Pharyngula

Lamprey skeletons

Bone is a sophisticated substance, much more than just a rock-like mineral in an interesting shape. It’s a living tissue, invested with cells dedicated to continually remodeling the mineral matrix. That matrix is also an intricate material, threaded with fibers of a protein, type II collagen, that give it a much greater toughness—it’s like fiberglass, a relatively brittle substance given resilience and strength with tough threads woven within it. Bone is also significantly linked to cartilage, both in development and evolution, with earlier forms having a cartilaginous skeleton that is replaced by bone. In us vertebrates, cartilage also contains threads of collagen running through it.

These three elements—collagen, cartilage, and bone—present an interesting evolutionary puzzle. Collagen is common to the matrices of both vertebrate cartilage and bone, yet the most primitive fishes, the jawless lampreys and hagfish, have been reported to lack that particular form of collagen, suggesting that the collagen fibers are a derived innovation in chordate history. New work, though, has shown that there’s a mistaken assumption in there: lampreys do have type II collagen! This discovery clarifies our understanding of the evolution of the chordate skeleton.

Collagen in general is an ancient protein, thought to have evolved about 800 million years ago. It’s such a useful substance, though, that variants have evolved repeatedly, and there are about 27 different types of collage in at least a dozen different classes, and it’s the most common protein in your body, used in all of your connective tissues. One particular type, type II collagen, is an essential part of the matrix of your bones and cartilages.

Type II collagen was thought not to be present in lampreys, however, which led to the assumption that the lamprey skeleton evolved independently in that lineage. That led to some awkward problems in evolution, though. For instance, here’s an anaspid ostracoderm from the late Devonian, a very primitive jawless fish that lived around 370 million years ago. This is an animal that is thought to be much more closely related to us gnathostomes (jawed vertebrates) than lampreys or hagfish, so you might expect it to have a skeleton more like ours.

i-c1c5dca42fa356ba2ec87991433b646b-euphanerops.jpg
a, Specimen 2a from the Miguasha Museum of Natural History in Quebec, Canada. Scale bar, 10 mm. b, Reconstruction of the specimen in a. The specimen shows tarry imprints of cartilage that became calcified later in life.

But no, this specimen is well enough preserved that the structure of its skeleton could be seen, and it looks remarkably similar to calcified lamprey cartilage. There isn’t any way to tell if collagen was present in this animal, so it doesn’t answer the question of the order of evolution of the matrix components: collagen, cartilage, or bone. It implies that a lamprey-like organization could have been an early stage in evolution, but if lampreys possess a different cellular method of building a skeleton, one that lacks collagen, this organization in Euphanerops would be only an example of convergence.

i-ee07db5974726234640e0915f6a6c272-e_cartilage.jpg
a,Euphanerops displays large, rounded spaces for inclusion of chondrocytes, which are lined with calicified cartilage. b, Lamprey chondrocyte spaces in groups of two or four (‘cell nests’), as in Euphanerops. c, Lamprey cartilage calcified in vitro, showing the same calcification pattern as in Euphanerops. Images in b and c are reproduced from ref. 4. CN, cell nest; CS, chondrocyte space; IM, intervening matrix; TM, territorial matrix. Scale bars, 50 µm.

Zhang et al. did something interesting, though: they questioned the assumption that lampreys lack collagen II, and went looking for it. They screened a library of lamprey sequences and isolated two forms of collagen II, Col2α1a and Col2α1b. The presence of a collagen homolog related to our collagen II tells us that the gene arose before the lamprey-gnathostome split, but the real question is whether lamprey Col2α1 is used in constructing their skeleton…and yes, it is. It’s all over the developing branchial cartilaginous skeleton.

i-207312b0d1594ff6faa5f67d7605c0cf-col2a1a_branch.jpg
Col2α1a expression during lamprey development. Col2α1a is expressed throughout the branchial skeleton (boxed) and posterior
to the oral cavity.
i-2ddf7adaf0e03a2c866dcfa6c11063df-col2a1a_trunk.jpg
Col2α1a expression during lamprey development. Whole mount in situ hybridization of lamprey embryos at stage 23. Col2α1a expression is evident in
the somites and within the dermatome and sclerotome.

It’s also present in skeletal elements of the trunk.

I like this paper, because it doesn’t just stop there. Lamprey have collagen, OK, but as I’ve said before, the really interesting stuff is what goes on in the interactions between genes—regulatory networks are the key targets of change in evolution. In vertebrates, we know that an important upstream regulator of cartilage formation is a gene called Sox9, which is directly involved in turning on the collagen II gene. Is Sox9 also present in lampreys?

Sure is, and it’s expressed in the same areas that turn on collagen II and form the cartilaginous skeleton. At least a couple of pieces of the gene regulatory network for skeleton formation are present in the lamprey, suggesting that this might be another of those conserved kernels that define a central element of vertebrate anatomy.

i-1bafdd90ac7910a1f527f0ab4f79bbc6-sox9.jpg
Sox9 expression during lamprey development. (A and B) Expression
of lamprey Sox9 in embryos at stages 23 (A) and 24 (B). (B Inset) Sagittal section
through hindbrain and pharyngeal arches are shown. Arrows indicate Sox9
expression in streams of neural crest cells invading pharyngeal arches.

With sequence data in hand, the authors could also do comparative analyses and put together cladograms for the collagens and Sox molecules they identified. As predicted from evolutionary theory, the usual nested hierarchy emerges, with the lamprey as the most different.

i-c72dd3ecba8089bd502dae20a2a4bbea-col_phylo.gif
Minimum evolution phylogeny for fibril A collagen proteins as obtained with JTT plus Γ distances (α=0.906). Numbers indicate bootstrap scores for
each node, based on 1,000 replicates, whereas branch lengths are proportional to expected replacements per site. This tree is rooted by sea urchin ColP2α. Equally
and unequally weighted MP, ML, and BP also place the two lamprey sequences together at the base of the Col2α1 clade, with bootstrap scores or a posterior
probability of 93%, 77%, 71%, and 87%, respectively. In contrast to this consistent support for a Col2α1 grouping, this ME tree is unique among the different
phylogenies in its placement of the root along the Col5 clade.

It’s a pretty result that simplifies and clarifies our view of the evolution of the skeleton—lampreys aren’t just a weird and unique group, but representative of an earlier pattern of skeletal organization. It also suggests that collagen is a unifying substance that ties all vertebrates together—that what evolved first in the early history of chordates was a collagenous skeleton.

Comments

  1. #1 Darryl Pearce
    February 27, 2006

    I almost hope that in the future our genetic-manipulation will allow us to grow collagen, cartilage, and bone into any shape that we design. Wouldn’t that be a cool way to get beyond plastic?

  2. #2 pablo
    February 27, 2006

    I can still recall the day my son came home from school and told me that all of us have bones beneath our skin. “Even you, dad,” he assured me solemnly.

  3. #3 Ithika
    February 27, 2006

    Interesting stuff. I shall pass this along to my gf, who’s just started doing tissue processing for transplants. She gets to clean out bones and remove the marrow — and says it’s just like making stock (but without the vegetables or herbs).

  4. #4 Corey
    February 27, 2006

    On collagen… I’ve learned a great deal by looking into the research of Albert Harris at UNC. I don’t mean to toot someone elses horn, but you might take a look at what he’s done showing that collagen does not self-organize, but the direction of it’s fibers can be aligned by contractile forces of fibroblasts. I get the sense this is not well known, but I NEVER learned about that stuff in school. There’s a great interview in this month’s International Journal of Developmental Biology.

    Re: Darryl’s comment, we may look past genetic manipulation to cause shapes to form. It doesn’t seem that the answer would come from gene manipulation. Afterall, contrary to popular belief, genes don’t cause shapes to form.

    Great stuff. I got to pondering, I wonder if we’ll find out that lampreys are secondarily cartilageounous like sharks/rays?

  5. #5 Bachalon
    February 27, 2006

    Off-topic: found an essay that has some rather interesting assumptions about evolution/atheists/scientists (http://www.everystudent.com/wires/claypots.html).

    “Are we to believe that though a clay pot did not arise from natural means, the human eye just came about from elements in the atmosphere? Some would say that science demands such a conclusion, because to believe in God is not scientific. How is that different from finding the clay pot and starting with the assumption that people didn’t exist in that location, so scientists must now find out how that clay pot developed from the elements in the ground or air.”

    Oh, man. It’s too easy isn’t it?

  6. #6 Torbjorn Larsson
    February 27, 2006

    “we may look past genetic manipulation to cause shapes to form”

    Which is BTW a use of collagen, for print-on-demand tissue replacements.

    http://www.newscientist.com/article.ns?id=dn3292 :
    “Mironov and Boland hope it will be possible to print the entire network of arteries, capillaries and veins that nourish organs. But to keep cells alive, the organs would have to be completed within a couple of hours and a growth medium circulated through the fragile new vessels.

    Large structures might not be strong enough to hold together if the gel is removed after such a short period. However, the team is already experimenting with adding substances such as the skin protein collagen to speed fusion and reinforce structures.”

    And in the same article:
    “Printing is not the only promising new technique for creating entire organs. It might one day be possible to grow them in situ. In December, scientists in Israel reported that they had managed to grow miniature but fully functional kidneys by implanting fetal pig or human cells into immunodeficient mice.”

  7. #7 SEF
    February 27, 2006

    in the future our genetic-manipulation will allow us to grow collagen, cartilage, and bone into any shape that we design

    Some people would vote for the genetic-manipulation techniques which would prevent the growing of more bone, eg:
    http://www.ifopa.org/

  8. #8 Ebonmuse
    February 27, 2006

    I almost hope that in the future our genetic-manipulation will allow us to grow collagen, cartilage, and bone into any shape that we design.

    Actually, we can already do that. Five couples in the UK are having wisdom teeth extracted, from which a group of tissue engineers plans to extract stem cells and use them to grow discs of bone that will be turned into personalized wedding rings.

    http://news.bbc.co.uk/1/hi/sci/tech/4070522.stm

    (A bit creepy, I agree, but very cool nonetheless!)

  9. #9 Daniel
    February 28, 2006

    OK, I’m not a scientist and have no idea of how genes are named, but I couldn’t help noticing “Sox9.” Is this gene intentionally named in honor of Ted Williams?

  10. #10 Dave Godfrey
    February 28, 2006

    “Great stuff. I got to pondering, I wonder if we’ll find out that lampreys are secondarily cartilageounous like sharks/rays?”

    The internal cartilaginous skeleton seems to be primitive to the vertebrates. Hagfish don’t (and probably never did) have any kind of bone. However almost all the early vertebrates (Ostracoderms and so on) have dermal bone. Lampreys almost certainly lost dermal bone at some point in their ancestry (provided that the morphology-based trees are correct, and the rather controversial molecular data is wrong).

  11. #11 Roman
    February 28, 2006

    Although this is about lampreys, it is a bit off topic. http://sailinganarchy.com/ scroll down to the posting “Snake Wake” from 24/2/2006

    now why would this happen?

  12. #12 Nix
    March 4, 2006

    Hm, this doesn’t quite have your usual lucidity. Paras 1 and 3, and 2 and 4, are in large part repeats.

    (Perhaps an artifact of repeated revision?)

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