Sonic Hedgehog is not a limb morphogen but acts as a trigger to specify all digits

Abstract

SummaryLimb patterning by Sonic hedgehog (Shh) is among the most highly touted and studied models of “morphogen” function1. Yet how Shh instructs distinct digit types (index to little finger) remains controversial. Both spatial concentration gradients2,3and temporal signal integration4–6have been proposed to explain how Shh patterns different digits, yet genetic studies in mouse suggested that Shh acts over a limited interval to specify digits7. Here, we replaced the cell survival function of Shh during limb bud outgrowth and demonstrate that a transient, early pulse of Shh activity is necessary and sufficient for normal limb development. Our lineage tracing of Shh response shows that Shh signals at very short-range during this time frame and patterns digits indirectly. We demonstrate that Gli3, the major Shh nuclear transducer8,9, is functionally unaltered and cryptic pathway re-activation doesn’t occur. Our findings are incompatible with either spatial or temporal signal integration models and indicate Shh initiates a relay mechanism. Using a genetic test for relay signaling, we unexpectedly discovered that Shh is required indirectly to specify digit 1 (thumb), previously thought to be exclusively Shh-independent10,11. Our results uncover a unique digit 1 regulatory hierarchy, implicating Shh in digit 1 evolutionary adaptations, such as an opposable thumb. These findings illuminate Shh function in the related contexts of limb development, regeneration, and evolutionary adaptation, and lay the groundwork for elucidating how Shh triggers a relay network that becomes rapidly self-sustaining.