The tragedy of the axolotl mutant and its mystery gene
Anyone who has suffered the misfortune of an amputation, and others with the imagination to conceive of such a horrible loss, might wish humans shared the famed ability of the Mexican axolotl ( Ambystoma mexicanum) to regenerate their limbs.
The axolotl is a species of salamander (lizard-like amphibians) originally found in Lake Xochimilco, near Mexico City. Sadly, they are now almost extinct in the wild. Their gene pool survives among individuals bred in captivity for the pet trade and for aquaria.
Even though they are amphibians, axolotls remain aquatic throughout their lives. In 1965, the American biologist Rufus R. Humphrey wrote:
“The common name, ‘axolotl’, of Aztec origin, has been variously interpreted as ‘water dog’, ‘water twin’, ‘water sprite’, or ‘water slave’. The last interpretation (“slave of the water”) is in one sense particularly appropriate: Since the Mexican axolotl does not … become adapted to a terrestrial existence, it must spend its life in water, in contrast with its many relatives of the genus Ambystoma.”
Today, a small number of scientists study how axolotls manage to quickly regenerate lost limbs, gills, tail, even their eyes, and parts of the head. The hope for such research is that by understanding how axolotls regenerate lost body parts, we might gather clues on how to increase our own chances of doing the same thing.
In the mid-1960s, Dr. Humphrey mated a pair of sibling axolotls in his laboratory at Indiana University, Bloomington. The mating produced larvae that crowded the laboratory glass bowl – and began to “chew each other’s legs off”, in Dr. Humphrey’s words. He noticed that one-quarter of the larvae that had lost their limbs failed to properly regenerate the chewed legs.
Dr. Humphrey isolated the poor regenerators, grew them to maturity, and mated them. He found that the males were sterile while the females produced eggs that failed to develop, even if they were fertilised by sperm from normal males. In 1966, he hypothesised that both members of the original brother-sister pair carried a mutation in one copy of a gene that he called o (for “ova deficient”). The other copy of the gene was functional, however.
Axolotls, like humans, contain two copies of every gene – one inherited from the father and the other from the mother. The cell created as a result of an axolotl sperm fertilising an axolotl egg is called a zygote. The zygotes develop into larvae, which go on to become adults.
Dr. Humphrey found that half of the sperm made by the male in the brother-sister pair carried the o mutation, as did half of the eggs from the female. Consequently, 25% of the fertilisation (i.e. ½ x ½) involved the fusion of a mutant sperm and a mutant egg. The resulting zygotes lacked a functional copy of the o gene, and developed to become poor limb regenerators.
Later, these axolotls become sterile males and females whose eggs did not develop after fertilisation.
This meant that the o gene coded for something that axolotls needed to develop normally as well as to regenerate damaged or absent appendages.
In the remaining 75% of fertilisations, the sperm, the egg, or both contained the non-mutated version of the o gene. The products of these fertilisations subsequently developed into normal larvae that regenerated injured limbs and turned into fertile adults.
A mystery component
Another American scientist named Robert W. Briggs confirmed Dr. Humphrey’s findings. In 1972, Dr. Briggs found that he could correct the developmental defect of a mutant axolotl female’s eggs by injecting them with a sap drawn from the eggs of normal females. He then fertilised the mutant eggs with sperm from male axolotls that carried one mutated and one functional copy of the o gene.
All the resulting zygotes responded equally at first, and grew to an advanced developmental stage. But at this point, 50% of the zygotes that contained no functional copy of the o gene stopped developing further. The other 50%, which contained one functional copy of the o gene from the father, continued to develop.
This indicated to Dr. Briggs that the father’s copy of the o gene did not have an effect on the zygote’s early development, but began to do so in more advanced stages. Instead, in the early stages, the zygote depended on the o gene product deposited by the mother in her eggs. Or – as in Dr. Briggs’s experiment – in the sap transferred from normal eggs.
Remarkably, even the sap from the nuclei of frog eggs worked.
Given that the product of the o gene was required for normal development and to regenerate damaged appendages, the next step was to identify the component of the sap that essentially ‘rescued’ mutant eggs. A later step would be to test the component’s effects on wound-healing and regeneration in humans.
Unfortunately, maintaining the o mutation turned out to be hard. The mutant’s effect was discernible only in individuals that lacked a functional copy of the o gene. But such individuals were sterile and did not produce progeny. So the researchers needed to fall back on sibling individuals that contained one functional and one mutant gene copy. But these individuals were indistinguishable from those with two functional copies and no mutant copy.
As a result, researchers had to set up multiple sibling matings in each generation, find broods such that 25% were poor regenerators, and then set up new sibling matings to produce the next generation.
Generation after generation of sibling mating results in increasingly inbred individuals that begin to develop other abnormalities. Eventually, tragedy struck. The o mutant was lost before the analytical tools required to zero into the sap component became available. This rendered the wonderful papers of Dr. Humphrey and Dr. Briggs effectively scientifically worthless.
Today, regenerative biologists may be willing to figuratively give an arm to rediscover it.
The author is a retired scientist.