Regulatory divergence modifies limb length between mammals

Check out: This paper by Cretekos et al.

The diversity of vertebrate limb morphology epitomizes our notion of natural selection and evolution by successive slight modifications of a conserved fundamental pattern. Differences in limb morphology between whale flippers, bat and bird wings, and humans certainly show how variation on a theme allows a species to exploit a niche. Take mice and bat forelimbs, for example: the total length of the limb is drastically different between these groups of animals. Certainly, if we could find the evolutionary mechanism for limb morphology divergence between animals, we’d have serious insight into the mechanism of vertebrate evolution in general. Following the observation that bat forelimbs are relatively much longer in late-stage fetuses (and ultimately, in adults) than in mice, these researchers set out to find a reason for this difference.

Vertebrate limbs: Variation on a theme

The present study was set around a premise that is gaining traction in the evolutionary biology world:  “Changes in DNA sequence that alter existing noncoding regulatory elements or create new one [are] enabling events for natural selection and consequently morphological evolution.”  These authors, along with the many proponents of these views, embrace the following, reasonable claim: “[Regulatory] mutations can modify specific aspects of the patterns and/or levels of gene expression, in contrast to coding sequence mutations that cause global alterations of gene function in all expressing tissues.” They assert that the former is more likely due to the later being more likely to cause a serious problem somewhere else in the organism. Let’s take a look at this study and see how well it supports these assertions.

Methods: Replace a regulatory region (a few short stretches of DNA sequence) in mice with the corresponding regulatory region in bats. This region controls the expression of a transcription factor termed PRX1, which has been previously shown to affect limb development in mice; specifically PRX1-null (deleted) mice have extremely short forelimbs…but die at birth. Thus, if changes in the timing or location of expression of the gene PRX1 – as controlled by the regulatory regions – results in the limb length difference between the two species, the transgenic mice will have relatively longer limbs. (Though it is possible to modify the genome of mice, genetic engineering techniques are lacking for most species, so the reciprocal experiment could not be accomplished).

Results: researchers found that PRX 1 expression is increased and a forelimb elongation phenotype develops in mice when combined with the bat enhancer region; mice fetuses that are homozygous (have two copies) for the bat enhancer have 6% longer forelimb length shortly before birth. They also found that complete deletion of the enhancer region in mice results in mice developing completely normally, i.e. forelimbs of normal length throughout all stages. Finally, even mice with two copies of the bat enhancer (whose forelimbs were 6% longer right before birth), end up with limbs of normal length by adulthood. They conclude with the following statement: “Since limb modification reported here accounts for a portion of the relative increase in forelimb length of bat compared to mouse, this may be one of Darwin’s predicted ‘successive slight modifications’ required to a evolve a mammalian wing.”

Ok, ok, so the bat-enhanced mice have slightly longer limbs as measured in a late-stage fetus. Interesting.  But, are the claims really supported by the study? Did they even ask the right question?

Here are some problems with the study, from my point of view:

  1. The authors made the a priori assertion that regulatory changes were the cause of a portion of the difference in limb length. They invoked these non-coding changes because the PRX1 amino acid sequence of mouse and bat differed by only 2 amino acids. I increased taxon sampling to see how conserved these changes are, adding opossum, cow, horse, human and macaque to their measly 2-species alignment of PRX1. Results: These two changes  are ONLY in bat, and not in any of the other species. To me, this would be good evidence that the two amino acid changes are not insignificant and perhaps implicated in limb-length divergence (heck, it takes only 1 amino acid change in hemoglobin to cause sickle-cell anemia).
  2. The PRX1 mRNA levels were increased almost 2-fold in forelimb of mouse under the control of the bat enhancer, but the change in forelimb length is almost insignificant. If regulatory change is so buffered, how can it really effect evolutionary change? What would have happened if the coding region was replaced?

    Closer to mice

  3. Mouse and Bat are not close cousins. They’re separated by about 160 million years of evolutionary time. The background, meaning the rest of the genome and all of its genes and products, are likely to be vastly different between these two animals.  The analysis might have been more informative with the enhancer from the much more closely related orangutan inserted into mouse, for example. These lanky beasts have forelimbs up to 2-m long!

For me, however, the most disappointing part of the study was the subjective choice of the gene chosen for the experiment, and the wide-reaching conclusions of the paper that were largely not supported by the findings.  Remember, PRX1 was chosen as the focus of this study because PRX1-null mice have a short-limb phenotype, not because of any evidence pointing to a role for this gene in limb-length divergence between species.  The question asked by this study was not “what is the mechanism of limb-length divergence between species of mammals”, but instead “does PRX1 enhancer from a bat affect limb-length when put into a mouse.”  To ask the question “how could a trait have evolved”, in my view, is categorically different than the question “how did a trait evolve.”

Finally, I was disappointed that the transgenic mice couldn’t fly (not even a little bit).

-Paul Cziko


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