Chick Module

The chick (Gallus gallus) is an excellent model system to study vertebrate embryogenesis and organ formation. It is one of the major amniote model systems used in developmental biology, the other being the mouse.  Unlike anamniotes (amphibians and fishes), amniotes generally produce very few eggs, which they protect either by a shell or by keeping the embryo inside the mother. The amniotic membrane envelops the embryo to provide a fluid environment protecting the embryo from drying out, while the evolution of the yolk sac endoderm allows the embryo to take up nutrients and oxygen from outside the embryo. These ‘amniote inventions’ allow embryos to grow for a long time in their protected environment before hatching or birth, and unlike anamniotes, to activate the zygotic genome immediately. Unlike the mouse, the early anatomy and tissue organization of chick embryos is very similar to that of human embryos: both initially develop as a flat disc of cells, the blastoderm, and gastrulation occurs through the primitive streak rather than a blastopore.

Fertilized chick eggs are readily available and the equipment needed is minimal – a humidified incubator (380C, no CO2 required), a dissecting microscope, simple microsurgical tools, and either a hand-held mouth pipette or a micromanipulator and picospritzer are used for electroporation and labelling. The eggs can be stored (usually at 150C) for up to 1 week before use.

The chick embryo is easily accessible and comes encased in its own container, the hard eggshell. Through a hole in the eggshell, the embryo can be visualized and easily manipulated with microsurgical tools or gene constructs, the egg is then sealed and allowed to continue development in ovo to determine the consequences of the experimental manipulation. Alternatively, various culture methods are available for early embryos that allow ex ovo growth from stages just after laying to embryonic day 3-5 (or longer) depending on the method.

The main strength of the chick is that the embryos are fairly large, making tissue transplantation and manipulation experiments possible at many different stages, and that gene manipulation can be performed in a temporally and spatially controlled way. “Cut-and-paste” experiments have been used very successfully to determine e.g. tissue interactions controlling organ development, time of commitment etc. paving the way for many fundamental concepts about development. These “cut-and-paste” techniques can be learned relatively easily, and can be combined with molecular gain and loss of function experiments to test both sufficiency and necessity for a molecule of interest or with reporters for gene expression and signaling readouts.

The most important advantage of the chick is that molecular manipulations can be done in a temporally and spatially controlled manner. Gene misexpression or knock down is achieved by electroporation to introduce antisense oligonucleotides (morpholinos), sh-RNA or plasmids containing a cDNA of interest into a particular tissue at a particular time. This allows us to study gene functions relevant to the process under investigation, even for genes that are used multiple times at different developmental stages. This is different from systems where constructs have to be injected into 2-4 cell stage embryos, or from gene inactivation using genetics, where the phenotypes observed at late stages can represent the cumulative effect of many processes gone wrong at different times and in different places. Overall, the speed at which these experiments can be done, owing to the ease of manipulation and the large numbers of eggs that can be obtained, makes the chick embryo a powerful system to test gene function.

More recently, the chick has emerged as a very rapid system to test the activity of regulatory regions, without the need to generate transgenic lines. Electroporation of cell specific enhancer-GFP constructs are also a powerful tool to label specific cell populations and e.g. to isolate such cells for transcriptome analysis or similar molecular approaches.

The genomic resources available for the chick have also increased rapidly. The chick genome has been sequenced and large collections of chicken ESTs are available (http://www.chick.manchester.ac.uk, http://www.chickest.udel.edu). Affymetrix sells chick DNA microarray chips for transcriptome profiling and RNAseq technology is possible from dissected tissue or FACS-sorted cells, making gene discovery much easier. There are also public resources providing useful links to many databases and cataloguing gene expression (http://geisha.arizona.edu/geisha/). With only 1×109 nucleotides the chick genome is more compact than the mouse or human genome (1/3 of the size) containing less repetitive regions and spacers. The chicken evolved 85-90 million years ago from a galliform ancestor (van Tuinen M, Dyke GJ. Mol Phylogenet Evol. 2004, 30:74- 86), yet the tissue and molecular interactions are highly conserved with mammals. Thus, genomic comparisons that include chicken sequence data aid tremendously e.g. in the search for evolutionarily conserved regulatory elements.

A disadvantage of the chick embryo is the relative lack of genetics. Although it has been possible to make transgenic chickens, the time to sexual maturity and the space required to maintain a flock of chickens places it beyond the normal capacity of a lab to rear their own experimental stock. However, GFP-transgenic chicken eggs can now be obtained, different transgenic reporter lines are now available (e.g. Notch-reporter) and several different quail transgenic lines are now available in the USA.

During the course, students will learn in ovo and ex ovo methods, some of the most commonly used transplantation experiments, and various ways of manipulating gene expression. Results will be analyzed using imaging, video time lapse movies, antibody staining, in situ hybridization, or through many other  techniques.