© 2006 Nature Publishing Group Evidence that mechanisms of fin development evolved in the midline of early vertebrates Renata Freitas 1 , GuangJun Zhang 1 & Martin J. Cohn 1,2 The origin of paired appendages was a major evolutionary innovation for vertebrates, marking the first step towards fin- (and later limb-) driven locomotion. The earliest vertebrate fossils lack paired fins but have well-developed median fins 1,2 , suggesting that the mechanisms of fin development were assembled first in the midline. Here we show that shark median fin development involves the same genetic programs that operate in paired appen- dages. Using molecular markers for different cell types, we show that median fins arise predominantly from somitic (paraxial) mesoderm, whereas paired appendages develop from lateral plate mesoderm. Expression of Hoxd and Tbx18 genes, which specify paired limb positions 3,4 , also delineates the positions of median fins. Proximodistal development of median fins occurs beneath an apical ectodermal ridge, the structure that controls outgrowth of paired appendages 5–7 . Each median fin bud then acquires an anteroposteriorly-nested pattern of Hoxd expression similar to that which establishes skeletal polarity in limbs 8,9 . Thus, despite their different embryonic origins, paired and median fins utilize a common suite of developmental mechanisms. We extended our analysis to lampreys, which diverged from the lineage leading to gnathostomes before the origin of paired appendages 2,10 , and show that their median fins also develop from somites and express orthologous Hox and Tbx genes. Together these results suggest that the molecular mechanisms for fin develop- ment originated in somitic mesoderm of early vertebrates, and that the origin of paired appendages was associated with re-deployment of these mechanisms to lateral plate mesoderm. Outgrowth of paired fins and limbs is maintained by the apical ectodermal ridge (AER) at the distal margin of the buds 5,6 , and members of the Fgf family synergistically mediate its signalling activity 7,11 . In catsharks, median fins develop from a continuous finfold extending along the dorsal and ventral midlines (Supplemen- tary Fig. 1). Outgrowth of the median finfold occurs beneath an AER-like structure that produces Fgf8 and Dlx proteins (Supplemen- tary Fig. 2). The AER then becomes an apical ectodermal fold (AEF), as in the paired fins of teleosts 6 . The similar embryology of median and paired fins raised the possibility that a common set of mecha- nisms regulates their development, but their anatomical positions suggested distinctive embryonic origins. Transplantation experi- ments in amphibians have led to the idea that median finfolds are neural crest derived, and the zebrafish caudal fin was shown to originate, at least in part, from trunk neural crest 12,13 . Recent fate- mapping studies, however, demonstrated that somitic mesoderm contributes to amphibian median finfold development 14 . We there- fore set out to determine the embryonic origin of catshark median fins. Studies in several model systems have shown that Foxc2 and Zic1 are expressed in the sclerotome, and that they remain in these cells as they migrate dorsally around the neural tube to form neural arches and spinous processes 15,16 . Neither of these genes is expressed by migratory trunk neural crest or differentiated myotome 15,16 , making them suitable for distinguishing sclerotomal cells during median fin development. We cloned and examined the expression of catshark Foxc2 and Zic1 and found that, as in tetrapods, both are expressed throughout the sclerotome (Fig. 1a, b). During median fin develop- ment, their expression domains extend dorsally and ventrally into the median finfolds (Fig. 1a, b; Supplementary Figs 3a, b and 4a, b). We also examined Scleraxis (sclerotome-related helix-loop-helix type transcription factor), a marker of the sclerotomal sub-compartment (syndetome) that forms axial tendons in chick and mouse 17,18 . Catshark Scleraxis marks a similar subset of the sclerotome and, like Foxc2 and Zic1, its expression domain extends into the median finfolds (Fig. 1c; Supplementary Figs 3c and 4c). Strong expression of all three sclerotomal markers persisted during differentiation of the fin radials and neural arches. To determine whether cells from the dermomyotome and neural crest also participate in median fin development, we examined Pax7, a marker of these cell types in other vertebrate embryos 19 . Pax7 was initially expressed in the catshark dermomyotome and dorsal neural tube, but Pax7-expressing cells were not detected in the median fin before stage 31 (Fig. 1d; Supplementary Figs 3d and 4d). Pax7 expression then extended from the dermomyotome into the median fins, in the muscle projections lateral to the developing skeleton (Fig. 1d; Supplementary Fig. 3d). Immunolocalization of Zn12, a neural crest marker 20 , revealed that a limited number of neural crest cells also invaded these fins, but most of the mesenchyme was negative for this marker (Fig. 1e; Supplementary Fig. 3e). By stage 31, Zn12 had localized predominantly to the space within the AEF, where dermal rays develop, and subjacent to the distal ectoderm (Fig. 1e; Supplementary Fig. 3e). Together our results suggest that the bulk of the median fin mesenchyme is derived from sclerotome, although cells from dermomyotome and neural crest also contribute to median fin development (Fig. 1f; Supplementary Fig. 3f). If technical challenges can be overcome, cell labelling in shark embryos will further address the contributions of these cell types. During limb development, lateral plate mesoderm is regionalized into limb-forming and non-limb-forming domains by differential expression of Hox and Tbx genes 3,4 . We investigated whether ante- roposterior regionalization of the median finfold into dorsal, anal and caudal regions involves similar mechanisms. In catsharks, median fins lie posterior to the cloaca, suggesting that, if Hox genes are involved in their development, then the most likely candidates would be AbdB-related Hox9–Hox13 genes. Therefore, we cloned 5 0 Hoxd genes from catsharks and examined their expression during median fin development (Fig. 2 and Supplemen- tary Figs 5 and 6). Prior to the extension of sclerotome towards the dorsal and ventral finfolds, we observed collinear expression of Hoxd9, Hoxd10, Hoxd12 and Hoxd13 in the somitic mesoderm (Supplementary Fig. 6). The Hoxd9 domain extended anterior to the cloaca, marking the region in which median fin outgrowth was LETTERS 1 Department of Zoology, 2 Department of Anatomy and Cell Biology, University of Florida, PO Box 118525, Gainesville, Florida 32611, USA. Vol 442|31 August 2006|doi:10.1038/nature04984 1033