Sonic hedgehog is Essential for Proximal-Distal Outgrowth of the Limb Bud in Salamanders

Abstract

The developing forelimb has been a foundational model to understand how specified progenitor cells integrate genetic information to produce the tetrapod limb bauplan. Although the reigning hypothesis is that all tetrapods develop limbs in a similar manner, recent work suggests that urodeles have evolved a derived mode of limb dvelopment. Here, we demonstrate through pharmacological and genetic inactivation of <i>Sonic hedgehog</i> (<i>Shh</i>) signaling in axolotls that <i>Shh</i> directs expansion and survival of limb progenitor cells in addition to patterning the limb across the proximodistal and antero-posterior axis. In contrast to inactivation of <i>Shh</i> in mouse or chick embryos where a humerus, radius, and single digit develop, <i>Shh</i> crispant axolotls completely lack forelimbs. In rescuing limb development by implanting SHH-N protein beads into the nascent limb field of <i>Shh</i> crispants, we show that the limb field is specified in the absence of <i>Shh</i> and that hedgehog pathway activation is required to initiate proximodistal outgrowth. When our results are examined alongside other derived aspects of salamander limb development and placed in a phylogenetic context, a new hypothesis emerges whereby the ability for cells at an amputation plane to activate morphogenesis and regenerate a limb may have evolved uniquely in urodeles.