Fig. 6. Limb hairy2 expression as a temporal and spatial output of joint FGF and SHH signaling.

(A) High levels of instructive AER-derived FGF signaling, together with the Gli3-A/Gli3-R>1 permissive state established by ZPA/SHH, define the required conditions for hairy2 expression. SHH acts as a long-term, long-range signal, ensuring a Gli3-A/Gli3-R>1 permissive state for hairy2 expression. In these conditions, short-term/short-range FGF signaling is capable of inducing hairy2 expression, acting as an instructive signal. (B) hairy2 is expressed in the mesenchyme adjacent to AER/FGF and presents distinct expression domains: hairy2 is persistently expressed in the posterior region overlapping the ZPA/SHH (PPD), is absent from the anterior limb (AND) and is cyclically expressed in the DCD. We describe FGF acting through Erk/MAPK and Akt/PI3K as an instructive signal for hairy2 induction, in a short-term, short-range fashion throughout the distal limb mesenchyme. However, a permissive state mediated by SHH signaling is required for the tissue to respond to FGF inductive signal. SHH permissive signal is mediated by Gli3 activity and acts in a long-term, long-range manner and patterns hairy2 expression along the distal limb AP axis. PPD and DCD present Gli3-A/Gli3-R above one (yellow dots), allowing sustained or oscillatory hairy2 expression. In the AND, AER/FGFs can no longer induce hairy2 since this tissue is in a non-permissive state: Gli3-A/Gli3-R below one (grey dots).