The ability to turn off a gene of interest in a temporally and spatially controlled manner is key to the analysis of gene function in developing organisms. However, the induction of temporally controlled loss-of-function phenotypes is very difficult. In the postgenomic era, the elucidation of the physiological function of genes has in fact become the rate-limiting step in the quest for understanding the development and function of living organisms. Although genes expressed under given conditions, such as healthy versus diseased, can be identified with high-throughput methods, their functional analysis often require the entire organism and is therefore far more challenging and labour-intensive. In this report we show that the Olympus SZX12 stereo microscope with fluorescence illumination proves its worth in such investigations.
For decades the chicken embryo was a classical model system for developmental studies in vertebrates due to its easy accessibility for in vivo studies (Fig. 1). However, its major drawback for functional gene analysis has been the lack of tools to modify gene activity. Now, with the possibility of gene silencing by in ovo RNAi, the chicken embryo has once again evolved as an excellent vertebrate model system. Functional gene analysis has become possible and comes with the added advantage of precise temporal control.
The chicken embryo as model to study gene function during the development of the nervous system
Due to its easy accessibility the chicken embryo has become a favoured model system to study central nervous system development in some research groups (Stoeckli, personal communication). One focus, for example, is on the molecular mechanisms of axonal pathfinding (Perrin et al. 2001, Pekarik et al., 2003; and Bourikas et al., 2005).
Commissural axons in the spinal cord cross the ventral midline, the floor plate, and then turn rostrally along the longitudinal axis. To tackle the question why commissural axons turn rostrally rather than caudally after midline crossing, a screen based on subtractive hybridisation was used to identify candidate guidance cues. Floor-plate cells from embryos of different stages were dissected to isolate two pools of mRNA. Subtractive hybridisation of the derived cDNAs resulted in a multitude of differentially expressed genes representing potential guidance cues. In order to characterise these candidates an assay was developed that enabled the selection of those genes among the candidates that are functionally involved in commissural axon guidance. When RNAi was discovered, it seemed like a perfect tool for this purpose.
Figure 1: In this five day-old chicken embryo imaged with an Olympus SZX12 stereo microscope equipped with fluorescence optics GFP can easily be detected in the spinal cord. Electroporation of a plasmid encoding EGFP under the β-actin promoter was carried out on the third day of incubation.
Due to its tube-like structure the spinal cord forms a reservoir for nucleic acids injected into its lumen. The consistency of the tissue in early chicken embryos allows for diffusion of injected molecules throughout the neural tube, but the basal lamina that forms a tight barrier after the third day of incubation prevents the injected molecules from leaving the neural tube. Therefore, injected molecules are kept at relatively high concentrations around the cells that are to be transfected. Electroporation as transfection method was shown to be effective for gene transfer in chicken embryos (Muramatsu et al. 1997). Using green fluorescent protein (GFP) as a marker showed that 60% of the cells in electroporated areas of the spinal cord expressed the transgene (Fig. 1) (Pekarik et al., 2003). Furthermore, the expression of the transgene could be targeted to specific areas of the spinal cord and to selected cell types by varying the position of the electrodes and the time point of electroporation.
In contrast to reports with cell lines or postnatal mammalian tissue where the transfection of dsRNA resulted in unspecific effects on protein synthesis, no such effects were seen in chicken embryos after in ovo RNAi (Pekarik et al. 2003). Downregulation is efficient and specific for the targeted gene, as for instance other members of the same protein family are not affected.
Gene silencing by in ovo RNAi results in specific loss-of-function phenotypes
The specificity of in ovo RNAi was demonstrated by the functional analysis of known guidance cues for commissural axons in the embryonic chicken spinal cord (Pekarik et al., 2003). The roles of axonin-1, NrCAM, and NgCAM in commissural axon guidance that were known based on in vivo loss-of-function assays at the protein level were confirmed (Stoeckli and Landmesser, 1995). The loss-of-function phenotypes induced by function-blocking antibodies and by in ovo RNAi were identical (Fig. 2). The fact that axonin-1, NrCAM, and NgCAM are related cell adhesion molecules, together with the finding that in ovo RNAi was capable of reflecting relatively subtle differences in growth behaviour of commissural axons demonstrates best the specificity of gene silencing.
Figure 2: In ovo RNAi silences targeted genes specifically and efficiently. For the analysis of axonal pathfinding phenotypes spinal cords are dissected and cut open at the dorsal midline (A). The trajectory of commissural axons can be traced by the lipophilic dye, DiI that is applied to the cell bodies. The boxed area is shown in B-D. In control embryos (B), commissural axons cross the floor plate (indicated by dashed lines) before they turn rostrally along the contralateral floor-plate border (open arrow). When the function of the axon guidance cue Axonin-1 is perturbed either at the protein level by the injection of function-blocking antibodies (C) or by in ovo RNAi commissural axons turn erroneously along the ipsilateral floor-plate border (arrow in C and D) and fail to cross the midline. Figure adapted from Pekarik et al., Nature Biotechnology 21(2003)93-96.
The analysis of pathways taken by commissural axons in experimental versus control embryos can be done in so-called open-book preparations. These are obtained by dissecting out the spinal cord from the embryo and cutting the dorsal midline to open the neural tube like a book. The trajectory of groups of commissural axons was then visualized by the application of the fluorescent dye DiI to the cell bodies (Perrin and Stoeckli, 2000). Mounting the open-book preparations between two cover slips allows the visualisation of commissural axons with an Olympus BX51 microscope equipped with the appropriate filters to detect DiI and GFP.
As an alternative method to analyse axonal pathfinding, especially in the peripheral nervous system, chicken embryos can be stained as whole mounts. Axons can be visualized by antibody staining for neurofilaments, the characteristic cytoskeletal elements of neurons, followed by a fluorescent secondary antibody. Embryos can be analysed after tissue clearing using an Olympus SZX12 stereomicroscope equipped with fluorescence optics (Fig. 3).
The possibility of silencing genes in chicken embryos by in ovo RNAi strongly supports the view that the avian embryo is an excellent model system for developmental studies and a powerful tool for functional genomics. The former limitations in genetic manipulation methods have been resolved by combining in ovo RNAi with electroporation and their application to the easily accessible chicken embryo. This combination allows for the temporal and spatial control of gene expression and is therefore a unique opportunity for developmental studies.
Figure 3: Axonal pathfinding in the peripheral nervous system can be visualized by anti-neurofilament staining of intact embryos. Here an antibody recognizing the 160-kD subunit of neurofilament was used followed by a Cy3-labeled secondary antibody. Tissue was cleared in a graded series of methanol and imaged after transfer to a mixture of benzyl alcohol/benzyl benzoate using an Olympus SZX12 equipped with the appropriate filters.
Temporal control of gene silencing opens new possibilities
In the above mentioned subtractive hybridisation approach and using in ovo RNAi to characterize candidates, Sonic hedgehog (Shh) was identified as a guidance cue that directs axons rostrally along the longitudinal axis of the spinal cord after they have passed the midline (Bourikas et al. 2005). Further analysis indicated that the Hedgehog interacting protein (Hip), rather than the formerly described Shh receptor Patched (Ptc) and Smoothened (Smo), was the mediator of the Shh signal on post-commissural axons. A graded expression of Shh, with high levels in the caudal-most region of the spinal cord, suggested a repulsive signal. Because Shh is a morphogen in early development, then acts as an attractant for commissural axons (Charron and Tessier-Lavigne, 2005) before it switches to being a repellent for post-commissural axons within a short period of time, these studies demonstrate clearly the importance of temporal and spatial control of gene silencing and therefore the power of in ovo RNAi.
References
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