KURATANI, S*; NAGASHIMA, H; KURAKU, S; USUDA, R; CDB RIKEN, Kobe, Japan: Nature and function of the carapacial ridge in the turtle embryo

The turtle carapace is an evolutionary novelty acquired through the modification of rib patterning. In the turtle embryo, the lateral margin of carapacial anlage is seen as the carapacial ridge (CR), which has been thought to play an essential role in the patterning of rib growth. To reveal the developmental origin and function of CR, we picked up the Chinese soft-shelled turtle, Pelodiscus sinensis, and performed cell labeling, and surgical experiments on the CR precursor. We found that the CR mesenchyme is derived from the lateralmost part of the dermomyotome, and the CR arises as the lateralmost axial structure just medial to the Wolffian ridge. Thus the CR does not belong to the laterall body wall. Removal of CR did not alter the rib growth, only inhibiting the turtle-specific fan-shaped pattern of the ribs. To identify the molecular mechanisms to differentiate CR, we performed a comprehensive screening of genes expressed specifically in the CR, using microbead-based differential cDNA analysis and real-time RT-PCR. We identified the turtle orthologues of Sp5, CRABP-I, APCDD1 and LEF-1 to be expressed in the CR with high intensity. Although these genes have been maintained in genomes throughout the major vertebrate lineages, they appeared to have acquired novel expressions in the CR in the turtle lineage. In relation to the CR-specific expression of LEF-1, we detected the nuclear localization of beta-catenin protein in the CR epidermis, suggesting that the canonical Wnt signalling is involved in carapace evolution. Overexpression of dominant-negative form of LEF-1 protein in the CR resulted in the local inhibition of CR. We conclude that the acquisition of the shell did not necessarily involve the creation of novel genes, but may have been based on the co-option of a pre-existing developmental module.