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.

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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 revealed CM-DiI–labeled cells in occasional CM-DiI–positive ameloblasts (Fig. S2 and Table S1). expected trunk neural crest derivatives, such as dorsal root ganglia While tooth ameloblasts are generally regarded as an exclusively (Fig. 3C) and melanocytes (Fig. 3D), as well as in the odontoblast ectodermally derived cell type (6), one genetic lineage-tracing layer of trunk dermal denticles. study using P0-Cre/R26R mice previously suggested that a small In cartilaginous fish dermal denticles, the odontoblast layer is number of tooth ameloblasts might, in fact, derive from neural morphologically distinct within the pulp cavity and consists of crest cells (27). Our observation of CM-DiI–positive ameloblasts

Gillis et al. PNAS | December 12, 2017 | vol. 114 | no. 50 | 13201 Downloaded by guest on September 26, 2021 Fig. 2. Trunk neural crest cell migration in the little skate. (A) Wholemount mRNA in situ hybridization for the neural crest specifier FoxD3 at embryonic stage 18 reveals neural tube expression in the cranial and rostral trunk dorsal midline, as well as in the pharyngeal and gut endoderm. At this stage, cross- sections through the rostral trunk (Ai and Aii) reveal no emigrating neural crest cells, although premigratory neural crest cells may be recognized by their expression of FoxD3 within the dorsal neural tube. (B) At stage 19, FoxD3 expression persists in the developing central nervous system and endoderm, and cross-sections through the rostral trunk (Bi and Bii) now reveal FoxD3-expressing neural crest cells emigrating from the dorsal neural tube. (C) By stage 22, FoxD3 expression in the cranial region localizes to the mandibular (m), hyoid (h), and branchial (b) streams of cranial neural crest cells, while expression persists in the dorsal midline of the trunk. A row of trunk neural crest-derived dorsal root ganglia (DRG, *) may now be recognized in the rostral trunk, and cross-sections through the trunk just beyond the front of DRG differentiation (Ci and Cii) reveal continued emigration of FoxD3-expressing trunk neural crest cells from the dorsal neural tube. (Scale bars: A–C, 375 μm; Ai, Aii, Bi, Bii, Ci, and Cii,10μm.)

in chondrichthyan trunk dermal denticles could be indicative of a Discussion neural crest contribution to the ameloblast layer of dental organs It has been demonstrated that the trunk neural crest cells of within the vertebrate dermal skeleton. However, we do observe mouse (28), chick (29), and axolotl (30) may be pushed toward a signs of ectodermal contamination in embryos with CM-DiI– skeletogenic fate through transplantation or recombination with labeled neural tubes (a result of colabeling of some surface ec- cranial epithelia in vitro, and it has been suggested that this re- toderm at the site of trunk neural tube injection), and we flects the vestigial skeletogenic potential of trunk neural crest therefore cannot rule out ectodermal contamination as the basis cells that is suppressed in tetrapods. Our demonstration of a of these labeled ameloblasts. neural crest origin of trunk dermal denticles during normal de- When labeling the trunk neural tube with CM-DiI, we would velopment in a cartilaginous fish is consistent with this view and sometimes contaminate adjacent paraxial mesodermal cells (as points to the likely primitive skeletogenic potential of trunk evidenced by the presence of some CM-DiI–positive chon- neural crest cells in vertebrates. Given the mesodermal origin of drocytes within the axial skeleton of S33 embryos). Given the teleost scales (a likely derivative of the basal-most bony layer paraxial mesodermal origin of teleost scales (10–12), we sought of the ostracoderm dermal skeleton), we hypothesize that the to specifically test for mesodermal contributions to skate trunk multilayered dermal armor of stem gnathostomes may have consisted of a basal, mesodermally derived osteogenic compo- dermal denticles. In three S18 skate embryos, we focally labeled nent and a superficial, neural crest-derived odontogenic com- the presomitic paraxial mesoderm of the trunk (Fig. S3A), which ponent (Fig. 4), with each of these layers being variously sits ventrolateral to the neural tube (Fig. S3B) and again left – retained, reduced, or lost in different vertebrate lineages. The embryos to develop until S33. Upon examination, no CM-DiI scales of some nonteleost actinopterygian fishes, such as Poly- labeled odontoblasts or pulp cavity cells were observed in the pterus, appear to have retained both the osteogenic and odon- denticles of any of these embryos, despite extensive labeling of togenic components of the postcranial dermal skeleton (15), other paraxial mesodermal derivatives, such as chondrocytes of rendering them an important model system with which to further the axial skeleton (Fig. S3C) and pectoral girdle (Fig. S3D), and test this hypothesis. trunk musculature (Fig. S3E). These experiments therefore It has recently been demonstrated that several well-established provide no evidence of a paraxial mesodermal contribution to neural crest derivatives in both the head (e.g., tooth odontoblasts— the trunk denticles of the little skate. ref. 31) and the trunk (e.g., melanocytes, parasympathetic ganglia,

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Fig. 3. A trunk neural crest origin of skate dermal denticles. (A) Premigratory trunk neural crest cells were labeled by microinjection of CM-DiI into the lumen of the trunk neural tube at stage 18. (B) This approach effectively labeled trunk neural crest cells, and at 5 d postinjection, CM-DiI–labeled neural crest cells were observed emigrating from the trunk neural tube. By stage 33, histological analysis revealed CM-DiI–labeled cells within anticipated trunk neural crest derivatives, such as (C) dorsal root ganglia (Ci is the same section as C, subsequently stained with Masson’s trichrome) and (D) melanocytes (Di is the same section as D, subsequently imaged with light). Following labeling of trunk neural crest cells, (E and F) CM-DiI–labeled odontoblasts were also recovered within trunk dermal denticles, indicating their neural crest origin. Ei and Eii and Fi and Fii are images of the same sections, imaged initially with fluorescence (CM-DiI with DAPI counterstain) and subsequently with Masson’s trichrome. CM-DiI–labeled odontoblasts are indicated with white arrows and dashed outline. (Scale bars: A, 375 μm; B,20μm; C and Ci,20μm; D and Di,10μm; E,60μm; Ei and Eii,20μm; F,75μm; Fi and Fii,15μm.)

and enteric neurons—refs. 26 and 32−34) arise from neural postcranial skeleton of cartilaginous fishes, our current findings crest-derived Schwann cell precursors associated with nerve fi- support the notion that the odontoblast is a potentially uniquely bers. If trunk denticle odontoblasts share a similar Schwann neural crest-derived skeletal cell type (35) and highlights the neural cell precursor origin, such a cell lineage bottleneck (along with crest as the source of the dentine throughout the primitive verte- dilution of CM-DiI through extensive proliferation) could ac- brate dermal skeleton. count for the small number of CM-DiI–positive odontoblasts observed here. While further cell lineage-tracing experiments Materials and Methods using indelible labels (e.g., genomically integrated fluorescent Embryo Collection and Fate Mapping. Skate (Leucoraja erinacea)eggs reporters) will eventually allow us to more accurately assess the were obtained at the Marine Biological Laboratory and maintained in a flow- precise nature and extent of the neural crest contribution to the through seawater system at ∼16 °C. CM-DiI was prepared for microinjection

Gillis et al. PNAS | December 12, 2017 | vol. 114 | no. 50 | 13203 Downloaded by guest on September 26, 2021 Fig. 4. Hypothesis of embryonic origin and evolution of the vertebrate dermal skeleton. The primitive vertebrate dermal skeleton, as seen in heterostracans, was composed of a mesodermally derived osteogenic layer (Ost.) and a neural crest-derived odontogenic layer (Odont.). This dermal skeleton has undergone re- ductions and modifications through vertebrate phylogeny, including the loss of the osteogenic layer in cartilaginous fishes (e.g., skate) and the loss of the odontogenic layer in teleosts (e.g., zebrafish). Certain nonteleost bony fishes, such as the bichir, have retained both the osteogenic and odontogenic layers in their scales, and we predict that the former would be mesodermal in origin, while the latter would be neural crest-derived.

as described (36). For injection of S18 skate embryos, eggs were removed develop in a flow-through seawater system until S33 (∼4–5 mo post- from the water, briefly dried, and a small window was cut in the top injection), at which point embryos were euthanized with an overdose of surface of the egg case with a razor blade. CM-DiI was microinjected MS-222 (1 g/L), fixed in 4% paraformaldehyde in phosphate-buffered sa- through the jelly layer into the lumen of the neural tube or the paraxial line (PBS) overnight at 4 °C, rinsed three times in PBS, and stored in PBS + mesoderm using a Picospritzer pressure injector, and the window was then 0.02% sodium azide at 4 °C. All animal work complied with protocols sealed completely using a piece of donor egg shell adhered with a cya- approved by the Institutional Animal Care and Use Committee at the noacrylate adhesive (“Krazy Glue”). All injected embryos were left to Marine Biological Laboratory (MBL).

13204 | www.pnas.org/cgi/doi/10.1073/pnas.1713827114 Gillis et al. Downloaded by guest on September 26, 2021 Skeletal Preparation, Histology, and mRNA in Situ Hybridization. Whole-mount ACKNOWLEDGMENTS. We acknowledge Dr. Kate Rawlinson, Prof. Richard skeletal preparations and paraffin sections were produced as previously Behringer, Prof. Alejandro Sánchez Alvarado, Prof. Jonathan Henry, the MBL described (37, 38), and histochemical staining of paraffin sections was per- embryology community and the staff of the MBL Marine Resources Center formed according to the Masson’s trichrome protocol of Witten and Hall (39) for facilitating this research, and Dr. Clare Baker for helpful comments on or according to a standard hematoxylin and eosin (H&E) staining protocol. the manuscript. We also acknowledge technical support and microscope Sections of CM-DiI–injected embryos were coverslipped with Fluoromount loans from Leica Microsystems at MBL. This research was supported by Royal G + DAPI, imaged with fluorescence, and then decoverslipped (by soaking Society University Research Fellowship UF130182 (to J.A.G.) and by the Uni- overnight in water) and stained with Masson’s trichrome. Wholemount and versity of Cambridge Isaac Newton Trust Grant 14.23z (to J.A.G.). K.E.C. was paraffin section mRNA in situ hybridization for FoxD3 (GenBank Accession supported by Royal Society Shooter International Postdoctoral Fellowship Number MF281542) was performed as previously described (36). NF160762.

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