Evolution and Development of the Sympathetic Nervous System

In Gnathostomata, the sympathetic nervous system is a branch of the autonomic nervous system and it is responsible for the unconscious control of the inner organs. It mainly conveys stress responses, the so called “flight or fight” reaction. Sympathetic neurons are derivatives of the neural crest, a pluripotent embryological cell lineage that has migratory capabilities. Neural crest cells give rise to various tissues, for example sensory neurons, autonomic neurons, glia, melanocytes, cells of the adrenal medulla as well as the bone, cartilage, connective tissue and muscle cells of the head. Lampreys are jawless basal vertebrates, Agnatha, that resemble Cambrian era fossils. Lampreys do have neural crest cells and most of their derivatives, but lack a commissural sympathetic chain. Despite the absence of a definite sympathetic ganglion chain, different cell types of endocrine cells were reported to implement sympathetic functions in lampreys. To shed light on the evolution and development of the sympathetic nervous system, immunohistochemistry, DiI cell tracing and in-situ hybridization experiments were carried out. Staining with a pan-neuronal antibody as well as the DiI cell labeling in lampreys confirmed the presence of dorsal root and enteric ganglia. In-situ hybridizations with probes against the sympathetic nervous system marker genes Ascl1, dHand and Phox2b, visualized transcripts of these genes in various tissues that correspond to the known expression of these genes in gnathostomes, except for expression in sites where sympathetic ganglia would be expected. None of the three different techniques detected sympathetic ganglia cells or cells that could be regarded as sympathetic precursor cells. The absence of a commissural sympathetic chain in lampreys led to the hypothesis that redeployment of genes within the gene regulatory network for sympathetic neuron differentiation contributed to the appearance of the sympathetic nervous system. To test this hypothesis the cis-regulatory activity of three conserved non-coding elements was compared in lamprey and chicken using a reporter expression assay. A conserved non-coding element lying directly proximal to the chicken Phox2b promoter showed expression in the chicken. In contrast, this element was silent when the same three sequences were tested in lamprey. The only element which gave rise to expression in lamprey was a different one, which is spanning the first intron. Surprisingly the two different conserved non-coding elements drove expression in corresponding tissues in both species. None of the tested elements showed expression in sympathetic neurons or ganglia. The interspecies experiment showed that chicken genomic regions can successfully recruit transcription factors and drive expression in the lamprey. This study confirms that a commissural sympathetic chain and peripheral sympathetic neurons are absent in lampreys. It is a plausible scenario that changes in cis-regulatory linkages led to the evolutionary deployment of neural crest cells as precursors for sympathetic neurons. Hence, the evolution of the sympathetic nervous system is discussed based on the presented results and in the light of previous publications

Expression of Sympathetic Nervous System Genes in Lamprey Suggests Their Recruitment for Specification of a New Vertebrate Feature

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

Expression of the helix-loop-helix transcription factor Hand in lamprey embryos.

<p>(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).</p

Expression of the transcription factor Phox2 in embryos of <i>P. marinus</i>.

<p>(A) Phylogenetic analysis of a putative Phox2 fragment places it at the base of the gnathostome Phox2a and Phox2b gene families. (B) Expression of Phox2 is initially observed on the hindbrain (red arrow) and on a group of cells of the ventral mesenchyme (black arrow). (C) This expression pattern is maintained at day 8.5, as Phox2 positive cells migrate ventrally (black arrow). (D) At day 10, a stream of positive cells is observed above the heart and appears to be migrating posteriorly (black arrow). At this stage, Phox2 transcripts are first detected on the epibranchial ganglia (red arrow). (E) At day 21, the expression domain of Phox2 expands to 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).</p

Expression of Ascl1 during embryonic development of the lamprey.

<p>(A) Phylogenetic analysis places the putative lamprey Ascl1 ortholog with gnathostome Ascl1 genes. (B) Onset of Ascl1 expression is apparent at day 7, when transcripts are detected in the anterior lip mesoderm. Shortly afterwards (C), faint expression is observed in the lens (arrow). (D) On day 12, lens expression is elevated (arrow), and staining on the hypophysis and trigeminal ganglia (red arrow) are also observed. (E) Finally, on day 14, Ascl1 expression is observed in the notochord (red arrow).</p