Pattern formation and regulation of gene expressions in chick recombinant limbs

Mechanisms of Development 90 (2000) 167±179 www.elsevier.com/locate/modo

Pattern formation and regulation of gene expressions in chick recombinant limbs M. Elisa Piedra 1, F. Borja Rivero 1, Marian Fernandez-Teran, Maria A. Ros* Departamento de AnatomõÂa y BiologõÂa Celular, Facultad de Medicina, Universidad de Cantabria, 39011 Santander, Spain Received 8 June 1999; received in revised form 10 September 1999; accepted 15 September 1999

Abstract Recombinant limbs were performed by ensembling dissociated-reaggregated wing bud mesoderm inside an ectodermal hull. The zone of polarizing activity was excluded from the mesoderm used to perform the recombinant limbs (non-polarized recombinants), and grafted when desired (polarized recombinants). Reorganization of patterning progressively occurred in the newly formed progress zone under the in¯uence of the apical ectodermal ridge (AER), explaining the proximo±distal gradient of morphogenesis observed in developed recombinant limbs. The AER, without the in¯uence of the polarizing region (ZPA), was suf®cient to direct outgrowth and appropriate proximo±distal patterning, as observed in the expression of the Hoxa-11 and Hoxa-13 genes. The development of the recombinant limbs coursed with symmetric AER and downregulation of Bmp expression in the mesoderm supporting a negative effect of Bmp signaling upon the apical ridge. The recombinant ectoderm maintained previously established compartments of gene expressions and organized a correct dorso-ventral patterning in the recombinant progress zone. Finally, the ZPA effect was only detected on Bmp expression and pattern formation along the antero-posterior axis. q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Limb development; Recombinant limbs; Hoxa; Fgf; Bmp; Lmx1; Apical ectodermal ridge; polarizing region; Dorso-ventral pattern

1. Introduction The development of the vertebrate limb is among the best known developmental biology models. Although the initial emergence of the limb bud continues to be poorly understood, the molecular bases underlying patterning and growth once the limb bud emerges are rapidly becoming known (reviewed in Johnson and Tabin, 1997; and Schwabe et al., 1998). The early limb bud emerges as a bulge composed of a central core of mesodermal mesenchyme covered by the ectoderm. It is the continuous interaction between these two components, the ectoderm and the mesoderm, that drives further development. Three axes can be distinguished in relation to the three-dimensional elongation of the limb bud: the proximo±distal (shoulder to ®nger tip); the anterior±posterior (thumb to little ®nger); and the dorsal±ventral (knuckle to palm) axes. Patterning in each of these three axes is controlled by a specialized signaling center. The * Corresponding author. Tel.: 134-942-201-933; fax: 134-942-201903. E-mail address: [email protected] (M.A. Ros) 1 These two authors equally contributed to this work.

apical ectodermal ridge (AER), a specialized epithelium rimming the distal tip of the limb, constitutes the signaling center for the proximo±distal (P/D) axis (Saunders, 1948). AER action is mediated by the secretion of several ®broblast growth factors (Fgfs; Niswander et al., 1993; Fallon et al., 1994; Mahmood et al., 1995; Crossley et al., 1996; Vogel et al., 1996). The zone of polarizing activity (ZPA), a group of mesodermal cells in the posterior margin of the bud, controls patterning in the anterior±posterior (A/P) axis through its production of Sonic hedgehog (Shh; Riddle et al., 1993; Chang et al., 1994; LoÂpez-MartõÂnez et al., 1995). And ®nally, the non-AER ectoderm of the bud regulates patterning in the dorso-ventral (D/V) axis. The dorsal ectoderm secretes Wnt-7a, which controls dorsalization of the limb bud through the induction of its downstream gene Lmx1 in the dorsal mesoderm (Parr and McMahon, 1995; Riddle et al., 1995; Vogel et al., 1995). Engrailed-1 (En-1) expression in the ventral AER and ventral ectoderm is essential for proper ventral patterning through the restriction of Wnt-7a expression (Logan et al., 1997; Loomis et al., 1996, 1998). It remains to be seen whether the ventral pattern is a default pathway or whether there is a speci®c molecular pathway controlling ventralization (Johnson and Tabin, 1997). It should be noted that these three signaling

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centers are not independent but their coordinated action is crucial for normal limb development to occur (reviewed in Johnson and Tabin, 1997). An exhaustive experimental analysis performed over the past 50 years created an inestimable background of knowledge of limb development in different species (Johnson and Hinchliffe, 1980). This background has certainly contributed to the rapid advance in identifying the molecular basis of limb development. In particular, the experimental system of recombinant limbs permitted the identi®cation of the speci®c roles played by the ectoderm and mesoderm in patterning by the limb bud (Zwilling 1956, 1964; MacCabe, 1973b; MacCabe et al., 1974) and more recently it has also proved useful at the molecular level for studies of gene regulation (Ros et al., 1994; Hardy et al., 1995; Wada et al., 1998). A recombinant limb can be brie¯y described as a limb-like structure created by assembling limb bud mesoderm inside a limb bud ectodermal jacket. This experimental situation was originally devised to analyze the interactions between the ectodermal and mesodermal components of the limb bud (Zwilling, 1956, 1964), but since it permits multiple variations in the type and conditions of the components, it can be applied to a variety of analyses (Fernandez-Teran et al., 1999). When grafted to an appropriate site of a host embryo (e.g. somites or dorsal limb bud), the recombinant limbs develop into limbs or limb-like structures whose morphogenesis depends on the characteristics of the recombination performed. In this report we have used recombinant limbs performed with dissociated-reaggregated anterior wing mesoderm to gain insights into the tissue and molecular interactions that are responsible for patterning of the limb. For this purpose, we have performed a detailed and systematic analysis of the expression of different genes implicated in limb patterning in recombinant limbs in the absence or presence of the ZPA. We have found that the AER, in the absence of the ZPA, is suf®cient to organize appropriate outgrowth in the P/D axis, as indicated by the expression of the most 5 0 Hoxa genes. Interestingly, the development of the recombinant limbs was accompanied by loss of AER asymmetry and downregulation of Bmp expression in the mesoderm, whereas normal expression was maintained in the AER. We also show that the ectoderm in the recombinant maintains the previously established compartments of gene expression, regardless of the disorganization of the mesoderm, and that a D/V pattern according to the polarity of the ectoderm is generated in the newly formed progress zone. Finally, the presence of the ZPA induces Bmp expression in the recombinant mesoderm but does not in¯uence Hoxa expression or patterning in the D/V axis.

third of the limb mesoderm, which mainly corresponds to the ZPA, was excluded because it has been shown that polarizing cells mixed with the recombinant mesoderm had a negative effect in its further development (Crosby and Fallon, 1975; Frederick and Fallon, 1982). Therefore, the mesoderm in the recombinant limbs was randomly aggregated and lacked the ZPA, thus permitting an independent analysis of the action of the AER in organizing limb patterning. When desired, the action of the ZPA was also analyzed by introducing a small fragment of ZPA (about 150 mm) into the posterior margin of the recombinant before grafting. The piece of ZPA was placed to coincide with the posterior border of the ectodermal hull (see Section 4) and its position de®ned the posterior border of the recombinant. It should be noted that, after grafting, the axes of the recombinant may not coincide with those of the host (see for example Fig. 3F).

2. Results

Fig. 1. Development of recombinant limbs. (A) pattern of cartilage of a non-polarized recombinant limb exhibiting two symmetrical digits. (B) shows the cartilage pattern of another non-polarized recombinant limbs exhibiting four symmetrical digits. (C) Pattern of cartilage of a polarized recombinant limb showing clearly identi®able digits II, III and IV. (D) Skeletal pattern in a control wing for comparison.

For this work, the recombinant limbs were performed with dissociated-reaggregated mesoderm from the anterior two-thirds of stage 19±21 HH wing buds. The posterior

2.1. Morphogenesis of the recombinant limbs When allowed to develop, the recombinant limbs formed structures clearly identi®able as limbs. The skeletal pattern of several types of recombinant limbs has been reported previously (Zwilling, 1964; MacCabe et al., 1973a; Ros et al., 1994; Hardy et al., 1995; Wada et al., 1998). Our recombinant limbs formed limb-like structures in which the distal segment, the autopod, exhibited a variable number of wellformed digits (Fig. 1A±C). Proximally a cartilage element, corresponding to the humerus, and a short sometimes broad element at the zeugopod level, were generally observed. In many specimens, the joint corresponding to the elbow was missing (Fig. 1A±C). In every case analyzed the autopod showed a good morphology, whereas the proximal elements were less well formed (Hardy et al., 1995). Some polarized recombinant limbs exhibited a better

M.E. Piedra et al. / Mechanisms of Development 90 (2000) 167±179

proximal morphogenesis than non-polarized recombinant in that the length and shape of proximal skeletal elements were closer to normal (compare Fig. 1A,B with C); however, this was not a consistent feature and at present it remains unclear whether the ZPA improved P/D development in the recombinants. Along the A/P axis the patterning of the skeletal elements depended on the presence of the ZPA. Polarity in this axis was mainly observed in the distal elements, the digits. Nonpolarized recombinant limbs gave outgrowths with A/P symmetrical digits (Fig. 1A,B) (MacCabe et al., 1973a; Ros et al., 1994; Hardy et al., 1995; Wada et al., 1998). In the best cases, four symmetrical digits developed (Fig. 1B). It is possible that the ®nal number of digits depended on the initial size of the recombinant, bigger aggregates giving rise to outgrowths with more digit number. Grafts containing a ZPA implant developed autopods in which wing digits II, III and IV were clearly identi®able (Fig. 1e) and close to normal (Fig. 1D). D/V polarity in the recombinant limbs, indicated by the feather pattern, was dif®cult to distinguish at proximal levels. Some recombinants exhibited a bidorsal pattern of feathers proximally while a distinct D/V polarity was progressively identi®ed towards the distal tip (Fig. 1C). The pattern of muscles and tendons in normal limbs differs from dorsal to ventral, which makes it useful to de®ne the D/ V pattern (Parr and McMahon, 1995; Riddle et al., 1995; Vogel et al., 1995). However, muscle development in the recombinant limbs is rudimentary and poorly organized (Fernandez-Teran et al., 1999) and does not allow the analysis of D/V polarity, which that can only be assessed by the feather pattern. It is notable that there was no difference in the D/V patterning of non-polarized and polarized recombinant limbs. We performed a sequential histological analysis during recombinant limb development to search for an explanation for the observed proximo±distal gradient of morphology. Routine histological analysis permitted the identi®cation, soon after grafting, of several cell aggregates in the core of the recombinant in a way that reminded the initial steps on nodule formation in micromass cultures (not shown; Solursh, 1986). Initially the mesoderm in the recombinant was a uniform mixture of cells of variable proximo±distal origin but the AER respeci®ed the proximal cells that came to lie under its region of in¯uence, creating a new progress zone (Saunders and Gasseling, 1959; Krabbenhoft and Fallon, 1989; Davidson et al., 1991; Ros et al., 1994). Therefore, the cells removed from the AER developed their chondrogenic potential, forming condensations comparable to micromass cultures (Schramm and Solursh, 1990). The initial aggregates observed in the core of the recombinant will probably contribute to the proximal cartilage elements that show poor morphogenesis, while more distal elements will develop from the newly formed progress zone, under the in¯uence of the AER and the ZPA, if present, and consequently will exhibit a much better morphogenesis.

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2.2. Hoxa-11 and Hoxa-13 patterns of expression were properly established in the recombinant limbs The pattern of expression of the most 5 0 Hoxa genes, Hoxa-11 and Hoxa-13, during limb development, together with the phenotypes when these genes are mutated in the mouse, support their involvement in the coordination of the P/D axis. The pattern of expression at both mRNA and protein level have been analyzed during normal limb development (Davis et al., 1995; Yokouchi et al., 1995; Nelson et al., 1996; Yamamoto et al., 1998). Around stage 20, when the mesoderm is taken to perform the recombinant, Hoxa-11 is expressed in most of the anterior mesoderm (Yokouchi et al., 1995; Nelson et al., 1996). In the recombinant limbs, Hoxa-11 expression was observed in the whole recombinant mesoderm during the ®rst day of development (Fig. 2A). Progressively, Hoxa-11 expression was excluded from the central chondrogenic region and con®ned to the distal part of the recombinant (Fig. 2C). During further development the pattern of expression of Hoxa-11 in the recombinant mesoderm matured in a way similar to normal, eventually becoming restricted to the zeugopod level from 72 h after grafting (Fig. 2E,G). Hoxa-13 expression is speci®c to the autopod region (Yokouchi et al., 1995; Nelson et al., 1996). Hoxa-13 is not detected in limb buds at stage 20, when the mesoderm is taken to perform the recombinant. In the recombinant limb, Hoxa-13 expression ®rst became activated in a thin stripe of mesoderm under the AER at about 24±30 h after grafting (Fig. 2B,D). The domain of expression of this gene progressively expanded as the autopod region formed and the expression persisted in this region, paralleling the normal pattern (Fig. 2F,H). From the third day of development, the domains of expression of Hoxa-11 and Hoxa-13 were restricted to the zeugopod and autopod segments of the recombinant limb respectively (Fig. 2G,H) in a pattern equivalent to the normal pattern (Fig. 2I,J). Thus, Hoxa-11 and Hoxa-13 expression patterns were properly established in the recombinant limb mesoderm, suggesting that the P/D axis has developed appropriately in the recombinant limb autopod and zeugopod mesoderm. Finally, it is of interest that the pattern of expression described for the Hoxa genes in the recombinant limbs was identical in non-polarized and polarized limbs (compare Fig. 2E,F with Fig. 2G,H), suggesting that the presence of the ZPA did not in¯uence the Hoxa pattern of expression. This result indicates that the AER is suf®cient for adequate reorganization of growth in the proximo±distal axis. 2.3. AER morphology and gene expression in the recombinant limbs The normal AER consists of a pseudostrati®ed columnar epithelium that is asymmetric along the A/P axis in that it is

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Fig. 2. Expression patterns of Hoxa genes in recombinant limbs. (A) and (B) are consecutive frontal sections of the same non-polarized recombinant limb hybridized 24 h after grafting respectively with Hoxa-11 and Hoxa-13 probes. Hoxa-11 expression is observed throughout the recombinant while activation of Hoxa-13 is starting under the ridge. (C) and (D) illustrate expression of Hoxa-11 and Hoxa-13, respectively, in two non-polarized recombinant limbs 48 h after grafting. (E) and (F) illustrate expression of Hoxa-11 and Hoxa-13 respectively in two consecutive frontal sections of a polarized recombinant limb 60 h after grafting. Note that the Hoxa pattern of expression is not modi®ed by the presence of the ZPA. In (E) a lower level of Hoxa -11 expression is observed in the distal part of the recombinant. (G) and (H) illustrates the Hoxa-11 and Hoxa-13 pattern of expression in non-polarized recombinant limbs from 72 h after grafting. These patterns are similar to normal (I) and (J).

Fig. 3. Fgf expression in the AER of the recombinant limbs. The probe or probes used for the hybridizations are indicated at the top-right of each panel. (A) Transverse section through the AER of a recombinant limb 24 h after grafting showing its tall morphology. (B) Shows high expression of Fgf-8 in the AER of a non-polarized recombinant limb 24 h after grafting. (C) Illustrates a non-polarized recombinant limb (indicated by arrows) in which Fgf-4 and Shh expressions are not detectable 24 h after grafting. (D) and (E) show clear Fgf-4 expression in the AER (red arrowheads) of nonpolarized recombinant limbs 24 (D) and 48 (E) h after grafting respectively. (F) Symmetrical Fgf-4 expression is observed in a polarized recombinant limb (red arrowheads). The red arrowheads in C-D indicate Fgf-4 expression in the recombinant ridge. The black arrowhead in (F) indicates Shh expression in the grafted piece of ZPA. (G) Non-polarized recombinant limb showing Fgf-10 expression in the subridge mesoderm.

taller posteriorly (Todt and Fallon, 1984). It is assumed that the taller the AER, the higher its growth promoting activity (Rubin and Saunders, 1974; Pizette and Niswander, 1999). Asymmetry in the AER is also manifested by the posterior restriction of Fgf-4 expression (Niswander et al., 1993). The morphology of the recombinant AER was normal, corresponding to a tall AER all along the A/P axis (Fig. 3A,B). This prominent morphology is consistent with the rapid induction of a subjacent progress zone marked by the expression of Msx1 (Ros et al., 1994). Thus, the normal AER asymmetry along the A/P axis is lost in the recombinant situation. The interface between the AER and the subjacent mesoderm was generally smooth (Figs. 3 and 4)

although frequently the AER appeared irregular or showed indentations (see Fig. 4F) probably re¯ecting local points of poor recombination with the subjacent mesoderm. We have extended earlier analysis of the AER function and maintenance in the recombinant limbs by analyzing the expression patterns of Fgf-8 and Fgf-4 in the AER and Fgf-10 in the underlying mesoderm. As expected, expression of Fgf-8, considered a marker of AER cells, was always observed in the recombinant AER (Fig. 3B) at a very high level and regardless of the presence of the ZPA. Fgf-4 is normally expressed in the posterior AER during

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Fig. 4. Expression patterns of Bmp2, Bmp4 and Bmp7 genes in recombinant limbs. (A) Bmp2 expression in a non-polarized recombinant limb 24 h after grafting is observed in a thin stripe under the AER. (B) By 48 h after grafting, Bmp2 expression is undetectable in the mesoderm of non-polarized recombinant limbs and barely discernible in the recombinant ridge. The arrowheads point to the location of the recombinant. (C) and (D) illustrates Bmp2 pattern of expression in two polarized recombinant limbs 24 h (C) and 48 h (D) after grafting respectively. (E) Bmp4 expression in a non-polarized recombinant limb 24 h after grafting is observed in the distal half of the recombinant. (F) However, by 48 h after grafting, Bmp4 expression becomes undetectable in the non-polarized recombinant mesoderm, while it is clearly observed in the AER. (G) Pattern of Bmp4 expression in a polarized recombinant limb 48 h after grafting. (H) Bmp7 expression in a non-polarized recombinant limb 24 h after grafting is observed in the distal half of the recombinant. (I) 48 h after grafting, Bmp7 expression becomes undetectable in the non-polarized recombinant mesoderm, while it is clearly observed in the AER. (J) Pattern of Bmp7 expression in a polarized recombinant limb 48 h after grafting. The red arrow indicates the location of the grafted ZPA when present. Note that the A/P axis of the recombinant in (D) is inverted with respect to that of the host

limb development and has been shown to establish a positive feed-back loop with Shh (Laufer et al., 1994; Niswander et al., 1994). We analyzed Fgf-4 expression in polarized (ZPA added) and non-polarized (no ZPA added) recombinant limbs and found that Fgf-4 was expressed in all of polarized (4 out of 4) but in half (2 out of 4) of the non-polarized recombinant limbs. Because of the positive feed-back loop between Fgf-4 and Shh, another set of specimens were conjointly hybridized with probes for both Fgf-4 and Shh. Our results showed that Shh expression was never detected in non-polarized recombinant limbs (n ˆ 8; Fig. 3C) while Fgf4 was clearly detectable in half of them (4 out of 8 cases; Fig. 3C±F). Polarized recombinant limbs showed Shh expression in the grafted ZPA and Fgf-4 in the AER (Fig. 3F). Thus, Fgf4 was maintained in some recombinant AERs in the absence of detectable Shh expression. Interestingly, when Fgf-4 expression was detected in the recombinant AER, its expression was symmetrical all along the length of the AER without the normal posterior bias (Fig. 3D,E). In some cases, as in Fig. 3D, expression appeared stronger at one end of the AER but this was not

typical. Since it is known that AER morphology and Fgf-4 expression depend on the underlying mesoderm (Zwilling, 1956; Laufer et al., 1994; Niswander et al., 1994), our results indicate that the symmetric Fgf-4 expression in the recombinant re¯ects the uniformity in the mesoderm along the A/P axis and that the AER in the recombinant has lost normal asymmetric morphology and gene expression. It was also noticeable that when Fgf-4 expression was detected in the AER, the level of expression was always lower than in the control wing (Fig. 2). A proposed candidate for the AER maintenance factor is Fgf-10 (Zwilling, 1956; Ohuchi et al., 1997). During normal development, Fgf-10 expression is detected in lateral plate mesoderm within the limb ®eld and then continues in the limb mesoderm under the AER, where it later localizes mainly posteriorly (Ohuchi et al., 1997). In the recombinants, Fgf-10 expression localized in the subridge mesoderm all along the A/P axis, independently of the presence of the ZPA, thus providing a possible explanation for the uniform AER maintenance capacity exhibited by the anterior mesoderm in the recombinant limbs (Fig. 3G).

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2.4. Bmp expression is downregulated in the recombinant mesoderm but maintained in the AER Patterning regulation along the A/P axis of the limb depends on the ZPA through its production of Shh (Riddle et al., 1993; Chang et al., 1994; LoÂpez-MartõÂnez et al., 1995). Non-polarized recombinant limbs developed in the absence of Shh signaling as Shh expression was never detected in these recombinants (see for example Fig. 3C). Accordingly, the outgrowths obtained from these limbs lacked polarity in the A/P axis (MacCabe et al., 1973a; Ros et al., 1994; this report). Therefore, in non-polarized recombinant limbs, growth along the proximo±distal axis occurred in the absence of A/P patterning making them a valuable system to examine the pattern of expression of the genes that are considered to play a role in A/P patterning. Three of the bone morphogenetic (Bmp) genes, Bmp2, Bmp4 and Bmp7, are expressed in the developing limb and have been related to A/P patterning, probably acting downstream of Shh (Francis et al., 1994; Francis-West et al., 1995; Duprez et al., 1996a). During limb bud development Bmps are also implicated in the regulation of chondrogenesis and programmed cell death (GanÄan et al., 1996; Duprez et al., 1996b; Zou and Niswander, 1996; Macias et al., 1997; Zou et al., 1997). During chick wing development, Bmp2 expression overlaps the Shh area of expression and extends more anteriorly (Francis et al., 1994). Bmp4 and Bmp7 are expressed in the posterior margin of the bud but also in the anterior margin (Francis-West et al., 1995). Here we have analyzed the expression of these three Bmps during the development of both non-polarized and polarized recombinant limbs searching for clues for their role in the speci®cation of A/P patterning. Non-polarized recombinant limbs showed expression of Bmp2, Bmp4 and Bmp7 in the recombinant mesoderm during the ®rst day of development (Fig. 4A,E,H). Bmp2 expression was detected at very low levels in a relatively thin strip of mesoderm under the AER (Fig. 4A). The specimen shown in Fig. 4A is among the non-polarized recombinant limbs with highest Bmp2 expression level. In most specimens, Bmp2 level of expression was lower. Bmp4 and Bmp7 were expressed at higher levels in the distal half of the non-polarized recombinant mesoderm (Fig. 4E±H). For the three Bmps, the pattern of expression was in clumps but symmetric along the A/P axis. The anterior two-thirds of the mesoderm used to perform the recombinant normally express Bmp4 and Bmp7 but not Bmp2, whose mesodermal domain of expression is posterior only. Thus, the observed pattern of expression of these three genes in the recombinant mesoderm probably re¯ects maintenance of Bmp4 and Bmp7 expression under AER in¯uence but it is likely that Bmp2 expression has been induced de novo. We can not discard the possibility, however, that some cells from the most anterior part of the normal Bmp2 domain of expression were taken to perform the recombinant. We tried to address this possibility by checking for Bmp2 expression in the

recombinant limbs at time 0, before grafting. Unfortunately, we were unable to ascertain whether there was a low level of Bmp2 expression or an elevated background due to probe trapping in the just reaggregated mesoderm (not shown). Interestingly, during the second day of recombinant development Bmp expression was downregulated and became undetectable (Fig. 4B,F,I). This may indicate that Shh, as well as the AER, is required for Bmp maintenance of expression. In contrast, polarized recombinant limbs exhibited a Bmp pattern of expression closer to that of normal limb buds (Fig. 4C,D,G,J). The piece of ZPA grafted into the recombinant normally expressed these three genes and furthermore induced their expression in the adjacent mesoderm, thus reproducing a pattern closer to normal. It has been shown that ZPA grafts induce Bmp2 expression in the adjacent mesoderm when implanted at the anterior limb margin (Francis et al., 1994). To further demonstrate induction of Bmp expression by the ZPA we used two approaches. Firstly we used ZPA of quail origin which together with the speci®c antiquail QCPN antibody, allowed us to con®rm that the grafted ZPA induced Bmp2, Bmp4 and Bmp7 expression in the surrounding chick mesoderm (not shown). Secondly, we performed polarized recombinant limbs using a bead soaked in Shh instead of the ZPA graft. Induction of Bmp2 adjacent to the Shh-bead was observed after 24 h (Fig. 5A). Bmp2 expression in the margin where the bead was placed persisted and increased (Fig. 5B), reproducing the pattern observed with the ZPA grafts. In contrast, recombinants performed with the same pellet of mesodermal cells but with no Shh-bead lacked detectable Bmp2 expression (Fig. 5C). However, the anterior domains of Bmp4 and Bmp7 expression were never clearly reestablished (Fig. 4G,J), indicating that the grafted ZPA was not able to reconstitute the normal situation along the A/P axis particularly at the anterior border. At later stages, when the digits were being speci®ed, Bmp expression was observed in the interdigital spaces (not shown). Therefore, non-polarized recombinant limbs expressed Bmp2, Bmp4 and Bmp7 during the ®rst day of development but rapid downregulation of expression also occurred. However, when a piece of ZPA was present in the recombinant, an asymmetric pattern of expression of these genes was observed. Regardless of the downregulation of Bmp expression in the mesoderm, both non-polarized and polarized recombinant limbs showed expression of the three Bmps studied here in the AER (Fig. 4). As in normal development, Bmp2 expression was downregulated earlier than Bmp4 and Bmp7 (compare Fig. 4B with Fig. 4F and Fig. 4I). This observation further indicates that the AER is normally functioning in the recombinant limbs. Expression in the AER continued even when mesodermal expression was downregulated (Fig. 4B,F,I), indicating that the ectodermal domain of expression of the Bmps genes does not depend on

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Fig. 5. Bmp2 expression is induced in the recombinant mesoderm by a Shh-bead. (A) shows a recombinant limb bud polarized by a Shh-bead (arrow), demonstrating induction of Bmp2 expression by Shh in the adjacent mesoderm 24 h after grafting. (B) Forty-eight hours after grafting, the Shh-induced Bmp2 expression has increased in the margin where the Shh-bead (arrow) was implanted. (C) Shows a recombinant limb performed with the same mesodermal pellet as those shown in (A) and (B) but with no Shh-loaded bead. Note that Bmp2 expression in undetectable in this recombinant. rec, Recombinant limb.

their mesodermal domains of expression and that both domains appear to be regulated independently. Finally, Bmp4 expression in the non-AER ectoderm was also maintained during recombinant development (Fig. 4F,G). 2.5. Organization of the dorso-ventral axis in the recombinant limbs Lmx1 is a LIM-homeodomain protein whose expression in the limb is normally restricted to the dorsal mesoderm under the control of Wnt7a, a secreted protein produced by the dorsal ectoderm (Riddle et al., 1995; Vogel et al., 1995). Misexpression experiments in chick and targeted disruption in mice have revealed that Lmx1 is necessary and suf®cient to specify mesodermal dorsal fate (Rodriguez-Esteban et al., 1998). The ventral limb ectoderm expresses En-1, a transcription factor that is also implicated in the control of dorso-ventral polarity probably by restricting Wnt7a expression to the dorsal ectoderm (Loomis et al., 1996, 1998; Logan et al., 1997; Cygan et al., 1998). Experiments using recombinant limbs demonstrated that at the beginning of limb development D/V speci®cation is controlled by the mesoderm but that this control is soon transferred to the ectoderm (MacCabe et al., 1973b, 1974). More recent experiments have challenged this view so that the way the ectoderm and mesoderm acquire their D/ V polarity remains controversial (for a review see Chen and Johnson, 1999). Nevertheless, at the stages used to perform the recombinants, the ectoderm controls D/V polarization. We have sequentially analyzed the expression pattern of Wnt7a, En-1 and Fgf-8 genes in the recombinant ectoderm. The ectodermal hulls used to perform the recombinant have previously established domains of gene expression with Wnt7a expressed in the dorsal ectoderm, Fgf-8 in the AER and En-1 in the ventral ectoderm and ventral half of the ridge. Consecutive adjacent sections of the same recombinant were hybridized with these probes and the results obtained demonstrated that the ectodermal hull maintained the established dorsal and ventral compartments throughout the experiment regardless of the subjacent disorganized

mesoderm or site of grafting (Fig. 6A,B,D). This indicates that the expression domains of Wnt7a, Fgf-8 and En-1 in the limb ectoderm, once established, are stable and autonomous, or at least not dependent upon organized signals from the subjacent mesoderm. To analyze whether signaling from contiguous ectoderms was required, the recombinant limbs were grafted to different sites (somites or dorsal limb) and in different orientations. The recombinant limb shown in Fig. 6A±D, was grafted to the somites but in an inverted D/V orientation in relation to that of the host. Wnt7a expression continued in the graft dorsal ectoderm independently of the acquired ventral position (Fig. 6A) and En-1 expression continued in the graft ventral ectoderm independently of the now dorsal position (Fig. 6B). This result also indicates that these ectodermal domains of gene expression are not dependent upon contiguous ectoderms. The temporo-spatial patterns of Wnt7a expression in the recombinant ectoderm developed as in normal limb buds (Akita et al., 1996). The procedure of dissociation to single cell level and reaggregation of the mesoderm results in the mixing up of dorsal and ventral mesodermal cells. Accordingly, a random pattern of Lmx1 expressing cells was observed in the recombinant mesoderm at time 0, indicating that dorsal cells expressing Lmx1 were intermingled with ventral cells not expressing it (not shown). Shortly afterwards Lmx1 expression was downregulated from the central core of the recombinant (Fig. 6C) but the random pattern of Lmx1 expression persisted in the peripheral mesoderm. As the recombinant limb elongated during further development, this random Lmx1 expression pattern persisted at proximal levels while distally a normal pattern was organized (Fig. 6E,F). In the recombinant progress zone, Lmx1 became activated only in the mesoderm beneath the original dorsal ectoderm expressing Wnt7a and consequently the distal part of the recombinant showed a normal dorso-ventral pattern of Lmx1 expression (Fig. 6F±H). This was most effectively seen when the recombinant limbs were observed longitudinally (Fig. 6F). Observation from the tip of the bud (Fig. 6G,H) also showed the clear dorsal restriction of Lmx1 expression at the distal level while proximally a random pattern of

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Fig. 6. Dorso-ventral organization in recombinant limbs. (A±D) are serial consecutive sections of a non-polarized recombinant limb (rec) 24 h after grafting. The dorso-ventral axis of the recombinant is inverted with respect to that of the host. Wnt7a (A) and En-1 (B) expressions are maintained in the original ectoderm compartments independently of the inverted position in the host. Lmx1 expression (C) is observed in a random pattern in the whole recombinant mesoderm except in the central core. The arrowhead in A±D indicates the AER. (E) Lmx1 expression in a recombinant limb 48 h after grafting. (F) longitudinal view of the same recombinant shown in (E) to appreciate the distal dorso-ventral organization of Lmx1 expression accordingly to the ectodermal compartments. (G) and (H) are views from the tip of two recombinants hybridized respectively with the Lmx1 and Wnt7a probes to further show how the dorsal Lmx1 pattern of expression is appropriately organized in the distal tip of the recombinant while a random pattern persist at more proximal levels. The red arrow in (F) and (G) indicates the approximate boundary between the proximal Lmx1 random pattern of expression and the distal correct pattern.

Lmx1 expression persisted (arrows in Fig. 6G). We observed a rather sharp transition between the proximal (random) and the distal (dorsally restricted) pattern of Lmx1 expression with absence of Lmx1-positive cells at ventral-distal levels. This result may re¯ect some sorting out of cell expressing and not expressing Lmx1. In fact, sorting out of cells with different developmental origin has been demonstrated in other types of recombinant limbs (Wada et al., 1993). Overall our results show organization of D/V patterning in the recombinant progress zone under ectoderm in¯uence and supports the hypothesis that D/V patterning takes place in the progress zone (Akita, 1996). D/V pattern in developed recombinant limbs can only be assessed by the feather patterns, because other elements such as muscle and tendons are rudimentary and poorly organized (Fernandez-Teran et al., 1999). The observation that only the distal part of the recombinant organizes a D/V pattern of Lmx1 expression explains why a clear dorsoventral polarity in the pattern of feathers (Fig. 1E) is observed in the autopod but becomes progressively more dif®cult to distinguish at more proximal levels. This ®ts well with the expression pattern of Lmx1 in recombinant limb at limb bud stages. There were no differences in the pattern described above between non-polarized and polarized recombinant limbs. It has been proposed that Wnt7a maintains appropriate levels of Shh expression (Parr and McMahon, 1995; Yang and Niswander, 1995), however, Shh does not appear to be necessary for Wnt7a or En-1 maintenance of expression since their patterns of expression are maintained in the

non-polarized recombinant limbs where Shh expression is undetectable. Consequently, Shh does not appear to in¯uence patterning in the D/V axis. 3. Discussion The model of recombinant limbs demonstrates the great organizing abilities of the limb mesoderm. After complete dissociation to single cell level and reaggregation, this mesoderm under the in¯uence of the ectoderm is able to reorganize and reestablish appropriate positional information and patterning in the P/D and D/V axes. To reestablish patterning in the A/P axis grafting of a ZPA piece into one of the margins of the recombinant is required. 3.1. The AER respeci®es a progress zone and is suf®cient to organize P/D patterning and growth in the recombinant limb Elongation in the proximo±distal axis occurs in a normal proximo±distal sequence with the consecutive formation of the stilopod, zeugopod and autopod regions. A constant feature of developed recombinant limbs was that they exhibited a clear proximo±distal gradient of morphogenesis. Proximal elements showed de®cient development while the distal elements, the digits, were well formed. This gradient was observed in both polarized and non-polarized recombinant limbs. We propose an explanation for this morphogenetic gradient. After performing the recombination, the uniform pellet

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of mesodermal cells becomes exposed to different ectodermal signals depending on the position. The distal mesoderm is under AER in¯uence while the proximal mesoderm is not. AER signaling will very soon respecify a new progress zone clearly marked by the expression of Msx1 (Saunders and Gasseling, 1959; Krabbenhoft and Fallon, 1989; Davidson et al., 1991; Ros et al., 1994). In this new progress zone, patterning events are reestablished and further elongation of the recombinant originates from this region. At proximal levels there is a certain sorting out of cells, with for example myogenic precursors arranging peripherally around a central chondrogenic core (Wada et al., 1993; FernandezTeran et al., 1999). This proximal chondrogenesis forms the proximal skeletal elements that consequently show a de®cient patterning. The most 5 0 Hoxa genes have been implicated in the speci®cation of the P/D axis during limb development (Yokouchi et al., 1995; Nelson et al., 1996). The pattern of expression of Hoxa-11 and Hoxa-13 genes during the development of the recombinant limbs parallels that of normal limbs, their expression being con®ned to the zeugopod and autopod levels, respectively. Accordingly it can be concluded that appropriate speci®cation in the P/D axis has occurred in the recombinant. Wada et al. (1998) reported similar patterns of expression for the Hoxa genes in recombinant limbs performed with stage 20 progress zone mesoderm. However, these authors did not observe the restriction of Hoxa-11 expression to the zeugopod region, but this was probably because they did not extend their analysis late enough, as this pattern is seen from the third day of development. Since the ZPA did not in¯uence the pattern of Hoxa-11 and Hoxa-13 in the recombinants, it can be concluded that the AER, without the coordinated action of the ZPA, appears suf®cient to regulate gene expressions implicated in P/D patterning of the bud that de®ne or accompany the different P/D segments of the limb. 3.2. Downregulation of Bmp expression can explain the loss of AER asymmetry in the recombinant limbs During normal development the AER is clearly asymmetric along the A/P axis in that it is taller posteriorly (Todt and Fallon, 1984). This is accompanied by the normally posterior restriction of Fgf-4 expression in the AER in relation to Shh signaling (Laufer et al., 1994; Niswander et al., 1994). Other gene expressions in the AER are symmetrical along its A/P length. Interestingly, the AER in the recombinants exhibited a prominent tall morphology and Fgf-4 expression, when detected, was symmetric all along its length. Thus, anterior mesoderm unable to maintain the ridge during normal development acquires this capability in the recombinant situation. In some way the process used to perform the recombinant has either increased the mesoderm AER maintenance capacity or, alternatively, has released a previously existing repression. The maintenance of Fgf-4 expression in the

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recombinant AER after Bmp2 expression becomes undetectable may appear dif®cult to reconcile with previous observations indicating Bmp2 involvement in Fgf-4 induction or maintenance of expression (Duprez et al., 1996a; Laufer et al., 1994; Niswander et al., 1994). However, recently Pizette and Niswander (1999) have unraveled a new activity of BMPs in the inhibition of AER morphology and function. Our results ®t very well with their model. Since expression of Bmp is considerably reduced in the recombinant mesoderm, the repressive signal to the AER should also be attenuated; this allows the tall shape and Fgf-4 activation in the anterior AER and also explains the Fgf-4 expression in the absence of Shh. However expression of Fgf-4 in the recombinant AER was not detected in all the specimens. Although we can not explain this variability at present, there could be a relationship between the level of Bmp expression in the recombinant mesoderm during the ®rst day of development and Fgf-4 expression in the AER. A suf®cient downregulation of Bmp expression will lead to activation of Fgf-4 expression in the AER (Pizette and Niswander, 1999). 3.3. Bmp expression and chondrogenesis in the recombinant limbs It is not clear why downregulation of Bmp expression occurs during recombinant development, but since it is apparently caused by the dissociation of the mesoderm, the extracellular matrix and associated molecules may be implicated. Bmp expression depends on the cellular context and little is known about upstream regulation of expression. It has been shown that the complete blocking of Bmps caused by overexpression of noggin increases Bmp expression (Capdevila and Johnson, 1998; Pizette and Niswander, 1999) indicating a possible negative self-control of Bmp expression. Such autoregulatory mechanisms was not evident in the recombinant mesoderm regardless of the decrease in the level of Bmp expression, this probably indicates that this mechanism only operates in extreme conditions. It would be interesting to analyze the expression of noggin and chordin during the development of the recombinant limbs. Complete blocking of Bmp signaling by overexpression of noggin is followed by inhibition of chondrogenesis (Capdevila and Johnson, 1998; Pizette and Niswander, 1999). Bmp expression becomes undetectable in non-polarized recombinant limbs, but this is not accompanied by inhibition of chondrogenesis. It is possible that residual levels of Bmp expression, although below the level of detection of the in situ hybridization, are suf®cient to permit chondrogenesis whereas blocking by noggin is total. In the polarized recombinant limbs expression of the three Bmps was observed around the grafted ZPA or Shh bead in a pattern closer to normal. However the correct expression of Bmp4 and Bmp7 anteriorly was never reestablished. The question of the control of the normal ante-

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rior Bmp pattern of expression remains open at this time. Polarized expression of Bmp2, Bmp4 and Bmp7 around the ZPA was followed by the formation of an adequate pattern of digits along the A/P axis. These results support a role for Shh in the regulation of Bmp pattern of expression during normal limb development, at least at the posterior border, and also allows a link to be made between asymmetric Bmp expression and normal asymmetries in the skeletal pattern. The possible implication of Bmps in A/P patterning during limb bud development remains controversial (Francis et al., 1994; Duprez et al., 1996a,b; Zou et al., 1997; Capdevila and Johnson, 1998). Our results support the notion that appropriate Bmp2, Bmp4 and Bmp7 expression may be required for adequate modulation and ®ne patterning of the different chondrogenic elements (Duprez et al., 1996b). The skeletal pattern observed in non-polarized recombinant limbs can be interpreted as the result of the lack of Shh signaling. However, Shh mutant mice show an extreme phenotype with a dramatic truncation of the limbs (Chiang et al., 1996). This difference could be explained by the absence of Shh signaling in the mutant which could in¯uence the feed-back loop with the AER from early development and consequently affect the A/P axis as well, whereas in the recombinant limb the ectoderm is normal. Furthermore, in contrast to Shh mutant limbs, which are never exposed to Shh, the mesoderm used for the recombinant has experienced normal Shh signaling up to the stage when the experiment was performed. Non-polarized recombinant limbs have been compared with talpid limbs since they present comparable skeletal patterns and similar pattern of Hoxd gene expression (Krabbenhoft and Fallon, 1992; IzpisuÂa-Belmonte et al., 1992; Coelho et al., 1992; Ros et al., 1994; Hardy et al., 1995). Talpid (talpid 2 and talpid 3) limbs are polydactylous and syndactylous (Dvorak and Fallon, 1991) and although the Shh domain of expression is normal, other proposed downstream components such as the most 5 0 Hoxd genes are expressed under the AER uniformly along the A/P axis (IzpisuÂa-Belmonte et al., 1992; Francis-West et al., 1995). However, non-polarized recombinant limbs differ from talpid limbs in their pattern of Bmp expression, which is uniform along the A/P axis in talpid (Francis-West et al., 1995; Rodriguez et al., 1998) and absent in non-polarized recombinant limbs. This difference strongly indicates that the constitutive activation of Shh signaling proposed in talpid 2 or the bifurcation in the Shh pathway proposed in talpid 3 does not occur in the recombinant limb mesoderm (Caruccio et al., 1999; Lewis et al., 1999). 3.4. Ectodermal domains of Bmp expression are independent of mesodermal domains Bmp expression in the recombinant AER was normal regardless of downregulation in the mesoderm. Consequently, the proposed bene®t in AER morphology and function caused by the suppression of Bmp signaling must

originate exclusively in the mesoderm with no effect from the ridge itself. This may also be the case in the normal limb bud (Pizette and Niswander, 1999) and ®ts well with the known dependence of AER shape on the underlying mesoderm (Zwilling 1956). Our results also indicate that the ectodermal and mesodermal domains of Bmp expression during limb development are independently regulated and that Bmp expression by the AER does not require Bmp signaling from the mesoderm. 3.5. The recombinant ectoderm maintains previously established domains of expression and organizes D/V patterning in the progress zone To perform the recombinant the mesoderm is randomized with respect to positional information, so that position speci®c patterns of gene expression are disrupted. However, the process of isolation and recombination does not modify the well-de®ned dorsal, ventral and AER compartments of gene expression in the limb ectoderm. In the recombinant limbs these ectodermal domains of gene expression are maintained independently of the subjacent mesoderm and site of grafting, indicating ectodermal self-autonomy for their maintenance. Further, signaling from the ectoderm establishes appropriate D/V patterning in the recombinant progress zone, con®rming previous work (Akita et al., 1996; for review see Chen and Johnson, 1999), and also indicating the independence of ectodermal compartments from Shh signaling since no differences regarding D/V patterning were observed in polarized versus non-polarized recombinant limbs. 4. Materials and methods 4.1. Formation of recombinant limbs Fertilized hen and quail eggs were routinely incubated and opened and the embryos staged according to Hamburger and Hamilton (1951). The recombinant limbs were performed with limb bud ectodermal hulls and the anterior two-thirds of wing bud mesoderm pelleted after dissociation to single cell level. The procedure used to make a recombinant limb was based on protocols already described with some modi®cations (Frederick and Fallon, 1982; Ros et al., 1994; for a detailed protocol see Ros et al., 1999). Brie¯y, the anterior two thirds of stage 19±21 wing buds were dissected from the embryo and the ectoderm peeled and discarded after incubation in 0.5% trypsin for 10±15 min at 388C. The mesodermal pieces were dissociated to single cell level by incubation for 10 min at 228C in 0.4% collagenase (750 units/ml) in saline G (140 mM NaCl, 5.4 mM KCl, 1.1 mM KH2PO4, 1.1 mM Na2HPO4, 0.1% glucose, 0.5% phenol red), gently pipetted, and pelleted by low speed centrifugation. The ectodermal jackets were obtained from whole stage 20 to 22 leg buds by mild trypsin

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digestion. The ectoderms were transferred to the plate containing the mesoderm pellet, and the recombinant limbs assembled by introducing a fragment of the pelleted mesoderm into the ectodermal hulls. To obtain polarized recombinant limbs, a small fragment (about 150 mm) of polarizing zone (from stage 20 embryos) was introduced into the posterior margin of the recombinant limb. Since the D/V and A/P axis of the ectodermal hull are easily discernible under the dissecting microscope, the piece of ZPA was always placed to coincide with the posterior border of the ectoderm. Occasionally, the ZPA used was of quail origin and in some cases a heparin acrylic bead (Sigma, H5263) soaked in Shh protein (3mg/ml) was implanted instead of the ZPA. In this report, the recombinant limbs containing a fragment of ZPA are called polarized recombinant limbs versus non-polarized recombinant limbs, without a ZPA fragment. Most of the recombinant limbs were grafted to the somites of host embryos (stages 20±22). In general we did not keep track of the axes of the recombinant limb and consequently, in grafting, the axes of the recombinant might not coincide with those of the host (see for example Fig. 4D). In some recombinants we marked the D/V axis and inverted it with respect to the host when grafting (see Fig. 6A±D); this was done to analyze possible in¯uences of contiguous ectoderms on the organization of the D/V axis of the recombinant. Finally, for some experiments, half of the recombinants were grafted to the dorsal surface of the host wing (Hardy et al., 1995) and half to the somites (Ros et al., 1994). The results obtained regarding outgrowth and patterns of gene expression were similar independently of the graft site and because it is easier in our hands, most of the recombinants were grafted to the somites. The specimens were sequentially ®xed in 4% paraformaldehyde for analysis of gene expression. Some recombinants were allowed to develop for 10 days to analyze the skeleton pattern. These were ®xed in 10% formalin, stained with Victoria blue and cleared in methyl salicylate. 4.2. In situ hybridization technique Hybridization with digoxigenin-labeled riboprobes was performed in whole mount as described in Nieto et al. (1996). Following hybridization, some embryos were paraf®n-embedded and serially sectioned. For hybridization in tissue sections 35S-labelled riboprobes were prepared and used following the protocol described in Wilkinson and Nieto (1993). The probes used were for Fgf-8, Fgf-10, Fgf-4, Hoxa-11, Hoxa-13, Shh, Bmp2, Bmp4, Bmp7, En-1, Wnt-7a, Lmx-1, kindly provided by C. Tabin, T. Jessel, J.C. Izpisua-Belmonte, S. Noji, B. Houston and A. Joyner. Acknowledgements We are grateful to J.F. Fallon for his critical reading of an earlier version of the manuscript and to Ian Williams for

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revising the ®nal English version. We also thank C. Tabin, T. Jessel, J.C. IzpisuÂa-Belmonte, B. Houston, S. Noji and A. Joyner for providing the cDNA clones for the probes used in this study and Chin Chiang for the gift of Shh protein. This work was supported by grant from the Spanish Ministry of Education and Science (DGICYT-PM95-0088). M.E.P. was the recipient of predoctoral fellowships (P.F.P.I.) from the Spanish Ministry of Education and Culture. References Akita, K., 1996. The effect of the ectoderm on the dorsoventral pattern of epidermis, muscles and joints in the developing chick leg: a new model. Anat. Embryol. 193, 377±386. Akita, K., Francis-West, P., Vargesson, N., 1996. The ectodermal control in chick limb development: Wnt-7a. Shh, Bmp2 and Bmp-4 expression and the effect of FGF-4 on gene expression. Mech. Dev. 60, 127±137. Capdevila, J., Johnson, R.L., 1998. Endogenous and ectopic expression of noggin suggest a conserved mechanisms for regulation of BMP function during limb and somite patterning. Dev. Biol. 197, 205±217. Caruccio, N.C., Martinez-Lopez, A., Harris, M., Dvorak, L., Bitgood, J., Simandl, B.K., Fallon, J.F., 1999. Constitutive activation of Sonic Hedgehog signaling in the chicken mutant talpid 2: Shh independent outgrowth and polarizing activity. Dev. Biol. 212, 137±149. Chang, D.T., LoÂpez, A., Von Kessler, D.P., Chang, C., Simandl, B.K., Zhao, R., Selding, M.F., Fallon, J.F., Beachy, P.A., 1994. Products, genetic linkage and limb patterning activity of a murine hedgehog gene. Development 120, 3339±3353. Chen, H., Johnson, R.L., 1999. Dorsoventral patterning of the vertebrate limb: a process governed by multiple events. Cell Tissue Res. 296, 67± 73. Chiang, C., Litingtung, Y., Lee, E., Young, K.E., Corden, J.L., Westphal, H., Beachy, P.A., 1996. Cyclopia and defective axial patterning in mice lacking Sonic hedgehog gene function. Nature 383, 407±413. Coelho, C.N.D., Upholt, W.B., Kosher, R.A., 1992. Role of the chicken homeobox-containing gene expressed in a fashion consistent with a role in patterning events during embryonic chick limb development. Differentiation 49, 85±92. Crosby, G.M., Fallon, J.F., 1975. Inhibitory effect on limb morphogenesis by cells of the polarizing zone coaggregated with pre- or postaxial wing bud mesoderm. Dev. Biol. 46, 28±39. Crossley, P.H., Minowada, G., MacArthur, C.A., Martin, G.R., 1996. Roles for FGF8 in the induction, initiation and maintenance of chick limb development. Cell 84, 127±136. Cygan, J.A., Johnson, R.L., McMahon, A.P., 1998. Novel regulatory interactions revealed by studies of murine limb pattern in Wnt7a and En-1 mutants. Development 124, 5021±5032. Davidson, D.R., Crawley, A., Hill, R.E., Tickle, C., 1991. Position dependent expression of two related homeobox genes in developing vertebrate limbs. Nature 352, 429±431. Davis, A.P., Witte, D.P., Hsieh-Li, H.M., Potter, S.S., Capecchi, M.R., 1995. Absence of radius and ulna in mice lacking Hoxa-11 and Hoxd-11. Nature 375, 791±795. Dvorak, L., Fallon, J.F., 1991. Talpid 2 mutant chick limb has anteroposterior polarity and altered patterns of programmed cell death. Anat. Rec. 231, 251±260. Duprez, D.M., Kostakopoulou, E.J., Francis-West, P.H., Tickle, C., Brickell, P.M., 1996a. Activation of expression of FGF-4 and HoxD gene expression by BMP-2 expressing cells in the developing chick limb. Development 122, 1821. Duprez, D.M., Bell, K., Richardson, M.K., Archer, C.W., Wolpert, L., Brickell, P.M., Francis-West, P.H., 1996b. Overexpression of BMP-2 and BMP-4 alters the size and shape of developing skeletal elements in the chick limb. Mech. Dev. 57, 145.

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