Expression of Sympathetic Nervous System Genes in Lamprey Suggests Their Recruitment for Specification of a New Vertebrate Feature Daniela Ha¨ ming . , Marcos Simoes-Costa., Benjamin Uy, Jonathan Valencia, Tatjana Sauka-Spengler, Marianne Bronner-Fraser* Division of Biology, California Institute of Technology, Pasadena, California, United States of America Abstract The sea lamprey is a basal, jawless vertebrate that possesses many neural crest derivatives, but lacks jaws and sympathetic ganglia. This raises the possibility that the factors involved in sympathetic neuron differentiation were either a gnathostome innovation or already present in lamprey, but serving different purposes. To distinguish between these possibilities, we isolated lamprey homologues of transcription factors associated with peripheral ganglion formation and examined their deployment in lamprey embryos. We further performed DiI labeling of the neural tube combined with neuronal markers to test if neural crest-derived cells migrate to and differentiate in sites colonized by sympathetic ganglia in jawed vertebrates. Consistent with previous anatomical data in adults, our results in lamprey embryos reveal that neural crest cells fail to migrate ventrally to form sympathetic ganglia, though they do form dorsal root ganglia adjacent to the neural tube. Interestingly, however, paralogs of the battery of transcription factors that mediate sympathetic neuron differentiation (dHand, Ascl1 and Phox2b) are present in the lamprey genome and expressed in various sites in the embryo, but fail to overlap in any ganglionic structures. This raises the intriguing possibility that they may have been recruited during gnathostome evolution to a new function in a neural crest derivative. Citation: Ha¨ming D, Simoes-Costa M, Uy B, Valencia J, Sauka-Spengler T, et al. (2011) Expression of Sympathetic Nervous System Genes in Lamprey Suggests Their Recruitment for Specification of a New Vertebrate Feature. PLoS ONE 6(10): e26543. doi:10.1371/journal.pone.0026543 Editor: Ferenc Mueller, University of Birmingham, United Kingdom Received April 29, 2011; Accepted September 28, 2011; Published October 27, 2011 Copyright: 2011 Ha¨ming et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the National Institutes of Health (Grant #R01 DE017911). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: mbronner@caltech.edu . These authors contributed equally to this work. Introduction Lampreys are agnathans (jawless vertebrates) that have many essential vertebrate characteristics but lack the sympathetic nervous system and jaws. Morphologically, they resemble Cambrian era fossils [1,2], suggesting a resemblance to the common ancestor of jawless (Agnatha) and jawed (Gnathostomata) vertebrates. Lamprey and hagfish, the only modern agnathans, are likely to be monophyletic, though there remains controversy on this point [3,4]. As basal vertebrates, both occupy a critical phylogenetic position for understanding emergence of vertebrate traits. However, lamprey offers a significant advantage for developmental studies due of the accessibility and ease of obtaining embryos for experimental manipulation. Neural crest cells are one of the defining features of vertebrates. This population of multipotent cells gives rise to a variety of different tissues and cell types including cartilage and bone of the facial skeleton, pigment cells, sensory and peripheral ganglia, among other derivatives [5,4]. The peripheral nervous system of jawed vertebrates is comprised of sensory, parasympathetic, sympathetic and enteric ganglia that form clusters of neurons that innervate peripheral structures and relay information back to the central nervous system. All of these sensory and autonomic ganglia are derived from the neural crest, together with a contribution of cranial placodes to the sensory ganglia of the head [6]. Previous studies have shown that lampreys possess neural crest cells and many neural crest derivatives [7,8,9], like cartilage, pigment cells and neurons. In fact, the gene regulatory network underlying the formation and differentiation of neural crest cells is remarkably similar to that of higher vertebrates [10–12]. Interestingly, however, lampreys lack some key neural crest structures including dentine, bone and sympathetic neurons. The sympathetic nervous system is a branch of the autonomic nervous system, responsible for the physiological modulation of inner organs in the absence of conscious control by the central nervous system (CNS). Lampreys and hagfishes lack the chain of sympathetic ganglia chain observed in gnathostomes [13]. Instead, their sympathetic innervation comes from preganglionic fibers that extend directly to the terminal plexus, similar to what is observed in amphioxus [14]. Nevertheless, lamprey and hagfish have been reported to have scattered chromaffin-like cells along blood vessels, the heart and cloaca [15]. Although these cells have been described as analogous to postganglionic neurons [14], it is not yet clear if they connect with the central nervous system and/or represent an evolutionary precursor to the gnathostome sympathetic nervous system [15]. PLoS ONE | www.plosone.org 1 October 2011 | Volume 6 | Issue 10 | e26543