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.

Why the long face?

As was predicted long ago, and is becoming increasingly apparent, many anatomical differences between individuals are due not so much to the DNA coding for specific proteins (“genes”), but rather to the DNA that helps regulate when, where and how much these genes are expressed. A recent paper by Catia Attanasio and colleagues have identified thousands of these latter regions that appear to influence the development of facial shape, using a mélange of modern molecular, microscopic & morphometric methods. This is an exciting step toward understanding the genetic bases of facial variation within, and probably between, species.

Attanasio and colleagues identified “enhancers,” bits of DNA that enhance or increase the transcription of certain genes, relating to the embryonic development of the face. One interesting thing about these enhancers is that they aren’t usually found within the genes they enhance, but may be as far away as a few hundred thousand nucleotides. This is part of why these regulatory elements can be so hard to ascertain. What’s more, in the researchers’ own words, enhancers “often control the expression of their target genes in a modular fashion, where different enhancers activate the expression of the same gene in different cell types, anatomical regions, or at different developmental time points.” So in addition to the difficulty in finding enhancers, their varied ‘behavior’ makes it difficult to figure out exactly what each one does.

I won’t get into the methods they used to do this, but basically they were able to visualize when and where many of these enhancers were active in the developing face of mouse embryos. They also showed that tinkering with these enhancers had characteristic effects on bony facial shape in adults. The results are amazing:

Figure 5 from the paper. Blue/red indicate presence of a given enhancer. The white/blue images are actual mouse embryos, from younger (left) to older (right). Each green/red image is a 3D reconstruction of the blue/white embryo above, based on optical projection tomography.

Figure 5 from the paper. Blue/red indicate presence of a given enhancer. The white/blue images are actual mouse embryos, from younger (left) to older (right). Each green/red image is a 3D reconstruction of the blue/white embryo above, based on optical projection tomography.

Science has also made a very informative and visually stunning video to accompany the paper. Check it out. NOW.

So. Facial shape is the result of massively complex interactions between not just numerous genes, but also the coordination of thousands enhancers and other types of non-coding DNA regulating gene expression. Many other studies have tried to uncover the genetic bases of complex phenotypes (usually diseases) via genome wide association studies (GWAS), scanning genomes for shared genetic variants between individuals with similar phenotypes (I discussed this approach briefly Friday). In contrast to GWAS, what I really like about this study by Attanasio and colleagues is that they not only identify specific stretches of DNA as enhancers, but they also mapped their activity in developing embryos. Thus they could actually see how genetic variants contribute to phenotypes.

This is an important step toward understanding exactly how various genetic diseases affecting the face manifest. In addition, this and other studies uncovering the complex molecular interactions influencing facial shape could form the bases for computational models of development, to predict the genetic and developmental origins of facial evolution.

The paper: Attanasio C et al. 2013. Fine tuning of craniofacial morphology by distant-acting enhancers. Science 342: 1241006.

We like turtles (‘s genomes)

June 2013, Volume 45 No 6 pp 579-714

Jonathan the zombie isn’t the only one who likes turtles. These heroes-in-a-half-shell adorn the cover of the current Nature Genetics, as two species of turtle have just joined the Genome Club (Wang et al. 2013; paper’s free!).

This definitely not one of those genome sequencing studies alluded to recently by John Hawks, that’s “too boring for journals.” Wang and colleagues didn’t just sequence the genomes of soft-shell and green sea turtles ‘just cuz.’ Rather, they use these copious data to address several questions, most interesting of which relate to embryonic development.

First, analysis of gene expression during embryonic development supports what the authors refer to as a “nested hourglass model” of development and gene expression. The hourglass shape serves as analogy for variation across related species over developmental time: there is great variation (in both morphology and gene expression) in the earliest stages of development, then species are more similar at a given developmental stage (the “phylotypic period”), and thereafter variation increases again. This phylotypic period (which I don’t believe is unanimously agreed upon) is arguably the most conserved developmental stage in evolution – all vertebrates, for example, simply must pass through this stage to become good vertebrates. Plus, several studies have found that evolutionarily younger genes tend to be expressed before and after this amorphous phylotypic stage, while more ancient genes are expressed during this time. As the authors state

“According to the recently supported developmental hourglass model … the changes underlying major adult morphological evolution occurred primarily in the developmental stages after the period … that serves as the basic vertebrate body plan.”

So the turtle data generally support this model. However they mention a nested hourglass, because they found evidence of an additional bottleneck, a second hourglass, of conserved gene expression when comparing turtles with their close relative the chicken. In other words, “the most conserved developmental stage changes depending on distantly related species are that are being compared.” So since turtles and chickens are more closely related to one another than to many other vertebrates, they might share another conserved developmental stage. Incidentally, both also make for good soup.

Wang and colleagues also looked for genes relating to some of the unique aspects of turtle anatomy, examining what parts of the genome seem to get kicked up after the phylotypic period. It doesn’t take a trained eye to see that these animals are kinda weird in that their bodies are encased in a flagrant shell, with a carapace on top and plastron on the bottom. Now it turns out this carapace is actually formed from what should, in most other vertebrates, become vertebrae and ribs. So by studying the earliest development of these structures, Wang and colleagues could examine the molecular bases of this carapacial deviation.

Fig. 5 from Wang et al., showing Wnt protein expression in turtle embryos. In a), only Wnt5a is expressed in the ‘carapacial ridge’ during its earliest development. Fig c) is a cross-section indicated in b) showing this expression. NT=neural tube, NC=  notochord. The scale bar is 0.5 mm. Tiny!
The authors were able to identify over 200 miRNAs, and implicate the signalling protein Wnt5a, in the development of the “carapacial ridge” (see the arrows in fig. c above), the embryonic precursors to the carapace. Interestingly, Wnt5a is involved in the development of limb buds (e.g, those big purple circles in the red square in a) above). The precise role of Wnt5a and the miRNAs in turtle shell development has yet to be determined, so this study really sets the stage for future investigations.
ResearchBlogging.orgSo there you have it, a pretty cool paper combining genomics with developmental biology, among other things. And so to close, for your bemusement, here’s a video I shot last week at the awesome Kansas City Zoo, of a turtle attempting to make embryos like in the figure above (sorry for the poor quality). Hang in there, little buddy!
They like tuhtles!
Wang Z, Pascual-Anaya J, Zadissa A, Li W, Niimura Y, Huang Z, Li C, White S, Xiong Z, Fang D, Wang B, Ming Y, Chen Y, Zheng Y, Kuraku S, Pignatelli M, Herrero J, Beal K, Nozawa M, Li Q, Wang J, Zhang H, Yu L, Shigenobu S, Wang J, Liu J, Flicek P, Searle S, Wang J, Kuratani S, Yin Y, Aken B, Zhang G, & Irie N (2013). The draft genomes of soft-shell turtle and green sea turtle yield insights into the development and evolution of the turtle-specific body plan. Nature genetics, 45 (6), 701-6 PMID: 23624526

An end to Ediacaran embryology?

The things people can do these days. Therese Huldtgren and colleagues reported in last week’s Science that they identified nucleus-like structures in 570 million year old fossilized cells from China. These date to the Ediacaran period, before the “Cambrian explosion” of animal life forms. Superficially, these fossilized balls of cells rather resemble the early stages of animal embryos (see A in the figure below), in which cells are dividing and increasing in number but the overall embryo size stays the same. To get the “inside story” (…sorry), Huldtgren and team used very fancy “synchrotron x-ray computed tomography” to look at the insides of these fossilized cells. The resulting images have micrometer resolution – that’s one thousandth* of a millimeter. The things people can do these days.

Fig. 2 from Huldtgren et al. 2011

And lo! each of these fossilized cells contains a small, globular structure that looks like a nucleus (left; if you cross your eyes you can merge the 2 halves of fig. C to make it look even more 3D).

Could these really be the earliest animal embryos? Probably not – some of these balls-of-cells had what resemble budding spores, unlike animals but similar to “nonmetazoan [non-animal] holozoans.” In other words, something neat and old, but not one of our earliest ancestors.

I’m really impressed with the biological applications of computed tomography (CT). Recall that a while ago, I posted about the potential to use synchrotron tomography to examine the small-scale, internal structure of bone (e.g. Cooper et al. 2011). Such non-destructive, high-resolution imaging techniques could be used to compare near-cellular-level growth in living and fossil animals. This is really significant because it adds a completely new kind of information we can get from fossils, which before now could only be studied well at the gross, macroscopic level (though scanning electron microscopy of teeth has been very informative about diet; see for example Ungar and Sponheimer 2011). Indeed, one of the most common applications of CT imaging in anthropology is making 3D computer models of body parts for morphometric (shape) analysis.

But high-resolution, synchrotron CT imaging opens up a whole new world of paleontology, new questions that can be asked. For example, many researchers have examined the microscopic appearance of bone surfaces to determine whether bone was being added or removed during growth, and comparing different species (Bromage 1989, O’Higgins et al. 2001, McCollum 2008, Martinez-Mata et al. 2010). These have been very informative studies, but it is not totally clear how growth at the cellular level relates to growth at visible level. Moreover, fossil surfaces are often abraded, obfuscating surface details. So, I can envision using synchrotron microscopy similar to Cooper et al. (2011) and Huldtgren et al. (2011), to examine bone growth in fossil hominids, at and beneath the surface. This can help us understand how facial growth was modified over the course of human evolution, from the snouty visage of Australopithecus afarensis to the tiny, starry-eyed faces we have today. People could also examine how activities like chewing, running or even talking affect (and effect) bone growth. There is much work to be done.

ResearchBlogging.orgNeat as these projects would be, it’s pretty humbling to consider that we have the technology to analyze microscopic fossils hundreds of millions of years old, and shed light on the developmental processes in our earliest ancestors.

Read those things I’d mentioned

BROMAGE, T. (1989). Ontogeny of the early hominid face Journal of Human Evolution, 18 (8), 751-773 DOI: 10.1016/0047-2484(89)90088-2

Cooper, D., Erickson, B., Peele, A., Hannah, K., Thomas, C., & Clement, J. (2011). Visualization of 3D osteon morphology by synchrotron radiation micro-CT Journal of Anatomy, 219 (4), 481-489 DOI: 10.1111/j.1469-7580.2011.01398.x

Huldtgren, T., Cunningham, J., Yin, C., Stampanoni, M., Marone, F., Donoghue, P., & Bengtson, S. (2011). Fossilized Nuclei and Germination Structures Identify Ediacaran “Animal Embryos” as Encysting Protists Science, 334 (6063), 1696-1699 DOI: 10.1126/science.1209537

Martinez-Maza, C., Rosas, A., & Nieto-Diaz, M. (2010). Brief communication: Identification of bone formation and resorption surfaces by reflected light microscopy American Journal of Physical Anthropology, 143 (2), 313-320 DOI: 10.1002/ajpa.21352

McCollum, M. (2008). Nasomaxillary remodeling and facial form in robust Australopithecus: a reassessment Journal of Human Evolution, 54 (1), 2-14 DOI: 10.1016/j.jhevol.2007.05.013

O’Higgins, P., Chadfield, P., & Jones, N. (2001). Facial growth and the ontogeny of morphological variation within and between the primates Cebus apella and Cercocebus torquatus Journal of Zoology, 254 (3), 337-357 DOI: 10.1017/S095283690100084X

Ungar, P., & Sponheimer, M. (2011). The Diets of Early Hominins Science, 334 (6053), 190-193 DOI: 10.1126/science.1207701

A poor depiction, indeed

As I’ve alluded to in some previous posts, in the Spring semester of 2012, I’ll be teaching “Anthrbio 297: Human Evo-devo” at the University of Michigan. It should be a really fun and interesting class, examining the role of development in human evolution.

Ernst Haeckel’s drawing of embryonic stages in some vertebrates. Taken from Richardson et al. 1997

My department recommends I create a flier that can be posted around campus. One of my first ideas was to adapt a Haeckel’s classic illustration of embryos of different animals passing through similar stages in utero (which we know today isn’t exactly correct; Richardson et al. 1997), but spin it to include primates and fossil humans. I started sketching it out (very crudely), but kept getting distracted with my pitiful attempts at multitasking. When I stopped zoning out, I was aghast to find my adaptation had taken a peculiar turn.

ResearchBlogging.orgI won’t quit my day job.
More about vertebrate embryology
Richardson, M., Hanken, J., Gooneratne, M., Pieau, C., Raynaud, A., Selwood, L., & Wright, G. (1997). There is no highly conserved embryonic stage in the vertebrates: implications for current theories of evolution and development Anatomy and Embryology, 196 (2), 91-106 DOI: 10.1007/s004290050082