Embryo mounts, whole

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. 

Lyn, S., Giguere, V. 1994. Localisation of CRABP-I and CRABP-II mRNA in the early mouse embryo by whole-mount in situ hybridisation Implications for teratogenesis and neural development. Dev. Dyn. 199, 280-291. 

Chapters 28 and 29 describe the use of nonradioactive probes to detect gene transcripts in thin sections in situ. Chapter 30 describes the use of nonradioactive probes on whole embryo mounts. The probes used in this section are digoxigenin-labeled RNA probes, and the detection is colorimetric, revealing the cell and tissue types that are expressing particular genes. 

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. 

Figure 9.4. Confocal micrographs of wild-type Drosophila embryos. (A) Whole-mount Drosophila embryos stained with propidium iodide to visuahze the nuclear division cycle. Shown are syncytial embryos in nuclear division cycles 1-13 and interphase of nuclear cycle 14 (cellularization). Bar, 100 pm. (B) Surface views of Drosophila embryos prepared by hand devitellinization and double-stained for actin (fluorescein phalloidin, green) and DNA (propidium iodide, red). Interphase actin caps (top panel) and actin-based metaphase furrows middle panel) are shown for embryos in nuclear division cycle 13. Bottom panel) Embryo at cellularization. Actin-based cellularization furrows surround each of the nuclei. Bar, 10 pm. (C) Surface view of a Drosophila embryo in late metaphase/early anaphase of nuclear division cycle 13. The embryo was prepared by hand devitellinization and triple-stained for actin (fluorescein phaUoidin,green), DNA (propidium iodide,pi/rp/e), and the furrow component, Dah (anti-Dah, Cy5-labeled secondary, red). Both actin and Dah locaUze to the furrows and appear to colocalize in some regions yellow staining, inset). Bar, 10 pm. Inset) 2X magnification.

Knratani, S., Tanaka, S., Ishikawa, Y., andZnkeran, C. (1988) Early development of the hypoglossal nerve in the chick embryo as observed by whole-mount nerve staining method. Am. J. Anal. 182,155-168. 

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

Photography and Image data of whole mount embryos after WISH assays can... 

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

Yabu, T., S. Todoriki and M. Yamashita. Stress-induced apoptosis by heat shock, UV and y-ray irradiation in zebrafish embryos detected by increased caspase activity and whole-mount TUNEL staining. Fish. Sci. 

Drosophila embryos are protected both by an outer layer called chorion and an impermeable and opaque vitelline membrane. Therefore preparation of whole mount Drosophila embryos for staining with antibodies and/or other fluorescent markers must go through the following steps chorion removal, fixation, vitelline membrane removal, and membrane permeabilization. The next subsection introduces the basic procedures for embryo collection and chorion removal that are common to all protocols described here, as well as the two most common fixation methods with or without methanol (the latter requiring hand devitellinization of embryos). The first one works well for immunostaining, while the second is ideal for F-actin staining with phalloidin. 

Fig. 1. Examples of fluorescence preparations of Drosophila whole mounts using the protocols is described in this chapter. All confocal images were obtained with a LeicaTCS4D confocal microscope, (a) Confocal optical section of a D. melanogaster embryo whole mount at blastoderm stage double stained with phalloidin—rhodamine (red) and DAPI (blue) to allow simultaneous visualization of nuclei and cortical actin around cell membranes. Anterior is to the left.

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

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... 

Patel N (1994) Imaging neuronal subsets and other cell types in whole-mount Drosophila embryos and larvae using antibody probes. Methods Cell Biol 44 445-187... 

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. 

Electroporation. Whole embryo electroporation in vitro was performed as previously described.1 For targeted regional electroporation (TREP) chick embryos were stained (neutral red) and staged according to Hamburger and Hamilton (HH).3 The vitelline membrane overlying the embryo was removed and a small slit was cut on the yolk membrane near the heart. Platinum electrodes (0.3 mm diameter, at 2.5 mm distance) were mounted on a micromanipulator and positioned parallel to the embryo. 

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