Transgenic reporters for studying development in the chick

The chick embryo has been a major model for the study of vertebrate development since the nineteenth century, particularly as a model for human development as human embryos cannot be studied using any manipulative techniques. The chick embryo is very easy to access as development takes place in the shelled egg, which can be opened to allow observation and manipulation as the embryo develops during incubation. The chick has been used to study the very earliest stages of vertebrate development during which the primitive streak forms, the beginning of the body axis. Other developmental processes for which studies in the chick have been particularly informative, are the development of limbs, the development of muscle and the development of the nervous system, including the brain. The major tissues and organs develop in the chick within a few days of the egg being laid, stages when it is much easier to investigate developmental processes than in other models, for example the mouse. These studies have been aided by the development of a range of techniques for manipulating chick embryos. A major approach has been to study cells in early embryos and follow them as they develop to see which cell types and tissues they generate as the embryo becomes more complex. This has been possible using methods that mark individual cells and the descendants of those cells for a few cell divisions. The disadvantage of this approach is that the mark is gradually lost. A very useful method has been to take small groups of cells from a quail embryo and graft them into a stage-matched chick embryo, replacing the equivalent cells in the chick embryo with the quail cells. The grafted chick embryo is then incubated and the embryo develops normally, incorporating the quail cells. The fate of the quail cells can be determined by staining sections of the grafted embryo to differentiate the quail cells from the chick. This has allowed, for example, the identification of the founder cells for red blood cells. The quail/chick method has limitations as the quail cells can only be detected at the end of the experiment. We have established transgenic chickens that carry a gene that leads to expression of green fluorescent protein in all the cells of the bird, which can be visualised under fluorescent light with no affect on the birds. Cells from embryos from this transgenic line can be used in grafting experiments, as in the quail/chick system, with many advantages over the established method. These include the visualisation of grafted cells in vivo, where the cells can be followed for days, potentially using time-lapse microscopy. There is a significant interest in access to embryos from these transgenic birds from labs in the UK that use the chick model system. We propose to establish a supply of fertile eggs from these transgenic birds to UK chick developmental biologists, who will use this material as a replacement for the quail/chick system. We will generate additional transgenic lines carrying transgenes that express in vivo markers, that will allow further sophistications of the approach outlined above. Firstly we will generate transgenic birds that express ubiquitously a form of GFP that is fused to a peptide sequence that will result in the GFP localising to the cell membrane (mem-GFP). Expression of mem-GFP will allow similar developmental studies but specifically will facilitate observation of cell shape changes during development, often key to developmental process but difficult to observe in fixed material. Secondly, we will generate transgenic lines that express a form of GFP that is activated by laser light. This will allow activation of GFP in single cells of developing embryos, without any possibility of embryo damage, and be very useful for studies of, for example, potential stem cells. Finally, we will generate transgenic birds that express GFP using the regulatory sequences of a gene that is critically involved in early embryo development.

The aim of this project is to develop transgenic lines of chickens that carry fluorescent protein reporters that are expressed ubiquitously in the birds, specifically in embryos. The embryos from the transgenic lines will be used in a wide range of developmental studies in the chick, by ourselves and by other members of the UK chicken developmental biology community. We have established transgenic lines carrying a transgene that includes the CAG enhancer/promoter driving ubiquitous expression of green fluorescent protein (GFP). We will set up a regular breeding programme that will supply approximately 80 dozen CAG-GFP eggs per week. Fertile eggs will be supplied to other laboratories that have expressed an interest in using the embryos for grafting and lineage analysis studies. We will clone a second transgene using CAG to express a membrane-localised form of GFP into an HIV vector and produce germline transgenic birds. Approximately 20 G0 transgenic birds will be raised to sexual maturity and selected G0 males will be bred. Their offspring will be screened to identify 10 hemizygous transgenic G1 birds. The eggs from G1 birds will be examined to determine the level and pattern of memGFP expression. We will then breed for G2 birds to use for production of fertile eggs for the user community. The second transgenic line will use a form of GFP that is photoactivatable (PA-GFP) that can be activated to fluoresce using a laser, enabling single cell labelling in a transgenic embryo. Finally, we will generate a transgenic line that expresses GFP under control of the Brachyury gene regulatory sequences. The chick sequence equivalent to the sequence regulating Brachyury expression identified in several other species, including Ciona, mouse and Xenopus, has been identified in the chicken genome. This reporter line will be used in our laboratories to investigate FGF and Wnt signalling during development.