Figure 6.

PCA results based on semilandmarks representing the associated shape of the limb and the gene expression domains. Solid lines depict the ontogenetic trajectories, whereas the shapes associated with negative and positive extremes of PC1 describe the start and ending points of the ontogenetic trajectory. a) Limb and Hoxa11 high expression domain. PC1 and PC2, which accounted for more than 83% of morphological variation, describe a complex ontogenetic trajectory with an inflexion point occurring around E11. The PC1 axis mainly expressed allometric shape changes through time (with 85.3% of shape variation explained by limb centroid size); whereas PC2 axis reflected changes in limb asymmetry. The ontogenetic trajectory starts with the first developmental stage (E10:11–E10:22) occupying the negative extreme of PC1 and exhibiting a wide range of variation both on PC1 and PC2. The shape associated with the most negative values of PC1 and the most positive values of PC2 shows that the earliest limbs are fairly symmetrical and present a posteriorly located Hoxa11 gene domain. During the second half of embryonic day 10 an incipient asymmetry is established. Over time, the limb bud grows, elongates and achieves a maximum asymmetry at the first half of day 11. Afterward, the second (E10:23–E11:10) and third (E11:11–E11:22) developmental stages considerably overlap at average values of PC1, showing that during embryonic day 11 shape remains fairly constant. From the second half of day 11, the limb bud keeps growing and reestablishing the symmetry. Finally, the ontogenetic trajectory finishes at the first half of day 12 with another period of morphological differentiation occurring at embryonic day 12 (E11:23–E12:10), in which limbs show dissimilar shapes, with the first appearance of digits and a proximally located Hoxa11 domain, and not overlapping with limbs from previous stages and occupying the positive extreme of PC1. b) Limb and Hoxa11 moderate expression domain. PC1 accounted for 69.5% of total morphological variation and was highly and significantly associated with limb growth and allometric shape changes. No other PCs separated any other known groups and the associated shape changes are similar to those earlier described for Hoxa11 high expression. c) Limb and Hoxa13 high expression domain. Results show a simple ontogenetic trajectory starting with an asymmetric limb and a large distal posteriorly located Hoxa13 domain. While the limb grows and changes shape, the Hoxa13 domain expands toward the anterior region following the limb contour. d) Limb and Hoxa13 moderate expression domain. A similar shape pattern was displayed by the moderate expression domain of Hoxa13. 189 × 189 mm (300 × 300 DPI).