How the snake lost its legs – Hox genes & Epigenetics switches?

Reptile Image Gallery
Spot the difference? Is it a snake or a legless lizard?
This article will work best if you also read the “Quotes” in ‘Evolution: Will the Real Ancestor Please Stand UP’ The Free E-book version can be downloaded elsewhere – much nicer than pdf. I’ll have details soon, but contact me in the meantime if you can’t wait at: digging up the future @ gmail dot com – thanks

Before getting into how snakes may have lost their legs seemingly via Hox genes and epigenetic processes and not by Neo-Darwinian means, I’ll begin with an interesting article about legless lizards and snakes and what the difference apparently is.

The article starts with the above image and describes it as follows:: “This legless lizard (an Ophisaurus apodus) is a different beast from its slithery look-alike, the snake” The image is from Joel Sartore/National Geographic/Getty Images

Link to view the whole article here: http://animals.howstuffworks.com/snakes/legless-lizard-vs-snake.htm or below is an excerpt:

What’s the difference between a snake and a legless lizard?

by

“In early 2008, in just one month of scouring the savanna of central Brazil, scientists discovered 14 new species. One of those new species was a type of legless lizard no one knew existed. A legless lizard? Wouldn’t that be called a snake?

Nope — they’re two entirely different animals from separate evolutionary lines. Legless lizards evolved from the legged lizards with which most of us are familiar; legless snakes evolved from four-legged snakes that most of us have never seen.

But the two do look an awful lot alike. Both have long, slender, cylindrical bodies; forked tongues; scaly exteriors and can often be found slithering through sand. And then, of course, there’s the leglessness. It’s tough for the casual observer to tell them apart. It’s not impossible, though.

[…] you’re out hiking and you come across a snake-looking creature, anywhere from 10 inches (25 cm) to 4 feet (122 cm) long. It has the typical reptile coloring, tan or brown, green, bronze or yellowish, and maybe it sports a dark stripe along its back. Is it a snake or a legless lizard?”

Apparently there is a difference…
Anyway that becomes a case of semantics I believe:: “Legless lizards evolved from the legged lizards […]; legless snakes evolved from four-legged snakes”  as most of us have never seen a legless snake, and this distinction also becomes rather interesting when we actually look at the fossil record and draw on more recent molecular data to attempt to understand these creatures that once walked, whether in their serpentine or more lizard-like form. So let’s start at the beginning, a way back in the deep mists of time, when according to the Bible: “The Lord God said unto the Serpent, or maybe we won’t…
So, why did some naughty lizards lose their legs while, others did not?  I found the following article interesting in the light of what I am about to discuss in terms of snakes losing their legs via non-genetic means:
(based on Nicholas Bakalar, Snakes Evolved on Land, New Fossil Find Suggests, National Geographic, April 19, 2006)
In 2003, biologists from Argentina found a 90 million year old snake fossil in a terrestrial deposit in the Río Negro province of northern Patagonia. The snake fossil had a well-defined sacrum supporting a pelvis and functional hind legs outside of its ribcage. This snake was “made for walking.” The discovery of Najash rionegrina is consistent with the concept that snakes evolved on land and that they evolved from long-bodied lizards.
Anyway, moving on to the most recent findings and how this is beginning to be understood in rather non-Neo-Darwinian terms, I will attempt to explain how some reptiles actually may have come to lose their legs and why it certainly doesn’t appear to be via natural selection and far from gradual or genetically driven as promoted in typical mainstream articles as that outlined above. And of course, how this has major implications for our current model that proclaims that it can explain past evolutionary development and how species became species in the first place. For instance, we also have evidence of “Reversion of Limbs in Snakes” as the title of this next quote reveals, which is  both very non-biblical and un-Neo-Darwinian.:
“An interesting instance of reversion of limbs in snakes is inferred from the study of a 95-million-year-old fossil snake from the Middle East. It represents the most extreme hindlimb development seen so far in snakes. The limb consists of tibia, fibula, tarsals, metatarsals, and phalanges. The snake is nested with basal snakes, macrostomatans, which retained rudimentary hind limbs and represents a reversion to the ancestral limbed state (Tchernov et al., 2000)”
.- In Epigenetics comes of Age website dedicated to epigenetic research and developmental evolutionary biology – compiled and edited by highly credentialed biologist Nelson R. Cabej and published in the book Epigenetic Principles of Evolution (2012) link to
There are several answers to serpents losing their legs and becoming legless lizards/snake and regaining them again and becoming a non-snake or legless lizard, but I will keep it as simple as possible. 1. Environmentally-driven  habitat dependent and general influences of the environment during (evolutionary) development of the reptile-type creature. 2, Epigenetic (meaning that it is beyond/above the genes) and this causes real change and adaptation via molecular processes that don’t change the genes, only how they are expressed and 3, Saltation (jumps) evolution which is rather rapid and involves whole and large changes in an organism. (all supported by evidence presented in books relating to Lamarckian-type evolution which Charles Darwin came to support along with natural selection and even believed that in some cases Lamarckian evolution was a better explanation. Plus, the updated form of Lamarckian evolutionary principles in its updated developmental evolutionary biological and genetic form around the turn of the 20th century  see publications by O’Hare 2014 The Epigenetic caterpillar: An Alternative to the neo-Darwinian view of the Peppered Moth Phenomenon and/or Lamarck: and the Sad Tale of the Blind Cave-Fish – Spring 2015 and the quote book: But Old Mr. Darwin wasn’t to Blame: The Little Book of Evolutionary Quotes.
Yes, it doesn’t sound very Neo-Darwinian like, because it isn’t genetically driven, or gradual and slow, it is an EVO-DEVO   (short for evolutionary developmental biology with its underlying saltationist – leaps of evolutionary change) and an epigenesis/epigenetic explanation, which were fully naturalistic and testable evolutionary ideas which were heavily marginalised and suppressed by the formation of an ideologically driven form of gene-centric, selection theory – a far cry from what Darwin had ever envisaged, but is now finding full scientific validation by our most up-to-date scientific techniques and investigations as documented in the above publications by this present writer.
The updated genetic and non-genetic epigenetic explanation is given in a recent article entitled: Learn Genetics on the topic of Genetic science which should give you an idea of what I mean by genetics and then I will apply this to the studies and specific epigenetic studies relating directly to loss of limbs in serpents.
… The epigenome, on the other hand, can change rapidly in response to signals from the environment. And epigenetic changes can happen in many individuals at once. Through epigenetic inheritance, some of the experiences of the parents may pass to future generations. At the same time, the epigenome remains flexible as environmental conditions continue to change. Epigenetic inheritance may allow an organism to continually adjust its gene expression to fit its environment – without changing its DNA code. http://learn.genetics.utah.edu/content/epigenetics/inheritance/
This encapsulates what Lamarck proposed over 200 years ago in a pre-genetic age and what they were beginning to discover in developmental studies over 100 years ago and now being solidly confirmed by our most recent molecular data. In other words, the same genome/genes within the organism do not account for the rather dramatic difference in how the species looks or acts. It is epigenetics and what is going on in the epigenome that makes the difference in the end. Think of a caterpillar. Now think about its adult form – a butterfly or moth. Can you tell me what makes the difference in its dramatically distinctive form (phenotype)? Why does the adult have wings, and antenna and even spindly legs etc? If you think the difference is genetic (its genotype), then you are incorrect. The Neo-Darwinian explanation of different phenotypes: same genotype does not have an explanation for this as our current model believes that the genotype (genes) and their mutations according to selection is the cause of variation seen in the species. The answer is its epigenome that is environmentally sensitive particularly during development and depending upon the season, that causes the exact same genome of the juvenile to be expressed, epigenetically, after metamorphosis in a profoundly different way. (See The Epigenetic Caterpillar: An Alternative to the Neo-Darwinian view of the Peppered Moth Phenomenon).
The lizard  losing its limbs is no different in principle. The Neo-Darwinian explanation would say it is a genetic phenomenon, but this is fully inadequate to explain the evidence as you will see further on. On the other hand, the evidence is strongly pointing to an epigenetic explanation where, environmental-drivers during development ( and this can be easily extrapolated to evolutionary development when the species were less defined as proposed by Lamarck himself), determine the expression or degree of activation (buds of the limbs and degree of developmental expression of existing genes). In other words, the snake became an elongated  (serpentine) creature whose body got too long to be supported by legs and the hind legs remaining on some of these basal snakes (primitive forms) is probably due to a change of habit back in the mists of time (when not fully developed as a fixed and specialised species) when they began burrowing for a living and the hind legs were useful in this pursuit in giving the pushing power to burrow further as the evidence clearly indicates.

Essentially, Lamarck developed the concept of disuse or use of organs, characters and features according to environmental adaptation particularly when organisms were less defined as noted above as one of the main means of adaptive change in the species, or acquired characters as it was termed. For example, the loss of limbs in animals such as snakes and blindness in cave-fish. This is what Lamarck wrote well over 200 years ago:

“The permanent disuse of any organ imperceptibly weakens and deteriorates it, and progressively diminishes its functional capacity, until finally it disappears”.
This quote is given in Cabej’s detailed review of research on epigenetic principles of evolution as referred to above. He goes on to outline Darwin’s own views on such phenomenon as the loss of organs via disuse (note that vestigialization refers to organs/limbs becoming redundant and metazoans is a very broad term for all animal forms, even the simplest amoeba like   animals) and Cabej continues to present the evidence for loss of limbs in reptiles (snakes) as an epigenetic phenomenon and the evidence against a Neo-Darwinian genetically driven one.
“Commonly evolutionary loss of structures is not an “All-or- none” process but the end result of an orderly process of vestigialization of the part or organ. From a Darwinian view, useless organs would not be lost if they would not be disadvantageous to the species. Charles Darwin believed that natural selection was not involved in vestigialization of organs in metazoans since useless organs would not be selected for or against:Rudimentary organs, from being useless, are not regulated by natural selection, and hence are variable. (Darwin, 1872c) […]
There are about 3,000 known reptile squamates (snakes and lizards) on Earth, which have repeatedly and independently experienced limb-reduction in every major continental region […]. Limb-reduced reptile squamates have snake-like body form and may be grouped in two ecomorphs: long-tailed surface dwellers and short-tailed burrowers (Wiens et al., 2006).Loss of Tetrapod LimbsLoss of limbs has occurred in three of four tetrapod groups (amphibians, reptiles, and mammals). It represents one of the most extreme morphological changes in the history of tetrapods (Lande, 1978) and has been associated especially with elongation of the body and increase in the number of vertebrae. It is believed that the loss of limbs occurred in response to new ways of locomotion as a result of a change in the life style of tetrapods. This seems to have been the case with transition of reptiles to a burrowing life style and reptant locomotion, which made their limbs useless. The loss and reduction in size of limbs in tetrapods was thought to have been a gradual process of sequential loss of limb components in the reverse order (distal-to-proximal) of their formation during the individual development (proximal-to-distal).Latter studies, however, have shown that often evolutionary processes of body elongation, reduction of limb size, and reduction of digits, occurred almost simultaneously (Wiens and Slingluff 2001).[…]What scientifically matters in this case is the fact that certain proportions of individuals of the same genotype, under the same environmental conditions, display different phenotypes. This clearly contradicts the basic tenet of the neoDarwinian paradigm that evolution of limblessness, as any other evolutionary change, requires accumulation of favorable mutations in relevant genes. Logically, this suggests that a nongenetic mechanism is inducing the loss of limbs in this snake species.

In other words, it is non-genetic and epigenetic in nature”.

– Taken from Epigenetic Principles of Evolution (2012) link to http://www.epigeneticscomesofage.com/Chapter_16.html

To explain the mechanism for such a radical makeover from walking tetrapod to snake in one simultaneous fell-swoop, or a bit slower in some cases as the fossil snake with the hind legs shows as outlined earlier, I will review another study by Gilbert, entitled: Hox Genes: Descent with Modification, has as is title suggests, fairly profound implications for our view of how species change and indeed offers an explanation in terms an underlying set of instructions common to all organisms in various scales of complexity depending upon their fundamental form (invertebrate/vertebrate etc). Gilbert gives a very solid example of just what these Hox complexes (that act like master switches during development giving instructions to build the fundamental body plan of the animal form according to its complexity) can end up activating (genetically expressing) or not, certain genes in specific order. Gilbert gives a very interesting example of the evolutionary history of how the snake lost its legs and reiterates the fact that snakes lose their forelimbs first and also makes reference to the more derived features of some less primitive type of snakes compared to other more primitive forms in the following. Although,  I should add that Gilbert does not discuss specifically the epigenetic mechanism of Hox complexes, i.e the genes are still present, it is their expression that makes a huge difference in the end, but I’ll return to several studies below that have dealt specifically with Hox gene switches and their epigenetic control:

Both paleontological and embryological evidence supports the view that snakes first lost their forelimbs and later lost their hindlimbs [refs] Fossil snakes with hindlimbs, but no forelimbs, have been found. Moreover, while the most derived snakes (such as vipers) are completely limbless, more primitive snakes (such as boas and pythons) have pelvic girdles and rudimentary femurs. The missing forelimbs can be explained by the Hox expression pattern in the anterior portion of the snake. In most vertebrates, the forelimb forms just anterior to the most anterior expression domain of Hoxc-6 […].
– Gilbert SF. Developmental Biology. 6th edition. Sunderland (MA): Sinauer Associates; 2000. Hox Genes: Descent with Modification. Available from: http://www.ncbi.nlm.nih.gov/books/NBK9978/
More specifically, regarding the epigenetic aspect of Hox gene control during development, in an article relating to the NOVA channel: Ghosts in your genes for example:
“[…] Gene switches such as Ubx make the initial decisions of which genes to turn on or off in
different body regions and cell types. .. This highly evolved, highly orchestrated ability to make
genes active or inactive—both genetically and epigenetically—is the key to the success of
multicellular plants and animals, including the most complex and mysterious of all, us”.
An article entitled: Epigenetic temporal control of mouse Hox genes in vivo is self-explanatory in linking the Hox gene switches to epigenetic processes during development. Another science paper entitled: Epigenetic control of Hox genes during neurogenesis, development, and disease (2010) outlines some of these epigenetic mechanisms and their role in the activation of Hox genes. and the epigenetic control (environmental) of Hox switches is clearly explained in: Epigenetic regulation of vertebrate Hox genes: a dynamic equilibrium, by Soshnikova and Duboule in the abstract they state the following:
 Temporal and spatial control of Hox gene expression is essential for correct patterning of many
animals. In both Drosophila and vertebrates, Polycomb and Trithorax group complexes control
the maintenance of Hox gene expression in appropriate domains. In vertebrates, dynamic
changes in chromatin modifications are also observed during the sequential activation of Hox
genes in the embryo, suggesting that progressive epigenetic modifications could regulate collinear
gene activation.
–  Epigenetics. 2009 Nov 16;4(8):537-40. Epub 2009 Nov 21
http://www.ncbi.nlm.nih.gov/pubmed/19923920
 Another paper discusses the molecular pathways and epigenetic processes and control of Hox genes during mammalian development and states:
The outcome is profoundly influenced by the role of epigenetics through transcriptional
regulation of key developmental genes. Epigenetics refer to changes in gene expression that are
inherited through mechanisms other than the underlying DNA sequence, which control cellular
morphology and identity. It is currently well accepted that epigenetics play central roles in
regulating mammalian development and cellular differentiation by dictating cell fate decisions via
regulation of specific genes.
Among these genes are the Hox family members, which are master regulators of embryonic
development and stem cell differentiation and their mis-regulation leads to human disease and
cancer. The Hox gene discovery led to the establishment of a fundamental role for basic genetics
in development. Hox genes encode for highly conserved transcription factors from flies to
humans that organize the anterior-posterior body axis during embryogenesis. Hox gene
expression during development is tightly regulated in a spatiotemporal manner, partly by
chromatin structure and epigenetic modifications. Here, we review the impact of different
epigenetic mechanisms in development and stem cell differentiation with a clear focus on the
regulation of Hox genes.
in, Ann Anat. 2010 Sep 20;192(5):261-74. doi:
10.1016/j.aanat.2010.07.009. Epub 2010 Aug 6.
http://www.ncbi.nlm.nih.gov/pubmed/20739155

Now returning to how quickly these epigenetically controlled (expressed or not) genetic switches (Hox genes) can operate and how they relate specifically to how the snake became legless, we’ll review some of the most pertinent parts of Cabje’s epigenetic study:

[…]Pythons have no forelimbs but they develop reduced hind limbs. Anatomical transformations in python limbs have been sudden rather than gradual and are related to the progressive expansion of Hox gene expression patterns (Cohn and Tickle, 1999).

The loss of forelimbs in pythons is believed to be related to an anterior expansion of expression pattern of Hox genes. Hind limb buds are initiated in pythons but the ZPA (zone of polarizing activity) does not develop and the ectoderm does not form an AER (apical ectodermal ridge) in the region where the limb bud emerges in tetrapods, even though all the signaling genes responsible for their development are present. This is believed to be caused by changes in mesodermal Hox gene expression:

Progressive expansion of Hox gene expression domains along the body axis can account for the major morphological transitions in snake evolution. (Cohn and Tickle, 1999)

[… ] With changes in genes excluded as cause of vestigialization and loss of limbs in pythons, the remaining alternative explanation is an epigenetic regulatory mechanism. […]

Epigenetic Explanation
Genes for enzymes for RA synthesis in vertebrates have not changed. What has changed is the spatio-temporal pattern of expression of RA in limbed and in limbless tetrapods […] This change is clearly nongenetic (all the limb-inducing genes are present and functional in both limbed and limbless species).Where may be the source of the epigenetic information that is used for these adaptive changes in expression patterns of Hox and other genes involved in the development of limbs or leading to limblessness in tetrapods?[…]In the process of vertebrate limb loss and reduction are also involved mechanisms of programmed cell death, which are epigenetically regulated as well […]
The fact that no changes have occurred in genes for the programmed cell death in limb tissues of tetrapods with reduced limbs, or that have lost their limbs, unequivocally shows that the cause of the programmed cell death is not genetic. […] the programmed cell death during the individual development is epigenetically determined via signal cascades that ultimately originate in the nervous system. Hence, evolution of the programmed cell death in limbless tetrapods has to start with changes in the activity or properties of neural circuits that produce signals that activating signal cascades for the programmed cell death.
So to summarise all this rather technical jargon about loss of limbs or organs or underused parts or change in habitats particularly during the earlier stages of evolutionary development, it would seem that nature has an in-built mechanism (an underlying set of instructions) to build using the genetic instructions, but with a level of flexibility (epigenetic/molecular and ultimately environmentally driven) that many variations can be derived from depending upon the habits, needs of a developing organism. Epigenetic inheritance, or acquired characters derived from these experiences and encounters in different environments passes on via tags/markers (chemical signals more than just genetic information but) epigenetic information. The signals and cascades during development will alter the control of master switches of the larger body plan and adapt, shape and sculpt an organism to its needs. Some of this signalling is informational and will allow for cell-death to take place of budding limbs and inhibit the full formation to continue to build that limb. Indeed, it would seem that nature wastes nothing and the extension of the spine by building more vertebrate, is a redirection of resources that would have otherwise been used to build bone and digits for the limbs. Therefore, if the serpentine creature is getting on much better as a burrowing animal – then the legs (appendages) are going to get in the way a little (particularly the front ones) and the extension of the vertebrate would necessitate only hind-limbs as it is easier to learn to slither and burrow with those hind legs to help, whereas, a set of four limbs would be under a lot of strain from an elongated body.
This is an entirely different form of evolutionary thought to the one we are used to but I believe we should let science speak for itself. And perhaps Mr. Darwin’s support of Lamarck and belief that selection didn’t play a part in the disuse of organs/limbs etc means that he and Mr. Lamarck were not that far off when they believed the loss of limbs was to do with environmental adaptation. And specifically, from disuse or epigenetic processes as it is known today and indeed was understood in the framework of updated Lamarckian evolution in its saltationist less genetically driven form of the early 20th and late 19th century before its ridicule, marginalisation and downright suppression in some cases by the Neo-Darwinian movement.
Lamarckian evolutionary principles may perhaps be contrasted with the Neo-Darwinian version of selection in being randomly genetically driven evolution (natural selection in its genetically modified form) by referring to it as NATURAL CORRECTION, as epigenetic adaptation according to environmental needs and niche specialisation as the species developed (as proposed in principle by Lamarck) is actually a highly adaptive self-correcting molecular system. If a snake doesn’t need it’s legs, well nature shrinks them until they disappear or at lease become inhibited from being fully expressed. This different way of seeing evolution certainly begins to explain the fossil record more clearly and why there are truly no transitional fossils when you look closely at it. Maybe it’s time to begin re-assessing everything we thought we knew about evolution in the light of Lamarckian epigenetic principles. Just a thought. So there concludes my epic tale of how the snake lost its legs, or at least, that is what the science is strongly suggesting how it might have happened….
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