Generation of Polydactyly Through Interplay of EGFR Signaling and AP Patterning Genes
Current models for establishment of limb AP patterning involve complex interaction between Shh, Gli3, dHand, and 5′ members of the HoxD genes (reviewed in Panman and Zeller, 2003, see also Chen et al., 2004; Zakany et al., 2004). It is thought that early expression of 5′HoxD genes in posterior mesoderm activates both Shh and dHand (Zakany et al., 2004), which down-regulate Gli3 expression or prevent its conversion to the active repressor form (Litingtung et al., 2002, te Welscher et al., 2002a, b). This process enables subsequent posterior expression of the genes and signals required for elaboration and specification of the digits, as well as the establishment of the Shh/Gremlin/BMP/FGF signal relay that promotes distal progression (te Welscher et al., 2002b, Khokha et al., 2003). It has been suggested that polydactylous phenotypes result from loss of anterior Gli3 expression or Gli3 repressor function (Wang et al., 2000b; Litingtung et al., 2002; Chen et al., 2004), either through genetic perturbation of Gli3 (Litingtung et al., 2002; te Welscher et al., 2002a) or by means of ectopic anterior expression of Shh, dHand, or 5′HoxD genes (Charite et al., 2000; Fernandez-Teran et al., 2000; te Welscher et al., 2002b; Chen et al., 2004; Zakany et al., 2004). The presence of ectopic Shh and dHand in the anterior mesoderm of RCAS-caEGFR limbs suggests polydactyly in response to activated EGFR signaling results from Shh- and dHand-mediated repression of anterior Gli3 expression or function. Consistent with this finding, in limbs in which expression of both Shh and Gli3 were analyzed, down-regulation of Gli3 occurred only in the presence of ectopic Shh. The comparatively mild ectopic expression of Hoxd13/Hoxd11 in the anterior mesoderm of RCAS-caEGFR limbs suggests that this is a later event in generation of the polydactylous phenotype, occurring downstream of ectopic anterior Shh and dHand expression.
Studies place the role of dHand in AP patterning as acting in parallel with Shh (Zakany et al., 2004) or upstream of it (Charite et al., 2000; te Welscher et al., 2002b, Panman and Zeller, 2003). Ectopic anterior dHand achieves the same effects in AP patterning as ectopic Shh: mirror-image digit duplication; induction of anterior Ptc, Bmp2, Gremlin, Shh, and 5′HoxD expression; and suppression of anterior Gli3 transcription (Charite et al., 2000; Fernandez-Teran et al., 2000; Yelon et al., 2000; McFadden et al., 2002; te Welscher et al., 2002b). In our study, the frequency of ectopic Shh expression in RCAS-caEGFR limbs (9% or 5 of 59), is somewhat less than what would be expected based on the frequency of mirror-image–type duplications obtained (14% or 7 of 50). Of interest, the frequency of ectopic dHand expression in RCAS-caEGFR limbs is much higher than that of ectopic Shh, as dHand is present in the anterior mesoderm in nearly three fourths (72% or 8 of 11) of the limbs analyzed simultaneously for dHand and Shh expression, and in no case was ectopic Shh also induced. Although it is possible that Shh is present in these limbs but below our level of detection, ectopic expression of Ptc, an extremely sensitive indicator of Shh signaling (Marigo et al., 1996), is seen in RCAS-caEGFR limbs only in the presence of ectopic Shh. These observations raise the intriguing possibilities that activated EGFR signaling may be capable of inducing either dHand or Shh, and/or that dHand may be the primary target of activated EGFR signaling, with subsequent induction of ectopic Shh.
During normal development, ErB1 (EGFR) transcripts are expressed in the mesoderm of the lateral plate, where prepatterning of the limb AP axis occurs, and in the budding limbs during establishment of the ZPA (Dealy et al., 1998). Thus, endogenous EGFR is present in the right time and place to potentially play a role in AP patterning and/or regulation of Shh or dHand expression. The mechanism whereby EGFR signaling may regulate Shh or dHand expression is not clear, but it has been shown that dHand phosphorylation and transcriptional activity is regulated by means of the Akt pathway, one of the pathways activated by EGFR signaling (Murakami et al., 2004). A role for EGFR signaling in vertebrate limb AP pattering is also consistent with functions of this receptor in the Drosophila wing imaginal disc, where critical interactions between the EGFR and Hh signaling pathways regulate AP patterning (Crozatier et al., 2002), and perturbation of EGFR signaling causes mirror-image wing duplications and alters expression of patterning genes, including Cubitus interruptus (Baonza et al., 2000), the functional equivalent of vertebrate Gli3.
The ligand mediating EGFR activities in limb AP patterning is not obvious. EGF and TGF-α are both present in the limb forming mesoderm of the lateral plate (Dealy et al., 1998) and in distal and peripheral mesoderm of the limb bud, including the posterior limb mesoderm, but are not specifically localized to the ZPA (Dealy et al., 1998). EGF or TGF-α could play a role in AP patterning, even acting at a distance or in low amounts, as amplification of the EGFR signal occurs after ligand binding by means of various mechanisms, including receptor clustering on the cell surface (Ichinose et al., 2004) and formation of autocrine loops with positive feedback (Shvartsman et al., 2002). The availability of ligand for EGFR activation is determined by local protease activity, which releases the ligands from the cell surface (Sahin et al., 2004), and subsequently by the ability of the ligand to reach the receptor by means of diffusion. Notably, direct measurement of the diffusion of EGF, a relatively small peptide, indicates that it diffuses much more readily through cells than larger peptides, and can reach a given concentration hundreds of microns further (Thorne et al., 2004). This finding suggests that an EGF source could be relatively far from target cells and still be able to elicit a biological response (Thorne et al., 2004). Alternately, other ligands for the EGFR also exist, including HB-EGF, Amphiregulin, Betacellulin, and Epiregulin (Normanno et al., 2001), but their expression patterns in chick limbs have not been reported.
EGFR Signaling in AER Formation and Position
The presence of ectopic AERs in response to activated EGFR signaling suggests a role for EGFR signaling in AER formation. The AER is induced from presumptive AER-forming ectoderm (Altabef et al., 1997; Michaud et al., 1997) in response to signals from the prospective limb forming mesoderm (Kieny, 1960; Douailly and Kieny, 1972; Saunders and Reuss, 1974). Mesodermal signals in induction of the AER include FGF10 (Ohuchi et al; 1997) and, more recently, BMPs (Ahn et al., 2001; Pizette et al., 2001; Soshnikova et al., 2003), which are required for induction of β-catenin/Wnt3a in the overlying ectoderm, which subsequently induce the AER and activate expression of AER genes, including FGF8 (Kawakami et al., 2001; Barrow et al., 2003; Soshnikova et al., 2003). Our study does not allow us to distinguish between mesodermal or ectodermal functions of EGFR signaling in AER formation. EGFR signaling could regulate expression or activity of a mesodermal or ectodermal factor involved in AER formation, similar to its ability to regulate Bmp2 or FGF8 (as shown in Figs. 2, 3). An endogenous role for EGFR signaling in AER induction is supported by our previous observations that the EGF receptor and its ligands, particularly TGF-α, are colocalized in prospective limb-forming mesoderm and AER-forming ectoderm (Dealy et al., 1998) and that exogenous TGF-α induces the formation of a thickened AER-like ectoderm in mutant limbless and wingless limbs (Dealy et al., 1998), which lack an AER.
Mispositioned or excessively widened AERs form in limb ectoderm in response to manipulation of ectodermal Wnt3a/B-catenin (Kengaku et al., 1998; McQueeney et al., 2002; Barrow et al., 2003; Soshnikova et al., 2003), FGF10/FGF8 (Ohuchi et al., 1997), radical fringe (Laufer et al., 1997; Rodriguez-Esteban et al., 1997), En1 (Loomis et al., 1998; Pizette et al., 2001), and the BMP antagonist Noggin (Ahn et al., 2001; Pizette et al., 2001; Khokha et al., 2003; Wang et al., 2004), suggesting roles for these pathways in positioning of the AER and/or in defining its boundaries. The boundaries of the AER are defined by lineage restrictions between the AER and nonridge ectoderm (Kimmel et al., 2000). Activation of EGFR signaling in the ectoderm in the present study could disrupt these lineage boundaries, leading to ectopic AER formation. A role for EGFR signaling in defining lineage boundaries in limb ectoderm would be consistent with the role of EGFR signaling in the Drosophila wing imaginal disc to direct the cell fate decisions that specify the wing-forming from non–wing-forming cells (Baonza et al., 2000; Zecca and Struhl, 2002a) and that establish the DV cell lineage-restricted compartment boundary (Zecca and Struhl, 2002b) along which forms the wing margin, an outgrowth-promoting signaling center analogous to the vertebrate AER (Dahman and Basler, 1999).
Maintenance of the ectodermal lineage boundaries that define the AER is suggested to require BMPR signaling by the AER itself (Wang et al., 2004), as antagonism of BMPR signals by means of ectodermal Noggin expression expands the AER boundaries and induces ectopic AERs (Pizette et al., 2001; Wang et al., 2004). EGFR signals are antagonistic to BMPR signals (Kretzschmar et al., 1997). Opposing activities of the EGFR and BMPR are involved in patterning of the Drosophila head and wing (Croatzier et al., 2002; Chang et al., 2003), and in vertebrate heart and skeletal development (Nonaka et al., 1999; Jackson et al., 2003). Thus, as in the case of Noggin (Wang et al., 2004), EGFR-mediated antagonism of BMPR signals within the AER could disrupt the AER/nonridge ectoderm boundary, resulting in ectopic AER formation. That ectopic expression of the activated EGF receptor is indeed present within the duplicated AERs of an RCAS-caEGFR limb (as determined by viral gag distribution) supports this idea (see Fig. 2H).
We have shown previously that, during normal development, ErbB1 (EGFR) is expressed by nonridge ectoderm but not by the AER and have suggested that exclusion of EGFR expression from the AER may be required for some aspect of AER formation and/or function (Dealy et al., 1998). Based on the observations above, exclusion of EGFR signaling from the AER may be necessary to permit BMPR-mediated repression of the conversion of nonridge ectoderm to AER. In turn, the repressive effects of AER-BMPs might be counterbalanced by EGFR signaling in nonridge ectoderm, which may promote conversion of nonridge ectoderm into AER, perhaps by means of interaction with other ectodermal factors such as Wnt3a/β-catenin, radical fringe, or FGFs.
EGFR Signaling in PD Outgrowth
Limb outgrowth and patterning is coordinated by reciprocal signals between the distal mesoderm, AER, and ZPA (Zwilling, 1961; Saunders, 1977; Laufer et al., 1994; Niswander et al., 1994; Boulet et al., 2004). BMP signals in distal mesoderm suppress the expression of FGFs by the overlying AER, and antagonism of this activity is required for maintenance of the AER and subsequent maintenance of Shh expression by the ZPA (Zuniga et al., 1999). Gremlin has been identified as the BMP antagonist expressed by posterior distal mesoderm that maintains this FGF/Shh signaling loop (Khokha et al., 2003). The ectopic anterior and distal expression of Bmp2 and Gremlin in RCAS-caEGFR limbs likely reflects their induction by ectopic Shh (Merino et al., 1999, Zuniga et al., 1999; Drossopoulu et al., 2000) and/or ectopic dHand (te Welscher et al., 2002b) and is consistent with the existence of an expanded and/or additional outgrowth and patterning center in the anterior distal mesoderm of RCAS-caEGFR limbs. Induction of ectopic Gremlin is also consistent with subsequent expansion of overlying FGF8 and FGF4 expression domains in response to activated EGFR signaling (Zuniga et al., 1999; Khokha et al., 2003). Expansion of FGF8 and FGF4 could also result from direct antagonism of BMPR signals by means of activated EGFR signaling in distal mesoderm.
We have suggested previously that endogenous EGFR signaling may be involved in reciprocal interactions required for PD limb outgrowth, as EGF and TGF-α are present in the AER and distal limb mesoderm, can suppress the differentiation and promote the proliferation of subridge mesoderm, and can maintain the thickness and activity of the normal AER in vitro (Dealy et al., 1998). Recent studies in Drosophila reveal that EGFR signaling is also required for PD outgrowth of the fly leg (Campbell, 2002; Galindo et al., 2002) and suggest that the tip of the leg provides a source of an EGFR ligand that promotes PD outgrowth in a manner similar to the vertebrate AER (Campbell, 2002).
The extension of the domains of FGF4 and FGF8 expression in the AER of RCAS-caEGFR limbs may explain some of the more mild cases of polydactyly, and/or the postaxial polydactyly, produced in response to activated EGFR signaling. An ectopic FGF signal produced by an extended AER has been suggested to cause non–mirror-image pre- and postaxial polydactyly due to recruitment of normally non–limb-forming cells from flank mesoderm located anterior and posterior of the limb bud (Tanaka et al., 2000). Postaxial polydactyly is also caused by expansion of the AER and of the FGF domains within it as a result of inhibition of BMPR signaling by Noggin (Guha et al., 2002; Wang et al., 2004). Finally, because one major function of the AER is to promote the survival and proliferation of underlying mesoderm (Kosher et al., 1979; Rowe et al., 1982), the extended AERs induced by activated EGFR signaling may promote local accumulation of limb bud mesoderm with intrinsic digit-forming potential (Omi et al., 2002).
EGFR Signaling in Regulation of DV Pattern
The formation of ectopic dorsal structures (ectopic dorsal scales and duplicated nail beds) and the loss of ventral ones (missing ventral scales and ventral tendon) in limbs in which activated EGFR is expressed indicate that EGFR signaling dorsalizes the limb. Dorsalization of RCAS-caEGFR limbs is confirmed by the early presence of ectopic Lmx and Wnt7a expression in ventral mesoderm and ectoderm, respectively, and by the loss of expression of En1. BMPR signaling is upstream of Lmx, Wnt7a, and En1 signals in limb DV patterning, as loss of ectodermal BMPR signaling achieved by conditional knockout of BMPRIA or misexpression of the BMP antagonist Noggin results in limb dorsalization (Ahn et al., 2001; Pizette et al., 2001; Barrow et al., 2003; Soshnikova et al., 2003; Wang et al., 2004). Accordingly, in the current model for limb DV patterning, ectodermal BMPs positively regulate expression of En1 by the ventral ectoderm, whereas En1, in turn, represses Wnt7a expression and limits it to the dorsal ectoderm, where it subsequently induces expression of Lmx by underlying dorsal mesoderm (see Fig. 8 of Pizette et al., 2001). In the present study, expression of activated EGFR in ventral ectoderm could dorsalize the limb by means of antagonism of BMPR signals, similar to the dorsalized limbs obtained after misexpression of the BMP antagonist Noggin.
We have shown previously that, in the normal limb, ErbB1 (EGFR) is expressed by both dorsal and ventral nonridge ectoderm but EGF is only present in ventral ectoderm (Dealy et al., 1998). The presence of an endogenous EGFR signal in ventral ectoderm could serve to counterbalance the ventralizing signals provided by BMPs, similar to our suggestion that nonridge EGFR signals may counterbalance AER-BMPs (see above). Remarkably, EGFR signaling directs dorsal cell fate decisions in the Drosophila wing imaginal disc, where it regulates expression of the Lmx homolog apterous by providing a permissive signal, active only in the presence of the fly EGFR ligand Vein (Zecca and Struhl, 2002a, b). A role for EGFR signals in limb dorsalization is also supported by our observation that the EGFR ligand EGF is ectopically localized throughout the bidorsal ectoderm of mutant limbless limb buds (Dealy et al., 1998).
EGFR Signaling Negatively Regulates Interdigital Regression
In the normal autopod during the time at which interdigital regression is occurring, ErbB1 (EGFR) and EGF are colocalized by the mesoderm adjacent to the digit rays, which does not participate in the cell death occurring in the adjacent central interdigital mesoderm (Ganan et al., 1996). This finding suggests that ligand-activated EGFR may function endogenously as a survival signal to prevent apoptosis of tissue not scheduled to regress. Endogenous EGFR signaling in the tissue adjacent to the digit ray may also suppress the pro-apoptotic activities of BMPs present in the adjacent interdigital mesoderm, thus confining regression to the central interdigital mesoderm. That activation of EGFR signaling prevents cell death and regression in the central interdigital mesoderm while suppressing Bmp4 expression is consistent with this idea. The loss of cell death and Bmp4 expression in the posterior necrotic zone of the RCAS-caEGFR limbs also suggests that its gross broadening may result from perturbation of the cell death and proliferative processes that normally refine the limb contours (Chen and Zhao, 1998). Inhibition of cell death and stimulation of proliferation as a consequence of activated EGFR signaling is consistent with the known involvement of ErbB survival pathways in development, tissue remodeling, and tumorigenesis (Danielson and Maihle, 2002), as well as in the regulation of cell growth and cell cycle progression (Grant et al., 2002).
Complexity of EGFR Signaling
EGFR signaling activates multiple distinct intracellular signaling cascades (Yarden and Sliwkowski, 2001), depending on which ligand is involved (Sweeney et al., 2001) and whether signaling is occurring from the homodimeric EGFR or from an EGFR-ErbB heterodimer (Olayioye et al., 1998). EGFR signaling is further diversified through interaction with other signaling networks such as those mediated by Wnts (Civenni et al., 2003), BMPs (Nonaka et al., 1999; Jackson et al., 2003), insulin-like growth factors (Hallak et al., 2002; Adams et al., 2004), and FGFs (Wu et al., 2003), which have themselves been implicated in a wide array of roles in vertebrate limb development (reviewed in Capedevila and Izpisua Belmonte, 2001; see also Dealy and Kosher, 1995, 1996; Allan et al., 2000; Dahn and Fallon, 2000; Ahn et al., 2001; Pizette et al., 2001; Church and Francis-West, 2002; Barrow et al., 2003). The diverse limb phenotypes obtained in response to activated EGFR expression in the present study likely reflect the nature of the EGFR signaling network as a paradigm for signal diversification (reviewed in Hackel et al., 1999; Gschwind et al., 2001; Prenzel et al., 2001). Specificity of the cellular responses resulting from activation of various signaling cascades is determined by the spectrum of intracellular mediators expressed within the responding cells (reviewed in Chang and Karin, 2001; Dumont et al., 2002; Tanoue and Nishida, 2003). Recently, it has been shown that overexpression of an FGF-regulated intracellular signaling intermediate that relays signals between the Ras/MAPK and PI(3)K/Akt pathways inhibits limb outgrowth, induces apoptosis, and causes limb truncations (Eblaghie et al., 2003; Kawakami et al., 2003). The markedly different phenotypes observed in the present study support the existence of distinct EGFR and FGFR signaling mechanisms in the limb, and suggest that the limb patterning defects caused by activation of EGFR signaling are unlikely to be mediated by means of alteration of FGFR signaling.
The results of this study suggest novel and previously unappreciated roles for EGFR signaling in vertebrate limb morphogenesis. Questions still to be addressed include identification of the EGFR ligands involved in various limb patterning processes, the potential roles of other ErbB family members in limb development, and relationship of EGFR signaling pathways to other signaling networks, particularly the BMPR network. Further studies in this area are clearly warranted.