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 Sonic hedgehog (Shh) signaling in axolotls that Shh 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 Shh in mouse or chick embryos where a humerus, radius, and single digit develop, Shh crispant axolotls completely lack forelimbs. In rescuing limb development by implanting SHH-N protein beads into the nascent limb field of Shh crispants, we show that the limb field is specified in the absence of Shh 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.

Molecular basis for positional memory and its reprogrammability in limb regeneration

“…3a ) 43 . The expanded Shh lineage in axolotls is consistent with the known, more proximal location of Shh cells during development and could explain why the Shh mutant phenotype is more severe in axolotl limbs than in mouse 20,37,44,45 . Our data support that expression level impacts on Hand2 ’s ability to induce Shh .…”

“…3c , n = 9). This phenotype is reminiscent of that of Shh CRISPant axolotls 37 . We obtained a range of digit number phenotypes, as expected from mosaic inactivation ( Extended Data Fig.…”

“…The modified assay resulted in the same outcome as the original cell injection assay; anterior cells acquired posterior identity ( Hand2 :EGFP) in the blastema. We then performed the assay while injecting test axolotls intraperitoneally at the conical blastema stage with a single dose of BMS-833923 (a Shh pathway inhibitor that more potently blocks Shh signalling in axolotls than the commonly used cyclopamine 37 ). Our data show that BMS-833923 completely prevented transplanted anterior cells from acquiring posterior identity ( Hand2 :EGFP) ( Fig.…”

Rebuilding limbs, one cell at a time

“…Surprisingly, recent work suggests that salamander limb development may deviate from classically studied mouse and chick limbs. 92,100 Efforts have already been made to compare limb development in mouse and chick using scRNA-seq data 101 and undoubtedly more of these important comparisons are underway. This suggests the potential for how a limb develops may influence its future regenerative capacity.…”

“…Most of the work comparing limb development to limb regeneration has been performed under the premise that limb development is a highly conserved process across species. Surprisingly, recent work suggests that salamander limb development may deviate from classically studied mouse and chick limbs 92,100 . Efforts have already been made to compare limb development in mouse and chick using scRNA‐seq data 101 and undoubtedly more of these important comparisons are underway.…”