How the embryo makes a limb: determination, polarity and identity

Abstract

The vertebrate limb with its complex anatomy develops from a small bud of undifferentiated mesoderm cells encased in ectoderm. The bud has its own intrinsic polarity and can develop autonomously into a limb without reference to the rest of the embryo. In this review, recent advances are integrated with classical embryology, carried out mainly in chick embryos, to present an overview of how the embryo makes a limb bud. We will focus on how mesoderm cells in precise locations in the embryo become determined to form a limb and express the key transcription factors Tbx4 (leg/hindlimb) or Tbx5 (wing/forelimb). These Tbx transcription factors have equivalent functions in the control of bud formation by initiating a signalling cascade involving Wnts and fibroblast growth factors (FGFs) and by regulating recruitment of mesenchymal cells from the coelomic epithelium into the bud. The mesoderm that will form limb buds and the polarity of the buds is determined with respect to both antero-posterior and dorso-ventral axes of the body. The position in which a bud develops along the antero-posterior axis of the body will also determine its identity - wing/forelimb or leg/hindlimb. Hox gene activity, under the influence of retinoic acid signalling, is directly linked with the initiation of Tbx5 gene expression in the region along the antero-posterior axis of the body that will form wings/forelimbs and determines antero-posterior polarity of the buds. In contrast, Tbx4 expression in the regions that will form legs/hindlimbs is regulated by the homeoprotein Pitx1 and there is no evidence that Hox genes determine antero-posterior polarity of the buds. Bone morphogenetic protein (BMP) signalling determines the region along the dorso-ventral axis of the body in which both wings/forelimbs and legs/hindlimbs develop and dorso-ventral polarity of the buds. The polarity of the buds leads to the establishment of signalling regions - the dorsal and ventral ectoderm, producing Wnts and BMPs, respectively, the apical ectodermal ridge producing fibroblast growth factors and the polarizing region, Sonic hedgehog (Shh). These signals are the same in both wings/forelimbs and legs/hindlimbs and control growth and pattern formation by providing the mesoderm cells of the limb bud as it develops with positional information. The precise anatomy of the limb depends on the mesoderm cells in the developing bud interpreting positional information according to their identity - determined by Pitx1 in hindlimbs - and genotype. The competence to form a limb extends along the entire antero-posterior axis of the trunk - with Hox gene activity inhibiting the formation of forelimbs in the interlimb region - and also along the dorso-ventral axis.

Keywords: Hox genes; Pitx1; Sonic hedgehog; Tbx4/5; Wnts; antero-posterior polarity; apical ectodermal ridge; bone morphogenetic proteins; dorso-ventral polarity; embryo; fibroblast growth factors; lateral plate mesoderm; limb; limb bud; polarizing region; retinoic acid.

Figure 1

Diagram of a chick wing bud; on the left, a dorsal view with a dotted line showing where the section shown on the right was taken. The signalling regions together with the key signalling molecules produced are indicated. The three main axes of the wing bud are shown at top right. A, anterior; D, dorsal; P, posterior; Prox, proximal; V, ventral. Staging according to Hamburger and Hamilton (reprinted 1992).

Figure 2

Diagram illustrating fate maps showing the location of the cells that will form the chick wing bud at different stages in early embryos together with a timeline showing when determination takes place and the polarity of the bud is fixed. Expression of some of the key molecules is indicated. s, somite. 2-s and 13-s fate maps looking down on the embryo according to Chaube (1959); fate maps 19/20-s and stage 16 according to Michaud et al. (1997). 19/20-s upper diagram looking down on the wing-forming region on the right side of embryo and below a transverse section in this region, stage 16 tranverse section through wing-forming region. The time between the 2-s stage and stage 16 is approximately 24–30 h. A, anterior; D, dorsal; L, lateral; M, medial; P, posterior; V, ventral.

Figure 3

Diagram showing a side view of the wing-forming region of the lateral plate mesoderm (19/20-s stage in Fig.2) together with a scheme outlining the interactions with neighbouring tissues along the antero-posterior axis involving retinoic acid signalling downstream of 3′ Hox genes that lead to Tbx5 being expressed in the wing/forelimb-forming region at this stage. Inhibitory interactions indicated by barred lines. RA, retinoic acid; A, anterior; P, posterior.

Figure 4

Diagram showing a transverse section through the wing/forelimb bud-forming region expressing Tbx5 (19/20-s stage in Fig.2) together with a scheme outlining the interactions with overlying ectoderm that lead to formation of the apical ectodermal ridge and with the underlying coelomic epithelium that lead to an epithelial–mesenchymal transition (EMT) and recruitment of cells to the bud (indicated by dotted line). Dashed lines indicate suggested interactions. M, medial; D, dorsal; L, lateral; V, ventral.

Figure 5

Diagram showing a side view of an early wing/forelimb bud (equivalent to stage 17 in Fig.1), showing a scheme outlining the molecular interactions that lead to the formation of the polarizing region expressing Shh at the posterior of the wing/forelimb bud. Not all known components of the regulatory pathway are illustrated (see Osterwalder et al. for more details). A, anterior; P, posterior; RA, retinoic acid.

Figure 6

(A) The result of Balinsky’s experiment in the newt used as a logo for his textbook in Embryology (Balinsky, 1965). (B) Induction of an additional limb (leg) in a chick embryo following insertion of a bead soaked in FGF to the flank (courtesy Martin Cohn). (C) Cow with additional limb arising dorsal to the forelimb; higher magnification of additional limb shown below. (courtesy Ruth Bellairs). Additional limbs in (B) and (C) indicated by arrows.