Fig. 1: Description of the CALM assay.

(Left boxes) Nerve deviations (ND) are performed by surgically deviating the branchial nerve bundle (yellow) to a lateral full thickness skin wound on the limb, which leads to the formation of a blastema (Endo et al.26). In the case of an anterior positioned ND (ND-A), within 7-days of surgery, there is a significant increase in the expression of “anterior” limb patterning genes, such as Alx4. Posterior positioned NDs (ND-P), exhibit a significant increase in the expression of “posterior” limb patterning genes, including Shh (Vieira et al.20). In both ND-A and ND-Ps, the blastema eventually regresses. (Right boxes) The Competency Accessory Limb Model (CALM) utilizes retinoic acid treatment to assess whether a ND-A or ND-P wound site has achieved competency to pattern limb structures. The achievement of patterning competency of the ND wound tissue can be identified in two ways; (1) location-specific changes in A/P patterning gene expression and (2) (in anterior-located tissue only) the formation of ectopic limb patterns (Vieira et al.20).