Figure 6.

Transgenic Analysis of Embryos Carrying Mutant ZRS Sequences

(A) The sequences of the wild-type Hoxsites 1–3 and the mutated sequences (designated MutHoxsite) that were used in the transgenic constructs.

(B–E) Limb buds from transgenic embryos (E11.5) carrying the following ZRS sequences driving LacZ expression: the wild-type ZRS sequences (B); MutHoxsite 1 (C), MutHoxsite 2 (D), and MutHoxsite 3 (E). (C)–(E) show no effect on expression of mutating single Hoxsites.

(F–H) Mutating combinations of sites results in lower LacZ expression: MutHoxsite2+3 (F), MutHoxsite 1+3 (G), and MutHoxsite 1,2+3 (H).

(I) The low level of expression in MutHoxsite 1,2+3 is reproduced in the WMSΔ110 construct.

(J–L) Addition of the Cu point mutation (J) or deletion of WMSΔ5 (K) results in distal and ectopic anterior expression, whereas deletion of WMSΔ20 (L) returns expression to wild-type levels.

(M–O) The Cu mutation in combination with mutant Hox sites: MutHoxsite 3+Cu (M), MutHoxsite 2+Cu (N), and MutHoxsite 2+3+Cu (O).

(P) Graphical representation of the LacZ expression patterns resulting from mutations within the ZRS. The width of the expression domain was divided by the width of the limb and expressed as a percentage. One spot represents the extent of reporter gene (LacZ) expression for each individual limb from a set of transient transgenic embryos. Data were subjected to one-way ANOVA and Tukey HSD test, and those that differ significantly from the wild-type are indicted (^∗p ≤ 0.05, ^∗∗∗p ≤ 0.001, ^∗∗∗∗p ≤ 0.0001).

Scale bars, 100 μm.