Learning Objectives:
- Understand why C. elegans is a good model organisms for studying development and disease-associated genes.
- Understand early stages of embryonic development in C. elegans.
- Recognize stages of the C. elegans life cycle.
- Understand how RNAi works.
- Record and interpret Experimental results.
· The nematode Caenorhabditis elegans is one of the organisms commonly studied by biological researchers.
· C. elegans is a microscopic roundworm. Although some roundworms are parasitic, C. elegans is a free-living worm that feeds on soil bacteria.
· These worms grow quickly, developing from embryos to adult in 3 days.
· C. elegans is a simple animal with only about 1000 somatic cells.
· Unlike the other animals we have studied in this course we know exactly how each of the somatic cells develops from the fertilized egg.
· Since there is no variation in C. elegans, the fate map can be precise down to the cellular level (Figure 7.1)
. For most embryo types there is some local mixing of similar cells and therefore the fate maps cannot be quite this precise
· C. elegans was the first multicellular organism to have its entire genome sequenced, with the surprising finding that 40% of its genes have human matches.
· Because of this, worms are a good system to study the biology of developmental and disease-associated genes.
· It is easier to control mating, isolate genes, and introduce foreign DNA in C. elegans than in more complex animals.
· All of these features make C. elegans a great model for studying how cells divide, develop, and take on specialized tasks
· Instead of males and females in a population of C. elegans, most are hermaphrodites (having 2 X chromosomes), and a few are males having a single X chromosome and no Y chromosome) (Figure 7.2).
· Although being a hermaphrodite might sound odd to you many nematode species are. Some nematodes go their entire life without encountering another member of their species so being a hermaphrodite is very helpful if the species is to go on
Although C. elegans hermaphrodites have both male and female structures they do not make sperm and eggs at the same time.
· They produce sperm first while they are larvae.
· The sperm are stored in paired structures called spermatheca.
· Once they become adults, they switch to producing exclusively eggs for the rest of their life.
· The number of offspring a C. elegans hermaphrodite can produce in isolation is limited by the number of sperm produced during the larval stages (about 300).
· When males are present in the population they mate with the hermaphrodites and their sperm is preferentially used over the hermaphrodite’s own sperm.
· This helps increase genetic variability in the population.
· One more fun fact about C. elegans, their sperm look very different than the sperm we are used to thinking of. They are ameboid and crawl instead of swim.
· Even their fertilization is different! The oocytes are fertilized even before they start the first meiotic division.
· The point of sperm entry marks the future posterior of the worm.
· After meiosis 1 and meiosis II are completed the worm enters a brief period of cytoplasmic rearrangements.
· During this time most of the cytoplasm and RNA containing P-granules move to the posterior.
· Because of these cytoplasmic rearrangements’ cleavage divisions are asymmetrical.
· The first cleavage forms 2 cells (Figure 7.3) called AB and P1.
· When AB divides during the second cleavage division it forms Aba (a= anterior) and ABp (p= posterior).
· When P1 divides during the second cleavage division it forms P2 and EMS.
· ABa gives rise to neurons, hypodermis and anterior pharynx.
· ABp gives rise to only neurons and hypodermis.
· EMS gives rise to muscle and the gut cells.
· P1 gives rise to the germline.
· The gene we will be investigating in this experiment is apx-1, which is a maternal gene that encodes for a membrane bound DSL-ligand that binds to the receptor Glp-1.
· Apx-1 is first expressed at the 2-cell stage in the P1 blastomere. It is inherited By P2 where it acts as a signal to induce the neighboring ABp blastomere to adapt a different fate than ABa (an inductive interaction).
· Apx-1 mutant embryos fail to produce cell types characteristic of the ABp cell lineage. These cells instead adopt the ABa characteristic.
· The embryos are therefore not viable.
· After the embryonic stage C. elegans, like most nematodes undergo four larval stages (L1-L4) (Figure 7.4) before reaching adulthood.
· At the end of each larval stage the worm undergoes a molt during which they shed their old cuticle and develop a new stage-specific one.
· If conditions are not ideal, such as overcrowding, starvation, or high temperature, during the L1 stage the worms will enter a state called dauer.
· In the dauer state the worms can exist without food for 4-6 months.
· Once conditions become optimal again the worms exit the dauer state and continue development as L4 larvae
. Notice in Figure 7.4B that as the larvae move from one stage to the next, they grow larger.
· The L4 larvae are almost as big as the adult
. You can distinguish the L4 larvae by a clear crescent moon shaped vulva forming in the middle of the body.
· Adult hermaphrodites have a pointed tail and 2 uterine arms filled with developing embryos.
· The males lack the developing embryos and have a hook-shaped tail.
• Studies to identify the function of a gene typically rely on mutating a particular gene, then looking for physical or behavioral change in the organism.
• Mutagenesis (creating mutations) is very time consuming and generally cannot target specific genes.
• This makes it difficult to study the function of a particular gene if no mutation in the gene already exists.
• A powerful alternative is to decrease the expression of the genes using RNA interference (RNAi).
• RNAi is a mechanism that likely evolved to protect organisms from infection by RNA viruses. This is the worm’s primitive immune system.
• Introduction of double-stranded RNA (dsRNA) of the same sequence as the normal mRNA can be a very effective method for mRNA destruction.
• RNAi can be activated in C. elegans by simply feeding them bacteria that express dsRNA corresponding to part of the gene you wish to silence.
• The dsRNA enters cells through the intestine and is recognized by the RNAi machinery, leading to silencing of the specific gene.
• In C. elegans the triggering of the RNAi mechanism can spread from cell to cell, eventually turning off the selected gene in the entire body.
• Once the dsRNA is taken up by cells it is cleaved by an enzyme called dicer into short 21-23 base pair length fragments (Figure 7.5).
• These enter a silencing complex that can bind to, unwind, and usually cleave the mRNAs that contain complementary sequences.
• Large libraries of dsRNA exist directed against every known gene in many organisms now exist, so this method is now being used instead of chemical mutagenesis to conduct screens.
• Many genes that are required for early developmental processes also have functions later in development or in adulthood.
• These are easier to study using this method. The dsRNA can be fed, and RNAi activated at any stage of development allowing us to see the effects of silencing a specific gene at different times.
• The experiment performed in this lab demonstrates how RNAi can be used to figure out the function of a gene.
• It is also a demonstration of how interfering with the function of one gene can dramatically affect development of an organism.
• At the start of this experiment wild-type worms (N2) were grown on a plate containing worm media (NGM).
• The plates were seeded with a strain of E.coli (OP50) as a food source.
• Worms were then transferred, using the "chucking” method (Figure 7.6) and allowed to grow and reproduce for 3 days.
• You will select 5 adult worms from the plate and placed them on a “control” plate seeded with OP50 as the food source.
• You will select another 5 adult worms to transfer to each of the “experimental” plates seeded with the RNAi “feeding strain” of bacteria designed to turn down a specific genes (bli-1 & dpy-11)) through the RNAi mechanism.
• The RNAi feeding strain contains a plasmid with a piece of DNA containing a sequence from the gene it is designed to silence. The plasmid also contains a gene for ampicillin resistance.
• The RNAi feeding strains must be grown on NGM/amp+IPTG plates.
• The ampicillin in the plates selects for bacteria containing the plasmid.
• The double-stranded RNA (dsRNA) needed for the RNAi was created when transcripts from both the coding and non-coding strands of the gene sequence cloned into the plasmid hybridize to each other. The transcription of these two RNAs begins from promotors on either side of the gene sequence and is activated by IPTG in the media.
• These promotors specifically use T7 RNA polymerase in the feeding strains of bacteria to induce transcription.
• The plates will be looked at again in 5 days to see if the RNAi had any effect on development.