Cell biology and Genetics - course module Cell biology 1

Practical director 2012: Vilma.Rraklli@ki.se

Laboratory exercise in developmental biology

Aim of exercise

To introduce the students to the use of experimental animal models in developmental cell biology. There is an animal free alternative (3D embryo) for those who do not wish to participate in the exercise with chicken embryos.

Goal of exercise

• To gain practical experience in the analysis of chick embryos.
• To analyse chick embryos at different developmental stages.
• To analyse tissue sections of chick embryos which have been stained for the detection of certain proteins.
• To gain practical experience in the documentation of such experiments by hand-drawn illustrations.

Materials

• Chicken embryos at different developmental stages.
• Tissue sections of chicken embryos stained with different methods.
• Light, fluorescence and stereo microscopes.

The chick embryos that are to be used are not prepared especially for this course but are part of on-going research at the department. This is done in order to minimize the number of experiments conducted.

Procedure

The laboratory will be organized in stations with different microscope analysis to be performed at each station. This compendium describes the background and the methodology of the experiments that you will perform. The specific descriptions of the embryos studied on the course including instructions for microscopy will be handed out previous to the laboratory exercise.

Remember to sign the list of presence in the lab (närvarolistan) every day!

Examination

The practical is examined by active presence in the practical.

General introduction

The adult animal originates from a fertilized egg, which is a totipotent (totally undifferentiated) cell. After fertilisation, the egg starts to divide and through a fine balance between proliferation (cell division), cell death and differentiation, the different organs and tissues are formed according to a strict schedule. In this laboratory exercise, we will study chicken embryos that are in this case used in research concerning the development of the nervous system.

The chicken embryo has been historically and is still today an important model organism suitable for experimental embryology. The main reason for the wide usage of the chick is its comparably large size and robustness as well as the fact that it is easily accessible during all stages of development. Other advantages in using chicken eggs are that these are plentiful, inexpensive and extraordinarily convenient compared to for example mice. Embryos from a precise developmental stage can be obtained merely by incubation for a certain time period.

The experimental repertoire of the chick embryo includes surgical manipulations, tissue grafting, explant techniques (culture of tissue in vitro) and electroporation of developing embryos, the latter comprising the basis of this practical exercise. With these unique experimental advantages the chick has made major contributions in our understanding of many basic processes. For example in, early embryonic patterning such as axis determination and left-right asymmetry (e.g. of the heart) and in the formation of the various tissues and organs such as in skeletogenesis, myogenesis, angiogenesis and neurogenesis.

However, studies in chicken cannot meet all requirements in modern research. There is for example no means of creating stable transgenic or knockout animal strains. Also, the genome is not even close from being fully sequenced. Today the genome of the mouse, fruitfly (Drosophila elegans), worm (C.elegans), bacteria (e.g. Streptococcus pyogenes), parasites (e.g. C. fasciculate) and plants (e.g. Tobacco) has been fully sequenced and are all amenable to transgenic/knockout techniques. Moreover, as a model for human disease chick is a very poor candidate as both pig and mouse are more closely related. Note however that the certain advantages stated above make the chicken an important complement to the other model organisms.

Ectopic expression of genes in chicken embryos

Many times one is interested in understanding the function of a certain gene(s) in the development of the biological system in study. This can be achieved by the creation of transgenic models. Through this technique it is possible to completely eliminate the function of a gene, or to induce expression where it is not normally present (ectopic gene expression). Since the complete elimination of overall ectopic expression of genes in the entire organisms may be lethal, several methods have been developed in order to restrain these changes to specific cell types, tissues or organs. In mice or Drosophila this can be achieved by using cell- or tissue-specific promoters to drive gene expression.

In chicken, an efficient method for local gene manipulation has been developed, called electroporation. This method is based on the electric properties of the DNA molecule. As the DNA is negatively charged, it will migrate towards the anode (positive pole) when placed in an electric field. In the case of neural tube studies, if one injects the DNA inside the tube and place the electric poles parallel to it, only half will incorporate the DNA (the tissue close to the anode).

The cathode side of the neural tube is unaffected, thus creating an excellent wild type control (see Figure 1).

Figure 1. The basic principle of electroporation. On the left hand side, the neural tube of the chicken embryo is shown. DNA is injected into the neural tube and electroporated. The DNA moves towards the anode, creating a natural negative control experiment, as shown in the sections on the right.

With the more recent development of RNA interference techniques, which allow the elimination/reduction of gene expression, it is also possible to eliminate the function of genes through electroporation techniques in chicken.

Despite the fact that with this technique no stable transgenic animal is generated, it has the clear advantage of being easy and very fast.

The method of electroporation

In principle, electroporation can be performed at any stage of development depending on the tissue and process being studied.

Unlike the eggs that you can buy in a supermarket, the eggs that are used for these experiments are fertilized. The eggs are stored at 16º C before incubation since this storage temperature will temporarily stop evelopment. This allows us to know exactly when development of the chick embryo will proceed. This also makes sure that all the embryos will be around the same stage of development. The fertilized eggs are incubated at 38º C in a humid standard egg incubator (Figure 2) until they each the desired developmental stage. We usually incubate the eggs for 36-40 h, until the eggs reach what is called stage10.

Figure 2. Egg incubator.

The manipulations of the eggs are done under a stereomicroscope. A hole is opened in the egg to reveal the embryo (Figure 3). Since the embryo itself is transparent, we inject ink underneath it to be able to exactly localize its position. After this, a small amount of DNA is injected into the neural tube using a mouth-pipetting device.

Figure 3. A partially opened egg showing the spot with embryo.

After the DNA injection, the embryo is electroporated (Figure 4). The shell is sealed again and the egg incubated for some time allowing for the expression of the injected DNA. The surviving embryos are dissected and prepared for microscopy. There will be a separate instruction for that on the course.

Figure 4. The electroporation device.