The European honeybee (Apis mellifera L.) exhibits various social behaviors. The molecular and neural mechanisms underlying these behaviors have long been explored, but causal relations between genes ...or neurons and behaviors remain to be elucidated because effective gene manipulation methods in the honeybee have not been available until recently. We recently established a basic technology to produce mutant honeybee drones using CRISPR/Cas9. Here we produced mutant drones using CRISPR/Cas9 targeting mKast, which is preferentially expressed in a certain subtype of class I Kenyon cells that comprise the mushroom bodies in the honeybee brain. By immunoblot analysis, we showed that mKast protein expression was completely lost in the mutant drone heads. In addition, during the production process of homozygous mutant workers, we demonstrated that heterozygous mutant workers could be produced by artificial insemination of wild-type queens with the sperm of mutant drones, indicating that mKast mutant drones were sexually mature. These results demonstrate that mKast is dispensable for normal development and sexual maturation in drone honeybees, and allow us to proceed with the production of homozygous mutant workers for the analysis of a particular gene by gene knockout in the future.
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.
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.
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.
Endoplasmic reticulum (ER) stress responses have been demonstrated to play important roles in maintaining various cellular functions and to underlie many tissue dysfunctions. In this study, we ...identified Sec16B as an ER stress-inducible gene by microarray analysis of brefeldin A (BFA)-inducible genes in a mouse neuroblastoma cell-line, Neuro2a. Sec16B mRNA was induced by treatment with the ER stress-inducing reagents thapsigargin (Tg) and brefeldin A in a time-dependent manner. In the genomic sequence of the mouse Sec16B gene, we found an unfolded protein response element (UPRE), which is well conserved between humans and mice. Using luciferase reporter analyses, we showed that the UPRE in the mouse Sec16B gene was functional and responded well to ER stress-inducing stimuli and spliced XBP1 (sXBP1)-overexpression. In addition, a unique ATF4-responsive sequence within the first intron of the mouse Sec16B gene was characterized. Our study may help to elucidate the regulation of trafficking through the ER–Golgi apparatus and the biogenesis of ER-derived intracellular organelles.
The hemodynamic and exercise capacity performance of the Jarvik 2000 left ventricular assist device (LVAD), which is generally used in patients with small body size and relatively preserved cardiac ...function, is not well understood. We retrospectively examined 18 patients implanted with the Jarvik 2000 LVAD. Pump rotation speed was optimized by the hemodynamic ramp test one year after implantation based on the criteria of mean pulmonary capillary wedge pressure (PCWP) < 18 mmHg, mean right atrial pressure (RAP) < 12 mmHg, and cardiac index (CI) > 2.2 L/min/m
2
as well as echocardiographic parameters. Exercise capacity was assessed by cardiopulmonary exercise test in an optimized setting. To investigate the impacts of larger body surface area (BSA) and extremely impaired pre-implantation cardiac function on hemodynamics and exercise capacity, two correlation analyses based on BSA and original CI were performed. At a pump speed of 9500 ± 707 rpm, the mean pulmonary artery pressure, PCWP, RAP, and CI were 17 ± 5 mmHg, 9 ± 5 mmHg, 6 ± 4 mmHg, and 2.82 ± 0.54 L/min/m
2
, respectively. Only one patient failed to achieve the hemodynamic criteria. The peak VO
2
and VE/VCO
2
slope were 12.9 ± 3.1 mL/min/kg and 37.7 ± 15.0, respectively. There was an inverse correlation between original CI and heart rate (
r
= −0.60,
p
= 0.01), and a weak correlation between BSA and PCWP (
r
= 0.43,
p
= 0.08). Based on this study, the overall performance of the Jarvik 2000 device was acceptable, and the patients’ body size and original cardiac function had minimum effect on the performance of this device.
The European honey bee is a model organism for studying social behaviors. Comprehensive analyses focusing on the differential expression profiles of genes between the brains of nurse bees and ...foragers, or in the mushroom bodies—the brain structure related to learning and memory, and multimodal sensory integration—has identified candidate genes related to honey bee behaviors. Despite accumulating knowledge on the expression profiles of genes related to honey bee behaviors, it remains unclear whether these genes actually regulate social behaviors in the honey bee, in part because of the scarcity of genetic manipulation methods available for application to the honey bee. In this review, we describe the genetic methods applied to studies of the honey bee, ranging from classical forward genetics to recently developed gene modification methods using transposon and CRISPR/Cas9. We then discuss future functional analyses using these genetic methods targeting genes identified by the preceding research. Because no particular genes or neurons unique to social insects have been found yet, further exploration of candidate genes/neurons correlated with sociality through comprehensive analyses of mushroom bodies in the aculeate species can provide intriguing targets for functional analyses, as well as insight into the molecular and neural bases underlying social behaviors.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Mushroom bodies (MBs), a higher-order center in the honeybee brain, comprise some subtypes/populations of interneurons termed as Kenyon cells (KCs), which are distinguished by their cell body size ...and location in the MBs, as well as their gene expression profiles. Although the role of MBs in learning ability has been studied extensively in the honeybee, the roles of each KC subtype and their evolution in hymenopteran insects remain mostly unknown. This mini-review describes recent progress in the analysis of gene/protein expression profiles and possible functions of KC subtypes/populations in the honeybee. Especially, the discovery of novel KC subtypes/populations, the "middle-type KCs" and "KC population expressing FoxP," necessitated a redefinition of the KC subtype/population. Analysis of the effects of inhibiting gene function in a KC subtype-preferential manner revealed the function of the gene product as well as of the KC subtype where it is expressed. Genes expressed in a KC subtype/population-preferential manner can be used to trace the differentiation of KC subtypes during the honeybee ontogeny and the possible evolution of KC subtypes in hymenopteran insects. Current findings suggest that the three KC subtypes are unique characteristics to the aculeate hymenopteran insects. Finally, prospects regarding future application of genome editing for the study of KC subtype functions in the honeybee are described. Genes expressed in a KC subtype-preferential manner can be good candidate target genes for genome editing, because they are likely related to highly advanced brain functions and some of them are dispensable for normal development and sexual maturation in honeybees.
IRE1 is the most conserved endoplasmic reticulum (ER)-resident stress sensor. Its activation not only splices XBP1 but also participates in a variety of cell signaling. We elucidated the role of ...IRE1α in Neuro2a cells by establishing IRE1α-deficient cells and applying four IRE1 inhibitors. IRE1α deficiency prevented almost all spliced XBP1 (sXBP1) protein expression by treatment with thapsigargin (Tg) and tunicamycin (Tm); these phenomena paralleled the values measured by our two Nanoluciferase-based IRE1 assays. However, cell viability and protein expression of other ER stress-responsive factors in the IRE1α-deficient cells were comparable to those in the parental wild-type cells with or without Tm treatment. Next, we elucidated the IRE1 inhibitory actions and cytotoxicity of four compounds: STF083010, KIRA6, 4μ8C, and toyocamycin. KIRA6 attenuated IRE1 activity in a dose-dependent manner, but it showed severe cytotoxicity even in the IRE1α-deficient cells at a low concentration. The IRE1α-deficient cells were slightly resistant to KIRA6 at 0.1 μM in both the presence and absence of ER stress; however, resistance was not observed at 0.02 μM. Treatment with only KIRA6 at 0.1 μM for 12 h remarkably induced LC3 II, an autophagic marker, in both parental and IRE1α-deficient cells. Co-treatment with KIRA6 and Tm induced LC3 II, cleaved caspase-9, and cleaved caspase-3; however, IRE1α-deficiency did not abolish the expression of these two cleaved caspases. On the other hand, KIRA6 prohibited Tm-induced ATF4 induction in an IRE1-independent manner; however, co-treatment with KIRA6 and Tm also induced LC3 II and two cleaved caspases in the ATF4-deficient Neuro2a cells. Thus, we demonstrate that IRE1α deficiency has little impact on cell viability and expression of ER stress-responsive factors in Neuro2a cells, and the pharmacological actions of KIRA6 include IRE1-independent ways.
In the honey bee, the mushroom bodies (MBs), a higher-order center in insect brain, comprise interneurons termed Kenyon cells (KCs). We previously reported that Mblk-1, which encodes a transcription ...factor involved in ecdysteroid-signaling, is expressed preferentially in the large-type KCs (lKCs) in the pupal and adult worker brain and that phosphorylation by the Ras/MAPK pathway enhances the transcriptional activity of Mblk-1 in vitro. In the present study, we performed immunoblotting and immunofluorescence studies using affinity-purified anti-Mblk-1 and anti-phosphorylated Mblk-1 antibodies to analyze the distribution and phosphorylation of Mblk-1 in the brains of pupal and adult workers. Mblk-1 was preferentially expressed in the lKCs in both pupal and adult worker brains. In contrast, some Mblk-1 was phosphorylated almost exclusively in the pupal stages, and phosphorylated Mblk-1 was preferentially expressed in the MB neuroblasts and lKCs in pupal brains. Immunofluorescence studies revealed that both Mblk-1 and phosphorylated Mblk-1 are located in both the cytoplasm and nuclei of the lKC somata in the pupal and adult worker brains. These findings suggest that Mblk-1 plays a role in the lKCs in both pupal and adult stages and that phosphorylated Mblk-1 has pupal stage-specific functions in the MB neuroblasts and lKCs in the honey bee brain.
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