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.

Analysis of the Differentiation of Kenyon Cell Subtypes Using Three Mushroom Body-Preferential Genes during Metamorphosis in the Honeybee (Apis mellifera L.).

The adult honeybee (Apis mellifera L.) mushroom bodies (MBs, a higher center in the insect brain) comprise four subtypes of intrinsic neurons: the class-I large-, middle-, and small-type Kenyon cells (lKCs, mKCs, and sKCs, respectively), and class-II KCs. Analysis of the differentiation of KC subtypes during metamorphosis is important for the better understanding of the roles of KC subtypes related to the honeybee behaviors. In the present study, aiming at identifying marker genes for KC subtypes, we used a cDNA microarray to comprehensively search for genes expressed in an MB-preferential manner in the honeybee brain. Among the 18 genes identified, we further analyzed three genes whose expression was enriched in the MBs: phospholipase C epsilon (PLCe), synaptotagmin 14 (Syt14), and discs large homolog 5 (dlg5). Quantitative reverse transcription-polymerase chain reaction analysis revealed that expression of PLCe, Syt14, and dlg5 was more enriched in the MBs than in the other brain regions by approximately 31-, 6.8-, and 5.6-fold, respectively. In situ hybridization revealed that expression of both Syt14 and dlg5 was enriched in the lKCs but not in the mKCs and sKCs, whereas expression of PLCe was similar in all KC subtypes (the entire MBs) in the honeybee brain, suggesting that Syt14 and dlg5, and PLCe are available as marker genes for the lKCs, and all KC subtypes, respectively. In situ hybridization revealed that expression of PLCe is already detectable in the class-II KCs at the larval fifth instar feeding stage, indicating that PLCe expression is a characteristic common to the larval and adult MBs. In contrast, expression of both Syt14 and dlg5 became detectable at the day three pupa, indicating that Syt14 and dlg5 expressions are characteristic to the late pupal and adult MBs and the lKC specific molecular characteristics are established during the late pupal stages.

A neural mechanism underlying mating preferences for familiar individuals in medaka fish.

Social familiarity affects mating preference among various vertebrates. Here, we show that visual contact of a potential mating partner before mating (visual familiarization) enhances female preference for the familiarized male, but not for an unfamiliarized male, in medaka fish. Terminal-nerve gonadotropin-releasing hormone 3 (TN-GnRH3) neurons, an extrahypothalamic neuromodulatory system, function as a gate for activating mating preferences based on familiarity. Basal levels of TN-GnRH3 neuronal activity suppress female receptivity for any male (default mode). Visual familiarization facilitates TN-GnRH3 neuron activity (preference mode), which correlates with female preference for the familiarized male. GnRH3 peptides, which are synthesized specifically in TN-GnRH3 neurons, are required for the mode-switching via self-facilitation. Our study demonstrates the central neural mechanisms underlying the regulation of medaka female mating preference based on visual social familiarity.

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.

Induction of c-fos transcription in the medaka brain (Oryzias latipes) in response to mating stimuli.

Immediate-early genes (IEGs) are useful for mapping active brain regions in various vertebrates. Here we identified a c-fos homologue gene in medaka and demonstrated that the amounts of c-fos transcripts and proteins in the medaka brain increased in relation to an artificially evoked seizure, suggesting that the homologue gene has the characteristics of IEGs, which are used as markers of neural activity. Next, quantitative reverse-transcription-polymerase chain reaction revealed that female mating behaviors upregulated c-fos transcription in some brain regions including the telencephalon, optic tectum, and cerebellum. In addition, we performed in situ hybridization with a c-fos intron probe to detect the de novo synthesis of c-fos transcripts and confirmed induction of c-fos transcription in these brain regions after mating. This is the first report of IEG induction in response to mating stimuli in teleost fish. Our results indicated that c-fos expression was induced in response to behavioral stimuli in the medaka brain and that medaka c-fos could be a useful marker of neural activity.

Transient and permanent gene transfer into the brain of the teleost fish medaka (Oryzias latipes) using human adenovirus and the Cre-loxP system.

In this study, we demonstrated that human type-5 adenovirus infected the brain of the teleost fish, medaka (Oryzias latipes), in vivo. Injection of adenoviral vector into the mesencephalic ventricle of medaka larvae induced the expression of reporter genes in some parts of the telencephalon, the periventricular area of the mesencephalon and diencephalon, and the cerebellum. Additionally, the Cre-loxP system works in medaka brains using transgenic medaka carrying a vector containing DsRed2, flanked by loxP sites under control of the beta-actin promoter and downstream promoterless enhanced green fluorescent protein (EGFP). We demonstrated that the presence of green fluorescence depended on injection of adenoviral vector expressing the Cre gene and confirmed that EGFP mRNA was transcribed in the virus-injected larvae.

Morph-dependent expression and subcellular localization of host serine carboxypeptidase in bacteriocytes of the pea aphid associated with degradation of the endosymbiotic bacterium Buchnera.

Pea aphids form a mutualistic association with the endosymbiotic bacterium Buchnera, which is harbored in specialized host cells called bacteriocytes. The adult aphids display dimorphism In which there are winged and wingless morphs. We previously reported that the Buchnera density in bacteriocytes of the winged morph (alate) decreases around final ecdysis, whereas that in the wingless morph (aptera) does not decrease; the decrease in density in alatae is accompanied by activation of the host lysosomal system and by Buchnera degradation. In the present study, we performed a proteomic analysis to clarify the molecular mechanisms underlying the decrease in Buchnera density. By comparing the protein expression profiles of bacteriocytes in alatae and apterae Just after final ecdysis, we identified three and one protein spots that were preferentially expressed in alatae and apterae, respectively. Among the three alate-preferential spots, two were an identical aphid protein, carboxypeptidase vitellogenic-like (CPVL), whereas the other was a mixture of four proteins: gamma-glutamyl hydrolase, acyl-CoA dehydrogenase, aphid short chain acyl-CoA dehydrogenase, and Buchnera S-adenosylmethionine synthetase. The aptera-preferential spot was Buchnera outer membrane protein A. Immunoblot and immunohistochemical analyses using aphid bacteriocytes Just after final ecdysis revealed that expression of aphid CPVL was preferentially upregulated in alatae and was localized around Buchnera cells in the bacterlocytes, suggesting the involvement of CPVL in Buchnera degradation in alatae.

Suppression of the immune response potentiates tadpole tail regeneration during the refractory period.

Regenerative ability varies depending on animal species and developmental stage, but the factors that determine this variability remain unclear. Although Xenopus laevis tadpole tails possess high regenerative ability, this is transiently lost during the ;refractory period'. Here, we show that tail amputation evokes different immune responses in wound tail stumps between the ;refractory' and ;regeneration' periods: there was delayed or prolonged expression of some immune-related genes in the refractory period, whereas there was no obvious or transient expression of other immune-related genes in the regeneration periods. In addition, immune suppression induced by either immunosuppressant treatment or immune cell depletion by knockdown of PU.1 significantly restored regenerative ability during the refractory period. These findings indicate that immune responses have a crucial role in determining regenerative ability in Xenopus tadpole tails.

A trophic role for Wnt-Ror kinase signaling during developmental pruning in Caenorhabditis elegans.

The molecular mechanism by which neurites are selected for elimination or incorporation into the mature circuit during developmental pruning remains unknown. The trophic theory postulates that local cues provided by target or surrounding cells act to inhibit neurite elimination. However, no widely conserved factor mediating this trophic function has been identified. We found that the developmental survival of specific neurites in Caenorhabditis elegans largely depends on detection of the morphogen Wnt by the Ror kinase CAM-1, which is a transmembrane tyrosine kinase with a Frizzled domain. Mutations in Wnt genes or in cam-1 enhanced neurite elimination, whereas overexpression of cam-1 inhibited neurite elimination in a Wnt-dependent manner. Moreover, mutations in these genes counteracted the effect of a mutation in mbr-1, which encodes a transcription factor that promotes neurite elimination. These results reveal the trophic role of an atypical Wnt pathway and reinforce the classical model of developmental pruning.

Age- and morph-dependent activation of the lysosomal system and Buchnera degradation in aphid endosymbiosis.

Endosymbiosis in aphids is maintained through a mutualistic association between the host and a symbiotic bacterium, Buchnera, which is harbored in specialized host cells called bacteriocytes. Here, we examined the changes in the Buchnera density in bacteriocytes in relation to the development and polyphenism of the host aphid. Buchnera density in the winged morph aphids, alatae, decreased drastically around the final ecdysis, whereas in the wingless morph aphids, apterae, Buchnera density decreased after the final ecdysis. Thereafter, in both apterae and alatae, Buchnera density was maintained at a constant level until 10 days and then again decreased gradually until 18 days after the final ecdysis. Cytochemical analysis with LysoTracker reagent and quantitative RT-PCR analysis revealed that the number of lysosome-like acidic organelles and the amount of lysosome-related gene (lysozyme and cathepsin L) transcripts increased drastically in the bacteriocytes of alatae around the final ecdysis. Electron microscopy of alatae bacteriocytes around the final ecdysis revealed many Buchnera with irregular electron-dense areas in their cytoplasm that were enclosed by a distended symbiosome membrane. These findings indicated that age- and morph-dependent decreases in Buchnera density coincided with activation of the host lysosomal system and the increased degradation of Buchnera.

The extracellular adenosine deaminase growth factor, ADGF/CECR1, plays a role in Xenopus embryogenesis via the adenosine/P1 receptor.

Adenosine deaminase-related growth factors (ADGF), also known as CECR1 in vertebrates, are a novel family of growth factors with sequence similarity to classical cellular adenosine deaminase. Although genes for ADGF/CECR1 have been identified in both invertebrates as well as vertebrates, their in vivo functions in vertebrates remain unknown. We isolated cDNA clones for two cerc 1s from Xenopus laevis. Both recombinant Xenopus CECR1s exhibited adenosine deaminase and growth factor activity, and the adenosine deaminase activity was found to be indispensable for growth factor activity. The Xenopus cerc 1s are expressed in the somites, pronephros, eyes, cement gland, neural tube, and neural floor plate of the embryos. Knock-down of these two genes using morpholino oligonucleotides caused a reduction in the body size and abnormalities of the body axis in the Xenopus embryos, accompanied by selective changes in the expression of developmental marker genes. Injection of adenosine, agonists for adenosine/P1 receptors, or adenosine deaminase inhibitor into late gastrula archenteron embryos resulted in developmental defects similar to those caused by morpholino oligonucleotide injection. These results show, for the first time, the involvement of CECR1s via the adenosine/P1 receptors in vertebrate embryogenesis via regulation of extracellular adenosine concentrations.

Novel insect picorna-like virus identified in the brains of aggressive worker honeybees.

To identify candidate genes involved in the aggressive behavior of worker honeybees, we used the differential display method to search for RNAs exclusively detected in the brains of aggressive workers that had attacked a hornet. We identified a novel, 10,152-nucleotide RNA, termed Kakugo RNA. Kakugo RNA encodes a protein of 2,893 amino acid residues that shares structural features and sequence similarities with various picorna-like virus polyproteins, especially those from sacbrood virus, which infects honeybees. The Kakugo protein contains several domains that correspond to the virion protein, helicase, protease, and RNA-dependent RNA polymerase domains of various picorna-like virus polyproteins. When the worker bee tissue lysate was subjected to sucrose density gradient centrifugation, Kakugo RNA, except for the material at the bottom, was separated into two major peaks. One of the peaks corresponded to the position of Kakugo mRNA, and the other corresponded to the position of the poliovirus virion. These results suggest that the Kakugo RNA exists as an mRNA-like free RNA and virion RNA in the honeybee. Furthermore, injection of the lysate supernatant from the attacker heads into the heads of noninfected bees resulted in a marked increase in Kakugo RNA. These results demonstrate that Kakugo RNA is a plus-strand RNA of a novel picorna-like virus and that the brains of aggressive workers are infected by this novel virus. Kakugo RNA was detected in aggressive workers but not in nurse bees or foragers. In aggressive workers, Kakugo RNA was detected in the brain but not in the thorax or abdomen, indicating a close relation between viral infection in the brain and aggressive worker behaviors.

Identification of genes expressed preferentially in the honeybee mushroom bodies by combination of differential display and cDNA microarray.

To clarify the molecular basis underlying the neural function of the honeybee mushroom bodies (MBs), we identified three genes preferentially expressed in MB using cDNA microarrays containing 480 differential display-positive candidate cDNAs expressed locally or differentially, dependent on caste/aggressive behavior in the honeybee brain. One of the cDNAs encodes a putative type I inositol 1,4,5-trisphosphate (IP(3)) 5-phosphatase and was expressed preferentially in one of two types of intrinsic MB neurons, the large-type Kenyon cells, suggesting that IP(3)-mediated Ca(2+) signaling is enhanced in these neurons.