Journal of Anatomy
J. Anat. (2017) doi: 10.1111/joa.12579
Evolutionary aspects of the development of teeth and baleen in the bowhead whale J. G. M. Thewissen,1 Tobin L. Hieronymus,1 John C. George,2 Robert Suydam,2 Raphaela Stimmelmayr2,3 and Denise McBurney1
1Department of Anatomy and Neurobiology, Northeast Ohio Medical University, Rootstown, OH, USA 2Department of Wildlife Management, North Slope Borough, Barrow, AK, USA 3Institute of Arctic Biology, University of Alaska Fairbanks, Fairbanks, AK, USA
Abstract
In utero, baleen whales initiate the development of several dozens of teeth in upper and lower jaws. These tooth germs reach the bell stage and are sometimes mineralized, but toward the end of prenatal life they are resorbed and no trace remains after birth. Around the time that the germs disappear, the keratinous baleen plates start to form in the upper jaw, and these form the food-collecting mechanism. Baleen whale ancestors had two generations of teeth and never developed baleen, and the prenatal teeth of modern fetuses are usually interpreted as an evolutionary leftover. We investigated the development of teeth and baleen in bowhead whale fetuses using histological and immunohistochemical evidence. We found that upper and lower dentition initially follow similar developmental pathways. As development proceeds, upper and lower tooth germs diverge developmentally. Lower tooth germs differ along the length of the jaw, reminiscent of a heterodont dentition of cetacean ancestors, and lingual processes of the dental lamina represent initiation of tooth bud formation of replacement teeth. Upper tooth germs remain homodont and there is no evidence of a secondary dentition. After these germs disappear, the oral epithelium thickens to form the baleen plates, and the protein FGF-4 displays a signaling pattern reminiscent of baleen plates. In laboratory mammals, FGF-4 is not involved in the formation of hair or palatal rugae, but it is involved in tooth development. This leads us to propose that the signaling cascade that forms teeth in most mammals has been exapted to be involved in baleen plate ontogeny in mysticetes. Key words: baleen; baleen whales; bowhead whale; Cetacea; embryology; FGF; keratin; mysticetes; ontogeny; tooth development.
Introduction which is mostly composed of the protein keratin (Werth, 2000; Lambertsen et al. 2005). The inside (lingual) of the About 34 million years ago, a macroevolutionary event plates frays into a matted mass of hair-like filaments that took place in which the ancestors of baleen whales (Mys- are used to filter food from water taken into the mouth. ticeti, Cetacea, Mammalia) lost their teeth and replaced Individual plates grow throughout life, and wear along them with baleen, allowing them to feed efficiently on their lingual border and their (ventral) tip. The rack is plankton (Demer e et al. 2008; Marx, 2011). In all modern implanted more or less where the tooth rows would be, mysticetes, the baleen rack is implanted on the left and but there is no trace of teeth (Demer e et al. 2008). right side of the upper jaw (Fig. 1), leaving a gap rostrally Interestingly, tooth development is initiated in baleen in the median plane in some (balaenids, eschrichtiids, whale embryos, and proceeds through several develop- neobalaenids), but not all species (balaenopterids). Each mental stages (Karlsen, 1962). At least in some species, rack is composed of a row of several hundred baleen plates, some mineralization of teeth occurs (Dissel-scherft & Ver- and each of those plates consists of a flat sheet of baleen, voort, 1954), but enamel never forms (Demer e et al. 2008). Later in fetal life, all traces of teeth and tooth develop- ment disappear (Ishikawa & Amasaki, 1995; Demer eetal. Correspondence 2008). Saint Hilaire (1807) was the first to describe tooth J. G. M. Thewissen, Department of Anatomy and Neurobiology, Northeast Ohio Medical University, 4209 State Route 44, Rootstown, germs in a mysticete studying the bowhead whale OH 44242, USA. (Balaena mysticetus L., 1758). Baleen development in this T: (330) 3256295; F: (330) 3255913; E: [email protected] species commences late in fetal life, they are born with
Accepted for publication 16 November 2016 baleen plates 10 cm long and, after approximately 10
© 2017 Anatomical Society 2 Tooth and baleen development in bowhead whales, J. G. M. Thewissen et al.
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Fig. 1 (A) Palate of bowhead whale showing left and right sides of baleen rack, each consisting of approximately 320 plates. Note the narrow palate and baleen plates flaring laterally from it. The lingual side of the plates is frayed and makes a matted surface. (B) Single baleen plate, medial to right. (C) Minor plates from the posterior side of the palate, arranged from lateral to medial on the palate, to the same scale as (B). (D) Enlarged view of (C), showing flat, bilateral symmetrical plates (1, 2, 3); radially symmetrical plates (4, 5, 6) and baleen hair (7). (E) Cranial view of baleen plate (cut off) and the minor plates medial to it (bottom of image). The oral cavity is to the right in this image, and the full depth of gingiva (gums) is seen (white material). Red lines indicate sectional planes of (F) and (G). (F) Cut section through (E), occlusal view, with minor plates cut off to show pattern of implantation, medial to bottom. (G) Section through gingiva, showing cross-sections of plate and minor plates as embedded in palate, medial to bottom.
years, the longest plates are more than 2 m in length flattened platelets with terminal hairs. The last element in (Lubetkin et al. 2008). After that, as the whale keeps this series is the asymmetrical baleen plate, much larger growing, growth of the plates exceeds their wear, and in than any of the structures medial to it. In addition to size an old, large whale, plates may be 4 m in length. Bowhead and complexity of individual elements, the implantation of whales appear to keep growing throughout life, and may the accessory plates and bristles in the gingiva is complex. reach 200 years (George, 2009). Elements are implanted in rostro-caudal rows, but they also Baleen takes on different forms in different species of form rows that are oblique at approximately 45 ° and 315 ° cetaceans, as well as in different parts of the mouth. with the sagittal plane (Fig. 1F,G). Adding further complex- Lambertsen et al. (1989, 2005) provided a full description of ity, larger elements are spaced more widely than smaller the baleen rack in the bowhead whale, and the histology elements. The implantation pattern is best seen if the pro- of baleen was described in great detail for balaenopterid truding part of the plates and bristles is cut off, leaving only whales (Fudge et al. 2009). In bowhead whales, about 320 stubs protruding from the gingiva (Fig. 1F) or when the gin- baleen plates are implanted along the length of the upper giva is cut off the maxilla, and that cut face is studied from jaw (Lambertsen et al. 2005). For most of the length of the dorsal (Fig. 1G). jaw, there is only a single bilateral large plate on each pala- In contrast to baleen, mammal teeth consist of inorganic tal cross-section, although, as an anomaly, a second row of hydroxyl-apatite (mostly calcium phosphate) and do not small plates may occur. grow after mineralization, except in special cases such as Near the posterior edge of the palate, bowheads have an the hypsodont teeth of elephants and beavers. Mammal array of keratinous elements medial to the large, lateral teeth are also limited in number (precisely 11 teeth per jaw baleen plate. Collectively these smaller structures are quadrant in ancestral placentals; isodonty; Bown & Kraus, referred to as accessory plates and bristles (Fudge et al. 1979), and are replaced just once (diphyodonty; Van Nievelt 2009). These elements increase in size and complexity from & Smith, 2005). The typical mammalian dentition is differen- medial to lateral (Fig. 1), and include, respectively: baleen tiated into tooth classes (incisors, canines, premolars and hairs, radially symmetrical columns with terminal hairs, and molars), each of which consists of several teeth that are
© 2017 Anatomical Society Tooth and baleen development in bowhead whales, J. G. M. Thewissen et al. 3 similar to each other but different from teeth in other tooth classes (heterodonty), and in the caudal part of the jaw (sometimes called the distal part), the molars have a complex crown morphology with multiple cusps (elevated areas) and crests. The position of these cusps is highly stable within species. In plesiomorphic placental mammals, there are three incisors, one canine, four premolars, and three molars on each side, both in upper and lower jaw. This is often expressed as a dental formula: 3.1.4.3/3.1.4.3. The dental formula of the deciduous dentition of these placen- tals lacks molars: 3.1.4/3.1.4 (Bown & Kraus, 1979). Adult archaic placental mammals thus have four times 11 teeth per jaw quadrant, 44 teeth in total. This number is reduced in many placental lineages, but increases of tooth counts, polydonty, are rare among extant mammals and occur only in odontocete cetaceans (Miyazaki, 2002) and the armadillo Priodontes. Cetacea (whales, dolphins and porpoises) are descended from terrestrial artiodactyls, with Hippopotamidae and Raoellidae, respectively, the modern and fossil family most closely related to cetaceans (Gatesy et al. 2013). Raoellids, as well as basal whales (pakicetids) are diphyodont, hetero- dont, have complex molar crown morphologies, and their dental formula is 3.1.4.3/3.1.4.3 (Fig. 2A). Hippopotamids and raoellids have upper molars with four elevated areas (cusps) arranged in a square (Fig. 2C), whereas pakicetids have three large cusps on their upper molars (Fig. 2D; Thewissen et al. 2001, 2007; Cooper et al. 2009). Raoellids have lower molars with a high anterior part (trigonid) and a low posterior part (talonid), each with two cusps (Fig. 2E). In pakicetids and some younger whales (protocetids), the lower molars also have a high trigonid and low talonid, but Fig. 2 Teeth of fossil cetaceans. (A) Right upper and lower dentition each is dominated by a single large cusp (Fig. 2F; Cooper of Pakicetus, the oldest known whale, in labial (lateral) view. Incisors et al. 2009; Gatesy et al. 2013). Late Eocene whales, basilo- not shown, first premolar indicated as alveolus. (B) Right upper and saurids, are heterodont with four tooth classes (Fig. 2B). lower dentition of Dorudon, a late Eocene whale, in labial view. (C, D) Their lower molars do not show a distinct trigonid and talo- Right upper molars of Indohyus and Pakicetus, rostral to right, with nid (Fig. 2G; Uhen, 2004), but the molars have several acces- homologous cusps identified by numbers. Lower molars of Indohyus (E), a middle Eocene protocetid whale (F), and late Eocene Zygorhiza sory cusps. (G), in lingual view. (H) Labial view of three cheek teeth (rostral to left) Mysticetes are generally thought to be derived from basi- of toothed mysticete (aetiocetid), notice that the last tooth appears to losaurids (Boessenecker & Fordyce, 2015; Marx & Fordyce, consist of two teeth that are fused. Drawings from Thewissen (2014). 2015). The oldest fossil mysticetes have teeth and are het- erodont (Clementz et al. 2014). In the family Aetiocetidae, The pattern of dental evolution in mysticetes is thus coun- there are three incisors and one canine, and cheek teeth terintuitive, first the number of teeth increases in evolution increase in complexity posteriorly (Barnes et al. 1994). Some but then teeth disappear altogether suddenly. Around the archaic mysticetes are isodont (Janjucetus; Fitzgerald, 2006), time that the teeth disappeared, mysticetes developed but others have up to 14 teeth per half jaw, three more baleen (Demer e et al. 2008). Skull anatomy of the Oligo- than the original 11 teeth (e.g. Mammalodon; Fitzgerald, cene mysticete Waharoa indicates that it had baleen, but it 2009; aetiocetids; Barnes et al. 1994). The fossil mysticete also retained alveoli rostrally in the upper and lower jaw with the most teeth is Aetiocetus polydentatus,inwhich (Boessenecker & Fordyce, 2015) and was not polydont. tooth numbers on the left and right differ (Barnes et al. Because of the vast differences between them, teeth and 1994). In aetiocetids (Barnes et al. 1994), cheek teeth baleen are usually considered to be independently evolved increase in complexity caudally: they are triangular in labial structures, and baleen is often thought to be derived from view with one main cusp, but one or more small additional the transverse keratinous ridges on the palate, the palatal cusps may occur on the slopes of the large cusp (Fig. 2H). rugae (Werth, 2000; Fudge et al. 2009, a translated and This is similar to basilosaurids (Fig. 2G). annotated version of Tullberg, 1883).
© 2017 Anatomical Society 4 Tooth and baleen development in bowhead whales, J. G. M. Thewissen et al.
Developmentally, teeth and baleen, as well as feathers, baleen (Fudge et al. 2009). Baleen development commences hair and palatal rugae, are epithelial organs: they form at long after tooth development in ontogeny, and is morpho- the interface of an ectodermal placode that overlies mes- logically simpler (Ishikawa & Amasaki, 1995), although enchyme, and much of the signaling involved in forming nothing is known about the genetic pathways involved. these is self-contained. Many proteins are involved in signal- Tooth development and signaling related to it is well ing related to formation of these organs, and we will not studied in laboratory models, especially the mouse, review them here. Instead, we here discuss only those that although tooth development in land-living artiodactyls is we were able to investigate in bowhead whales. probably a better model for whales, given that cetaceans The fibroblast growth factor (FGF) family of signaling are the sister group of one of the terrestrial artiodactyls proteins plays an important role in the formation of ecto- (Gatesy et al. 2013) and we use some comparative samples dermal organs, and different FGFs are involved in the of developing pig teeth, mostly from the work of Armfield ontogeny of different organs. While nothing is known (2010). about signaling related to the development of baleen, First, we study the arrangement of tooth buds in bow- signaling in other keratinized tissues can involve FGF-7 head whale fetuses, determining the number of teeth that and FGF-10 (Mitsui et al. 1997; Nakatake et al. 2001; are formed (isodonty or polydonty), what their shape is Porntaveetus et al. 2010a,b), whereas FGF-4 and FGF-8 along the tooth row (homodonty or heterodonty), whether have not been shown to be involved in signaling related there is evidence of a secondary dentition (monophyodonty to the formation of keratinized structures (Rosenquist & or diphyodonty), and how complex their crowns are. Sec- Martin, 1996; Mitsui et al. 1997; Porntaveetus et al. ond, we report on the ontogeny of baleen, with particular 2010a). interest in signaling related to it. The embryology of teeth and baleen shows limited simi- larities. Recent reviews of tooth development were pro- Materials and methods vided by Caton & Tucker (2009) and Jernvall & Thesleff (2012). Tooth development begins early in the fetal period The acquisition of mysticete embryos is difficult. Mysticetes are pro- when an epithelial placode on the palate invaginates into tected under US laws, and no individuals are in captivity in the USA. the underlying mesenchyme (bud stage). Relevant to our Stranded specimens are usually in a state of decomposition that does not allow meaningful histological study of their embryos, and study, FGF-8 is expressed in what eventually will be the there are no complete ontogenetic series of mysticetes in museums. molar field of the jaw (Tucker & Sharpe, 2004; Mitsiadis & However, a small number of bowhead whales, Balaena mysticetus, Smith, 2006). The epithelium then folds into shapes that are harvested annually by Alaskan natives, Inupiat~ Eskimos. This foreshadow the shape of the occlusal surface of the tooth harvest is permitted under the U.S. Marine Mammal Protection Act, (the inner enamel epithelium in the cap and bell stages). as well as approved by the International Whaling Commission. Tips of cusps on a tooth are initiated partly as a result of Occasionally and accidentally, a pregnant female is harvested allow- sonic hedgehog (SHH) signaling deep to the inner enamel ing extraction of an embryo or fetus under NOAA-NMFS permit 17350. This hunt takes place only twice per year and, therefore, no epithelium, and area called the enamel knot (Jernvall & complete ontogenetic series can be collected as females of this spe- Thesleff, 2000; Thesleff, 2000). The number of enamel knots cies tend to get pregnant in the same season and the gestation time per tooth cap determines the number of cusps on a tooth. is slightly longer than 1 year. Embryos and fetuses collected this FGF-4 is also expressed in the enamel knot (Niswander & way will be in similar stages: about 8 weeks, 7 months and full-term Martin, 1992; Kettunen & Thesleff, 1998; Tucker et al. (George et al. 2016). Our study is based on six prenatal specimens 1998). After folding of the inner enamel epithelium, hydro- xyl-apatite is deposited on both sides of this surface in the form of enamel (on the epithelial side) and dentin (on the Table 1 Bowhead whale prenatal specimens available. mesenchymal side). In this paper, we investigate several aspects of the devel- ID TL Tooth Baleen opment and evolution of teeth and baleen in mysticetes. number (cm) Stage stage stage Characteristic Very little is known about the signaling that forms teeth in 1999B7F 8.7 18 Dental None Tail conical cetaceans. Ishikawa et al. (1999) and Armfield et al. (2013) lamina studied the signaling that gives rise to the loss of tooth 1999B6F 16.6 20 Early None Tail lanceolate classes in dolphins. Morphological studies of the embryol- cap ogy of odontocete teeth are also rare. Mi sek et al. (1996) 2000B3F 40.3 21 Bell None Fluke fully studied tooth anlagen, and Sterba et al. (2000) described formed, hair tooth formation as part of a monograph on dolphin onto- on snout geny. 2007B16F 159 23 None Placode Full hair pattern 2009KK1F 163 23 None Placode Full hair pattern Baleen development also begins as an epithelial placode, 2015B9F 422 23 None Plates Full term but that placode does not invaginate into the mesenchyme, present and instead evaginates directly into the oral cavity as
© 2017 Anatomical Society Tooth and baleen development in bowhead whales, J. G. M. Thewissen et al. 5
(Table 1), one embryo and five fetuses, four from the spring hunt ABCD I and two from the fall hunt, collected between 1999 and 2015. Additional fetuses from this hunt were described by Durham (1980). Armfield et al. (2011) published several of these fetuses, and Drake G et al. (2015) published a photo of the hair pattern on the face of one of them. We staged specimens following the criteria of Thewis- E F sen & Heyning (2007) for dolphins. For small specimens, the entire head was processed in a dehydra- tion series embedded in paraffin, and cut on a microtome at 7 lm. For larger specimens, subsampled specimens were excised out of H fetal heads. Histological stains used included hematoxylin/eosin and thionin. We also employed immunohistochemical methods with J commercial antibodies. Because these antibodies were not designed N for bowhead whales, caution is required in interpreting them. For each staining session, we used negative controls to make sure that no staining takes place if the primary antibody is omitted from the protocol. Specimens stained with antibodies were counterstained with thionin. We used an FGF-4 antibody (Santa Cruz Biotechnology, O sc-1361) at 1 : 150 dilution (except for Fig. 6J,K, where it was 1 : 100), and an FGF-7 antibody (Santa Cruz Biotechnology, sc-1365) at 1 : 100. The concentration for our FGF-8 antibody (Santa Cruz Biotechnology sc-6958) was 1 : 250. This antibody has been shown to be competent in cetacean tissues (Armfield et al. 2013). The concentration of the SHH antibody (Santa Cruz Biotechnology, sc-9024) was 1 : 100 and this antibody was used before in ceta- ceans by Thewissen et al. (2006). Our BMP-4 antibody (Santa Cruz Biotechnology, sc-6896) was used at 1 : 100. The SP-6 antibody K (epiprofin) was used at 1 : 100 (Santa Cruz Biotechnologies, SC-134025). ML All specimens are part of the collection of the North-Slope Bor- ough, Department of Wildlife Management in Barrow, Alaska (NSB-DWM). We used the computer program AMIRA to make three-dimensional reconstructions based on photographs of histo- logical sections.
Results Fig. 3 Histological sections through tooth germs and hair follicle of Dental lamina stage bowhead whale (A–I, K–O) and pig (only J). (A–D) Dental lamina stage (NSB-DWM 1999B7F); (E–H) cap stage (NSB-DWM 1999B6F); (K–O) bell Stratified cuboidal epithelium lines the oral cavity of this stage (NSB-DWM 2000B3F); staining indicated by arrows, absence of embryo (NSB-DWM 1999B7F). For much of its extent, the arrows indicates absence of staining. (A) Rostral part of upper dental oral epithelium is two-layered, but the dental placode is lamina (slide 83, FGF-8 stain). (B) Caudal part of upper dental lamina thicker, consisting of multiple layers. The dental lamina pro- (slide 125, FGF-8 stain). (C) Intermediate area of upper dental lamina jects into the mesenchyme and its aboral edges (Fig. 3A,B) (slide 103, SP-6 stain for epiprofin). (D) Rostral part of lower dental lamina (slide 81, FGF-8 stain). (E) Upper tooth cap (slide 111, SHH stain). are thickened, but there is no morphological sign of individ- (F) Upper tooth cap (slide 149, SHH stain). (G) Upper tooth cap (slide ual tooth buds (Fig. 4A). In the upper jaw, the dental lam- 195, SP-6 stain). (H) Lower tooth cap (slide 111, SHH stain). (I) Hair folli- ina extends caudally much farther than in the lower jaw cle of NSB-DWM 2003B3F, showing competency of antibody in bow- (approximately as far as slide 140 in the upper jaw, vs. slide heads (slide 29, SP-6 stain). (J) Bell stage upper tooth germ of pig, 115 in the lower jaw). The cranioventral extent of the den- showing complete dental lamina and germ for permanent tooth. (K, L) tal lamina is greater in the upper than in the lower jaw Bell stage upper tooth germ 4 (slide 49 and 77, no antibody stain). (M) (compare Fig. 3A,D). Bell stage upper tooth germ 5 (slide 22, SHH stain). (N) Mandible with tooth germ of lower tooth 15 (slide c.30, no antibody stain), lingual to We investigated the dental lamina for expression of shh left. (O) Bell stage lower tooth germ, enlarged from (N). dl, dental lam- using an antibody competent in this species, but this experi- ina; dp, dental placode; iee, inner enamel epithelium; oee, outer enamel ment failed to show presence of the protein. An antibody epithelium; sec, secondary tooth germ for permanent tooth. for FGF-8 indicates that this protein is not present in the dental lamina or the dental placode of rostral slides (slide can be detected in the upper and lower dental placode, 33, not shown), although it does occur in oral epithelium and occasionally in the dental lamina of the upper teeth. In elsewhere in the mouth. Farther caudally (Fig. 3A,B), FGF-8 general, its expression is weaker in the lower jaw. Staining
© 2017 Anatomical Society 6 Tooth and baleen development in bowhead whales, J. G. M. Thewissen et al.
Fig. 4 Three-dimensional reconstructions of tooth germs of bowhead whale, made using AMIRA, based on histological sections. The green areas indicate the presence of antibody stain. (A) Dental lamina stage in bowhead whale (NSB-DWM 1999B7F); (B–I) cap stage (NSB-DWM 1999B6F); (J–R) bell stage (NDB-DWM 2000B3F). Scale bars are approximate because of foreshortening of images, and magnification of images of the same specimen are similar. (A) Rostral part of upper dental lamina, caudolingual view (slides 73–93). (B) Cap stage upper tooth germs 4 and 5, dorsolin- gual view, rostral to right (slides 64–81). (C–E) Cap stage upper tooth germs 9 (C, dorsolingual view, slides 104–114), 10 (D, dorsolabial view, slides 115–124) and 14 (E, dorsal view, slides 146–153); the green areas in (C) and (E) indicate antibody stain for SHH. (F–H) Cap stage upper tooth germs 23 and 24 in dorsorostral, lateral and dorsal views, respectively, showing topography of inner enamel epithelium; the numbers iden- tify folds in the inner enamel epithelium as discussed in the text (slides 249–274). (I) Cap stage lower tooth germs 19 and 20 in ventrolingual view (slides 241–259). (J) Bell stage upper tooth germ 4 (slides P1:41–83) in dorsocaudal view. (K, L) Bell stage upper tooth germ 5 in lingual and dorsal view (slides P1:1–27). (M, N) Bell stage upper tooth germ 24 (slides P4:4–42) in lingual and dorsolabial view. (O, P) Bell stage lower tooth germ 15 (slides C:13–40) in ventral and lingual view. (Q, R) Bell stage lower tooth germ 40 (slides G:24–45) in labial and lingual view.
© 2017 Anatomical Society Tooth and baleen development in bowhead whales, J. G. M. Thewissen et al. 7 for epiprofin (SP-6) occurs in upper and lower dental lamina (Fig. 3C).
Cap stage
Developing teeth in this bowhead fetus (NSB-DWM 1999B6F) are mostly in the early cap stage: they show a cap shape, but lack stellate reticulum. There are 41 upper and 35 lower caps. Upper tooth caps are in general larger than lower tooth caps (Fig. 3E–H), and the upper dental lamina extends farther caudal then the lower dental lamina: the last tooth bud occurs in slide 455 in the upper, and in slide 425 in the lower jaw. In most mammals the dental lamina has the shape of a sheet that invaginates from the oral epithelium into the underlying mesenchyme and remains there until long after the cap stage. For instance, the dental lamina of the pig is connected to the oral epithelium in the early cap stage and remains attached in some regions until the bell stage (Fig. 3J; Armfield, 2010). In contrast, the bow- head dental lamina is an irregular bar that extends between the tooth caps without being clearly distinct from them (Fig. 4F) and is not connected to the oral epithelium. The dental lamina does connect all tooth caps, and extends well beyond the last tooth. Three-dimensional reconstruction shows that the topog- raphy of the inner enamel epithelium of upper and lower tooth caps differs (Fig. 4B–H). In the upper jaw, tooth caps 4, 9, 10, 14, 23 and 24 (counting from rostral to cau- dal) were reconstructed. The inner enamel epithelium of all of these caps is similar: they display a more or less cir- cular outline in dorsal view (Fig. 4H), with a raised central Fig. 5 Palate and head of bowhead fetus (NSB-DWM 2000B3F). area that is surrounded by three or four depressed areas (A) Palatal epithelium, as removed from skull, backlit showing trans- that are connected, arranged in a square around the cen- mitted light to show developing tooth germs (in the bell stage). (B) tral area. This is best seen in oblique views, such as Head of fetus showing great extent of the lower lip. (C) Clear/stain Fig. 4C–E. In the lower jaw, the outline of the tooth cap, preparation of head of the same fetus, showing the narrow dentary in ventral view, is elongate, and there are two depres- that does not match the outline of the lower lip. Cartilage is blue and sions, a deeper one rostrally, and a shallower one cau- bone is red in this preparation. dally (Fig. 4I). We stained some of these sections with an antibody for Most of the upper tooth germs are simple bowl- or bell- SHH. SHH is present in the upper tooth caps (Figs 3E,F and shaped structures (Fig. 4K–N), separated from their neigh- 4C,E), but not in the lower tooth caps (Fig. 3H). In the upper bors by undifferentiated mesenchyme. However, a few teeth, only the central depressed area of each germ shows germs are in contact with the neighboring germ and such a SHH signaling (Fig. 4C,E). Staining with an antibody for epi- pair could be interpreted as a single, elongate, bicuspid profin indicated that this protein is not present in the bow- germ (Fig. 4J). Macroscopically, recognizable joined pairs head dental caps at this age (Fig. 3G), whereas the ability of are numbers 26–27, 28–29, 30–31, 32–33 and 34–35 on the this antibody to recognize the protein is shown by staining right side, and 22–23, 24–25, 26–27 and 29–30 on the left in the dental lamina (Fig. 3C) and hair follicles (Fig. 3I) of side. Microscopic examination after sectioning reveals that bowhead whales. some additional pairs of germs that look unpaired macro- scopically are in reality also paired. For instance, germ 4 on the right side appears externally to be single, and was Bell stage counted as such, but is in fact paired (Fig. 4J). The individual This fetus (NSB-DWM 2000B3F) has a row of bell stage cusps in this germ are lined up craniocaudally, with the tooth germs in the upper jaw (Fig. 5A), evenly spaced in a anterior cusp being lower in elevation than the posterior more or less straight line. Macroscopically, 40 germs can be cusp.Ontheleftside,themostrostralbellsoccurwellcau- distinguished on the right side and 32 on the left side. dal to the most rostral bells in the right side (Fig. 5A).
© 2017 Anatomical Society 8 Tooth and baleen development in bowhead whales, J. G. M. Thewissen et al.
In the lower jaw, tooth germs are embedded in the dor- Baleen placode stage solingual part of the dentary (Fig. 3N), which is ossifying at The palate of these fetuses (NSB-DWM 2007B16F and this stage. On the left side, 28 lower tooth bells are present, 2009KK1F) is narrow, and displays no remnants of teeth. and the most rostral of these is located well caudal to the The palatal surface consists of four gross morphologically tip of the dentary. The most caudal germs are located well distinct zones. From medial to lateral these are: a median rostral to the coronoid process. Lower tooth germs thus vascularized area that is convex and narrows caudally, the have a relation to bone, whereas upper tooth germs do corpus cavernosum maxillaris (Zone 1; Figs 6I and 7A); a not. As a result, lower tooth germs are not close to the oral band of undifferentiated epithelium that narrows rostrally epithelium, as the edge of the lower lip at this stage flares (Zone 2); a white placode that also narrows rostrally (Zone dorsally, foreshadowing the shape of the lower jaw after 3); and a second band of undifferentiated epithelium (Zone birth. This is best appreciated when comparing a lateral 4). The white placode is the area from which the baleen view of a fetal head with a cleared/stained fetal head develops. Near the caudal edge of the palate, the Zone 2 (Fig. 5B,C) of the same age. Unlike the lower jaw, upper broadens. tooth bells are close to the oral epithelium, and are not in FGF-4 signaling can be demonstrated in much of the close contact to the bones of the upper jaw. palatal epithelium. In Zone 2, such signaling occurs Histologically, upper and lower tooth germs are in the mostly superficial to vessels that are located in dermal early bell stage in which the inner enamel epithelium con- papillae (Fig. 6J,K). In Zone 3, FGF-4 signaling shows an sists of columnar epithelium. In lower tooth bells (Fig. 3N, iterative pattern: signaling occurs in dorsoventral bands O), the inner enamel epithelium consists of cuboidal cells, that span most of the depth of the epithelium and is visi- whereas the outer enamel epithelium consists of squamous ble in cross- and parasagittal sections (Fig. 6L,M), with cells. The cells that form the tip of the dental papilla are bands separated by areas lacking staining. In horizontal elongating, forming odontoblasts. In the upper jaw, there sections of some areas of Zone 3, these bands fuse into is no remnant of the dental lamina, whereas in the lower linearfeaturesthatextendmediolaterally, and are lined jaw the dental lamina is weak, connects subsequent tooth up craniocaudally, an orientation similar to postnatal germs, but is not connected to the oral epithelium (Fig. 3O). baleen plates (Fig. 6N). There is no trace of secondary tooth buds in the upper den- Staining for FGF-8 occurs in the area medial to Zone 3, tition, but in the lower dentition a flange of the dental lam- mostly in epithelial areas away from dermal papillae ina projects lingual to the primary tooth bud, and may (Fig. 6O), and is very faint to absent in the placode. We did represent a secondary bud (Fig. 3O). In the pig, the dental not detect FGF-7 in the palatal sections, including the lamina at this stage shows clear lingual invaginations that baleen-forming placode, whereas the competency of this will give rise to the teeth of the permanent dentition antibody in this species is shown by staining in the hair folli- (Fig. 3J). cle of a bowhead whale (Fig. 6H). Investigated upper teeth (Fig. 3L–O) are similar in size and shape along the tooth row (except for those that are paired). Lower tooth germs are smaller and of more diverse Full term shape (Fig. 3O–R). More anterior teeth are elongate The four zones distinguished on the palate can still be rec- (Figs 3O and 4O,P), whereas more posterior teeth are more ognized in this fetus (Fig. 7B,C). The median vascular area globular but less regular and smaller than upper bells has not changed, but the undifferentiated epithelium is (Fig. 4Q,R). now a narrow strip. Lateral to this zone extends a wide field At this stage, there are no morphological signs of baleen of thin baleen hairs (Fudge et al. 2009) that are anchored formation in the epithelium (Fig. 6A). Many small blood into the gingiva by small accessory columns and platelets vessels occur in the region medial to the tooth bells, and that are topographically in the same area as the undifferen- deep to it are the major palatine artery, vein and nerve. tiated epithelium of the previous stage. These accessory The area immediately adjacent to the median plane is the plates (Fudge et al. 2009) are not keratinized and increase area where, in adult bowhead whales, a large subcutaneous in cross-sectional area and length from medial to lateral. plexus of blood vessels occurs that will develop into the cor- Further lateral yet, accessory plates are subsumed in and pus cavernosum maxillaris of Ford et al. (2013). SHH signal- constitute a single large sheet of barely keratinized tissue ing occurs in the stellate reticulum of the upper and lower that does not project into the oral cavity and is totally teeth (Fig. 4M; lower jaw, slide C-57, not shown). Lateral to embedded into the gingiva (Zone 3). This area grades later- this region, immunohistochemistry indicates that FGF-4, ally into the baleen plate, which projects approximately FGF-7 and FGF-8 signaling occurs in the epithelium, but 6 cm into the oral cavity in the middle of the baleen rack. In there is no iterative pattern of intensity fluctuations that palatal view (Fig. 7C), the baleen hairs and frayed inner sur- could be associated with initiation of individual baleen face of the baleen plate give the appearance of a matted plates (Fig. 6B,E–G), although all three proteins are absent field of hair. near the median plane (Fig. 6C,D).
© 2017 Anatomical Society Tooth and baleen development in bowhead whales, J. G. M. Thewissen et al. 9
A
B C D
Fig. 6 Histological sections showing baleen formation. (A) Cross-section through half of E FG H the palate of fetus NSB-DWM 2000B3F, showing bell-shaped tooth germ and epithelium that will form baleen (slides P6:36). Medial to left, letters indicate approximate location of figures (B)–(G) in antibody-stained sections of this figure. (B–D) Details of medial palatal epithelium of NSB- I DWM 2000B3F (B, slides P6:32, FGF-4 stain; C, slides P6:33, FGF-7 stain; D, slides P6:31, FGF-8 stain). (E–G) Details of palatal epithelium near where baleen will form of NSB-DWM 2000B3F (E, slides P6:32, FGF-4 stain; F, slides P6:33, FGF-7 stain; G, slides M P6:31, FGF-8). (H) Hair follicle of bowhead whale 2011B8 (slide 2, FGF-7 stain). (I) Cross- section through half of the palate of fetus L NSB-DWM 2009KK1F, same orientation as (A) J (slides P12:26). (J, K) Detail of Zone 2 of the palatal epithelium of NSB-DWM 2009KK1F (J, slides P12:24, cross-section; K, slides P13:47, horizontal section; FGF-4 stain). (L, M) Detail of Zone 3 of palatal epithelium of NSB-DWM 2009KK1F with FGF-4 stain (L, slides P12:24, ONK cross-section; M, slides P6:3, parasagittal section). (N) Detail of Zone 3 of palatal epithelium of NSB-DWM 2009KK1F (slides P13:78) with FGF-4 stain, horizontal section, labial to top. (O) Detail of Zone 2 of palatal epithelium of NSB-DWM 2009KK1F (slides P12:29, cross-section, FGF-8 stain). Scale bars: 0.1 mm, unless otherwise indicated.
later ones. Karlsen (1962) reanalyzed Kukenthal’s€ data and Discussion based his analysis on a collection that also included younger embryos than were available to Kukenthal.€ Karlsen (1962) Tooth counts concluded that some tooth germs fuse occasionally in onto- Our bowhead specimens display polydonty, and this was geny, but that the fusion pattern is consistent neither along also documented in other mysticete fetuses (Julin, 1880; the tooth row, nor between left and right tooth row, nor Kukenthal,€ 1891; Dissel-scherft & Vervoort, 1954; Karlsen, between different individuals. Karlsen (1962) also observed 1962). Embryologists have speculated on the origin of that crown complexity does not increase toward the caudal polydonty in cetaceans. Kukenthal€ (1893) proposed that part of the tooth row as is common in heterodont denti- polydonty was the result of splitting of multicuspid teeth, tions of other mammals. We concur that fusion observed in whereas Julin (1880) suggested the opposite: that individ- tooth crowns of our specimens is not related to the patterns ual, conical tooth germs fuse in development, implying that that determine tooth counts and crown shape in hetero- earlier embryos would have even more tooth germs than dont mammals. Consistent with the view of Karlsen (1962),
© 2017 Anatomical Society 10 Tooth and baleen development in bowhead whales, J. G. M. Thewissen et al.
postnatally (Fig. 2H), and it is possible that these were formed by the same process as is documented in the mys- ticete fetuses we studied. Karlsen (1962) pointed out that, in Balaenoptera,the dental lamina continues to produce new germs caudal to existing teeth for longer than in other mammals. The exten- sion of the bowhead dental lamina caudally, long after it has disappeared in other mammals, would lengthen the period of tooth formation and thus create more tooth germs. In terrestrial mammals (Yamanaka et al. 2007) the dental lamina develops buds soon after its formation, and this appears to be the case in dolphins too (Mi sek et al. 1996). It is not the case in bowhead whales, where forma- tion of tooth buds is delayed. It has been shown experimen- tally that a persistent dental lamina can result in polydonty in lab animals (Rodrigues et al. 2011). Fetal embryonic poly- donty in bowhead whales may be the result of the early, strong and extended development of the dental lamina. This type of heterochronic development of the dental lam- ina is taken to an extreme in the manatee Trichechus, where the dental lamina persists throughout life, and new molar-shaped teeth are formed nearly indefinitely long after birth. Nakamura et al. (2006, 2008) found that a loss-of-func- tion mutation of the transcription factor epiprofin causes polydonty and simple crown morphologies in mice. In mice with functional copies of the gene (+/+), this protein is extensively expressed through the enamel epithelium from the bud through the enamel formation stage (Nakamura et al. 2006). Epiprofin development in bowhead whales is truncated, our specimens showed evidence in the dental lamina (Fig. 3C), but not at the cap stage, consistent with polydonty and simple crown morphologies of our later
Fig. 7 Palate and baleen in fetus. (A) Palate of fetus NSB-DWM fetuses. It is possible that changes in epiprofin signaling 2007B16F, rostral to left. (B) Rostral cross-section through the poste- underlie the morphological changes in the dental lamina. rior part of the unilateral palate of NSB-DWM 2015B9F. (C) Ventral Our working hypothesis is that the increased variability in view of the posterior palate (same individual as B), showing baleen tooth numbers as well as polydonty in early mysticetes is hairs and their root in the gingiva of the palate. Zones identify the result of a release of the selection pressures related to homologous areas in Figs 6 and 7. accurate occlusion as occlusion was not critical to food acquisition. The ontogenetic fingerprints thereof are still we consider the high tooth number in fetal bowhead present in mysticete embryos. Reduced expression, in time whales to be the result of dental patterning earlier in onto- or concentration, of epiprofin may be part of the mecha- geny, not of divisions in already formed dental germs long nism by which this was accomplished. after they form. One of the geologically youngest toothed mysticetes is Molar crown morphology Aetiocetus polydentatus. This species has the highest num- bers of excess teeth of any fossil mysticete (Barnes et al. At the cap stage, bowhead whalesdisplayfoldsoftheinner 1994), and has different numbers of teeth in the left and enamel epithelium that resemble those that are associated right jaw. Cobourne & Sharpe (2010) discussed differences with secondary enamel knots in land mammals (Jernvall & betweenleftandrightjawsassymptomsofareleaseofsta- Thesleff, 2000). In land mammals, these folds eventually bilizing selection, and Salazar-Ciudad & Jernvall (2010) indi- give rise to the complex molar crown morphology of these cated that the lack of strict occlusion in seals was correlated taxa (Fig. 2C). Lower bowhead tooth germs resemble the to higher dental variation. The same may be true for pattern of the lower teeth of the Eocene whale Pakicetus toothed mysticetes. Indeed, fused crowns that complicate with a high part rostrally (trigonid) and low part caudally precise occlusion do occur in some fossil cetaceans (talonid; Fig. 2A,E). The folding pattern of the inner enamel
© 2017 Anatomical Society Tooth and baleen development in bowhead whales, J. G. M. Thewissen et al. 11 epithelium in upper teeth resembles the cusp pattern of the anterior teeth at the cap stage, suggesting that they are artiodactyl relatives of cetaceans, hippopotamids and raoel- not related to the original heterodonty of the mysticete lids (Fig. 2C; Gatesy et al. 2013), with four folds that match ancestors. However, this does not explain the discrepancy the arrangement of the original four cusps (Fig. 4F,H). between upper and lower tooth bells. At present, it is not Dissel-scherft & Vervoort (1954) studied the mineralized possible to test these two evolutionary scenarios as speci- fetal teeth of a Balaenoptera fetus of 102 cm, a stage for mens of the relevant developmental stages are not avail- which we do not have bowhead fetuses. They found that able. posterior lower teeth have a complex three-cusped crown, similar to the trigonid of raoellid relatives of cetaceans, Tooth shape along the tooth row whereas anterior teeth have single-cusped crowns similar to incisors and canines of heterodont mammals. These teeth Signaling that is important in defining mammalian tooth were composed of dentin and lacked enamel, consistent classes occurs at the dental lamina stage, when morpho- with the study of Demer e et al. (2008) and Meredith et al. genetic fields are created in the jaw (Caton & Tucker, 2009; (2009) that detected nonsense mutations in the genes Jernvall & Thesleff, 2012). These guide the formation of dif- involved with amelogenesis of mysticetes. It would be ferent tooth classes. Such signaling occurs long before the remarkable if the embryonic germs resembled molar crowns signaling that determines the size, spacing and number of of species that lived more than 40 million years ago. teeth, suggesting that these processes are independent to To investigate this, we used the signaling protein SHH to some degree. In investigated mammals (mice, shrews and further explore the folds of the inner enamel epithelium. minks), BMP-4 signaling sets up the incisor field of the jaw, SHH is a marker for the enamel knot, the signaling center whereas FGF-8 sets up the molar field. The canine and pre- for cusp tips (Jernvall & Thesleff, 2000; Thesleff, 2000). Our molar fields may result, in part, on the interaction of these experiments show that the protein is not expressed in the signaling proteins along a gradient in the jaw. Armfield tips of the folds but instead in the valley between them in et al. (2013) studied the dolphin Stenella, and showed that the upper teeth. This pattern of expression foreshadows the signaling field of BMP-4 has expanded caudally and the single-cusped germs of the bowhead bell stage, but overlaps with the FGF-8 field. Both Stenella and bowhead does not support the interpretation of the enamel folds as whales retain the FGF-8 field in the caudal part of the jaw. original cusps. With our embryos, we could not document In bowhead whales the signaling is more distinct in the any SHH in the lower teeth at the cap stage. Furthermore, upper than in the lower jaw. All teeth in Stenella are simi- in our specimens, even anterior upper tooth germs (germs lar, and have incisor-like morphologies. Experimentally, it 1–8) have complex morphologies of the inner enamel has been determined that, in mice, BMP-4 forces posterior epithelium at the cap stage, while the artiodactyl relatives teeth into incisor-like morphologies (Murashima-Suginami of cetaceans have simple crowns for these teeth (Thewissen et al. 2008; Munne et al. 2010), a mechanism that is consis- et al. 2007). tent with the Stenella data. Unfortunately, our BMP-4 anti- At least two interpretations of these data are possible. body appears to be incompetent in bowhead whales, and Thefirstisthatthefoldingpatternsoftheinnerenamel we cannot determine the protein’s signaling traces in this epithelium of bowhead whales are induced by secondary species. enamel knots (and are thus cusp homologs) at some stage In our specimens, there is clear morphological variation but that our collection does not include specimens of that of teeth along a cline in the lower jaw, but not in the upper stage. The heterodonty of mineralized teeth described by jaw. Lower crowns of rostral teeth are longer and more Dissel-scherft&Vervoort(1954)isconsistentwiththisinter- pointed, whereas those of caudal teeth are more globular pretation, and so is the heterodonty of lower tooth crowns in the 40.3-cm fetus. Karlsen (1962) also found this in Balae- in our bell stage fetus. The homodonty of the upper tooth noptera. Julin (1880) studied the lower jaw of a Balaenop- bells then provides a discordant datum. tera fetus with 41 tooth germs, where teeth 1–9had The second possible explanation is that these folds are simple, one-cuspid tooth bells, whereas teeth 10–41 had not related to secondary enamel knots at all. Nakamura two or three cusps per tooth, sometimes of the same et al. (2008) found that the inner enamel epithelium dis- height, sometimes of different heights. In the Balaenoptera plays incipient folding that is not related to enamel knot mandible described by Dissel-scherft & Vervoort (1954), the formation in mice that do not express epiprofin. These mice anterior 36 right and 38 left teeth were simple conical also show a deficiency in shh expression. This is consistent teeth, and teeth more posterior showed molar trigonid with the disruption of epiprofin signaling in the bowhead morphologies. The lower jaw of our 40.3-cm specimen dis- whale. In fact, it is possible that the small accessory cusps plays heterodonty too, although it is less pronounced, and that occur on the anterior and posterior slope of the main clearerinthelowerthaninthe upper jaw. Karlsen (1962) cusp in fossil mysticetes and their archaeocete ancestors indicated that Julin’s fetus may have been 45 cm in length, (Fig. 2B,G,H) are caused by truncated expression of epipro- indicating that it was at a later ontogenetic time than our fin. We observed inner enamel epithelium folds even in bowhead fetus.
© 2017 Anatomical Society 12 Tooth and baleen development in bowhead whales, J. G. M. Thewissen et al.
Heterodonty occurs in all extinct toothed mysticetes, with that projects lingually from the monophyodontic teeth of distinct incisors and canine, and a gradual transition of sim- terrestrial mammals (mouse, hedgehog, pig) is a likely rem- ple one-cusped teeth to more complex teeth in premolars nant of a developing permanent tooth. This is similar to the and molars (Barnes et al. 1994; Fitzgerald, 2006, 2009). The crest we observed in the lower dentition of bowhead most consistent interpretation of this pattern is that the whales, and we interpret the bowhead tooth bells as part polydont and heterodont fetal lower dentition of mysticete of the primary (deciduous) dentition, and hypothesize that fetuses is a hold-over of their Oligocene ancestors, whereas these tooth germs represent the single tooth generation of morphological remnants of the fetal upper dentition have fossil toothed mysticetes such as aetiocetids. The lingual been erased. buds would then be remnants of the permanent dentition. Whitlock & Richman (2013) reviewed tooth replacement mechanisms. The dental lamina plays a key role in this pro- Tooth replacement cess (Jarvinen€ et al. 2009). In mammals, the loss of a connec- Julin (1880) did not find evidence of a deciduous dentition tion between dental lamina and oral cavity stops new tooth in the (single) early fetus of Balaenoptera that he studied. generations from forming (Buchtova et al. 2012). In Kukenthal€ (1893) interpreted patterns observed in his col- humans, the dental lamina begins to break up at the bell lection of prenatal Balaenoptera to indicate that there were stage by detaching from the oral epithelium (Bhussry, three waves of dentition development. In his view, the sec- 1980), whereas in bowheads this connection is lost by the ond of these waves proceeds to develop furthest and is cap stage. homologous to the deciduous dentition of other mammals. We hypothesize that the early loss of a connection He identified remnants of a predeciduous dentition lateral between dental lamina and oral epithelium in bowhead to, and germs of the adult dentition medial to, the decidu- prenatal development plays a role in the loss of diphyo- ous dentition. Karlsen (1962) studied a Balaenoptera donty. Traces of a second tooth generation can still be embryo with early dental caps and disputed the presence of found in the lower, but not in the upper dentition. a predeciduous dentition, instead interpreting the lateral clusters of epithelial cells as remnants of the lateral dental Developmental tooth loss lamina. The dental lamina occurs in many groups of mam- mals, and disappears before the bell stage is reached. Karl- Complete tooth loss in bowheads takes place between the sen (1962) interpreted the lingual clusters of cells as stages of fetuses of a total length 40.3 and 159 cm, and is emanating from the dental lamina, not from other tooth not documented by our specimens. Several aspects of the buds, and part of the same tooth generation as the larger process of tooth loss are known for mysticetes. Karlsen buds. In Karlsen’s material (Karlsen, 1962), these clusters (1962) found evidence of dentin formation in Balaenoptera, occasionally developed into tooth caps. It is possible that but not enamel formation, consistent with the absence of such a process in related taxa with teeth actually results in enamel formation gene expression shown by Demer eetal. the formation of erupted tooth crowns. Fordyce (1982) (2008). Dissel-scherft & Vervoort (1954) describe mineralized reported supernumery single-rooted teeth in a fossil odon- crowns in detail, noting several histological features that tocete that may be the result of such a process. are indicative of resorption. Davit-Beal et al. (2009) Karlsen (1962) noted the presence of conspicuous folds in reviewed the little that is known about tooth loss in tetra- the outer enamel epithelium in a 56-cm fetus, and that pods in a developmental context. Comparing ontogenetic smaller germs in the cap stage are located somewhat lin- data for different taxa, it is clear that different developmen- gually from larger more developed germs in the lower, but tal processes underlie tooth loss in different taxa (Louchart not in the upper jaw. & Viriot, 2011; Tokita et al. 2013). It is also clear that the Dissel-scherft & Vervoort (1954) described in some detail developmental processes that arrest tooth development are how, in the lower jaw of Balaenoptera, tooth bells of multi- unrelated to and overprint the processes related to tooth ple sizes and shapes seem to co-occur in a cranio-caudal pat- formation. This implies that, in cladistics analyses, the tern that shows no discernable regularity or trend. These absence of teeth in a clade should be considered a character authors did not go so far as to interpret these as different independent from that related to the precise number of tooth generations, taking them instead for evidence of a teeth in its toothed relatives. gradually disappearing heterodont dentition. Our evidence is consistent with the view of Karlsen (1962) and Dissel- Developmental origin of baleen scherft & Vervoort (1954): the tooth germs that reach the bell stage in modern bowhead fetuses all arise from a single The first morphological signs of baleen formation occur tooth generation. There is evidence of anlagen for teeth of when the teeth are at the bell stage. When the palate is a second tooth generation in the form of lingual buds pro- backlit, a dense area is visible medial to the tooth germs truding from the lower, but not the upper tooth bells (Fig. 5A). This area is underlain by a neurovascular bundle (Fig. 3O). Dosedelov a et al. (2015) found that a weak crest (Fig. 6A) and develops into the baleen placode later in
© 2017 Anatomical Society Tooth and baleen development in bowhead whales, J. G. M. Thewissen et al. 13 ontogeny (Fig. 6I), when all traces of teeth disappear. FGF-4 upper and lower dentition are related to the lack of this signaling occurs in an iterative pattern in the placode and function in the lower teeth. could be related to eventual iterative pattern of the baleen The fossil record is also consistent with this hypothesis. plates. In mammals with teeth, FGF-4 is expressed in the There is a trend in the earliest fossil mysticetes toward enamel knot (Niswander & Martin, 1992; Kettunen & The- homodonty of the molars and premolars, but polydonty sleff, 1998; Tucker et al. 1998), but the protein is not was limited, with no more than a single additional tooth involved in hair formation (Mitsui et al. 1997) or nail forma- (beyond the original 11 per quadrant) in most clades and tion. four additional teeth in one taxon (Aetiocetus polydenta- FGF-8 plays an important role in the initiation of tooth tus). It is puzzling that modern mysticete embryos have development in mammals (Niswander & Martin, 1992; more than 40 tooth germs if known members related to Kettunen & Thesleff, 1998; Nadiri et al. 2004), including their ancestors never had more than 15 teeth, unless those cetaceans (Armfield et al. 2013), but plays no role in the germs serve a function beyond that of teeth. This situation development of hair (Rosenquist & Martin, 1996) or palatal differs from odontocetes, where polydonty is widespread rugae (Porntaveetus et al. 2010a). By contrast, FGF-7 is across fossil and modern clades. We hypothesize that mys- involved in signaling in the mammalian hair follicle (Mitsui ticetesthatwerehighlypolydontasadultsneverexisted, et al. 1997; Nakatake et al. 2001), and FGF-10 in palatal and that the iterative signaling that underlies the formation rugae development (Porntaveetus et al. 2010a), and these of so many tooth germs is related to the signaling that is are not involved in tooth signaling (Porntaveetus et al. important in baleen formation later in ontogeny, but was 2010b). We could not detect signaling of FGF-7 and FGF-10 not engaged until later in evolution, when postnatal teeth relatedtobaleenformation. had been lost already. Involvement of FGF-4 in baleen development instead sug- The pattern of signaling in the palate coincides with gests that some of the original signaling related to tooth results presented by Fudge et al. (2009) regarding baleen formation now partakes in the formation of baleen. There development: initially undifferentiated epithelium is are other indications that this may be the case. In most induced to form accessory plates and, as the fetus grows, mammals, upper and lower teeth follow similar ontoge- these structures are incorporated into the main baleen netic trajectories, but in bowheads they deviate early in plate. The pattern of implantation of baleen plates, acces- ontogeny. The upper dental lamina differs morphologically sory plates and bristles on the back of the palate has no from the lower dental lamina: it is longer (rostrocaudally) matchinmammals,butisreminiscent of other ectodermal and higher (dorsoventrally) and creates more tooth germs. structures in the mouth of other vertebrates. Generations Those tooth germs are more homodont than those in the of replacement teeth in reptiles, for instance, are arranged lower jaw. In some sense then, the tooth germs of the in patterns that show a maturation gradient from medial to upper jaw appear to foreshadow baleen development, lateral, but implantation of individual elements in rows that where many, near-identical plates form. Lower tooth germs are oblique to the mediolateral plane (Westergaard & Fer- develop close to the dentary, and they are far from the guson, 1990; Whitlock & Richman, 2013). lower lip because the lip bulges dorsally (Fig. 5B,C). Thus, It might seem strange that two structures that are very the distance between the lower mandibular epithelium and different in composition, teeth and baleen, are formed by the developing tooth germs is great. This epithelium is similar signaling pathways. However, it should be remem- known to play a role in tooth development (Jarvinen€ et al. bered that enamel and keratin have shared the oral cavity 2009), and that topography may affect lower tooth devel- since the earliest vertebrates, as evidenced by the kerati- opment. While this may explain the differences in upper nous teeth of cyclostomes (Trott & Lucow, 1964), although and lower dentition development in bowhead whales, the the formation of these has no relation to tooth origins lower lip of other fossil and modern mysticetes (e.g. bal- (Smith & Coates, 2000; Huysseune et al. 2009). The link aenopterids) is closer to the mandible with its tooth germs. between keratin and the oral cavity also virtually character- A different mechanism may underlie the loss of tooth izes birds. Louchart & Viriot (2011) discussed the evolution germs in these taxa. of tooth loss and the formation of a rhamphoteca (keratin Ishikawa et al. (1999) noted similarities in extracellular beak) in birds, noting that local keratinization may be a matrix proteins involved in baleen development to those cause for arrest of tooth formation. Wu et al. (2004) noted involved in tooth development, and Davit-Beal et al. (2009) the developmental similarity in signaling among morpho- speculated that baleen development was functionally logically very different keratinized epithelial appendages, related to tooth loss based on developmental timing. These and teeth too share some of these pathways. Clearly, there observations support our interpretation of the signaling evi- is great plasticity in epithelial appendages, both develop- dence. Our working hypothesis is that development of the mentally and evolutionarily (Chuong, 1998; Dhouailly, upper dentition in the bowheads is a necessary step in the 2009), and common developmental pathways underlie all development of baleen, and that differences between of them.
© 2017 Anatomical Society 14 Tooth and baleen development in bowhead whales, J. G. M. Thewissen et al.
Some of the significant differences in developmental pro- upper jaw is higher dorsoventrally (Fig. 8.1) and longer cesses forming teeth and baleen may also be bridged by cranio-caudally (Fig. 8.2) than the dental lamina of the minor changes in patterning. For instance, mammalian lower jaw. FGF-8 signaling occurs in the posterior regions of teeth form by invagination of epithelium into underlying both, but is stronger in the upper jaw than in the lower jaw mesenchyme, whereas baleen forms by evagination into (Fig. 8.3). Such signaling is the basis for molar morphologies the oral cavity. The protein IKKa is involved with the devel- of posterior teeth in generalized mammals, and is also opment of hair and whiskers, and, possibly, in the regula- found in homodont dolphins. In dolphins, FGF-8 signaling tion of other ectodermal–mesenchymal interactions (Karin in the posterior jaw is overprinted by BMP-4 signaling, & Delhase, 2000; Ohazama et al. 2010). In Ikka mutant mice, which may induce the incisor-like morphologies of the incisors and whiskers form by evagination, not invagina- cheek teeth (Armfield et al. 2013), as it is known to do in tion. Furthermore, in the talpid2 mutant of chicks, tooth mutant mice (Munne et al. 2010). It is likely that the FGF-8 germs form by evagination, not invagination (Harris et al. signaling causes the heterodontic features of the lower 2006). There are many other genes that play a role both in jaw of bowhead fetuses, as well as the limited degree of the development of teeth and keratin structures. For heterodonty in extinct toothed mysticetes (e.g. Janjucetus, instance, fgf-20 is involved in tooth and hair development, Mammalodon and aetiocetids; Barnes et al. 1994; Fitzger- and its subtle effects on the morphology of teeth could be ald, 2006, 2009). a potent agent in evolutionary changes in tooth morphol- At the dental cap stage, the dental lamina has lost con- ogy (Ha€ar€ a€ et al. 2012). Obviously, shifts in single genes are tact with the oral epithelium (Fig. 8.4), an event that takes insufficient to turn teeth into baleen in ontogeny, but it place much later in generalized mammals. The dental lam- does challenge the assumption that the morphological dif- ina is necessary for the formation of subsequent tooth gen- ference between these processes is unbridgeable. erations, and we propose that its loss underlies the absence Our evidence suggests a greater level of homology of a second tooth generation even in fossil, toothed between teeth and baleen than their homology as epithe- mysticetes. lial organs (sensu Chuong, 1998), namely that the particular At the bell stage, upper and lower dentitions vary signifi- gene expression pathway that leads to two generations of cantly. The upper tooth germs are homodont (Fig. 8.8), teeth in most mammals has been co-opted to be involved there is no evidence of primordia for a second tooth gener- in baleen formation in cetaceans. Several approaches can ation, and individual bells are not connected by the dental be used to investigate this in greater depth. First, signaling lamina. In the lower jaw, there are fewer, more variably that underlies the number of palatal rugae that forms and developed germs (Fig. 8.5 and 8.6) and they are smaller the spacing between them is suggestive (Pantalacci et al. than the upper teeth, there is a gradient of heterodonty 2008) and may elucidate the developmental basis for along the jaw (Fig. 8.9), while the dental lamina remains. baleen plate spacing. Second, a detailed understanding of This is consistent with the findings of Karlsen (1962). A lin- the development of the compounds that compose baleen gual crest protrudes from the tooth bells that may be the could help to understand selective powers at work remnant of the secondary dental lamina that is involved in (Szewciw et al. 2010). These compounds may include a the formation of permanent teeth. We interpret these dif- large diversity of keratins (Bragulla & Homberger, 2009), ferences in the context of the imposition of selection forces and their developmental pathways may be quite different on the upper dentition, leading to greater polydonty, from each other. homodonty and monophyodonty. In contrast, the lower teeth maintain some of the ancestral patterns of hetero- donty and diphyodonty, while they are underdeveloped in Conclusion general. We integrate our data and discussion into a working Individual tooth caps show patterns of folds in the inner hypothesis that comprises the development of teeth and enamel epithelium that resemble cusps of the Eocene baleen in bowhead whales. Until the bell stage, bowhead ancestors of whales in the upper molars, and lower molar dental ontogeny is easily recognizable as a variation on the morphology of the earliest whales in the lower teeth dental ontogeny of generalized mammals. We interpret dif- (Fig. 8.7). Although this pattern is suggestive, there is only ferences that do exist as precursors of events that happen a single area of SHH signaling in these teeth, and this area after the bell stage: the complete loss of teeth and the probably is related to the single cusps that occur in the development of baleen. Here, we summarize that process bell stage of these teeth. Folding of the inner enamel and our interpretation of it and provide an evolutionary epithelium could also be caused by absence of epiprofin context (Fig. 8). signaling and, indeed, epiprofin signaling is reduced in The first differences between dental development of Balaena. Mice with disrupted epiprofin signaling display bowheads and generalized mammal development already polydonty and simple crown morphologies along the occur at the dental lamina stage, and were observed before tooth row (Nakamura et al. 2008). The dental patterns of by Karlsen (1962) in Balaenoptera: the dental lamina of the teeth in À/À epiprofin mice suggest that if enamel
© 2017 Anatomical Society Tooth and baleen development in bowhead whales, J. G. M. Thewissen et al. 15
Fig. 8 Diagrams showing tooth and baleen development in bowhead whale. Upper and lower oral epithelium and structures derived from it are shown, mesenchyme is not shown. For baleen placode and postnatal stage, the lower jaw is not shown. Numbers refer to important features dis- cussed in the text. (1) Upper dental lamina is higher than lower dental lamina. (2) Upper dental lamina extends further caudal than lower dental lamina. (3) FGF-8 expression in caudal (molar) region of dental lamina. (4) Dental lamina lacks contact with oral epithelium. (5) More tooth caps occur in the upper than in the lower jaw. (6) Lower tooth caps are more variable in shape and size than upper tooth caps. (7–8) Inner enamel epithelium of tooth caps shows complex folds, whereas that of upper tooth bells (8) shows single fold that corresponds to single cusp of tooth. (9) Lower tooth bells are heterodont. (10) Secondary tooth buds develop in lower, but not upper jaw. (11) Dental lamina persists longer in lower than upper jaw. (12) FGF-4 expression in upper jaw epithelium. The pattern of expression of fgf-4 foreshadows the footprint of baleen plates.
© 2017 Anatomical Society 16 Tooth and baleen development in bowhead whales, J. G. M. Thewissen et al.
formation were to take place in these tooth caps, small whales and collection of samples. We thank all of those involved, cuspules would develop on these teeth, a feature that is especially Director Taqulik Hepa and Deputy Director Harry Brower characteristic for the posterior cheek teeth of toothed mys- Jr. Funding was provided by the North Slope Borough and NEOMED. ticetes such as Mammalodon, Janjucetus and Aetiocetus. All authors declare that there is no conflict of interest that might Jarvinen€ et al. (2006) showed that simple crowns and poly- affect their objectivity in preparing this paper. donty are not independent developmentally. It is possible that lack of epiprofin contributes to both effects in bow- heads. Many other genes are involved in the determina- Author’s contributions tion of cusp patterns (e.g. Eda, Spry, Sostdc; Jernvall & Thewissen, George, Suydam and Stimmelmayr collected the Thesleff, 2012), and further investigations are needed to samples; Hieronymus and McBurney prepared the samples test these hypotheses. and did the experiments; and Thewissen, Hieronymus and The first signs of homodonty, monophyodonty, poly- George did the writing. donty and simple molar crown patterns all appear in the fossil record closely spaced, near the origin of mysticetes. Evidence presented by us suggests that multiple factors may References play a role in the evolution of these features. These include Armfield BA (2010) The evolution and development of mam- the disruption of epiprofin signaling and the lengthening malian tooth class. PhD dissertation, Kent State University: of the dental lamina and its early detachment from the oral Kent, Ohio, USA. epithelium. 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