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.     

BMPs restrict the position of premuscle masses in the limb buds by influencing Tcf4 expression

Previous studies have shown the distally retreating source of Scatter factor/Hepatocyte growth factor (SF/HGF) can account for the distal migration of myogenic precursor cells in the limb bud mesenchyme. However, the normal expression pattern of Sf/Hgf alone does not explain the distribution of muscle precursor cells. Hence, the position of the dorsal and ventral premuscle masses suggests the presence of additional patterning factors. We present evidence that BMP2 and 4 can act as such factors by inhibiting the expression of Tcf4, a downstream element of the canonical Wnt pathway. The normal position of muscle cells depends on the correct distribution of BMP and SF/HGF throughout the limb bud mesenchyme. Removal or inhibition of the BMP signals within the limb margins leads to a shift in position resulting in the fusion of the dorsal and ventral premuscle masses towards the manipulated areas. In the absence of BMPs, mispositioning requires the presence of SF/HGF. Consequently, ectopic application of exogenous SF/HGF in the presence of BMP signals does not change muscle positioning. We conclude that correct positioning of the premuscle masses in the limb buds is controlled by the combined influence of SF/HGF signals--guiding cells mainly in the proximo-distal axis--and BMP signals that restrict the positioning to the dorsal and ventral central portions of the limb buds.     

Morphogens in chick limb development

Retinoic acid is a good candidate for a morphogen in chick limb bud development. The challenge now is to determine how retinoic acid interacts with limb bud cells and how the retinoic acid signal is integrated with other signals to mould and pattern the developing limb.     

Dorsal-ventral signaling in limb development

In both Drosophila wings and vertebrate limbs, signaling between dorsal and ventral cells establishes an organizer that promotes limb formation. Significant progress has been made recently towards characterizing the signaling interactions that occur at the dorsal—ventral limb border. Studies of chicks have indicated that, as in Drosophila, this signaling process requires the participation of Fringe. Studies of Drosophila have indicated that Fringe functions by inhibiting the ability of Notch to be activated by one ligand, Serrate, while potentiating the ability of Notch to be activated by another ligand, Delta. Recent studies of both Drosophila and vertebrates have also shed new light on the signaling activity of the dorsal—ventral boundary limb organizer, and have highlighted how this organizer is maintained by feedback mechanisms with neighboring cells.     

Constructive antagonism in limb development

Several advances have been made in our understanding of the control of the growth and patterning of embryonic limbs. Development of the vertebrate limb is dependent on reciprocal interactions between the ectoderm and mesoderm that regulate the structure and function of the apical ectodermal ridge. One key component of this regulatory program appears to be the precise control of signaling by members of the bone morphogenetic protein family via multiple antagonistic interactions.     

Vertebrate limb development and possible clues to diversity in limb form

Chick embryos are good models for vertebrate development. The principles that underlie chick wing development have been discovered and there is increasing knowledge about the molecules involved. The importance of identifying molecules is that this provides a direct link to understanding the genetic basis of diversity in form. Chick wing development will be compared with limb development in other vertebrates. Possible mechanisms that could lead to variations in form, including limb reductions and limblessness, differences between fore- and hindlimbs, limb proportions, and interdigital webbing can be suggested.     

Vertical regulation of En-2 expression and eye development by FGFs and BMPs

The formation of the midbrain region depends mainly on the activity of a signalling center located in the isthmus region, on the border between the prospective mesencephalon and metencephalon. FGF-8 has been proposed as a signalling molecule responsible for this specification because of its expression pattern and its ability to elicit duplication of the midbrain region when expressed ectopically in the neuroepithelium. Here we present evidence that members of the FGF family of growth factors when released in the cephalic mesenchyme are able to extend the expression of the mesencephalic marker En-2 to both the anterior and the posterior regions of its original landmark. This alteration in the expression pattern of En-2 is not accompanied by a significant alteration in the later development of the midbrain-cerebellar anlage, although the eye development is severely altered. Members of the bone morphogenetic protein family ectopically released from the mesenchyme down-regulate the expression of En-2 and also have an effect on the development of the eye. These results demonstrate that growth factor molecules produced in the mesenchyme (vertical signalling) participate in the correct establishment of the antero-posterior patterning of the cephalic nervous system during development.     

Control of vertebrate limb outgrowth by the proximal factor Meis2 and distal antagonism of BMPs by Gremlin

The mechanisms controlling growth and patterning along the proximal-distal axis of the vertebrate limb are yet to be understood. We show that restriction of expression of the homeobox gene Meis2 to proximal regions of the limb bud is essential for limb development, since ectopic Meis2 severely disrupts limb outgrowth. We also uncover an antagonistic relationship between the secreted factors Gremlin and BMPs required to maintain the Shh/FGF loop that regulates distal outgrowth. These proximal and distal factors have coordinated activities: Meis2 can repress distal genes, and Bmps and Hoxd genes restrict Meis2 expression to the proximal limb bud. Moreover, combinations of BMPs and AER factors are sufficient to distalize proximal limb cells. Our results unveil a novel set of proximal-distal regulatory interactions that establish and maintain outgrowth of the vertebrate limb.     

Multiple functions of BMPs in chondrogenesis

The ability of bone morphogenetic proteins (BMPs) to promote chondrogenesis has been investigated extensively over the past two decades. Although BMPs promote almost every aspect of chondrogenesis, from commitment to terminal differentiation is well known, the mechanisms of BMP action in discrete aspects of endochondral bone formation have only recently begun to be investigated. In this review, we focus on in vivo studies that have identified interactions between BMP signaling pathways and key downstream targets of BMP action in chondrogenesis. We also discuss evidence regarding the potential roles of BMP receptors in mediating distinct aspects of chondrogenesis, and studies investigating the intersection of BMP pathways with other pathways known to coordinate the progression of chondrocytes through the growth plate. These studies indicate that both Smad-dependent and -independent BMP pathways are required for chondrogenesis, and that BMPs exert essential roles via regulation of the Indian hedgehog (IHH)/parathyroid hormone-related protein (PTHrP) and fibroblast growth factor (FGF) pathways in the growth plate.     

The vasculature and limb development

The developing vascular pattern of the embryonic chick limb results from a combination of two properties: the intrinsic self-assembly and branching properties of the vascular cells and the extrinsic information associated with the expanding mitotic population of mesenchymal cells; and the inhibitory factors which restrict the entrance of vessels into particular domains and/or decrease the branching frequency of such vessels. It is hypothesized that an important component of limb pattern formation is the interplay between the dividing population of mesenchymal cells and the intrinsic properties of the vascular cells. It is further asserted that the presence of particular vascular elements may, indeed, be 'positional information'. Two examples are cited involving aspects of limb duplication to support this possibility; it is suggested that vascular vessel size of a host limb may dictate the polarity of duplication events. The presented hypothesis emphasizes that the interplay between the intrinsic properties of self-assembly into tissues and extrinsic factors which establish boundaries and morphologies is involved in both vascular and limb pattern formation.     

Wnt signalling during limb development

Wnts control a number of processes during limb development--from initiating outgrowth and controlling patterning, to regulating cell differentiation in a number of tissues. Interactions of Wnt signalling pathway components with those of other signalling pathways have revealed new mechanisms of modulating Wnt signalling, which may explain how different responses to Wnt signalling are elicited in different cells. Given the number of Wnts that are expressed in the limb and their ability to induce differential responses, the challenge will be to dissect precisely how Wnt signalling is regulated and how it controls limb development at a cellular level, together with the other signalling pathways, to produce the functional limb capable of coordinated precise movements.     

Function of FGF-4 in limb development

The apical ectodermal ridge plays a central role in limb development through its interactions with the underlying mesenchyme. Removal of the AER results in cessation of limb outgrowth and leads to truncation of the limb along the proximo-distal axis. The many functions attributed to the ridge include maintenance of the progress zone mesenchyme. Here, cells are stimulated to proliferate, are maintained in an undifferentiated state, and are assigned progressively more distal positional values as the limb grows. The AER also functions to maintain the activity of the polarizing region, a region of mesenchyme which is thought to provide the primary signal for patterning along the antero-posterior axis. We have begun to explore the function of fibroblast growth factor-4 (FGF-4) during limb development. FGF-4, which encodes an efficiently secreted protein, is expressed in the AER. We have previously demonstrated that FGF-4 protein can stimulate limb mesenchyme proliferation and can induce the expression of a downstream homeobox gene, Evx-1 (homologue of the Drosophila even-skipped gene), that is normally regulated by a signal from the AER. To determine to what extent FGF-4 protein can substitute for the AER to allow normal limb outgrowth, we performed experiments on the developing chick limb in ovo. Remarkably, we find that after AER removal, the FGF-4 protein can provide all the signals required for virtually normal outgrowth and patterning of the limb. Further studies indicate that proliferation of progress zone cells is not sufficient, and that an additional signal is produced by the posterior mesenchyme in response to FGF-4 which enables progress zone cells to acquire progressively more distal fates. Thus FGF-4 maintains progress zone activity through a combination of at least two signals—one that acts directly on progress zone cells to stimulate their proliferation, and one that acts indirectly by maintaining the production of patterning signal(s) by the posterior mesenchyme. We further show that failure of the posterior mesenchyme to produce this signal correlates with failure to maintain polarizing activity. This raises the possibility that the signal produced by the posterior mesenchyme and required for progressive proximo-distal limb patterning is identical to the polarizing activity. Further experiments demonstrate that retinoic acid, which mimics the activity of the polarizing region, can supply this signal. In conclusion, the finding that a single growth factor can serve as both the direct and indirect signals required to maintain progress zone activity provides a simple mechanism for ensuring that growth and pattern formation are linked in the developing limb. © 1994 Wiley-Liss, Inc.     

Retinoid receptors in vertebrate limb development.

Although the precise role of retinoids in limb development remains obscure, the finding that retinoic acid can produce major alterations in limb patterning suggests that this ligand might be involved in the process of limb morphogenesis. Here we describe the patterns of expression of retinoic acid receptors and cytosolic retinoid binding proteins during the course of limb morphogenesis. Examining the distribution of these molecules in the limb and correlating their presence with important processes in limb development could help elucidate their possible functions.