Inductive activity and enduring cellular constitution of a supernumerary apical ectodermal ridge grafted to the limb bud of the chick embryo.

Apical ectodermal ridges (AERs) isolated from 3- to 4-day chick and quail embryos were prepared by means of trypsinization and microdissection and then were grafted to the dorsal or ventral side of a host chick wing bud. They induced supernumerary limb outgrowths from the host bud showing, respectively, a bidorsal or biventral organization, as determined by the patterns of feather germs. The grafted ridge cells persisted, as revealed by histological sections of supernumerary chick limb parts growing under the influence of quail AERs, whose cells are readily distinguished after application of the Feulgen reagent.These results show that the AER induces limb outgrowth regardless of whether it is associated with dorsal or ventral limb ectoderm and that its continued existence is not dependent on contributions of ectodermal cells from the opposed ectodermal faces of the limb bud. The AER is pictured as maintaining the subjacent mesoderm in a condition of developmental plasticity without specifying its differentiation with respect to the proximodistal axis. It remains uncertain whether the positional values of cells that develop under the influence of the AER arise within these cells themselves or appear in response to influences from proximal sources.     

Beta-catenin-dependent Wnt signaling in apical ectodermal ridge induction and FGF8 expression in normal and limbless mutant chick limbs

The fibroblast growth factor (FGF) and beta-catenin-dependent Wnt signaling pathways are key regulators of vertebrate limb development. FGF10 induces expression of Wnt3a, which regulates the formation and FGF8 expression of the apical ectodermal ridge (AER). In amelic limbless limbs, an AER fails to form and FGF8 is not expressed, despite expression of FGF10. It has been found that Wnt3a is initially expressed in limbless ectoderm, although subsequently is drastically reduced. In addition, changes in the expression pattern or level of several Frizzled receptors, Axin, Lef1/Tcf1 and beta-catenin have been found in limbless limbs. Notably, while normal wing buds respond to LiCl-stimulated activation of beta-catenin-dependent signaling by forming ectopic, FGF8-expressing AER, LiCl was unable to induce an AER in limbless wing buds. The results of this study suggest that the limbless gene is required for beta-catenin-dependent Wnt signaling in limb ectoderm leading to FGF8 expression and AER formation.     

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.     

Apical ectodermal ridge induction by the transplantation of En-1-overexpressing ectoderm in chick limb bud

In the early chick embryo, the dorsal–ventral (DV) boundary organizes the apical ectodermal ridge (AER) structure in the limb bud field. Here it is reported that Engrailed-1 ( En-1 ), a homolog of the Drosophila segment polarity gene engrailed expressed in the ventral limb ectoderm, participates in AER formation at the DV boundary of the limb bud. Restricted ectopic expression of En-1 in the dorsal side of the limb bud by transplantation of En-1 -overexpressing ectoderm induces ectopic AER at the boundary of En-1 -positive and -negative cells. The results suggest that En-1 is involved in AER formation at the DV boundary of the limb bud.     

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.     

Smad1/Smad5 signaling in limb ectoderm functions redundantly and is required for interdigital programmed cell death

Bone morphogenetic proteins (BMPs) are secreted signals that regulate apical ectodermal ridge (AER) functions and interdigital programmed cell death (PCD) of developing limb. However the identities of the intracellular mediators of these signals are unknown. To investigate the role of Smad proteins in BMP-regulated AER functions in limb development, we inactivated Smad1 and Smad5 selectively in AER and ventral ectoderm of developing limb, using Smad1 or/and Smad5 floxed alleles and an En1(Cre/+) knock-in allele. Single inactivation of either Smad1 or Smad5 did not result in limb abnormalities. However, the Smad1/Smad5 double mutants exhibited syndactyly due to a reduction in interdigital PCD and an increase in interdigital cell proliferation. Cell tracing experiments in the Smad1/Smad5 double mutants showed that ventral ectoderm became thicker and the descendents of ventral En1(Cre/+) expressing ectodermal cells were located at dorsal interdigital regions. At the molecular level, Fgf8 expression was prolonged in the interdigital ectoderm of embryonic day (E) 13 Smad1/Smad5 double mutants, suggesting that the ectopic Fgf8 expression may serve as a survival signal for interdigital epithelial and mesenchymal cells. Our result suggests that Smad1 and Smad5 are required and function redundantly as intracellular mediators for BMP signaling in the AER and ventral ectoderm. Smad1/Smad5 signaling in the AER and ventral ectoderm regulates interdigital tissue regression of developing limb. Our mutants with defects in interdigital PCD could also serve as a valuable model for investigation of PCD regulation machinery.     

Dual requirement of ectodermal Smad4 during AER formation and termination of feedback signaling in mouse limb buds

BMP signaling is pivotal for normal limb bud development in vertebrate embryos and genetic analysis of receptors and ligands in the mouse revealed their requirement in both mesenchymal and ectodermal limb bud compartments. In this study, we genetically assessed the potential essential functions of SMAD4, a mediator of canonical BMP/TGFß signal transduction, in the mouse limb bud ectoderm. Msx2‐Cre was used to conditionally inactivate Smad4 in the ectoderm of fore‐ and hindlimb buds. In hindlimb buds, the Smad4 inactivation disrupts the establishment and signaling by the apical ectodermal ridge (AER) from early limb bud stages onwards, which results in severe hypoplasia and/or aplasia of zeugo‐ and autopodal skeletal elements. In contrast, the developmentally later inactivation of Smad4 in forelimb buds does not alter AER formation and signaling, but prolongs epithelial‐mesenchymal feedback signaling in advanced limb buds. The late termination of SHH and AER‐FGF signaling delays distal progression of digit ray formation and inhibits interdigit apoptosis. In summary, our genetic analysis reveals the temporally and functionally distinct dual requirement of ectodermal Smad4 during initiation and termination of AER signaling. genesis 51:660–666. © 2013 Wiley Periodicals, Inc.     

Fate map of mouse ventral limb ectoderm and the apical ectodermal ridge

The apical ectodermal ridge (AER) is a critical signaling center at the tip of the limb that promotes outgrowth. In mouse, formation of the AER involves a gradual restriction of AER gene expression from a broad ventral preAER domain to the tip of the limb, as well as progressive thickening of cells to form a multilayered epithelium. The AER is visible from embryonic day 10.5 to 13.5 (E10.5-E13.5) in the mouse forelimb. Previous short-term fate mapping studies indicated that, once a cell is incorporated into the AER, its descendents remain within the AER. In addition, some preAER cells appear to become incorporated into the ventral ectoderm. In the present study, we used an inducible CreER/loxP fate mapping approach in mouse to examine the long-term contribution of preAER cells to limb ventral ectoderm, as well as the ultimate fate of the mature AER cells. We used a CreER transgene that contains Msx2 regulatory sequences specific to the developing AER, and demonstrate by marking preAER cells that, at stage 2 of mouse limb bud development, the majority of the ventral ectoderm that protrudes from the body wall later covers only the paw. Furthermore, when Msx2-CreER-expressing preAER cells are marked after the onset of preAER gene expression, a similar domain of paw ventral ectoderm is marked at E16.5, in addition to the AER. Strikingly, mapping the long-term fate of cells that form the mature AER showed that, although this structure is indeed a distinct compartment, AER-derived cells are gradually lost after E12.5 and no cells remain by birth. A distinct dorsal/ventral border nevertheless is maintained in the ectoderm of the paw, with the distal-most border being located at the edge of the nail bed. These studies have uncovered new aspects of the cellular mechanisms involved in AER formation and in partitioning the ventral ectoderm in mouse limb.     

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.     

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.     

BMPR-IA signaling is required for the formation of the apical ectodermal ridge and dorsal-ventral patterning of the limb.

We demonstrate that signaling via the bone morphogenetic protein receptor IA (BMPR-IA) is required to establish two of the three cardinal axes of the limb: the proximal-distal axis and the dorsal-ventral axis. We generated a conditional knockout of the gene encoding BMPR-IA (Bmpr) that disrupted BMP signaling in the limb ectoderm. In the most severely affected embryos, this conditional mutation resulted in gross malformations of the limbs with complete agenesis of the hindlimbs. The proximal-distal axis is specified by the apical ectodermal ridge (AER), which forms from limb ectoderm at the distal tip of the embryonic limb bud. Analyses of the expression of molecular markers, such as Fgf8, demonstrate that formation of the AER was disrupted in the Bmpr mutants. Along the dorsal/ventral axis, loss of engrailed 1 (En1) expression in the non-ridge ectoderm of the mutants resulted in a dorsal transformation of the ventral limb structures. The expression pattern of Bmp4 and Bmp7 suggest that these growth factors play an instructive role in specifying dorsoventral pattern in the limb. This study demonstrates that BMPR-IA signaling plays a crucial role in AER formation and in the establishment of the dorsal/ventral patterning during limb development.     

Distribution and possible function of an adrenomedullin-like peptide in the developing chick limb bud

Adrenomedullin (AM) is a multifunctional peptide that exhibits discrete domains of expression during mouse embryogenesis consistent with a role in regulating growth and differentiation during morphogenesis. Here we report that AM immunoreactivity is present at high levels throughout the apical ectodermal ridge (AER) of the chick limb bud as the AER is directing the outgrowth and patterning of underlying limb mesoderm. Immunostaining is particularly strong along the surfaces of the contiguous cells of the AER. AM immunoreactivity attenuates as the AER regresses and is absent from the distal apical ectoderm of stage 20 limbless mutant limb buds which fail to develop an AER. To explore the possible role of AM in AER activity, we examined the effect of exogenous AM and an AM inhibitor on the in vitro morphogenesis of limb mesoderm, cultured in the presence and absence of the AER. Although exogenous AM cannot substitute for the AER in promoting outgrowth of limb mesoderm in vitro, a specific AM antagonist, AM(22-52), impairs the outgrowth and proliferation of limb mesoderm cultured in the presence of the AER. This is consistent with the possibility that inhibition of endogenous AM activity in the AER impairs the ability of the AER to promote limb morphogenesis. Taken together, these studies suggest that an AM-like molecule may function in an autocrine fashion to regulate some aspect of AER activity.     

Regulation by members of the transforming growth factor beta superfamily of the digital and interdigital fates of the autopodial limb mesoderm

Embryonic limb outgrowth is accomplished by the proliferation of mesodermal cells in the progress zone. In this region, mesodermal cells are maintained in an undifferentiated and proliferating state by the action of the apical ectodermal ridge (AER). Differentiation of these cells into individual skeletal elements occurs when the cells are displaced proximally and leave the influence of the AER as a consequence of the accumulation of cells in that region. Here we review the evidence obtained in the last few years showing that members of the transforming growth factor beta (TGFbeta) subfamily and bone morphogenetic proteins (BMPs) act as proximal signals in the autopod regulating the fate of the progress zone cells towards chondrogenesis or apoptosis. Our findings show that apoptosis is regulated by BMPs while chondrogenesis requires the interaction of TGFbetas and BMPs. Fibroblast growth factors (FGFs) produced by the AER exert an opposite function to both TGFbetas and BMPs, maintaining the progress zone cells in an undifferentiated state.     

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.     

The apical ectodermal ridge is a timer for generating distal limb progenitors

The apical ectodermal ridge (AER) is a transient embryonic structure essential for the induction, patterning and outgrowth of the vertebrate limb. However, the mechanism of AER function in limb skeletal patterning has remained unclear. In this study, we genetically ablated the AER by conditionally removing FGFR2 function and found that distal limb development failed in mutant mice. We showed that FGFR2 promotes survival of AER cells and interacts with Wnt/beta-catenin signaling during AER maintenance. Interestingly, cell proliferation and survival were not significantly reduced in the distal mesenchyme of mutant limb buds. We established Hoxa13 expression as an early marker of distal limb progenitors and discovered a dynamic morphogenetic process of distal limb development. We found that premature AER loss in mutant limb buds delayed generation of autopod progenitors, which in turn failed to reach a threshold number required to form a normal autopod. Taken together, we have uncovered a novel mechanism, whereby the AER regulates the number of autopod progenitors by determining the onset of their generation.