Lack of membrane sex steroid receptors for mediating rapid endocrine responses in molluscan nervous systems.

Despite the lack of endogenous synthesis and relevant nuclear receptors, several papers have been published over the decades claiming that the physiology of mollusks is affected by natural and synthetic sex steroids. With scant evidence for the existence of functional steroid nuclear receptors in mollusks, some scientists have speculated that the effects of steroids might be mediated via membrane receptors (i.e. via non-genomic/non-classical actions) - a mechanism that has been well-characterized in vertebrates. However, no study has yet investigated the ligand-binding ability of such receptor candidates in mollusks. The aim of the present study was to further trace the evolution of the endocrine system by investigating the presence of functional membrane sex steroid receptors in a mollusk, the great pond snail (Lymnaea stagnalis). We detected sequences homologous to the known vertebrate membrane sex steroid receptors in the Lymnaea transcriptome and genome data: G protein-coupled estrogen receptor-1 (GPER1); membrane progestin receptors (mPRs); G protein-coupled receptor family C group 6 member A (GPRC6A); and Zrt- and Irt-like protein 9 (ZIP9). Sequence analyses, including conserved domain analysis, phylogenetics, and transmembrane domain prediction, indicated that the mPR and ZIP9 candidates appeared to be homologs, while the GPER1 and GPRC6A candidates seemed to be non-orthologous receptors. All candidates transiently transfected into HEK293MSR cells were found to be localized at the plasma membrane, confirming that they function as membrane receptors. However, the signaling assays revealed that none of the candidates interacted with the main vertebrate steroid ligands. Our findings strongly suggest that functional membrane sex steroid receptors which would be homologous to the vertebrate ones are not present in Lymnaea. Although further experiments are required on other molluscan model species as well, we propose that both classical and non-classical sex steroid signaling for endocrine responses are specific to chordates, confirming that molluscan and vertebrate endocrine systems are fundamentally different.

Characterization of corazonin signaling in a molluscan model species, Lymnaea stagnalis.

In recent years, new concepts have emerged regarding the nomenclature, functions, and relationships of different peptide families of the gonadotropin-releasing hormone (GnRH) superfamily. One of the main driving forces for this originated from the emerging evidence that neuropeptides previously called molluscan GnRH are multifunctional and should be classified as corazonin (CRZ). However, research articles still appear that use incorrect nomenclature and attribute the same function to molluscan CRZs as vertebrate GnRHs. The aim of the present study was to further support the recent interpretation of the origin and function of the GnRH superfamily. Towards this goal, we report the characterization of CRZ signaling system in the molluscan model species, the great pond snail (Lymnaea stagnalis). We detected a CRZ-receptor-like sequence (Lym-CRZR) by homology-searching in the Lymnaea transcriptomes and the deduced amino acid sequence showed high sequence similarity to GnRH receptors and CRZ receptors. Molecular phylogenetic tree analysis demonstrated that Lym-CRZR is included in the cluster of molluscan CRZRs. Lym-CRZR transiently transfected into HEK293 cells was found to be localized at the plasma membrane, confirming that it functions as a membrane receptor, like other G protein-coupled receptors. The signaling assays revealed that the previously identified Lym-CRZ neuropeptide stimulated intracellular Ca2+ mobilization in a dose-dependent manner, but not cyclic AMP production, in HEK293 cells transfected with Lym-CRZR. Finally, we demonstrated a wide tissue distribution of Lym-CRZR. These results suggest that Lym-CRZ is a multifunctional peptide and provide further insights into the evolution of the GnRH neuropeptide superfamily. The present study also supports the notion that previously termed molluscan "GnRH" should be classified as "CRZ".

Ovarian Follicle Development in Ascidians.

Ovarian follicle development is an essential process for continuation of sexually reproductive animals, and is controlled by a wide variety of regulatory factors such as neuropeptides and peptide hormones in the endocrine, neuroendocrine, and nervous systems. Moreover, while some molecular mechanisms underlying follicle development are conserved, others vary among species. Consequently, follicle development processes are closely related to the evolution and diversity of species. Ciona intestinalis type A (Ciona rubusta) is a cosmopolitan species of ascidians, which are the closest relative of vertebrates. However, unlike vertebrates, ascidians are not endowed with the hypothalamus-pituitary-gonadal axis involving pituitary gonadotropins and sexual steroids. Combined with the phylogenetic position of ascidians as the closest relative of vertebrates, such morphological and endocrine features suggest that ascidians possess both common and species-specific regulatory mechanisms in follicle development. To date, several neuropeptides have been shown to participate in the growth of vitellogenic follicles, oocyte maturation of postvitellogenic follicles, and ovulation of fully mature follicles in a developmental stage-specific fashion. Furthermore, recent studies have shed light on the evolutionary processes of follicle development throughout chordates. In this review, we provide an overview of the neuropeptidergic molecular mechanism in the premature follicle growth, oocyte maturation, and ovulation in Ciona, and comparative views of the follicle development processes of mammals and teleosts.

Distribution of cionin, a cholecystokinin/gastrin family peptide, and its receptor in the central nervous system of Ciona intestinalis type A.

The cholecystokinin (CCK)/gastrin family peptides are involved in regulation of feeding and digestion in vertebrates. In the ascidian Ciona intestinalis type A (Ciona robusta), cionin, a CCK/gastrin family peptide, has been identified. Cionin is expressed exclusively in the central nervous system (CNS). In contrast, cionin receptor expression has been detected in the CNS, digestive tract, and ovary. Although cionin has been reported to be involved in ovulation, its physiological function in the CNS remains to be investigated. To elucidate its neural function, in the present study, we analyzed the expression of cionin and cionin receptors in the CNS. Cionin was expressed mainly in neurons residing in the anterior region of the cerebral ganglion. In contrast, the gene expressin of the cionin receptor gene CioR1, was detected in the middle part of the cerebral ganglion and showed a similar expression pattern to that of VACHT, a cholinergic neuron marker gene. Moreover, CioR1 was found to be expressed in cholinergic neurons. Consequently, these results suggest that cionin interacts with cholinergic neurons as a neurotransmitter or neuromodulator via CioR1. This study provides insights into a biological role of a CCK/gastrin family peptide in the CNS of ascidians.

A Novel Hemocyte-Derived Peptide and Its Possible Roles in Immune Response of Ciona intestinalis Type A.

A wide variety of bioactive peptides have been identified in the central nervous system and several peripheral tissues in the ascidian Ciona intestinalis type A (Ciona robusta). However, hemocyte endocrine peptides have yet to be explored. Here, we report a novel 14-amino-acid peptide, CiEMa, that is predominant in the granular hemocytes and unilocular refractile granulocytes of Ciona. RNA-seq and qRT-PCR revealed the high CiEma expression in the adult pharynx and stomach. Immunohistochemistry further revealed the highly concentrated CiEMa in the hemolymph of the pharynx and epithelial cells of the stomach, suggesting biological roles in the immune response. Notably, bacterial lipopolysaccharide stimulation of isolated hemocytes for 1-4 h resulted in 1.9- to 2.4-fold increased CiEMa secretion. Furthermore, CiEMa-stimulated pharynx exhibited mRNA upregulation of the growth factor (Fgf3/7/10/22), vanadium binding proteins (CiVanabin1 and CiVanabin3), and forkhead and homeobox transcription factors (Foxl2, Hox3, and Dbx) but not antimicrobial peptides (CrPap-a and CrMam-a) or immune-related genes (Tgfbtun3, Tnfa, and Il17-2). Collectively, these results suggest that CiEMa plays roles in signal transduction involving tissue development or repair in the immune response, rather than in the direct regulation of immune response genes. The present study identified a novel Ciona hemocyte peptide, CiEMa, which paves the way for research on the biological roles of hemocyte peptides in chordates.

The neuroendocrine system of Ciona intestinalis Type A, a deuterostome invertebrate and the closest relative of vertebrates.

Deuterostome invertebrates, including echinoderms, hemichordates, cephalochordates, and urochordates, exhibit common and species-specific morphological, developmental, physiological, and behavioral characteristics that are regulated by neuroendocrine and nervous systems. Over the past 15 years, omics, genetic, and/or physiological studies on deuterostome invertebrates have identified low-molecular-weight transmitters, neuropeptides and their cognate receptors, and have clarified their various biological functions. In particular, there has been increasing interest on the neuroendocrine and nervous systems of Ciona intestinalis Type A, which belongs to the subphylum Urochordata and occupies the critical phylogenetic position as the closest relative of vertebrates. During the developmental stage, gamma-aminobutylic acid, D-serine, and gonadotropin-releasing hormones regulate metamorphosis of Ciona. In adults, the neuropeptidergic mechanisms underlying ovarian follicle growth, oocyte maturation, and ovulation have been elucidated. This review article provides the most recent and fundamental knowledge of the neuroendocrine and nervous systems of Ciona, and their evolutionary aspects.

PTBP2 binds to a testis-specific long noncoding RNA, Tesra, and activates transcription of the Prss42/Tessp-2 gene.

Long noncoding RNAs (lncRNAs) have recently been proved to be functional in the testis. Tesra, a testis-specific lncRNA, was suggested to activate the transcription of Prss42/Tessp-2, a gene that is involved in meiotic progression, in mouse spermatocytes. To reveal the molecular mechanism underlying the activation, we searched for Tesra-binding proteins by a Ribotrap assay followed by LC-MS/MS analysis and identified polypyrimidine tract binding protein 2 (PTBP2) as a candidate. Analysis of public RNA-seq data and our qRT-PCR results indicated that Ptbp2 mRNA showed an expression pattern similar to the expression patterns of Tesra and Prss42/Tessp-2 during testis development. Moreover, PTBP2 was found to be associated with Tesra in testicular germ cells by RNA immunoprecipitation. To evaluate the effect of PTBP2 on the Prss42/Tessp-2 promoter, we established an in vitro reporter gene assay system in which Tesra expression could be induced by the Tet-on system and thereby Prss42/Tessp-2 promoter activity could be increased. In this system, the Prss42/Tessp-2 promoter activity was significantly decreased by the knockdown of PTBP2. These results suggest that PTBP2 contributes to Prss42/Tessp-2 transcriptional activation by Tesra in spermatocytes. The finding provides a precious example of a molecular mechanism of testis lncRNA functioning in spermatogenesis.

Anthriscus sylvestris Deoxypodophyllotoxin Synthase Involved in the Podophyllotoxin Biosynthesis.

Tetrahydrofuran ring formation from dibenzylbutyrolactone lignans is a key step in the biosynthesis of aryltetralin lignans including deoxypodophyllotoxin and podophyllotoxin. Previously, Fe(II)- and 2-oxoglutarate-dependent dioxygenase (2-ODD) from Podophyllum hexandrum (Himalayan mayapple, Berberidaceae) was found to catalyze the cyclization of a dibenzylbutyrolactone lignan, yatein, to give deoxypodophyllotoxin and designated as deoxypodophyllotoxin synthase (DPS). Recently, we reported that the biosynthesis of deoxypodophyllotoxin and podophyllotoxin evolved in a lineage-specific manner in phylogenetically unrelated plant species such as P. hexandrum and Anthriscus sylvestris (cow parsley, Apiaceae). Therefore, a comprehensive understanding of the characteristics of DPSs that catalyze the cyclization of yatein to deoxypodophyllotoxin in various plant species is important. However, for plant species other than P. hexandrum, the isolation of the DPS enzyme gene and the type of the enzyme, e.g. whether it is 2-ODD or another type of enzyme such as cytochrome P-450, have not been reported. In this study, we report the identification and characterization of A. sylvestris DPS (AsDPS). Phylogenetic analysis showed that AsDPS belonged to the 2-ODD superfamily and shared moderate amino acid sequence identity (40.8%) with P. hexandrum deoxypodophyllotoxin synthase (PhDPS). Recombinant protein assay indicated that AsDPS and PhDPS differ in terms of the selectivity of substrate enantiomers. Protein modeling using AlphaFold2 and site-directed mutagenesis indicated that the Tyr305 residue of AsDPS probably contributes to substrate recognition. This study advances our understanding of the podophyllotoxin biosynthetic pathway in A. sylvestris and provides new insight into 2-ODD involved in plant secondary (specialized) metabolism.

Characterization of a novel species-specific 51-amino acid peptide, PEP51, as a caspase-3/7 activator in ovarian follicles of the ascidian, Ciona intestinalis Type A.

Invertebrates lack hypothalamic-pituitary-gonadal axis, and have acquired species-specific regulatory systems for ovarian follicle development. Ascidians are marine invertebrates that are the phylogenetically closest living relatives to vertebrates, and we have thus far substantiated the molecular mechanisms underlying neuropeptidergic follicle development of the cosmopolitan species, Ciona intestinalis Type A. However, no ovarian factor has so far been identified in Ciona. In the present study, we identified a novel Ciona-specific peptide, termed PEP51, in the ovary. Immunohistochemical analysis demonstrated the specific expression of PEP51 in oocyte-associated accessory cells, test cells, of post-vitellogenic (stage III) follicles. Immunoelectron microscopy revealed that PEP51 was localized in the cytosol of test cells in early stage III follicles, which lack secretory granules. These results indicate that PEP51 acts as an intracellular factor within test cells rather than as a secretory peptide. Confocal laser microscopy verified that activation of caspase-3/7, the canonical apoptosis marker, was detected in most PEP51-positive test cells of early stage III. This colocalization of PEP51 and the apoptosis marker was consistent with immunoelectron microscopy observations demonstrating that a few normal (PEP51-negative) test cells reside in the aggregates of PEP51-positive apoptotic test cells of early stage III follicles. Furthermore, transfection of the PEP51 gene into COS-7 cells and HEK293MSR cells resulted in activation of caspase-3/7, providing evidence that PEP51 induces apoptotic signaling. Collectively, these results showed the existence of species-specific ovarian peptide-driven cell metabolism in Ciona follicle development. Consistent with the phylogenetic position of Ciona as the closest sister group of vertebrates, the present study sheds new light on the molecular and functional diversity of the regulatory systems of follicle development in the Chordata.

Kobayashi Award 2021: Neuropeptides, receptors, and follicle development in the ascidian, Ciona intestinalis Type A: New clues to the evolution of chordate neuropeptidergic systems from biological niches.

Ciona intestinalis Type A (Ciona robusta) is a cosmopolitan species belonging to the phylum Urochordata, invertebrate chordates that are phylogenetically the most closely related to the vertebrates. Therefore, this species is of interest for investigation of the evolution and comparative physiology of endocrine, neuroendocrine, and nervous systems in chordates. Our group has identified>30 Ciona neuropeptides (80% of all identified ascidian neuropeptides) primarily using peptidomic approaches combined with reference to genome sequences. These neuropeptides are classified into two groups: homologs or prototypes of vertebrate neuropeptides and novel (Ciona-specific) neuropeptides. We have also identified the cognate receptors for these peptides. In particular, we elucidated multiple receptors for Ciona-specific neuropeptides by a combination of a novel machine learning system and experimental validation of the specific interaction of the predicted neuropeptide-receptor pairs, and verified unprecedented phylogenies of receptors for neuropeptides. Moreover, several neuropeptides were found to play major roles in the regulation of ovarian follicle development. Ciona tachykinin facilitates the growth of vitellogenic follicles via up-regulation of the enzymatic activities of proteases. Ciona vasopressin stimulates oocyte maturation and ovulation via up-regulation of maturation-promoting factor- and matrix metalloproteinase-directed collagen degradation, respectively. Ciona cholecystokinin also triggers ovulation via up-regulation of receptor tyrosine kinase signaling and the subsequent activation of matrix metalloproteinase. These studies revealed that the neuropeptidergic system plays major roles in ovarian follicle growth, maturation, and ovulation in Ciona, thus paving the way for investigation of the biological roles for neuropeptides in the endocrine, neuroendocrine, nervous systems of Ciona, and studies of the evolutionary processes of various neuropeptidergic systems in chordates.

Two O-Methyltransferases from Phylogenetically Unrelated Cow Parsley (Anthriscus sylvestris) and Hinoki-Asunaro (Thujopsis dolabrata var. hondae) as a Signature of Lineage-Specific Evolution in Lignan Biosynthesis.

O-Methyltransferases (OMTs) play important roles in antitumor lignan biosynthesis. To date, six OMTs catalyzing the methylation of dibenzylbutyrolactone lignans as biosynthetic precursors of antitumor lignans have been identified. However, there is still no systematic understanding of the diversity and regularity of the biosynthetic mechanisms among various plant lineages. Herein, we report the characterization of two OMTs from Anthriscus sylvestris and Thujopsis dolabrata var. hondae [designated as AsSecoNorYatein (SNY) OMT and TdSNYOMT] together with the six known OMTs to evaluate their diversity and regularity. Although A. sylvestris 5-O-methylthujaplicatin (SecoNorYatein) and 4-O-demethylyatein (NorYatein) OMT (AsSNYOMT) and TdSNYOMT accept 5-O-methylthujaplicatin and 4-O-demethylyatein as substrates, phylogenetic analysis indicated that these two OMTs shared low amino acid sequence identity, 33.8%, indicating a signature of parallel evolution. The OMTs and the six previously identified OMTs were found to be diverse in terms of their substrate specificity, regioselectivity and amino acid sequence identity, indicating independent evolution in each plant species. Meanwhile, two-entropy analysis detected four amino acid residues as being specifically acquired by dibenzylbutyrolactone lignan OMTs. Site-directed mutation of AsSNYOMT indicated that two of them contributed specifically to 5-O-methylthujaplicatin methylation. The results provide a new example of parallel evolution and the diversity and regularity of OMTs in plant secondary (specialized) metabolism.

Impact of Machine Learning-Associated Research Strategies on the Identification of Peptide-Receptor Interactions in the Post-Omics Era.

BACKGROUNDS: Elucidation of peptide-receptor pairs is a prerequisite for many studies in the neuroendocrine, endocrine, and neuroscience fields. Recent omics analyses have provided vast amounts of peptide and G protein-coupled receptor (GPCR) sequence data. GPCRs for homologous peptides are easily characterized based on homology searching, and the relevant peptide-GPCR interactions are also detected by typical signaling assays. In contrast, conventional evaluation or prediction methods, including high-throughput reverse-pharmacological assays and tertiary structure-based computational analyses, are not useful for identifying interactions between novel and omics-derived peptides and GPCRs. SUMMARY: Recently, an approach combining machine learning-based prediction of novel peptide-GPCR pairs and experimental validation of the predicted pairs have been shown to breakthrough this bottleneck. A machine learning method, logistic regression for human class A GPCRs and the multiple subsequent signaling assays led to the deorphanization of human class A orphan GPCRs, namely, the identification of 18 peptide-GPCR pairs. Furthermore, using another machine learning algorithm, the support vector machine (SVM), the peptide descriptor-incorporated SVM was originally developed and employed to predict GPCRs for novel peptides characterized from the closest relative of vertebrates, Ciona intestinalis Type A (Ciona robusta). Experimental validation of the predicted pairs eventually led to the identification of 11 novel peptide-GPCR pairs. Of particular interest is that these newly identified GPCRs displayed neither significant sequence similarity nor molecular phylogenetic relatedness to known GPCRs for peptides. KEY MESSAGES: These recent studies highlight the usefulness and versatility of machine learning for enabling the efficient, reliable, and systematic identification of novel peptide-GPCR interactions.

Key Amino Acids for Transferase Activity of GDSL Lipases.

The Gly-Asp-Ser-Leu (GDSL) motif of esterase/lipase family proteins (GELPs) generally exhibit esterase activity, whereas transferase activity is markedly preferred in several GELPs, including the Tanacetum cinerariifolium GDSL lipase TciGLIP, which is responsible for the biosynthesis of the natural insecticide, pyrethrin I. This transferase activity is due to the substrate affinity regulated by the protein structure and these features are expected to be conserved in transferase activity-exhibiting GELPs (tr-GELPs). In this study, we identified two amino acid residues, [N/R]208 and D484, in GELP sequence alignments as candidate key residues for the transferase activity of tr-GELPs by two-entropy analysis. Molecular phylogenetic analysis demonstrated that each tr-GELP is located in the clusters for non-tr-GELPs, and most GELPs conserve at least one of the two residues. These results suggest that the two conserved residues are required for the acquisition of transferase activity in the GELP family. Furthermore, substrate docking analyses using ColabFold-generated structure models of both natives and each of the two amino acids-mutated TciGLIPs also revealed numerous docking models for the proper access of substrates to the active site, indicating crucial roles of these residues of TciGLIP in its transferase activity. This is the first report on essential residues in tr-GELPs for the transferase activity.

The TRP channel PKD2 is involved in sensing the mechanical stimulus of adhesion for initiating metamorphosis in the chordate Ciona.

Metamorphosis is the dramatic and irreversible reconstruction of animal bodies transitioning from the larval stage. Because of the significant impact of metamorphosis on animal life, its timing is strictly regulated. Invertebrate chordate ascidians are the closest living relatives of vertebrates. Ascidians exhibit metamorphosis that converts their swimming larvae into sessile adults. Ascidian metamorphosis is triggered by a mechanical stimulus generated when adhesive papillae adhere to a substrate. However, it is not well understood how the mechanical stimulus is generated and how ascidian larvae sense the stimulus. In this study, we addressed these issues by a combination of embryological, molecular, and genetic experiments in the model ascidian Ciona intestinalis Type A, also called Ciona robusta. We here showed that the epidermal neuronal network starting from the sensory neurons at the adhesive papillae is responsible for the sensing of adhesion. We also found that the transient receptor potential (TRP) channel PKD2 is involved in sensing the stimulus of adhesion. Our results provide a better understanding of the mechanisms underlying the regulation of the timing of ascidian metamorphosis.

Mass spectrometry of short peptides reveals common features of metazoan peptidergic neurons.

The evolutionary origins of neurons remain unknown. Although recent genome data of extant early-branching animals have shown that neural genes existed in the common ancestor of animals, the physiological and genetic properties of neurons in the early evolutionary phase are still unclear. Here, we performed a mass spectrometry-based comprehensive survey of short peptides from early-branching lineages Cnidaria, Porifera and Ctenophora. We identified a number of mature ctenophore neuropeptides that are expressed in neurons associated with sensory, muscular and digestive systems. The ctenophore peptides are stored in vesicles in cell bodies and neurites, suggesting volume transmission similar to that of cnidarian and bilaterian peptidergic systems. A comparison of genetic characteristics revealed that the peptide-expressing cells of Cnidaria and Ctenophora express the vast majority of genes that have pivotal roles in maturation, secretion and degradation of neuropeptides in Bilateria. Functional analysis of neuropeptides and prediction of receptors with machine learning demonstrated peptide regulation of a wide range of target effector cells, including cells of muscular systems. The striking parallels between the peptidergic neuronal properties of Cnidaria and Bilateria and those of Ctenophora, the most basal neuron-bearing animals, suggest a common evolutionary origin of metazoan peptidergic nervous systems.

Characterization and localization of relaxin-like gonad-stimulating peptide in the crown-of-thorns starfish, Acanthaster cf. solaris.

In starfish, a relaxin-like gonad-stimulating peptide (RGP) is the gonadotropin responsible for final gamete maturation. RGP comprises two different peptides, A- and B-chains with two interchain and one intrachain disulfide bonds. The existence of two isomers of RGP in the crown-of-thorns starfish, Acanthaster planci, has been reported previously, but it was recently shown that A. planci represents a species complex with four different species. Here we elucidated the authentic sequence of the Pacific species, Acanthaster cf. solaris, RGP (Aso-RGP). The Aso-RGP precursor encoded by a 354 base pair open reading frame was composed of 117 amino acids (aa). The amino acid identity of Aso-RGP to Patiria pectinifera RGP (Ppe-RGP) and Asterias amurensis RGP (Aam-RGP) was 74% and 60%, respectively. Synthetic Aso-RGP induced spawning of ovarian fragments from A. cf. solaris. Ppe-RGP and Aam-RGP also induced spawning by A. cf. solaris ovaries. In contrast, Ppe-RGP and Aso-RGP induced spawning by P. pectinifera ovaries, but Aam-RGP was inactive. Notably, anti-Ppe-RGP antibodies recognized Aso-RGP as well as Ppe-RGP. Localization of Aso-RGP was observed immunohistochemically using anti-Ppe-RGP antibodies, showing that Aso-RGP was mainly present in the radial nerve cords of A. cf. solaris. Aso-RGP was distributed not only in the epithelium of the ectoneural region but also in the neuropile of the ectoneural region. These results suggest that Aso-RGP is synthesized in the epithelium of the ectoneural region, then transferred to fibers in the neuropile of the ectoneural region in radial nerve cords.

Evidence for a functional role of Start, a long noncoding RNA, in mouse spermatocytes.

A mouse testis-specific long noncoding RNA (lncRNA), Start, is localized in the cytosol of Leydig cells and in the nucleus of pachytene spermatocytes. We previously showed that Start regulates steroidogenesis through controlling the expression of Star and Hsd3b1 genes in Leydig cells, but its function in germ cells was not known. Here we verified that a spermatocyte-specific protease gene, Prss43/Tessp-3, was downregulated in Start-knockout testes. To investigate the transcriptional regulatory activity of Start in spermatocytes, we first performed a series of reporter gene assays using a thymidine kinase promoter in spermatocyte-derived GC-2spd(ts) cells. A 5.4-kb genome sequence encompassing Start exhibited enhancer activity for this promoter, and the activity was decreased by knockdown of Start. Deletion of the Start promoter and replacement of the Start sequence abolished the enhancer activity and, consistently, the activity was detected in further experiments only when Start was actively transcribed. We then examined whether the Prss43/Tessp-3 gene could be a target of Start. A reporter gene assay demonstrated that the 5.4-kb sequence exhibited enhancer activity for a Prss43/Tessp-3 promoter in GC-2spd(ts) cells and that the activity was significantly decreased by knockdown of Start. These results suggest that Start functions in transcriptional activation of the Prss43/Tessp-3 gene in spermatocytes. Given that Start is presumed to regulate steroidogenic genes at the posttranscriptional level in Leydig cells, the function in spermatocytes is a novel role of Start. These findings provide an insight into multifunctionality of lncRNAs in the testis.

Expression analysis of peptidergic enteroendocrine cells in the silkworm Bombyx mori.

Enteroendocrine cells (ECs) in the insect midgut respond to physiological changes in the intestine by releasing multiple peptides to control food intake, gastrointestinal activity and systemic metabolism. Here, we performed a comprehensive mapping of ECs producing different regulatory peptides in the larval midgut of Bombyx mori. In total, we identified 20 peptide genes expressed in different ECs in specific regions of the midgut. Transcript-specific in situ hybridisation combined with antibody staining revealed approximately 30 subsets of ECs, each producing a unique peptide or a combination of several different peptides. Functional significance of this diversity and specific roles of different enteroendocrine peptides are largely unknown. Results of this study highlight the importance of the midgut as a major endocrine/paracrine source of regulatory molecules in insects and provide important information to clarify functions of ECs during larval feeding and development.

Draft Genome of Tanacetum Coccineum: Genomic Comparison of Closely Related Tanacetum-Family Plants.

The plant Tanacetum coccineum (painted daisy) is closely related to Tanacetum cinerariifolium (pyrethrum daisy). However, T. cinerariifolium produces large amounts of pyrethrins, a class of natural insecticides, whereas T. coccineum produces much smaller amounts of these compounds. Thus, comparative genomic analysis is expected to contribute a great deal to investigating the differences in biological defense systems, including pyrethrin biosynthesis. Here, we elucidated the 9.4 Gb draft genome of T. coccineum, consisting of 2,836,647 scaffolds and 103,680 genes. Comparative analyses of the draft genome of T. coccineum and that of T. cinerariifolium, generated in our previous study, revealed distinct features of T. coccineum genes. While the T. coccineum genome contains more numerous ribosome-inactivating protein (RIP)-encoding genes, the number of higher-toxicity type-II RIP-encoding genes is larger in T. cinerariifolium. Furthermore, the number of histidine kinases encoded by the T. coccineum genome is smaller than that of T. cinerariifolium, suggesting a biological correlation with pyrethrin biosynthesis. Moreover, the flanking regions of pyrethrin biosynthesis-related genes are also distinct between these two plants. These results provide clues to the elucidation of species-specific biodefense systems, including the regulatory mechanisms underlying pyrethrin production.