Development of the limbs

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CHAPTER 51 Development of the limbs

Development of the limb depends on a series of complex cell–cell interactions that have been identified in experiments on amphibian, avian and reptilian species; the range demonstrates the remarkable conservation of developmental processes. Fate maps have revealed which structures in the limb arise from particular cells or groups of cells in the embryo and considerable progress has been made in identifying the genetic basis of limb development. The finding that the same molecules are involved in developing embryos of both model organisms and humans has promoted an increasing convergence between developmental biology and clinical genetics.

The limbs develop from somatopleuric mesenchyme in the lateral body wall. Regions of the somatopleuric mesenchyme at specific positions along the main body axis proliferate extensively to give rise to limb buds. These buds are the first visible signs of limb development and are rimmed by a longitudinal ridge of high columnar epithelial cells, the apical ectodermal ridge (Fig. 51.1).

Fig. 51.1 Scanning electron micrographs to show the development of the upper limb. A, An early limb bud viewed from the postaxial border. B, Limb bud viewed from postaxial border; the apical ectodermal ridge (arrowed) can be seen. C, Limb bud, dorsolateral view; the shoulder and elbow region can be discerned, and a hand plate has formed. The apical ectodermal ridge is still obvious at the margin of the hand plate. D, Digital rays are present in the hand plate and the margin of the plate is becoming notched. E, The fingers are nearly separated and proliferation is commencing at the distal end of each digit to form the nail bed. F, The fingers each have tactile pads distally, and nail development continues.

(Photographs by P Collins; printed by S Cox, Electron Microscopy Unit, Southampton General Hospital.)

The early limb bud contains a mixed population of mesenchymal cells: somatopleuric mesenchyme gives rise to the connective tissues of the limb, including cartilage, bone, tendon and loose connective tissue, paraxial mesenchyme from the somites gives rise to the myogenic cells of the muscles, and angiogenic mesenchyme produces an extensive vascular network in the early limb bud. Motor and sensory nerves and the neural crest derived Schwann cells and melanocytes of the skin, migrate into the developing limb somewhat later.

AXES OF LIMBS

For descriptive, experimental and conceptual purposes, it has been necessary to define and name various ‘axes’, borders, surfaces and lines in relation to the developing limb bud (Fig. 51.2). An imaginary line from the centre of the elliptical base of the bud, through the centre of its mesenchymal core, to the centre of the apical ectodermal ridge, defines the proximodistal axis of the limb bud (previously known in descriptive embryology simply as the axis). Named in relation to the latter, the cranially placed limb border is the preaxial border, and the caudally placed limb border is the postaxial border. (In tetrapods and birds, the last two are termed anterior and posterior borders, respectively.) Any line that passes through the limb bud from preaxial to postaxial border, orthogonal to the proximodistal axis, constitutes a craniocaudal axis. The dorsal and ventral ectodermal surfaces thus clothe their respective aspects from preaxial to postaxial borders, and any line that passes from dorsal to ventral aspect, orthogonal to both proximodistal and craniocaudal axes, constitutes a dorsoventral axis. It should be noted here that the terms dorsal and ventral axial lines are to be used exclusively in relation to developing and definitive patterns of cutaneous innervation of the limbs and their associated levels of the trunk.

Fig. 51.2 Axes of the developing limb and specification of structures along the different axes. A, Proximodistal and B, craniocaudal; limb bud (approximately stage 14) viewed from dorsal side; C, dorsoventral; limb bud viewed from distal end. The pattern of development of limb structures and the ectodermal specializations are controlled by the limb mesenchyme. Outgrowth of limb development is controlled by the apical ectodermal ridge and this is accompanied by the laying down of structures along the proximodistal axis; craniocaudal pattern is controlled by zone of polarizing activity (polarizing region); dorsoventral pattern is controlled by signals from dorsal and ventral ectoderm, shown in cross section, section taken in plane shown by dotted line.

The three developmental axes (proximodistal, craniocaudal, and dorsoventral) can be identified in the developing limb bud by stage 13. Experiments in chick embryos have shown that development of structures in relation to each of these three principal axes seem to be specified by different mechanisms (Fig. 51.2). Outgrowth along the proximodistal axis is controlled by the apical ectodermal ridge and subjacent somatopleuric mesenchyme; outgrowth of the limb bud is accompanied by the sequential formation of limb structures along the proximodistal axis. The craniocaudal axis is controlled by a small population of mesenchymal cells on the postaxial border of the limb bud; this region of mesenchyme is termed the zone of polarizing activity or polarizing region. The dorsoventral axis of the limb appears to be controlled by the surface ectodermal covering of the limb bud.

Early differential growth of parts of the limb bud results in two main changes to the originally symmetrical axes of the limb. The dorsal aspect of the limb grows faster than the ventral, which causes the limb bud to curve around the body wall. The ventral surface of the limb (closest to the body wall) remains relatively flat, but the dorsal surface bulges into the amniotic cavity. The originally laterally facing apical ectodermal ridge becomes increasingly directed ventrally.

APICAL ECTODERMAL RIDGE

The outgrowth of a limb bud is controlled by the apical ectodermal ridge. For many years it was thought that a timing mechanism that specified proximodistal pattern operated in the zone of proliferating undifferentiated mesenchyme cells at the tip of a developing limb bud. This zone was therefore called the progress zone. It was assumed that as cells left the progress zone their proximal distal value became fixed. Cells that spent a short time at the tip of the limb bud would form proximal structures, while cells that spent a longer time at the tip of the limb bud would form distal structures. However, it has been suggested more recently that proximodistal pattern is specified in the very early limb bud and that structures are elaborated as the bud grows out.

The fundamental epithelial–mesenchymal interactions seen in limb development are discussed in detail in Hinchcliffe & Johnson (1980) and the more recently discovered genetic basis of these interactions in Ferretti & Tickle (2006). The knowledge of limb embryology gained from studying chicks may be summarized as follows (Fig. 51.3). The apical ectodermal ridge provides the orientating influence for limb bud outgrowth. Its removal results in cessation of limb development, whereas grafting a second apical ectodermal ridge results in two axes of development, and duplication of distal structures (Fig. 51.3). There is evidence that the limb mesenchyme beneath the apical ectodermal ridge provides an apical ectodermal ridge maintenance factor that is essential to the function of the ridge. Thus, when leg bud cells are grafted to the tip of a wing bud beneath the apical ectodermal ridge, the ridge is maintained and bud outgrowth continues (it should be noted that the leg cells, even though placed in a wing bud, still form distal leg structures) (Fig. 51.3). In addition, the leg mesenchyme will pass information to the local ectoderm eliciting the appropriate epidermal development, which in this case is the formation of scales rather than feathers.

Fig. 51.3 A–C, Experimental manipulations that identified fundamental interactions involved in patterning the three main axes of the limb in the chick.

ZONE OF POLARIZING ACTIVITY

The zone of polarizing activity is a small region of somatopleuric mesenchyme on the postaxial border of the limb. When a second zone of polarizing activity is grafted beneath or adjacent to the apical ectodermal ridge at the preaxial border of a chick wing bud, duplication of distal limb structures occurs, generating a mirror-image pattern of six digits (Fig. 51.3). The digit closest to the zone of polarizing activity is always the most postaxial digit (in the chick wing this is digit 4), while more preaxial digits develop further away from the zone of polarizing activity. Thus signalling from the zone of polarizing activity appears to control both digit number and digit pattern. It has been suggested that digit number is controlled by regulation of the production of the apical ectodermal ridge maintenance factor, while digit pattern is regulated by production of a diffusible morphogen that acts in a concentration dependent fashion.