Evolution of the new head by gradual acquisition of neural crest regulatory circuits

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Abstract

The neural crest, an embryonic stem-cell population, is a vertebrate innovation that has been proposed to be a key component of the ‘new head’, which imbued vertebrates with predatory behaviour1,2. Here, to investigate how the evolution of neural crest cells affected the vertebrate body plan, we examined the molecular circuits that control neural crest development along the anteroposterior axis of a jawless vertebrate, the sea lamprey. Gene expression analysis showed that the cranial subpopulation of the neural crest of the lamprey lacks most components of a transcriptional circuit that is specific to the cranial neural crest in amniotes and confers the ability to form craniofacial cartilage onto non-cranial neural crest subpopulations3. Consistent with this, hierarchical clustering analysis revealed that the transcriptional profile of the lamprey cranial neural crest is more similar to the trunk neural crest of amniotes. Notably, analysis of the cranial neural crest in little skate and zebrafish embryos demonstrated that the transcriptional circuit that is specific to the cranial neural crest emerged via the gradual addition of network components to the neural crest of gnathostomes, which subsequently became restricted to the cephalic region. Our results indicate that the ancestral neural crest at the base of the vertebrate lineage possessed a trunk-like identity. We propose that the emergence of the cranial neural crest, by progressive assembly of an axial-specific regulatory circuit, allowed the elaboration of the new head during vertebrate evolution.

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Fig. 1: Lamprey cranial NC lacks most components of a chick ‘cranial crest circuit’.
Fig. 2: Nodes of an early cranial NC-specific circuit were acquired in the cranial NC progressively throughout gnathostome evolution.
Fig. 3: Tissue-specific RNA sequencing comparisons between lamprey and chicken reveal that the ancestral NC had a more trunk-like identity.
Fig. 4: Model of the evolution of NC axial levels during vertebrate evolution.

Data availability

All raw sequencing data for all RNA-seq libraries (Fig. 3) and merged reference transcriptomes are available online (NCBI BioProject# PRJNA497902). Sequences of in situ probe templates for Figs. 1b, c, 2a, c are available through GenBank accession codes (see Methods).

Code availability

Code used to analyse sequencing datasets are available from the corresponding author upon request.

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Acknowledgements

We thank J. Tan-Cabugao and E. Grossman for technical assistance; D. Mayorga and R. Fraser for help with fish husbandry; B. Martik for illustrating the adult animals for our expression matrices; the Caltech Millard and Muriel Jacobs Genetics and Genomics Laboratory and in particular I. Antoshechkin for sequencing of our RNA-seq libraries; and R. Diamond, J. Tijerina, D. Perez, and P. Cannon of the The Caltech Flow Cytometry Cell Sorting Facility for cell sorting assistance. This work is supported by NIH grants R01NS086907, R01DE024157, and R35NS111564 to M.E.B. M.L.M. is supported by a Helen Hay Whitney Foundation postdoctoral fellowship. S.G. is supported by a graduate fellowship from the American Heart Association (18PRE34050063).

Author information

Project and analysis conception were designed by M.L.M., M.S.-C., and M.E.B. Writing and interpretation were performed by M.L.M., S.G., B.R.U., J.A.G., S.A.G., M.S.-C., and M.E.B. Lamprey orthologue cloning and all in situ hybridization, imaging, and analysis were performed by M.L.M. Bioinformatics and chicken RNA-seq were performed by S.G. Phylogenetic analysis and lamprey embryo acquisition were performed by S.A.G. Cloning of skate orthologues and skate embryo acquisition were performed by J.A.G. Lamprey embryo dissections and library preparations were performed by B.R.U. and M.S.C.

Correspondence to Marianne E. Bronner.

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Competing interests

The authors declare no competing interests.

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Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Peer review information Nature thanks Robert Cerny and Jeremiah Smith for their contribution to the peer review of this work.

Extended data figures and tables

Extended Data Fig. 1 Heterochronic shifts of cranial specific gene regulatory nodes from later neural crest derivatives to an early specification program throughout gnathostome evolution.

ad, Expression of lamprey orthologues of amniote cranial NC-specific genes at T21 (cranial) and T23 (trunk) in cross-section. e, Pharyngeal NC derivative expression in T26 P. marinus frontal section (based on ref. 40). fl, Cranial circuit orthologues are expressed in pharyngeal arch derivatives, with the exception of Brn3, which is expressed in the NC-derived cranial sensory ganglia41 in lamprey frontal sections. m, Gene expression matrix summarizing the heterochronic shift of cranial crest specific circuit nodes. nc, neural crest; nt, neural tube; n, notochord. Scale bars, 100 µm. Cryosections of in situ hybridizations were reproducible on n ≥ 5 embryos per time point for n ≥ 2 experiments.

Extended Data Fig. 2 P. marinus Dmbx is homologous to gnathostome Dmbx genes.

a, Truncated alignment of Dmbx protein sequences. An alignment of full-length Dmbx protein sequences was assembled using TCoffee and contiguous regions tagged by the program as poorly or moderately well-aligned were removed, leaving 218 well-aligned residues. b, Bayesian consensus phylogenetic tree, with posterior probabilities shown at corresponding nodes.

Extended Data Fig. 3 Expression of cranial circuit genes in the neural crest of the little skate.

a, Schematic of a stage 18 L. erinacea embryo with the NC illustrated as blue (cranial) and red (trunk). bf, Cross-sections as depicted in a. Scale bar, 50 µm. Cryosections of in situ hybridizations were reproducible on n ≥ 2 embryos for n ≥ 2 experiments.

Extended Data Fig. 4 Pharyngeal neural crest derivative expression of cranial circuit orthologues in stage 25 L. erinacea embryos.

a, Schematic of head of L. erinacea embryo. Dashed box represents the region of the head for each embryo shown in bi (left); purple dashed line depicts the location of the frontal section for bi (right). bf, Right, pharyngeal NC derivative expression of cranial circuit orthologues. gi, Right, Dmbx1, Lhx5, and Brn3c are absent in pharyngeal arch derivatives at stage 25. bi, Scale bars, 500 µm (left); 100 µm (right). In situ hybridizations were reproducible on n ≥ 2 embryos.

Extended Data Fig. 5 Expression of cranial circuit genes in the NC of the zebrafish.

a, Schematic of a 14ss D. rerio embryo with the NC illustrated as blue (cranial) and red (trunk). bh, Cross-sections as shown in a. Scale bars, 50 µm. In situ hybridizations were reproducible on n ≥ 10 embryos.

Extended Data Fig. 6 Expression of cranial circuit orthologues in pharyngeal NC derivatives of D. rerio embryos at 3 days post fertilization.

a, Schematic of head of zebrafish embryo at 3 days post fertilization (dpf). Purple dashed line depicts the location of the frontal sections for the insets in eh. eh, Expression of cranial circuit orthologues in pharyngeal arches. bd, Dmbx1, Lhx5, and Brn3c are absent from pharyngeal arch derivatives at 3 dpf. Scale bar, 150 µm. In situ hybridizations were reproducible on n ≥ 10 whole-mount and cryosectioned embryos.

Supplementary information

Reporting Summary

Supplementary Table 1

Alignment percentage scores for lamprey/chicken alignments

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Martik, M.L., Gandhi, S., Uy, B.R. et al. Evolution of the new head by gradual acquisition of neural crest regulatory circuits. Nature 574, 675–678 (2019) doi:10.1038/s41586-019-1691-4

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