It’s just a stage. A phylotypic stage. Part III: Fish and more.

by Stephen F. Matheson
Originally posted on Panda's Thumb, December 2010.

Given that disputes over the existence and meaning of the phylotypic stage and the hourglass
model have simmered in various forms for a century and a half, the remarkable correspondence
between the hourglass model and gene expression divergence discovered by Kalinka and Varga and
colleagues would be big news all by itself. But amazingly, that issue of Nature included two distinct
reports on the underpinnings of the phylotypic stage. The other article involved work in another
venerable model system in genetics, the zebrafish.
The report is titled "A phylogenetically based transcriptome age index mirrors ontogenetic
divergence patterns" and is co-authored by Tomislav Domazet-Loso and Diethard Tautz. To
understand how their work has shed light on the phylotypic stage and the evolution of
development, we’ll need to look first at an approach to the analysis of evolutionary genetics that
these two scientists pioneered: phylostratigraphy.
The authors first described phylostratigraphy in 2007 and have since used the approach to examine
genes that cause human genetic disease and cancer. They define it as:
a statistical approach for reconstruction of macroevolutionary trends based on the
principle of founder gene formation and punctuated emergence of protein families.

The idea is that every gene has a birthday, a point at which it is first identifiable in evolutionary
history. Some genes are ancient, having arisen before there were even complex cells, and others are
relative juveniles, having arisen much more recently. Genes present today, then, can (in principle)
be assigned an "age." Domazet-Loso and Tautz represent the "age" of a gene by the evolutionary
"epoch" in which it appeared, by analogy with the identification of the appearance of biological
lineages with stratigraphic epochs in earth’s history. So for example, some genes appear with the
development of true animals (metazoa), and so these genes are assigned to that "stratum" of
biological history. In fact, the authors call each epoch a ‘phylostratum’ to reinforce that metaphor.
So how does this work? To do phylostratigraphic analysis, you need two major sets of tools. First,
you need a pretty solid phylogeny, or family tree, of your organism(s) of interest. Second, you need
complete or nearly-complete genome sequences of the organism of interest and of organisms that
can represent the major branch points (or nodes) in the family tree. The procedure from there
seems clear enough: using a well-known alignment program, you search through the family tree for
each of the genes in your organism of interest, to see where it is first recognizable in the phylogeny.
That point is the phylostratum from which that gene arises. With that data, you could look at the
contributions of various phylostrata to various body parts or processes. Or conversely, you could
look at the relative age of the sets of genes associated with those body parts or processes. Or you
could look at the relative age of the sets of genes associated with different stages of development.
And that’s what Domazet-Loso and Tautz did in their Nature paper on the hourglass model.
Specifically, the authors took their phylostratigraphic data and merged it with expression data at
various stages of zebrafish development; they called the resulting parameter the transcriptome age
index (TAI). Basically, they calculated a relative age of the genes that are turned on at each stage of
development, corrected for the extent to which particular genes are being used at those stages.
Then they mapped the TAI onto the timeline of zebrafish development. And this is what they saw.

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Does that look familiar? Like, say, half an hourglass? In the earliest stages of development, active
genes are young-ish, as they are in the juvenile and the adult. In between, the genes that are active
are older – a lot older. And the low point, where genes are oldest? It’s the end of segmentation and
the beginning of the pharyngula stage. That’s the stage that is considered the phylotypic stage in
vertebrates. And so we see that hourglass again, this time traced out by the evolutionary age of the
genes that are active during the phylotypic stage.
As you look at the graph, you might notice some other interesting periods in the life of a fish.
There’s a prominent peak of gene youthfulness at 6 hours of development; this corresponds to
gastrulation, that wonderful time in your life when you established yourself as a three-layered
animal. That peak is due to the activation of a lot of animal-specific genes, namely those that date
to the metazoan phylostratum. This includes genes that control cell-cell interactions, certainly a
hallmark of animal-building. Those might seem like incredibly basic functions, but they’re
relatively young compared to even more basic cellular processes, and the genes that control those
processes are the ones that predominate during the later phylotypic stage. (The authors showed, in
fact, that extremely ancient genes are active uniformly throughout development, whereas the
younger gene sets display the hourglass pattern: high-low-high.)
And notice that gene youthfulness declines during aging (after adulthood). Now why would that be?
The authors propose that the most recent innovations (facilitated by relatively young genes) are
likely to have resulted from adaptation, and so:

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 The fact that ageing animals revert to older transcriptomes is in line with the notion
that animals beyond the reproductive age are not ‘visible’ to natural selection and can
therefore not be subject to specific adaptations any more.

There’s a lot more: the study found differences between males and females (look at the dotted lines
in the figure), for example. But they also extended their analysis to other animals with known
genomes: fruit fly, roundworm and mosquito. In every case they saw the same pattern: young-old-
young. Their fly graph displays a pattern strikingly similar to that in the fish, and nicely dovetails
with the distinct analysis done by Pavel Tomancak’s group:

Look at the low point, where the genes are the oldest. It’s the germband elongation stage – the
recognized phylotypic stage for insects, and the same point singled out in the fly paper.
Remarkable.
So to summarize, the two papers, reported separately but simultaneously, strongly support the
hourglass model of development, in which embryos are seen to converge on an evolutionarily-
ancient form, after diverse beginnings and followed by radical divergence into the wonderful
variety of animals seen today and in the past. Domazet-Loso and Tautz explain how these new
results make sense of the hourglass:
These consistent overall patterns across phyla, as well as the detailed analysis within
zebrafish, suggest that there is a link between evolutionary innovations and the
emergence of novel genes. Adaptations are expected to occur primarily in response to

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 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.

And as they note, Darwin himself made this connection, reflecting on von Baer’s earlier
observations. Ideas, like genes, can have a long and productive history.

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