sign in sign up
<<
Home , Bichir, Gill slit, Polypterus, Polypterus senegalus

Journal of Applied J. Appl. Ichthyol. 26 (2010), 179–182 Received: September 01, 2009 Ó 2010 Blackwell Verlag, Berlin Accepted: December 29, 2009 ISSN 0175–8659 doi: 10.1111/j.1439-0426.2010.01400.x

Short communication Mouth development in the senegalus does not involve the oropharyngeal membrane: possible implications for the ecto-endoderm boundary and tooth initiation By M. Kralovic, I. Hora´ cˇ ek and R. Cerny

Department of Zoology, Charles University in Prague, Czech Republic

Summary advocates that the cells of neural crest origin rather than any Polypterid fishes are considered the basal-most group of extant epithelium constitute a major agent of dental development actinopterygians and thus may serve as a direct link to (Soukup et al., 2008). understand the of the first bony fishes. Embryonic We decided to analyse early oral and dental morphogenesis and larval specimens, however, are extremely scarce, making it of the Senegal bichir () with special difficult to study their developmental patterns and processes. attention to the ecto-endoderm boundary and tooth initiation. During the past few years we collected many embryos and , as the basal-most lineage of Actinopterygian fishes larvae of the Senegal bichir Polypterus senegalus and in this (Noack et al., 1996; Kikugawa et al., 2004) occupy a key paper we describe some novel observations concerning the position in phylogeny and therefore are uniquely supposed ecto-endoderm border in the mouth and consecutive well suited for assessing the ancestral states of initiation of dental development. The mouth of the Senegal and , as well as the divergence of developmental bichir does not develop via a classical oropharyngeal mem- patterns between e.g. fishes and (Takeuchi et al., brane but instead, opening the mouth occurs via a separation 2009). Moreover, as the extant chondrichthyans (Neoselachii of the upper and lower that are connected by epithelial and ) are derived and their dental system is distant bridges. These structures are bilaterally symmetrical and are from the presumed primitive state (Carroll, 1988; Janvier, found invariantly at places of the earliest tooth bud develop- 1996), bichirs might be considered the most pertinent taxon for ment. It is suggested that the epithelial bridges may represent revealing the Gnathostome Bauplan and its respective devel- the ecto-endoderm bordering zone, are both structurally and opmental dynamics. In this paper, we will briefly describe some functionally homologous to the oropharyngeal membrane and novel observations concerning the supposed ecto-endoderm consequently it is hypothesized that the epithelial bridges and border in the mouth of the Senegal bichir and consecutive the developmental factors producing them play a key role in initiation of dental development. initiation and early distribution of the particular dental domains. Materials and methods Embryos of the Senegal bichir (Polypterus senegalus) were Introduction obtained from our breeding colony at Department of Zoology, The oral cavity of is generally thought to arise as Charles University in Prague. Embryos were anaesthetized in a an ingrowth of the oral ectoderm called the stomodeum solution of tricaine methane-sulfonate (MS-222, Sandoz) and (Balinsky, 1975; Kardong, 1995; Liem et al., 2000). Between fixed in 4% paraformaldehyde in 0.1 M phosphate-buffered the stomodeum and the lumen of the foregut alimentary canal, saline (PFA ⁄ PBS) at least overnight. After washing in PBS, a transient barrier, known as the oral, stomopharyngeal or specimens were dehydrated through a graded series of ethanol buccopharyngeal membrane forms. It is generally believed that and embedded in JB4 (Polysciences, Inc.). Sections (5 lm) opening the mouth in vertebrates proceeds by thinning of this were cut with a Reichert-Jung microtome (Biocut 2035), membrane, cell-intercalation and finally by breaking-through stained with Azure B–Eosin (Serva) and mounted in DPX (Dickinson and Sive, 2006). It has been recognized, however, (Serva). Sections were investigated under Olympus BX 51 that after the rupture and disappearance of this membrane it is microscope with a SPOT camera and optimized in Adobe not possible to locate the ecto-endoderm border (Soukup Photoshop software. et al., 2008). The ecto-endoderm boundary zone, however, is In total, 28 bichir embryos at particular stages were utilised often considered to play an important role in dentition in this study. Since bichir staging tables (Kerr, 1907; Bartsch initiation and ⁄ or patterning (e.g. Smith and Coates, 2001; et al., 1997) are incomplete and not detailed, we have prepared Smith, 2003; Huysseune et al., 2009) what is consistent with our own embryonic and larval staging system, which, however, the classical experiments in amphibian developmental biology will be comprehensively published elsewhere. Here, we will showing that epithelia of both ectoderm and endoderm origin only briefly describe those stages utilized for this study. All our are required for the production of teeth (e.g. Sellman, 1946; stages roughly correspond to BartschÕs yolk-sac larva (free Wilde, 1955; Graveson et al., 1997). Other evidence, however, embryonic stage, day 6) (Bartsch et al., 1997). The external

U.S. Copyright Clearance Centre Code Statement: 0175–8659/2010/2602–0179$15.00/0 180 M. Kralovic et al. distinguishing characteristics for the stages utilised here are the (a) following: Stage 1: total length (TL) between 5.5–6 mm; the mandibular arch is still not distinguishable from the ventral view. Stage 2: TL = 6.5–7 mm; opercular fold become visible; stage 3: TL = 7–7.5 mm, lower jaws begins to elongate anteriorly, cement glands become reduced.

Results and discussions For the purpose of this study, 28 embryos of the Senegal bichir at particular stages were thoroughly analysed in frontal, horizontal as well as on paramedial plastic sections (see (b) Material and methods). However, from the earliest stages investigated (embryonic period, pre-hatchling step of Bartsch et al., 1997), the mouth of the Senegal bichir was closed and no cavitation was observed (not shown). At stage 1 (Fig. 1a,b) the first, mandibular arch is already prominent, mouth epithelia are obviously thickened and mesenchyme cells are oriented perpendicularly to the oral epithelia (Fig. 1a). Epithelia at these early embryonic stages are double-layered as a rule, however, just in the mouth, only the basal epithelial layers can be observed with no apical layers (c) distinguishable (Fig. 1b, asterisks). At the following stage the mandibular arch is even more prominent and many more mesenchymal cells are observed to condense under the oral epithelium (Fig. 1c). Inside the mouth, the zone in between basal layers of the oral epithelia become more spaced and several small cavities can be seen (Fig. 1d; arrows). This process of the oral cavity opening by means of stretching the oral epithelia apart can be better observed on frontal sections (Fig. 2). At stage 2 there are only few small cavities in between the Fig. 2. Mouth cavitation and epithelial bridges as seen at stages 2 (a), 2–3 (b) and 3 (c). Frontal sections, the right side of the head is shown upper and lower oral epithelium (Fig. 2a; arrows). However, only. Arrows (a) indicate developing cavities in the mouth, arrows (b) when epithelia of the upper and lower parts of the mouth have point to protracting epithelial bridges. Arrowheads (b, c) point to separated further, the vacuities enlarge and epithelial strands developing tooth buds. cg, cement gland; den.t.b., dentary tooth buds; interconnecting the epithelial surfaces of the lower and upper epi, epithelium; max.t.b., maxillary tooth bud; mes, mesenchyme. jaws appear. We call these structures epithelial bridges bars, 100 lm (Fig. 2b; arrows). Finally, when the mouth is completely open (Fig. 2c), it is noticeable that the epithelial bridges are situated In total three epithelial bridges can be observed at each right just at the positions of the earliest oral tooth buds (compare and left half of the head at stage 2–3 (Fig. 3). The first one, Fig. 2b,c; arrowheads). situated at the rostro-lateral edge of the mandibular arch

(a) (b)

(c) (d) Fig. 1. The earliest stages of mouth opening in the Senegal bichir, as seen on parasagittal sections, head to the left, at stages 1 (a+b) and 2 (b+c). Asterisks (b) indicate non-distinguish- able apical epithelial layers in the mouth. Arrows (d) indicate developing cavities in between the upper and lower mouth parts. cg, cement gland; e, eye; epi, epithelium; mes, mesenchyme; og, outer ; ot, otic vesicle; y, yolk. Scale bars, 100 lm a+c; 50 lm b+d Mouth development in Polypterus senegalus 181

(a) (b)

(c) (d)

Fig. 3. Relative topography of the epithelial bridges and tooth initiation as seen on parasagittal sections taken from one particular specimen at stage 2–3, head to the left. The sections are ordered from the most lateral (a) to the most medial (d). ahy, adenohypophy- sis; cg, cement gland; cor.t.b., coro- noid tooth bud; den.t.b., dentary tooth bud; dpt.t.b., dermopalatinum tooth bud; h.a., hyoid arch; m.a., mandibu- lar arch; max.t.b., maxillary tooth bud; y, yolk. Scale bars, 100 lm

(Fig. 3a), represents the area of future maxillary teeth (notice the ectoderm (the anterior layer) and endoderm (the posterior the budding tooth on the upper ; arrowhead). The second layer). We therefore suggest that these epithelial bridges epithelial bridge, situated more posteriorly and medially represent the ecto-entodermal boundaries that are structurally (Fig. 3b) represents the area of future dentary tooth field homologous to the classical oropharyngeal membrane. Instead (notice the budding dentary tooth epithelium on the lower jaw; of ÔruptureÕ of a single membrane, however, mouth opening in arrowhead). On Fig. 3c, there are two epithelial bridges visible: the bichir occurs by schizocoelic-like separation of both mouth the anterior one represents some remnants of the dentary parts via fissure-like vacuities temporarily connected by the epithelial bridge (compare Fig. 3b, c) and the third bridge is above reported epithelial bridges. We put forward a hypothesis situated at the prospective coronoid tooth area (arrowhead). here that the epithelial bridges and ⁄ or the developmental Fig. 3d shows a section very close to the midline of the embryo factors producing them play a key role in initial topographic where, at the posteriormost edge of the mandibular arch, the constitution of the particular dental domains (maxillary, posterior part of the third epithelial bridge is seen (Fig. 3d; dermopalatium, dentary and coronoid tooth fields). arrowhead). Just in the midline of the embryo, however, there The process of early mouth formation described here for the is clearly a free entrance to the alimentary canal (60–80 lm Senegal bichir is quite different from the expected mode in thick; not shown). These epithelial bridges clearly represent other vertebrates (e.g. Balinsky, 1975; Kardong, 1995; Dick- precisely bilateral characteristics, i.e. they are situated accu- inson and Sive, 2006). Bichir mouth development, however, rately at the identical positions on both sides of the embryo. resembles the developmental mode known from urodele During the process of mouth development in the Senegal amphibians (e.g. Johnston, 1910; Adams, 1924; de Beer, bichir, no ectodermal ingrowth was observed that would 1947; Takahama et al., 1988). In the Mexican axolotl, for develop into a classical stomodeum, as seen e.g. in frogs example, the oral ectoderm does not invaginate, but instead, (Dickinson and Sive, 2006). Consequently, no clear oropha- only the basal layer of the oral ectoderm moves inwards to ryngeal membrane arises that would represent a single ecto- cover the surface of the mouth endoderm as the so called endoderm border and opening the mouth therefore does not stomodeal collar. Opening the mouth then leads to the happen via breaking of a vertical membrane. Instead, from the formation of epithelia, which consist of the ectodermal basal earliest stages investigated, the upper and lower parts of the layer and the endodermal apical layer, however, intercalation mouth are closely apposed. Later, as a consequence of of cells from different germ-layers was also observed (Soukup separating the upper and lower mouth parts, several narrow et al., 2008). Mouth formation via stomodeal collar also takes epithelial columns are developing which are described here as place in lungfishes, according to many authors (e.g. Kerr, the epithelial bridges. These bridges seem to be formed from 1902; Adams, 1924), In the South American lungfish Lepid- the apical lining of the oral epithelium and during the process osiren paradoxa, Kerr (1902) did not observe a true stomo- of mouth opening are protracting to finally tear across. It is of deum, the epithelium of the oral cavity seemed to him to be a great interest, however, that these epithelial bridges are formed from the endoderm layer and opening the mouth invariantly positioned at places of the earliest tooth bud cavity appeared by a process of Ôdegeneration and breaking development (Fig. 3). downÕ of some anterior cells (Kerr, 1902). It is, however, quite As yet, we are unable to establish the germ layer origin of difficult to get a more detailed view on lungfish mouth the oral epithelia for sure. The contextual topographic settings, development, simply because there are no such data available. however, suggest that oral epithelial linings derive from both Even more interestingly, in Ascaphus truei, a basal frog 182 M. Kralovic et al. with unusual ventrally placed sucker mouths, it was observed Graveson, A. C.; Smith, M. M.; Hall, B. K., 1997: Neural crest that there are some ectodermal bands running back along the potential for tooth development in an urodele amphibian: devel- gut wall from the mouth corners, which together with a opmental and evolutionary significance. Dev. Biol. 188, 34–42. Huysseune, A.; Van der heyden, C.; Verreijdt, L.; Wautier, K.; Van shallow stomodeum, clearly resembles a situation in Urodeles Damme, N., 2002: dentitions as paradigms for odontogenic (Reiss, 1997). Moreover, a comparable morphodynamics was questions. Connect. Tissue Res. 43, 98–102. also observed during slit formation in Huysseune, A.; Sire, J.-Y.; Witten, P. E., 2009: Evolutionary and (Edwards, 1929). Here, during the opening of the branchial developmental origins of the vertebrate dentition. J. Anat. 214, 465–476. membranes the ectoderm lining spreads into the whole Janvier, P., 1996: Early vertebrates. Oxford Univ. Press, Oxford. UK, oropharyngeal cavity over the surface of the endoderm pp. 390; ISBN-0198526466. epithelium resulting in the formation of double-germ-layer Johnston, J. B., 1910: The limit between ectoderm and endoderm in the epithelium with the apical ectoderm layer and basal endoderm mouth and the origin of taste buds in amphibians. Am. J. Anat. layer (Huysseune et al., 2002), i.e. a reciprocal situation to the 10, 41–67. Kardong, K. V., 1995: Vertebrates. Comparative , function, axolotl oral epithelium (Soukup et al., 2008). evolution. Brown. In any case, the mode of the earliest development of the Kerr, J. G., 1902: The development of Lepidosiren paradoxa III. mouth cavity in the Senegal bichir differs from the situation in Development of the skin and its derivatives. Q. J. Microsc. Sci. 46, the other vertebrate taxa studied (e.g. Dickinson and Sive, 418–459. Kerr, J. G. 1907: The development of Polypterus senegalus Cuv. by 2006). The essential differences are as following: (i) mouth J. Graham Kerr, University of Glasgow. In: The work of John opening forms by a schizocoelic-like split of the epithelial Samuel Budgett, Balfour Student of the University of Cambridge: layers forming simultaneously the epithelial bridges, (ii) there Being a Collection of His Zoological Papers, together with a is a clear topographical correlation between the bridges (i.e. Biographical Sketch by A. E. Shipley, F.R.S., and Contributions the presumptive ectodem-endoderm bordering zone) and the by Richard Assheton, Edward J. Bles, Edward T. Browne, J. Herbert Budgett and J. Graham Kerr. J. G. Kerr (Ed.). earliest tooth buds that constitute the first elements of the Cambridge University Press, Cambridge, pp. 195–290. respective tooth fields. At the moment we do not know which Kikugawa, K.; Katoh, K.; Kuraku, S.; Sakurai, H.; Ishida, O.; Iwabe, of the observed phenomena relate to a true plesiomorphic N.; Miyata, T., 2004: Basal jawed vertebrate phylogeny inferred conditions of the gnathostome dental development. However, from multiple nuclear DNA-coded genes. BMC Biol. 2, 1–11. Liem, K. F.; Grande, L.; Bemis, W. E. 2000: Functional anatomy of in respect to the prominent phylogenetical position of bichir, the vertebrates: an evolutionary perspective. 3rd edn. Thomson even these preliminary data are undoubtedly worth of atten- Brooks ⁄ Cole, Belmont, CA, pp. 703. ISBN: 0030223695. tion. Noack, K.; Zardoya, R.; Meyer, A., 1996: The complete mitochondrial DNA sequence of the bichir (Polypterus ornatipinis), a basal ray- finned fish: ancient establishment of the consensus vertebrate gene Acknowledgements . Genetics 144, 1165–1180. Reiss, J. O., 1997: Early development of chondrocranium in the tailed We would like to thank organizers of the IAFSB meeting in frog Ascaphus truei (Amphibia: Anura): implications for anuran Tavira for the invitation and organizing of this meeting; we are palatoquadrate homologies. J. Morphol. 231, 63–100. grateful to Vladimir Soukup, David Jandzik and two anon- Sellman, S., 1946: Some experiments on the determination of the larval tooth in Amblystoma mexicanum. Odontol. Tidskr. 54, 1–128. ymous reviewers for their helpful comments on the manu- Smith, M. M., 2003: Vertebrate dentitions and the origin of jaws: when script. The research reported here was supported by MSˇ MT and how pattern evolved. Evol. Dev. 5, 394–413. project 0021620828 and GACR 206 ⁄ 09 ⁄ 1007. Smith, M. M.; Coates, M. I., 2001: The evolution of vertebrate dentitions: Phylogenetic pattern and developmental models. In: Major Events in Early Vertebrate Evolution (Paleontology, References Phylogeny, Genetics and Development). P. E. Ahlberg (Ed.). Taylor and Francis, London and New York, pp. 223–240. Adams, A. E., 1924: An experimental study of the development of the Soukup, V.; Epperlein, H. H.; Hora´ cek, I.; Cerny, R., 2008: Dual mouth in the amphibian embryo. J. Exp. Zool. 40, 311–379. epithelial origin of vertebrate oral teeth. Nature 455, 795–798. Balinsky, B. I., 1975: An introduction to , 4th edn. W.B. Takahama, H.; Sasaki, F.; Watanabe, K., 1988: Morphological Saunders, Philadelphia. changes in the oral (buccopharyngeal) membrane in Urodelan Bartsch, P.; Gemballa, S.; Piotrowski, T., 1997: The embryonic and embryos: development of the mouth opening. J. Morphol. 195, larval development of Polypterus senegalus Cuvier, 1829: its 59–69. staging with reference to external and skeletal features, behaviour Takeuchi, M.; Okabe, M.; Aizawa, S. 2009: The Polypterus and locomotory habits. Acta. Zool. 78, 309–328. (Bichirs). A fish group diverged at the stem of Ray-finned fishes de Beer, G. R., 1947: The differentiation of neural crest cells into (Actinopterygii). in: Emerging model organisms: a laboratory visceral cartilages and odontoblasts in Amblystoma, and a re- manual Vol. 1. D.A. Crotty, A. Gann (Eds). Cold Spring Harbor examination of the germ-layer theory. Proc. R. Soc. Lond. B Biol. Laboratory Press, Cold Spring Harbor, New York, pp. 447–467. Sci. 134, 337–406. Wilde, C. E., 1955: The urodele neuroepithelium: I. The differentiation Carroll, R. L., 1988: Vertebrate paleontology and evolution. W.H. in vitro of the cranial neural crest. J. Exp. Zool. 130, 573–591. Freeman, New York. Dickinson, A. J. G.; Sive, H., 2006: Development of the primary AuthorÕs address: Robert Cerny, Department of Zoology, Charles mouth in Xenopus laevis. Dev. Biol. 295, 700–713. University in Prague, Vinicna 7, 128 44 Prague, Edwards, L. F., 1929: The origin of the of the carp Czech Republic. (Cyprinus carpio Linnæus). Ohio. J. Sci. 29, 93–130. E-mail: [email protected]