Have a close look: vulva and gonad
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To do
wild type animals
First, learn how to use the microscope. Then take a plate with Pristionchus pacificus in good shape, ranging from J2 to J4. Prepare a slide with 30-50 "mixed stage" worms and observe adult animals. Easy to identify are e.g. the two bulbs of the pharynx, the vulval opening and the anus. Try also to follow the two arms of the gonad, which have a pretzel-like shape (you will have to go through different focus planes to find the arms; not always easy..) You might also compare Pristionchus with Caenorhabditis elegans: How do they differ? Who would eat whom, if it would come to the worse ?
Then proceed to smaller worms: During J3 stage you will see vulvae differentiated at various degrees. The vulva is made by three Vulva Precursor Cells(VPCs), called P5.p, P6.p and P7.p. These cells divide always the same way, P5.p and P7.p having 7 progeny and P6.p, which is the central cell, six (check the "To read" section to learn more). In early J3s you can observe the VPCs before they divide. Also try to find P8.p, a Pn.p cell that is not part of the vulva equivalence group in wild type animals. If you are patient, follow the lineage by observing individual worms every 30 min - 1h. Rescue them in between on a OP50 plate with the Schnürpfel (takes some practice...)
mutants
Get the following mutants:
Ppa-lin-39
Ppa-ced-3
Ppa-mab-5
Ppa-lin-17
Try to find out which of the phenotypes described below correspond to these mutants:
- apoptosis defect: P1.p - P4.p and P9.p-P11.p , cells in the ventral hypodermis that usually die in P. pacificus, are suriviving
- generation vulvaless VPC die of programmed cell death.
- P7.p lineage reversal, leading to a posterior bivulva
- P8.p differentiates ectopically
Explore the literature and find out which genes are affected. Ask questions like
What is the role of lin-39?
" How might a lin-39/ced-3 double mutant look like?
Where does the inductive signal come from?
...
To read
P. Sternberg Vulva development in "wormbook.org": [1]
A. Eizinger & R.J. Sommer The homeotic gene lin-39 and the evolution of nematode epidermal cell fates Science 278,452ff (1997)
B. Jungblut & R.J. Sommer The Pristionchus pacificus mab-5 gene is involved in the regulation of ventral epidermal cell fates Current Biology 8:775-778 (1998)
D. Rudel, M. Riebesell & R.J. Sommer Gonadogenesis in Pristionchus pacificus and organ evolution: development, adult morphology and cell-cell interactions in the hermaphrodite gonad. Dev. Biol. 277, 200-221 (2005)
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