Interdigital webbing retention in bat illustrates genetic changes underlying amniote 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 of the interdigi- tal mesenchyme, leading to freed digits, whereas in , 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 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 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 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 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 , 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 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

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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 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 (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. 2 E–H). Bmp4 expression appears to be regression (Fig. 2 M–P). Thus, in Carollia, Msx expression reduced or absent in bat forelimb and hindlimb interdigits domains are similar to the patterns observed in chick and mouse compared with chicks and ducks (2, 3, 11) but is similar to Bmp4 limbs but contrast with the pattern in the duck webbed foot. patterns in mice (10). Bmp7 expression in bat forelimbs initiates Although Msx activity is implicated in interdigital cell death, as strong expression in every interdigit, as well as anterior to digit expression of Msx RNA or protein is apparently not an accurate I and posterior to digit V (Fig. 2I). Later, the strongest expres- indicator of whether interdigital apoptosis will occur. sion is observed flanking the forelimb digits, with lower levels in the interdigits (Fig. 2 J). In the hindlimbs, Bmp7 expression is Bat Wings Express Gremlin and Display Unique Expression of Fgf8. In found in the proximal interdigit and subjacent to the AER at the webbed duck foot, proximal expression of the Bmp antag- stage 16 (Fig. 2K), similar to chicks and ducks but different from onist Gremlin is thought to prevent Bmp-mediated cell death and mice, where Bmp7 is expressed in all except the most distal restrict this activity to the distal region of the interdigits (8). We interdigit (10). During hindlimb interdigit regression, Bmp7 is examined the expression of Gremlin to test whether it might play most strongly expressed flanking the digits and in distal interdigit a similar role in the retention of in bat wings. tissue (Fig. 2L). Despite some differences in bat Bmp expression By stage 16, Gremlin is highly expressed in the two anteriormost compared with mice and birds, Bmp2 and Bmp7 are expressed interdigits in the forelimb and at lower levels in the posterior in both the forelimb and the hindlimb, although bat hindlimb forelimb, whereas it is expressed proximally in all interdigit

15104 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0604934103 Weatherbee et al. regions of the hindlimb (Fig. 3 A and C). At stage 17, Gremlin is expressed in all interdigits of the forelimb, but it is largely A B C D excluded from the most distal portions of the limb (Fig. 3B). By this time, the interdigital expression in the hindlimb is lost, and Gremlin is largely found flanking the digits and at their tips (Fig. 3D). Because Gremlin is a potent Bmp inhibitor, the expression in the forelimb interdigits places Gremlin in a position to block Bmp-mediated cell death. Intriguingly, Msx gene expression in bat wings suggests that Gremlin is not able to inhibit all Bmp E signaling in the forelimb interdigits or that another factor activates Msx expression there. Fgfs have been proposed to be survival signals for a variety of tissues (15), and Fgf application has been shown to transiently block cell death in chick limb interdigits (16–19). In mice, expression of a gain-of-function allele of Fgf4 results in persistent webbing between the digits (20). Similarly, human mutations that activate Fgf receptors block cell death between the digits Fig. 4. Functional analysis of Fgf and Bmp signaling in bat limbs. (A–D) (21). Together, these experiments suggested that Fgf signaling Control (A and C) and Bmp- and SU5402-treated (B and D) cultured bat limbs (stage 16 late) after a 23-h incubation. (C and D) Active caspase-3 immuno- might be a good candidate for repressing cell death in bat wing fluorescence on longitudinal bat forelimb sections shows interdigit region interdigits. Thus, we examined the expression of a key Fgf gene IV–V (boxed regions in A and B). Yellow circles are sections through beads, and in the limb, Fgf8. As in mice (22) and chicks (23), Fgf8 is asterisks mark the position of beads that are not in the plane of section or that expressed throughout the AER of bat forelimbs and hindlimbs fell out after sectioning of the limbs. (E) Schematic of the differences in gene (Fig. 3 E and G; data of earlier stages not shown). However, Fgf8 expression in free-toed mouse limbs and webbed duck and bat limbs. Mouse is also expressed in limb mesenchyme at early stages and in the forelimbs show proximally restricted Gremlin expression (red) and high levels forelimb during the time when interdigit regression should occur of Bmp signaling (yellow) throughout the interdigit, which results in extensive (Fig. 3 E and G; data of earlier stages not shown). The early cell death of interdigit tissue and free digits. Duck hindlimbs have strong mesenchyme expression is similar to that reported in axolotls proximal expression of Gremlin, which blocks Bmp-induced gene expression and apoptosis. Bat forelimbs exhibit Bmp signaling, but cell death is blocked, (24) but is strikingly different from other amniotes. Fgf8 was likely because of the widespread expression of Gremlin and the unique expressed at high levels in the interdigit region of bat forelimbs, domain of Fgf8 signaling (blue) in forelimb interdigit regions. most strongly in the posterior two interdigits (Fig. 3E) and at lower levels in hindlimb interdigits. In contrast to axolotl, Fgf8 is maintained in bat forelimb interdigits at the time of interdigital tissue in the bat wing is due, at least in part, to inhibition of Bmp regression and lost in the bat hindlimb. (Fig. 3 F and H). The signaling (possibly through Gremlin expression) and activation of interdigit expression of Fgf8 might also account for Msx expres- Fgf signaling by means of a previously uncharacterized domain sion in the forelimb, because Fgfs have been shown to induce of Fgf8. Msx2 expression in avian interdigit tissue (19). The interdigit expression of Fgf8 in the forelimb interdigits was largely mir- Discussion rored by Sprouty2 (Spry2) (Fig. 3 I–L), which is activated in Here, we reveal a previously uncharacterized mechanism that is response to Fgf signaling (25). This differential expression of used in the bat wing to prevent apoptosis and interdigital Fgf8 led us to hypothesize that Fgf signaling in the bat forelimb regression (Fig. 4E). In the bat wing, inhibition of Bmp and interdigit may act to specifically maintain the survival of bat wing activation of Fgf signaling are required to prevent interdigital interdigital tissue. cell death. Specifically, we show that high levels of Gremlin expression in the forelimb interdigits were not reflected by a Functional Disruption of Fgf Signaling and Enhancement of Bmp difference in Msx expression in forelimbs and hindlimbs. In Signaling Induces Interdigital Cell Death in Bat Wings. Comparative addition, increasing the levels of Bmps in interdigits was not gene expression served as a starting point for understanding how sufficient to induce cell death, suggesting an additional mecha- the bat interdigital webbing is maintained. In other systems, nism for maintenance of interdigital tissue in the bat wing. The coapplication of Fgf and a Bmp inhibitor has been shown to strong and maintained expression of Fgf8 in bat forelimb inter- significantly suppress interdigital apoptosis (19). To functionally digits is intriguing and suggestive of an evolutionarily unique role test the role of Fgf and Bmp signaling on bat wing interdigital cell death, we performed the converse experiment. We implanted in the maintenance of interdigital webbing. Experimentally, we into bat forelimb interdigit regions beads soaked in a Fgf show that perturbation of this balance by increasing Bmp and receptor inhibitor (SU5402) and Bmp protein (Fig. 4 A and B). decreasing Fgf signaling results in extensive forelimb interdigital The limbs were then cultured and subsequently assayed for cell apoptosis. We therefore propose that formation of the webbed death by an active caspase-3 antibody or by TUNEL. Analysis of bat forelimb requires Fgf signaling in the forelimb interdigit freshly dissected uncultured forelimbs shows there is little cell domains acting in combination with reduced Bmp signaling death in the bat forelimb interdigit region, although there is cell (Fig. 4E). death in the AER. We quantified the results of our culture The evolution of flight in bats is a matter of conjecture. The experiments and found a highly significant increase in cell death paucity of intermediate forms in the fossil record has made it compared with controls (P ϭ 0.0003; n ϭ 17; Fig. 4 C and D). difficult to ascertain the order of events that led to flight in the The few active caspase-3-positive mesenchyme cells in the order Chiroptera. One hypothesis is that bat ancestors first control limb (Fig. 3C) are a result of culturing the limbs. Thus, glided by means of the lateral wing membranes (plagiopata- the combination of decreased Fgf signaling and increased Bmp gium), similar to flying squirrels (subfamily Pteromyinae), flying signaling is able to cause the regression of the bat forelimb lemurs (order Dermoptera), and marsupial gliders (genus Petau- interdigital tissue. Single treatments with Bmp- or SU5402- rus). This evolutionary change would have required an out- soaked beads did not result in significantly increased cell death growth of tissue from the flank of the body or limbs, and the BIOLOGY

compared with controls. Taken together with the expression molecular mechanisms regulating plagiopatagium initiation and DEVELOPMENTAL pattern data, our studies suggest that retention of the interdigital development in any of these groups are not known. In bats, this

Weatherbee et al. PNAS ͉ October 10, 2006 ͉ vol. 103 ͉ no. 41 ͉ 15105 tissue forms in a very different manner than the chiropatagium. centrations of growth factor. Human recombinant Bmp-4 and The plagiopatagium grows out from the flank starting at stage Bmp-2 (R & D Systems, Minneapolis, MN) were used inter- 14 (9). At stage 17, the plagiopatagium has begun to connect to changeably at concentrations of 0.3 and 0.5 mg͞ml, respectively the proximal portion of digit V of the forelimb. Fusion of the (1). Control beads were incubated in 4 mM HCl plus 0.1% BSA. plagiopatagium to digit V is not complete until late stage SU5402 was used at a concentration of 4 mg͞ml diluted in 19͞early stage 20. Because the outgrowth of the plagiopatagium DMSO (19), and control beads were soaked in DMSO alone. is not due to blocking apoptosis, we assume that another mechanism regulates its morphogenesis. Gene Expression Analysis. In situ hybridization was performed on The development of the chiropatagium depends partly on whole-mount specimens by using digoxygenin-labeled RNA the retention of early interdigit tissue. Our data suggest that probes derived from bat or mouse sequences. Using degenerate the modulation of Bmp and Fgf signaling plays a critical role primers (5Ј-TCTyTAACCTCAGCAGCATCC-3Ј and 5Ј- in this process and may have been involved in the evolution of CCCCTCyACyACCATCTCCTG-3Ј), we cloned a 784-bp frag- the wing membrane, with one of the key events being acqui- ment of the Bmp4 gene from Carollia genomic DNA and a sition of a unique expression domain of Fgf8 signaling. Our 467-bp fragment of the Carollia Gremlin gene (5Ј-GGAAT- data provide molecular insight into the evolution of powered TCAAAGGkTCCCAAGGwGCC-3Ј and 5Ј-TGCGGC- flight in bats. Retention of interdigital webbing in the bat CGCrTCGATGGATATGCAACG-3Ј). These sequences have would have been an important step to developing true powered been submitted to GenBank (accession nos. DQ855011 and flight and likely contributed to the success of this widespread DQ855012). We cloned a 410-bp fragment of Carollia Bmp7 and diverse order. from RNA extracted from a stage-13 embryonic head by using Another step in the evolution of bat wings was the elongation the primers that are described in ref. 26. We used mouse of the forelimb digits to support the wing membrane. Flying riboprobes, which also recognize the bat transcripts, to test for lemurs possess both plagiopatagia and webbing between the the expression of Fgf8 (27), Bmp2 (28), Msx2 (29), and Sprouty2 digits but lack the elongated digits found in bat wings. It would (25). The monoclonal antibody 4G1 (which recognizes Msx1 and be interesting to determine whether a similar mechanism for Msx2), developed by Thomas M. Jessell and Susan Brenner- interdigital retention operates in flying lemurs. In bats, the Morton, was obtained from the Developmental Studies Hybrid- length of the forelimb digits appears to be controlled by an oma Bank (Iowa City, IA). increase in Bmp activity within the cartilage (26). It is exciting that the modulation of both of these aspects of bat wing Cell Death Assays and Statistical Analysis. Active caspase-3 expres- development depends partly on changes in Bmp signaling: sion was assayed by an antibody that recognizes the cleaved increased Bmp in the digits and reduced Bmp in the interdigits. active form of caspase-3 (Promega). The TUNEL assay was Thus, changes in the temporal expression, spatial expression, and performed as recommended by the manufacturer (Roche). levels of expression of key developmental regulators such as Bmp Apoptotic cell numbers were quantified by counting the and Fgf appear to be important in driving the evolution of percentage of dying cells in a 100-␮m square distal to the vertebrate limbs. implanted beads in the interdigital space between digits IV and V. Using contralateral limbs as a control and the Wilcoxon Methods signed rank test, the average difference between Bmp- or Animal Use and Experimental Embryology. Carollia embryos were SU5402-treated and control-treated limbs was not significant collected from wild-caught, pregnant females on the island of (n ϭ 6, P ϭ 0.345 and n ϭ 7, P ϭ 0.6121, respectively), whereas Trinidad. Limbs were removed from stage 15–18 embryos (9), limbs treated with Bmp and SU5402 beads did show a statis- and beads soaked in control or experimental solutions were tically significant (n ϭ 17, P ϭ 0.0003) increase in the implanted in the third and fourth interdigit regions of the left and percentage of dying cells. right limbs from the same embryo. The limbs were cultured for 18–72 h and then fixed, photographed, and assayed for cell death We thank Chris Cretekos, Karen Sears, Irene Zohn, and Kathryn either by immunofluorescence using an active anti-caspase-3 Anderson for support, discussions, and comments on the manuscript; antibody (Promega, Madison, WI) or TUNEL assay (Roche, Simeon Williams for field assistance; the Department of Life Sciences, Indianapolis, IN). University of the West Indies (St. Augustine, Trinidad) and, particularly, Dr. Indira Omah-Maharaj for assistance and use of departmental Preparation of Beads. Affi-Gel Blue or formate-derivatized facilities during the course of the fieldwork; and the Wildlife Section, AG1-X2 beads (Bio-Rad, Hercules, CA) were used as carriers Forestry Division, Ministry of Agriculture, Land, and Marine Resources (currently in the Ministry of Public Utilities and the Environment) of the for the administration of Bmps or SU5402 (Calbiochem, Not- Republic of Trinidad and Tobago for providing required collecting and tingham, U.K.), respectively. Beads with a diameter ranging export licenses. This work was supported by a National Research Service between 50 and 150 ␮m were selected, depending on the stage Award fellowship (to S.D.W.), the National Science Foundation of the embryo. The beads were washed in PBS or DMSO and (R.R.B.), and the National Institutes of Health (L.A.N.). L.A.N. is an then incubated for1hatroom temperature in different con- Investigator of the Howard Hughes Medical Institute.

1. Ganan Y, Macias D, Basco RD, Merino R, Hurle JM (1998) Dev Biol 196:33–41. 9. Cretekos CJ, Weatherbee SD, Chen CH, Badwaik NK, Niswander L, Behringer 2. Yokouchi Y, Sakiyama J, Kameda T, Iba H, Suzuki A, Ueno N, Kuroiwa A RR, Rasweiler JJ, IV (2005) Dev Dyn 233:721–738. (1996) Development (Cambridge, UK) 122:3725–3734. 10. Jiang R, Lan Y, Chapman HD, Shawber C, Norton CR, Serreze DV, 3. Zou H, Niswander L (1996) Science 272:738–741. Weinmaster G, Gridley T (1998) Genes Dev 12:1046–1057. 4. Macias D, Ganan Y, Sampath TK, Piedra ME, Ros MA, Hurle JM (1997) 11. Laufer E, Pizette S, Zou H, Orozco OE, Niswander L (1997) Science 278:305. Development (Cambridge, UK) 124:1109–1117. 12. Chen Y, Zhao X (1998) J Exp Zool 282:691–702. 5. Guha U, Gomes WA, Kobayashi T, Pestell RG, Kessler JA (2002) Dev Biol 13. Lallemand Y, Nicola MA, Ramos C, Bach A, Cloment CS, Robert B (2005) 249:108–120. Development (Cambridge, UK) 132:3003–3014. 6. Wang CK, Omi M, Ferrari D, Cheng HC, Lizarraga G, Chin HJ, Upholt WB, 14. Merino R, Ganan Y, Macias D, Rodriguez-Leon J, Hurle JM (1999) Ann NY Dealy CN, Kosher RA (2004) Dev Biol 269:109–122. Acad Sci 887:120–132. 7. Zuzarte-Luis V, Montero JA, Rodriguez-Leon J, Merino R, Rodriguez-Rey JC, 15. Eswarakumar VP, Lax I, Schlessinger J (2005) Cytokine Growth Factor Rev Hurle JM (2004) Dev Biol 272:39–52. 16:139–149. 8. Merino R, Rodriguez-Leon J, Macias D, Ganan Y, Economides AN, Hurle JM 16. Buckland RA, Collinson JM, Graham E, Davidson DR, Hill RE (1998) Mech (1999) Development (Cambridge, UK) 126:5515–5522. Dev 71:143–150.

15106 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0604934103 Weatherbee et al. 17. Macias D, Ganan Y, Ros MA, Hurle JM (1996) Anat Embryol 193: 23. Crossley PH, Minowada G, MacArthur CA, Martin GR (1996) Cell 84:127–136. 533–541. 24. Han MJ, An JY, Kim WS (2001) Dev Dyn 220:40–48. 18. Ganan Y, Macias D, Duterque-Coquillaud M, Ros MA, Hurle JM (1996) 25. Minowada G, Jarvis LA, Chi CL, Neubuser A, Sun X, Hacohen N, Krasnow Development (Cambridge, UK) 122:2349–2357. MA, Martin GR (1999) Development (Cambridge, UK) 126:4465–4475. 19. Montero JA, Ganan Y, Macias D, Rodriguez-Leon J, Sanz-Ezquerro JJ, 26. Sears KE, Behringer RR, Rasweiler JJ, IV, Niswander LA (2006) Proc Natl Merino R, Chimal-Monroy J, Nieto MA, Hurle JM (2001) Development Acad Sci USA 103:6581–6586. (Cambridge, UK) 128:2075–2084. 27. Tanaka A, Miyamoto K, Minamino N, Takeda M, Sato B, Matsuo H, 20. Lu P, Minowada G, Martin GR (2006) Development (Cambridge, UK) 133: Matsumoto K (1992) Proc Natl Acad Sci USA 89:8928–8932. 33–42. 28. Dickinson ME, Kobrin MS, Silan CM, Kingsley DM, Justice MJ, Miller DA, 21. Wilkie AO, Slaney SF, Oldridge M, Poole MD, Ashworth GJ, Hockley AD, Ceci JD, Lock LF, Lee A, Buchberg AM, et al. (1990) Genomics 6: Hayward RD, David DJ, Pulleyn LJ, Rutland P, et al. (1995) Nat Genet 505–520. 9:165–172. 29. Zhang H, Hu G, Wang H, Sciavolino P, Iler N, Shen MM, Abate-Shen C (1997) 22. Crossley PH, Martin GR (1995) Development (Cambridge, UK) 121:439–451. Mol Cell Biol 17:2920–2932. BIOLOGY DEVELOPMENTAL

Weatherbee et al. PNAS ͉ October 10, 2006 ͉ vol. 103 ͉ no. 41 ͉ 15107