ABSTRACT
It has been suggested that the reciprocal expression of the chicken homeobox-containing genes GHox-8 and GHox-7 by the apical ectodermal ridge and subjacent limb mesoderm might be involved in regulating the proximodistal outgrowth of the developing chick limb bud. In the present study the expression of GHox-7 and GHox-8 has been examined by in situ and dot blot hybridization in the developing limb buds of limbless mutant chick embryos. The limb buds of homozygous mutant limbless embryos form at the proper time in development (stage 17/18), but never develop an apical ectodermal ridge, fail to undergo normal elongation, and eventually degenerate. At stage 18, which is shortly following the formation of the limb bud, the expression of GHox-7 is considerably reduced (about 3-fold lower) in the mesoderm of limbless mutant limb buds compared to normal limb bud mesoderm. By stages 20 and 21, as the limb buds of limbless embryos cease outgrowth, GHox-7 expression in limbless mesoderm declines to very low levels, whereas GHox-7 expression increases in the mesoderm of normal limb buds which are undergoing outgrowth. In contrast to GHox-7, expression of GHox-8 in limbless mesoderm at stage 18 is quantitatively similar to its expression in normal limb bud mesoderm, and in limbless and normal mesoderm GHox-8 expression is highly localized in the anterior mesoderm of the limb bud. In normal limb buds, GHox-8 is also expressed in high amounts by the apical ectodermal ridge. However, expression of GHox-8 is severely impaired in the ectoderm of limbless mutant limb buds, which lack an apical ectodermal ridge. The severely impaired expression of GHox-7 in the mesoderm of limbless limb buds suggests that the normal expression of GHox-7 by limb mesoderm during limb bud outgrowth may be regulated by the apical ectodermal ridge, and further suggests the possibility that expression of GHox-7 by subridge mesenchymal cells may be required for normal limb outgrowth to occur. Furthermore, the severely impaired expression of GHox-8 by limbless ectoderm suggests GHox-8 may indeed be involved in ridge function. These observations also raise the interesting possibility that normal GHox-8 expression by the apical ectodermal ridge may be required for normal expression of GHox-7 by the subridge mesenchymal cells.
Introduction
Homeobox-containing (HOX) genes, which encode transcription factors containing a highly conserved 61 amino acid DNA-binding motif, have been implicated in a variety of patterning events during vertebrate limb development (see Tabin, 1991 for review). We have recently described the pattern of expression in the developing chick limb bud of the chicken HOX genes, GHox-8 (Coelho et al. 1991) and GHox-7 (Coelho, Sumoy, Kosher and Upholt, unpublished data), two members of the Drosophila msh-like family of vertebrate HOX genes. The spatial pattern of expression of GHox-8 and GHox-7 along the anterior-posterior axis of the developing chick limb bud has suggested the possible involvement of these genes in the specification of anterior positional identity, and their expression in discrete regions of the limb bud in which programmed cell death is occurring has suggested their possible role in the regulation of the programmed cell death involved in shaping the contours of the limb bud (Coelho et al. 1991; Coelho et al. unpublished data). In addition, GHox-7 (Coelho et al. unpublished data) and its mouse cognate Hox-7 (Hill et al. 1989; Robert et al. 1989) are expressed by the mesenchymal cells of the limb bud directly subjacent to the apical ectodermal ridge. This suggests that GHox-7 might be involved in the response of limb mesenchymal cells to the influence of the ridge, which is required for the outgrowth of the limb bud and the formation of the various skeletal elements of the limb in their appropriate sequence along the proximo-distal axis. Furthermore, GHox-8 is expressed in high amounts by the apical ectodermal ridge itself, suggesting its possible involvement in ridge function (Coelho et al. 1991).
In order to gain further insight into the possible roles of GHox-7 and GHox-8 in patterning events during chick limb development, in the present study we have examined the expression of these genes in the developing limb buds of limbless mutant chick embryos. Limbless is a simple Mendelian autosomal recessive mutation which, in the homozygous condition, yields chicks lacking fore- and hindlimbs (Prahlad et al. 1979; Fallon et al. 1983). Homozygous mutant limbless embryos form limb buds at the proper time (stage 17–18) in development that appear grossly normal (Fallon et al. 1983; Carrington and Fallon, 1988). However, the limb buds of limbless embryos never develop an apical ectodermal ridge, and fail to undergo normal outgrowth. By late stage 22, the mesodermal cells of limbless limb buds undergo massive necrosis, and the limb buds disappear by stage 24 (Fallon et al. 1983). The ectoderm is the limb bud component affected by the limbless gene. Microsurgical recombination experiments have shown that the mesoderm of limbless mutant embryos forms a normal limb in response to normal apical ectodermal ridge-containing limb ectoderm, whereas limbless ectoderm is unable to support the growth and development of normal limb mesoderm (Fallon et al. 1983; Carrington and Fallon, 1988).
In the present study, we have found that the expression of GHox-8 is severely impaired in the ectoderm of limbless limb buds, which lack an apical ectodermal ridge, and that the expression of GHox-7 is severely impaired in the mesoderm of limbless limb buds. These observations suggest that the reciprocal expression of GHox-8 and GHox-7 by the apical ectodermal ridge and subjacent limb mesoderm is involved in the regulation of normal limb bud elongation.
Materials and methods
Preparation of tissue
Limbless mutant embryos were obtained from mating members of a heterozygous flock maintained at the University of Wisconsin, Madison, WI. After stage 19 (Hamburger and Hamilton, 1951), the limb buds of homozygous limbless embryos are morphologically distinguishable from normal limb buds (see Carrington and Fallon, 1988). Prior to this stage, however, the limb buds of homozygous limbless embryos appear grossly normal. Therefore, to be sure that limb buds being used for analysis at these early stages possessed the mutant genotype, the following procedure was utilized. Windows were cut into the shells of the limbless 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, 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 until their genotype was established. Limb buds to be used for determination of steady-state mRNA levels by cytoplasmic dot hybridization were individually stored at -70°C in 10mM Tris-HCl, pH7.0,1 mM EDTAcontaining 20 U RNAsin (Promega). Following the establishment of genotype, individual samples corresponding to either limbless or normal limb buds were pooled and processed as described below. In some experiments, the ectoderm and mesoderm of the limb buds were separated following treatment for 15 min at 37 °C with dispase II (Boehringer-Mannheim) diluted 1:2 with Earles’ balanced salt solution, and processed separately.
Determination of mRNA levels and in situ hybridization
To prepare specific GHox-7 and GHox-8 hybridization probes that exhibit little sequence similarity, the polymerase chain reaction was used to amplify regions of GHox-7 and GHox-8 cDNAs located 3’ to the homeobox that consist primarily of nontranslated sequence (Coelho et al. 1991; Coelho et al. unpublished data). The probes were labeled with 32P-dCTP by the random oligonucleotide primer procedure of Feinberg and Vogelstein (1984).
Cytoplasmic levels of GHox-7 and GHox-8 mRNAs were determined by the cytoplasmic dot hybridization procedure (White and Bancroft, 1982) as previously described (Kosher et al. 1986), and quantified as described (Coelho et al. 1991). Ghox-7 and GHox-8 mRNA levels were normalized to the total poly(A)+ mRNA content of samples by hybridizing portions of the same RNA samples used for determination of specific mRNAs with 32P-labeled oligo(dT)20 as described by Harley (1987). In situ hybridization was performed on serially sectioned limb buds as described by Mallein-Gerin et al. (1988), except that sections were treated with 10μgml-1 of Proteinase K.
Results
Expression of GHox-7 in the limb buds of normal and limbless mutant embryos
In situ hybridization analyses of the pattern of expression of GHox-7 during the early development of the limb buds of normal and limbless mutant embryos are shown in Fig. 1. At stages 17-19 in normal limb buds (Fig. 1A-D), GHox-7 transcripts are present throughout most of the mesenchyme of the limb, with only the mesoderm at the extreme posterior border exhibiting little detectable expression. GHox-7 expression is also detectable in the mesenchyme of the limb buds of limbless embryos at stages 17-19 (Fig. 1G-J), although its level of expression (Fig. 1G-J) appears to be lower than in normal limb bud mesenchyme (Fig. 1A-D). At subsequent stages of development (stages 20-21), when the limb buds of limbless embryos have ceased outgrowth, GHox-7 expression declines to very low levels in limbless mesenchyme (Fig. 1K,L). In contrast, GHox-7 continues to be expressed in high amounts at stage 20/21 in normal limb mesenchyme, with an expression domain extending in an asymmetric arc from the anterior border of the limb bud to the cells directly subjacent to the ridge (Fig. 1E,F).
In situ hybridization analysis of GHox-7 expression during the early development of the limb buds of normal (A-H) and limbless (G-L) embryos. Sections were hybridized with a 32P-labeled GHox-7-specific cDNA probe. Dark field and corresponding bright light autoradiographs of frontal sections through stage 17 normal (A,B) and limbless (G-H) limb buds; stage 18/19 normal (C,D) and limbless limb buds (I,J); and, stage 20/21 normal (E,F) and limbless (K,L) limb buds. The anterior (a) and posterior (p) borders of the limb buds are indicated on the bright light photographs. At stages 17-19 in normal (A-D) and limbless (G-J) limb buds, GHox-7 transcripts are present throughout most of the mesenchyme of the limb, with only the mesoderm at the extreme posterior border exhibiting little detectable expression. However, GHox-7 expression at stages 17-19 in limbless mesenchyme (G-J) appears to be lower than in normal (A–D) mesenchyme. By stage 20/21, when the limb buds of limbless embryos have ceased outgrowth, GHox-7 expression declines to very low levels in limbless mesenchyme (K,L). In contrast, GHox-7 continues to be expressed in high amounts at stage 20/21 in normal limb mesenchyme, with an expression domain extending in an asymmetric arc from the anterior border of the limb bud to the cells directly subjacent to the apical ectodermal ridge (E,F). The rim of silver grains along the edges of the sections are not over tissue, and presumably result from the accumulation of emulsion between the edge of the sections and the slides.
In situ hybridization analysis of GHox-7 expression during the early development of the limb buds of normal (A-H) and limbless (G-L) embryos. Sections were hybridized with a 32P-labeled GHox-7-specific cDNA probe. Dark field and corresponding bright light autoradiographs of frontal sections through stage 17 normal (A,B) and limbless (G-H) limb buds; stage 18/19 normal (C,D) and limbless limb buds (I,J); and, stage 20/21 normal (E,F) and limbless (K,L) limb buds. The anterior (a) and posterior (p) borders of the limb buds are indicated on the bright light photographs. At stages 17-19 in normal (A-D) and limbless (G-J) limb buds, GHox-7 transcripts are present throughout most of the mesenchyme of the limb, with only the mesoderm at the extreme posterior border exhibiting little detectable expression. However, GHox-7 expression at stages 17-19 in limbless mesenchyme (G-J) appears to be lower than in normal (A–D) mesenchyme. By stage 20/21, when the limb buds of limbless embryos have ceased outgrowth, GHox-7 expression declines to very low levels in limbless mesenchyme (K,L). In contrast, GHox-7 continues to be expressed in high amounts at stage 20/21 in normal limb mesenchyme, with an expression domain extending in an asymmetric arc from the anterior border of the limb bud to the cells directly subjacent to the apical ectodermal ridge (E,F). The rim of silver grains along the edges of the sections are not over tissue, and presumably result from the accumulation of emulsion between the edge of the sections and the slides.
In order to confirm and quantify the differences in GHox-7 expression detectable by in situ hybridization in limbless and normal limb buds, cytoplasmic dot hybridization was used to compare the steady-state cytoplasmic levels of GHox-7 mRNA in limbless and normal limb buds. As shown in Table 1, the amount of GHox-7 mRNA is about 3-fold lower in the limb buds of limbless embryos at stage 18 than in normal limb buds and 8- to 9-fold lower in limbless limb buds than in normal limb buds at stage 20. The greater difference in the levels of expression between limbless and normal limb buds at stage 20 reflects both a decline in GHox-7 mRNA levels in limbless limb buds at stage 20 compared to stage 18 and an increase in GHox-7 mRNA in normal limb buds at stage 20 compared to stage 18.
It is important to note that although GHox-7 mRNA levels are considerably reduced in the limb buds of limbless embryos at stage 18 (Table 1), the amount of GHox-8 mRNA in limbless mesenchyme at stage 18 is comparable to its levels in normal limb mesenchyme (Table 2). Furthermore, stage 18 limbless and normal limb buds possess comparable amounts of total poly(A)+ RNA. Thus, there appears to be a selective reduction in GHox-7 expression in the limb buds of limbless mutant embryos at stage 18. It is also significant that the reduction in GHox-7 expression in limbless limb buds compared to normal limb buds at stage 18 is detectable before the limbless phenotype is obvious and prior to the onset of degenerative changes in limbless mesoderm. Even at stage 20, GHox-7 expression is reduced to a considerably greater extent (8- to 9-fold) in limbless limb buds compared to normal limb buds (Table 1) than is the expression of GHox-8, which is only about 2-fold lower in limbless than in normal limb buds (Table 2).
Expression of GHox-8 in the limb buds of normal and limbless mutant embryos
In normal limb buds at stage 17/18, GHox-8 is expressed at high amounts in the anterior mesenchyme of the limb mesoderm, and high expression does not extend into the mesenchyme directly subjacent to the apical ectodermal ridge (Fig. 2A,B). GHox-8 transcripts are also highly localized in the anterior mesenchyme of limbless limb buds at these stages (Fig. 2G,H), and the level of GHox-8 expression in the anterior limbless mesenchyme appears to be comparable to its level of expression in normal limb mesenchyme (Fig. 2A,B). At subsequent stages of development (stages 19-20), GHox-8 continues to be expressed at moderately high levels in the anterior mesenchyme of limbless limb buds (Fig. 2I–L), although its level of expression at these later stages appears to be somewhat lower than in normal anterior limb bud mesenchyme (Fig. 2C-F). In normal limb buds, in addition to being expressed in the anterior mesenchyme, GHox-8 is expressed in high amounts by the apical ectodermal ridge (Fig. 2A-D and Fig. 3A). In contrast, little or no GHox-8 transcripts are detectable by in situ hybridization in the distal ectoderm of the limb buds of limbless embryos at any stage of development (Fig. 2G-L and Fig. 3B).
In situ hybridization analysis of GHox-8 expression during the early development of the limb buds of normal (A–F) and limbless (G-L) embryos. Sections were hybridized with a 32P-labeled GHox-8-specific cDNA probe (see Coelho et al. 1991). Dark field and corresponding bright light autoradiographs of frontal sections through stage 17/18 normal (A,B) and limbless (G-H) limb buds; stage 19 normal (C,D) and limbless limb buds (I,J); and, stage 20 normal (E,F) and limbless (K,L) limb buds. The anterior (a) and posterior (p) borders of the limb buds are indicated on the bright light photographs. At stage 17/18, in limbless (G,H) and normal (A,B) limb buds GHox-8 transcripts are present in similar amounts in the anterior mesoderm of the limb buds. At subsequent stages of development, GHox-8 continues to be expressed at moderately high levels in the anterior mesenchyme of limbless limb buds (I–L), although its level of expression appears to be somewhat lower than in normal anterior limb mesenchyme (C-F). In normal limb buds, in addition to being expressed in the anterior mesenchyme, GHox-8 is expressed in high amounts by the apical ectodermal ridge (see arrows in A,C). (Note that in the section of the normal stage 20 limb bud shown in E,F the apical ectodermal ridge was lost during processing. A stage 20 apical ectodermal ridge exhibiting high level GHox-8 expression has previously been published; see Fig. 9 in Coelho et al. 1991). In contrast, little or no GHox-8 transcripts are detectable by in situ hybridization in the distal ectoderm of the limb buds of limbless embryos (G–L).
In situ hybridization analysis of GHox-8 expression during the early development of the limb buds of normal (A–F) and limbless (G-L) embryos. Sections were hybridized with a 32P-labeled GHox-8-specific cDNA probe (see Coelho et al. 1991). Dark field and corresponding bright light autoradiographs of frontal sections through stage 17/18 normal (A,B) and limbless (G-H) limb buds; stage 19 normal (C,D) and limbless limb buds (I,J); and, stage 20 normal (E,F) and limbless (K,L) limb buds. The anterior (a) and posterior (p) borders of the limb buds are indicated on the bright light photographs. At stage 17/18, in limbless (G,H) and normal (A,B) limb buds GHox-8 transcripts are present in similar amounts in the anterior mesoderm of the limb buds. At subsequent stages of development, GHox-8 continues to be expressed at moderately high levels in the anterior mesenchyme of limbless limb buds (I–L), although its level of expression appears to be somewhat lower than in normal anterior limb mesenchyme (C-F). In normal limb buds, in addition to being expressed in the anterior mesenchyme, GHox-8 is expressed in high amounts by the apical ectodermal ridge (see arrows in A,C). (Note that in the section of the normal stage 20 limb bud shown in E,F the apical ectodermal ridge was lost during processing. A stage 20 apical ectodermal ridge exhibiting high level GHox-8 expression has previously been published; see Fig. 9 in Coelho et al. 1991). In contrast, little or no GHox-8 transcripts are detectable by in situ hybridization in the distal ectoderm of the limb buds of limbless embryos (G–L).
High magnification bright light autoradiographs through the distal ectoderm of stage 18 normal (A) and limbless (B) limb buds hybridized with a GHox-8-specific cDNA probe. Hybridization is detectable throughout the distal apical ectoderm of the normal limb bud (A) (arrow), but little, or no hybridization is detectable in the distal ectoderm of the limbless limb bud (B) (arrow), m indicates the subridge mesenchyme.
High magnification bright light autoradiographs through the distal ectoderm of stage 18 normal (A) and limbless (B) limb buds hybridized with a GHox-8-specific cDNA probe. Hybridization is detectable throughout the distal apical ectoderm of the normal limb bud (A) (arrow), but little, or no hybridization is detectable in the distal ectoderm of the limbless limb bud (B) (arrow), m indicates the subridge mesenchyme.
Since our in situ hybridization studies indicated that GHox-8 is expressed in high amounts by the apical ectodermal ridge of normal limb buds but not in detectable amounts by the distal ectoderm of limbless limb buds, the ectoderm and mesoderm of limbless and normal limb buds were analyzed separately by cytoplasmic dot hybridization for steady-state GHox-8 mRNA levels. At stage 18, the amount of GHox-8 mRNA in whole limbless limb buds (Table 2) or in limbless mesoderm (Table 4) is similar to the amount of GHox-8 mRNA in the limb buds or mesoderm of normal embryos. In contrast, at stage 18, the amount of GHox-8 mRNA in the ectoderm of limbless limb buds is 3- to 7-fold lower than its expression in the ectoderm of normal limb buds (Table 3). Thus, our combined in situ and dot blot hybridization analyses indicate that initially following the formation of the limb bud, the spatial pattern and level of expression of GHox-8 is relatively normal in limbless mesoderm, but that GHox-8 expression is severely impaired in the ectoderm of limbless limb buds, which lack a functional apical ectodermal ridge. The ectoderm of limbless limb buds continues to exhibit a severely impaired expression of GHox-8 at stage 20 (Table 3). At stage 20, the amount of GHox-8 mRNA is 2- to 4-fold lower in whole limbless limb buds (Table 2) or in limbless mesoderm (Table 4) than in normal limbs or normal limb bud mesoderm.
Relative steady-state cytoplasmic GHox-8 mRNA levels in limbless and normal limb bud ectoderm

Discussion
Involvement of GHox-7 in the outgrowth of limb mesoderm in response to the apical ectodermal ridge
The severely impaired expression of GHox-7 in the mesoderm of limbless limb buds which lack an apical ectodermal ridge suggests that normal expression of GHox-7 in limb mesoderm during limb bud outgrowth may be regulated by the ridge. Consistent with this possibility, Davidson et al. (1991) have recently shown that the 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. The altered expression of GHox-7 in limbless limb bud mesoderm also raises the possibility that the expression of GHox-7 by subridge mesenchymal cells may be required for normal limb bud elongation to occur.
Although GHox-7 expression is severely reduced in the mesoderm of limbless limb buds at stage 18, it is not completely absent. This indicates that the apical ectodermal ridge does not regulate the onset of GHox-7 expression by limb mesoderm. Rather, it appears that the ridge is required for maintenance and amplification of GHox-7 expression following the formation of the limb bud. These data suggest that in limbless limb buds, which lack an apical ectodermal ridge, GHox-7 expression cannot be maintained or amplified, and parallel to this limb outgrowth fails to occur.
Involvement of GHox-8 in apical ectodermal ridge function
The severely impaired expression of GHox-8 in the ectoderm of limbless limb buds, which lack an apical ectodermal ridge, suggests that GHox-8 may indeed be involved in ridge differentiation or function. Since GHox-8 is a homeobox-containing gene, it probably acts as a transcription factor that regulates the expression of other genes. Therefore, it is possible that GHox-8 is involved in regulating the expression of a molecule or molecules synthesized and secreted by the apical ectodermal ridge involved in directing the outgrowth of underlying limb mesenchymal cells. Furthermore, the concomitant deficient expression of GHox-8 in the distal ectoderm and of GHox-7 in the subridge mesoderm of limbless limb buds suggests the interesting possibility that normal GHox-8 expression by the ridge may be required for normal expression of GHox-7 by the subridge mesenchymal cells.
Although GHox-8 expression is severely impaired in limbless ectoderm, initially following the formation of the limb bud its expression in the mesoderm of normal and limbless limb buds is comparable. Furthermore, in limbless and normal mesoderm, high GHox-8 expression is localized in the anterior mesoderm. The relatively normal expression of GHox-8 in the mesoderm of limbless limb buds suggests that its highly localized anterior domain of mesodermal expression is not regulated by the apical ectodermal ridge, and provides further support for the notion that GHox-8 may be involved in the specification of anterior positional identity (Coelho et al. 1991). It should be noted, however, that Davidson et al. (1991) have reported that a low level expression of mouse Hox-8.1, as well as a high level expression of mouse Hox-7.1, is induced in proximal mouse limb mesoderm implanted subjacent to the apical ridge of chick limb buds.
Implications concerning the nature of genes involved in regulating apical ectodermal ridge function
The ectoderm is the limb bud component affected by the limbless gene, not limb bud mesoderm (Carrington and Fallon, 1988). It is significant, therefore, that although GHox-8 expression is severely impaired in limbless ectoderm, its expression in limb bud mesoderm is relatively normal. Thus, the expression of GHox-8 in the apical ectodermal ridge might be regulated by the normal product of the limbless gene, while the expression of GHox-8 in anterior limb bud mesoderm is regulated by some other means. Alternatively, a cisacting regulatory sequence of the GHox-8 gene that is specifically involved in regulating its expression in the apical ectodermal ridge might be defective in limbless.
A model concerning the roles of GHox-7 and GHox-8 in directing limb outgrowth
We propose the following model to serve as the basis for future experimentation on the possible roles of GHox-7 and GHox-8 in the reciprocal interaction between the apical ectodermal ridge and subjacent limb mesoderm that is required for normal limb outgrowth. The expression of GHox-8 by the apical ectodermal ridge is regulated by the product of the limbless gene. The GHox-8 expressed by the apical ectodermal ridge, in turn, regulates directly or indirectly the expression of a molecule or molecules synthesized and secreted by the ridge involved in directing the outgrowth of the subridge mesenchymal cells. One of the effects of this molecule or molecules is to maintain and amplify the expression of GHox-7 by the subjacent mesenchymal cells. The expression of GHox-7 by the subridge mesoderm is required for elongation of the limb bud.
ACKNOWLEDGEMENTS
This work was supported by NIH grants HD22610 to RAK and WBU and HD20743 to JFF. This work represents a collaborative effort between two Program Project grants (‘Genetic Control of Limb Development’, HD22610 and ‘Extracellular Molecular Interaction in Limb Development’, HD20743) supported by the National Institute of Child Health and Human Development. KMK was supported by Predoctoral Fellowships T32HD07118 and T32GM07215.