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Gilbert SF. Developmental Biology. 6th edition. Sunderland (MA): Sinauer Associates; 2000.

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Developmental Biology. 6th edition.

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Generating the Proximal-Distal Axis of the Limb

The apical ectodermal ridge: The ectodermal component

The proximal-distal growth and differentiation of the limb bud is made possible by a series of interactions between the limb bud mesenchyme and the AER (Figure 16.8; Harrison 1918; Saunders 1948). These interactions were demonstrated by the results of several experiments on chick embryos:

Figure 16.8. Summary of experiments demonstrating the effect of the apical ectodermal ridge (AER) on the underlying mesenchyme.

Figure 16.8

Summary of experiments demonstrating the effect of the apical ectodermal ridge (AER) on the underlying mesenchyme. (Modified from Wessells 1977.)

1.

If the AER is removed at any time during limb development, further development of distal limb skeletal elements ceases.

2.

If an extra AER is grafted onto an existing limb bud, supernumerary structures are formed, usually toward the distal end of the limb.

3.

If leg mesenchyme is placed directly beneath the wing AER, distal hindlimb structures (toes) develop at the end of the limb. (However, if this mesenchyme is placed farther from the AER, the hindlimb mesenchyme becomes integrated into wing structures.)

4.

If limb mesenchyme is replaced by nonlimb mesenchyme beneath the AER, the AER regresses and limb development ceases.

Thus, although the mesenchyme cells induce and sustain the AER and determine the type of limb to be formed, the AER is responsible for the sustained outgrowth and development of the limb (Zwilling 1955; Saunders et al. 1957; Saunders 1972; Krabbenhoft and Fallon 1989). The AER keeps the mesenchyme cells directly beneath it in a state of mitotic proliferation and prevents them from forming cartilage. Hurle and co-workers (1989) found that if they cut away a small portion of the AER in a region that would normally fall between the digits of the chick leg, an extra digit emerged at that place* (Figure 16.9).

Figure 16.9. Cross section through the distal region of a chick limb 3 days after a wedge of the AER was removed from an area that would normally form interdigital tissue.

Figure 16.9

Cross section through the distal region of a chick limb 3 days after a wedge of the AER was removed from an area that would normally form interdigital tissue. Instead of degenerating, the remaining interdigital tissue formed an extra digit (marked by (more...)

The progress zone: The mesodermal component

The proximal-distal axis is defined only after the induction of the apical ectodermal ridge by the underlying mesoderm. The limb bud elongates by means of the proliferation of the mesenchyme cells underneath the AER. This region of cell division is called the progress zone, and it extends about 200 μm in from the AER. Molecules from the AER are thought to keep the progress zone mesenchyme cells dividing, and it is now thought that FGFs are the molecules responsible. When the AER is removed from an early limb bud, only the most proximal parts of the stylopod are made. However, if an FGF-containing bead is placed in the hole left by the removal of the AER, a normal limb will form (see Figure 16.8; Niswander et al. 1993; Fallon et al. 1994; Crossley et al. 1996).

When the mesenchyme cells leave the progress zone, they differentiate in a regionally specific manner. The first cells leaving the progress zone form proximal (stylopod) structures; those cells that have undergone numerous divisions in the progress zone become the more distal structures (Saunders 1948; Summerbell 1974). Therefore, if the AER is removed from an early-stage wing bud, the cells of the progress zone stop dividing, and only a humerus forms. If the AER is removed slightly later, humerus, radius, and ulna form (Figure 16.10; Iten, 1982; Rowe et al. 1982).

Figure 16.10. Dorsal view of chick skeletal pattern after removal of the entire AER from the right wing bud of chick embryos at various stages.

Figure 16.10

Dorsal view of chick skeletal pattern after removal of the entire AER from the right wing bud of chick embryos at various stages. The last photo (E) is of a normal wing skeleton. (From Iten 1982; photographs courtesy of L. Iten.)

Proximal-distal polarity resides in the mesodermal compartment of the limb. If the AER provided the positional information—somehow instructing the undifferentiated mesoderm beneath it as to what structures to make—then older AERs combined with younger mesoderm should produce limbs with deletions in the middle, while younger AERs combined with older mesoderm should produce duplications of structures. This was not found to be the case, however (Rubin and Saunders 1972). Rather, normal limbs form in both experiments. But when the entire progress zone, including both the mesoderm and AER, from an early embryo is placed on the limb bud of a later-stage embryo, new proximal structures are produced beyond those already present. Conversely, when old progress zones are added to young limb buds, distal structures immediately develop, so that digits are seen to emerge from the humerus without the intervening ulna and radius (Figure 16.11; Summerbell and Lewis 1975).

Figure 16.11. Control of proximal-distal specification by the cells of the progress zone.

Figure 16.11

Control of proximal-distal specification by the cells of the progress zone. (A) Extra set of ulna and radius formed when an early-bud progress zone was transplanted to a late wing bud that had already formed ulna and radius. (B) Lack of intermediate structures (more...)

The mitotic state of the progress zone is maintained by interactions between the FGF proteins of the progress zone and of the AER. FGF10 secretion by the mesenchyme cells induces the AER, and it also induces the AER to express FGF8 (Figure 16.12). The FGF8 secreted by the AER reciprocates by maintaining the mitotic activity of the progress zone mesenchyme cells (Figure 16.13; Mahmood et al. 1995; Crossley et al. 1996; Vogel et al. 1996; Ohuchi et al. 1997).

Figure 16.12. FGF8 in the AER.

Figure 16.12

FGF8 in the AER. (A) In situ hybridization showing expression of Fgf8 message in the ectoderm as the limb bud begins to form. (B) Expression of Fgf8 RNA in the apical ectodermal ridge, the source of mitotic signals to the underlying mesoderm. (C) In normal (more...)

Figure 16.13. A molecular model for the initiation of the limb bud.

Figure 16.13

A molecular model for the initiation of the limb bud. FGF10 secreted by the lateral plate mesoderm induces FGF8 expression in the competent ectoderm at the dorsal-ventral boundary. The anterior-posterior boundary is present at stage 16 (and perhaps earlier). (more...)

Hox genes and the specification of the proximal-distal axis

The type of structure formed along the proximal-distal axis is specified by the Hox genes. The products of the Hox genes have already played a role in specifying the place where the limbs will form. Now they will play a second role in specifying whether a particular mesenchymal cell will become stylopod, zeugopod, or autopod. The 5´ (AbdB-like) portions (paralogues 9–13) of the HoxA and HoxD complexes appear to be active in the forelimb buds of mice. Based on the expression patterns of these genes, and on naturally occurring and gene knockout mutations, Davis and colleagues (1995) proposed a model wherein these Hox genes specify the identity of a limb region (Figure 16.14). For instance, when they knocked out all four loci for the paralogous genes Hoxa-11 and Hoxd-11, the resulting mice lacked the ulna and radius of their forelimbs (Figure 16.14A,Figure 16.14B,). Similarly, knocking out all four Hoxa-13 and Hoxd-13 loci resulted in loss of the autopod (Fromental-Ramain et al. 1996). Humans homozygous for a HOXD13 mutation show abnormalities of the hands and feet wherein the digits fuse, and human patients with homozygous mutant alleles of HOXA13 also have deformities of their autopods (Figure 16.14C; Muragaki et al. 1996; Mortlock and Innis 1997). In both mice and humans, the autopod (the most distal portion of the limb) is affected by the loss of function of the most 5´ Hox genes.

Figure 16.14. Deletion of limb bone elements by the deletion of paralogous Hox genes.

Figure 16.14

Deletion of limb bone elements by the deletion of paralogous Hox genes. (A) Wild-type mouse forelimb. (B) Forelimb of mouse made doubly mutant such that it lacked functional Hoxa-11 and Hoxd-11 genes. The ulna and radius are absent. (C) Human synpolydactyly (more...)

The mechanism by which Hox genes could specify the proximal-distal axis is not yet understood, but one clue comes from the analysis of chicken Hoxa-13. Ectopic expression of this gene (which is usually expressed in the distal ends of developing chick limbs) appears to make the cells expressing it stickier. This, in turn, would cause the cartilaginous nodules to condense in specific ways (Yokouchi et al. 1995; Newman 1996).

As the limb grows outward, the pattern of Hox gene expression changes. When the stylopod is forming, Hoxd-9 and Hoxd-10 are expressed in the progress zone mesenchyme (Figure 16.15; Nelson et al. 1996). When the zeugopod bones are being formed, the pattern shifts remarkably, displaying a nested sequence of Hoxd gene expression. The posterior region expresses all the Hoxd genes from Hoxd-9 to Hoxd-13, while only Hoxd-9 is expressed anteriorly. In the third phase of limb development, when the autopod is forming, there is a further redeployment of Hox gene products. Hoxd-9 is no longer expressed. Rather, Hoxa-13 is expressed in the anterior tip of the limb bud and in a band marking the boundary of the autopod. Hoxd-13 products join those of Hoxa-13 in the anterior region of the limb bud, while Hoxa-12, Hoxa-11, and Hoxd-10–12 are expressed throughout the posterior two-thirds of the limb bud.

Figure 16.15. Hox gene expression changes during the formation of the tetrapod limb.

Figure 16.15

Hox gene expression changes during the formation of the tetrapod limb. (A) During the formation of the stylopod (phase I), Hoxd-9 and Hoxd-10 are expressed in the newly formed limb bud. (B) During zeugopod formation (phase 2), there is a nested expression (more...)

Hox Genes and the Evolution of the Tetrapod Limb

Macroevolution, the generation of morphological novelties in the evolution of new species and higher taxa, results from alterations of development. One of the most obvious macroevolutionary changes is that from the fish fin to the amphibian leg. As Richard Owen (1849) pointed out, there is considerable homology between the bones of the fish fin and the tetrapod limb, the pectoral and pelvic fins of the fish being homologous to the tetrapod forelimb and hindlimb, respectively. While specific homologies were able to be made between the proximal elements of the fin and the limb, the homologies proposed between the autopod of the limb (the hand or foot at the distal end) and the rays of the fins “did not hold water.” This was true even when one compared the tetrapod limb with the fins of the crossopterygian (lobe-finned) fishes thought to have been closely related to the ancestors of the amphibians (see Coates 1994; Hinchliffe 1994). While there seems to be homology for the proximal and central elements of the limb, the autopod seems to be something new—what evolutionary biologists call a neomorphic structure.

Recent studies have strongly suggested that the expression of the 5´ genes of the Hoxd group may be crucial in the change from fin to limb. Tetrapods and fishes share the first two phases of the Hox expression pattern in their appendages. Thus, both groups form stylopods and zeugopods. However, the phase III pattern of Hox gene expression is unique to tetrapods and is not found in fishes. Moreover, this change in Hox gene expression is mediated by a single enhancer element that is not found in fishes (Gerard et al. 1993; van der Hoeven et al. 1996). This phase III change represents an inversion of gene expression, placing the most 5´ Hox gene products in the anterior of the limb bud. Instead of being restricted to the posterior of the limb bud, the expression of the 5´ Hox genes sweeps across the distal mesenchyme, just beneath the AER. This band of expression is coincident with the “digital arch” from which the digits form (Figure 16.16; Morgan and Tabin 1994; Sordino et al. 1995; Nelson et al. 1996).

Thus, while the Hox gene expression pattern is homologous between fish and tetrapod limbs in the proximal regions, the expression pattern in the late-bud distal mesenchyme is new. These studies also confirm the paleontological interpretations of Shubin and Alberch (1986; Shubin et al. 1997), who proposed that the path of digit formation was not (as previously believed) through the fourth digit (making the fin rays homologous to the other digits), but through an arch of distal wrist condensations (metapterygia) that begins posteriorly and turns anteriorly across the distal mesenchyme (Figure 16.16). Thus, the border of 5´ HoxD gene expression follows the metapterygial axis that Shubin and Alberch hypothesized as being the origin of digits. The foot and hand, then, appear to be new structures in evolution, and they appear to have been formed by the repositioning of HoxD gene expression during fin development. The use of the same enhancer to generate both fingers and toes also helps solve the problem of how these structures were evolved at the same time. ▪

WEBSITE

16.4 Dinosaurs and the origin of birds. Whether birds are descendants of certain dinosaurs is a controversial subject that concerns hip structure, and whether the autopod of the dinosaur is made of the same digits as the bird autopod. http://www.devbio.com/chap16/link1604.shtml

Figure 16.16. Differences in Hoxd-11 and Hoxd-13 expression in fish and tetrapod embryonic appendages.

Figure 16.16

Differences in Hoxd-11 and Hoxd-13 expression in fish and tetrapod embryonic appendages. (A) Fin of a fish, wherein Hoxd-11 expression is distal to Hoxd-13 expression. The fin axis extends distally. (B) In tetrapods, Hoxd-13 expression becomes distal (more...)

Footnotes

*

When referring to the hand, one has an orderly set of names to specify each digit (digitus pollicis, d. indicis, d. medius, d. annularis, and d. minimus, respectively, from thumb to little finger). No such nomenclature exists for the pedal digits, but the plan proposed by Phillips (1991) has much merit. The pedal digits, from hallux to small toe, would be named porcellus fori, p. domi, p. carnivorus, p. non voratus, and p. plorans domi, respectively.

By agreement with the publisher, this book is accessible by the search feature, but cannot be browsed.

Copyright © 2000, Sinauer Associates.
Bookshelf ID: NBK10102

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