(a–n) HH20 wing- (a) and leg-polarizing regions (h) grafted with pins to posterior and anterior of HH24 wing- and leg-polarizing regions express Shh after 32 h (arrows—b,e,i,l, n=8/8), endogenous Shh undetectable (asterisks—b,e,i,l). Wings with posterior grafts normal (c,d, n=2/2), wings with anterior grafts have an additional digit 1* (f,g, n=2/2), legs with posterior leg grafts gave rise to digit IV (j,k, n=3/3) and anterior leg grafts to digit IV* and induce a digit I* (m,n, n=2/2). (o–p) Cell cycle parameters of wing- (o) and leg-polarizing regions (p)—bars indicate s.e.m. (q–s) HH20-polarizing regions grafted in place of HH24 wing- and leg-polarizing regions (q). After 24 h, 61 and 65% of cells in grafted wing (n=10, r) and leg (n=11, s) polarizing regions, respectively in G1 compared with 79 and 76% of cells in equal numbers of contralateral limb bud-polarizing regions. In both experiments there is a significant difference in G1 numbers between host and donor polarizing regions (Pearson’s χ² test—P<0.05) consistent with graft behaving intrinsically. All scale bars, 1 mm.

(a–h) HH27 wing-polarizing region grafted to posterior and anterior of HH20 wings (a) express Shh after 32 h (arrows, b,e, n=11/13). Wings with posterior grafts normal (c,d, n=2/3—one lost digit 3), wings with anterior graft duplicated* (f,g, n=10/12). After 24 h, 62% of cells in G1 phase in grafted polarizing regions (n=10) compared with 60% in contralateral wings (h, n=10). (i–p) HH24 wing-polarizing region serially grafted (twice) to posterior and anterior of HH20 wings (i) express Shh after 32 h (arrows, j,m, n=3/3). Wings with posterior grafts normal (k,l, n=2/2), wings with anterior grafts duplicated* (n,o, n=2/3). After 24 h, 62% of cells in G1 phase in grafted polarizing regions (n=10) compared with 59% in contralateral wings (p, n=10). (q–x) HH27 wing-polarizing region grafted to posterior or anterior of HH24 wings (q), Shh not expressed after 16 h (arrows, r,u, n=15/15). Wings with posterior graft normal (s,t, n=3/3), wings with anterior graft have duplicated digit 1* (v,w, n=2/4—two not duplicated). After 24 h, 79% of cells in polarizing regions (n=12) in G1 phase compared with 78% in contralateral wing bud-polarizing regions (x, n=12). In h,p and x there is a significant difference in G1 numbers between host values and expected values for the stage of the donor polarizing region (Pearson’s χ² test—P<0.05) consistent with cell cycle parameters of the graft being reset close to host levels (see also ). Scale bars, 500 μm (b,e,j,m), 750 μm (r,u), 1 mm (c,d,f,g,k,l,n,o,s,t,v,w).

(a–c) HH27 leg-polarizing region grafted to posterior of HH20 host-polarizing region (a) give rise to digit IV (b,c, n=3/4; one case no digit from graft). (d–f) HH27 leg-polarizing region grafted to posterior of HH18/19 leg-polarizing region treated with cyclopamine (d) gives rise to digit IV (e,f, n=3/4 one case limb truncated). (g–i) HH27 leg-polarizing region grafted to anterior margin of HH21 leg treated with cyclopamine (g) give rise to digit IV *, a duplicate digit I* from host (h,i, n=2/3, one case no duplicated host digits, note extra digit IV* (i) is out of plane with the other digits and appears posterior although it is an anterior digit. (j–l) HH27 leg-polarizing regions grafted to anterior margins of HH21 wing treated with cyclopamine (j) give rise to digit IV* (k,l n=2/2). All scale bars, 1 mm.

(a,b) HH27 wing-polarizing region grafted to anterior margin of BMS-493 bead-treated HH20 wings (a) have reduced (or undetectable) Shh expression after 16 h (arrow, b, n=10/16—compare with untreated wings; and ). (c,d) HH27 wing-polarizing region grafted to anterior margin of retinoic acid (RA) bead-treated HH24 wings (c) express Shh after 16 h (arrow, d, n=3/3—compare with untreated wings; ). (e–g) HH18 wing buds treated with BMS-493 terminate Shh expression 4 h earlier (arrows, f, n=5/7) than in untreated wings (asterisks, f). After 48 h, 71% of polarizing region cells in treated wing buds (n=11) compared with 67% in untreated buds (n=11, g). (h–k) HH20 wings treated with retinoic acid beads (h) express Shh at high levels 10–12 h longer (arrows, i) than untreated wings (asterisks, i, n>5 for each time-point). After 24 h (j) 62% of polarizing region cells in G1 phase in treated wing buds (n=12) compared with 64% in untreated buds (n=12) and after 48 h (k) 62% of polarizing region cells in treated wing buds (n=12) compared with 74% in untreated buds (n=12). (l–m) Shh-expressing limb bud cells measure time by an intrinsic cell cycle clock. In early buds (l) retinoic acid promotes limb initiation and is suggested to specify proximal structures (that is, humerus) and be required for Shh transcription in the polarizing region (oval, l). Shh then contributes to clearing retinoic acid from the early bud and our data suggest that this starts an intrinsic cell cycle clock (red circle, m) that times Shh duration. Graded paracrine Shh signalling specifies digits 1, 2 and 3 (ref. ) while autocrine Shh signalling specifies digit 4 in the chick leg (also digit 5 in the mouse limb, m). The cell cycle clock enables polarizing region cells to measure time of Shh expression and thus irreversibly acquire antero-posterior positional values. For g,j and k, Pearson’s χ² tests reveal a significant difference (P-value<0.05) in G1 phase cell numbers between treated and untreated buds. Scale bars, 500 μm (b,i; left panels), 750 μm (d,f,i; right panels).