The Parapineal Is Incorporated into the Habenula during Ontogenesis in the Medaka Fish

2015 ◽  
Vol 85 (4) ◽  
pp. 257-270 ◽  
Author(s):  
Yuji Ishikawa ◽  
Keiji Inohaya ◽  
Naoyuki Yamamoto ◽  
Kouichi Maruyama ◽  
Masami Yoshimoto ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Developmental Stages ◽  
Medaka Fish ◽  
Interpeduncular Nucleus ◽  
Fasciculus Retroflexus ◽  
Roof Plate ◽  
Green Fluorescent ◽  
Rostral Region ◽  
The Right ◽  
Early Developmental

The parapineal is present in many teleost families, while it is absent in several others. To find out why the parapineal is absent at adult stages in the latter families, the development of the epithalamus was examined in the medaka fish (Oryzias latipes). For this purpose, a green fluorescent protein-transgenic medaka line, in which the pineal complex (pineal and parapineal) is visible fluorescently, was used. We found that a distinct parapineal was present in the roof plate at early developmental stages. Subsequently, however, the parapineal and the associated roof plate began to be incorporated into the habenula between embryonic stages 28 and 29. Between embryonic stages 29 and 30, the entire parapineal was incorporated into the habenula. That is, the parapineal became a small caudomedial region (termed the ‘parapineal domain') within the left habenula in the majority of embryos, resulting in the left-sided asymmetry of the epithalamus. Thereby the left habenula became larger and more complex than its right counterpart. In the minority of embryos, the parapineal was incorporated into the right habenula or into the habenulae on both sides. In the majority of embryos, the parapineal domain projected a fiber bundle to a subnucleus (termed the ‘rostromedial subnucleus') in the left habenula. The rostromedial subnucleus sent axons, through the left fasciculus retroflexus, to the rostral region of the left half of the interpeduncular nucleus. We further found that the ratio of the left-sided phenotype was temperature dependent and decreased in embryos raised at a high temperature. The present study is the first demonstration that the supposed lack of a distinct parapineal in adult teleost fishes is due to ontogenetic incorporation into the habenula.

scholarly journals RNAi-Mediated Knockdown of Imaginal Disc Growth Factors (IDGFs) Genes Causes Developmental Malformation and Mortality in Melon Fly, Zeugodacus cucurbitae

2021 ◽  
Vol 12 ◽  
Author(s):  
Shakil Ahmad ◽  
Momana Jamil ◽  
Muhammad Fahim ◽  
Shujing Zhang ◽  
Farman Ullah ◽  
...  
Keyword(s):  
Growth Factors ◽  
Imaginal Disc ◽  
Fluorescent Protein ◽  
Developmental Stages ◽  
Small Body ◽  
Functional Characterization ◽  
Oral Feeding ◽  
Developmental Defects ◽  
Melon Fly ◽  
Green Fluorescent

This study reports the first successful use of oral feeding dsRNA technique for functional characterization of imaginal disc growth factors (IDGFs) genes (IDGF1, IDGF3_1, IDGF4_0, IDGF4_1, and IDGF6) in melon fly Zeugodacus cucurbitae. Phylogenetic and domain analysis indicates that these genes had high similarity with other Tephritidae fruit flies homolog and contain only one conserved domain among these five genes, which is glyco-18 domain (glyco-hydro-18 domain). Gene expression analysis at different developmental stages revealed that these genes were expressed at larval, pupal, and adult stages. To understand their role in different developmental stages, larvae were fed dsRNA-corresponding to each of the five IDGFs, in an artificial diet. RNAi-mediated knockdown of IDGF1 shows no phenotypic effects but caused mortality (10.4%), while IDGF4_0 caused malformed pharate at the adult stage where insects failed to shed their old cuticle and remained attached with their body, highest mortality (49.2%) was recorded compared to dsRNA-green fluorescent protein (GFP) or DEPC. Silencing of IDGF3_1 and IDGF4_1 cause lethal phenotype in larvae, (17.2%) and (40%) mortality was indexed in Z. cucurbitae. IDGF6 was mainly expressed in pupae and adult stages, and its silencing caused a malformation in adult wings. The developmental defects such as malformation in wings, larval–larval lethality, pupal–adult malformation, and small body size show that IDGFs are key developmental genes in the melon fly. Our results provide a baseline for the melon fly management and understanding of IDGFs specific functions in Z. cucurbitae.

scholarly journals Effect of 5-lipoxygenase on the development of pulmonary hypertension in rats

2004 ◽  
Vol 286 (5) ◽  
pp. H1775-H1784 ◽  
Author(s):  
John E. Jones ◽  
Jennifer L. Walker ◽  
Yanli Song ◽  
Norbert Weiss ◽  
Wellington V. Cardoso ◽  
...  
Keyword(s):  
Pulmonary Hypertension ◽  
Fluorescent Protein ◽  
Recombinant Adenovirus ◽  
Critical Role ◽  
Vascular Endothelial Cell ◽  
Weight Ratio ◽  
Right Heart Catheterization ◽  
Heart Catheterization ◽  
Green Fluorescent ◽  
The Right

5-Lipoxygenase (5-LO) and its downstream leukotriene products have been implicated in the development of pulmonary hypertension. In this study, we examined the effects of 5-LO overexpression in rat lungs on pulmonary hypertension using a recombinant adenovirus expressing 5-LO (Ad5-LO). Transthoracic echocardiography and right heart catheterization data showed that 5-LO overexpression in the lung did not cause pulmonary hypertension in normal rats; however, it markedly accelerated the progression of pulmonary hypertension in rats treated with monocrotaline (MCT). An increase in pulmonary artery pressure occurred earlier in the rats treated with MCT + Ad5-LO (7–10 days) compared with those treated with control vector, MCT + adenovirus expressing green fluorescent protein (AdGFP), or MCT alone (15–18 days). The weight ratio of the right ventricle to left ventricle plus septum was higher in the MCT + Ad5-LO group than that of the MCT + AdGFP or MCT group (0.45 ± 0.08 vs. 0.35 ± 0.03 or 0.33 ± 0.06). Lung tissue histological sections from MCT + Ad5-LO rats exhibited more severe inflammatory cell infiltration and pulmonary vascular muscularization than those from MCT + AdGFP- or MCT-treated rats. Administration of 5-LO inhibitors, zileuton or MK-886, to either MCT- or MCT + Ad5-LO-treated rats prevented the development of pulmonary hypertension. These data suggest that 5-LO plays a critical role in the progression of pulmonary hypertension in rats and that the detrimental effect of 5-LO is manifest only in the setting of pulmonary vascular endothelial cell dysfunction.

scholarly journals Nanoluciferase-Based Method for Detecting Gene Expression in Caenorhabditis elegans

Genetics ◽  
2019 ◽  
Vol 213 (4) ◽  
pp. 1197-1207 ◽  
Author(s):  
Ivana Sfarcic ◽  
Theresa Bui ◽  
Erin C. Daniels ◽  
Emily R. Troemel
Keyword(s):  
Gene Expression ◽  
Caenorhabditis Elegans ◽  
Promoter Activity ◽  
Fluorescent Protein ◽  
Developmental Stages ◽  
Fluorescent Reporters ◽  
C Elegans ◽  
Link Type ◽  
Highly Sensitive ◽  
Green Fluorescent

Genetic reporters such as the green fluorescent protein (GFP) can facilitate measurement of promoter activity and gene expression. However, animal autofluorescence limits the sensitivity of GFP and other fluorescent reporters in whole-animal settings like in the nematode Caenorhabditis elegans. Here, we present a highly sensitive Nanoluciferase (NanoLuc)-based method in a multiwell format to detect constitutive and inducible gene expression in C. elegans. We optimize detection of bioluminescent signals from NanoLuc in C. elegans and show that it can be detected at 400,000-fold over background in a population of 100 animals expressing intestinal NanoLuc driven by the vha-6 promoter. We can reliably detect signal in single vha-6p::Nanoluc-expressing worms from all developmental stages. Furthermore, we can detect signal from a 1/100 dilution of lysate from a single vha-6p::Nanoluc-expressing adult and from a single vha-6p::Nanoluc-expressing adult “hidden” in a pool of 5000 N2 wild-type animals. We also optimize various steps of this protocol, which involves a lysis step that can be performed in minutes. As a proof-of-concept, we used NanoLuc to monitor the promoter activity of the pals-5 stress/immune reporter and were able to measure 300- and 50-fold increased NanoLuc activity after proteasome blockade and infection with microsporidia, respectively. Altogether, these results indicate that NanoLuc provides a highly sensitive genetic reporter for rapidly monitoring whole-animal gene expression in C. elegans.

scholarly journals Habenular Kiss1 Neurons Modulate the Serotonergic System in the Brain of Zebrafish

Endocrinology ◽  
2012 ◽  
Vol 153 (5) ◽  
pp. 2398-2407 ◽  
Author(s):  
Satoshi Ogawa ◽  
Kai We Ng ◽  
Priveena Nair Ramadasan ◽  
Fatima Megala Nathan ◽  
Ishwar S. Parhar
Keyword(s):  
Fluorescent Protein ◽  
Serotonergic System ◽  
Raphe Nuclei ◽  
Transgenic Zebrafish ◽  
Mrna Levels ◽  
Interpeduncular Nucleus ◽  
Hypothalamic Nuclei ◽  
Green Fluorescent ◽  
Raphé Nuclei ◽  
The Brain

The Kiss1/KISS1 gene has recently been implicated as a potent hypothalamic regulator of reproductive functions, in particular, the onset of puberty in mammals. In zebrafish (Danio rerio), there are two kiss1 homologues (kiss1 and kiss2) expressed in the brain: Kiss2-expressing neurons in the hypothalamic nuclei are considered potent regulators of reproduction, whereas the role of Kiss1-expressing neurons in the habenula remains unknown. We first analyzed the expression of kiss1 mRNA in a transgenic zebrafish, in which the habenula-interpeduncular nucleus (IPN) pathway is labelled with green fluorescent protein, and our application of a biocytin neural tracer into the habenula showed the presence of neuronal projections of Kiss1 neurons to the ventral IPN. Therefore, we speculated that kiss1 neurons might regulate the serotonergic system in the raphe. However, laser microdissection followed by real-time PCR revealed the expression of Kiss1 receptor (kissr1) mRNA in the habenula and the ventral IPN but not in the dorsal IPN or the serotonergic neurons in the raphe nuclei. Dual-fluorescent in situ hybridization revealed the coexpression of kiss1 and kissr1 mRNA in the habenula. Administration of Kiss1 significantly decreased the level of kiss1 mRNA (0.3- to 0.5-fold, P < 0.001), but the level of c-fos mRNA was increased (∼3-fold, P < 0.05) in the ventral habenula, suggesting that there is autocrine regulation of the kiss1 gene. Kiss1 administration significantly increased the c-fos mRNA levels in the raphe nuclei (2.5-fold, P < 0.001) and genes involved in the regulation of serotonin levels (pet1 and slc6a4a; 3.3- and 2.2-fold, P < 0.01). These findings suggest that the autocrine-regulated habenular Kiss1 neurons indirectly regulate the serotonergic system in the raphe nuclei through the IPN in the zebrafish.

scholarly journals Plasma Cell Ontogeny Defined by Quantitative Changes in Blimp-1 Expression

2004 ◽  
Vol 200 (8) ◽  
pp. 967-977 ◽  
Author(s):  
Axel Kallies ◽  
Jhagvaral Hasbold ◽  
David M. Tarlinton ◽  
Wendy Dietrich ◽  
Lynn M. Corcoran ◽  
...  
Keyword(s):  
Plasma Cell ◽  
B Lymphocyte ◽  
Plasma Cells ◽  
Fluorescent Protein ◽  
Developmental Stages ◽  
Maturation Protein ◽  
Quantitative Changes ◽  
Green Fluorescent

Plasma cells comprise a population of terminally differentiated B cells that are dependent on the transcriptional regulator B lymphocyte–induced maturation protein 1 (Blimp-1) for their development. We have introduced a gfp reporter into the Blimp-1 locus and shown that heterozygous mice express the green fluorescent protein in all antibody-secreting cells (ASCs) in vivo and in vitro. In vitro, these cells display considerable heterogeneity in surface phenotype, immunoglobulin secretion rate, and Blimp-1 expression levels. Importantly, analysis of in vivo ASCs induced by immunization reveals a developmental pathway in which increasing levels of Blimp-1 expression define developmental stages of plasma cell differentiation that have many phenotypic and molecular correlates. Thus, maturation from transient plasmablast to long-lived ASCs in bone marrow is predicated on quantitative increases in Blimp-1 expression.

scholarly journals The Variable Transmembrane Domain of Drosophila N-Cadherin Regulates Adhesive Activity

2006 ◽  
Vol 26 (17) ◽  
pp. 6598-6608 ◽  
Author(s):  
Shinichi Yonekura ◽  
Chun-Yuan Ting ◽  
Guilherme Neves ◽  
Kimberly Hung ◽  
Shu-ning Hsu ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Fluorescent Protein ◽  
Developmental Stages ◽  
Transmembrane Domain ◽  
Cell Aggregation ◽  
Transmembrane Domains ◽  
Molecular Architecture ◽  
Cell Type Specific ◽  
Green Fluorescent

ABSTRACT Drosophila N-cadherin (CadN) is an evolutionarily conserved classic cadherin which has a large, complex extracellular domain and a catenin-binding cytoplasmic domain. The CadN locus contains three modules of alternative exons (7a/b, 13a/b, and 18a/b) and undergoes alternative splicing to generate multiple isoforms. Using quantitative transcript analyses and green fluorescent protein-based cell sorting, we found that during development CadN alternative splicing is regulated in a temporal but not cell-type-specific fashion. In particular, exon 18b is predominantly expressed during early developmental stages, while exon 18a is prevalent at the late developmental and adult stages. All CadN isoforms share the same molecular architecture but have different sequences in their extracellular and transmembrane domains, suggesting functional diversity. In vitro quantitative cell aggregation assays revealed that all CadN isoforms mediate homophilic interactions, but the isoforms encoded by exon 18b have a higher adhesive activity than those by its alternative, 18a. Domain-swapping experiments further revealed that the different sequences in the transmembrane domains of isoforms are responsible for their differential adhesive activities. CadN alternative splicing might provide a novel mechanism to fine-tune its adhesive activity at different developmental stages or to restrict the use of high-affinity 18b-type isoforms at the adult stage.

scholarly journals Differential Expression of the Aspergillus fumigatus pksP Gene Detected In Vitro and In Vivo with Green Fluorescent Protein

2001 ◽  
Vol 69 (10) ◽  
pp. 6411-6418 ◽  
Author(s):  
Kim Langfelder ◽  
Bruno Philippe ◽  
Bernhard Jahn ◽  
Jean-Paul Latgé ◽  
Axel A. Brakhage
Keyword(s):  
Green Fluorescent Protein ◽  
Aspergillus Fumigatus ◽  
Polyketide Synthase ◽  
Fluorescent Protein ◽  
Developmental Stages ◽  
Gene Encoding ◽  
Immunocompromised Mice ◽  
Green Fluorescent

ABSTRACT Aspergillus fumigatus is an important pathogen of immunocompromised hosts, causing pneumonia and invasive disseminated disease with high mortality. To be able to analyze the expression of putative virulence-associated genes of A. fumigatus, the use of the enhanced green fluorescent protein (EGFP) as a reporter was established. Two 5′ sequences, containing the putative promoters of thepyrG gene, encoding orotidine-5′-phosphate decarboxylase, and the pksP gene, encoding a polyketide synthase involved in both pigment biosynthesis and virulence ofA. fumigatus, were fused with the egfpgene. The PpksP-egfp construct was integrated via homologous recombination into the genomicpksP locus. EGFP production was analyzed by fluorescence spectrometry, Western blot analysis, and fluorescence microscopy. Differential gene expression in A. fumigatus was observed. Fluorescence derived from the PYRG-EGFP fusion protein was detected during all developmental stages of the fungus, i.e., during germination, during vegetative growth, in conidiophores, and weakly in conidia. In addition, it was also detected in germinating conidia when isolated from the lungs of immunocompromised mice. By contrast, PKSP-EGFP-derived fluorescence was not found in hyphae or stalks of conidiophores but was found in phialides and conidia in vitro when the fungus was grown under standard conditions, indicating a developmentally controlled expression of the gene. Interestingly,pksP-egfp expression was also detected in hyphae of germinating conidia isolated from the lungs of immunocompromised mice. This finding indicates that thepksP gene can also be expressed in hyphae under certain conditions and, furthermore, that the pksP gene might also contribute to invasive growth of the fungus.

scholarly journals A new transgenic reporter line reveals Wnt-dependent Snail2 reexpression and cranial neural crest differentiation in Xenopus

2019 ◽  
Author(s):  
Jiejing Li ◽  
Mark Perfetto ◽  
Christopher Materna ◽  
Rebecca Li ◽  
Hong Thi Tran ◽  
...  
Keyword(s):  
Wnt Signaling ◽  
Neural Crest ◽  
Fluorescent Protein ◽  
Developmental Stages ◽  
Egfp Expression ◽  
Cranial Neural Crest ◽  
Canonical Wnt Signaling ◽  
Reporter Line ◽  
Green Fluorescent ◽  
Canonical Wnt

AbstractDuring vertebrate embryogenesis, the cranial neural crest (CNC) forms at the neural plate border and subsequently migrates and differentiates into many types of cells. The transcription factor Snail2, which is induced by canonical Wnt signaling to be expressed in the early CNC, is pivotal for CNC induction and migration in Xenopus. However, snail2 expression is silenced during CNC migration, and its roles at later developmental stages remain unclear. We generated a transgenic X. tropicalis line that expresses enhanced green fluorescent protein (eGFP) driven by the snail2 promoter/enhancer, and observed eGFP expression not only in the pre-migratory and migrating CNC, but also the differentiating CNC. This transgenic line can be used directly to detect deficiencies in CNC development at various stages, including subtle perturbation of CNC differentiation. In situ hybridization and immunohistochemistry confirm that Snail2 is reexpressed in the differentiating CNC. Using a separate transgenic Wnt reporter line, we show that canonical Wnt signaling is also active in the differentiating CNC. Blocking Wnt signaling shortly after CNC migration causes reduced snail2 expression and impaired differentiation of CNC-derived head cartilage structures. These results suggest that Wnt signaling drives the reexpression of snail2 in the post-migratory CNC and regulates CNC differentiation.

Effect of epigenetic modification with trichostatin A and S-adenosylhomocysteine on developmental competence and POU5F1–EGFP expression of interspecies cloned embryos in dog

Zygote ◽  
2014 ◽  
Vol 23 (5) ◽  
pp. 758-770
Author(s):  
M. Mousai ◽  
S.M. Hosseini ◽  
M. Hajian ◽  
F. Jafarpour ◽  
V. Asgari ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Developmental Stages ◽  
Epigenetic Modification ◽  
Trichostatin A ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Egfp Expression ◽  
Beneficial Effects ◽  
Pre Treatment ◽  
Green Fluorescent

SummaryAdult canine fibroblasts stably transfected with either cytomegalovirus (CMV) or POU5F1 promoter-driven enhanced green fluorescent protein (EGFP) were used to investigate if pre-treatment of these donor cells with two epigenetic drugs [trichostatin A (TSA), or S-adenosylhomocysteine (SAH)] can improve the efficiency of interspecies somatic cell nuclear transfer (iSCNT). Fluorescence-activated cell sorting (FACS), analyses revealed that TSA, but not SAH, treatment of both transgenic and non-transgenic fibroblasts significantly increased acetylation levels compared with untreated relatives. The expression levels of Bcl2 and P53 were significantly affected in TSA-treated cells compared with untreated cells, whereas SAH treatment had no significant effect on cell apoptosis. Irrespective of epigenetic modification, dog/bovine iSCNT embryos had overall similar rates of cleavage and development to 8–16-cell and morula stages in non-transgenic groups. For transgenic reconstructed embryos, however, TSA and SAH could significantly improve development to 8–16-cell and morula stages compared with control. Even though, irrespective of cell transgenesis and epigenetic modification, none of the iSCNT embryos developed to the blastocyst stage. The iSCNT embryos carrying CMV–EGFP expressed EGFP at all developmental stages (2-cell, 4-cell, 8–16-cell, and morula) without mosaicism, while no POU5F1–EGFP signal was observed in any stage of developing iSCNT embryos irrespective of TSA/SAH epigenetic modifications. These results indicated that bovine oocytes partially remodel canine fibroblasts and that TSA and SAH have marginal beneficial effects on this process.

scholarly journals Cardiac cholinergic NO-cGMP signaling following acute myocardial infarction and nNOS gene transfer

2008 ◽  
Vol 295 (3) ◽  
pp. H990-H998 ◽  
Author(s):  
T. A. Dawson ◽  
D. Li ◽  
T. Woodward ◽  
Z. Barber ◽  
L. Wang ◽  
...  
Keyword(s):  
Myocardial Infarction ◽  
Gene Transfer ◽  
Guinea Pigs ◽  
Fluorescent Protein ◽  
Cholinergic Neurons ◽  
Right Atrial ◽  
Ach Release ◽  
Green Fluorescent ◽  
The Right

Myocardial infarction (MI) is associated with oxidative stress, which may cause cardiac autonomic impairment. We tested the hypothesis that acute MI disrupts cardiac cholinergic signaling by impairing nitric oxide (NO)-cGMP modulation of acetylcholine (ACh) release and whether the restoration of this pathway following cardiac neuronal NO synthase (nNOS) gene transfer had any bearing on the neural phenotype. Guinea pigs underwent four ligature coronary artery surgery ( n = 50) under general anesthesia to induce MI or sham surgery ( n = 32). In a separate group, at the time of MI surgery, adenovirus encoding nNOS ( n = 29) or enhanced green fluorescent protein (eGFP; n = 30) was injected directly into the right atria, where the postganglionic cholinergic neurons reside. In vitro-evoked right atrial [3H]ACh release, right atrial NOS activity, and cGMP levels were measured at 3 days. Post-MI 24% of guinea pigs died compared with 9% in the sham-operated group. Evoked right atrial [3H]ACh release was significantly ( P < 0.05) decreased in the MI group as was NOS activity and cGMP levels. Tetrahydrobiopterin levels were not significantly different between the sham and MI groups. Infarct sizes between gene-transferred groups were not significantly different. The nNOS transduced group had significantly increased right atrial [3H]ACh release, right atrial NOS activity, cGMP levels, and decreased cAMP levels. Fourteen percent of the nNOS transduced animals died compared with 31% mortality in the MI + eGFP group at 3 days. In conclusion, cardiac nNOS gene transfer partially restores the defective NO-cGMP cholinergic pathway post-MI, which was associated with a trend of improved survival at 3 days.

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