Expression of limb initiation genes and clues to the morphological diversification of threespine stickleback

Populations of threespine stickleback (Gasterosteus aculeatus) differ widely in spine number, extent of body armour, body depth, jaw length as well as many other traits [1] and provide an opportunity to gain insight into mechanisms of morphological diversification. The phenotypic differences are heritable but the gene(s) involved have not yet been identified [2]. We focused on the pelvic girdle and associated spines (modified pelvic fins [3]) in two extreme phenotypic classes. The expression of genes involved in vertebrate limb initiation suggests that Pitx1 and/or an upstream regulator is involved in pelvic spine deficiency. The spined phenotype has a substantial pelvic girdle with well developed pelvic spines, three dorsal spines and lateral body plates (Figure 1A,B). By contrast, the spine-deficient phenotype has only two dorsal spines, no plates or pelvic spines (Figure 1C); in some fish, the pelvic girdle is absent (Figure 1D), while in others, it consists of small remnants of the anterior processes [1] (Figure 1C,E) often showing striking left/right asymmetry (left larger than right 17/19 cases; Figure 1E) (see also [4]). In spined fish, transparent pelvic fin buds first appear approximately 3 weeks after hatching at 5 mm total body length (TL) [5] (Figure 1 G,H, compare with Figure 1F); these become denser and form tiny spines pointing posteriorly (Figure 1I). Scanning EM reveals small areas of raised cells which form discrete ‘tucks’ (Figure 1J,K). By contrast, no sign of buds could be detected in spine-deficient fish, even by scanning EM (Figure 1L,M), at any stage from hatching to 12 mm TL (n = 22). Thus, lack of pelvic spines is due to a failure of fin bud initiation rather than of subsequent skeletogenesis. We examined expression of genes associated with vertebrate hindlimb/pelvic fin initiation in both phenotypes from hatching to 12 mm TL. PCR fragments of stickleback genes were obtained using primers designed against conserved regions of such genes in other species. Tbx4, which can induce ectopic hindlimbs in chick embryos [6], is first detectable in pelvic fin buds in spined fish at 5 mm TL, as in zebrafish [7] (Figure 2A–C; n = 18). By contrast, no Tbx4 transcripts could be detected in the pelvic regions of spine-deficient fish (Figure 2D; n = 11). Tbx4 is thought to be controlled by Pitx1 in mouse and chick embryos [8–10] and although Pitx1 is expressed in pelvic fin buds in spined fish (Figure 2E,F) (n = 12), no Pitx1 transcripts can be detected in the pelvic region in spine-deficient fish (Figure 2G) (n = 16). Pitx1 transcripts are abundant in both phenotypes in other regions (Figure 2E,G). The finding that Pitx1 is not expressed in the pelvic region of spinedeficient fish suggests that Tbx4 is not directly responsible for fin loss. In mice, Pitx2 compensates partially for absence of Pitx1 in hindlimb development [9], but because Pitx2 is asymmetrically expressed in early mouse embryos, right limbs in Pitx1 knockout mice have more severe defects. It is striking that the right is also more affected in stickleback pelvic reduction (this