Regulative Development in Axolotl Embryos; Splitting the Heart Field
Objective/Background Materials/Procedure Results Discussion Acknowledgments Works Cited	Discussion From our observation of a single distinct heartbeat, it appeared that the attempt to divide the heart field was not successful. Additionally the observed direction of blood flow and apparent synchronous flow through the left and right gills seemed consistent with normal amphibian heart development (Rugh, 1951). However, the ability to observe the heart beats was hindered by the opacity of the axolotl skin, as well as the position of the embryo in the operating dish, so these observations may not accurately reflect the true condition of the heart. In addition, the heart appeared to be somewhat more posterior than would normally be expected. The presence of this irregularity leads us to believe that the heart field was affected in some way. If this were the case, the heat could actually be split, or partially split, but have two heart beats that happen to appear synchronous. The fact that the grafted tissue was observed erupting from the belly/ventral region of the embryo may suggest that the graft, which was intended to split the heart region, was not incorporated into the embryo enough to sufficiently block communication between the two halves of the heart field (Figures 3, 4,&5). It should be noted that there are other explanations for these data that might also be evidence for regulative development. For instance, the gill tissue could be transforming into heart tissue through cell-cell interactions. However, the relatively advanced developmental stage of the donor tissue, and the fact that the photos seem to clearly show tissue rejection, do not support this conclusion. In the end, these results are at best inconclusive in regards to regulative development in the formation of the axolotl heart. It should be noted however, that previous experiments using small pieces of sterile foil as a barrier rather than gill tissue, did yield results supporting the existence of regulative development in the axolotl heart. Specifically, two hearts, one on either side of the foil barrier, were seen to develop and clearly beat asynchronously (Vélez and Krsmanovic, 2004). Other examples of the manipulation of morphogenetic fields, and therefore evidence of regulative development, have been shown in limb regeneration studies. Axolotls are actually able to compensate for damage to the limb morphogenetic fields throughout their lives, to the point where entire limbs may be regenerated from the remaining tissue of a limb stump (Gardiner et al 2002). Since studies show that there is ample evidence for regulative development in axolotls it would therefore seem that the our results likely have more to do with experimental technique than absence of regulative development in axolotls. While this tissue graft procedure was not particularly complex, and was the method originally suggested in Hamburger's Manual of Experimental Embryology, in retrospect it does not seem well suited to those who have had little to no practice doing microsurgery. Additionally, it should be noted that while the embryos were largely at the appropriate stage at the beginning of the procedure, as time went on, the warmth of the room allowed them to develop more quickly than we had anticipated, leading to fewer suitable embryos. It is important to remember that there is only so much time before the heart primordia fuse, and grafts must be done before this happens in order to be sucessful. In the future it would be advisable to keep the embryos cold whenever they are not actually being worked on, rather than letting a number of them sit out at room temperature.