Corsi_2015: A transparent window into biology: a primer on caenorhabditis elegans
- keywords
A transparent window into biology: a primer on caenorhabditis elegans
Summary
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”
http://labs.bio.unc.edu/Goldstein/movies.html
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
Allows researchers to track the fate of every cell between fertilization and adulthood
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
The bleach decontaminates by killing everything but embryos
Can also by used to isolate eggs with are resistant to 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
Indicating that hermaphrodite-produced sperm is a limiting factor in self-fertilization
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
i.e., populations of hermaphrodites tend to lose heterozygotes (because hermaphrodites cannot mate with other hermaphrodites)
Strains htat are mutagenized are essentially isogenic
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
selfing reduces greatly the effort needed to find such mutants.
4. Mutants with neuromuscular defects that impair the ability to mate can still be maintained in the laboratory
Only 11 of the 302 nerve cells of the hermaphrodite causes it to become dauer larvae
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
Which allows the embryo to develop completely independent of the mother
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)
L1 stage is ~16 hr long
All others are ~12 hr
Each stage ends with a sleep-like period call
lethargus, where a new cuticle is made, and it ends with the molting of the old cuticle
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
completely surrounds the animal and plugs the mouth preventing the animal from eating and thereby arresting development
Gives it greater protection against evironmental stresses and caustic agents
Most commonly encountered form in the wild
When they're tranfered onto plates with bateria they shed the mouth plugs, molt and continue as slightly different L4 larvae
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
Process can be done in a number of weeks
transformation rescue (or complementation testing) provides evidence that the correct gene has been identified
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)
The use of double-stranded RNA (dsRNA) to reduce gene activity
Can soak the animals in a soltion of dsRNA
Or by feeding the animals bacter that generate specific dsRNA
4. Why choose C. elegans?
C. elegans has many inherent advantages as a model for eukaryotic biology
Small size
Large brood size
Ease of cultivation
Low maintenance expense
Long-term cryopresevation
Quick generation time
Transparency
Invariant Cell number and devlopment
The ability to reduce gene activity using feeding RNAi
C. elegans is the organisms are quite benign to humans
Because they cannot grow at body temperatures, they cannot grow in humans
Allergic reactions to C. elegans have not been documented
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
allowing the application of various compounds or other agents while simultaneously monitoring fluorescent readout of gene regulation or electrophysiological activity by microscopy
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
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Some C. elegans cells can be studied in vitro
embryonic glial cells
larval muscle
neuronal cells
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
The chromosomes do not contain traditional centromeres
During mitosis the microtubule spindle attaches to more than one position along the chromosome (these attachments are said to be holocentric or polycentric)
a specific sequence does not seem to be required for attachment since extrachromosomal DNA-containing transgenes can be inherited throughout many cell divisions
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
When compared to vertebrate genes
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