Evolution of the avian digital pattern

Abstract

Variation in digit number has occurred multiple times in the history of archosaur evolution. The five digits of dinosaur limbs were reduced to three in bird forelimbs, and were further reduced in the vestigial forelimbs of the emu. Regulation of digit number has been investigated previously by examining genes involved in anterior-posterior patterning in forelimb buds among emu (Dromaius novaehollandiae), chicken (Gallus gallus) and zebra finch (Taeniopygia guttata). It was described that the expression of posterior genes are conserved among these three birds, whereas expression of anterior genes Gli3 and Alx4 varied significantly. Here we re-examined the expression pattern of Gli3 and Alx4 in the forelimb of emu, chicken and zebra finch. We found that Gli3 is expressed in the anterior region, although its range varied among species, and that the expression pattern of Alx4 in forelimb buds is broadly conserved in a stage-specific manner. We also found that the dynamic expression pattern of the BMP antagonist Gremlin1 (Grem1) in limb buds, which is critical for autopodial expansion, was consistent with the digital pattern of emu, chicken and zebra finch. Furthermore, in emu, variation among individuals was observed in the width of Grem1 expression in forelimb buds, as well as in the adult skeletal pattern. Our results support the view that the signalling system that regulates the dynamic expression of Grem1 in the limb bud contributes substantially to variations in avian digital patterns.

Figure 1

Expression patterns of Gli3 and Alx4 in limb buds of emu, chicken and zebra finch embryos. The distal domain of Gli3 expression is posteriorly extended in limb buds of emu, chicken and zebra finch (a, n = 5; d, n = 9; g, n = 8), although it is most extensively expressed in the emu forelimb buds. Alx4 show a similar anterior expression in limb buds of all species (b, j, n = 5; e, l, n = 17; h, n, n = 3) at stage 25. Additional posterior expression of Alx4 is detected in both fore- and hindlimb buds of emu, chicken and zebra finch embryos at stage 26 (c, k, n = 6; f, m, n = 13; i, o, n = 7). The shapes of the limb bud are similar, but not exactly identical among species at the same stage^,,. c, d, j, k, Left limb buds flipped horizontally.

Figure 2

Expression patterns of Grem1 in limb buds is consistent with the resulting skeletal pattern. (a−g) Distal Grem1 expression in forelimb and hindlimb buds of stage 25 emu, chicken and zebra finch embryos is correlated with the digital skeletal patterns shown on the left (a, b, e n = 9; c, f, n = 10; d, g, n = 3). a, b Left limb buds flipped horizontally. Note that size of the distal Grem1-positive area (bracket) varies among emu embryos at the same stage.

Figure 3

Digital pattern of the forelimb of emu adults and embryos. (a–c) Three-dimensional rendering from CT images of the digital plate of adult emu forelimb skeletons (left; ventral view) and their transverse sections taken from the limb at the level of the dashed line (right). Arrowheads indicate the vestigial digit 4 (4*). Medullar cavities are seen in the vestigial digit 4. (c) A right limb flipped horizontally. Scale bars, 20 mm (left) and 10 mm (right). (d–f) Alcian blue staining of wings of emu embryos at stages 36 (d), 37 (e) and 40 (f). Note that individual variation was recognised in the cartilage pattern of developing forelimbs (stage 36, n = 4; stage 37, n = 3; stage 40, n = 3). Left wings flipped horizontally. 2−4, digits 2−4; 2*, 4*, rudiment of digit 2 and 4, respectively. Scale bars, 2 mm.