LAB 1

                                             THYROXIN AND XENOPUS DEVELOPMENT

I. Introduction

Amphibian metamorphosis is a dramatic transformation of a herbivorous, aquatic tadpole into a carnivorous, terrestrial adult.  This complex process requires a precise orchestration of individual events in the developing tadpole, influenced by triiodothyronine, a secretion of the thyroid gland. The triiodothyronine molecule is composed of two iodinated tyrosine residues.  It is therefore a dipeptide, but small size allows it to traverse the cell membrane and perform as a steroid hormone in modifying gene activity.

The effects of triiodothyronine on urodeles (salamanders) are more subtle than they are on anurans (frogs and toads).  In former, the metamorphic changes effected by this hormone include tail resorption, gill degeneration, and skin alterations.  Anuran metamorphic changes are considerably more striking, including physiological and structural alteration in the locomotory, respiratory, circulatory, digestive, nervous, and excretory systems.  Metamorphosis, in this case must be a carefully coordinated process.

As the concentration of thyroid hormone rises, different events occur.  For example, in water containing dilute amounts of hormone, tadpole may show only a shortening of the intestines and accelerated hindlimb growth.  At higher concentrations of hormone however, tail regression may occur before hindlimb formation.  These observations suggest that metamorphic events occur as a result of organ-specific responses; that is, the response to triiodothyronine is an intrinsic property of the organ itself and does not depend on other factors (such as other organs).

This laboratory exercise tests the hypothesis that exposure to various concentrations of triiodothyronine or tetraidothyronine will elicit different metamorphic changes in the tadpoles of Xenopus (or we can use Rana catesbeiana). This lab utilizes one of the more famous amphibian develop­mental systems, that of Xenopus laevis.  The common name is the South African clawed frog.  Its native home is southern Africa, and probably more is known about this vertebrate than any other.  It is a favored amphibian because the female ovary contains oocytes of all stages.  Injection of gonadotropic hormones at any time of the year will cause egg laying.  The frog is easily cultured in aquaria containing dechlorinated water and can subsist well on a diet of uncooked beef liver.

In this lab, several groups of tadpoles will be constantly exposed, for the duration of the experiment, to different levels of thyroid hormone. The objective of the experiment is to identify the threshold levels for the metamor­phic changes that are observable. These changes should be noticeable in such externally visible structures as the tail and limbs. Tadpoles will be dissected at the end of the ex­periment to determine any changes in their gills and intes­tines.

An important component of this lab exercise is the coop­eration among the different students who will be performing specific segments of the experiment.  Co­ordination of feeding, water-changing, and tadpole-measur­ing chores is necessary for the success of the experiment, which will run for 4 weeks.  The first lab is shorter to allow time over the next few weeks for students to maintain the tadpoles. Measurements of  tadpoles will occur once a week.  A great deal of common courtesy and trust is re­quired for this lab because water pails have to be filled and labeled accurately, thyroxine solutions have to be stored properly, and so on.  If not, all the elaborate preparations could yield un-interruptible results-the ultimate horror for any experimentalist!

All observations should be recorded.  These will include measurements, descriptions of any visible morphological or behavioral changes in the tad­poles, and any variations in their care and feeding.  At the end of the experiment, the class will pool the data. 

II. Objectives

            1.         To observe the effect, if any, of different concentrations of T3 or T4 on amphibian                                  development.

III. Procedure

1.         Divide the tadpoles into four groups: I, II,  III, and IV.

2.         Each group should have available: 1 aquaria with 5 liters of dechlorinated tap water in each, 5 sheets of millimeter graph paper, 2 finger bowls, large pipette to remove food and waste from tank and 1 small aquarium net to use for tadpoles.

3.         Mark the 5-liter and 4-liter water level (full tank and 20% waste water removal level) on the outside of each aquarium with a permanent marker so that the amount of water will not have to be measured each time the contents are changed.  Label each of the aquaria.

4.         Use an aquarium net to transfer 10 tadpoles to each aquarium.

5.         Time=0 data collection: For the first lab, each student in a group should do a measurement to a tadpole. Each of the four groups of students should measure 4 tadpoles every week. To do the measurements take out 1 tadpole from either aquarium and transfer it into a finger bowl with dechlorinated water.  Slowly cool the water by adding ice cubes made from dechlorinated water. The cold water will anesthetize the tadpole, slowing it down so that observations can be made.

            Important: Gradually cool the water, if they are placed in cold water too rapidly, the shock could kill the tad­poles.) Slide a sheet of millimeter graph paper under the finger bowl to serve as a measuring grid.  An alternative way to measure the tadpoles is to use a short ruler cali­brated in millimeters and hold it near the tadpole while it is in the finger bowl.

6.         Using a net, return the tadpole to its aquarium. 

7.         A stock solution of thyroxin will be available, we will call this a 1X solution (it is actually a 1:100,000 solution). Keep the stock solution in foil-wrapped or opaque glass bottles in the refrigerator. Shake each bottle well, but carefully, before taking out the correct amount of stock solution for each aquarium, as indicated.  Re­member to return the thyroxin bottle to the refrigerator when you are done.

Group I             =          control (no thyroxine)

Group II            =          1:20 dilution of stock solution (250 ml stock to 2250 ml water)

Group III            =          1:100 diltuion of stock solution (25 ml stock to 2475 ml water)

Group IV           =          1:1000 dilution of stock solution (2.5 ml stock to 2.5L water)

8.         Observa­tions must be made next week on the same day.  Record observations in your notebook.  The experiment runs 4 weeks, or longer if necessary.

9.         The success of this experiment depends in large part on how the tadpoles are maintained. If their water is fouled by too much food or the buildup of wastes, high mortality will result. This is the procedure for maintaining your tadpoles throughout the experiment:

a.                     Feed tadpoles ever other day, Monday and Friday on the weekends. It is very important not to overfeed, as it will foul the water and they will die. Allow them to eat for one hour, then remove any leftover food from the aquaria. Use net or large pipette to remove excess food.

b.                     The day after feeding change 20% of the tank water (500ml) with the appropriate dilution of thyroxin. When changing the tank water don’t just dump in 500ml of fresh water with thyroxin. First use the large mouthed pipette to remove any waste material and fouled water from the bottom of the tank first and remove 20% of the water (refer to the mark you made on the side of the tank), then add the 20% fresh water with thyroxine.

10.        It is your responsibility to record the fol­lowing observations for your group of tadpoles: (a) head width (mm); (b) tail length (mm); (c) total body length (mm); (d) appearance of limbs, tail, and so on; (e) activ­ity level; and (f) deaths, if any.  At the end of the experi­ment, the class will collate the data and discuss the re­sults.

11.        In the event that a tadpole dies, the following should be noted when and how it died (your best guess).  The tad­pole should be fully described in your report, and any metamorphic changes (described in the introduction to this lab exercise) should be recorded.  This means dis­secting the tadpoles as soon as possible.  Tadpoles may be preserved in 70 % ethanol if dissection cannot be per­formed immediately.

12.        At the conclusion of the experiment, surviving a will be sacrificed and dissected in order to characterize metamorphic changes occurring in visceral organs result of tadpole exposure to different concentrations of thyroxin.

IV. Questions/Data Collection

1.         How would you describe the emergence and growth of the limbs? The changes in the tadpoles' tails? Did limb changes precede tail changes?

2.         Make a graph to represent each set of measurements.  Devise a way of reporting the limb emergence data.  What trends, if any, are noticeable in your graphs?

3.         Do the data support the threshold concept? Explain your answer.  In your report, include a discussion of the threshold concept as it applies to this experiment.

4.         In any experiment compromises must be made to take into account time, materials and expertise of the experimenters. How could you improve the design of this experiment, or putting it another way, what problems are there with the current design of this experiment?

Note:    Class data should be pooled and analyzed at the end of the experiment.

Data Collection – Week 1/Your Group

Data Collection – Week 2/Your Group

Data Collection – Week 3/Your Group

Data Collection – Week 4/Your Group

Lab I, Part II (optional) Chronic Gonadotrophin and Ovulation in Xenopus

This is an optional part of this lab, I don’t think we will be doing it this year. But if we want to and have some Xenopus females on hand (they are expensive) we can examine the effect of human chroionic gonadotrophin (HCG) on egg production in Xenopus. I originally planned to do this exercise first and use the tadpoles produced for the first part of this lab. But tadpole production is not guaranteed. Injection of commercial chorionic gonadotropins into the dorsal lymph sac of female frogs causes egg laying in 6-12 hours.  Egg-laying females will be available in the laboratory.

Fertilization can be achieved at a precisely determined time for experimental purposes by sacrificing a male and removing the testis.  Fertilization can also be achieved by injecting the females and then placing them in a tank with breeding males, we will utilize the latter procedure. The time scale of developmental events leading to first cleavage is important to you. One sign of a successful fertilization is the change of random orientation of unfertilized eggs to the uniform pigmentation orientation of fertilized ones.

Three gravid Xenopus females will be injected by student volunteers  with 500-800 I.U. commercial human chorionic gonadotropin (hCG) the night before the scheduled lab. Note that it takes at least twice as long if gonadotropin from preg­nant horse serum is used. Injections are made at an oblique angle just under the skin of the back, using a hypodermic syringe and needle of about 26 gauge.

Make a frog burrito with paper towels as described by your instructor.  Inject sample, wait 5 seconds then withdraw needle. You will inject just outside the stitch marks with the needle going under the marks. You will initially feel some resistance, which will stop after you have penetrated properly.

All individual male and female frogs should be kept in separate tanks away from any disturbances. At least one female should be placed in a tank with high salt concentration. This should keep any eggs laid overnight viable for the next day’s lab.

V. Materials

1.         Hind-leg-emergent bullfrog or Xenopus tadpoles (the latter are supposed to respond to better to the hormone) are available from most biological supply companies, such as Ward's Natu­ral Science Establishment, Inc. (5100 West Henrietta Road, P.O. Box 92912, Rochester, New York 14692­9012) and Carolina Biological Supply Company (2700 York Road, Burlington, North Carolina 2 7 215). Tim at Carolina extension 4381.

2.         Use carbon-filtered dechlorinated tap water and to ensure that it will be at room temperature when used, allow it to stand for about 12 hr in an open container. Also have available carbon-filtered dechlorinated tap water in the refrigerator, which will be used to slow the tadpoles down to be able to take measurements.

3.         L-thyroxine is available in powder form from Sigma Chemical Company (P.O. Box 14508, St. Louis, Missouri 63178-9916).  It should first be dissolved in a small Volume of 9 5 % ethyl alcohol, then slowly mixed into water to a final stock concentration of 0.1 M. If thyroxine precipitates out of the solution, add by drops, 1 M NAOH to increase the pH and improve solubility. 

            Another method is to use 10 mg of thyroxine in 5 ml of 1% NaOH, then dilute to 1 liter of distilled water.

The stock solution should be stored in a dark or foil-wrapped glass bottle and refrigerated. it should be shaken thoroughly before using.

4.         5 aquaria for maintaining the 5 groups of 10-12 tadpoles/            Tadpole food. Because this lab exercise            runs over many weeks and the aquarium water has to be changed every two days, students    should have off-hours access to the lab and to such materials as tadpole food, thyroxine stock         solution, and dechlorinated water.

5.          Transparent Rulers and finger bowls, large pipette to remove food and waste from tanks

VI. References

Biroc, S. L. (1986).  Developmental Biology: A Laboratory Course with Readings, pp. 197-198.  New York: Macmillan Publishing Co.

Gilbert, S. F. (1991).  Developmental Biology, 3rd ed., pp. 686-699.  Sunderland, Massachusetts: Sinauer Associ­ates.

Kollross, J. J. (1961).  Mechanisms of amphibian metamorphosis: Hormones.  Amer.  Zool. 1, 107-114.

Cruz, Y.P. (1993). Laboratory Exercises in Developmental Biology. Academic Press, New York.