Specification of the limb fields: Hox genes and retinoic acid

Limbs will not form just anywhere along the body axis. Rather, there are discrete positions where limb fields are generated. Using the techniques described in Chapter 3, researchers have precisely localized the limb fields of many vertebrate species. Interestingly, in all land vertebrates, there are only four limb buds per embryo, and they are always opposite each other with respect to the midline. Although the limbs of different vertebrates differ with respect to which somite level they arise from, their position is constant with respect to the level of Hox gene expression along the anterior-posterior axis (see Chapter 11). For instance, in fishes (in which the pectoral and pelvic fins correspond to the anterior and posterior limbs, respectively), amphibians, birds, and mammals, the forelimb buds are found at the most anterior expression region of Hoxc-6, the position of the first thoracic vertebra^* (Oliver et al. 1988; Molven et al. 1990; Burke et al. 1995). The lateral plate mesoderm in the limb field is also special in that it will induce myoblasts to migrate out from the somites and enter the limb bud. No other region of the lateral plate mesoderm will do that (Hayashi and Ozawa 1995).

Retinoic acid appears to be critical for the initiation of limb bud outgrowth, since blocking the synthesis of retinoic acid with certain drugs prevents limb bud initiation (Stratford et al. 1996). Bryant and Gardiner (1992) suggested that a gradient of retinoic acid along the anterior-posterior axis might activate certain homeotic genes in particular cells and thereby specify them to become included in the limb field. The source of this retinoic acid is probably Hensen's node (Hogan et al. 1992). The specification of limb fields by retinoic acid-activated Hox genes might explain a bizarre observation made by Mohanty-Hejmadi and colleagues (1992) and repeated by Maden (1993). When the tails of tadpoles were amputated and the stumps exposed to retinoic acid during the first days of regeneration, the tadpoles regenerated several legs from the tail stump (Figure 16.2). It appears that the retinoic acid caused a homeotic transformation in the regenerating tail by respecifying the tail tissue as a limb-forming pelvic region (Müller et al. 1996).

Figure 16.2

Legs regenerating from retinoic acid-treated tadpole tail. (A) The tail stump of a balloon frog tadpole treated with retinoic acid after amputation will form limbs from the amputation site. (B) Normal tail regeneration in a Rana temporaria tadpole 4 weeks (more...)

Induction of the early limb bud: Fibroblast growth factors

Limb development begins when mesenchyme cells proliferate from the somatic layer of the limb field lateral plate mesoderm (limb skeletal precursors) and from the somites (limb muscle precursors; Figure 16.3) These cells accumulate under the epidermal tissue to create a circular bulge called a limb bud. Recent studies on the earliest stages of limb formation have shown that the signal for limb bud formation comes from the lateral plate mesoderm cells that will become the prospective limb mesenchyme. These cells secrete the paracrine factor FGF10. FGF10 is capable of initiating the limb-forming interactions between the ectoderm and the mesoderm. If beads containing FGF10 are placed ectopically beneath the flank ectoderm, extra limbs emerge (Figure 16.4) (Ohuchi et al. 1997; Sekine et al. 1999).

Figure 16.3

Limb bud formation. (A) Proliferation of mesodermal cells from the somatic region of the lateral plate mesoderm causes the limb bud in the amphibian embryo to bulge outward. These cells generate the skeletal elements of the limb. Contributions of cells (more...)

Figure 16.4

FGF10 expression and action in the developing chick limb. (A) FGF10 becomes expressed in the lateral plate mesoderm in precisely those positions where limbs normally form. (B) When cells genetically constructed to secrete FGF10 are placed into the flanks (more...)

Specification of forelimb or hindlimb: Tbx4 and Tbx5

The limb buds have to be specified as being those of either the forelimb or the hindlimb. How are these distinguished? In 1996, Gibson-Brown and colleagues made a tantalizing correlation: The gene encoding the Tbx5 transcription factor is transcribed in mouse forelimbs, while the gene encoding the closely related transcription factor Tbx4 is expressed in hindlimbs.^† Could these two transcription factors be involved in directing forelimb versus hindlimb specificity? The loss-of-function data were equivocal: humans heterozygous for the TBX5 gene have Holt-Oram syndrome, characterized by abnormalities of the heart and upper limbs (Basson et al. 1996; Li et al. 1996). The legs are not affected, but neither are the arms transformed into a pair of legs.

In 1998 and 1999, however, several laboratories (Ohuchi et al. 1998; Logan et al. 1998; Takeuchi et al. 1999; Rodriguez-Esteban et al. 1999, among others) provided gain-of-function evidence that Tbx4 and Tbx5 specify hindlimbs and forelimbs, respectively. First, if FGF-secreting beads were used to induce an ectopic limb between the chick hindlimb and forelimb buds, the type of limb produced was determined by the Tbx protein expressed. Those buds induced by placing FGF beads close to the hindlimb (opposite somite 25) expressed Tbx4 and became hindlimbs. Those buds induced close to the forelimb (opposite somite 17) expressed Tbx5 and developed as forelimbs (wings). Those buds induced in the center of the flank tissue expressed Tbx5 in the anterior portion of the limb and Tbx4 in the posterior portion of the limb. These limbs developed as chimeric structures, with the anterior resembling a forelimb and the posterior resembling a hindlimb (Figure 16.5). Moreover, when a chick embryo was made to express Tbx4 throughout the flank tissue (by infecting the tissue with a virus that expressed Tbx4), limbs induced in the anterior region of the flank often became legs instead of wings (Figure 16.6). Thus, Tbx4 and Tbx5 appear to be critical in instructing the limbs to become hindlimbs and forelimbs, respectively.

Figure 16.5

Specification of limb type by Tbx4 and Tbx5. (A) During normal chick development, in situ hybridizations show that Tbx5 is found in the anterior lateral plate mesoderm, while Tbx4 is found in the posterior lateral plate mesoderm. Tbx5-containing limb (more...)

Figure 16.6

Respecification of forelimb into hindlimb by ectopic expression of Tbx4. (A) An FGF-secreting bead opposite somite 21 usually induces a Tbx5-expressing limb bud that forms a new wing. (B) If the entire flank is experimentally made to express Tbx4 (by (more...)

Induction of the apical ectodermal ridge

As mesenchyme cells enter the limb region, they secrete factors that induce the overlying ectoderm to form a structure called the apical ectodermal ridge (AER) (Figure 16.7; Saunders 1948; Kieny 1960; Saunders and Reuss 1974). This ridge runs along the distal margin of the limb bud and will become a major signaling center for the developing limb. Its roles include (1) maintaining the mesenchyme beneath it in a plastic, proliferating phase that enables the linear (proximal-distal) growth of the limb; (2) maintaining the expression of those molecules that generate the anterior-posterior (thumb-pinky) axis; and (3) interacting with the proteins specifying the anterior-posterior and dorsal-ventral axes so that each cell is given instructions on how to differentiate.

Figure 16.7

Scanning electron micrograph of an early chick forelimb bud, with its apical ectodermal ridge in the foreground. (Courtesy of K. W. Tosney.)

The factor secreted by the mesenchyme cells to induce the AER is probably FGF10 (Xu et al. 1998; Yonei-Tamura et al. 1999). (Other FGFs, such as FGF2, FGF4, and FGF8, will also induce an AER to form; but FGF10 appears to be the FGF synthesized at the appropriate time and in the appropriate places.) FGF10 is capable of inducing the AER in the competent ectoderm between the dorsal and ventral sides of the embryo. This junction is important. In mutants in which the limb bud is dorsalized and there is no dorsal-ventral junction (as in the chick mutant limbless), the AER fails to form, and limb development ceases (Carrington and Fallon 1988; Laufer et al. 1997; Rodriguez-Esteban et al. 1997; Tanaka et al. 1997).

Interestingly, the Hox gene expression pattern in at least some snakes (such as Python) creates a pattern in which each somite is specified to become a thoracic (ribbed) vertebra. The patterns of Hox gene expression associated with limb-forming regions are not seen (Cohn and Tickle 1999; see Chapter 22).

Tbx stands for T-box. The T (Brachyury) gene and its relatives have a sequence that encodes this specific DNA-binding domain.

Footnotes

*

Interestingly, the Hox gene expression pattern in at least some snakes (such as Python) creates a pattern in which each somite is specified to become a thoracic (ribbed) vertebra. The patterns of Hox gene expression associated with limb-forming regions are not seen (Cohn and Tickle 1999; see Chapter 22).

†

Tbx stands for T-box. The T (Brachyury) gene and its relatives have a sequence that encodes this specific DNA-binding domain.