Corsi_2015: A transparent window into biology: a primer on caenorhabditis elegans


C. elegans

A transparent window into biology: a primer on caenorhabditis elegans


1. Introduction

In 1963, Sydney Brenner sent a letter to Max Perutz

"classical problems of molecular biology have either been solved or will be solved in the next decade” and proposing that the future of molecular biology lies in the extension to other fields, “notably development and the nervous system”

Lots of Fluorsecnt protein use

These features have helped to make C. elegans a powerful model of choice for eukaryotic genetic studies

C. elegans has a rapid life cycle (3 days at 25° from egg to egg-laying adult)

exists primarily as a self-fertilizing hermaphrodite, although males arise at a frequency of <0.2%

Has an invariant number of somatic cells

Reconstructed the shape of all C. elegans cells from electron micrographs, including each of the 302 neurons of the adult hermaphrodite

Invariant wild-type cell lineage and neuroanatomy of C. elegans, mutations that give rise to developmental and behavioral defects are readily identified in genetic screens

C. elegans was the first multicellular organism with a complete genome sequence

experimental strengths and the similarities between the cellular and molecular processes present in C. elegans and other animals across evolutionary time

At least 38% of the C. elegans protein-coding genes have predicted orthologs in the human genome

60-80% of human genes have an ortholog in the C. elegans genome

40% of genes known to be associated with human diseases have clear orthologs in the C. elegans genome

2. C. elegans basics

2.1. Growth and maintenance

Isolated from rotting vegetable matter, which contains an ample supply of their bacterial food source

They're not actually a soil nematode

Grown on agar plates containing a lawn of the bacterium Escherichia coli

Once they deplete the bacteria they utilize their fat supply

Without food, the development of young larval stage animals is arrested

As a result of entering this stasis, animals can survive for at least a month (often starved plates can be usefully kept for up to six months at 15°), and as stocks, they do not require constant feeding.

Features that greatly facilitate the maintenance of C. elegans stocks

1. Self-fertilizing hermaphrodite, a single animal can populate a plate
2. Animal populations can be frozen for years and revived when needed
3. Animal's small size means that many can be grown in a small space
4. Animals can be grown at temperatures ranging from 12° to 25°
This means you can use temperature-sensitive mutants
growth above 25° is not possible because the animals become sterile
Shorter exposures to higher temperatures are possible for heat shock experiments and to increase production of males
5. Animals can be synchronized by isolating newly hatched larvae or by treating gravid adults with bleach
6. To facilitate biochemical studies animals can be grown in bulk in liquid medium

2.2. Sexual forms and their importance

Wild-type C. elegans has two sexual forms: self-fertilizing hermaphrodites and males

Hermaphrodites are females whose gonads temporarily produce sperm before they produce oocytes
Hermaphrodites can produce up to 300 self-progeny
If mated with males, hermaphrodites are capable of producing ~1000 offspring
Sexes differ in that hermaphrodites have two X chromosomes and males have a single X chromosome

Males are reffered to as XO

Self-fertilizing hermaphrodites provide several advantages for genetic analysis

1. Self-fertilization (often referred to as selfing) simplifies maintaining stocks because a single animal can give rise to an entire population
2. The animals are driven to homozygosity
3. Selfing follows the standard Mendelian rules of segregation, so a parent that is heterozygous for a recessive trait will produce the standard 1:2:1 pattern of segregation, such that 25% of the progeny will be homozygous for the mutant allele and display the autosomal recessive trait
4. Mutants with neuromuscular defects that impair the ability to mate can still be maintained in the laboratory
5. The viability of even severely defective mutants and their ability to self-fertilize allows for easy screens for modifier (enhancer and suppressor) mutations

lin-12 lin-12 mutants are defective in vulval development and components of the LIN-12/Notch signaling pathway have been identified with both suppressor and enhancer screens

TODO Box 1. C. elegans Nomenclature

2.3. Life cycle

C. elegans embryogenesis takes approximately 16 hours at 20°

A virtually impermeable eggshell is made after fertilization

Embryos are usually retained within the hermaphrodite until about the 24-cell stage at which time they are laid

Hermaphrodite embryo hatches with 558 nuclei

The animals begin to eat and develop through four larval stages (L1-L4)

12 hr after the L4 molt, adult hermaphrodites begin producing progeny for a period of 2-3 days

After the reproductive period, hermaphrodites can live several more weeks before dying of senescence

Aka getting old

Alternative L3 larval stage called the “dauer” larva

Dauer larva cuticle

3. C. elegans genetics

Self-fertilization means that after hermaphrodites (P0s) are mutagenized, any mutant alleles (except dominant lethals) can be maintained through self-propagation in first-generation (F1) progeny, second-generation (F2) progeny, etc. without mating

Genetics in C. elegans uses forward genetics

A variety of mutagens have been used

Ethane Methylsulfonate (EMS)

An alkylating reagent that causes principally GC-to-AT transition mutations and small deletions

The entire process of cloning a gene could easily take a year using classical gentic tools

Back in the day

The process of connecting a mutant phenotype to a gene is much more rapid due to advances in whole-genome sequencing

Researchers can also use a known gene sequence to obtain mutant strains, a process called "reverse genetics"

The generation of strains that deleted all or part of a target gene

Million mutation project

Recent advances in efficient genome-editing methods (TALEN and CRISPR/Cas9) in C. elegans now allow investigators to create targeted mutations in nearly any location in the genome in any genetic background

Mutant-like phenotypes can also be obtained using RNA interference (RNAi)

4. Why choose C. elegans?

C. elegans has many inherent advantages as a model for eukaryotic biology

C. elegans is the organisms are quite benign to humans

Studies of cell and developmental biology that use C. elegans are greatly aided by the transparency of the animal

Greater control of the animal's position and environment can be accomplished by microfluidic devices in which individual worms are mounted in custom-designed channels
Figure 4. Anatomy and study of the C. elegans nervous system

Sharing large amounts of genetic and cellular information has been central to the success of C. elegans research

C. elegans has some limitations

Not all metazoan genes are found in the C. elegans genome
Hedgehog (Hh) signaling is important in vertebrates for the patterning of various organs during development, but C. elegans lacks many of the genes in the regulatory cascade


Some C. elegans cells can be studied in vitro
No C. elegans cell culture lines exist
Small size of the animal and its cells also provides a challenge since experimental manipulation in individual tissues of an organism that is less than a millimeter long is difficult
Electrophysiology of C. elegans neurons and muscle is possible, but demanding

Indirect measurements of neuronal activity, such as calcium imaging are often used instead

Biochemical approaches in C. elegans have lagged behind the genetic approaches
The development of an axenic culture medium for C. elegans has meant that biochemical studies can be done on animals under defined conditions.

TODO 5. C. elegans tissues

TODO 5.1. Epidermis: a model for extracellular matrix production, wound healing, and cell fusion

TODO 5.2. Muscles—controlling animal movement

TODO 5.3. The digestive system—a model for organogenesis and pathogenesis

TODO 5.4. The nervous system—small yet complex

TODO 5.5. Reproductive tissue—sex-specific anatomy

6. The C. elegans genome

C. elegans was the first multicellular eukaryotic organism to have its genome sequenced

The entire C. elegans genome is 100 Mb and has 20,444 protein-coding genes

Both C. elegans sexes contain five autosomal chromosomes named linkage group (LG ) I, II, III, IV, and V and the X chromosome

Individual genes of C. elegans are arranged in conventional eukaryotic fashion with 5’ untranslated regions, open reading frames (ORFs) containing exons and introns, and 3’ untranslated regions

C. elegans genes are relatively small with the average gene size of 3 kb due primarily to the presence of very small introns

The C. elegans genome has two unusual aspects

Most protein-coding mRNAs are trans-spliced

Trans-splicing is the addition of one of two 22-nucleotide leader sequences (SL1 and SL2) at the 5’ end of mRNA
The leader sequence is believed to aid in translational initiation
Because SL1/2 sequences are known, can be used experimentally to identify the sequence at the 5’ end of mRNAs
Some C. elegans mRNAs are formed from multigenic transcripts with the first mRNA spliced to SL1 and subsequent mRNAs to SL2

Some genes are organized in operons

The genes that code for these transcripts are closely spaced together in tandem and are transcribed under the control of a single promoter

These transcripts are similar to those produced by bacterial operons and code for gene products that are co-expressed

They differ, however, in that the processed transcripts in C. elegans generate multiple mRNAs
Past experiments have indicated that DNA is not methylated in C. elegans, but recent, higher resolution studies have suggested that some methylation does occur
Aspects of gene regulation such as transcription, translation, chromatin remodeling, and post-transcriptional modifications (ubiquitination, phosphorylation, histone methylation, glycosylation) have all been studied using the genetic tools of C. elegans

TODO 7. Caenorhabditis ecology and evolution

TODO 8. Brief history of C. elegans research and key discoveries

TODO 9. The C. elegans community