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Hmx gene conservation identifies the origin of vertebrate cranial ganglia

Abstract

The evolutionary origin of vertebrates included innovations in sensory processing associated with the acquisition of a predatory lifestyle1. Vertebrates perceive external stimuli through sensory systems serviced by cranial sensory ganglia, whose neurons arise predominantly from cranial placodes; however, the understanding of the evolutionary origin of placodes and cranial sensory ganglia is hampered by the anatomical differences between living lineages and the difficulty in assigning homology between cell types and structures. Here we show that the homeobox transcription factor Hmx is a constitutive component of vertebrate sensory ganglion development and that in the tunicate Ciona intestinalis, Hmx is necessary and sufficient to drive the differentiation programme of bipolar tail neurons, cells previously thought to be homologues of neural crest2,3. Using Ciona and lamprey transgenesis, we demonstrate that a unique, tandemly duplicated enhancer pair regulated Hmx expression in the stem-vertebrate lineage. We also show notably robust vertebrate Hmx enhancer function in Ciona, demonstrating that deep conservation of the upstream regulatory network spans the evolutionary origin of vertebrates. These experiments demonstrate regulatory and functional conservation between Ciona and vertebrate Hmx, and point to bipolar tail neurons as homologues of cranial sensory ganglia.

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Fig. 1: Hmx expression in chordates.
Fig. 2: Hmx regulation and downstream target genes in Ciona.
Fig. 3: Vertebrate Hmx locus evolution and CNE identification.
Fig. 4: Lamprey Hmx CNE activity in transgenic lamprey and Ciona embryos.

Data availability

Cloned Hmx gene sequences have been deposited in Genbank accessions MN264670MN264672. RNA-seq data have been deposited in SRA accession GSE141046. Original data underlying Fig. 4b, c of this manuscript can be accessed from the Stowers Original Data Repository at http://odr.stowers.org/websimr/.

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Acknowledgements

We thank A. Stolfi and R. Zeller for sharing plasmids used in the Ciona CRISPR and overexpression studies, respectively; H. Escriva for hosting C.P. and for access to his amphioxus facility; and S. Green for lamprey husbandry assistance. V.P. was supported by a Natural Motion scholarship. V.P. and S.M.S. acknowledge the Elizabeth Hannah Jenkinson fund for financial support. V.P. also thanks T. Manousaki and C. Tsigenopoulos for their support while based in HCMR. A.P. was supported by the Accademia Nazionale dei Lincei while working in Oxford and by the H2020 Marie Sklodowska-Curie COFUND ARDRE to U.R. while working in Innsbruck. C.P. was supported by an EMBO Long Term Fellowship while working in Oxford. M.E.B. acknowledges support from award R35NS111564 from the NIH. H.J.P. was supported by funds from the Stowers Institute (grant no. 1001).

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Contributions

V.P., C.P and S.M.S. conceived the study. V.P. conducted lamprey gene expression analysis, CNE identification, analysis of lamprey reporter gene experiments, Ciona Hmx expression analysis, Ciona Hmx overexpression analysis and RNA-seq and the molecular phylogenetic analyses. A.P. conducted Ciona CNE identification and reporter gene experiments, tests of lamprey CNE activity in Ciona, CRISPR and overexpression reporter analyses, and analysis of Ciona Hmx and Ngn gene expression. C.P. conducted the amphioxus in situ hybridization and participated in RNA-seq data analysis. H.J.P. conducted the lamprey reporter construct injections and analysis. U.R., M.E.B. and S.M.S. supervised the work. All authors contributing to drafting and editing the manuscript.

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Correspondence to Sebastian M. Shimeld.

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Extended data figures and tables

Extended Data Fig. 1 Expression of jawed vertebrate Hmx genes in neural derivatives.

The summaries show expression by gene cluster, by genome duplication paralogue (as in the associated diagram), or overall.

Extended Data Fig. 2 Schematics of experimental strategies for reporter assays and Ciona Hmx CNE identification.

a. Hmx overexpression in Ciona. b. Hmx or Ngn CRISPR Cas9 knockout in Ciona. c. Ciona Hmx CNE identification. Approximately 2 Kbp 5′ to the first Hmx exon in Ciona intestinalis (Type A) (Ciona robusta) scaffold KhS563 is shown, with conservation to the Hmx locus in Ciona savignyi shown below. d. Ciona Hmx CNE analysis in Ciona.

Extended Data Fig. 3 CRISPR-Cas9 knockout of Ciona Hmx and Ciona Ngn.

a. Placement of sgRNA guides for Hmx CRISPR knockout and primers used for validation, relative to gene structure. Guide and primer sequences in Methods and Supplementary Table 1. b. Predicted engineered outcome of Hmx CRISPR knockout. c. PCR amplification of Ciona intestinalis (Type B) genomic DNA from wild type, CRISPR control and Hmx CRISPR embryo DNA (as well as from additional sgRNAs that were tested but not used in further experiments). The guide used in further experiments is marked in yellow. Sizes of bands in the DNA ladder (100bp DNA-Ladder, extended: Carl Roth) are given in base pairs (bp). d. Sequencing of amplified bands with sequence identity matching the predicted outcomes in (b). e. Placement of sgRNA guides for Ngn CRISPR knockout and primers used for validation, relative to gene structure. Guide and primer sequences in Methods and Supplementary Table 1. f. Predicted engineered outcome of Ngn CRISPR knockout. g. PCR amplification of Ciona intestinalis (Type B) genomic DNA from CRISPR control and Ngn CRISPR embryo DNA (as well as from additional sgRNAs that were tested but not used in further experiments). The guide used in further experiments is marked in yellow. Sizes of bands in the DNA ladder (100 bp DNA-Ladder, extended: Carl Roth) are given in base pairs (bp). h. Sequencing of amplified bands with sequence identity matching the predicted outcomes in (f).

Extended Data Fig. 4 Early developmental expression of Hmx and Ngn in C. intestinalis (Type B).

Gene expression was analysed by whole mount in situ hybridisation. Only posterior BTNs (arrowheads) are marked by faint Hmx expression during neurula stages, while Ngn is expressed in posterior BTNs (arrowheads) and anterior BTNs (arrows) and the CNS. Scale bars 100 μM.

Extended Data Fig. 5 Molecular phylogenetic analysis of chordate Hmx sequences and alignment of lamprey Hmx sequences.

a. This phylogenetic analysis includes Hmx sequences from amphioxus and Ciona. The analysis was conducted using the Maximum Likelihood method and numbers indicate percentage node support out of 1000 bootstraps. b. Lamprey HmxA, HmxB and HmxC nucleotide sequence alignment. The translation shows the identical homeodomain amino-acid sequence encoded by all three genes. HmxA and HmxC share additional nucleotide sequence identity before and after the homeodomain encoding sequence. Nucleotide sequences are from the lamprey Lethenteron camtschaticum.

Extended Data Fig. 6 Model of evolution of vertebrate Hmx uCNE and dCNE from an ancestral udCNE.

CNE activity is shown in green on the embryo diagrams in the Central Nervous System (CNS) and Cranial Sensory Ganglia (CSG).

Extended Data Fig. 7 Assessment of background deriving from the vector used to generate lamprey transgenics.

Embryos were injected with vector only (which includes the zebrafish krt4 minimal promoter and reporter gene but no cloned enhancer), allowed to develop then fixed and labelled for DNA (DAPI, blue), GFP (green) and Hu/ELAV (red) before analysis by confocal microscopy. Each embryo was scored for expression in multiple tissues, as shown in the table at the top right of the picture, with a focus on tissues overlapping with Hmx expression. D and A indicate dorsal and anterior orientations for each image. CSG, cranial sensory ganglia. G, geniculate ganglion. VA, vestibuloacoustic ganglion. P, petrosal ganglion. Spinal cord expression was confined to isolated cells and distinct from the consistent column of expression generated by Hmx enhancers (see Fig. 4, main text). Brain expression appeared in the dorsal hindbrain and midbrain and was also distinct from Hmx and Hmx enhancer expression. In two embryos (1 and 2 below: also in high magnification in top left focused on the otic area) close examination revealed scattered cells around some cranial ganglia, including a few co-expressing Hu/ELAV. These differed from those labelled with Hmx enhancers in that GFP staining did not penetrate into axons. Scale bars 100μM except for the high magnification views of the otic region where they are 10μM.

Extended Data Table 1 Quantification of BTN signal in control and Hmx CRISPR knockout embryos using the Ngn or Asic markers
Extended Data Table 2 Quantification of Ciona Hmx CNE activity in control and Ngn CRISPR knockout embryos
Extended Data Table 3 Full count data for Ciona Hmx -2kb transgenic reporter analysis (Fig. 2e–g) and for dCNE reporter analysis in Ciona transgenic embryos (Fig. 4h)

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Papadogiannis, V., Pennati, A., Parker, H.J. et al. Hmx gene conservation identifies the origin of vertebrate cranial ganglia. Nature 605, 701–705 (2022). https://doi.org/10.1038/s41586-022-04742-w

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