Developmental Expression Patterns Research Articles

Evolutionary redesign of the Atlantic cod (Gadus morhua L.) Toll-like receptor repertoire by gene losses and expansions.

Genome sequencing of the teleost Atlantic cod demonstrated loss of the Major Histocompatibility Complex (MHC) class II, an extreme gene expansion of MHC class I and gene expansions and losses in the innate pattern recognition receptor (PRR) family of Toll-like receptors (TLR). In a comparative genomic setting, using an improved version of the genome, we characterize PRRs in Atlantic cod with emphasis on TLRs demonstrating the loss of TLR1/6, TLR2 and TLR5 and expansion of TLR7, TLR8, TLR9, TLR22 and TLR25. We find that Atlantic cod TLR expansions are strongly influenced by diversifying selection likely to increase the detectable ligand repertoire through neo- and subfunctionalization. Using RNAseq we find that Atlantic cod TLRs display likely tissue or developmental stage-specific expression patterns. In a broader perspective, a comprehensive vertebrate TLR phylogeny reveals that the Atlantic cod TLR repertoire is extreme with regards to losses and expansions compared to other teleosts. In addition we identify a substantial shift in TLR repertoires following the evolutionary transition from an aquatic vertebrate (fish) to a terrestrial (tetrapod) life style. Collectively, our findings provide new insight into the function and evolution of TLRs in Atlantic cod as well as the evolutionary history of vertebrate innate immunity.

Developmental and adult expression patterns of the G-protein-coupled receptor GPR88 in the rat: Establishment of a dual nuclear-cytoplasmic localization.

GPR88 is a neuronal cerebral orphan G-protein-coupled receptor (GPCR) that has been linked to various psychiatric disorders. However, no extensive description of its localization has been provided so far. Here, we investigate the spatiotemporal expression of the GPR88 in prenatal and postnatal rat tissues by using in situ hybridization and immunohistochemistry. GPR88 protein was initially detected at embryonic day 16 (E16) in the striatal primordium. From E16-E20 to adulthood, the highest expression levels of both protein and mRNA were observed in striatum, olfactory tubercle, nucleus accumbens, amygdala, and neocortex, whereas in spinal cord, pons, and medulla GPR88 expression remains discrete. We observed an intracellular redistribution of GPR88 during cortical lamination. In the cortical plate of the developing cortex, GPR88 presents a classical GPCR plasma membrane/cytoplasmic localization that shifts, on the day of birth, to nuclei of neurons progressively settling in layers V to II. This intranuclear localization remains throughout adulthood and was also detected in monkey and human cortex as well as in the amygdala and hypothalamus of rats. Apart from the central nervous system, GPR88 was transiently expressed at high levels in peripheral tissues, including adrenal cortex (E16-E21) and cochlear ganglia (E19-P3), and also at moderate levels in retina (E18-E19) and spleen (E21-P7). The description of the GPR88 anatomical expression pattern may provide precious functional insights into this novel receptor. Furthermore, the GRP88 nuclear localization suggests nonclassical GPCR modes of action of the protein that could be relevant for cortical development and psychiatric disorders. J. Comp. Neurol. 524:2776-2802, 2016. © 2016 Wiley Periodicals, Inc.

The LSD1 inhibitor RN-1 recapitulates the fetal pattern of hemoglobin synthesis in baboons (P. anubis)

Increased fetal hemoglobin levels lessen the severity of symptoms and increase the lifespan of patients with sickle cell disease. Hydroxyurea, the only drug currently approved for the treatment of sickle cell disease, is not effective in a large proportion of patients and therefore new pharmacological agents that increase fetal hemoglobin levels have long been sought. Recent studies identifying LSD-1 as a repressor of γ-globin expression led to experiments demonstrating that the LSD-1 inhibitor RN-1 increased γ-globin expression in the sickle cell mouse model. Because the arrangement and developmental stage-specific expression pattern of the β-like globin genes is highly conserved between man and baboon, the baboon model remains the best predictor of activity of fetal hemoglobin-inducing agents in man. In this report, we demonstrate that RN-1 increases γ-globin synthesis, fetal hemoglobin, and F cells to high levels in both anemic and non-anemic baboons with activity comparable to decitabine, the most potent fetal hemoglobin-inducing agent known. RN-1 not only restores high levels of fetal hemoglobin but causes the individual 5' Iγ- and 3' Vγ-globin chains to be synthesized in the ratio characteristic of fetal development. Increased fetal hemoglobin was associated with increased levels of acetylated Histone H3, H3K4Me2, H3K4Me3, and RNA polymerase II at the γ-globin gene, and diminished γ-globin promoter DNA methylation. RN-1 is likely to induce clinically relevant levels of fetal hemoglobin in patients with sickle cell disease, although careful titration of the dose may be required to minimize myelotoxicity.

Molecular and functional characterization of the vitellogenin receptor in oriental river prawn, Macrobrachium nipponense

A complementary DNA (cDNA) that encodes the vitellogenin receptor (VgR) in the oriental river prawn, Macrobrachium nipponense, was cloned using expressed sequence tag analysis and a rapid amplification of cDNA ends approach. The coding region consists of 5920 base pairs (bp) that encode a 1902 amino acid protein, with a predicted molecular mass of 209kDa. The coding region is flanked by a 45bp 5ʹ-untranslated region (UTR) and a 166bp 3ʹ-UTR. The deduced amino acid sequence of the M. nipponense VgR cDNA had typically conserved domains, such as an extracellular, lipoprotein-binding domain, epidermal growth factor-like and O-glycosylation domains, a transmembrane domain and a short C-terminal, cytosolic tail. Quantitative real-time PCR (qPCR) indicated that Mn-VgR is highly expressed in the female ovary. Expression analysis by qPCR demonstrated the larval and ovarian developmental stage-specific expression pattern. As the ovaries developed, the expression level of Mn-VgR gradually increased during the reproductive cycle (stage I), to reach a peak in stage III. Levels then dropped as a new development cycle was entered after reproduction molting. Eyestalk ablation led to a significant increase in the expression of Mn-VgR during the ovarian development stages (P<0.05), when compared with the eyestalk-intact group. The investigation revealed that eyestalk ablation initially affected Mn-VgR expression and then influenced vitellogenesis. In adult females, VgR RNA interference (RNAi) dramatically delayed the maturation of the ovary, in accordance with the gonad somatic index. In addition, Mn-VgR RNAi led to vitellin depletion in the oocytes and the accumulation of vitellin in the hepatopancreas.

Dysregulation of sirtuins and key metabolic genes in skeletal muscle of pigs with spontaneous intrauterine growth restriction is associated with alterations of circulating IGF-1

Prenatal and early postnatal life determines future health, and intrauterine growth restriction (IUGR) – associated low birth weight predisposes to metabolic syndrome in adulthood. We hypothesize here that IUGR might induce hormonal and gene expression alterations predisposing to metabolic disease.Using a porcine model of spontaneous IUGR, we determined in utero (71, 112days post-conception) and early-postnatal (2days post-birth) IGF-1, insulin and leptin levels, and in parallel we investigated, in skeletal muscle, the developmental expression patterns of sirtuins and metabolic and signaling genes IRS1, GLUT4, HK2 and GAPDH.IUGR was associated with impaired IGF-1 plasmatic levels. Gene expression of sirtuin 1, 5, 6, 7, GLUT4 and HK2 exhibited significant correlations with gestational age or body weight. SIRT1 and HK2 expression displayed an age- and weight-dependent downregulation in controls, which was lost in IUGR pigs. Conversely, SIRT2 and GLUT4 were upregulated in IUGR pigs. Within the set of genes studied, we found a significant correlation between IGF-1 levels and gene expression in control, but not IUGR samples, indicating that lower IGF-1 may be a limiting factor in IUGR. IUGR-dependent gene alterations were partly linked to epigenetic changes on histone H3 acetylation and methylation.Overall, our data indicate that several sirtuins and metabolic genes display specific gene expression trajectories during fetal and early postnatal life. Gene expression alterations observed in IUGR are correlated to IGF-1 dysregulation. Given the importance of the genes studied in metabolic control, their perinatal alterations might contribute to the predisposition to metabolic disease of adulthood.

Gene expression and enzyme activity of lipoprotein lipase correlate with intramuscular fat content in Guangxi san-huang and Arbor Acres chickens.

Lipoprotein lipase (LPL) is a key enzyme in lipid metabolism. This study investigated LPL gene expression, LPL enzyme activity, and the correlation of each with intramuscular fat (IMF) in Chinese Guangxi san-huang (GXSH) and Arbor Acres (AA) chickens. The results showed that age and breed had significant effects on LPL expression and enzyme activity. Correlation analyses showed significant positive correlations between LPL expression levels and IMF contents in the breast and thigh tissues of both GXSH (r = 0.712, P = 0.001; r = 0.792, P < 0.001, respectively) and AA (r = 0.644, P < 0.001; r = 0.545, P < 0.001, respectively) chickens. The results also indicated a significant positive correlation between LPL enzyme activity and IMF contents in the breast and thigh tissues of both GXSH (r = 0.615, P = 0.001; r = 0.685, P < 0.001, respectively) and AA (r = 0.600, P = 0.001; r = 0.528, P = 0.003, respectively) chickens. The results indicated that the LPL gene was significantly correlated with IMF in these two breeds. The results presented here could contribute to knowledge of LPL mRNA developmental expression patterns and enzyme activity, and it could facilitate further research on the molecular mechanisms underlying IMF deposition in chickens.

Evolution of brain elaboration.

Large, complex brains have evolved independently in several lineages of protostomes and deuterostomes. Sensory centres in the brain increase in size and complexity in proportion to the importance of a particular sensory modality, yet often share circuit architecture because of constraints in processing sensory inputs. The selective pressures driving enlargement of higher, integrative brain centres has been more difficult to determine, and may differ across taxa. The capacity for flexible, innovative behaviours, including learning and memory and other cognitive abilities, is commonly observed in animals with large higher brain centres. Other factors, such as social grouping and interaction, appear to be important in a more limited range of taxa, while the importance of spatial learning may be a common feature in insects with large higher brain centres. Despite differences in the exact behaviours under selection, evolutionary increases in brain size tend to derive from common modifications in development and generate common architectural features, even when comparing widely divergent groups such as vertebrates and insects. These similarities may in part be influenced by the deep homology of the brains of all Bilateria, in which shared patterns of developmental gene expression give rise to positionally, and perhaps functionally, homologous domains. Other shared modifications of development appear to be the result of homoplasy, such as the repeated, independent expansion of neuroblast numbers through changes in genes regulating cell division. The common features of large brains in so many groups of animals suggest that given their common ancestry, a limited set of mechanisms exist for increasing structural and functional diversity, resulting in many instances of homoplasy in bilaterian nervous systems.

Differential developmental competence and gene expression patterns in buffalo (Bubalus bubalis) nuclear transfer embryos reconstructed with fetal fibroblasts and amnion mesenchymal stem cells

The developmental ability and gene expression pattern at 8- to 16-cell and blastocyst stages of buffalo (Bubalus bubalis) nuclear transfer (NT) embryos from fetal fibroblasts (FFs), amnion mesenchymal stem cells (AMSCs) and in vitro fertilized (IVF) embryos were compared in the present studies. The in vitro expanded buffalo FFs showed a typical "S" shape growth curve with a doubling time of 41.4h and stained positive for vimentin. The in vitro cultured undifferentiated AMSCs showed a doubling time of 39.5h and stained positive for alkaline phosphatase, and these cells also showed expression of pluripotency markers (OCT 4, SOX 2, NANOG), and mesenchymal stem cell markers (CD29, CD44) and were negative for haematopoietic marker (CD34) genes at different passages. Further, when AMSCs were exposed to corresponding induction conditions, these cells differentiated into adipogenic, chondrogenic and osteogenic lineages which were confirmed through oil red O, alcian blue and alizarin staining, respectively. Donor cells at 3-4 passage were employed for NT. The cleavage rate was significantly (P<0.05) higher in IVF than in FF-NT and AMSC-NT embryos (82.6±8.2 vs. 64.6±1.3 and 72.3±2.2%, respectively). However, blastocyst rates in IVF and AMSC-NT embryos (30.6±2.7 and 28.9±3.1%) did not differ and were significantly (P<0.05) higher than FF-NT (19.5±1.8%). Total cell number did not show significant (P>0.05) differences between IVF and AMSC-NT embryos (186.7±4.2, 171.2±3.8, respectively) but were significantly (P<0.05) higher than that from FF-NT (151.3±4.1). Alterations in the expression pattern of genes implicated in transcription and pluripotency (OCT4, STAT3, NANOG), DNA methylation (DNMT1, DNMT3A), histone deacetylation (HDAC2), growth factor signaling and imprinting (IGF2, IGF2R), apoptosis (BAX, BCL2), metabolism (GLUT1) and oxidative stress (MnSOD) regulation were observed in cloned embryos. The transcripts or expression patterns in AMSC-NT embryos more closely followed that of the in vitro derived embryos compared with FF-NT embryos. The results demonstrate that multipotent amnion MSCs have a greater potential as donor cells than FFs in achieving enhanced production of cloned buffalo embryos.

The LSD1 Inhibitor RN-1 Recapitulates the Fetal Pattern of Hemoglobin Synthesis in Anemic and Normal, Non-Anemic Baboons

Increased fetal hemoglobin (HbF) levels lessen the severity of symptoms and increase the lifespan of patients with sickle cell disease (SCD). Hydroxyurea (HU), the only drug approved for the treatment of sickle cell disease, increases HbF levels, reduces pain crises, and increases the lifespan of patients. Because HU is not effective in a large proportion of patients, new pharmacological agents that increase HbF levels have long been sought. Recent studies identifying LSD-1 as a repressor of γ-globin expression led to experiments demonstrating that the LSD-1 inhibitor RN-1 increased γ-globin expression in SCD mice. As the arrangement and developmental stage-specific expression pattern of the β-like globin genes is highly conserved between man and baboon, the baboon remains the best predictor of the activity of HbF-inducing agents in man. Therefore, experiments were performed to test the effect of RN-1 on HbF in anemic baboons. Anemia (Hct=20) was induced by repeated phlebotomies for 14 days prior to administration of RN-1. Five anemic baboons were treated with varying doses of RN-1(0.125-2.5 mg/kg/d; 5d; sc). Dose-dependent increases in HbF, F cells, F retics, and γ-globin chain synthesis in reticulocytes were associated with decreased neutrophils and increased monocytes. Globin chain synthesis analysis following RN-1 treatment showed that the 5' Iγ and 3' Vγ-globin genes were expressed in the ratio characteristic of fetal development (5'Iγ/3'Vγ>2) rather than that induced in adults by erythropoietic stress (5'Iγ/3'Vγ<0.5) or decitabine treatment where changes in the Iγ/Vγ ratio are observed but the fetal ratio is not attained. RT-PCR analysis of pre-and post-treatment BM showed that RN-1 increased γ-globin mRNA nearly 5 fold (p<0.05) with no effect on ε-globin mRNA. ChIP analysis of pre- and post-treatment BM erythroid cells showed increased levels of pol II, H3K9ac, H3K4me2 and H3K4me3 associated with the γ-globin but not with the ε- or β-globin promoter and IVS II regions consistent with increased γ-globin transcription. Levels of H3K9me2 were increased at the β-globin promoter and IVSII regions. Bisulfite sequence analysis showed a small decrease in level of DNA methylation of the γ-globin promoter in post-treatment (0.68+0.07% total cytosine) compared to pre-treatment (0.79+0.05%; p<0.05). To investigate whether induction of γ-globin expression was dependent on erythropoietic stress, four normal, non-anemic baboons were treated with varying doses and schedules of RN-1 (0.2-0.5mg/kg/d; 5d/wk; 1-10wks; sc). The effect of RN-1 was measured by analysis of F cells and globin chain synthesis in peripheral blood. The results (Table) show that RN-1 increases γ-globin expression and F cells in normal, non-anemic baboons in a dose-dependent manner. Analysis of globin chain synthesis showed predominant synthesis of the Iγ-globin chain with an Iγ/Vγ ratio exceeding the fetal ratio in all individuals.Effect of RN-1 in Normal, Non-Anemic Baboons TableAnimalRN-1 Dose (mg/kg/d)ScheduleGlobin chain synthesis (γ/γ+β)Iγ/Vγ chain synthesisF cells (%)ANC (/μl)Plt(X103/μl)Mono(%)PrePostPrePostNadirNadirPeak85490.55d0.030.714.63NDND191016921.685490.255d0.010.264.47NDND191021914.586980.255d/wk/2 wks0.010.2020.12.09.21210687.986960.2-0.255d/wk/10 wksND0.106.633.526.81440646.1Neutropenia following RN-1 treatment of anemic baboons (mean ANC nadir=746±156) was reduced in non-anemic animals (2 of 4 baboons >1500; mean ANC nadir=1617±350; p<0.02). Increased monocytes and decreased platelet counts were observed. Differences between anemic and non-anemic baboons may reflect perturbation of hematopoietic differentiation by phlebotomy. We conclude that RN-1 is a powerful in vivo inducer of HbF in both anemic and non-anemic baboons that preferentially increases synthesis of the 5' Iγ-globin gene and recapitulates the fetal pattern of hemoglobin synthesis. Our data predicts that RN-1 treatment will induce clinically relevant levels of HbF in SCD patients, although careful titration of dose may be required to minimize effects on hematopoiesis. DisclosuresNo relevant conflicts of interest to declare.

Developmental Expression Patterns of KCC2 and Functionally Associated Molecules in the Human Brain.

Work on rodents demonstrated that steep upregulation of KCC2, a neuron-specific Cl- extruder of cation-chloride cotransporter (CCC) family, commences in supraspinal structures at around birth, leading to establishment of hyperpolarizing GABAergic responses. We describe spatiotemporal expression profiles of the entire CCC family in human brain. KCC2 mRNA was observed already at 10th postconceptional week (PCW) in amygdala, cerebellum, and thalamus. KCC2-immunoreactive (KCC2-ir) neurons were abundant in subplate at 18 PCW. By 25 PCW, numerous subplate and cortical plate neurons became KCC2-ir. The mRNA expression profiles of α- and β-isoforms of Na-K ATPase, which fuels cation-chloride cotransport, as well of tropomyosin receptor kinase B (TrkB), which promotes developmental upregulation of KCC2, were consistent with data from studies on rodents about their interactions with KCC2. Thus, in human brain, expression of KCC2 and its functionally associated proteins begins in early fetal period. Our work facilitates translation of results on CCC functions from animal studies to human and refutes the view that poor efficacy of anticonvulsants in the term human neonate is attributable to the lack of KCC2. We propose that perinatally low threshold for activation of Ca2+-dependent protease calpain renders neonates susceptible to downregulation of KCC2 by traumatic events, such as perinatal hypoxia ischemia.

Expression and Function of mARC: Roles in Lipogenesis and Metabolic Activation of Ximelagatran.

Recently two novel enzymes were identified in the outer mitochondrial membrane, mARC1 and mARC2. These molybdenum containing enzymes can reduce a variety of N-hydroxylated compounds, such as N-hydroxy-guanidines and sulfohydroxamic acids, as well as convert nitrite into nitric oxide (NO). However, their endogenous functions remain unknown. Here we demonstrate a specific developmental pattern of expression of these enzymes. mARC1, but not mARC2, was found to be expressed in fetal human liver, whereas both, in particular mARC2, are abundant in adult liver and also expressed in omental and subcutaneous fat. Caloric diet restriction of obese patients caused a decreased expression of mARC2 in liver, similar to that seen in the livers of starved rats. Knock down of mARC2 expression by siRNA in murine adipocytes had statistically significant effect on the level of diglycerides and on the fatty acid composition of some triglycerides, concomitantly a clear trend toward the reduced formation of most of triglyceride and phospholipid species was observed. The involvement of mARC2 in the metabolism of the hepatotoxic drug ximelagatran was evaluated in hepatocytes and adipocytes. Ximelagatran was shown to cause oxidative stress and knock down of mARC2 in adipocytes prevented ximelagatran induced inhibition of mitochondrial respiration. In conclusion, our data indicate that mARC1 and mARC2 have different developmental expression profiles, and that mARC2 is involved in lipogenesis, is regulated by nutritional status and responsible for activation of ximelagatran into a mitotoxic metabolite(s).

Expression of myostatin in the spotted rose snapper Lutjanus guttatus during larval and juvenile development under cultured conditions.

In this study, the developmental expression pattern of myostatin (mstn) in the spotted rose snapper Lutjanus guttatus under culture conditions is presented. The full coding sequence of mstn from L. guttatus was isolated from muscle tissue, obtaining 1134 nucleotides which encode a peptide of 377 amino acids. The phylogenetic analysis indicated that this sequence corresponds to mstn-1. mstn expression was detected in embryonic stages, and maintained at low levels until 28 days post-hatch, when it showed a significant increase, coinciding with the onset of metamorphosis. After that, expression was fluctuating, coinciding probably with periods of rapid and slow muscle growth or individual growth rates. mstn expression was also analysed by body mass with higher levels detected in smaller animals, irrespective of age. mstn was also expressed in other tissues from L. guttatus, presenting higher levels in brain, eye and gill. In brain for instance, two variants of mstn were isolated, both coding sequences were identical to muscle, except that one of them contained a 75 nucleotide deletion in exon 1, maintaining the reading frame but deleting two conserved cysteine residues. Phylogenetic analysis indicated that this brain variant was also mstn-1. The function of this variant is not clear and needs further investigation. These results indicate that mstn-1 participates in different physiological processes other than muscle growth in fishes.

Identification of bolting-related microRNAs and their targets reveals complex miRNA-mediated flowering-time regulatory networks in radish (Raphanus sativus L.).

MicroRNAs (miRNAs) play vital regulatory roles in plant growth and development. The phase transition from vegetative growth to flowering is crucial in the life cycle of plants. To date, miRNA-mediated flowering regulatory networks remain largely unexplored in radish. In this study, two small RNA libraries from radish leaves at vegetative and reproductive stages were constructed and sequenced by Solexa sequencing. A total of 94 known miRNAs representing 21 conserved and 13 non-conserved miRNA families, and 44 potential novel miRNAs, were identified from the two libraries. In addition, 42 known and 17 novel miRNAs were significantly differentially expressed and identified as bolting-related miRNAs. RT-qPCR analysis revealed that some miRNAs exhibited tissue- or developmental stage-specific expression patterns. Moreover, 154 target transcripts were identified for 50 bolting-related miRNAs, which were predominately involved in plant development, signal transduction and transcriptional regulation. Based on the characterization of bolting-related miRNAs and their target genes, a putative schematic model of miRNA-mediated bolting and flowering regulatory network was proposed. These results could provide insights into bolting and flowering regulatory networks in radish, and facilitate dissecting the molecular mechanisms underlying bolting and flowering time regulation in vegetable crops.

Neuropeptide Arginine Vasotocin Positively Affects Neurosteroidogenesis in the Early Brain of Grouper, Epinephelus coioides.

The neuropeptide arginine vasotocin (AVT) has versatile physiological functions in non-mammalian vertebrates. However, the functional association between AVT and neurosteroidogenesis in the early brain of teleosts remains elusive. We thus studied the developmental expression patterns of the avt gene and their V1 type receptor (avt-rv1 ) at various stages of development [90-150 days after hatching (dah)] in relation to neurosteroidogenesis and oestrogen signalling in the early brain of the orange-spotted grouper (Epinephelus coioides). avt and avt-rv1 mRNAs displayed a significantly increase in expression at 110 dah in the telencephalon and diencephalon. Further, avt mRNAs were localised in three magnocellular neuronal populations of the preoptic area, such as parvocellular, magnocellular and gigantocellular preoptic neurones. Intriguingly, the avt transcripts in those neurones were more abundant in 110 dah compared to other ages. Subsequently, dual fluorescence in situ hybridisation analysis showed that the avt and avt-rv1 genes were highly coexpressed with cyp11a1, hsd3b1, cyp17a1, erα, erβ and gpr30, which indicates their potential for functional association. Cyp19a1b-immunoreactive positive fibres were found in close proximity to avt-expressing neurones. Moreover, our results showed that exogenous Avt caused a significant increase in the cellular and gene levels of steroidogenic enzymes and oestrogen receptors (ers), whereas the administration of an Avt-rv1 antagonist caused a decrease in the expression of both steroidogenic enzymes and ers genes in the brain. Furthermore, exogenous oestradiol (E2 ) strongly up-regulated avt mRNAs in the grouper brain. Taken together, the present studies suggest that avt and steroidogenesis may positively work together to increase both E2 biosynthesis and early brain development.

The Essential Role of Vitellogenin Receptor in Ovary Development and Vitellogenin Uptake in Bactrocera dorsalis (Hendel)

The vitellogenin receptor (VgR) functions as an essential component in uptaking and transporting vitellogenin (Vg) in female adults, which is involved in ovary development and oviposition. This study aimed to clarify the molecular characteristics and function of VgR in the oriental fruit fly Bactrocera dorsalis (Hendel). Here, we identified the full-length of BdVgR (GenBank Accession No. JX469118), encoding a 1925 residue (aa) protein with a 214.72 kDa molecular mass and several typical motifs of low-density lipoprotein receptor superfamily (LDLR). Phylogenic analysis suggested that BdVgR was evolutionary conserved with other Dipteran VgRs. The expression of BdVgR was exclusively detected in the ovaries rather than head, thorax or other tissues. The developmental expression patterns showed that the signal of BdVgR was detectable in very beginning of adult stage, and positively correlated with the growth rate of ovaries and the expression levels of its ligands. In addition, we also demonstrated that the expression level of BdVgR, and ovary development were significantly suppressed after being injected with BdVgR-targeted dsRNA. Together, all of these results indicated that BdVgR was critical for yolk protein absorption and ovary maturation in B. dorsalis, playing a vital role in female reproduction.

Imaging early stages of the female reproductive structure of Arabidopsis by confocal laser scanning microscopy.

The gynoecium is the female reproductive structure and probably the most complex plant structure. During its development, different internal tissues and structures are formed. Insights in gene expression or hormone localization patterns are key to understanding gynoecium development from a molecular biology point of view. Imaging with a confocal laser scanning microscope (CLSM) is a widely used strategy; however, visualization of internal developmental expression patterns in the Arabidopsis gynoecium can be technically challenging. Here, we present a detailed protocol that allows the visualization of internal expression patterns at high resolution during gynoecium development. We demonstrate the applicability using a cytokinin response marker (TCS::GFP), an auxin response marker (DR5::VENUS), and a SEPALLATA3 marker (SEP3::SEP3:GFP). The detailed protocol presented here allows the visualization of fluorescence signals in internal structures during Arabidopsis gynoecium development. This protocol may also be adapted for imaging other challenging plant structures or organs.

The multicopper oxidase gene family in the brown planthopper, Nilaparvata lugens

The multicopper oxidase (MCO) family of enzymes includes laccases, ascorbate oxidases, bilirubin oxidases and a subgroup of metal oxidases. On the basis of a bioinformatics investigation, we identified 7 genes encoding putative multicopper oxidase proteins in the genome of the brown planthopper (BPH), Nilaparvata lugens (Hemiptera: Delphacidae). MCO1 and MCO2 are conserved, while others diverse in insects. Analysis of developmental and tissue-specific expression patterns revealed the following: NlMCO2 was mainly expressed in the integument, and its expression peaked periodically during molting; NlMCO3 was an ovary-specific MCO gene with a high expression level only at the adult stage; NlMCO4 was a salivary gland-specific MCO gene that was expressed at all developmental stages; NlMCO5 only had short-term expression in the middle of the fourth instar stage and was expressed mainly in the gut; NlMCO6 had a developmental expression pattern similar to that of NlMCO2 and was expressed in most N. lugens tissues; and NlMCO1 was expressed in most N. lugens tissues except for the testis, whereas NlMCO7 was mainly expressed in the gut and the Malpighian tube. BPHs injected with double-stranded RNA (dsRNA) targeting NlMCO2 failed to pigment and sclerotize, were colorless and soft-bodied and subsequently died in a short time. Lethal phenotypes were also observed in insects challenged by dsRNA targeting NlMCO6. However, no observable morphological or internal structural abnormality was obtained in the insects treated with dsRNA for NlMCO1, NlMCO3, NlMCO4, NlMCO5 or NlMCO7.

Expression of insulin-like factor 3 hormone-receptor system in the reproductive organs of male goats.

Relaxin-like factor (RLF), generally known as insulin-like factor 3 (INSL3), is essential for testis descent during fetal development. However, its role in adult males is not fully understood. We investigate the function of INSL3 in male Saanen goats by identifying cell types expressing its receptor, relaxin/insulin-like family peptide receptor (RXFP)2 and by characterizing the developmental expression pattern of INSL3 and RXFP2 and the binding of INSL3 to target cells in the male reproductive system. A highly specific RXFP2 antibody that co-localizes with an anti-FLAG antibody in HEK-293 cells recognizes RXFP2-transcript-expressing cells in the testis. INSL3 and RXFP2 mRNA expression is upregulated in the testis, starting from puberty. INSL3 mRNA and protein expression has been detected in Leydig cells, whereas RXFP2 mRNA and protein localize to Leydig cells, to meiotic and post-meiotic germ cells and to the epithelium and smooth muscle of the cauda epididymis and vas deferens. INSL3 binds to all of these tissues and cell types, with the exception of Leydig cells, in a hormone-specific and saturable manner. These results provide evidence for a functional intra- and extra-testicular INSL3 ligand-receptor system in adult male goats.

Two variants of selenium-dependent glutathione peroxidase from the disk abalone Haliotis discus discus: Molecular characterization and immune responses to bacterial and viral stresses.

Glutathione peroxidase (GPx) is an essential member of the antioxidant systems of living organisms and may be involved in immune defense against pathogenic invasion. In the current study, two selenium-dependent glutathione peroxidases (AbSeGPxs) that shared 54.3% identity were identified from the disk abalone Haliotis discus discus. The open reading frames (ORFs) of AbSeGPx-a and AbSeGPx-b coded for 222 and 220 amino acids, respectively, with a characteristic selenocysteine residue encoded by an opal stop codon (TGA). The conserved selenocysteine insertion sequence (SECIS) element was predicted in the 3' untranslated region (UTR) of both isoforms, and they were found to form two stem-loop structures. Amino acid comparison and phylogenetic studies revealed that the AbSeGPxs were closely related to those in other mollusk species and were evolutionarily distinct from those of other taxonomic groups. The SYBR Green qPCR was employed in investigating the transcripts of AbSeGPxs. The expression of AbSeGPxs mRNA was examined in different embryonic developmental stages and differential expression patterns for AbSeGPx-a and AbSeGPx-b were noted. Meanwhile, the highest expression of AbSeGPxs was detected in the hepatopancreas of healthy adult animals. Next, transcriptional levels were profiled in hemocytes of adults to determine the immune responses of AbSeGPxs to microbial infections. The results revealed the significant up-regulation of AbSeGPx-a in a time-dependent manner after bacterial (Listeria monocytogenes and Vibrio parahaemolyticus) and viral (viral hemorrhagic septicemia virus) infections. Consequently, these findings indicate that AbSeGPx-a and AbSeGPx-b might be involved in the embryonic development of disk abalone and the regulation of immune defense system of adult animals.

Messenger ribonucleic acid expression of KiSS-1 and serum level of kisspeptin in rat at different developmental stages

Purpose KiSS-1 and its product, kisspeptin is necessary for pubertal onset and proper adult gonadal function due to its stimulatory effect on the secretion of gonadotropinreleasing hormone (GnRH). Although the pathophysiological importance of KiSS-1 and kisspeptin is well known, the developmental patterns of expression of KiSS-1 genes and serum level of kisspeptin have not been explored to date. We report herein the expression profile of KiSS-1 genes and serum level of kisspeptin in the rat at different developmental stages.