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Neural regeneration is very limited in humans but extremely efficient in echinoderms. The brittle star Amphiura filiformis can regenerate both components of its central nervous system as well as the peripheral system, and understanding the molecular mechanisms underlying this ability is key for evolutionary comparisons not only within the echinoderm group, but also wider within deuterostomes. Here we characterise the neural regeneration of this brittle star using a combination of immunohistochemistry, in situ hybridization and Nanostring nCounter to determine the spatial and temporal expression of evolutionary conserved neural genes. We find that key genes crucial for the embryonic development of the nervous system in sea urchins and other animals are also expressed in the regenerating nervous system of the adult brittle star in a hierarchic and spatio-temporally restricted manner.
Figure 1. Nervous system in the brittle star arm. (A) Schematic diagram of the distribution of the nervous system in a mature arm segment of a brittle star, adapted from  under the terms of Creative Commons Attribution 4.0 International Licence. (B) SynB staining in regen-erating 50% proximal segments of A. filiformis. (C) SynB staining in regenerating 50% distal seg-ments of A. filiformis. (C’) Magnification of (C). (D) SynB staining in mature podium base of A. fili-formis. (D’) Tip of the same podium from (D). am—aboral intervertebral muscle; amn—aboral mixed nerve; en—epineural epithelium, hn—hyponeural epithelium, hin—horizontal intermuscular hyponeural nerve; ljn—lateral juxtaligametal node; ojn—oral juxtaligamental node; om—oral in-tervertebral muscle; p—podium; pg—podial ganglion; pmn—proximal muscle nerve; rnc—radial nerve cord; rwc—radial water-vascular canal; sg—spine ganglion.
Figure 2. Whole mount in situ hybridisation of Afi-elav, Afi-soxB1, Afi-soxB2, Afi-soxC, Afi-pax6, and Afi-six3 in A. filiformis at early (stage 2/3 and stage 4/5) and late (50% proximal and distal segments) stages of arm regeneration and podia showing a variety of patterns within the different regions of the regenerating nervous system. OV—oral view, OLV—oral-lateral view, LV—lateral view. Scale bar 100 um.
Figure 3. Dynamics of gene expression during arm regeneration of the brittle star Amphiura filiformis. (A) Schematic diagram representing the tissue collection procedure for generating quantitative expression data using the nanoString nCounter for different stages of arm regeneration. Red dashed lines indicate the tissue being collected at each stage. (B) Heatmap and clustering analysis of quantitative data for nervous system gene expression at different stages of regeneration relative to the non-regenerating arm segments obtained using the nSolver package. (C) Heatmap of nanoString data for proliferation and stem cell gene expression at different stages of regeneration relative to the non-regenerating arm segments. (D) Comparison of the two main observed patterns of expression changes during regeneration using stem cell/proliferation genes Afi-mycb, Afi-piwi and Afi-vasa and nervous system differentiation genes Afi-elav, Afi-neuroD and Afi-pax6. Note the opposite trend in expression suggestive of the different functionality of the genes during regeneration. Expressed as abundance relative to maximum expression (%). NR—non-regenerating, hpa—hours post amputation, St—stage, prox—proximal, dist—distal.
Figure 4. Summary of neurogenic gene expression patterns during early and late regenerative stages of the brittle star A. filiformis.