Interdigital webbing retention in bat wings illustrates genetic changes underlying amniote limb diversification Scott D. Weatherbee*†, Richard R. Behringer‡, John J. Rasweiler IV§, and Lee A. Niswander†¶ *Department of Developmental Biology, Memorial Sloan–Kettering Cancer Center, New York, NY 10021; ‡Department of Molecular Genetics, University of Texas M. D. Anderson Cancer Center, Houston, TX 77030; §Department of Obstetrics and Gynecology, State University of New York, Brooklyn, NY 11203; and ¶Department of Pediatrics, Section of Developmental Biology, and Howard Hughes Medical Institute, University of Colorado Health Sciences Center, Aurora, CO 80045 Edited by Kathryn V. Anderson, Sloan–Kettering Institute, New York, NY, and approved August 24, 2006 (received for review June 13, 2006) Developmentally regulated programmed cell death sculpts the limbs and other embryonic organs in vertebrates. One intriguing A B example of species-specific differences in apoptotic extent is ob- served in the tissue between the digits. In chicks and mice, bone morphogenetic proteins (Bmps) trigger apoptosis of the interdigi- tal mesenchyme, leading to freed digits, whereas in ducks, Bmp antagonists inhibit the apoptotic program, resulting in webbed feet. Here, we show that the phyllostomid bat Carollia perspicillata utilizes a distinct mechanism for maintaining interdigit tissue. We find that bat forelimb and hindlimb interdigital tissues express Bmp signaling components but that only bat hindlimbs undergo interdigital apoptosis. Strikingly, the retention of interdigital web- bing in the bat forelimb is correlated with a unique pattern of Fgf8 Fig. 1. Differential forelimb morphology in mice and bats. (A) An adult expression in addition to the Bmp inhibitor Gremlin. By using a mouse, Mus musculus.(B) An adult bat, Carollia perspicillata. Digits are functional assay, we show that maintenance of interdigit tissue in numbered from anterior (I) to posterior (V). Bat digits are elongated com- the bat wing depends on the combined effects of high levels of Fgf pared with mouse digits (Inset) and maintain webbing between the posterior digits. signaling and inhibition of Bmp signaling. Our data also indicate that although there is not a conserved mechanism for maintaining interdigit tissue across amniotes, the expression in the bat forelimb To determine the molecular mechanism underlying the main- interdigits of Gremlin and Fgf8 suggests that these key molecular tenance of the interdigital tissue in bat wings, we compared the changes contributed to the evolution of the bat wing. expression of Gremlin, Bmps, and their downstream targets during development of the bat forelimb (where there is little cell bone morphogenetic protein ͉ Carollia perspicillata ͉ Fgf ͉ Gremlin death) and the hindlimb (where there is significant apoptosis, resulting in free digits). We also discovered a unique domain of he morphological diversification of homologous structures is Fgf8 expression in the bat forelimb. To explore the importance Ta common trend in animal evolution. All tetrapod limbs of these signals in the regulation of interdigital cell death in the derive from a common ancestral appendage, yet great diver- bat limb, we devised an ex vivo culture system to test the effects gence is evident in form and function. One of the most produc- of manipulation of these signals. Our data demonstrate that, in tive terrestrial adaptations is for flight. Although bats are the the bat wing, inhibition of Bmp signaling and activation of Fgf only mammalian order that evolved powered flight, they make signaling cooperate to prevent interdigital cell death. up Ͼ20% of mammalian species. Bat wings are highly specialized structures with unique features, such as elongated autopod Results skeletal elements and membranous wing surfaces (Fig. 1). The Bmp Expression in Bat Forelimbs and Hindlimbs. We examined the evolution of the wing membranes in the forelimb autopod region expression of Bmp genes before (stage 16) and at the start of (chiropatagium) and powered flight must have depended on (stage 17) hindlimb interdigit regression (9). At stage 16, Bmp2 mechanisms to retain and elaborate interdigit tissue. Although is expressed throughout the hindlimb interdigits (Fig. 2C) but is much progress has been made in our understanding of the restricted distally during regression of the mesenchyme (Fig. mechanisms that regulate interdigital apoptosis in mice, chicks, 2D). At early stages in the forelimb, Bmp2 expression is strongest and ducks, the molecular mechanisms underlying the retention in interdigit III–IV, with lower levels in anterior and posterior of interdigit tissue in bats is not understood. Current data argue that interdigital cell death is largely Author contributions: S.D.W. and L.A.N. designed research; S.D.W., R.R.B., J.J.R., and L.A.N. regulated by bone morphogenetic protein (Bmp) signaling. performed research; S.D.W. and R.R.B. contributed new reagents͞analytic tools; S.D.W. Bmps are expressed in the interdigit regions during mouse and analyzed data; and S.D.W. and L.A.N. wrote the paper. chick limb development, and inhibition of Bmp signaling sup- The authors declare no conflict of interest. presses cell death (1–7). Although Bmps are expressed similarly This paper was submitted directly (Track II) to the PNAS office. in developing webbed duck feet and in the free-toed chick, cell Abbreviations: Bmp, bone morphogenetic protein; AER, apical ectodermal ridge. death and Bmp targets such as Msx2 are restricted to the distal Data deposition: The sequences reported in this paper have been deposited in the GenBank region of duck feet. Duck feet show significant expression of database (accession nos. DQ855011 and DQ855012). Gremlin, a Bmp inhibitor, in the interdigit region, which appears †To whom correspondence may be addressed. E-mail: [email protected] or BIOLOGY [email protected]. to restrict the action of Bmps to the distal portion of the duck DEVELOPMENTAL foot (8). © 2006 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0604934103 PNAS ͉ October 10, 2006 ͉ vol. 103 ͉ no. 41 ͉ 15103–15107 ABCD A B CD EF G H EFGH I J KL I J KL M N O P Fig. 3. Fgf signaling and Gremlin expression in bat limbs. (A–D) Gremlin expression in forelimbs (A and B) and hindlimbs (C and D) at stage 16 (A and C) and stage 17 (B and D). Roman numerals in A and C indicate digit number. (E–H) Fgf8 expression in forelimbs (E and F) and hindlimbs (G and H) at stage 16 (E and G) and stage 17 (F and H). Fgf8 is expressed throughout the hindlimb AER, in the forelimb AER between digits I-III, and in the interdigits of the forelimb. Interdigital expression persists in the forelimb, but AER expression Fig. 2. Bmp pathway gene expression in developing bat limbs. Analysis of is restricted to the tips of digits II and III, and expression in the hindlimbs is Bmp signaling components in Carollia forelimbs and hindlimbs is shown. (A–D) found in remnants of the AER at the tips of all digits. (I–L) Spry2 expression in Bmp2 expression in forelimbs (A and B) and hindlimbs (C and D) at stage 16 (A bat forelimbs (I and J) and hindlimbs (K and L) at stage 16 (I and K) and stage and C) and stage 17 (B and D). Roman numerals in A and C indicate digit 17 (J and L). Spry2 expression correlates with the domains of Fgf8 expression. number. (E–H) Bmp4 expression in forelimbs (E and F) and hindlimbs (G and H) Anterior is up in all images. (Scale bars, 1 mm. The scale bar in B also applies at stage 16 (E and G) and stage 17 (F and H). (I–L) Bmp7 expression in forelimbs to F and J. The scale bar in A also applies to C–E, G–I, K, and L.) (I and J) and hindlimbs (K and L) at stage 16 (I and K) and stage 17 (J and L). (M and O) Msx1͞2 protein expression on longitudinal sections of stage-15 fore- limbs (M) and hindlimbs (O). (N and P) Msx2 RNA expression in forelimbs (N) interdigits will undergo regression, whereas bat forelimb inter- and hindlimbs (P) at stage 17. Anterior is up in all images. (Scale bars, 1 mm. digits will form the chiropatagium. The scale bar in B also applies to F, J, and N. The scale bar in A also applies to Msx genes, which are downstream targets of Bmp signaling, are C–E, G–I, K–M, O, and P.) expressed in interdigits before and during regression in chicks and mice and appear to play a role in interdigital apoptosis (12, 13). In ducks, interdigital cell death in the webbed foot is interdigits (Fig. 2A). At stage 17, Bmp2 is strongest flanking the correlated with the restriction of Msx2 to the distal edge of the tips of digits III–V, with lower levels in the posterior interdigits limbs (14). If repression of Bmp targets is a common mechanism (Fig. 2B). The bat Bmp2 expression differs from Bmp2 expres- to restrict cell death and generate webbed limbs, Msx gene sion patterns in mice (10) and birds (2, 3, 11), where Bmp2 is expression in bat wings should reflect this repression and hence expressed throughout most of each interdigit in both forelimbs be absent or greatly reduced compared with bat hindlimbs. We and hindlimbs. examined the expression of Msx1 and Msx2 by using a cross- In contrast to Bmp2, Bmp4 shows little interdigit expression in reactive monoclonal antibody as well as a mouse Msx2 riboprobe. forelimbs and hindlimbs but is localized within the distal mes- Correlating well with the early domains of Bmp7 gene expres- enchyme, apical ectodermal ridge (AER), and digit tips and sion, we found Msx genes to be highly expressed throughout the flanking the digits dorsally and ventrally, possibly in the pre- interdigits in both forelimbs and hindlimbs before and during sumptive tendons (Fig.