Limb Development

Limbs are remarkable structures that are designed almost solely for mechanical functions: motion and force. These functions are achieved through the coordinated development of various tissue components. No single tissue in the limb takes shape without reference to the other tissues with which it is associated. The limb as a whole develops according to a master blueprint that reveals itself sequentially with each successive stage in limb formation. Many of the factors that control limb development cannot be seen by examining morphology alone, but rather must be shown by experimental means or through the localization of molecules. Despite remarkable progress in understanding the molecular basis of the tissue interactions that control limb development, many fundamental questions remain poorly understood. Limb anomalies are common and highly visible. Many of these anomalies are now known to be reflections of disturbances in specific cellular or molecular interactions that are fundamental to limb development. These are discussed in Clinical Correlation 10.1 at the end of the chapter.

Initiation of Limb Development

Limb formation begins relatively late in embryonic development (at the end of the fourth week in humans) with the activation of a group of mesenchymal cells in the somatic lateral plate mesoderm (Fig. 10.1). The initial stimulus for limb development is incompletely understood. Experimental evidence suggests that signals from the paraxial mesoderm (probably based on the Hox code and ultimately dependent on retinoic acid signaling) initiates a level-specific expression of two T-box transcription factors in the lateral plate mesoderm. Tbx5 in the area of the future forelimb and Tbx4 (along with Pitx-1) in the area of the future hindlimb stimulate the expression and secretion of fibroblast growth factor-10 (FGF-10) by the local mesodermal cells (Fig. 10.2A). FGF-10 stimulates the overlying ectoderm to produce FGF-8. Soon a feedback loop involving FGF-10 and FGF-8 is established, and limb development begins.

Fig. 10.1 Cross section through the trunk at an early stage of limb bud development that shows the position of the limb bud in relation to that of the somite (dermatome) and other major structures. The limb bud is an outgrowth of the body wall (lateral plate mesoderm).

The Tbx transcription factors appear to be the initial local driving forces of limb development. If Tbx5 expression in a mouse is prevented, forelimbs fail to develop (Fig. 10.2B). Similarly, in FGF-10 knockout mice, limbs (and lungs) do not form. Conversely, if a bead soaked with FGF-10 is implanted within the future flank region of a chick embryo, a supernumerary limb develops at the spot. Once the interaction between ectoderm and mesoderm has begun, the limb primordium contains sufficient developmental information to form a limb even if isolated from the rest of the body (a so-called self-differentiating system).

The primacy of the early limb mesoderm was shown long ago by transplantation experiments on amphibian embryos. If early limb mesoderm is removed, a limb fails to form. If the same mesoderm is transplanted to the flank of an embryo, however, a supernumerary limb grows at that site. In contrast, if the ectoderm overlying the normal limb mesoderm is removed, new ectoderm heals the defect, and a limb forms. If the original ectoderm that was removed is grafted to the flank, no limb forms. These experiments show that in early limb development, mesoderm is the primary bearer of the limb blueprint, and ectoderm is only secondarily co-opted into the system.

In rare instances, individuals are born without one or sometimes all limbs (amelia) (Fig. 10.3). In some cases, this situation may reflect a disturbance in the production of the transcription factors or signaling molecules that initiate limb development or the cellular receptors for these molecules.

Fig. 10.3 Amelia of the right leg in an infant.
Despite the absence of a foot, the left leg contains an upper and a lower leg segment. (From Connor JM, Ferguson-Smith MA: *Essential medical genetics,* ed 3, Oxford, 1991, Blackwell Scientific.)

Regulative Properties and Axial Determination

The early limb primordium is a highly regulative system, with properties similar to those described for the cleaving embryo (see p. 45). These properties can be summarized with the following experiments (Fig. 10.4):

Fig. 10.4 Experiments showing regulative properties of limb disks in amphibian embryos.
A, Combining two identical halves of limb disks results in a single limb. B, Separation of two halves of a limb disk by a barrier causes each half to form a normal limb of the same polarity. C, After various types of tissue removal, the remaining limb tissue regulates to form a normal limb. D, Combining two disks results in the formation of a single normal limb. E, Mechanical disruption of a limb disk is followed by reorganization of the pieces and the formation of a normal limb. A, anterior; D, dorsal; P, posterior; V, ventral. (Data from Harrison RG: *J Exp Zool* 32:1-136, 1921; and Swett FH: *Q Rev Biol* 12:322-339, 1937.)

1. If part of a limb primordium is removed, the remainder reorganizes to form a complete limb.

2. If a limb primordium is split into two halves, and these are prevented from fusing, each half gives rise to a complete limb (the twinning phenomenon).

3. If two equivalent halves of a limb primordium are juxtaposed, one complete limb forms.

4. If two equivalent limb disks are superimposed, they reorganize to form a single limb (see the section on tetraparental embryos [p. 45]).

5. In some species, disaggregated limb mesoderm can reorganize and form a complete limb.

The organization of the limb is commonly related to the Cartesian coordinate system. The anteroposterior * axis runs from the first (anterior) to the fifth (posterior) digit. The back of the hand or foot is dorsal, and the palm or sole is ventral. The proximodistal axis extends from the base of the limb to the tips of the digits.

Experiments involving the transplantation and rotation of limb primordia in lower vertebrates have shown that these axes are fixed in a sequential order: anteroposterior to dorsoventral to proximodistal. Early fixation of the anteroposterior axis may result from the expression of the transcription factors Gli-3 in the anterior and Hand-2 in the posterior part of the early limb field (see Fig. 10.2A). These two molecules mutually oppose each other’s actions. Before all three axes are specified, a left limb primordium can be converted into a normal right limb simply by rotating it with respect to the normal body axes. These axes are important as reference points in several aspects of limb morphogenesis. Evidence indicates a similar sequence of axial specifications in certain other primordia, such as those of the retina and inner ear.

Outgrowth of Limb Bud

Shortly after its initial establishment, the limb primordium begins to bulge from the body wall (late in the first month for the human upper extremity [Fig. 10.5]). At this stage, the limb bud consists of a mass of similar-looking mesodermal cells covered by a layer of ectoderm. Despite its apparently simple structure, the limb bud contains enough intrinsic information to guide its development because if a mammalian limb bud is transplanted to another region of the body or is cultured in vitro, a recognizable limb forms.

Fig. 10.5 Scanning electron micrograph of a 4-week human embryo (5 mm), with 34 pairs of somites. Toward the *lower left,* the right arm bud protrudes from the body. (From Jirásek JE: *Atlas of human prenatal morphogenesis,* Amsterdam, 1983, Martinus Nijhoff.)

A distinctive feature is the presence of a ridge of thickened ectoderm (apical ectodermal ridge [AER]) located along the anteroposterior plane of the apex of the limb bud (Fig. 10.6). During much of the time when the AER is present, the hand-forming and foot-forming regions of the developing limb bud are paddle shaped, with the apical ridge situated along the rim of the paddle (Fig. 10.7). Experiments have shown that the AER interacts with the underlying limb bud mesoderm to promote outgrowth of the developing limb. Other aspects of limb development, such as morphogenesis (the development of form), are guided by information contained in the mesoderm.

Fig. 10.6 Scanning electron micrograph of the flattened limb bud of a human embryo that shows the prominent apical ectodermal ridge traversing the apical border. (From Kelley RO, Fallon JF: *Dev Biol* 51:241-256, 1976.)

This section outlines many of the ways in which the limb bud mesoderm and ectoderm interact to control limb development. Recognition of these developmental mechanisms is important in understanding the genesis of many limb malformations.

Apical Ectodermal Ridge

The earliest limb bud begins to form before an AER is present, but soon a thickened AER appears along the border between dorsal and ventral limb ectoderm. Molecular studies have shown that the position of the AER corresponds exactly to the border between dorsal ectoderm, which expresses the signaling molecule radical fringe, and ventral ectoderm, which expresses the transcription factor Engrailed-1 (En-1) (see Fig. 10.17A).

Although the AER had been recognized for years, its role in limb development was not understood until it was subjected to experimental analysis. Removal of the AER results in an arrest of limb development, thus leading to distal truncation of the limb (Fig. 10.8). In the limbless mutant in chickens, early limb development is normal; later, the AER disappears, and further limb development ceases. If mutant ectoderm is placed over normal limb bud mesoderm, limb development is truncated, whereas combining mutant mesoderm with normal ectoderm results in more normal limb development. These findings suggest that the ectoderm is defective in this mutant.

Fig. 10.8 *Top three,* Effect of removing the apical ectodermal ridge at successively later stages on the development of the avian wing bud. The more mature the wing bud, the more skeletal elements form after apical ridge removal. Missing structures are shown in *light gray. Bottom,* Normal development of an untouched wing bud. (Based on Saunders JW: *J Exp Zool* 108:363-403, 1948.)

Further analysis has shown that in the limbless mutant, the entire ectoderm of the limb bud displays a dorsal character; that is, radical fringe and other “dorsal” molecules are expressed in dorsal and ventral ectoderm. Correspondingly, En-1 is not expressed in ventral ectoderm. In the absence of the juxtaposition of ectoderm with dorsal and ventral properties, an AER cannot be maintained.

The power of the AER is shown by experiments or mutants that result in the formation of two AERs on the limb bud. This situation leads to a supernumerary limb, as is illustrated by the mutants eudiplopodia in chickens and diplopodia in humans (Fig. 10.9).

Fig. 10.9 A, Duplicated wing bud in a chick with eudiplopodia. Under the influence of a secondary apical ectodermal ridge, a supernumerary limb bud forms. B, Diplopodia in a human. Dorsal and ventral views of the right foot, in which duplication has occurred along the anteroposterior axis. (A, From Goetinck P: *Dev Biol* 10:71-79, 1964, B, Courtesy of D. Hootinck, Buffalo, N.Y.)

The outgrowth-promoting signal produced by the AER is FGF. In the earliest stages of limb formation, the lateral ectoderm begins to produce FGF-8 as it thickens to form an AER. As the limb bud begins to grow out, the apical ridge also produces FGF-4, FGF-9, and FGF-17 in its posterior half. If the AER is removed, outgrowth of limb bud mesoderm can be supported by the local application of FGFs. Other studies have shown that in mutants characterized by deficient or absent outgrowth of the limb, the mutant ectoderm fails to produce FGF. The effects of the FGF produced by the apical ectoderm on the underlying mesoderm are discussed later in this chapter.

Mesoderm of Early Limb Bud

Structure and Composition

The mesoderm of the early limb bud consists of homogeneous mesenchymal cells supplied by a well-developed vascular network. The mesenchymal cells are embedded in a matrix consisting of a loose meshwork of collagen fibers and ground substance, with hyaluronic acid and glycoproteins prominent constituents of the latter. There are no nerves in the early limb bud.

It is impossible to distinguish different cell types within the early limb bud mesenchyme by their morphology alone. Nevertheless, mesenchymal cells from several sources are present (Fig. 10.10). Initially, the limb bud mesenchyme consists exclusively of cells derived from the lateral plate mesoderm. These cells give rise to the skeleton, connective tissue, and some blood vessels. Mesenchymal cells derived from the somites migrate into the limb bud as precursors of muscle and endothelial cells. Another population of migrating cells is that from the neural crest; these cells ultimately form the Schwann cells of the nerves, sensory nerves, and pigment cells (melanocytes).

Fig. 10.10 The different types of cells that enter the limb bud.

Mesodermal-Ectodermal Interactions and the Role of Mesoderm in Limb Morphogenesis

Limb development occurs as the result of continuous interactions between the mesodermal and ectodermal components of the limb bud. The apical ectoderm stimulates outgrowth of the limb bud by promoting mitosis and preventing differentiation of the distal mesodermal cells of the limb bud. Although the AER promotes outgrowth, its own existence is reciprocally controlled by the mesoderm. If an AER from an old limb bud is transplanted onto the mesoderm of a young wing bud, the limb grows normally until morphogenesis is complete. If old limb bud mesoderm is covered by young apical ectoderm, however, limb development ceases at a time appropriate for the age of the mesoderm and not that of the ectoderm.

Polydactyly is a condition characterized by supernumerary digits and exists as a mutant in birds. Reciprocal transplantation experiments between mesoderm and ectoderm have shown that the defect is inherent in the mesoderm and not the ectoderm. Polydactyly in humans (Fig. 10.11) is typically inherited as a genetic recessive trait and is commonly found in populations such as certain Amish communities in the United States in which the total genetic pool is relatively restricted (see Clinical Correlation 10.1 for further details).

Fig. 10.11 Ultrasound images of normal (A) and polydactylous (six digit) (B) hands of human fetuses at 16 and 31 weeks. In both cases, the digits are imaged in cross section. *Arrow* indicates the thumb. F, face; 1, thumb; 2 to 6, fingers on polydactylous hand. (A, From Bowerman R: *Atlas of normal fetal ultrasonographic anatomy,* St. Louis, 1992, Mosby; B, from Nyberg D and others: *Diagnostic ultrasound of fetal anomalies,* St. Louis, 1990, Mosby.)

Zone of Polarizing Activity and Morphogenetic Signaling

During experiments investigating programmed cell death in the avian limb bud, researchers grafted mesodermal cells from the posterior base of the avian wing bud into the anterior margin. This manipulation resulted in the formation of a supernumerary wing, which was a mirror image of the normal wing (Fig. 10.12). Much subsequent experimentation has shown that this posterior region, called the zone of polarizing activity (ZPA), acts as a signaling center along the anteroposterior axis of the limb. The signal itself has been shown to be sonic hedgehog (shh) (see Fig. 10.16), a molecule that mediates a wide variety of tissue interactions in the embryo (see Table 4.4). As seen later in this chapter, shh not only organizes tissues along the anteroposterior axis, but also maintains the structure and function of the AER. In the absence of the ZPA or shh, the apical ridge regresses.

Fig. 10.12 A, Grafting of the zone of polarizing activity (ZPA) into the anterior border of the avian limb bud results in the formation of a secondary apical ectodermal ridge and a supernumerary limb. B, Implantation of a bead soaked in retinoic acid into the anterior border of the limb bud also stimulates the formation of a supernumerary limb. AER, apical epidermal ridge.

Cross-species grafting experiments have shown that mammalian (including human) limb buds also contain a functional ZPA. A transplanted ZPA acts on the AER and elicits a growth response from the mesenchymal cells just beneath the part of the ridge adjacent to the transplanted ZPA. As few as 50 cells from the ZPA can stimulate supernumerary limb formation. Other structures, such as pieces of Hensen’s node, notochord, and even feather germs have been shown to stimulate the formation of supernumerary limbs if grafted into the anterior margin of the limb. Since these experiments were conducted, all the effective implanted tissues have been found to be sources of shh.

The ZPA is already established by the time the limb bud begins to grow out from the body wall. There is evidence that in the forelimb the position of the ZPA is determined by the location of the highest concentration of Hoxb8 expression along the body axis. Experiments have shown that in response to the localized application of retinoic acid along the anterior margin of the forelimb bud, Hoxb8 expression is induced within 30 minutes. This suggests a cascade beginning with retinoic acid signaling, leading to Hoxb8 expression, which determines the location of the ZPA.

Shh induces the expression of the signaling molecule gremlin, which has two inhibitory functions. Gremlin inhibits the action of mesodermal bone morphogenetic protein-2 (BMP-2), which in itself inhibits the expression of FGF-4 in the AER. Such an inhibition of a BMP inhibitor is reminiscent of the sequence of events involved in primary neural induction (see p. 84). In contrast, gremlin, which is localized in the posterior part of the limb bud, inhibits the action of Gli-3 so that Gli-3 functions only in the anterior part. Within the anterior part of the limb bud, Gli-3 inhibits the expression of shh. In Gli-3 mutants, shh becomes expressed ectopically in the anterior limb bud, and preaxial polydactyly results.

As the limb bud elongates, the ZPA becomes translocated more distally, and it becomes surrounded by an increasingly large zone of formerly shh-producing cells that were derived from the ZPA. Later, these cells become heavily involved in the formation of digits and in events leading to the termination of limb development.

Morphogenetic Control of Early Limb Development

Control of Proximodistal Segmentation

As a limb (e.g., an arm) grows out from a simple bud, it eventually forms three structural segments: the stylopodium (upper arm), the zeugopodium (forearm), and the autopodium (hand). Over the years, several hypotheses concerning the control of proximodistal segmentation have been proposed, but only more recently has a hypothesis been supported by strong experimental data. During development, more proximal segments differentiate first, followed successively by the more distal segments. The mesenchymal cells at the distal tip of the limb bud are kept in a proliferative state through the actions of FGFs and Wnts, whereas cells in the proximal part of the limb bud, under the influence of retinoic acid and possibly other molecules, undergo differentiation into proximal components of the limb (Fig. 10.13). The balance between retinoic acid and the FGFs and Wnts is thought to determine the course of segmental differentiation. In the early limb bud, the proximal mesenchymal cells are exposed to a high concentration of retinoic acid because they are near the source (somites), and they differentiate into tissues of the stylopod. As the limb bud grows out, the remaining undifferentiated cells are exposed to a lesser concentration of retinoic acid because outgrowth has taken them farther from the source of retinoic acid. Thus, those remaining mesenchymal cells in later limb buds first differentiate into the zeugopodial segment and finally, in the late limb bud, into the autopodial segment. This balance between the differentiation-promoting effects of retinoic acid and the proliferation-maintaining effects of FGF is similar to that occurring in the posterior end of the early embryo (see Figs. 6-5 and 6.9A).

Cells in the distal mesenchyme are characterized by their expression of Msx-1, a marker of undifferentiated cells, and as they leave that region, expression of that gene ceases (Fig. 10.14A). Something about the distal mesenchymal environment stimulates Msx-1 expression because if mesenchymal cells that have left that region (and consequently cease production of Msx-1) are transplanted back into the distal region, they express that molecule again (Fig. 10.14B).

Fig. 10.14 Msx-1 expression and the distal mesenchyme.
A, If Msx-1–expressing tissue from the distal mesenchyme is transplanted into more proximal regions of the limb bud, it soon stops expressing that molecule. B, If proximal mesenchyme that has already stopped expressing Msx-1 is transplanted back into the distal mesenchymal region, it expresses the molecule again.

Molecular Signals in Limb Development

As discussed earlier, initial development of the limbs involves the establishment of a limb field by the actions of a Hox gene combinatorial code that acts through yet unidentified axial signals to stimulate the expression of Tbx5 in the area of the future forelimb and Tbx4 in the hindlimb. Even in later development, Tbx5 is expressed exclusively in the forelimb, and Tbx4 is expressed exclusively in the hindlimb (Fig. 10.15). Because of these exclusive expression regions, it was originally assumed that these two genes determined the identity of the forelimb and hindlimb. More recent research has shown this not to be the case, however, and the search for the factors that determine limb identity continues. Pitx-1, which is also expressed in the hindlimb, may play a more important role than Tbx-4 as a determinant of hindlimb identity. The main functions of Tbx-4 and Tbx-5 appear to be the initiation of development in a limb-specific manner.

Fig. 10.15 Whole mount in situ hybridization preparations of stage 29 chick embryos showing the localized expression of the mRNAs to Tbx4 in the hindlimb and Tbx5 in the forelimb. (Courtesy of H.-G. Simon, Northwestern University Medical School, Chicago.)

When the limb bud takes shape, its further development depends to a great extent on the actions of three signaling centers, one for each of the cardinal axes of the limb. As already discussed, outgrowth along the proximodistal axis is largely under the control of the apical ectodermal ridge and the FGFs that it produces. FGF-8 is produced along the entire length of the AER, and FGF-4 is produced only along the posterior half. FGF-4, in particular, is an integral part of a feedback loop linking the growth center in the AER to that of the ZPA.

The second major signaling center, this time along the anteroposterior axis, is the ZPA, and the signaling molecule is shh (Fig. 10.16). Although shh is a diffusible molecule, it functions through its effects on BMP-2 and the inhibitor of BMP-2, gremlin (Fig. 10.17). Gremlin has two major functions. First, it antagonizes Gli-3, confining Gli-3 activity to the anterior part of the limb bud, where it represses the expression of posterior limb genes. As mentioned earlier, gremlin also inhibits the inhibitory action of BMP-4 on the AER, thus promoting the activity of FGF-4. FGF-4 is necessary for maintaining the activity of the ZPA.