If cell culture has been and remains the preferred material of the cell biologist – essentially because of its ease of use – the cells cultured lose some of their properties: and acquire others. In addition, these same cells represent only a part of a tissue and a fortiori of an organ, and the results obtained under culture conditions cannot be extrapolated to more complex conditions.
Consequently, the researcher had to call upon animal models to solve the problems which were posed to him, the question becoming that of the choice of the model. The main existing systems are described in this text in a non-exhaustive manner, in the form of examples, illustrating their contributions in cell biology.
Why the animal cell?
The privileged material of the cell biologist is the cell in culture, easy to handle and maintain in the laboratory. Cell cultures have made great strides in understanding animal cell architecture, cell division mechanisms, intracellular transport and the organization of different cell organelles. However, these cells, most often immortalized, have adopted a particular behavior which is accompanied, among other things, by the loss of a certain number of properties such as strict control of the cell cycle and the distribution of chromosomes or the setting intercellular communications.
Certain results obtained on these lines may therefore not be found in the much more complex context of a tissue. It then becomes necessary to use a more sophisticated animal model, the choice of which depends on the objective. The history of cell biology research is interspersed with milestones obtained through the development of animal models. This text intends to provide a quick overview of the main existing systems. For each of them, an example is chosen to illustrate the contribution of this model in this scientific discipline. The emergence of a new animal model first comes from the will of an experimenter to solve a problem for which this animal seems particularly suitable.
The survival of the model will depend on its effective capacity to lend itself to biological analysis, since the creation of a scientific community of “a certain weight” working on this model will allow it to become a reference. Originally, the animal model must meet a first essential criterion: to be adapted, at low cost, to laboratory breeding. Depending on the scientific objectives, a certain number of secondary criteria will appear. An example of the choice that the biologist must make in the face of a change in model is perfectly described by François Jacob when he found himself confronted with this problem in 1967: “How to choose among the favorite organisms of embryologists: sea urchin, frog, fly, mouse …? Each of them lent itself to a particular type of experiment, but little or no others.
One day I took a sheet of paper to write down all the properties that I thought it desirable to find in an animal to meet the type of research I wanted to undertake: ease of breeding, speed of reproduction, simplicity of genetic analysis, cell culture, developed physiological studies, easy biochemistry, possibility of studying behavior… It was clear that the animal did not exist. To meet these requirements, a hybrid of frog, sea urchin, fly, etc. was required! ” Today, most of the scientific community works with around twenty animal models of reference.
The mouse, chicken, xenopus and zebrafish represent the four major models for the study of vertebrates. The fruit fly and the Caenorhabditis elegans worm are the most commonly used invertebrate models. Yeast, algae Chlamydomonas or paramecium are the most worked single-cell models. Among the marine animals, we can mention the sea urchin, the starfish, the clam or even the sea squid. The comparison of the size of some young embryos belonging to these experimental systems is presented in Figure 1. The mouse constitutes a model of choice for the study of human diseases, in particular those of genetic origin.
It is not the closest mammal to humans, but one of the smallest and has one of the fastest reproductive cycles (9 weeks). Its breeding is therefore relatively easy, even if a “mouse pet store” is still a complex and expensive structure. The embryonic development is internal, as for all mammals, which does not facilitate its analysis. The mouse, however, lends itself to genetic analysis, in particular through the selective inactivation of certain genes. This technique makes it possible to study the consequences, at the level of an organism, of the suppression of a protein having a cellular function. A recent example concerns the study of the p53 protein.
The corresponding gene has been defined by studies in cultured cells as a tumor suppressor gene, the inactivation of which results in defects in cell cycle control and the development of cancers. The p53 protein is one of the many cellular players involved in verifying the proper segregation of genetic material during the cell cycle. Mice, deprived of this gene following genetic modifications, develop tumors very early. Conversely, animals in which the protein is produced in too large quantities are more resistant to the appearance of cancers.
However, they also show signs of premature aging, as if the cellular anti-cancer mechanisms are partly responsible for aging. This observation could not of course have been obtained by the sole study of these mechanisms in cultured cell lines: the analysis carried out at the level of a living organism therefore provides invaluable additional information. The xenopus is a frog of African origin easy to raise in the laboratory. This species was used in the 1940s in pregnancy tests.
A woman’s urine was injected into a female frog; if the woman was pregnant, the pregnancy hormones in her urine triggered the laying of xenopus. For the cell biologist, xenopus eggs are interesting because they are particularly large and easy to inject, but opaque under the microscope, which represents a handicap for the study of the development of the first embryonic cells. In addition, if embryonic development from fertilization is rapid, the maturation of eggs in the female is extremely long (10 to 12 months), preventing any recourse to genetic manipulation.
However, a single female lays a large quantity of eggs, which can be started to develop artificially, which makes it possible to harvest a perfectly synchronous population for a particular stage of embryonic development. This model is therefore ideal for biochemical experiments which require a significant amount of biological material. It is thanks to this abundant source of proteins that some of the actors of the regulation of the cell cycle (maturation promoting factor and cyclins) have been characterized by Tim Hunt, Nobel Prize in Medicine in 2001.
Drosophila is a fly from 2 to 3 millimeters long with a very rapid development cycle (around fifteen days). Its reproduction was adapted in the laboratory at the very beginning of the 20th century. Thomas Hunt Morgan adopted it as an experimental system in 1909 to clarify the basis for the transmission of different characters from one generation to the next. At that time, it was already suspected that chromosomes were responsible for inheritance, but the factors involved were completely unknown.
Thomas Hunt Morgan and his collaborators, gathered in their laboratory nicknamed “the room of flies”, began to collect individuals whose morphological features differed from those of the original strains. The first Drosophila mutant thus isolated was a fly with white eyes, not red as in the wild strain, a mutation widely used today as a genetic marker. Quickly, Morgan and his collaborators will advance the theory of the “gene”, unit responsible for a given trait, and will discover that these genes are grouped into four classes defined according to the properties of connections to the others, classes representing the four chromosomes of fly. All modern genetics were founded on the basis of this work, and it is no coincidence that Drosophila was one of the first organisms whose genome was fully sequenced; it is now possible to visualize directly at the DNA level the entire genetic map defined by Thomas Hunt Morgan.
Drosophila is a particularly interesting experimental system for biologists. For the geneticist, this system benefits from the possibility of manipulating gene segments by the use of mobile genetic elements, the transposons, used both to create mutations and to detect genes or gene activators. For the cell biologist, the huge collection of mutants available, as well as the possibility of identifying the factors sought by comparison with the genome sequence are precious assets. One of the “flaws” of Drosophila is, however, the difficulty of obtaining sufficient quantities of protein extracts for substantial biochemical analyzes. Two types of yeast share the role of reference model for single-celled cells, whose strength lies in their great capacity to lend themselves to genetic manipulation.
The most widely used is baker’s yeast, Saccharomyces cerevisiae, whose genome was fully sequenced more than five years ago. The project, now completed, to carry out the systematic inactivation of each gene was quickly put in place. This simple experimental system contributed significantly to a better understanding of many aspects of cellular and molecular biology, in particular through the genetic approach implemented by Leland Hartwell (Nobel Prize in Medicine 2001) from the 1970s and 1980s, which helped to better understand the regulation of the cell cycle.
Paul Nurse, third winner of the 2001 Nobel Prize in Medicine, uses Schizosaccharomyces pombe to decipher these same mechanisms of cell cycle control. This yeast has the characteristic of dividing by median fission, unlike S. cerevisiae which divides by budding. By this aspect, and by others such as the structure of its cell cycle, the large size of its chromosomes and their condensation during mitosis, S. pombe has more functional homologies with higher eukaryotes than S. cerevisiae and may, for certain applications, represent a better study model.
The nematode Caenorhabditis elegans represents a recent experimental system, introduced in 1965 by Sydney Brenner (Nobel Prize in Medicine 2002) to study the functioning of the nervous system within a simple organism. C. elegans is a small round worm, one millimeter long, which feeds on bacteria and can be relatively simple grown on a layer of gel in a Petri dish. In addition, the eggs and tissues of C. elegans are transparent, allowing identification and monitoring of cells during development. There are two sexes, male and hermaphrodite.
During their development, hermaphrodites start by producing sperm, which is stored in a spermathèque, then oocytes which will be fertilized on contact with the spermathèque. Males only produce sperm and can fertilize hermaphrodites. Thanks to an as yet unclear property, sperm from the male dominates sperm from the hermaphrodite. The animal’s life cycle is three days at room temperature. A hermaphrodite lays up to 300 fertilized eggs.
One of the goals of Sydney Brenner’s project was to accurately describe the fate of each cell, from the embryo to terminal differentiation. Fortunately, the cell line of this nematode species has been found to be invariant, which is not the case for its cousins: thus, a particular muscle or nervous system cell always comes from the same original cell. At the end of its development, hermaphrodite has exactly 959 somatic cells. Another facet of the project was the systematic description, by electron microscopy, of the architecture of the 302 neurons and the 56 associated cells making up the nervous system. In recent years, the genetics of C. elegans have grown considerably, providing a number of tools for manipulating the six chromosomes of this organism.
Recently, an international consortium has completed the sequencing of its genome. This considerable mass of information collected can advantageously be used thanks to the technique of “RNA interference”, discovered precisely in C. elegans. This method uses the inhibitory effect of the injection of double-stranded RNA (messenger RNA and its image) on the translation of the corresponding protein. This RNA interference technique was later adapted to Drosophila and cultured mammalian cells. More than in any other biological discipline, advances in cell biology depend on the development of techniques: the emergence of new study models, in particular animals, has already allowed, and will allow remarkable progress in the knowledge of the functioning of the cell.