The molecular foundation of the phylotypic stage


When last we left this subject, I had pointed out that the phenomenon of embryonic similarity within a phylum was real, and that the creationists were in a state of dishonest denial, arguing with archaic interpretations while trying to pretend the observations were false. I also explained that constraints on morphology during development were complex, and that it was going to take something like a thorough comparative analysis of large sets of gene expression data in order to drill down into the mechanisms behind the phylotypic stage.

Guess what? The comparative analysis of large sets of gene expression data is happening. And the creationists are wrong, again.

Again, briefly, here's the phenomenon we're trying to explain. On the left in the diagram below is the 'developmental hourglass': if you compare eggs from various species, and adults from various species, you find a diversity of forms. However, at one period in early development called the phylytypic stage (or pharyngula stage specifically in vertebrates), there is a period of greater similarity. Something is conserved in animals, and it's not clear what; it's not a single gene or anything as concrete as a sequence, but is instead a pattern of interactions between developmentally significant genes.


The diagram on the right is an explanation for the observations on the left. What's going on in development is an increase in complexity over time, shown by the gray line, but the level of global interactions does not increase so simply. What this means is that in development, modular structures are set up that can develop autonomously using only local information; think of an arm, for instance, that is initiated as a limb bud and then gradually differentiates into the bones and muscle and connective tissue of the limb without further central guidance. The developing arm does not need to consult with the toes or get information from the brain in order to grow properly. However, at some point, the limb bud has to be localized somewhere specific in relation to the toes and brain; it does require some sort of global positioning system to place it in the proper position on the embryo. What we want to know is what is the GPS signal for an embryo: what it looks like is that that set of signals is generated at the phylotypic stage, and that's why this particular stage is relatively well-conserved.

One important fact about the diagram above: the graph on the right is entirely speculative and is only presented to illustrate the concept. It's a bit fake, too—the real data would have to involve multiple genes and won't be reducible to a single axis over time in quite this same way.

Two recent papers in Nature have examined the real molecular information behind the phylotypic stage, and they've confirmed the molecular basis of the conservation. Of course, by "recent", I mean a few weeks ago…and there have already been several excellent reviews of the work. Matthew Cobb has a nice, clean summary of both, if you just want to get straight to the answer. Steve Matheson has a three part series thoroughly explaining the research, so if you want all the details, go there.

In the first paper by Kalinka and others, the authors focused on 6 species of Drosophila that were separated by as much as 40 million years of evolution, and examined quantitative gene expression data for over 3000 genes measured at 2 hour intervals. The end result of all that work is a large pile of numbers for each species and each gene that shows how expression varies over time.

Now the interesting part is that those species were compared, and a measure was made of how much the expression varied: that is, if gene X in Drosophila melanogaster had the same expression profile as the homologous gene X in D. simulans, then divergence was low; if gene X was expressed at different times to different degrees in the two species, then divergence was high. In addition, the degree of conservation of the gene sequences between the species were also estimated.

The prediction was that there ought to be a reduction of divergence during the phylotypic period. That is, the expression of genes in these six species should differ the least in developmental genes that were active during that period. In addition, these same genes should show a greater degree of evolutionary constraint.

Guess what? That's exactly what they do see.

Temporal expression divergence is minimized during the phylotypic period. a, Temporal divergence of gene expression at individual time points during embryogenesis. The curve is a second-order polynomial that fits best to the divergence data. Embryo images are three-dimensional renderings of time-lapse embryonic development of D. melanogaster using Selective Plane Illumination Microscopy (SPIM).

That trough in the graph represents a period of reduced gene expression variance between the species, and it corresponds to that phylotypic period. This is an independent confirmation of the morphological evidence: the similarities are real and they are an aspect of a conserved developmental program.

By the way, this pattern only emerges in developmental genes. They also examined genes involved in the immune system and metabolism, for instance, and they show no such correlation. This isn't just a quirk of some functional constraint on general gene expression at one stage of development, but realy is something special about a developmental and evolutionary constraint.

The second paper by Domazet-Loso and Tautz takes a completely different approach. They examine the array of genes expressed at different times in embryonic development of the zebrafish, and then use a comparative analysis of the sequences of those genes against the sequences of genes from the genomic databases to assign a phylogenetic age to them. They call this phylostratigraphy. Each gene can be dated to the time of its origin, and then we can ask when phylogenetically old genes tend to be expressed during development.

The prediction here is that there would be a core of ancient, conserved genes that are important in establishing the body plan, and that they would be expressed during the phylotypic stage. The divergence at earlier and later stages would be a consequence of more novel genes.

Can you guess what they saw? Yeah, this is getting predictable. The observed pattern fits the prediction.

(Click for larger image)

Transcriptome age profiles for the zebrafish ontogeny. a, Cumulative transcriptome age index (TAI) for the different developmental stages. The pink shaded area represents the presumptive phylotypic phase in vertebrates. The overall pattern is significant by repeated measures ANOVA (P = 2.4 3 10-15, after Greenhouse-Geisser correction P = 0.024). Grey shaded areas represent ± the standard error of TAI estimated by bootstrap analysis.

So what does this all tell us? That the phylotypic stage can be observed and measured quantitatively using several different techniques; that it represents a conserved pattern of development gene expression; and that the genes involved are phylogenetically old (as we'd expect if they are conserved.)

Domazet-Loso and Tautz propose two alternative explanations for the phenomenon, one of which I don't find credible.

Adaptations are expected to occur primarily in response to altered ecological conditions. Juvenile and adults interact much more with ecological factors than embryos, which may even be a cause for fast postzygotic isolation. Similarly, the zygote may also react to environmental constraints, for example, via the amount of yolk provided in the egg. In contrast, mid-embryonic stages around the phylotypic phase are normally not in direct contact with the environment and are therefore less likely to be subject to ecological adaptations and evolutionary change. As already suggested by Darwin, this alone could explain the lowered morphological divergence of early ontogenetic stages compared to adults, which would obviate the need to invoke particular constraints. Alternatively, the constraint hypothesis would suggest that it is difficult for newly evolved genes to become recruited to strongly connected regulatory networks.

They propose two alternatives, that the phylotypic stage is privileged and therefore isn't being shaped by selection, or that it is constrained by the presence of a complicated gene network, and therefore is limited in the amount of change that can be tolerated. The first explanation doesn't make sense to me: if a system is freed from selection, then it ought to diverge more rapidly, not less. I'm also baffled by the suggestion that the mid-stage embryos are not in direct contact with the environment. Of course they are…it's just possible that that mid-development environment is more stable and more conserved itself.

What we need to know more about is the specifics of the full regulatory network. A map of the full circuitry, rather than just aggregate measures of divergence, would be nice. I'm looking forward to it!

The creationists aren't, though.

Domazet-Loso, T., & Tautz, D. (2010). A phylogenetically based transcriptome age index mirrors ontogenetic divergence patterns. Nature 468 (7325): 815-818. DOI: 10.1038/nature09632

Kalinka, A., Varga, K., Gerrard, D., Preibisch, S., Corcoran, D., Jarrells, J., Ohler, U., Bergman, C., Tomancak, P. (2010). Gene expression divergence recapitulates the developmental hourglass model. Nature 468 (7325): 811-814 DOI: 10.1038/nature09634

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