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Procedure 1) Wash albino Axolotl amphibian gastrulae in a dish filled with 100% HEPES-buffered Modified Steinberg's Solution (HBSt). Using albino Axolotl's will make it easier to track the dye. 2) Place embryos in 10% HBSt and remove jelly coat using using fine forceps. 3) Transport embryos and 10% HBSt to black agarose dish for contrast. 4) Apply a nile blue sulfate dye chip to one end (animal pole or vegetal pole) of the gastrulae and a neutral red dye chip to the other end of the gastrulae (animal pole or vegetal pole) (figure 1). Replace 10% Steinberg's solution with fresh 10% HBSt. 5) Observe gastrulae in microscope and take a picture on day one of experiment. Take a picture each day for the next few days to observe the movement of the dyed cells of the gastrulae. (Note: To delay growth, place gastrulae at 14oC). Results and Conclusions Dye was applied to opposite ends of 6 Axolotl embryos (figure 2). Over a three day period, the elongation of the stained material was noted. The embryo just below the green star (figure 2) was dyed at opposite ends (left and right) of the blastopore. A dye-marked embryo similar to the star-marked embryo (figure 2) would be expected to give rise to the left and right side of the neural ectoderm (figure 3). Looking down on the day 2 dye-marked embryo, the blue and red dye appear as longitudinal stripes on opposite sides. The blue dye can be seen on the right side of the neural ectoderm and red dye on the left side of the neural ectoderm (figure 3a). The day 3 neurula, which is lying on its left side (which would be expected to contain red dye on the neural ectoderm), shows the blue dye on the right side of the neural ectoderm (figure 3b). It was noted that with time, it became harder to sustain the movement of the dye-marked populations of cells, where the dye became lighter and much more difficult to trace. Also, some embryos became sick and died. In the future, it would be a good idea to dye more embryos and possibly even take photos more frequently (i.e., twice a day).  Figure 2. Dye marked Axolotl embryos on day one. The embryo just below the star was dyed on the left and right side of the blastopore lip. That population of cells would be expected to follow the pathway through the left and right neural ectoderm shown below (figure 3).
Figure 3. (A) dye-marked embryo on day 2 showing a blue longitudeal stipe on the right side of the embryo and a red longitudal stripe on the right side of the embryo. The dyed region is on the left and the right side of the neural ectoderm. (B) day 3 dye marked neurula lying on its left side. The population of cells containing the blue dye can be seen along the right neural ectoderm of the neurula. Acknowledgements I would like to thank the Franklin and Marshall biology department for allowing me to conduct the experiment in their laboratory facilities and for the availability of their laboratory supplies. Another special thank you to my lab partners Matthews Banda and John DeLong for their assistance in dying the embryos. I would especially like to thank professor Cebra-Thomas, my developmental biology professor, for her assistnace in conducting the experiment and interpreting the results of the experiment. Literature Cited Gilbert, Scott. 2000. Developmental biology, 6th edition. Sinauer Associates, Inc., MA, pp. 10-12, 305-307. Hamburger, Viktor. (1960). A Manual of Experimental Embryology. The University of Chicago, Press, Chicago, pp. 61-68. Hardin, Jeff. November 25, 1995. "Gastrulation: Fate Mapping." http://worms.zoology.wisc.- edu/frogs/gast/gast_fatemap.html#agar |
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© Cebra-Thomas 2001 Last Modified: 24 April 2001
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