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Genes in Development
RE-READING THE MOLECULAR PARADIGM
Duke University Press
Copyright © 2006 Duke University Press
All right reserved. ISBN: 978-0-8223-3656-3
Chapter One
GENOME ANALYSIS AND DEVELOPMENTAL BIOLOGY The Nematode Caenorhabditis elegans as a Model System
THOMAS R. BÜRGLIN
A general and basic tenet of scientific research is to simplify a complex problem to smaller, more tractable units that can be studied and unraveled. Depending on the biological question, scientists choose particular organisms as model systems. Each model provides researchers with particular biological or experimental advantages that help them in their quest to understand fundamental biological principles and problems. In 1963 Sydney Brenner wanted to proceed from bacterial and viral genetics to a more complex, multicellular animal. He proposed studying a small nematode that he thought would be eminently suitable for investigating many aspects of cell and nervous system development. After careful deliberation he chose Caenorhabditis elegans, and with this model he succeeded in establishing a whole new research field. As a result, "founding father" Sydney Brenner and two other C. elegansresearchers, John Sulston and Bob Horvitz, were awarded the Nobel Prize in Physiology or Medicine in 2002. John Sulston made key contributions to elucidating the C. elegans cell lineage as well as to its genome project, and Bob Horvitz contributed significantly to the understanding of programmed cell death. I became attracted to this model system because of its elegance and other advantages I will outline below.
The little worm will serve here as an introduction to how researchers study genes and understand their function in the context of a living organism. This chapter will first introduce the advantages of the C. elegans model system (see also the key textbooks by Wood [1988] and Riddle et al. [1997]), and then will proceed to the C. elegans genome, where the principle of gene function via proteins is introduced (for a key textbook on molecular biology, see Alberts et al. 2002). Subsequently, I will talk about a particular group of genes that regulate other genes-the homeobox genes-and demonstrate how we study the function of particular genes in the worm. I hope that this exposition will remove at least some of the mystical connotations that the term gene has acquired in recent times and reveal the true beauty of the gene and the genome.
The Biology of Caenorhabditis elegans
C. elegans belongs to a group of animals called nematodes. Nematodes are roundworms or threadworms with smooth-skinned, unsegmented, long-cylindrical bodies. There are both free-living and parasitic forms, and they can be found in both aquatic and terrestrial environments. Quite a number of parasitic nematodes are known to afflict human beings: it has been estimated that as much as 25 percent of the world's population is infected by some type of parasitic nematode. C. elegans is a small, free-living nematode found in temperate regions in the soil, where it feeds on bacteria (fig. 1).There are two sexes: self-fertilizing hermaphrodite animals and male animals. Both are small, the adult hermaphrodite being a little larger and reaching a size of 1.2 mm when fully grown.
Because of its small size and simple food requirements, C. elegans can be easily reared in the laboratory on agar plates seeded with a lawn of bacteria such as Escherichia coli. The life cycle of C. elegans is extremely fast: it takes only about three days from the time a young adult starts to lay eggs until the next generation has grown and starts laying its own eggs. Development proceeds through several stages: embryogenesis, four larval stages (termed L1, L2, L3, and L4), and the adult stage. This extremely rapid reproductive rate is unique among multicellular animals. Each hermaphrodite animal can produce up to three hundred offspring, so a single animal on an agar plate can produce thousands of first- and second-generation offspring in about a week. C. elegans is thus an ideal model system for genetic studies because a large number of offspring can be analyzed in a very short time.
Apart from easy maintenance, small size, and fast and plentiful reproduction, C. elegans offers many other advantages that have made it an excellent model system for modern biologists. In part because of their small size, the embryos, larvae, and adult animals are transparent when viewed under a microscope, allowing researchers to identify all the different cells (in fact, the cell nuclei) and making it possible to follow the cell divisions of embryo-genesis and larval development. The fact that the cell division patterns are remarkably reproducible from embryo to embryo permitted John Sulston and his co-workers to establish the complete cell lineage for C. elegans. That means we know exactly how each cell divides during development, what it gives rise to (e.g., a neuron, a muscle cell, or a gut cell), and what its relatives are. Thus, we know that the adult hermaphrodite animal has 959 somatic cell nuclei, and the male animal has 1,031. These cells can be classified into about 150 different cell types. The greatest complexity of cell types is found in the nervous system, which consists of 302 neurons and 56 glia and associated cells in hermaphrodites, and 381 neurons and 92 glia and associated cells in males. The 302 neurons of the hermaphrodite include at least 118 different types. John White and his co-workers examined serial sections of complete animals by electron microscope and analyzed their neuronal connections, and succeeded in elucidating the complete wiring diagram of the nervous system. All of this information constitutes an unparalleled fund of knowledge about a complex multicellular animal.
Despite its small size, the nervous system of C. elegans has an astounding complexity which permits the animal to respond to many different environmental cues. The animal has a large set of behaviors that allow it to survive and propagate: it pumps food with its pharynx ("the throat"), expels digested food through the anus, retracts when it bumps into an object with the tip of its head (the nose), and moves forward or backward in sinusoidal waves to move toward or away from particular stimuli such as chemical attractants or repellents, touch, heat, or fine temperature differences. The hermaphrodite lays eggs through the vulva, while the male animal has a specialized set of behaviors that enable it to find hermaphrodites and fertilize them with its specialized tail structure. Researchers are now using genetic and molecular tools to unravel the function of the nervous system to understand how the worm's behavior is controlled by genes.
The Molecular Approach and the Genome of C. elegans
The size of the haploid genome of C. elegans-that is, the DNA contributed by one parent (an organism is usually diploid, having DNA from both parents)-is fairly small, consisting of 100 million base pairs distributed on five autosomes (i.e., "regular" chromosomes not involved in sex determination) and one X chromosome (i.e., the sex-determining chromosome). Short pieces (about 30,000 to 300,000 base pairs) of C. elegans DNA were cloned into bacterial or yeast vectors so that the DNA could be amplified, mapped, sequenced, and distributed to researchers. Clones covering the complete genome are available from the Genome Center, either as cosmids (essentially, special types of plasmids which are grown in E. coli bacteria) or as cloned YACS (yeast artificial chromosomes, which are grown in yeast). These clones were mapped and used to establish a physical map of the genome of C. elegans. In mapping, the relative order of these clones was established to determine where precisely each clone lies with respect to other clones and where these clones are located on the chromosomes. After the clones had been placed in order they were sequenced. Since the end of 1998 the virtually complete genomic sequence of C. elegans has been available; and the finalized, totally gap-free sequence has been available since late 2002. At the time of this writing C. elegans is the only multicellular animal for which this can be said. The delay in achieving the complete sequence lay in the fact that some pieces of DNA are particularly difficult to clone and sequence, a general problem not restricted to C. elegans. Usually such DNA does not contain many important genes; these hard to clone regions are often highly repetitive in nature and may be nonfunctional "junk" DNA, or may contribute to chromosomal structure.
Dispersed among the long strands of DNA that make up the chromosomes are the genes. Most genes are pieces of DNA that are transcribed and translated into proteins, although some genes are transcribed only into RNA. A protein-coding gene usually consists of several stretches of chromosomal DNA called exons that are separated by introns, although some genes have no introns (fig. 2). When a gene is to be activated in a particular cell, specific transcription factors sit down on the DNA in the regulatory region, also referred to as "gene control region." Transcription factors have the ability to recognize very specific short sequences of DNA, and each type of transcription factor has a specific sequence (called a "binding site" or "enhancer element") that it recognizes. The extent of a gene control region is defined by all the different binding sites and other regulatory DNA elements that are necessary for the correct regulation of the gene. Thus a control region can be small or large, depending how far the regulatory binding sites are spread out on the chromosome.
The transcription factors binding in the control region make the gene accessible so that the enzyme RNA polymerase can bind to the promoter and transcribe the DNA of the gene into precursor RNA (fig. 2). Thus, genetic information is converted from DNA into RNA. The precursor RNA has to be spliced so that the intron sequences, which do not code for proteins, are removed and only the exons are left. The spliced RNA-the messenger RNA, or MRNA-is then translated by the ribosome into a chain of amino acids: the protein. The ribosome binds to the start codon of the protein-coding region in the MRNA and translates the MRNA into a string of amino acids (the protein) until it reaches the stop codon. When a gene is "turned on" or "switched on" by the process outlined above, then this gene is said to be "expressed."
An enzyme called "reverse transcriptase" can convert MRNA back into DNA; this is mainly done in the laboratory for cloning purposes. The DNA thus formed is called complementary DNA (CDNA). Randomly cloned and sequenced CDNAS are also called expressed sequence tags (ESTS) because they can be used as "tags" for expressed genes.
A mutation in the chromosomal DNA can destroy or affect the function of the final gene product, the protein, at many levels. For example, mutations in the regulatory region will lead to levels of the gene product that are either too high or too low (too much or too little activity). Mutations in introns may cause inappropriate splicing that results in MRNA that will not be translated properly and thus produces aberrant proteins. Mutations within the exons can change individual residues in the protein or introduce a premature stop codon, while deletion mutations can remove whole parts of a gene. (This is done in the laboratory to produce gene knockout mutants in C. elegans, as discussed below). Large deletions often lead to a loss of function, but smaller deletions or changes in individual residues can lead to hyperactivity of the gene product.
Proteins can have many functions: some are structural components, some catalyze chemical reactions, some fight disease, and some transport molecules through cells. Examples of structural genes are the components of hair or of the minute fibers that constitute the skeleton of a cell. Catalysts of chemical reactions are called enzymes; among other things, enzymes digest food, generate energy, and synthesize signal molecules in neurons (neurotransmitters). Proteins such as insulin are hormonal signals, while proteins called antibodies are produced by the immune system to inactivate microorganisms such as bacteria or viruses. The well-known protein hemoglobin in blood transports oxygen through the body. Proteins may not be active immediately after they are synthesized; some need to be chemically modified (e.g., by attachment of sugars or phosphate) or cleaved into fragments, and some must bind to other proteins to form an active complex.
Although most genes produce proteins, there are some exceptions. A few produce only RNA molecules; the biochemically active entity is the RNA molecule itself, which is not translated into a protein. Examples are the ribosomal RNAS. The ribosome, mentioned above, is a large complex made up of many proteins and ribosomal RNA molecules.
Finding genes in genomic DNA sequences is not a trivial task. C. elegans has a considerable advantage over other organisms here, especially mammals, because the junctions between introns and exons (the splice sites) are quite highly conserved. In conjunction with other characteristic features, this allows the protein-encoding genes to be identified by computer methods (bioinformatics). While computer prediction alone is not always sufficient to identify genes, it is often an excellent starting point for experimental work. The analysis of thousands of ESTS has allowed the identification of expressed genes and helped refine the computer prediction programs for genes. We know now that C. elegans has about 20,000 genes in its genome.
How Cells Become Different
The basic question developmental biologists seek to answer is: How do the different cell types develop from a single cell? All of the cells of a particular organism, whether muscle cells, skin cells, or gut cells, have the same number of chromosomes and thus the same number of genes. And yet, differentiated cells do different things. Neurons produce specific neurotransmitters, muscle cells make proteins that can contract to produce movement, blood cells make hemoglobin, and so on. How is this achieved? Earlier I described how genes are turned on (or off) by specific transcription factors sitting down in the promoter region. Let us now consider a hypothetical case in which a "mother" cell divides into two daughter cells. The anterior one becomes a neuron, the other becomes a skin cell (fig. 3).
Within the mother cell there is a protein, say n, which is an inactive transcription factor waiting to be activated (through phosphorylation, for example). When the mother cell divides, the anterior most daughter cell touches other cells located anteriorly. This releases a surface signal which activates (in this case phosphorylates) the inactive transcription factor n. The active factor n can now bind to the specific binding sites it recognizes in the regulatory regions of its target genes. The genes activated by factor n could be neuronal growth factors, neurotransmitters, or other genes which turn the cell into a neuron. In the posterior daughter cell, factor n is not active, but perhaps another factor, S, that turns on skin cell-specific genes is activated (not shown in fig. 3). While this scenario is rather simplistic, it illustrates the principle of how cells become different. In reality, hundreds of factors regulate cell fate and cell differentiation, some factors regulate others, and transcription factors can have feedback loops to their own regulatory regions. Thus, complicated cascades of transcription factor events turn different genes on and off during the development of an organism to generate the vast diversity of cell types found in the adult animal.
(Continues...)
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