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.

ECTODERMAL INTERACTION

It is possible to remove the mesenchymal core of an early chick limb bud leaving the surface ectodermal epithelium. When the ectodermal epithelium from a left limb bud is recombined with the mesenchymal core from a right limb bud, keeping the cranio-caudal axis in register, the distal part of the limb that subsequently develops conforms to the polarity of the ectoderm rather than that of the mesenchyme (Fig. 51.3). This can be seen in the pattern of muscles, orientation of the joints and eventual characteristic differentiation of the epidermis. Thus signals from the ectoderm control the pattern along the dorso-ventral axis.

MOLECULAR BASIS OF CELL–CELL INTERACTIONS

The apical ectodermal ridge secretes fibroblast growth factors that act on the underlying mesenchyme and maintain the zone of polarizing activity. The latter expresses Sonic hedgehog (Shh), a gene encoding another class of secreted protein that mediates cell–cell signalling. Sonic Hedgehog protein acts on adjacent mesenchyme cells and maintains the expression of the gene encoding Gremlin (an extracellular bone morphogenetic protein antagonist), which functions as an apical ectodermal ridge maintenance factor. Sonic hedgehog also plays a pivotal role in digit patterning: it diffuses across the limb bud and responding cells transduce the signal through the Gli family of transcription factors. (The Gli proteins are bifunctional effectors of Shh signalling and can lead to either activation or repression of expression of gene targets.) Dorsal limb ectoderm expresses Wnt7a, which acts as a dorsalizing signal.

Among the genes expressed in response to these cell–cell signalling molecules are 5′ members of two Hox gene complexes. Hox-d gene expression in the distal limb bud shows a nested arrangement similar to that of Russian dolls: the postaxial border of the limb bud tip expresses five Hox-d genes (d-13, d-12, d-11, d-10 and d-9), while in the next preaxial portion only four genes are expressed (d-12, d-11, d-10 and d-9), this sequential loss of expression of Hox-d genes is repeated until only d-9 is expressed in the most preaxial regions of the limb bud. The fact that five different Hoxd expression domains can be distinguished has prompted the suggestion that this may be why we have five fingers (and toes), but the explanation is unlikely to be so simple.

SKELETAL ELEMENTS OF THE LIMB

It has been suggested that formation of the cartilaginous elements of a limb is related to the shape of the limb and the conditions necessary for chondrogenesis. An antichondrogenic zone beneath the ectoderm of a developing limb prevents chondrogenesis within the dermis and myogenic zones. Foci of chondrogenesis occur in the centre of the limb bud where cell density is greatest. One centre of chondrogenesis forms in the proximal region of an early limb bud. As the limb bud lengthens it also widens, forming next two centres of chondrogenesis and five centres that appear further distally. The Sox9 transcription factor is essential for chondrocyte differentiation: expression of the Sox9 gene is coincident with expression of the gene that encodes collagen type II, the collagen characteristic of cartilage extracellular matrix. Sox9 gene transcripts are expressed in the humeral and forearm skeletal elements of human embryos at approximately 44 days, and in carpals, metacarpals and phalanges, in addition to more proximal elements, at approximately 52 days (Fig. 51.4).

Fig. 51.4 Sox9 expression in the skeleton of developing limbs of human embryos. A, Stage 18; approximately 44 days postovulation. 1. Humerus. 2. Ribs. 3. Heart. 4. Forearm. 5. Vertebral body. 6. Scapula. B, Stage 21; approximately 52 days postovulation. 1. Carpal bone. 2. Metacarpal bone. 3. Phalanges. 4. Humerus. 5. Scapula. 6. Rib. Sox9 transcripts detected in sections with radioactively labelled probes followed by autoradiography, positive signal represented by silver grains that show up as white areas. Scale bars represent 500 μm in both A and B.

(The human tissue was provided by the joint MRC-Wellcome Human Developmental Biology Resource (http://www.hdbr.org) at the Institute of Human Genetics, Newcastle upon Tyne, UK. By courtesy of Dr Susan Lindsay, Institute of Human Genetics, University of Newcastle upon Tyne, UK.)

The apical ectodermal ridge controls the width of the digital plate, and the number of digits that develop is related to the width of the limb bud. Zones of preprogrammed cell death can be identified on the preaxial and postaxial borders of the limb which limit the length of the apical ectodermal ridge. (It should be noted that in the literature relating to work on animal embryos, pre- and post-axial borders are termed anterior and posterior borders respectively, and therefore the regions in which programmed cell death occurs are termed the anterior and posterior necrotic zones.) If the length of the apical ectodermal ridge is reduced, fewer digits will form (oligodactyly), whereas if the apical ectodermal ridge becomes longer, more digits will form (polydactyly). Supernumerary digits may develop on either the pre- or postaxial borders. Other regions of apoptotic cell death occur between the digits and result in digital separation, but these appear later than the anterior and posterior necrotic zones: the resultant debris in all of the necrotic zones is removed by macrophages.

The first evidence of bone formation is seen at the mid section of the diaphyses of long bones at 7–8 weeks in human embryos. Vascular invasion of the cartilage matrix precedes the formation of a periostial collar, and subsequently extends proximally and distally until it reaches the future epiphysial level, where a growth plate will be established. By 10 weeks, columns of chondrocytes can be seen at the epiphysial level of most bones. However, only the lower end of the femur and upper end of the tibia develop secondary ossification centres before birth.

For further details describing the development of cartilage see page 81, and the development of bone see page 92. For further details about the development of specific limb bones see the appropriate regional chapter.

DEVELOPMENT OF JOINTS

Regions of developing cartilage are easily recognized histologically in the developing limb because they consist of widely spaced cells surrounded by matrix. Somatopleuric mesenchymal cells are more condensed between the developing skeletal elements, and form plates of interzonal mesenchyme which mark the sites of future joints. Their subsequent development varies according to the type of joint that is formed.

In fibrous joints, the interzone is converted into collagen, which is the definitive medium connecting the bones involved. In synchondroses it becomes (growth) cartilage of the modified hyaline type, and in symphyses it is predominantly fibrocartilage. The interzonal mesenchyme of developing synovial joints becomes trilaminar when a more tenuous intermediate zone appears splitting the mesenchyme into two dense strata. As the skeletal elements chondrify, and in part ossify, the dense strata of the interzonal mesenchyme also become cartilaginous; subsequent cavitation of the intermediate zone establishes the cavity of the joint. The loose mesenchyme around the cavity forms the synovial membrane and probably also gives rise to all other intra-articular structures, such as tendons, ligaments, discs and menisci. In joints containing discs or menisci, and in compound articulations, more than one cavity may appear initially, sometimes merging later into a complex single cavity. As development proceeds, thickenings in the fibrous capsule can be recognized as the specializations peculiar to a particular joint. (In some joints, such accessions to the fibrous capsule are derived from neighbouring tendons, muscles or cartilaginous elements.) Although the initial stages in the process of cavitation of joints is independent of movements, a true joint cavity can form only in the presence of movements (Pitsillides 2006). The literature suggests that all musculoskeletal elements are in their appropriate positions by 10 weeks. For detailed accounts of the process, consult O’Rahilly & Gardner (1975) and Uhthoff (1990).

Musculature of the limb

It is now well established that all limb myogenic precursor cells originate from the somites. These cells are committed at an early stage and can be identified in the lateral halves of the somites. After the mesenchymal sclerotome cells have migrated from the epithelial somite, the remaining dorsolateral portion is termed the epithelial plate or dermomyotome of the somite (Fig. 44.3). Cells from the dorsomedial edge of the dermomyotomes form the axial musculature, whereas at limb levels cells de-epithelialize and migrate from the ventrolateral edges of the dermomyotome into the limb bud. These precursor limb myoblasts migrate through a non-random, structured network of extracellular fibrils. At their leading ends the migrating cells exhibit filopodia which are in contact with the extracellular fibrils or with other cells: it is believed that the orientation of the extracellular fibrils may direct the migration of the cells. The precursor muscle cells do not differentiate into muscle before their migration into the limb, probably in response to inhibitory signals produced by the somatopleuric mesenchyme.

The myogenic cells colonize the limb bud in a proximodistal direction, but never reach the most distal portion of the limb, where there seems to be a distal boundary for the muscle cells. Myogenic cells are still indifferent regarding their region-specific determination when they first enter the limb. When brachial or pelvic level somites are grafted opposite the leg-forming region instead of the wing, or opposite the wing instead of the leg, the precursor cells will give rise to normal leg or wing musculature respectively, i.e. the muscle cells, unlike the somatopleuric mesenchyme, have no ‘limbness’. Further, the muscle pattern developed in the limb reflects the pattern of the skeletal elements: duplication or lack of digits is accompanied by the duplication or lack of the corresponding muscles.

Two subpopulations of myogenic cells can be discerned in the limb bud. In the early buds, there are mainly replicating presumptive myoblasts, considered to be premitotic, whereas in later stages there are also postmitotic myoblasts. It is interesting that the invading myoblasts are still replicating; this may be a prerequisite for the formation of the considerable amount of skeletal muscular tissue that will develop in the limbs.

The first myogenic cells to arrive in the limb form the principal dorsal and ventral premuscle masses. It is believed that all classes of tetrapods begin limb muscle development with these blocks, which produce all the skeletal muscle in the limb. The blocks of premuscle undergo a spatiotemporal sequence of divisions and subdivisions as the limb lengthens, and this leads to the individualization of about 19 muscles in the upper limb and 14 muscles in the lower limb. The mechanisms that create gaps that lead to these divisions of the muscle masses are not known. In the upper limb, the premuscle masses first divide into three masses, the next division gives rise to the muscles attached to the carpus, and the final division produces the long muscles of the digits. A similar pattern is seen in the lower limb. Thus the patterning of the musculature of the limb is controlled by the somatopleuric mesenchyme.

Each anatomical muscle appears as a composite structure. The muscle cells and myosatellite cells are of somitic origin, and the connective tissue envelopes and the tendons are of somatopleuric origin. The precise way in which the muscles become anchored to the developing bones by the tendons is not clear.

For further development of skeletal muscle, see page 115.

EMBRYONIC MOVEMENTS

Embryonic movements are vital for development of the musculoskeletal system (reviewed by Pitsillides, 2006). They have effects on the developing muscle, and are necessary to align trabeculae within bones, and to ensure the correct attachments of the tendons and the appropriate coiling of the constituent collagen fibres within the tendons. Simple movements of an extremity have been observed sporadically as early as the seventh week of gestation. Combined movements of limb, trunk and head commence between 12 and 16 weeks of gestation in human embryos. Movements of the embryo and fetus encourage normal skin growth and flexibility, in addition to the progressive maturation of the musculoskeletal system.