Journal of Oral Biosciences 57 (2015) 148–156

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Journal of Oral Biosciences

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Original Article The sonic hedgehog signaling pathway and the development of pharyngeal arch Derivatives in piceatus, a

Sung-Won Cho a, Jacco C. van Rijssel b,1, Frans Witte b, Merijn A.G. de Bakker b, Michael K. Richardson b,n a Division of Anatomy and Developmental Biology, Department of Oral Biology, Yonsei University College of Dentistry, Seoul, Republic of Korea b Department of Integrative Zoology, Institute of Biology, Leiden University, Sylvius Laboratory, Sylviusweg 72, 2333BE Leiden, The Netherlands article info abstract

Article history: Objectives: Pharyngeal arches develop in the head and neck regions, and give rise to teeth, oral jaws, the Received 5 March 2015 hyoid bone, operculum, gills, and pharyngeal jaws in teleosts. In this study, the expression patterns of Received in revised form genes in the sonic hedgehog (shh), wnt, ectodysplasin A (eda), and bone morphogenetic protein (bmp) 7 April 2015 pathways were investigated in the pharyngeal arches of Haplochromis piceatus, one of the Lake Victoria Accepted 9 April 2015 . Furthermore, the role of the shh pathway in pharyngeal arch development in H. piceatus larvae Available online 17 July 2015 was investigated. Keywords: Methods: The expression patterns of lymphocyte enhancer binding factor 1 (lef1), ectodysplasin A shh receptor (edar), shh, patched 1 (ptch1), bmp4, sp5 transcription factor (sp5), sclerostin domain containing Development 1a (sostdc1a), and dickkopf 1 (dkk1) were investigated in H. piceatus larvae by in situ hybridization. The Pharyngeal arch role of the shh pathway was investigated through morphological phenotypic characterization after its Cichlid Haplochromis piceatus inhibition. Results: We found that lef1, edar, shh, ptch1, bmp4, dkk1, sostdc1a, and sp5 were expressed not only in the teeth, but also in the operculum and gill filaments of H piceatus larvae. After blocking the shh pathway using cyclopamine, we observed ectopic shh expression and the disappearance of ptch1 expression. After six weeks of cyclopamine treatment, an absence of teeth in the oral upper jaws and a poor outgrowth of premaxilla, operculum, and gill filaments in juvenile H. piceatus were observed. Conclusions: These results suggest that the shh pathway is important for the development of pharyngeal arch derivatives such as teeth, premaxilla, operculum, and gill filaments in H. piceatus. & 2015 Japanese Association for Oral Biology. Published by Elsevier B.V. All rights reserved.

1. Introduction of oral dentitions [5]. The structural diversity of jaws and teeth provides clues into the adaptive radiations of cichlids in the East- Pharyngeal arches develop on the ventrolateral side of the head in African great lakes, including Lake Malawi [6,7] and Lake Victoria [2,8]. vertebrates. Teleost fish have seven pharyngeal arches, while amniotes Genes in the sonic hedgehog (Shh), Wnt, ectodysplasin A (Eda), have five [1].Thefirst and second pharyngeal arches form the oral and bone morphogenetic protein (Bmp) pathways such as lym- jaws and operculum, respectively [1]. The remaining posterior arches phocyte enhancer binding factor 1 (Lef1), ectodysplasin A receptor of teleosts are involved in the formation of gills and gill-related (Edar), Dickkopf 1 (Dkk1), sp5 transcription factor (Sp5), Shh, skeletal structures [1]. An oral dentition is developed on the first Patched 1 (Ptch1), sclerostin domain containing 1 (Sostc1), and pharyngeal arch and, thus, forms the functional jaws in teleosts, while Bmp4 are known to be related to mouse tooth development a pharyngeal dentition is developed on the rostral margin of the [9–15]. Here, we investigated the expression pattern of lef1, edar, seventh pharyngeal arch [2–4]. Most teleosts have multiple rows of shh, ptch1, bmp4, sostdc1, dkk1, and sp5 during the development of teeth on both their oral and pharyngeal jaws, while zebrafish lost a set pharyngeal arch derivatives, specifically of teeth, operculum, and gill filaments, in the Lake Victoria cichlid Haplochromis piceatus, because this species is considered to be a morphologically and n Corresponding author. Tel.: þ31 71 527 5215; fax: þ31 71 527 4900. trophically generalized species [16]. E-mail address: [email protected] (M.K. Richardson). 1 Current address: Institute for Ecology & Evolution, University of Bern, Among the above pathways, a negative feedback loop mechan- Baltzerstrasse 6, CH-3012 Bern, Switzerland. ism between the Shh and Wnt pathways has been reported during http://dx.doi.org/10.1016/j.job.2015.04.001 1349-0079/& 2015 Japanese Association for Oral Biology. Published by Elsevier B.V. All rights reserved. S.-W. Cho et al. / Journal of Oral Biosciences 57 (2015) 148–156 149 mouse tooth development [14,15]. The Wnt pathway positively conditions. Treatment and control experiments were performed activates the Shh pathway, then, the Shh pathway induces the in Petri dishes kept at 26 1C on an oscillating platform in a culture expression of Sostdc1, which inhibits the Wnt pathway [15].In incubator (KS 130 basic, IKA Works GmbH & Co., Germany). teleost, a negative feedback loop between the shh and wnt Following DMSO or cyclopamine treatment for 24 h, cichlid larvae pathways has not been reported in developing pharyngeal arches. were fixed with 4% paraformaldehyde in phosphate buffered saline Here, we investigate the role of shh in the morphogenesis of for in situ hybridization analysis or kept for one day in aquaria pharyngeal arch derivatives in H. piceatus, and the relationship containing approximately 3 L of fresh water and then transferred between the shh and other pathways by comparing morphological to an aquarium containing 10 L of fresh water for six weeks for and molecular differences between cyclopamine-treated and con- morphological analysis. trol larvae. Cyclopamine is one of the chemical modulators used to experimentally manipulate the Shh signaling pathway. 3. Results

2. Materials and methods 3.1. lef1, edar, shh, ptch1, sostdc1, bmp4, sp5, and dkk1 are expressed in the oral jaws of the H. piceatus at 6 dpf 2.1. care The expression patterns of lef1, edar, shh, ptch1, sostdc1, bmp4, Adult H. piceatus were kept in aquaria of 100 50 50 cm3 sp5, and dkk1 were investigated in H. piceatus larvae at 5, 6, 6.5, with a water temperature of 24.170.5 1C. The wild-type stock was and 7 dpf. The developing premaxillae and mandibles of H. collected in 1984 in the Mwanza Gulf of Lake Victoria, East Africa. piceatus expressed lef1, edar, dkk1, sp5, shh, ptch1, and bmp4 at They were subsequently bred for 15–16 generations in our aquaria 6 dpf. All but ptch1 and shh showed a punctate expression pattern at Leiden University, Leiden, The Netherlands. They were kept in the oral jaws (Fig. 1A–D, H). In contrast, shh was expressed as a under a day–night light cycle of 12:12 h and were fed daily with continuous band (Fig. 1E) and ptch1 was widely expressed (Fig. 1F), commercial flake fish food (Landman, Hoevelaken, The Nether- which indicates the strong activity of the shh signaling pathway in lands) alternated with a range of frozen fish foods (Aquadistri BV, the oral jaws. Klundert, The Netherlands). All procedures were carried out with Two copies of sostdc1, sostdc1a and sostdc1b, have been found the approval of the local ethics committee. in H. piceatus. Here, we show only the expression pattern of sostdc1a. sostdc1a was found to be expressed in the oral jaws, as 2.2. Collection and storage of larvae well as in the lacrimal and nasal bones during development (Fig. 1G). These results indicate that these genes are involved in Haplochromis piceatus is a mouthbrooding cichlid. Larval ages tooth development. (in days post-fertilization, dpf) were determined using the first identification of mouthbrooding in a female as the starting point 3.2. lef1, edar, shh, ptch1, sostdc1, bmp4, sp5, and dkk1 are expressed (0 dpf). Larvae were then obtained from the mouths of brooding in the operculum and gills of H. piceatus at 6 dpf females by manual compression, and maintained in separate culture tanks provided with flowing water and oxygenation for Genes expressed during tooth development were also expr- further development. The oxygen concentration in the water was essed in the developing operculum and gill filaments. The devel- 8 mg/L. oping operculum of H. piceatus at 6 dpf showed striped expression of lef1, edar, dkk1, shh, and bmp4 (Fig. 2A–C, E, H). The operculum 2.3. Whole-mount in situ hybridization margin expressed sp5 and ptch1 (Fig. 2D, F). The tips of developing gill filaments showed expression of lef1, edar, dkk1, sp5, shh, ptch1, In situ hybridization was carried out as described in Vonk et al. and bmp4. These results indicate that these genes are involved not [17]. For probe synthesis, we isolated total RNA from H. piceatus only in tooth development but also in the development of the larvae with Trizol (Invitrogen, Breda, The Netherlands) and operculum and gill filaments. Interestingly, sostdc1a was expressed synthesized cDNA from the RNA by a reverse transcriptase (Super- in developing gill filaments at 5 dpf (Fig. 2G0) but not at 6 dpf Script III, Invitrogen or RevertAid, Fermentas, Leon-Roth, Ger- (Fig. 2G). many). Primers for the targeted genes were designed using alignments of fugu (Takifugu rubripes), medaka (Oryzias latipes), 3.3. Disruption of the shh signaling pathway alters shh and ptch1 stickleback (Gasterosteus aculeatus), tetraodon (Tetraodon nigrovir- expression patterns in developing teeth, operculum, and gill filaments idus), Nile tilapia (Oreochromis niloticus), and zebrafish (Danio rerio) nucleotide sequences. The resulting PCR products from the In normal cichlid larvae at 6 dpf, shh was expressed as multiple targeted genes were sequenced and the sequences were depo- odontogenic bands in the premaxilla, vomer, and palate (Fig. 3A). sited in GenBank (accession numbers KJ129615–KJ129622 and At 6.5 dpf, shh expression disappeared in the vomer, but was KJ705283, Table 1). We investigated the expression patterns of maintained in the premaxilla and palate (Fig. 3B). At 7 dpf, shh was lef1, edar, shh, ptch1, bmp4, sp5, and dkk1 in larvae at 5, 6, 6.5, and expressed as multiple dots on the rostral end of the premaxillary 7 dpf. At least three larvae of each stage were hybridized with odontogenic band (Fig. 3C). A caudal margin of shh expression in each probe. the premaxillary odontogenic band was evident; however, shh expression was not observed in the regions between the odonto- 2.4. Cyclopamine treatment genic bands of the premaxilla and palate (Fig. 3A–C). This band- like shh expression pattern was conserved in control larvae but Cichlid larvae were treated with 10 μM cyclopamine (Toronto changed in cyclopamine-treated larvae (Fig. 3D–F). While shh Research Chemicals, ON, Canada). The cyclopamine stock solution, expression in the odontogenic bands of the premaxilla and palate dissolved in pure dimethyl sulfoxide (DMSO), had a concentration was discrete in control larvae (Fig. 3D), its expression expanded to of 1 mM. This stock was diluted to 1:100 in the aquarium water the regions between the odontogenic bands of the premaxilla and to a final concentration of 10 μM cyclopamine and 1% DMSO. As palate after cyclopamine treatment (Fig. 3E, F). Although shh a control, we used 1% DMSO (vehicle only) under the same expression pattern was altered after cyclopamine treatment, its 150 S.-W. Cho et al. / Journal of Oral Biosciences 57 (2015) 148–156

Table 1 Probe of genes in Haplochromis piceatus

Gene Gene name Forward primer Reverse primer Probe NCBI Accession number of probe symbol length sequence

1 lef1 lymphocyte enhancer binding 50-Agacccgcacaag gagcagatct- 50-GCATGTGGAGCTG CCGTTCCTT- 899 bp KJ129618 factor 1 30’ 30 2 edar ectodysplasin A receptor 50-gccatgtcaaccat cttcatcatggc- 50-AGCCTCTCGATCTG CACCAG-30 863 bp KJ129616 30 3 dkk1 dickkopf 1 50-ctgtctgccatgc cgcaaga-30 50-CCCTTTGACAGTGT 472 bp KJ129617 GTAGGTTCCG-30 4 sp5 sp5 transcription factor 50-CCACGCACATCCA 50-CTCCTCATGAACCT 721 bp KJ129622 GAGCTCCTT-30 CTTGCAGCAGT-30 5 shh sonic hedgehog 50-CAGGGGCTACGGC 50-CGAGTGGCTGTCC AAGAGCCA- 1132 bp KJ129620 AGGAGAAG-30 30 6 ptch1 patched 1 50-TTCCAGCTGATGAC 50-TGGACTGCATGGTG 955 bp KJ129619 TCCCAAGCA-30 ATTTGGGTCT-30 7 sostdc1a sclerostin domain containing 1a 50-tgcatcctgctga ggagttgcca-30 50-CTTGCACTTGCAGG 456 bp KJ705283 AGGTCACC-30 8 sostdc1b sclerostin domain containing 1b 50-tccaccaagtacat atctgatggc-30 50-CTTGCACTTGCAGG 261 bp KJ129621 AGGTCACC-30 9 bmp4 bone morphogenetic protein 4 50-ttatatgccaagtc ctgctgggaga- 50-GGGTGCCACTATCC 938 bp KJ129615 30’ AGTCATTCCAG-30

expression persisted. However, expression of ptch1, one of the plays important roles during H. piceatus development, not only downstream genes of the shh signaling pathway, disappeared in during teeth development but also in the development of pre- the developing teeth, operculum, and gill filaments of larvae 24 h maxilla, operculum, and gill filaments. after treatment with cyclopamine. In contrast, ptch1 expression was observed in these structures in control larvae (Fig. 3G, H). This result suggests that the shh signaling pathway activity was bloc- 4. Discussion ked by cyclopamine treatment in our experiments. The expression pattern of lef1 did not significantly change in 4.1. The expression of lef1, edar, shh, ptch1, sostdc1, bmp4, sp5, and developing teeth, operculum, and gill filaments after cyclopamine dkk1 in the developing oral jaws, operculum, and gill filaments of treatment (Fig. 4A, B). The expression patterns of dkk1 and bmp4 in H. piceatus larvae H. piceatus did not change either in developing teeth, operculum, and gill filaments after cyclopamine treatment (Fig. 4C–F). The While there is only one Sostdc1 gene in mice and humans, there expression pattern of shh in the operculum and gill filaments are two copies of sostdc1 in medaka (Oryzias latipes) (NCBI gene showed no significant difference between cyclopamine-treated symbol: LOC101160585 and LOC101175090), zebrafish (Danio and control larvae (Fig. 4I, J). The expression pattern of sostdc1a rerio)(sostdc1a and sostdc1b), tilapia (Oreochromis niloticus) (NCBI in the oral jaw, and lacrimal and nasal bones was the same in gene symbol: LOC100701930 and LOC100694302), Lake Victoria cyclopamine-treated and control larvae (Fig. 4G, H). cichlid (Pundamilia nyererei) (NCBI gene symbol: LOC102210052 and LOC102209719), and Lake Tanganyika cichlid [Haplochromis 3.4. Development of tooth, operculum, and gill filaments is disturbed (Astatotilapia) burtoni] (NCBI gene symbol: LOC102305493 and by shh signaling pathway suppression in H. piceatus LOC102307311). The two orthologs of sostdc1 in medaka and tilapia have been named sostdc1a and sostdc1b in the Zebrafish H. piceatus larvae were treated with 10 μM cyclopamine at Model Organism Database (ZFIN, http://zfin.org). In our study, the 6 dpf, and morphological abnormalities were investigated six two copies of sostdc1 in H. piceatus were designated as sostdc1a weeks after treatment. After six weeks, no abnormality was and sostdc1b based on their sequence homology with sostdc1a and observed in control juvenile cichlids (after six week, cichlids are sostdc1b, respectively, in medaka and tilapia. The expression juvenile rather than larvae) (n¼40), but two of the cyclopamine- pattern of sostdc1a was similar to that of sostdc1 in the Lake treated juvenile cichlids (n¼45) showed abnormalities. Compared Malawi cichlid Maylandia zebra shown in a previous study [18]. with the control cichlids, the abnormal cichlids showed at least This similar expression pattern seems to be due to the fact that the four significant changes in morphometric parameters. First, the partial coding sequence of sostdc1 of M. zebra described in the distance from the eye to the rostral edge of the premaxilla was previous report is more similar to the sequence of sostdc1a rather shorter in cyclopamine-treated cichlids than in controls (Fig. 5A– than to that of sostdc1b in H. piceatus. D). Second, the operculum was too short to cover the gills in The developing premaxillae, mandibles, operculum, and gill cyclopamine-treated cichlids. Third, teeth did not develop in the filaments of H. piceatus showed dkk1 and sp5 expression at 6 dpf. premaxillae of the cyclopamine-treated cichlids (Fig. 5F). Though To the best of our knowledge, this is the first report showing that teeth were present in the mandibles, there were less teeth than in dkk1 and sp5 are expressed in the developing pharyngeal arches of the mandibles of the control larvae (Fig. 5H). All control larvae had cichlids. a full complement of unicuspid teeth in premaxillae and mand- ibles (Fig. 5E, G). These results show that cyclopamine treatment at 4.2. Altered gene expression patterns in developing teeth, operculum, 6 dpf inhibited teeth formation in the oral jaws. Fourth, each gill and gill filaments after disruption of the shh signaling pathway filament was shorter than in control cichlids. Particularly in the first and second gill arches, dorsal gill filaments appeared shorter Cyclopamine does not have a direct effect on Shh transcription than ventral gill filaments (Fig. 5I, J). In the third and fourth gill in mice [15] and, thus, shh expression persisted in cichlids. arches, whole filaments were much shorter than in the control However, the shh expression pattern changed in cichlids after group. This evidence indicates that the shh signaling pathway cyclopamine treatment. After treatment with cyclopamine, ptch1 S.-W. Cho et al. / Journal of Oral Biosciences 57 (2015) 148–156 151

Fig. 1. Gene expression patterns in H. piceatus oral jaws at 6 dpf. (A–D) Developing tooth germs (black arrows) strongly express lef1, edar, dkk1, and sp5. (E) shh (black arrows) is expressed in the odontogenic band of the oral jaws. (F) ptch1 is widely expressed around the tooth forming regions (black arrows). (G) sostdc1a is expressed weakly in developing teeth (black arrow), but strongly at the sites of lacrimal and nasal bone development (white arrow). (H) bmp4 is expressed in developing tooth germs (black arrows). Scale bar: 500 μm. expression disappeared in developing teeth, operculum, and gill in the premaxillae, and shorter gill filaments. These differences filaments. The expression patterns of lef1, dkk1, and bmp4 did suggest that the shh pathway plays important roles during not significantly change after cyclopamine treatment. Although H. piceatus development, not only during teeth development but Sostdc1 expression was reported to decrease after blocking Shh also during the development of the premaxilla, operculum, and gill activity in mice [15], sostdc1a expression did not change in cichlids filaments. after cyclopamine treatment. These results suggest that the shh The abnormal morphological characteristics caused by cyclo- pathway is not directly related to the wnt and bmp pathways, and pamine treatments were observed in regions such as the oral jaw, that sostdc1a in cichlids does not represent a link between the operculum, and gill filaments, which showed strong shh and ptch1 shh, wnt, and bmp pathways. Whether sostdc1b rather than expression at 6 dpf. The teeth absence caused by cyclopamine in sostdc1a is a downstream gene of the shh signaling pathway in our study is consistent with results from a previous report, in cichlids remains to be determined. which blocking of the hedgehog pathway in Lake Malawi cichlid larvae at 5 dpf by 50 μM cyclopamine treatments for 24 h impaired 4.3. The role of the shh signaling pathway in the development of dentition development and teeth were absent in the mandible of teeth, operculum, and gill filaments of H. piceatus cichlids at 12 dpf, one week after the cyclopamine treatments [19]. The short operculum length in our study is also similar to the Compared with control cichlids, cyclopamine-treated juvenile results of a previous report on zebrafish and Lake Malawi cichlids. cichlids showed at least four significant changes in morphometric The distance between the edge of the opercular flap and the edge parameters, including a shorter distance from the eye to the rostral of the eye was reduced significantly after blocking shh pathway edge of the premaxilla, a shorter operculum length, tooth agenesis activity using cyclopamine in our study. The interopercular bone 152 S.-W. Cho et al. / Journal of Oral Biosciences 57 (2015) 148–156

Fig. 2. Gene expression patterns in H. piceatus operculum and gill filaments. (A) lef1 is expressed in developing tooth germs (black arrowhead), operculum (black arrow), and gill filaments (white arrow) at 6 dpf. (B) edar shows a strong expression in tooth germs (black arrowhead) and operculum (black arrow), but weak expression in gill filaments (white arrow). (C) dkk1 expression is strong in the teeth and operculum, but weak in gill filaments. (D) sp5 is weakly expressed in the operculum and gill filaments. (E–F) shh and ptch1 expression is strong in tooth germs, operculum, and gill filaments at 6 dpf. (G–G0) sostdc1a is expressed in many gill filaments at 5 dpf (white arrow in G0), but only in the dorsal-most gill filaments at 6 dpf (white arrowheads in G). (H) bmp4 expression is clearly observed in developing operculum and gill filaments at 6 dpf. Scale bar: 500 μm. S.-W. Cho et al. / Journal of Oral Biosciences 57 (2015) 148–156 153

Fig. 3. Altered shh and ptch1 expression patterns in developing teeth after shh signaling pathway suppression. (A) Normal cichlid larvae show shh expression in the odontogenic bands of the premaxilla (white arrow), vomer (white arrowhead) and palate (black arrowhead) at 6 dpf. (B) At 6.5 dpf, shh expression is observed in the premaxilla and palate. (C) At 7 dpf, punctuated shh expressions appear on the rostral side of the odontogenic band (white arrow). (D) shh expression is observed in the odontogenic bands of premaxilla (white arrow) and palate (black arrowhead) in control larvae. (E–F) After cyclopamine treatment at 6 dpf, shh expression is detected not only in the odontogenic bands of the premaxilla (white arrow) and palate (black arrowhead) but also between these two odontogenic bands (yellow arrowheads). (G–H) From the ventral view, ptch1 expression in developing teeth, operculum and gill filaments is strong in control larvae at 6 dpf (F) but disappears in cichlid larvae 24 h after treatment with cyclopamine (G). Scale bars: 250 μminA–F; 500 μm in (G and H). 154 S.-W. Cho et al. / Journal of Oral Biosciences 57 (2015) 148–156

Fig. 4. Altered lef1, dkk1, bmp4, sostdc1a, and shh expression patterns in developing teeth, operculum, and gill filaments after shh signaling pathway suppression. (A–F) lef1, dkk1 and bmp4 show the same expression pattern in developing teeth, operculum, and gill filaments in both cyclopamine-treated and DMSO-treated larvae 24 h after treatment. Ventral views. (G–H) sostdc1a expression pattern in the oral jaws, and lacrimal and nasal bones does not differ between cyclopamine-treated and control larvae. (I–J) shh expression pattern in the operculum and gill filaments shows no significant difference between cyclopamine-treated and control larvae. Scale bar: 500 μm. S.-W. Cho et al. / Journal of Oral Biosciences 57 (2015) 148–156 155

Fig. 5. Abnormal development of teeth, operculum, and gill filaments after shh signaling pathway suppression in H. piceatus at 6 dpf. (A–D) The distance from the anterior boundary of the eye to the edge of the premaxilla (black star) is shorter in cyclopamine-treated cichlids (B and D) than in control juveniles (A and C). The gills are not fully covered by the operculum in cyclopamine-treated cichlids so that gill arches are exposed to the outside (arrows in B and D). (E and G) Control juveniles have teeth in the premaxilla (E) and mandible (G). (F and H) Teeth are not observed in the oral jaws in some cyclopamine-treated cichlids. There are no teeth in the upper jaw (F), while teeth are observed in the lower jaw (H) of the same individual. (I and J) The length of each gill filament (arrow) is shorter in cyclopamine-treated cichlids (J) than in control juveniles (I). Scale bars: 2 mm in (A–D); 200 μmin(E–H); 500 μmin(I–J). length of Lake Malawi cichlids decreased when larvae were observations were recorded six weeks after blocking shh pathway treated with cyclopamine at 6 dpf [20]. The lower jaw length activity, while in previous studies observations were carried out remained essentially unaffected by cyclopamine treatment in our one week after treatment. study, which is also consistent with previous reports [20,21]. The The complete absence of teeth in the upper jaw, the incomplete overall length of the lower jaw in Lake Malawi cichlids did not elongation of the operculum, and the short gill filaments might change after cyclopamine treatment of larvae at 6 dpf [20,21].It impair major functions such as food intake and respiration. These was suggested that the dentary and lower jaw bone might be characteristics could result in a competitive disadvantage for the determined primarily by the outgrowth of Meckel's cartilage that survival of cichlids over the six-week period and might cause only occurs prior to 6 dpf [21]. a small number of cichlids showing morphological abnormalities Not all the results of our study are consistent with previous to survive for six weeks after cyclopamine treatment in our study. findings. An absence of oral teeth was observed in the upper jaw in our study, but this absence was recorded in the lower jaw in a previous study [19]. This discrepancy could be due to differences 5. Conclusions in the developmental stages of the cichlid larvae treated with cyclopamine in the two studies. Cichlid larvae were treated with In the present study, we found that lef1, sp5, dkk1, edar, shh, cyclopamine at 5 dpf in the above mentioned study but at 6 dpf in ptch1, sostdc1a, and bmp4 are involved in the development of our study. These results could suggest that the shh signaling pharyngeal arch derivatives in H. piceatus. In particular, morpho- pathway is important for lower teeth development at an earlier logical changes induced by suppressing shh activity suggest that stage than for upper teeth development. In addition, morphologi- the shh signaling pathway plays a significant role in the develop- cal changes in premaxilla and gill filaments were observed in our ment of teeth, premaxilla, operculum, and gill filaments in H. study but not in previous reports. This difference might be caused piceatus, a type of Lake Victoria cichlid. Our experimental results by differences in the timing of the observations. 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