We report the region-specific expression of a novel gene, named mario, whose expression domain is in the distal tip of the presumptive and developing digit 2 region in the developing chick wing bud. The anterior region-specific expression of mario corresponds well with the presence of digit 2, and fate map analysis showed that mario expression at early stages represents the presumptive digit 2 region. Using mario expression as a region-specific marker for the digit 2 region, several surgical operations were performed to obtain insights into digit 2 development in the chick wing. Cell fate tracing concomitant with a zone of polarizing activity (ZPA) implantation revealed that an additional digit 2 in the ZPA implantation into the anterior or middle region of wing bud is derived from the original digit 2 region (mario-positive region). Surgical manipulations revealed that the anterior nondigit region has an inhibitory effect on digit 2 formation. Taken together, these results suggest that the most-anterior region, including the anterior necrotic zone, restricts the position of digit 2 region by limiting the anterior border of the digit 2 region and preventing its expansion. Developmental Dynamics 233:326–336, 2005. © 2005 Wiley-Liss, Inc.