The following paper was written by Craig Berman, as part of a developmental biology course at the University of Utah SLC. It has implicit relations to some of the phase space concepts as analyzed in recent texts on architecture by Greg Lynn, Sanford Kwinter, et al. Because of its implications for the lizard families, we at basilisk naturally look upon this with interest. -ed.

The question this text hypothetically investigates is as follows: develop a theory for the phenomenon of limb regeneration, which occurs in various species of lizards, as well as other animals.

The second rule of the polar coordinate model of limb regeneration is an extremely interesting form of regulation. In this model, distant--not proximal--structures are regenerated. Molecularly, this may be controlled by the effect of different cell types having different adhesive properties. Such adhesive properties, i.e. affinity, cause separate aggregation of cells to form. Additionally, the strength of the adhesive properties of the cells establishes the proximal and distal characteristics of the limb.

Upon amputation, a single layer of cells forms to make the apical ectodermal cap. Underneath this cap, cell dedifferentiation occurs, as does cell-cell detachment. This mass of dedifferentiated cells is called the regeneration blastema (Gilbert, p. 696).
It is possible that such cells establish and express their adhesive properties at this time in order to establish an adhesion affinity gradient within the blastema. Nardi and Stocum (1983) were able to determine that when the blastemas were formed at different levels and then experimentally mixed, the more proximal blastema cells surrounded the more distal blastema cells. These results lead one to conclude that the adhesive properties of the cells form a gradient along the proximo-distal axis. The adhesive properties are lowest proximally and are increased distally (Gilbert, p 85-6).

In order to continue, it is necessary to establish why the adhesive gradient is so important in limb regeneration. First, different adhesion properties can cause different genes to be expressed. Second, specific adhesive properties allow for homogeneous cell adhesion, which is ideal for cell-cell interactions. Gene expression and cell-cell interactions are largely responsible for cell differentiation and formation of tissue. To put it simply, the formation of tissue and organs is mediated by events occurring at the cell surfaces of adjacent cells (Gilbert p. 85).

Additionally, it is necessary to support why the adhesion gradient moves from a lower adhesive affinity in the proximal regions to a higher adhesive affinity distally. During the act of regeneration of the blastemas, the gradient of adhesive properties is established, and the cells naturally rearrange themselves into their most thermodynamically stable pattern. If cells types have different strengths of adhesion, separate aggregates of cells can form. This is known as the differential adhesion hypothesis (Gilbert, pp, 84-5).
The most thermodynamically stable pattern is that of homogenous cells. This stable pattern is crucial for proper cell-cell interaction to occur. Experimentally , it is interesting to observe the result of mixing together two groups of cell types with different adhesion properties. Upon mixing, the cells with the greater adhesion affinity are arranged homogeneously; the cells with the lower adhesion affinity, however, would be surrounding the cluster of homogenous cells. Clearly, this experiment produces one cluster of homogenous cells, and another of homogenous cells contaminated by the first cluster. This contamination prevents pure homogenous cell adhesion. This problem can be eliminated if the cluster of higher affinity cells were to migrate away from the other cluster. This would then yield two cells clusters, each with homogenous cells adhesion.

This mechanism of the higher adhesive affinity cell clusters migrating distally is the manner in which the regenerated blastema establishes the adhesive affinity gradient. This gradient-- low adhesion affinity proximally and high adhesion affinity distally--accounts for the manner in which limb regeneration occurs.

Conversely, a low adhesive property cluster could not exist homogeneously if located distally to a high adhesive property cluster. If this sort of migration were to occur, the high adhesive affinity cluster would form surrounded by the low adhesive affinity cluster, thus not yielding the optimal potential for cell-cell adhesion as mentioned earlier. It is the above mechanism that establishes why distal structures always regenerate as opposed to proximal.

Experimentally, there are a great many factors that are involved in producing a molecular model for limb regeneration.
There is a key experiment that could contribute important data. This new data would then help solidify a hypothesis for an accurate molecular model. It has already been established that, at different levels of blastema development, there are different adhesion affinities. (Gilbert p. 85-6). It has also been established that the formation of tissue and organs is mediated by events occurring at the cell surface of adjacent ells, i.e. gene expression and cell-cell interactions (Gilbert, p. 85).

This experiment must establish two things and, therefore, consist of two separate experiments. The first experiment will attempt to find the signal that occurs in the blastema which established the adhesion affinity gradient. The cells at the regenerated blastema should be tested for different adhesive properties at many stages of the limb regeneration. At coinciding times, the experiment should test for different signals that might be coming from extra-blastema cells, i.e. the apical ectodermal cap. In order to test the apical ectodermal cap's effect, one would monitor limb regeneration with the cap removed at different stages of development.

The second experiment will attempt to prove that if space is provided and homogeneous cell interactions are preferred, that a cluster of cells with different adhesive affinities will relocate in order to achieve separate homogeneous clusters. This experiment will test the hypothesis that the cluster of cells with the higher adhesive affinity will migrate away from the cluster of cells with the lower adhesive affinity. This experiment could be performed by creating a cell adhesion affinity gradient sample in vitro, containing clusters of cells ranging from a low adhesive affinity to a high adhesive affinity. The clusters of cells would be arranged progressing from low affinity to high affinity. Then, an external cluster of high adhesive affinity cells would be added to an area of the gradient which contains a lower adhesive affinity. I expect that the cluster of high adhesion affinity cells would migrate until it reached a place where the gradient is greater on one side and lower on the other.

I project that the two above experiments will generate much more evidence to accurately piece together a molecular model for limb regeneration.

| Craig Berman

(cf. Greg Lynn's piece, The Renewed Novelty of Symmetry -ed.)

zones |