Dual Bmp-negative feedback loops modulate both AER and ZPA function to buffer and constrain postaxial digit number

ABSTRACT Several lines of evidence indicate that posterior (postaxial) digit number in tetrapod vertebrates is constrained to the pentadactyl state by interactions between the 2 major signaling centers that organize digit pattern and growth, the Shh-expressing ZPA and the Fgf-expressing AER ectoderm. Negative short-range effects of Shh on the immediately overlying AER limit its posterior extent and function, either by direct Shh signaling or with Bmps playing a key role. How this strong inhibitory effect is counter-balanced to maintain pentadactyly in many vertebrate species remains less clear. In this study we used genetic approaches in mouse to re-evaluate the mechanism by which Shh signaling modulates AER function and demonstrate that this occurs via Shh-target Bmps that act as a relay signal, rather than by direct Shh action. Furthermore, we show that Shh-induced Bmps also act directly on the ZPA, in a negative feedback loop, to down-regulate Shh expression and ZPA extent. We provide evidence that these dual Bmp-driven negative feedback loops robustly balance total Bmp levels to constrain postaxial digit number. Significance Statement This study examines how vertebrates, including humans, are generally constrained to forming five fingers or toes (pentadactyly) during normal development. Two key signaling centers in the limb bud, producing Sonic hedgehog (Shh) in the posterior mesoderm (ZPA) and Fgfs in the marginal ectoderm (AER), interact in a positive feedback loop to direct and coordinate limb outgrowth. However, short-range negative interaction between these two centers limits posterior digit numbers. Using genetic approaches in mouse, we show that Bmps, rather than Shh, signal directly to the AER to act as the primary mediators of this constraint. Shh-induced target Bmps act directly on AER to inhibit its function and prevent posterior digit expansion, and they also act directly on ZPA in a negative feedback loop to inhibit Shh expression. These dual Bmp-driven feedback circuits act together to balance Bmp activity and robustly limit posterior digit number to the pentadactyl state. This finding enhances our understanding of how disrupted developmental regulation may lead to congenital limb malformations and how evolutionary constraints on digit number may be imposed.