Developmental basis for hind-limb loss in dolphins and origin of the cetacean bodyplan

Abstract

Among mammals, modern cetaceans (whales, dolphins, and porpoises) are unusual in the absence of hind limbs. However, cetacean embryos do initiate hind-limb bud development. In dolphins, the bud arrests and degenerates around the fifth gestational week. Initial limb outgrowth in amniotes is maintained by two signaling centers, the apical ectodermal ridge (AER) and the zone of polarizing activity (ZPA). Our data indicate that the cetacean hind-limb bud forms an AER and that this structure expresses Fgf8 initially, but that neither the AER nor Fgf8 expression is maintained. Moreover, Sonic hedgehog (Shh), which mediates the signaling activity of the ZPA, is absent from the dolphin hind-limb bud. We find that failure to establish a ZPA is associated with the absence of Hand2, an upstream regulator of Shh. Interpreting our results in the context of both the cetacean fossil record and the known functions of Shh suggests that reduction of Shh expression may have occurred approximately 41 million years ago and led to the loss of distal limb elements. The total loss of Shh expression may account for the further loss of hind-limb elements that occurred near the origin of the modern suborders of cetaceans approximately 34 million years ago. Integration of paleontological and developmental data suggests that hind-limb size was reduced by gradually operating microevolutionary changes. Long after locomotor function was totally lost, modulation of developmental control genes eliminated most of the hind-limb skeleton. Hence, macroevolutionary changes in gene expression did not drive the initial reduction in hind-limb size.

Fig. 1.

Hind-limb loss in embryos of the dolphin S. attenuata. (A and B) Embryo (LACM 94706, section 183a, coronal section) at the stage of largest hind-limb development (Carnegie Stage 15). Hind-limb bud (hl) with apical ectodermal ridge (aer) is visible on either side of the abdomen of the embryo, with notochord (no) and neural tube (nt) in the median plane. (C and D) Embryo (LACM 94747, section 238, cross section) with reduced hind limbs and missing AER (Carnegie Stage 16). Chondrification is taking place in the vertebral column (vc). Boxes in A and C indicate location of enlargements found in B and D, respectively.

Fig. 2.

Gene expression in Stenella embryos. Drawings of embryos at Carnegie Stages 12, 14, 15, and 16 are shown, and red lines indicate location of sections. Sections through fore-limb bud (A, C, D, G, H, J, and K) and hind-limb bud area (B, E, F, I, and L). (A and B) Hand2 expression in embryo LACM 94789 (sections 208c and 286b, respectively). (C–F) Fgf8 expression in embryo LACM 94594 (sections 72c, detail of 72c, 208a, detail of 208b, respectively) region labeled flank in E shows low epithelial background staining. (G–I) Shh expression in embryo LACM 94746 (three views of section 4b). (J and K) Myosin expression in embryo LACM 94770 (section 214a with detail and 481a, respectively). Boxes indicate areas enlarged in adjacent image, and arrows indicate areas of high expression.

Fig. 3.

Simplified phylogeny of cetaceans discussed here with evolutionary events indicated. Hind limbs represent fossil ambulocetid Ambulocetus (20, 21), fossil basilosauroid Basilosaurus (22, 23), and two modern mysticetes (Bowhead Whale and Sei Whale, respectively; ref. 24). In most odontocetes, the only hind-limb element preserved is the innominate, as in the Sei Whale (see supporting information). Labeled bony elements of the hind limb are innominate (inn.), femur (fem.), and tibia (tib).