DEVELOPMENT2649RESEARCH ARTICLEINTRODUCTIONVertebrate limbs arise bilaterally from the lateral plate mesoderm(LPM) of the embryo. Within the trunk LPM, interactions betweenthe limb bud mesenchyme and the overlying ectoderm result in theinitiation of local patterning and distal outgrowth that shape the foreand hindlimb buds at the appropriate levels along the anteroposterior(AP) axis (reviewed by Capdevila and Izpisua Belmonte, 2001;Logan, 2003; Tickle, 2003).Fibroblast growth factors (FGFs) have been shown to play animportant role in limb induction. When beads are soaked in a rangeof fibroblast growth factors and are placed in the flank of chickembryos, they induce the formation of ectopic limb buds (Cohn etal., 1995; Ohuchi et al., 1995). Competence of the LPM to respondto these signals is initially distributed along most of the AP axis ofthe LPM in chick and mouse embryos (Tanaka et al., 2000).The search for the axial sources of FGF expression in the chickembryo lead to the observation that the intermediate mesoderm(IM), situated between the somites and the LPM, is required for cellproliferation in the adjoining limb bud mesenchyme (Geduspan andSolursh, 1992; Smith et al., 1996); furthermore, FGF8 is producedin the intermediate mesoderm at the time of limb induction and,when applied to the chick flank, induces the development ofadditional limbs (Crossley et al., 1996; Vogel et al., 1996). On theother hand, when the induction of mesonephros, a component of IM,is blocked rostral to the future limb field, limb buds neverthelessform and develop normally (Fernandez-Teran et al., 1997). Inaddition, elimination of FGF8 from the IM does not affect limbinitiation or outgrowth (Boulet et al., 2004). In addition, genes of theWnt family are differentially expressed in the chick IM and LPMand are capable of inducing limb outgrowth (Kawakami et al., 2001).Specifically, forelimb initiation requires Wnt2b expression in the IMand/or LPM of the chick, while its source in zebrafish appears to bethe somites flanking the pectoral fin field (Ng et al., 2002). Thus, theidentity of axial signals required for limb development as well astheir source(s) remain to be determined.Retinoic acid (RA) is required for a variety of processes duringvertebrate embryonic development. Its effect is transmitted byretinoic acid receptors (RARs) at the level of regulating theexpression of target genes (reviewed by Morriss-Kay and Ward,1999). Three enzymes, Aldh1a1-3 (previously designated Raldh1-3), catalyze the final oxidative step by which vitamin A (retinol) isconverted to RA, but only Aldh1a2 has been shown to be responsiblefor RA synthesis during early stages of embryogenesis. Duringgastrulation, aldh1a2 is expressed in embryonic mesoderm and islater found in mesodermal derivatives, such as somites, IM and LPM(Niederreither et al., 1997; Berggren et al., 1999; Swindell et al.,1999; Begemann et al., 2001; Chen et al., 2001; Grandel et al.,2002).Mouse and zebrafish mutants in aldh1a2 have been used to showthat the somitic mesoderm acts a source of RA that patterns adjacentaxial tissues, such as the hindbrain and pancreas; importantly,aldh1a2 mutants also lack forelimb buds (Begemann et al., 2001;Grandel et al., 2002; Niederreither et al., 2002; Linville et al., 2004;Molotkov et al., 2005; Stafford et al., 2006). The absence of forelimbbuds in aldh1a2 mutants is associated with the loss of tbx5expression, the earliest known marker of the developing forelimbfield (Gibson-Brown et al., 1996; Tamura et al., 1999; Begemannand Ingham, 2000). In the mouse, Tbx5 first serves to establishforelimb bud outgrowth by initiating the expression of regulatoryloops of Wnt and Fgf proteins (reviewed by Capdevila and IzpisuaBelmonte, 2001; Logan, 2003); while in the zebrafish, tbx5 isrequired for the migration of mesenchymal LPM cells to the pectoralfin bud. As a result, mutants in both vertebrates do not form forelimbbuds (Ahn et al., 2002; Garrity et al., 2002; Agarwal et al., 2003;Induction and prepatterning of the zebrafish pectoral finbud requires axial retinoic acid signalingYann Gibert, Alexandra Gajewski, Axel Meyer and Gerrit Begemann*Vertebrate forelimbs arise as bilateral appendages from the lateral plate mesoderm (LPM). Mutants in aldh1a2 (raldh2), anembryonically expressed gene encoding a retinoic acid (RA)-synthesizing enzyme, have been used to show that limb developmentand patterning of the limb bud are crucially dependent on RA signaling. However, the timing and cellular origin of RA signaling inthese processes have remained poorly resolved. We have used genetics and chemical modulators of RA signaling to resolve theseissues in the zebrafish. By rescuing pectoral fin induction in the aldh1a2/neckless mutant with exogenous RA and by blocking RAsignaling in wild-type embryos, we find that RA acts as a permissive signal that is required during the six- to eight-somite stages forpectoral fin induction. Cell-transplantation experiments show that RA production is not only crucially required from flankingsomites, but is sufficient to permit fin bud initiation when the trunk mesoderm is genetically ablated. Under the latter condition,intermediate mesoderm alone cannot induce the pectoral fin field in the LPM. We further show that induction of the fin field isdirectly followed by a continued requirement for somite-derived RA signaling to establish a prepattern of anteroposterior fates inthe condensing fin mesenchyme. This process is mediated by the maintained expression of the transcription factor hand2, throughwhich the fin field is continuously posteriorized, and lasts up to several hours prior to limb-budding. Thus, RA signaling fromflanking somites plays a dual early role in the condensing limb bud mesenchyme.KEY WORDS: Retinoic acid, Raldh2, Limb, Pectoral fin, Somites, Spadetail, No tail, Neckless, ZebrafishDevelopment 133, 2649-2659 (2006) doi:10.1242/dev.02438Lehrstuhl für Zoologie und Evolutionsbiologie, Department of Biology, University ofKonstanz, Konstanz, Germany.*Author for correspondence (e-mail: [email protected])Accepted 10 May 2006DEVELOPMENT2650Rallis et al., 2003). The failure to induce tbx5 expression in theabsence of early RA signaling therefore indicates an essential earlyrole for RA during the establishment of the limb/fin field.In zebrafish, aldh1a2 is expressed in the posterior mesenchymeof the developing pectoral fin and in the LPM (Grandel et al., 2002;Emoto et al., 2005). RA has