Whole embryos, in-situ hybridization

The CK library membrane-associated embryonic cDNA clones are in general not full length, but have been analyzed by whole-embryo in situ hybridization. Data from this library can be queried for clones expressed in particular tissues. 

Large-Scale Whole Mount In Situ Hybridization of Mouse Embryos... 

Hume DA, Monkley SJ, Wainwright BJ. Detection of c-fms protooncogene in early mouse embryos by whole mount in situ hybridization indicates roles for macrophages in tissue remodelling. British Journal of Haematology 1995, 90, 939-942. 

Rosen B, Beddington R (1993) Whole-mount in situ hybridization in the mouse embryo gene expression in three dimensions. Trends Genet 9 162-167... 

Bell GW, Yatskievych TA, Antin PB (2004) GEISHA, a whole-mount in situ hybridization gene expression screen in chicken embryos. Dev Dyn 229 677-87. Sang H (2004) Prospects for transgenesis in the chick. Mech Dev 121 1179-1186. Petitte JN, Liu G, Yang Z (2004) Avian pluripotent stem cells. Mech Dev 121 1159-1168. 

As the notochord is crucial for the development of the surrounding tissues but can be replaced by the floor plate of the neural tube (Fig. IB and IE), once this structure is established, the effect of notochord ablations varies depending on the type and timing of the operation. It is therefore advisable to analyze the embryos for the process of interest, but in addition for a known phenotype of the particular manipulation. This can be achieved, for instance, by double whole mount in-situ hybridization (see Fig. 1), employing molecular markers... 

Fig. 2. In-ovo electroporation. A pair of electrodes held by a manipulator (A) is inserted intom a window opened on the shell (B). The electrode is placed on the vitelline membrane overlying the embryo (C), and a 25-V 50-ms pulse is charged five times. The entire procedure is monitored under a dissection microscope. Plasmid solution is injected to the E2 (HH stage 10) chick neural tube (D) prior to the pulse charge.The dimensions of the electrode are shown in E. Most of the electrode is insulated (black in the figure) so that only the tip is exposed (white area). One hour after electroporation, some embryos were fixed, processed for paraffin sectioning, and observed with a Nomarski interference microscope (F). The right-hand side of the figure corresponds to the right of the embryo, where injected plasmid was transfected. The morphology of the cells and the structure of neural tube were almost normal. The blue deposit inside the neural tube is a complex of plasmid and the color substrates not removed by washing in dimethylformamide after whole-mount in-situ hybridization. Twenty-four hours after electroporation, the development of yolk sac plexus, vitelline veins, and vitelline arteries are retarded in the area contacted on the electrodes (arrows in G). Bar is 2mm (C) 50 pm (F) 4mm (G). Source (3).

Jowett, T. and Lettice, L. (1994) Whole-mount in-situ hybridizations on zebrafish embryos using a mixture of digoxigenin-labeled and fluorescein-labeled probes. Trends Genet. 10,73,74. 

Wilkinson DG (1992) Whole mount in situ hybridization of vertebrate embryos. In Wilkinson DG (ed) In situ hybridization, a practical approach. IRL Press, Oxford University Press, Oxford, UK. 

Whole-mount in situ hybridization was first used to detect gene expression in Drosophila embryos (1). Various methods are now used to localize mRNAs in most species used for biological studies, and the methods have proven particularly useful when applied to vertebrate species. Here we provide a protocol commonly used for localizing transcripts in Xenopus embryos. The method is applicable to whole embryos and intact tissue explants. The methods are essentially as originally described by Hemmati-Brivanlou et al. (2) and then modified by HarlandfJ). 

Tautz, D. and Pfeifle, C. (1989) A non-radioactive in situ hybridization method for the localization of specific RNAs in Drosophila embryos reveals translational control of the segmentation gene hunchback. Chromosoma 98,81-85. Hemmati-Brivanlou, A., Frank, D., Bolce, M. B., Sive, H. L., and Harland, R. M. (1990) Localization of specific mRNAs in Xenopus embryos by whole mount in situ hybridization. Development 110,325-330. 

Hauptmann, G andGerster, T (1994) Two-color whole-mount in situ hybridization to vertebrate and Drosophila embryos. Trends Genet. 10, 226... 

Nonradioactive in Situ Hybridization Simplified Procedures for Use in Whole Mounts of Mouse and Chick Embryos... 

Stern CD (1998) Detection of multiple gene products simultaneously by in situ hybridization and immunohistochemistry in whole mounts of avian embryos. Curr Top Dev Biol 36 223-243... 

Singer and colleagues (68, 69) have successfully visualized cytoskeletal mRNAs and their associate proteins using biotinylated cDNA probes followed by antibodies to biotin and collodial gold-conjugated antibodies. Their method used in situ hybridization followed by whole mount TEM of Triton-extracted chicken embryo fibroblasts. Cytoskeletal mRNAs were found in close proximity to actin protein and further from tubulin filaments. While the whole mount technology does limit the technique to extracted cells, applications to thin sections will allow greater resolution. 

Peripheral axotomy caused a decrease of Y1 mRNA in the small neurons and an increase in large neurons. These results suggested that the DRG Y1 receptor is mosdy a prejunctional receptor in primary afferent neurons and that it may play a role in the modulation of somatosensory information (Zhang et al., 1994). Low levels of Y1 mRNA have also been reported for rat splenic lymphocytes as documented by a partial cDNA clone, PCR and RNase protection (Petitto et al., 1994). A developmental study by in situ hybridization of whole rat embryo sections (Jazin et al., 1993) detected the earliest Y1 mRNA around day 14, both in diencephalon and spinal cord. 

A EXPERIMENTAL FIGURE 15-19 Maternally derived bicoid mRNA is localized to the anterior region of early Drosophila embryos. All embryos shown are positioned with anterior to the left and dorsal at the top. In this experiment, In situ hybridization with a radloactively labeled RNA probe specific for bicoid mRNA was performed on whole-embryo sections 2.5-3.5 hours after fertilization. 

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