Of Frog Eyes and Jellyfish Tentacles

Author’s note: There were errors in the original version of this article. Please see the Post Scripts for more details.


In 1901, Hans Spemann revolutionized biology by doing something very strange. He had been watching various embryos grow, and he got bored. That’s the short version of his motivation.

Basically, the embryologists of the day had already spent oodles of time carefully documenting the normal development of various animals from egg to embryo to hatchling. They had established that most animals – vertebrates included – go through a succession of embryological stages called the morula, blastula and gastrula. The morula is just a dense ball of cells (In Latin, morula means “mullberry”), the blastula is a hollow ball of cells, and the gastrula is like a blastula with an indentation somewhere. The indentation keeps growing inward until it meets the other side and becomes a tube that runs through the embryo’s whole body. This tube becomes the digestive tract, which, if you think about it, is just a tube running through an animal’s whole body. In some species, called the protostomes, the original indentation becomes the mouth, but in other animals (deuterostomes) it becomes the anus. We are deuterostomes. In fact, it’s all very interesting, because the same program seems to occur in wildly different organisms, from worms to molluscs to starfish and humans.

But there was Spemann, watching his embryos, and he was thinking, “We know what patterns these animals form, but what mechanisms drive it?”

So he did that weird thing. He used very fine instruments to pinch out a few cells from the region near the eye of a frog embryo (or at least, where the eye would later form), and transplanted them somewhere else.

And the frog grew an lens on its belly.

This was the first demonstration of a phenomenon called “induction.” The cells from the head had somehow induced the cells of the frog’s belly to act like eye cells. He concluded that in the undisturbed frog, an eye knows where and when to grow because they receive signals from their neighbors.

We now know that these signals are substances somewhat like hormones, called morphogens. We call the regions that produce the morphogens “organizers” because they induce some new structure. To put it another way, the organizer secretes morphogens, and the morphogens get detected by nearby cells, and these target cells respond by growing faster or slower or making some new structure. Morphogens can even induce the production of other morphogens, to create very complex systems, with various regions of overlapping morphogen production, which we call “developmental fields”. Spemann had transplanted part of the eye organizer, so it made an eye.

The thing that makes these morphogens amazing, however, is that we can use the same basic idea to explain the development of all sorts of multicellular organisms – including plants! So, in general, the theory of developmental fields can be used to understand what makes various species different or similar. And that, in turn, is pretty much the central question of evolution. How do genetic changes translate into morphological differences?

Since Spemann’s time, we’ve discovered many morphogenic substances, and learned a lot about how they are made and how they work. They tend to be proteins that can activate or repress the production of other proteins from DNA. And, since proteins are all coded in DNA, this means that they are basically signals for communication between the DNA of one cell and the DNA of another.

Just this week, the story got even more interesting, because it seems that a certain set of morphogenic signals is more conservative than we thought. To understand it, we need to talk about one of the most important organizers that we’ve found: “blastopore lip” area of chordates. Let’s unpack that concept first.

Get some clay and roll it into a large number of tiny little balls. These represent cells. Now, take the balls and carefully put them together into a big hollow ball. A ball of balls. Imagine that it is about the size of your fist. This is the blastula stage that I mentioned earlier, only way bigger. Now, take one finger and carefully push gently inward on the ball, so that you have a sort of double-walled cup. This is the gastrula. The space you created with your finger is the blastopore, and the lip of the cup is the blastopore lip.

It seems that in chordates (that’s the group that includes us, other vertebrates, hagfish and sea-squirts) the blastopore lip is the organizer that sets up the basic axes of the body: which parts will become the head and tail, which the back and belly. This is extremely important.

Now, the gastrula is a stage of development that all multicellular animals have, except maybe sponges (depending on how you define a gastrula). But there are many different ways to form an embryo – and an adult organism – from the gastrula. Specifically, different kinds of organisms have different body axes and planes of symmetry. We humans have bilateral symmetry, as do all the vertebrates and chordates, and many other species that are grouped together as “bilaterians”. But some organisms have radial symmetry, and some (e.g. sea stars) have a sort of radial symmetry and bilateral symmetry (they start with one and then convert to the other).

Furthermore, as I mentioned before, different species do different things with their blastopores. We humans turn them into our anus and form a mouth secondarily. But insects and earthworms do the reverse. So, their axes are reversed.

Finally, in order to get to the gastrula stage, the cells have to divide rapidly, over and over again. This process is called cleavage, and it can take many forms, with many implied body axes.

The upshot so far is that although all animals have a gastrula stage, they might not all have the same gastrula stage, or develop along the same program thereafter. We think so, but it’s not obviously true. And it’s not at all clear what it tells us about the shape and development of the common ancestor of all animals.

But by studying the morphogens – which are of course the products of heritable genes – we can start to tease these questions apart.

Several authors in the 1990’s established that several non-chordates (e.g. the sea urchin) use the same organizer, and a similar system of morphogens. (Logan et. al. 1999; Ransick & Davidson 1993)  So far, however, only bilaterians have been shown to use this blastoporal axial organizer, and very little is known about the molecular signalling in protostomes, such as the arthropods – just that their blastopore has similar inducing abilities when transplanted (Itow et. al. 1999).

In this week’s issue of Nature Communications, a mixed Austrian and Russian team (Kraus, Aman, Technau and Genikhovich, 2016) report a series of experiments that bring us closer to understanding the development and common evolutionary history of nearly all animals. They confirmed, in detail, a whole spate of previous work (Kraus et al. 2007; Guder et al. 2006; Kusserow et.. al. 2005; Lee et. al. 2006; Rentzsch et al. 2006; Wikramanayake et. al. 2003; Lee et. al. 2007; Saina et al. 2009; Lengfeld et al. 2009; Fritzenwankeret al. 2007) that suggests that even Cnidarians (jelly fish, sea anemonies, corals and so forth) use the blastoporal organizer to establish their body axes. This is nifty because we don’t even know whether the common ancestor of Protostomes and Deuterostomes had this feature, but now it seems that the common ancestor of nearly all living animals had it (see cladogram, below)

FrogsAndJellyfish

A handy cladogram to show the relations between Deuterostomes, Protostomes and Cnidarians.

The body-plan of an adult Cnidarian is basically an overgrown gastrulla with tentacles around its mouth. It has no anus; food goes in the mouth, gets digested in a blind-ended pouch, and comes out the way it came in. Evolutionarily, Cnidarians are considered to be descended from an ancestor that came before the first bilaterian. We generally believe that the only multicellular animals more distantly related to us are the sponges.

Nevertheless, the team confirmed that not only do the Cnidarians use a blastoporal organizer; they use some of the same morphogens: β-catenin and the wnt family of proteins. And they did it using a combination of the same techniques that Spemann had used, plus modern gene editing and histology. By transplanting cells, as well as injecting the wnt and β-catenin morphogens (and morphogen blockers) in strategic locations, they were able to tweak the development of a small species of sea anemone, called Nematostella. They blocked normal development, made an animal that grew two “mouths” and made one that had tentacles on the wrong end. They gave the animals new body axes.

Twoheads

A normal anemone, left and one with two mouths, right. From Kraus, Aman, Technau and Genikhovich, 2016.

AboralTentacles

An anemone with tentacles at both ends. From Kraus, Aman, Technau and Genikhovich, 2016.

Transplanting

A diagram showing the location of the blastopore lip as well as the transplant procedures and results. From Kraus, Aman, Technau and Genikhovich, 2016.

 

All together, this work suggests that the common ancestor of jelly fish and humans – one of the fist animals – used the blastopore organizer. It further suggests that the basic body axes of all the animals (except maybe sponges) are in some way homologous, derived from that distant common ancestor. We are all variants on a theme that has been playing across the oceans and land for some 600 million years.

If Spemann could see this, he would not be bored.

 

Post Script 6/7/2016:

Since I posted this article, one of the authors of the jellyfish paper, Grigory Genikhovich, sent me some comments and corrections. To paraphrase him,

  1. The Cnidarian body plan is an overgrown gastrula, not an overgrown blastula, as I originally said. I have since corrected the article accordingly.
  2. I may have oversimplified the distribution of β-catenin and Wnt signalling systems across the various invertebrate phyla. In fact, only the sea urchin, among non-chordates, had been shown to have β-catenin signalling before it was found in Cnidarians.
  3. Very little is known about the molecular signalling in animals such as the horseshoe crab, although there have been transplantation experiments. The current version of the article reflects this correction as well – plus, I’ve iadded a cladogram to keep track of the relationship between protostomes and deuterostomes.

Thanks, Grigory!!

Post Post Script 6/8/2016:

Apparently, my summary or Grigory’s correction only made a bigger mess of things. Here are Grigory’s comments on my Post Script, in his own words:

  1. correct
  2. Wrong. Wnt/b-catenin signaling is extremely ancient and very important. It works in all animals from sponges and comb jellies to humans. Among many other things, it is required for the definition of the gastrulation territory and axis formation. It is also responsible for the anterior-posterior axis patterning in Bilateria, oral-aboral axis patterning in Cnidaria, segmentation in Drosophila and multiple other processes. What was not known before our paper was that organizer activity depends on the b-catenin signal outside deuterostomes. “Organizer activity” means that you take a bit of tissue, put it into an ectopic location, and the implanted bit is able to force the surrounding cells to form an axis.
  3. There is indeed not much information about molecular signaling in the horseshoe crab. This is not a problem, since a lot is known about signaling in other arthropods as well as in non-arthropod protostomes. However, to-date there is no information on the molecular underpinnings of organizer activity in the protostomes (horseshoe crab, spider, annelid) where transplantations were shown to induce axis formation.

Sorry Grigory!

 

Cited References

  • Kraus, Aman, Technau and Genikhovich, 2016. Pre-bilaterian origin of the blastoporal axial organizer. Nat. Commun. 7:11694 doi: 10.1038/ncomms11694.
  • Logan, C. Y., Miller, J. R., Ferkowicz, M. J. & McClay, D. R. 1999. Nuclear beta-catenin is required to specify vegetal cell fates in the sea urchin embryo. Development 126, 345–357
  • Ransick, A. & Davidson, E. H. 1993. A complete second gut induced by transplanted micromeres in the sea urchin embryo. Science 259, 1134–1138
  • Itow, T., Kenmochi, S. & Mochizuki, T. 1991. Induction of secondary embryos by intra- and interspecific grafts of center cells under the blastopore in horseshoe crabs. Dev. Growth & Differ 33, 251–258
  • Kraus, Y., Fritzenwanker, J. H., Genikhovich, G. & Technau, U. 2007. The blastoporal organiser of a sea anemone. Curr. Biol. 17, R874–R876.
  • Guder, C. et al. 2006 The Wnt code: cnidarians signal the way. Oncogene 25, 7450–7460. Kusserow, A. et al. 2006. Unexpected complexity of the Wnt gene family in a sea anemone. Nature 433, 156–160.
  • Lee, P. N., Pang, K., Matus, D. Q. & Martindale, M. Q. 2006. A WNT of things to come: evolution of Wnt signaling and polarity in cnidarians. Sem. Cell & Dev. Biol. 17, 157–167
  • Rentzsch, F. et al. 2006. Asymmetric expression of the BMP antagonists chordin andgremlin in the sea anemone Nematostella vectensis: implications for the evolution of axial patterning. Dev. Biol. 296, 375–387.
  • Wikramanayake, A. H. et al. An ancient role for nuclear beta-catenin in the evolution of axial polarity and germ layer segregation. Nature 426, 446–450
  • Lee, P. N., Kumburegama, S., Marlow, H. Q., Martindale, M. Q. & Wikramanayake, A. H. 2007. Asymmetric developmental potential along the animal-vegetal axis in the anthozoan cnidarian, Nematostella vectensis, is mediated by Dishevelled. Dev. Biol. 310, 169–186.
  • Saina, M., Genikhovich, G., Renfer, E. & Technau, U. 2009. BMPs and chordin regulate patterning of the directive axis in a sea anemone. Proc. Natl. Acad. Sci. U.S.A. 106, 18592–18597
  • Lengfeld, T. et al. 2009. Multiple Wnts are involved in Hydra organizer formation and regeneration. Dev. Biol. 330, 186–199
  • Fritzenwanker, J. H., Genikhovich, G., Kraus, Y. & Technau, 2007. U. Early development and axis specification in the sea anemone Nematostella vectensis. Dev. Biol. 310, 264–279

All figures taken with permission from Kraus, Aman, Technau and Genikhovich, 2016. Pre-bilaterian origin of the blastoporal axial organizer. Nat. Commun. 7:11694 doi: 10.1038/ncomms11694.

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