Interdigital webbing retention in bat wingsillustrates genetic changes underlyingamniote limb diversificationScott 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 ofTexas 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 thelimbs and other embryonic organs in vertebrates. One intriguingexample of species-specific differences in apoptotic extent is ob-served in the tissue between the digits. In chicks and mice, bonemorphogenetic proteins (Bmps) trigger apoptosis of the interdigi-tal mesenchyme, leading to freed digits, whereas in ducks, Bmpantagonists inhibit the apoptotic program, resulting in webbedfeet. Here, we show that the phyllostomid bat Carollia perspicillatautilizes a distinct mechanism for maintaining interdigit tissue. Wefind that bat forelimb and hindlimb interdigital tissues expressBmp signaling components but that only bat hindlimbs undergointerdigital apoptosis. Strikingly, the retention of interdigital web-bing in the bat forelimb is correlated with a unique pattern of Fgf8expression in addition to the Bmp inhibitor Gremlin. By using afunctional assay, we show that maintenance of interdigit tissue inthe bat wing depends on the combined effects of high levels of Fgfsignaling and inhibition of Bmp signaling. Our data also indicatethat although there is not a conserved mechanism for maintaininginterdigit tissue across amniotes, the expression in the bat forelimbinterdigits of Gremlin and Fgf8 suggests that these key molecularchanges contributed to the evolution of the bat wing.

bone morphogenetic protein � Carollia perspicillata � Fgf � Gremlin

The morphological diversification of homologous structures isa common trend in animal evolution. All tetrapod limbs

derive from a common ancestral appendage, yet great diver-gence is evident in form and function. One of the most produc-tive terrestrial adaptations is for flight. Although bats are theonly mammalian order that evolved powered flight, they makeup �20% of mammalian species. Bat wings are highly specializedstructures with unique features, such as elongated autopodskeletal elements and membranous wing surfaces (Fig. 1). Theevolution of the wing membranes in the forelimb autopod region(chiropatagium) and powered flight must have depended onmechanisms to retain and elaborate interdigit tissue. Althoughmuch progress has been made in our understanding of themechanisms that regulate interdigital apoptosis in mice, chicks,and ducks, the molecular mechanisms underlying the retentionof interdigit tissue in bats is not understood.

Current data argue that interdigital cell death is largelyregulated by bone morphogenetic protein (Bmp) signaling.Bmps are expressed in the interdigit regions during mouse andchick limb development, and inhibition of Bmp signaling sup-presses cell death (1–7). Although Bmps are expressed similarlyin developing webbed duck feet and in the free-toed chick, celldeath and Bmp targets such as Msx2 are restricted to the distalregion of duck feet. Duck feet show significant expression ofGremlin, a Bmp inhibitor, in the interdigit region, which appearsto restrict the action of Bmps to the distal portion of the duckfoot (8).

To determine the molecular mechanism underlying the main-tenance of the interdigital tissue in bat wings, we compared theexpression of Gremlin, Bmps, and their downstream targetsduring development of the bat forelimb (where there is little celldeath) and the hindlimb (where there is significant apoptosis,resulting in free digits). We also discovered a unique domain ofFgf8 expression in the bat forelimb. To explore the importanceof these signals in the regulation of interdigital cell death in thebat limb, we devised an ex vivo culture system to test the effectsof manipulation of these signals. Our data demonstrate that, inthe bat wing, inhibition of Bmp signaling and activation of Fgfsignaling cooperate to prevent interdigital cell death.

ResultsBmp Expression in Bat Forelimbs and Hindlimbs. We examined theexpression of Bmp genes before (stage 16) and at the start of(stage 17) hindlimb interdigit regression (9). At stage 16, Bmp2is expressed throughout the hindlimb interdigits (Fig. 2C) but isrestricted distally during regression of the mesenchyme (Fig.2D). At early stages in the forelimb, Bmp2 expression is strongestin interdigit III–IV, with lower levels in anterior and posterior

Author contributions: S.D.W. and L.A.N. designed research; S.D.W., R.R.B., J.J.R., and L.A.N.performed research; S.D.W. and R.R.B. contributed new reagents�analytic tools; S.D.W.analyzed data; and S.D.W. and L.A.N. wrote the paper.

The authors declare no conflict of interest.

This paper was submitted directly (Track II) to the PNAS office.

Abbreviations: Bmp, bone morphogenetic protein; AER, apical ectodermal ridge.

Data deposition: The sequences reported in this paper have been deposited in the GenBankdatabase (accession nos. DQ855011 and DQ855012).

†To whom correspondence may be addressed. E-mail: s-weatherbee@ski.mskcc.org orlee.niswander@uchsc.edu.

© 2006 by The National Academy of Sciences of the USA

A B

Fig. 1. Differential forelimb morphology in mice and bats. (A) An adultmouse, Mus musculus. (B) An adult bat, Carollia perspicillata. Digits arenumbered from anterior (I) to posterior (V). Bat digits are elongated com-pared with mouse digits (Inset) and maintain webbing between the posteriordigits.

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interdigits (Fig. 2 A). At stage 17, Bmp2 is strongest f lanking thetips of digits III–V, with lower levels in the posterior interdigits(Fig. 2B). The bat Bmp2 expression differs from Bmp2 expres-sion patterns in mice (10) and birds (2, 3, 11), where Bmp2 isexpressed throughout most of each interdigit in both forelimbsand hindlimbs.

In contrast to Bmp2, Bmp4 shows little interdigit expression inforelimbs and hindlimbs but is localized within the distal mes-enchyme, apical ectodermal ridge (AER), and digit tips andflanking the digits dorsally and ventrally, possibly in the pre-sumptive tendons (Fig. 2 E–H). Bmp4 expression appears to bereduced or absent in bat forelimb and hindlimb interdigitscompared with chicks and ducks (2, 3, 11) but is similar to Bmp4patterns in mice (10). Bmp7 expression in bat forelimbs initiatesas strong expression in every interdigit, as well as anterior to digitI and posterior to digit V (Fig. 2I). Later, the strongest expres-sion is observed flanking the forelimb digits, with lower levels inthe interdigits (Fig. 2 J). In the hindlimbs, Bmp7 expression isfound in the proximal interdigit and subjacent to the AER atstage 16 (Fig. 2K), similar to chicks and ducks but different frommice, where Bmp7 is expressed in all except the most distalinterdigit (10). During hindlimb interdigit regression, Bmp7 ismost strongly expressed flanking the digits and in distal interdigittissue (Fig. 2L). Despite some differences in bat Bmp expressioncompared with mice and birds, Bmp2 and Bmp7 are expressedin both the forelimb and the hindlimb, although bat hindlimb

interdigits will undergo regression, whereas bat forelimb inter-digits will form the chiropatagium.

Msx genes, which are downstream targets of Bmp signaling, areexpressed in interdigits before and during regression in chicksand mice and appear to play a role in interdigital apoptosis (12,13). In ducks, interdigital cell death in the webbed foot iscorrelated with the restriction of Msx2 to the distal edge of thelimbs (14). If repression of Bmp targets is a common mechanismto restrict cell death and generate webbed limbs, Msx geneexpression in bat wings should reflect this repression and hencebe absent or greatly reduced compared with bat hindlimbs. Weexamined the expression of Msx1 and Msx2 by using a cross-reactive monoclonal antibody as well as a mouse Msx2 riboprobe.Correlating well with the early domains of Bmp7 gene expres-sion, we found Msx genes to be highly expressed throughout theinterdigits in both forelimbs and hindlimbs before and duringregression (Fig. 2 M–P). Thus, in Carollia, Msx expressiondomains are similar to the patterns observed in chick and mouselimbs but contrast with the pattern in the duck webbed foot.Although Msx activity is implicated in interdigital cell death,expression of Msx RNA or protein is apparently not an accurateindicator of whether interdigital apoptosis will occur.

Bat Wings Express Gremlin and Display Unique Expression of Fgf8. Inthe webbed duck foot, proximal expression of the Bmp antag-onist Gremlin is thought to prevent Bmp-mediated cell death andrestrict this activity to the distal region of the interdigits (8). Weexamined the expression of Gremlin to test whether it might playa similar role in the retention of interdigital webbing in bat wings.By stage 16, Gremlin is highly expressed in the two anteriormostinterdigits in the forelimb and at lower levels in the posteriorforelimb, whereas it is expressed proximally in all interdigit

A B C D

E F G H

I J K L

Fig. 3. Fgf signaling and Gremlin expression in bat limbs. (A–D) Gremlinexpression in forelimbs (A and B) and hindlimbs (C and D) at stage 16 (A andC) 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 stage16 (E and G) and stage 17 (F and H). Fgf8 is expressed throughout the hindlimbAER, in the forelimb AER between digits I-III, and in the interdigits of theforelimb. Interdigital expression persists in the forelimb, but AER expressionis restricted to the tips of digits II and III, and expression in the hindlimbs isfound in remnants of the AER at the tips of all digits. (I–L) Spry2 expression inbat forelimbs (I and J) and hindlimbs (K and L) at stage 16 (I and K) and stage17 (J and L). Spry2 expression correlates with the domains of Fgf8 expression.Anterior is up in all images. (Scale bars, 1 mm. The scale bar in B also appliesto F and J. The scale bar in A also applies to C–E, G–I, K, and L.)

A B C D

E F G H

I J K L

M N O P

Fig. 2. Bmp pathway gene expression in developing bat limbs. Analysis ofBmp signaling components in Carollia forelimbs and hindlimbs is shown. (A–D)Bmp2 expression in forelimbs (A and B) and hindlimbs (C and D) at stage 16 (Aand C) and stage 17 (B and D). Roman numerals in A and C indicate digitnumber. (E–H) Bmp4 expression in forelimbs (E and F) and hindlimbs (G and H)at stage 16 (E and G) and stage 17 (F and H). (I–L) Bmp7 expression in forelimbs(I and J) and hindlimbs (K and L) at stage 16 (I and K) and stage 17 (J and L). (Mand O) Msx1�2 protein expression on longitudinal sections of stage-15 fore-limbs (M) and hindlimbs (O). (N and P) Msx2 RNA expression in forelimbs (N)and hindlimbs (P) at stage 17. Anterior is up in all images. (Scale bars, 1 mm.The scale bar in B also applies to F, J, and N. The scale bar in A also applies toC–E, G–I, K–M, O, and P.)

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regions of the hindlimb (Fig. 3 A and C). At stage 17, Gremlinis expressed in all interdigits of the forelimb, but it is largelyexcluded from the most distal portions of the limb (Fig. 3B). Bythis time, the interdigital expression in the hindlimb is lost, andGremlin is largely found flanking the digits and at their tips (Fig.3D). Because Gremlin is a potent Bmp inhibitor, the expressionin the forelimb interdigits places Gremlin in a position to blockBmp-mediated cell death. Intriguingly, Msx gene expression inbat wings suggests that Gremlin is not able to inhibit all Bmpsignaling in the forelimb interdigits or that another factoractivates Msx expression there.

Fgfs have been proposed to be survival signals for a variety oftissues (15), and Fgf application has been shown to transientlyblock cell death in chick limb interdigits (16–19). In mice,expression of a gain-of-function allele of Fgf4 results in persistentwebbing between the digits (20). Similarly, human mutationsthat activate Fgf receptors block cell death between the digits(21). Together, these experiments suggested that Fgf signalingmight be a good candidate for repressing cell death in bat winginterdigits. Thus, we examined the expression of a key Fgf genein the limb, Fgf8. As in mice (22) and chicks (23), Fgf8 isexpressed throughout the AER of bat forelimbs and hindlimbs(Fig. 3 E and G; data of earlier stages not shown). However, Fgf8is also expressed in limb mesenchyme at early stages and in theforelimb during the time when interdigit regression should occur(Fig. 3 E and G; data of earlier stages not shown). The earlymesenchyme expression is similar to that reported in axolotls(24) but is strikingly different from other amniotes. Fgf8 wasexpressed at high levels in the interdigit region of bat forelimbs,most strongly in the posterior two interdigits (Fig. 3E) and atlower levels in hindlimb interdigits. In contrast to axolotl, Fgf8 ismaintained in bat forelimb interdigits at the time of interdigitalregression and lost in the bat hindlimb. (Fig. 3 F and H). Theinterdigit expression of Fgf8 might also account for Msx expres-sion in the forelimb, because Fgfs have been shown to induceMsx2 expression in avian interdigit tissue (19). The interdigitexpression of Fgf8 in the forelimb interdigits was largely mir-rored by Sprouty2 (Spry2) (Fig. 3 I–L), which is activated inresponse to Fgf signaling (25). This differential expression ofFgf8 led us to hypothesize that Fgf signaling in the bat forelimbinterdigit may act to specifically maintain the survival of bat winginterdigital tissue.

Functional Disruption of Fgf Signaling and Enhancement of BmpSignaling Induces Interdigital Cell Death in Bat Wings. Comparativegene expression served as a starting point for understanding howthe bat interdigital webbing is maintained. In other systems,coapplication of Fgf and a Bmp inhibitor has been shown tosignificantly suppress interdigital apoptosis (19). To functionallytest the role of Fgf and Bmp signaling on bat wing interdigital celldeath, we performed the converse experiment. We implantedinto bat forelimb interdigit regions beads soaked in a Fgfreceptor inhibitor (SU5402) and Bmp protein (Fig. 4 A and B).The limbs were then cultured and subsequently assayed for celldeath by an active caspase-3 antibody or by TUNEL. Analysis offreshly dissected uncultured forelimbs shows there is little celldeath in the bat forelimb interdigit region, although there is celldeath in the AER. We quantified the results of our cultureexperiments and found a highly significant increase in cell deathcompared with controls (P � 0.0003; n � 17; Fig. 4 C and D).The few active caspase-3-positive mesenchyme cells in thecontrol limb (Fig. 3C) are a result of culturing the limbs. Thus,the combination of decreased Fgf signaling and increased Bmpsignaling is able to cause the regression of the bat forelimbinterdigital tissue. Single treatments with Bmp- or SU5402-soaked beads did not result in significantly increased cell deathcompared with controls. Taken together with the expressionpattern data, our studies suggest that retention of the interdigital

tissue in the bat wing is due, at least in part, to inhibition of Bmpsignaling (possibly through Gremlin expression) and activation ofFgf signaling by means of a previously uncharacterized domainof Fgf8.

DiscussionHere, we reveal a previously uncharacterized mechanism that isused in the bat wing to prevent apoptosis and interdigitalregression (Fig. 4E). In the bat wing, inhibition of Bmp andactivation of Fgf signaling are required to prevent interdigitalcell death. Specifically, we show that high levels of Gremlinexpression in the forelimb interdigits were not reflected by adifference in Msx expression in forelimbs and hindlimbs. Inaddition, increasing the levels of Bmps in interdigits was notsufficient to induce cell death, suggesting an additional mecha-nism for maintenance of interdigital tissue in the bat wing. Thestrong and maintained expression of Fgf8 in bat forelimb inter-digits is intriguing and suggestive of an evolutionarily unique rolein the maintenance of interdigital webbing. Experimentally, weshow that perturbation of this balance by increasing Bmp anddecreasing Fgf signaling results in extensive forelimb interdigitalapoptosis. We therefore propose that formation of the webbedbat forelimb requires Fgf signaling in the forelimb interdigitdomains acting in combination with reduced Bmp signaling(Fig. 4E).

The evolution of flight in bats is a matter of conjecture. Thepaucity of intermediate forms in the fossil record has made itdifficult to ascertain the order of events that led to flight in theorder Chiroptera. One hypothesis is that bat ancestors firstglided by means of the lateral wing membranes (plagiopata-gium), similar to flying squirrels (subfamily Pteromyinae), f lyinglemurs (order Dermoptera), and marsupial gliders (genus Petau-rus). This evolutionary change would have required an out-growth of tissue from the flank of the body or limbs, and themolecular mechanisms regulating plagiopatagium initiation anddevelopment in any of these groups are not known. In bats, this

E

A B C D

Fig. 4. Functional analysis of Fgf and Bmp signaling in bat limbs. (A–D)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-fluorescence on longitudinal bat forelimb sections shows interdigit regionIV–V (boxed regions in A and B). Yellow circles are sections through beads, andasterisks mark the position of beads that are not in the plane of section or thatfell out after sectioning of the limbs. (E) Schematic of the differences in geneexpression in free-toed mouse limbs and webbed duck and bat limbs. Mouseforelimbs show proximally restricted Gremlin expression (red) and high levelsof Bmp signaling (yellow) throughout the interdigit, which results in extensivecell death of interdigit tissue and free digits. Duck hindlimbs have strongproximal expression of Gremlin, which blocks Bmp-induced gene expressionand apoptosis. Bat forelimbs exhibit Bmp signaling, but cell death is blocked,likely because of the widespread expression of Gremlin and the uniquedomain of Fgf8 signaling (blue) in forelimb interdigit regions.

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tissue forms in a very different manner than the chiropatagium.The plagiopatagium grows out from the flank starting at stage14 (9). At stage 17, the plagiopatagium has begun to connect tothe proximal portion of digit V of the forelimb. Fusion of theplagiopatagium to digit V is not complete until late stage19�early stage 20. Because the outgrowth of the plagiopatagiumis not due to blocking apoptosis, we assume that anothermechanism regulates its morphogenesis.

The development of the chiropatagium depends partly onthe retention of early interdigit tissue. Our data suggest thatthe modulation of Bmp and Fgf signaling plays a critical rolein this process and may have been involved in the evolution ofthe wing membrane, with one of the key events being acqui-sition of a unique expression domain of Fgf8 signaling. Ourdata provide molecular insight into the evolution of poweredf light in bats. Retention of interdigital webbing in the batwould have been an important step to developing true poweredf light and likely contributed to the success of this widespreadand diverse order.

Another step in the evolution of bat wings was the elongationof the forelimb digits to support the wing membrane. Flyinglemurs possess both plagiopatagia and webbing between thedigits but lack the elongated digits found in bat wings. It wouldbe interesting to determine whether a similar mechanism forinterdigital retention operates in flying lemurs. In bats, thelength of the forelimb digits appears to be controlled by anincrease in Bmp activity within the cartilage (26). It is excitingthat the modulation of both of these aspects of bat wingdevelopment depends partly on changes in Bmp signaling:increased Bmp in the digits and reduced Bmp in the interdigits.Thus, changes in the temporal expression, spatial expression, andlevels of expression of key developmental regulators such as Bmpand Fgf appear to be important in driving the evolution ofvertebrate limbs.

MethodsAnimal Use and Experimental Embryology. Carollia embryos werecollected from wild-caught, pregnant females on the island ofTrinidad. Limbs were removed from stage 15–18 embryos (9),and beads soaked in control or experimental solutions wereimplanted in the third and fourth interdigit regions of the left andright limbs from the same embryo. The limbs were cultured for18–72 h and then fixed, photographed, and assayed for cell deatheither by immunofluorescence using an active anti-caspase-3antibody (Promega, Madison, WI) or TUNEL assay (Roche,Indianapolis, IN).

Preparation of Beads. Affi-Gel Blue or formate-derivatizedAG1-X2 beads (Bio-Rad, Hercules, CA) were used as carriersfor the administration of Bmps or SU5402 (Calbiochem, Not-tingham, U.K.), respectively. Beads with a diameter rangingbetween 50 and 150 �m were selected, depending on the stageof the embryo. The beads were washed in PBS or DMSO andthen incubated for 1 h at room temperature in different con-

centrations of growth factor. Human recombinant Bmp-4 andBmp-2 (R & D Systems, Minneapolis, MN) were used inter-changeably at concentrations of 0.3 and 0.5 mg�ml, respectively(1). Control beads were incubated in 4 mM HCl plus 0.1% BSA.SU5402 was used at a concentration of 4 mg�ml diluted inDMSO (19), and control beads were soaked in DMSO alone.

Gene Expression Analysis. In situ hybridization was performed onwhole-mount specimens by using digoxygenin-labeled RNAprobes derived from bat or mouse sequences. Using degenerateprimers (5�-TCTyTAACCTCAGCAGCATCC-3� and 5�-CCCCTCyACyACCATCTCCTG-3�), we cloned a 784-bp frag-ment of the Bmp4 gene from Carollia genomic DNA and a467-bp fragment of the Carollia Gremlin gene (5�-GGAAT-TCAAAGGkTCCCAAGGwGCC-3� and 5�-TGCGGC-CGCrTCGATGGATATGCAACG-3�). These sequences havebeen submitted to GenBank (accession nos. DQ855011 andDQ855012). We cloned a 410-bp fragment of Carollia Bmp7from RNA extracted from a stage-13 embryonic head by usingthe primers that are described in ref. 26. We used mouseriboprobes, which also recognize the bat transcripts, to test forthe expression of Fgf8 (27), Bmp2 (28), Msx2 (29), and Sprouty2(25). The monoclonal antibody 4G1 (which recognizes Msx1 andMsx2), developed by Thomas M. Jessell and Susan Brenner-Morton, was obtained from the Developmental Studies Hybrid-oma Bank (Iowa City, IA).

Cell Death Assays and Statistical Analysis. Active caspase-3 expres-sion was assayed by an antibody that recognizes the cleavedactive form of caspase-3 (Promega). The TUNEL assay wasperformed as recommended by the manufacturer (Roche).Apoptotic cell numbers were quantified by counting thepercentage of dying cells in a 100-�m square distal to theimplanted beads in the interdigital space between digits IV andV. Using contralateral limbs as a control and the Wilcoxonsigned rank test, the average difference between Bmp- orSU5402-treated and control-treated limbs was not significant(n � 6, P � 0.345 and n � 7, P � 0.6121, respectively), whereaslimbs treated with Bmp and SU5402 beads did show a statis-tically significant (n � 17, P � 0.0003) increase in thepercentage of dying cells.

We thank Chris Cretekos, Karen Sears, Irene Zohn, and KathrynAnderson for support, discussions, and comments on the manuscript;Simeon Williams for field assistance; the Department of Life Sciences,University of the West Indies (St. Augustine, Trinidad) and, particularly,Dr. Indira Omah-Maharaj for assistance and use of departmentalfacilities during the course of the fieldwork; and the Wildlife Section,Forestry Division, Ministry of Agriculture, Land, and Marine Resources(currently in the Ministry of Public Utilities and the Environment) of theRepublic of Trinidad and Tobago for providing required collecting andexport licenses. This work was supported by a National Research ServiceAward fellowship (to S.D.W.), the National Science Foundation(R.R.B.), and the National Institutes of Health (L.A.N.). L.A.N. is anInvestigator of the Howard Hughes Medical Institute.

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