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Neural crest origin of sympathetic neurons at the dawn of vertebrates

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Abstract

The neural crest is an embryonic stem cell population unique to vertebrates1 whose expansion and diversification are thought to have promoted vertebrate evolution by enabling emergence of new cell types and structures such as jaws and peripheral ganglia2. Although jawless vertebrates have sensory ganglia, convention has it that trunk sympathetic chain ganglia arose only in jawed vertebrates3,4,5,6,7,8. Here, by contrast, we report the presence of trunk sympathetic neurons in the sea lamprey, Petromyzon marinus, an extant jawless vertebrate. These neurons arise from sympathoblasts near the dorsal aorta that undergo noradrenergic specification through a transcriptional program homologous to that described in gnathostomes. Lamprey sympathoblasts populate the extracardiac space and extend along the length of the trunk in bilateral streams, expressing the catecholamine biosynthetic pathway enzymes tyrosine hydroxylase and dopamine β-hydroxylase. CM-DiI lineage tracing analysis further confirmed that these cells derive from the trunk neural crest. RNA sequencing of isolated ammocoete trunk sympathoblasts revealed gene profiles characteristic of sympathetic neuron function. Our findings challenge the prevailing dogma that posits that sympathetic ganglia are a gnathostome innovation, instead suggesting that a late-developing rudimentary sympathetic nervous system may have been characteristic of the earliest vertebrates.

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Fig. 1: Co-expression of sympathoadrenal fate-specifying genes in late embryonic lamprey.
Fig. 2: Expression of genes encoding catecholamine biosynthetic enzymes by lamprey sympathoblasts.
Fig. 3: Late-developing sympathetic neurons in the lamprey trunk.
Fig. 4: Neural crest origin of lamprey sympathoblasts.
Fig. 5: Conserved molecular characteristics of lamprey sympathetic neurons.

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Data availability

All raw RNA-sequencing data generated from this study are publicly available through the National Center for Biotechnology Information’s Gene Expression Omnibus database under the accession number GSE246248. RNA-sequencing data used for comparison to mouse sympathetic neurons were published previously41 and are available under the accession number GSE78845. Raw data can be made available upon request. Source data are provided with this paper.

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Acknowledgements

We thank A. Collazo and G. Spigolon for microscopy training and assistance; I. Antoshechkin for discussion and assistance with library preparation and sequencing; G. Shin and N. Pierce for discussion and assistance with HCR probe design; and J. Stundlova, R. Fraser and D. Mayorga for lamprey husbandry. This work was supported by the grants NIH R35NS111564 to M.E.B., NIH F32HD106627 to B.M.E. and NIH F31DE031154 to H.A.U. J.S. is supported by a Marie Skłodowska-Curie grant, agreement no. 897949.

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Authors and Affiliations

  1. Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, USA

    Brittany M. Edens, Jan Stundl, Hugo A. Urrutia & Marianne E. Bronner

  2. Faculty of Fisheries and Protection of Waters, University of South Bohemia in Ceske Budejovice, Vodnany, Czech Republic

    Jan Stundl

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Contributions

The project was conceived of and designed by B.M.E. and M.E.B. Descriptive and transcriptomic analyses were carried out by B.M.E. CM-DiI labelling was carried out by J.S. and H.A.U. Preparation and sectioning of all T21 and T30 CM-DiI-labelled embryos was carried out by J.S. Lamprey husbandry and spawning were led by J.S. Writing and interpretation were carried out by B.M.E. and M.E.B. All authors approved the manuscript.

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Correspondence to Marianne E. Bronner.

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Extended data figures and tables

Extended Data Fig. 1 Expression of sympathoadrenal genes at early embryonic stages.

(a-c) HCR detection of Phox2 (teal), Hand (red), and Ascl1 (white) in lamprey at T25. (d) The three transcripts are not co-expressed. Scale bar = 50 μm. n = 6 embryos across three independent replicates.

Extended Data Fig. 2 Co-expression of sympathoadrenal fate-specifying genes with catecholamine biosynthetic enzymes in post-embryonic lamprey.

HCR detection of Th (teal) and Dbh (red) with (a-d) Ascl1, (e-h) Phox2, and (i-l) Hand in white at T30+ in whole mount. The core sympathoadrenal transcription factors are persistently co-expressed with catecholamine synthesis enzymes. Scale bars = 20 μm. n = 9 embryos across three independent replicates.

Extended Data Fig. 3 Early expression of catecholamine biosynthetic enzymes.

(a-d) HCR detection of Th (teal) and Dbh (red) in lamprey at T27. Colocalization of the transcripts is seen in cells surrounding the heart and spanning the initial segment of the trunk in bilateral streams. Scale bars = 100 μm. Red asterisk denotes the heart. n = 6 embryos across three independent replicates.

Extended Data Fig. 4 Quantification of relative morphology of TH+ neuroblasts/neurons from T27 to T30.

Morphology of TH+ cells was compared from T27- and T30- staged embryos by plotting [neurite length/soma diameter]. This measure revealed increased neurite length in T30 as compared to T27, consistent with greater neuronal maturation. **** p < 0.0001 by two-tailed Mann-Whitney U-test. A total of fifty cells from five embryos were measured for each developmental stage. Error bars indicate SEM.

Source Data

Extended Data Fig. 5 Late neurogenesis of lamprey sympathoblasts.

(a-i) Immunohistochemical detection of TH (teal) and Neurofilament-M (Nf-M, red) at T30 (a-c), 2.5 month ammocete (d-f), and 4 month ammocete (g-i) stages. Co-expression of TH and Nf-M is not observed in late embryonic or earlier ammocete stages. Consistent co-labeling is observed by four months of age. n = 12 embryos, 12 ammocetes of each stage per marker across four independent replicates (j-o) Co-expression of TH with additional neuronal markers is observed in four month old ammocetes. HuC/D (j-l) and SCG10 (m-o) are shown. DAPI is shown in white. Scale bars = 50 μm. n = 9 ammocetes of each stage per marker across three independent replicates.

Extended Data Fig. 6 Lamprey sympathoblasts are neural crest-derived.

Lineage tracing of TH+ sympathoblasts. CM-DiI-labeled neural crest (red) localizes to TH+ cells (teal) at (a-c) T27 and (d, e) T30, indicating a neural crest origin. Arrowheads (b, c, and e) indicate CM-DiI-labeled cells. DAPI is shown in white. Scale bars = 20 μm. DA=dorsal aorta, E=esophagus, I=Intestine. Experimental details are shown in Extended Data Table 1.

Extended Data Fig. 7 Analysis of gene expression in lamprey sympathetic neurons.

(a) Representative image of a wild-caught lamprey ammocete used for selection of trunk sympathetic neurons. Scale bar = 1 cm. (b and c) Immunohistochemical detection of TH (teal) in transverse sections through ammocete trunks. DAPI is shown in white. Yellow box indicates the site of the sympathetic chain neurons. SC=spinal cord, NC=notochord. Scale bars = 250 μm (a) and 10 μm (b). n = 4 ammocetes across three independent replicates. (d and e) GO analyses of Metabolites and Cell Compartment for TH+ neurons. Analyses are performed using the top 500 significantly upregulated transcripts with an enrichment cutoff of 1.5 log2(Fold Change). p-values are determined using Fisher’s exact test. (f) Immunohistochemical detection of DBH (teal) at T28. Punctate expression is consistent with vesicular localization of DBH protein. Scale bar = 20 μm. n = 6 embryos across three independent replicates.

Extended Data Fig. 8 Comparison of gene expression between mouse and lamprey sympathetic neurons.

(a) GO analysis of Enrichr Protein-Protein Interaction (PPI) Hubs49 for mouse sympathetic neurons previously reported41. Analysis is performed using the 500 most abundantly expressed genes across all noradrenergic neuron subtypes. p-values are determined using Fisher’s exact test. (b) Venn diagram indicating PPI Hub terms implicated in both mouse and lamprey sympathetic neurons.

Extended Data Fig. 9 Analysis of noradrenergic subtypes in lamprey.

HCR detection of Th (teal) with (a-c) Npy (red), (d-g) Npy (red) and Ret (white), and (h-k) Gfra2 (red) and Ret (white) in whole mount ammocete trunks. Consistent co-expression of Th and Npy (a-c) suggests the presence of the NA3 noradrenergic subtype in lamprey. A lack of consistent co-expression of Th with Npy and Ret (d-g) and Gfra2 and Ret suggest the absence of noradrenergic subtypes NA2 and NA5, respectively. Scale bars = 20 μm. n = 12 ammocetes across four independent replicates.

Extended Data Table 1 Number of sympathoblasts labeled by CM-DiI injection at T21
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Edens, B.M., Stundl, J., Urrutia, H.A. et al. Neural crest origin of sympathetic neurons at the dawn of vertebrates. Nature (2024). https://doi.org/10.1038/s41586-024-07297-0

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