There are a handful of characteristic markers for sympathetic neurons, including Phox2b, Ascl1 (Ash1), and dHand (hand2). Phox2b is a homeodomain transcription factor expressed in several types of neurons in the developing nervous system [16]. The bHLH transcription factor achaete-scute homolog 1 (Ascl1 formerly ash1) is a proneural gene that influences neuronal fate. Ascl1 is expressed in some domains of the neuroepithelium of the forebrain and in precursors of sympathetic and enteric neurons [17]. dHand is a basic helix-loop-helix transcription factor that is essential for proliferation and noradrenergic differentiation of sympathetic neuron precursors during development [18]. Here, we asked whether this suite of genes exists in lamprey and if so, where they were expressed. To address this question, we isolated lamprey homologues of these genes and examined their expression patterns in embryos by in situ hybridization at various stages of development. The results show that all three genes are individually found in different areas of the head with only Phox2 expressed in cells at the trunk level. DiI labeling of presumptive neural crest cells failed to show a neural crest contribution to sites where sympathetic ganglia would be expected to coalesce. In contrast, DiI labeled neural crest cells contributed to both dorsal root ganglia and enteric ganglion cells of the gut. Taken together, the results raise the intriguing possibility that the transcriptional program responsible for migration to and/or differentiation within the site of sympathetic ganglion formation in gnathostomes was assembled through the recruitment of Phox2b, dHand and Ascl1 by precursors derived from the neural crest. Results While it has been established that lamprey lacks an organized sympathetic nervous system [14,19], some studies suggest that scattered sympathetic neurons and chromaffin cells might be associated with blood vessels, hindgut, cloaca and kidneys [15]. Furthermore, it has been suggested that lamprey has sympathetic innervation to the heart [13,20], as opposed to spinal adrenergic innervation as seen in other vertebrates. To investigate the organization of the sympathetic nervous system in lamprey embryos, we examined neural crest migration in the vagal and trunk regions, and mapped the expression pattern of markers characteristic of differentiating sympathetic neurons. DiI and neurofilament labeling of trunk neural crest in lamprey In the trunk region of gnathostomes, neural crest precursors to sensory and sympathetic ganglia migrate from the dorsal neural tube, along a ventral pathway to coalesce either next to the neural tube, to form dorsal root ganglia, or further ventrally adjacent to the dorsal aorta, to form sympathetic ganglia. To test whether lamprey neural crest cells migrate ventrally to contribute to sensory and/or sympathetic ganglia as in gnathos- tomes, we first performed focal injections of the lipophilic dye, DiI into the dorsal neural tube at trunk levels. In lamprey, the neural tube forms by secondary neurulation, where a solid rod-like structure transforms into a tube whose lumen forms by cavitation. At embryonic day 5, the neural rod elevates, gradually detaching from the dorsal epithelium. The first indication of neural crest precursors occurs at this time. The head morphologically extends and becomes visible at day 6, concomitant with cavitation. Neural crest primordia at this stage appear as bulges on the dorsal aspect of the newly formed neural tube [21]. Focal injections of DiI performed at any level of the trunk dorsal neural rod prior to cavitation (day 5.5 to day 6) (Figure 1A) failed to give rise to any SNS Marker Genes in Lamprey Embryonic Development Figure 1. DiI labeling of lamprey neural crest cells reveals absence of sympathetic ganglia during embryonic develop- ment. (A) Focal injections of DiI in the lamprey neural tube at day 5 results in labeling of migrating cephalic neural crest (arrow in B). However, focal injections into the posterior neural tube (C) fail to label trunk neural crest cells. D) Filling the lumen of the neural tube with DiI after cavitation produces labeled trunk neural crest cells in several neural crest derivatives (E). A section through an injected embryo (F) shows labeling of the dorsal root ganglia (DRG, arrow in G), the mesenchyme of the fin (H) and neurons surrounding the gut (F), but no structure that resembles sympathetic ganglia. Neurofilament staining (J) labels neural crest derivatives such as the DRG but also fails to reveal any sympathetic like structures. doi:10.1371/journal.pone.0026543.g001 migrating neural crest. In contrast, similar injections into the head neural folds at similar stages resulted in labeling of the migrating crest (Figure 1A and 1B). Accordingly, we performed DiI-labeling at later times, after cavitation at day 6.5 to day 7, by injecting dye into the lumen of the neural tube (Figure 1D). This approach resulted in labeling of migrating neural crest cells. In embryos receiving neural tube injections at day 6.5 to 7 and examined through day 34, the labeled cells contributed to several neural crest derivatives at trunk and vagal levels (Figure 1E). These include dorsal root ganglia (DRGs) (Figure 1F and 1G), the mesenchymal cells of the fin (Figure 1H), as well as enteric ganglia (Figure 1I). However, no structures resembling sympathetic ganglia were observed at any stage. These results suggest that lamprey neural crest cells contribute to dorsal root ganglia but fail to condense into sympathetic-like structures during embryonic development. To examine neuronal differentiation, we performed immuno- staining using antibodies against neuronal markers. At day 16, anti-neurofilament staining was observed in the neural tube, dorsal root ganglia (Figure 1J) and also in the ventral part of the gut, PLoS ONE | www.plosone.org 2 October 2011 | Volume 6 | Issue 10 | e26543