Centipedes, fruit flies and humans; three seemingly disparate animals at first glance. However probing their development reveals that all three undergo a process of segmentation, whereby repetitive units are generated along the axis of their bodies, running from front to back. Segmentation is common to three large groups of animals, namely arthropods (such as centipedes and fruit flies), vertebrates (from zebrafish to humans), and annelids (including earthworms and leeches). Decades of research have generated several models on how this fundamental developmental process occurs. In a recent study in BMC Biology, Carlo Brena and Michael Akam from the University of Cambridge, UK, explore the segmentation dynamics of the arthropod Strigamia maritima. Here Brena and Akam discuss what led them to focus their investigations on this centipede, what this model organism can tell us about segmentation and how their findings add to our current understanding.
What sparked your interest in studying segmentation in early development?
CB: I am interested in evolution and in particular in understanding how such an incredible variety of body shapes has possibly evolved from a common ancestor. One of the major aspects of the animal body plan is segmentation, with three out of the four most diverse and successful animal phyla typically recognised as being segmented (arthropods, annelids and vertebrates). To understand body shapes and patterns, one needs to look at early stages of development when major patterning occurs on the undifferentiated population of cells of an early embryo. This has been a general interest of mine that has been nurtured by the explosion of molecular tools and knowledge that has characterized biology in particular in the last 20 years, and which is the basis of the re-evaluation of development in the field of evolutionary studies – what has been called EvoDevo.
MA: It’s always a mixture of different things. I started working on fruit flies, on Drosophila, as a graduate student, but doing something very, very different from early development. I was introduced then, as a graduate student in Oxford, UK, to some of the wonderful phenomenology of the homeotic mutants – the bithorax mutants and so on that Ed Lewis had been working on. They seemed fascinating as biological phenomena that we didn’t understand at all. I’ve always been attracted to problems at the stage where they seem to be outside the range of understood explanations; I’m more interested in scoping out answers than dotting the i’s and crossing the t’s.
By the late 1970s, it was clear that it would soon be possible to find the DNA that was mutated in homeotic mutants. I wanted to work with the people who were doing that. I arranged to do a postdoc in Stanford, USA, with David Hogness, and in close collaboration with Ed Lewis at CalTech. That got me into the field of studying mutations that affect Drosophila development. While I was doing that, the Nüsslein-Volhard and Wieschaus screen was being published, revealing wonderful segmentation phenotypes – pair rule mutants, segment polarity mutants and so forth. The existence of phenotypes like that suggested that these mutants would provide some insight into the mechanism of segment formation. Peter Lawrence and Sydney Brenner had excited me about pattern formation, and these Drosophila mutants seemed to provide a way of getting at the molecular mechanisms that underlay pattern formation.
What is known about the basic mechanism of segmentation?
CB: Segmentation in an animal is basically the introduction of a reiterated pattern on a uniform – or broadly differentiated – field of cells along the anteroposterior axis. Researchers have now gathered a great deal of information on two thoroughly studied model systems, representing, in a certain way, the two extreme segmentation systems: the vertebrate embryo and the fruit fly Drosophila.
In Drosophila, segmental patterning is achieved simultaneously along the body axis by the combinatorial activation of segmentation genes by broadly overlapping expressed ‘gap’
genes. In contrast in vertebrates, segmentation is a temporally sequential process associated with germ band elongation, where a complex circuit of ‘segmentation clock’ genes, oscillating in their expression in an undifferentiated field of cells, transfer a periodicity in time into a periodicity in space.
MA: It was known that lots of different animals made segments; that three of the major phyla were segmented. But Drosophila was the only organism in which there was any hint of mechanism. It became clear in the 1980s and early 1990s that Drosophila used a process whereby gradients – and in Drosophila’s case it was maternally established gradients – were interpreted by transcription factors through a hierarchical mechanism to subdivide a pre-existing field of cells into a repetitive array of different cell states; a particular juxtaposition of cell states made a segment boundary. The rest of the process of segmentation came from that. It became a model, really, for hierarchical transcription factor interactions, and that was the only model in town.
Later, with the discovery of oscillating gene expression in the chick embryo by Olivier Pourquié and colleagues, the mechanism of segmentation was worked out in vertebrates. Vertebrate segmentation, making somites, involves gene expression in a whole population of cells oscillating together, more or less in phase. But at a particular wave front in the tissue – a position at a particular point in the growth of the embryo – that oscillation stops. Stopping the oscillation freezes cells in a series of alternative cell states, and those alternative cell states are translated into the signals that generate segment boundaries.
Once that was worked out, the question of the evolution of segmentation became even more interesting, because we had two completely different types of mechanism, in two different groups of animals, for building a segmented body.
Why are you working on arthropods, in particular short-germ insects?
CB: Arthropods are by far the most diversified and successful animals, and one could argue that part of their potential in diversifying different parts along their anteroposterior axis relies on the modularity of their body. Drosophila has proven to be a fantastic system to understand the basic function of segmentation genes, but it is now clear that it is a very derived arthropod whose segmentation system is not strictly extendible to other arthropods. The large majority of arthropods have in fact a short-germ kind of embryo and add most segments sequentially, even post-embryonically. This system requires a cyclic activation of genes which is reminiscent of what happens in vertebrates. To understand how segmentation has evolved throughout the animals, and what elements and genes of the segmentation system are homologous, we need to look at more basal insects or arthropods, i.e. possibly closer to the common ancestor.
MA: Drosophila, the fruit fly, is an unusual arthropod – in fact an unusual organism – in that it makes the whole body by subdividing a pre-existing field of cells. It’s like having an entire cake and making slices by just cutting up the cake, whereas most organisms couple segmentation and growth.
I wanted to see how the Drosophila segmentation mechanism was modified in an organism that made its segments while it was growing, rather than made its segments all at once in this extended field of cells. I’ve always been interested in the evolution of developmental mechanisms and it seemed to me that segmentation was an example of a developmental mechanism where we had a very good understanding in Drosophila of how it worked and a very clear question about how it must have evolved, or at least how it must be different in relatively closely related animals. That’s why I started looking at short germ insects initially – things like the locust – and then, more recently, in the centipede, which has become my favourite for the last few years.
Where does the centipede you work on (Strigamia maritima) come from?
CB: Strigamia maritima is a thin 2-3 cm long centipede which lives on the seashore under the shingle layer all around the British coastline. We collect our material in Brora along the North-East coast of Scotland because that’s the only place in the UK where we can find a large enough number of eggs.
Myriapods, the subphylum to which Strigamia maritima belongs, are pivotal in understanding arthropod segmentation evolution because of their highly segmented body, limited differentiation and phylogenetic position at the base of the arthropod tree, between the group including spiders and the group of crustaceans plus insects. Strigamia, in particular, belong to the geophilomorphs, soil dwelling centipedes.
Why is Strigamia maritima an interesting organism to probe for insights on segmentation?
MA: It has two important characteristics: one is that, despite having more segments than most centipedes, it makes them all in the embryo. Embryos are much more accessible to work with than the later juvenile stages. Actually, having an animal that makes about 50 segments in the embryo in a relatively short period of time was experimentally very convenient.
The group of centipedes to which Strigamia belongs is also very variable in segment number. Different species have a very wide range of segment numbers, from the high 20s up to 200 or so, and even within species, there is variability in final adult segment number. Not many arthropods that have been used for lab studies show such variability. Insects certainly don’t. So if you are interested in how segment number evolved, then Strigamia maritima is a good species to work with.
Then there’s a very pragmatic reason: S. maritima is an incredibly abundant centipede in environments that suit it. We can collect thousands of eggs in the wild in a couple of days. We’d previously worked with a centipede which you could grow in the lab – which is an advantage – but one of my graduate students who worked with that species, Louise Smith, managed to collect a only a couple of hundred eggs in the three years that she was doing her PhD. Just looking after the animals took a large fraction of her time. So Strigamia was an easier animal to work with.
How does your work on the centipede add to the historical debate on the evolution of body axis segmentation?
CB: Our work, although far from being fully understood, shows that a segmentation system which involved cyclic activation of genes spreading with waves of expression is indeed present not only in vertebrates but also in relatively primitive arthropods, like the centipedes. Even more interesting (this is the crux of the novelty of our BMC Biology article), is that it shows how although some of the segmentation genes involved may be the same, not all homogenous segments within the same animals are produced in the same way.
MA: By the time we started working on centipedes, it was already clear that aspects of the Drosophila mechanism were very special adaptations of the fast developing insects. It was also clear that one possible mode of making segments was this oscillator mechanism. We had some ideas about what genes might be involved in oscillatory processes and the work with the centipede let us test whether there were parallels between segmentation in this more basally branching arthropod group and what was known in vertebrates.
The first suggestions were that, yes indeed, there were parallels in terms of the genetic pathways used to make segments. My colleague Ariel Chipman found that the Notch signalling pathway, which was not known to have a role in Drosophila segmentation, but which was central to vertebrate segmentation, seemed to have a role in our centipede segmentation. That had been suggested by earlier work with spiders, but in our embryos it was much easier to get visual insight into what was going on, because the centipede embryo develops with a large field of cells in which we can see oscillatory waves of gene expression. We examine these waves in detail in the BMC Biology article.
The centipede work has provided strong evidence for an alternative and very different way of making segments, as compared to Drosophila. And yet at the same time, it has also provided convincing evidence for similarities in the gene networks. For example, work that my student Jack Green has done shows that almost all of the genes involved at the pair rule level of the cascade in Drosophila – genes that first reflect the repeating segment pattern – also seem to be involved in centipede segmentation. Indeed, they show very similar interactions, at least at the downstream end of the process. This work confirms that centipede and fly share a common origin or segmentation, and yet suggests that they use fundamentally different mechanisms to generate the initial periodicities.
What are the implications of your work for our future understanding of the mechanisms of segmentation and gene networks involved?
CB: Our data suggest that a partially different segmentation system could easily evolve from a common underling segmentation gene network. If that’s the case, we should be extremely cautious in inferring statements of general homology, and consequently of evolutionary history, based on a few genes or, worse, on similar dynamics.
MA: I think we – ‘we’ meaning the whole field – are only just beginning to get some insight into how gene networks evolve. There’s a lot of talk about ‘recruiting modules’ – the idea that a module that evolves in the context of one process of segmentation gets recruited wholesale to another biological context. There’s a lot of discussion about how specific interactions between genes – links in the network – might evolve in a way that is apparently without an effect on phenotype but then causes processes to respond in different ways to stresses in different organisms.
There are all sorts of things we would like to know about how gene networks can evolve both to generate changes in morphology and to cope with environmental stresses, to adapt for example, to different rates of development or requirements for different numbers of segments. Segmentation is a really good model to work out some of these questions.
There are some intriguing hints in the arthropod segmentation story that segment numbers can double in very closely related species. There’s a species of brine shrimp called Polyartemia, which looks almost exactly the same as an ordinary brine shrimp but has twice the number of segments. And there has recently been reported, by Sandro Minelli and colleagues, a centipede which, by all criteria, fits well into an established family of centipedes, except that it has almost twice as many segments as all of its close relatives. These data suggest that some unusual evolutionary events can result in a segmentation network adopting a different mode of activity that results in a substantial change in form.
Our work in Strigamia touches on that question, because we suggest that in Strigamia the segmentation network switches, during normal development of the embryo, between a process of making double segment units which are then subdivided, to a process of directly adding single segment units. That’s not something we’d expected to see. Carlo Brena noticed it when he was looking carefully at the process of segment addition. It will be very interesting to model how a gene network might make such a transition.
What organism do we need to work on to provide more answers?
CB: Given the apparent plasticity of the segmentation system, the short answer is that we just need more and more different species of any kind to look at. Crucially, though, we need to move from the ‘one gene-one evolutionary scenario’ approach of the early days of EvoDevo and take a deeper look at new, non-model systems, in particular from a functional point of view. Unfortunately, a functional molecular approach has not yet proved possible in any myriapod, hence the priority should be to look for any myriapod species that allows for those techniques (or to develop alternative tools with Strigamia). Further, we should endeavour to understand the animal evolution of segmentation from more basal groups, such as primitive insects like jumping bristletails, primitive non-malacostracans crustaceans like remipedes, and primitive chelicerates like scorpions.
MA: From the point of view of someone who works on arthropods, I would say without hesitation that the animal I would really like to understand is the velvet worm, the onychophoran. Onychophorans have been enigmatic for a long time. They are not arthropods – they don’t have a hard external skeleton – but they have a segmented body like arthropods and were for a long time seen as an intermediate between annelids and arthropods. We are now pretty sure that they are not closely related to annelids, but they do seem to be the sister group to the arthropods. They represent an arthropod-like body plan before arthropods adopted hardened body plates and jointed limbs.
Onychophorans are slightly exotic – they only occur now in the southern continents, in South Africa, Australia and New Zealand, so they’re not that easy to get hold of. But if you are lucky enough to look at their embryos, you could be forgiven for thinking that you weren’t looking at any relative of an arthropod at all. They look in some ways quite extraordinarily vertebrate-like. The first sign of segmentation is a beautiful band of mesodermal somites lying underneath the epidermis. Yet some of the genes that pattern segments, engrailed and wingless for example, seem to be used in the same way as they are in arthropods, to set up a boundary in the limbs. I would love to know how segmentation works in onychophorans.
One difference between arthropods and vertebrates is that in vertebrates segment patterning is in the mesoderm, whereas in arthropods it seems that the ectoderm – the outer layer – is primary for segmentation. In onychophorans, it’s not clear whether mesoderm or ectoderm is primary for segmentation. Onychophorans are sufficiently close to arthropods for us to be able to make comparisons sensibly, but sufficiently different to give real insight into how very different modes of segment patterning may evolve.
They are also lovely creatures. I don’t have too much difficulty in collecting centipedes and cutting them open, but cutting open an onychophoran is terribly difficult: they are just so beautiful.
Who is going to be interested in this/be affected by this research?
CB: Anyone interested in the evolution of animal body plans should be interested and the information gathered from this nodal point in the arthropod phylogenetic tree should affect how they interpret the evolutionary history leading to their studied species. Additionally, students who might not be normally exposed to this kind of article but who may be interested, such as modellers and theoretical biologists interested in understand how, given the changing cell/population conditions during development, changes in gene network dynamics fit in, as illustrated in late segmentation stages in Strigamia.
MA: I belong to that part of the EvoDevo community that comes closest to classical zoology – people who are interested in the big picture of animal evolution, in questions like: What did the last common ancestor of all animals look like? From what sort of animal did arthropods evolve? That community will, I think, be very interested in the work that we are doing. This is an area where modern comparative genomics is really opening up a lot of old questions to new ways of analysis. It used to be the case that we only had molecular insight into a very small number of examples of animal diversity, Now, comparative genomics is giving us genome sequences for all sorts of obscure creatures. The genome alone will not to tell us everything, but it certainly makes it easier to do experiments that test hypotheses about the evolution of limbs or the origin of nervous systems. That is one of the communities we’re talking to.
Another community who I hope will be interested are the people who model gene networks to understand in quantitative terms the way that these networks, composed of largely conserved components, can generate what appear to be very different developmental mechanisms and final body plans.
BMC Biology 2013, 11:112
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