likely staining enteric ganglia. Some embryos were allowed to develop until day 31 at which time the dorsal root ganglia continued to express neurofilament protein and appeared somewhat larger in size than at earlier stages (data not shown). However, at no time point did we note neurofilament staining in the vicinity of the dorsal aorta where sympathetic ganglia coalesce in gnathostomes. Expression pattern of transcription factors associated with sympathetic neuron differentiation in the lamprey In gnathostomes, several transcription factors have been implicated in sympathetic nervous system formation. These include the basic-helix-loop helix factors, dHand and Ascl1, as well as the homeodomain transcription factor Phox2b. To examine the presence and deployment of these lamprey genes during neural crest development, we cloned fragments of lamprey Hand, Phox2 and Ascl1 orthologues using 59RACE and determined their expression patterns by in situ hybridization. Phylogenetic analysis places the putative Phox2 lamprey ortholog at the base of the branch containing gnathostome Phox2a and Phox2b genes (Figure 2A). This suggests the duplication event that gave rise to these paralogs took place after cyclostomes diverged from the vertebrate lineage. Expres- sion of lamprey Phox2 was first observed in hindbrain motor neurons and the ventral branchial mesenchyme at day 7 and 8 (Figure 2B and C). By day 10, there was additional expression in the epibranchial ganglia and cranial nerves (Figure 2D). Expres- sion of Phox2 was also observed in individual cells in a medial stream reminiscent of cells migrating posteriorly (Figure 2D). On day 12, expression was maintained in the hindbrain, cranial nerves and ventral mesenchyme (Figure 2E and 2H). Moreover, the expression domain expanded to include the anterior epibranchial ganglia (arrow on Figure 2E). In the trunk, the medial expression first observed on day 10 appeared to expand posteriorly (Figure 2F and I). At later stages, Phox2 transcripts also were detected in cells surrounding the yolk (E14, arrow on Figure 2G) and in the notochord. The Hand ortholog isolated from lamprey clusters to the base of the branch that contains both the D-Hand and E-hand gnathostome genes in our phylogenetic analysis (Figure 3A). Lamprey Hand is first observed at day 5 in the bilateral precursors that form the cardiac field (data not shown). From day 7 to day 10, there is additional staining visible in the anterior portion of the ventral mesenchyme (Figure 3D and 3F). At day 12, in addition to the heart, the entire ventral mesenchyme that surrounds the endostyle and the notochord expresses Hand (Figure 3D and 3F). Hand transcripts were also detected at day 14 in the cardiac ganglia (Figure 3D and 3E) and in the posterior mesoderm (Figure 3G). The lamprey Ascl1 fragment was within the branch that contains the Ascl1 gnathostome orthologues (Figure 4A). Expres- sion of Ascl1 was first noted at day 6, with very faint expression observed in the pituitary gland (Figure 1B). This pattern was maintained at day 7, accompanied by additional expression in the lens (Figure 4C) By day 10, Ascl1 was upregulated in the anterior lip mesoderm (day 10) and by day 12, transcripts also were detected in the VI cranial ganglion (Figure 4D). From day 11 onward, Ascl1 also was expressed faintly in the notochord (Figure 4G). In addition, analysis of the expression of these three transcrip- tion factors in lamprey juveniles through in situ hybridization performed in tissue slices failed to reveal any co-expression in ganglionic structures (data not shown). Thus, we were unable to identify a structure resembling sympathetic ganglia in the lamprey SNS Marker Genes in Lamprey Embryonic Development embryo in which Hand, Ascl1 and Phox2 were co-expressed. Our results, in concert with the neurofilament and DiI data, demonstrate that the lamprey does not possess a sympathetic nervous system analogous to that in gnathostomes. We were also unable to identify any cells that might represent precursors to sympathetic postganglionic neurons of cyclostomes. Discussion We analyzed the expression patterns of three known marker genes of the gnathostome sympathetic nervous system for their deployment in lamprey embryos. At 9 and 11 days after fertilization, we found expression of Hand, Ascl1 and Phox2 in the head region, with only Phox2 showing additional expression in a stream of cells that migrate toward the trunk. These cells may correspond to lateral line cells derived from ectodermal placodes. In ganglia, we see only Hand expressed in the cardiac ganglia and Phox2 in the epibranchial ganglia. Interestingly, there was no overlapping expression of these ‘‘sympathetic’’ genes in any domain in the embryo. Consistent with this marker analysis, we found no contribution of DiI- labeled neural crest cells to structures adjacent to the dorsal aorta, where sympathetic ganglia arise in gnathostomes, despite finding robust labeling of other neural crest derivatives like dorsal root ganglia and enteric neurons. In addition, the absence of neuronal staining with a neurofilament antibody supports the lack of sympathetic ganglia, while revealing the presence of dorsal root and enteric ganglia. Taken together, our results suggest that the lamprey does not have a definitive sympathetic nervous system. In gnathostomes, Ascl1 is generally required for development of autonomic neurons, with expression initiating earlier than Phox2b. Phox2b also is required for autonomic neurogenesis and, in a feedback loop, is required for maintenance of Ascl1 expression [22]. Ascl1 induces expression of pan-neuronal genes in neural crest precursor cells of the peripheral nervous system, but does not specify subtype specific expression of tyrosine hydroxylase (TH) or dopamine-b-hydroxylase (DBH), the enzymes responsible for the catalyzing synthesis of the neurotransmitter, nor-epinephrine [23]. Loss-of-function of dHand, another determinant of the sympa- thetic lineage, blocks neural crest cell differentiation into noradrenergic neurons, whereas its over-expression upregulates Phox2b, TH and DBH. Expression of dHand depends on Phox2b, but not Ascl1 [22]. Classical literature regarding the lamprey sympathetic nervous system is scarce and contradictory. Lampreys possess several neurotransmitters including acetylcholine and noradrenaline in the central nervous system. In addition, sequenced fragments from lamprey DNA reveal the presence of two adrenergic receptors [24] that, in gnathostomes, are the most abundant in the sympathetic nervous system. While this suggests the presence of sympathetic activity in cyclostomes, ganglionic structures are absent in the trunk and tail of the lamprey. Only a sympathetic ganglion formed by small intensely fluorescent cells (SIF cells) has been described adjacent to the adult heart [25,26]. It is possible that such a ganglion would be innervated by the vagus nerve, which is cardio-inhibitory in all vertebrates, with the exception of the cyclostomes [27]. Additionally, it has been suggested that cardiovascular function is controlled by chromaf- fin cells, located on the wall of blood vessels, kidney and urogenital ducts [28]. SIF cells and chromaffin cells are closely related, and SIF cells are considered to be the intermediate in morphology between chromaffin cells and sympathetic neurons [28]. One intriguing PLoS ONE | www.plosone.org 3 October 2011 | Volume 6 | Issue 10 | e26543