Timing of positional information and self-organization in digit patterning. (A) In the chick wing, graded paracrine Shh signalling (numbers shaded blue) from the polarizing region (green) promotes antero-posterior expansion (red) and specifies cells with three positional values (PV) 1, 2 and 3. Cells specified with anterior positional values are promoted to more-posterior values every 4 h and give rise to three digits (d) by self-organization (SO). In parallel, non-graded autocrine Shh signalling (black numbers) specifies polarizing region cells with a positional value, which then fail to form a digit (X). In all cases, colours on digits indicate a different positional value with which cells were specified, which are interpreted into phalange number (metacarpals are shaded grey and limb buds not drawn to scale). (B) Chick wing treated with cyclopamine (Cyc) at HH20/21. Specification is stopped when cells have the positional value of a digit 1 and 2, then posteriorly extended AER (orange) promotes antero-posterior expansion to enlarge the field of cells specified with the positional value of a digit 2 (enlarged 2) to give rise to two digits by self-organization, note fused metacarpals. In addition, polarizing region cells specified with a digit 2 positional value give rise to a digit (green number). Note, 1-2-2-3 pattern (Fig. 2E) could be explained by asynchronous promotion by paracrine and autocrine Shh signalling (Towers et al., 2011). (C) In the mouse limb, patterning is as in the cyclopamine-treated chick wing (B), but the AER extends further and allows polarizing region cells specified with a digit 2 positional value (enlarged 2) to form two digits. Shh has a later role in promoting antero-posterior expansion to allow digits 2-5 to form. (D) In the chick leg, patterning is as in the wing (A), but autocrine Shh signalling continues to promote polarizing region cells with more-posterior positional values, which then produce a digit (Towers et al., 2011). (E) In the ancestral limb, patterning is as in chick leg (D), but most-posterior polarizing region cells specified with a digit 2 positional value could become refractory to Shh signalling at an early stage, as in the mouse limb (Ahn and Joyner, 2004).