Specification of cell fate along the proximal-distal axis in the developing chick limb bud

Pattern formation along the proximal-distal (PD) axis in the developing limb bud serves as a good model for learning how cell fate and regionalization of domains, which are essential processes in morphogenesis during development, are specified by positional information. In the present study, detailed fate maps for the limb bud of the chick embryo were constructed in order to gain insights into how cell fate for future structures along the PD axis is specified and subdivided. Our fate map revealed that there is a large overlap between the prospective autopod and zeugopod in the distal limb bud at an early stage (stage 19), whereas a limb bud at this stage has already regionalized the proximal compartments for the prospective stylopod and zeugopod. A clearer boundary of cell fate specifying the prospective autopod and zeugopod could be seen at stage 23, but cell mixing was still detectable inside the prospective autopod region at this stage. Detailed analysis of HOXA11 AND HOXA13 expression at single cell resolution suggested that the cell mixing is not due to separation of some different cell populations existing in a mosaic. Our findings suggest that a mixable unregionalized cell population is maintained in the distal area of the limb bud, while the proximal region starts to be regionalized at the early stage of limb development.     

Patterning of forelimb bud myogenic precursor cells requires retinoic acid signaling initiated by Raldh2

Limb skeletal muscle is derived from cells of the dermomyotome that detach and migrate into the limb buds to form separate dorsal and ventral myogenic precursor domains. Myogenic precursor cell migration is dependent on limb bud mesenchymal expression of hepatocyte growth factor/scatter factor (Hgf), which encodes a secreted ligand that signals to dermomyotome through the membrane receptor tyrosine kinase Met. Here, we find that correct patterning of Hgf expression in forelimb buds is dependent on retinoic acid (RA) synthesized by retinaldehyde dehydrogenase 2 (Raldh2) expressed proximally. Raldh2(-/-) forelimb buds lack RA and display an anteroproximal shift in expression of Hgf such that its normally separate dorsal and ventral expression domains are joined into a single anterior-proximal domain. Met and MyoD are expressed in this abnormal domain, indicating that myogenic cell migration and differentiation are occurring in the absence of RA, but in an abnormal location. An RA-reporter transgene revealed that RA signaling in the forelimb bud normally exists in a gradient across the proximodistal axis, but uniformly across the anteroposterior axis, with all proximal limb bud cells exhibiting activity. Expression of Bmp4, an inhibitor of Hgf expression, is increased and shifted anteroproximally in Raldh2(-/-) limb buds, thus encroaching into the normal expression domain of Hgf. Our studies suggest that RA signaling provides proximodistal information for limb buds that counterbalances Bmp signaling, which in turn helps mediate proximodistal and anteroposterior patterning of Hgf expression to correctly direct migration of Met-expressing myogenic precursor cells.     

The effect of vitamin A on limb regeneration in Rana temporaria

Previous experiments in which vitamin A has been administered to developing or regenerating limbs have shown that different limb axes are affected. In regenerating axolotl limbs, serial reduplications in the proximodistal axis are produced. In the developing chick limb bud, mirror-imaged reduplications in the anteroposterior axis are produced. Results reported here on Rana temporaria limb buds reveal that vitamin A causes both effects to occur. That is, limbs are both serially reduplicated in the proximodistal axis and mirror imaged in the anteroposterior axis. Time and concentration effects are explored and the significance of these results for our current understanding of axial organisation in limbs is discussed.     

A cellular lineage analysis of the chick limb bud

The chick limb bud has been used as a model system for studying pattern formation and tissue development for more than 50 years. However, the lineal relationships among the different cell types and the migrational boundaries of individual cells within the limb mesenchyme have not been explored. We have used a retroviral lineage analysis system to track the fate of single limb bud mesenchymal cells at different times in early limb development. We find that progenitor cells labeled at stage 19-22 can give rise to multiple cell types including clones containing cells of all five of the major lateral plate mesoderm-derived tissues (cartilage, perichondrium, tendon, muscle connective tissue, and dermis). There is a bias, however, such that clones are more likely to contain the cell types of spatially adjacent tissues such as cartilage/perichondrium and tendon/muscle connective tissue. It has been recently proposed that distinct proximodistal segments are established early in limb development; however our analysis suggests that there is not a strict barrier to cellular migration along the proximodistal axis in the early stage 19-22 limb buds. Finally, our data indicate the presence of a dorsal/ventral boundary established by stage 16 that is inhibitory to cellular mixing. This boundary is demarcated by the expression of the LIM-homeodomain factor lmx1b.     

A gradient of gap junctional communication along the anterior-posterior axis of the developing chick limb bud.

A modification of the scrape-loading/dye transfer technique was used to study gap junctional communication along the anterior-posterior (A-P) axis of embryonic chick wing buds at an early stage of development (stage 20/21) when positional values along the A-P axis are being specified. Extensive intercellular transfer of the gap junction-permeable dye, lucifer yellow, from scrape-loaded mesenchymal cells to contiguous cells occurs in the posterior mesenchymal tissue of the wing bud adjacent to the zone of polarizing activity, which is thought to be the source of a diffusible morphogen that specifies A-P positional identity according to its local concentration. Considerably less transfer of lucifer yellow dye occurs in scrape-loaded mesenchymal tissue in the middle of the limb bud compared to posterior mesenchymal tissue, and little or no transfer of lucifer yellow is observed in the mesenchymal tissue in the anterior portion of the limb bud. No intercellular transfer of the gap junction-impermeable dye, rhodamine dextran, occurs in any region of the limb bud. These results indicate that there is a gradient of gap junctional communication along the A-P axis of the developing chick wing bud. This gradient of gap junctional communication along the A-P axis might generate a graded distribution of a relatively low molecular weight intracellular regulatory molecule involved in specifying A-P positional identities.     

Experimental manipulation leading to induction of dorsal ectodermal ridges on normal limb buds results in a phenocopy of the eudiplopodia chick mutant

Elongation of chick limb buds depends on the presence of the apical ectodermal ridge which is induced by subjacent limb bud mesoderm. Recombination experiments have shown that the limb bud mesoderm loses the capacity to induce ridges by late stage 17. Moreover, in normal limb development only one ridge forms. However, in the eudiplopodia chick mutant accessory ectodermal ridges form on the dorsal surface of limb buds as late as stage 22. Tissue recombinant experiments show that the mutation affects the ectoderm, extending the time it responds to ridge induction (Fraser and Abbott, 1971a, Fraser and Abbott, 1971b while the mesoderm is normal. The result is polydactyly, with extra digits dorsal to the normal digits. Because eudiplopodia limb bud dorsal mesoderm can induce ridges at stage 22 but is unaffected by the gene, genetically normal dorsal limb bud mesoderm may also be able to induce ridges after stage 17. To test this possibility we grafted stages 14–18 flank ectoderm to normal limb bud dorsal mesoderm and found that mesoderm from stages 17 through 20 was able to induce a ridge and subsequently dorsal digits developed. Limbs with duplicate digits were similar to eudiplopodia limbs. In other experiments, stage 18, 19, and 20 leg bud dorsal ectoderm did not form ridges when grafted to leg bud dorsal mesoderm of the same stage, indicating a lack of response to the mesoderm. Finally, the inductive capacity of limb bud mesoderm appeared to be reduced compared to mesoderm at pre-limb bud stages. These experiments demonstrate a spatially generalized potential in limb bud dorsal mesoderm to induce ridges during the stages when the apical ridge is induced. The determination of where the ridge will form and the acquired inability of limb bud dorsal ectoderm to respond to induction by underlying mesoderm are necessary early pattern forming events which assure that a single proximodistal limb axis will form.     

Branching,segmentation and the metapterygial axis: pattern versus process in the vertebrate limb

Explanations of the patterns of vertebrate fin and limb evolution are improving as specific hypotheses based on molecular and developmental data are proposed and tested. Comparative analyses of gene expression patterns and functions in developing limbs, and morphological patterns in embryonic, adult and fossil limbs point to digit specification as a key developmental innovation associated with the origin of tetrapods. Digit development during the fin-to-limb transition involved sustained proximodistal outgrowth and a new phase of Hox gene expression in the distal fin bud. These patterning changes in the distal limb have been explained by the linked concepts of the metapterygial axis and the digital arch. These have been proposed to account for the generation of limb pattern by sequential branching and segmentation of precartilagenous elements along the proximodistal axis of the limb. While these ideas have been very fruitful, they have become increasingly difficult to reconcile with experimental and comparative studies of fin and limb development. Here we argue that limb development does not involve a branching mechanism, and reassess the concept of a metapterygial axis in limb development and evolution.     

Use of retinoids to analyze the cellular basis of positional memory in regenerating amphibian limbs

Cells of the amphibian limb regeneration blastema inherit memories of their level of origin (positional memory) along the limb axes. These memories serve as boundaries of what is to be regenerated, thus preventing regeneration of any but the missing structures. Because of its importance in determining the boundaries of regenerate pattern, it is essential to understand the cellular and molecular basis of positional memory. One approach to this problem is to look for position-related differences in a cell or molecular property along a limb axis and then show, using an agent that modifies regenerate pattern, that the cell or molecular property and the pattern are coordinately modified. We have done this using retinoic acid (RA) as a pattern-modifying agent and an in vivo assay that detects position-related differences in a cell recognition-affinity property along the proximodistal (PD) axis of the regenerating axolotl limb. RA proximalizes positional memory in the PD axis, posteriorizes it in the anteroposterior axis, and ventralizes it in the dorsoventral axis. The level-specific PD cell recognition-affinity property is proximalized by RA, indicating that this property and positional memory are causally related. The effects of RA on positional memory may be mediated through a cellular RA-binding protein (CRABP), since the concentration of unbound (apo) CRABP molecules is highest during early stages of regeneration when the proximalizing effects of RA are greatest.     

Involvement of the Chox-4 chicken homeobox genes in determination of anteroposterior axial polarity during limb development.

We have isolated and identified four chicken homeobox genes in the upstream region of the Chox-4 complex. The Chox-4g and -4f genes, at the 5' extremity of the complex, were expressed locally in the vicinity of the zone of polarizing activity (ZPA) at early stages of limb development, substantiating the involvement of the genes in anteroposterior axis determination. To confirm their function, we implanted a bead containing retinoic acid, or the ZPA itself, in the anterior margin of the limb bud, leading to formation of mirror-image duplicated digits, and observed the resultant change in gene expression. Expression of the Chox-4g and -4f genes was induced in the new digit-forming region. Those results suggest that positional information assigned by a ZPA morphogen is imprinted on cellular memory by expression of the Chox-4 genes to maintain positional signaling along the anteroposterior axis in the limb field.     

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.     

Positional signalling and the development of the humerus in the chick limb bud

The positional signal model for specification of the cartilaginous elements in limb development has been tested by examining the effect on the humerus of grafting a polarizing region to different positions along the anteroposterior axis of the limb bud at stage 16. The humerus between the host and grafted polarizing region was largely normal though there were variations in width, particularly the distal epiphysis. The humerus often showed mirror-image symmetry along the anteroposterior axis. When the grafted polarizing region was in a very anterior position, there were a few cases where a second humerus developed. Anterior to the graft an additional humerus often developed. This was associated with the splitting of the bud into two domains. It is suggested that these results are not consistent with a positional signal model and that an additional mechanism involving an isomorphic prepattern may be involved in the specification of the cartilaginous elements.     

Analysis of gene expressions during Xenopus forelimb regeneration

Xenopus laevis can regenerate an amputated limb completely at early limb bud stages, but the metamorphosed froglet gradually loses this capacity and can regenerate only a spike-like structure. We show that the spike formation in a Xenopus froglet is nerve dependent as is limb regeneration in urodeles, since denervation concomitant with amputation is sufficient to inhibit the initiation of blastema formation and fgf8 expression in the epidermis. Furthermore, in order to determine the cause of the reduction in regenerative capacity, we examined the expression patterns of several key genes for limb patterning during the spike-like structure formation, and we compared them with those in developing and regenerating limb buds that produce a complete limb structure. We cloned Xenopus HoxA13, a marker of the prospective autopodium region, and the expression pattern suggested that the spike-like structure in froglets is accompanied by elongation and patterning along the proximodistal (PD) axis. On the other hand, shh expression was not detected in the froglet blastema, which expresses fgf8 and msx1. Thus, although the wound epidermis probably induces outgrowth of the froglet blastema, the polarizing activity that organizes the anteroposterior (AP) axis formation is likely to be absent there. Our results demonstrate that the lost region in froglet limbs is regenerated along the PD axis and that the failure of organization of the AP pattern gives rise to a spike-like incomplete structure in the froglet, suggesting a relationship between regenerative capacity and AP patterning. These findings lead us to conclude that the spike formation in postometamorphic Xenopus limbs is epimorphic regeneration.     

Is the progress zone model a victim of progress?

The prevailing view has long been that mesenchymal cells at the apex of the vertebrate limb bud proliferate in a progress zone (PZ) under the influence of the apical ectodermal ridge (AER). According to the PZ model, proximodistal cell fates are specified according to the time spent in the PZ. New results, considered along with some earlier findings, fail to support this model.     

Opposing RA and FGF signals control proximodistal vertebrate limb development through regulation of Meis genes

Vertebrate limbs develop in a temporal proximodistal sequence, with proximal regions specified and generated earlier than distal ones. Whereas considerable information is available on the mechanisms promoting limb growth, those involved in determining the proximodistal identity of limb parts remain largely unknown. We show here that retinoic acid (RA) is an upstream activator of the proximal determinant genes Meis1 and Meis2. RA promotes proximalization of limb cells and endogenous RA signaling is required to maintain the proximal Meis domain in the limb. RA synthesis and signaling range, which initially span the entire lateral plate mesoderm, become restricted to proximal limb domains by the apical ectodermal ridge (AER) activity following limb initiation. We identify fibroblast growth factor (FGF) as the main molecule responsible for this AER activity and propose a model integrating the role of FGF in limb cell proliferation, with a specific function in promoting distalization through inhibition of RA production and signaling.     

An epidermal signal regulates Lmx-1 expression and dorsal-ventral pattern during Xenopus limb regeneration

The results of recent studies have supported the idea that the ability to organize the formation of axes such as the anteroposterior and proximodistal axes corresponds to limb regeneration ability in Xenopus. In this study, we investigated the mechanism by which the dorsoventral (D-V) axis of regenerating Xenopus limbs is established and the relationships between D-V patterning and regenerative ability. Transplantation experiments were performed to study which epidermis or mesenchyme is responsible for the D-V patterning in regenerating limbs. Naked mesenchyme of a donor limb was rotated and implanted on a host opposite-side limb stump to make a reversed recombination about the D-V axis. The resultant regenerates had a normal-looking D-V pattern, including Lmx-1 expression, muscle pattern, and joints, in stage 52 recombinants and a reversed D-V pattern in stage 55 recombinants. Further experiments in recombination at stage 52 and stage 55 showed that the epidermal signal is responsible for producing the D-V pattern in the regenerating blastema. These results, together with the finding that Lmx-1 expression is absent in the froglet forelimb blastema, suggest that D-V axis formation is a key step in understanding the loss of regenerative ability.     

Abnormal anteroposterior and dorsoventral patterning of the limb bud in the absence of retinoids.

We describe here how the early limb bud of the quail embryo develops in the absence of retinoids, including retinoic acid. Retinoid-deficient embryos develop to about stage 20/21, thus allowing patterns of early gene activity in the limb bud to be readily examined. Genes representing different aspects of limb polarity were analysed. Concerning the anteroposterior axis, Hoxb-8 was up-regulated and its border was shifted anteriorly whereas shh and the mesodermal expression of bmp-2 were down-regulated in the absence of retinoids. Concerning the apical ectodermal genes, fgf-4 was down-regulated whereas fgf-8 and the ectodermal domain of bmp-2 were unaffected. Genes involved in dorsoventral polarity were all disrupted. Wnt-7a, normally confined to the dorsal ectoderm, was ectopically expressed in the ventral ectoderm and the corresponding dorsal mesodermal gene Lmx-1 spread into the ventral mesoderm. En-1 was partially or completely absent from the ventral ectoderm. These dorsoventral patterns of expression resemble those seen in En-1 knockout mouse limb buds. Overall, the patterns of gene expression are also similar to the Japanese limbless mutant. These experiments demonstrate that the retinoid-deficient embryo is a valuable tool for dissecting pathways of gene activity in the limb bud and reveal for the first time a role for retinoic acid in the organisation of the dorsoventral axis.