During early stages of normal chick limb development, the homeobox-containing (HOX) gene GHox-4.6 is expressed throughout the posterior mesoderm of the wing bud from which most of the skeletal elements including the digits will develop, whereas GHox-8 is expressed in the anterior limb bud mesoderm which will not give rise to skeletal elements. In the present study, we have examined the expression of GHox-4.6 and GHox-8 in the wing buds of two polydactylous mutant chick embryos, diplopodia-5 and talpid2, from which supernumerary digits develop from anterior limb mesoderm, and have also examined the expression of these genes in response to polarizing zone grafts and retinoic acid-coated bead implants which induce the formation of supernumerary digits from anterior limb mesoderm. We have found that the formation of supernumerary digits from the anterior mesoderm in mutant and experimentally induced polydactylous limb buds is preceded by the ectopic expression of GHox-4.6 in the anterior mesoderm and the coincident suppression of GHox-8 expression in the anterior mesoderm. These observations suggest that the anterior mesoderm of the polydactylous limb buds is “posteriorized” and support the suggestion that GHox-8 and GHox-4.6, respectively, are involved in specifying the anterior non-skeletal and posterior digit-forming regions of the limb bud. Although the anterior mesodermal domain of GHox-8 expression is severely impaired in the mutant and experimentally induced polydactylous limb buds, this gene is expressed by the prolonged, thickened apical ectodermal ridges of the polydactylous limb buds that extend along the distal anterior as well as the distal posterior mesoderm. These findings indicate that the anterior mesodermal and apical ectodermal ridge domains of GHox-8 expression are independently regulated, and provide further support for the concept that GHox-8 expression is involved in apical ectodermal ridge function.

The development of the chick limb including the formation of the various skeletal elements of the limb in their appropriate position and sequence along the proximodistal and anterior-posterior axes involves a complex and reciprocal series of interactions between the ectoderm and the mesoderm of the developing limb bud. A thickened cap of pseudostratified epithelium at the distal apex of the limb bud known as the apical ectodermal ridge is required for the outgrowth of the mesodermal cells of the limb bud and the formation of the various skeletal elements by limb mesoderm in their appropriate sequence along the proximodistal axis (see Saunders, 1977 for review). In turn, the mesodermal cells of the developing limb bud may be required to maintain the apical ectodermal ridge in a thickened functional condition (Zwilling, 1961; Saunders and Gasseling, 1968). The patterning of the various parts of the chick limb in their appropriate sequence along the anterior-posterior axis is thought to be regulated by a group of mesenchymal cells at the posterior margin of the limb bud known as the zone of polarizing activity (Saunders and Gasseling, 1968). This polarizing zone has been suggested to be the source of a diffusible morphogen that defines anterior-posterior positional identities according to its local concentration (Brickell and Tickle, 1989; Eichele, 1989).

The chicken homeobox-containing (HOX) genes GHox-7 and GHox-8, members of the Drosophila msh-Vike family of vertebrate HOX genes, have been implicated in the regulation of a variety of patterning events during chick limb development (Coelho et al., 1991a,b, 1992; Robert et al., 1991; Suzuki et al., 1991; Yokouchi et al., 1991; Nohno et al., 1992). GHox-8 is expressed in high amounts by the apical ectodermal ridge of normal chick limb buds, and GHox-7 is expressed in high amounts by the mesoderm directly subjacent to the apical ridge (Coelho et al., 1991b, 1992). The expression of GHox-8 is severely impaired in the distal ectoderm of the limb buds of limbless mutant chick embryos, which lack an apical ectodermal ridge and fail to undergo normal limb outgrowth, suggesting the possibility that GHox-8 expressed by the ridge is involved in its function (Coelho et al., 1991a). Furthermore, the mesoderm of limbless mutant limb buds exhibits severely impaired expression of GHox-7 (Coelho et al., 1991a), but exhibits normal high expression of GHox-7 when it is recombined with normal apical ectodermal ridge-containing limb ectoderm (Robert et al., 1991). In addition, expression of the mouse Hox-7.1 gene is induced in proximal mouse limb mesoderm implanted subjacent to the apical ridge of chick limb buds (Davidson et al., 1991). These observations suggest that the reciprocal expression of GHox-8 by the ridge and GHox-7 by the subridge mesoderm is involved in the interaction between these tissues that is required for normal limb outgrowth to occur (Coelho et al., 1991a). GHox-7 and GHox-8 are also expressed in discrete regions of the developing chick limb bud, such as the posterior necrotic zone, anterior necrotic zone (Saunders et al., 1962), and interdigital mesenchyme, in which programmed cell death is occurring, suggesting the possible involvement of these genes in defining regions of cell death that are involved in shaping the contours of the developing limb bud (Coelho et al., 1991b, 1992).

GHox-8, as well as 5’ members of the HOX-4 cluster of chicken HOX genes such as GHox-4.6, have also been implicated in the specification of positional identities along the anterior-posterior axis of the developing limb. At early stages of limb development, when positional identities along the anterior-posterior axis are being specified, GHox-8 is expressed in high amounts in the anterior mesoderm of the chick limb bud (Coelho et al., 1991b), and, conversely, GHox-4.6 is expressed throughout the posterior (postaxial) mesoderm of the chick limb bud (Izpisua-Belmonte et al., 1991; Nohno et al., 1991; Rogina et al., 1992).

It is of particular interest that the posterior expression domain of GHox-4.6 (Izpisua-Belmonte et al., 1991 ; Nohno et al., 1991; Rogina et al., 1992) corresponds to the region of the limb mesoderm, which, based on fate maps (Hinchliffe et al., 1981), will give rise to most of the skeletal elements of the limb including the digits. Furthermore, the posterior expression domain of GHox-4.6 corresponds to those regions of the limb bud mesoderm that are subjacent to the regions of the apical ectodermal ridge that are required for the formation of digits (Rowe and Fallon, 1981). In contrast, the anterior expression domain of GHox-8 (Coelho et al., 1991b) corresponds predominantly, if not exclusively, to the region of the limb mesoderm that does not give rise to skeletal elements. In the present study, we have examined the expression of GHox-4.6 and GHox-8 in the limb buds of two mutant chick embryos, diplopodia-5 (Olympio et al., 1983) and talpid2 (Abbott et al., 1960), which develop supernumerary digits from anterior limb mesoderm. We have also examined the expression of these genes in response to experimental manipulations that induce the formation of supernumerary digits from anterior limb mesoderm. We have found that the formation of supernumerary digits from anterior limb mesoderm in each of these situations is preceded by the ectopic expression of GHox-4.6 in the anterior mesoderm and by the concomitant suppression of GHox-8 expression in the anterior mesoderm.

Procurement of mutant limb buds

Homozygous diplopodia-5 mutant embryos were obtained from mating members of a heterozygous flock maintained at the University of Connecticut, Storrs, CT. The limb buds of homozygous diplopodia-5 mutant embryos are distinguishable from normal limb buds at day 5 of development by their broadened apex and by the presence of a thickened preaxial, as well as postaxial, apical ectodermal ridge. To be sure that suspected diplopodia-5 limb buds at earlier developmental stages possessed the mutant genotype, the following procedure was utilized. Windows were cut into the shells of the diplopodia-5 flock eggs, and the right forelimbs of the embryos were removed and individually processed as described below. The windows in the shells of the donor eggs were sealed with Scotch tape, and the donor embryos were reincubated and allowed to develop until their phenotype was clearly expressed. Individual limb buds to be used for in situ hybridization analysis were fixed in Bouin’s solution, processed to 70% ethanol, and stored in 70% ethanol at 4°C until their genotype was established.

Homozygous talpid2 mutant embryos were obtained from mating members of a heterozygous flock maintained at the University of Wisconsin, Madison, WI. The limb buds of homozygous talpid2 embryos are clearly recognizable as early as stage 18 (Hamburger and Hamilton, 1951 ) of limb development, because the mutant limb buds are considerably wider than normal, do not exhibit the typical asymmetric contour of normal limb buds, and possess a broad extensive apical ectodermal ridge that extends continuously along the entire periphery of the limb buds (Abbott et al., 1960; Dvorak and Fallon, 1991).

Implantation of retinoic acid-coated beads and polarizing zone grafts

Implantation of retinoic acid-coated beads into the anterior margin of stage 20 (Hamburger and Hamilton, 1951) wing buds was performed essentially as described by Tickle et al. (1982, 1985). Windows were cut into the shells of eggs at 3 days of incubation, the windows sealed, and the eggs reincubated overnight until stage 20. The site of implantation in stage 20 host wing buds was prepared by teasing away a portion of the ectoderm at the anterior margin of the right wing bud from the mesoderm to create a space for the implant (see Tickle et al., 1982, 1985). AG1-X2 polystyrene beads (BioRad) about 200 pm in diameter were soaked for 20 minutes in a 0.01 mg/ml solution of all-tranr-retinoic acid (Sigma) in dimethyl sulfoxide (DMSO). Beads were then washed three times with phos-phate-buffered saline containing 0.01% phenol red, and inserted underneath the loop of ectoderm at the anterior margin of the host wing buds. Control beads soaked in DMSO without retinoic acid were also implanted in this manner.

To prepare polarizing zone grafts, wing buds were cut away from stage 20 embryos, and the limb ectoderm was removed following brief treatment with dispase II as previously described (Coelho et al., 1991a). The mesoderm making up the zone of polarizing activity at the posterior border of the stage 20 donor wing buds (Mac-Cabe et at., 1973) was dissected into 200 ×m 3 blocks, and the blocks of polarizing zone mesoderm were inserted underneath a loop of ectoderm at the anterior margin of stage 20 host wing buds as described above for retinoic acid bead implants. Control operations were performed by grafting 200μm 3 blocks of mesodermal tissue from the anterior border of stage 20 donor wing buds into the anterior margin of stage 20 host wing buds. Limb buds receiving anterior mesoderm grafts developed normally, and exhibited no duplications of digital elements.

In situ hybridization

The polymerase chain reaction was used to prepare a GHox-8-spe- cific hybridization probe located 3’ to the homeobox that consists primarily of 3’ nontranslated sequence (see Coelho et al., 1991b). A GHox-4.6-specific probe from a region 5’ to the homeobox was prepared as described by Rogina et al. ( 1992). The probes were labeled with [ 32P]dCTP by the random oligonucleotide primer procedure. In situ hybridization was performed on serially sectioned limb buds as described by Mallein-Gerin et al. (1988), except that sections were treated with 10 pg/ml of Proteinase K. At least 5 limb buds of each type were examined by in situ hybridization with each probe.

Expression o/GHox-4.6 and GHox-8 in the limb buds of diplopodia-5 mutant embryos

Diplopodia-5 is a simple autosomal recessive mutation (Olympic et al., 1983). The limb buds of homozygous diplopodia-5 embryos possess 2–4 supernumerary preaxial digits that apparently develop from preaxial (anterior) mesoderm, in addition to normal digits that develop from the postaxial (posterior) mesoderm (Fig. 1). The supernumerary preaxial digits of diplopodia-5 wing buds, which often share a common proximal phalanx, do not readily correspond to any normal digit although they appear to most closely resemble a normal digit 2 (Fig. 1). Homozygous diplopodia- 5 limb buds are distinguishable from normal limb buds by the presence in the mutant limb buds of a thickened apical ectodermal ridge that extends not only along the postaxial mesoderm, but also along the anterior (preaxial) mesoderm of the limb bud. By day 5 (stage 25), diplopodia-5 limb buds are characterized by a broad paddle-shaped distal tip.

Fig. 1.

Skeletal elements of the wings of 10-day normal (A) and diplopodia-5 (B) embryos; and, a limb resulting from the implantation of a retinoic acid-coated bead into the anterior margin of the wing bud at stage 20 (C). In each photograph, anterior is to the left and posterior to the right. Diplopodia-5 wings (B) possess 23 supernumerary preaxial digits that do not readily correspond to the normal postaxial digits. Limb buds that received a retinoic acid bead implant at stage 20 most frequently develop duplicated digits 4 and 3, so that the limbs possess a 43234 digit pattern (C).

Fig. 1.

Skeletal elements of the wings of 10-day normal (A) and diplopodia-5 (B) embryos; and, a limb resulting from the implantation of a retinoic acid-coated bead into the anterior margin of the wing bud at stage 20 (C). In each photograph, anterior is to the left and posterior to the right. Diplopodia-5 wings (B) possess 23 supernumerary preaxial digits that do not readily correspond to the normal postaxial digits. Limb buds that received a retinoic acid bead implant at stage 20 most frequently develop duplicated digits 4 and 3, so that the limbs possess a 43234 digit pattern (C).

In situ hybridization analyses of the pattern of expression of GHox-4.6 and GHox-8 during the early development of the wing buds of normal and diplopodia mutant embryos are shown in Fig. 2. At stage 20/21 in normal limb buds, GHox-4.6 transcripts are present throughout the posterior mesoderm of the limb bud, but no expression of GHox-4.6 is detectable in the preaxial (anterior) mesoderm (Fig. 2A,B). In contrast, in stage 20/21 diplopodia-5 limb buds, in addition to the normal expression throughout the posterior mesoderm, GHox-4.6 expression extends into the preaxial (anterior) mesoderm of the limb buds (Fig. 2C,D). Similarly, at stage 25 in diplopodia limb buds, GHox-4.6 is expressed throughout the distal anterior mesoderm from which the supernumerary digits will develop, as well as throughout the posterior mesoderm from which the normal digits will develop (Fig. 2G,H). In contrast, in normal stage 25 limb buds, GHox-4.6 expression is confined to the posterior mesoderm (Fig. 2E,F).

Fig. 2.

In situ hybridization analysis of GHox-4.6 (A-H) and GHox-8 (I-P) expression during the early development of the wing buds of normal (A,B,E,F; and diplopodia-5 (C,D,G,H; K,L,O,P) embryos. In each photograph the anterior border of the limb bud is on the left and the posterior border is on the right. (A-H) Dark-field and corresponding bright-light autoradiographs of frontal sections through stage 20/21 normal (A,B) and diplopodia (C,D) wing buds and stage 25 normal (E,F) and diplopodia (G,H) wing buds hybridized with a GHox-4.6-specific cDNA probe. In normal limbs buds (A,B,E,F), GHox-4.6 expression is limited to the posterior mesoderm, and does not extend into the anterior mesoderm. In contrast, in diplopodia (C,D,G,H) wing buds, GHox-4.6 expression extends into the distal anterior (preaxial) mesoderm from which supernumerary digits will develop. (I-P) Dark-field and corresponding bright-light autoradiographs of frontal sections through stage 20/21 normal (Id) and diplopodia (K,L) wing buds and stage 25 normal (M,N) and diplopodia (O,P) wing buds hybridized with a GHox-8-specific cDNA probe. In normal stage 20/21 limb buds (U), GHox-8 is expressed in high amounts in the anterior (preaxial) mesoderm and in the apical ectodermal ridge extending along the postaxial periphery of the wing bud (arrow); and, at stage 25 (M,N) GHox-8 is expressed in the mesoderm in the distal and proximal anterior periphery of the limb bud and in the apical ectodermal ridge (arrow), in diplopodia wing buds (K,L,O,P), the normal anterior mesodermal domain of GHox-8 expression is severely impaired or absent, although GHox-8 is expressed in the extensive apical ectodermal ridge of the diplopodia limb buds (arrows).

Fig. 2.

In situ hybridization analysis of GHox-4.6 (A-H) and GHox-8 (I-P) expression during the early development of the wing buds of normal (A,B,E,F; and diplopodia-5 (C,D,G,H; K,L,O,P) embryos. In each photograph the anterior border of the limb bud is on the left and the posterior border is on the right. (A-H) Dark-field and corresponding bright-light autoradiographs of frontal sections through stage 20/21 normal (A,B) and diplopodia (C,D) wing buds and stage 25 normal (E,F) and diplopodia (G,H) wing buds hybridized with a GHox-4.6-specific cDNA probe. In normal limbs buds (A,B,E,F), GHox-4.6 expression is limited to the posterior mesoderm, and does not extend into the anterior mesoderm. In contrast, in diplopodia (C,D,G,H) wing buds, GHox-4.6 expression extends into the distal anterior (preaxial) mesoderm from which supernumerary digits will develop. (I-P) Dark-field and corresponding bright-light autoradiographs of frontal sections through stage 20/21 normal (Id) and diplopodia (K,L) wing buds and stage 25 normal (M,N) and diplopodia (O,P) wing buds hybridized with a GHox-8-specific cDNA probe. In normal stage 20/21 limb buds (U), GHox-8 is expressed in high amounts in the anterior (preaxial) mesoderm and in the apical ectodermal ridge extending along the postaxial periphery of the wing bud (arrow); and, at stage 25 (M,N) GHox-8 is expressed in the mesoderm in the distal and proximal anterior periphery of the limb bud and in the apical ectodermal ridge (arrow), in diplopodia wing buds (K,L,O,P), the normal anterior mesodermal domain of GHox-8 expression is severely impaired or absent, although GHox-8 is expressed in the extensive apical ectodermal ridge of the diplopodia limb buds (arrows).

In normal limb buds at stage 20/21, GHox-8 is expressed in high amounts in the apical ectodermal ridge extending along the distal posterior periphery of the limb bud and in the anterior mesoderm of the limb bud (Fig. 21,J). The anterior domain of GHox-8 expression in normal limb buds appears to correspond to that region of the limb mesoderm which, based on fate maps (Hinchliffe et al., 1981 ) and ridge extirpation experiments (Rowe and Fallon, 1981), will not give rise to skeletal elements. In stage 20/21 diplopodia-5 limb buds, in which supernumerary digits will develop from anterior mesoderm, the normal anterior mesodermal domain of GHox-8 expression is severely impaired or absent (Fig. 2K,L). Similarly, at stage 25, the anterior mesodermal domain of GHox-8 expression present in normal limb buds (Fig. 2M,N) is severely impaired or absent in diplopodia-5 limb buds (Fig. 20,P). The preaxial regions of diplopodia-5 limb buds which exhibit an absence of the normal anterior domain of GHox-8 expression correspond to those regions which exhibit ectopic expression of GHox-4.6. However, in diplopodia-5 limb buds, GHox-8 is expressed in high amounts by the thickened apical ectodermal ridge which in mutant limb buds extends along the distal anterior, as well as posterior periphery (Fig. 2K,L,O,P).

Expression o/GHox-4.6 and GHox-8 in the limb buds of talpid 2 mutant embryos

Talpid2 is a simple autosomal recessive mutation which results in the formation of limbs possessing 6-8 digits which develop from the anterior as well as the posterior mesoderm of the limb bud (Abbott et al., 1960; Dvorak and Fallon, 1991). The digits which develop in talpid2 limb buds are extremely short and broad, morphologically similar to one another, and bear no resemblance to any normal digits (Dvorak and Fallon, 1991). The radius and ulna are also grossly abnormal in talpid limbs, being short, wide, and often fused (Dvorak and Fallon, 1991). talpid1 limb buds are distinguishable from normal limb buds as early as stage 18, at which time the mutant limb buds are wider than normal, do not exhibit the typical asymmetric contour of normal limb buds, and possess a thickened extensive apical ectodermal ridge that extends continuously along the postaxial and preaxial periphery of the limb buds (Abbott et al., 1960; Dvorak and Fallon, 1991).

As shown in Fig. 3, at stages 20/21 and 25, GHox-4.6 transcripts are present in high amounts throughout the anterior as well as the posterior mesoderm of talpid1 limb buds (Fig. 3C,D, G,H). In contrast, GHox-4.6 transcripts are confined to posterior mesoderm in normal limb buds (Fig. 3A,B, E,F).

Fig. 3.

In situ hybridization analysis of GHox-4.6 (A-H) and GHox-8 (I-P) expression during the early development of the wing buds of normal (A,B,E,F; I,J,M,N) and talpid1 (C,D,G,H; K,L,O,P) embryos. In each photograph the anterior border of the limb bud is on the left and the posterior border is on the right. (A-H) Dark-field and corresponding bright-light autoradiographs of frontal sections through stage 20/21 normal (A,B) and talpid (C,D) wing buds and stage 25 normal (E,F) and talpid (G,H) wing buds hybridized with a GHox-4.6- specific cDNA probe. GHox-4.6 is expressed in high amounts throughout the anterior as well as the posterior mesoderm of talpid limb buds (C,D,G,H), in contrast to normal limb buds (A,B,E,F) in which GHox-4.6 expression is confined to the posterior mesoderm. (I-P) Dark-field and corresponding bright-light autoradiographs of frontal sections through stage 20/21 normal (I,J) and talpid (K,L) wing buds and stage 25 normal (M,N) and talpid (O,P) wing buds hybridized with a GHox-8-spec\hc cDNA probe. The anterior mesoderm of talpid limb buds (K,L,O,P) exhibits a severe impairment or absence of the normal (I,J,M,N) anterior domain of GHox-8 expression. However, GHox-8 is expressed in high amounts in the extensive apical ectodermal ridge of talpid limb buds (K,L,O,P; arrows), as well as in the apical ectodermal ridge of normal limb buds (I,J,M,N; arrows).

Fig. 3.

In situ hybridization analysis of GHox-4.6 (A-H) and GHox-8 (I-P) expression during the early development of the wing buds of normal (A,B,E,F; I,J,M,N) and talpid1 (C,D,G,H; K,L,O,P) embryos. In each photograph the anterior border of the limb bud is on the left and the posterior border is on the right. (A-H) Dark-field and corresponding bright-light autoradiographs of frontal sections through stage 20/21 normal (A,B) and talpid (C,D) wing buds and stage 25 normal (E,F) and talpid (G,H) wing buds hybridized with a GHox-4.6- specific cDNA probe. GHox-4.6 is expressed in high amounts throughout the anterior as well as the posterior mesoderm of talpid limb buds (C,D,G,H), in contrast to normal limb buds (A,B,E,F) in which GHox-4.6 expression is confined to the posterior mesoderm. (I-P) Dark-field and corresponding bright-light autoradiographs of frontal sections through stage 20/21 normal (I,J) and talpid (K,L) wing buds and stage 25 normal (M,N) and talpid (O,P) wing buds hybridized with a GHox-8-spec\hc cDNA probe. The anterior mesoderm of talpid limb buds (K,L,O,P) exhibits a severe impairment or absence of the normal (I,J,M,N) anterior domain of GHox-8 expression. However, GHox-8 is expressed in high amounts in the extensive apical ectodermal ridge of talpid limb buds (K,L,O,P; arrows), as well as in the apical ectodermal ridge of normal limb buds (I,J,M,N; arrows).

The anterior mesoderm of talpid limb buds, in which GHox-4.6 is ectopically expressed and from which preaxial digits will develop, exhibits a severe impairment or absence of the normal anterior domain of GHox-8 expression (Fig. 3K,L, O,P). However, GHox-8 is expressed in high amounts in the extensive apical ectodermal ridge of talpid limb buds (Fig. 3K,L, O,P), as well as in the apical ectodermal ridge of normal limb buds (Fig. 31,J, M,N). Expression of GHox-4.6 and GHox-8 in response to retinoic acid-coated beads and polarizing zone implants Implantation of retinoic acid-coated beads or the zone of polarizing activity into the anterior margin of stage 20/21 wing buds results in the formation from the anterior mesoderm of duplicated digital elements that exhibit mirror-image symmetry to the normal digits that develop from posterior mesoderm. In the present study, we have found that limb buds receiving retinoic acid-coated beads or polarizing zone implants most frequently (13/17 cases) develop duplicated digits 4 and 3, so that the limbs possess a 43234 digit pattern (Fig. 1), although occasionally 432234 (2/17 cases) or (2/3)2234 (2/17 cases) digit patterns are observed.

We have confirmed the previous observations of Izpisua-Belmonte et al. ( 1991 ) and Nohno et al. ( 1991 ) that limb buds that received either a retinoic acid-coated bead or polarizing zone implant exhibit an ectopic domain of GHox-4.6 expression in the distal anterior mesoderm (data not shown). Limb buds receiving a retinoic acid-coated bead implant (Fig. 4C-H) lack the distal anterior domain of GHox-8 expression that is present in control limb buds (Fig. 4A,B) (see also Yokouchi et al., 1991). Similarly, limb buds receiving polarizing zone grafts (Fig. 5C,D) lack the distal anterior domain of GHox-8 expression that is present in control limb buds (Fig. 5A,B) which received grafts of anterior limb bud mesoderm. Thus, retinoic acid-coated bead or polarizing zone implants concomitantly elicit an induction of GHox-4.6 expression and a suppression of GHox-8 expression in the anterior mesoderm. Polarizing zone and retinoic acid-coated bead implants both result in the formation of an ectopic thickened apical ectodermal ridge along the anterior mesoderm of the limb bud. The thickened anterior apical ectodermal ridges (Figs 4E,F and 5C,D), as well as the posterior apical ectodermal ridges (Figs 4G,H and 5C,D) of limbs that received polarizing zone or retinoic acid bead implants exhibit high expression of GHox-8. This latter observation differs from that of Yokouchi et al. (1991) who recently reported that retinoic acid-coated bead implants which elicit mirror image duplications decrease GHox-8 expression in the apical ectodermal ridge.

Fig. 4.

Expression of GHox-8 in wing buds fixed 18 hours after receiving vehicle (DMSO)-coated (A,B) or retinoic acid-coated (C-H) bead implants into the anterior margin of the limb buds at stage 20. In each photograph the anterior border of the limb bud is on the left and the posterior border is on the right The retinoic acid-treated limb buds (C-H which represent-3 separate specimens) exhibit an absence of the distal anterior domain of GHox-8 expression that is present in control limb buds (A.B) that received a vehicle-coated bead implant. Note that in the retinoic acid-treated limb buds GHox-8 is expressed in the thickened apical ectodermal ridge that has formed along the anterior periphery of the limb bud (E,F) (arrow), as well as in the apical ectodermal ridge along the posterior periphery of the limb bud (G,H) (arrow).

Fig. 4.

Expression of GHox-8 in wing buds fixed 18 hours after receiving vehicle (DMSO)-coated (A,B) or retinoic acid-coated (C-H) bead implants into the anterior margin of the limb buds at stage 20. In each photograph the anterior border of the limb bud is on the left and the posterior border is on the right The retinoic acid-treated limb buds (C-H which represent-3 separate specimens) exhibit an absence of the distal anterior domain of GHox-8 expression that is present in control limb buds (A.B) that received a vehicle-coated bead implant. Note that in the retinoic acid-treated limb buds GHox-8 is expressed in the thickened apical ectodermal ridge that has formed along the anterior periphery of the limb bud (E,F) (arrow), as well as in the apical ectodermal ridge along the posterior periphery of the limb bud (G,H) (arrow).

Fig. 5.

Expression of GHox-8 in wing buds fixed 18 hours after receiving an anterior limb mesoderm implant (control; A, darkfield; B, bright-light) or polarizing zone implant (C, dark-field; D, bright-light) into the anterior margin of the limb bud at stage 20. In each photograph the anterior border of the limb bud is on the left and the posterior border is on the right. The limb bud that received a polarizing zone graft (C,D) exhibits an absence of the distal anterior domain of GHox-8 expression that is present in control (A,B) limb buds. Note that in the limb that received a polarizing zone graft (C,D) GHox-8 is expressed in the thickened apical ectodermal ridge that has formed along the anterior periphery of the limb bud (arrow), as well as in the apical ectodermal ridge along the posterior periphery of the limb bud (arrow).

Fig. 5.

Expression of GHox-8 in wing buds fixed 18 hours after receiving an anterior limb mesoderm implant (control; A, darkfield; B, bright-light) or polarizing zone implant (C, dark-field; D, bright-light) into the anterior margin of the limb bud at stage 20. In each photograph the anterior border of the limb bud is on the left and the posterior border is on the right. The limb bud that received a polarizing zone graft (C,D) exhibits an absence of the distal anterior domain of GHox-8 expression that is present in control (A,B) limb buds. Note that in the limb that received a polarizing zone graft (C,D) GHox-8 is expressed in the thickened apical ectodermal ridge that has formed along the anterior periphery of the limb bud (arrow), as well as in the apical ectodermal ridge along the posterior periphery of the limb bud (arrow).

Roles o/GHox-4.6 and GHox-8 in specification of skeletal and non-skeletal-forming regions of the limb bud

We have found that the formation of supernumerary digital elements from the anterior mesoderm of diplopodia-5 and talpuP- mutant limb buds is preceded by the ectopic expression of GHox-4.6 in the anterior mesoderm and the coincident severe impairment or cessation of GHox-8 expression in the anterior mesoderm. Furthermore, the induction of dupli-cated digital elements in anterior mesoderm by the implantation of the zone of polarizing activity or retinoic acid-coated beads is accompanied by a concomitant induction of GHox-4.6 expression and suppression of GHox-8 expression in the anterior mesoderm (see also Izpisua-Belmonte et al., 1991; Nohno et al., 1991; Yokouchi et al., 1991). These observations suggest that the anterior mesoderm of the polydactylous limb buds is “posteriorized” and support the suggestion that GHox-8 and GHox-4.6, respectively, are involved in specifying the anterior non-skeletal and posterior digitforming regions of the limb bud.

GHox-4.6 is ectopically expressed in the anterior mesoderm in diplopodia limb buds, talpid limb buds, and in response to polarizing zone or retinoic acid-coated bead implants, despite the fact that distinctly different morphological types of supernumerary digital elements are formed in each of these situations. This suggests that the expression of GHox-4.6 in and of itself does not specify the type of digit that develops. Rather, its expression may be required for the formation of any digit type, be it normal or abnormal. These observations provide support for the notion that the specific types of digits that develop might depend on the combinatorial expression of GHox-4.6 and other 5’ members of the HOX-4 cluster such as GHox-4.7 and GHox-4.8 (see Izpisua-Belmonte et al., 1991; Nohno et al., 1991).

Possible interrelationships between GHox-8, GHox-4.6, the zone of polarizing activity, the apical ectodermal ridge, and apical ectoderm maintenance activity

Implantation experiments have shown that the anterior mesoderm of talpid2 limb buds, which exhibits ectopic expression of GHox-4.6 and absence of GHox-8 expression, does not possess polarizing activity, and that the zone of polarizing activity is present in its normal posterior location in the mutant limb buds (MacCabe and Abbott, 1974). The anterior mesoderm of diplopodia-\ and -4 mutant limbs buds, which are phenotypically similar to the diplopodia-5 mutants used in the present study, also lacks polarizing activity (MacCabe and Abbott, 1974; MacCabe et al., 1975). Thus, the altered expression pattern of GHox-4.6 and GHox-8 in the anterior mesoderm of the polydactylous mutant limb buds, and rhe subsequent formation of supernumerary digits from the anterior mesoderm, apparently do not result from high concentrations of a morphogen emanating from the polarizing zone.

The regions of the mesoderm of normal and polydactylous mutant limb buds in which GHox-4.6 expression is present and GHox-8 expression is absent correspond to those regions of the mesoderm that have been suggested to be the source of a factor or activity (apical ectoderm maintenance factor) that is thought to be required to maintain the apical ectodermal ridge in a thickened functional condition, allowing it to direct limb outgrowth and the formation of skeletal elements (Zwilling, 1961; Saunders and Gasseling, 1968; MacCabe and Abbott, 1974; MacCabe et al., 1975). Indeed, the mutant and experimentally induced polydactylous limb buds, which express GHox-4.6 and fail to express GHox-8 in both the anterior and posterior mesoderm, possess thickened apical ectodermal ridges that extend along the anterior as well as the posterior periphery of the limb buds, and when the mesoderm of diplopodia or talpid limb buds is recombined with normal limb ectoderm, it elicits the formation of a thickened apical ectodermal ridge along the anterior ectoderm (Goetinck and Abbott, 1964; MacCabe et al., 1975). These observations suggest that there may be a regulatory relationship between GHox-4.6 expression, absence of GHox-8 expression, and the production of a factor or factors that maintain a thickened functional apical ectodermal ridge. Alternatively, the altered expression of GHox-4.6 and GHox-8 in the anterior mesoderm of the polydactylous limb buds might be regulated by the ectopic anterior apical ectodermal ridges, or the altered expression of these genes in the anterior mesoderm may be a consequence of posteriorization, and not necessarily causally related to it.

Independently regulated functional domains of /GHox-8 during limb development

Although the anterior mesodermal domain of GHox-8 expression is severely impaired in diplopodia-5, talpid2 and experimentally induced polydactylous limb buds, this gene is expressed by the ectopic thickened apical ectodermal ridges of the polydactylous limb buds, as well as the apical ectodermal ridge of normal limb buds. The correlation between the presence of thickened functional apical ectodermal ridges and the expression of GHox-8 by such ridges provides support for the notion that GHox-8 expressed by the ridge is involved in its function. We have previously found that GHox-8 expression is severely impaired in the distal ectoderm of limbless mutant limb buds which lack an apical ectodermal ridge and fail to undergo normal outgrowth (Coelho et al., 1991a).

Our studies also indicate that the anterior mesodermal and apical ectodermal ridge domains of GHox-8 expression are independently regulated. In diplopodia-5 and talpid1 mutant limb buds GHox-8 continues to be expressed by the apical ectodermal ridge, while its anterior mesodermal domain of expression is impaired, and, conversely, in limbless mutant limb buds the expression of GHox-8 by the apical ectodermal ridge is severely impaired, while its expression in the anterior mesoderm of limbless limb buds is normal (Coelho et al., 1991a). These two independently regulated spatial domains of GHox-8 expression appear to reflect different functions of the gene in the two locations, the ridge domain of expression being involved in limb outgrowth (Coelho et al., 1991a) and the anterior mesodermal domain being involved in specification of the non-skeletal-forming regions of the limb mesoderm. GHox-8 may also have yet another independently regulated functional domain of expression, since it is also expressed in discrete regions of the developing limb bud such as the posterior necrotic zone, anterior necrotic zone, and interdigital mesenchyme in which programmed cell death is occurring (Coelho et al., 1991b, 1992). In this regard, Whiting et al. (1991) have recently shown that spatially distinct regions of expression of the mouse HOX gene Hox-2.6 in the developing neural tube are independently regulated via multiple spatially specific enhancer elements in the Hox-2.6 gene. Similar spatially specific enhancers may conceivably play a role in regulating the distinct functional domains of GHox-8 expression during limb development.

This paper is dedicated to John W. Saunders, Jr. whose elegant studies on limb development have provided the foundation for much of our work. We would also like to thank Dr. Saunders for critically analyzing this paper, and providing numerous helpful suggestions. We thank Louis Pierro and Jean Haines for providing diplopodia-5 mutant embryos, John Fallon and Karen Krabbenhoft for providing the talpid2 embryos, and Blanka Rogina for providing the GHox-4.6 cDNA. This work was supported by NIH grant HD22610 to R.A.K. andW.B.U.

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