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whole mount in situ hybridization

Yoshinori Kawamura, Kiyoshi Matsumoto
Rabbits are valuable experimental animals used to evaluate the teratogenicity of chemical entities. If teratogenicity is recognized, it is very important to investigate the pathogenesis to assess the potential risk in humans. For this investigation, whole-mount in situ hybridization (WISH) is highly useful for visualizing spatial and temporal changes in the expression patterns of the targeted genes and or proteins. In this chapter, the WISH method in rabbit embryos, using a digoxigenin-labeled RNA probe, and the morphometric analysis of the expression pattern is described...
2018: Methods in Molecular Biology
Heithem M El-Hodiri, Lisa E Kelly
In situ hybridization performed using whole fixed embryos provides accurate and detailed visualization of gene expression patterns. These patterns are useful for investigating spatial patterns of gene expression in normally developing embryos but can also be useful in investigating the effects of genetic or environmental changes on expression of genetic markers characteristic of particular tissues, organs, or genetic pathways. Our lab's protocol for whole-mount in situ hybridization is presented.
2018: Methods in Molecular Biology
Camille Cosse-Etchepare, Isabelle Gervi, Isabelle Buisson, Laurent Formery, Michael Schubert, Jean-François Riou, Muriel Umbhauer, Ronan Le Bouffant
The POU (Pit-Oct-Unc) genes encode a large transcription factor family comprising 6 classes (pou1f to pou6f ) involved in many developmental processes, such as cell commitment and differentiation. The pou3f class contains four members (pou3f1, pou3f2, pou3f3, pou3f4) characterized by expression in ectodermal tissue derivatives, such as nervous system and otic vesicle, during mammalian development. In order to obtain insights into the potential conservation of this class of transcription factors in vertebrates, we carried out a phylogenetic analysis and a comprehensive comparative study of pou3f expression in the frog Xenopus laevis...
2018: International Journal of Developmental Biology
Jinfeng Liu, Limei Sun, Hongqin Zhang, Mengru Shi, Randy A Dahlgren, Xuedong Wang, Huili Wang
Triclosan (TCS), 2,4,6-trichlorophenol (2,4,6-TCP) and 2,4-dichlorophenol (2,4-DCP) frequently co-exist in real-world aquatic environments; the latter two contaminants contributing to TCS photolytic products or chlorinated derivatives. There is a paucity of information regarding their joint toxicity to aquatic organisms leading us to study their effects on the swimming behavior of zebrafish (Danio rerio). Herein, we reported that 0.28 mg/L TDT exposure (mixtures of TCS, 2,4,6-TCP and 2,4-DCP) enhanced 24-hpf embryonic spontaneous movement frequency, 96-hpf larval activity; however, the 0...
February 2018: Chemosphere
Michael Weber, Julia Wunderer, Birgit Lengerer, Robert Pjeta, Marcelo Rodrigues, Lukas Schärer, Peter Ladurner, Steven A Ramm
BACKGROUND: Along with sperm, in many taxa ejaculates also contain large numbers of seminal fluid proteins (SFPs). SFPs and sperm are transferred to the mating partner, where they are thought to play key roles in mediating post-mating sexual selection. They modulate the partner's behavior and physiology in ways that influence the reproductive success of both partners, thus potentially leading to sexual conflict. Despite the presumed general functional and evolutionary significance of SFPs, their identification and characterization has to date focused on just a few animal groups, predominantly insects and mammals...
May 30, 2018: BMC Evolutionary Biology
Chunjie Liu, Ruixue Cao, Yuejuan Xu, Tingting Li, Fen Li, Sun Chen, Rang Xu, Kun Sun
BACKGROUND: Heterotaxy (Htx) syndrome comprises a class of congenital disorders resulting from malformations in left-right body patterning. Approximately 90% of patients with heterotaxy have serious congenital heart diseases; as a result, the survival rate and outcomes of Htx patients are not satisfactory. However, the underlying etiology and mechanisms in the majority of Htx cases remain unknown. The aim of this study was to investigate the function of rare copy number variants (CNVs) in the pathogenesis of Htx...
May 30, 2018: Genome Medicine
Marta Vitorino, Nídia Cunha, Natércia Conceição, M Leonor Cancela
Atypical Rett syndrome is a child neurodevelopmental disorder induced by mutations in CDKL5 gene and characterized by a progressive regression in development with loss of purposeful use of the hands, slowed brain and head growth, problems with walking, seizures, and intellectual disability. At the moment, there is no cure for this pathology and little information is available concerning animal models capable of mimicking its phenotypes, thus the development of additional animal models should be of interest to gain more knowledge about the disease...
May 11, 2018: Molecular Biology Reports
Glenn P Lobo, Gayle Pauer, Joshua H Lipschutz, Stephanie A Hagstrom
Vitamin A/retinol (ROL) and its metabolites (retinoids) play critical roles in eye development and photoreception. Short-term dietary vitamin A deficiency (VAD) manifests clinically as night blindness, while prolonged VAD is known to cause retinal pigment epithelium (RPE) and photoreceptor degeneration. Therefore, sustained uptake of dietary vitamin A, for ocular retinoid production, is essential for photoreceptor health and visual function. The mechanisms influencing the uptake, storage, and supply of dietary vitamin A, for ocular retinoid production, however, are not fully understood...
2018: Advances in Experimental Medicine and Biology
Kana Ishii, Kusumika Mukherjee, Takashi Okada, Eric C Liao
Background: Craniofacial malformations are among the most common congenital anomalies. Cranial neural crest cells (CNCCs) form craniofacial structures involving multiple cellular processes, perturbations of which contribute to craniofacial malformations. Adhesion of cells to the extracellular matrix mediates bidirectional interactions of the cells with their extracellular environment that plays an important role in craniofacial morphogenesis. Talin (tln) is crucial in cell-matrix adhesion between cells, but its role in craniofacial morphogenesis is poorly understood...
March 2018: Plastic and Reconstructive Surgery. Global Open
Ruben Plöger, Christoph Viebahn
Attaining molecular and morphological axial polarity during gastrulation is a fundamental early requirement for normal development of the embryo. In mammals, the first morphological sign of the anterior-posterior axis appears anteriorly in the form of the anterior marginal crescent (or anterior visceral endoderm) while in the avian the first such sign is the Koller's sickle at the posterior pole of the embryonic disc. Despite this inverse mode of axis formation many genes and molecular pathways involved in various steps of this process seem to be evolutionary conserved amongst amniotes, the nodal gene being a well-known example with its functional involvement prior and during gastrulation...
April 26, 2018: Annals of Anatomy, Anatomischer Anzeiger: Official Organ of the Anatomische Gesellschaft
Caroline Borday, Karine Parain, Hong Thi Tran, Kris Vleminckx, Muriel Perron, Anne H Monsoro-Burq
Wnt proteins form a family of highly conserved secreted molecules that are critical mediators of cell-cell signaling during embryogenesis. Partial data on Wnt activity in different tissues and at different stages have been reported in frog embryos. Our objective here is to provide a coherent and detailed description of Wnt activity throughout embryo development. Using a transgenic Xenopus tropicalis line carrying a Wnt-responsive reporter sequence, we depict the spatial and temporal dynamics of canonical Wnt activity during embryogenesis...
2018: PloS One
Sujian Tan, Pin Huan, Baozhong Liu
Though the roles of BMP signaling in development is studied extensively in insects and vertebrates, our knowledge of BMP signaling in molluscan development is limited. In the present study, we performed a genome-based analysis of TGF-β receptors in the Pacific oyster Crassostrea gigas and revealed that C. gigas possessed all five canonical members of the gene family, including three type I and two type II receptors. Whole mount in situ hybridization revealed that four receptor genes exhibited universal expression at the gastrula stage but cgi-bmprII mRNA was only expressed at the anterior lip of blastopore, indicating the regulation of BMP signaling through restricted expression of TGF-β receptors...
July 15, 2018: Gene
Junchao Duan, Shuang Liang, Yang Yu, Yang Li, Lijing Wang, Zehao Wu, Yueyue Chen, Mark R Miller, Zhiwei Sun
Nowadays, nanotechnology environmental health and safety (nanoEHS) is gaining attention. We previously found that silica nanoparticles (SiNPs) could induce vascular endothelial damage. However, the subsequent toxicologic response to SiNPs-induced endothelial damage was still largely unknown. In this study, we explored the inflammation-coagulation response and thrombotic effects of SiNPs in endothelial cells and zebrafish embryos. For in vitro study, swollen mitochondria and autophagosome were observed in ultrastructural analysis...
June 2018: Nanotoxicology
Dan-Dan Guo, Guo-Dong Zheng, Shang-Ke Du, Bo Qin, Xia-Yun Jiang, Shu-Ming Zou
Midkine (Mdk) is a heparin-binding growth factor that is involved in regulating cell growth, differentiation and migration. Here, we report the isolation and characterization of duplicated mdk genes in blunt snout bream (Megalobrama amblycephala). The mdka and -b genes encode 146 aa and 147 aa peptides, respectively, sharing a sequence identity of 64%. During embryogenesis, mdka mRNA is detectable after 12 h post-fertilization (hpf) and mdkb mRNA can be detected after 8 hpf, about 4 h prior to mdka mRNA...
May 2018: Comparative Biochemistry and Physiology. Part B, Biochemistry & Molecular Biology
Kazuko Koshiba-Takeuchi
In situ hybridization is defined as one of the most useful and powerful methods to know where genes (e.g., mRNA, ncRNA) of interest are expressed in tissues. Expression of mRNA can be detected as blue or dark purple signals though hybridization, immunoreaction and coloring steps. Genome-wide approaches in various model animals have been conducted thoroughly, and have led to new research areas aimed at uncovering novel gene functions in cell differentiation and development. To elucidate gene function, spatiotemporal gene expression analysis is very important...
2018: Methods in Molecular Biology
Yuan Sun, Hong-Hong Guo, Dan-Dan Guo, Xia-Yun Jiang, Shu-Ming Zou
The proteins CITED belong to a family of non-DNA-binding transcriptional co-regulators involved in the regulation of various transcriptional responses. Previous studies suggest that members of CITED family may function in response to hypoxia in mammals. however, the molecular and functional information on CITED genes in aquaculture fish is unclear. Here, we characterized and examined the transcriptional patterns of CITED1 and CITED2 genes in the hypoxia-sensitive blunt snout bream ( Megalobrama amblycephala )...
2018: Frontiers in Physiology
Yana Li, Shuna Sun, Zhiwen Ding, Chunjie Yang, Guoping Zhang, Qiu Jiang, Yunzeng Zou
Fibroblast growth factor binding proteins (FGFBPs) are a class of secreted proteoglycans that function as an extracellular chaperone for locally stored FGFs and enhance FGF signaling. To date, all three human FGFBP genes have been identified and one orthologue fgfbp1a has been studied in zebrafish embryos. Here, we described the cloning and expression patterns of four novel FGFBP orthologues in zebrafish, fgfbp1b, fgfbp2a, fgfbp2b, and fgfbp3. Quantitative PCR and whole-mount in situ hybridization results showed that all transcripts except fgfbp2a are initially expressed in a maternal manner...
June 15, 2018: Gene
Rebecca L Cunningham, Kelly R Monk
In situ hybridization enables visualization of mRNA localization, and immunohistochemistry enables visualization of protein localization within a tissue or organism. Both techniques have been extensively utilized in zebrafish (Thisse et al., Development 119:1203-1215, 1993; Dutton et al., Development 128:4113-4125, 2001; Gilmour et al., Neuron 34:577-588, 2002; Lyons et al., Curr Biol 15:513-524, 2005) including for visualization of mRNA localization in Schwann cells (Lyons et al., Curr Biol 15:513-524, 2005; Monk et al...
2018: Methods in Molecular Biology
Diana Bauermeister, Tomas Pieler
Whole-mount in situ hybridization (WMISH) is a common approach that is used to visualize spatial and temporal gene expression in embryos. In this process, digoxygenin-labeled antisense RNA is hybridized to the complementary transcript of interest and RNA hybrids are immunohistochemically detected using an alkaline phosphatase-conjugated antibody against digoxigenin. During Xenopus laevis oogenesis, certain RNAs localize to the animal or vegetal pole laying the foundation for germ cell development and germ layer formation of the future embryo...
March 1, 2018: Cold Spring Harbor Protocols
Xiaoming Zhang, Xiaozhuo Liu, Chengzhang Liu, Jiankai Wei, Haiyan Yu, Bo Dong
BACKGROUND: Metamorphosis takes place within the life cycle of most marine invertebrates. The marine ascidian is a classical model to study complex cellular processes and underlying molecular mechanisms involved in its larval metamorphosis. The detailed molecular signaling pathways remain elusive, though extracellular signal-regulated kinases (ERKs) and c-Jun N-terminal kinase (JNK) have been revealed to regulate cell migration, differentiation, and apoptosis in ascidian larval organ regression and juvenile organ development...
March 1, 2018: BMC Genomics
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