lamina have been formed in the presence of an inductive endoderm as one part of the internal visceral skeleton. This would be remarkably different to the development of external denticles, which are only under the influence of an inductive ectoderm [39]. Our Runt expression pattern supports the classical view that teeth and placoid scales have a common evolutionary origin, at least on the level of the molecular pathway underlying their development. In other words, the hypothesis that teeth and placoid scales evolved from a common developmental module, which might have been shifted and extended in its expression topology [41] is supported by the striking similarity of the Runt expression patterns in teeth and placoid scales. Conservation of molecular pathways in skeletogenesis The gut appears to be an ancient expression domain of Runt. This expression in the chordate stem species can be reconstructed as Runt genes are expressed in the gut in representatives of the outgroup (sea urchins, nematode [16,17] and the lancelet (this study, Figures 7 and 8). The Runt expression in the gill bars, structures that stabilize the gill gut, might be linked to the later role of Runt genes in the evolution of the pharyngeal skeleton. In zebrafish Runx3 was shown to promote cartilage formation via the endodermal expression of Runx3 in pharyngeal pouch cells [23]. However, in vertebrates most of the branchial arch cartilage, the cranial bone forming cells (osteoblasts), as well as the cells that deposit dentin (odontoblasts) are derived from the neural crest [42]. It was previously proposed that the neural crest acquired chondrogenic ability by recruiting proto-chondrogenic gene programs from the notochord, neural tube and gill gut [4,5,28,43–45] Strikingly, we found high Hh expression together with high Runt expression in exactly these three sites indicating that the described interaction between the Runt and Hh pathways is of relevance for chordate cartilage evolution. Whereas the homology of the gill gut in lancelets and vertebrates is well established [28] little is known about the molecular machinery necessary for development and maintenance of the skeletal-like structures of the pharyngeal gill slits in lancelets. The gill bars are stabilized by 15 nm thick filaments aligned parallel to the long axis of the rods, and are covered by a single layered epithelium, that can be morphologically distinguished into atrial, lateral and pharyngeal epithelium [46]. Gill bars gave a positive signal when stained with an antibody against type II collagen [28] indicating a cartilage-like structure, which appears to be acellular. To get deeper insights into the molecular machinery underlying the early evolution of the skeleton we analyzed Runt and SoxE gene expression in adult lancelets. Our analyses revealed that both genes were highly expressed in the gill bar region (Figure 8). Furthermore our in situ hybridization results revealed that the lancelet Runt gene is expressed in atrial, lateral, and pharyngeal epithelium of ectodermal and endodermal origin (Figure 8D and 8E), but not in the mesodermal coelomic cells of the primary gill bars. It has recently been reported that the lancelet gills contains lymphocyte-like cells most likely located between the cells of the lateral and pharyngeal epithelia [47]. We cannot resolve these cells in our in situ hybridizations and thus cannot detect if Runt is expressed in these cells of the gill bars (Figure 8D and 8E). The finding of endodermal Runt expression supports the model in which endodermal secretion was the ancestral mode of making cartilage [5]. Since in deuterostomes the endoderm is a plesiomorphic Runt expression domain, Runt is likely to be present also in the endoderm of the gill gut in hemichordates. Other crucial genes for mammalian skeletogenesis are Ihh and Shh. For Ihh a direct regulation by Runx2 has been shown and Runt Gene Evolution Runx2 influences Shh signaling in tooth development [10,11]. Furthermore, Runt and Ihh genes are coexpressed during skeletogenesis in zebrafish [23,25,48]. We observed Runt expres- sion in the midgut and foregut of lancelet larvae, similar to a recent study [49]. The exon 1 variant, however, showed additional expression in the notochord and neural tube (Figure 7A, 7C, and 7E). These expression domains were still detected in adult lancelets together with high Hh expression (Figure 8A and 8B). The observation that the single Runt and Hh genes of lancelets are co- expressed in the notochord, neural tube and in the adult lancelet gill gut (Figures 7 and 8 and reference [50]) prompted us to investigate if also the lancelet Runt protein might regulate lancelet Hh gene expression. In our Hh promoter studies the lancelet Runt protein bound directly to Runt binding sites in the lancelet Hh promoter and regulated the reporter gene driven by this promoter (Figure 9). The highest Hh expression together with Runt co- expression was found in the notochord, the neural tube, and the gill gut, all of which were previously proposed to be involved in the evolution of chordate cartilage [4,5,28,44,45]. It is thus likely, that the direct regulation of Hh by Runt was a relevant mechanism in chordate evolution. This suggests that the core gene network involved in vertebrate cartilage, bone and tooth formation was present prior to the divergence of cephalochordates and vertebrates and the duplication of the Runt and Hh genes. Further research is needed to determine if a small cell group directly adjacent to both sites of the acellular matrix, with high Runt and Hh expression (arrows in Figure 8D–8G), is of special importance for cartilage formation in lancelets. Another interest- ing aspect will be to determine if a direct regulatory interaction between the Runt and Hh pathways is also present in hemichor- dates and whether a direct interaction between Runt and Hh pathways was maintained during vertebrate evolution in other important developmental processes, such as vertebrate hemato- poiesis [29,51]. Materials and Methods Materials Lancelets (B. floridae) were collected by shovel and sieve in water of 1 m in depth in Tampa Bay, Florida and in vitro fertilization, embryo culture and fixation were performed as previously described [52]. Adult B. lanceolatum were obtained from the Biologische Anstalt Helgoland. Hagfish (M. glutinosa) were collected by S.E. Material from adult dogfish (S. canicula) was obtained from the Biologische Anstalt Helgoland and dogfish embryos from the Aquazoo (Du¨ sseldorf). Oligonucleotides All primers and oligonucleotides employed in our study are given as supporting information. Primers for dogfish sequences can be found in Table S1. Primers for hagfish sequences are given in Table S2, and primers for amphioxus are listed in Table S3. Oligonucleotides employed for EMSAs are given in Table S4. Analysis of Runt gene sequence and number Total RNA was isolated as described previously [17] from B. floridae (larvae), B.lanceolatum (adult), M. glutinosa (adult), S. canicula (embryos 4,5 cm, 6,5 cm, and 9,5 cm as well as adult animals). Runt genes were amplified by a strategy reported previously, using degenerated primers to amplify the conserved Runt domain followed by RACE PCRs to amplify the full length Runt genes [17]. The only exception was the amplification of the hagfish MgRunxB 59 end which was obtained by inverse PCR with gene specific primers [53] PLoS Genetics | www.plosgenetics.org 10 March 2008 | Volume 4 | Issue 3 | e1000025