Evolutionary aspects of the development of teeth andbaleen in the bowhead whaleJ. 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, USA2Department of Wildlife Management, North Slope Borough, Barrow, AK, USA3Institute 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

About 34 million years ago, a macroevolutionary event

took place in which the ancestors of baleen whales (Mys-

ticeti, Cetacea, Mammalia) lost their teeth and replaced

them with baleen, allowing them to feed efficiently on

plankton (Dem�er�e et al. 2008; Marx, 2011). In all modern

mysticetes, the baleen rack is implanted on the left and

right side of the upper jaw (Fig. 1), leaving a gap rostrally

in the median plane in some (balaenids, eschrichtiids,

neobalaenids), but not all species (balaenopterids). Each

rack is composed of a row of several hundred baleen plates,

and each of those plates consists of a flat sheet of baleen,

which is mostly composed of the protein keratin (Werth,

2000; Lambertsen et al. 2005). The inside (lingual) of the

plates frays into a matted mass of hair-like filaments that

are used to filter food from water taken into the mouth.

Individual plates grow throughout life, and wear along

their lingual border and their (ventral) tip. The rack is

implanted more or less where the tooth rows would be,

but there is no trace of teeth (Dem�er�e et al. 2008).

Interestingly, tooth development is initiated in baleen

whale embryos, and proceeds through several develop-

mental stages (Karlsen, 1962). At least in some species,

some mineralization of teeth occurs (Dissel-scherft & Ver-

voort, 1954), but enamel never forms (Dem�er�e et al. 2008).

Later in fetal life, all traces of teeth and tooth develop-

ment disappear (Ishikawa & Amasaki, 1995; Dem�er�e et al.

2008). Saint Hilaire (1807) was the first to describe tooth

germs in a mysticete studying the bowhead whale

(Balaena mysticetus L., 1758). Baleen development in this

species commences late in fetal life, they are born with

baleen plates 10 cm long and, after approximately 10

Correspondence

J. G. M. Thewissen, Department of Anatomy and Neurobiology,

Northeast Ohio Medical University, 4209 State Route 44, Rootstown,

OH 44242, USA.

T: (330) 3256295; F: (330) 3255913; E: thewisse@neomed.edu

Accepted for publication 16 November 2016

© 2017 Anatomical Society

J. Anat. (2017) doi: 10.1111/joa.12579

Journal of Anatomy

years, the longest plates are more than 2 m in length

(Lubetkin et al. 2008). After that, as the whale keeps

growing, growth of the plates exceeds their wear, and in

an old, large whale, plates may be 4 m in length. Bowhead

whales appear to keep growing throughout life, and may

reach 200 years (George, 2009).

Baleen takes on different forms in different species of

cetaceans, as well as in different parts of the mouth.

Lambertsen et al. (1989, 2005) provided a full description of

the baleen rack in the bowhead whale, and the histology

of baleen was described in great detail for balaenopterid

whales (Fudge et al. 2009). In bowhead whales, about 320

baleen plates are implanted along the length of the upper

jaw (Lambertsen et al. 2005). For most of the length of the

jaw, there is only a single bilateral large plate on each pala-

tal cross-section, although, as an anomaly, a second row of

small plates may occur.

Near the posterior edge of the palate, bowheads have an

array of keratinous elements medial to the large, lateral

baleen plate. Collectively these smaller structures are

referred to as accessory plates and bristles (Fudge et al.

2009). These elements increase in size and complexity from

medial to lateral (Fig. 1), and include, respectively: baleen

hairs, radially symmetrical columns with terminal hairs, and

flattened platelets with terminal hairs. The last element in

this series is the asymmetrical baleen plate, much larger

than any of the structures medial to it. In addition to size

and complexity of individual elements, the implantation of

the accessory plates and bristles in the gingiva is complex.

Elements are implanted in rostro-caudal rows, but they also

form rows that are oblique at approximately 45 ° and 315 °

with the sagittal plane (Fig. 1F,G). Adding further complex-

ity, larger elements are spaced more widely than smaller

elements. The implantation pattern is best seen if the pro-

truding part of the plates and bristles is cut off, leaving only

stubs protruding from the gingiva (Fig. 1F) or when the gin-

giva is cut off the maxilla, and that cut face is studied from

dorsal (Fig. 1G).

In contrast to baleen, mammal teeth consist of inorganic

hydroxyl-apatite (mostly calcium phosphate) and do not

grow after mineralization, except in special cases such as

the hypsodont teeth of elephants and beavers. Mammal

teeth are also limited in number (precisely 11 teeth per jaw

quadrant in ancestral placentals; isodonty; Bown & Kraus,

1979), and are replaced just once (diphyodonty; Van Nievelt

& Smith, 2005). The typical mammalian dentition is differen-

tiated into tooth classes (incisors, canines, premolars and

molars), each of which consists of several teeth that are

A

G E F

B D

C

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.

© 2017 Anatomical Society

Tooth and baleen development in bowhead whales, J. G. M. Thewissen et al.2

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

each is dominated by a single large cusp (Fig. 2F; Cooper

et al. 2009; Gatesy et al. 2013). Late Eocene whales, basilo-

saurids, are heterodont with four tooth classes (Fig. 2B).

Their lower molars do not show a distinct trigonid and talo-

nid (Fig. 2G; Uhen, 2004), but the molars have several acces-

sory cusps.

Mysticetes are generally thought to be derived from basi-

losaurids (Boessenecker & Fordyce, 2015; Marx & Fordyce,

2015). The oldest fossil mysticetes have teeth and are het-

erodont (Clementz et al. 2014). In the family Aetiocetidae,

there are three incisors and one canine, and cheek teeth

increase in complexity posteriorly (Barnes et al. 1994). Some

archaic mysticetes are isodont (Janjucetus; Fitzgerald, 2006),

but others have up to 14 teeth per half jaw, three more

than the original 11 teeth (e.g. Mammalodon; Fitzgerald,

2009; aetiocetids; Barnes et al. 1994). The fossil mysticete

with the most teeth is Aetiocetus polydentatus, in which

tooth numbers on the left and right differ (Barnes et al.

1994). In aetiocetids (Barnes et al. 1994), cheek teeth

increase in complexity caudally: they are triangular in labial

view with one main cusp, but one or more small additional

cusps may occur on the slopes of the large cusp (Fig. 2H).

This is similar to basilosaurids (Fig. 2G).

The pattern of dental evolution in mysticetes is thus coun-

terintuitive, first the number of teeth increases in evolution

but then teeth disappear altogether suddenly. Around the

time that the teeth disappeared, mysticetes developed

baleen (Dem�er�e et al. 2008). Skull anatomy of the Oligo-

cene mysticete Waharoa indicates that it had baleen, but it

also retained alveoli rostrally in the upper and lower jaw

(Boessenecker & Fordyce, 2015) and was not polydont.

Because of the vast differences between them, teeth and

baleen are usually considered to be independently evolved

structures, and baleen is often thought to be derived from

the transverse keratinous ridges on the palate, the palatal

rugae (Werth, 2000; Fudge et al. 2009, a translated and

annotated version of Tullberg, 1883).

Fig. 2 Teeth of fossil cetaceans. (A) Right upper and lower dentition

of Pakicetus, the oldest known whale, in labial (lateral) view. Incisors

not shown, first premolar indicated as alveolus. (B) Right upper and

lower dentition of Dorudon, a late Eocene whale, in labial view. (C, D)

Right upper molars of Indohyus and Pakicetus, rostral to right, with

homologous cusps identified by numbers. Lower molars of Indohyus

(E), a middle Eocene protocetid whale (F), and late Eocene Zygorhiza

(G), in lingual view. (H) Labial view of three cheek teeth (rostral to left)

of toothed mysticete (aetiocetid), notice that the last tooth appears to

consist of two teeth that are fused. Drawings from Thewissen (2014).

3

© 2017 Anatomical Society

Tooth and baleen development in bowhead whales, J. G. M. Thewissen et al.

Developmentally, teeth and baleen, as well as feathers,

hair and palatal rugae, are epithelial organs: they form at

the interface of an ectodermal placode that overlies mes-

enchyme, and much of the signaling involved in forming

these is self-contained. Many proteins are involved in signal-

ing related to formation of these organs, and we will not

review them here. Instead, we here discuss only those that

we were able to investigate in bowhead whales.

The fibroblast growth factor (FGF) family of signaling

proteins plays an important role in the formation of ecto-

dermal organs, and different FGFs are involved in the

ontogeny of different organs. While nothing is known

about signaling related to the development of baleen,

signaling in other keratinized tissues can involve FGF-7

and FGF-10 (Mitsui et al. 1997; Nakatake et al. 2001;

Porntaveetus et al. 2010a,b), whereas FGF-4 and FGF-8

have not been shown to be involved in signaling related

to the formation of keratinized structures (Rosenquist &

Martin, 1996; Mitsui et al. 1997; Porntaveetus et al.

2010a).

The embryology of teeth and baleen shows limited simi-

larities. Recent reviews of tooth development were pro-

vided by Cat�on & Tucker (2009) and Jernvall & Thesleff

(2012). Tooth development begins early in the fetal period

when an epithelial placode on the palate invaginates into

the underlying mesenchyme (bud stage). Relevant to our

study, FGF-8 is expressed in what eventually will be the

molar field of the jaw (Tucker & Sharpe, 2004; Mitsiadis &

Smith, 2006). The epithelium then folds into shapes that

foreshadow the shape of the occlusal surface of the tooth

(the inner enamel epithelium in the cap and bell stages).

Tips of cusps on a tooth are initiated partly as a result of

sonic hedgehog (SHH) signaling deep to the inner enamel

epithelium, and area called the enamel knot (Jernvall &

Thesleff, 2000; Thesleff, 2000). The number of enamel knots

per tooth cap determines the number of cusps on a tooth.

FGF-4 is also expressed in the enamel knot (Niswander &

Martin, 1992; Kettunen & Thesleff, 1998; Tucker et al.

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

mesenchymal side).

In this paper, we investigate several aspects of the devel-

opment and evolution of teeth and baleen in mysticetes.

Very little is known about the signaling that forms teeth in

cetaceans. Ishikawa et al. (1999) and Armfield et al. (2013)

studied the signaling that gives rise to the loss of tooth

classes in dolphins. Morphological studies of the embryol-

ogy of odontocete teeth are also rare. Mi�sek et al. (1996)

studied tooth anlagen, and �St�erba et al. (2000) described

tooth formation as part of a monograph on dolphin onto-

geny.

Baleen development also begins as an epithelial placode,

but that placode does not invaginate into the mesenchyme,

and instead evaginates directly into the oral cavity as

baleen (Fudge et al. 2009). Baleen development commences

long after tooth development in ontogeny, and is morpho-

logically simpler (Ishikawa & Amasaki, 1995), although

nothing is known about the genetic pathways involved.

Tooth development and signaling related to it is well

studied in laboratory models, especially the mouse,

although tooth development in land-living artiodactyls is

probably a better model for whales, given that cetaceans

are the sister group of one of the terrestrial artiodactyls

(Gatesy et al. 2013) and we use some comparative samples

of developing pig teeth, mostly from the work of Armfield

(2010).

First, we study the arrangement of tooth buds in bow-

head whale fetuses, determining the number of teeth that

are formed (isodonty or polydonty), what their shape is

along the tooth row (homodonty or heterodonty), whether

there is evidence of a secondary dentition (monophyodonty

or diphyodonty), and how complex their crowns are. Sec-

ond, we report on the ontogeny of baleen, with particular

interest in signaling related to it.

Materials and methods

The acquisition of mysticete embryos is difficult. Mysticetes are pro-

tected under US laws, and no individuals are in captivity in the USA.

Stranded specimens are usually in a state of decomposition that

does not allow meaningful histological study of their embryos, and

there are no complete ontogenetic series of mysticetes in museums.

However, a small number of bowhead whales, Balaena mysticetus,

are harvested annually by Alaskan natives, I~nupiat Eskimos. This

harvest is permitted under the U.S. Marine Mammal Protection Act,

as well as approved by the International Whaling Commission.

Occasionally and accidentally, a pregnant female is harvested allow-

ing extraction of an embryo or fetus under NOAA-NMFS permit

17350. This hunt takes place only twice per year and, therefore, no

complete ontogenetic series can be collected as females of this spe-

cies tend to get pregnant in the same season and the gestation time

is slightly longer than 1 year. Embryos and fetuses collected this

way will be in similar stages: about 8 weeks, 7 months and full-term

(George et al. 2016). Our study is based on six prenatal specimens

Table 1 Bowhead whale prenatal specimens available.

ID

number

TL

(cm) Stage

Tooth

stage

Baleen

stage Characteristic

1999B7F 8.7 18 Dental

lamina

None Tail conical

1999B6F 16.6 20 Early

cap

None Tail lanceolate

2000B3F 40.3 21 Bell None Fluke fully

formed, hair

on snout

2007B16F 159 23 None Placode Full hair pattern

2009KK1F 163 23 None Placode Full hair pattern

2015B9F 422 23 None Plates

present

Full term

© 2017 Anatomical Society

Tooth and baleen development in bowhead whales, J. G. M. Thewissen et al.4

(Table 1), one embryo and five fetuses, four from the spring hunt

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

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-

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

fetal heads. Histological stains used included hematoxylin/eosin and

thionin. We also employed immunohistochemical methods with

commercial antibodies. Because these antibodies were not designed

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,

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

(epiprofin) was used at 1 : 100 (Santa Cruz Biotechnologies,

SC-134025).

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

Dental lamina stage

Stratified cuboidal epithelium lines the oral cavity of this

embryo (NSB-DWM 1999B7F). For much of its extent, the

oral epithelium is two-layered, but the dental placode is

thicker, consisting of multiple layers. The dental lamina pro-

jects into the mesenchyme and its aboral edges (Fig. 3A,B)

are thickened, but there is no morphological sign of individ-

ual tooth buds (Fig. 4A). In the upper jaw, the dental lam-

ina extends caudally much farther than in the lower jaw

(approximately as far as slide 140 in the upper jaw, vs. slide

115 in the lower jaw). The cranioventral extent of the den-

tal lamina is greater in the upper than in the lower jaw

(compare Fig. 3A,D).

We investigated the dental lamina for expression of shh

using an antibody competent in this species, but this experi-

ment failed to show presence of the protein. An antibody

for FGF-8 indicates that this protein is not present in the

dental lamina or the dental placode of rostral slides (slide

33, not shown), although it does occur in oral epithelium

elsewhere in the mouth. Farther caudally (Fig. 3A,B), FGF-8

can be detected in the upper and lower dental placode,

and occasionally in the dental lamina of the upper teeth. In

general, its expression is weaker in the lower jaw. Staining

A B

E

J

O

N

M L

K

F

G

H

C D I

Fig. 3 Histological sections through tooth germs and hair follicle of

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

stage (NSB-DWM 2000B3F); staining indicated by arrows, absence of

arrows indicates absence of staining. (A) Rostral part of upper dental

lamina (slide 83, FGF-8 stain). (B) Caudal part of upper dental lamina

(slide 125, FGF-8 stain). (C) Intermediate area of upper dental lamina

(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).

(F) Upper tooth cap (slide 149, SHH stain). (G) Upper tooth cap (slide

195, SP-6 stain). (H) Lower tooth cap (slide 111, SHH stain). (I) Hair folli-

cle of NSB-DWM 2003B3F, showing competency of antibody in bow-

heads (slide 29, SP-6 stain). (J) Bell stage upper tooth germ of pig,

showing complete dental lamina and germ for permanent tooth. (K, L)

Bell stage upper tooth germ 4 (slide 49 and 77, no antibody stain). (M)

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

left. (O) Bell stage lower tooth germ, enlarged from (N). dl, dental lam-

ina; dp, dental placode; iee, inner enamel epithelium; oee, outer enamel

epithelium; sec, secondary tooth germ for permanent tooth.

5

© 2017 Anatomical Society

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.6

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

area that is surrounded by three or four depressed areas

that are connected, arranged in a square around the cen-

tral area. This is best seen in oblique views, such as

Fig. 4C–E. In the lower jaw, the outline of the tooth cap,

in ventral view, is elongate, and there are two depres-

sions, a deeper one rostrally, and a shallower one cau-

dally (Fig. 4I).

We stained some of these sections with an antibody for

SHH. SHH is present in the upper tooth caps (Figs 3E,F and

4C,E), but not in the lower tooth caps (Fig. 3H). In the upper

teeth, only the central depressed area of each germ shows

SHH signaling (Fig. 4C,E). Staining with an antibody for epi-

profin indicated that this protein is not present in the bow-

head dental caps at this age (Fig. 3G), whereas the ability of

this antibody to recognize the protein is shown by staining

in the dental lamina (Fig. 3C) and hair follicles (Fig. 3I) of

bowhead whales.

Bell stage

This fetus (NSB-DWM 2000B3F) has a row of bell stage

tooth germs in the upper jaw (Fig. 5A), evenly spaced in a

more or less straight line. Macroscopically, 40 germs can be

distinguished on the right side and 32 on the left side.

Most of the upper tooth germs are simple bowl- or bell-

shaped structures (Fig. 4K–N), separated from their neigh-

bors by undifferentiated mesenchyme. However, a few

germs are in contact with the neighboring germ and such a

pair could be interpreted as a single, elongate, bicuspid

germ (Fig. 4J). Macroscopically, recognizable joined pairs

are numbers 26–27, 28–29, 30–31, 32–33 and 34–35 on the

right side, and 22–23, 24–25, 26–27 and 29–30 on the left

side. Microscopic examination after sectioning reveals that

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

counted as such, but is in fact paired (Fig. 4J). The individual

cusps in this germ are lined up craniocaudally, with the

anterior cusp being lower in elevation than the posterior

cusp. On the left side, the most rostral bells occur well cau-

dal to the most rostral bells in the right side (Fig. 5A).

Fig. 5 Palate and head of bowhead fetus (NSB-DWM 2000B3F).

(A) Palatal epithelium, as removed from skull, backlit showing trans-

mitted light to show developing tooth germs (in the bell stage). (B)

Head of fetus showing great extent of the lower lip. (C) Clear/stain

preparation of head of the same fetus, showing the narrow dentary

that does not match the outline of the lower lip. Cartilage is blue and

bone is red in this preparation.

7

© 2017 Anatomical Society

Tooth and baleen development in bowhead whales, J. G. M. Thewissen et al.

In the lower jaw, tooth germs are embedded in the dor-

solingual part of the dentary (Fig. 3N), which is ossifying at

this stage. On the left side, 28 lower tooth bells are present,

and the most rostral of these is located well caudal to the

tip of the dentary. The most caudal germs are located well

rostral to the coronoid process. Lower tooth germs thus

have a relation to bone, whereas upper tooth germs do

not. As a result, lower tooth germs are not close to the oral

epithelium, as the edge of the lower lip at this stage flares

dorsally, foreshadowing the shape of the lower jaw after

birth. This is best appreciated when comparing a lateral

view of a fetal head with a cleared/stained fetal head

(Fig. 5B,C) of the same age. Unlike the lower jaw, upper

tooth bells are close to the oral epithelium, and are not in

close contact to the bones of the upper jaw.

Histologically, upper and lower tooth germs are in the

early bell stage in which the inner enamel epithelium con-

sists of columnar epithelium. In lower tooth bells (Fig. 3N,

O), the inner enamel epithelium consists of cuboidal cells,

whereas the outer enamel epithelium consists of squamous

cells. The cells that form the tip of the dental papilla are

elongating, forming odontoblasts. In the upper jaw, there

is no remnant of the dental lamina, whereas in the lower

jaw the dental lamina is weak, connects subsequent tooth

germs, but is not connected to the oral epithelium (Fig. 3O).

There is no trace of secondary tooth buds in the upper den-

tition, but in the lower dentition a flange of the dental lam-

ina projects lingual to the primary tooth bud, and may

represent a secondary bud (Fig. 3O). In the pig, the dental

lamina at this stage shows clear lingual invaginations that

will give rise to the teeth of the permanent dentition

(Fig. 3J).

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

shape (Fig. 3O–R). More anterior teeth are elongate

(Figs 3O and 4O,P), whereas more posterior teeth are more

globular but less regular and smaller than upper bells

(Fig. 4Q,R).

At this stage, there are no morphological signs of baleen

formation in the epithelium (Fig. 6A). Many small blood

vessels occur in the region medial to the tooth bells, and

deep to it are the major palatine artery, vein and nerve.

The area immediately adjacent to the median plane is the

area where, in adult bowhead whales, a large subcutaneous

plexus of blood vessels occurs that will develop into the cor-

pus cavernosum maxillaris of Ford et al. (2013). SHH signal-

ing occurs in the stellate reticulum of the upper and lower

teeth (Fig. 4M; lower jaw, slide C-57, not shown). Lateral to

this region, immunohistochemistry indicates that FGF-4,

FGF-7 and FGF-8 signaling occurs in the epithelium, but

there is no iterative pattern of intensity fluctuations that

could be associated with initiation of individual baleen

plates (Fig. 6B,E–G), although all three proteins are absent

near the median plane (Fig. 6C,D).

Baleen placode stage

The palate of these fetuses (NSB-DWM 2007B16F and

2009KK1F) is narrow, and displays no remnants of teeth.

The palatal surface consists of four gross morphologically

distinct zones. From medial to lateral these are: a median

vascularized area that is convex and narrows caudally, the

corpus cavernosum maxillaris (Zone 1; Figs 6I and 7A); a

band of undifferentiated epithelium that narrows rostrally

(Zone 2); a white placode that also narrows rostrally (Zone

3); and a second band of undifferentiated epithelium (Zone

4). The white placode is the area from which the baleen

develops. Near the caudal edge of the palate, the Zone 2

broadens.

FGF-4 signaling can be demonstrated in much of the

palatal epithelium. In Zone 2, such signaling occurs

mostly superficial to vessels that are located in dermal

papillae (Fig. 6J,K). In Zone 3, FGF-4 signaling shows an

iterative pattern: signaling occurs in dorsoventral bands

that span most of the depth of the epithelium and is visi-

ble in cross- and parasagittal sections (Fig. 6L,M), with

bands separated by areas lacking staining. In horizontal

sections of some areas of Zone 3, these bands fuse into

linear features that extend mediolaterally, and are lined

up craniocaudally, an orientation similar to postnatal

baleen plates (Fig. 6N).

Staining for FGF-8 occurs in the area medial to Zone 3,

mostly in epithelial areas away from dermal papillae

(Fig. 6O), and is very faint to absent in the placode. We did

not detect FGF-7 in the palatal sections, including the

baleen-forming placode, whereas the competency of this

antibody in this species is shown by staining in the hair folli-

cle of a bowhead whale (Fig. 6H).

Full term

The four zones distinguished on the palate can still be rec-

ognized in this fetus (Fig. 7B,C). The median vascular area

has not changed, but the undifferentiated epithelium is

now a narrow strip. Lateral to this zone extends a wide field

of thin baleen hairs (Fudge et al. 2009) that are anchored

into the gingiva by small accessory columns and platelets

that are topographically in the same area as the undifferen-

tiated epithelium of the previous stage. These accessory

plates (Fudge et al. 2009) are not keratinized and increase

in cross-sectional area and length from medial to lateral.

Further lateral yet, accessory plates are subsumed in and

constitute a single large sheet of barely keratinized tissue

that does not project into the oral cavity and is totally

embedded into the gingiva (Zone 3). This area grades later-

ally into the baleen plate, which projects approximately

6 cm into the oral cavity in the middle of the baleen rack. In

palatal view (Fig. 7C), the baleen hairs and frayed inner sur-

face of the baleen plate give the appearance of a matted

field of hair.

© 2017 Anatomical Society

Tooth and baleen development in bowhead whales, J. G. M. Thewissen et al.8

Discussion

Tooth counts

Our bowhead specimens display polydonty, and this was

also documented in other mysticete fetuses (Julin, 1880;

K€ukenthal, 1891; Dissel-scherft & Vervoort, 1954; Karlsen,

1962). Embryologists have speculated on the origin of

polydonty in cetaceans. K€ukenthal (1893) proposed that

polydonty was the result of splitting of multicuspid teeth,

whereas Julin (1880) suggested the opposite: that individ-

ual, conical tooth germs fuse in development, implying that

earlier embryos would have even more tooth germs than

later ones. Karlsen (1962) reanalyzed K€ukenthal’s data and

based his analysis on a collection that also included younger

embryos than were available to K€ukenthal. Karlsen (1962)

concluded that some tooth germs fuse occasionally in onto-

geny, but that the fusion pattern is consistent neither along

the tooth row, nor between left and right tooth row, nor

between different individuals. Karlsen (1962) also observed

that crown complexity does not increase toward the caudal

part of the tooth row as is common in heterodont denti-

tions of other mammals. We concur that fusion observed in

tooth crowns of our specimens is not related to the patterns

that determine tooth counts and crown shape in hetero-

dont mammals. Consistent with the view of Karlsen (1962),

A

B

E

I

M

LJ

O N K

F G H

C D

Fig. 6 Histological sections showing baleen

formation. (A) Cross-section through half of

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-

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

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

NSB-DWM 2009KK1F, same orientation as (A)

(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,

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.

9

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Tooth and baleen development in bowhead whales, J. G. M. Thewissen et al.

we consider the high tooth number in fetal bowhead

whales to be the result of dental patterning earlier in onto-

geny, not of divisions in already formed dental germs long

after they form.

One of the geologically youngest toothed mysticetes is

Aetiocetus polydentatus. This species has the highest num-

bers of excess teeth of any fossil mysticete (Barnes et al.

1994), and has different numbers of teeth in the left and

right jaw. Cobourne & Sharpe (2010) discussed differences

between left and right jaws as symptoms of a release of sta-

bilizing selection, and Salazar-Ciudad & Jernvall (2010) indi-

cated that the lack of strict occlusion in seals was correlated

to higher dental variation. The same may be true for

toothed mysticetes. Indeed, fused crowns that complicate

precise occlusion do occur in some fossil cetaceans

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

fetuses. It is possible that changes in epiprofin signaling

underlie the morphological changes in the dental lamina.

Our working hypothesis is that the increased variability in

tooth numbers as well as polydonty in early mysticetes is

the result of a release of the selection pressures related to

accurate occlusion as occlusion was not critical to food

acquisition. The ontogenetic fingerprints thereof are still

present in mysticete embryos. Reduced expression, in time

or concentration, of epiprofin may be part of the mecha-

nism by which this was accomplished.

Molar crown morphology

At the cap stage, bowhead whales display folds of the inner

enamel epithelium that resemble those that are associated

with secondary enamel knots in land mammals (Jernvall &

Thesleff, 2000). In land mammals, these folds eventually

give rise to the complex molar crown morphology of these

taxa (Fig. 2C). Lower bowhead tooth germs resemble the

pattern of the lower teeth of the Eocene whale Pakicetus

with a high part rostrally (trigonid) and low part caudally

(talonid; Fig. 2A,E). The folding pattern of the inner enamel

Fig. 7 Palate and baleen in fetus. (A) Palate of fetus NSB-DWM

2007B16F, rostral to left. (B) Rostral cross-section through the poste-

rior part of the unilateral palate of NSB-DWM 2015B9F. (C) Ventral

view of the posterior palate (same individual as B), showing baleen

hairs and their root in the gingiva of the palate. Zones identify

homologous areas in Figs 6 and 7.

© 2017 Anatomical Society

Tooth and baleen development in bowhead whales, J. G. M. Thewissen et al.10

epithelium in upper teeth resembles the cusp pattern of the

artiodactyl relatives of cetaceans, hippopotamids and raoel-

lids (Fig. 2C; Gatesy et al. 2013), with four folds that match

the arrangement of the original four cusps (Fig. 4F,H).

Dissel-scherft & Vervoort (1954) studied the mineralized

fetal teeth of a Balaenoptera fetus of 102 cm, a stage for

which we do not have bowhead fetuses. They found that

posterior lower teeth have a complex three-cusped crown,

similar to the trigonid of raoellid relatives of cetaceans,

whereas anterior teeth have single-cusped crowns similar to

incisors and canines of heterodont mammals. These teeth

were composed of dentin and lacked enamel, consistent

with the study of Dem�er�e et al. (2008) and Meredith et al.

(2009) that detected nonsense mutations in the genes

involved with amelogenesis of mysticetes. It would be

remarkable if the embryonic germs resembled molar crowns

of species that lived more than 40 million years ago.

To investigate this, we used the signaling protein SHH to

further explore the folds of the inner enamel epithelium.

SHH is a marker for the enamel knot, the signaling center

for cusp tips (Jernvall & Thesleff, 2000; Thesleff, 2000). Our

experiments show that the protein is not expressed in the

tips of the folds but instead in the valley between them in

the upper teeth. This pattern of expression foreshadows

the single-cusped germs of the bowhead bell stage, but

does not support the interpretation of the enamel folds as

original cusps. With our embryos, we could not document

any SHH in the lower teeth at the cap stage. Furthermore,

in our specimens, even anterior upper tooth germs (germs

1–8) have complex morphologies of the inner enamel

epithelium at the cap stage, while the artiodactyl relatives

of cetaceans have simple crowns for these teeth (Thewissen

et al. 2007).

At least two interpretations of these data are possible.

The first is that the folding patterns of the inner enamel

epithelium of bowhead whales are induced by secondary

enamel knots (and are thus cusp homologs) at some stage

but that our collection does not include specimens of that

stage. The heterodonty of mineralized teeth described by

Dissel-scherft & Vervoort (1954) is consistent with this inter-

pretation, and so is the heterodonty of lower tooth crowns

in our bell stage fetus. The homodonty of the upper tooth

bells then provides a discordant datum.

The second possible explanation is that these folds are

not related to secondary enamel knots at all. Nakamura

et al. (2008) found that the inner enamel epithelium dis-

plays incipient folding that is not related to enamel knot

formation in mice that do not express epiprofin. These mice

also show a deficiency in shh expression. This is consistent

with the disruption of epiprofin signaling in the bowhead

whale. In fact, it is possible that the small accessory cusps

that occur on the anterior and posterior slope of the main

cusp in fossil mysticetes and their archaeocete ancestors

(Fig. 2B,G,H) are caused by truncated expression of epipro-

fin. We observed inner enamel epithelium folds even in

anterior teeth at the cap stage, suggesting that they are

not related to the original heterodonty of the mysticete

ancestors. However, this does not explain the discrepancy

between upper and lower tooth bells. At present, it is not

possible to test these two evolutionary scenarios as speci-

mens of the relevant developmental stages are not avail-

able.

Tooth shape along the tooth row

Signaling that is important in defining mammalian tooth

classes occurs at the dental lamina stage, when morpho-

genetic fields are created in the jaw (Cat�on & Tucker, 2009;

Jernvall & Thesleff, 2012). These guide the formation of dif-

ferent tooth classes. Such signaling occurs long before the

signaling that determines the size, spacing and number of

teeth, suggesting that these processes are independent to

some degree. In investigated mammals (mice, shrews and

minks), BMP-4 signaling sets up the incisor field of the jaw,

whereas FGF-8 sets up the molar field. The canine and pre-

molar fields may result, in part, on the interaction of these

signaling proteins along a gradient in the jaw. Armfield

et al. (2013) studied the dolphin Stenella, and showed that

the signaling field of BMP-4 has expanded caudally and

overlaps with the FGF-8 field. Both Stenella and bowhead

whales retain the FGF-8 field in the caudal part of the jaw.

In bowhead whales the signaling is more distinct in the

upper than in the lower jaw. All teeth in Stenella are simi-

lar, and have incisor-like morphologies. Experimentally, it

has been determined that, in mice, BMP-4 forces posterior

teeth into incisor-like morphologies (Murashima-Suginami

et al. 2008; Munne et al. 2010), a mechanism that is consis-

tent with the Stenella data. Unfortunately, our BMP-4 anti-

body appears to be incompetent in bowhead whales, and

we cannot determine the protein’s signaling traces in this

species.

In our specimens, there is clear morphological variation

of teeth along a cline in the lower jaw, but not in the upper

jaw. Lower crowns of rostral teeth are longer and more

pointed, whereas those of caudal teeth are more globular

in the 40.3-cm fetus. Karlsen (1962) also found this in Balae-

noptera. Julin (1880) studied the lower jaw of a Balaenop-

tera fetus with 41 tooth germs, where teeth 1–9 had

simple, one-cuspid tooth bells, whereas teeth 10–41 had

two or three cusps per tooth, sometimes of the same

height, sometimes of different heights. In the Balaenoptera

mandible described by Dissel-scherft & Vervoort (1954), the

anterior 36 right and 38 left teeth were simple conical

teeth, and teeth more posterior showed molar trigonid

morphologies. The lower jaw of our 40.3-cm specimen dis-

plays heterodonty too, although it is less pronounced, and

clearer in the lower than in the upper jaw. Karlsen (1962)

indicated that Julin’s fetus may have been 45 cm in length,

indicating that it was at a later ontogenetic time than our

bowhead fetus.

11

© 2017 Anatomical Society

Tooth and baleen development in bowhead whales, J. G. M. Thewissen et al.

Heterodonty occurs in all extinct toothed mysticetes, with

distinct incisors and canine, and a gradual transition of sim-

ple one-cusped teeth to more complex teeth in premolars

and molars (Barnes et al. 1994; Fitzgerald, 2006, 2009). The

most consistent interpretation of this pattern is that the

polydont and heterodont fetal lower dentition of mysticete

fetuses is a hold-over of their Oligocene ancestors, whereas

morphological remnants of the fetal upper dentition have

been erased.

Tooth replacement

Julin (1880) did not find evidence of a deciduous dentition

in the (single) early fetus of Balaenoptera that he studied.

K€ukenthal (1893) interpreted patterns observed in his col-

lection of prenatal Balaenoptera to indicate that there were

three waves of dentition development. In his view, the sec-

ond of these waves proceeds to develop furthest and is

homologous to the deciduous dentition of other mammals.

He identified remnants of a predeciduous dentition lateral

to, and germs of the adult dentition medial to, the decidu-

ous dentition. Karlsen (1962) studied a Balaenoptera

embryo with early dental caps and disputed the presence of

a predeciduous dentition, instead interpreting the lateral

clusters of epithelial cells as remnants of the lateral dental

lamina. The dental lamina occurs in many groups of mam-

mals, and disappears before the bell stage is reached. Karl-

sen (1962) interpreted the lingual clusters of cells as

emanating from the dental lamina, not from other tooth

buds, and part of the same tooth generation as the larger

buds. In Karlsen’s material (Karlsen, 1962), these clusters

occasionally developed into tooth caps. It is possible that

such a process in related taxa with teeth actually results in

the formation of erupted tooth crowns. Fordyce (1982)

reported supernumery single-rooted teeth in a fossil odon-

tocete that may be the result of such a process.

Karlsen (1962) noted the presence of conspicuous folds in

the outer enamel epithelium in a 56-cm fetus, and that

smaller germs in the cap stage are located somewhat lin-

gually from larger more developed germs in the lower, but

not in the upper jaw.

Dissel-scherft & Vervoort (1954) described in some detail

how, in the lower jaw of Balaenoptera, tooth bells of multi-

ple sizes and shapes seem to co-occur in a cranio-caudal pat-

tern that shows no discernable regularity or trend. These

authors did not go so far as to interpret these as different

tooth generations, taking them instead for evidence of a

gradually disappearing heterodont dentition. Our evidence

is consistent with the view of Karlsen (1962) and Dissel-

scherft & Vervoort (1954): the tooth germs that reach the

bell stage in modern bowhead fetuses all arise from a single

tooth generation. There is evidence of anlagen for teeth of

a second tooth generation in the form of lingual buds pro-

truding from the lower, but not the upper tooth bells

(Fig. 3O). Dosed�elov�a et al. (2015) found that a weak crest

that projects lingually from the monophyodontic teeth of

terrestrial mammals (mouse, hedgehog, pig) is a likely rem-

nant of a developing permanent tooth. This is similar to the

crest we observed in the lower dentition of bowhead

whales, and we interpret the bowhead tooth bells as part

of the primary (deciduous) dentition, and hypothesize that

these tooth germs represent the single tooth generation of

fossil toothed mysticetes such as aetiocetids. The lingual

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-

cess (J€arvinen et al. 2009). In mammals, the loss of a connec-

tion between dental lamina and oral cavity stops new tooth

generations from forming (Buchtov�a et al. 2012). In

humans, the dental lamina begins to break up at the bell

stage by detaching from the oral epithelium (Bhussry,

1980), whereas in bowheads this connection is lost by the

cap stage.

We hypothesize that the early loss of a connection

between dental lamina and oral epithelium in bowhead

prenatal development plays a role in the loss of diphyo-

donty. Traces of a second tooth generation can still be

found in the lower, but not in the upper dentition.

Developmental tooth loss

Complete tooth loss in bowheads takes place between the

stages of fetuses of a total length 40.3 and 159 cm, and is

not documented by our specimens. Several aspects of the

process of tooth loss are known for mysticetes. Karlsen

(1962) found evidence of dentin formation in Balaenoptera,

but not enamel formation, consistent with the absence of

enamel formation gene expression shown by Dem�er�e et al.

(2008). Dissel-scherft & Vervoort (1954) describe mineralized

crowns in detail, noting several histological features that

are indicative of resorption. Davit-B�eal et al. (2009)

reviewed the little that is known about tooth loss in tetra-

pods in a developmental context. Comparing ontogenetic

data for different taxa, it is clear that different developmen-

tal processes underlie tooth loss in different taxa (Louchart

& Viriot, 2011; Tokita et al. 2013). It is also clear that the

developmental processes that arrest tooth development are

unrelated to and overprint the processes related to tooth

formation. This implies that, in cladistics analyses, the

absence of teeth in a clade should be considered a character

independent from that related to the precise number of

teeth in its toothed relatives.

Developmental origin of baleen

The first morphological signs of baleen formation occur

when the teeth are at the bell stage. When the palate is

backlit, a dense area is visible medial to the tooth germs

(Fig. 5A). This area is underlain by a neurovascular bundle

(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.12

ontogeny (Fig. 6I), when all traces of teeth disappear. FGF-4

signaling occurs in an iterative pattern in the placode and

could be related to eventual iterative pattern of the baleen

plates. In mammals with teeth, FGF-4 is expressed in the

enamel knot (Niswander & Martin, 1992; Kettunen & The-

sleff, 1998; Tucker et al. 1998), but the protein is not

involved in hair formation (Mitsui et al. 1997) or nail forma-

tion.

FGF-8 plays an important role in the initiation of tooth

development in mammals (Niswander & Martin, 1992;

Kettunen & Thesleff, 1998; Nadiri et al. 2004), including

cetaceans (Armfield et al. 2013), but plays no role in the

development of hair (Rosenquist & Martin, 1996) or palatal

rugae (Porntaveetus et al. 2010a). By contrast, FGF-7 is

involved in signaling in the mammalian hair follicle (Mitsui

et al. 1997; Nakatake et al. 2001), and FGF-10 in palatal

rugae development (Porntaveetus et al. 2010a), and these

are not involved in tooth signaling (Porntaveetus et al.

2010b). We could not detect signaling of FGF-7 and FGF-10

related to baleen formation.

Involvement of FGF-4 in baleen development instead sug-

gests that some of the original signaling related to tooth

formation now partakes in the formation of baleen. There

are other indications that this may be the case. In most

mammals, upper and lower teeth follow similar ontoge-

netic trajectories, but in bowheads they deviate early in

ontogeny. The upper dental lamina differs morphologically

from the lower dental lamina: it is longer (rostrocaudally)

and higher (dorsoventrally) and creates more tooth germs.

Those tooth germs are more homodont than those in the

lower jaw. In some sense then, the tooth germs of the

upper jaw appear to foreshadow baleen development,

where many, near-identical plates form. Lower tooth germs

develop close to the dentary, and they are far from the

lower lip because the lip bulges dorsally (Fig. 5B,C). Thus,

the distance between the lower mandibular epithelium and

the developing tooth germs is great. This epithelium is

known to play a role in tooth development (J€arvinen et al.

2009), and that topography may affect lower tooth devel-

opment. While this may explain the differences in upper

and lower dentition development in bowhead whales, the

lower lip of other fossil and modern mysticetes (e.g. bal-

aenopterids) is closer to the mandible with its tooth germs.

A different mechanism may underlie the loss of tooth

germs in these taxa.

Ishikawa et al. (1999) noted similarities in extracellular

matrix proteins involved in baleen development to those

involved in tooth development, and Davit-B�eal et al. (2009)

speculated that baleen development was functionally

related to tooth loss based on developmental timing. These

observations support our interpretation of the signaling evi-

dence. Our working hypothesis is that development of the

upper dentition in the bowheads is a necessary step in the

development of baleen, and that differences between

upper and lower dentition are related to the lack of this

function in the lower teeth.

The fossil record is also consistent with this hypothesis.

There is a trend in the earliest fossil mysticetes toward

homodonty of the molars and premolars, but polydonty

was limited, with no more than a single additional tooth

(beyond the original 11 per quadrant) in most clades and

four additional teeth in one taxon (Aetiocetus polydenta-

tus). It is puzzling that modern mysticete embryos have

more than 40 tooth germs if known members related to

their ancestors never had more than 15 teeth, unless those

germs serve a function beyond that of teeth. This situation

differs from odontocetes, where polydonty is widespread

across fossil and modern clades. We hypothesize that mys-

ticetes that were highly polydont as adults never existed,

and that the iterative signaling that underlies the formation

of so many tooth germs is related to the signaling that is

important in baleen formation later in ontogeny, but was

not engaged until later in evolution, when postnatal teeth

had been lost already.

The pattern of signaling in the palate coincides with

results presented by Fudge et al. (2009) regarding baleen

development: initially undifferentiated epithelium is

induced to form accessory plates and, as the fetus grows,

these structures are incorporated into the main baleen

plate. The pattern of implantation of baleen plates, acces-

sory plates and bristles on the back of the palate has no

match in mammals, but is reminiscent of other ectodermal

structures in the mouth of other vertebrates. Generations

of replacement teeth in reptiles, for instance, are arranged

in patterns that show a maturation gradient from medial to

lateral, but implantation of individual elements in rows that

are oblique to the mediolateral plane (Westergaard & Fer-

guson, 1990; Whitlock & Richman, 2013).

It might seem strange that two structures that are very

different in composition, teeth and baleen, are formed by

similar signaling pathways. However, it should be remem-

bered that enamel and keratin have shared the oral cavity

since the earliest vertebrates, as evidenced by the kerati-

nous teeth of cyclostomes (Trott & Lucow, 1964), although

the formation of these has no relation to tooth origins

(Smith & Coates, 2000; Huysseune et al. 2009). The link

between keratin and the oral cavity also virtually character-

izes birds. Louchart & Viriot (2011) discussed the evolution

of tooth loss and the formation of a rhamphoteca (keratin

beak) in birds, noting that local keratinization may be a

cause for arrest of tooth formation. Wu et al. (2004) noted

the developmental similarity in signaling among morpho-

logically very different keratinized epithelial appendages,

and teeth too share some of these pathways. Clearly, there

is great plasticity in epithelial appendages, both develop-

mentally and evolutionarily (Chuong, 1998; Dhouailly,

2009), and common developmental pathways underlie all

of them.

13

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Tooth and baleen development in bowhead whales, J. G. M. Thewissen et al.

Some of the significant differences in developmental pro-

cesses forming teeth and baleen may also be bridged by

minor changes in patterning. For instance, mammalian

teeth form by invagination of epithelium into underlying

mesenchyme, whereas baleen forms by evagination into

the oral cavity. The protein IKKa is involved with the devel-

opment of hair and whiskers, and, possibly, in the regula-

tion of other ectodermal–mesenchymal interactions (Karin

& Delhase, 2000; Ohazama et al. 2010). In Ikkamutant mice,

incisors and whiskers form by evagination, not invagina-

tion. Furthermore, in the talpid2 mutant of chicks, tooth

germs form by evagination, not invagination (Harris et al.

2006). There are many other genes that play a role both in

the development of teeth and keratin structures. For

instance, fgf-20 is involved in tooth and hair development,

and its subtle effects on the morphology of teeth could be

a potent agent in evolutionary changes in tooth morphol-

ogy (H€a€ar€a et al. 2012). Obviously, shifts in single genes are

insufficient to turn teeth into baleen in ontogeny, but it

does challenge the assumption that the morphological dif-

ference between these processes is unbridgeable.

Our evidence suggests a greater level of homology

between teeth and baleen than their homology as epithe-

lial organs (sensu Chuong, 1998), namely that the particular

gene expression pathway that leads to two generations of

teeth in most mammals has been co-opted to be involved

in baleen formation in cetaceans. Several approaches can

be used to investigate this in greater depth. First, signaling

that underlies the number of palatal rugae that forms and

the spacing between them is suggestive (Pantalacci et al.

2008) and may elucidate the developmental basis for

baleen plate spacing. Second, a detailed understanding of

the development of the compounds that compose baleen

could help to understand selective powers at work

(Szewciw et al. 2010). These compounds may include a

large diversity of keratins (Bragulla & Homberger, 2009),

and their developmental pathways may be quite different

from each other.

Conclusion

We integrate our data and discussion into a working

hypothesis that comprises the development of teeth and

baleen in bowhead whales. Until the bell stage, bowhead

dental ontogeny is easily recognizable as a variation on the

dental ontogeny of generalized mammals. We interpret dif-

ferences that do exist as precursors of events that happen

after the bell stage: the complete loss of teeth and the

development of baleen. Here, we summarize that process

and our interpretation of it and provide an evolutionary

context (Fig. 8).

The first differences between dental development of

bowheads and generalized mammal development already

occur at the dental lamina stage, and were observed before

by Karlsen (1962) in Balaenoptera: the dental lamina of the

upper jaw is higher dorsoventrally (Fig. 8.1) and longer

cranio-caudally (Fig. 8.2) than the dental lamina of the

lower jaw. FGF-8 signaling occurs in the posterior regions of

both, but is stronger in the upper jaw than in the lower jaw

(Fig. 8.3). Such signaling is the basis for molar morphologies

of posterior teeth in generalized mammals, and is also

found in homodont dolphins. In dolphins, FGF-8 signaling

in the posterior jaw is overprinted by BMP-4 signaling,

which may induce the incisor-like morphologies of the

cheek teeth (Armfield et al. 2013), as it is known to do in

mutant mice (Munne et al. 2010). It is likely that the FGF-8

signaling causes the heterodontic features of the lower

jaw of bowhead fetuses, as well as the limited degree of

heterodonty in extinct toothed mysticetes (e.g. Janjucetus,

Mammalodon and aetiocetids; Barnes et al. 1994; Fitzger-

ald, 2006, 2009).

At the dental cap stage, the dental lamina has lost con-

tact with the oral epithelium (Fig. 8.4), an event that takes

place much later in generalized mammals. The dental lam-

ina is necessary for the formation of subsequent tooth gen-

erations, and we propose that its loss underlies the absence

of a second tooth generation even in fossil, toothed

mysticetes.

At the bell stage, upper and lower dentitions vary signifi-

cantly. The upper tooth germs are homodont (Fig. 8.8),

there is no evidence of primordia for a second tooth gener-

ation, and individual bells are not connected by the dental

lamina. In the lower jaw, there are fewer, more variably

developed germs (Fig. 8.5 and 8.6) and they are smaller

than the upper teeth, there is a gradient of heterodonty

along the jaw (Fig. 8.9), while the dental lamina remains.

This is consistent with the findings of Karlsen (1962). A lin-

gual crest protrudes from the tooth bells that may be the

remnant of the secondary dental lamina that is involved in

the formation of permanent teeth. We interpret these dif-

ferences in the context of the imposition of selection forces

on the upper dentition, leading to greater polydonty,

homodonty and monophyodonty. In contrast, the lower

teeth maintain some of the ancestral patterns of hetero-

donty and diphyodonty, while they are underdeveloped in

general.

Individual tooth caps show patterns of folds in the inner

enamel epithelium that resemble cusps of the Eocene

ancestors of whales in the upper molars, and lower molar

morphology of the earliest whales in the lower teeth

(Fig. 8.7). Although this pattern is suggestive, there is only

a single area of SHH signaling in these teeth, and this area

probably is related to the single cusps that occur in the

bell stage of these teeth. Folding of the inner enamel

epithelium could also be caused by absence of epiprofin

signaling and, indeed, epiprofin signaling is reduced in

Balaena. Mice with disrupted epiprofin signaling display

polydonty and simple crown morphologies along the

tooth row (Nakamura et al. 2008). The dental patterns of

teeth in �/� epiprofin mice suggest that if enamel

© 2017 Anatomical Society

Tooth and baleen development in bowhead whales, J. G. M. Thewissen et al.14

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.

15

© 2017 Anatomical Society

Tooth and baleen development in bowhead whales, J. G. M. Thewissen et al.

formation were to take place in these tooth caps, small

cuspules would develop on these teeth, a feature that is

characteristic for the posterior cheek teeth of toothed mys-

ticetes such as Mammalodon, Janjucetus and Aetiocetus.

J€arvinen et al. (2006) showed that simple crowns and poly-

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-

tion of cusp patterns (e.g. Eda, Spry, Sostdc; Jernvall &

Thesleff, 2012), and further investigations are needed to

test these hypotheses.

The first signs of homodonty, monophyodonty, poly-

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

play a role in the evolution of these features. These include

the disruption of epiprofin signaling and the lengthening

of the dental lamina and its early detachment from the oral

epithelium. Selection for precise occlusion may have been

released at this time, similar to the mechanism proposed for

phocid seal cheek tooth evolution proposed by Salazar-Ciu-

dad & Jernvall (2010) and for odontocete tooth crown mor-

phology by Armfield et al. (2013). In contrast, Tsai &

Fordyce (2014) reached the opposite conclusion for the evo-

lution of mysticete cranial morphology; they studied hete-

rochrony in late-term fetal and postnatal mysticete skulls

and showed highly constrained heterochonic evolution in

some clades.

In the ontogeny of one tooth germ, several developmen-

tal genes are used multiple times during development

(Tucker & Sharpe, 2004). That same developmental cassette

is also replayed for topographically sequential teeth in the

tooth row, as well as for chronologically subsequent teeth

such as the deciduous and permanent dentitions of mam-

mals, or the polyphyodont dentitions of other vertebrates.

One of the proteins involved in this cassette is FGF-4, a

member of its family that does not play a great role in

other tissues of the head.

Based on these findings, we formulate a working hypoth-

esis that the tooth development cascade was adapted and

plays a role in baleen development in mysticetes. This is an

example of exaptation in baleen whale evolution, and

implies that teeth and baleen are homologous to some

degree. Obviously, there are many differences, including

developmental ones. Homology is not an absolute concept,

baleen is homologous with teeth as an epithelial organ,

and as an organ formed by the iterative signaling sequence

that initiates teeth. However, this level of homology is less

deep than the homology of the first molar of a pakicetid to

that of a dorudontid cetacean.

Acknowledgements

The authors thank the captains of Barrow, Alaska, and the Alaska

Eskimo Whaling Commission for allowing for examination of their

whales and collection of samples. We thank all of those involved,

especially Director Taqulik Hepa and Deputy Director Harry Brower

Jr. Funding was provided by the North Slope Borough and

NEOMED.

All authors declare that there is no conflict of interest that might

affect their objectivity in preparing this paper.

Author’s contributions

Thewissen, George, Suydam and Stimmelmayr collected the

samples; Hieronymus and McBurney prepared the samples

and did the experiments; and Thewissen, Hieronymus and

George did the writing.

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