STUDIES WITH XENOPUS

For experiments on Xenopus, see the reference book: Xenopus laevis: practical used in cell and molecular biology, eds: BK Kay, HB Peng in Methods in Cell Biol v.36 1991. On reserve, and in lab. It has extensive appendices with solutions, techniques, and the chapters give detailed protocols.

 

You will want to decide what kind of experiment you will do, so you can xerox articles from the reference. Look through your textbook to see experiments with explantation, transplantation, cell recombination, cell injection, chemical treatment that you are interested in, get help from instructor about what animals are available for egg production. We usually have Xenopus.

Remember for every experiment you must have a control of embryos untreated but of the same stage to start with, so you can compare them with experimentals.

 

Xenopus develops very rapidly: we have albino Xenopus, so the eggs will not be pigmented, so it will be hard to tell animal from vegetal pole. If they are fertile, they will rotate with an pole up

1st cleavage- 80 min at 20 degrees, 3 hr at 15 degrees

For the centrifugation studies:

1.      determine what stage they are by observing under the dissection microscope with the light coming from the top, not the mirror. Separate out 12 of the same stage

2.      keep them on ice after what stage they are, separate out several tubes of 12 embryos. Then centrifuge them at different speeds for varying periods of time. Use settings 1,2,3 on the centrifuge for less than 5 min.

3.      Make sure you have a control tube that you only put on ice and take in and out of the tube.

4.      Carefully remove the eggs from the centrifuge tube and observe in a petri dish of dechlor tapwater. Try taking a video photo of them. Then place the 12 eggs from each setting and time into labeled separate culture dishes with lids (with holes in it) in about 1/2-1 inch of dechlorinated tap water. Observe them next lab period to see how far they developed and how they developed.

 

To understand the purpose of this experiment, consider what happens when you centrifuge cells, what kinds of localized materials and organelles may be in eggs, how they can be disrupted by centrifugation, and what this may do to processes of development such as setting of axes, parcelling out of a mosaic cytoplasm, or cytoskeleton elements.

 

For many experiments, you may need to remove the jelly and vitelline membrane. Two techniques are used in this book:

  1. Chemical-one in the appendix uses cysteine (how can a -SH reagent be useful to dissolve jelly or fertilizaion membrane?)Their technique is make solution 2% cysteine hydrochloride, adjust pH to 8.1 with 10 M NaOH. Place eggs in it for 15-20 min swirl, when eggs get close together showing jelly is gone, rinse 4x in 1/3 strength Holtfreter solution.Place at 18 degrees until needed.
  2.  The other uses mercaptoethanol. What does it have in common with cysteine?
  3. mechanical- dissect off in a wax lined dish with the egg in a bed of permaplast (clay) to hold it in position, then with 2 pair of fine forceps- The vitelline envelope is hard to see, just take fine forceps and grab at the egg surface. It is best to make a hole in the embryo while doing this and then grab the envelope with other forceps and pull it off and place far from embryo. The puncture has little effect, and besides you are going to remove something or use vital stain anyway.

 

Vital staining: nile blue sulfate gel made as in the appendix of this book can be collected hydrated with water and then placed on a glass needle and the placed on the surface of the egg to show which end was uppermost, to show how rotation has an effect on axis formation. This could also be done in the presence of microtubule disruptors to show MT involvement.

 

Isolate animal and vegetal halves to run in electrophoresis or TLC:

freeze embryos on foil over dry ice, cut in 2 with scalpel or razor blade. Place all halves in one tube, light in another.

    What is the difference of fate in the 2 halves? What difference in growth factors or receptors or cytoskeleton? Do you expect their components to be different as detected in electrophoresis or TLC?

 

Animalization, vegetalization, reajjustment of DV axis:

 

Exposing early cleavage stages to UV light or deuterium water can alter DV development by inhibiting development of mesodermal derivatives and body axis. (p.272, Kao and Danilchik). DV axis disruption can also occur by exposing blastula stage briefly to 0.3M lithium chloride p.275,280. It reduces anterior structures when done at this stage whereas treament of early cleavage stages enhances dorsoanterior structures. How could you explain this? Injection of trypan blue (polysulfonated) into blastocoel interferes with gastrulation cell movements.

 

Transplantation and explantation: See other experiments in this lab manual and consider doing the surgery on some of the permaplast (clay) found in your drawer to hold the eggs in position before, during and after surgery. Always do surgery in 100% Holtfreter's solution and allow them to heal for 30 minutes before disturbing them. Use demembranated embryos. Keep all surgery embryos separate in 10% Holtfreter’s with penicillin and streptomycin.

 

Fixation and sectioning can also be done on successful experiments, where abnormal development results. (be sure to do some controls as well!)