Regeneration in Animals

Orientation.—Applying the technique of grafting to animals (see GRAFTING IN ANIMALS) it has been possible to test experi mentally whether a structure already determined to form a special organ can be influenced by the place to which it has been trans planted, in regard to its orientation. If regenerating limbs are simply left growing, nobody would expect them to be oriented differently from the original limb they are replacing, therefore no special interest attaches to the fact that the regenerates always are in accord with the whole animal in respect to their orientation. But when young regenerating buds were first grafted without their fate being known, the question arose if a regulation would set in to give the bud the proper orientation towards the host independently of the way in which it had been artificially oriented to the host's stump. The answer seemed to be in the affirmative, when it was found that not only the orientation but also the differentiation itself conformed to the place where the graft had been attached. Only when older buds yielded the result already mentioned, was the observation made that with the determination to form a spe cial organ the orientation had also been stamped on them. When, for instance, an old regenerating bud is taken from a newt's limb it will develop a dorsal surface looking ventrally if it has been grafted upsidedown ; such a limb will, of course, be of no use to its bearer. Any sort of oblique and twisted limb can thus be pro duced by transplanting at different angles of rotation. The direc tion of growth always remains restricted to the long axis which runs proximodistally along the member. It remains restricted, but it need not proceed in the original direction. If a bud, already determined as to the sort of differentiation it is to undergo, be taken from a regenerating limb, its tip be cut off and this new cut surface adapted to the stump of the host's limb, it is of course prevented from growing in the right direction by the contact of the stump. Its free surface now grows in an opposite direction and something occurs that could be taken for a regulation : the differentiation proceeds as if the body were dictating the wanted orientation. Yet this is only apparent. One need only to change the dorso-ventral orientation, together with the reversion of the bud, to find the lack of influence on the part of the body as a whole, for no regulation towards the normal position will assert itself regarding the limb's back and palm. Further insight is given by experiments in which regenerating buds are not severed at their base but amputated together with a portion of old tissue. In grafting, this latter tissue is fixed to the host-stump and regen eration always proceeds along the lines originally laid down by the orientation of the old portion of tissue. Not only does this apply to cases in which the regenerating bud was old at the time of grafting, but also to young buds. These must then be determined by the orientation inherent in the old tissue immediately adjoining the regenerating cells, no influence from the body of the host com ing into play. Moreover, it is not necessary that any regenerating bud be present at the time of transplantation for the same result to appear. When a portion of a newt's limb is grafted in a reversed position, so that the originally distal cut surface becomes prox imal, outgrowth of all distal joints of the member will occur at the free surface, which before the amputation had been in con tact with the proximal part of the limb. Here again, since the superfluous leg is only orientated by the direction of its original outgrowth and free end, we see that there is no regulation exerted by the body itself. If we want then to arrive at an explanation of the phenomena in reversed grafts, it is necessary to take into account the faculties residing in the parts and set free, when opportunity arises, of growing in the opposite direction by the ex posure of cut surfaces. Not all parts are able to keep living when they have been totally separated from the animal, so that in many cases it is necessary to resort to grafting for keeping the parts alive long enough to watch their behaviour in regeneration. In these cases it is often difficult to decide if a given result has been influenced by the host or if the graft has exclusively used its inherent faculties.

Heteromorphosis.—On the other hand, animals which may be cut into several parts transversely to the length of the body with out dying, afford the opportunity of observing structures growing from cut surfaces in a reversed direction. Let a flatworm be cut into three pieces, the head-end with the eyes, the body to where it narrows in to the tail, and the tail. Then the middle piece will regenerate a head with eyes at its anterior, and a tail at its posterior cut surface. The head-piece will not regenerate a tail posteriorly but a second head will appear directed backwards. Similarly, the tail will grow no head anteriorly but develop a second tail. This example of the flatworm shows conclusively that the faculty of producing a structure opposite in direction to that of normal growth is inherent in the head-piece and in the tail-piece independently of the organism as a whole, for the latter is not present in the experiment. Similar results have been obtained with hydroids and also with annelid worms.

The regrowth of tissue, which does not exhibit the form of the missing part but substitutes another familiar form of the same species instead, is termed heteromorphic regeneration (see HETEROMORPHOgIS) ; reproduction of the opposite body-end is called axial heteromorphosis. When a transverse cut is made near the eyes of a flatworm without totally severing the head from the body, the latter may remain attached to the trunk by a small connection, the borders of the wound gaping. From each of its

free surfaces a bud of regeneration will arise. The one from the anterior margin of the trunk will of course develop a head looking in the same direction as the old one. But the bud from the posterior margin of the head behaves as if it were arising from a piece totally severed from the body, developing into a head point ing in the opposite direction to the other two. By taking a tri angular piece out of the body-side near the eye, the tip of the triangle reaching in to the middle line, the two cuts will again try to produce heads. These will grow at right angles to the edges of the wound, but not having room enough they will impede each other in their development and more or less coalesce.

Triplicity.

We are now in a position to give a more satis factory interpretation of monstrosities which consist of the tripli cation of parts and conform to Bateson's rule—viz., that the three components lie in the same plane, the middle one being the mirror-image of the other two. Such triplications have not only been found in lower animals but also in arthropods and verte brates. Generally, they affect in these types not the long axis of the body but lateral appendages. Only in one instance, the tail of the lizard, the monstrosity is not rarely found in the form of a threefold ending of the long axis. Now we may remember that long ago these abnormal tails were ascribed to accident and in our days their regenerative origin has been proved by experiment. It is of course not possible to keep the lizard's tail alive by itself, but the orientation of the three tails in the monstrosities conforms so exactly to what would be expected, if a tail nearly broken off regenerated, one in a forward direction and a second one growing backward, that there can be no doubt of the analogy between the faculty of the tail in flatworms and lizards of producing axial heteromorphosis by themselves. Passing over to the lateral ap pendages, we find triplicity occurring most often in animals with rigid external skeleton, such as crayfish and beetles, or with in ternal skeleton of hard bone, like frogs, birds and mammals. This correlation indicates the origin by breakage, because plastic, soft parts will easily join up again after having been wounded, whereas rigid structures will remain gaping when they have been broken, thereby giving opportunity for regrowth from the two lips of the wound. In several instances it has been possible to observe the two supplementary components arising in this way; triplicated horns have arisen after centrifuging the unicellular Ceratium; treble legs appear in lobster or praying mantis after appropriate injury; trefoil form has been induced in the newt's leg by pre venting a cut from reuniting by insertion of a thread and in the arm of the sea-lily Antedon by a deep rupture. These observations all refer to animals and stages capable of regeneration. When specimens of frogs with a supernumerary pair of legs are found in nature, they cannot have arisen by regeneration of the frog after its metamorphosis because, as we have mentioned before, this stage is incapable of regrowing members. Here experiments on tadpoles had to be performed in their early stage, when the hind-legs were beginning to appear and they were still capable of regeneration. By an appropriate operation this rudiment was made to develop endings in two different directions, a pair of supernumerary legs, mirror-images of each other, being the result. There can scarcely be a doubt that triplications of a mem ber in other animals incapable of regeneration in their developed state are also due to the severing of cells in the larval or embryonic stage. The fruit fly Drosophila has yielded a race in which not unfrequently treble legs appear in the imago. As the maggot has no legs that could break, this deformity must be ascribed to a weakness in the compound of cells developing into the imago's leg, which makes them fall apart, thus making multiple growth possible. In warm-blooded animals the artificial production of triplications has not yet been achieved. Nevertheless, all cases found in nature, to be seen at shows or in museums, conform exactly to Bateson's rule, so that we need not hesitate to see their cause in the rupture of embryonic tissues. In the last few years biologists have begun to make operations on embryos inside the maternal uterus, and will by this technique be able to decide at what stage a given mammal will be able to regenerate an arm or a leg. The few experiments so far conducted on rats indicate that this period lies relatively far before the day of birth. In fowls, the method of grafting pieces from early developmental stages to the chorio-alantoic membrane of the egg, has enabled us to see what becomes of these pieces nourished by the host's blood. This method will make it possible even in birds to test the faculty of embryonic limbs to produce terminal structures at both ends. Everybody is acquainted with the fact of our limbs being mirror-images of those on the other side of the body. It was therefore thought that when supernumerary pairs of legs are found in vertebrates they might represent a bud containing a leg of the left and one of the right side of the body. Apart from the improbability that a left and right member should arise without interposition of the vertebral column, we have been able to collect proofs of both supernumerary members belonging to the same body-side.