Cell to Cell Signalling during Vertebrate Limb Development

The vertebrate limb is one of the most relevant experimental models in which to analyse cell-cell signalling during patterning of embryonic fields and organogenesis. Recently, the combination of molecular and genetic studies with experimental manipulation of developing limb buds has significantly advanced our understanding of the complex molecular interactions coordinating limb bud outgrowth and patterning. Certain of these studies indicate the need to revise some of the “textbook views” of vertebrate limb development. In this review, we focus our discussion on how signalling by the polarizing region is established and how limb bud morphogenesis is controlled by both long-range and signal relay mechanisms. Furthermore, we discuss recent results showing that differential mesenchymal responsiveness to SHH signalling is established prior to its expression by the polarizing region. 1. A brief history of the limb bud mesenchymal organiser called polarising region or ZPA and the apical ectodermal ridge (AER) A few decienia ago the seminal experiments by Saunders (1948) and Saunders and Gasseling (1968) led to the discovery of two major signalling centres controlling vertebrate limb pattern formation. The first one is a group of cells located in the posterior limb bud mesenchyme, the so-called polarising region or zone of polarising activity (ZPA, reviewed by Johnson and Tabin, 1997). The second signalling centre is a ridge of cells running along the distal margin of the limb bud ectoderm called apical ectodermal ridge (AER). The AER maintains limb bud outgrowth in a proximal to distal direction by keeping the cells of the progress zone (layer of cells directly underlying the AER) in a proliferating, undifferentiated state (reviewed by Capdevila and Izpisua Belmonte, 2001). Removal of the AER results in truncation of the limb. The level of truncation along the proximal-distal axis of the limb depends on the time of AER removal. Ablation of the AER early during limb bud development truncates the limb at a proximal, while removal at a later stage during development only affects the digits (Summerbell et al., 1973). This observation can be explained by two controversial models. According to the progress zone model, a model that has been accepted for more than 30 years, cells measure the time they have spent in the progress zone by a socalled internal clock. This internal clock, which is controlled by the AER, is active as long the cells stay in the progress zone. Cells that leave the progress zone will adopt a fate according to the time they have spent in the progress zone. Cells that leave the progress zone early will adopt a proximal fate, while cells that have stayed a longer period in the progress zone will differentiate in more distal structures. Once the AER is removed, the clock will be arrested, resulting in a truncation of the limb (Summerbell et al., 1973). However recently, the progress zone model has been challenged by the early specification model (Dudley et al., 2002; Sun et al., 2002). According to this model, limb segments are already specified in broad domains from an early stage. These domains will expand during development and subsequently become determined to form skeletal elements. Removal of the AER will give rise to cell death in the mesenchyme, resulting in truncation of the limb