Developmental Biology 273 (2004) 361–372 www.elsevier.com/locate/ydbio The roles of Fgf4 and Fgf8 in limb bud initiation and outgrowth Anne M. Bouleta, Anne M. Moonb,c, Benjamin R. Arenkiela, Mario R. Capecchia,* aDepartment of Human Genetics, Howard Hughes Medical Institute, University of Utah School of Medicine, Salt Lake City, UT 84112, USA bProgram in Human Molecular Biology and Genetics, University of Utah School of Medicine, Salt Lake City, UT 84112, USA cDepartment of Pediatrics, University of Utah School of Medicine, Salt Lake City, UT 84112, USA Received for publication 12 May 2004, revised 16 June 2004, accepted 21 June 2004 Abstract Although numerous molecules required for limb bud formation have recently been identified, the molecular pathways that initiate this process and ensure that limb formation occurs at specific axial positions have yet to be fully elucidated. Based on experiments in the chick, Fgf8 expression in the intermediate mesoderm (IM) has been proposed to play a critical role in the initiation of limb bud outgrowth via restriction of Fgf10 expression to the appropriate region of the lateral plate mesoderm. Contrary to the outcome predicted by this model, ablation of Fgf8 expression in the intermediate mesoderm before limb bud initiation had no effect on initial limb bud outgrowth or on the formation of normal limbs. When their expression patterns were first elucidated, both Fgf4 and Fgf8 were proposed to mediate critical functions of the apical ectodermal ridge (AER), which is required for proper limb bud outgrowth. Although mice lacking Fgf4 in the AER have normal limbs, limb development is severely affected in Fgf8 mutants and certain skeletal elements are not produced. By creating mice lacking both Fgf4 and Fgf8 function in the forelimb AER, we show that limb bud mesenchyme fails to survive in the absence of both FGF family members. Thus, Fgf4 is responsible for the partial compensation of distal limb development in the absence of Fgf8. A prolonged period of increased apoptosis, beginning at 10 days of gestation in a proximal–dorsal region of the limb bud, leads to the elimination of enough mesenchymal cells to preclude formation of distal limb structures. Expression of Shh and Fgf10 is nearly abolished in double mutant limb buds. By using a CRE driver expressed in both forelimb and hindlimb ectoderm to inactivate Fgf4 and Fgf8, we have produced mice lacking all limbs, allowing a direct comparison of FGF requirements in the two locations. D 2004 Elsevier Inc. All rights reserved. Keywords: FGF; Limb development; Mouse; Intermediate mesoderm; Limb bud initiation; Apical ectodermal ridge; Cell survival; Apoptosis Introduction 2001). Due to their localization in the apical ectodermal ridge (AER), a structure that plays a critical role in Numerous factors have been proposed to be required for maintenance of limb bud development, members of the the processes of limb bud initiation and outgrowth in the FGF family were proposed to be involved in the control of mouse and in the chick (Johnson and Tabin, 1997; Martin, limb bud outgrowth and patterning. Moreover, experiments 1998). Recent gene knockout experiments in the mouse in the chick demonstrated that FGFs could indeed substitute have confirmed the importance of some of these molecules for the AER (Crossley et al., 1996; Fallon et al., 1994; and have identified others with unexpected functional roles Niswander et al., 1993). (Niswander, 2003; Tickle, 2003; Tickle and Munsterberg, Fgf8 is expressed in the ventral limb ectoderm when the limb bud can first be detected, before the appearance of transcripts for any other FGF family member, and is * Corresponding author. Department of Human Genetics, Howard subsequently expressed throughout the AER. Expression of Hughes Medical Institute, University of Utah School of Medicine, 15 North 2030 East, Suite 5400, Salt Lake City, UT 84112-5331. Fax: +1 801 585 Fgf8 has also been detected in the intermediate mesoderm 3425. (IM) of both chick and mouse embryos before limb bud E-mail address: [email protected] (M.R. Capecchi). initiation (Agarwal et al., 2003; Crossley et al., 1996; Vogel et 0012-1606/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ydbio.2004.06.012 362 A.M. Boulet et al. / Developmental Biology 273 (2004) 361–372 al., 1996). Studies in the chick system have pointed to the IM In this manuscript, we investigate the various roles as a potential source of the endogenous limb bud inducer suggested for Fgf4 and Fgf8 in mouse limb bud outgrowth. (Martin, 1998). When the mesonephros is ablated or when To determine whether Fgf8 is required for limb bud communication between the mesonephros and the lateral initiation, we have eliminated expression of Fgf8 in the plate mesoderm is blocked, wing development is severely IM. To test the hypothesis that the formation of distal affected (Geduspan and Solursh, 1992; Stephens and forelimb structures in Fgf8 conditional limb mutants is due McNulty, 1981; Stephens et al., 1993). These studies to compensation by Fgf4, we produced mice that lack suggested a model in which a signal from the IM or expression of both Fgf4 and Fgf8 in the forelimb. More mesonephros is required to initiate limb bud formation. importantly, we show that production of CRE from the Fgf8 expression in the IM of the chick correlates spatially and AP2a locus (AP2-Cre) results in recombination-mediated temporally with limb bud initiation from the lateral plate inactivation of Fgf4 and Fgf8 in both forelimb and hindlimb mesoderm. Based on expression of Fgf8 in the IM at the ectoderm. Finally, utilization of the AP2-Cre driver has appropriate developmental time, FGF8 became the favorite allowed us to address the question of differences in FGF candidate for such an inducer (Crossley et al., 1996; Vogel et requirement in formation of forelimb versus hindlimb. al., 1996). Specifically, Fgf8 has been proposed to be required for restriction of Fgf10 to the limb bud-forming region of the lateral plate mesoderm (Ohuchi et al., 1997), and Materials and methods FGF8 induction of Fgf10 is thought to be mediated by WNT2b signaling in the chick (Kawakami et al., 2001). Generation and genotyping of mutant mice Therefore, because Fgf10 is absolutely required for limb bud outgrowth (Min et al., 1998; Sekine et al., 1999), the model The Fgf4 alleles used to generate double mutant mice predicts that Fgf8 is essential for initiation or positioning of were the Fgf4 conditional allele (Fgf4AP) described the forelimb bud as the first step in the cascade. Although it is previously (Moon et al., 2000) and a bminimalQ Fgf4 often cited in the literature, not all experimental evidence conditional allele lacking both the human placental alkaline supports this model (Fernandez-Teran et al., 1997). phosphatase (PLAP) reporter and the NEO gene (Fgf4FRT). TheroleofFgf8 expressedintheAERinthe Both are referred to interchangeably as Fgf4c (Fgf4 condi- maintenance of limb bud outgrowth has been well estab- tional). The Fgf8 conditional alleles (Fgf8AP and Fgf8GFP, lished (Lewandoski et al., 2000; Moon and Capecchi, 2000). called Fgf8c) were described previously (Macatee et al., Interestingly, although limb development is abnormal in 2003; Moon and Capecchi, 2000). The RAR-Cre driver and mouse embryos lacking Fgf8 expression in the forelimb the AP2-Cre driver were previously described (Moon et al., AER, limb bud outgrowth nevertheless continues and the 2000; Macatee et al., 2003; Arenkiel et al., 2004). ulna and four digits are formed in a relatively normal The Lefty2-Cre transgene was constructed by placing the manner (Moon and Capecchi, 2000). Similar findings are 5.5 kb Lefty2 promoter fragment that was shown to obtained when Fgf8 expression is ablated from the hindlimb reproduce the expression pattern of the endogenous gene AER (Lewandoski et al., 2000). The sustained development (Saijoh et al., 1999) upstream of coding sequences for CRE of distal limb elements suggests that the loss of Fgf8 recombinase (Sauer and Henderson, 1988). expression is partially compensated by the activity of another FGF family member in the AER. Immunofluorescence on cryosections and whole embryos Mice lacking limb expression of Fgf4 have normal forelimbs and hindlimbs (Moon et al., 2000; Sun and Frozen 10 Am sections of mutant and control embryos Martin, 2000). Nevertheless, the ability of Fgf4 to provide were subjected to the TUNEL reaction using the In Situ AER function in in vitro assays and the observation that Cell Death Detection Kit (TMR red, Roche), or stained Fgf4 expression is altered in Fgf8 limb mutants suggest that with anti-pHH3 (anti-phospho-Histone H3, rabbit IgG, it may compensate for the lack of Fgf8. The fact that Upstate Biotechnology), anti-Sox10/9 (kindly provided by production of FGF4 by the AER is delayed relative to FGF8 M. Wegner), or anti-cleaved Caspase-3 (rabbit anti-cleaved by approximately 1 day readily explains the failure to rescue Caspase-3 (Asp175), Cell Signaling Technology) anti- development of the humerus or the femur. This prediction bodies. The anti-Sox10/9 monoclonal antibody was gen- has been tested for the hindlimb, demonstrating that erated against Sox10, but cross-reacts with Sox9. Primary outgrowth and skeletal development fail in the absence of antibodies were detected with goat anti-mouse or anti- both Fgf4 and Fgf8 (Sun et al., 2002). However, forelimb rabbit Alexa Fluor 488 or 594 antibodies (Molecular expression of Fgf8 and Fgf4 was not completely ablated in Probes). this system, and elements of all three segments of the For whole mount staining of embryos, an anti-GFP forelimb skeleton were still present. Thus, the characteristics primary antibody (rabbit anti-green fluorescent protein, of the CRE driver used in these experiments precluded a Molecular Probes) and a goat anti-rabbit secondary antibody comprehensive evaluation of the roles of FGF4 and FGF8 in (Alexa Fluor 488, Molecular Probes) were used.