Journal of Applied Ichthyology 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 Senegal bichir Polypterus 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 evolution 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 (Polypterus senegalus) 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 Bichirs, 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 vertebrate phylogeny and therefore are uniquely supposed ecto-endoderm border in the mouth and consecutive well suited for assessing the ancestral states of Osteichthyes initiation of dental development. The mouth of the Senegal and Actinopterygii, as well as the divergence of developmental bichir does not develop via a classical oropharyngeal mem- patterns between e.g. fishes and tetrapods (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 jaws that are connected by epithelial and Holocephali) 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 vertebrates 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. Scale 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 gills; 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 jaw; 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.