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tracking step using a minimal movement algorithm and local neighborhood
search. Nuclei that do not satisfy the strict minimal movement condition (i.e.,
those that move less than their radius from t to t+1, usually less than 5% of
nuclei), are flagged for manual curation. Nuclear divisions are also curated
manually. At each time point the correct set of nuclei is annotated, preventing
the accumulation of annotation errors. Our software does not search for nuclear
radii as an additional free parameter. Our initial manual lineaging confirmed the
initial observation (
Bao et al.,2006
) that all nuclei in each of the major subli-
neages (AB, C, D, E, MS, and P4) show distinct radii values that linearly decrease
with each round of division. Hence, our software prescribes nuclear radii values to
all nuclei depending on their position in the lineage. Using this semiautomated
approach it is possible to lineage essentially all nuclei up to the 1.5-fold stage (566
live nuclei, 103 cell deaths).
B. Protocol 2: Post-Embryonic Cell-Lineage Analysis
1. Worms to be lineaged must be in healthy, unstarved condition.
2. Prepare a standard slide mount agar pad (cf. Sulston and Hodgkin methods
appendix in C. elegans
I). The agar should have been freshly prepared or
melted.
3. Using a drawn-out capillary and mouth pipette pick up the worm(s) to be lineaged
in a few microliters of M9 or S basal. Deposit the larva onto the agar pad together
with a small volume of buffer. [If you are very dextrous, it is possible to do this
with a worm pick, but small larvae are very easily injured. We recommend the
mouth pipette.] Remove excess buffer by wicking with lens paper.
4. Using lens paper, wipe clean a small coverslip (18
18 mm OK, 12
12 mm best
but can be hard to find). Using aworm pick, smear a small amount of OP50 E. coli
onto the center of the coverslip. Place the coverslip gently over the buffer + worm
so that the bacterial blob is within a couple of mm of the worm.
5. After 1-5 min the worm should become active and head toward the bacteria and
start browsing contentedly. Under optimal circumstances, the wormwill continue
eating for hours, with occasional bouts of movement. If the worm does not move
or begin eating within 10 min of mounting, it may be damaged.
6. If the worm appears healthy, trim the agar around the coverslip with a razor
blade. Seal the edges of the coverslip with immersion oil or vaseline. Some
brands of immersion oil are toxic and can interfere with long-term observation.
Vaseline works fine unless your worm swims into it or you get some on the
objective.
7. Find the area of interest in the animal and draw out everything you see as often as
you can, identifying nuclei by reference to the standard maps in the papers in.
With practice, multiple animals can be lineaged at a time, depending on the
complexity of the lineage being traced. It is usually best to keep one animal
per slide to avoid confusion.
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