Axolotls as models in neoteny and secondary differentiation

Axolotl Basics

The axolotl (AXE-uh-lot-uhl) (Ambystoma mexicanum) is a neotenic salamander congeneric with the tiger salamanders. For a general introduction to these creatures, and information on the availability of transgenic stocks and tips on care, check the permanent colonies housed at Indiana University and the University of Kentucky.

Wild-type melanistic axolotl (Ambystoma mexicanum). From the Indiana University Axolotl Colony website.

Albino axolotl. From the Indiana University Axolotl Colony website.

Neoteny is defined as the retention of conspicuously juvenile characteristics into adulthood, and it is most evident in the axolotl’s prominent gills and fins, which most other amphibians lose in their adult stage. Adapted to live their whole lives in water, they also do not develop eyelids and are fully capable of reproducing in their ‘larval’ state. For more on neoteny in a variety of species (including humans), check the work of Girish Chandra. This does beg the question, though: what is the molecular basis for the abolition of the adult life stage in the axolotl (a similar mechanism turns out to be responsible in other neotenic amphibians like the Amphiumidae.)

Early Hormonal Studies

Preliminary studies undertaken about a century ago to determine the source of the hormone signals cueing amphibian metamorphosis were crude yet revealing. J.F. Gudernatsch made simple extracts from various macerated hormonal organ tissues from several different animals, then added them to aquaria containing young tadpoles in Prague’s unfiltered tap water (which, he notes, was teeming with microbes, as a food source.) Most organ extracts did not significantly differ from controls (no extract added) in their ability to induce metamorphosis. However, thyroid tissue from cats and horses was indeed able halt growth and induce metamorphosis at incredible rates, no matter whether the animal had earlier been fed a different tissue extract. Below is an excerpt from Gudernatsch’s notebook:

(From Gudernatsch 1912)

The Role of Thyroid Hormones

Interestingly, in the reverse situation, amphibians (and fish) have demonstrated sensitivity to goitrogens at early stages of development. A goitrogen is a factor that inhibits the action of THs, resulting in exposure phenotypes identical to mutants with inactive TH signaling. In human adults, the main symptom of thyroid dysfunction is swelling of the thyroid gland in the neck, termed a goiter.

The figure below shows dorsal (A) and lateral (B) views of larval zebrafish with (top) no treatment; (middle) 1mM methimazole, a potent goitrogen; and (C) 1mM methimazole and 30 nM  T4. The top and bottom fish show normal development of their paired fins, while the middle fish show retarded growth. This was replicated in the axolotl, but the data were not presented.

(From Brown 1997.)

Further chemical extractions had by the middle of the century characterized the major thyroid hormones (THs) acting across the Chordata: thyroxine (T4)and triiodothyronine (T3). This spurred a dramatic increase in research into amphibian metamorphosis as then the addition of an easily obtainable chemical to media is all that was required to rapidly induce metamorphosis.

Schematic of general TH levels across metamorphosing amphibians. (From Brown & Cai 2007.)

It Gets Complicated

So, with the advent of more advanced molecular technologies, scientists were enabled to ask probing questions about possible changes in the regulation of THs in the axolotl and other neotenic systems. Unlike in mammals, who have a primordial TH source from their mother, tadpoles hatch externally and are born without any TH present in their bodies. This means that the initial state for TH receptors (TR) is unbound in all tissues until prometamorphosis. In this way, genes responding to TH addition can be identified by differential hybridization of cDNA pools to elucidate downstream effects. TRs are transcription factors having a DNA binding domain (DBD) and a ligand binding domain (LBD) for TH and cofactors, including RXR (unnecessary but facultative). An important finding is that two TRs are found in amphibians: TRα and TRβ. TRα is expressed in newly hatched embryos, and is responsible for the initial binding of TH, while TRβ is largely expressed downstream of TRα signaling, and has a slightly different DBD. So, TRα initiates early metamorphic genes like BTEB and Fra2 (generate adult tissues), while TRβ activates late metamorphic genes like FAP and CN3 (degenerate larval tissues).

The first TR is native, the second is inactive due to a C-terminal deletion, the third is constitutionally active due to a promiscuous viral promoter, and the fourth has an LBD for Vitamin D instead of TR. (From Buchholz et al. 2005.)

Molecular Consequences

Conformational changes occur in the TR on TH binding, changing its binding with other protein partners. Corepressors and histone deacetylases deactivate transcription when TH is unbound, but the histone acetyltransferase complex binds in the presence of TH. This action results in loose histone binding and so greater access to genes with TR promoter sequences.

(From Buchholz et al. 2005.)

Interestingly, axolotls have been experimentally demonstrated to express fully functional isoforms of TRα and TRβ, which bind all necessary cofactors for transcription. Cloning the sequences for the transcripts in the presence of appropriate DNA sequences and T3 produces effective transcription, as shown below.

(From Safi et al. 2004.)

The Secret to Eternal Youth

However, the presence of alternative transcripts for both TRs with deletions mapped to their LBDs provides a potential explanation. If in many axolotl cells only these short, nonfunctional transcripts are expressed, TH signaling should be abolished. This leads to no downstream metamorphosis signals, and juvenile traits are retained.

(From Safi et al. 2004.)

So, experimentation in the axolotl has uncovered the fundamental mechanism underlying their neoteny. Further exploration into the regulation of TR and TH action in other neotenic organisms (including humans) might uncover similar or alternative mechanisms leading to the retention of juvenile characters and explain the evolutionary pressures that favor them.

Return to Amphibians page.

References:
Brown DD. (1997). The role of thyroid hormone in zebrafish and axolotl development. PNAS. 94: (24) 13011-13016.

Brown DD & Cai L. (2007). Amphibian metamorphosis. Developmental Biology. 306: 20–33.

Buchholz, D.R., Paul, B.D., Fu, L., Shi, Y-B., 2006. Molecular and developmental analyses of thyroid hormone receptor function in Xenopus laevis, the African clawed frog. Gen. Comp. Endocrinol. 145, 1–19.

Gudernatsch, J.F., 1912. Feeding experiments on tadpoles: I. The influence of specific organs given as food on growth and differentiation. A contribution to the knowledge of organs with internal secretion. Wilhelm Roux Arch. Entwicklungsmech. Organismen. 35, 457–483.

R. Safi, S. Bertrand, O. Marchand, M. Duffraisse, A. De Luze, J.-M. Vanacker, M. Maraninchi, A. Margotat, B.A. Demeneix and V. Laudet, The axolotl (Ambystoma mexicanum), a neotenic amphibian, expresses functional thyroid hormone receptors, Endocrinology 145 (2004), pp. 760–772.

http://www.ambystoma.org/AGSC/

http://www.axolotl.org/index.htm

http://www.iaszoology.com/neoteny/

http://cairnarvon.rotahall.org/2007/01/27/neoteny-in-humans/

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