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Shared features of blastula and neural crest stem cells evolved at the base of vertebrates

Nature Ecology & Evolution volume 8, pages 1680–1692 (2024)Cite this article

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Abstract

The neural crest is a vertebrate-specific stem cell population that helped drive the origin and evolution of vertebrates. A distinguishing feature of these cells is their multi-germ layer potential, which has parallels to another stem cell population—pluripotent stem cells of the vertebrate blastula. Here, we investigate the evolutionary origins of neural crest potential by comparing neural crest and pluripotency gene regulatory networks of a jawed vertebrate, Xenopus, and a jawless vertebrate, lamprey. We reveal an ancient evolutionary origin of shared regulatory factors in these gene regulatory networks that dates to the last common ancestor of extant vertebrates. Focusing on the key pluripotency factor pou5, we show that a lamprey pou5 orthologue is expressed in animal pole cells but is absent from neural crest. Both lamprey and Xenopus pou5 promote neural crest formation, suggesting that pou5 activity was lost from the neural crest of jawless vertebrates or acquired along the jawed vertebrate stem. Finally, we provide evidence that pou5 acquired novel, neural crest-enhancing activity after evolving from an ancestral pou3-like clade. This work provides evidence that both the neural crest and blastula pluripotency networks arose at the base of the vertebrates and that this may be linked to functional evolution of pou5.

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Fig. 1: Lamprey animal pole cells co-express neural crest and pluripotency GRN components.
Fig. 2: Neural crest and pluripotency GRN components are both expressed in the blastula, neural plate border and neural crest in lamprey.
Fig. 3: Comparative transcriptomics of neural crest and pluripotency GRNs.
Fig. 4: pou5 is absent from the neural crest of jawless vertebrates but can enhance neural crest formation in a jawed vertebrate.
Fig. 5: pou5 is essential for neural crest formation and evolved neural crest-enhancing activity from a pou3-like ancestor involved in neurogenesis.
Fig. 6: Neural crest evolution by deployment of pluripotency GRN components.

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Data availability

RNA-seq data have been deposited at NCBI (GSE205436). All other data are available in the main text or Supplementary Information. Source data are provided with this paper.

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Acknowledgements

We thank S. Miehls and the staff of the Hammond Bay Biological Station for shipment of lampreys. We also thank D. McCauley, D. Medeiros, T. Sauka-Spengler and M. Bronner for clones and reagents; R. Braun for statistical advice; and participants in the Woods Hole Embryology course and members of the LaBonne laboratory for helpful discussions. Funding for the study was received from Life Sciences Research Foundation postdoctoral fellowship (J.R.Y.), National Institutes of Health (grant nos. R01GM116538 (C.L.) and F32DE029113 (E.N.S.)), National Science Foundation (grant no. 1764421 to C.L.) and Simons Foundation (grant no. SFARI 597491-RWC to C.L.). We dedicate this work to Dr. J. Walder (deceased), founder of Integrated DNA Technologies and co-founder of the Walder Foundation, which generously underwrote J.R.Y.’s Life Sciences Research Foundation fellowship.

Author information

Authors and Affiliations

  1. Department of Molecular Biosciences, Northwestern University, Evanston, IL, USA

    Joshua R. York, Paul B. Huber, Elizabeth N. Schock, Andrew Montequin, Sara Rigney & Carole LaBonne

  2. Research Department, Gilead Sciences, Foster City, CA, USA

    Anjali Rao

  3. National Institute for Theory and Mathematics in Biology, Chicago, IL, USA

    Carole LaBonne

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  1. Joshua R. York
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Contributions

J.R.Y. and C.L. established the research concept and designed the study methodology. J.R.Y., A.R., P.B.H., A.M., E.N.S. and S.R. undertook the study investigations. J.R.Y. visualized the results. C.L., J.R.Y. and E.N.S. acquired the funding. J.R.Y. and C.L. were responsible for administration of the project and supervising the study. J.R.Y. wrote the original draft manuscript. All authors reviewed and edited the manuscript.

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Correspondence to Carole LaBonne.

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Extended data

Extended Data Fig. 1 Neural crest and pluripotency regulatory genes are expressed in animal pole stem cells in Xenopus.

Neural crest genes are in (a) and pluripotency genes are in (b). Reproducible on n = 10 embryos for n ≥ 3 experiments for each panel. Scale bar: 250 μm.

Extended Data Fig. 2 Neural crest and pluripotency regulatory genes are expressed in the blastula and gradually resolve to the neural plate border and neural crest in Xenopus.

Pluripotency genes are in (a) and neural crest genes are in (b). Arrowheads in sox3 panels show absence of sox3 expression from the neural crest at late neurula stages. Scale bar: 250 μm. Abbreviations: ant = anterior, post = posterior, npb = neural plate border. Reproducible on n = 10 embryos per time point for n ≥ 3 experiments for each panel.

Extended Data Fig. 3 Colocalization of neural crest and pluripotency regulatory genes in lamprey embryos.

a) Coronal sections showing colocalization of pax3 protein (neural crest marker) and myc mRNA expression (pluripotency marker) in the gastrula ectoderm, neural plate border, and neural folds (arrowheads) of the early neural crest. Dorsal is up and ventral is down. (b) Validation of immunofluorescent staining for pax3 expression in the early neural crest of lamprey embryos. Reproducible on n = 10 embryos per time point for n ≥ 3 experiments for each panel in (a, b). Abbreviations: ant = anterior, epi = epidermal ectoderm, npb = neural plate border, np = neural plate, post = posterior. Scale bars: 100 μm for (a) and 250 μm for (b).

Extended Data Fig. 4 Neural crest and pluripotency regulatory genes are expressed along the antero-posterior axis in lamprey embryos.

Scale bar: 250 μm. Dorsal is up and ventral is down. Embryos are shown in posterior view. Reproducible on n = 10 embryos for n ≥ 3 experiments for each panel.

Extended Data Fig. 5 Analysis of RNA-Seq data in Xenopus and lamprey.

(a) Example dissection of lamprey animal caps. (b-e) Comparisons of principal components analyses (PCA) of samples used in this study and elsewhere indicate minimal batch effects. (f) Heatmap of z-scored expression values (by stage) for lamprey neural crest and pluripotency genes. (g, h) Reproducibility of RNA-Seq data generated in this study for Xenopus. (i) Lamprey animal caps express canonical pluripotency genes but not markers of germ layer differentiation. (j) Genes involved in vertebrate pluripotency that are expressed in blastula-stage lamprey embryos are not expressed in Xenopus animal pole cells. (k) Several genes involved in mammalian pluripotency are not expressed in animal pole cells of Xenopus and lamprey. Transcript abundance of soxB1 genes are included for both Xenopus (sox3) and lamprey (soxB1a) for context. (l) Validation of RNA-Seq comparisons showing species-specific differences in expression of neural crest regulatory genes in the blastula. Reproducible on n = 10 embryos per time point for n ≥ 3 experiments for each panel. Lamprey soxE2 and pax3/7 are expressed in both animal pole cells and neural crest, whereas these genes are only expressed in the neural crest of Xenopus. (m) Full results of k-means analysis. Abbreviations: Pm = Petromyzon marinus, Xl = Xenopus laevis, uninj9 = uninjected caps, st9; wntchd9 = wnt8a/chordin-injected caps, st9; uninj13 = uninjected caps, st13; wntchd13 = wnt8a/chordin-injected caps, st13; uninj17 = uninjected caps, st17; wntchd17 = wnt8a/chordin-injected caps, st17. Scale bar: 250 μm. All analyses conducted in this figure were reproducible on n ≥ 3 experiments. No statistical comparisons were made. Data in (i, j, k) are presented as mean values ± SEM. Box-whisker plots in (m) depict minima and maxima at the whiskers, median values (50th percentile) at the middle, and lower (25th percentile) and upper (75th percentile) quartiles bounding the top and bottom of each box.

Source data

Extended Data Fig. 6 Phylogenetic analysis and characterization of lamprey pou5 and Xenopus pou5f3.

(a) Bayesian (left) and maximum likelihood (right) phylogenies of pou-family transcription factors. (b) Synteny comparisons of pou5 loci between lamprey and Xenopus. (c) Quantification of transcript abundance (TPM) by RSEM for lamprey pou5 and Xenopus pou5f3 in animal pole cells and neural crest. Data were obtained from ≥100 lamprey animal caps for n = 3 biological replicates and ≥10 Xenopus animal caps at each stage for n = 3 biological replicates. (d) in situ hybridization of Xenopus pou5f3 paralogs. Orientation of embryos is the same as Extended Data Fig. 2. (e) HCR-FISH showing colocalization of pou5f3.2 with snai2 transcripts in Xenopus neural crest. Embryos are shown in anterior view. Dorsal is up and ventral is down. Reproducible on n = 10 embryos for n ≥ 3 experiments for panels (d, e). Data in (c) are presented as mean values ± SEM. Scale bars: 250 μm.

Extended Data Fig. 7 pou5f3 gain-of-function experiments in lamprey embryos.

(a) Western blot showing matched protein levels for gain-of-function experiments. (b) Overexpression of Xenopus pou5f3 factors in lamprey causes loss or no change of expression of multiple neural plate border and neural crest markers. Embryos are shown in dorsal view. Anterior is up and posterior is down. Asterisk denotes the injected side of the embryo. Reproducible on n = 10 embryos per time point for n ≥ 3 experiments for each panel in (a, b). Scale bar: 250 μm.

Extended Data Fig. 8 Morpholino-mediated knockdown of Xenopus pou5f3.1 and pou5f3.2 factors does not result in a compensatory increase in pou5f3.3 expression.

Embryo is shown in anterior view. Dorsal is up and ventral is down. Reproducible on n = 10 embryos per time point for n ≥ 3 experiments for this panel. Scale bar: 250 μm.

Extended Data Fig. 9 Wildtype expression of pou3 in Xenopus and lamprey embryos.

(a) Xenopus (Xl) pou3f1 transcripts are first detectable in neural folds and enriched in the neural plate, whereas lamprey (Pm) pou3 transcripts are expressed in animal pole blastula cells, gastrula ectoderm, neural plate and neural plate border, and early neural crest. (b) Coronal sections show lamprey pou3 transcripts in the neural crest of early and mid-neurulae (arrowheads). By late neurula stages, pou3 expression is largely absent from the neural crest (arrowheads) while being enriched in the rest of neural tube. Dorsal is up and ventral is down. Reproducible on n = 10 embryos per time point for n ≥ 3 experiments for each panel. Scale bar: 250 μm for (a) and 100 μm for (b).

Extended Data Fig. 10 Sequence comparisons of chordate pou5 and pou3 proteins.

(a) Alignment of Xenopus pou5 and pou3 proteins. The box on the C-terminus shows a conserved region in the transactivation domain of pou5 that is highly conserved across most vertebrates (asterisks in b). (c) Alignment of chordate pou3 and vertebrate pou5 proteins reveals a conserved potential SUMOylation site (first box) within the pou-s domain of pou3 that is absent from pou5. There are also highly conserved residues among pou3 proteins (second and third boxes) representing potential MAPK phosphorylation sites in the linker domain and pou-hd that are also absent from pou5. Full alignment is in Supplementary Fig. 2.

Supplementary information

Supplementary Information

Supplementary Figs. 1 and 2.

Reporting Summary

Supplementary Data 1

Statistical data for Fig. 3b containing log-transformed TPMs used for correlation analyses.

Supplementary Data 2

Statistical data for Fig. 3c containing full output of k-means analyses.

Supplementary Data 3

Statistical data for Fig. 3d containing output from DESeq2 analysis for Xenopus and lamprey.

Source data

Source Data Extended Data Fig. 5

Statistical source data for Extended Data Fig. 5m containing full output of k-means analyses.

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York, J.R., Rao, A., Huber, P.B. et al. Shared features of blastula and neural crest stem cells evolved at the base of vertebrates. Nat Ecol Evol 8, 1680–1692 (2024). https://doi.org/10.1038/s41559-024-02476-8

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  • DOI: https://doi.org/10.1038/s41559-024-02476-8

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