SNS Marker Genes in Lamprey Embryonic Development include cranial (black arrow) and epibranchial ganglia (arrowhead). A section of this embryo (H) reveals staining in the motor neurons of the hindbrain, epibranchial ganglia and ventral mesenchyme (red, black and blue arrows, respectively). (F) At posterior axial levels, Phox2b expressing cells are observed adjacent to the yolk sac (section on I). (G) At later stages, a larger number of cells around the yolk start expressing Phox2 (black arrow), and there is strong expression in cranial nerves (arrowhead). doi:10.1371/journal.pone.0026543.g002 possibility is that there may have been a shift from chromaffin and SIF cells to sympathetic neurons during gnathostome evolution, such that chromaffin and SIF cells represent the evolutionary precursor to sympathetic neurons. These three cell types are closely related lineage-wise, sharing a sympathoadrenal progenitor which co-expresses markers characteristic of both chromaffin cells and sympathetic neurons [28,29]. Our DiI labeling experiments show a discrete neural crest stream that migrates towards the heart (the cardiac neural crest; data not shown) which could be the source of the progenitors that Figure 3. Expression of the helix-loop-helix transcription factor Hand in lamprey embryos. (A) Phylogenetic analysis suggests lamprey has one ortholog of both the dHand and eHand gnathostome genes. (B) Lamprey Hand expression is first observed in the cardiac field, and is conspicuous after 7 days of development. (B) At day 10, two domains of expression are clearly present: the heart (black arrow) and a part of the anterior mesenchyme (red arrow). (C) Expression in the cardiac ganglia (black arrow) is first detected at day 12 days; transverse sections reveal high abundance of transcript in the mesenchyme flanking the pharynx (black arrows on F). (E) Strong expression is observed in the ventral mesenchyme of the lamprey head (read arrow), as well as the heart at day 14 (black arrow). (G) Posterior expression appears to be restricted to the unsegmented mesoderm of the tail (black arrow). doi:10.1371/journal.pone.0026543.g003 PLoS ONE | www.plosone.org 5 October 2011 | Volume 6 | Issue 10 | e26543