unknown. In the present study, we established a simple behavioral paradigm to evaluate the swimming proximity to conspecifics based on visual cues in an inter-individual interaction of two medaka fish throughout life. When two fish were placed separately in a cylindrical tank with a concentric transparent wall, the two fish maintained close proximity to each other. A normal fish inside the tank maintained proximity to an optic nerve-cut fish outside of the tank, while the converse was not true. This behavioral paradigm enabled us to quantify visually-induced motivation of a single fish inside the tank. The proximity was detected from larval/juvenile to adult fish. Larval fish, however, maintained close proximity not only to conspecifics, but also to heterospecifics. As the growth stage increased, the degree of proximity to heterospecifics decreased, suggesting that shoaling preferences toward conspecifics and/or visual ability to recognize conspecifics is refined and established according to the growth stage. Furthermore, the proximity of adult female fish was affected by their reproductive status and social familiarity. Only before spawning, adult females maintained closer proximity to familiar males rather than to unfamiliar males, suggesting that proximity was affected by familiarity in a female-specific manner. This simple behavioral paradigm will contribute to our understanding of the neural basis of the development of visually-mediated social behavior using medaka fish.

Production of Knockout Mutants by CRISPR/Cas9 in the European Honeybee, Apis mellifera L.

The European honeybee (Apis mellifera L.) is used as a model organism in studies of the molecular and neural mechanisms underlying social behaviors and/or advanced brain functions. The entire honeybee genome has been sequenced, which has further advanced molecular biologic studies of the honeybee. Functions of genes of interest, however, remain largely to be elucidated in the honeybee due to the lack of effective reverse genetic methods. Moreover, genetically modified honeybees must be maintained under restricted laboratory conditions due to legal restrictions, further complicating the application of reverse genetics to this species. Here we applied CRISPR/Cas9 to the honeybee to develop an effective reverse genetic method. We targeted major royal jelly protein 1 (mrjp1) for genome editing, because this gene is predominantly expressed in adult workers and its mutation is not expected to affect normal development. By injecting sgRNA and Cas9 mRNA into 57 fertilized embryos collected within 3 h after oviposition, we successfully created six queens, one of which produced genome-edited male offspring. Of the 161 males produced, genotyping demonstrated that the genome was edited in 20 males. All of the processes necessary for producing these genome-edited queens and males were performed in the laboratory. Therefore, we developed essential techniques to create knockout honeybees by CRISPR/Cas9. Our findings also suggested that mrjp1 is dispensable for normal male development, at least till the pupal stage. This new technology could pave the way for future functional analyses of candidate genes involved in honeybee social behaviors.

Splicing variants of NOL4 differentially regulate the transcription activity of Mlr1 and Mlr2 in cultured cells.

Mlr1 (Mblk-1-related protein-1) and Mlr2 are mouse homologs of transcription factor Mblk-1 (Mushroom body large-type Kenyon cell-specific protein-1), which we originally identified from the honeybee brain. In the present study, aiming at identifying coregulator(s) of Mlr1 and Mlr2 from the mouse brain, we used yeast two-hybrid screening of mouse brain cDNA library to search for interaction partners of Mlr 1 and Mlr2, respectively. We identified nucleolar protein 4 (NOL4) splicing variants as major interaction partners for both Mlr1 and Mlr2. Among the three murine NOL4 splicing variants, we further characterized NOL4-S, which lacks an N-terminal part of NOL4-L, and NOL4-SΔ, which lacks nuclear localization signal (NLS)-containing domain of NOL4-S. A GST pull-down assay revealed that Mlr1 interacts with both NOL4-S and NOL4-SΔ, whereas Mlr2 interacts with NOL4-S, but not with NOL4-SΔ. These results indicate that the NLS-containing domain of NO4-S Is necessary for in vitro binding with Mlr2, but not for that with Mlr1. Furthermore, a luciferase assay using Schneider's Line 2 cells revealed that transactivation activity of Mlr1 was significantly suppressed by both NOL4-S and NOL4-SΔ, with almost complete suppression by NOL4-SΔ. In contrast, transactivation activity of Mlr2 was significantly suppressed by NOL4-S but rather activated by NOL4-SΔ. Our findings suggest that transactivation activities of Mlr1 and Mlr2 are differentially regulated by splicing variants of NOL4, which are expressed in a tissue-selective manner.

Unique spatially and temporary-regulated/sex-specific expression of a long ncRNA, Nb-1, suggesting its pleiotropic functions associated with honey bee lifecycle.

Honey bees are social insects, and each colony member has unique morphological and physiological traits associated with their social tasks. Previously, we identified a long non-coding RNA from honey bees, termed Nb-1, whose expression in the brain decreases associated with the age-polyethism of workers and is detected in some neurosecretory cells and octopaminergic neurons, suggesting its role in the regulation of worker labor transition. Herein, we investigated its spatially and temporary-regulated/sex-specific expression. Nb-1 was expressed as an abundant maternal RNA during oogenesis and embryogenesis in both sexes. In addition, Nb-1 was expressed preferentially in the proliferating neuroblasts of the mushroom bodies (a higher-order center of the insect brain) in the pupal brains, suggesting its role in embryogenesis and mushroom body development. On the contrary, Nb-1 was expressed in a drone-specific manner in the pupal and adult retina, suggesting its role in the drone visual development and/or sense. Subcellular localization of Nb-1 in the brain during development differed depending on the cell type. Considering that Nb-1 is conserved only in Apidae, our findings suggest that Nb-1 potentially has pleiotropic functions in the expression of multiple developmental, behavioral, and physiological traits, which are closely associated with the honey bee lifecycle.

Proteomic analysis of the royal jelly and characterization of the functions of its derivation glands in the honeybee.

To identify candidate royal jelly (RJ) proteins that might affect the physiologic status of honeybee colony members, we used shotgun proteomics to comprehensively identify the RJ proteome as well as proteomes of the hypopharyngeal gland (HpG), postcerebral gland (PcG), and thoracic gland (TG), from which RJ proteins are assumed to be derived. We identified a total of 38 nonredundant RJ proteins, including 22 putative secretory proteins and Insulin-like growth factor-binding protein complex acid labile subunit. Among them, 9 proteins were newly identified from RJ. Comparison of the RJ proteome with the HpG, PcG, and TG proteomes revealed that 17 of the 22 putative secretory RJ proteins were derived from some of these glands, suggesting that the RJ proteome is a cocktail of proteins from these three glands. Furthermore, pathway analysis suggested that the HpG proteome represents the molecular basis of the extremely high protein-synthesizing ability, whereas the PcG proteome suggests that the PcG functions as a reservoir for the volatile compounds and a primer pheromone. Finally, to further characterize the possible total RJ proteome, we identified putative secretory proteins in the proteomes of these three glands. This will be useful for predicting novel RJ protein components in future studies.

Expression analysis of XPhyH-like during development and tail regeneration in Xenopus tadpoles: possible role of XPhyH-like expressing immune cells in impaired tail regenerative ability.

Xenopus tadpoles have high regenerative ability of amputated tails except during the 'refractory period', when the ability is transiently lost. We previously demonstrated that distinct immune responses occur in tail stumps between the refractory and pre/post-refractory regeneration periods. Furthermore, treatment with an immunosuppressant, FK506, restores the tail regenerative ability during the refractory period. Based on these findings, we previously proposed that autoreactive immune cells infiltrate the tail stumps to attack blastema cells as 'non-self' during the refractory period, resulting in the impaired regenerative ability. The immune cells that attack the blastema cells, however, remained unclear. Here we screened for genes whose expression in the tail stumps was altered by FK506 treatment during the refractory period and identified a Xenopus homolog of phytanoyl-CoA dioxygenase (PhyH)-like. XPhyH-like expression transiently increased in tail stumps after amputation during the refractory period, and was reduced by FK506 treatment. XPhyH-like expression in the whole tadpole body specifically increased during the refractory period and was enriched in the blood cell fraction. These findings suggest that XPhyH-like is expressed in autoreactive immune cells that are distributed in the whole body during the refractory period and transiently infiltrate the tail stumps to attack the blastema cells as 'non-self'.

Evolutionary dynamics of mushroom body Kenyon cell types in hymenopteran brains from multifunctional type to functionally specialized types.

Evolutionary dynamics of diversification of brain neuronal cell types that have underlain behavioral evolution remain largely unknown. Here, we compared transcriptomes and functions of Kenyon cell (KC) types that compose the mushroom bodies between the honey bee and sawfly, a primitive hymenopteran insect whose KCs likely have the ancestral properties. Transcriptome analyses show that the sawfly KC type shares some of the gene expression profile with each honey bee KC type, although unique gene expression profiles have also been acquired in each honey bee KC type. In addition, functional analysis of two sawfly genes suggested that the functions in learning and memory of the ancestral KC type were heterogeneously inherited among the KC types in the honey bee. Our findings strongly suggest that the functional evolution of KCs in Hymenoptera involved two previously hypothesized processes for evolution of cell function: functional segregation and divergence.

Identification of ecdysone receptor target genes in the worker honey bee brains during foraging behavior.

Ecdysone signaling plays central roles in morphogenesis and female ovarian development in holometabolous insects. In the European honey bee (Apis mellifera L.), however, ecdysone receptor (EcR) is expressed in the brains of adult workers, which have already undergone metamorphosis and are sterile with shrunken ovaries, during foraging behavior. Aiming at unveiling the significance of EcR signaling in the worker brain, we performed chromatin-immunoprecipitation sequencing of EcR to search for its target genes using the brains of nurse bees and foragers. The majority of the EcR targets were common between the nurse bee and forager brains and some of them were known ecdysone signaling-related genes. RNA-sequencing analysis revealed that some EcR target genes were upregulated in forager brains during foraging behavior and some were implicated in the repression of metabolic processes. Single-cell RNA-sequencing analysis revealed that EcR and its target genes were expressed mostly in neurons and partly in glial cells in the optic lobes of the forager brain. These findings suggest that in addition to its role during development, EcR transcriptionally represses metabolic processes during foraging behavior in the adult worker honey bee brain.

p53 Mutation suppresses adult neurogenesis in medaka fish (Oryzias latipes).

Tumor suppressor p53 negatively regulates self-renewal of neural stem cells in the adult murine brain. Here, we report that the p53 null mutation in medaka fish (Oryzias latipes) suppressed neurogenesis in the telencephalon, independent of cell death. By using 5-bromo-29-deoxyuridine (BrdU) immunohistochemistry, we identified 18 proliferation zones in the brains of young medaka fish; in situ hybridization showed that p53 was expressed selectively in at least 12 proliferation zones. We also compared the number of BrdU-positive cells present in the whole telencephalon of wild-type (WT) and p53 mutant fish. Immediately after BrdU exposure, the number of BrdU-positive cells did not differ significantly between them. One week after BrdU-exposure, the BrdU-positive cells migrated from the proliferation zone, which was accompanied by an increased number in the WT brain. In contrast, no significant increase was observed in the p53 mutant brain. Terminal deoxynucleotidyl transferase (dUTP) nick end-labeling revealed that there was no significant difference in the number of apoptotic cells in the telencephalon of p53 mutant and WT medaka, suggesting that the decreased number of BrdU-positive cells in the mutant may be due to the suppression of proliferation rather than the enhancement of neural cell death. These results suggest that p53 positively regulates neurogenesis via cell proliferation.

Identification of kakusei, a nuclear non-coding RNA, as an immediate early gene from the honeybee, and its application for neuroethological study.

The honeybee is a social insect that exhibits various social behaviors. To elucidate the neural basis of honeybee behavior, we detected neural activity in freely-moving honeybee workers using an immediate early gene (IEG) that is expressed in a neural activity-dependent manner. In European honeybees (Apis mellifera), we identified a novel nuclear non-coding RNA, termed kakusei, as the first insect IEG, and revealed the neural activity pattern in foragers. In addition, we isolated a homologue of kakusei, termed Acks, from the Japanese honeybee (Apis cerana), and detected active neurons in workers fighting with the giant hornet.

Xenopus laevis il11ra.L is an experimentally proven interleukin-11 receptor component that is required for tadpole tail regeneration.

Xenopus laevis tadpoles possess high regenerative ability and can regenerate functional tails after amputation. An early event in regeneration is the induction of undifferentiated cells that form the regenerated tail. We previously reported that interleukin-11 (il11) is upregulated immediately after tail amputation to induce undifferentiated cells of different cell lineages, indicating a key role of il11 in initiating tail regeneration. As Il11 is a secretory factor, Il11 receptor-expressing cells are thought to mediate its function. X. laevis has a gene annotated as interleukin 11 receptor subunit alpha on chromosome 1L (il11ra.L), a putative subunit of the Il11 receptor complex, but its function has not been investigated. Here, we show that nuclear localization of phosphorylated Stat3 induced by Il11 is abolished in il11ra.L knocked-out culture cells, strongly suggesting that il11ra.L encodes an Il11 receptor component. Moreover, knockdown of il11ra.L impaired tadpole tail regeneration, suggesting its indispensable role in tail regeneration. We also provide a model showing that Il11 functions via il11ra.L-expressing cells in a non-cell autonomous manner. These results highlight the importance of il11ra.L-expressing cells in tail regeneration.

Mblk-1/E93, an ecdysone related-transcription factor, targets synaptic plasticity-related genes in the honey bee mushroom bodies.

Among hymenopteran insects, aculeate species such as bees, ants, and wasps have enlarged and morphologically elaborate mushroom bodies (MBs), a higher-order brain center in the insect, implying their relationship with the advanced behavioral traits of aculeate species. The molecular bases leading to the acquisition of complicated MB functions, however, remains unclear. We previously reported the constitutive and MB-preferential expression of an ecdysone-signaling related transcription factor, Mblk-1/E93, in the honey bee brain. Here, we searched for target genes of Mblk-1 in the worker honey bee MBs using chromatin immunoprecipitation sequence analyses and found that Mblk-1 targets several genes involved in synaptic plasticity, learning, and memory abilities. We also demonstrated that Mblk-1 expression is self-regulated via Mblk-1-binding sites, which are located upstream of Mblk-1. Furthermore, we showed that the number of the Mblk-1-binding motif located upstream of Mblk-1 homologs increased associated with evolution of hymenopteran insects. Our findings suggest that Mblk-1, which has been focused on as a developmental gene transiently induced by ecdysone, has acquired a novel expression pattern to play a role in synaptic plasticity in honey bee MBs, raising a possibility that molecular evolution of Mblk-1 may have partly contributed to the elaboration of MB function in insects.