Download C. elegans Germline Stem Cells Lab

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BY440 STEM CELL BIOLOGY -
C. elegans Germline Stem Cells Lab
One of the best described stem cell niches, both molecularly
as well as ultrastructurally, is the germline stem cell niche in
the C. elegans gonad. The niche is created by the distal tip cell
(DTC), a somatic cell that is shaped somewhat like an octopus
with a cell body at the distal end of the gonad tube and with
cytoplasmic projections that extend like arms around the
germline stem cells. The molecular interaction between the
DTC and the germ cells is a Notch signaling mechanism
where the DTC produces a membrane-bound ligand called
LAG-2 that signals through the GLP-1/Notch transmembrane
receptor on the germ cells. Activation of this pathway
promotes mitotic proliferation of germline stem cells and
inhibits the meiotic differentiation pathway. Cells that are not
in contact with the DTC enter into meiosis and differentiate
into sperm or oocytes. Other somatic cells such as the sheath
cells are involved in signaling to the meiotic cells to
differentiate and contractile sheath cells are involved in oocyte
ovulation.
C. elegans Gonad
The DTC and germline stem cells by SEM.
(Hall et al., 1999; Dev Biol).
The Germ Line
The Somatic Gonad
Goal: The goal of this lab is to use differential interference contrast and fluorescence microscopy to investigate
the stem cell niche in C. elegans and to observe the phenotype caused by various mutations that disrupt molecular
pathways that influence germline development in the worm. You will receive several mutations labeled A, B, C…
and you will attempt to determine the identity of the mutations based on descriptions of the mutant phenotypes
from published papers and from descriptions on www.wormbase.org - a ridiculously fabulous database of C.
elegans genetics.
GFP-labeled DTC
To observe the niche-providing DTC it has been labeled with
GFP. The lag-2 gene is expressed specifically in the DTC
and thus the lag-2 promoter, when fused to the GFP coding
sequence, will drive GFP expression in the DTC allowing it
to be observed in living animals with fluorescence
microcopy. This transgene is described as Plag-2::GFP. In
addition to the DTC, the somatic gonad sheath cells that line
the gonad tube are also labeled with GFP using the lim-7
promoter that is expressed in the sheath cells.
DTC
Sheath
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Preparing Slides for Observation of DTC GFP Expression
1. Prepare a slide for mounting worms (see figure on the right). Line up one
slide between 2 other slides that have a piece of lab tape running along them.
Add a very small drop of molten 2% agarose onto the middle slide and then
quickly place another slide on top of it but perpendicular and press down on
it lightly with a finger. The agarose drop will flatten into a pad roughly the
thickness of the tape. It will polymerize in a few seconds and you can slide
the two slides apart and the pad will stick to one or the other.
2. Pipette 5 microliters of the 800uM levamisole solution onto the agarose
pad.
3. Pick several worms (strain GC678) from the Petri plate using a platinum
wire (“worm pick”) and place them in the drop of levamisole.
4. Place a cover slip on the worms and label the slide and you are ready to image the worms.
DAPI Staining for Germ Cell Nuclear Morphology
4',6-diamidino-2-phenylindole (DAPI) is a fluorescent stain that passes through cell membranes and binds
strongly to A-T rich regions in DNA. Thus, DAPI staining is a great way to visualize cell nuclei and nuclear
morphology in intact specimens. DAPI staining C. elegans is extremely easy and reveals a great deal about the
germ cells – whether they are germline stem cells, transitioning into meiosis, or in one of several various stages of
meiosis I or II. See the figure of a DAPI-stained gonad below.
Germline Stem Cells
Meiotic Transition
(crescent shaped)
Meiotic Pachytene
Sperm
(tiny dots with compacted DNA)
Oocytes
(you can see all 6 chromosomes)
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DAPI Staining Protocol
1. For each strain, pipette 1.5ml of water directly onto the Petri plate and gently rock back and forth to get the
worms off the surface of the plate and into the water. Use the dissecting scope to see that they are swimming or
thrashing about in the water.
2. Use a pipette and collect the water and worms and add to a 1.5ml centrifuge tube. Get as much as you can and
if the tube is not full, add fresh water to fill it.
3. Spin the worms down in the tabletop centrifuge at full speed for 1 minute. Believe it or not, the worms are fine
even at high speeds.
4. Carefully remove the supernatant with a pipette – be sure not to suck up the worms. There should be a little
water left in the bottom with many worms thrashing about. Use the dissecting scope to be sure that this is the case.
5. Fill the tube with 95% ethanol. This will desiccate and “fix” the worms. Let the tube sit upright in a tube rack
for about 10 minutes. During this time the worms will slowly settle to the bottom. We will not centrifuge them
again because they are too fragile in ethanol so they must settle to the bottom with gravity.
6. Remove the ethanol with a pipette and discard leaving the worms and as little ethanol as possible.
7. Add 10 microliters of DAPI to the tube. Pipette the DAPI slowly; it is in a glycerol-mounting medium and thus
is very thick or viscous.
8. Prepare a slide for mounting worms (see above).
9. Pipette 5 microliters of the DAPI + worms directly onto the agarose pad and cover with a cover slip. Label the
slide. The worms are now ready to image.
The Mutants
I have gathered several mutants that display defects in germline stem cell development/differentiation of meiotic
cells. Each of the mutants is described below but you will receive plates labeled A, B, C… and your job will be to
discern which plate corresponds to which mutation based on the phenotype (see questions on next page).
glp-1(null)
Fails to maintain germline stem cells and thus is sterile with no apparent germ cells at all or just a few
fully differentiated sperm at most.
glp-1(ts)
This mutation is similar to the null mutation but is temperature sensitive; at lower temperatures
(permissive) the allele behaves as WT and the worms are normal but at higher temperatures (restrictive)
the allele behaves as a null and all germ cells enter into meiosis. The phenotype will vary depending on
the temperature and when the worms are shifted from the permissive to restrictive temperature.
glp-1(oz112)
This gain-of-function mutation produces a constitutively active receptor and thus all germ cells are
germline stem cells at the expense of meiotic cells. Essentially, there is a tumor of germline stem cells.
glp-1(ar202)
This partial gain-of-function mutation allows for some degree of germ cell differentiation but is
characterized by an extended stem cell population and a germline tumor in the proximal region (where
sperm normally reside).
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puf-8(lf)
This mutation affects differentiation. Germline stem cells are maintained and daughters try to differentiate
but fail to make proper gametes leading to gonads that do not contain (or contain few) sperm and oocytes.
The mutation is partially temperature sensitive such that normal gametes form at lower temperatures and
the animals are fertile, but gametogenesis fails at higher temperature and the animals are sterile.
gld-1(null)
This mutation affects a downstream member of the GLP-1 signaling pathway in germ cells. Mutants have
germ cells that continue to proliferate and fail to differentiate because the normal function of gld-1 is to
promote differentiation and inhibit proliferation in the absence of GLP-1 signaling. GLP-1 signaling
inhibits GLD-1 activity.
CC – BIOLOGY
KILLIAN
BY440 STEM CELL BIOLOGY C. elegans germline Stem Cell Lab – Questions
1. Within the germline stem cell population there are three different end results of a mitotic division: both
daughter cells remain germline stem cells, both daughter cells differentiate via meiosis and gametogenesis, one
daughter cell remains a stem cell and the other enters meiosis. What is the most likely determinant for which of
these three options happens when any one given stem cell divides? How does the system ensure that there is an
appropriate balance between stem cell maintenance and supply of gametes?
2. The C. elegans germline is a syncytium. How does this fact complicate how we think about the DTC/germline
stem cell signaling pathway? Think about the LAG-2/GLP-1 signaling pathway and the events that occur
“downstream” of receptor activation.
3. Match the mutant strain (A, B, C…..) to the following genotypes using your DAPI images, mutant descriptions
in the lab manual and www.wormbase.org.
_________ = glp-1(null)
_________ = glp-1(temperature senstive) and shifted to non-permissive temperature
_________ = glp-1(oz112gain-of-function)
_________ = glp-1(ar202Pro)
_________ = puf-8(loss-of-function)
_________ = gld-1(null)
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4. Some of these strains were maintained as heterozygotes and some are homozygous. Which ones are
heterozygotes and which are homozygous? Why is it the case that some cannot be maintained as homozygotes
while others can?
5. Suppose that one tinkers with the lag-2 gene and re-engineers it such that it lacks its transmembrane domain
and thus the LAG-2 protein is secreted. How will this affect germline development in an otherwise WT animal?
How will this affect germline development in glp-1(null) and glp-1(oz112gf) worms?
6. The lag-2(null) mutant phenotype might be expected to be equivalent to the glp-1(null) phenotype but it is
surprisingly different; it is embryonic lethal. Which of the following genetic phenomenon could explain this:
incomplete penetrance, variable expressivity, pleiotropy, or epigenetics? Briefly explain.
7. DAPI stains DNA and thus reveals some degree of nuclear morphology in stained samples. DAPI is listed as a
possible mutagen although it is of minimal concern to the scientist so long as he/she is not licking it off his/her
fingers. Briefly explain why DAPI is likely to be a mutagen if present in living cells.
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8. For the glp-1(null); gld-1(null) double mutant, describe the germline phenotype that you would expect to see
taking into account the phenotype of each single mutant and the knowledge of how the molecular signaling
pathway works.
9. What would you expect to happen to the germline if the DTC were laser ablated in a worm that is homozygous
gld-1(null)? Briefly explain.
10. Would you expect that there is a greater rate of transciption occuring in germline stem cells or oocytes? What
is it about the specific nuclear mor phologies of these cell types is a big clue here?