Figure 4: Retinoic acid influences the chick wing-polarizing region timer.
From: Sonic hedgehog-expressing cells in the developing limb measure time by an intrinsic cell cycle clock
(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; Fig. 2e and Supplementary Fig. 5). (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; Fig. 2u). (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 initiation10,11 and is suggested to specify proximal structures (that is, humerus)7,8 and be required for Shh transcription in the polarizing region (oval, l)9,17. Shh then contributes to clearing retinoic acid from the early bud28 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. 21) while autocrine Shh signalling specifies digit 4 in the chick leg21 (also digit 5 in the mouse limb18, 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).
