Cheryl Tickle 1 Introduction

The apical ectodermal ridge (AER; also known as the apical ridge) and the zone of polarizing activity (ZPA; also known as the polarizing region) are two major signaling regions in developing vertebrate limbs. Limbs arise as small buds of undifferentiated mesenchyme cells encased in ectoderm. The apical ridge and the polarizing region were first identified in limb buds of chick embryos, and functions of these regions were explored by traditional "cut and paste" experiments by Saunders (reviewed in ref. 1). Similar signaling regions are also found in embryonic limb buds of other vertebrate species, including mice, rats, snapping turtles, and humans (2). Signals from apical ridge and polarizing region act together with signal(s) from the ectoderm in a region of undifferentiated cells called the progress zone (3). The progress zone is found at the tip of the bud as it grows out and is maintained by the apical ridge. As cells leave the progress zone, they lay down the structures of the limb in sequence with proximal structures being formed first and distal structures later (4). Cellular responses to polarizing and ectoderm signals in the progress zone establish the limb plan, and subsequently cartilage, bone, muscle, tendon, and other specialized cell types differentiate and become organized into tissues.

The apical ridge is the thickened rim of epithelium at the tip of the limb bud and is required for bud outgrowth (Fig. 1). Apical ridge cells are elongated, tightly packed, and linked by extensive gap junctions. When the apical ridge is removed, bud outgrowth ceases and a truncated limb develops; in contrast, grafting a second apical ridge to the surface of a limb bud induces a second outgrowth (1,5). The signal from the apical ridge can be substituted by beads soaked in fibroblast growth factors (FGF-2, FGF-4, FGF-8; 6,7). Several members of the FGF family are expressed in tissues at the tip of the limb bud, in-

From: Methods in Molecular Biology, Vol. 97: Molecular Embryology: Methods and Protocols Edited by: P. T. Sharpe and I. Mason © Humana Press Inc., Totowa, NJ

Fig. 1. Diagram to show early chick limb with signaling tissues and the three axes, A-Po (anterior-posterior), P-Di (proximal-distal), D-V (dorsal-ventral).

eluding apical ridge (reviewed in ref. 8). The polarizing region is a group of mesenchyme cells at the posterior edge of the bud (Fig. 1; the posterior edge is that nearest the tail end of the embryo). Signaling by the polarizing region controls antero-posterior limb pattern (reviewed in refs. 1 and 5). Cells of the polarizing region of chick wing buds cannot be distinguished morphologically, but have a remarkable effect on the pattern when grafted to the anterior margin of a second wing bud. The normal chick wing has three digits arranged from anterior to posterior 2 3 4 (Fig. 2). The limb develops six digits following a polarizing region graft, and these are arranged in the mirror-image symmetrical pattern 4 3 2 23 4 (Fig. 2). The additional digits arise from anterior mesenchyme in response to a signal from the graft. Tissues from different regions of the limb bud and from other regions of embryos have been assayed for polarizing activity. This has led to construction of maps showing distribution of cells with polarizing activity or potential polarizing activity (9-12; Table 1).

The extent of digit duplication depends on both the number of polarizing region cells in the graft and the length of time that the graft is left in place (reviewed in ref. 5). With small numbers of polarizing region cells (around 30),only an additional 2 are produced giving the pattern 2234; with more polarizing region cells (60 cells) an additional digit 3 is formed (patterns, such as 32234, 3234, 334) and about 120 cells are required to give an additional digit 4 (ref. 21). Signaling by the polarizing region can be attenuated by X-irradiation and various chemicals. All of these treatments probably effectively reduce the number of signaling cells. When the graft is left in place for short periods of time (14 h) and then removed, only an additional digit 2 is formed,

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