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Development of the Limbs
Summary
The upper limb buds appear on day 24 as small bulges on the lateral body wall at about the level of C5 to T1. By the end of the fourth week, the upper limb buds have grown to form pronounced structures protruding from the body wall, and the lower limb buds first appear, forming at about the level of L1 to S1. Limb morphogenesis takes place from the fourth to the eighth weeks, with development of the lower limbs lagging slightly behind that of the upper limbs. Each limb bud consists of a mesenchymal core of mesoderm covered by an epithelial cap of ectoderm. Along the distal margin of the limb bud, the ectoderm thickens to form an apical ectodermal ridge . This structure maintains outgrowth of the limb bud along the proximal-distal axis .
By 33 days, the hand plates are visible at the distal end of the lengthening upper limb buds, and the lower limb buds have begun to elongate. By the end of the sixth week, the segments of the upper and lower limbs can be distinguished. Digital rays appear on the handplates and footplates during the sixth (upper limbs) and seventh (lower limbs) weeks. A process of programmed cell death occurs between the rays and accompanies freeing of the fingers and toes. By the end of the eighth week, all components of the upper and lower limbs are distinct.
The skeletal elements of the limbs develop by endochondral ossification in a proximodistal sequence from mesodermal condensations that first appear along the long axis of the limb bud during the fifth week. The cartilaginous precursors of the limb bones chondrify within this mesenchymal condensation, starting in the sixth week. Ossification of these cartilaginous precursors begins in the 7th to 12th weeks.
The bones, tendons, and other connective tissues of the limbs arise from the lateral plate mesoderm, but the limb muscles and endothelial cells arise in the somitic mesoderm and migrate into the limb buds. In general, the muscles that form on the ventral side of the developing long bones become the flexors and pronators of the upper limbs and the flexors and adductors of the lower limbs. These muscles are innervated by ventral branches of the ventral primary rami of the spinal nerves. The muscles that form on the dorsal side of the long bones generally become the extensor and supinator muscles of the upper limbs and the extensor and abductor muscles of the lower limbs. These muscles are innervated by dorsal branches of the ventral primary rami. However, some muscles of the limbs shift their position dramatically during development, either by differential growth or by passive displacement during lateral rotation of the upper limb and medial rotation of the lower limb.
Clinical Taster
Freddie Musena M’tile (Musena means friend in Kenyan) was born in 2004 in Kenya with a condition called tetra-amelia (absence of all four limbs; Fig. 20.1 ). Children with birth defects are shunned in some cultures, and Freddie’s biologic mother gave him up for adoption, fearing that her husband would kill him. A British charity worker and her Kenyan husband adopted him and brought him to the UK for treatment. The case received notoriety after Freddie was, for a time, denied a British visa. With donations obtained through Thalidomide UK, he was fitted with prosthetic devices to help him sit up, with future plans to fit him with artificial limbs. Unfortunately, Freddie died of a fungal infection after returning to Africa. By the time of his death, Freddie had become a national symbol in Kenya.
Although the cause of Freddie’s birth defects is not certain, his biologic mother took medicine that was believed to have been thalidomide . Once banned after causing an estimated 12,000 cases of limb defects like Freddie’s in the late 1950s and early 1960s, use of thalidomide is on the rise again. Originally prescribed in Europe and the UK to treat morning sickness during pregnancy, thalidomide is now used to treat leprosy, AIDS, and certain cancers (e.g., multiple myeloma). It is widely available in developing countries, and Freddie’s case helped raise awareness of the risks of thalidomide exposure during pregnancy, especially in countries where literacy rates are low.
The thalidomide epidemic that occurred now over 50 years ago led to concerns about methods used to validate the safety of new drugs. This resulted in new Food and Drug Administration (FDA) guidelines for drug testing, including the requirement to test drugs in several animal models, guidelines that remain in effect today.
Thalidomide is a potent teratogen that causes defects at single exposures as low as 100 mg. Several mechanisms by which thalidomide causes amelia (absent limbs) or phocomelia (hands or feet projecting directly from the shoulder or hip, respectively) have been proposed. The drug’s ability to inhibit angiogenesis (blood vessel formation) is a strong potential mechanism. Disruption of blood supply has long been hypothesized to play a role in similar limb reduction defects. Additionally, it has been recently shown that thalidomide enhances the degradation of the Sall4 transcription factor. Mutations in SALL4 result in Duane Radial Ray and Holt-Oram syndromes, both characterized by limb defects that phenotypically overlap with those induced by thalidomide. Thus, thalidomide-induced degradation of Sall4 provides another strong potential mechanism for thalidomide teratogenicity. These studies also revealed that the ability to modify Sall4 is species-specific, occurring in rabbits, primates, and humans, but not in rodents and fish. This explains why its teratogenic effects were missed in the 1950s when drug screening was typically carried out on one species and emphasizes the importance of testing drugs in several species.
Epithelial-Mesenchymal Interactions Control Limb Outgrowth
Animations are available online at StudentConsult .
Limb development takes place over a 5-week period from the fourth to the eighth weeks. The upper limbs develop slightly in advance of the lower limbs, although by the end of the period of limb development, upper and lower limbs are nearly synchronized. Initiation of limb development starts with continued proliferation of the somatic lateral plate mesoderm in the limb regions of the lateral body wall ( Fig. 20.2 ). The upper limb bud appears in the lower cervical region at 24 days, and the lower limb bud appears in the lower lumbar region at 28 days. The origin of the limb buds is reflected in their final innervation (see later in this chapter and Chapter 10 ). Each limb bud consists of an outer ectodermal cap and an inner mesodermal core.
As each limb bud forms, the ectoderm along the distal tip of the bud is induced by the underlying somatic mesoderm to form a ridge-like thickening called the apical ectodermal ridge (AER) (see Fig. 20.2 ). This structure forms at the dorsal-ventral boundary of the limb bud and plays an essential role in the outgrowth of the limb.
In the Research Lab
Overview of Patterning of Limb Bud
Once the limb bud has formed, it differentiates with respect to three axes ( Fig. 20.3 ). The proximal-distal axis runs from the shoulder or hip to the fingers or toes and consists of the stylopod (humerus or femur), zeugopod (radius and ulna or tibia and fibula), and autopod (the carpals and metacarpals or tarsal and metatarsals and the phalanges). Along the cranial-caudal axis (often called the anterior-posterior axis), the thumb (or big toe) is the most cranial digit, whereas the little finger (or little toe) is the most caudal digit. Along the dorsal-ventral axis , the knuckle side of the hand or the top of the foot is dorsal, whereas the palm of the hand or the sole of the foot is ventral. The limb bud develops from an apparently homogeneous cell population; thus, a cell in the limb bud must respond appropriately to its position relative to all three axes. Several questions arise: How does one part of the upper limb bud form the shoulder and another the forearm? How does one digital ray in the hand plate form an index finger and another the thumb? How do dorsal and ventral sides of the limb become differentiated from each other? Significant advances have been made toward answering these questions. We now know the key players that pattern the limb bud, and we can link these key players to mutations that cause human birth defects.
The cranial-caudal (anterior-posterior) axis is determined by signals from a small region of mesenchyme in the caudal part of the limb bud known as the zone of polarizing activity (ZPA), and this activity is mediated by sonic hedgehog (Shh). Signals from the dorsal ectoderm (Wnt7a) determine the dorsal-ventral axis, whereas Fgfs and Wnts from the AER, together with retinoic acid in the lateral plate mesoderm, pattern the proximal-distal axis. These signals do not act in isolation to control patterning along the individual axes but are interdependent. For example, Shh maintains expression of Fgfs in the AER and Wnt7a in the dorsal ectoderm. Conversely, Fgfs and Wnt7a maintain Shh expression in the ZPA, resulting in a positive feedback loop , promoting and coordinating patterning along each of the axes. During limb bud initiation, establishment of the dorsal-ventral axis and AER are coordinated to correctly position the AER at the distal tip of the limb, whereas expression of Fgf8 in the AER acts together with other signals within the limb bud mesenchyme to initiate expression of Shh in the ZPA.
The position of the limb buds along the cranial-caudal axis of the body is specified by the expression of Hox genes in the lateral plate mesenchyme (see Chapter 8 for further coverage of Hox genes and craniocaudal patterning). The identity of the limb (arm vs. leg) is specified in the lateral plate mesoderm before the formation of the limb bud is initiated (leg versus arm defects are covered in the following “In the Clinic” in the section entitled “Congenital Anomalies of Limb”). The skeleton patterns the developing musculature, as in other regions of the body (e.g., the developing face; see Chapter 17 ).
The following paragraphs in this “In the Research Lab” detail development of the limb along the proximal-distal axis, including initiation of limb outgrowth. Development along the other two axes is covered in the next “In the Research Lab” in the sections entitled “Specification of Cranial-Caudal Axis” and “Specification of Dorsal-Ventral Axis.”
Growth and Patterning Along Proximal-Distal Axis
Classic embryologic experiments have shown that proximal-distal outgrowth is controlled by the AER. Removal of the AER results in the arrest of limb development, with the degree of development determined by the stage of development at which the AER was removed ( Fig. 20.4 ). For example, in the chick, removal at stage 20 of development results in the formation of a limb truncated at the elbow joint, whereas removal slightly later at stage 24 leads to a limb lacking just the digits. Furthermore, in chick wingless and mouse limb deformity mutants, in which the AER develops initially but is not maintained, the limbs are truncated. Initiation of limb development without further maintenance occurs naturally in some species of snakes, whales, and dolphins. In these species, small hindlimb buds initially form. However, they do not develop, as the AER either does not form or is not maintained.
Several members of the Fgf family are expressed in the AER (Fgf4, 8, 9, and 17) ( Fig. 20.5 ). These factors are key regulators of limb outgrowth. Beads soaked in Fgfs and transplanted to the tip of a limb bud following AER removal can proxy for the AER and maintain limb outgrowth. Moreover, there is redundancy in their function, such that several different Fgfs can proxy for the AER and for each other. For example, limbs lacking Fgf4, 9, or 17 are normal. Gene inactivation of Fgf8 in mice results in the formation of a slightly smaller limb bud, affecting growth of all limb segments. In these limbs, Fgf8 function is rescued by Fgf4, which is upregulated/sustained, but gene inactivation of both Fgf8 and Fgf4 results in increased apoptosis of the limb mesenchymal cells and no limb outgrowth. Therefore, Fgf4 and Fgf8 are the key effectors of AER function in limb outgrowth.
Fgf signaling is also essential for the initiation of limb development. Strikingly, application of an Fgf-soaked bead to the interlimb flank of an early chick embryo induces the formation of an extra limb ( Fig. 20.6 ). At the forelimb level, Tbx5 induces Fgf10 expression in the presumptive forelimb mesenchyme. Fgf10 signaling in the mesoderm then induces Wnt3a (in the chick) in the overlying ectoderm. Wnt3a in turn induces Fgf8 in the presumptive AER, which maintains Fgf10 expression in the underlying mesenchyme and establishes a feedback loop between Fgf8 and Fgf10 to maintain limb outgrowth. The interplay between Wnt3a and Fgf8 continues throughout development of the limb, with misexpression of Wnt3a resulting in the induction of ectopic AER formation. Parallel processes occur in the mouse, where Wnt3/β-catenin signaling is required for both AER formation and maintenance.
How patterning is specified along the proximal-distal axis is still uncertain. One model that has been used for 40 years to explain this patterning is called the progress zone model . The progress zone is defined as a narrow zone of mesenchyme about 300 μm in width underlying the AER, where cells are thought to acquire positional information that will inform them of their final positional address along the proximal-distal axis. Cells that exit the progress zone after a short residence are destined to form proximal structures such as the humerus or femur (i.e., elements of the stylopod). Cells with longest residence in the progress zone become the most distal structures, that is, the phalanges (i.e., elements of the autopod). How cells actually acquire positional information during residence in the progress zone is unknown. However, a timing mechanism in which a cell counts its number of mitotic divisions has been proposed.
Removal of the AER results in differences in the extent of cell death and cell proliferation in the limb bud mesenchyme depending on the stage of removal. Removal early in development results in cell death encompassing the autopod and zeugopod (i.e., the digits, carpus or tarsus, and the radius and ulna or the tibia and fibula) progenitors, whereas later removal does not induce significant cell death but significantly decreases cell proliferation. Furthermore, reducing the levels of Fgf expression in the AER affects all skeletal elements. If the number of cell cycles determines cell fate, as suggested by the timing mechanism of positional information covered earlier, then the sequential reduction in Fgf signaling, which controls cell proliferation, should preferentially affect the autopod; it does not.
This conundrum is resolved with the two-signal model ( Fig. 20.7 ). According to this model, limb bud cells are initially exposed to a proximal signal from the trunk (possibly retinoic acid produced by the enzyme Raldh2 in the flank mesenchyme) and a distal signal from the AER (Fgfs and Wnts). At an early stage of limb development, the entire limb bud is exposed to both signals; as a consequence, it expresses markers of the stylopod (the homeobox transcription factors Meis1 and Meis2). As the limb grows out from the flank, only the proximal part of the limb continues to be exposed to retinoic acid and maintains Meis1/2 expression, whereas only the distal region is exposed to Fgf/Wnt signaling from the AER, which keeps the cells in an undifferentiated state to generate the zeugopod and autopod. The cells exposed to prolonged Fgf and Wnt signaling form the autopod. An alternative model in the chick combines the two-signal and progress zone model. Fgfs also induce Cyp26b1 expression in the distal mesenchyme, which degrades retinoic acid. In Cyp26b1 mouse mutants, the distal markers are absent, whereas Meis1/2 expression is extended along the proximal-distal axis. In this model, the proximal domain (i.e., up to the elbow or knee) is specified by a signal from the trunk, and the zeugopod and autopod are specified by an intrinsic timing mechanism.
Insight into the molecular mechanisms that regulate growth of each region of the limb bud has also been obtained. As described in Chapter 17, Chapter 5, Chapter 8 , four clusters of Hox genes are sequentially activated in vertebrates (including humans) following the 3' to 5' sequence along the DNA of the four respective chromosomes. Moreover, the most 5' members of the Hoxd and Hoxa clusters (9 to 13) are initially coordinately expressed in nested cranial-caudal and proximal-distal domains within the growing limb bud ( Fig. 20.8 ). The more 3' members of the Hox gene family are expressed first, followed sequentially by more 5' members. This temporal and nested expression is known as temporal and spatial colinearity , and it is also seen during Hox expression and patterning of the developing somites (see Chapter 8 ). Hox gene expression is required for growth and patterning of the limb bud. Hox genes 8 to 11 are required for formation of the AER, whereas Hox genes 11 to 13 activate Shh expression in the ZPA. This first wave of Hox expression is followed by a second wave of Hoxd regulation that expands and reverses the domains of Hoxd11–13 expression across the anterior-posterior axis of the developing autopod ( Fig. 20.9 ). This also results in a domain lacking Hoxd expression that corresponds to the developing mesopod (wrist or ankle) (see Fig. 20.9 ). Hox expression is further refined (e.g., Hoxa13 and Hoxd13 downregulate Hoxa11 expression in the autopod) so that ultimately, expression of each of the 5' Hoxd genes (along with those of the Hoxa group) can be correlated with development of specific skeletal elements of upper and lower limb segments (see Fig. 20.9 ). For example, in the forelimb, Hoxd9 is expressed within the segment forming the scapula; Hoxd9 and Hoxd10 are largely restricted to the arm (containing the humerus); Hoxd11 paralogs are restricted to the forearm; and Hoxd13 paralogs are restricted to the hand (containing metacarpals and phalanges; see Fig. 20.9 ).
The requirement for Hox genes in regional growth of the limb bud is directly shown by the knockouts of multiple paralogs of Hox genes in mice. For example, in forelimbs lacking Hoxa11 and Hoxd11 genes, the radius and ulna are severely affected ( Fig. 20.10 ), whereas knockout of both Hoxa13 and Hoxd13 results in loss of the digits. Analysis of the Hoxa11/Hoxd11 mutants has shown reduced Fgf signaling, resulting in smaller skeletal condensations and delayed chondrocyte differentiation. Once these cartilaginous elements have formed, a growth plate defect significantly contributes to hypoplasia/aplasia of these elements at birth.
Morphogenesis of Limb
Once the AER has been established, the limb continues to grow, with development occurring predominantly along the proximal-distal axis. Proliferation and growth are also slightly higher on the dorsal side of the limb bud, resulting in a ventral curvature of the developing limbs. Later development takes place as follows ( Fig. 20.11 ):
Day 33. In the upper limb, the hand plate , forearm , arm , and shoulder regions can be distinguished. In the lower limb, a somewhat rounded proximal part can be distinguished from a more tapering distal part that will form the foot.
Day 37. In the hand plate of the upper limb, a central carpal region is surrounded by a thinner crescentic rim, the digital plate , which will form the fingers. In the lower limb, the thigh, leg , and foot have become distinct.
Day 38. Finger rays (more generally, digital rays) are visible as radial thickenings in the digital plate of the upper limb. The tips of the finger rays project slightly, producing a crenulated rim on the digital plate. A process of programmed apoptotic cell death between the digital rays will gradually sculpt the digital rays out of the digital plate by removing intervening tissue. This will free the fingers and toes. The lower limb bud has increased in length, and a clearly defined footplate has formed on the distal end of the limb.
Day 44. In the upper limb, the distal margin of the digital plate is deeply notched and the grooves between the finger rays are deeper. The bend where the elbow will form along the proximal-distal axis is becoming defined. Toe rays are visible in the digital plate of the foot, but the rim of the plate is not yet crenulated.
Day 47. The entire upper limb has undergone ventral flexion ( Fig. 20.12A ; see also Fig. 20.11 ). The lower limb has also begun to flex toward the midline. The toe rays are more prominent, although the margin of the digital plate is still smooth (see Fig. 20.11 ).
Day 52. The upper limbs are bent at the elbows, and the fingers have developed distal swellings called tactile pads (see Fig. 20.12B ; see also Fig. 20.11 ). The hands are slightly flexed at the wrists and meet at the midline in front of the cardiac eminence. The legs are longer, and the feet have begun to approach each other at the midline. The rim of the digital plate is notched.
Day 56. All regions of the arms and legs are well defined, including the tactile pads on the toes (see Fig. 20.12C ). The fingers of the two hands overlap at the midline.
In the Research Lab
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