Introduction to C. Elegans
C. elegans as a Model System
Several approaches can be used to study how a biological system functions. You will use a type of genetics during the course of this project. You will use a technique called RNAi (which we will discuss later) to reduce (or eliminate) the function of a single gene, and look to see how that reduction affects the development of the worms. It is just like removing (or breaking!) part of a car and seeing if the car will move. If development is disrupted, you have identified a gene that is required for normal development. Thus, you have defined a function of your gene. Next you would want to do additional experiments to find out what the role of the gene is in development, what it actually does to control or participate in the process. This "second step" can be years or a lifetime of work.
Why study C. elegans?
There are several attractive features that make Caenorhabditis elegans (C. elegans) an ideal organism for the study of gene regulation and function:
C. elegans is a eukaryote, which means that it shares cellular and molecular structures (membrane bound organelles; DNA complexed into chromatin and organized into discreet chromosomes, etc.) and control pathways with higher organisms. C. elegans is a multicellular organism, which means that it goes through a complex developmental process, including embryogenesis, morphogenesis, and growth to an adult. Thus, biological information that we learn from C. elegans may be directly applicable to more complex organisms, such as humans, which are somewhat more refractory to laboratory scrutiny. About 35% of C. elegans genes have human homologs. A homolog is a DNA or protein sequence that is similar to another DNA or protein sequence because the sequences have common ancestry. The definition of a homolog includes common ancestry, but, in practice, we often INFER that two sequences are homologs by similarity of sequence. We believe that the only plausible way for two sequences to share extensive similarity is by having a common ancestry. Remarkably, it has been shown repeatedly that human genes replace their C. elegans homologs when introduced into C. elegans. Conversely, many C. elegans genes can function similarly to mammalian genes.
The C. elegans genome size is relatively small (9.7 x 107 base pairs or 97 Megabases), when compared to the human genome which is estimated to consist of 3 billion base pairs (3 X 109 bp or 3000 Megabases). The entire C. elegans genome has been sequenced. Furthermore, several novel molecular biological techniques exist for C. elegans experimentation that are not available to researchers studying higher eukaryotes. For example, manipulation of the genome by adding, removing, or altering specific genes occurs by relatively routine procedures.
C. elegans is easy to maintain in the laboratory (in petri dishes) and has a fast and convenient life cycle. Embryogenesis occurs in about 12 hours, development to the adult stage occurs in 2.5 days, and the life span is 2-3 weeks. The development of C. elegans is known in great detail because this tiny organism (1 mm in length) is transparent and the developmental pattern of all 959 of its somatic cells has been traced.
General Biology of C. elegans
The nematode Caenorhabditis elegans is a multicellular organism that is utilized as a model system to address fundamental questions in developmental biology, neurobiology and behavioral biology. One of the key strengths of the model system is that sophisticated genetic approaches can be applied to address questions of interest.
Features of the animal.
C. elegans is a small (about 1 mm long as an adult), free living (as opposed to parasitic) round worm. The normal habitat of the animal is in soil, where it feeds on bacteria and fungi. In the lab, it is grown by spreading a layer of E. coli onto a plate and letting the animal feed on the bacterial lawn.
Because C. elegans is so small and because its anatomy is invariant from one animal to the next, it has been possible to describe the anatomy in exquisite detail by performing serial section electron microscopy through the entire animal. By stacking together more than 200,000 minute sections, a three-dimensional picture of the entire animal has been reconstructed! For example, all synaptic connections made by each of the 302 neurons of the animal are known. This is the only animal for which the entire "wiring diagram" has been determined and it makes C. elegans an excellent model for study of neurodevelopment.
Basic Hermaphrodite Anatomy:
Click to enlarge the image.
Sex and the single nematode.
C. elegans exists either as a hermaphrodite or a male. The predominant sexual form of C. elegans is the hermaphrodite — this animal produces both sperm and eggs. Thus, it can self-fertilize. When it does, each animal produces about 300 progeny. The standard lab strain of C. elegans has beem propagated by self-fertilization for many generations. Self-fertilization leads to homozygosity of alleles; therefore, individual worms are considered to be genetically identical (as long as mutations have not occurred).
Click to enlarge the image.
Note that in C. elegans hermaphrodite development there is a developmental switch — first sperm are produced and stored in the spermatheca and then oocytes are produced. Oocyte nuclei are produced by meiotic cell division at the distal end of the gonad. They mature in a syncytium--without complete plasma membranes that separate them from one another. The nucleus and cytoplasm are completely enclosed in a plasma membrane later, just prior to fertilization. After fertilization, the eggshell is added. Fertilization takes place as maturing oocytes are squeezed through the spermatheca. Eggs develop in the hermaphrodite body briefly and then are laid through the vulva at about the 40-cell stage.
Here is a closer look at the organization of the hermaphrodite gonad:
Hermaphrodites have about 10 eggs inside--the
older eggs are laid about as fast as new eggs are made.
Oocytes pass into the spermatheca and are fertilized. Embryos develop in the uterus, and have a few cleavages before the eggs are laid, so the embryos have a few dozen cells when the embryos are laid. The embryos develop into worms over the next 8-18 hours (depending on temperature).
Chromosomes and males.
There are six chromosomes in C. elegans — five pairs of autosomes (chromosomes I, II, III, IV, V) and the sex chromosome, X (this is the letter X, not the Roman numeral ten). Hermaphrodites have two X chromosomes (designated XX). Males have one X chromosome (designated XO); having only one chromosome instead of a pair is called the hemizygous state. This state can be produced by the loss of one X chromosome or by mating. Males cannot produce progeny on their own. However, they can cross-fertilize hermaphrodites. They are commonly used in C. elegans genetics for making genetic combinations.
One of the advantages of working with C. elegans is that it has a short life cycle. The life cycle is temperature-dependent. C. elegans goes through a reproductive life cycle (egg to egg-laying parent) in 5.5 days at 15°C, 3.5 days at 20°C, and 2.5 days at 25°C.
C. elegans eggs are fertilized within the adult hermaphrodite and laid a few hours afterward--at about the 40 cell stage. Eggs hatch and animals proceed through 4 larval stages, each of which ends in a molt. When animals reach adulthood, they produce about 300 progeny each. They live a total of about 2 weeks.
Note that C. elegans can adopt an alternative life form, called the dauer larval stage, if plates are too crowded or if food is scarce. Dauer larvae are thin and can move but their mouths are plugged and they cannot eat. Interestingly, dauers can remain viable for three months. They appear to be non-aging: dauer larvae can roam around for months and then reenter the L4 stage when they encounter a food source and live about 15 more days! Think about it--those worms can live nearly 10 times their normal lifespan!
C. elegans development.
C. elegans development is characterized better than any multicellular organism the complete cell lineage of the animal has been recorded. A cell lineage is a description of all the cell divisions that occur to generate a specific group of differentiated cells (in the case of C. elegans, the entire animal!). In other words, the developmental pattern of each somatic cell is known, from the zygote to the adult worm. Thus, a scientist can identify any cell at any point in development, and know the fate of that particular cell.