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



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.

An un-hominid foot in hominid times

This post was chosen as an Editor's Selection for ResearchBlogging.orgThough my better sense tells me not to say this, researchers announced in Nature today the discovery of a 3.4 million-year-old foot that doesn’t “toe the hominid line.” Dammit I regret that already. Anyway, Ethiopian paleoanthropologist Yohannes Haile-Selassie and colleagues have found the foot of a creature whose big toe was oriented away from the rest of the foot and capable of grasping, like all primates (including Ardipithecus ramidusexcept hominids. See for yourself:

BRT-VP-2/73 foot bones. Look at that fat, abducted hallux! And too-long 4th metatarsal! (fig. 1 from the paper)
World’s greatest left foot.

To help you orient yourself, the left third of the above figure (labeled with a tiny “a”) is a top-view of the ‘articulated’ right foot of this mystery animal. To the right is an X-ray (or “roentgenogram,” if you’re so inclined) of my left foot. This is from two years ago – I’ve been running in Vibrams for about a year now, so I’d really like to see what this X-ray would look like today. And just look at my big toe, having an identity crisis and trying to get away from the rest of the foot.

This is an immensely exciting find. The fossils are from a site in Ethiopia called Burtele dating to around 3.4 million years old. This is 1 million years after Ardipithecus ramidus from Aramis (also in Ethiopia), and contemporaneous with Australopithecus afarensis (also Ethiopian, viz. sites like Maka, Dikika and the earlier parts of the Hadar formation). With its divergent, grasping big toe, we can be pretty sure this foot did not belong to Au. afarensis, the maker of the famous Laetoli Footprints which are a few hundred thousand years older than the Burtele foot. Other aspects of the foot, however, like the round, “domed” heads of the metatarsals and the upward-angling of the proximal toe-bones do suggest this thing may have been bipedal in light of its grasping big toe (or shall we say, “foot-thumb”). Now, this upward canting of proximal toe bones’ proximal ends is associated with bipedalism, but what it most basically reflects is hyper-dorsiflexing (or hyperextension) of the toes – this movement doesn’t necessarily have to come solely during bipedalism, and we have some baboon proximal toe bones in our lab that have slight angling (admittedly, though, not as strongly as in humans).

From the metric analyses of the foot, a few major things stick out. First, where the Burtele foot is similar to humans, both species are also extremely similar to gorillas. The plots at right, from the paper, show the height of the first metatarsal’s (foot-thumb’s) base relative to its length (a), and relative to the base height of the second metatarsal (b). The first plot shows that, compared with chimpanzees and Old World monkeys, the foot-thumb’s base is fairly tall relative to its length. Here, the fossil is smack within the highly-overlapping human and gorilla ranges. The second plot shows that, compared with monkeys, all apes (including humans) and the fossil have tall first metatarsal bases relative to the height of the second metatarsal. Notice that the human and gorilla ranges overlap, though humans are a little higher; here the fossil is at the far end of the human range with a very tall foot-thumb base. Finally, in a principle components analysis of foot bone ratios, humans and gorillas overlap a bit, to the exclusion of chimpanzees and monkeys, and the fossil plots within the gorilla (but not human) range. What really gets me here is the remarkable similarity between humans and gorillas. Since metric analyses indicate that the gorilla-human similarities are largely confined to the aspects foot-thumb, I’d imagine the similarity is due to (1) humans’ putting greater force on our big toes because we walk on two legs, and (2) gorillas’ putting lots of force on their foot-thumbs because they are massive, massive animals. It’s not clear why, though, the Burtele foot-thumb is so similar to both of us.

Another interesting thing revealed by Haile-Selassie et al.’s analyses is that Burtele’s fourth metatarsal is extremely long, unlike African apes (including humans), but more similar to Old World monkeys and the 20 million-year-old early ape Proconsul. The authors take this to suggest that a long 4th metatarsal is the primitive condition for apes, which is quite reasonable. But another question you could raise is, why can’t this mean that Burtele is a giant monkey and not an ape or hominid at all? After all, some hand bones that turned out to belong to a giant colobus monkey were initially thought to belong to the type specimen of Homo habilis (OH 7). I’m certainly not saying this is what I think about the fossil, and it’s very possible that this question is quashed somewhere in the paper’s 35-page online supplement. Nevertheless, you’ll notice that throughout this post, I’ve refrained from referring to BRT-VP-2/73 as an ape, a hominid, or a monkey. In the absence of other parts of the skeleton I don’t think we can be too sure what we have here.

And so what I think is so exciting and important about the Burtele fossils is that they further demonstrate that we have a ton to learn about human (and other apes’) evolution via the fossil record (not that the recent Ardipithecus ramidus, Australopithecus sediba and the Woranso-Mille A. afarensis skeletons haven’t told us this, too). The authors say the Burtele fossils demonstrate a second kind of bipedalism in a hominid lineage separate from the contemporaneous A. afarensis. But since we have no idea what the rest of this animal looked like, it raises the intriguing possibility that we may finally (F*ING FINALLY!) have a fossil ancestor to a living African ape. I’ve long been suspicious that nearly every single ape-like (including humans) fossil found in Africa younger than 7 million years is attributed to the hominid line. I’d be very pleased if this turned out to be a non-hominid ape. (though again I don’t necessarily think that’s what the Burtele fossils are)

Put this in your pipe and read it. Then smoke it.

Haile-Selassie, Y., Saylor, B., Deino, A., Levin, N., Alene, M., & Latimer, B. (2012). A new hominin foot from Ethiopia shows multiple Pliocene bipedal adaptations Nature, 483 (7391), 565-569 DOI: 10.1038/nature10922

Why Lucy, what sweet kicks you had

For decades people have debated whether Australopithecus afarensis was an obligate biped like us, or whether our ancestor was a little less lithe in life on land. They asked, sort of, “Would Lucy have rocked some sweet Air Jordans, or would she have put some flat-foot orthotics in her new kicks?”

Carol Ward and colleagues report on a new fourth metatarsal of Australopithecus afarensis from Hadar in Ethiopia, over 3.2 million years old. The foot bone shows that A. afarensis had the two foot arches that we humans enjoy today.
Metatarsals are the longbones comprising much of the foot right before your silly-looking toes. One exceptional thing about our metatarsals compared to our ape cousins is that they contribute to two arches, one running front-to-back and another side-to-side. The arches provide critical support to our foot for bipedal stance, and a little Fred-Astaire-springiness as our foot hits the ground and then lifts off again when walking and running and sashaying.
The new A. afarensis metatarsal (AL 333-160, right) shows that by 3.2 million years ago, our ancestors had these arches, too. The twisting and angulation of the shaft relative to the base show these arches are similar to humans and our later fossil ancestors, whereas apes’ MT4s tend to be less twisted and angled. Such morphology was hinted at by the famous Laetoli footprints from Tanzania, around 3.7 million years ago, also attributed to A. afarensis. Other evidence from the skeleton suggested Lucy was a biped and nothing else, and so this new find from Hadar further solidifies the idea that some of our skeletal adaptations to bipedalism are ancient indeed.
UPDATE: Thinking about this finding in the shower this morning, I recalled that buddies Jerry DeSilva and Zach Throckmorton recently published a study where they concluded, based on the morphology of the end of the tibia, that A. afarensis probably had at least a rear-foot arch. Interestingly, though, they found some hominid specimens probably had “asymptomatic flatfoot.” Lucy (AL 288) was among these, so maybe she’d be sporting orthoticized Jordans after all.
The Papers
DeSilva JM, & Throckmorton ZJ (2010). Lucy’s flat feet: the relationship between the ankle and rearfoot arching in early hominins. PloS one, 5 (12) PMID: 21203433

Ward, C., Kimbel, W., & Johanson, D. (2011). Complete Fourth Metatarsal and Arches in the Foot of Australopithecus afarensis Science, 331 (6018), 750-753 DOI: 10.1126/science.1201463

Ardipithecus: Foot

I’ve fallen a bit behind in posting about the Ardipithecus remains. Here are some things about the foot, which is quite an interesting piece of the puzzle.

What they have of the foot is nothing too shabby: a talus, medial and intermediate cuneiforms, a cuboid, the first three metatarsals (MT1-3), and some phalanges.

I mentioned a few weeks ago that one of the most striking features of the foot is the abducted hallux–Ardi had a grasping big toe, like apes. They can tell this from the orientation of the medial cuneiform’s articular facet for the MT1. In addition, the joint surfaces of the two bones show that the MT1 could rotate about the joint. In humans and other bipedal hominins, these surfaces are more or less flat. Ardi’s foot is a big deal because what we have of hominin feet up to this point suggest a more-or-less human-like ability to transmit forces from bipedal walking, and MT1 plays a major role in this (that’s why our big toes are so big and stupid looking).

But Ardi couldn’t have walked bipedally this way. Rather, the authors posit that walking-forces were largely transmited through MT2-3. They point out some interesting features to support this: relative to their length, the bases of these bones are fairly tall; the sphericity of their heads (especially on the superior surface) hint that the toes were hyperextended. Additionally, notches on the dorsal aspects of both the MT2 base and the intermediate cuneiform (which articulates with the MT2) were probably caused by habitual pressure caused by the tarso-metatarsal joint capsule, possibly from “upright walking and running” (Lovejoy et al. 2009, p. 72e9). Well, I don’t know about running. One thing I noticed about the MT3 head is that, while it is fairly “domed” as in humans, with a transverse depression just behind the dorsal surface as in humans, it also has a large tuberosity behind the depression, which looks fairly similar when viewed from the side (but not totally) to the transverse ridges on African ape metacarpals. In apes, these ridges prevent the fingers from hyperextending–this similar-looking feature in Ardi’s foot could also have prevented hyperextension of the toe (?), not very biped-like. Of course, who says there’s only one way to be bipedal?

The talus–the bone sitting the center of your ankle–is variously ape- or monkey-like in how the joint contacts the tibia, suggesting the tibia was fairly obliquely oriented on the joint (in us bipeds it’s more or less perpendicular) (DeSilva 2009). Ardi’s cuboid also gets a lot of air-time in the paper. The cuboid is a little wedged bone sitting on the side of your foot, and because of this lateral position, the tendon of the fibularis longus muscle crosses over the side and inserts into the base of the MT1 (and of the medial cuneiform in humans). In humans and Old World monkeys, this surface also bears an articular surface for the ‘os peroneum,’ a small bone sitting within the tendon. Apparently this articular surface is lacking in apes. For Ardi, the authors discover a fairly monkey-like morphology for this surface. Ardi’s cuboid, then, indicates a monkey-like ability to adduct the big toe, but also support the structural integrity of the middle foot.

In sum, like in the pelvis paper, the authors posit more bipedal functionality than I’d be comfortable making. The foot of Ardipithecus ramidus was certainly something interesting, capable of ape-like grasping, and probably a non-trivial amount of dorsiflexion at the ankle, as well (read: ‘capable climber’). At the same time, it is not clear to what extent it was used for ape-like climbing and/or monkey-like quadrupedalism. And if it was walking bipedally, it was doing it quite differently from any human or other fossil hominin. Hey, maybe this is the foot you need to be bipedal in the trees?

Just one more thing: I’m not terribly familiar with what feet are available from Miocene apes–a decent outgroup for comparing the functional/phylogenetic morphology of fossil feet. But, again like in the pelvis paper, this paper doesn’t mention Oreopithecus, which may be a good comparison to make. Oreopithecus was an Italian insular ape some 7 or so million years ago. It has also been argued, fairly recently, to be bipedal based on a lordotic lumbar spine, presence on the innominate of an anterior inferior iliac spine, and the architecture of the internal trabecular bone of the ilium (Rook et al. 1999; Kohler and Moya-Sola 1997). For the longest time, the enigmatic morphological convergences between Oreopithecus and hominins have been attributed to the former’s unique insular habitat: absence of predators in this unique enviroment allowed for upright posture, and possibly even bipedalism, to evolve in this now-extinct ape. But seeing what all this shares with Ardipithecus–hominin or not–it may be that such upright, “arboreal bipedal” positional behavior is the ancestral hominoid condition.

DeSilva J. 2009. Functional morphology of the ankle and the likelihood of climbing in early hominins. Proceedings of the National Academy of Sciences 106: 6567-6572.

Kohler M and Moya-Sola S. 1997. Ape-like or hominid-like? The positional behavior of Oreopithecus bambolii reconsidered. Proceedings of the National Academy of Sciences 94: 11747-11751.

Lovejoy CO, Latimer B, Suwa g, Asfaw B, and White TD. 2009. Combining Prehension and Propulsion: The Foot of Ardipithecus ramidus. Science 326: 72e1-72e8.

Rook L, Bondioli L, Kohler M, Moya-Sola S, and Macchiarelli R. 1999. Oreopithecus was a bipedal ape after all: evidence from the iliac cancellous architecture. Proceedings of the National Academy of Sciences 96: 8795-8799.

Australopithecus africanus (?) foot bone, and a small rant







