Noncanonical Hox, Etv4, and Gli3 gene activities give insight into unique limb patterning in salamanders

Limb development in salamanders is unique among tetrapods in significant ways. Not only can salamanders regenerate lost limbs repeatedly and throughout their lives, but also the preaxial zeugopodial element and digits form before the postaxial ones and, hence, with a reversed polarity compared to all other tetrapods. Moreover, in salamanders with free-swimming larval stages, as exemplified by the axolotl (Ambystoma mexicanum), each digit buds independently, instead of undergoing a paddle stage. Here, we report gene expression patterns of Hoxa and d clusters, and other crucial transcription factors during axolotl limb development. During early phases of limb development, expression patterns are mostly similar to those reported for amniotes and frogs. Likewise, Hoxd and Shh regulatory landscapes are largely conserved. However, during late digit-budding phases, remarkable differences are present: (i) the Hoxd13 expression domain excludes developing digits I and IV, (ii) we expand upon previous observation that Hoxa11 expression, which traditionally marks the zeugopodium, extends distally into the developing digits, and (iii) Gli3 and Etv4 show prolonged expression in developing digits. Our findings identify derived patterns in the expression of key transcription factors during late phases of salamander limb development, and provide the basis for a better understanding of the unique patterning of salamander limbs.