When sculpting evolutionary histories—when telling the stories of change over time—the developmental biologist is often drawn to similarity. She wants to figure out what that last common ancestor was like; what do these extant representatives have in common that illuminates the primitive condition? But a good scientist and theorist also knows to keep her eyes open. In some cases, the differences in two lineages may be just as informative as the similarities when explaining evolutionary trajectories.
For instance, many scientists have sought out the similarities between fish fins and tetrapod limbs. Fins and limbs are homologous structures; the fossil record beautifully illustrates acquisition of limb-type characters and loss of fin rays over evolutionary time as tetrapods evolved from lobe-finned fish ancestors . The lobe-finned fishes and tetrapods together comprise the sister group to the ray-finned fishes. While there are definitely striking similarities in the development of fins and limbs, it turns out that a key difference may allow us to uncover the developmental changes that transformed a fin-like structure to a limb-like structure.
The initial steps of limb development are basically identical in fish and tetrapods: a combination of signals in the lateral plate mesoderm creates a limb-forming region where a bulge of mesodermal cells form the first visible sign of a limb, the limb bud. In both fish and tetrapods, a ridge of ectodermal tissue, the apical ectodermal ridge (AER), initially forms at the distal apex of the bud. The AER persists throughout limb outgrowth in tetrapods, acting both to maintain a zone of proliferating mesodermal cells at the distal end of the limb and to provide important patterning signals. In fish, however, the AER is later transformed into a different structure, the apical fold (AF), which is morphologically distinct from the AER.
One model for the evolution of limbs, the clock model (fig. 1), suggests that a heterochronic shift in timing of the AER-to-AF transition may have been the main developmental process driving the fin-to-limb transition . The AF is thought to pattern fin ray outgrowth whereas the AER is thought to regulate endoskeletal patterning and promote endochondral outgrowth.
To make a limb from a fin, one needs to do two important things (among others, of course): lose fin rays and gain well-patterned endochondral elements. A trend of less time with an AF structure, while maintaining the AER for a longer amount of time would produce the limb-type morphology. And in fact, the AER-to-AF transformation occurs relatively early in the ray-finned fishes, at a later time point in lobe-finned fishes, and not at all in the tetrapods.
The basic idea is that ray-finned fish have a limited amount of developmental time spent with an AER, thus the endoskeletal region is relatively short. Then the AF drives the majority of limb outgrowth, resulting in elaborated fin rays. In the lobe-finned fishes, the signals from the AER are maintained for a longer amount of time, resulting in elaboration of the endoskeletal pattern. Subsequent AF formation results in some fin rays. In tetrapods, the AER is maintained through the entirety of limb outgrowth and an AF never forms, resulting in an elongated endoskeleton and a limb with no fin rays (see fig. 1). While this model fits the fossil evidence for the fin-to-limb transition well, little evidence from embryological and developmental studies support this hypothesis. A recent paper in Development sought to change this.
An Exciting Find
Yano et al.,  described the structure of the AF in detail and investigated the timing of the AER-to-AF transformation in the zebrafish. They found that after AF formation, the majority of pectoral fin outgrowth resulted from growth of the AF region; the endoskeletal region only modestly increases in length. These data support the idea that endoskeletal outgrowth is mainly moderated by the AER; when the AER is no longer around to signal, the endoskeleton grows little. To investigate this further, Yano et al. took advantage of microsurgical techniques in the zebrafish to remove the AF from developing limb buds. Zebrafish have amazing regenerative capabilities and upon removal of the AF the endoskeletal region slightly increased in length and an AER was regenerated within six hours. The surgically manipulated fin then underwent the normal AER-to-AF transformation and outgrowth proceeded normally.
This slight extension of the endoskeletal elements may mean that prolonged AER-exposure leads to elongation of the endoskeleton. Repeated removal of the AF exposed the endoskeletal mesoderm to the AER for a longer amount of time, imitating a delay in the AER-to-AF transition. In support of the clock model, after three removals of the AF, the endoskeletal elements of the removal fin were elongated distally compared to fin on the control side (Fig. 2) Furthermore, there was an abundance of proliferating cells at the distal edge of the removal fin after AER exposure compared to the control fin, suggesting the maintained AER exposure promotes proliferation and outgrowth.
When these fish completed limb development, the endoskeletal region of the removal fin was altered, losing some normal fin bones and in some cases gaining structures distally. These alterations can be interpreted to be more limb-like, but much more work should be done characterizing these phenotypes to make such a claim. Even so, these data do suggest that exposure to AER signaling controls the outgrowth and morphology of the endoskeleton. The AER-to-AF transition might indeed restrict the growth and shape of the endoskeletal region.
This study is a great example of evolutionary developmental biology designed to experimentally test a proposed model. The clock model rests on the assumption that the AER and AF have different functions patterning distinct morphological structures. This paper demonstrated different skeletal morphologies for fins exposed only to endogenous AER signals versus those exposed to the AER for a longer amount of time, lending embryological support to the clock model. We may not be able to literally turn back time to examine the common ancestor, but by experimentally “manipulating the clock” we may be able to get a pretty good idea as to how extant representatives gained their characteristic limb features. Read the paper here.
1. Coates, M.I. (1994). The origin of vertebrate limbs. Dev Suppl, 169-180.
2. Thorogood, P. (1991). The development of the teleost fin and implications for our understanding of tetrapod limb evolution. In Developmental patterning of the vertebrate limb. Springer, pp. 347-354.
3. Yano, T., Abe, G., Yokoyama, H., Kawakami, K., and Tamura, K. (2012). Mechanism of pectoral fin outgrowth in zebrafish development. Development 139, 2916-2925.
4. Long, J.A., Young, G.C., Holland, T., Senden, T.J., and Fitzgerald, E.M. (2006). An exceptional Devonian fish from Australia sheds light on tetrapod origins. Nature 444, 199-202.
This post results from the discussion of Yano et al., 2012 by the Development, Regeneration, and Stem Cell Biology Journal Club at the University of Chicago. It was authored by Haley K. Stinnett, Department of Organismal Biology and Anatomy, University of Chicago, Chicago, IL 60637.