Control of Vertebrate Limb Outgrowth by the Proximal Factor Meis2 and Distal Antagonism of BMPs by Gremlin

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1 Molecular Cell, Vol. 4, , November, 1999, Copyright 1999 by Cell Press Control of Vertebrate Limb Outgrowth by the Proximal Factor Meis2 and Distal Antagonism of BMPs by Gremlin Javier Capdevila,* Tohru Tsukui,* Concepción Rodríguez Esteban,* Vincenzo Zappavigna, and Juan Carlos Izpisúa Belmonte* * The Salk Institute for Biological Studies Gene Expression Laboratory North Torrey Pines Road La Jolla, California Laboratory of Gene Expression Department of Molecular Pathology and Medicine DIBIT-H San Raffaele Via Olgettina Milano Italy Summary The mechanisms controlling growth and patterning along the proximal distal axis of the vertebrate limb are yet to be understood. We show that restriction of expression of the homeobox gene Meis2 to proximal regions of the limb bud is essential for limb develop- ment, since ectopic Meis2 severely disrupts limb outgrowth. We also uncover an antagonistic relationship between the secreted factors Gremlin and BMPs re- quired to maintain the Shh/FGF loop that regulates distal outgrowth. These proximal and distal factors have coordinated activities: Meis2 can repress distal genes, and Bmps and Hoxd genes restrict Meis2 ex- pression to the proximal limb bud. Moreover, combina- tions of BMPs and AER factors are sufficient to distalize proximal limb cells. Our results unveil a novel set of proximal distal regulatory interactions that establish and maintain outgrowth of the vertebrate limb. Introduction (Saunders, 1948; Summerbell, 1974; Todt and Fallon, 1984). The AER maintains the cells in the PZ in a proliferative, undifferentiated state (reviewed by Martin, 1998), and cells that exit the PZ first adopt proximal cell fates, whereas the cells that spend more time in the PZ adopt more distal fates. Thus, the limb bud forms following a P/D progression, so that the more proximal structures of the limb (humerus in the forelimb or femur in the hindlimb) form first, followed by the middle structures, and finally the more distal structures (digits). Insights into the mechanisms of P/D development have also been provided by the characterization of secreted factors that mediate the activities of the zone of polarizing activity (ZPA) and the AER. The ZPA is the region of the posterior mesenchyme of the limb bud that organizes growth and patterning along the A/P axis (Saunders and Gasseling, 1968). In particular, the product of the Sonic hedgehog (Shh) gene has been shown to mediate this A/P organizing activity of the ZPA. As far as the AER is concerned, several fibroblast growth factors (FGFs) can substitute for its activity when the AER is surgically removed (reviewed by Johnson and Tabin, 1997; Martin, 1998; Schwabe et al., 1998). It is known that development along the A/P and P/D axes is coordinated, so that, for example, Shh and Fgf-4 expres- sion are mutually dependent (Laufer et al., 1994; Niswander et al., 1994). Thus, a Shh/FGF regulatory loop ensures coordination of growth and patterning along the A/P and P/D axes of the limb bud. In spite of these discoveries, a conceptual framework that explains how particular P/D coordinates are laid out in the limb bud is still lacking. The putative instructive role for FGFs in patterning the P/D axis has been ob- scured by their requirement in limb initiation and in the maintenance of the proliferative state of PZ cells. More- over, and although Shh is a key regulator of A/P growth and patterning in the limb bud, it does not appear to be directly involved in limb initiation nor in the initial establishment of A/P polarity (Grieshammer et al., 1996; Noramly et al., 1996; Ros et al., 1996). Mice lacking the Shh gene develop limbs that have the more proximal skeletal elements (humerus in the forelimbs and femur in the hindlimbs), whereas the rest of the skeletal ele- ments are absent (Chiang et al., 1996). This indicates Much is already known about the cellular and molecular mechanisms that control development of the vertebrate limb bud along its anterior posterior (A/P) and dorsal ventral (D/V) axes (reviewed by Johnson and Tabin, 1997; Martin, 1998; Schwabe et al., 1998), but the spe- that Shh function is only absolutely required for the decific mechanisms that control growth and patterning velopment of the distal elements of the limb, and, along along the proximal distal (P/D) axis are still largely un- with the considerations about the exact role of FGFs known. According to the progress zone model (Sum- outlined above, it raises the question of what factors merbell et al., 1973), cell fate along the P/D axis of the actually control the development of the proximal elelimb bud is specified by the time cells spend in the so- ments of the limb. called progress zone (PZ), the region of distal mesen- There is considerable evidence to support the idea chyme that underlies the apical ectodermal ridge (AER), that Drosophila appendages are divided into a proximal a specialized epidermal structure that runs along the and a distal compartment, defined by specific patterns A/P axis at the interface between dorsal and ventral ter- of gene expression (reviewed by Morata and Sánchezritories (Saunders, 1948). If the AER is surgically re- Herrero, 1999). For example, in the primordia of the adult moved, outgrowth is affected and distal truncations result leg, the leg imaginal disk, the homeobox gene extradenticle (exd) is exclusively required for the development To whom correspondence should be addressed ( belmonte@ of proximal structures, although it is transcribed in the salk.edu). whole imaginal disk (González-Crespo and Morata, 1995, These authors contributed equally to this work. 1996; Rauskolb et al., 1995). Conversely, patterning of the

2 Molecular Cell 840 Figure 1. Expression of Meis2, BMPs, Gremlin, and Spalt in the Chick Limb Bud Whole-mount in situ hybridizations (probes indicated to the left). Meis2 is detected in the lateral mesoderm of the trunk (data not shown) and in the whole limb bud mesenchyme at the initial stages of limb bud development (stage 17 [A]). Later, Meis2 becomes restricted to a proximal domain of the limb bud mesenchyme, shown at stages 20 (B), 21 (C), 23 (D), and 25 (E). BMP-2, -4, and -7 are all expressed in the AER ([F H], stage 22). BMP-2 is also expressed in a posterior mesenchymal domain (F), and BMP-4 and BMP-7 show distal region of the leg requires signaling by the secreted factors Wingless (Wg, present in ventral cells) and Decapentaplegic (Dpp, present in dorsal cells). Transcription of wg and dpp genes is controlled by Hedgehog (Hh), itself a secreted factor. The wg and dpp genes act antagonistically through mutual repression to establish a D/V subdivision in the imaginal disk (reviewed by Morata and Sánchez-Herrero, 1999). The Exd protein has to be localized to the nucleus in order to be active, and another homeoprotein, the product of the homothorax (hth) gene, which is restricted to the proximal region of the imaginal disk, controls Exd function by promoting its nuclear transport (Rieckhof et al., 1997; Pai et al., 1998; Abu-Shaar et al., 1999; Berthelsen et al., 1999). In the proximal domain of the leg, nuclear Exd protein prevents cells from responding to the distal signals Wg and Dpp, and in the distal domain, nuclear localization of Exd (and hence its activity) is prevented by Wg and Dpp through transcriptional repression of hth (Abu-Shaar and Mann, 1998; González-Crespo et al., 1998). This interplay between proximal (Exd/Hth) and distal (Wg/Dpp) factors effectively divides the developing Drosophila leg into two antagonistic domains along the P/D axis. Although orthologs of the exd and hth genes have already been described in vertebrates (Pbx and Meis, respectively), and some of them have expression patterns suggestive of their involvement in P/D determination in the limb bud (for example, see González-Crespo et al., 1998), their exact roles in this process remain largely unexplored. Orthologs of Drosophila wg (Wnts) and dpp (Bmps) also play important roles in vertebrate limb development, but their possible interactions with Meis and Pbx1 genes remain unexplored. Here, we describe a novel set of regulatory interactions that controls growth and patterning along the P/D axis of the vertebrate limb bud. We show that proximal restriction of the chick hth ortholog Meis2 is essential for limb outgrowth, since its ectopic expression results in severe alterations of distal development. In the distal limb bud, antagonism of BMP activity by the secreted factor Gremlin modulates AER activity and regulates distal outgrowth. Proximal and distal factors are interdependent, since ectopic Meis2 causes generalized repression of distal genetic programs and, in turn, Bmps and Hoxd genes of the Abdominal B subclass operate to restrict Meis2 expression to the proximal limb bud. Moreover, a combination of BMPs and AER signals distalizes proximal limb bud cells. Taken together, our results describe a complex network of regulatory interactions that generate, allocate, and maintain a system of interdependent proximal and distal genetic factors that controls outgrowth along the P/D axis of the developing vertebrate limb. the limb bud, being stronger distally ([J], stage 22; [K], stage 23; [L], stage 25), until expression is detected in the interdigital spaces ([M], stage 27). Spalt is always observed in the AER and the distal-most anterior and posterior mesenchymal domains of expression (G and part of the mesenchyme ([N], stage 17; [O], stage 21; [P], stage 23), H). Gremlin is detected in dorsal and ventral aspects of the mesen- until it starts fading distally at stages 24/25 (Q). (A E) and (I M) show chyme of stage 17 limb buds, in a pattern that is maintained during hindlimb buds, and (F H) and (N Q) show forelimb buds. All are the fist stages of limb budding ([I] shows a stage 19 limb), being dorsal views, and limbs are oriented so that anterior is up and characteristically excluded from the posterior margin. Later, it is posterior is down in these and all subsequent panels, unless otherexpressed in a wide domain that surrounds the posterior margin of wise indicated.

3 Control of Limb Outgrowth by Meis2 and Gremlin/BMPs 841 Results Vertebrate Limb Outgrowth Requires Proximal Restriction of the Homeobox Gene Meis2 An effort to clone chick orthologs of the Drosophila hth and the mouse Meis genes resulted in the isolation of a gene that shows 98% homology to the mouse Meis2 gene. The pattern of expression of this newly isolated chick Meis2 gene was studied by whole-mount in situ hybridization (Figures 1A 1E). At the initial stages of limb budding, Meis2 transcripts are expressed throughout the entire lateral plate mesoderm (data not shown), including the prospective limb bud cells (Figure 1A). Later on, around stages 18/19, Meis2 transcripts begin to be excluded from the most distal cells of the limb bud. At stages 20 23, Meis2 expression encompasses the proximal half of the developing bud and is absent from the distal part (Figures 1B 1D). As limb bud outgrowth proceeds, Meis2 transcripts become even more restricted to the most proximal regions of the limb (Figure 1E). Similar patterns of expression are observed in both the fore- and hindlimb. We decided to investigate a possible role for this gene in determining proximal cell fates by analyzing the effects of the ectopic expression of Meis2 in the limb bud. To this end, we obtained an adenoviral construct encoding the chick Meis2 gene (Ad-Meis2, see the Experimental Procedures) and infected presumptive forelimb and hindlimb regions of stage 10 embryos. The limb phenotypes obtained after Ad-Meis2 infection were quite variable, ranging from a slight widening of the limb bud, detectable at stages (Figures 2A and 2B) to marked deletions of distal limb tissue where the AER was divided into two or more domains (Figures 2C and 2D). Approximately 70% of the Ad-Meis2- injected embryos (n 576) displayed recognizable limb alterations. When the embryos were allowed to develop further (up to 12 days), alterations in cartilage pattern were found that correlated well with the different morphological perturbations seen in the early limb buds. In some embryos, the limbs were found to be slightly reduced in length, and, in some cases, the nails and the most distal phalanges were absent (Figure 2E). In other embryos, the limbs were found to be more severely truncated, mainly lacking digits (Figures 2F 2H). We never observed alterations of the more proximal skeletal elements of the limb (humerus or femur). The variability of the observed phenotypes was most likely due to variations in the extent of viral infection, since limb buds with a mild phenotype generally showed a rather patchy viral infection, while limb buds lacking distal limb tissue consistently showed a uniform extent of viral infection, as assayed by detection of adenoviral transcripts by in situ hybridization (data not shown). From these results, we conclude that restriction of Meis2 expression to the proximal domain of the limb bud is essential for normal limb outgrowth. Similar conclusions have been obtained by Mercader et al. (1999). Meis2 Represses Distal Gene Expression We next explored the molecular basis of the alterations of limb outgrowth caused by Meis2 misexpression in the limb. Embryos were harvested at different time Figure 2. Limb Outgrowth Requires Proximal Restriction of Meis2 Expression Phenotypes caused by infection of right-side stage 10 presumptive limb regions with an adenovirus expressing Meis2 (Ad-Meis2). (A) Contralateral (uninfected) control hindlimb bud photographed at stage 25, and (B) Ad-Meis2-infected hindlimb bud showing a mild phenotype consisting of slight widening of the limb (arrowheads, compare with [A]). (C) Contralateral (uninfected) control forelimb bud (stage 24), and (D) Ad-Meis2-infected forelimb bud showing a stronger phenotype of severe disruption of the AER (arrowhead). (E) Day 10 legs that developed from Ad-Meis2-infected hindlimb buds, showing distal truncations of toes (asterisks) and shortening of tibia and fibula (black arrow); compare with the normal length of the control (white arrow). (F) Alcian blue staining of a control day 10 wing that shows the normal cartilage pattern. (G and H) Same staining of day 10 wings that developed from Ad-Meis2-infected forelimb buds, showing truncations of cartilage elements, predomi- nantly distal (arrowheads). H, humerus; R, radius; U, ulna; II, III, and IV, digits (asterisks indicate pattern alterations in skeletal elements). points after injection with Ad-Meis2, and whole-mount in situ hybridization was performed to detect expression of several genes known to be involved in limb growth and patterning (Figure 3). We focused our attention on genes known to be involved in distal outgrowth, since Meis2 overexpression appears to selectively affect this process. Thus, we analyzed the expression of Fgf-4 and Fgf-8 (expressed in the AER), Shh (expressed in the posterior mesenchyme of the limb bud), Bmp-2 and Bmp-7 (both expressed in the AER and posterior mesenchyme, and Bmp-7 also in the anterior mesenchyme), Hoxd-13 (in posterior and distal mesenchyme), and the distal markers Hoxa-13 and Spalt (see below). We also included in our analysis the Tbx2 and Msx-1 genes, which have been shown to be targets of Bmps in the

4 Molecular Cell 842 Figure 4. Chick Meis2 Promotes Nuclear Translocation of Pbx1 The coexpression of chick Meis2 induces nuclear localization of mouse Pbx1 protein in Drosophila Schneider cells, which were transiently transfected with expression constructs for Pbx1 and Meis2 and processed for indirect immunofluorescence using anti-pbx1 polyclonal antibodies. Pbx1 protein (in red [A]) is localized to the cytoplasm of expressing Schneider cells, and coexpression of Meis2 with Pbx1 causes nuclear localization of Pbx1 (in red [B]). The arrowhead indicates nuclear accumulation of Pbx1 in one of the doubly transfected cells. limb. It is clear that the effect of Meis2 overexpression is a severe repression of the genes examined. For example, single or multiple gaps in the AER correlated with reduced or absent expression of the posterior AER marker Fgf-4 (Figures 3A and 3B, the latter being a more severe phenotype) and the AER marker Fgf-8 (Figures 3C and 3D, the latter being a stronger phenotype). The effect was observed even in cases where the morphological alterations of the infected limbs appeared to be subtle (for example, see Shh [Figure 3E], Tbx2 [Figure 3F], Bmp-7 [Figure 3H], Hoxd-13 [Figure 3J], and Msx-1 [Figure 3K]). Thus, ectopic Meis2 is able to cause a generalized repression of genetic programs in the distal limb bud, and we conclude that this is the most likely mechanism by which ectopic Meis2 impairs limb outgrowth. We further characterized the activity of chick Meis2 by studying whether the chick Meis2 protein can interact with Pbx1 proteins and promote their nuclear transloca- Figure 3. Ectopic Meis2 Causes Generalized Repression of Distal tion. To this end, we used Drosophila Schneider cells Limb Bud Gene Expression where, as previously reported (Berthelsen et al., 1999), Ad-Meis2 infection of stage 10 presumptive limb regions. The gene transiently expressed Pbx1 protein is located mainly in detected in each in situ hybridization is indicated at the bottom of the cytoplasm (see also Figure 4A). Coexpression with each panel. (A and B) Fgf-4 ([A], mild phenotype, with only a small proteins of the MEINOX family, such as Prep1, Hth, or gap in the Fgf-4 pattern, indicated by an arrowhead; [B], severe phenotype, with a big gap in the AER and almost complete disapof Pbx1 in these cells via a mechanism analogous to that Meis1, has been shown to induce nuclear localization pearance of Fgf-4). (C and D) Fgf-8 ([C], mild, with a gap in the AER; [D], severe, with multiple gaps). (E) Shh is almost completely absent, involved in the nuclear localization of Exd in proximal even in limbs with no detectable morphological alterations in the regions of the Drosophila leg imaginal disk (Rieckhof et AER. Note the altered shape (shorter and wider) of the infected al., 1997; Abu-Shaar et al., 1999; Berthelsen et al., 1999). limb bud when compared to the contralateral noninjected limb bud. As shown in Figure 4B, coexpression of chick Meis2 Similar downregulation is observed for Tbx2 (F), Bmp-2 (G), and causes localization of Pbx1 to the nuclei, and we con- Bmp-7 (H). (I) Hoxa-13 is severely repressed (the picture shows an clude that the chick Meis2 protein behaves similarly to extreme phenotype), as are Hoxd-13 (J), Msx-1 (K), and Spalt ([L], extreme phenotype). Infected limbs are to the right, and the arrowheads indicate sites of changes in gene expression in all panels. Meis1 in its interaction with Pbx1 proteins. The limb buds shown constitute a representative example of the Distal Outgrowth Requires Modulation various phenotypes obtained that range from mild (e.g., [A]) to ex- of BMP Activity treme (e.g., [I]). As indicated above, Meis2 is initially expressed throughout the nascent limb bud, but it begins to restrict proximally around the time when Shh appears in the posterior

5 Control of Limb Outgrowth by Meis2 and Gremlin/BMPs 843 Figure 5. Modulation of BMP Activity by the Secreted Antagonist Gremlin Regulates Distal Limb Outgrowth (A E) Infection of stage 10 presumptive limb regions with RCAS Gremlin results in a number of phenotypical alterations that include repression of PCD in the anterior margin of the limb ([A], revealed by Nile blue staining, arrowheads) and in the interdigital spaces ([B], note the presence of interdigital membranes in the infected leg, which are already absent from the contralateral noninjected leg, arrowheads). Also, in the day 10 specimen shown in (B), the toes are truncated. (C) RCAS-Gremlin-injected limb buds are initially wider (arrowheads), but, later on, distal outgrowth is inhibited and the limbs end up being shorter, displaying distal truncations. (D and E) Overexpression of Gremlin in the forelimb bud results in distal truncations. (D) Alcian blue staining of the contralateral control wing, and (E) same staining of the wing that developed from a RCAS-Gremlin-injected forelimb bud. Note the truncation of the digits (asterisks) and the shortening of radius (R*) and ulna (U*). (F and G) In RCAS-Gremlin-injected limbs, Fgf-4 (F) and Fgf-8 (G) expression is reinforced, and Fgf-4 expands into the anterior AER (asterisk in [F]). Expression of Shh and Hoxd-13 is also initially reinforced (H and I), but, later on, transcripts for both genes begin to fade in the injected limbs (K and L), along with other distal markers such as Msx-1 (M). Injected limbs display severe hyperplasia of the AER (arrowheads in [J], stage 28), and they do not develop distal structures (C) or, in the case of mildly affected limbs, they show distal truncations (E). (N Q) Gremlin expression is regulated by Shh and BMPs. (N) Mouse Gremlin fails to be maintained in Shh / limbs. (O) RCAS-Shh induces Gremlin in the chick limb (arrowhead points to anterior expansion of the domain). (P) RCAS Noggin downregulates Gremlin (arrowhead). (Q) A bead soaked in BMP-2 protein implanted in the anterior margin of the limb bud induces Gremlin expression along the whole P/D axis of the limb (arrowhead). Infected limbs are to the right when not indicated, and the arrowheads point to sites of changes in gene expression in all panels. margin of the mesenchyme and the AER is induced (stages 17/18). Thus, we reasoned that Meis2 might be repressed by some component(s) of the Shh/FGF loop. If this is indeed the case, the Meis2 repressor is also expected to play a role in controlling distal outgrowth in the limb bud. Recently, Bmps, which are expressed in the AER and anterior and posterior portions of the mesenchyme (Figures 1F 1H), have been proposed to act as negative regulators of the AER, based on two sets of results. First, application of beads soaked in BMP protein is able to antagonize AER structure and function (Gañán et al., 1998). Second, inhibition of BMP signaling by ectopic expression of the BMP antagonist Noggin inhibits regression of the AER and prolongs its inductive capacity, resulting in overgrowth of soft limb tissues (Pizette and Niswander, 1999). BMP inhibition in the limb reinforces the expression of Fgf-8 and Fgf-4 and expands Fgf-4 into the anterior AER. However, it is unlikely that Noggin is the primary factor that antagonizes the repression of the AER by BMPs, since its pattern of expression in the limb is not closely related to BMPs until the stages of cartilage formation (Capdevila and Johnson, 1998; Brunet et al., 1998; Merino et al., 1998). Thus, we decided to look for alternative BMP antagonists that might be involved in the maintenance of the AER and, therefore, that could mediate and implement the genetic interactions involved in the function of the Shh/FGF loop. The Secreted Factor Gremlin Regulates Limb Outgrowth by Antagonizing BMPs We cloned (see the Experimental Procedures) and analyzed the pattern of expression of the chick Gremlin gene, shown in Xenopus to encode an antagonist of BMPs (Hsu et al., 1998). We have found that its spatiotemporal pattern of expression during limb bud outgrowth is very suggestive of a possible involvement in

6 Molecular Cell 844 AER maintenance. From limb induction on, Gremlin tran- is a target of BMPs; Figure 5M), are severely downregulated scripts are detected in the superficial dorsal and ventral or lost. Nonetheless, the AER is still present and mesenchyme of the limb bud, always excluding the ante- proliferation under the AER is not reduced in limbs where rior and posterior margins and appearing stronger in the BMP signaling has been abolished, such as RCAS-Nog- most posterior and distal cells of the domain (Figures gin- orrcas-gremlin-injected limbs (for example, see 1I 1L). Around stage 27, Gremlin is detected in the proxibeing Pizette and Niswander, 1999). Injected limbs end up mal interdigital mesoderm (Figure 1M). In general, exures shorter and lacking the most distal structures (Fig- pression of Gremlin until stage 27 appears to be complementary 5E and 5J). We conclude that a certain amount of to that of Bmps (compare, for example, Figures BMP signaling, fine-tuned by Gremlin antagonism, is 1J and 1F 1H). We reasoned that the presence of Gremis absolutely required for distal outgrowth and that Gremlin lin in mesenchymal cells close to the AER could potensive able to maintain the AER by antagonizing the repres- tially antagonize the repressive effect of BMPs on the activity of BMPs on the AER. Independently, Zeller AER. In order to test this hypothesis, we overexpressed and collaborators (Zuniga et al., 1999) and Hurlé and the chick Gremlin gene in the limb bud with an RCAS collaborators (Merino et al., 1999) have obtained similar retrovirus and analyzed the resultant phenotypes. conclusions about Gremlin. We found that Gremlin overexpression represses prosion. Finally, we analyzed the control of Gremlin expresgrammed cell death (PCD) in the anterior necrotic zone Using a probe kindly provided by Richard Harland (Figure 5A) and in the interdigital mesenchyme of the (University of California, Berkeley), we determined that limb bud, resulting in interdigital webbing and soft tissue mouse Gremlin, although initially present in the limb overgrowths, along with truncations of distal cartilage buds of Shh-deficient mice, fails to be maintained and elements (Figure 5B). These phenotypes, related to all eventually disappears (Figure 5N; Chiang et al., 1996; the processes BMPs are known to regulate in the limb, Zuniga et al., 1999; the wild-type pattern is very similar were observed in approximately 70% of the injected to the chick counterpart). Also, ectopic Shh delivered to embryos (n 226). The phenotypes are remarkably simi- the chick limb bud with a RCAS-Shh retrovirus expands lar to the ones causes by viral overexpression of Noggin Gremlin expression, although it fails to induce the gene (Capdevila and Johnson, 1998; Pizette and Niswander, in the posterior margin (where we assume a repressor 1999), as indicated above, and are consistent with both of Gremlin may exist). The control of Gremlin by Shh Noggin and Gremlin proteins acting as extracellular an- appears to be mediated by BMPs (which are targets of tagonists of BMP activity. RCAS-Gremlin-infected limbs Shh), since inhibition of BMP signaling with a RCAS are wider initially (for example, see Figures 5A and 5C), Noggin virus results in disappearance of Gremlin (Figure but as development proceeds, distal outgrowth is inhibinduce 5P), and a bead soaked in BMP-2 protein is able to Gremlin expression (generally at some distance ited, the limbs end up being shorter, and distal skeletal elements are extremely reduced (compare Figure 5E from the bead) when implanted in the anterior margin with Figure 5D) or almost completely absent. Proximal of the limb bud (Figure 5Q). limb elements are typically unaffected. Thus, antagorole of Gremlin is to act as a controller of BMP activity From all these data, we conclude that the endogenous nism of BMP signaling by Gremlin specifically disrupts distal outgrowth. via extracellular antagonism. Since Gremlin itself is a Given this phenotype, how can Gremlin be postulated target of BMP-2, it follows that BMPs regulate their own as an AER maintenance factor? The analysis of gene activity by inducing the expression of an extracellular antagonist in responsive cells. Thus, the Gremlin/BMP expression in the infected limb buds revealed that regulatory loop is an integrated component of the Shh/ Fgf-4 is reinforced and expanded into the anterior AER FGF loop that is essential for proper distal limb out- (Figure 5F) in RCAS-Gremlin-infected limb buds, and growth. Fgf-8 is also reinforced (Figure 5G), which is similar to the previously reported effect of ectopic Noggin (Pizette Regulatory Interactions between Proximal and Niswander, 1999). Consistently, the AER in the and Distal Factors RCAS-Gremlin-infected limbs is taller and abnormally We decided to further explore possible regulatory interexpanded anteriorly and posteriorly, giving the distal actions between proximal and distal factors in the limb part of the limb an almost symmetrical shape (Figure bud by performing ectopic expression experiments (in- 5J). Thus, ectopic Gremlin indeed maintains the AER volving RCAS retroviruses, bead implants, or both). and Fgf-4 expression, most likely by antagonizing the First, in order to discover the mechanisms that restrict repressive effect of BMPs, but, at the same time, an Meis2 expression to the proximal limb bud, we ectopiexcess of Gremlin causes distal truncations, most likely cally expressed in the whole limb bud several secreted because BMP signaling is also required for distal factors known to be required for distal development, growth. This suggests that antagonism of BMP signaling using RCAS retroviruses, but we were unable to detect by Gremlin plays a major role in fine-tuning BMP activity any alteration in Meis2 transcription after overexpresin the vertebrate limb bud. sion of Shh, Wnt3a, orfgf-8 (data not shown). In con- There are two phases in the response to ectopic trast, we observed that BMP-2 beads were able to Gremlin. Initially, expression of Shh (Figure 5H), Hoxd- downregulate Meis2 (Figure 6A; n 10). It has been 13 (Figure 5I), and other genes involved in distal out- previously shown by others that BMP beads implanted growth is reinforced, probably due to the reinforcement in the proximal anterior mesenchyme cause shoulder of the AER and expanded Fgf expression. However, girdle defects (Hofmann et al., 1998), but the effects of as development proceeds, Shh (Figure 5K), Hoxd-13, a more severe exposition of proximal limb cells to BMP (Figure 5L) and other distal genes, such as Msx-1 (which beads have not been examined.

7 Control of Limb Outgrowth by Meis2 and Gremlin/BMPs 845 Regarding the last result, it has been previously shown that misexpression of several HOM-C genes or mouse Hoxd-10 (an Abdominal-B ortholog) in the eye antenna disk of Drosophila blocks the nuclear localization of Exd by suppressing hth transcription (Azpiazu and Morata, 1998; Casares and Mann, 1998; Yao et al., 1999). Thus, we explored a possible role of the distal genes of the Hoxd complex in suppressing expression of Meis2 in the chick limb bud. RCAS-Hoxd-11 (Morgan et al., 1992) and RCAS-Hoxd-13 (Morgan et al., 1992; Goff and Tabin, 1997) viruses (a kind gift of Cliff Tabin, Harvard Medical School, Boston) were injected in the presumptive forelimb and hindlimb regions at stage 10, and embryos were harvested at different time points to detect Meis2 transcripts. We observed that both ectopic Hoxd-11 and Hoxd-13 repress Meis2 transcription (Figures 6E and 6F; effects were observed in approximately 60% of the injected embryos, n 48 for each retrovirus). We conclude that the mechanism of transcriptional repression of Meis/hth genes by Hox genes is conserved among invertebrates and vertebrates. Taken together, our results suggest that Bmps and Hoxd genes are involved in the proximal restriction of Meis2 expression and that the concerted action of BMPs plus AER factors (Wnt-3a or FGFs) confers distal molec- Figure 6. Regulatory Interactions between Proximal and Distal Fac- ular identity to limb bud cells. tors in the Vertebrate Limb (A) A bead soaked in BMP-2 protein represses proximal expression Discussion of Meis2. (B) A BMP-2 bead, when applied to previously injected RCAS-Wnt3a limb buds, induces in proximal limb bud cells the distal marker Spalt. In this paper, we unveil three absolute requirements for (C) Similar induction is achieved by BMP-2 plus RCAS-Fgf-8. the normal establishment of the P/D axis in the ver- (D) BMP-2 plus RCAS-Fgf-8 induces Hoxd-13 proximally. tebrate limb. First, the homeobox gene Meis2 must nec- (E and F) Hoxd-11 and Hoxd-13 both repress Meis2 when ectopically essarily be restricted to the proximal limb bud, since expressed using an RCAS retrovirus. Arrowheads point to sites of ectopic Meis2 abolishes distal growth. Second, an anchanges in gene expression, and the injected limbs in (E) and (F) tagonistic relationship between the secreted factors are to the right. Gremlin and BMPs operates to maintain the Shh/FGF loop that integrates A/P and P/D development and regulates distal outgrowth. Third, Meis2 and the components We then asked whether the Meis2 repression caused by ectopic BMP was accompanied by an effective distalrefine each other s activities. of the Shh/FGF loop act antagonistically to restrict and ization of proximal cells in response to BMP. In order to monitor this at the molecular level, we isolated a novel distal marker, Spalt (see the Experimental Procedures). Involvement of Meis2 in Proximal Limb Development Expression of the chick Spalt gene (Figures 1N 1Q) is The phenotypic consequences of ectopic Meis2 expresfound in the most distal part of the limb mesenchyme sion in the limb bud are better understood in the context and the AER (Figures 1N 1P), until around stage 24/25, of the proposed biochemical functions of Meis proteins, when expression starts shifting proximally (Figure 1Q). which are intimately linked to the patterning activities By analogy with Drosophila, where the spalt gene is a of Hox genes. There is considerable evidence that Hox target of dpp, we expected chick Spalt to be a target proteins interact with several cofactors. For example, of BMPs. However, this particular Spalt gene is not a the Meis/Prep1/Hth homeodomain proteins form com- target of BMPs, since BMP beads fail to induce it (data plexes with another group of homeoproteins, Pbx1/Exd, not shown). This experiment also indicates that ectopic and these complexes (Meis/Pbx1, for instance) bind BMP (which is sufficient to repress Meis2) is not suffi- DNA with a subset of Hox proteins that contain tryptophan dimerization motifs, thus modulating Hox function cient to activate distal markers in proximal limb bud cells. We then implanted BMP-2 beads in the proximal (reviewed by Mann and Affolter, 1998). Meis/Prep1/Hth mesenchyme of limb buds previously injected with controls the activity of Pbx1/Exd proteins by promoting RCAS-Wnt3a (Kengaku et al., 1998) or RCAS-Fgf-8 vi- their nuclear transport, which is absolutely required for ruses. In these limb buds, ectopic Spalt was observed their function (Mann and Abu-Shaar, 1996; Rieckhof et in the vicinity of the bead (Figures 6B and 6C; n 11 al., 1997; Pai et al., 1998; Abu-Shaar et al., 1999; Berthelsen et al., 1999). In the mouse, Pbx1 is detected in for [B] and n 9 for [C]), indicating that either BMP-2 plus Wnt-3a or BMP-2 plus FGF-8 is able to distalize nuclei of proximal limb bud cells and in the cytoplasm proximal limb bud cells. Other genes involved in distal of distal limb bud cells (González-Crespo et al., 1998), outgrowth are also induced by this procedure, including consistent with the proximal pattern of expression of the Hoxd-13 (Figure 6D). highly related mouse Meis genes, mmeis1 and mmeis2

8 Molecular Cell 846 Figure 7. Model of P/D Determination during Vertebrate Limb Outgrowth (A) Meis2 is expressed in the flank region of the embryo prior to limb induction (stage 15) and initially in the mesenchyme of the whole nascent limb bud (stages 17/18). Concomitantly with the initiation of Shh expression in the posterior margin of the limb bud and the induction of the AER, Meis2 begins to be restricted proximally (stage 19). Shh expression is depicted in pink and the AER in yellow. (B) Proximal confinement of Meis2 is absolutely required for distal gene expression in the limb bud, since Meis2 is able to repress AER genes (such as Fgf-8, Bmps, and presumably Wnt3a, all indicated in yellow) and mesenchymal genes (such as Shh, Tbx2, Bmps, Hoxa-13, Hoxd-13, Msx-1, and Spalt) (only Bmps are indicated in the figure for clarity and to stress their roles in distal development). Gremlin (in red) is a target of Bmp-2 (and possibly of other Bmps) and encodes an extracellular antagonist of BMPs that acts by preventing AER repression by BMPs, as shown by the anterior expansion of Fgf-4 and the prolonged maintenance of the AER caused by ectopic Gremlin. Red lines indicate the types of antagonistic regulatory relationships between Meis2 and distal genes described in the main text. Green arrows depict the maintenance of Fgf-4 (in green) in the posterior AER by the antagonistic interaction of Gremlin with BMPs. These schemes are not intended to depict all the interactions involved in P/D determination, and the domains of gene expression are simplified for clarity. See main text for details. (Moskow et al., 1995; Nakamura et al., 1996; Cecconi et al., 1997; Oulad-Abdelghani et al., 1997). Also, Meis proteins have been shown to promote nuclear transport of Pbx1 in mouse fibroblasts (Mercader et al., 1999) and Drosophila Schneider cells (this paper). From all these data, it has been proposed that Pbx1 is active exclusively in the proximal limb and that Meis/Pbx1 modulate the activity of Hox genes in the proximal domain of the limb. Our results certainly reveal similarities between the mechanisms of limb outgrowth in vertebrates and invertebrates, but they also uncover important differences that are likely to result from the very different modes of development of, for instance, the vertebrate limb bud and the Drosophila imaginal disk. In Drosophila, ectopic expression of hth in the distal portion of the leg imaginal disk disrupts distal development by interfering with the activation of targets that depend on Wg and Dpp signals. However, the expression of the wg and dpp genes and the diffusion of their products are not affected (reviewed by Morata and Sánchez-Herrero, 1999). The situation is very different in the vertebrate limb bud, where the severe inhibition of distal outgrowth observed after deregulated expression of Meis2 in the limb bud is most likely due to a generalized repression of genes involved in the Shh/FGF regulatory loop, and not only of BMP and Wnt targets. There are several possible explanations for this strong repressive effect of Meis2 on the distal program of gene expression. Meis2 ectopic expression could, for example, interfere with the initiation of Shh expression, thus impairing (or severely attenuating) the Shh/FGF loop from the very beginning. Alternatively, ectopic Meis2 could directly affect some other component of the loop, including the Hoxd-11 and Hoxd-12 genes, which, besides their role in the development of the autopod, have also been proposed to be involved in the maintenance of Shh expression (reviewed by Mackem and Knezevic, 1999). Interestingly, Hoxd-11, -12, and -13 proteins (which do not interact with Pbx1) can interact directly with Meis proteins in vitro (Shen et al., 1997). Therefore, it is conceivable that Meis2/Hox or Meis2/ Pbx1/Hox repressive complexes may form and that, as speculated above, this might result in repression of distal gene programs. Gremlin/BMP Antagonism Controls Distal Outgrowth Together with previous reports, our results demonstrate that BMP deregulation is incompatible with limb development. We propose that the BMP antagonist Gremlin plays a key role in protecting the limb bud from an excess of BMP activity. Gremlin is transcribed in a wide domain surrounding the Shh and Bmp-2 domains in the posterior margin of the limb bud. The maintenance of Gremlin expression depends on Shh activity, but this regulatory interaction is most likely mediated by Bmp-2, itself a target of Shh. BMP-2 beads implanted in the anterior margin of the limb (where Gremlin is normally not expressed) induce Gremlin transcription, but at a certain distance from the bead. This, together with the observation that BMP-2 beads implanted in the normal domain of Gremlin repress its transcription (data not shown; Merino et al., 1999), indicates that Gremlin may be activated by intermediate levels of BMP but repressed by high levels of BMP. This is consistent with the mutually exclusive patterns of expression of Gremlin and Bmps (compare Figures 1F 1H with 1J). Thus, Bmp-2 (and probably other Bmps) controls the production of Gremlin protein that, in turn, antagonizes the repressive activity of BMPs on the AER (thus maintaining the AER) and the mesenchyme (presumably protecting cells from PCD). The Gremlin/BMP-2 regulatory loop is an example of a recurring theme in the development of many structures that require epithelial mesenchymal interactions: a diffusible factor that restricts its own range of activity through induction of an extracellular antagonist. However, the question remains of how the absence of BMPs impairs distal outgrowth. There are two phases

9 Control of Limb Outgrowth by Meis2 and Gremlin/BMPs 847 BMP proteins, in the context of the Shh/FGF loop, acts as a distal determinant. The mechanisms that first re- strict Meis2 to the proximal limb bud are still unknown, but they could conceivably be linked to the mechanisms that induce the limb buds (Figure 7A). As limb outgrowth proceeds, the establishment of the Shh/FGF loop stabi- lizes the antagonistic interaction between Meis2 and distal genes (Figure 7B). In evolutionary terms, the for- mation of appendages is a relative novelty that has in- volved the recruitment of a molecular module (or set of specific genetic interactions) that includes Shh, BMPs, Wnts, FGFs, and other secreted factors, in order to con- stitute a distal organizer that controls growth and pat- terning along the P/D axis. Remarkably, besides being antagonized transcriptionally by the proximal factor Meis2, each secreted factor involved in distal outgrowth of the vertebrate limb bud seems to restrict its own activity by inducing the expression of extracellular antagonists in their responsive cells. In here, we describe how the antagonism of BMP activity by Gremlin is con- trolled by BMPs themselves. Previous reports indicate that similar mechanisms seem to operate to restrict the range or activity of Shh (which induces its antagonist Hip; Chuang and McMahon, 1999) and FGFs (which in- duce the FGF antagonist Sprouty; Minowada et al., 1999). All these results allow us to propose that a remarkable degree of self-restraint is a fundamental property of the distal organizer that controls vertebrate limb bud outgrowth. In conclusion, our results describe an integrated set of genetic interactions between the proximal factor Meis2 and the antagonistic modulation of BMPs by the se- creted factor Gremlin, which is required for normal distal outgrowth. Proximal and distal factors antagonize each other, thus ensuring the division of the developing ap- pendage into proximal and distal territories of differential gene expression, which seems to be a common theme in the development of invertebrate and vertebrate ap- pendages. in the molecular response to ectopic Gremlin: first, expression of factors that are known to depend on AER input expands, concomitantly with the reinforcement of gene expression in the AER (including ectopic Fgf-4 anteriorly). As development proceeds, the AER is maintained but there is generalized repression of distal gene programs, which results in distal truncations. We find it unlikely that the excess of FGF observed in RCAS-Noggin-orRCAS-Gremlin-injected limbs causes distal truncations, since an excess of FGF (applied in beads) results in shortening of the whole limb, but it does not result in distal truncations (data not shown). Our preliminary results indicate that ectopic Shh fails to induce pattern duplications if the responding cells (for example, in the anterior margin of the limb bud) are also exposed to BMP antagonists such as Noggin or Gremlin. This seems to indicate that the activation of some Shh targets may require active BMP signaling in the limb bud. Several examples of BMPs acting as distal growth factors have also been described in other developing tissues and organs that require epithelial mesenchymal interactions, such as branchial arches, tooth buds, and lungs. It will be interesting to see if extracellular modes of regulation similar to the Gremlin/BMP regulatory loop described here also operate in other embryonic structures. Interactions between Proximal and Distal Factors in the Limb Bud We have uncovered several specific interactions between distal factors and Meis2. First, BMP alone is able to repress Meis2, but it is not sufficient to activate expression of distal genes (such as Spalt) in proximal regions of the limb bud. Second, distal genes can be induced proximally by ectopic application of at least two combinations of signals: BMP plus FGF-8 and BMP plus Wnt3a. This indicates that BMPs can repress Meis2, but signals from the AER appear to be needed in order to induce distalization of the proximal limb bud cells. Third, Hoxd-11 and Hoxd-13 are able to repress Meis2, which is reminiscent of the repression of hth by several Drosophila Hox genes (reviewed by Morata and Sánchez-Herrero, 1999). These results indicate that Meis2 repression can be elicited by Bmps and Hoxd genes independently (ectopic BMP only induces Hoxd expres- sion near the AER) and that distal limb fates are probably not determined by a single factor, but rather by specific combinations of distal factors. While in the Drosophila leg the combined action of Wg and Dpp is required to activate distal genes proximally, in the vertebrate limb bud, either Wnt3a plus BMP-2 or FGF-8 plus BMP-2 is able to activate distal genes proximally. It appears as if the role played by the dorsal (Dpp) ventral (Wg) interaction that directs distal growth in the single-layered epithelium of the leg imaginal disk, is performed in the vertebrate limb bud by an epithelial (AER signals) mesenchymal (BMPs) interaction. Still, in both cases, there is an interplay between two populations of cells that produce diffusible signals that act in combinatorial ways to direct distal outgrowth. These and previously published data allow us to pro- pose a model for the control of P/D outgrowth of the vertebrate limb (Figure 7) where Meis2/Pbx1 act as proximal factors and the antagonism between Gremlin and Experimental Procedures Cloning of Chick Meis2, Gremlin, and Spalt The chick Meis2 gene was isolated by screening a stage chick cdna library with a probe derived from the mouse Meis2 gene, using standard conditions (42% formamide, 4 SSC, 0.1% SDS, and 10 mg/ml denatured salmon sperm DNA at 42 C). In order to amplify by PCR the entire ORF of the chick Gremlin gene (GenBank accession number AF045799), the following oligos were used on cdna synthesized from limb bud mrna: 5 -CCGGAACCATGGTCC GCACACTGTATGCC-3 and 5 -GGCCGAATTCGCGTCCAAGTCGA TAGATATACACCG-3. A fragment of the chick Spalt gene was am- plified using the following degenerate oligos based on the comparison between Drosophila and human sequences: 5 -GARAAYCARAT GAARATGAT-3 and 5 -TTCCACATRTGNGTNCCCATRTG-3. The amplified 970 base pair fragment was cloned into the pbluescript vector for sense and antisense mrna probe synthesis. Viral Infection and Bead Implantation RCAS (subtype A) retroviral stocks containing full-length chick Ffg-8 and Gremlin were produced as described (Vogel et al., 1996). Adeno- virus-meis2 was produced as described (Miyake et al., 1996). Viruses were injected in the embryos by air pressure. The extent of viral infection was monitored in a few embryos from each experiment by detecting the viral mrnas by in situ hybridization. Beads were soaked in BMP-2 (provided by Genetic Institute, Cambridge, MA) as described (Hofmann et al., 1998). Experimental and control beads

10 Molecular Cell 848 were placed in small openings cut vertically or horizontally at various Chiang, C., Litingtung, Y., Lee, E., Young, K.E., Corden, J.L., Westphal, limb bud stages (stages 17 23; according to Hamburger and Hamilton, H., and Beachy, P.A. (1996). Cyclopia and defective axial patlimb 1951). terning in mice lacking sonic hedgehog gene function. Nature 383, In Situ Hybridization, Cartilage Staining, and Detection of PCD Chuang, P.T., and McMahon, A.P. (1999). Vertebrate Hedgehog sig- Embryos were processed for whole-mount in situ hybridization as naling modulated by induction of a Hedgehog-binding protein. Nadescribed (Ryan et al., 1998). Alcian blue cartilage staining was ture 397, performed according to Vogel et al. (1996). Nile blue staining was Gañán, Y., Macías, D., Basco, R.D., Merino, R., and Hurlé, J.M. performed as described (Zou et al., 1997). The entire ORFs of Meis2 (1998). Morphological diversity of the avian foot is related with the and Gremlin and the 970 bp PCR fragment of Spalt were used for pattern of msx gene expression in the developing autopod. Dev. riboprobe synthesis. The mouse Gremlin probe was kindly provided Biol. 196, by Richard Harland. The rest of the probes were described else- Goff, D.J., and Tabin, C.J. (1997). Analysis of Hoxd-13 and Hoxdwhere, and details may be provided upon request. 11 misexpression in chick limb buds reveals that Hox genes affect both bone condensation and growth. Development 124, Transfection and Immunostaining of Schneider Cells González-Crespo, S., and Morata, G. (1995). Control of Drosophila For expression in Drosophila Schneider cells, the cdnas for mouse adult pattern by extradenticle. Development 121, Pbx1 and chick Meis2 were cloned into the BamHI site of the pac5c González-Crespo, S., and Morata, G. (1996). Genetic evidence for actin promoter driven expression vector. Drosophila SL-2 Schnei- the subdivision of the arthropod limb into coxopodite and telopodite. der cells were cultured as described (Berthelsen et al., 1999) and Development 122, transfected using SuperFect (QIAGEN) according to the manufactur- González-Crespo, S., Abu-Shaar, M., Torres, M., Martínez-A, C., er s instructions. The polyclonal antibody against Pbx1 (Pbx1 C-20; Mann, R.S., Morata, G., Healy, C., Uwanogho, D., and Sharpe, P.T. Santa Cruz Biotechnology, Santa Cruz, CA) recognizes an epitope (1998). Antagonism between extradenticle function and Hedgehog located at the carboxyl terminus of Pbx1 and was used according signaling in the developing limb. Nature 394, to the manufacturer s instructions. Cells were treated for immunostaining as previously described (Berthelsen et al., 1999). Grieshammer, U., Minowada, G., Pisenti, J.M., Abbott, U.K., and Martin, G.R. (1996). 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