An in vitro Analogue of Early Chick Limb Bud Outgrowth

Our culture system appears to represent an in vitro analogue of early chick limb morphogenesis. Organized mesodermal cell accumulations resembling limb buds were derived from a monolayer of limb mesoderm cells when covered by limb ectoderm which included the apical ectodermal ridge (AER). The ridge retained its normal configuration when grown over a limb mesoderm monolayer and the mesoderm cells accumulated under the ridge to form a multilayered structure (10–25 cells in thickness) with the characteristic shape of a limb bud. Ectoderm which did not include the ridge failed to promote the formation of limb-like mesodermal accumulations thus the action of the ridge appears to be specific. The AER-elicited expression of mesodermal cell behaviour leading to early limb outgrowth is discussed in terms of possible morphogenetic mechanisms involved i.e. differential mitosis, cell migration, changes in cell shape and especially the adhesive properties of the cells.     

FGF7 and FGF10 directly induce the apical ectodermal ridge in chick embryos.

During vertebrate limb development, the apical ectodermal ridge (AER) plays a vital role in both limb initiation and distal outgrowth of the limb bud. In the early chick embryo the prelimb bud mesoderm induces the AER in the overlying ectoderm. However, the direct inducer of the AER remains unknown. Here we report that FGF7 and FGF10, members of the fibroblast growth factor family, are the best candidates for the direct inducer of the AER. FGF7 induces an ectopic AER in the flank ectoderm of the chick embryo in a different manner from FGF1, -2, and -4 and activates the expression of Fgf8, an AER marker gene, in a cultured flank ectoderm without the mesoderm. Remarkably, FGF7 and FGF10 applied in the back induced an ectopic AER in the dorsal median ectoderm. Our results suggest that FGF7 and FGF10 directly induce the AER in the ectoderm both of the flank and of the dorsal midline and that these two regions have the competence for AER induction. Formation of the AER of the dorsal median ectoderm in the chick embryo is likely to appear as a vestige of the dorsal fin of the ancestors.     

Cell migration and chick limb development: chemotactic action of FGF-4 and the AER.

Experiments have been carried out to investigate the role of the apical ectodermal ridge (AER) and FGF-4 on the control of cell migration during limb bud morphogenesis. By coupling DiI cell labeling with ectopic implantation of FGF-4 microcarrier beads we have found that FGF-4 acts as a potent and specific chemoattractive agent for mesenchymal cells of the limb bud. The response to FGF-4 is dose dependent in both the number of cells stimulated to migrate and the distance migrated. The cell migration response to FGF-4 appears to be independent of the known inductive activity of FGF-4 on Shh gene expression. We investigated the role of the AER in controlling cell migration by characterizing the migration pattern of DiI-labeled subapical cells during normal limb outgrowth and following partial AER removal. Subapical cells within 75 micrometer of the AER migrate to make contact with the AER and are found intermingled with nonlabeled cells. Thus, the progress zone is dynamic with cells constantly altering their neighbor relationships during limb outgrowth. AER removal studies show that cell migration is AER dependent and that subapical cells redirect their path of migration toward a functional AER. These studies indicate that the AER has a chemoattractive function and regulates patterns of cell migration during limb outgrowth. Our results suggest that the chemoattractive activity of the AER is mediated in part by the production of FGF-4.     

Msx1 expressing mesoderm is important for the apical ectodermal ridge (AER)-signal transfer in chick limb development

The apical ectodermal ridge (AER) is a specialized thickening of the distal limb ectoderm, and its signals are known to support limb morphogenesis. The expression of a homeobox gene, Msx1 , in the distal limb mesoderm depends on signals from the AER. In the present paper it is reported that Msx1 expression in the distal mesoderm is necessary for the transfer of AER signals in chick limb buds. Interruption of AER-mesoderm interaction by insertion of a thick filter led to the inhibition of pattern specification in the mesoderm just under the filter. In such cases, the expression of Msx1 disappeared in the mesoderm under the filter, suggesting that AER is able to signal over short ranges. In advanced limb buds, Msx1 is also expressed in the proximal mesoderm under the anterior ectoderm. However, it was found that a grafted antero-proximal mesoderm shows no inhibitory effects on pattern specification of the host mesoderm, as is the case with the distal mesoderm. On the other hand, grafted mesoderms without potent Msx1 re-expression, even underneath AER, disturbed normal limb development. In such cases, the expression of Msx1 disappeared in the mesoderm under the grafts, whereas Fgf-8 expression was maintained in the AER above the graft. These results indicate that the expression of Msx1 in the mesoderm is important for the transfer of AER signals.     

Pattern formation and regulation of gene expressions in chick recombinant limbs

Recombinant limbs were performed by ensembling dissociated-reaggregated wing bud mesoderm inside an ectodermal hull. The zone of polarizing activity was excluded from the mesoderm used to perform the recombinant limbs (non-polarized recombinants), and grafted when desired (polarized recombinants). Reorganization of patterning progressively occurred in the newly formed progress zone under the influence of the apical ectodermal ridge (AER), explaining the proximo–distal gradient of morphogenesis observed in developed recombinant limbs. The AER, without the influence of the polarizing region (ZPA), was sufficient to direct outgrowth and appropriate proximo–distal patterning, as observed in the expression of the Hoxa-11 and Hoxa-13 genes. The development of the recombinant limbs coursed with symmetric AER and downregulation of Bmp expression in the mesoderm supporting a negative effect of Bmp signaling upon the apical ridge. The recombinant ectoderm maintained previously established compartments of gene expressions and organized a correct dorso-ventral patterning in the recombinant progress zone. Finally, the ZPA effect was only detected on Bmp expression and pattern formation along the antero-posterior axis.     

MSX2 expression in the apical ectoderm ridge is regulated by an MSX2 and Dlx5 binding site.

The apical ectodermal ridge (AER) is a specialized ectodermal region essential for limb outgrowth. Msx2 expression patterns in limb development strongly suggest an important role for Msx2 in the AER. Our previous studies identified a 348-bp fragment of the chicken Msx2 gene with AER enhancer activity. In this study, the functions of four potential homeodomain binding TAAT sites in this enhancer were studied using transgenic mice and in vitro protein-DNA interactions. Transgenic studies indicate that the four TAAT sites are not redundant and that only the B-TAAT site is critical for AER enhancer activity. The expression patterns of Msx2 and Dlx5 genes in the AER suggest that they might be involved in the regulation of Msx2. In support of this hypothesis, we found that Msx2 and Dlx5 can bind to the B-TAAT site as well as to a fragment containing the D- and E-TAAT sites in the Msx2 AER enhancer sequences. (c)2002 Elsevier Science (USA).     

Mitogenic property of the apical ectodermal ridge

While the apical ectodermal ridge (AER) is well known for its required role in the development of distal parts of the limb and for its ability to stimulate limb duplications, the mechanism of its action is unknown. In this study we use a culture system previously developed by M. Globus and S. Vethamany-Globus (1976, Differentiation6, 91–96) in which an AER grafted onto a high-density cell culture of limb mesenchyme stimulates the formation of an outgrowth. Time-lapse movies taken during the outgrowth period demonstrated no cellular activities other than cell division. Both the mitotic index and labeling index in the mesenchyme were significantly elevated under the AER as compared to that without AER, indicating that the AER provides a growth-promoting stimulus which increases the proportion of dividing cells. On the other hand, nonridge ectoderm had no detectable effect on the mitotic index. Treatment of cultures with cytosine arabinoside both inhibited DNA synthesis and prevented AER-induced outgrowth. These results demonstrate a mitogenic capacity of AER tissue and suggest that mesenchymal outgrowth requires this activity. The mitogenic property of the AER is considered in relation to limb outgrowth in situ.     

Initial limb budding is independent of apical ectodermal ridge activity; evidence from a limbless mutant

Outgrowth of normal chick limb bud mesoderm is dependent on the presence of a specialized epithelium called the apical ectodermal ridge. This ectodermal ridge is induced by the mesoderm at about the time of limb bud formation. The limbless mutation in the chick affects apical ectodermal ridge formation in the limb buds of homozygotes. The initial formation of the limb bud appears to be unaffected by the mutation but no ridge develops and further outgrowth, which is normally dependent on the ridge, does not take place. As a result, limbless chicks develop without limbs. In the present study, which utilized a pre-limb-bud recombinant technique, limbless mesoderm induced an apical ectodermal ridge in grafted normal flank ectoderm. However, at stages when normal flank ectoderm is capable of responding to ridge induction, limbless flank ectoderm did not form a ridge or promote outgrowth of a limb in response to normal presumptive wing bud mesoderm. We conclude from this that the limbless mutation affects the ability of the ectoderm to form a ridge. In addition, because the limbless ectoderm has no morphological ridge and no apparent ridge activity (i.e. it does not stabilize limb elements in stage-18 limb bud mesoderm), the limbless mutant demonstrates that the initial formation of the limb bud is independent of apical ectodermal ridge activity.     

Limb outgrowth in the chick embryo induced by dissociated and reaggregated cells of the apical ectodermal ridge.

Mesodermal cores of the stage 19 chick leg bud were capped with an intact apical ectodermal ridge (AER) or with strips cut from centrifugal pellets formed from Pronase-dissociated AERs. They were then covered with embryonic back-skin ectoderm and grown as grafts to the somite region of a host embryo. Control mesoderms were capped with centrifugal aggregates of nonridge limb ectoderm or similarly treated back-skin ectoderm, with ethanol-killed AERs or with no ectodermal cells other than the enveloping back-skin ectoderm.Controls were vascularized slowly and atypically and showed little outgrowth, forming only proximal skeletal structures. Recombinants equipped with AER cells were vascularized more fully and promptly and began vigorous growth after brief delay, forming legs with all skeletal segments represented, including claw-tipped toes. The latter were arranged in anteroposterior order corresponding to the original polarity of the mesoderm.Histological sections of recombinants made with cytologically distinctive quail AERs reveal that the cap of ridge cells, whether initially intact or reaggregated beneath the back-skin envelope, undergo a period of reorganization, forming a typical AER at the apex of the chimeric appendage after 48 hr. Meanwhile vigorous growth of the recombinant continues.These results show that the AER can cooperate with nonlimb ectoderm in promoting the morphogenesis of successively more distal levels of the limb skeleton. They also show that dissociated ridge cells can reorganize a typical AER at the apex of the limb mesoblast, meanwhile exercising their inductive effect on it.     

Bmp2, Bmp4 and Bmp7 are co-required in the mouse AER for normal digit patterning but not limb outgrowth

Outgrowth and patterning of the vertebrate limb requires a functional apical ectodermal ridge (AER). The AER is a thickening of ectodermal tissue located at the distal end of the limb bud. Loss of this structure, either through genetic or physical manipulations results in truncation of the limb. A number of genes, including Bmps, are expressed in the AER. Previously, it was shown that removal of the BMP receptor Bmpr1a specifically from the AER resulted in complete loss of hindlimbs suggesting that Bmp signaling in the AER is required for limb outgrowth. In this report, we genetically removed the three known AER-expressed Bmp ligands, Bmp2, Bmp4 and Bmp7 from the AER of the limb bud using floxed conditional alleles and the Msx2-cre allele. Surprisingly, only defects in digit patterning and not limb outgrowth were observed. In triple mutants, the anterior and posterior AER was present but loss of the central region of the AER was observed. These data suggest that Bmp ligands expressed in the AER are not required for limb outgrowth but instead play an essential role in maintaining the AER and patterning vertebrate digits.     

Cyclic AMP derivatives stimulate the chondrogenic differentiation of the mesoderm subjacent to the apical ectodermal ridge of the chick limb bud.

Recent studies indicate that one of the major functions of the apical ectodermal ridge (AER) of the embryonic chick limb bud is to maintain mesenchymal cells directly subjacent to it (i.e., cells extending 0.4-0.5 mm from the AER) in a labile, undifferentiated condition. Furthermore, when mesenchymal cells are freed from the AER's influence, either artifically or as a result of normal polarized proximal-to-distal limb outgrowth, they are freed to commence cytodifferentiation. In a preliminary attempt to investigate at a molecular level the mechanism by which the AER exerts its "negative" effect on the cytodifferentiation of subridge mesenchymal cells, we have examined the effect of a variety of agents that elevate cyclic AMP levels on the chondrogenic differentiation of the unspecialized subridge mesoderm of the limb bud in an organ culture system. Dibutyryl- and 8-hydroxy-cyclic AMP elicit a dose-dependent increase in the rate and amount of cartilage matrix formation and a corresponding dose-dependent increase in sulfated glycosaminoglycan accumulation by subridge mesoderm explants. The stimulatory effect of suboptimal concentrations of cyclic AMP derivatives is potentiated by the addition of theophylline. The stimulatory effect is limited to cyclic AMP derivatives, since dibutyryl-cyclic GMP and 5'-AMP have no effect. Thus agents that elevate intracellular cyclic AMP levels stimulate the chondrogenic differentiation of the unspecialized subridge mesoderm of the embryonic chick limb bud.     

In the limb AER Bmp2 and Bmp4 are required for dorsal-ventral patterning and interdigital cell death but not limb outgrowth

The apical ectodermal ridge (AER) in the vertebrate limb is required for limb outgrowth and patterning. To investigate the role BMP ligands expressed in the AER play in limb development we selectively inactivated both Bmp2 and Bmp4 in this tissue. The autopods of mice lacking both of these genes contained extra digits, digit bifurcations and interdigital webbing due to a decrease in programmed cell death and an increase in cell proliferation in the underlying mesoderm. Upon removal of Bmp2 and Bmp4 in the AER, no defects in proximal-distal patterning were observed. At the molecular level, removal of Bmp2 and Bmp4 in the AER caused an increase in Fgf expression, which correlated with an increase in both the width and length of the AER. Investigation of Engrailed-1 (En1) expression in the AER of limb buds in which Bmp2 and Bmp4 had been removed indicated that En1 expression was absent from this tissue. Our data suggests that AER expression of Bmp2 and Bmp4 is required for digit and dorsal-ventral patterning but surprisingly not for limb outgrowth.     

Evidence that the limb bud ectoderm is required for survival of the underlying mesoderm

The limb forms from a bud of mesoderm encased in a hull of ectoderm that grows out from the flank of the embryo. Coordinated signaling between the limb mesoderm and ectoderm is critical for normal limb outgrowth and patterning. The apical ectodermal ridge (AER), found at the distal tip, is a rich source of signaling molecules and has been proposed to specify distal structures and maintain the survival of cells in the underlying distal mesoderm. The dorsal and ventral non-AER ectoderm is also a source of signaling molecules and is important for dorsal–ventral patterning of the limb bud. Here we determine if this ectoderm provides cell survival signals by surgically removing the dorsal or ventral ectoderm during early chicken limb bud development and assaying for programmed cell death. We find that, similar to the AER, removal of the dorsal or ventral non-AER ectoderm results in massive cell death in the underlying mesoderm. In addition, although a re-epithelialization occurs, we find perturbations in the timing of Shh expression and, for the case of the dorsal ectoderm removal, defects in soft tissue and skeletal development along the proximal–distal axis. Furthermore, ectoderm substitution experiments show that the survival signal produced by the dorsal limb ectoderm is specific. Thus, our results argue that the non-AER ectoderm, like the AER, provides a specific survival signal to the underlying mesoderm that is necessary for normal limb development and conclusions drawn from experiments in which the non-AER ectoderm is removed, need to take into consideration this observation.     

Retinoic acid synthesis controlled by Raldh2 is required early for limb bud initiation and then later as a proximodistal signal during apical ectodermal ridge formation

We present evidence for the existence of two phases of retinoic acid (RA) signaling required for vertebrate limb development. Limb RA synthesis is under the control of retinaldehyde dehydrogenase-2 (Raldh2) expressed in the lateral plate mesoderm, which generates a proximodistal RA signal during limb outgrowth. We report that Raldh2(-/-) embryos lack trunk mesodermal RA activity and fail to initiate forelimb development. This is associated with deficient expression of important limb determinants Tbx5, Meis2, and dHand needed to establish forelimb bud initiation, proximal identity, and the zone of polarizing activity (ZPA), respectively. Limb expression of these genes can be rescued by maternal RA treatment limited to embryonic day 8 (E8) during limb field establishment, but the mutant forelimbs obtained at E10 display a significant growth defect associated with a smaller apical ectodermal ridge (AER), referred to here as an apical ectodermal mound (AEM). In these RA-deficient forelimbs, a ZPA expressing Shh forms, but it is located distally adjacent to the Fgf8 expression domain in the AEM rather than posteriorly as is normal. AER formation in Raldh2(-/-) forelimbs is rescued by continuous RA treatment through E10, which restores RA to distal ectoderm fated to become the AER. Our findings indicate the existence of an early phase of RA signaling acting upstream of Tbx5, Meis2, and dHand, followed by a late phase of RA signaling needed to expand AER structure fully along the distal ectoderm. During ZPA formation, RA acts early to activate expression of dHand, but it is not required later for Shh activation.     

Limbiting outgrowth: BMPs as negative regulators in limb development

Rapid progress is being made in understanding how integrated signaling pathways direct patterned outgrowth of the vertebrate limb. In contrast, the mechanisms that constrain limb outgrowth, and thus delimit adult morphology, remain poorly understood. Two recent pioneering reports have implicated bone morphogenetic proteins (BMPs) in negatively regulating the function of the apical ectodermal ridge (AER), an inductive structure required for continued proximodistal specification of limb elements.^(1,2) These studies provide the first insights into how the termination of a limb bud signaling center is accomplished, and intriguingly suggest how distinct aspects of limb morphogenesis are regulated. BioEssays 21:721–725, 1999. © 1999 John Wiley & Sons, Inc.