Trunk neural crest origin of dermal denticles in a cartilaginous fish J. Andrew Gillisa,b,1, Els C. Alsemaa,c, and Katharine E. Criswella,b aDepartment of Zoology, University of Cambridge, Cambridge CB2 3EJ, United Kingdom; bWhitman Center, Marine Biological Laboratory, Woods Hole, MA 02543; and cKarolinska Institutet, SE-171 77 Stockholm, Sweden Edited by Marianne Bronner, California Institute of Technology, Pasadena, CA, and approved October 26, 2017 (received for review August 4, 2017) Cartilaginous fishes (e.g., sharks and skates) possess a postcranial odontogenic in nature (14, 15), they do not exhibit key histo- dermal skeleton consisting of tooth-like “denticles” embedded within logical features of dentine (e.g., dentine tubules), and their mode their skin. As with teeth, the principal skeletal tissue of dermal den- of development and growth is more similar to that of acellular ticles is dentine. In the head, cranial neural crest cells give rise to the bone (16, 17). We therefore sought to test the odontogenic po- dentine-producing cells (odontoblasts) of teeth. However, trunk neu- tential of trunk neural crest cells in a taxon that has retained ral crest cells are generally regarded as nonskeletogenic, and so the typical dentine in their postcranial dermal skeleton—a cartilag- embryonic origin of trunk denticle odontoblasts remains unresolved. inous fish, the little skate, Leucoraja erinacea. Here, we use expression of FoxD3 to pinpoint the specification and emigration of trunk neural crest cells in embryos of a cartilaginous Results fish, the little skate (Leucoraja erinacea). Using cell lineage tracing, The neural crest is a multipotent cell population that undergoes we further demonstrate that trunk neural crest cells do, in fact, give epithelial-to-mesenchymal transition from the developing central rise to odontoblasts of trunk dermal denticles. These findings expand nervous system of vertebrate embryos and that migrates exten- the repertoire of vertebrate trunk neural crest cell fates during nor- sively to give rise to a diversity of skeletal and nonskeletal cell mal development, highlight the likely primitive skeletogenic poten- types (18). Histological studies of shark (Scyliorhinus torazame) tial of this cell population, and point to a neural crest origin of embryos have revealed that neural crest cells begin to emigrate dentine throughout the ancestral vertebrate dermal skeleton. from the cranial neural tube at developmental stage (S)18 and from the trunk neural tube at approximately stage S19 (ref. 19, skate | neural crest | denticle | evolution | skeleton with staging according to ref. 20). To verify the timing of trunk neural crest cell emigration in skate embryos, we conducted a awed vertebrates (gnathostomes) primitively possessed an series of mRNA in situ hybridization experiments to characterize Jextensive postcranial dermal skeleton, consisting of both a expression of the transcription factor, FoxD3—a neural crest basal osteogenic component (composed of acellular bone and/or specifier that is expressed in nascent and early migratory neural lamellar bone-like tissues) and a superficial odontogenic com- crest cells (21). At S18 (Fig. 2A), we observed FoxD3-expressing ponent (composed of dentine overlain by enamel or enameloid) cells (i.e., premigratory neural crest cells) in the dorsal trunk (1). Elaboration of this dermal skeleton is closely tied to the neural tube. We observed that, as in the shark, trunk neural crest diversification of gnathostomes, but the developmental and cells begin emigrating in skate embryos at S19, based on the evolutionary origins of this skeletal system remain unresolved. A appearance FoxD3-expressing cells undergoing epithelial-to- postcranial dermal skeleton is present in several extant gna- mesenchymal transition from the dorsal trunk neural tube (Fig. 2B). thostome lineages—e.g., in the form of scales, denticles, or — scutes of fishes (2) although these elements exhibit consider- Significance able histological variation and are often much reduced relative to the dermal armor of stem gnathostomes. For example, the vast The earliest mineralized skeleton of vertebrates was the der- majority of teleost fishes possess lightly mineralized bony scales mal skeleton: superficial armor of tooth-like skeletal units that lack typical dentine or enamel and that likely derive from composed of dentine and basal bone of attachment. Remnants the osteogenic component of the primitive gnathostome dermal of this dentinous armor have been retained as teeth in the skeleton. Conversely, cartilaginous fishes (sharks, skates, and head of all jawed vertebrates and as dermal denticles in the rays) possess well-mineralized dermal denticles (Fig. 1) that are skin of cartilaginous fishes (sharks and skates). Cranial neural composed of typical dentine and enameloid, but that lack basal crest cells (NCCs) give rise to dentine-secreting odontoblasts of bone of attachment (3). teeth. However, trunk NCCs are regarded as nonskeletogenic, Bone and dentine are both mesenchymal in origin and are raising questions about the embryonic origin of postcranial secreted by osteoblasts and odontoblasts, respectively, while denticles in cartilaginous fishes. Here, we show that trunk enamel matrix is secreted by epithelial ameloblasts (with NCCs give rise to trunk denticle odontoblasts in the skate, “ ” enameloid resulting from the mineralization of mixed dentine Leucoraja erinacea. This finding expands the repertoire of and enamel matrices; ref. 4). Based on comparison with the trunk NCC fates, highlighting the primitive skeletogenic po- cranial dermal skeleton, in which much of the dermal bone (i.e., tential of this cell population. of the skull vault) and all dentine (i.e., of oral teeth) is neural crest-derived (5–7), it has long been assumed that these tissues Author contributions: J.A.G. designed research; J.A.G. and K.E.C. performed research; share a similar neural crest origin in the trunk (8, 9). However, J.A.G., E.C.A., and K.E.C. analyzed data; and J.A.G. wrote the paper. recent fate-mapping studies in teleosts have called this assump- The authors declare no conflict of interest. tion into question: cell lineage-tracing experiments in zebrafish This article is a PNAS Direct Submission. (10, 11) and medaka (12) have demonstrated a paraxial meso- Published under the PNAS license. dermal origin of scale-producing osteoblasts, leading to sugges- Data deposition: The sequence reported in this paper has been deposited in the GenBank tions that trunk neural crest cells lack skeletogenic potential in database (accession no. MF281542). fishes (10, 12, 13). However, the embryonic origin of the denti- 1To whom correspondence should be addressed. Email: [email protected]. nous component of the postcranial dermal skeleton remains This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. unresolved. While it has been suggested that teleost scales are 1073/pnas.1713827114/-/DCSupplemental. 13200–13205 | PNAS | December 12, 2017 | vol. 114 | no. 50 www.pnas.org/cgi/doi/10.1073/pnas.1713827114 Downloaded by guest on September 26, 2021 BIOLOGY DEVELOPMENTAL Fig. 1. Dermal denticles of the little skate. (A) A skate hatchling and (B) skeletal preparation exhibit prominent dorsal median (arrow) and paired dorso- lateral rows (arrowheads) of dermal denticles extending the length of the trunk. (C) These denticles (*) are recurved, with a tooth-like morphology. (D)A sagittal section through a differentiated dermal denticle (preeruption) at embryonic stage 33 reveals a pulp cavity filled with mesenchyme (Pulp in Di), a morphologically distinct layer of dentine-secreting odontoblasts (Odont. in Di) lining the wall of the pulp cavity, and a layer of cuboidal enamel-secreting ameloblasts (Amel. in Di). (Scale bars: A and B, 1 cm; C,750μm; D,20μm; Di,10μm.) This migration continues through S22 (Fig. 2C), at which point polarized secretory cells with characteristic cell processes lying ad- we also begin to observe rostral differentiation of trunk neural jacent to newly secreted dentine matrix (3) (Fig. 1D). In total, we crest derivatives (e.g., dorsal root ganglia). recovered 15 CM-DiI–labeled trunk denticle odontoblasts in em- To label premigratory trunk neural crest cells in skate embryos, bryos that were examined histologically (n = 8; Fig. 3 E and F— we microinjected the lipophilic dye CM-DiI into the lumen of the additional examples provided in Fig. S1), indicating their neural trunk neural tube at S18 (Fig. 3A). This strategy ensures focal la- crest origin. Seven out of eight examined embryos possessed CM- beling of the neural tube (including premigratory neural crest cells) DiI–labeled odontoblasts, with each positive embryo possessing and has been used extensively in other systems to test neural crest between one and four labeled cells (Table S1). CM-DiI–labeled cell fates (22–26). Five days postinjection, labeled neural crest cells odontoblasts always occurred singly, with their rarity likely due to could be observed emigrating from the trunk neural tube of CM- dilution of the label through substantial proliferation between DiI–injected skate embryos (Fig. 3B). Embryos with CM-DiI–la- labeling and analysis. beled neural tubes were left to develop for 4–5 mo (to S33, when Some embryos (n = 4) possessed CM-DiI–labeled cells within dermal denticles have differentiated within the trunk), at which tissue of the denticle pulp cavity, and some (n = 4) also possessed point, histological examination