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. 2019 Sep 27:19:1248-1259.
doi: 10.1016/j.isci.2019.06.003. Epub 2019 Jul 25.

Evolution and Developmental Diversity of Skin Spines in Pufferfishes

Takanori Shono  1 , Alexandre P Thiery  2 , Rory L Cooper  2 , Daisuke Kurokawa  3 , Ralf Britz  4 , Masataka Okabe  5 , Gareth J Fraser  6
Affiliations

Evolution and Developmental Diversity of Skin Spines in Pufferfishes

Takanori Shono et al. iScience. .

Abstract

Teleost fishes develop remarkable varieties of skin ornaments. The developmental basis of these structures is poorly understood. The order Tetraodontiformes includes diverse fishes such as the ocean sunfishes, triggerfishes, and pufferfishes, which exhibit a vast assortment of scale derivatives. Pufferfishes possess some of the most extreme scale derivatives, dermal spines, erected during their characteristic puffing behavior. We demonstrate that pufferfish scale-less spines develop through conserved gene interactions that underlie general vertebrate skin appendage formation, including feathers and hair. Spine development retains conservation of the EDA (ectodysplasin) signaling pathway, important for the development of diverse vertebrate skin appendages, including these modified scale-less spines of pufferfish. Further modification of genetic signaling from both CRISPR-Cas9 and small molecule inhibition leads to loss or reduction of spine coverage, providing a mechanism for skin appendage diversification observed throughout the pufferfishes. Pufferfish spines have evolved broad variations in body coverage, enabling adaptation to diverse ecological niches.

Keywords: Biological Sciences; Evolutionary Biology; Evolutionary Developmental Biology.

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Conflict of interest statement

The authors declare no competing interests.

Figures

None
Graphical abstract
Figure 1
Figure 1
Skin Appendage Diversity in the Order Tetraodontiformes Cleared and stained spinoid scales and spines of larvae/juveniles and adults in representative species of tetraodontiform families. (A) Triacanthodidae, Hollardia sp., Standard Length (SL); 5mm (A and A′) and Paratriacanthodes herrei, SL; 42.5 mm (A″). (B) Triacanthidae, Tripodichthys oxycephalus, SL; 4, 26 mm (B and B′) and Pseudotriacanthus strigilifer SL; 88 mm (B″). (C) Balistidae, Balistes vetula, SL; 10 mm (C and C′) and B. capriscus, SL; 128 mm (C″). (D and E) (D) Monacanthidae, Stephanolepis sp., SL; 5 mm (D and D′) and S. hispidus SL; 94.5 mm. (E) Ostraciidae, Ostracion sp., SL; 6.2 mm (E and E′) and Ostracion trigonus, SL; 330 mm (E″). (F) Triodontidae, Triodon macropterus, SL; 20 mm (F and F′) and T. macropterus SL; 315 mm (F″). (G) Tetraodontidae, Takifugu niphobles, 10 mm (G and G′) and T. niphobles, SL; 102.3 mm (G″). (H) Diodontidae, Diodon holocanthus, SL; 12.5 mm (H and H′) and D. holocanthus SL; 101 mm (H″). (I) Molidae, Ranzania laevis, SL; 1.7 mm (I and I′) and R. laevis, SL; 620 mm (I″). Phylogenetic relationships of tetraodontiform families follow a published dataset (Santini and Tyler, 2003), which combined data from both extinct and extant members of the Tetraodontiformes to build the phylogeny. Scale bars, 2 mm (A, C, D, E, G, and H), 500 μm (A′, A″, B″, G″, and I) 5 mm (B and F), 100 μm (B′, C′, D′, F′, G′, H′, and I′), 1 mm (C″, E″, F″, H″, and I″), and 200 μm (D″ and E′).
Figure 2
Figure 2
Spine Diversity of the Tetraodontidae Computed tomography (CT) renders of Tetraodontidae species, Lagocephalus guentheri (B), Canthigaster punctatissima (C), Chelonodon pleurospilus (D), Lagocephalus lunaris (F), Sphoeroides testudineus (G), Dichotomyctere fluviatilis (H), Tylerius spinosissimus (J), Takifugu oblongus (K) and Tetraodon lineatus (L). The coverage of spines in the species are ranging from (A) ventrally restricted in B–D, (E) dorsal and ventral in F and G, and (I) complete in H, J, K, and L. All CT data acquired from MorphoSource digital 3D media repository.
Figure 3
Figure 3
Location and Histological Structure of Spines of Takifugu (A) Scanning electron microscopy of ventral bossy surface of Takifugu niphobles, 12 dpf. (B) Close-up of spine regions. (C) Sagittal sections of spine regions in 13 dpf of an embryo stained with Hematoxylin and Alcian Blue staining. (D) Sagittal section of spines in 46 dpf embryo stained with Hematoxylin. Black dotted lines indicate spines. (E) Sagittal section of spines in an adult stained with Alizarin Red. Scale bars, 500 μm (A), 20 μm (B), 50 μm (C and D), and 200 μm (E).
Figure 4
Figure 4
Gene Expression in Embryonic Spine Primordia of Takifugu at 12 dpf Gene expression for β-catenin (A), lef1 (B), wnt7a (C), shh (D), sostdc1 (E), fgf3 (F), fgf10a (G), bmp2 (H), bmp4 (I), and fst (J). Right each panel (A′–J′) shows images of spine primordia in sagittal section. White dotted line indicates a boundary line between spine primordia and epithelium layer. Scale bars, 100 μm (A–J) and 10 μm (A′–J′).
Figure 5
Figure 5
BMP Signaling Genes Demarcate Spine Competent Region in Takifugu during Early Development Ventral view of 10 dpf embryos. During initiation of spine development, bmp2 (A) and bmp4 (B) are expressed in a specific region of ventral (abdominal) mesenchyme. Scale bar, 200 μm.
Figure 6
Figure 6
Manipulation of Hh, Notch, BMP, and FGF Pathways during Spine Development (A–C) Alcian Blue–stained ventral spine of T. niphobles embryos treated with small molecules for 72 h and subsequent recovery process until 14 dpf. The treatment is 1% DMSO as a control (A), 50 μM Cyclopamine (B), and 25 μM DAPT (C), respectively. (A′-C′) Ventral spines in each sample marked with dotted red line. (D and E) Gene expression of notch3 (D) and shh (E) in the ventral region where spines will form by whole-mount RNA in situ hybridization of embryos at 10 dpf. (F–K) Comparison of gene expression in the spines for chemically treated embryos between 1% DMSO control (F and G), 50 μM Cyclopamine (H and I), and 25 μM DAPT (J and K) by whole-mount RNA in situ hybridization of embryos past from 72 h treatment. Gene expression for bmp2 (F, H, and J) and lef1 (G, I, and K). (L) Quantitative comparison for the spine total number of 14-dpf embryos between treatment with 1% DMSO control (n = 5), 50 μM Cyclopamine (n = 6), 25 μM DAPT (n = 9), 5 μM LDN193189 (n = 9), and 50 μM SU5402 (n = 10). Data are represented as mean ± SEM. Scale bar, 100 μm.
Figure 7
Figure 7
Schematic Representation of Hypothesized Evolution of Tetraodontid and Diodontid Skin Spines from Scales (A) Basic cycloid scale, of squamous type without ornamentations observed in teleost species. (B and C) (B) Ctenoid scale with tooth-like spines projecting on the posterior side separate from the main cycloid compartment (Roberts, 1993) (C) Plesiomorphic condition of tetraodontiform spinoid scale, composed of cycloid scale and single projecting spine posteriorly as in larval stages of extant Triacanthodidae, Triacanthidae, Balistidae, Monacanthidae (see Figure 1). During evolution of ancient lineage of Tetraodontidae and Diodontidae (or Molidae; Gymnodontes), cycloid compartment has been lost, but single spines are retained. (D) Because the EDA signaling pathway has been implicated in the normal formation of the cycloid scale compartment in teleost fishes, it seems plausible that modification of gene pathways such as EDA may have contributed to the developmental changes by which derivative skin appendages can form without the typical teleost “cycloid” scale compartment.
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