Transcriptomics of digit loss in salamander development and evolution

Student: Danielle Beier* and Samantha Balestrieri
Mentor: Geffrey Stopper
Major: Biology

Tetrapods typically have four or five digits on their forelimbs and five digits on their hindlimbs.  Amphibians have forelimbs typically reduced to four digits, but retain five digits on their hindlimbs.  Amphibian forelimbs represent an example of evolutionary reduction in digit number.  It has been hypothesized that instances of evolved digit reduction are due to evolutionary changes in the Sonic hedgehog (Shh) signaling pathway.  Shh is a diffusible morphogen that is expressed in the posterior of tetrapod limbs and patterns the anterior-posterior axis of the limb; limbs with no Shh develop no more than one digit.  Blocking Shh function with cyclopamine during forelimb development in salamanders can result in the loss of one digit.  Here we block Shh function in salamander forelimb and hindlimb development to reduce forelimbs from 4 digits to 3 digits, and to reduce hindlimbs from 5 digits to 4 digits.  We follow this with transcriptomic analysis of RNA expression levels in normal forelimbs and hindlimbs and limbs with a digit removed experimentally through blocking Shh. Expression changes that are similar between experimental and evolved digit reductions can support the hypothesis that evolved digit reductions are due to reductions in Shh signaling pathway function.  Salamanders with normal forelimbs, normal hindlimbs, forelimbs reduced to 3 digits, and hindlimbs reduced to 4 digits have been successfully developed for morphological analysis, and larvae exposed at the same stages were preserved in RNAlater for transcriptomic analysis.  Blocking hedgehog signaling at particular stages of limb development predictably produces digit numbers deviating from a full complement, suggesting simple evolution of hedgehog regulation could produce reduced digit phenotypes. Generation of transcriptomic data is in progress.

*Honors Senior