ESPECTRO TRÓFICO

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Chapter 12

152 Part II Systems-Based Embryology

in irregular bones, such as the vertebrae, one or more primary centers of ossifi cation and usually several secondary centers are present.

Synovial joints between bones begin to form at the same time that mesenchymal con-densations initiate the process of forming cartilage.

Thus, in the region between two chondrify-ing bone primordia, called the interzone (for example between the tibia and femur at the knee joint), the condensed mesenchyme dif-ferentiates into dense fi brous tissue. This fi brous tissue then forms articular cartilage, cov-ering the ends of the two adjacent bones; the synovial membranes; and the menisci and ligaments within the joint capsule (e.g., the anterior and posterior cruciate ligaments in the knee). The joint capsule itself is derived from mesenchyme cells surrounding the interzone region. Fibrous joints (e.g., the sutures in the

skull) also form from interzone regions, but in this case the interzone remains as a dense fi brous structure.

LIMB MUSCULATURE

Limb musculature is derived from dorsolateral cells of the somites that migrate into the limb to form muscles and, initially, these muscle components are segmented according to the somites from which they are derived (Fig. 12.6).

However, with elongation of the limb buds, the muscle tissue fi rst splits into fl exor and extensor components (Fig. 12.7) and then additional split-tings and fusions occur, such that a single muscle may be formed from more than one original seg-ment. The resulting complex pattern of muscles is determined by connective tissue derived from lateral plate mesoderm.

A B C

Figure 12.1 Development of the limb buds in human embryos. A. At 5 weeks. B. At 6 weeks. C. At 8 weeks. Hindlimb development lags behind forelimb development by 1 to 2 days.

Ectoderm Apical ectodermal ridge

(AER) Ectoderm Apical ectodermal ridge

(AER)

A B

Figure 12.2 A. Longitudinal section through the limb bud of a chick embryo, showing a core of mesenchyme covered by a layer of ectoderm that thickens at the distal border of the limb to form the AER. In humans, this occurs during the fi fth week of development. B. External view of a chick limb at high magnifi cation showing the ectoderm and the specialized region at the tip of the limb called the AER.

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Chapter 12 Limbs 153

Upper limb buds lie opposite the lower fi ve cervical and upper two thoracic segments (Fig. 12.6), and the lower limb buds lie opposite the lower four lumbar and upper two sacral

segments. As soon as the buds form, ventral primary rami from the appropriate spinal nerves penetrate into the mesenchyme. At fi rst, each ven-tral ramus enters with dorsal and venven-tral branches derived from its specifi c spinal segment, but soon branches in their respective divisions begin to unite to form large dorsal and ventral nerves (Fig. 12.7). Thus, the radial nerve, which sup-plies the extensor musculature, is formed by a combination of the dorsal segmental branches, whereas the ulnar and median nerves, which supply the fl exor musculature, are formed by a combination of the ventral branches. Immediately after the nerves have entered the limb buds, they establish an intimate contact with the differenti-ating mesodermal condensations, and the early contact between the nerve and muscle cells is a prerequisite for their complete functional differentiation.

Spinal nerves not only play an important role in differentiation and motor innervation of the limb musculature, but also provide sensory innervation for the dermatomes. Although the original dermatomal pattern changes with growth and rotation of the extremities, an orderly sequence can still be recognized in the adult (Fig. 12.8).

A

C

B

Areas of cell death

Areas of cell death

Figure 12.3 Schematic of human hands. A. At 48 days.

Cell death in the AER creates a separate ridge for each digit. B. At 51 days. Cell death in the interdigital spaces produces separation of the digits. C. At 56 days. Digit separation is complete.

Tibia

Pubis

Femur

Ilium Femur Fibula

Tarsal cartilages

Tibia

Ilium Pubis

Fibula Footplate cartilages

Tarsal cartilages Metatarsal cartilages

Pubis

Ilium

Ischium A

B

C

Figure 12.4 A. Lower extremity of an early 6-week embryo, illustrating the fi rst hyaline cartilage models. B,C. Complete set of cartilage models at the end of the sixth week and the beginning of the eighth week, respectively.

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A B C D

Cartilage Osteoblasts

Bone

Growth plate Proliferating

chondrocytes

Secondary ossification center

Mesenchyme

Figure 12.5 Endochondral bone formation. A. Mesenchyme cells begin to condense and differentiate into chondrocytes.

B. Chondrocytes form a cartilaginous model of the prospective bone. C,D. Blood vessels invade the center of the cartilagi-nous model, bringing osteoblasts (black cells) and restricting proliferating chondrocytic cells to the ends (epiphyses) of the bones. Chondrocytes toward the shaft side (diaphysis) undergo hypertrophy and apoptosis as they mineralize the surrounding matrix. Osteoblasts bind to the mineralized matrix and deposit bone matrices. Later, as blood vessels invade the epiphyses, secondary ossifi cation centers form. Growth of the bones is maintained by proliferation of chondrocytes in the growth plates.

Occipital myotomes Cervical

myotomes

Thoracic myotomes

T1

C1

I IVIII

II

Pharyngeal arch muscles

Eye muscles

Eye Limb axis Mesenchymal

condensation of limb bud

Epithelial ridge

Figure 12.6 Muscle cells for the limbs are derived from somites at specifi c segmental levels. For the upper limb these segments are C5–T2; for the hind limb they are L2–S2. Ultimately, muscles are derived from more than one segment and so the initial segmentation pattern is lost.

Back (epaxial) muscles

Dorsal primary ramus Ventral primary

ramus Body wall

muscles

Hypaxial muscles

Flexor muscle of limb

Extensor muscle

of limb

Figure 12.7 As muscle cells move into the limb, they split into dorsal (extensor) and ventral (fl exor) compart-ments. Muscles are innervated by ventral primary rami that initially divide to form dorsal and ventral branches to these compartments. Ultimately, branches from their respective dorsal and ventral divisions unite into large dorsal and ventral nerves.

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Chapter 12 Limbs 155

Molecular Regulation of Limb Development

Positioning of the limbs along the craniocau-dal axis in the fl ank regions of the embryo is regulated by the HOX genes expressed along this axis. These homeobox genes are expressed in overlapping patterns from head to tail (see Chapter 6, p. 81), with some having more cranial limits than others. For example, the cranial limit of expression of HOXB8 is at the cranial border of the forelimb, and misexpression of this gene alters the position of these limbs.

Once positioning along the craniocaudal axis is determined, growth must be regulated along the proximodistal, anteroposterior, and dorsoventral axes (Fig. 12.9). Limb outgrowth, which occurs fi rst, is initiated by TBX5 and FGF10 in the forelimb and TBX4 and FGF10 in the hindlimb secreted by lateral plate mesoderm cells (Fig. 12.9A).

Once outgrowth is initiated, bone morphoge-netic proteins (BMPs), expressed in ventral ecto-derm, induce formation of the AER by signaling through the homeobox gene MSX2. Expression of Radical fringe (a homologue of Drosophila fringe), in the dorsal half of the limb ectoderm, restricts the location of the AER to the distal tip of the limbs.

This gene induces expression of SER2, a homo-logue of Drosophila serrate, at the border between cells that are expressing Radical fringe and those that are not. It is at this border that the AER is estab-lished. Formation of the border itself is assisted by expression of Engrailed-1 in ventral ectoderm cells,

because this gene represses expression of Radical fringe. After the ridge is established, it expresses FGF4 and FGF8, which maintain the progress zone, the rapidly proliferating population of mes-enchyme cells adjacent to the ridge (Fig. 12.9A).

Distal growth of the limb is then affected by these rapidly proliferating cells under the infl uence of the FGFs. As growth occurs, mesenchymal cells at the proximal end of the progress zone become farther away from the ridge and its infl uence and begin to slow their division rates and to differentiate.

Patterning of the anteroposterior axis of the limb is regulated by the zone of polarizing activity (ZPA), a cluster of cells at the posterior border of the limb near the body wall (Fig. 12.9B). These cells produce retinoic acid (vitamin A), which initi-ates expression of sonic hedgehog (SHH), a secreted factor that regulates the anteroposterior axis.

Thus, for example, digits appear in the proper order, with the thumb on the radial (anterior) side. As the limb grows, the ZPA moves distal-ward to remain in proximity to the posterior bor-der of the AER. Misexpression of retinoic acid or SHH in the anterior margin of a limb containing a normally expressing ZPA in the posterior bor-der results in a mirror image duplication of limb structures (Fig. 12.10).

The dorsoventral axis is also regulated by BMPs in the ventral ectoderm, which induce expression of the transcription factor EN1. In turn, EN1 represses WNT7a expression, restrict-ing it to the dorsal limb ectoderm. WNT7a is a

C3 C5 C4

C6 C7

C8

T1 T2

T3 T4

C6

C6 C7

C7 C8

C8 T1

T2 T3 T4 C4

C5

Posterior view Anterior view

C3 C4 C5

C6

C7

C8

T1 T2

T3 T4 C4

C5

C6

C7 C8

T1 T2 T3 T4

Figure 12.8 Forelimbs with their sensory innervation to the dermatomes represented. Note that sensory innervation to the limb maintains a segmental pattern refl ecting the embryological origin of each dermatome and its innervation.

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Proximodistal