Fig. 2. ROSA reporter analyses demonstrated that Cre-mediated recombination occurred in the limb ectoderm prior to hindlimb bud formation, but after the forelimb bud had begun to form. Cre-mediated rearrangement of the ROSA reporter results in activation of lacZ expression and elucidates the temporal and spatial domain of ectopic Brn4-Cre gene expression in the embryonic limb. (A) This panel depicts a 9.75 dpc embryo that is doubly transgenic for the ROSA reporter and the Brn4-Cre transgene. The initial expression of the ROSA reporter was detected in ventrolateral ectoderm at a time in which the forelimb bud had begun to form (white arrowhead), but before initial hindlimb bud formation, which occurred several hours later. Arrows designate major axes: D, dorsal; V, ventral; R, rostral; C, caudal. (B) Vibratome section (100 µm) of a 10.5 dpc hindlimb demonstrated that Cre-mediated lacZ expression was restricted to the ectoderm of the limb. Cre-mediated lacZ expression was highest in the AER, although significant levels of expression were found in the ventral ectoderm. Limited induction of expression occurred in the dorsal limb ectoderm. Arrows designate major axes: D, dorsal; V, ventral; Di, distal; P, proximal (C-E). These panels demonstrate Cre-mediated expression of lacZ from the earliest stages of hindlimb formation in embryos sacrificed at 10.0 dpc. As the hindlimb bud grows, expression becomes particularly high in the pre-AER region at the distal tip of the limb. The hindlimbs depicted represent the typical variability in age of embryos sacrificed at 10.0 dpc; hindlimbs were ordered progressively based upon limb bud size. (F) By 12.0 dpc, the hindlimb expresses the activated lacZ throughout much of the dorsal ectoderm, as well as the ventral ectoderm and AER. (G) Both the extent and timing of ROSA reporter activation are different in the forelimb. At 10.25 dpc, the forelimb attains a more advanced limb bud stage than the hindlimb, but the degree of ROSA reporter activation is reduced when compared with earlier limb bud stages of the hindlimb (compare with E). (H) At 12.0 dpc, the degree of ROSA reporter is reduced compared to the hindlimb (compare with F). However, there is considerable induction of the ROSA reporter gene in the AER at this stage of embryogenesis.
