Teaching |
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| at the Institute of Evolutionary Biology, University of Edinburgh | ||
Informatics MSc |
Bioinformatics MSc |
Developmental Biology 3 2007
Caenorhabditis elegans
PRACTICAL: Vulval development
The post-embryonic lineages of the P-cells [a larger version of this image]
This page is at http://www.nematodes.org/teaching/devbio3/practical.html
Lecture Notes: Development of the C. elegans vulva | References for lecture
The purpose of this practical is to introduce you to C. elegans as a living organism, to let you examine the different morphologies of male and female hermaphrodite worms, to observe directly embryo cleavages, and to examine some of the mutants used by Bob Horvitz and Paul Sternberg in their groundbreaking work on the developmental biology of the hermaphrodite vulva.
2 Introduction to 'worm-wrangling'
You will be provided (in pairs) with 5 cm diameter petri dishes containing a base of agar medium, seeded with E. coli food, and then with various strains of Caenorhabditis elegans. Each petri dish is labelled on the bottom with the name of the strain.
** This year, the E. coli food got slightly contaminated with a yeast that C. elegans does not like to eat. Sorry about this, but the worms seem just as happy...**
STRAIN NAME GENOTYPE*
PHENOTYPE N2 wild-type This culture has been selected to maintain an artifically high number of males. It is otherwise equivalent to the standard wild type N2. This strain was grown at 25°C. CB1489 him-8(e1489)IV High incidence of males. A mutant in the pathway of chromosome disjunction that causes a high rate of X-chromosome non-disjunction, and thus the development of nullo-X sperm, and subsequently XO, male nematodes. CB indicates that the strain was isolated in Cambridge. This strain was grown at 25°C. MT2123 let-23(n1045)II let indicates that some alleles at this locus cause embryonic lethality. The MT prefix indicates that this strain was established in the MIT lab of Bob Horvitz. This strain was grown at 15°C. MT4866 let-60(n2021)IV let indicates that some alleles at this locus cause embryonic lethality. This strain was grown at 25°C. MT309 lin-15AB(n309)X lin indicates that mutants at this locus cause lineage defects. This strain was grown at 25°C. CB1370 daf-2(e1370)III daf indicates that this locus when mutated affects the formation of dauer larvae. Dauer larvae are an alternative L3 stage that is resistant to stressful environmental conditions, and is one way that C. elegans manages to survive poor conditions in the wild. This strain was grown at 15°C. * Genotype notation in C. elegans
All C. elegans strains are presumed to be homozygous unless otherwise indicated. The first part of the genotype of any allele is the locus name, which is three (sometimes four) letters long, followed by a dash and a number. The letters usually act as a mnemonic to help you remember the phenotype induced by mutation of the locus. The locus name is italicised. Following this is the allele name, eg "e1489" in round brackets, and then the chromosome to which the locus has been mapped. The letter preceeding the allele number indicates the laboratory where the allele was generated: "e" alleles derive from the Brenner lab in Cambridge, for example.
Other materials:
pipette and tips
[worm picks, a few, are also available]
microscope slides and coverslips
anaesthetic solution (marked “A”: 0.5 % phenoxypropanol in water)
compound and dissection microscopesNote on growth temperatures: Some mutant alleles have temperature-sensitive phenotypic effects. Often, mutants are isolated that are defective at 25°C, but normal (or at least viable) at 15°C. It so happens that one of the two temperature sensitive alleles you will be looking at is cold-sensitive, i.e. it exprersses a phenotype at 15°C and not 25°C.
N2 nematodes on an E. coli lawn (Image from Mario De Bono)
Introduction to worm-wrangling
Caenorhabditis elegans at home in their (un)natural habitat (the petri dish).
C. elegans on the surface of the agar in a petri dish culture can be observed quite well under a dissection microscope. It is not usually necessary to take the lids off of the petri plates to observe them, unless there is condensation on the underside of the lid. Any condensation can be removed by a sharp shake of the lid, or by wiping it. Adjust the light level until you get a good clear contrast between the nematodes and their substrate.
Observe the following
* nematodes of different sizes. The smallest, just visible are the L1 larvae. The larger, fatter ones, up to 1 mm in length, are the females. Males (if present) are about 0.8 mm long, and thinner than females. Note that the larval nematodes have much the same body form as the adults: while C. elegans moults between larval stages, there is no metamorphosis. See the lifecycle of C. elegans at http://www.nematodes.org/Caenorhabditis/caenorhabditis_lifecycle.shtml for developmental timings.
* how the nematodes move. The sinusoidal body movements are mediated by the elegant simple hydrostatic skeleton. The nematodes are actually lying on their sides. Note that the nematodes often stop, back up for a short distance, and then head off in anther direction. The head (or just the front part of the head) is more mobile than the rest of the body, moving from side to side.
* eggs. Eggs are only 60 microns long, and so you may not notice them at first. They tend to be laid by the hermaphrodites just off the bacterial lawn. Can you see pre-hatch larval nematodes curled up in their eggs?
Up close: Anaesthetised nematodes under higher power microscopy.
To look at the nematodes’ morphology in more detail under the compound microscope it is necessary to transfer them to a microscope slide. Once on the slide, as they are transparent, you should be able to identify all the major organs and structures. Live nematodes move vigorously, and in order to be able to focus in on them it is necessary to anaesthetise them first.
There are two ways to get nematodes onto slides:
1 The nematodes can be picked up using a fine platinum wire mounted in a pasteur pipette while observing them under the dissection microscope. The tip of the wire can be used to deftly pick nematodes from the plate and transfer them to a small drop (~40 µl) of anaesthetic solution on a slide. Once on the slide, place a coverslip gently over the top of the drop - use the platinum wire to support the coverslip as you lower it down. It takes a little practice to get the technique going, so you can expect to have a few false starts and damaged worms. Different people find different methods best, but two favoured ones are:
(a) placing the pick beside and below the worm in the midbody region and quickly raising it
(b) getting the pick coated with E. coli and picking the worm up by deftly touching the loaded pick to the worm
Method (b) is preferred for young (egg-L4) worms.
We dont have a lot of picks, but if you want to have a go, pick one up from the front bench.2 The other much simpler but less elegant way of getting nematodes onto the slide is to place a drop of anaesthetic solution (~50 µl) onto the agar plate, over some nematodes. Then suck up the anaesthetic and nematodes, and place them on the slide. This doesn’t pick up single nematodes, but is much easier for the first-time nematode wrangler. Once the nematodes are on the slide, place a coverslip gently over the top of the drop.
Basic compound microscope instructions: You should remember how to set up and use a compound microscope, but as a reminder.... To get the best image, you need to regulate the light coming to the specimen on the slide. This is done using both the variable power supply to the light, and the diaphragm on the condenser below the stage. Too much light, and the specimen will be 'bleached out' and details will be hard to see. Too little light and, well, everything is grey. Use the condenser to limit the diameter of the beam of light hitting the specimen. Often a quite well-closed condenser duiaphragm will give the image with the highest contrast. You can use phase contrast if you remember how to... basically phase enhances the appearance of structures with different optical density, and can assist in visualising some structures.
Male and female (hermaphrodite) nematodes
An adult hermaphrodite Caenorhabditis elegans
Adult female anatomy
Pick an adult female nematode to a small drop of 10% anaesthetic/water to a slide. Carefully lower on a coverslip - balance the coverslip over the drop of anaesthetic using a yellow tip, and lower it carefully on. Observe under the compound microscope. You should be able to distinguish the head and tail ends, the pharynx, the gut, the gonad and the vulva, which is on the ventral side of the animal. There will be fertilised embryos in the uterus, and you should be able to distinguish early embryos from those which are undergoing morphogenesis.
Draw a female nematode and label its anatomy. Pay particular attention to the gonad and its constituent parts.
Adult male anatomy
You can either pick males from the N2 plate, or from the CB1489 (him-8) plates. The male nematodes are distinguished by the copulatory bursa at the tail end. males are very active on the plate, moving rapidly, with frequent reversals and changes of direction.
Pick a male to a slide as before and check for differences compared to the female.
Draw a male nematode and label its anatomy. Pay particular attention to the gonad and its constituent parts. How do male C. elegans differ from females?
An adult male Caenorhabditis
For your entertainment and education, I have put a gallery of a few mutant worm photos on the world wide web at
http://www.nematodes.org/Caenorhabditis/mutants/mutants.html
C. elegans embryos develop really rather quickly. For one of your adult hermaphrodites from the N2 plate, check if she has embryos in utero. The stage to look for is the first cleavage: most embryos are not laid untl they have reached ~60 or so cells, and so all the embryos in the uterus should be in the early stages of development.
Mark on the slide where the female and embryos are, and return to them periodically during the practical. You should be able to note the early cleavages of the embryo, occurring once every 30 minutes or so.
Images of early C. elegans embryos
a-d: Migration and fusion of pronuclei; zygote formation;
d: zygote.e-i: early cleavage divisions.
f: two-cell stage. g: four-cell stage. h: eight-cell stage. Formation of germline cells P1-P4 (P4=primordial germcell). i: Beginning of gastrulation with immigration of two gut precursor cells (E).j-k: different morphogenesis stages.
j: lima bean stage. l: two-fold stage.(Image and text from Einhardt Schierenberg at http://www.uni-koeln.de/math-nat-fak/zoologie/expmorph/agschier/schier2e.html)
Now you will observe and try to understand some mutations which have been isolated because of their effects on hermaphrodite vulval development. Unfortunately, there's not enough time for you to do any real genetics on these (for example, to set up matings, etcetera) but hopefully you will get an idea of the sorts of observations, made on these mutants, which underlie the models of the lineage-based dynamics and genetic determination of vulval morphogenesis.
Each of the nematode strains we will supply is homozygous for one particular vulval development mutant: the genetic background is otherwise wild type.
Muv, Vul and pVul Phenotypes
Compare adult hermaphrodites of the different strains (N2, CB1489, CB1370, MT309, MT2123 and MT4866). You should aim to describe a vulval phenotype for each strain.
The three classes of phenotype you should be able to distinguish from the wild type pattern of the N2 hermaphrodites are
Vul vulvaless. These nematodes have no vulva, but do have a fully formed gonad, and have fertilised eggs in the uterus. As these eggs continue development, and hatch as normal, but cannot escape from their mother's cuticle, the mother ultimately becomes a 'bag of worms', filled with her offspring (which proceed to eat her until they escape).
pVul protruding vulva. These nematodes do have a (single) vulva) but it protrudes from the body surface. Under the microscope, these nematodes look as if they have a blob on their ventral surface, and close examination reveals that it 'replaces' the vulva. pVul animals may still be able to lay eggs, though they can also 'bag'.
Muv multivulva. These animals have more than one protrusion or vulva-like structure on their ventral surfaces. It is unusual for any of these vulva-like structures to be functional, and so the animals usually 'bag'.
Please Note: Rather obviously, larval nematodes and male nematodes will not have a vulva (and thus could be scored, trivially, as Vul). For working out what the phenotype of a strain is, check that the nematode you have picked is female (ie it has a two armed gonad) and that it is mature (ie that it has eggs in the uterus, and oocytes in the ovary).
For each of three strains, we will collectively score the phenotype (using quantitative measures if relevant) for at least 50 adult hermaphrodites. YOU dont have to count 50 for each strain (phew!) - aim to look at five hermaphrodites of each of the five strains. We will then pool results by adding them to a table on the chalkboard at the end of the practical.
What proportion of the animals show the vulval defects?
For the Muv strains, you can provide a quantitative measure of the degree of "Muv-ness" by counting the number of blobs (vulvae, protruding vulvae and pseudovulvae) on their ventral sides. Why do you think different individuals have different numbers of vulva-like protrusions?
For the Muv strain, observe pre-adult L3 and L4 nematodes: Can you identify the patterns of cell division that result in the adult phenotype?
Are there any other phenotypic changes apart from the vulval defects?
Muv multivulva phenotype
'bag of worms' phenotype resulting from failure to develop a vulva (Vul)
![[a larger version of this image]](images/Celegans_vulva_for_prac.jpg){kind=link}