3-DIMENSIONAL REPRESENTATION OF WNT PATHWAY GENE EXPRESSION IN THE MOUSE EMBRYO
Wnt signalling pathways are involved in controlling patterning and growth in a wide variety of developing systems (reviewed in Cadigan and Nusse, 1997; Nusse, 2005). Understanding their mode of action is relevant to birth defects, regeneration and oncogenesis. To analyse the role of any signalling pathway in these contexts it is necessary to bridge the gap between the study of the individual genes in the pathway and a more integrated view of multiple components; at the level of gene expression this includes how the localisation of the relevant gene products relate to the changing morphology of the embryo across time.
These pages archive and present the results of a gene expression survey focussed on components of the Wnt signalling pathway across key stages of mouse development. The study, funded by Science Foundation Ireland, presents full 3D data in carefully stage-matched embryos that allow cross comparison between specimens and gene patterns. The full 3D data are entered in the Edinburgh Mouse Atlas of Gene Expression (EMAGE).
Navigate the entries through the page links at the top that categorise the data based on gene family; Wnts and Frizzleds (Tcf/Lef and Sfrp pages are under assembly). From there you can select a gene of interest. Data is currently presented for Theiler stages (Ts) 15, 17 and 19. In addition, each gene page gives details of the hybridisation probe used to reveal expression and a list of expression sites. The passages below give some information about the methodology used to generate the data and a description of how the data is presented.
In situ hybridisation was carried out on whole embryos using digoxygenin labelled RNA probes largely as per Xu and Wilkinson, (1998), detailed in Summerhurst et al (under revision). A minimum of two independent hybridisations with 5 embryos per probe were carried out for each gene where the expression patterns were very clear; for more difficult patterns up to 6 hybridisations were carried out often altering the probe used (probe details given with each entry). The staining procedure was optimised for use in 3D analysis by Optical Projection Tomography (Sharpe et al 2002 and described below). For the best OPT reconstruction data the intensity of colourometric stain should be moderate. Also a low level of background staining of the tissue (so the tissue appears vaguely pink) was found to be helpful in viewing OPT data captured in the visible channel alone as this allows the tissue context to be just visible when the full spectrum of grey level data is viewed. Staining components were diluted to 175 µg/ml 4-Nitro blue tetrazolium chloride and 62.5 µg/ml 5-Bromo-4-chloroindolyl-phosphate as a standard staining solution and staining was allowed to develop slowly with careful monitoring. For strong signals the above solution was diluted up to 1/10. Different intensities of staining were tested for each gene to ensure maximum capture of the data.
The CD1 outbred mouse strain was chosen for the establishment of this Wnt pathway gene expression database in order to represent the normal expression pattern of the genes. No obvious variability in expression pattern was noted between specimens.
OPT scanning and 3D reconstruction: At least two perfectly intact specimens from each hybridisation, representative of the externally visible pattern, were selected for OPT scanning. They were embedded in 1% low melting point agarose, dehydrated in MeOH overnight and cleared in Benzyl benzoate/ Benzyl alcohol (1:2) (BABB) for at least 5 hours (as previously described, Sharpe et al. 2002). Projection images of the specimens were captured in a prototype OPT scanner constructed at the MRC Human Genetics Unit, Edinburgh (Sharpe et al., 2002) and installed in the Zoology Department Trinity College Dublin. At least two scans were performed for each specimen using visible light either with or without a 700nm longpass filter, depending on staining intensity, to capture the expression pattern and under UV light using either a TXR filter (560/40nm excitation, 610LP nm emission) or a GFP1 filter (425/60nm excitation, 480 nm emission) to capture autoflouresence from the tissue to reconstruct embryo morphology. The raw data (400 projected images) from each of the scans were loaded onto a Linux workstation, reconstructed using a set of programmes provided by the Edinburgh Mouse Atlas Project (EMAP) and analysed using custom made software (MA3DView and MAPaint), again provided by EMAP.
Data presentation:
For each gene entry, external views of the full 3D specimen rotated around the anterior-posterior axis are denoted as "3D movies". For all entries the whole embryo data is presented as grey level where the expression sites are visible as white/light grey domains within the just visible dark grey representation of the morphology. In some cases the data is also presented as a merge of two scans, one recorded in visible light (pseudocoloured in red or blue) showing the expression domains and the other recoding autoflouresence under UV light (pseudocoloured in green or yellow) showing embryo morphology. These methods can emphasise different aspects of the pattern. Simple grey level representation of expression shows subtle details such as differences in level of intensity and very fine spatial restriction that is sometimes obscured in images that use strong pseudocoloration. Pseudocolored images, however, often show more clearly the morphological context of expression. In each case the method of presentation was chosen to best show particular features of the pattern.
For genes showing specific limb bud expression a separate movie focussing on the cropped forelimb is also shown.
References:
Cadigan, K.M. and Nusse, R. (1997) Wnt signaling: a common theme in animal development. Genes Dev 11, 3286-305.
Nusse, R. (2005) Wnt signaling in disease and in development. Cell Res 15, 28-32.
Sharpe, J., Ahlgren, U., Perry, P., Hill, B., Ross, A., Hecksher-Sorensen, J., Baldock, R. and Davidson, D. (2002) Optical projection tomography as a tool for 3D microscopy and gene expression studies. Science 296, 541-5.
Summerhurst K., Stark M., Sharpe J., Davidson D., Murphy P. (2008) 3D representation of of Wnt and Frizzled gene expression patterns in the mouse embryo at embryonic day 11.5 (Ts 19). Gene Expression Patterns 8, 331-348.
Xu, Q. and Wilkinson, D. (1998) In situ hybridisation of mRNA with hapten labelled probes. In Wilkinson, D.G. (ed.), In Situ Hybridisation: A Practical Approach, 2nd edition, Oxford University Press, Oxford, pp. 87-106.