Vertebrate Embryo: Limb Development

Abstract

Vertebrate limbs develop from small buds of mesoderm cells encased in ectoderm. Limb development is an excellent system for studying morphogenesis and pattern formation. Growth plays a major role in morphogenesis, which shapes the limb, while pattern formation ensures that the parts of the limb arise in the proper places and depends mainly on positional information. Both processes are governed by cell–cell interactions involving signalling centres that operate along each of the three limb axes but are functionally interconnected. The main proliferative and positional signals are the secreted molecules, WNTs, fibroblast growth factors, Sonic Hedgehog and bone morphogenetic proteins. The transcription factors encoded by Hox genes are involved in multiple steps in limb development and together with other transcription factors are expressed in response to positional signals in different regions of the limb bud. But how this positional information is interpreted to generate the detailed limb anatomy is largely unknown.

Key Concepts

  • The limb develops from a bud of mesoderm cells encased in ectoderm which grows out from the body wall.
  • The limb bud mesoderm is made up of cells with two different origins; cells of the lateral plate mesoderm which give rise to the connective tissues and cells that have migrated from the somites which give rise to the myogenic cells of the limb muscles and some of the endothelial cells that line the blood vessels.
  • Three sets of cell–cell interactions specify positional information; one set for each of the three axes of the limb.
  • The apical ectodermal ridge at the tip of the limb bud produces FGFs which are required for bud outgrowth and laying down the pattern along the proximodistal axis.
  • The dorsal and ventral ectoderm of the limb bud produce WNT7a and BMPs, respectively, which are involved in specifying dorsoventral positional information.
  • The polarising region, a mesodermal signalling region at the posterior margin of the limb bud, produces Sonic Hedgehog (SHH) which specifies anteroposterior positional information and controls growth across this axis.
  • Hox genes are involved in specifying forelimb position and in establishing their initial anteroposterior polarity. Interactions between the signalling regions ensure that pattern formation is integrated along all three axes of the developing limb.
  • 5′ genes in the HoxA and HoxD clusters are expressed in early and late limb buds under the control of long‐range enhancers located, respectively, 3′ and 5′ of the cluster, with the early phase of activity specifying proximo‐distal regional identity, the later phase, controlling the development of the digits.
  • Differences in forelimb and hindlimb anatomy depend on the interpretation of positional information, with the transcription factor PITX1 being a major hindlimb determinant.

Keywords: limb; pattern formation; morphogenesis; apical ectodermal ridge; polarising region (zone of polarising activity); dorsoventral pattern; limb identity; cell–cell signalling molecules; Hox genes

Figure 1. (a) Origin of tissue types within the limb. The cartilage, tendon and other connective tissues of the limb arise from the mesoderm derived from the lateral plate somatopleure (shown by a bracket in the diagram of a prelimb bud stage chick embryo (Hamilton–Hamburger stage 14) on the left side). Muscle cell progenitors arise from the somite (purple), where they delaminate from the lateral aspect of the dermomyotome and migrate into the early limb bud (see diagram of Hamilton–Hamburger stage 20 chick embryo on the right side). The nerves (green) arise from the spinal cord and migrate towards the developing limb to form a plexus; the nerve axons then enter the developing limb once the tissues have started to differentiate. The ectoderm will give rise to the epidermis of the skin. The apical ectodermal ridge (AER; see stage 20 diagram) forms at the boundary between the dorsal and ventral limb ectoderm. (b,c) Experimental determination of the dorsoventral (D–V) signalling centres. (b) Prelimb bud stage chick embryo (stage 14). Rotation of the ectoderm 180° relative to the lateral plate somatopleure results in a limb bud with D–V polarity that corresponds to the mesenchyme. This indicates that D–V patterning information resides in the mesenchyme at this early stage. (c) Early limb bud stage chick embryo (stage 20). Rotation of the ectoderm 180° relative to the limb bud mesenchyme results in formation of distal skeletal elements with D–V polarity that corresponds to the ectoderm. This indicates that D–V patterning information resides in the ectoderm by this stage. (d) Molecular determinants of D–V signalling. The early mesenchymal D–V signal is likely to involve BMP signalling which is graded from ventral to dorsal. After stage 15, the dorsal ectoderm expresses Wnt7a (purple). WNT7a induces Lmx1 (green) in the mesenchyme. WNT7a and LMX1 are required for specification of dorsal cell fates. BMPs produced by the ventral ectoderm induce the expression of Engrailed‐1 (En‐1; blue) which prevents Wnt7a expression, thus allowing the development of ventral limb structures. AER, apical ectodermal ridge; D, dorsal; DE, dorsal ectoderm; DM, dorsal mesenchyme; V, ventral; VE, ventral ectoderm; VM, ventral mesenchyme.
Figure 2. (a) The proximodistal signalling centre: the AER. On the left is a diagram of a stage 20 chick wing bud and on the right is the normal pattern of skeletal elements. The AER signals (arrows) to the underlying mesenchyme (red stipple) to maintain it in an undifferentiated and proliferative state. This region of mesenchyme has been proposed to act as a progress zone in proximodistal pattern formation. The pattern of the skeletal elements of the chick wing from proximal to distal is humerus (H), radius (R) and ulna (U), and the three digits (traditionally numbered 2, 3, 4 from anterior to posterior, but now considered to be 1, 2, 3). (b,c) Experimental demonstration of the proximodistal signalling centre. Surgical removal of the AER results in truncation of the limb along the proximodistal axis. Stages indicated are Hamburger–Hamilton stages of chick wing development. (c) Approximate level of truncation following removal of the AER at the indicated stages. (d) Molecules produced by the proximodistal signalling centre. Left: FGF signalling initiates limb development. Fgf8 (red) and Fgf10 (purple) are expressed in the mesonephros (M), but FGF signalling by the mesonephros is not required for limb initiation. Fgf10 is expressed in the lateral plate mesenchyme in the limb‐forming region and leads to induction of Fgf8 expression in the surface ectoderm. Right: FGF signalling from the AER signalling centre. Fgfs are expressed in the AER and can substitute for the AER to allow proximodistal outgrowth of the limb. Fgf4 is expressed in the posterior half of the AER (green), whereas Fgf8 is expressed throughout the AER (red). Fgf2 is present in the AER and mesenchyme. Fgf10 is expressed in the undifferentiated mesenchyme at the tip of the bud. A, anterior; FGF, fibroblast growth factor; H, humerus; LP, lateral plate; M, mesonephros; NT, neural tube; P, posterior; R, radius; S, somite; SE, surface ectoderm; U, ulna; 2, 3, 4, digits 2, 3 and 4, respectively.
Figure 3. (a) Experimental demonstration of the anteroposterior signalling centre: the polarising region or zone of polarising activity (ZPA). Transplantation of mesenchyme from the posterior margin of an early donor chick wing bud to the anterior region of an early host chick wing bud results in additional digits forming in a mirror image polarity compared with the normal digits. Note that two ulnae develop in the forewing, rather than an anterior radius and posterior ulna. (b) Molecules produced by the ZPA signalling centre. Sonic Hedgehog (SHH) is produced by cells in the posterior region of the wing bud and, when introduced anteriorly into the wing bud, results in the same mirror‐image skeletal duplications as those which result from a polarising region graft. A, anterior; H, humerus; NT, neural tube; P, posterior; S, somite; SHH, Sonic Hedgehog; U, ulna; 2, 3, 4, digits 2, 3 and 4, respectively.
Figure 4. Interactions between signalling centres in the developing limb. Shh expression in the polarising region is maintained by both the signalling molecule encoded by the Wnt7a gene expressed in the overlying dorsal ectoderm and FGF4 produced by the AER. The effects of FGF signalling in the underlying mesenchyme are mediated through control of expression of genes encoding ETS transcription factors involved in regulating ZRS activity. Fgf4 expression in the apical ectodermal ridge is maintained by the apical ridge maintenance factor, the BMP antagonist, GREMLIN. At early stages in limb development, Gremlin expression is maintained by SHH produced by the polarising region and GREMLIN antagonises BMP4 signalling, which would otherwise inhibit Fgf4 expression in the apical ectodermal ridge.
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Further Reading

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Tickle, Cheryll, and Towers, Matthew(Jul 2020) Vertebrate Embryo: Limb Development. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0000728.pub3]