The apical ectodermal ridge plays a central role in limb development through its interactions with the underlying mesenchyme. Removal of the AER results in cessation of limb outgrowth and leads to truncation of the limb along the proximo-distal axis. The many functions attributed to the ridge include maintenance of the progress zone mesenchyme. Here, cells are stimulated to proliferate, are maintained in an undifferentiated state, and are assigned progressively more distal positional values as the limb grows. The AER also functions to maintain the activity of the polarizing region, a region of mesenchyme which is thought to provide the primary signal for patterning along the antero-posterior axis. We have begun to explore the function of fibroblast growth factor-4 (FGF-4) during limb development. FGF-4, which encodes an efficiently secreted protein, is expressed in the AER. We have previously demonstrated that FGF-4 protein can stimulate limb mesenchyme proliferation and can induce the expression of a downstream homeobox gene, Evx-1 (homologue of the Drosophila even-skipped gene), that is normally regulated by a signal from the AER. To determine to what extent FGF-4 protein can substitute for the AER to allow normal limb outgrowth, we performed experiments on the developing chick limb in ovo. Remarkably, we find that after AER removal, the FGF4 protein can provide all the signals required for virtually normal outgrowth and patterning of the limb. Further studies indicate that proliferation of progress zone cells is not sufficient, and that an additional signal is produced by the posterior mesenchyme in response to FGF4 which enables progress zone cells to acquire progressively more distal fates. Thus FGF-4 maintains progress zone activity through a combination of at least two signals-one that acts directly on progress zone cells to stimulate their proliferation, and one that acts indirectly by maintaining the production of patterning signal(s) by the posterior mesenchyme. We further show that failure of the posterior mesenchyme to produce this signal correlates with failure to maintain polarizing activity. This raises the possibility that the signal produced by the posterior mesenchyme and required for progressive proximo-distal limb patterning is identical to the polarizing activity. Further experiments demonstrate that retinoic acid, which mimics the activity of the polarizing region, can supply this signal. In conclusion, the finding that a single growth factor can serve as both the direct and indirect signals required to maintain progress zone activity provides a simple mechanism for ensuring that growth and pattern formation are linked in the developing limb. (C) 1994 Wiley-Liss, Inc.
