Evolution and Developmental Diversity of Skin Spines in Pufferfishes.

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.

Introduction

Vertebrates possess a huge diversity of skin appendages, including teleost fish scales, reptilian scales, avian feathers, and mammalian hair (Pispa and Thesleff, 2003). Although these units are not structurally homologous, the genetic control of their early development appears to be highly conserved between diverse vertebrate clades (Musser et al., 2015, Di-Poï and Milinkovitch, 2016, Cooper et al., 2017). Vertebrate skin appendages broadly originate from a conserved anatomical placode (Di-Poï and Milinkovitch, 2016), before divergence in morphogenesis gives rise to a plethora of disparate adult structures. This process is underpinned by inductive epithelial-mesenchymal interactions, mediated by a conserved set of signaling molecules. These include the hedgehog (Hh), Wnt, bone morphogenetic protein (BMP), fibroblast growth factor (FGF), Notch, and ectodysplasin A (EDA) pathways (Biggs and Mikkola, 2014).

Although the diversity of skin ornaments in teleost fishes is vast, our knowledge of their development is limited. Skin appendages in tetrapod lineages arise from epithelial cells of ectodermal origin (Di-Poï and Milinkovitch, 2016), whereas teleost scales primarily arise from underlying mesodermal layers of the dermis (Shimada et al., 2013, Mongera and Nüsslein-Volhard, 2013). Previous research has demonstrated that the Hh, Fgf, Bmp, Wnt/β-catenin and Eda pathways are involved in teleost scale development (Kondo et al., 2001, Sire and Akimenko, 2004, Harris et al., 2008, Iwasaki et al., 2018, Albertson et al., 2018, Aman et al., 2018). Signaling of eda and its receptor, edar, also control the morphology and patterning of the body plate armor of marine and freshwater stickleback (Colosimo et al., 2005, O'Brown et al., 2015). It is likely that even the most extremely modified teleost skin ornaments share elements of these core signaling pathways, known to underpin skin appendage development throughout diverse vertebrate groups.

Tetraodontiformes include a range of remarkable teleost fishes, such as ocean sunfishes (Molidae), triggerfishes (Balistidae), and pufferfishes (Tetraodontidae). The ∼350 species of this highly derived order exhibit extreme morphological diversification, both in terms of their craniofacial and dermal skeletons (Santini and Tyler, 2003, Fraser et al., 2012, Thiery et al., 2017). Members of each of the tetraodontiform clades possess striking mineralized integumentary appendages, ranging from spinoid scales to individual spines and thick, plate-like armor (Figure 1). Dermal scale development in the Tetraodontiformes is thought to be associated with evolution and modification of the typical squamous scale types (e.g., cycloid, spinoid, or ctenoid) found in most teleost fishes (Roberts, 1993). However, evolutionary developmental approaches have not been used to test this hypothesis.

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′).

Pufferfishes (Tetraodontidae) possess a diverse range of body spines, which provide a powerful defense mechanism. The Takifugu pufferfish lineage is well known from recent genome sequencing efforts that demonstrated that pufferfishes represent vertebrates with the smallest known, most compact genome. Furthermore, this lineage has undergone a relatively recent explosive radiation (Venkatesh et al., 2000, Jaillon et al., 2004). During this radiation, pufferfish spines diversified in their size and shape, as well as their patterning across the body, which ranges from a complete lack of spines to a dense coverage (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.

Here, we present (1) information on the diversity and modification of skin ornaments in this particularly morphologically varied order of teleost fishes, the Tetraodontiformes; (2) describe the development of the dermal spines in pufferfish (Takifugu niphobles) as a model for skin appendage novelty and diversification; (3) report on the results of developmental and genetic manipulation of the spines that cover the ventral surface of Takifugu; and finally (4) suggest an evolutionary mechanism for how morphological diversity of skin ornament in this group may have arisen.

Results

Tetraodontiform Skin Appendages Exhibit Highly Varied Morphologies

There are 10 tetraodontiform families with a diverse array of dermal ornamentations, which vary both in terms of their structure and spatial distribution (Santini and Tyler, 2003, Matsuura, 2014). To examine this morphological diversity, we cleared and stained both juvenile and adult specimens representing extant tetraodontiform families (Figure 1). In the families Triodontidae, Tetraodontidae, and Diodontidae, the morphology and localization of ornamentation are similar between both juvenile and adult specimens (Figure 1). The juveniles of both basal spikefishes (family Triacanthodidae) and the more derived three-toothed pufferfish (family Triodontidae) exhibit mineralized units in the skin that consist of a scale-like plate with one (in triacanthodids) or several (in triodontids) individual spines at the posterior margin. The anterior scale-like plate strongly resembles a typical cycloid scale with concentric ridges or circuli (Figures 1A and 1F). The “cycloid” base in these two groups is still present, although less obvious, in the spinoid scales observed in the adults.

Juveniles of the tripod fishes (family Triacanthidae), the triggerfishes (family Balistidae), and the filefishes (family Monacanthidae) also exhibit a single-spined unit on a “cycloid” base, which appears to lack circuli. In contrast, adults of these three families possess a more complex structure, with spines or spinules sometimes covering the entire visible scale surface (Figures 1C and 1D). Boxfishes (family Ostraciidae) do not possess any spines but instead have a covering of separate hexagonal bony plates on the body surface of juveniles, which grow together to form a thick armor in adults (Figure 1E).

Pufferfishes (family Tetraodontidae) and porcupine fishes (family Diodontidae) possess individual spines that vary in their patterning between different species. Porcupine fishes have particularly long spines covering their entire body, whereas the smaller spines of pufferfishes range from forming a complete coverage of the entire body (seen in Carinotetraodon; Britz et al., 2012) to being restricted to specific ventral and/or dorsal regions (Figure 2). In Takifugu niphobles, these spines are restricted to the dorsal head and ventral abdominal regions. Individual spines of pufferfishes are composed of a basal multi-pronged root embedded into the dermis and a distally pointed single spine tip (Figures 1G and 1H). Juvenile ocean sunfish (family Molidae) are covered with a network of jagged-edged pyramidal spines covering the skin, whereas adults have no spines, but instead their entire body is covered with polygonal scale-like plates (Figure 1I) (Katayama and Matsuura, 2016). Overall, Tetraodontiformes show a huge diversity in dermal ornamentations (see Table S1 for a summary of observations). For this reason, they are particularly well suited for the study of the evolutionary and developmental basis of the diversification of body ornamentation.

Pufferfish Spines Develop from Mesenchymal Condensations

Morphological descriptions of spinoid scales, plates, and spines have previously been reported, predominantly for adult Tetraodontiformes (Roberts, 1993, Santini and Tyler, 2003, Gauldie, 1992). However, the developmental basis of their diversity remains poorly understood. Therefore, we investigated the development of spines of the Japanese Grass Pufferfish (Takifugu niphobles; Tetradontidae).

Scanning electron microscopy revealed a localized region of skin protrusions between the ventral pectoral girdle and the abdominal region at 12 days post fertilization (dpf) (Figure 3A). The ectodermal units that project from the body surface at this stage mark the spines' initial site of development (Figure 3B). Sagittal sections of embryos at 13 dpf demonstrated that developing spine regions are composed of two tissue layers, epithelium and mesenchyme, and associated pigment cells, including melanocytes. The spine primordia are derived from mesenchyme (dermis) and stain strongly with hematoxylin (and Alcian Blue; Figure 3C). In juvenile stages (at 46 dpf), developing spines extend from a fibrous layer of the dermis (Figure 3D). In adult stages, Alizarin red staining shows functional mineralized spines protruding through the outer layers of the skin (Figure 3E). Overall, these results demonstrate that spine development in Takifugu initiates from the mesenchyme (dermal mesoderm) during early ontogeny. This bears similarity to the scale development of medaka and zebrafish, although in zebrafish this process begins later in development, at approximately 1 month post fertilization (Kondo et al., 2001, Le Guellec et al., 2004, Sire and Akimenko, 2004, Shimada et al., 2013, Mongera and Nüsslein-Volhard, 2013).

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).

Conserved Molecular Pathways Underlie in Spine Development in the Pufferfish

Having observed this similarity between Takifugu spine development and scale development of other teleost fishes (Shimada et al., 2013; Mongera and Nüsslein-Volhard, 2013), we sought to investigate the genetic pathways that underlie spine formation in this pufferfish. Therefore, we selected a suite of important signaling pathways (including Wnt/β-catenin, Hh, FGF, and BMP) that underlie ectodermal appendage development throughout diverse vertebrates and chartered their expression during Takifugu spine development (Pispa and Thesleff, 2003; Di-Poï and Milinkovitch, 2016).

β-Catenin is a signal transducer of canonical Wnt signaling, a pathway known to play important roles in cell proliferation and differentiation during organogenesis of various structures (Grigoryan et al., 2008). We observed expression of β-catenin within both the epithelium and mesenchyme of the spine primordium, with stronger expression at the boundary region between primordia and the overlying epithelium (Figure 4A). We also observed expression of the transcription factor lef1 (Lymphoid enhancer binding factor-1), which is associated with the canonical Wnt pathway by interaction with β-catenin (Hovanes et al., 2001), in the distal region of the primordium epithelium (Figure 4B). Expression of lef1 is consistent throughout the early regional specification that demarcates spine competent ectoderm, with its expression upregulated at the border of the ventrally restricted spine-forming region (Figure 4B). The Wnt pathway ligand, wnt7a, of the frizzled family of transmembrane receptors (Vogel et al., 1995), is expressed throughout both the mesenchymal and epithelial compartment, although its expression is stronger at the distal tip (at the apex of the spine) of the epithelium where the spine forms (Figure 4C).

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′).

shh, a ligand of the Hh pathway, has a critical role in cell division, specification, and patterning of organs (Varjosalo and Taipale, 2008). We observed shh expression in the epithelium adjacent to spine primordia (Figure 4D). This expression pattern bears similarity to zebrafish scale development, where shh is expressed in the epithelium adjacent to the scale anlagen (Sire and Akimenko, 2004).

Sostdc1 (Sclerostin domain-containing 1, also known as Wise, ectodin, and USAG-1) encodes an N-glycosylated secreted protein, associated with the BMP pathway as an antagonist, and mediates regulation of the network for both the Hh and Wnt pathways (Yanagita, 2004, Ahn et al., 2010). We observed expression of sostdc1 in the ventral spine region and restricted to the distal end of the spine primordium (Figure 4E).

FGF ligands play a key role in the processes of proliferation and differentiation in various organs, interacting with Hh, Wnt, and BMP signaling pathways (Neubüser et al., 1997). fgf3 is expressed in the epithelial cells adjacent to the distal tip of spine primordia in Takifugu (Figure 4F). In contrast, fgf10a is expressed extensively in the epithelium, with stronger expression observed in the epithelium adjacent to the distal tip of spines (Figure 4G). The BMP pathway is involved in a variety of cellular interactions during various organogenic processes, including antagonism of the FGF signaling pathway (Neubüser et al., 1997, Shi and Massagué, 2003). The Bmp ligands bmp2 and bmp4 are both expressed in the spine primordium and, importantly, in a pre-patterned ventral region before spine placode formation, demarcating the restricted field where spines will form (Figures 4H and 4I). This suggests Bmp signaling may be important in initiating a spine competent dermis in Takifugu. Follistatin (FST) is an activin-binding protein known to antagonize BMP signaling (Bauer et al., 1998, Thompson et al., 2005). We observed fst in the mesenchyme surrounding each spine primordia (Figure 4J). Overall, these results highlight that conserved markers of vertebrate ectodermal appendage formation are expressed and active during the initiation, development, and organization of the highly derived spine ornament of pufferfishes (Figure S1).

Gene Knockdown Assays Suggest the BMP Pathway Controls Patterning of Takifugu Spines

BMP signaling is known to regulate the number, pattern, and sizes of diverse vertebrate skin appendages (Jung et al., 1998, Jiang et al., 1999, Mou et al., 2006, Plikus et al., 2008). Therefore, we investigated the role of the Bmp pathway in regulating dermal spine patterning in Takifugu. At 10 dpf, before spine initiation, bmp2 and bmp4 are expressed in the ventral mesenchyme, defining the restricted region in which spines can form (Figures 5A and 5B). To identify the functional role of the Bmp pathway in spine development, we undertook Bmp gene knockdown assays through injection of an antisense morpholino (MO), in fertilized Takifugu eggs at the one-cell stage.

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.

Although injections with bmp2 or bmp4-MO resulted in mortality at the hatchling stage (6 dpf), morphants injected with fst-MO, an antagonist of BMP, continued to develop. These fst-MO morphants exhibited perturbed jaw development at 12 dpf (Figures S2A and S2B). Similar to experiments undertaken on zebrafish (Dal-Pra et al., 2006), Alcian blue staining revealed abnormal arrangement of Meckel's cartilages in fst-MO Takifugu, with the formation of only four branchial arches, rather than the normal set of five (Figures S2C and S2D). Treatment with fst-MO also perturbed normal development of Takifugu spines. Spines are normally present in the ventral region of Takifugu. However, in morphant embryos injected with fst-MO, hyperplasia of spines was observed (Figures S2E and S2G). Furthermore, the distal tip of spines developed earlier in fst-MO morphants than in control embryos (Figures S2F and S2H). We also documented a reduction in spine number in the ventral region (per 2 mm2) in fst-MO morphants, compared with wild-types (Figure S2I). Despite an expansion of the ventral spine-competent region Takifugu morphants, the overall number of spines was reduced. Overall, these results suggest that Bmp pathway–associated fst controls pufferfish spine patterning, influencing both the size of the spine-competent field and the patterning of spines within this region.

Small-Molecule Gene Inhibition Experiments Reveal Functional Conservation of Signaling Pathways

Pufferfish spine development involves members of the Hh, Bmp, Wnt, and Fgf signaling pathways (Figures 4 and S3). To understand the function of these pathways, and also the potential role of Notch, a transmembrane receptor that mediates cell communication and fate (Lai, 2004), we undertook small-molecule gene inhibition experiments to inhibit specific gene signaling. After hatching, embryos were exposed to small molecules for 72 h, covering the initiation, patterning, and primordium formation stages of spine development. Control embryos were treated with 1% DMSO. Following treatment, embryos developed under standard conditions for a further 14 days, before Alcian blue staining was used to observe phenotypic shifts (Figure 6A).

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.

Treatment with the Hh antagonist Cyclopamine (Incardona et al., 1998) (50 μM) severely repressed spine formation in the ventral region (Figure 6B), indicating Hh signaling is essential for normal development. Additionally, treatment with the BMP inhibitor LDN193189 (Cuny et al., 2008) (5 μM) and the FGF receptor inhibitor SU5402 (Mohammadi et al., 1997) (50 μM) resulted in a small reduction in ventral spine number (Figure 6L). Treatment with the Wnt inhibitor, IWR-1-endo, mostly resulted in lethality of embryos (1 μM); however, a single individual survived the treatment and displayed a severe reduction in the number of spines (Table S2). Furthermore, treatment with the γ-secretase complex and Notch pathway inhibitor, DAPT (Geling et al., 2002) (25 μM), resulted in abnormal spine morphogenesis and a 40% reduction in spine number in the ventral region of Takifugu (Figures 6C and 6L). This suggests the Notch pathway is required for normal spine development and led us to examine the expression of Notch pathway members. We observed the Notch ligand notch3 in the ventral spine-forming region of Takifugu at 10 dpf, although expression was not specifically restricted to spine primordia (Figure 6D). This same region was also found to strongly express shh (Figure 6E).

In situ hybridization following small-molecule treatments revealed that cyclopamine and DAPT affected the activity of other important signaling pathways, including the Bmp and Wnt pathways, 72 h post treatment (Figures 6F–6K). Cyclopamine-treated embryos exhibited reduced expression patterns of bmp2 and lef1 compared with control embryos (Figures 6F–6J). Treatment with DAPT reduced bmp2 expression, whereas lef1 was significantly upregulated in spine primordia, compared with control embryos (Figures 6H–6K).

Overall, these results suggest that conserved signaling pathways associated with the development of other diverse vertebrate ectodermal appendages are also involved in the development of pufferfish spines. The Hh pathway plays a prominent role in the initiation and patterning of spines in Takifugu and is important for the regulation of Bmp and Wnt signaling.

edar Is Required for Development and Patterning of Pufferfish Spines

edar is a cell surface receptor for eda and plays an important role during the development of diverse vertebrate skin appendages (Kondo et al., 2001, Colosimo et al., 2005, Fessing et al., 2006, Harris et al., 2008). This gene is well characterized in relation to the evolution of stickleback armor patterning (Colosimo et al., 2005, O'Brown et al., 2015). To determine whether edar is involved in the development of the related, yet distinct, dermal armor of spines in pufferfishes, we examined edar gene expression from embryonic to juvenile stages of Takifugu. In the embryonic stage at 12 dpf, edar was expressed in the ventral region of the protruding epithelium overlying the spine primordium (Figures S4A–S4C). At the later juvenile stages, expression of edar was present in the epidermis, especially in cells adjacent to each distal spine tip (Figure S4D).

To understand the function of edar in the development of spines, we next performed gene knockout mutagenesis by CRISPR-Cas9 targeted edar. Two guide RNAs (edar#1 and edar#2) were designed targeting exons of edar (Figure S4E). The designed RNAs were co-injected with CAS9 nuclease protein into fertilized eggs. The sequence analyses of DNA fragment harboring target site from injected embryos demonstrated the insertion/deletion (Figures S4F and S4G). We compared expression patterns of shh between wild-type and edar mutated G0 embryos at 12 dpf. In wild-type embryos, we saw expression of shh specific to the spine-forming region, whereas expression patterns for edar mutated embryos (edar#1 and edar#2) were irregular (Figure S4H). We also documented reduction in the number of shh-positive spines in edar mutated embryos, when compared with the number of spines in wild-type (Figure S4I). These results suggest that edar has an important role in the proper arrangement and development of the spine competent territory in Takifugu.

Discussion

Overall, our results demonstrate that the unique skin ornamentation of the pufferfishes (Tetraodontidae) has likely evolved through changes to the initiatory process of skin appendage patterning. This includes early regional restriction of signaling molecules demarcating competent fields of spine formation. It differs from the typical signaling that instigates an initiatory row from which other appendages propagate, such as zebrafish scales, shark dermal denticles, and avian feather tracts (Sire and Akimenko, 2004, Pan et al., 2004, Cooper et al., 2018). Instead, spine primordia form simultaneously within the territories established by the Hh, Wnt, and Bmp signaling molecules (Figure 4). Similar to feather patterning, where genes associated with these pathways are up-regulated in regions destined to become feather tracts (Jung et al., 1998, Jiang et al., 1999, Pan et al., 2004), we see their expression restricted to the spine-forming region of the pufferfish, albeit in a larger non-sequential domain. Alterations to these expression patterns may have facilitated the modification of this region, enabling diverse species-specific spine patterning to arise (Figure 2). Spine patterning in pufferfishes ranges from a complete coverage of spines in groups such as Carinotetraodon to the more restricted coverage of spines in Takifugu niphobles. Pufferfishes provide interesting developmental models for studying the regional restriction and elaboration of vertebrate skin appendages.

Morphological variation of tetraodontiform dermal ornamentation is substantial (Figures 1 and 2). Triacanthodidae, the most basal family in the Tetraodontiform phylogeny, possess numerous spines with complex adult morphologies. However, juvenile stages exhibit a single spine that projects from the cycloid compartment of the scale (Figures 1A–1C). Therefore, the common ancestor of the group may have had one (or several) spine(s) attached to a scale with a cycloid base. This hypothesized primitive condition for tetraodontiforms is similar to spinoid scales observed in other teleost groups, where spines project out from the main body of the scale posteriorly as continuations of the cycloid base (Roberts, 1993). Based on our findings and conclusions, we propose a new hypothesis for the morphological evolution of the dermal skeleton in Tetraodontiformes (Figure 7). Members of different families exhibit loss of either the cycloid base (i.e., Tetraodontidae and Diodontidae) or spines (i.e., Molidae and Ostraciidae) from the scale. In molids and ostraciids, we would hypothesize that the cycloid base has been modified into a thick armor of polygonal plates, which may provide increased protection from predators. When looking at the diversification of spines, the Triacanthodidae, Balistidae, Monacanthidae, and Triodontidae show a “cycloid” base adorned with a varying number of spines, whereas we would suggest that the cycloid compartment has been lost entirely in the Tetraodontidae and Diodontidae. We therefore conclude that spines in the Tetraodontidae and Diodontidae are highly derived dermal elements that are homologous to the standard scales of other teleost fishes (Figure 7) and not neoformations.

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.

The transition to spines in pufferfish and the associated loss of the cycloid compartment in the Tetraodontidae and Diodontidae could have occurred by the modification of the conserved genetic network that operates during cycloid scale development (Colosimo et al., 2005, Harris et al., 2008, O'Brown et al., 2015, Aman et al., 2018, Iwasaki et al., 2018). Specifically, the Eda (ectodysplasin) pathway may have played a crucial role during the diversification of tetraodontiform scales. This pathway is important in refining the size, spacing, and shape of various vertebrate skin appendages (Sadier et al., 2014), including the density and patterning of bony armor derived from modified scales of several stickleback species (Colosimo et al., 2005, O'Brown et al., 2015), and the number of scales in Medaka (Kondo et al., 2001). The Eda pathway interacts with other important signaling pathways, including Hh, Wnt, FGF, and BMP, throughout skin appendage development of diverse vertebrates (Mikkola, 2008, Häärä et al., 2011, Häärä et al., 2012). Therefore, a shift in Eda signaling may likely underlie the morphological evolution of the dermal skeleton in teleost groups, including the pufferfishes.

Conclusion

Members of the highly derived superorder Acanthopterygii possess astonishingly diverse scale morphologies. This diversity has arisen through acquisition of various ornamentations to the posterior region of the cycloid scale, such as tubercles, ridges, serrations, cteni, and spines (Sire, 1986). Tetraodontiformes constitute an exceptional example of scale evolution, particularly with respect to the spines observed in Tetraodontidae and Diodontidae. We suggest these spines are formed through the loss of the cycloid scale compartment present in the tetraodontiform ancestor, resulting in a reduced single spine, driven through modification of gene pathway signaling during scale development (such as Eda signaling; Figure 7). Tetraodontiform fishes have utilized this dermal system to develop a remarkable diversity of spine and scale morphologies. Together, our data suggest that Eda signaling may play a role in the evolutionary transition from spinoid scale to spine. Furthermore, shifts in expression of signaling pathways (such as Bmp) at various stages of appendage development have likely produced tremendous diversity in patterning and unit morphology in this group of derived teleost fishes. Future work on comparative expression patterns or transcriptome analyses may uncover a network of genes related to scale formation that can be directly compared with developing spines, potentially highlighting the genes lost or gained during the scale-to-spine transition.

Limitation of the Study

The Takifugu pufferfish breeding seasons are short, and this work could be performed only during 2 months of the summer season. We are now working on several pufferfish species to increase our knowledge of the development of these fishes.

Methods

All methods can be found in the accompanying Transparent Methods supplemental file.