In conclusion, our results indicate that no structures homolo- gous to sympathetic ganglia arise during lamprey embryogenesis. While lamprey may possess a blueprint for the sympathetic nervous system comprised of chromaffin and SIF cells, a more detailed analysis of the molecular identity of such cells would be necessary to establish them as true phylogenetic precursors of sympathetic neurons. Thus, our data suggests that sympathetic neurons are a gnathostome innovation, and that the recruitment of Phox2, Hand and Ascl1 into a new gene battery allowed for the emergence of this new neural crest derivative. Materials and Methods DiI labeling in lamprey embryos 5 –7 day old embryos were dechorionated in 0.1xMMR and placed into agarose-coated petridishes. DiI solution (0.5 mg/ml, prepared in 0.3M sucrose) was filled into glass needles and injected into distinct neural crest population or into the entire neural tube. Embryos were analyzed for the injection into a discrete location or into the neural tube by fluorescence microscopy. Embryos were raised in petri dishes containing 0.16MMR at 18uC and the migration of DiI stained cells was analyzed every day. Once the embryos had reached the desired stage they were fixed using 4%Paraformaldehyde in PBS at room temperature for 1hr. Obtention of lamprey orthologs through 59 Rapid Amplification of cDNA ends (RLM-59 RACE) Orthologs of Phox2b, dHand and Asc1 were identified by bioinformatic survey of the lamprey genomic sequences and cloned using RACE. Total RNA was extracted from embryos using the Ambion: RNAquous kit. RLM-59 RACE was conducted on the total mRNA in accordance with Invitrogen: GeneRacer Kit. Total RNA was dephosphorylated through Calf Intestinal Phosphatase (CIP) treatment, decapped via Tobacco Acid Pyrophosphatase (TAP), ligated with the GeneRacer RNA oligo, and finally reverse transcribed using random hexamer priming to form the cDNA template. The genes of interest were amplified using touch down PCR and cloned with TOPO TA Cloning Kit (Invitrogen). Immunostaining of lamprey embryos Immunostaining of lamprey embryos was performed as previously described [30]. Neurofilament (NF-M) antibody was used 1:200 in blocking solution. As a secondary antibody Alexa 488 anti mouse IgG2a was used 1:1000 in blocking solution. Sections were degelatinized in 42uC PBS for 10 minutes and washed with PBSTr 2 times for 5 minutes. Afterwards the sections were blocked in 10% goat serum in PBSTr at 4uC for 5 hrs. The blocked sections were incubated overnight at 4uC with the neurofilament (NF-M) antibody 1:200 in blocking solution. To remove unbound antibody the sections were washed 5 times for 10 minutes with PBSTr. As a secondary antibody Alexa 488 anti mouse IgG2a 1:1000 in blocking solution was used for 2 hrs at room temperature. Unbound secondary antibody was washed of 3 times for 10 minutes in PBSTr followed by 2 washes for 10 References SNS Marker Genes in Lamprey Embryonic Development minutes in PBS. For mounting, sections were dipped into distilled water a few times and mounted with Permaflour. Whole-mount in situ hybridization on lamprey embryos Whole-mount lamprey in-situ was performed as previously described [21]. Plasmid DNA templates used for digoxigenin- labeled RNA probes were Hand, Ascl1 and Phox2. Embryos fixed in MEMFA for 1 hr at room temperature. For detection of transcripts, embryos were bleaches with a 10% hydrogen peroxide solution for 10 minutes, washed with PBSTw (DEPC) 3 times for 5 minutes, and were treated with 20 mg/ml proteinase K in PBSTw (DEPC) for 10 minutes. Following this incubation the proteinase K solution was replaced with 2 mg/ml glycine in PBSTw (DEPC) for 10 minutes. After this treatment the embryos were postfixed with 4% PFA for 20 minutes. Afterwards the embryos were pre-hybridized for 3 hrs in hybridization solution at 70uC, and incubated in hybridization solution containing 1–10 mg/ml labeled RNA probe for 16 hrs at 70uC. To remove unbound probe the embryos were washed twice for 15 minutes with hybridization solution at 70uC and then 4 times for 45 minutes. Afterwards the embryos were washed with hybridiza- tion solution and MABT 1:1 at 70uC for 30 minutes followed by four washes in MABT only at room temperature for 30 minutes wash. Blocking of the embryos was done for 4 hrs in blocking solution. The embryos were subsequently incubated with the Anti- DIG-AP antibody 1:2000 in blocking solution over night at 4uC. To remove unbound antibody, embryos were washed with MABT for 2 times 5 minutes, 2 times 30 minutes, 6 times 1 hr and an overnight wash at 4uC. For the color reaction the embryos were adjusted to NTMT buffer by 4 washes for 15 minutes. For the color reaction we used BM purple. The embryos were incubated in BM purple, covert from light, until the desired staining intensity was reached. To stop the color reaction the embryos were washed in PBSTw 3 times for 5 minutes and fixed again in 4% paraformaldehyde for 2 hrs at room temperature. Embedding and sectioning of lamprey embryos Lamprey embryos were washed with PBSTr 3 times 15 min. Subsequently they were incubated in 15% sucrose in PBS for 3 hrs at room temperature, 7.5% gelatin and 15% sucrose for 12 at 37uC, and 20% gelatin for 4 hrs at 37uC. Subsequently embryos were positioned in 20% gelatin and frozen with liquid nitrogen. Embryos were sectioned at 8 – 10 mm with a Microm HM550 cryostat. Phylogenetic Analysis Alignments were built with the coding sequences retrieved from GenBank. Neighbor Joining (NJ) tree were constructed using ClustalX protocol from the DNA STAR package. The trees were visualized using Tree View v. 0.5.0. Author Contributions Conceived and designed the experiments: MSC TSS MBF. Performed the experiments: DH MSC BU JV MBF. Analyzed the data: DH MSC TSS MBF. Wrote the paper: DH MSC MBF. 1. Gess RW, Coates MI, Rubidge BS (2006) A lamprey from the Devonian period of South Africa. Nature 443: 981–984. 2. Janvier P (2006) Palaeontology: modern look for ancient lamprey. Nature 443: 921–924. 3. Kuraku S, Hoshiyama D, Katoh K, Suga H, Miyata T (1999) Monophyly of lampreys and hagfishes supported by nuclear DNA-coded genes. Journal of molecular evolution 49: 729–735. 4. Osorio J, Retaux S (2008) The lamprey in evolutionary studies. Development Genes and Evolution 218: 221–235. 5. Le Douarin N (1982) The neural crest. Cambridge University Press. 6. 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