Molecular Regulation Of Limb Development

Positioning of the limbs along the craniocaudal axis in the flank regions of the embryo is regulated by the HOX genes expressed along this axis. These

Figure 8.15 Endochondral bone formation. A. Mesenchyme cells begin to condense and differentiate into chondrocytes. B. Chondrocytes form a cartilaginous model of the prospective bone. C and D. Blood vessels invade the center of the cartilaginous model bringing oteoblasts (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 ossification centers form. Growth of the bones is maintained by proliferation of chondrocytes in the growth plates (D).

homeobox genes are expressed in overlapping patterns from head to tail (see Chapter 5), 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. 8.16). Limb outgrowth, which occurs first, is initiated by FGF-10 secreted by lateral plate mesoderm cells (Fig. 8.16A). Once outgrowth is initiated, bone morphogenetic proteins (BMPs), expressed in ventral ectoderm, 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 Ser-2, a homologue of Drosophila serrate, at the border between cells expressing Radical fringe and those that are not. It is at this border that the AER is established. Formation of the border itself is assisted by expression of Engrailed-1 in ventral ectoderm cells, since this gene represses expression of Radical fringe. After the ridge is established, it expresses FGF-4 and FGF-8, which maintain the progress zone, the rapidly proliferating population of mesenchyme cells adjacent to the ridge (Fig. 8.16A). Distal growth of the limb is then effected by these rapidly proliferating cells under the influence of the FGFs. As growth occurs, mesenchymal cells at the proximal end of the progress zone become farther away from the ridge and its influence 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 flank (Fig. 8.16B). These cells produce retinoic acid (vitamin A), which initiates 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 distalward to remain in proximity to the posterior border of the AER. Misexpression of retinoic acid or SHH in the anterior margin of a limb containing a normally expressing ZPA in the posterior border results in a mirror image duplication of limb structures (Fig. 8.17).

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 restricting it to the dorsal limb ectoderm. WNT7a is secreted factor that induces expression of LMX1, a transcription factor containing a homeodomain, in the dorsal mesenchyme (Fig. 8.16C). LMX1 specifies cells to be dorsal, establishing the dorsoventral components. In addition, WNT7a maintains SHH expression in the ZPA and therefore indirectly affects anteroposterior patterning as well. These two genes are also intimately linked in signaling pathways in Drosophila, and this interaction is conserved in vertebrates. In fact, all of the patterning genes in the limb have feedback loops. Thus, FGFs in the AER activate SHH in the ZPA, while WNT7a maintains the SHH signal.

Although patterning genes for the limb axes have been determined, it is the HOX genes that regulate the types and shapes of the bones of the limb (Fig. 8.15D). Thus, HOX gene expression, which results from the combinatorial expression of SHH, FGFs, and WNT7a, occurs in phases in three places in the limb that correspond to formation of the proximal (stylopod), middle (zeugo-pod), and distal (autopod) parts. Genes of the HOXA and HOXD clusters are the primary determinants in the limb, and variations in their combinatorial patterns of expression may account for differences in forelimb and hindlimb structures. Just as in the craniocaudal axis of the embryo, HOX genes are nested in overlapping patterns of expression that somehow regulate patterning

(Fig. 8.16D). Factors determining forelimb versus hindlimb are the transcription factors TBX5 (forelimbs) and TBX4 (hindlimbs).

CLINICAL CORRELATES Bone Age

Radiologists use the appearance of various ossification centers to determine whether a child has reached his or her proper maturation age. Useful information about bone age is obtained from ossification studies in the hands and wrists of children. Prenatal analysis of fetal bones by ultrasonography provides information about fetal growth and gestational age.

Limb Defects

Limb malformations occur in approximately 6/10,000 live births, with 3.4/10,000 affecting the upper limb and 1.1/10,000, the lower. These defects are often associated with other birth defects involving the craniofacial, cardiac, and genitourinary systems. Abnormalities of the limbs vary greatly, and they may be represented by partial (meromelia) or complete absence (amelia) of one or more of the extremities. Sometimes the long bones are absent, and rudimentary hands and feet are attached to the trunk by small, irregularly shaped bones (phocomelia, a form of meromelia) (Fig. 8.18, A and B). Sometimes all segments of the extremities are present but abnormally short (micromelia).

Although these abnormalities are rare and mainly hereditary, cases of teratogen-induced limb defects have been documented. For example, many children with limb malformations were born between 1957 and 1962. Many mothers of these infants had taken thalidomide, a drug widely used as a sleeping pill and antinauseant. It was subsequently established that thalidomide causes a characteristic syndrome of malformations consisting of absence or gross deformities of the long bones, intestinal atresia, and cardiac anomalies.

Figure 8.16 (opposite page). Molecular regulation of patterning and growth in the limb. A. Limb outgrowth is initiated by FGF-10 secreted by lateral plate mesoderm in the limb forming regions. Once outgrowth is initiated, the AER is induced by BMPs and restricted in its location by the gene radical fringe expressed in dorsal ectoderm. In turn, this expression induces that of SER2 in cells destined to form the AER. After the ridge is established, it expresses FGF-4 and FGF-8 to maintain the progress zone, the rapidly proliferating mesenchyme cells adjacent to the ridge. B. Anteroposterior patterning of the limb is controlled by cells in the ZPA at the posterior border. These cells produce retinoic acid (vitamin A), which initiates expression of sonic hedgehog, regulating patterning. C. The dorsoventral limb axis is directed by WNT7a, which is expressed in the dorsal ectoderm. This gene induces expression of the transcription factor LMX1 in the dorsal mesenchyme, specifying these cells as dorsal. D. Bone type and shape are regulated by HOX genes whose expression is determined by the combinatorial expression of SHH, FGFs, and WNT7a. HOXA and HOXD clusters are the primary determinants of bone morphology.

Figure 8.17 Experimental procedure for grafting a new ZPA from one limb bud into another using chick embryos. The result is the production of a limb with mirror image duplication of the digits (chicks have only three digits, numbered II, III, and IV), indicating the role of the ZPA in regulating anteroposterior patterning of the limb. Sonic hedgehog protein is the molecule secreted by the ZPA responsible for this regulation.

Phocomelia Babies
Figure 8.18 A. Child with unilateral amelia. B. Patient with a form of meromelia called phocomelia. The hands and feet are attached to the trunk by irregularly shaped bones.

Figure 8.19 Digital defects. A. Polydactyly, extra digits. B. Syndactyly, fused digits. C Cleft foot, lobster claw deformity.

Since the drug is now being used to treat AIDS and cancer patients, there is concern that its return will result in a new wave of limb defects. Studies indicate that the most sensitive period for teratogen-induced limb malformations is the fourth and fifth weeks of development.

A different category of limb abnormalities consists of extra fingers or toes (Polydactyly) (Fig. 8.19A). The extra digits frequently lack proper muscle connections. Abnormalities with an excessive number of bones are mostly bilateral, while the absence of a digit such as a thumb (ectrodactyly) is usually unilateral. Polydactyly can be inherited as a dominant trait but may also be induced by teratogens. Abnormal fusion is usually restricted to the fingers or toes (syndactyly). Normally mesenchyme between prospective digits in the handplates and footplates breaks down. In 1/2000 births this fails to occur, and the result is fusion of one or more fingers and toes (Fig. 8.19B). In some cases the bones actually fuse.

Cleft hand and foot (lobster claw deformity) consists of an abnormal cleft between the second and fourth metacarpal bones and soft tissues. The third metacarpal and phalangeal bones are almost always absent, and the thumb and index finger and the fourth and fifth fingers may be fused (Fig. 8.19C). The two parts of the hand are somewhat opposed to each other and act like a lobster claw.

The role of the HOX genes in limb development is illustrated by two abnormal phenotypes produced by mutations in these genes: Mutations in HOXA13 result in hand-foot-genital syndrome, characterized by fusion of the carpal bones and small short digits. Females often have a partially (bicornuate) or completely (didelphic) divided uterus and abnormal positioning of the urethral orifice. Males may have hypospadias. Mutations in HOXD13 result in a combination of syndactyly and polydactyly (synpolydactyly).

Clubfoot usually accompanies syndactyly. The sole of the foot is turned inward, and the foot is adducted and plantar flexed. It is observed mainly in males and in some cases is hereditary. Abnormal positioning of the legs in utero may also cause clubfoot.

Congenital absence or deficiency of the radius is usually a genetic abnormality observed with malformations in other structures, such as craniosynostosis-radial aplasia syndrome. Associated digital defects, which may include absent thumbs and a short curved ulna, are usually present.

Amniotic bands may cause ring constrictions and amputations of the limbs or digits (Fig. 8.20). The origin of bands is not clear, but they may represent adhesions between the amnion and affected structures in the fetus. Other investigators believe that bands originate from tears in the amnion that detach and surround part of the fetus.

Wnt7a Engrailed
Figure 8.20 Digit amputations resulting from amniotic bands.

Congenital hip dislocation consists of underdevelopment of the acetabulum and head of the femur. It is rather common and occurs mostly in females. Although dislocation usually occurs after birth, the abnormality of the bones develops prenatally. Since many babies with congenital hip dislocation are breech deliveries, it has been thought that breech posture may interfere with development of the hip joint. It is frequently associated with laxity of the joint capsule.

Vertebral Column

During the fourth week of development, cells of the sclerotomes shift their position to surround both the spinal cord and the notochord (Fig. 8.1). This mesenchymal column retains traces of its segmental origin, as the sclerotomic blocks are separated by less dense areas containing intersegmental arteries (Fig. 8.21 A).

During further development the caudal portion of each sclerotome segment proliferates extensively and condenses (Fig. 8.21 B). This proliferation is so extensive that it proceeds into the subjacent intersegmental tissue and binds the caudal half of one sclerotome to the cephalic half of the subjacent sclerotome (arrows in Fig. 8.21,A and B). Hence, by incorporation of the intersegmental tissue into the precartilaginous vertebral body (Fig. 8.21 B), the body of the

Notochord

Nucleus pulposus and intervertebral disc

Intervertebral disc

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  • Alessandra
    How hox genes regulate type and shape of bones in human embryo limb development?
    8 years ago

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