Did GDF6 “gene tweak” allow humans to become upright?

The short answer is, “Not really.” But as is often the case, the real story behind so many headlines last week is a bit more complicated.


smh. Links to the first, second, third, and fourth stories.

What are they talking about, Willis?

These headlines, each saying something slightly different, are referring to a study by Indjeian and colleagues published in Cell.  Researchers identified a stretch of DNA that is highly conserved across mammals, or in other words, it is very similar between very different organisms. In humans, however, this conserved region is actually missing (“hCONDEL.306”):

Fig. 4A from Indjeian et al. 2016. A stretch of DNA, "hCONDEL.306" is completely missing in humans (as is another stretch, hCONDEL.305) but otherwise very similar between chimpanzees, monkeys and mice.

Fig. 4A from Indjeian et al. 2016. A stretch of DNA on Chromosome 8, “hCONDEL.306,” is very similar between chimpanzees, macaque monkeys, and mice, but is completely missing in humans (as is another stretch, hCONDEL.305).

That a stretch of DNA should be highly conserved across diverse animal groups suggests purifying natural selection has prevented any mutations from occurring here – alterations to this stretch of DNA negatively affected fitness. But that humans should be missing such a highly conserved region suggests that this deletion came under positive natural selection at some point in human evolution. This strategy, of seeking stretches of DNA that are similar between many animals but very different in humans, has led to the identification of hundreds of genetic underpinnings of human uniqueness. Some of these, such as the case in question, involve deleted sequences and have been termed “hCONDELs,” for “regions with high sequence conservation that are surprisingly deleted in humans” (McLean et al., 2011: 216). Others involve the accumulation of mutations where other animals show few or none (e.g., HACNS1; Prabhakar et al. 2008). In many (most?) cases these are “non-coding” sequences of DNA.

How can “non-coding” DNA help make humans upright?

As was predicted 30 years ago (King and Wilson, 1975), what makes humans different from other animals isn’t so much in the protein-coding DNA (the classical understanding of the term, “genes”), but rather in the control of these protein-coding genes. “Non-coding” means that a stretch of DNA may get transcribed into RNA but is not then translated into proteins. But even though these sequences themselves don’t become anything tangible, many are nevertheless critical in regulating gene expression – when, where and how much a gene gets used. It’s wild stuff. Indeed, “Many human accelerated regions are developmental [gene] enhancers” (Capra et al., 2013).

In the present case, hCONDEL.306 refers to the human-specific deletion of a developmental enhancer located near the GDF6 gene, which is a bone morphogenetic protein. The major finding of the paper, as stated succinctly in the Highlights title page, is that “Humans have lost a conserved regulatory element [hCONDEL.306] controlling GDF6 expression…. Mouse phenotypes suggest that [this] deletion is related to digit shortening in human feet.”

How do they link this “gene tweak” to digit shortening?

Since humans have lost this gene enhancer that is highly conserved in other mammals, Indjeian and team reasoned that the chimpanzee DNA sequence associated with this deletion, retaining the enhancer sequence, is likely the ancestral condition from which the human version evolved. They inserted the chimpanzee version into mouse embryos and watched what happened as they developed. The enhancer was only active in the mice’s back legs, specifically in the cartilage that would later become the lateral toe bones and cells that would become a muscle of the big toe (abductor hallucis). These are areas where humans and chimpanzees differ: our lateral toes are shorter than chimps’, and we only have one abductor hallucis muscle whereas chimpanzees have an additional, longer abductor hallucis  (Aiello and Dean, 2002). So, we’re on our way to seeing how hCONDEL.306 might relate to our big toe or upright walking, as the headlines say.

But this still doesn’t explain how this deletion affects GDF6 gene expression, and therefore what this does for our feet. Pressing onward, the scientists compared the size of certain bones in mice with a normal Gdf6 gene, and those in which the Gdf6 gene was completely turned off (or “knocked out”).  The Gdf6 knock-out mice had shorter lateral toe bones than regular mice, but they also had shorter big toes as well – the previous experiment staining mouse embryos showed the ancestral enhancer was expressed more in the latter toes, not so much the big toe.

Figures 5-6 from Indjeian et al. (2016) sum up the findings. Figure 5 (left) shows that the ancestral version of the GDF6 enhancer (blue staining) is most strongly expressed in the lower limb, especially the fifth toe bone. Figure 6 (right) shows that a lack of GDF6 expression (black bars) results in shorter skull and toe bones. Combining these findings, humans lack a gene enhancer associated with the development of long lateral toes.

Figures 5-6 from Indjeian et al. (2016) sum up the findings. Figure 5 (left) shows that the ancestral version of the GDF6 enhancer (blue staining) is most strongly expressed in the lower half of the body, especially the fifth toe bone. Figure 6 (right) shows that a lack of Gdf6 expression (black bars) results in shorter skull and toe bones. Combining these findings, humans lack a gene enhancer associated with the development of long lateral toes.

hCONDEL.306 doesn’t completely turn off GDF6, so this second experiment doesn’t really tell us exactly what the hCONDEL does. But the results are highly suggestive. Indjeian and team showed that Gdf6 affects toe length, among other skeletal traits, in mice. The ancestral enhancer that humans are missing seems to affect GDF6 activity in the leg/foot only. This illustrates a mechanism of modularity – as the authors state, “Loss of this enhancer would thus preserve normal GDF6 functions in the skull and forelimbs, while confining any … changes to the posterior digits of the hindlimb.” In other words, developmental enhancers allow different parts of the body to evolve independently despite being made by some of the same genes (such as GDF6).

As with any good study, results are intriguing but they raise more questions for future studies. The researchers conducted two experiments to investigate the function of hCONDEL.306: first putting the chimp version in mouse embryos to see where the ancestral enhancer is expressed, and then turning off Gdf6 completely in mice to see what happens. A more direct way to see what hCONDEL.306 does might be to put a longer stretch of the human sequence surrounding GDF6 containing (or rather missing) the ancestral enhancer into mouse embryos. I’m not a molecular biologist so maybe this isn’t possible. But this is important because the ancestral (chimpanzee) enhancer appeared to be most strongly expressed in the little toe, but of course this isn’t our only toe that is short compared to chimps. Similarly, how hCONDEL.306 relates to the abductor hallucis muscle remains in question – does it reduce the size of the intrinsic muscle present in both humans and chimps, or does it prevent development of the longer muscle that chimps have but we lack? We can expect to find hCONDEL.306 in the genomes of Neandertals (and Denisovans?), since they also have short toes, but what would it mean if they retained the ancestral enhancer?

So how does this gene tweak help with upright walking?

This is a really cool paper with important implications for human evolution, but something seems to have been lost in translation between the paper and the headlines (the news pieces themselves are good, though). The upshot of the study is that humans lack a stretch of non-coding DNA, which in chimpanzees (or chimp-ified mice) promotes embryonic development of the lateral toes and a big toe muscle. This may be a genetic basis for at least some aspects of our unique feet that have evolved under natural selection for walking on two legs.

But the headlines misrepresent this result, with words like “led to,” “allowed,” and “caused,” especially when these are followed by “big toe” or “upright walking.” hCONDEL.306 doesn’t really have anything to the big toe bone itself, although it might relate to a muscle affecting our big toe. The only sense in which the “Gene tweak led to humans’ big toe” (first title above) is that hCONDEL.306 might be responsible for our short lateral toes, which make our first toe look big by comparison. The other headlines are misleading since we know from fossil evidence that hominins walked upright long before we have evidence for short toes:

These little piggies get none. Fourth toe bones of living apes and humans (left) and possible hominins from 3-5 million years ago (right).

These little piggies get none. Fourth toe bones of living apes and humans (left) and (probable) hominins from 3-5 million years ago (right). I did my best to get all images to scale.

“Epigenetic,” from the fourth article headline, is simply wrong. Modern day epigenetics is a field concerned with the chemical alterations to the structure of DNA. Even the broad concept of epigenetic as originally devised by Conrad Waddington was about how environments (cellular or outside the body) influence development.

ResearchBlogging.orgIt’s hard to fit all the important and interesting information from scientific papers into news headlines. Still, it would be good if headlines more accurately portrayed scientific findings, especially avoiding such definitive verbs as “caused.” Especially in the realm of biology, people should know that there’s a lot that we still don’t know, that there’s lots more important work left to be done.


Aiello and Dean, 2002. Human Evolutionary Anatomy. Academic Press.

Capra et al., 2013. Many human accelerated regions are developmental enhancers. Philosophical Transactions of the Royal Society B 368: 20130025.

Indjeian et al. 2016. Evolving new skeletal traits by cis-regulatory changes in bone morphogenetic proteins. Cell http://dx.doi.org/10.1016/j.cell.2015.12.007

King and Wilson, 1975. Evolution at two levels in humans and chimpanzees. Science 188: 107-116 DOI: 10.1126/science.1090005

McLean et al., 2011. Human-specific loss of regulatory DNA and the evolution of human-specific traits. Nature 471: 216-219.

Prabhakar et al., 2008. Human-specific gain of function in a developmental enhancer. Science 321: 1346-1350.

Results of the toe-tally easy lab activity

Alternate title: Dorsal canting in primate PPP4s

Earlier this year I suggested a classroom activity in which students can scrutinize the evidence used to argue that the >5 million year old (mya) Ardipithecus kadabba was bipedal. To recap: Ar. kadabba is represented by some teeth, a broken lower jaw, and some fragmentary postcrania. The main piece of evidence that it is a human ancestor and not just any old ape is from a single toe bone, and the orientation of its proximal joint. In Ar. kadabba and animals that hyperdorxiflex their toes (i.e., humans and other bipeds when walking), this joint faces upward, whereas it points backward or even downward in apes. This “dorsal canting” of the proximal toe joint has also been used as evidence that the 4.4 mya Ardipithecus ramidus and 3.5 mya owner of the mystery foot from Burtele are bipedal hominins. A question remains, though – does this anatomy really distinguish locomotor groups such as bipeds from quadrupeds?

Use ImageJ to measure the canting angle between the proximal joint and plantar surface. Proximal to the right, distal to the left.

STUDENT SCIENTISTS TO THE RESCUE! Use ImageJ to measure the canting angle between the proximal joint and plantar surface, as I’ve done on this Japanese macaque monkey (they are not bipedal). Proximal to the right, distal to the left Note I changed the measured angle from the March post.

I sicked my students in Ant 364 (Human Evolutionary Developmental Biology) here at NU on this task. I had students look at only 11 modern primates from the awesome KUPRI database. Most groups are only represented by 1 (Homo sapiens, Hylobates lar and Macaca fuscata) or two (Pongo species and Gorilla gorilla) specimens, all adults. For chimpanzees (Pan troglodytes) there is one infant and four adults. The database has more individuals, and it would be better to include more specimens to get better ideas of species’ ranges of variation, but this is a good training sample for a class assignment. The fossil group includes one Ardipithecus ramidus, one Ar. kadabba, one Australopithecus afarensis, and the PPP4 of the mystery foot from Burtele. The human and all fossils except Ar. kadabba are based off of lateral photographs and not CT scans like for the living primates, meaning there may be some error in their measurements, but we’ll assume for the assignment this is not a problem. Here are their results:

Dorsal canting angle of the fourth proximal pedal phalanx in primates.

Dorsal canting angle of the fourth proximal pedal phalanx in primates. The lower the angle, the more dorsally canted the proximal joint surface. The “Fossil” group includes specimens attributed to ArdipithecusAustralopithecus and something unknown.

Great apes have fairly high angles, meaning generally not dorsally canted proximal joint surfaces. The two gorillas fall right in the adult chimpanzee (adult) range of variation, while chimp infant and orangutans have much higher angles (≥90º means they’re actually angled downward or plantarly). The gibbon (Hylobates) is slightly lower than the chimpanzee range. The macaque has an even more dorsally canted joint, and the human even more so. The fossils, except the measurement for Ar. ramidus (see note above), have lower angles than living apes, but higher than the human and the monkey. If dorsal canting really is really a bony adaptation to forces experienced during life, then the fossil angles suggest these animals’ toes were dorsiflexed more so than living great apes (but not as much as the single monkey and human).

This lab helps students become familiar with CT data, the fossil record, taking measurements (students also measure maximum length of the toe bones and look at the relationship between length and canting), analyzing data, and hypothesis testing. You can also have fun exploring inter-observer error by comparing students’ measurements.

Here’s the full lab handout if you want to use or modify it for your own class: Lab 5-Toe instructions and report

What big teeth you have, indeed

If our friend Little Red Riding Hood was dumb enough to’ve thought a wolf in babushka threads was her grandma, well, she probably would have played Bingo with a grandmother-mimicking Australopithecus anamensis.
Australopithecus anamensis is the earliest undisputed hominid, found in deposits ranging from 4.2 – 3.9 million years ago in Ethiopia and Kenya (Leakey et al. 1995, White et al. 2006). Now, hominids are allegedly distinguished from other apes by having relatively short canine teeth distinguished by having relatively tall ‘shoulders,’ creating a diamond-shape in front view. Nevertheless, compared with humans these early australopiths had pretty murdersome canines, within the range of female chimpanzee species. (my dictionary is trying to tell me ‘murdersome’ isn’t a word, but I learned long ago not to learn right and wrong from a book)
Such canine form – relatively small with tall shoulders – was important in diagnosing Ardipithecus ramidus (> 4.4 million years) as a hominid back in the roaring 1990s (White et al. 1994). Of course, we learned in the 1980s that many ancient fossil apes looked superficially like hominids because of dental similarities, the result of either parallel evolution or hominids’ retention of primitive features. Indeed, even in light of the recently described Ardipithecus ramidus skull and skeleton, the main similarities with later, undisputed hominids are dental.
With this in mind, I’m struck by the canine of Nakalipithecus nakayamai, an ape from Kenya dating to nearly 10 million years ago (Kunimatsu et al. 2007). This is ape was a pretty important discovery because it began to fill in a rather lonesome Late Miocene ape fossil record in Africa. So, below is a picture of Nakali and anamensis canines, which I’ve tried to properly scale with the cutting-edge techniques of Microsoft Powerpoint (that is absolutely not a plug for Microsoft). On the left is Nakalipithecus, and the 2 on the right are Au. anamensis. The middle one is anamensis from Asa Issie in Ethiopia, and is the largest canine found of any hominid, ever I think. On the right is anamensis from Kanapoi in Kenya, not as big but sharp as shi…
…sh kabob skewers. Well crap, the “hominid feature” of short canine crown with nice shoulders is found in this 10 million year-old ape!
Two mutually exclusive scenarios could explain this similarity: [1] this canine morphology truly is a shared-derived feature of hominids, but hominids and Nakalipithecus just happened to evolve the same morphology independently for no better reason than, say, ennui. [2] This morphology is the ancestral condition for hominids (and chimpanzees and possibly gorillas). The fanciest cladistic methods won’t resolve this issue, only the discover of more badass fossils will. But if [2] is correct, that would deal a tough blow to the case of Ar. ramidus (and Sahelanthropus) behing a hominid. Really, it seems like the distinguishing feature of early hominids was their deplorable lack of distinguishing features.
Oy, if bones and teeth are prone to homoplasy (similarity due to parallel evolution and not because of common ancestry), could paleoanthropologists have a special proclivity for it, too (that is, in naming dental hominids)?

Further reading!
Kunimatsu, Y., Nakatsukasa, M., Sawada, Y., Sakai, T., Hyodo, M., Hyodo, H., Itaya, T., Nakaya, H., Saegusa, H., Mazurier, A., Saneyoshi, M., Tsujikawa, H., Yamamoto, A., & Mbua, E. (2007). A new Late Miocene great ape from Kenya and its implications for the origins of African great apes and humans Proceedings of the National Academy of Sciences, 104 (49), 19220-19225 DOI: 10.1073/pnas.0706190104
Leakey, M., Feibel, C., McDougall, I., & Walker, A. (1995). New four-million-year-old hominid species from Kanapoi and Allia Bay, Kenya Nature, 376 (6541), 565-571 DOI: 10.1038/376565a0
Ward, C. (2001). Morphology of Australopithecus anamensis from Kanapoi and Allia Bay, Kenya Journal of Human Evolution, 41 (4), 255-368 DOI: 10.1006/jhev.2001.0507
White, T., Suwa, G., & Asfaw, B. (1994). Australopithecus ramidus, a new species of early hominid from Aramis, Ethiopia Nature, 371 (6495), 306-312 DOI: 10.1038/371306a0
White, T., WoldeGabriel, G., Asfaw, B., Ambrose, S., Beyene, Y., Bernor, R., Boisserie, J., Currie, B., Gilbert, H., Haile-Selassie, Y., Hart, W., Hlusko, L., Howell, F., Kono, R., Lehmann, T., Louchart, A., Lovejoy, C., Renne, P., Saegusa, H., Vrba, E., Wesselman, H., & Suwa, G. (2006). Asa Issie, Aramis and the origin of Australopithecus Nature, 440 (7086), 883-889 DOI: 10.1038/nature04629