Thomas Hunt Morgan

There is a constant interchange of material and of energy that takes place between a plant or an animal and its surroundings, and this interchange may be influenced by such physical conditions as temperature, light, gravity, etc., or by such chemical conditions as the composition of the atmosphere or of the water surrounding the organism. We can study the process of regeneration either by keeping the regenerating organism under the same conditions that it is subject to in its natural environment, or else we can change the surrounding physical or chemical conditions. In this way we can determine how far the regeneration is affected by external changes, and how far it is independent of them. If a change in the external conditions produces a definite change in the regeneration, then the new condition is called an external factor of regeneration.

TEMPERATURE

That the rate at which regeneration takes place can be influenced by temperature has been shown by Trembley, Spallanzani, Bonnet, and by many more recent writers. In fact, so familiar is the process to every one who has studied regeneration, that it is usually taken for granted that such is the case.

In general it may be stated that the limits of temperature under which normal growth may take place represent also the limits of temperature for regeneration. Lillie and Knowlton (’97) have determined the limits of temperature within which regeneration takes place in Planaria torva. The worm was cut in two transversely through the pharynx, and the time required at different temperatures to produce a new head on the posterior piece was recorded. The lowest temperature at which regeneration was found to take place was 3°C. Of six individuals kept at this temperature only one regenerated at all, and in this one the eyes and brain were still incomplete after six months. The optimum temperature, or at least that at which regeneration takes place most rapidly, was found to be 29.7°C.; a new head developed in 46 days at this temperature. At 31.5°C. regeneration was slower, requiring 8.5 days to make a new head. At 32°C. incomplete regeneration sometimes took place, but death occurred in about six days. At 33°C. regeneration was very slight, and the animals died within three days. At 34°C., and above this point, no regeneration took place, and death soon occurred.

In Hydra viridis, Peebles (’98) has found that regeneration is quicker at 26°-27°C. than at 28°-30°C. At the former temperature regeneration takes place in 48 hours. If kept at 12°C. pieces may regenerate in 96 hours, but not all the pieces had regenerated in this case until 168 hours.

INFLUENCE OF FOOD ON REGENERATION

While the growth of an animal or of a plant is, in most cases, and, of course, within certain limits, directly connected with the amount of food that is obtainable, nevertheless extensive regeneration may take place in an animal, or part of an animal, entirely deprived of food. In this case the material for the new part is derived from the excess of material in the old part, and not only surplus food material, but even the protoplasm itself appears to be drawn upon to furnish material to the new part. The relation between regeneration and the amount of food present in the old part is well shown by experiments with planarians. If a planarian is kept for several months without food, it will decrease very much in size. In fact, the volume of a starved worm of Planaria lugubris compared with that of a fully fed individual may be only one-thirteenth of the latter (Fig. 13, A, B). If a starved worm is cut in two pieces, each piece will regenerate, although less quickly than in a well-fed worm. The new part will continue to increase in size at the expense of the old piece that is already in a starved condition. On the other hand, an excess of food does not necessarily produce a hastening of the regeneration, for, as Bardeen (’01) has shown, worms that have been for several days without food may regenerate more quickly than worms that have been fed just before they were cut into pieces.

[Image unavailable.]

Fig. 13.—Drawn by N. M. Stevens. A. Large well-fed individual of Planaria lugubris. B. Same after being kept without food for 4 mos. 13 days. Both drawn to same scale.

The growth of the new part at the expense of the old tissues is a phenomenon of the greatest importance, an explanation of which will involve, I think, the most fundamental questions pertaining to

[Image unavailable.]

Fig. 13½.—Planaria lugubris. Dotted line indicates where the worm was cut in two lengthwise. Upper three figures show how a half, that is being fed, regenerates. Lower three figures show other half kept without food.

growth. The results show that growth is connected with a structural factor, and is not simply a physiological phenomenon, although no doubt physiological factors are involved. But the physiological factors that are here at work seem to be different from what is ordinarily understood; for the fact that a tissue that is slowly starving to death should be reduced still further, and at a more rapid rate, in order to supply material to a new part, is certainly a remarkable phenomenon. At present we are not in a position to offer any explanation that rests on observation, or experiment, as to how the transfer of material takes place, or as to how the new tissue manages to get hold of the material from other parts. It is possible to protect the old part to a large extent by keeping the regenerating piece well supplied with food. If a well-fed planarian is cut in two along the middle line of the body as indicated in Fig. 13½, A, there develops, in the course of five or six days after the operation, new material along the cut-side of each piece, and a new pharynx appears at the border between the old and the new parts. If one of the pieces is fed at intervals, it is found that the new part grows more rapidly than does the new part in the piece without food. The old tissue in both pieces has shortened somewhat after the operation, and has also decreased somewhat in size as the first new material developed along the cut-side, but in the piece that is fed the old half begins to increase again until it reaches its former size, and may even surpass the latter. A large full-sized worm is produced from this piece, as shown in Fig. 13½, B, C, D. In the starved piece the old part continues to grow small, due to the lack of food and also to the increase in the new side. This increase takes place very slowly, but ultimately a small symmetrical worm may be produced, as shown in Fig. 13½, E, F, G. It will be seen that the starved piece needs to produce relatively less and less new material in order to become symmetrical, because as the old material diminishes, the pharynx comes to lie nearer to the middle line.

EFFECT OF LIGHT ON REGENERATION

Although few experiments have been made to test the effect of light on regeneration, it is certain that in many cases light has no effect on the process, neither as to the quality nor the quantity of the result. In one form, a tubularian hydroid, Eudendrium racemosum, it has been shown by Loeb that the regeneration of the hydranth takes place only when the animal is exposed to light. When a colony of eudendrium is brought into the laboratory and placed in an aquarium, the hydranths soon die; but if the colony is kept in a lighted aquarium, new hydranths are regenerated in a few days. If, on the other hand, the colony is kept in the dark, new hydranths do not appear; but if it is brought back again into the light the hydranths appear. In one experiment one lot of pieces was kept in diffuse daylight, and another lot in the dark. The former produced fifty new hydranths in a few days; those in the dark had not made any hydranths after seventeen days. They were then brought into the light, and in a few days several hydranths had developed on each piece.

Loeb also tried the effect of different colored light on the regeneration of eudendrium. Dishes containing pieces of the hydroid were put into a box that was covered by colored glass plates. Pieces subjected to dark red and to dark blue light gave the following results. The old hydranths, as is generally the case, were absorbed in the course of three days. The first new hydranths appeared in the blue light on the fourth day, and during the following days the hydranths in this lot steadily increased. Eight days after the beginning of the experiment there were eighty hydranths under the blue glass, but not one had developed in the red light. On the ninth day the red glass was replaced by a dark blue one. Two days later hydranths began to appear, and on the following day thirty-two hydranths had appeared, and in a few days more as many as sixty had developed.[19] Loeb concluded that only in the more refrangible (blue) rays does the regeneration of the hydranth take place, while the less refrangible (red) rays act as darkness does.[20] This hydroid is the only animal yet found that shows the effect of light on regeneration, and it is interesting to find that it is one of the few animals known in which light has an influence on the growth, if the heliotropism, or turning towards the light, of the hydranth is looked upon as a phenomenon of growth.

There is another series of experiments made to test the effect of light on regeneration, which gave, however, negative results. Herbst observed that when the eye of certain crustacea[21] is cut off, sometimes an eye and sometimes an antenna is regenerated. A number of individuals from which the eyes had been removed were kept in the light, and others in the dark, in order to see if the presence or absence of light is a factor in determining the kind of regeneration that takes place. It was found that as many individuals regenerated eyes in the dark as in the light. It was discovered later by Herbst and myself, independently, that, when the end only of the eye-stalk is cut off, an eye regenerates, but when the eye-stalk is cut off at the base, an antenna regenerates. The difference in the result has therefore no connection with the presence or absence of light.

GRAVITY

The only case known amongst animals, in which regeneration is influenced by the action of gravity,[22] is that of the hydroid Antennularia antennina. This hydroid lives attached to the bottom of the sea several metres below the surface. The hydroid consists of a single, vertical, central stem, or axis, with two or four series of lateral branches along which the hydranths arise (Fig. 14, A). The stem is attached by so-called stolons, or roots. In its normal growth at the free end the hydroid has been shown by Loeb to exhibit marked geotropic changes. If, for instance, the stem is bent over to one side the new growth that takes place at the apex of the stem directs the new part upwards in a vertical direction.

If pieces are cut from the stem of antennularia and suspended in

Fig. 14.—After Loeb. Normal stalk of Antennularia antennina. B. Piece regenerating in vertical, normal position. C. Piece regenerating in inverted position. D. Piece regenerating in inclined, vertical position. E. Piece regenerating in inclined, inverted position. F. Piece regenerating in horizontal position.

the water, regeneration takes place at the cut-ends. If a piece is suspended with its apical end upwards (Fig. 14, B), a new stem develops at the upper cut-end, and new roots from the lower cut-end. If a piece is suspended with its basal end upwards (Fig. 14, C), there is formed at its upper (basal) end a new stem with its branches also slanting upwards as shown in the figure. Roots appear at the lower (apical) end. Since gravity is the only force that acts in a vertical direction under the conditions of the experiment, Loeb concluded that it plays an important rÔle in determining the kind of regeneration that takes place. Its action is of such a nature that a new stem develops from the upper cut-end, and roots from the lower end, regardless of whether the upper end is the basal or the apical end of the piece. Similar results are also obtained, according to Loeb, if the pieces are suspended obliquely. In a piece of this sort, it is found that new stems arise along the upper surface of the old stem, and roots from the lower surface as well as from the lower cut-end (Fig. 14, D, E). If a piece of the stem is placed horizontally on the bottom of an aquarium, the branches that come off from the under surface of the stem begin to grow downwards at their ends, and where they come in contact with a solid body they fasten themselves to it, thus showing that they are true roots (Fig. 14, F). One or more stems may arise from the upper side of the main stem. These stems grow vertically upwards, and produce lateral branches. Only in one case did a new stem, or stem-like structure, arise from one of the vertical branches, as shown to the left in Fig. 14, F.

Loeb found it also possible to change the character of the growth of the apex of the normal stem and to transform it into a root. A long piece of the hydroid was cut off and suspended vertically with the basal end upwards. From the upper end a new stem began to grow, and then the entire piece was reversed, so that the new stem pointed downwards. Under these circumstances the young stem did not bend around and begin to grow upwards, as a young plant might have done, but it ceased to grow as a stem, and at its apex one or more roots developed. Loeb concludes: “I cannot imagine by what means the place of the formation of organs in antennularia is determined in connection with the orientation of the animal except by means of gravity.”

The response of antennularia to the action of gravity is, I think, conclusively demonstrated by Loeb’s results, but that the phenomenon may be complicated by other factors is shown, I think, by the following experiments. Driesch found that if pieces of antennularia are cut off and placed between horizontal plates, so that both ends are free, roots are produced by the basal end.[23] If the basal end with its new roots is cut off, new roots may appear, but sometimes a thin stem also. If the end is again cut off, a larger stem, and also one or two roots, may appear, and if the operation is repeated again only a stem is formed. The factor that brings about this change is not shown by the experiment. The piece had been kept in a horizontal position throughout the whole time. The apical end died in most cases without producing roots, but it is not stated whether or not roots appear on the stem between the plates of glass. If they develop they may affect the result, as certain experiments that I have made seem to show.

In my experiments, made at a different time of year from that at which Loeb’s experiments were made, pieces of the stem were suspended vertically,—some with the apical end upwards, others with the basal end upwards. In nearly all cases roots were formed by both the upper and lower ends. In a few cases, in which the apical end was upwards, a new stem developed at that end. Pieces suspended in a horizontal position also produced roots at both ends. After removing the ends with their new roots from the pieces suspended vertically, I found that roots again appeared at both ends in nearly every case. The difference between these results and those of Loeb may be due to the time of the year at which the experiments were made, or possibly to some other difference, but the results show that the response to gravity is not always so constant as Loeb’s results indicate.

In a few cases in my experiments the basal end of the hydroid was left attached to the stem on which it had grown, and the piece was put into the same aquarium used for the preceding experiments. In those pieces that lay on the bottom of the aquarium, with the stem standing vertically, a new shoot, and not new roots, appeared on the upper end. Other pieces were hung at the top of the water of the aquarium with the stem turned downwards, and the basal, attached end of the piece upwards. These pieces produced neither a stem nor roots from the apical end. The results show that the presence of roots at one end has an influence on the regeneration at the other end. The same thing was shown in one case in which a short piece sank to the bottom of the dish and, developing roots at its basal end, became fixed: a stem grew out of the apical end.

A number of other experiments that I made, in which pieces of antennularia were fixed to a rotating wheel, gave negative results, since neither roots nor stems appeared on the pieces. The rubbing of the ends of the piece against the water as the wheel turned round, or else the agitation of the water, prevented, most probably, the regeneration from taking place.

How gravity acts on antennularia has not as yet been determined. The only suggestion that we can offer at present is that it brings about a rearrangement of the lighter and heavier parts of the tissues. A rearrangement of this sort has been demonstrated when the egg of the frog is inverted, and in consequence certain changes are brought about in the development that will be described in another chapter.

EFFECT OF CONTACT

The contact of a newly forming part with a solid body has been shown by Loeb in a few cases, at least, to be a factor in regeneration. If a piece is cut from the stem of the tubularian hydroid Tubularia mesembryanthemum, and the piece held so that its basal end comes in contact with a solid body, a root develops at that end. If a piece is held in a similar position, but with its apical end in contact with a solid body, a root does not develop from this end. Evidently the development of a root in this form is also connected with an internal factor; but that there is in reality a reaction in this case, and not simply the development of a root at the basal end, is shown by the following experiment: If a piece is cut from the stem and suspended so that both ends are surrounded by water—it makes no difference whether the piece is vertical or horizontal—a hydranth develops first on the apical end, and then another on the basal end (Fig. 15, B). When the apical end of a piece is stuck in the sand, leaving the basal end free, a hydranth develops on the latter, but not on the end in the sand.

In another hydroid, Margelis carolinensis, studied by Loeb, the effect of contact is more easily demonstrated. If a branch of margelis is put into a dish of water and is kept from all motion, the parts that come in contact with the dish produce roots that attach themselves. Even the apical end of the stem may grow out as a root, as shown in Fig. 15, A. Those parts of the branch that are not in contact with any solid object give rise to new hydranths. Another hydroid, Pennaria tiarella, also shows, according to Loeb, the same response to contact. In this connection it is interesting to find that a growing hydranth of pennaria, if brought in contact with a solid body, turns away from the region of contact and bends at right angles to the body which it touches. We find, once more, that a factor having an influence on the growth of the animal has also a similar influence on the regeneration.

Loeb has found that if pieces of the hydroid Campanularia are cut off and placed in a dish filled with sea water, all the hydranths that touch the bottom of the dish are absorbed and transformed into the substance of the stem. The coenosarc may creep out of the stem wherever it comes in contact with the glass, and produce stolons that give rise to new polyps on their upper surfaces. Loeb shows that growth takes place at the end of the stolon that pushes out of the perisarc, and this growing region draws the rest of the coenosarc after it. If a new hydranth appears along the old piece, the coenosarc is drawn towards the hydranth.

EFFECT OF CHEMICAL CHANGES IN THE ENVIRONMENT

Temperature, light, gravity, and contact are the most familiar kinds of external physical agencies that have a direct influence upon the growth of organisms. Food, though coming from the outside, yet acts only after it has entered the body. Organisms that live in water may be affected by the quantity and the kinds of the salts contained in the water, and also by the dissolved gases. The only experiments that have been made to show the influence of this last class of agents on animals are those made by Loeb. He placed pieces of the stem of tubularia in sea water of different degrees of concentration. After eight days the pieces, that had meanwhile produced hydranths, were measured. It was found that the maximum growth in length takes place, not in normal sea water, but in a much diluted solution. Loeb interprets this result to mean that the cells of tubularia must have a certain amount of turgidity in order to grow, and this is possible so long as the concentration does not pass a certain limit. This limit is reached by the addition of 1.6 grams of sodium chloride to each 100 c.c. of sea water. With a decrease in the concentration, the cells become more turgid, the maximum point corresponding to the maximum amount of growth. Below this point the solution is supposed to act as a poison. The most important result of this experiment is to show that the maximum growth does not take place in sea water in which the animal is accustomed to live, but in a much more dilute solution. Normal sea water contains about 3.8 per cent of salts; the maximum growth takes place in a solution containing only 2.2 per cent. Not only is the length of the stem greater in the latter solution, but the thickness of the stem is also greater. The stem is smaller in a solution containing more salt than that contained in ordinary sea water.

There is another variant in these solutions which Loeb takes into account. With the increase in concentration of the solution its power of absorbing oxygen decreases, but the difference is too slight to affect the main result.

Not only does the amount of salts in solution affect the osmotic condition of the cells, but the salts also play a part in the metabolism of the animal. As the result of a series of experiments, the details of which may be here omitted, Loeb has shown that the regeneration of tubularia takes place only when the salts of potassium and of magnesium are present. A very little of the potassium salt is necessary, too much retards, and still more prevents regeneration.

There must be also a certain amount of oxygen dissolved in sea water in order that regeneration may take place. If a piece of the stem of tubularia is cut off and one end pushed into a small tube that fits the stem closely, and if the tube is then stuck into the sand at the bottom of an aquarium, a hydranth develops only at the free end of the piece, and none at the end in the tube. The result appears to be due to the lack of oxygen. If the piece is then taken from the tube, a hydranth may appear at the end that has been in the tube.

Another experiment shows the same result even more clearly. If a piece of the stem is suspended freely in the water, so that its lower end is almost in contact with the surface of the sand, but does not quite touch it, no regeneration takes place at the lower end. This result is interpreted by Loeb as due to the lack of oxygen in the water near the surface of the sand.[24]

GENERAL CONSIDERATIONS

In connection with the action of external factors on regeneration it is evident that in some cases they may not be in themselves necessary for the growth of a new part, yet when growth takes place they may determine what sort of a part is produced. For instance, if gravity determines the kind of regeneration in antennularia, it is possible that if the regenerating piece were placed on a rotating wheel, the piece might still produce a new stem at the apical end, and roots at the lower end. In an experiment of this sort that I made, the pieces did not, it is true, regenerate at all, but this was probably due not to the change of position in regard to gravity, but to agitation of the water, or to the rubbing of the cut-end against the water. It is also possible that in this form the attachment of the piece at one end may be a factor that may counterbalance the action of gravity. Other factors, such as food, or temperature, or oxygen, appear not to determine the kind of product that results, but only the rapidity with which the change takes place. The salts in solution seem also to act on the rate and extent of the new growth, but possibly other cases may be found in which the kind of regeneration may also be affected by the salts.

It is important to find that those animals whose growth and regeneration are influenced by such external factors as light, gravity, and contact are attached animals that stand in a constant relation to these physical agents. They form only a very small part of the entire number of animals in which regeneration takes place. Animals that constantly move about are not, as a rule, influenced during their growth and regeneration by gravity and contact, and under natural circumstances they are always changing their position in regard to these agents. Temperature, and food, and substances in solution act alike on fixed and free forms, and they are, it appears, both influenced in the same way by these agents. The most significant fact that has been discovered in connection with the influence of external factors on regeneration is that the same factors that influence the normal growth of the organism also affect in the same way the regeneration.

As yet an analysis of the external factors that influence growth has not been made out as completely for animals as for plants, especially in those cases in which the result is determined by several factors at the same time. An examination of the factors that influence regeneration in plants will be made in a later chapter. First, however, the internal factors of regeneration in animals will be considered.