University of Kentucky Research on Amphibian Limb Development Published in eLife – via Herp Digest

By Jenny Wells-Hosley Press Release, University of Kentucky 10/10/19

A new study out of UK's biology department examines how forelimbs develop in the axolotl, a type of salamander originally from Mexico. The new findings open up questions for evolutionary biologists regarding the evolution of limbs.

LEXINGTON, Ky. (Oct. 10, 2019) — A new paper by University of Kentucky researchers was recently published in the journal eLife, offering new insights and implications into the study of limb development and the evolution of vertebrate limbs.

Sruthi Purushothaman, a biology doctoral student in the UK College of Arts and Sciences and lead author of the study, examined how forelimbs develop in the axolotl, a type of salamander originally from Mexico. The study presents new information showing how salamanders develop limbs in a strikingly different manner compared to frogs, chickens and mice.  

Most four-limbed animals (known as tetrapods) develop forelimbs as embryos in response to two major cell-signaling centers. These centers are called "the zone of polarizing activity" (located in what's known as the limb bud mesenchyme) and "the apical ectodermal ridge” (located at the tip of the limb in the overlying ectoderm). Both of these centers release signaling molecules that act on limb bud cells to control growth and patterning as the limb takes shape. 

The zone of polarizing activity produces a molecule commonly referred to in the science community as "Sonic hedgehog," or Shh for short. The apical ectodermal ridge produces another group of signals called fibroblast growth factors, or Fgfs.

“The cross-talk between these two centers/signaling molecules is what drives limb bud outgrowth," Purushothaman said.

While older studies indicated that salamander embryos did not have an apical ectodermal ridge (suggesting that these amphibians' limbs may form differently to other tetrapods), contemporary research has treated salamander limbs like those of other tetrapods, such as chicks and mice.

"The integrative model for limb development is based on classic studies in chicken and mouse that do not regenerate their limbs," Purushothaman said. "We asked if this model could be extended to axolotls that not only lack a morphological signaling center but also have the ability to regenerate lost limbs.”

The new experiments by Purushothaman et al. showed that, along with lacking an apical ectodermal ridge, axolotls did not produce Fgfs normally found in this tissue. Instead, Fgfs were only found in the limb bud mesenchyme. The team also found that Fgfs played a different role during salamander limb development than previously reported in other tetrapod embryos. However, the pattern and function of Shh activity in the axolotl limb bud was similar to that previously observed in chicks and mice.

“We found that all the apical ectodermal ridge-specific Fgfs surprisingly reside in the limb mesenchyme during limb development which coincides with previous limb regeneration studies," Purushothaman said. "Upon functional inhibition we found that these Fgfs were not important for limb bud outgrowth although their role during regeneration awaits functional testing.”

The new findings show that not all limbs develop in the same way and open up questions for evolutionary biologists regarding the evolution of limbs. Future studies that examine limb development in other animals that regenerate tissues, such as other amphibians and lungfish, will help answer these questions.

Biology’s beloved amphibian — the axolotl — is racing towards extinction- Although abundant in captivity, the salamander has nearly disappeared from its natural habitat, and that’s a problem. – via Herp Digest

NATURE NEWS FEATURE  by Erik Vance, 11/15/17

Axolotls inhabit thousands of labs and home aquariums around the world, but are vanishing from their natural habitat.Credit: Brett Gundlock for Nature

When biologist Luis Zambrano began his career in the late 1990s, he pictured himself working miles from civilization, maybe discovering new species in some hidden corner of Mexico’s Yucatán Peninsula. Instead, in 2003, he found himself counting amphibians in the polluted, murky canals of Mexico City’s Xochimilco district. The job had its advantages: he was working minutes from his home and studying the axolotl (Ambystoma mexicanum), a national icon in Mexico and arguably the world’s most recognizable salamander. But in that first year, Zambrano couldn’t wait for it to be over.

“Let me tell you, I hated the project at the beginning,” he says. For one thing, “I couldn’t catch anything”.

Over time, however, he did catch some axolotls. What he found surprised him — and changed the course of his career. In 1998, the first robust study to count axolotls estimated that there were about 6,000 of them per square kilometre in Xochimilco1. Zambrano — who now is a professor at the National Autonomous University of Mexico (UNAM) in Mexico City — discovered in 2000 that the number had dropped to about 1,000 animals per square kilometre. By 2008, it was down to 100; today, thanks to pollution and invasive predators, there are fewer than 35 animals per square kilometre1. 

The axolotl is on the brink of annihilation in the canals of Mexico City, its only natural habitat. But although there might be just a few hundred individuals left in the wild, tens of thousands can be found in home aquariums and research laboratories around the world. They are bred so widely in captivity that certain restaurants in Japan even serve them up deep-fried

“The axolotl is a complete conservation paradox,” says Richard Griffiths, an ecologist at the University of Kent in Canterbury, UK, who recruited Zambrano to the project. “Because it’s probably the most widely distributed amphibian around the world in pet shops and labs, and yet it’s almost extinct in the wild.” 

This creates a problem for biologists. Thanks to its unique physiology and remarkable ability to regenerate severed limbs, the axolotl has become an important lab model for everything from tissue repair to development and cancer. But after centuries of inbreeding, captive populations are vulnerable to disease. And the loss of genetic diversity in wild axolotls — owing to their diminishing population — means that scientists lose out on learning all they can about the animal’s biology. 

As lab scientists continue to study the captive animal and its large and complex genome, Zambrano and a handful of other researchers are doing their best to preserve the wild version. They are breeding and releasing axolotls into control ponds and canals in and around Xochimilco to see how they fare, and hopefully to retain some of their natural genetic diversity. The task of saving them is difficult, but should be doable given the animal’s hardiness — if the Mexican government would only engage with the process.

“I’ve seen that in other places in the world, these kinds of huge tasks are possible,” Zambrano says. “If they can do it, why can’t we?”

Axolotls evolved relatively recently compared to other salamander species in the region, and they thrived along the banks of Lake Texcoco in the mountains of central Mexico. They are neotenic, meaning that the adults retain traits seen only in juveniles of similar species. Although other salamanders metamorphose into terrestrial creatures, axolotls hold on to their feathery gills and stay in the water for their entire lives. It’s as if they never grow up.

Sometime in the thirteenth century, Lake Texcoco was settled by the Mexica (the people that Europeans dubbed Aztecs). They built a powerful empire controlled by an island city built in the middle of the lake. As the empire grew, so did the land, expanding much faster after the Spanish conquest in 1521. Today, all that remains of the axolotl’s habitat are about 170 kilometres of canals criss-crossing Xochimilco, a district in the southern part of Mexico City (see maps at https://www.nature.com/articles/d41586-017-05921-w).

The species might have perished entirely under colonial rule, except that its odd inability to grow up caught the attention of European scientists, who puzzled over it in the late nineteenth century. 

Visitors to Mexico brought the creatures back and began breeding them. The animal turned out to be ideal for research: it reproduces readily in the lab, is a hardy survivor and is easy to care for. Axolotls have large cells that simplify investigations into development. Their eggs are almost 30 times larger than a human’s. And in an axolotl embryo, the neural plate cells — a precursor to the brain and spinal cord — are almost 600 times larger by volume. 

Also, the pigmentation of axolotls varies greatly from one cell to the next, unlike in humans or other animals, in which cell traits tend to be uniform. This can help researchers to track which tissues in an embryo become which organs. Yet it has a large genome — roughly ten times the size of a human’s — which can make it challenging to study in some respects.

“It is not a good genetic model organism, but it does regenerate — and that makes it an awesome biological model,” says David Gardiner, a developmental biologist at the University of California, Irvine, who has studied axolotl regeneration for decades. 

In the early twentieth century, axolotls were central to understanding how organs develop and function in vertebrates. They helped scientists to unpick the causes of spina bifida in humans — a birth defect in which the spine doesn’t form properly. And they played a part in the discovery of thyroid hormones: in the 1920s, scientists fed thyroid tissue from livestock to axolotls. If the tissue had been secreting hormone, the axolotls would metamorphose, losing their gills and shedding their larval skin.

In the 1980s, axolotls helped scientists to develop a model explaining how cells take on different forms in embryos. The ‘cell state splitter’ model proposes that many stem cells turn into specific tissues in the body through waves of pulling and stretching as embryos. Scientists found that they could watch the axolotl’s cells squeeze and stretch before they formed tissues. More recently, in 2011, extract from axolotl oocytes has been used to stop breast-cancer cells multiplying by switching on a tumour-suppressor gene2.

But perhaps the most fascinating contribution of the axolotl to science has been in regenerative medicine. The animals can grow back missing limbs, tails, organs, parts of the eye and even portions of the brain. Many scientists have presumed this is because, being neotenic, they retain some trait from their embryonic stages, although other salamanders seem to regenerate even as adults. 

Biologists have been trying to identify the mechanisms behind their regenerative abilities for decades, says Tatiana Sandoval Guzmán, a regeneration researcher at the Technical University of Dresden, Germany. “How do they do it? What is it that they have that we don’t? Or maybe the opposite — what in mammals is stopping that?”

Sandoval Guzmán is interested in bone and muscle regeneration and has taken over a long-standing axolotl laboratory in Dresden. A Mexican national who went to school not far from Xochimilco, she never thought much about the animal and certainly never considered studying it until she came to Germany. Today she is fascinated by the creature, and has shown3 that many of the mechanisms in axolotl regeneration — such as those involving muscle-tissue stem cells — are not so different from those found in humans.

Most regeneration research focuses on the stub — or blastema — that forms over the wound of a severed limb. Whereas such a wound in humans gets covered with skin tissue, axolotls transform nearby cells into stem cells and recruit others from farther away to gather near the injury. There, the cells begin forming bones, skin and veins in almost the same way as when the animal was developing inside the egg. Each tissue contributes its own stem cells to the effort. 

Researchers showed that a protein called transforming growth factor-β is key both in axolotl regeneration and in preventing scar tissue in injured human embryos during the first trimester. Adult mice and humans can regenerate digit tips, although humans lose this ability with age, suggesting that regenerative abilities could be reawakened in mammals. 

“There will be a day when we as humans can regenerate,” says Gardiner. His studies are not focused on rebuilding limbs, but on curing paralysis, growing healthy organs and even reversing ageing by repairing damaged and worn-out tissues. “And when they write that story, it will go back to these model organisms,” he says.

By the time that day comes, however, the wild axolotl may be gone. That worries Gardiner and Sandoval Guzmán because the animals that they study, like many lab animals, are highly inbred. Scientists use an ‘inbreeding coefficient’ to measure how small a gene pool is. Identical twins have a coefficient of 100%; totally unrelated individuals would score close to zero. A score above 12% indicates a population in which individuals are mostly breeding with their first cousins, and is considered a serious concern by ecologists and geneticists. The famously inbred and unhealthy Spanish Habsburg kings of the seventeenth century often had a coefficient somewhere above 20%. The average axolotl inbreeding coefficient is 35%. 

“These animals that we have, they still work just fine, they regenerate just fine. But they are so inbred. It’s a bottleneck,” Gardiner says. “Populations are very vulnerable to disease when inbred.” 

Their high level of inbreeding is partly a result of the bizarre historical path captive axolotls have taken. Most laboratory specimens trace their heritage back to a single group of 34 animals that were taken out of Xochimilco by a French-funded expedition in 1863. They sparked an axolotl-breeding craze across Europe by museums and naturalists.

In 1935, some of the animals travelled from a Polish laboratory back to North America, where they eventually became a breeding stock at the University of Buffalo, New York. Here, scientists brought in a series of wild axolotls to mix up the gene pool and at one point even added in tiger salamanders (Ambystoma tigrinum). The Buffalo population thrived and eventually moved to the University of Kentucky in Lexington, which is now the centre of global axolotl breeding. This means that, in addition to being inbred, almost all of the axolotls in labs and aquariums today are actually part tiger salamander. 

“They got bottlenecked in Europe for sure and then they got bottlenecked again,” says Randal Voss, head of the programme in Kentucky, which holds some 2,000 adults and 3,000–5,000 larvae. 

Voss says that axolotl research today is expanding throughout the world, thanks to modern genetics and stem-cell research. In 2015, he and his group published an initial assembly of the axolotl genome4, a Herculean task given its large size, estimated to be about 32 billion bases. But it is incomplete — the size and complexity of the genome proved too much for the computational power Voss’s group could throw at it. Scientists in several centres continue to work on completing the picture. 

But as they work on that, the creature’s vulnerability to disease has already caused mysterious massive die-offs in Voss’ facility. Scientists worry that if a new infectious disease were to race around labs worldwide, it might force them to abandon the axolotl, potentially setting research back by years. 

What’s more, no one can be sure that lab axolotls haven’t already diverged so much from their wild counterparts that they have lost key elements of regeneration. “Going back to study the wild population can give you a different mechanism or different genes,” says Sandoval Guzmán. “Losing the genetic diversity — of course it’s a loss for science.” 

“I can’t always know for sure, but the axolotls from Kentucky do have some differences,” says Arturo Vergara Iglesias, staring into a tank of axolotls lazily crawling about. “They have a lot of malformations. For example, they often have too many fingers.”

Vergara Iglesias is a biologist at the Centre for Biological and Aquaculture Research (CIBAC), an axolotl breeding facility near Xochimilco that is hoping to preserve a few wild lines. On the side, he breeds his own wild axolotls to sell to labs and pet distributors. He is standing over a salamander tank on a traditional Xochimilco farm plot, or chinampa, that is used as an educational facility for tourists. These animals, and the others he sells, were bred from a group of 32 pulled out of the water not far from the plot. In Mexico, the axolotl is a prized pet and a source of national pride. It’s the subject of countless Mexican memes and souvenirs, and is even the official emoji for Mexico City.

It’s hard to know exactly how many axolotls are left in the wild there. Zambrano guesses that during his last survey, in 2014, there were fewer than 1,000 in total, and perhaps fewer than 500. But he can’t be more specific — in the past two years, he’s been unable to raise the money to do any follow-up studies. That he can’t obtain funding for a simple census does not bode well for conservation efforts.

Students supervised by biologist Luis Zambrano release an axolotl into a protected pond near the National Autonomous University of Mexico in Mexico City. Credit: Brett Gundlock for Nature

Zambrano says that to save the wild axolotl, policymakers must address its two primary threats. The first is non-native fish such as the common carp (Cyprinus carpio) and tilapia (Oreochromis niloticus). Ironically, these were introduced to Xochimilco in the 1970s and 1980s through programmes run by the Food and Agriculture Organization of the United Nations, with the aim of getting more protein into local diets. Zambrano says he has mapped the areas where axolotls still remain; he envisions a team of local fishers being paid to sweep them of fish on an ongoing basis. Although this wouldn’t remove all the fish, for a few hundred thousand dollars it might give the salamanders a window in which to re-establish themselves. His work has shown that axolotls are most vulnerable to carp when they are at the egg stage, and to tilapia when they are juveniles, but reveals that if they can grow beyond a certain size, they might still thrive5.

The second threat is trickier. Every time a powerful storm fills the city’s ageing sewer system, treatment facilities release human waste into Xochimilco, carrying with it ammonia, heavy metals and untold other toxic chemicals. Amphibians, which breathe in part through their highly permeable skin, are vulnerable to these regular pollution dumps. It’s a testament to the animal’s resilience that it exists in the wild at all. 

These are complex issues, but they are not unsolvable. So far, however, there have been no efforts to save the wild axolotl beyond a few halfhearted outreach programmes and some photo opportunities. In 2013, CIBAC released a few thousand axolotls for a behavioural study; some of them survived and even seemed to breed the following year. This suggests that lab-bred salamanders might be able to thrive in the wild if they are raised in captivity to a certain size. But biologists caution that this doesn’t mean Mexico should start releasing them into canals. 

“There’s probably not much point in doing releases into the wild until you can neutralize the threats,” says Griffiths. “You just might be increasing the fish population by just chucking out more fish food.”

When Griffiths first started working in Xochimilco in 2000, his plan was to create a breeding programme aimed at releasing axolotls into the wild. But he and his Mexican partners quickly abandoned the idea once they saw the condition of the ecosystem, which was polluted and teeming with predators. It seemed pointless to send axolotls off to their deaths. Successful reintroductions, such as those of the pool frog (Pelophylax lessonae) in Britain or the hellbender salamander (Cryptobranchus alleganiensis) in the United States, require managing the ecosystem as a whole and working with the community. 

“If we had a million dollars per year for ten years, we would save Xochimilco. Which is nothing compared with the amount of money that is spent in this city,” says Zambrano.

One afternoon in October, Zambrano and a group of volunteers gather by the ponds near the UNAM campus to release ten lab-raised wild axolotls into a protected pond. If the animals survive and breed, they might someday act as a sort of genetic bank for the organism. Zambrano has been sporadically releasing and tracking animals here over two years to understand their behaviour and habitat preferences. His work so far suggests that the salamanders prefer fairly dirty ponds over the most pristine ones — another sign that axolotls might still thrive in Xochimilco if other pressures are removed. Similarly, CIBAC is breeding wild-type animals in an effort to preserve the axolotl’s genetic diversity. But if axolotls do not have a suitable home, most researchers say that their extinction in the wild might be inevitable, no matter what they do. 

“I would be frustrated if I saw it in that way,” says Zambrano. “I see it with another view — that I am doing my best to keep that from happening.”

From Sci Fi to reality: Unlocking the secret to growing new limbs

August 5, 2016

The axolotl, or Mexican salamander, is one of the three regenerative species described in a new paper that identified common genetic regulators governing limb regeneration in all three species. The findings suggest that these regulators …more

Many lower organisms retain the miraculous ability to regenerate form and function of almost any tissue after injury. Humans share many of our genes with these organisms, but our capacity for regeneration is limited. Scientists at the MDI Biological Laboratory in Bar Harbor, Maine, are studying the genetics of these organisms to find out how regenerative mechanisms might be activated in humans.

The ability of animals to regenerate body parts has fascinated scientists since the time of Aristotle. But until the advent of sophisticated tools for genetic and computational analysis, scientists had no way of studying the genetic machinery that enables regeneration. Using such tools, scientists at the MDI Biological Laboratory have identified genetic regulators governing regeneration that are common across species.

In a paper published in the journal PLOS ONE, MDI Biological Laboratory scientists Benjamin L. King, Ph.D., and Voot P. Yin, Ph.D., identified these common genetic regulators in three regenerative species: the zebrafish, a common aquarium fish originally from India; the axolotl, a salamander native to the lakes of Mexico; and the bichir, a ray-finned fish from Africa.

The discovery of genetic mechanisms common to all three of these species, which diverged on the evolutionary tree about 420 million years ago, suggests that these mechanisms aren't specific to individual species, but have been conserved by nature through evolution.

"I remember that day very well—it was a fantastic feeling," said King of the discovery. "We didn't expect the patterns of genetic expression to be vastly different in the three species, but it was amazing to see that they were consistently the same."

Read more at:

Axolotl found in Mexico City lake after scientists feared it only survived in captivity

The amphibian is important in scientific research because of its ability to regenerate severed limbs

KASHMIRA GANDER

Monday 24 February 2014

A rare, salamander-like amphibian has been spotted in its only known natural habitat, after researchers feared the creature had disappeared from the wild.

Mexican biologists have seen, but not caught, two axolotls during a second attempt to find them in the Xochimilco network of lakes and canals of Mexico City.

The researchers took to the muddy waters of lake Xochimilco in small boats last year, and searched for weeks for the amphibian, but to no avail.

But biologist Armando Tovar Garza, of Mexico's National Autonomous University, said that members of the team carrying out the search had seen two axolotls during the first three weeks of a second survey expected to conclude in April.

“We weren't able to capture them...because the behaviour of the axolotl makes them very difficult to capture,” he said.

“But we have had two sightings. That's important, because it tells us we still have a chance.”

Continued

Mexico's 'water monster' disappears

Mexico's salamander-like axolotl may have disappeared from its only known natural habitat in Mexico City's few remaining lakes.

It's disturbing news for an admittedly ugly creature, which has a slimy tail, plumage-like gills and mouth that curls into an odd smile.

The axolotl is known as the "water monster" and the "Mexican walking fish," and its only natural habitat is Lake Xochimilco, which is suffering from pollution and urban sprawl.

Biologist Luis Zambrano of Mexico's National Autonomous University says the most recent three-month attempt to net axolotls found not one of the creatures. He says researchers are planning a second three-month hunt for the creatures, which still survive in labs and breeding tanks.

Read on

Missing Parts? Salamander Regeneration Secret Revealed

Tanya Lewis, LiveScience Staff Writer

Date: 20 May 2013 Time: 03:00 PM ET

Salamanders can regrow entire limbs and regenerate parts of major organs, an ability that relies on their immune systems, research now shows.

axolotl

A study of the axolotl (Ambystoma mexicanum), an aquatic salamander, reveals that immune cells called macrophages are critical in the early stages of regenerating lost limbs. Wiping out these cells permanently prevented regeneration and led to tissue scarring. The findings hint at possible strategies for tissue repair in humans.

"We can look to salamanders as a template of what perfect regeneration looks like," lead study author James Godwin said in a statement. "We need to know exactly what salamanders do and how they do it well, so we can reverse-engineer that into human therapies," added Goodwin, of the Australian Regenerative Medicine Institute (ARMI) at Monash University in Melbourne. 

Read on

Lens regeneration in axolotl: new evidence of developmental plasticity

Rinako Suetsugu-Maki, Nobuyasu Maki, Kenta Nakamura, Saulius Sumanas, Jie Zhu, Katia Del Rio-Tsonis and Panagiotis A Tsonis

For all author emails, please log on.

BMC Biology 2012, 10:103 doi:10.1186/1741-7007-10-103

Published: 17 December 2012

Abstract (provisional)

Background

Among vertebrates lens regeneration is most pronounced in newts, which have the ability to regenerate the entire lens throughout their lives. Regeneration occurs from the dorsal iris by transdifferentiation of the pigment epithelial cells. Interestingly, the ventral iris never contributes to regeneration. Frogs have limited lens regeneration capacity elicited from the cornea during pre-metamorphic stages. The axolotl is another salamander which, like the newt, regenerates its limbs or its tail with the spinal cord, but up until now all reports have shown that it does not regenerate the lens.

Results

Here we present a detailed analysis during different stages of axolotl development, and we show that despite previous beliefs the axolotl does regenerate the lens, however, only during a limited time after hatching. We have found that starting at stage 44 (forelimb bud stage) lens regeneration is possible for nearly two weeks. Regeneration occurs from the iris but, in contrast to the newt, regeneration can be elicited from either the dorsal or the ventral iris and, occasionally, even from both in the same eye. Similar studies in the zebra fish concluded that lens regeneration is not possible.

Conclusions

Regeneration of the lens is possible in the axolotl, but differs from both frogs and newts. Thus the axolotl iris provides a novel and more plastic strategy for lens regeneration.