New Limbs from Old Fins, Part 4: Gene Expression

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September 29, 2011 Tags: History of Life

Today's entry was written by Stephen Matheson. Please note the views expressed here are those of the author, not necessarily of The BioLogos Foundation. You can read more about what we believe here.

In the previous post, we tackled the concept of homology, and further explored why biologists consider tetrapod limbs to be homologues, both with each other and with the fins of fish. Observations from the fossil record, from comparative anatomy, and from developmental biology all strongly suggest that tetrapod limbs are built based on a pattern inherited from a common ancestor – and specifically from the fins of a fish.

We saw developmental homology in the basic signaling network that controls the growth of the limb bud. But what about the shape of that bud, and of the limb that it will become? The final structure will have a front and a back, a top and a bottom, an end and a base. And different things will happen in each of those areas – for example, the digits are at the end, and the humerus is at the base. And furthermore, why do the limbs form where they do? These are questions concerning what developmental biologists call pattern formation. And pattern formation in developing embryos is orchestrated by an elegant genetic system, a dance of genes in time and space.

Before we look at this system, let's review two concepts in genetics that are important to us here: 1) the process of gene expression; and 2) the function of transcription factors.

Genetics: turning genes on and off

You probably know that the complete collection of genes in an organism is its genome, and that every cell in the body of the organism has a complete copy of that genome. (Two copies, in fact.) So, your brain cells and your skin cells, though very different in form and function, both contain your entire genetic endowment. The difference between brain cells and skin cells lies largely in which various genes are turned on and off. Brain cells have brain-specific genes turned on, and just about everything else turned off. Genes that are turned on are said to be expressed, and the overall process is called gene expression.

The control of gene expression is critical in the patterning of a developing body, determining where the parts will be made, and directing those parts to take on their specific characteristics. Gene expression is under the control of proteins called transcription factors that specialize in turning genes on and off. Transcription factors exert this control by docking with sites in the genome that are near to the genes they seek to control. These sites are pretty specific, so that only particular transcription factors can control a particular gene. The transcription factor proteins are often members of large families of closely-related proteins, distinguished by the way they stick to sites in the genome.

Biologists have learned a lot about the network of gene expression that leads to the patterning of animals during development. One remarkable discovery is this: the transcription factor networks that direct basic development display extraordinary evidence of homology. Animals as different as fish and mice are sketched out in form by the expression of the same genes. And the patterning of tetrapod limbs has become a classic example.

Gene expression during limb development

Recall from the last post that biologists first learned about limb development through extensive studies of the chick. The process begins with a tiny bulge called a limb bud. That bud grows and takes shape under the influence of chemical signals released by cells in the limb bud or its vicinity. But how does the limb bud know where to start growing? And how do the different parts of the limb know what they should become? In both cases, the patterning decisions are controlled to a large extent by members of one family of transcription factors. That family is the Hox family.

Hox proteins are named after one of their defining characteristics: they contain a so-called homeobox, which is the part of the protein that enables it to dock with specific sites in the genome. (The homeobox is one type of genome-binding part; there are several other types that are employed by other families of transcription factors.) The Hox proteins are famous for controlling something called “segment identity,” meaning that they often help to tell a part of the body what it is, and specifically where that body part lies on the front-to-back scheme of things. So, for example, Hox proteins tell one particular part of the embryo that it is the tail.

As you might have guessed, Hox proteins tell the chick embryo where to grow its limbs. We will focus on the forelimb (which is a wing in the chick). The limb bud that will form a wing emerges from a boundary region in the embryo: the boundary between the cervical and thoracic levels. That boundary is delineated precisely by the expression of two Hox family members: B8 and C6. The limb bud grows just ahead of the boundary, meaning just ahead of the region that expresses Hox protein B8, in a region expressing Hox protein C6.

Patterning proteins in the limb: initial outgrowth

That's in the chick. Are these systems homologous to those in other animals? Strikingly, they are. In a well-known comparative study, scientists in Cliff Tabin's lab at Harvard Medical School showed that the Hox-based patterning system is the same throughout the vertebrates. Most notably, the B8/C6 boundary marks the position of the forelimb in animals with widely varying structures. In the diagram below, taken from that paper, the B8 expression range is marked by the shaded circles and the position of the limb bud is indicated by a bud-shaped arc. The limb bud's position is determined by the same Hox proteins in the chick and in the mouse. And in the fish and in the frog. And even in the goose, which has a ridiculous number of added vertebrae to hold up its ridiculously long neck. The key simple point here is this: in all of these animals, the Hox expression pattern underlying the point of emergence of the limb bud is the same. Note that there are several other transcription factors known to be involved in limb emergence, including two that are specific for a forelimb vs. a hindlimb, and those are the same across the board as well. It's a remarkable example of homology in developmental genetics.

Patterning proteins in the limb: specialization

But once the limb starts to grow, how do the different regions of the limb become specialized? Is there a Hox expression pattern behind that, too? Indeed there is, and again we'll start with the chick and focus on the forelimb.

The development of the limb bud seems to proceed in three phases, each generating one of the three basic zones of the limb. Those zones are the upper arm/wing, the lower arm/wing, and the digits. In the diagram below, you'll see those anatomical regions (e.g. the humerus, which is the upper arm/wing) and you'll see some technical terms for the region (e.g. stylopod). What matters to us is the pattern of Hox protein expression. Phase I involves two Hox proteins, D9 and D10. This is followed by Phase II, which is significantly more complex. Now there are a few more proteins involved (D11, D12, and D13), and they are expressed in a more interesting pattern. Finally, Phase III introduces a new pattern, generated by some additional players. To recap: Phase I generates the upper limb, Phase II generates the lower limb, and Phase III generates the stuff at the end. Each phase is characterized by a particular pattern of expression of Hox proteins.

The patterns of Hox expression provide clear evidence of homology. Phases I and II are the same in chicks, mice, and fish. (Even sharks.) And again, those observations are coherently explained by common descent. Moreover, the confluence of the various observations we have discussed in this series – shared structural features, fossil intermediates, identical signaling systems, and now identical genetic control systems – very strongly suggests common ancestry. Common descent provides a singular explanation for a large collection of related phenomena.

But what about Phase III? Is that phase conserved? It was long thought that the answer was “no.” For one thing, the structures at the ends of animal limbs are wildly different: fingers, hooves, flippers, wings. And more importantly, early observations from various disciplines (anatomy, genetics, development) suggested that the Phase III structures in tetrapods were something completely new. Specifically, those early findings led most experts to conclude that while Phases I and II were modified from a fish ancestor, Phase III – the part that seems to generate all the diversity – was a new invention.

Next time we'll look specifically at the structures made by Phase III, and at some new findings that strongly suggest that even those most tetrapod-like of structures – hands and feet, fingers and toes – have their roots in the fins of fish.

Further Reading

Brian K. Hall, editor (2007) Fins into Limbs. Chicago: The University of Chicago Press.

Scott F. Gilbert (2000) “Chapter 16. Development of the Tetrapod Limb.” In Developmental Biology, 6th Edition. Sunderland (MA): Sinauer Associates.

John Gerhart and Marc Kirschner (1997) Cells, Embryos, and Evolution. Malden (MA): Blackwell Scientific.

Laura Hoopes, editor (2011) Gene Expression and Regulation at Nature's Scitable site. Part of a large and excellent educational resource in genetics.

Image credits:

Hox genes and limb buds in various vertebrates from Figure 11 of Burke et al. (1995) Development 121:333-346.
Phases of Hox expression in limb buds is Figure 16.15 in Gilbert (2000) Developmental Biology, 6th Edition. Sunderland (MA): Sinauer Associates.

Stephen Matheson is an author, editor, and developmental cell biologist, formerly at Calvin College in Grand Rapids, Michigan. He writes regularly on his blog “Quintessence of Dust”, which explores issues of science and Christian faith, focusing on genetics, development, evolution, neuroscience, and related topics, regularly discussing intelligent design, creationism, and other scientific issues that worry evangelical Christians.

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beaglelady - #65250

September 29th 2011

I’m really enjoying this series. Thanks!

sfmatheson - #65263

September 29th 2011

Thanks for the thumbs-up. It’s great stuff to write about, although it’s hard to avoid going way over my number-of-words budget. There’s so much more detail that could be fun to present and discuss.

mjblyth - #65296

September 30th 2011

I for one would be happy to have you go over the limit!

Peter Hickman - #65275

September 30th 2011

Thanks for explaining terminology and processes in a way that non-specialists (like me) can readily understand.

Ronnie - #65276

September 30th 2011

This is a very interesting article. I am amazed at how precise the gene works, in this case, to produce a limb. Not only does it have to produce “a” limb, but another, mirror copy on the other side of the body, plus all the individual parts of the limbs, bones, muscles, tendons, joints, blood vessels, nerves, skin, etc. All must be formed in an extremely precise order and sequence to successfully produce two complementary limbs, not to mention all the other parts of the body. How a microscopic genome “knows” what to do, how to do it and when, is truly fascinating.

Darrel Falk - #65284

September 30th 2011


You have just defined the joys of being a biologist. 

 For those of you who don’t get to spend your days as a biologist but would like to learn more about how all of this comes about, I would especially recommend Sean Carroll’s “Endless Forms Most Beautiful.”

KevinR - #65277

September 30th 2011

“The patterns of Hox expression provide clear evidence of homology. Phases I and II are the same in chicks, mice, and fish. (Even sharks.) And again, those observations are coherently explained by common descent.”
One of course needs to take great care in reaching conclusions to support one’s own particular kind of belief. Here the writer wants the alleged homology [from Hox expression] to support common descent, but leaves untouched the possibility [ and I might say more a appropriate explanation ] of common design at the level of Hox expression. All the suggestions that things have evolved from fish are simply that - suggestions to support the writer’s own belief system.

In the big scheme of things, because evolutionists cannot explain where life comes from in the first place and therefore cannot account for the FORM in which it arrived here on earth, they cannot make definite statements about all lifeforms having descended from one single ancestor.

The physical evidence I see point to there having been one designer of lifeforms and that those lifeforms were fully developed in the various KINDS we find today - no evolution required.
If evolution were true, we should have many more fossils of FAILUREs instead of the much more definite successful fossil lifeforms we encounter today. This being a direct inference from evolution being a randomly driven process. In short there just are not enough[i dare say ANY] transitional forms to warrant the conclusion that all life descended from one common ancestor.

Darrel Falk - #65283

September 30th 2011

Thanks, Kevin.   

For readers who are interested in learning more about the points Kevin raises in his final paragraph, please see

For readers interested in learning more about the issues raised in Kevin’s first paragraph please see, Dr. Matheson’s Part 3—the final full paragraph.

beaglelady - #65289

September 30th 2011

If evolution were true, we should have many more fossils of FAILUREs
instead of the much more definite successful fossil lifeforms we
encounter today.

Most species that have ever lived have become extinct. 

John - #65300

September 30th 2011

“The physical evidence I see point to there having been one designer of lifeforms…”

What physical evidence have you seen for yourself, Kevin?

Can you distinguish between mere similarity and nested hierarchies?

beaglelady - #65285

September 30th 2011

Curiously, hind limb buds appear in the whale and dolphin fetus and subsequently degenerate before birth.  And occasionally a whale will be found with atavistic hind legs!  This is best explained by evolution. A designer can go back to the drawing board, but evolution doesn’t have that luxury.

glsi - #65301

September 30th 2011

You’re talking about looking at patterns in bone structure (as well as the way they develop) and then making conclusions based on your own logic about how animals look they way they do today.  It’s a good theory and makes a lot of sense until you go looking at whole of the fossil record that we have available to us.  It’s as though you cannot see the forest for the trees.

You would need to be able to show at least one good case of gradualism in the fossil record to bear  your theory out.  Of course, there are none.  In a recent blog, Dr. Falk said no one should be surprised because fossilization is such a rare event.  I don’t know who is surprised at this point.  Fossils are found all the time but they are generally the same as others which have been previously discovered.  When new species are found they cannot be shown to fill any “gaps” in a chronologically linear evolution.  

I see no gaps.  I see individual species with similar bone patterns and means of development.  This is based upon looking at the factual evidence in its entirety.  

beaglelady - #65302

October 1st 2011

Did you bother to watch the giraffe video I pointed you to?

btw, evolution isn’t linear.  Have you considered fossil horses?  And what is your natural history museum? 

glsi - #65303

October 1st 2011

Bush or tree, it makes no difference.  There aren’t  fossils to show either one.  With the bush you can more easily claim you can’t find the connection points. 

Fossil horses are the best example of an actual continuum I’ve seen many times in Morrill Hall at UNL.  They’re horses.  They’re not changing into camels and they didn’t hatch out from a salamander.  It’s linear by the way.

I haven’t seen the giraffe video yet, but I’ll try to.  That way folks here will be able to say I haven’t seen it.

beaglelady - #65305

October 1st 2011

Horse evo is bushy, just like human evo.  

Originally you said “You would need to be able to show at least one good case of gradualism in the fossil record to bear  your theory out.” Are you saying that for horse evolution to be true they now must be shown to change into camels or hatch out of salamanders?

glsi - #65306

October 1st 2011

You’re right about the horses being “bushy”, if by bushy you mean they aren’t actually descendants of each other.  The Morrill Hall exhibit shows it linearly as if it flows directly from one to the next.  Thanks for pointing that out.

But, yeah, Darwinism has never  been able to demonstrate one species changing into some other species.  As new fossils are discovered there’s only increasingly flimsy excuses for why it can’t be demonstrated in the fossil record.  

beaglelady - #65308

October 1st 2011

You’re right about the horses being “bushy”, if by bushy you mean they aren’t actually descendants of each other. 

No, bushy doesn’t mean they aren’t actually descendants of each other. What an idea! And I said that horse evo was bushy, not that horses are bushy.   Millions of years ago multiple horse species lived at the same time. 

The Morrill Hall exhibit shows it linearly as if it flows directly from one to the next.

If that is true they are badly in need of an update.  Or maybe they can only afford to show a simplified version of horse evo. 

But, yeah, Darwinism has never  been able to demonstrate one species changing into some other species.

But that isn’t how evolution works. 

glsi - #65310

October 1st 2011

Can’t show it as a bush, can’t show it as a tree.  Pretty mysterious.  It’s almost as if new species just appear in the fossil record.

beaglelady - #65311

October 1st 2011

Described it as bushy as opposed to linear.

Here’s a diagram:

What’s your explanation? Some species disappeared or were killed off by God and God then poofed new ones into existence?

Did you watch the giraffe video yet?

glsi - #65316

October 1st 2011

I checked out your horse chart.  Then I checked out one from “Evolution of Tertiary Mammals of NA…”, by Janis, etc.

They’re different.  These things are always disputed and ever changing.  It’s easy to draw diagram showing anything you’d like it to show.

beaglelady - #65317

October 1st 2011

If it has changed it’s because of new research. Science is a work in progress. 

It’s still evolution.  What’s your alternative story? Does God keep releasing herds of new species into history?

glsi - #65326

October 1st 2011

I have absolutely no idea.  That’s why I’m here because it’s such a fascinating question and I’ll try to pick up clues anywhere I can.  I don’t expect God uses “poofs”, but then again God does stuff all the time that I don’t expect.  It seems like you need a miracle though and why not?  To get the Alpha you’re going to need a miracle.  I bet even Dawkins knows that, but he can’t bring himself to admit it.

I don’t believe anyone can answer the question.  It seems to me that the more that new information is revealed (such as the ever more complicated workings of the cell and the micro molecular machines within it), the more preposterous any human conception of life becomes.

beaglelady - #65327

October 2nd 2011

All the clues are there.  Look at the split bones in horses—evolutionary leftovers from when they had 3 toes.  Similar to the dewclaws in dogs, cows, and goats. 

How did you find the giraffe video?

glsi - #65330

October 2nd 2011

That might be a clue and there are others, but I don’t see that they add up to mutation and natural selection.  It’s very unconvincing.  

I think people only believe in it because there’s nothing else science has to offer right now excepting some other fringe theories like symbiogenesis which are equally unconvincing.  Clues are not enough when evidence is contradictory.  Fossil evidence from the Cambrian and preCambrian is certainly contradictory and I believe falsifies Darwinism.

beaglelady - #65331

October 2nd 2011

A goofy God could be handing out useless digits simply to fool us.  Just like hiccups and hernias.

glsi - #65332

October 2nd 2011

Could you give the giraffe link again?  I can’t remember where it is.

beaglelady - #65335

October 2nd 2011

Sure. Here you go:  It’s in several parts.

glsi - #65340

October 2nd 2011

Yes, well, I watched it.  Having butchered many deer myself the only frightening part was seeing Richard Dawkins in his dissection suit.  Very creepy!

Not sure what you thought would be convincing in this video.  There was some awesome footage of the animals and facts about the workings of their bodies which I enjoyed.  In terms of evolution it was long on assertions and speculation and little to nothing of factual evidence.  They said the neck grew gradually longer, but had nary a fossil to show.  They subbed in fancy computer graphics drawn by their artist.  Then Dawkins says there’s some design flaws that no engineer would have conceived.  That’s it?  Like I look to Dawkins as an authority on how God would design something?  My assumption is that Dawkins has no clue whatsoever why things are designed the way they are.

beaglelady - #65341

October 2nd 2011

How about the recurrent laryngeal nerve and its odd detour, making a nerve more than 15 feet long, down the neck and back up, to go from the brain to the larynx.
Easy to explain with evolution, a tad difficult to explain by design, unless the designer is a drinker.  What is your take on this?

btw, why go for the giraffe neck, which has the same number of vertebrae as our own necks? Why not go for sauropod dinos, since they have long tails also?

glsi - #65342

October 2nd 2011

I think giraffe are perfect in every way.   I suppose if Dawkins were God the nerves would be made of titanium and the design would be subcontracted out to I.M. Pei.

beaglelady - #65343

October 2nd 2011

As they explained in the video,  over half of newborn giraffes die in their 1st 6 months

the adult giraffe being dissected died from acute unexplained paralysis—
turns out it was from a simple fall.

glsi - #65344

October 2nd 2011

I guess to an evolutionist the world is a pretty miserable place.

beaglelady - #65353

October 3rd 2011

I think these facts would be a bit sad to any animal lover. I don’t see what being an “evolutionist” has to do with it.

sfmatheson - #65366

October 3rd 2011

glsi writes, in a conversation about the recurrent laryngeal nerve in giraffes, “I think giraffe are perfect in every way.  I suppose if Dawkins were God the nerves would be made of titanium and the design would be subcontracted out to I.M. Pei.”

I think that this response is notable in two ways. First, it ignores the context of the conversation, miscasting the issue of the nerve away from the specific point that Dawkins and others are making, which is the fact that the nerve’s configuration is remarkable, even bizarre, and that the best explanation for this is not “design” in any plain sense of that word. That is the context. To assert that the giraffe is “perfect in every way” is to assert that issues of design don’t actually matter. And there are more straightforward ways to make that assertion.

Which leads to the second important aspect of glsi’s response. She/he should not be constrained by scientific explanation when it comes to expressing awe and wonder at God’s creation, nor should any believer feel hesitant to proclaim God’s creations to be “perfect.” I think it’s appropriate, even laudable, for glsi to refer to giraffes as “perfect.” But I think it’s a mistake to do that in the context of a discussion of claims regarding “design,” whenever those claims aim to challenge the plain effectiveness of common descent as an explanation for some of the stranger aspects of our anatomy. Like the recurrent laryngeal nerve.

beaglelady - #65368

October 3rd 2011

Yes, he “missed the point” quite deliberately by dodging it.  The video makes it perfectly clear that this nerve is best explained by our fishy ancestry.  Nothing intelligent about plugging in a toaster with a 10 foot extension chord when an outlet is nearby.

btw, that video is one in a whole series of dissecting “nature’s giants.”  There’s also a whale video where you can see the vestigial hind limbs.

beaglelady - #65370

October 3rd 2011

oops I meant “extension cord”

Ronnie - #65372

October 3rd 2011

Perhaps this nerve serves other parts of the body as it makes its way through the body, or there may be some as of yet unknown benefit to it taking this long route.

To assume this is a ‘bad design’ ignores a possible purpose for this nerve.

sfmatheson - #65373

October 3rd 2011

Ronnie, the nerve’s anatomy and function are well known. Its circuitous path makes sense in light of its developmental and evolutionary history, but is a hindrance to its function. The point is not specifically that it’s a “bad design,” but that it’s clearly not a good one. More basically, the point is that the nerve’s anatomy makes no sense from an engineering perspective.

One can always say, about anything, that “maybe there’s something we don’t know,” but that’s a poor reason to ignore evidence and explanation. That’s my perspective on the “design” of the recurrent laryngeal nerve. Those who prefer supernatural creation are likely to see things differently.

glsi - #65378

October 3rd 2011

Dr. Matheson,

The context of the conversation was originally and, for me, always about the monumental lack of fossil evidence which might document a gradual lengthening of the giraffe neck in a Darwinian fashion.  As you know, Stephen Jay Gould also underscored this problem in his paper.   It was in this context that beaglelady steered me to the Dawkins video on giraffes.  So if there’s any moving target here it would be the recurrent laryngeal nerve.  That’s about as straightforward as I can make it. 

If you’ve seen the video you’ll know that they don’t  mention a solitary thing about the missing fossil evidence.  Is it any wonder that  evolutionists are accused of only teaching the part of the science which serves their theory?  Instead they used high production value  computer graphics and sound effects to gloss over the evidence which they do not possess or know where to get.  I’d call that obfuscation.

The length and routing of the recurrent laryngeal nerve is a curiosity I’ll give you that much.  To claim it proves evolution is true because God wouldn’t have made it that way is just a weak argument that doesn’t impress.   It’s exactly the same type of argument that evolutionists condemn when ID’ers claim that that the molecular machines in a cell are so tiny and so elegant and so efficient that they had to have been designed by God rather than natural selection.

sfmatheson - #65379

October 3rd 2011

glsi, just two quick responses to your very disappointing comment. First, a full-length research report on giraffe paleontology was posted here. You have chosen instead to cite Gould, writing years before in an essay on a different topic. The experts who wrote the technical report contradict every claim you have made about giraffe fossils. And yet you have failed to address the article at all. It’s not clear to me that you are interested in giraffe paleontology.

Second, no one has argued as you claim in your final paragraph. I’m disappointed by your use of that strawman, and by your penchant for ridiculing scientists.

In this fins-to-limbs series, I hope you have been able to learn about how evolutionary creationists see the explanatory power of common descent in multiple areas of biology. While I strongly respect your preference for miraculous creation of animals, I have been unable to understand your animosity toward science and your strategy for reading and critiquing scientific research. I guess we just see the world differently. And that’s okay with me. You can have the last word in this conversation.

beaglelady - #65385

October 4th 2011

Here again is the technical report on giraffe evolution that Steve Matheson had posted on his previous thread:

And glsi, the video did discuss giraffe evolution. 

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