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Step-wise evolution of neural patterning by Hedgehog signalling in chordates

Nature Ecology & Evolution volume 4, pages 1247–1255 (2020)Cite this article

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Abstract

In vertebrate embryos, Hedgehog (Hh) is expressed in some anterior basal plate domains and by notochord and floorplate cells, and ventral neural cells are patterned by the activities of Hh-regulated transcription factors. Hh signalling is antagonized by signals from the dorsal neural tube and loss of Hh leads to loss of ventral patterning as dorsal pattern expands. These mechanisms are critical for producing the neurons that implement motor responses to sensory inputs but understanding how they evolved has been hindered by lack of insight from commonly studied invertebrates where nervous system morphology and genetic mechanisms are non-conserved with vertebrates. The invertebrate chordate amphioxus, which expresses Hh in its notochord and floorplate, provides a window into the prevertebrate condition. We examined amphioxus neural development by manipulating Hh and downstream genes involved in neural pattern and cell identity. We show that Hh signalling regulates the differentiation of some neurons in amphioxus, including a subset of motor neurons. This demonstrates some conservation of mechanism between vertebrates and amphioxus. However, other aspects of neural patterning differ between the lineages. We suggest the complexity of Hh-dependent neural patterning in vertebrates evolved in a step-wise manner. Alongside other previously described regulatory changes, initial recruitment of Hh along the length of the axis occurred in an ancestor to the chordates to regulate the differentiation of a subset of neurons. This was followed, in the vertebrate lineage, by additional changes to the downstream gene regulatory network of transcription factors, giving Hh a broader role in dorsal–ventral neural patterning.

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Fig. 1: A schematic of the Hedgehog signalling pathway and effects of Hh and Ptch mutations on amphioxus embryo neural tube patterning.
Fig. 2: Double fluorescent in situ hybridization of OligA and Nkx6 in late neurula and schematic diagram of genes expression changes in mutants.
Fig. 3: The expression of Nkx6, OligA, Mnxa, Lhx3 and Islet in the central nervous system through neurula stages in amphioxus Hh+/+, Hh+/– and Hh–/– embryos.
Fig. 4: Hh mRNA injection restores expression of motor neuron genes in Hh–/– amphioxus.
Fig. 5: Analysis of cell identity in Hh, Ptch, Mnxa, and Err mutant embryos and Mnxa mRNA injected embryos.
Fig. 6: Model for the acquisition of Hh signalling in the patterning of the chordate rhombospinal/spinal cord.

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All data generated or analysed during this study are in the main text, Extended Data figures and Supplementary material.

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Acknowledgements

We thank Z. Zuo and Y. Zhou for their help with qrtPCR. Collaboration between G.L. and S.M.S. was supported by an International Exchanges 2017 Cost Share award funded by The Royal Society (grant no. IEC\NSFC\170126) and the National Natural Science Foundation of China (grant no. 31811530298). The work is also supported by grants from the National Natural Science Foundation of China (nos. 31872186, 31672246 and 31471986).

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  1. These authors contributed equally: Qiongqiong Ren, Yanhong Zhong.

Authors and Affiliations

  1. State Key Laboratory of Cellular Stress Biology, School of Life Sciences, Xiamen University, Xiamen, China

    Qiongqiong Ren, Yanhong Zhong, Xin Huang, Chaofan Xing, Hui Wang, Guangwei Hu, Yiquan Wang & Guang Li

  2. Department of Zoology, University of Oxford, Oxford, UK

    Brigid Leung & Sebastian M. Shimeld

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Contributions

G.L., S.M.S., Q.R. and Y.W. designed experiments. Q.R., Y.Z., X.H., G.L., C.X. and G.H. conducted experiments and collected data. G.L., S.M.S., Q.R., Y.W., Y.Z., X.H., C.X., B.L., H.W. and G.H. analysed the data. S.M.S., G.L. and Q.R. wrote the manuscript.

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Correspondence to Yiquan Wang, Sebastian M. Shimeld or Guang Li.

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Extended data

Extended Data Fig. 1 Ptch expression in Hh-/- and Smo-/- embryos.

Whole mounts with anterior to the left and the scale bars are 50 µm, with the bars in A-F also applying to A’-F’ respectively. All the embryos are in lateral view. Expression of Ptch in (a–c) control (Hh+/+ and Hh+/-) and (a’–c’) Hh-/- embryos at neurula (8 S and 14 S) and larval stages. Expression of Ptch in (d–f) control (Smo+/+ and Smo+/-) and (d’–f’) Smo-/- embryos at neurula and larval stages. 8 S: embryos with 8 somites; 14 S: embryos with 14 somites. Numbers in the bottom right corner of a panel show the number of times the phenotype was seen, out of the number of embryos of that genotype analysed.

Extended Data Fig. 2 The expression of neural tube marker genes in Hh-/- and Ptch-/- embryos at 3-somite neurula stage.

Anterior is to the left and the scale bar is 50 µm, the images are in lateral views except for a, a’, e, f’, l, l’, M and M’, which are ventral views focused on the dorsal side. Expression patterns of neural tube marker gene Dlx, IrxA, IrxB, IrxC, Pax3/7, Pax4/6 and Nkx2.2 in (a–g) control (Hh+/+ and Hh+/-) and (a’–g’) Hh-/- embryos at early neurula stage. Expression patterns of neural tube marker gene Dlx, IrxA, IrxB, IrxC, Pax3/7, Pax4/6 and Nkx2.2 in (H-N) control (Ptch+/+ and Ptch+/-) and (h’–n’) Ptch-/- embryos at early neurula stage. Numbers in the bottom right corner of a panel show the number of times the phenotype was seen, out of the number of embryos of that genotype analysed.

Extended Data Fig. 3 The expression of neural tube marker genes in Hh-/- and Ptch-/- embryos at 5-somite neurula stage.

The scale bar in A is 50 μm and applies to other embryos, the scale bar in a is 20 μm and applies to other sections. Embryos are shown with anterior to the left and in lateral view except for a, a’, e, e’, h, h’, l, l’. Sections are transverse with dorsal to the top, and at the point indicated by the black line in the embryo depicted to the left of each section. Numbers in the bottom right corner of a panel show the number of times the phenotype was seen, out of the number of embryos of that genotype analysed. Embryos and sections in a row show the expression of the same gene, indicated at the far left of the row.

Extended Data Fig. 4 The expression of neural tube marker genes in Hh-/- and Ptch-/- embryos at 14-somite neurula stage.

The scale bar in A is 50 μm and applies to other embryos, the scale bar in a is 20 μm and applies to other sections. Embryos are shown with anterior to the left and in lateral view except for a, a’, e, e’, h, h’, l, l’. Sections are transverse with dorsal to the top, and at the point indicated by the black line in the embryo depicted to the left of each section. Numbers in the bottom right corner of a panel show the number of times the phenotype was seen, out of the number of embryos of that genotype analysed. Embryos and sections in a row show the expression of the same gene, indicated at the far left of the row.

Extended Data Fig. 5 Double in situ hybridization of Mnxa and OligA.

Confocal images of WT amphioxus at 11 somites stage in (a–c) lateral views and in (a’–c’) ventral views focused on the dorsal side. (a) Merge of (b) Mnxa+ neurons (green) and (c) OligA+ neurons (red), the white arrows in A mark the coexpresssion positions of Mnxa and OligA (yellow). (a’) Merge of (b’) Mnxa+ neurons (green) and (c’) OligA+ neurons (red), the white arrows in A’ mark the coexpresssion positions of Mnxa and OligA (yellow) in anterior neural tube. Whole mounts are shown with anterior to the left and the scale bar is 50 µm.

Extended Data Fig. 6 Nkx6, OligA or Mnxa mRNA injection could not restore the motor neuron defects to normal in Hh-/- amphioxus.

Whole mounts with anterior to the left and scale bar is 50 µm. All the embryos are in lateral views and at 9 somites stage (middle neurula stage). The expression of Mnxa in the neural tube of (a) control (Hh+/+ and Hh+/-) and (b) Hh-/- embryos. (c, d) The expression of Mnxa in embryos injected with Nkx6 mRNA. (e, f) The expression of Mnxa in the neural tube of embryos injected with OligA mRNA. (g, h) The expression of Mnxa in the neural tube of embryos injected with Nkx6 and OligA mRNA. The expression of Lhx3 in the neural tube of (i) control (Hh+/+ and Hh+/-) and (j) Hh-/- embryos. (k, l) The expression of Lhx3 in the neural tube of embryos injected with Mnxa mRNA. (m, n) The expression of Lhx3 in the neural tube of embryos injected with OligA, Nkx6 and Mnxa mRNA. Numbers in the bottom right corner of a panel show the number of times the phenotype depicted was confirmed, out of the total number of embryos of that genotype on which the experiment was performed.

Extended Data Fig. 7 OligA, Nkx6, Lhx3, Mnxa, Islet and Vacht expression in OligA-/- embryos.

Whole mounts with anterior to the left and the scale bar is 50 µm. Ventral views focused on the dorsal side except for e and e’, which are lateral views. (a, a’, d and d’) The expression of OligA and Mnxa in control (OligA+/+ and OligA+/-) and OligA-/- embryos at 11 somites stage. (b, c’ and e, f’) The expression of Nkx6, Lhx3, Islet and Vacht in control (OligA+/+ and OligA+/-) and OligA-/- embryos at 14 somites stage. Numbers in the bottom right corner of a panel show the number of times the phenotype was seen, out of the number of embryos of that genotype analysed.

Extended Data Fig. 8 The expression of Lhx and Islet in Mnxa-/- amphioxus embryos at 14 somites stage.

Top panels show digestion products from PCR across the mutated region: mutation abolishes the restriction site, so the higher band (uncut) indicates presence of the mutated allele, the lower band (cut) the wild-type allele. Lower panels show Lhx and Islet expression in embryos analysed. Embryo numbers and genotypes are shown on the top left corner of each image, with -/- highlighted in red. Whole mounts with anterior to the left. Scale bars are 50 µm. The embryos stained with Lhx probes are all in ventral view, focused on the dorsal side, except number #4 embryo which is in lateral view; the embryos stained with Islet probes are all in lateral view.

Extended Data Fig. 9 The expression of Vglut and Vgat in Hh-/- amphioxus embryos at 14 somites stage.

Whole mounts with anterior to the left. Scale bar is 50 µm. The embryos are all in ventral view, focused on the dorsal side. Hh+/+;+/- and Hh-/- embryos are distinguished according to symmetry of somites as indicated by red dotted lines (oblique lines show asymmetrical arrangement of somites in Hh+/+ and Hh+/- embryos, vertical lines show symmetrical arrangement of somites in Hh-/- embryos).

Extended Data Fig. 10 Overlapping expression of Err, Vacht and Mnxa revealed by double in suit hybridization.

Whole mounts with anterior to the left and the scale bars are 50 µm. (a) Merge of (b) Err+ neurons (black) and (c) Vacht+ neurons (red) in WT amphioxus neural tube at 14 somites stage in ventral view, focused on the dorsal side. (d) Merge of (e) Mnxa+ neurons (red) and (f) Vacht+ neurons (Green) in WT amphioxus neural tube at 11 somites stage in lateral views, the white arrows in D mark the coexpresssion positions of Mnxa and Vacht in amphioxus neural tube. (g) Merge of (h) Mnxa+ neurons (red) and (I) Vacht+ neurons (Green) in WT amphioxus neural tube at 11 somites stage in ventral view, focused on the dorsal side, the white arrows in G mark the coexpresssion positions of Mnxa and Vacht in amphioxus neural tube. Panels a–c are each collages of two separate photographs, with the boundary between the images identified in the figure.

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Ren, Q., Zhong, Y., Huang, X. et al. Step-wise evolution of neural patterning by Hedgehog signalling in chordates. Nat Ecol Evol 4, 1247–1255 (2020). https://doi.org/10.1038/s41559-020-1248-9

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