Development of bat flight: Morphologic and molecular evolution of bat wing digits Karen E. Sears*, Richard R. Behringer†, John J. Rasweiler IV‡, and Lee A. Niswander*§ *Howard Hughes Medical Institute, Department of Pediatrics, Section of Developmental Biology, University of Colorado at Denver and Health Sciences Center, 12800 East 19th Avenue, Aurora, CO 80045; †Department of Molecular Genetics, University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030; and ‡Department of Obstetrics and Gynecology, State University of New York Downstate Medical Center, Brooklyn, NY 11203 Edited by Cliff Tabin, Harvard Medical School, Boston, MA, and accepted by the Editorial Board March 14, 2006 (received for review November 8, 2005) The earliest fossil bats resemble their modern counterparts in used morphometric analyses to quantitatively demonstrate the possessing greatly elongated digits to support the wing mem- similarity of the wing supportive digits between the earliest fossil brane, which is an anatomical hallmark of powered flight. To and modern bats. quantitatively confirm these similarities, we performed a morpho- Because of the similarity between the forelimb digits of the metric analysis of wing bones from fossil and modern bats. We earliest preserved and modern bats, the fossil record currently found that the lengths of the third, fourth, and fifth digits (the can provide little evidence of the evolutionary transitions that primary supportive elements of the wing) have remained constant led to the elongation of bat forelimb digits and the associated relative to body size over the last 50 million years. This absence of evolution of powered flight in mammals, although this situation transitional forms in the fossil record led us to look elsewhere to may change with the discovery of additional fossil material. The understand bat wing evolution. Investigating embryonic develop- fossil record, as well as molecular-clock studies (2, 9), suggest ment, we found that the digits in bats (Carollia perspicillata) are that bats achieved powered flight in a few million years, which initially similar in size to those of mice (Mus musculus) but that, is a relatively short span of geologic time. However, these data subsequently, bat digits greatly lengthen. The developmental do not preclude the morphological prerequisites of powered timing of the change in wing digit length points to a change in flight (e.g., elongated digits) having evolved by means of gradual longitudinal cartilage growth, a process that depends on the processes. relative proliferation and differentiation of chondrocytes. We Several studies (10, 11–15) have shown that major morpho- found that bat forelimb digits exhibit relatively high rates of logical transitions can be accommodated by a few key develop- chondrocyte proliferation and differentiation. We show that bone mental genetic changes. Here, we provide functional and mo- morphogenetic protein 2 (Bmp2) can stimulate cartilage prolifer- lecular comparisons of the development of the forelimb digits of ation and differentiation and increase digit length in the bat the short-tailed fruit bat Carollia perspicillata with the digits of embryonic forelimb. Also, we show that Bmp2 expression and Bmp the bat hind limb and the digits of the forelimb of a more signaling are increased in bat forelimb embryonic digits relative to generalized quadruped, the mouse Mus musculus. Using these mouse or bat hind limb digits. Together, our results suggest that an data, we identified uniquely derived developmental features of up-regulation of the Bmp pathway is one of the major factors in the bat wing digits. Doing so allows us to highlight a key develop- developmental elongation of bat forelimb digits, and it is poten- mental genetic change and suggest evolutionary mechanisms tially a key mechanism in their evolutionary elongation as well. underlying bat digit elongation. Developmental elongation of the digits (and other long bones) ͉ ͉ Chiroptera Bmp cartilage is achieved by means of the relative rates of proliferation and differentiation of cartilage cells (chondrocytes) in the growth s a consequence of their achievement of powered flight, bats plate. Within the growth plate of developing digits, chondrocytes A(order Chiroptera) underwent one of the greatest adaptive go through the following series of maturation steps: resting (in radiations in the history of mammalian evolution and now the Resting zone), proliferation (Proliferative zone), early dif- constitute one of every five mammalian species (1). A key ferentiation (Prehypertrophic zone), and terminal differentia- innovation that enabled this extraordinary radiation is the bat tion (Hypertrophic zone) (16). Upon differentiation, chondro- wing. The bat wing consists of a membrane of skin stretched cyte cell division ceases. Subsequently, the hypertrophic between dramatically elongated third, fourth, and fifth forelimb chondrocytes secrete extracellular matrix in which ossification digits. Because of their importance in wing support, understand- begins. When the cartilage matrix ossifies, the hypertrophic ing the mechanisms that are responsible for the elongation of bat chondrocytes undergo apoptosis. Several genes that have a role forelimb digits is key to understanding the evolutionary tempo in chondrocyte maturation have been identified (i.e., Ihh, Ffg and morphological transitions that underlie this major mamma- genes, Bmp genes, and Pthrp, etc.). The bone morphogenetic lian radiation. protein (Bmp) family of secreted growth factors is of particular Molecular phylogenetic evidence suggests that Chiroptera interest. Members of the Bmp family are involved in almost every (consisting of both microbats and megabats) is a monophyletic aspect of chondrogenesis, from chondrocyte commitment to clade that is nested within the Laurasiatheria, which is a group terminal differentiation (17). In the mouse limb, Bmps are that comprises carnivorans, pangolins, ungulates, and ‘‘core’’ insectivores (or, eulipotyphlans; e.g., shrews, hedgehogs, moles, and solenodons) (2–5). There is a general consensus that the Conflict of interest statement: No conflicts declared. common ancestor of bats was a small, quadrupedal mammal, This paper was submitted directly (Track II) to the PNAS office. C.T. is a guest editor invited with a limb morphology that was similar to that of mice (6, 7). by the Editorial Board. The earliest known bats appear in the fossil record Ϸ50 million Abbreviations: Bmp, bone morphogenetic protein; En, embryonic day n; PC, principal years ago, and they appear suddenly and already possessing the component. anatomical hallmarks of powered flight (including elongated Data deposition: The sequences reported in this paper have been deposited in the GenBank third, fourth, and fifth forelimb digits) (6–8). Thus, it seems to database (accession nos. DQ279782–DQ279785). EVOLUTION be likely that the earliest known fossil bats were already capable §To whom correspondence should be addressed. E-mail: [email protected]. of powered flight (3–5). In the first component of this study, we © 2006 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0509716103 PNAS ͉ April 25, 2006 ͉ vol. 103 ͉ no. 17 ͉ 6581–6586 Downloaded by guest on September 27, 2021 weakly expressed within the growth plate in proliferating (Bmp7), prehypertrophic (Bmp4), and hypertrophic (Bmp2) cells, and they are moderately expressed in the perichondrium (a dense fibrous connective tissue) that surrounds the growth plate (Bmp2–Bmp5 and Bmp7) (18). Proliferation and maturation of chondrocytes are the result of many complex interactions be- tween the perichondrium and the growth plate. Bmp from both the perichondrium and the growth plate itself interacts with other factors within the growth plate and, thus, mediates chon- drocyte maturation (17). Intriguingly, it has been shown that mouse and rat limbs that are cultured in the presence of Bmp2 protein increase in overall length, whereas mouse and rat limbs that are cultured in the presence of the Bmp antagonist Noggin result in stunted digits (19, 20). Bmp2 protein affects these changes by stimulating proliferation and the transition to hyper- trophic differentiation and by inhibiting the most terminal stages of hypertrophic differentiation (19). To determine the developmental basis of bat digit elongation, we compared the embryonic development of the digits of the bat forelimb and hind limb and mouse forelimb in terms of (i) their gross and cellular morphology throughout development; (ii) their relative rates of chondrocyte proliferation and differenti- ation during digit elongation; (iii) the response of the limbs to culture in the presence of Bmp2 protein or a Bmp antagonist (Noggin); and (iv) the expression of many important develop- mental genes, including several members of the Bmp family. Our results demonstrate that bat digit length can be altered in response to changes in Bmp. Also, we show that, in the bat forelimb, there is a dramatic change in the intensity of Bmp2 expression and Bmp signaling relative to the bat hind limb and mouse forelimb. This species- and limb-specific change suggests that Bmp2 has a major role in the developmental elongation of bat wing digits. By linking small changes in molecular patterning to dramatically different phenotypes, we provide a potential explanation for the evolution of the wings of bats, which is a key innovation in mammalian history. Results Forelimb Digits of Fossil