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 beexpressed, 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 factorsthat 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.
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.
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.