Journal of Hard Tissue Biology 26[4] (2017) 399-404 2017 The Hard Tissue Biology Network Association Printed in Japan, All rights reserved. CODEN-JHTBFF, ISSN 1341-7649 Original Nature of Apatite Crystals in the Tooth of Eusthenopteron from Devonian

Hiroyuki Mishima1), Mitsuo Kakei2), Ichiro Sasagawa3) and Yasuo Miake4)

1) Department of Dental engineering, Tsurumi University School of Dental Medicine, Kanagawa, Japan 2) Tokyo Nishinomori Dental Hygienist College, Tokyo, Japan 3) Advanced Research Center, The Nippon Dental University School of Life Dentistry at Niigata, Niigata, Japan 4) Department of Histology and Developmental Biology, Tokyo Dental College, Tokyo, Japan (Accepted for publication, August 28, 2017) Abstract: Eusthenopteron come under the rhipidistians. Little information is available regarding the ultrastructure and properties of tooth in Eusthenopteron. The purpose of the present study is to examine the nature of apatite crystals in the tooth of Eusthenopteron. Backscattered electron image of SEM revealed the tooth consisted of two layers, tentatively named as the bright surface layer and the dark inner dentin layer, respectively. The surface layer was more calcifi ed than the inner dentin layer. The incremental lines were not observed in the surface layer. Narrow dentinal tubules were confi rmed in the inner dentin layer. TEM study demonstrated the crystals of surface layer were not bearing the central dark lines (CDL-free type) in its structures. By contrast, the crystals of the inner dentin layer possessed the central dark lines (CDL-bearing type). X-ray diff raction analysis suggested that the crystal was fl uorapatite in the surface layer, and a mixture of hydroxyapatite and fl uorapatite in the inner dentin layer. The presence of fl uorapatite in the dentin was estimated to be the infl uence of the fossilization. Using EPMA, F, Al, Si, Ca, and P were detected in the surface layer, and F, Na Mg, Si, Ca, and P were detected in dentin layer. The weight % F of the surface layer was 3.07, and 3.35 in the inner dentin layer. Raman spectrum analysis demonstrated that the phosphate peaks of 965 cm-1 assigned for hydroxyapatite in the inner dentin layer and 967 cm-1 assigned for fluorapatite in the surface layer were detected, respectively. Taking the crystallographic viewpoint and histological feature into consideration, the surface layer was regarded as enameloid and the inner dentin layer was orthodentin including plicidentin.

Key words: Tooth, Eusthenopteron, Fluoraptite, Enameloid

Introduction Materials and Methods Eusthenopteron foodi, a Devonian lobe-fi nned fi sh lived 385 million The tooth and the jaw bone (premaxilla, maxilla, and dentary) of years ago, come under the rhipidistians1). Shellis described that the Eusthenopteron foodi (Miguasha Formation, Devonian, Quebec, Canada) Rhipidistia are a very important group of the main line of vertebrates, were studied in this study. The ground sections and ultrathin sections because they are a transition between placoderms and amphibians2). were prepared from these samples. These specimens were examined Eusthenopteron foodi is the tetrapod stem group in early tetrapod using a transmission electron microscopy (TEM, JEM 100CX, JEOL) body plan evolution3). The dermal exoskeleton or scale was classified and a scanning electron microscopy (SEM, S-2380N, Hitachi Co, Toyo, into the thin outer layer of ganoin (enameloid), cosmine (dentin), the Japan and JSM-6340, JEOL Ltd, Tokyo Japan). vascular layer (spongy bone), and the isopedin layer (laminar bone)1,4). Analysis of specimens was conducted by using a laser Raman The cosmine, which is equivalent to the scales of some ancient fish microprobe spectrometry (Raman rxn systems, Kasier optical systems), (Megalichthys), is considered to be a continuous layer of dentin1,5). But, an electron-probe microanalyzer (EPMA, JXA-8200, JEOL), and the Zylberberg et al. stated that the disappearance of both enamel/enameloid x-ray diff raction method (RINT2000, RIGAKU, Tokyo, Japan). and dentin from scales might be related to the evolutional trend towards a To prepare the samples for TEM observation, they were dissected lightening of scales in Eusthenopteron6), although some features, such as into small pieces, dehydrated by passage through a series of ascending the structure of dermal bone, remain in argument in Eusthenopteron. ethanol concentrations, and embedded in Araldite 502 resin. Thin The tooth of rhipidistians are characterized by the presence of the sections (about 10nm thickness) were obtained using a Porter-Blum MT- complex folding of the dentin 1,2,4,7) However, little information is 2B ultramicrotome (Sorvall, USA.) equipped with a diamond knife. available regarding the ultrastructure and properties of tooth crystal The sections were examined under a JEM 100CX transmission electron in Eusthenopteron so far8). It has been reported that enamel might be microscope (JEOL) at an accelerating voltage of 80 kV. covering the surface of Eusthenopteron tooth8,9). Smith reported that Single-side ground sections were applied for SEM study. Polished enameloid was a more recent phylogenetic development than enamel10). sections were fi rst subjected for SEM observation. Subsequently, sections However, the phylogenesis origin of the enamel and enameloid, is still were etched for 30 seconds in 5% HCl, then they were again subjected to under discussion11-13). Furthermore, the ultrastructure of the enamel apatite SEM study. in Eusthenopteron is not well known9). The purpose of the present study Using the EPMA equipped with Scanning Electron Microscope- is to examine the nature of apatite crystals in the tooth of Eusthenopteron. Energy Dispersive Spectrometry (SEM-EDS) and Scanning Electron Microscope-Wavelength Dispersive X-ray Spectroscopy (SEM-WDS), Correspondence to: Dr. Hiroyuki Mishima, Department of Dental Engineering, the chemical compositions were analyzed. Spot mode analysis to clarify Tsurumi University School of Dental Medicine, 2-1-3 Tsurumi, Tsurumi-ku, Yokohama, 230-8501, Japan; Tel: 81-45-580-8369; Fax: 81-45-573-9599; the elements was performed by SEM-EDS at an accelerating voltage of E-mail: [email protected] 15kV and the measuring time was 60 seconds. Elemental mapping of 399 J Hard Tissue Biology Vol. 26(4):399-404, 2017

Figure 1. Micrographs of ground sections of tooth of Eusthenopteron foodi. Longitudinal section shows the dentinal tubules (arrows) of the tooth (a), and cross section reveals the complex folding of the dentin at the base of tooth (b). The surface layer (S) and the inner dentin layer (D) were observed. (a): bar = 500 μm, (b): bar = 100 μm.

Figure 2. Secondary electron images of SEM. After polishing, longitudinal Figure 3. Backscattered electron image of SEM. The surface layer (S) is more section shows the socketed tooth. After polishing, 30 seconds etching in 5% calcifi ed than the inner dentin layer (D). Longitudinal ground section shows the HCl. dentinal tubules and its lateral branches.

area (396 × 375 μm) was carried out by using SEM-WDS with 15kV accelerating voltage, and the measuring time was 3 hours. The laser Raman microprobe analysis was carried out under the condition of a wave length of 532 nm, and 1μm spot. The analytical time was 10 seconds. The x-ray diffraction analysis was performed under conditions of 40kV, 200mA, using a fi lter kβ. The irradiation time of the x-ray was 300 seconds. The collimator diameter was 100μm.

Results On the longitudinal ground section, the dentinal tubules (arrows) were confi rmed in the inner dentin layer of the tooth (Fig. 1a). On the cross ground section, the complex folding of the dentin was recognized at the base of the tooth (Fig. 1b). The surface layer (S) and the inner dentin layer were observed in the tooth (Fig. 1b). In the secondary electron images of SEM, the socketed tooth was appeared in the jaw bone (premaxilla, maxilla, and dentary). The tooth was tightly attached to the Figure 4. Secondary electron images of SEM of the surface layer. Longitudinal spongy bone (Fig. 2). ground section reveals the tooth consisting of two layers (a). The crystals and The backscattered electron image of SEM revealed the tooth consisted the stomas (arrows) were observed in the surface layer (b and c). The crystal was running almost vertically (c). S: the surface layer, D: the inner dentin layer. of two layers in the longitudinal ground section (Fig. 3). The surface After polishing, 30 seconds etching in 5% HCl. layer (S) showed higher electron density than the inner dentin layer. The inner dentin layer possessed many dentinal tubules. The dentinal tubules showed an average diameter of 1 μm. The lateral branches of dentinal 400 Hiroyuki Mishima et al.: Apatite Crystals in the Tooth of Eusthenopteron

1000 a 800 b

s 800 s

t t 600 n n u u o o

c 600 c

y y

t t 400 i i s s

n 400 n e e t t n n

I I 200 200

0 0 20 30 40 50 60 70 20 30 40 50 60 70 2 2 Figure 5. X-ray diff raction patterns of the surface layer and dentin. Arrows show fl uorapatite (a) and hydroxyapatite (b): a: the surface layer, b: dentin.

980000 a a

975000 s t n u o c

970000 y t i s n e t 965000 n I

960000 860 880 900 920 940 960 980 1000 cm-1 710000

b s t 705000 n u o c

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i 700000 s n e t n I 695000

690000 860 880 900 920 940 960 980 1000 cm-1

3- Figure 6. Backscattered electron image and element mapping images of Figure7. Raman spectrum of the surface layer and inner dentin. A PO4 peak -1 3- ground section of teeth. The teeth consisted of 2 layers (surface layer of 967cm (arrow) was detected in the surface layer (a), and a PO4 peak of and dentin) (a). CP: backscattered electron image, F: F mapping, Ca: Ca 965cm-1 (arrow) was detected in the inner dentin (b). mapping. P: P mapping.

Figure 8. Electron micrographs of the crystals of the surface layer and dentin, respectively. The crystals of surface layer show no CDL (a), while the crystals of dentin possess the CDLs (b). Arrows indicate CDL. Bar = 10 nm. tubules were also confirmed. Secondary electron image of SEM, the towards the surface. The stomas (arrows) were observed in the surface surface layers (S) showed an average thickness of 4.5 μm (Fig. 4a). The layer, and the diameters of stomas were estimated to 0.3-0.5μm (Fig. 4b, incremental lines were not observed in the surface layer (Fig. 4b, c). The c). crystals were linearly arranged from the boundary of the inner dentin X-ray diff raction analysis revealed that the crystal of the surface layer 401 J Hard Tissue Biology Vol. 26(4): 399-404, 2017 formation16). One is octacalcium phosphate (OCP) pathway appeared in the early Cambrian period, and the other is CDL pathway developed around the Silurian period16). In general, the involvement of odontoblast process into enameloid is taken place2,17). The stomas present in the surface layer, which were considered to be the marks of odontoblast processes, disappeared during the fossilization. The crystals of dentin and bone possess the CDL- bearing crystals18-19). Kakei et al. reported that the microstructure of apatite crystals might remain considerably stable during the course of fossilization20-21). X-ray diff raction analysis revealed that the crystal was fl uorapatite in the surface layer of tooth of Eusthenopteron foodi. The present study showed that the weight % F of the surface layer was 3.07 in tooth. F in natural mineral of fluorapatite showed 3.831 ± 0.061 wt%22). Enameloid of blue sharks showed 3.047 ± 0.360 wt % of F content11,22), though all sharks have fl uorapatite crystal23). Mature tooth 23) Figure 9. Electron micrograph of bone crystal of Eusthenopteron foodi. T of cichlid enameloid crystals showed 3.2% fl uoride content . F content The central dark line can be seen (arrow). Bar =10 nm. was 3.35 wt% on average in the inner dentin layer. In the process of fossilization, it is presumed that F in the seawater penetrated in dentin through the dentinal tubule. was fl uoraptite. (Fig. 5a). The crystal of the inner dentin layer mainly was Raman microprobe analysis showed two peaks of 965 cm-1 and 967 hydroxyapatite (Fig. 5b). But, the peaks of fl uorapatite also coexisted in cm-1, assigned for phosphate ion 11,22,24). In general, the PO 3- value of the dentin. 4 hydroxyapatite is around 960-964cm-1, and the PO 3- value of fl uorapatite From SEM-EDS and SEM-WDS analyses of the surface layer, F, 4 in general is higher than those of hydroxyapatite11,22,25-26). Therefore, Al, Si, Ca, and P were detected. Ca/P ratio was 1.97 on average, and a peak of 967cm-1 in the surface layer was judged for phosphate of F content was 3.07 wt % on average. In the inner dentin layer, F, Na, fl uorapatite, while a peak of 965cm-1 in the inner dentin layer was judged Mg, Si, Ca, and P were detected. Ca/P ratio was 2.10 on average, and F for phosphate of hydroxyapatite. content was 3.35 wt% on average. Backscattered electron image, and the From the crystallographic viewpoint and histological feature, the image element mapping of Ca and P, revealed that the tooth was divided surface layer of tooth was regarded as enameloid like Selachii and into two layers, the surface (S) and the inner dentin layers (D) (Fig. 6a, c, Actinoptergii, because this layer was occupied by fluorapatite. The d). The image of F mapping was not clearly divided into two layers (Fig. crystal of dentin consists generally of hydroxyapatite23,26), therefor the 6b). The dentinal tubules were observed in the inner dentin layer (Fig. presence of fluorapatite in the inner dentin layer might be the result 6a). 22) -1 of fossilization . The inner dentin layer might be considered to be From Raman analysis, a peak of 967cm (arrow) assigned for orthodentin with branching dentinal tubules and including plicidentin2,27). phosphate was detected in the surface layer (Fig. 7a), while a peak of -1 In the future, we need to conduct and verify genetic and molecular 965cm (arrow) assigned for phosphate was detected in the inner dentin analyses on the tooth of Eusthenopteron foodi. layer (Fig. 7b). Electron micrographs showed that the surface layer contained crystals Acknowledgements without the central dark line (CDL-free crystals) (Fig. 8a), while the inner This study was performed under the cooperative research program dentin layer consisted of CDL-bearing crystals (Fig. 8b, arrows). The of the Center for Advanced Marine Core Research (CMCR), Kochi lattice (100) were present in the crystal. Also, CDL-bearing crystals could University (10A011, 10B011, 11A004, 11B004, 12B035, 13A018, be seen in the bone (Fig. 9, arrow). 13B015, 14A027, 14B025, 15A021, 15B018, 16A006, 16B006). This work was supported by JSPS KAKENHI Grant Number JP15K11034. Discussion The infolded dentin (plicidentin) exists around the pulp cavity Confl ict of Interest at the base of a tooth in sarcopterygians, basal tetrapods, and 1,4,7) The authors have declared that no COI exists. actinopterygian . The presence of the complex folding of the dentin (labyrinthodont teeth) at the base of tooth was also confi rmed in the tooth References of Eusthenopteron. The two types of tooth attachment of the jaw such 14) 1. Carrol RL. Vertebrate paleontology and evolution. WH Freeman and as sub-thecodont and thecodont are reported in mosasaurids . The sub- Company, New York, 1988, pp 136-148. thecodont is anykylosis and has a shallow socket. On the other hand, 7) 2. Shellis P. Comparative histology of dental tissues. In: Dental the thecodont is not anykylosis and is deeply rooted in the socket . On Anatomy and Embryology, ed by Osborn J W, Blackwell Scientifi c the basis of the present fi ndings, all teeth of Eusthenopteron foodi were Publications, Oxford, 1981, pp 58-165. socketed and directly joined to the bone, suggesting that the type of tooth 3. Pardo JD, Szostakiwskyj M, Ahlberg PE and Anderson JS. Hidden attachment of Eusthenopteron foodi might be the sub-thecodont. morphological diversity among early tetrapods. Nature 546: 642-645, The incremental lines of surface layer, commonly seen in tooth enamel 15) 2017 and not in enameloid , could not be confi rmed in Eusthenopteron foodi. 4. Lumsden AGS. Evolution and adaptation of the vertebrate mouth. Further, the crystals of surface layer lacked of CDLs in its structures. In: Dental Anatomy and Embryology, ed by Osborn JW, Blackwell Regarding the difference between enameloid and enamel crystals, Scientifi c Publications, Oxford, 1981, pp 88-117. TEM studies of fossil hard tissues revealed that two different types of 5. Meinke DK and Thomson KS. The distribution and signifi cance of biologically-induced apatite crystals have evolved independently during and enameloid in the dermal skeleton of osteolepiform rhipidistian the early geological periods, suggesting two diff erent pathways for apatite 402 Hiroyuki Mishima et al.: Apatite Crystals in the Tooth of Eusthenopteron fi shes. Paleobiology 92: 138-149, 1983 underlying crystal structure defects, and the pathological calcifi cation 6. Zylberberg L, Meunier FJ and Laurin M. A microanatomical and event. J Fossil Res 48: 53-65, 2016 histological study of the postcranial dermal skeleton in the Devonian 17. Sasagawa I and Ishiyama M. The structure and development of the sarcopterygian Eusthenopteron foordi. Acta Palaeontol Pol 55: 459- collar enameloid in two teleost fi shes, halichoeres poecilopterus and 470, 2010 pagrus major. Anat Embryol 178: 499-511, 1988 7. Maxwell EE, Caldwell MW and Lamoureux DO. The structure and 18. Kakei M, Sakae T and Yoshikawa M. Electron microscopy of phylogetic distribution of amniote plicidentine. J Vertebr Paleontol octacalcium phosphate in the dental calculus. J Electron Microsco 31: 553-561, 2011 58: 393-398, 2009 8. Smith MM. Distribution and variation in enamel structure in the 19. Kakei M, Sakae T, Mishima H and Yoshikawa M. Ultrastructure of oral teeth of sacopterygians: its significance for the evolution of a apatite crystals formed during vascular calcification in Humans. J protoprismatic enamel. Historical Biol 3: 97-126, 1989 Hard Tissue Biol 18: 135-140, 2009 9. Matton O, Cloutier R and Stevenson R. Apatite for destruction: 20. Kakei M, Nakahara H, Kumegawa M, Mishima H and Kozawa Y. Isotopic and geochemical analyses of bioapatites and sediments High-resolution electron microscopy of the crystallites of fossil from the upper devonian escuminac formation (Miguasha, Quebec). enamels obtained from various geological ages. J Dent Res 80: 1560- Palaeogeogra Palaeoclimatol Palaeoecol 361-362:73-83, 2012 1564, 2001 10. Smith MM and Coates MI. Evolutionary origins of teeth and Jaws: 21. Kakei M, Nakahara H, Kumegawa M, Mishima H and Kozawa developmental modes and phylogenetic pattern. In: Development, Y. Ultrastuctural study on the lattice images of calcium phosphate Function and Evolution of Teeth. ed by Teaford MF, Smith MM and minerals in fossil tooth. In: Biomineralization (BIOM2001); Ferguson MWJ, Cambridge University Press, Cambridge, 2000. pp Formation, Diversity, Evolution and Application, ed by Kobayashi I 133-151. and Ozawa H, Tokai Univ Press, Kanagawa, 2004, pp 364-368. 11. Mishima H,Kakei M, Yasui T, Miyamoto S, Miake Y and Yanagisawa 22. Mishima H, Kakei M, Kitahara M, Yasui T and Miake Y. Analysis T. Apatite crystal in hard tissue of conodont fossils. Front Mater Sci of structure on hard tissue of conodont fossil from Silurian to China 2: 179-181, 2008 Carboniferous. J Fossil Res 42: 193-200, 2010 12. Kawasaki K and Amemiya CT. SCPP Genes in the Coeacanth: Tissue 23. Lowenstam HA, Weiner S. On biomineralization. Oxford University by Sarcopterygians. J Exp Zool (Mol Dev Evol) 322B: 390-402, Press, Oxford, 1989, pp 180-186. 2014 24. Mishima H, Oono Y, Nakaishi Y, Waziki S and Nomura K. 13. Qu Q, Haitina T, Zhu M and Ahlberg PE. New genomic and fossil Apatite crystal in dentin among monophyodont, diophyodont and data illuminate the origin of enamel. Nature 526: 108-111, 2015 polyphyodont teeth. J Oral Tissue Engin 4: 43-50, 2006 14. Caldwell MW, Budney LA and Lamoureux DO. Histology of tooth 25. Penel G, Delfosse C, Descamps M and Leroy G. Composition of attachment tissues in the late Cretaceous mosasaurid Platecarpus. J bone and apatitic biomaterials as revealed by intravital Raman Vertebr Paleontol 23: 622-630, 2003 microspectroscopy. Bone 36: 893-901, 2005 15. Sasagawa I, Ishiyama M and Kodera H. Fine structure of the 26. Elliott JC. Structure and chemistry of the apatite and other calcium pharyngeal teeth in the coelacanthid fish (Latimeria chalumnae). orthophosphates. Elsevier, Amsterdam, 1994, pp 259-298. In: Tooth Enamel IV, ed by Feranhead EW and Suga S, Elsevier 27. Smith MM and Sansom IJ. Evolutionary origins of dentine in the Publishers BV, Amsterdam, 1984, pp 462-466. fossil record of early evolution of teeth, ed by Teaford MF, Smith 16. Kakei M., Yoshikawa M. and Mishima H. Aspects of the apatite MM and Ferguson MWJ, Cambridge University Press, Cambridge, crystal: Two pathways for apatite formation, the mechanisms 2000, pp 65-81.

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