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 anteroposterior 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.

Limb patterning genes and heterochronic development of the emu wing bud

“…In the chicken, mis-expression of Shh in the limb bud induces digit anomalies consistent with its role as an anterior–posterior organizer [22]. Genes 5′ in the HOXD cluster also play a role in anterior–posterior patterning of the limb bud (reviewed in [23]).…”

The chick limb: embryology, genetics and teratology

“…Transplantation experiments in chick embryos were instrumental in showing that limb -forming regions and their antero-posterior polarity are determined long before any limb buds are visible. Further cut and paste experiments indicated that signals from neighbouring tissues are involved in specifying the position of the limb-forming regions at the sides of the body (reviewed Tickle, 2015).…”

Homeotic transformation of tails into limbs in anurans

“…It is necessary to investigate the expression of hindlimb development-related genes, for example, Shh, Tbx4, and Apical ectodermal ridge of the limb bud is formed at the trunk dorsoventral (D-V) boundary (Cohn & Tickle, 1999;Rodriguez-Esteban et al, 1999;Tickle, 2015). It is necessary to investigate the expression of hindlimb development-related genes, for example, Shh, Tbx4, and Apical ectodermal ridge of the limb bud is formed at the trunk dorsoventral (D-V) boundary (Cohn & Tickle, 1999;Rodriguez-Esteban et al, 1999;Tickle, 2015).…”

“…Pitx1 activates other hindlimb development-related genes such as Tbx4 expression of genes such as Engrailed-1 (Nishimoto & Logan, 2016;Tickle, 2015). The tadpole in this panel is depicted as a representative of tetrapods.…”

“…Therefore, it is reasonable to assume that vitamin A treatment changes the anterior-posterior (A-P) positional information of segments in regenerating tails (Figure 3a). Positional values along the A-P axis are determined by the expression pattern of genes, for example, Hox genes (Cohn & Tickle, 1999;Nishimoto & Logan, 2016;Tickle, 2015) ( Figure 6). In normal tail regeneration, Hox gene expression patterns should induce a positional value posterior to amputation lines ( Figure 3a).…”