morphology and Gdf5/6/7 expression we previously proposed an evolutionary relationship between gnathostome joint tissue and lamprey mucocartilage in the ventral pharynx[72]. Our current results showing that mucocartilage in the ventral pharynx does not express Runx and Barx lend support this hypothesis. We speculate that gnathostome joints may have evolved by the redeployment of a primitive mucocartilage-like skeletal tissue to the regions between rigid cellular cartilage condensations (Figure 8D). Materials and Methods Ethics Statement All methods were reviewed and approved by the University of Colorado, Boulder IACUC protocol 08-07-MED-02. We performed an exhaustive search of the 5.9X coverage, preassembly Petromyzon marinus genome for homologs of gnathos- tome Runx, Barx and Alx genes by repeated BLAST[87] searching with gnathostome, amphioxus, and sea urchin protein sequences. We then designed exact-match PCR primers for the two Runx paralogs (RunxA and RunxB), and the single Barx and Alx genes. Runx and Barx exons were PCR amplified from adult lamprey genomic DNA according to standard methods. Alx was isolated from embryonic cDNA using the GeneRacer kit (Invitrogen). A SoxE1 fragment corresponding to nucleotides 211-619 of the published SoxE1 nucleotide sequence[47] was amplified from embryonic cDNA for use as a riboprobe template. Embryos and early larvae for in situ hybridization were obtained from adult spawning phase sea lampreys (Petromyzon marinus) as previously described[88]. 5–10 cm pre-metamorphic ammocoete larvae, and newly metamorphosed juvenile lamprey were collected from streams feeding Lake Huron in the Fall with a backpack electroshocker and kept in chilled holding tanks until fixed in MEMFA (MOPS buffer, EGTA, MgSO4, Formaldehyde). Thick cross sections of 100–200 microns were then cut through the pharynx using a vibratome. In situ hybridization on embryos, larvae, and vibratome sections of juveniles, was performed with 300–500 bp riboprobes against coding regions and/or 59 untranslated regions using a high- stringency hybridization protocol [49,89]. Key parameters of this protocol include post-hybridization washes at 70uC, and the use of a low salt, low pH hybridization buffer (50% formamide; 1.3X SSC, pH 5.0; 5 mM EDTA, pH 8.0; 50 mg/ml tRNA; 0.2% Tween-20; 0.5% CHAPS; 100 mg/ml heparin). In situ-hybridized embryos were then cryostat sectioned and counterstained using Nuclear Fast Red (Vector Laboratories, Burlingame, CA). Alcian blue staining was as previously described [37] with the addition of a 2 hour bleaching step in 1%H2O2, 5% formaminde, and.5% SSC on a fluorescent light box. References 1. Gans C, Northcutt RG (1983) Neural Crest and the Origin of Vertebrates - a New Head. Science 220: 268–274. 2. 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Meulemans D, Bronner-Fraser, M (2007) Insights from Amphioxus into the Evolution of Vertebrate Cartilage. PLoS ONE 2: e787. Vertebrate Cartilage Evolution Supporting Information Figure S1 Phylogenetic analysis of lamprey Runx and Alx genes. Lamprey RunxA and RunxB group with deuterostome Runx/Runt homologs with high confidence values using either the Neighbor-Joining (A) or Maximum Likelihood (B) methods. Lamprey Alx groups with deuterostome Alx/Cart homologs with high confidence values using either the Neighbor-Joining (C) or Maximum Likelihood (D) methods. The related homeobox gene Rx (retinal homeobox) from Drosophila melanogaster serves as an outgroup. Gene names are prefixed with the initials of their respective species names. Bf, Branchiostoma floridae, Pm, Petromyzon marinus, Mm, Mus Musculus, Sc, Scyliorhinus canicula, Dr, Danio rerio, Dm, Drosophila melanogaster, Sp, Strongylocentrotus purpuratus, Pl, Paracentrotus lividus, Ce, Caenorhabditis elegans, Mg, Myxine glutinosa. (TIF) Figure S2 Clustal alignments of Runx (A) and Alx (B) genes used to generate the trees in Figure S1. Only the sequences spanning the highly conserved DNA binding domains are shown for each alignment. (TIF) Figure S3 Expression of RunxA, RunxB, Barx, and Alx in larvae at 35 days post-fertilization. Expression patterns are essentially the same as those seen earlier at st. 30. Side view (A) and ventral view (B) of RunxA mRNA distribution showing weak expression in pharyngeal endoderm. Side view (C) and ventral view (D) of RunxB mRNA distribution showing weak expression in pharyngeal endoderm. Side view (E) and ventral view (F) of Barx mRNA distribution showing persistent expression in the medial aspect of the posterior pharyngeal arches (arrowhead) and the ventral portion of the first pharyngeal arch (arrow). Side view (G) and ventral view (H) of Alx expression in the dorsal and ventral aspects of the branchial basket (asterisks), dorsal fin (arrowhead), and dorsal first arch (arrow). (TIF) Acknowledgments We thank Alex Marsh, Charles Ventriglia, and Jacob Doherty for excellent technical assistance, Roger Bergstedt, Deborah Winkler, Nikolas Rewald, and Kathy Jones at the Hammond Bay biological station for generously supplying adult, larval, and juvenile lampreys. We also thank David Jandzik and two anonymous reviewers for their insightful comments. Author Contributions Conceived and designed the experiments: DMM. Performed the experiments: MC SL RC DMM. Analyzed the data: RC DMM. Wrote the paper: DMM. 8. Yan YL, Miller CT, Nissen RM, Singer A, Liu D, et al. (2002) A zebrafish sox9 gene required for cartilage morphogenesis. Development 129: 5065–5079. 9. 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Biochemical and Biophysical Research Communications 345: 495–501. PLoS ONE | www.plosone.org 11 July 2011 | Volume 6 | Issue 7 | e22474