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.

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.

References

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.

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Taking back Epigenetics

If I’m good at anything, it’s looking into one topic and then getting distracted by something else during my search. In a recent case, I was scouring the literature on growth and life history. One ribald thing led to another, and next thing I know I’ve stumbled upon Gunter Wagner’s recent review of the book Epigenetics: Linking Genotype and Phenotype in Development and Evolution. WTF is epigenetics, you ask? That’s actually a pretty good question (see here). In the past several years, the term has most often been associated with the causes/effects of structural modifications to chromatin (the DNA-containing stuff that makes up chromosomes). For sure, coincident with Wagner’s review, a paper in last week’s Nature Reviews Genetics defines epigenetics as “the study of mitotically and/or meiotically heritable changes in gene function that cannot be explained by changes in DNA sequence.” (Feil and Fraga 2012).

This is an extremely narrow focus for a term that was originally meant to be about basically everything besides genes that contribute to an organism’s phenotype (this idea was developed by the great, rather underrated, 20th century biologist Conrad Waddington). Lotsa epigenetics research by the narrow definition (i.e. modifications to histones and chromatin) focuses on how cells – not organisms – retain their identity/function (or, phenotype). Epigenetics in the narrow sense are important determinants of an organism’s phenotype, but these alone are insufficient to understand how and why organisms’ become the way they are. Yes, the narrow definition leaves room for environmental influences on gene expression (though “environment” could refer to the state of affairs within a cell or an organism, in addition to the outside world). But it nevertheless imparts agency solely to genomes in affecting an organism becomes.

And this is what the aforesaid book and review are about. Wagner asks, “what would be lost if the original perspective of epigentics [as defined by Waddington] was lost to science?” This is important because an organism is not simply a robotic readout of its genes, but people cannot seem to shake this centuries-old biological determinism.

Is that a homunculus
in your [sperm’s]
pocket?

In the early days of ‘modern’ (or let’s say ‘recent’) biology, there was a popular idea of “Preformationism,” that animals grew from these pre-formed miniature versions of themselves (homunculi) in germ cells. It did not take long for this idea to be quashed, but the underlying idea persisted. Wagner recounts, “With the rise of genetics during the 20th century, however, a new form of quasi-preformism arose, basically replacing the old homunculus with the genome, whereas the developmental process creating the phenotype was put in a black box” (emphasis mine). [See Gilbert et al. (1996) for a nice historical overview describing how the rise of population genetics in the early 20th century left embryology and developmental biology by the wayside of the Modern Evolutionary Synthesis]

This latent desire to essentialize biology to some singular determinant (be it an homunculus or a gene) is something people just can’t get away from. Srsly, there’s a persistent sentiment in biology that Real Science is only the high-profile, lab-coated work in genetics. Along these lines, even I adopted the recently popular narrow view of “epigenetics” a while back when I dated a woman who worked at an epigenetics lab, in hindsight probably so I would sound more like a capital-S Scientist (below).

Hipster scientist. H3S10 phosphorylation correlates with 
decreased levels of heterochromatin, possibly regulating
chromosome condensation (Chenet al 2008). Image: bit.ly/zEfPaq

Of course, genes code for how a cell should behave, but we have this tendency to want to extrapolate from the cell to the organism, and this is where developmental biology becomes a critical link. And this is what the new Epigenetics book is about (so far as I can tell, I haven’t yet had a chance to read it all).

It’s abundantly clear that phenotypes arise out of an inextricably complex series of interactions – between genes, proteins, cells, tissues, environments, etc. These interactions do not occur solely at the genetic (or narrow-sense epigenetic) level. Developmental biology helps ‘connect the dots’ between genes and morphology, but cannot do so by focusing solely on genes and chromatin.

ResearchBlogging.org

References
Chen, E., Zhang, K., Nicolas, E., Cam, H., Zofall, M., & Grewal, S. (2008). Cell cycle control of centromeric repeat transcription and heterochromatin assembly. Nature, 451 (7179), 734-737 DOI: 10.1038/nature06561

Feil, R., & Fraga, M. (2012). Epigenetics and the environment: emerging patterns and implications. Nature Reviews Genetics DOI: 10.1038/nrg3142

Gilbert, S. (1996). Resynthesizing Evolutionary and Developmental Biology. Developmental Biology, 173 (2), 357-372 DOI: 10.1006/dbio.1996.0032

Hallgrímsson B and Hall BK, eds. 2011. Epigenetics: Linking Genotype and Phenotype in Development and Evolution. Berkeley: University of California Press.

Wagner, G. (2011). Epigenetics in all its beauty Trends in Ecology & Evolution DOI: 10.1016/j.tree.2011.09.003