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whole mount in situ hybridization

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https://www.readbyqxmd.com/read/29462671/expression-of-the-adhesion-g-protein-coupled-receptor-a2-adgra2-during-xenopus-laevis-development
#1
Franziska A Seigfried, Petra Dietmann, Michael Kühl, Susanne J Kühl
The adhesion G protein-coupled receptor A2 (Adgra2) is a seven transmembrane receptor that has been described to be a regulator for angiogenesis in mice. Furthermore, the zebrafish ouchless mutant is unable to develop dorsal root ganglia through a disrupted trafficking of Adgra2. Besides RNA sequencing data, nothing is reported about Adgra2 in the south African crawled frog Xenopus laevis. In this study, we investigated for the first time the spatio-temporal expression of adgra2 during early Xenopus embryogenesis in detail...
February 17, 2018: Gene Expression Patterns: GEP
https://www.readbyqxmd.com/read/29425878/the-klotho-related-protein-klph-lctl-has-preferred-expression-in-lens-and-is-essential-for-expression-of-clic5-and-normal-lens-suture-formation
#2
Jianguo Fan, Joshua Lerner, M Keith Wyatt, Phillip Cai, Katherine Peterson, Lijin Dong, Graeme Wistow
KLPH/lctl belongs to the Klotho family of proteins. Expressed sequence tag analyses unexpectedly revealed that KLPH is highly expressed in the eye lens while northern blots showed that expression is much higher in the eye than in other tissues. In situ hybridization in mouse localized mRNA to the lens, particularly in the equatorial epithelium. Immunofluorescence detected KLPH in lens epithelial cells with highest levels in the germinative/differentiation zone. The gene for KLPH in mouse was deleted by homologous recombination...
February 6, 2018: Experimental Eye Research
https://www.readbyqxmd.com/read/29353190/characterization-of-acrylamide-induced-oxidative-stress-and-cardiovascular-toxicity-in-zebrafish-embryos
#3
Mengmeng Huang, Jingjing Jiao, Jun Wang, Zhidan Xia, Yu Zhang
Acrylamide (AA) is a high production volume chemical in industrial applications and widely found in baked or fried carbohydrate-rich foods. In this study, we unravelled that AA induced developmental toxicity associated with oxidative stress status and disordered lipid distribution in heart region of developing zebrafish. Treatment with AA caused a deficient cardiovascular system with significant heart malformation and dysfunction. We also found that AA could reduce the number of cardiomyocytes through the reduced capacity of cardiomyocyte proliferation rather than cell apoptosis...
January 8, 2018: Journal of Hazardous Materials
https://www.readbyqxmd.com/read/29346450/cloning-and-spatiotemporal-expression-of-xenopus-laevis-apolipoprotein-ci
#4
Jyotsna Sridharan, Tomomi Haremaki, Daniel C Weinstein
Apolipoprotein CI (ApoCI) belongs to the Apolipoprotein superfamily, members of which are involved in lipid transport, uptake and homeostasis. Excessive ApoCI has been implicated in atherosclerosis and Alzheimer's disease in humans. In this study we report the isolation of Xenopus laevis apoCI and describe the expression pattern of this gene during early development, using reverse transcription polymerase chain reaction and whole mount in situ hybridization. Xenopus apoCI is enriched in the dorsal ectoderm during gastrulation, and is subsequently expressed in sensory placodes, neural tube and cranial neural crest...
2018: PloS One
https://www.readbyqxmd.com/read/29322524/a-comprehensive-analysis-of-neurotrophins-and-neurotrophin-tyrosine-kinase-receptors-expression-during-development-of-zebrafish
#5
Valeria Nittoli, Rosa M Sepe, Ugo Coppola, Ylenia D'Agostino, Elena De Felice, Antonio Palladino, Quirino A Vassalli, Annamaria Locascio, Filomena Ristoratore, Antonietta Spagnuolo, Salvatore D'Aniello, Paolo Sordino
Neurotrophins (NTF) are a family of secreted nerve growth factors with affinity for tyrosine kinase (Ntrk) and p75 receptors. To fully understand the variety of developmental roles played by NTFs, it is critical to know when and where genes encoding individual ligands and receptors are transcribed. Identification of ntf and ntrk transcripts in zebrafish development remains to be fully characterized for further uncovering the potential function(s) of the NTF signal transduction pathway. Here, we conducted a systematic analysis of the expression profiles of four ntf and five ntrk genes during zebrafish development using whole-mount in situ hybridization...
January 11, 2018: Journal of Comparative Neurology
https://www.readbyqxmd.com/read/29311262/multidimensional-quantitative-analysis-of-mrna-expression-within-intact-vertebrate-embryos
#6
Vikas Trivedi, Harry M T Choi, Scott E Fraser, Niles A Pierce
For decades, in situ hybridization methods have been essential tools for studies of vertebrate development and disease, as they enable qualitative analyses of mRNA expression in an anatomical context. Quantitative mRNA analyses typically sacrifice the anatomy, relying on embryo microdissection, dissociation, cell sorting and/or homogenization. Here, we eliminate the trade-off between quantitation and anatomical context, using quantitative in situ hybridization chain reaction (qHCR) to perform accurate and precise relative quantitation of mRNA expression with subcellular resolution within whole-mount vertebrate embryos...
January 8, 2018: Development
https://www.readbyqxmd.com/read/29299324/impaired-caudal-fin-fold-regeneration-in-zebrafish-deficient-for-the-tumor-suppressor-pten
#7
Alexander James Hale, Ali Kiai, Jelte Sikkens, Jeroen den Hertog
Zebrafish are able to completely regrow their caudal fin-folds after amputation. Following injury, wound healing occurs, followed by the formation of a blastema, which produces cells to replace the lost tissue in the final phase of regenerative outgrowth. Here we show that, surprisingly, the phosphatase and tumor suppressor Pten, an antagonist of phosphoinositide-3-kinase (PI3K) signaling, is required for zebrafish caudal fin-fold regeneration. We found that homozygous knock-out mutant (ptena-/-ptenb-/- ) zebrafish embryos, lacking functional Pten, did not regenerate their caudal fin-folds...
August 2017: Regeneration
https://www.readbyqxmd.com/read/29286368/visualizing-multiciliated-cells-in-the-zebrafish-through-a-combined-protocol-of-whole-mount-fluorescent-in-situ-hybridization-and-immunofluorescence
#8
Amanda N Marra, Marisa Ulrich, Audra White, Meghan Springer, Rebecca A Wingert
In recent years, the zebrafish embryo has emerged as a popular model to study developmental biology due to traits such as ex utero embryo development and optical transparency. In particular, the zebrafish embryo has become an important organism to study vertebrate kidney organogenesis as well as multiciliated cell (MCC) development. To visualize MCCs in the embryonic zebrafish kidney, we have developed a combined protocol of whole-mount fluorescent in situ hybridization (FISH) and whole mount immunofluorescence (IF) that enables high resolution imaging...
November 18, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/29276941/rnf152-is-essential-for-neurod-expression-and-delta-notch-signaling-in-the-zebrafish-embryos
#9
Ajeet Kumar, Tae-Lin Huh, Joonho Choe, Myungchull Rhee
We report the biological functions of a zebrafish homologue of RING-finger protein 152 (rnf152) during embryogenesis. rnf152 was initially identified as a brain-enriched E3 ligase involved in early embryogenesis of zebrafish. Expression of rnf152 was ubiquitous in the brain at 24 hpf but restricted to the eyes, midbrain-hindbrain boundary (MHB), and rhombomeres at 48 hpf. Knockdown of rnf152 in zebrafish embryos caused defects in the eyes, MHB, and rhombomeres (r1-7) at 24 hpf. These defects in rnf152-deficient embryos were analyzed by whole-mount in situ hybridization (WISH) using neuroD, deltaD, notch1a, and notch3 probes...
December 22, 2017: Molecules and Cells
https://www.readbyqxmd.com/read/29203198/phylogeny-and-expression-patterns-of-two-apolipoprotein-e-genes-in-the-flatfish-senegalese-sole
#10
Javier Roman-Padilla, Ana Rodríguez-Rúa, Carlos Carballo, Manuel Manchado, Ismael Hachero-Cruzado
The apolipoprotein E (ApoE) is a key component of several lipoproteins involved in lipid homeostasis. In this study, two cDNA sequences encoding ApoE (referred to as apoEa and apoEb) were characterized in the flatfish Solea senegalensis. The predicted peptides contained conserved structural blocks related with their capacity for lipid binding and lipoprotein receptor interaction. At genomic level, both genes contained five exons and four introns and they were organized into two tandem arrays with apoA-IV gene copies...
December 1, 2017: Gene
https://www.readbyqxmd.com/read/29196253/involvement-of-lypge-in-the-formation-of-eye-and-pineal-gland-in-zebrafish
#11
Dongrui Ji, Su Wang, Mingyue Li, Shicui Zhang, Hongyan Li
The proteins of Ly-6 (lymphocyte antigen-6) family are involved in the regulation of immunoreaction, cell migration and adhesion, and neuronal excitability. However, little is known about the function of Ly-6 proteins in embryogenesis. Herein, we identified a GPI anchored Ly-6 member named ly6 expressed in pineal gland and eye (lypge). Dynamic expression pattern of lypge was revealed by whole mount in situ hybridization. It was strikingly expressed in the pineal gland and cone photoreceptor, and its expression was regulated by orthodenticle homolog 5 (otx5) which has been shown to control the expression of many pineal genes...
November 28, 2017: Gene
https://www.readbyqxmd.com/read/29130196/super-resolution-single-molecule-fish-at-the-drosophila-neuromuscular-junction
#12
Joshua S Titlow, Lu Yang, Richard M Parton, Ana Palanca, Ilan Davis
The lack of an effective, simple, and highly sensitive protocol for fluorescent in situ hybridization (FISH) at the Drosophila larval neuromuscular junction (NMJ) has hampered the study of mRNA biology. Here, we describe our modified single molecule FISH (smFISH) methods that work well in whole mount Drosophila NMJ preparations to quantify primary transcription and count individual cytoplasmic mRNA molecules in specimens while maintaining ultrastructural preservation. The smFISH method is suitable for high-throughput sample processing and 3D image acquisition using any conventional microscopy imaging modality and is compatible with the use of antibody colabeling and transgenic fluorescent protein tags in axons, glia, synapses, and muscle cells...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29075432/establishment-of-molecular-genetic-approaches-to-study-gene-expression-and-function-in-an-invasive-hemipteran-halyomorpha-halys
#13
Yong Lu, Mengyao Chen, Katie Reding, Leslie Pick
Hemiptera is a large clade of insects understudied in terms of developmental biology. Halyomorpha halys, the Brown Marmorated Stink Bug (BMSB, referred to throughout as H. halys), is an invasive hemipteran pest of the mid-Atlantic region of the USA that has rapidly spread to other regions in recent years, devastating a wide range of crops using a piercing and sucking mechanism. Its phylogenetic position, polyphagous habits, and rapid spread in the USA suggested that H. halys would be an ideal system to broaden our knowledge of developmental mechanisms in insects...
2017: EvoDevo
https://www.readbyqxmd.com/read/29052208/fluorescence-in-situ-hybridization-fish-and-immunolabeling-on-3d-preserved-nuclei
#14
Till David Bey, Maria Koini, Paul Fransz
The spatial distribution of genes in the nucleus emerges as an important factor in gene regulation and epigenetics. The position of loci relative to each other, to nuclear landmarks such as the nucleolus and chromocenters, as well as to chromatin proteins is therefore highly interesting. With fluorescent in situ hybridization (FISH) specific DNA sequences can be stained and antibodies allow the detection of specific proteins. Here, we present two protocols that preserve the 3D structure of nuclei. With whole-mount FISH, specific sequences can be stained in intact tissues and, secondly, a combined immunolabeling and FISH protocol on acrylamide-embedded nuclei makes it possible to stain DNA sequences and proteins simultaneously...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29030842/microrna-detection-by-whole-mount-in-situ-hybridization-in-c-elegans
#15
Yoshiki Andachi, Yuji Kohara
MicroRNAs (miRNAs) loaded on argonaute proteins guide RNA-induced silencing complexes to target mRNAs. An excellent method to decipher the spatiotemporal expression patterns of miRNAs is whole-mount in situ hybridization (WISH), which has been successfully used in vertebrate embryos but still remains unavailable for many animal species. Here, we describe a WISH method for miRNA detection in Caenorhabditis elegans at both embryonic and post-embryonic stages. Strategies devised for detection include fixation of animals with carbodiimide at a high temperature and subsequent partial digestion of the fixed animals with an extremely high concentration of proteinase...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29024245/hace1-influences-zebrafish-cardiac-development-via-ros-dependent-mechanisms
#16
Babak Razaghi, Shelby L Steele, Sergey V Prykhozhij, Matthew R Stoyek, Jessica A Hill, Matthew D Cooper, Lindsay Mcdonald, William Lin, Mads Daugaard, Nicolas Crapoulet, Simi Chacko, Stephen M Lewis, Ian C Scott, Poul H B Sorensen, Jason N Berman
BACKGROUND: In this study, we reveal a previously undescribed role of the HACE1 (HECT domain and Ankyrin repeat Containing E3 ubiquitin-protein ligase 1) tumor suppressor protein in normal vertebrate heart development using the zebrafish (Danio rerio) model. We examined the link between the cardiac phenotypes associated with hace1 loss of function to the expression of the Rho small family GTPase, rac1, which is a known target of HACE1 and promotes ROS production via its interaction with NADPH oxidase holoenzymes...
October 11, 2017: Developmental Dynamics: An Official Publication of the American Association of Anatomists
https://www.readbyqxmd.com/read/28987646/molecular-cloning-and-expression-of-octamer-binding-transcription-factor-oct4-in-the-large-yellow-croaker-larimichthys-crocea
#17
Yonghua Jiang, Kunhuang Han, Shihai Chen, Mingyi Cai, Yilei Wang, Ziping Zhang
Octamer-binding transcription factor 4 (Oct4) is a crucial pluripotent transcription factor in controlling pluripotency of embryonic stem cells (ESCs), formation of primordial germ cells (PGCs), early embryonic development, and gonadal germ cells. To understand the roles of Oct4 in the large yellow croaker (Larimichthys crocea) (Lc-Oct4), the full-length cDNA of Oct4 was cloned and analyzed. Lc-Oct4 includes a 104-bp 5' untranslated region (UTR), a 567-bp 3' UTR and a 1431-bp coding region encoding a protein of 476 amino acids (aa) with a predicted molecular mass (Mm) of 52...
October 4, 2017: Gene Expression Patterns: GEP
https://www.readbyqxmd.com/read/28964895/functions-of-the-vasa-gene-in-schistosoma-japonicum-as-assessed-by-rna-interference
#18
Siyu He, Lulu Zhu, Fengchun Liu, Quan Liu, Yanjing Shao, Mengqing Hua, Han Ding, Wei Shao, Yinan Du, Xin Hou, Cuiping Ren, Miao Liu, Jijia Shen
Vasa, an enzyme belonging to the helicase family, contributes to the regulation of reproductive system development in many species. Thus, we hypothesized that the Vasa3 gene may function in the reproductive system of the parasite Schistosoma japonicum (S. japonicum), which is a major causative agent of schistosomiasis. It is a severe disease globally affecting humans and animals. To test this hypothesis, we firstly conducted whole mount in situ hybridization analyses and found that the S. japonicum Vasa3 (SjVasa3) gene was expressed mainly in the reproductive organs...
January 5, 2018: Gene
https://www.readbyqxmd.com/read/28936657/whole-mount-rna-fish-on-ovules-and-developing-seeds
#19
Andrea Bleckmann, Thomas Dresselhaus
A key element to understand developmental and reproductive processes like germline development, double fertilization, and embryogenesis is the study of cell-specific gene expression patterns which is best analyzed by RNA in situ hybridization. Different visualization techniques have been established to mark either the region of mRNA production (using the classical chromogenic detection system) or the specific localization of mRNAs (using fluorescent labeled probes). In this chapter, we describe and compare whole mount RNA in situ hybridization techniques on ovules and young developing seeds from Arabidopsis thaliana using three different detection systems...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28891223/promoter-architecture-and-transcriptional-regulation-of-musculoskeletal-embryonic-nuclear-protein-1b-mustn1b-gene-in-zebrafish
#20
Paula Suarez-Bregua, Chien-Ju Chien, Manuel Megias, Shaojun Du, Josep Rotllant
BACKGROUND: Mustn1 is a specific musculoskeletal protein that plays a critical role in myogenesis and chondrogenesis in vertebrates. Whole-mount in situ hybridization revealed that mustn1b mRNAs are specifically expressed in skeletal and cardiac muscles in Zebrafish embryos. However, the precise function and the regulatory elements required for its muscle-specific expression are largely unknown. RESULTS: The purpose of this study was to explore and uncover the target genomic regions that regulate mustn1b gene expression by in vivo functional characterization of the mustn1b promoter...
September 11, 2017: Developmental Dynamics: An Official Publication of the American Association of Anatomists
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