Experimental Production of Atrophied and Partly Deficient Limbs (Phocomelias) in the Axolotl Embryo^*

In an earlier paper (Brandt, 1940) the author has shown that supernumerary limbs such as reduplications and some higher degrees of multiplications can be produced experimentally in the frog embryo by special methods. Contrasting with the group of supernumerary limbs is the group showing stunted growth or even absence of the limb as a whole. On the other hand, there may be absence of parts of a single limb such as phocomelias and stunted growth or abnormal position of the digits.

The following experiments are the first which have been published concerning the problem of experimental production of various types of atrophied limbs in the amphibian embryo.

The methods of production of atrophied or partly deficient limbs in the embryo are different from those which can be applied to the adult animal. The removal of parts of a primordium of the limb or of the whole limb bud in the amphibian embryo is followed by its perfect regeneration. The larva which develops later from such an embryo shows no trace of the operation as a rule, but it was found (Brandt, 1924) that after removal of the limb bud in the embryo newt there was no regeneration of the limb in 6% of the cases.

Implantation of foreign bodies, such as small pieces of coal, wax, or glass, into the primordium of the limb bud is followed either by their elimination in the course of a few hours after the operation or by a large hydrops of the affected area and its adjacent parts which usually causes death of the animal.

The author succeeded in implanting small pieces of ectoderm cut from the tip of the tail of another embryo. This implant was never eliminated and readily healed wherever it was implanted. It was possible to apply the implant at any given spot on the limb bud, and produce different types of atrophied limbs.

Three different methods of implantation were used :

1. Deep implantation into the base of the primordium of the limb, allowing the ectoderm of the host to grow over the implant.

2. Superficial implantation into the outer zone of the primordium after removal of a part of the overlying ectoderm of the host.

3. Removal of a vertical zone of ectoderm from the limb bud running through the whole length of the limb bud and replacing it by the ectoderm of the donor embryo.

In methods (2) and (3) a part only of the ectoderm of the limb bud is replaced by the ectoderm of the donor. If the whole extent of the ectoderm of the limb bud is replaced by the ectoderm of the tail, then a tail-like structure develops and regeneration of the limb is suppressed.

The three methods were employed on sixty embryos of the axolotl (Amblystoma mextcanum) in the straight tail-bud stage and they refer to the cases 950–1010 of the author. For an exact determination of the stage of development, the length of each embryo from the vertex to the tip of the tail and the greatest breadth of the body of the embryo were measured by the aid of an oculo-micrometer; the average of the length-breadth index of the operated embryos was calculated at each day.

The above table shows the details of measurements and will be referred to in the description of the experiments.

Exp. 959, operation 16 May. Six weeks after the operation the right limb consisted of a short pointed stump; the left limb showed rudimentary digits. After a further 4 weeks the right stump did not show any finger-like processes, and it remained at this stage of development without showing any further growth during the next few weeks. The left limb was normal and well differentiated.

Exp. 960, operation 17 May. The right limb bud developed a short stump with a swelling at its caudal border 3 weeks after operation. No further differentiation took place later.

Exp. 963, operation 17 May. Twenty-six days after the operation a normal right limb was fully developed, the length of its upper arm was 1·3 mm., the length of its forearm was 2·4 mm. The total length of the operated left limb was 0·6 mm., and its distal end possessed instead of fingers a series of four very small finger-like elevations (Pl. 4, fig. 1). ^*

Exp. 975, operation 17 May. Six weeks after the operation the normal left limb was fully developed ; the operated right limb consisted of a pointed stump, the proximal end of which was swollen and oedematous (Pl. 4, fig. 2).

Exp. 989, operation 22 May. The limb 3 weeks after the operation was represented by a stump, the distal end of which was bent ventrally. In the course of the next 5 weeks no further growth took place, and the distal end of the limb persisted as a knoblike protuberance.

Exp. 990, operation 22 May. Four weeks after the operation the operated limb was shorter than normal and showed an abnormal position of its digits. The distal end of the limb was bifurcated and showed two digits which were longer than normal. Two other digits of unequal length were inserted along the ulnar border of the limb.

Exp. 1007, operation 27 May. Five weeks after the operation a small stump had developed, the distal end of which was curved downwards (Pl. 4, fig. 3).

Exp. 983, operated 19 May. The left limb showed two digits $512$ weeks after the operation. The development of the third digit was suppressed and in its place the implanted ectoderm was visible. The limb was normal in length (Pl. 4, fig. 4).

Exp. 999, operated 23 May. Five weeks after the operation a limb of normal length had developed, showing a deformed distal end which was formed by the implanted ectoderm and three deformed digits. Two of these consisted of small knob-like elevations, one of which had grown to an abnormal length.

Exp. 1009, operated 26 May. Four weeks after the operation a limb of normal length had developed, the distal end of which was bifurcated and showed two finger-like structures which were longer than normal.

Exp. 993, operated 22 May. Four weeks after the operation the operated limb consisted of a short pointed stump, the proximal end of which showed some oedema, the distal end was formed by the implanted ectoderm.

Exp. 1000, operated 23 May. Five weeks after the operation the implanted ectoderm was attached along the whole length of the radial border of the limb. The distal end of the limb showed three fingerlike elevations. The digits of the unoperated hmb were fully developed. The total length of the operated limb was half the length of the normal limb (Pl. 4, fig. 5).

Exp. 1001, operated 24 May. Five weeks after the operation the implanted ectoderm was attached along the whole length of the radial border of the operated right limb. The pigmentation of the implanted ectoderm contrasted with that of the normal part of the limb which was half the length of the normal limb. A pointed distal end replaced the digits (Pl. 4, fig. 6).

Exp. 1002, operated 24 May. Four weeks after the operation the implanted ectoderm was attached to the side of the body of the larva 1 mm. distant from the root of the limb. The limb itself was normal m size and shape.

Exp. 1005, operated 25 May. Nineteen days after the operation the implanted ectoderm was attached to the dorsum of the proximal part of the limb. The distal end of the operated limb showed normal digits. The limb was stunted and there were no movements visible in the shoulder joint.

1. The results obtained indicate the development of some types of atrophied and locally deficient limbs as related to the method applied.

2. The larvae operated after method 3 (implantation along a vertical line) show the implanted ectoderm still attached along the radial border of the operated limbs (figs. 5 and 6). The length of these limbs is half as long as normal and the distal ends show rudiments of fingers, the number of which is reduced.

3. The length of the limbs, the primordia of which were operated after method 2 (superficial implantation), was always normal, but the digits of those limbs are either deformed (case 999) or they show the implanted ectoderm replacing a digit (case 983) or they possess an abnormal number of digits or some of the digits are abnormally long (case 1009).

4. All cases developing after application of method 1 (deep implantation) showed abnormally short and atrophied limbs. The type of deformity developing after this method is the so-called phoco-mehas. In the phocomelias the proximal parts of the limb are essentially reduced in length and the distal parts show normal or slightly reduced lengths of the digits.

5. Phocomelias is not uncommon in the human newborn and it is considered as a deformity developing on a genetical basis. It has not yet been proved that phocomelias can be produced experimentally by non-genetical methods. We can conclude from the experimental method applied that the ectoderm implanted into the deep area of the embryonic primordium of the limb bud acts as a factor preventing the normal efficiency of developmental potencies.