Recent Techniques

Throughout this century, many techniques have been used in an attempt to discover the mechanisms of limb regeneration—denervation, transplantation of blastemas to ectopic sites, organ culture, radiolabeling, gel electrophoresis, administration of compounds, such as retinoic acid, and so forth. However, recently three technical advances have been made that are certain to have an important influence on future discoveries.

One is the advent of whole-mount in situ hybridization for use with probes to genes expressed at very low levels, such as the homeobox genes (26). Gardiner et al. have cloned 17 homeobox genes from the axolotl, and work such as this is already beginning to answer some question, such as: Are these genes expressed in the same domain and in the same sequence during regeneration as they were during development?

The second advance is the establishment of long-term cultures of blastemas cells, allowing for their genetic manipulation by transfection and grafting back into the blastema. In contrast to normal, untransformed cells from mammalian tissues, both blastemal cells and a muscle cell line established from a dissociated limb have generated permanent cell lines, showing no senescence (27).

Fig. 3. (continued) complete limb as the missing portion, the intercalary regenerate represented by the gap between the solid line and the dotted line (I), is filled in by cells from the proximal level stump according to the law of distal transformation. This phenomenon is also shown here because the distal blastema was from a black animal (melanophores can be seen in the digits and wrist) and it was grafted to a white animal. The intercalary regenerate (I) is white, since it has no melanophores showing that it came from stump tissue. (F) The result of treating a distal level regenerate (solid line marks the amputation plane through the wrist) with retinoids. Instead of just regenerating the missing elements, a complete limb has been produced from distal level blastemal cells. Retinoids respecify blast-emal cells in a manner that breaks the law of distal transformation.

Law Distal Transformation

Fig. 4. Drawing of a Biolistics Particle Delivery System for transfecting a regenerating limb. Helium at high pressure is passed into the gas acceleration tube, and the ruptured disk breaks. This releases a shock wave, which propels the macrocarrier disk and DNA-coated gold microprojectiles downward. The screen is halted by a stopping screen, but the gold particles continue on toward the target. The anesthetized animal is supported against the bottom of the assembly and the regenerate inserted into the hole, so that it can serve as a target (from ref. 29).

Fig. 4. Drawing of a Biolistics Particle Delivery System for transfecting a regenerating limb. Helium at high pressure is passed into the gas acceleration tube, and the ruptured disk breaks. This releases a shock wave, which propels the macrocarrier disk and DNA-coated gold microprojectiles downward. The screen is halted by a stopping screen, but the gold particles continue on toward the target. The anesthetized animal is supported against the bottom of the assembly and the regenerate inserted into the hole, so that it can serve as a target (from ref. 29).

This again emphasizes a unique feature of blastemal cells, and the ability of limb tissues to dedifferentiate and turn into blastemal cells.

The third advance has to some degree obviated the need for cultures, because it is a remarkable technique by which cells can be transfected both in vitro and in vivo. It involves the use of a biolistic gun (28), which fires 1.6-pm gold particles coated with the DNA of one's choice into cells in a Petri dish or into the cells of the regenerate (Fig. 4). If the gun is fired at the external surface of the blastema then cells in the epidermis are transfected at a frequency of about 10%, and their altered behavior can be assessed in various ways. If the blastema is cut off the limb stump and the cut edge exposed to the gold particles, then the mesenchymal cells of the blastema are trans-fected. Transfection with constructs, such as chimeric retinoic acid receptors (RARs), has provided a wealth of valuable data on which receptors perform which functions

(29). At least six different receptors are expressed in the limb blastema and in addition to the positional respecification referred to above, RA induces many other phenotypic changes in the cells. In order to examine which receptor mediates which effect, Schilthuis et al. (30) constructed chimeric receptors by exchanging each of the newt RAR ligand binding domains with that from the Xenopus thyroid hormone receptor a. The genes activated by each RAR then became responsive, not to the RA, but to thyroid hormone. In this way, they showed, for example, that the RARa was responsible for growth inhibition, one of the established effects of RA on blastemal cells. It is hard to imagine how such a result would have been obtained without these important technical advances.

Hopefully, these and further advances will ultimately provide answers to those fundamental questions posed at the beginning of this chapter, the most profound of which is: Why can urodeles regenerate and mammals cannot and will we ever be able to stimulate regeneration in humans?

Was this article helpful?

0 0

Post a comment