Phylogenetic analysis Alignments were obtained with ClustalW from 28 full length Runt amino acid sequences [54]. Ambiguously aligned proportions were omitted using Gblocks ver. 0.91b [55] with following parameters: minimum number of sequences for a conserved/ flanking position (15/15), maximum number of contiguous nonconserved positions (8), minimum length of a block (5), allowed gap positions (all). The phylogenetic analysis was performed using MrBayes 3.1.5 [56], employing JTT+G+I as substitution model and running eight chains for 1.000.000 generations. Trees were sampled every 1000 generations and according to a saturation curve of likelihood values the first 500 trees were discarded as burn-in. Analysis was performed with Runt sequences from O. dioica (AAS21356.1), C. intestinalis (ci0100131551, ci010013155, ciad013o19, cinc013i02 and cies003n20), B. lanceolatum and B. floridae (AAN08567.1, AAN08565.1), M. glutinosa (DQ990008, DQ990009), S. canicula (DQ990010, DQ990012, DQ990014), D. rerio (NP_571678.1, AAS02047.1, AAQ88389.1, AAO85550.1). T. rubripes (BAF36011.1, BAF36001.1, AB280005.1, NP_001092121), G. aculeatus (Ensemble Gene Id: ENSGACP00000020145, EN- SGACG00000012322, ENSGACG00000011721, EN- SGACG00000007301), M. musculus (EDL03777.1, BAA03485.1, EDL29993.1) and H. sapiens (NP_001001890.1, EAX04278.1, NP_004341.1), while using the sea urchin Runt genes from S. purpuratus (U41512.2, XM_776533.1) as an outgroup. In situ hybridizations Whole mount in situ hybridizations with lancelet larvae were performed as previously described [43]. Radioactive in situ hybridizations on paraffin embedded tissue sections were performed as reported in [57] with the exception of using lower hybridization and washing temperatures of 50uC, and using 0,26 SSC instead of 26SSC for washing of B. lanceolatum tissue sections. Non-radioactive in situ hybridization on cryo-sections of B. lanceolatum was carried out using the GenePaint System [58]. Probes for MmIhh and ScRunx3 were used as hybridization controls for B. lanceolatum. Expression profiling of Runt genes in M. glutinosa, S. canicula and B. lanceolatum by qRT-PCR QRT-PCR was performed on an ABIPrism 7900HT Cycler (Applied Biosystems, Forster City, USA) using SYBR Green PCR Master Mix (Applied Biosystems). TaqMan Reverse Transcription Reagents (Applied Biosystems) were used to synthesize the cDNA and primers were generated using the Primer Express software (Applied Biosystems). Quantification was performed using the standard curve method with dilutions of plasmids containing the sequence to be amplified in a known copy number as a standard. For the analysis of SoxE expression by qRT-PCR first a SoxE cDNA fragment was amplified by employing primers which were designed according to a SoxE sequence of B. floridae. Expression of target genes was normalized using 18S rRNA as reference. Immunohistology For immunohistochemistry on paraffin sections citrate antigen retrieval was performed. Anti-human Ihh antibody (Santa Cruz) was applied 1:50 over night. Secondary antibody (biotinylated anti- goat, Sigma-Aldrich) was applied 1:500 for one hour. Subsequent staining was performed with the Vectastain ABC kit from Vector laboratories according to the manufacturers9 instructions. EMSA Electrophoretic mobility shift assays for putative binding sites were performed as described in [59] with nuclear extracts from Runt Gene Evolution chicken DF-1 cells infected with a RCAS-virus expressing the Runt cDNA from B. lanceolatum. Specific binding was confirmed with a labeled oligo containing the putative binding site and using either wild type oligos or oligos with mutated binding sites as competitors. Luciferase reporter assays PCR amplified fragments of the B. floridae Hh promoter (AC150424) were cloned into the pGL3-basic reporter vector. NIH3T3 cells were transfected in 24-well plates with the reporter constructs (250 ng per well) together with an expression vector containing either the cDNA for BlRunt or MmRunx2 or an empty vector as control (100 ng per well). 5 ng per well of pRL-CMV were co-transfected for normalization. Cells were lysed with 100 ml passive lysis buffer (Dual Luciferase Assay Kit; Promega, Madison, USA). 5 ml of the lysate were measured using the Dual- Glo Luciferase Assay Kit (Promega) with 25 ml of the assay reagents each. Measurements were performed on a 1450 MicroBeta Scintillation and Luminescence Counter (Perkin Elmer, Waltham, USA). The result of a representative experiment is shown which was confirmed five times independently. Data deposition The sequences reported in this paper have been deposited in the GenBank databases. Dogfish: MASNS-like-promoter variant 1, ScRunx1 Acc-Nr DQ990011, ScRunx2 DQ990013, ScRunx3 DQ990015 and MRIPV-like-motifs promoter variant 2, ScRunx1 DQ990010, ScRunx2 DQ990012, ScRunx3 DQ990014. Hagfish: MgRunxA DQ990008, MgRunxB DQ990009. Lancelet: SoxE EF051347. Supporting Information Figure S1 Alignment used for Phylogenetic Analysis. Alignment (ClustalW, BioEdit: http://www.mbio.ncsu.edu/BioEdit/bioedit. html) of newly detected Runt genes in hagfish (MgRunxA and B, DQ990008, DQ990009) and dogfish (ScRunx1-3, DQ990010, DQ990012, DQ990014) with other deuterostome Runt genes. The conserved sequence blocks used for the phylogenetic analysis are underlined with #. Parameters used with Gblocks 0.91b were: Minimum number of sequences for a conserved / flanking position: 15/15; Maximum number of contiguous nonconserved positions: 8; minimum length of a block: 5; allowed gap positions: all. 338 (52%) of the original 645 alignment positions were used in the phylo- genetic analysis. Abbreviations: B.l.: Branchiostoma lanceolatum; C.i.: Ciona intestinalis; D.r.: Danio rerio; G.a.: Gasterosteus aculeatus; H.s.: Homo sapiens; M.m.: Mus musculus; M.g.: Myxine glutinosa; O.d.: Oikopleura dioica; S.p.: Strongylocentrotus purpuratus; S.c.: Scyliorhinus canicula; T.r.: Takifugu rubripes. Found at: doi:10.1371/journal.pgen.1000025.s001 (0.09 MB DOC) Figure S2 Synteny Analysis. A search for cross-species con- served gene orders was performed as previously described [1]. We compared a larger contig of the FrRunt locus (Ensemble: Scaffold 39) than previously analyzed (Ensemble: Scaffold 835[2]) to the zebrafish genome and detected a synteny region between the 39 genomic region of the FrRunt gene and chromosome 1 of zebrafish comprising Fstl1 and Gja5 (A). Furthermore we detected in the stickleback (G. aculeatus) genome a FrRunt orthologous gene with a genomic environment almost identical to the FrRunt gene locus (B). The gene orthologous to Clic 5 located 39 of Runx2a in the zebrafish genome was found by Blast searches on group 1 in the stickleback genome. Together these results suggest that a translocation between a region of the 39 end of the FrRunt locus PLoS Genetics | www.plosgenetics.org 11 March 2008 | Volume 4 | Issue 3 | e1000025