Manifestation of the limb prepattern: limb development in the absence of sonic hedgehog function

The secreted protein encoded by the Sonic hedgehog (Shh) gene is localized to the posterior margin of vertebrate limb buds and is thought to be a key signal in establishing anterior-posterior limb polarity. In the Shh(-/-) mutant mouse, the development of many embryonic structures, including the limb, is severely compromised. In this study, we report the analysis of Shh(-/-) mutant limbs in detail. Each mutant embryo has four limbs with recognizable humerus/femur bones that have anterior-posterior polarity. Distal to the elbow/knee joints, skeletal elements representing the zeugopod form but lack identifiable anterior-posterior polarity. Therefore, Shh specifically becomes necessary for normal limb development at or just distal to the stylopod/zeugopod junction (elbow/knee joints) during mouse limb development. The forelimb autopod is represented by a single distal cartilage element, while the hindlimb autopod is invariably composed of a single digit with well-formed interphalangeal joints and a dorsal nail bed at the terminal phalanx. Analysis of GDF5 and Hoxd11-13 expression in the hindlimb autopod suggests that the forming digit has a digit-one identity. This finding is corroborated by the formation of only two phalangeal elements which are unique to digit one on the foot. The apical ectodermal ridge (AER) is induced in the Shh(-/-) mutant buds with relatively normal morphology. We report that the architecture of the Shh(-/-) AER is gradually disrupted over developmental time in parallel with a reduction of Fgf8 expression in the ridge. Concomitantly, abnormal cell death in the Shh(-/-) limb bud occurs in the anterior mesenchyme of both fore- and hindlimb. It is notable that the AER changes and mesodermal cell death occur earlier in the Shh(-/-) forelimb than the hindlimb bud. This provides an explanation for the hindlimb-specific competence to form autopodial structures in the mutant. Finally, unlike the wild-type mouse limb bud, the Shh(-/-) mutant posterior limb bud mesoderm does not cause digit duplications when grafted to the anterior border of chick limb buds, and therefore lacks polarizing activity. We propose that a prepattern exists in the limb field for the three axes of the emerging limb bud as well as specific limb skeletal elements. According to this model, the limb bud signaling centers, including the zone of polarizing activity (ZPA) acting through Shh, are required to elaborate upon the axial information provided by the native limb field prepattern.     

Modification of the zone of polarizing activity signal by trypsin

Patterning of the developing vertebrate limb along the anterior‐posterior axis is controlled by the zone of polarizing activity (ZPA) via the expression of Sonic hedgehog (Shh) and along the proximal‐distal axis by the apical ectodermal ridge (AER) through the production of fibroblast growth factors (FGFs). ZPA grafting, as well as ectopic application of SHH to the anterior chick limb bud, demonstrate that digit patterning is largely influenced by these secreted factors. Although signal transduction pathways have been well characterized for SHH and for FGFs, little is known of how these signals are regulated extracellularly in the limb. The present study shows that alteration of the extracellular environment through trypsin treatment can have profound effects on digit patterning. These effects appear to be mediated by the induction of Shh in host tissues and by ectopic AER formation, implicating the extracellular matrix in regulating the signaling activities of key patterning genes in the limb.     

Hox-4 gene expression in mouse/chicken heterospecific grafts of signalling regions to limb buds reveals similarities in patterning mechanisms.

The products of Hox-4 genes appear to encode position in developing vertebrate limbs. In chick embryos, a number of different signalling regions when grafted to wing buds lead to duplicated digit patterns. We grafted tissue from the equivalent regions in mouse embryos to chick wing buds and assayed expression of Hox-4 genes in both the mouse cells in the grafts and in the chick cells in the responding limb bud using species specific probes. Tissue from the mouse limb polarizing region and anterior primitive streak respecify anterior chick limb bud cells to give posterior structures and lead to activation of all the genes in the complex. Mouse neural tube and genital tubercle grafts, which give much less extensive changes in pattern, do not activate 5'-located Hox-4 genes. Analysis of expression of Hox-4 genes in mouse cells in the grafted signalling regions reveals no relationship between expression of these genes and strength of their signalling activity. Endogenous signals in the chick limb bud activate Hox-4 genes in grafts of mouse anterior limb cells when placed posteriorly and in grafts of mouse anterior primitive streak tissue. The activation of the same gene network by different signalling regions points to a similarity in patterning mechanisms along the axes of the vertebrate body.     

Sonic hedgehog (SHH) specifies muscle pattern at tissue and cellular chick level, in the chick limb bud.

Development of the musculature in chick limbs involves tissue and cellular patterning. Patterning at the tissue level leads to the precise arrangement of specific muscles; at the cellular level patterning gives rise to the fibre type diversity in muscles. Although the data suggests that the information controlling muscle patterning is localised within the limb mesenchyme and not in the somitic myogenic precursor cells themselves, the mechanisms underlying muscle organisation have still to be elucidated. The anterior-posterior axis of the limb is specified by a group of cells in the posterior region of the limb mesenchyme, called the zone of polarizing activity (ZPA). When polarizing-region cells are grafted to the anterior margin of the bud, they cause mirror-image digit duplications to be produced. The effect of ZPA grafts can be reproduced by application of retinoic acid (RA) beads and by grafting sonic hedgehog (SHH)-expressing cells to the anterior margin of the limb. Although most previous studies have looked at changes of the skeletal patterning, ZPA and RA also affect muscle patterning. In this report, we investigated the role of SHH in tissue and cellular patterning of forearm wing muscles. Ectopic application of a localised source of SHH to the anterior margin of the wing, leading to complete digit duplication, is able to transform anterior forearm muscles into muscles with a posterior identity. Moreover, the ectopic source of SHH induces a mirror image duplication of the normal posterior muscles fibre types in the new posterior muscles. The reorganisation of the slow fibres can be detected before muscle mass cleavage has started; suggesting that the appropriate fibre type arrangement is in place before the splitting process can be observed.     

Control of the Expression of AV-1 Protein, a Position-Specific Antigen, by ZPA (Zone of Polarizing Activity) Factor in the Chick Limb Bud

AV-1 protein is a molecule which shows position-specific expression during chick limb development, and is expected to have some important roles in limb pattern formation. In this study, to examine whether the ZPA (Zone of polarizing activity) effects the expression of the AV-1 protein, we have removed or grafted the ZPA in chick limb buds and observed AV-1 expression. Anterior halves of the limb buds which lack a ZPA were used as hosts. In such anterior halves, AV-1 expression was initially observed in distal mesodermal cells including the cut surface. These anterior halves were combined with ZPA fragments, anterior fragments, posterior half limb buds, or left to develop alone, and the distribution of AV-1 expression was examined. The results of these experiments show that AV-1 expression requires the ZPA, and that expression occurs in the distal mesodermal cells certain distance from the ZPA.     

The control of the anteroposterior and dorsoventral axes in embryonic chick limbs constructed of dissociated and reaggregated limb-bud mesoderm

In the 3- to 4-day embryonic avian limb bud, a unique zone of mesodermal tissue is located posteriorly at the junction of bud and body wall. Appropriately grafted to a host limb bud, it induces the formation of a supernumerary limb outgrowth from preaxial tissue and determines that its posterior side will face the graft. It is called the zone of polarizing activity (ZPA).When limb-bud mesoderm is isolated, dissociated, reaggregated centrifugally, jacketed in the mesoderm-free hull of another limb bud, and grown as a graft on a host embryo, the recombinant frequently forms a limb-like structure terminating in digits that fail to show differentiation with respect to the anteroposterior axis. When, however, a bit of ZPA tissue is implanted in the recombinant subjacent to the anterior or posterior margin of the ectoderm, the resulting outgrowth shows a characteristic anteroposterior order of digits that corresponds to the placement of the implant, regardless of its relationship with the anteroposterior axis of the ectoderm or of the host embryo.Dorsoventral differentials have been recognized only in limbs formed from reaggregated leg-bud mesoderm. The direction of the dorsoventral axis always corresponds to the original axis of the ectodermal jacket regardless of the orientation of the recombinant on the host.     

Differential expression of chicken CYP26 in anterior versus posterior limb bud in response to retinoic acid

Multiple studies indicate that quantitative control of the levels of all-trans-retinoic acid (RA) in the vertebrate embryo is necessary for correct development. The function of RA in cells is regulated by a number of coordinated mechanisms. One of those mechanisms involves controls on the rate of RA catabolism. Recently, enzymes capable of catabolizing RA were found to constitute a new family, called CYP26, within the cytochrome P450 superfamily. CYP26 homologues have been isolated from human, mouse, zebra fish, and recently from the chick. In this study, we examined the regulation of chicken CYP26 (cCYP26) expression by RA during the early phase of chick limb outgrowth. In the anterior limb mesenchyme and apical ectodermal ridge (AER), cCYP26 expression was induced in a concentration dependent manner by implanting beads soaked in 0.1, 1, and 5 mg/ml RA. The RA-induced expression of cCYP26 in anterior limb mesenchyme and the AER was detected as early as 1 hr after treatment and was not affected by the presence of cycloheximide. In contrast to the anterior limb, the induction of cCYP26 was dramatically reduced (or absent) when RA beads were implanted in the posterior limb mesenchyme. Furthermore, induction of cCYP26 expression in the anterior mesenchyme was inhibited by transplantations of the zone of polarizing activity (ZPA) and by Shh-soaked beads. Our data suggest that different mechanisms regulate retinoid homeostasis in the AER and mesenchyme during limb bud outgrowth. J. Exp. Zool. 290:136-147, 2001.     

SF/HGF is a mediator between limb patterning and muscle development.

Scatter factor/hepatocyte growth factor (SF/HGF) is known to be involved in the detachment of myogenic precursor cells from the lateral dermomyotomes and their subsequent migration into the newly formed limb buds. As yet, however, nothing has been known about the role of the persistent expression of SF/HGF in the limb bud mesenchyme during later stages of limb bud development. To test for a potential role of SF/HGF in early limb muscle patterning, we examined the regulation of SF/HGF expression in the limb bud as well as the influence of SF/HGF on direction control of myogenic precursor cells in limb bud mesenchyme. We demonstrate that SF/HGF expression is controlled by signals involved in limb bud patterning. In the absence of an apical ectodermal ridge (AER), no expression of SF/HGF in the limb bud is observed. However, FGF-2 application can rescue SF/HGF expression. Excision of the zone of polarizing activity (ZPA) results in ectopic and enhanced SF/HGF expression in the posterior limb bud mesenchyme. We could identify BMP-2 as a potential inhibitor of SF/HGF expression in the posterior limb bud mesenchyme. We further demonstrate that ZPA excision results in a shift of Pax-3-positive cells towards the posterior limb bud mesenchyme, indicating a role of the ZPA in positioning of the premuscle masses. Moreover, we present evidence that, in the limb bud mesenchyme, SF/HGF increases the motility of myogenic precursor cells and has a role in maintaining their undifferentiated state during migration. We present a model for a crucial role of SF/HGF during migration and early patterning of muscle precursor cells in the vertebrate limb.     

The effect of the zone of polarizing activity (ZPA) on the anterior half of the chick wing bud

Removal of the posterior half of the chick wing bud between stages 17-22 results in failure of the anterior distal tissue to survive and differentiate. This observation has been interpreted in terms of a requirement by the anterior half of a factor supplied by the posterior half of the limb containing the zone of polarizing activity (ZPA). This relationship has been tested by grafting ZPA tissue to the posterior surface of the anterior half after posterior half removal. Grafts made proximally on the cut surface did not significantly improve survival and development, nor did the ZPA prevent the expansion of the cell death in the ANZ beyond its normal boundaries into the distal mesenchyme. However, when grafted distally the ZPA inhibited cell death in the apical mesenchyme and caused the anterior mesenchyme to change its normal prospective fate (radius and digit 2). In all these cases, in addition to digit 2, digit 3 and frequently also digit 4 differentiated. The anterior half went on to develop a full set of digits and zeugopod parts in almost 50% of cases, although no skeleton resulting from this regulation of the anterior half had totally size regulated. These results demonstrate a developmental 'rescue' effect by the ZPA, and further support the view that the ZPA has a central and unique function in normal limb bud development, controlling survival and differentiation of the mesenchyme along the anteroposterior axis.     

Temporal pattern of posterior positional identity in mouse limb buds

This study describes the temporal pattern of posterior positional identity in mouse limb bud cells. To do this wedges of tissue from the posterior edge of mouse limb buds at various stages (limb stages: Wanek et al., 1989b. J. Exp. Zool. 249, 41-49) were grafted to the anterior edge of a host chick embryo wing bud. Grafts of mouse posterior cells are able to induce the formation of supernumerary digits every time when they are taken from buds from stage 3 through stage 6. At stage 7, the frequency declines and by stage 8 the chick cells no longer respond. The results indicate a change in tissue properties at stage 7, which progresses by stage 8 to the point at which posterior positional identity is no longer detectable by this assay. These temporal changes in this aspect of limb pattern formation can be used as an additional criterion to guide the identification of genes involved in the specification of posterior positional identity.     

The role of the polarizing zone in the pattern of experimental chondrogenesis in the chick embryo interdigital space

We have previously shown that removal of the apical ectodermal ridge of the third interdigital space of the chick leg bud at stages 28 and 29 is followed by the appearance of ectopic cartilage, which in the course of development gives rise to extra digits. These in vivo studies suggest that the pattern of skeletal morphogenesis in the limb depends on the inhibitory effect of the ectoderm. In the present study we tested whether zone polarizing activity (ZPA) exerted an effect on the pattern of experimental chondrogenesis in the interdigital space of the leg bud in stage 29 HH chick embryos. A small fragment of tissue from the ZPA in chick embryos in which ZPA activity was most intense was grafted onto the interdigital space in which chondrogenesis had previously been experimentally induced. No significant changes were observed in the course of differentiation of the recipient interdigital spaces with ZPA grafts, leading us to conclude that the graft failed to modify the morphogenetic fate of interdigital tissue.     

Sonic hedgehog signaling during digit pattern duplication after application of recombinant protein and expressing cells

HoxD expression and cartilage pattern formation were compared after application of a recombinant amino-terminal peptide of Sonic hedgehog protein (Shh-N) and implantation of cells expressing the Sonic hedgehog (Shh) gene. During digit duplication after implantation of a Shh-N-soaked bead, BMP-2 and Patched expression was transiently induced in the anterior limb mesenchyme 20 h after grafting, but was reduced to the basal level 48 h after grafting. On the contrary, when Shh-expressing cells were grafted to the anterior limb bud, expression domains of the BMP-2 and Patched genes were initially induced in the restricted region in close proximity to the grafted cells. Induced expression of BMP-2 and Patched was maintained in the anterior-peripheral region of the limb bud for 42 h after grafting. In either case, HoxD12 and HoxD13 were consistently induced in the anterior-distal limb mesenchyme, accompanying mirror-image duplication of the digit pattern. Induction and maintenance of HoxD expression were consistent with the resultant digit pattern. A steep gradient of Shh activity provided by Shh-expressing cells is most adequate to induce complete digit pattern, as compared to the shallow gradient provided by Shh-N protein released from a bead. These results suggest that positional identity is respecified by Shh-N activity within the first 24 h during digit duplication, and that Shh-N on its own is not acting as a long-range signaling molecule to determine positional identity at a distance in the limb bud.     

Retinoic acid respecifies limb bud cells in vitro.

Retinoic acid (RA) is known to have dramatic effects on limb pattern formation and has been shown to exert its effects on limbs by converting anterior limb bud cells into cells with posterior positional properties. In this study we find that dissociated posterior limb bud cells from chick and mouse embryos cultured at high density (micromass cultures) are able to stimulate the formation of supernumerary digits when grafted into developing wing buds and that the positional identity of both chick and mouse limb bud cells can be maintained for finite periods of time in vitro. Furthermore, using this assay system we have tested whether anterior cells from mouse and chick limb buds can be converted into cells with posterior identity by exposure to RA in vitro. We find that anterior limb bud cells acquire posterior properties after culture in the presence of RA.     

Distinct signaling molecules control Hoxa-11 and Hoxa-13 expression in the muscle precursor and mesenchyme of the chick limb bud.

The limb muscles, originating from the ventrolateral portion of the somites, exhibit position-specific morphological development through successive splitting and growth/differentiation of the muscle masses in a region-specific manner by interacting with the limb mesenchyme and the cartilage elements. The molecular mechanisms that provide positional cues to the muscle precursors are still unknown. We have shown that the expression patterns of Hoxa-11 and Hoxa-13 are correlated with muscle patterning of the limb bud (Yamamoto et al., 1998) and demonstrated that muscular Hox genes are activated by signals from the limb mesenchyme. We dissected the regulatory mechanisms directing the unique expression patterns of Hoxa-11 and Hoxa-13 during limb muscle development. HOXA-11 protein was detected in both the myogenic cells and the zeugopodal mesenchymal cells of the limb bud. The earlier expression of HOXA-11 in both the myogenic precursor cells and the mesenchyme was dependent on the apical ectodermal ridge (AER), but later expression was independent of the AER. HOXA-11 expression in both myogenic precursor cells and mesenchyme was induced by fibroblast growth factor (FGF) signal, whereas hepatocyte growth factor/scatter factor (HGF/SF) maintained HOXA-11 expression in the myogenic precursor cells, but not in the mesenchyme. The distribution of HOXA-13 protein expression in the muscle masses was restricted to the posterior region. We found that HOXA-13 expression in the autopodal mesenchyme was dependent on the AER but not on the polarizing region, whereas expression of HOXA-13 in the posterior muscle masses was dependent on the polarizing region but not on the AER. Administration of BMP-2 at the anterior margin of the limb bud induced ectopic HOXA-13 expression in the anterior region of the muscle masses followed by ectopic muscle formation close to the source of exogenous BMP-2. In addition, NOGGIN/CHORDIN, antagonists of BMP-2 and BMP-4, downregulated the expression of HOXA-13 in the posterior region of the muscle masses and inhibited posterior muscle development. These results suggested that HOXA-13 expression in the posterior muscle masses is activated by the posteriorizing signal from the posterior mesenchyme via BMP-2. On the contrary, the expression of HOXA-13 in the autopodal mesenchyme was affected by neither BMP-2 nor NOGGIN/CHORDIN. Thus, mesenchymal HOXA-13 expression was independent of BMP-2 from polarizing region, but was under the control of as yet unidentified signals from the AER. These results showed that expression of Hox genes is regulated differently in the limb muscle precursor and mesenchymal cells.