ARTICLE IN PRESS

ZOOLOGY
Zoology 110 (2007) 231–251
www.elsevier.de/zool

Embryonic development of Python sebae – II: Craniofacial microscopic

anatomy, cell proliferation and apoptosis
Marcela Buchtováa, Julia C. Boughnera,1, Katherine Fua,
Virginia M. Diewertb, Joy M. Richmana,
a
Department of Oral Health Sciences, Life Sciences Institute, University of British Columbia, Life Sciences Centre, 2350 Health
Sciences Mall, Vancouver, BC, Canada V6T 1Z3
b
Department of Oral Health Sciences, Faculty of Dentistry, University of British Columbia, 2199 Wesbrook Mall, Vancouver, BC,
Canada V6T 1Z3

Received 24 August 2006; received in revised form 21 January 2007; accepted 23 January 2007

Abstract
This study explores the microscopic craniofacial morphogenesis of the oviparous African rock python (Python sebae)
spanning the first two-thirds of the post-oviposition period. At the time of laying, the python embryo consists of largely
undifferentiated mesenchyme and epithelium with the exception of the cranial base and trabeculae cranii, which are
undergoing chondrogenesis. The facial prominences are well defined and are at a late stage, close to the time when lip
fusion begins. Later (11–12 d), specializations in the epithelia begin to differentiate (vomeronasal and olfactory epithelia,
teeth). Dental development in snakes is different from that of mammals in several aspects including an extended dental
lamina with the capacity to form 4 sets of generational teeth. In addition, the ophidian olfactory system is very different
from the mammalian. There is a large vomeronasal organ, a nasal cavity proper and an extraconchal space. All of these
areas are lined with a greatly expanded olfactory epithelium. Intramembranous bone differentiation is taking place at
stage 3 with some bones already ossifying whereas most are only represented as mesenchymal condensations. In
addition to routine histological staining, PCNA immunohistochemistry reveals relatively higher levels of proliferation in
the extending dental laminae, in osseous mesenchymal condensations and in the olfactory epithelia. Areas undergoing
apoptosis were noted in the enamel organs of the teeth and osseous mesenchymal condensations. We propose that
localized apoptosis helps to divide a single condensation into multiple ossification centres and this is a mechanism
whereby novel morphology can be selected in response to evolutionary pressures. Several additional differences in head
morphology between snakes and other amniotes were noted including a palatal groove separating the inner and outer
row of teeth in the upper jaw, a tracheal opening within the tongue and a pharyngeal adhesion that closes off the
pharynx from the oral cavity between stages 1 and 4. Our studies on these and other differences in the python will
provide valuable insights into in developmental, molecular and evolutionary mechanisms of patterning.
r 2007 Elsevier GmbH. All rights reserved.

Keywords: Snakes; Chondrogenesis; Tooth development; Mesenchymal condensation; Vomeronasal organ

Corresponding authors. Tel.: +1 604 822 3568; fax: +1 604 822 3562.
E-mail address: richman@interchange.ubc.ca (J.M. Richman).
1
Current address: Department of Cell Biology and Anatomy, Faculty of Medicine, Heritage Medical Research Centre, 3330 Hospital Drive NW,
Calgary, Alta., Canada T2N 4N1.

0944-2006/$ - see front matter r 2007 Elsevier GmbH. All rights reserved.
doi:10.1016/j.zool.2007.01.006
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232 M. Buchtová et al. / Zoology 110 (2007) 231–251
Introduction
The study of craniofacial anatomy in the fossil record
and in extant gnathostomes has for years provided
excellent insights into the adaptation of skeletal patterns
(De Beer, 1937; Carroll, 1988). In the last 20 years,
molecular studies on extant gnathostomes representing
primitive vertebrates have provided insights into how genes
and subsequent phenotypes have been modified through
evolution. Some of these newer studies in lamprey showed
not only conservation of the key stages of facial develop-
ment (neural crest cell migration in defined streams) but
also in the types of genes expressed in these primitive jaws.
The differences lie with the expression domains such that
subtle shifts in gene expression in embryos result in
divergent jaw morphology (Kuratani, 2005a, b).
Facial development in gnathostomes begins with a
specialized group of neuroectodermal cells called the
neural crest. Neural crest cells carry the patterning
information for the jaws (Le Lievre, 1978; Noden, 1978;
Couly et al., 1993; Kontges and Lumsden, 1996;
Schneider and Helms, 2003) and go on to form all the
skeletal tissues of the face. In addition, the frontal bones
and the chondrocranium rostral to the pituitary fossa are
from the neural crest (Noden, 1978; Couly et al., 1993;
Morriss-Kay, 2001). The mesenchymal-derived parts of
teeth (at least in mammals) are also neural crest-derived
(Chai et al., 2000). The patterning of the majority of
craniofacial bones and cartilages depends on later cues
received from the local environment (Richman and Lee,
2003; Richman et al., 2006). It is likely that reptiles have
similar neural crest origins for the skull and facial bones
but this idea is untested.
Neural crest cells and parasomitic mesoderm form the
head mesenchyme, which later fills the facial processes
that surround the primitive mouth. The facial buds or
prominences are quite conserved amongst amniotes and
the fate of the prominences is known mainly from work
on avian embryos. The facial prominences consist of a
central frontonasal mass or medial nasal prominences
between the nasal pits and these buds merge to form the
midline of the face (Richman and Tickle, 1989). The
lateral nasal prominences (lateral to the nasal pits) form
the nasal cavities (MacDonald et al., 2004). The paired
maxillary prominences flank the stomodeum and give rise
to the upper jaw bones (Lee et al., 2004), while the
mandibular prominences form the entire lower jaw and
parts of the jaw joint (Richman and Tickle, 1989). The
earliest time it is possible to discern the species-specific
form is just prior to fusion of the facial prominences. In
birds, there are differences in size and shapes of the facial
prominences that translate into differences in beak shapes
(Abzhanov et al., 2004; Wu et al., 2004, 2006). We would
therefore be interested in seeing whether or not the early
snake face resembles other amniotes, especially those
with a close phylogenetic relationship.
The craniofacial skeleton forms by two mechanisms.
There is either direct formation of bone from mesench-
yme (intramembranous ossification) or the replacement
of cartilage elements with bone (endochondral ossifica-
tion). The early stages of both types of bones are
characterized by condensations of mesenchyme. It is
thought that such condensations are the building blocks
of the skull and that these can be modified during
evolution to form different architectures (Atchley and
Hall, 1991). Differences in the morphology and/or
number of bones may occur by several mechanisms:
(1) loss of condensations; (2) fusion of once separate
condensations; or (3) increasing the number of condensa-
tions through subdivision (Richman et al., 2006).
Recognizing which process has occurred to which bones
in adult animals is problematic (Donoghue and Sansom,
2002). Thus, one of the goals of the present study is to
capture development of the python at precisely the stages
when condensations form up until the characteristic
morphology of bones can be recognized.
Another goal of our study is to examine cell proliferation
and apoptosis around the skeletal condensations and other
regions of the craniofacial complex. Relative differences in
cell proliferation have been shown to promote the
formation of skeletal condensations (Hall and Miyake,
2000), the sizes of beak prominences (Wu et al., 2004, 2006)
and the folding of tooth germs (Jernvall et al., 1998;
Shigemura et al., 1999). Similarly, programmed cell death is
essential for proper morphogenesis. For example, apopto-
sis is required to sculpt the embryonic face during
formation of the lip (McGonnell et al., 1998; Ashique
et al., 2002a). We wanted to see whether some of these
patterns of proliferation and apoptosis were conserved in
non-avian reptiles, e.g. Python sebae.
Materials and methods
Python egg acquisition and incubation
African rock python (P. sebae) eggs were obtained
from the Rainforest Reptile Refuge in Surrey, B.C. as
described (Boughner et al., 2007). All animal work was
reviewed and approved by the UBC Animal Ethics
Committee, Certificate No. A04-0271.
Fixation, processing histological staining
Embryos were removed from the egg and immersed in
ice-cold phosphate-buffered saline (PBS) immediately.
They were then placed directly into 4% paraformalde-
hyde (PFA), processed through ethanol and xylene into
wax, and sectioned for histological analyses. Stage 4 and
older embryos were stored in decalcifying solution (10%
formaldehyde and 5% ethylenediaminetetraacetic acid)
for 1 week to several months prior to dehydration and
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M. Buchtová et al. / Zoology 110 (2007) 231–251
embedding in paraffin. No histological data were avail-
able for stage 9 due to damage during transport of the
single collected specimen.
The specimens were sectioned (7 mm) either in the
transverse plane from the tip of the upper jaw to the
pharynx or in the sagittal plane. Alternate sections were
left unstained or were stained with a combination of
Picrosirius red and Alcian blue that stains bone and type
I collagen-rich areas red and cartilage (sulfated proteo-
glycans) blue (Ashique et al., 2002b). We selected this dye
since it also nicely highlights early mesenchymal con-
densations several days prior to ossification.
PCNA staining and TUNEL reaction
In order to label proliferating cells, PCNA (proliferat-
ing cell nuclear antigen) was detected immunohisto-
chemically. After de-paraffinization and rehydration,
epitope recovery was done as follows: 0.01 M citric acid,
in microwave oven (10 min); 0.05% trypsin in PBS
(2 min); 0.1% saponin (10 min); 50 mM NH4Cl (5 min).
Sections were incubated for 1 h at room temperature with
primary mouse anti-PCNA antibody (Vector). The
secondary biotinylated anti-mouse antibody (1:500,
ABC kit, Vectastain) and avidin–biotin complex (ABC
kit, Vectastain) were applied for 1 h each. Products of the
reaction were visualized with diaminobenzidine (DAB).
TUNEL reaction was carried out on serial sections
with ApopTag plus Peroxidase In situ Apoptosis Detec-
tion Kit (Chemicon, S7101). Counterstaining was done
with 0.5% methyl green in 0.1 M sodium acetate, pH 4.0
for one minute. Controls for the TUNEL reaction were
sections treated with DNAse I before TUNEL detection
and sections to which no enzyme was added. Sections
were photographed under bright field illumination with a
Zeiss compound microscope.
Results
Microscopic craniofacial development of Python
sebae
Very little histological anatomy of snake embryos has
been published except for a limited set of structures
(vomer nasal organ, olfactory epithelium, oral glands:
Kochva, 1978; Holtzman and Halpern, 1991). Further-
more, even fewer studies of snakes included sections of
peri-ovipositional embryos; the exception is a study on
venom gland development in Natrix tessellata (Gygax,
1971) in which a number of craniofacial sections were
presented. Thus, one of the aims of the present study is to
provide microscopic information on soft-tissue and
skeletal structures as they are developing. The relatively
slow development of reptiles is an advantage when trying
to understand the complex tissue interactions that
233
characterize craniofacial and tooth development. We
describe development at each of the stages described in
the preceding paper (Boughner et al., 2007).
Stage 1 (day 1–3 post-oviposition)
We were able to collect eggs immediately after
oviposition, a fortunate circumstance, since it is not a
trivial matter to remove eggs from the clutch at this time.
Our first observation was that the facial prominences
closely resembled those of other amniotes. The fronto-
nasal mass was flanked by the nasal passages (Fig. 1A).
There were two invaginations for the nasal cavity
(Fig. 1A). The vertical nasal slit forms the nasal cavity
proper, whereas the more inferior invagination gives rise
to the medially positioned vomeronasal organ. In our
specimens, the epithelium lining this inferior cavity was
thicker than the nasal slit epithelium (Fig. 1A). The
maxillary prominences and frontonasal mass had formed
an epithelial contact, which is a necessary step prior to
fusion of the primary palate (Figs. 1B and C). The
maxillary prominences had started to develop medial
swellings that would later form the palatal shelves. Closer
examination of the maxillary prominences revealed slight
thickenings where the dental lamina was forming. This
first lamina will later form the outer row of teeth (Figs.
1G and H). Dental laminae for the upper, inner tooth
row or lower teeth were not yet seen. The oral cavity was
continuous with the pharynx at stage 1; however, just
caudal to the tongue, the lateral edges of the pharynx had
partly fused (not shown). The pharynx was therefore
narrower at this axial level compared to more caudal
regions.
Stage 1 embryos were well into organogenesis by the
time eggs were laid. Chondrocytes had started to
differentiate in Meckel’s cartilage, but without distinct
extracellular matrix deposition (Fig. 1D). Trabeculae
cranii were developed as early-stage cartilage condensa-
tions rostral to the notochord (Fig. 1E). These condensa-
tions were areas of increased cell density, with some
extracellular matrix stained with Alcian blue. Further
caudally, these embryos had cartilages around the
notochord and in the cranial base (Fig. 1F). In summary,
with the exception of early-stage chondrogenesis, there
was little histodifferentiation at stage 1.
Stage 3 (days 11 and 12 post-oviposition)
Morphological changes at stage 3 included the forma-
tion of palatal shelves and increased complexity of the
nasal passages. The skin folds of the head, which later
become scales, had started to form at stage 3 (Figs. 2A,
C, D); these early scales were not yet visible macro-
scopically.
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234 M. Buchtová et al. / Zoology 110 (2007) 231–251

Fig. 1. Transverse sections of a stage 1 embryo. (A) The nasal capsule is composed of two parts, the main nasal cavity and a medial
invagination into the frontonasal mass that gives rise to the vomeronasal organ. (B) Fusion of frontonasal mass and maxillary
prominences (arrow), (C) enlarged view of fusion area. (D) Chondrogenesis of Meckel’s cartilage in the lower jaw has already started.
(E) Trabeculae cranii are less developed and the deposition of Alcian blue intercellular matrix in the condensation is only just starting.
(F) In contrast, the cartilage of the basicranium is fully differentiated. (G, H) Outer dental laminae in the maxillary prominences are
starting to appear. Scale bar for all panels ¼ 100 mm. Abbr.: ah – adenohypophysis; bc – basicranium; dl – dental lamina; fnm –
frontonasal mass; lnp – lateral nasal prominence; mc – Meckel’s cartilage; mdp – mandibular prominence; mxp – maxillary prominence;
nc – nasal cavity proper; nt – notochord; vno – vomeronasal organ.
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M. Buchtová et al. / Zoology 110 (2007) 231–251 235

Fig. 2. Stage 3 embryo. (A–G) are transverse sections, (H, I) are parasagittal sections. Transverse sections are arranged in a rostro-caudal
sequence. (A) Skin folds are forming on the exterior surface of the head. The olfactory epithelium and dorsal vomeronasal epithelium are
thickened as compared to the ventral, respiratory epithelium. (B,C) Two dental laminae are present in the upper jaw. The extraconchal space
is forming ventral to the main nasal cavity. Trabeculae cranii are fully differentiated. (D) Eyelids are forming but have not yet covered the
eyes. The palatine bone is differentiating within the palatal shelves and the Picrosirius red stain marks early mineralized bone. (E). The
prearticular process of the compound bone and pterygoid are forming. The nasal cavities are lined with thickened, specialized epithelium.
(F, G) The dentary bones are forming close to Meckel’s cartilage. The mandibular dental lamina is also visible. (H) A pharyngeal adhesion is
at the cranial-most end of the foregut. The position of the laryngotracheal duct relative to the tongue is also shown. Planes of section for
panels (A) and (C) are indicated with dashed lines. (I) Midline section showing the adenohypophysis connected to the oral epithelium. Scale
bars ¼ 300 mm. Abbreviations: ad – adhesion; ah – adenohypophysis; d – dentary bone; dl – dental lamina; dlt – ductus laryngotrachealis; ecs
– extraconchal space; ed – endoderm; el – eyelid; h – hyoid; idl – inner dental lamina; mc – Meckel’s cartilage; nc – nasal cavity proper; ncs –
nasal capsule; nh – neurohypophysis; nt – notochord; oc – oral cavity; odl – outer dental lamina; on – olfactory nerve; pa – prearticular
process of the compound bone; pl – palatine bone; ps – palatal shelf; pt – pterygoid; re – respiratory epithelium; sf – skin fold; tc – trabeculae
cranii; vno – vomeronasal organ.
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236 M. Buchtová et al. / Zoology 110 (2007) 231–251
Skeletal development
At stage 3, several neural crest-derived intramembra-
nous bones were beginning to form directly from
condensing mesenchyme, which is typical for fish,
amphibians and amniotes. The earliest upper jawbones
to ossify were the palatine and pterygoid, both of which
were forming within the palatal shelves, which previously
had grown out from the maxillary prominences (Figs. 2C
and D). The palatine and pterygoid ossification centres
were discreet although they shared a mesenchymal
condensation (not shown). The caudal part of the
pterygoid ended at the rostral level of the notochord
(not shown). The nasal capsular cartilage was either
present only as a condensation or, in some cases, the
medial and dorsal parts were already differentiated
(Fig. 2A). Some parts of the chondrocranium including
the trabeculae cranii (Figs. 2C–E) were more complete
than others.
In the mandible, the adult compound bone consists of
two parts – the medial prearticular process and the lateral
surangular process. Both processes of the compound
bone arise from separate ossification centres and only
later grow together. In stage 3 embryos only the
prearticular process had formed (Fig. 2E). The dentary
bone also had initiated at stage 3, lateral to Meckel’s
cartilage (Fig. 2F). Meckel’s cartilage (Figs. 2E–G) and
the hyoid cartilage were fully differentiated (Figs. 2G
and H) as compared to stage 1 where only mesenchymal
condensations were detected.
Development of soft tissues and teeth
The head mesenchyme and epithelium was further
differentiated at stage 3. Many specialized cell types were
visible, including myoblasts, olfactory cells and olfactory
supporting cells. However, the surface epithelium was not
yet keratinized or pigmented.
The vomeronasal organ (also known as Jacobson’s
Organ; Jacobson, 1811) was one of the most prominent
features of the stage 3 embryo. The epithelium of this
organ houses the sensory neurons, and the forked snake
tongue transports external molecules such as pheromones
through the open palate to the oral opening of the
vomeronasal ducts (Ernst and Zug, 1996). Furthermore,
the vomeronasal organ is an important chemosensory
structure that facilitates hunting and mating (Ernst and
Zug, 1996). In line with the important functions of this
organ, we and others (Holtzman, 1998) found that the
dorsal sensory epithelium is hypertrophied compared to
the epithelium lining the ventral, respiratory side
(Fig. 2A).
The nasal passages were significantly changed when
compared to stage 1 embryos. The external nares were
plugged with a mass of epithelial cells (not shown). The
nares were connected to the nasal cavity proper rostrally
but did not connect to the more laterally positioned
extraconchal space (Fig. 9A). Instead, the extraconchal
space connected to the oral cavity (Fig. 2C). In addition
there was a communication between the two nasal
cavities, just caudal to the vomeronasal organ (not
shown). Nasal conchae were not yet formed.
In several areas of the head, epithelial tissue connec-
tions persisted at relatively late stages of organogenesis in
python compared to other amniotes. There was a multi-
layered-epithelial adhesion or seam in the rostral pharynx
(Fig. 2H). Unlike stage 1, the pharynx was completely
obliterated and therefore separated from the oral cavity.
The pituitary gland or hypophysis had a more primitive
appearance in python than in other amniotes. In stage 3
python, Rathke’s pouch was rostral to the pharynx
(Fig. 2H), however, there was still a connection to the
oral epithelium (Fig. 2I). In contrast, the connection
between the pituitary gland and the oral epithelium is lost
much earlier in humans, by the end of the second month
(Arey, 1965).
The lower respiratory system consists of the larynx,
trachea, and lungs. The snake is interesting since the
laryngotracheal tube orifice is positioned in the centre of
the tongue rather than posterior to the tongue as
in mammals and birds. In stage 3 python embryos,
dense precartilagenous mesenchymal condensations
had formed around the laryngotracheal tube periphery
(Fig. 2G).
Dental development was underway at stage 3. The
upper jaw had two rows of teeth, one outer lateral row
and a medial inner row caudolateral to and near the
palatine bone (Figs. 2A and B). In the dental lamina of
the outer row, the first tooth generation was beginning to
form, with some teeth invaginated to form a cap, partly
enclosing the dental mesenchyme (not shown). The short
dental lamina continued medially and in later stages
would give rise to the second tooth generation. The inner
tooth row was developed only as far as an epithelial
thickening (initiation stage). In the lower jaw, a single
row of teeth arose from a dental lamina located lateral to
Meckel’s cartilage. Here, tooth formation ranged from
dental lamina to cap stage (Fig. 2F). Ventral to the
ossifying premaxilla, two connected epithelial thickenings
had begun to form in the rostral part of the oral cavity
(not shown). These thickenings would later develop into a
single, midline egg tooth, which is used by the hatchling
to tear the leathery shell.
Stage 4 (day 18 post-oviposition)
In general, histodifferentiation at stage 4 was similar to
stage 3 embryos; however, odontoblasts were detected for
the first time in the first-generation teeth. Skin folds had
advanced further in a cranial direction along the sides of
the head as compared to stage 3 embryos (Figs. 3B
and C). Other morphological changes included a deep
groove beginning to form between the inner and outer
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M. Buchtová et al. / Zoology 110 (2007) 231–251 237

Fig. 3. Transverse sections of a stage 4 embryo in a rostral to caudal sequence. (A) The premaxilla can be seen along with the
premaxillary teeth in the rostral-most section of the upper jaw. These teeth do not have a deeply invaginating dental lamina.
(B) Ossification of the vomer is just visible. The palatal groove (black arrowheads) is deep and narrow and lies between the two
upper dental laminae. (C) Externally there are numerous skin folds that will later contribute to the scales. The olfactory nerve is
medial to the nasal cavity proper. There are two openings into the oral cavity (asterisks), one from the nasal cavity proper and one
connected to the extraconchal space. The single nasal concha is just forming at stage 4. The palatal groove is much shallower in
caudal sections (black arrowheads). Ossifying maxillary and palatine bones can be seen. (D) Section through the eyes and posterior
mandible. In this section the posterior oral cavity is superior to the pharynx and is separated by pink-stained epithelium adhering to
the oral cavity. The oral cavity is irregular and contains a central ridge of mesenchyme separating the recesses on the right and left
sides. The cartilaginous tracheal ring and proximal end of the hyoid are visible. (E) Detail of the ossifying parietal bone and
pterygoid. (F) Predentin is forming in the first generation cap-stage teeth and the second-generation tooth is at the dental lamina
stage. (G–I) Mandibular bones present from rostral to caudal include the dentary, surangular and prearticular. Well-developed
tongue musculature is visible close to the ductus laryngotrachealis, approximately midway through the tongue. The mandibular
nerve is lateral to Meckel’s cartilage and is surrounded by mandibular bones. Thick collagen bundles are present in the dermis (white
arrowheads, see also D). Scale bars for A–E, G–I ¼ 300 mm, F ¼ 100 mm. Abbreviations: 1 g – first tooth generation; 2 g – second
tooth generation; ad – adhesion; c – concha; d – dentary bone; dl – dental lamina; dlt – ductus laryngotrachealis; ecs – extraconchal
space; h – hyoid; idl – inner dental lamina; lm – lingual muscles; mc – Meckel’s cartilage; mx – maxilla; nc – nasal cavity proper; np –
nasal plug; nv – nerve; odl – outer dental lamina; oc – oral cavity; on – olfactory nerve; p – parietal bone; pa – prearticular process of
the compound bone; pl – palatine bone; pm – premaxilla; ps – palatal shelf; pt – pterygoid; pth – premaxillary teeth; px – pharynx; sf
– skin fold; sp – splenial bone; su – surangular process of the compound bone; t – tongue; tr – trachea.

rows of teeth in the upper jaw. This groove was more
prominent in the middle part of the jaw (Fig. 3B) as
compared to extreme rostral (Fig. 3A) or caudal (Fig. 3C)
regions. The palatal shelves were more prominent than at
stage 3 and projected into the oral cavity (Fig. 3C).
Skeletal development
Several new bones were initiating at stage 4 including
the premaxilla, maxillary, nasal, vomer and parietal
bones. The premaxilla was ossifying as a small plate-like
structure located rostroventrally to the nasal capsule
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238 M. Buchtová et al. / Zoology 110 (2007) 231–251
(Fig. 3A). One small ossification centre for the maxillary
bone was visible at the lateral edges of the upper jaw
(Fig. 3C). Paired vomers were present as single rod-like
ossification centres, medial to each vomeronasal organ
(Fig. 3B). Ossification of the nasal bones had begun on
either side of the midline (not shown). Of the cranial vault
bones, the prefrontal (not shown) and parietal (Fig. 3E)
had just begun to ossify bilaterally.
None of the lower jawbones contacted each other at
stage 4. Rostrally, the dentary bone was ossifying lateral
to Meckel’s cartilage (Figs. 3G and H). Caudally, the
splenial (Fig. 3H) as well as the surangular and
prearticular processes of the compound bone (Fig. 3I)
had formed. The surangular process and the splenial
bone circumscribed Meckel’s cartilage dorsolaterally and
ventromedially, respectively (Figs. 3H and I). The
prearticular process of the compound bone wrapped
around Meckel’s cartilage medioventrally (Fig. 3I). At
this stage, the prearticular process extended further
caudally than did the surangular process. The hyoid
cartilage was visible inferior to the muscle condensations
(Fig. 3I).
Development of soft tissues and teeth
The structure of the nasal cavity was similar to stage 3
although the single nasal concha had just begun to form
(Fig. 3C). In addition, the blind-ended sac that is the
termination of the extraconchal space was expanded
and lined with thicker epithelium compared to stage 3
(Fig. 3C). The ventral epithelium of the vomeronasal
organ was much thinner (30 mm) relative to the dorsal
epithelium (160 mm, measured across its greatest height;
Fig. 3B). The olfactory nerves were clearly present
adjacent to the dorsomedial and dorsolateral parts of
the nasal cavity and connected to the olfactory bulbs in
the telencephalon (Figs. 3B and C and data not shown).
At stage 4, the pharyngeal adhesion persisted, separat-
ing the oral cavity and the pharynx (Fig. 3D). The caudal
oral cavity had formed two recesses, separated by a ridge
of mesenchyme. The multi-layered epithelial adhesion
contacted the oral cavity mesenchyme on one side
and the pharynx on the other. The pituitary gland had
begun to lose its connection with the oral epithelium
(data not shown). The laryngotracheal tube was con-
nected to the oral cavity at the surface of the tongue,
although the tube was not yet opened to the oral cavity
(Fig. 3H). Further proximally, the laryngotracheal duct
was below the tongue surface and had a central lumen
(not shown).
Ventral to the ossifying premaxilla, two premaxillary
teeth were at the cap stage of development in the rostral
part of oral cavity (Fig. 3A). In general, the dental
laminae that form the majority of the teeth were more
mature in the rostral half of the oral cavity, with first
and second tooth generations underway in the outer row
(Fig. 3F). While the first tooth generation in the outer
dental lamina was forming dentin (Fig. 3F), the
second tooth generation had progressed to the bud stage
(Fig. 3F). The outer tooth row of the upper jaw and the
single tooth row of the lower jaw were at comparable
advanced stages of development relative to the inner row
of the upper jaw. The close spatial relationship between
the palatine bones and the dental laminae was consistent
with the later attachment of teeth to these bones
(Fig. 3C).
Stage 6 (day 33 post-oviposition)
Skeletal development
All of the upper and lower jawbones had formed by
stage 6 and the relationships between the skeletal
elements and nerves were more defined. The premaxilla
was ossified – unlike stage 4 embryos – and the palatal
processes flanked the trabecular cartilage (Fig. 4A). The
maxillary bone had the most complex pattern of
ossification centres. There was one ossification centre
rostrally (Fig. 4B) and three centres caudally, surround-
ing the maxillary nerve (Figs. 4G and H). For the first
time, ossification of the ectopterygoid was observed,
dorsal to the maxillary bone and lateral to the pterygoid
bones (Fig. 4E). Two additional new upper jaw/skull
bones observed at this stage included the septomaxilla
just lateral to the vomeronasal organ (Fig. 4C) and
frontal bone (not shown). The nasal capsule was there-
fore supported by the nasal bones dorsally, the prefrontal
bones laterally (Fig. 4D) and the vomers ventrally
(Fig. 4C). The vomeronasal organ was supported by the
vomeronasal cartilages and vomer bone on the ventral
side (Fig. 4C).
In the lower jaw, the mandibular bones surrounded
Meckel’s cartilage and the inframandibular nerve in semi-
overlapping layers. The angular and coronoid bones
(Figs. 4E and L) were visible for the first time in stage 6
embryos. The dentary bone varied from a single
ossification centre to more complex bi- and tripartite
anatomy in the centre of the jaw (Figs. 4E, I–K). The
surangular and prearticular processes of the compound
bone continued to ossify lateral and medial to Meckel’s
cartilage, respectively (Figs. 4E and L). Elsewhere in the
lower jaw, the tracheal opening was located in the dorsal
half of the tongue above the lingual muscles (Fig. 4L) and
was surrounded by well-differentiated cartilages.
Development of soft tissues and teeth
Many features noted at earlier stages also persisted at
stage 6 including plugged nasal passages (Fig. 4A). The
surface of the pituitary gland was more lobular than at
earlier stages (not shown). The palatal shelves were
formed; however, the relative size and position of the
shelves was unchanged compared to stage 4 embryos. The
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M. Buchtová et al. / Zoology 110 (2007) 231–251 239

Fig. 4. Transverse sections of a stage 6 embryo in rostral to caudal sequence. Specimen was hemisected prior to sectioning. (A) The
rostral-most section shows the presence of premaxilla and premaxillary teeth in close relationship to the trabeculae cranii. The
epithelial plug is visible in the nasal passage and the early stages of the supralabial gland lamina. (B) An ossification centre for the
maxillary bone is visible. The supralabial gland is forming lateral to the outer dental lamina. (C) Multiple ossification centres for the
maxillary bone. The septomaxillary and vomer bones flank the vomeronasal organ. (D) The single nasal concha projects into the
nasal cavity proper. The three bones supporting the nasal cavity are present, nasal, prefrontal and vomer. (E) The ectopterygoid is
seen for the first time at stage 4, medial to the palatal groove (black arrowhead). (F) The ectoderm is one cell layer thick comprised
of tall columnar epithelial cells with apically positioned nuclei. (G, H) Maxilla is composed of three ossification centres surrounding
the maxillary nerve; all three centres merge in more caudal sections. Dentin is beginning to form in the first generation of teeth
(arrowhead). (I–L) Mandibular bones surround the mandibular nerve and Meckel’s cartilage. The tongue musculature is dorsal to
the trachea and then drops ventral to the trachea in caudal sections. Scale bars for A–L ¼ 300 mm, Inset in F ¼ 20 mm.
Abbreviations: 2g – second tooth generation; a – angular; c – concha; co – coronoid; d – dentary bone; dl – dental lamina; ec –
ectopterygoid; ecs – extraconchal space; ect – ectoderm; h – hyoid; idl – inner dental lamina;lm – lingual muscles; mc – Meckel’s
cartilage; mx – maxilla; n – nasal bone; nc – nasal cavity proper; np – nasal plug; nv – nerve; odl – outer dental lamina; pa –
prearticular process of the compound bone; pf – prefrontal bone; pl – palatine bone; pm – premaxilla; pth – premaxillary teeth; slg –
supralabial gland; sm – septomaxilla; sp – splenial bone; su – surangular process of the compound bone; t – tongue; tc – trabeculae
cranii; th – tooth; tr – trachea; v – vomer; vno – vomeronasal organ.

unfused secondary palate (Fig. 4C) is a characteristic of ectodermal specializations (salivary glands and teeth).
lizards, snakes and birds (Richman et al., 2006). The pharyngeal adhesion present at stages 3 and 4 had
The epithelial tissues with the most significant changes disappeared and instead the oral cavity (ectoderm) was
were the pharyngeal lining, the epidermis and the continuous with the gut/pharyngeal endoderm (not
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240 M. Buchtová et al. / Zoology 110 (2007) 231–251
shown). The extra-oral epidermal cells had polarized.
The basal epidermal layer was formed by high columnar
cells, with apically positioned nuclei (Fig. 4F, inset).
Keratin was not yet deposited on the surface of the
ectoderm, nor was pigmentation present. For the first
time fully formed eyelids were visible across the corneas
(not shown).
In the rostral-most part of the head lateral to the
premaxillary teeth, the epithelial lamina invaginated to
form the supralabial gland (Figs. 4A, B, H). The
glandular and tooth laminae were close to each other
rostrally but separated more caudally (Fig. 4H). Of the
two generations of upper teeth in the outer and inner
rows, the first generation had formed dentin (not shown);
the second-generation teeth were producing predentin
(Fig. 4G). The groove separating the inner and outer row
was much deeper than in the earlier staged embryos (Figs.
4C, E, G). In the lower jaw, the single row of teeth had
developed as far as the dentin stage (first generation) and
predentin production (second generation; data not
shown).
Stage 7 (day 44 post-oviposition)
A single stage 7 specimen was collected for histological
analysis and, although the heart was beating, this was not
a completely healthy specimen as judged by later TUNEL
analysis. Nonetheless, tissue morphology was good and
histochemical staining was of sufficient quality to be
included in our study.
Skeletal development
In the upper and lower jaw, all membranous bones
were well established with a similar pattern to their post-
natal morphology. The maxillary bone had developed
two processes rostrally (Fig. 5C) and enveloped the
maxillary nerve more caudally (Fig. 5D). The most
caudal upper jawbones, the ectopterygoid and pterygoid
bones, were not yet in contact with each other but were
surrounded by a mesenchymal condensation (Fig. 5G). In
the mandible, even though the dentary bone and
Meckel’s cartilage arose independently, they were now
partly fused (Fig. 5E). The nature of the connection and
whether or not there was intermingling of the cells was
not clear from this type of analysis. At all other levels,
these lower jaw elements were clearly separated (Fig. 5F).
The surangular and prearticular are connected at the
proximal end (Fig. 5H). More distally, the coronoid
(dorsal), prearticular (medial), angular (ventral), and
surangular (ventrolateral) were separate (Fig. 5G). Else-
where in the head, endochondral ossification in the
central body of the quadrate, the basisphenoid and
prootic bone was underway (Figs. 5G, H and data not
shown).
Development of soft tissues and teeth
In general, the appearance of stage 7 connective tissues
and epidermis was similar to those at stage 6. The nasal
cavity was still obstructed with vacuolated epithelial cells
(Fig. 5A). Many macrophages were found in deeper parts
of the lateral connective tissue of the upper and lower
jaws (Fig. 5B). Compared to stage 6, there was a
significant expansion in the thickness of the as yet
unpigmented epidermis (Fig. 5B). In the rostral part of
the upper jaw, salivary laminae were lateral to the
premaxillary teeth (Fig. 5A), whereas more caudally the
glandular laminae were lateral to the outer tooth row. In
the lower jaw, infralabial gland laminae were lateral to
the dental lamina (not shown).
Tooth development was the most advanced in the outer
row of the upper jaw where three generations of teeth
were present (not shown). In the inner, upper row and the
lower row there were only two generations of teeth, of
which only the first generation had developed dentin. The
groove between the inner and outer dental lamina was
shallow near the premaxillary bone (Fig. 5A) and
gradually increased in depth towards the middle of the
palate (Figs. 5C and D).
Stage 8 (day 54 post-oviposition)
Skeletal development
Since there were few changes in the skeletal pattern
between stage 7 and 8, these will not be discussed in detail
here. The processes of the maxillary and palatine bones
extended further towards each other in the centre of the
jaw where they will later articulate. With increased
maturity of the bones, it was possible to recognize canals
for neurovascular bundles in the maxillary and the
palatine bones. The orbit was more fully supported by
the prefrontal, post-orbital and newly formed supraorbi-
tal bone (Fig. 6E). The basisphenoid had reached a
similar stage of ossification to the other bones of the skull
base (not shown). Perichondral ossification in the
trabeculae cranii was beginning at the most proximal
end, where these elements will ultimately join with the
basisphenoid (Fig. 6F). The mandibular bones (dentary
splenial, angular, coronoid and compound, not shown)
remained separate, just as they are in the adult skull
(Frazzetta, 1959).
Development of soft tissues and teeth
The main difference that distinguished stage 7 from
stage 8 embryos was the presence of keratin and pigment
cells in the epidermis (Fig. 6H). Underneath the dermis
was composed of dense bundles of collagenous fibrils.
Another distinguishing feature of stage 8 embryos was
that luminization of the nostril had at last begun in the
deeper areas of the epithelial plug (Fig. 6A). Numerous
capillaries entered deep to the olfactory epithelium of the
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M. Buchtová et al. / Zoology 110 (2007) 231–251
vomeronasal organ (Fig. 6G). Supralabial salivary glands
opened into the oral cavity near the outer dental lamina
(Fig. 6B). The infralabial glands opened into the vestibule
of the lower jaw (Fig. 6D). In addition, the development
of parenchymal (secretory) tissue of the salivary gland
had begun. There was no evidence of secretions; however,
salivary gland structure was more complex and the
alveolae had begun to form (Fig. 6A). Lumina were
absent in the salivary ducts.
241
By stage 8, the single egg tooth contained dentin
(Fig. 6A). In addition, there was a space where the enamel
had been removed during demineralization of the tissues
(Fig. 6A). Three generations of teeth were present in upper
and lower jaws with only the first showing an enamel space
(Figs. 6C, D, F). The palatal groove was narrow and deep in
the plane of the septomaxillary bone (Fig. 6C), wider in the
caudal part of the nasal cavity (Fig. 6D) and much shallower
and wider at the level of the eyes (not shown). The palatal
groove was completely lined with high, cylindrical epithelial
cells and separated the ridges bearing the outer teeth and the
inner row of teeth.
Interestingly, at this stage the dental ridges contained
mainly teeth and connective tissue but no bone, although
ultimately teeth attach directly to bone. The connective
tissue of the dental ridges picked up the Alcian blue
demonstrating that the extracellular matrix was rich in
sulfated proteoglycans (Fig. 6B). This region had less
dense collagen fibers compared to the dense collagen
bundles found in the subepidermal areas. This distinct
matrix may be important for the formation of the future
junction between the jawbone and the teeth. Consistent
Fig. 5. Transverse sections through a stage 7 embryo. (A) The
supralabial gland is more developed, adjacent to the premax-
illary teeth. A nasal plug is blocking the vestibulum nasi and the
nasal capsular cartilage is well differentiated. (B) In the surface
epithelium, the number of epidermal layers is increased and
basal cells are columnar. A collection of multinucleated white
blood cells is present in the connective tissue. (C) The
septomaxillary bone lies between the nasal cavity proper and
is separated from the vomer by the nasal capsule. The shape of
the maxilla is changed at different levels of the upper jaw. There
are distinct dorsal processes for muscle attachments whereas it
forms a tube in more caudal sections. (D) The palatal groove is
larger in the centre of the upper jaw (arrowhead). The nasal
cavity is composed of the nasal cavity proper and the
extraconchal space, which are connected to each other in
deeper sections. The outer row of teeth is approaching the
maxillary bone, the point of their attachment at later stages.
(E) Detail of the lower jaw with apposition of Meckel’s cartilage
to dentary bone. (F) More caudal level in the lower jaw.
(G) Endochondral ossification of basisphenoid is beginning.
The ectopterygoid is beginning to articulate with the pterygoid
bone. (H) The compound bone is formed by the unification of
the surangular and prearticular processes. Scale bar ¼ 300 mm.
Abbreviations: a – angular; bs – basisphenoid; c – concha;
co – coronoid; d – dentary bone; dl – dental lamina; e – ear; ec –
ectopterygoid; ecs – extraconchal space; ep – epidermis;
idl – inner dental lamina; mc – Meckel’s cartilage; mx – maxilla;
nc – nasal cavity proper; ncs – nasal capsule; np – nasal plug;
odl – outer dental lamina; ocs – otic capsule; p – parietal bone; p
– parietal bone; pa – prearticular process of the compound
bone; pl – palatine bone; pt – pterygoid; pth – premaxillary
teeth; slg – supralabial gland; sm – septomaxilla; su – surangular
process of the compound bone; v – vomer; vnc – vomeronasal
cartilage; wbc – white blood cell.
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M. Buchtová et al. / Zoology 110 (2007) 231–251 243

with this idea, the lower jaw had a similar Alcian blue- epithelium and were never observed in the oral connective
stained matrix surrounding the teeth (Fig. 6D). tissue. The epidermis was fully keratinized at this stage
and had well-formed scales (Fig. 7C).

Stage 10 (day 75 post-oviposition)

These data were collected from a specimen that had
been damaged during transport; thus, the connective
tissue had separated into sheets. Nonetheless, some
details were distinct. For the first time the nostrils were
unplugged and open to the external surface (Fig. 7A).
Lateral to the dental lamina in the upper and lower jaws,
tuboalveolar salivary glands were fully differentiated and
contained mucous-filled lumina (Figs. 7A and B).
Further, we observed minor mucous-secreting intrae-
pithelial oral glands (Fig. 7B). Similar to stage 8 embryos,
pigment cells were present in the epidermis and the deeper
layers of connective tissue epithelium. In addition, these
melanocytes were observed in the nasal vestibulum (not
shown). Pigment cells were rarely present in the oral
Fig. 7. Transverse sections of stage 10 embryo. Arrangement of
collagen bundles is an artifact of poor preservation. (A) The
nostrils are unplugged and open to the external surface.
(B) Infralabial salivary glands were fully differentiated with
mucous-filled lumina in the mandible. There were also minor
mucous glands within the ectoderm. (C) The maxillary bone as
well as a fully formed scale are visible. Scale bar ¼ 500 mm.
Abbreviations: ilg – infralabial gland; mgl – minor salivary
gland; mx – maxilla; sc – scales; slg – supralabial gland; vn –
vestibulum nasi.
Cellular dynamics in the craniofacial tissues of
Python sebae
Cell apoptosis
One of the many functions of apoptosis is to define the
shapes of body structures by selectively deleting tissue. In
the embryonic face and teeth there are many restricted
areas of increased apoptosis in other vertebrates so we
were interested in knowing whether some of these
patterns were conserved in reptiles.
We observed apoptosis in both mesenchymal and
epithelially derived structures. At stage 1 there was
mesenchymal apoptosis in the lateral nasal, frontonasal
and maxillary prominences. The apoptotic cells were
mainly concentrated in the centre of the mesenchyme
(Fig. 8A). There are some similarities to chicken embryo
results at stage 28 (Richman et al., unpublished data).
One of the most striking mesenchymal apoptotic areas
was near the early condensing membranous bones in
stage 3 embryos. There were numerous TUNEL-positive
cells around the rostral and caudal ends of the
mesenchymal condensation for the palatine bone. There
were clusters of positive cells between the palatine bone
and pterygoid (Figs. 8D and E) and within the palatine
bone (Fig. 8F). A similar pattern was seen in stage 4
embryos (Figs. 8K and L) with many positive cells
between the palatine bone and pterygoid. At stage 3, the
prefrontal bone condensation also contained apoptotic
cells (data not shown) and by stage 4 these cells were
concentrated on the surface facing the brain. The vomer
condensation also contained a group of apoptotic cells at
stage 3 and subsequently at stage 4, TUNEL-positive
cells were surrounding the differentiating bone (data not
shown).

Fig. 6. Transverse sections in rostral to caudal sequence of the upper jaw in a hemisected stage 8 specimen. (A) Nasal plug still
obliterates the vestibular opening, but luminization is just beginning (arrow). Supralabial salivary glands beneath the epidermis will
connect and drain into the oral cavity near the egg tooth. The egg tooth has formed enamel (space) and dentin. (B) Further caudally,
the supralabial glands are close to the premaxillary teeth. A zone of sulfated proteoglycan-rich extracellular matrix (distinctly
stained with Alcian blue) surrounds the maxillary bone and the teeth. Scales are present on the surface of the embryo. (C) A very
deep palatal groove separates the two rows of upper teeth (arrowhead). The teeth are close but not yet contacting either the
maxillary or palatine bone. (D) The infralabial gland lies lateral to the lower teeth. Several generations of differentiated teeth in the
outer and inner row are visible. (E) The supraorbital bone is forming adjacent to the frontal bone and is one of the last membranous
bones to form. (F) The caudal-most end of the trabeculae cranii underlying the brain is beginning to undergo perichondrial
ossification (arrows). (G) Numerous vessels have perforated the expanded sensory epithelium of the vomeronasal organ (H). Scale
bars for A–F ¼ 300 mm, G ¼ 200 mm and H ¼ 100 mm. Abbreviations: d – dentary bone; dl – dental lamina; et – egg tooth; f –
frontal; ilg – infralabial gland; mc – Meckel’s cartilage; mx – maxilla; nc – nasal cavity proper; ncs – nasal capsule; np – nasal plug; pf
– prefrontal bone; pg – pigment; pl – palatine bone; pth – premaxillary teeth; sc – scales; slg – supralabial gland; sm – septomaxilla;
so – supraorbital; th – tooth; tc – trabeculae cranii; vnc – vomeronasal cartilage; vno – vomeronasal organ; vsl – blood vessel.
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244 M. Buchtová et al. / Zoology 110 (2007) 231–251

Fig. 8. Cellular apoptosis in several stages of python embryos. (A) Stage 1, (B–F) stage 3 and (G–L) stage 4 embryos. (A) A stage 1
embryo showing a group of apoptotic cells in the mesenchyme underlying the furrow between the lateral nasal and maxillary
prominences. Inset shows location of section in relation to the frontonasal mass. (B) Apoptotic cells are dispersed throughout the
olfactory epithelium. (C) Apoptosis in the stellate reticulum of the cap-stage tooth. (D) Apoptosis in the palatal shelf mesenchyme
(shown at higher power in E). (E) Mesenchymal condensation rostral to the palatine bone. (F) Apoptotic cells in the palatine bone
and in the periosteal mesenchyme. (G, H) Apoptotic cells are dispersed in the olfactory epithelium and in the perichondrium of the
nasal capsule. (I) Positive cells in the stellate reticulum of the first generation tooth (arrowhead). (J) Apoptosis in the palatal shelf
mesenchyme (shown at higher power in K, L). (K) Cells undergoing programmed cell death (arrowheads) between the caudal end of
the palatine and pterygoid bone. (L) Apoptotic cells (arrowheads) between the pterygoid and ectopterygoid condensation. Scale
bars: Inset in A, D, G, J ¼ 500 mm; A, C, E, F, H, I ¼ 100 mm; B, K, L ¼ 50 mm. Abbreviations: 1g – first tooth generation;
2g – second tooth generation; dl – dental lamina; fnm – frontonasal mass; lnp – lateral nasal prominence; oe – olfactory epithelium;
mcs – mesenchymal condensation; mxp – maxillary prominence; pl – palatine bone; pt – pterygoid; S – stage; sr – stellate reticulum;
th – tooth.
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M. Buchtová et al. / Zoology 110 (2007) 231–251
At stages 3 and 4, several epithelial structures had
apoptotic cells, including the olfactory and respiratory
epithelia lining the nasal passages (Figs. 8B, G, H) and
the enamel organs of the teeth (Fig. 8C). The inner
enamel organ and the dental lamina connecting the tooth
to the oral epithelium contained many positive cells
(Fig. 8C).
At stages 6 and 8 very few apoptotic cells were seen.
One of the few areas that remained positive was the
surface of the maxillary bone (data not shown). In
comparison to the upper jaw, there were fewer apoptotic
cells in the mandible. The main positive areas were the
medial side of the compound bone facing Meckel’s
cartilage (data not shown) and the tongue. Evidence of
programmed cell death around the dentary bone was far
less obvious than in the upper jawbones of stages 6 and 8
(data not shown).
Cell proliferation
Relatively higher and lower intensities of cell prolifera-
tion within an area of the embryo can lead to elongation
of parts of the embryo and shape changes of individual
structures. In addition, differential cell proliferation is
one mechanism by which condensations of mesenchyme
develop. Proliferating cells were found in the highest
numbers and densest concentrations early in development
(stages 3 and 4). PCNA-labeled cells were most notable in
the condensing cartilages, ossification centres and mus-
cles of the face and jaws (data not shown). Later, at stage
6, many PCNA-positive cells were localized in Meckel’s
cartilage (data not shown) and nasal capsules (Figs. 9D
and E) but not in the perichondrium (data not shown).
There were numerous PCNA-positive cells in the tongue
muscles and in the surrounding perimysium at this stage
(data not shown).
Cell proliferation was also high in selective areas of the
olfactory epithelium. At stage 3, PCNA-positive cells
were seen in the superficial layers of the thick olfactory
epithelium where the supporting cells will form (Figs. 9A
and B). In the vomeronasal organ, proliferating cells were
located in the superficial epithelial layers and also near
the basal membrane (Figs. 9A and C). At stage 6,
proliferation in the vomeronasal epithelium was limited
to a few cells at the basal layer and by stage 8 there was
no staining with PCNA antibodies (not shown). In
contrast, the stage 6 olfactory epithelium in both the
nasal cavity proper and the extraconchal space (Figs. 9D
and E) had abundant proliferating cells at the luminal
and basal sides. At stage 8, cell proliferation in the
olfactory epithelium was reduced to a few cells close
to the basement membrane or in Bowman’s glands
(Figs. 9G and H). We found no proliferating cells in
the respiratory epithelium at any of the stages examined
(Figs. 9C, D, E, H).
In stages 3 and 4 tooth germs, the highest proliferation
activity was observed in the growing dental lamina, the
245
outer enamel epithelium as well as the dental papilla (Fig.
9A and data not shown). This trend continued in stage 6
and 8 embryos; however, there was decreased prolifera-
tion in the dental lamina that connected the first
generation teeth to the oral cavity. Instead, staining was
concentrated in the lamina forming the second and the
third generation teeth (Figs. 9D, F, I). In the stage 8
embryo, while PCNA staining was generally decreased
throughout the head, the second and third generation
teeth had some of the most concentrated labeling
(Fig. 9I).
Discussion
The goal of this work was to compare the development
of craniofacial structures in the python compared to
other amniotes. Areas studied in detail included the
chondrocranium, dermatocranium, olfactory organs,
teeth and salivary glands. We did not include
the eye and brain in these studies. The range of
embryo development spanned morphogenesis from
early organogenesis until full adult morphology was
established. We have learned that the general processes
of skeletal differentiation are similar amongst amniotes
studied thus far; however, there are some interesting
differences in the way the teeth, salivary glands,
olfactory and upper respiratory system form that
invite further study. The unique python craniofacial
morphology could be ascribed to a variety of
factors including early differences in neural crest
cell contributions, expression patterns of signaling
molecules in pharyngeal endoderm and facial
ectoderm, differential cell proliferation and apoptosis
and changes in the positions and sizes of skeletal
condensations.
Early facial morphogenesis is different in reptiles and
mammals
The eggs of P. sebae are laid long after gastrulation,
neurulation and neural crest cell migration have been
completed. Thus, these processes cannot be studied in this
type of snake. A viviparous model such as the garter
snake is more suitable for early embryo studies (Zehr,
1962). However, the python is suitable for studying the
shape of the facial prominences. In the present study and
in previous work, we have found that appearance and
relative proportions of the facial prominences are very
similar in python and chicken (Richman and Lee, 2003;
Richman et al., 2006). There are differences, however,
between mammals and reptiles. The frontonasal mass in
both reptilian orders is flat, while in the mouse there is a
deep midline furrow between the medial nasal promi-
nences. In birds, formation of a continuous upper beak is
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M. Buchtová et al. / Zoology 110 (2007) 231–251 247

the result of contact and then fusion between the
frontonasal mass and the maxillary prominences
(Ashique et al., 2002a). We did not observe fusion in
python, since by stage 3 the mesenchyme was completely
merged; however, the initial zone of contact was
identical in pythons and chickens suggesting the
steps are conserved. In contrast, lip formation in
mammals requires an additional contribution from the
lateral nasal prominence (Wang et al., 1995). Another
important part of the oral cavity is the secondary
palate. Initially, the palatal shelves grow medially out
from the maxillary prominences in snakes and all
amniotes. Shortly afterwards, in mammals, palatal
shelves grow down beside the tongue, and later the
mandible drops, moving the tongue away from the
palatal shelves allowing midline fusion to take place.
This never happens in birds, snakes or lizards. Instead the
shelves do not progress any further and the secondary
palate remains naturally cleft (Richman et al., 2006). In
addition, the snake palate develops a deep groove
separating the two dental ridges. This is a unique
adaptation only found in the upper jaw of snakes and
could be considered a primitive rather than a derived trait
since palatal teeth are present in many primitive amniotes
(Carroll, 1988).
There are also some features in the early facial
prominences that are characteristic of snakes but not of
birds. The vomeronasal organ forms as an invagination
into the frontonasal mass. A similar origin for the
vomeronasal organ was reported in garter snakes (Zehr,
1962; Holtzman, 1993). In mammals and birds, it is clear
that morphogenesis of the vomeronasal organ does not
involve the frontonasal mass; however, little is known
about the origins of this organ in either taxon. Thus,
several questions arise from our work: (1) How widely
conserved are the frontonasal mass origins of the
vomeronasal organ amongst reptilian taxa? (2) Does the
nasal placode contain the precursor cells for the
vomeronasal organ? This second question is most easily
addressed in the chicken embryo with the following
caveat: In Aves the vomeronasal organ is only rudimen-
tary (Romanoff, 1960); thus we predict that, similar to
the snake limb, the initiation of the vomeronasal organ
takes place but signals supporting development must be
lacking in birds.
Amniote vomeronasal proliferation patterns are
conserved in snakes
We found that the majority of proliferating cells in the
python vomeronasal organ was on the luminal side of the
olfactory epithelium where supporting cells arise. There
were also proliferating cells on the basal side of the
epithelium where vomeronasal receptor cells are born.
The patterns of proliferation in the garter snake are
similar, as detected using an antibody to a cyclin-
dependent kinase (Holtzman, 1998). Cell proliferation
in mouse vomeronasal organs has recently been described
and here there are also dividing cells in the basal,
neuronal precursors and supporting cells which arise
elsewhere in the epithelium (Wakabayashi and Ichikawa,
2007). The evolutionary origins of the vomeronasal organ
are thought to begin with tetrapods and specific receptor
genes that are expressed in the vomeronasal system as
compared to the olfactory system proper have been
identified (Grus and Zhang, 2006; Wakabayashi and
Ichikawa, 2007). Some of the G-protein-coupled recep-
tors have been found in teleosts, thus representing a
precursor to the signaling systems in the tetrapod
vomeronasal system (Grus and Zhang, 2006). Taken
together with the large expansion of the vomeronasal
organ in snakes, we predict that there will be an increased
number of vomeronasal receptor types as compared to
mammals.
Additional condensations and reorganization of late
condensations give rise to additional bones in python
The divergence in the number and shape of the bones
of snakes as compared to birds and mammals is of great
interest from developmental and evolutionary points of
view. In the present study we have shown that the
developmental mechanisms are largely conserved. The
staining we used was sensitive enough to detect con-
densations and the early bone matrix prior to miner-
alization. We acknowledge, however, that using
molecular markers would tell us more about whether
the well-characterized osteogenic and chondrogenic
signaling pathways are employed during the course of
python skeletal differentiation.

Fig. 9. Cellular proliferation using the PCNA antigen in python. Transverse sections of the upper jaw show more proliferating cells
in younger than older embryos. (A–D, G) The luminal side of the olfactory epithelium in the nasal cavity and vomeronasal organ is
strongly labeled showing two distinct regions where cell division is occurring. This pattern is preserved between stage 3 and 6 (B, E).
(F) The mesenchymal cells surrounding membranous bones were also labeled. Teeth had distinct regions undergoing proliferation,
with the highest levels in the dental papilla, and elongating tips of the enamel organ (A, D, F, I). The level of proliferation was
similarly high in the second and third generation teeth, but had decreased in the first generation teeth by stage 8 (I). (G, H) At stage
8, the main cells undergoing proliferation in the nasal cavity are the epithelial cells of Bowman’s gland (arrows). Scale bar ¼ 200 mm.
Abbreviations: 1g – first tooth generation; 2g – second tooth generation; 3g – third tooth generation; dp – dental papilla; nc – nasal
cavity proper; ncs – nasal capsule; oe – olfactory epithelium; on – olfactory nerve; pl – palatine bone; re – respiratory epithelium; S –
stage; th – tooth; vno – vomeronasal organ.
 ARTICLE IN PRESS
248 M. Buchtová et al. / Zoology 110 (2007) 231–251
Despite similar developmental mechanisms, several
additional bones are present in python and other non-
avian reptiles such as the septomaxilla, supratemporal
and ectopterygoid (Richman et al., 2006). The embryonic
basis for morphology changes could include early
differences in neural crest cell contributions to the facial
prominence. However, effects on neural crest cells are
unlikely due to high conservation of the early stages in
neural crest cell formation and migration among
gnathostomes (Kuratani, 2005a). A second mechanism
could work through differences in expression domains of
epithelial signals required for setting up the bone
condensations (Hall and Miyake, 1992; Shigetani et al.,
2002). This could lead to supernumerary condensations
or differences in sizes and positions of evolutionarily
conserved condensations. Finally, a third possibility
would be the later reorganization of mesenchymal
condensations. We found evidence for an independent
condensation giving rise to the septomaxillary bone,
separate from the condensations forming the vomer, the
maxillary and other surrounding bones. The septomaxilla
is generally present in Sauropsida (i.e. extinct and extant
crocodilians, turtles, birds, snakes and lizards), but
mostly absent in Synapsida (i.e. extinct and extant
mammals), with a couple of exceptions. Early mammals
from the Permian and Triassic periods did have a
septomaxilla (Hillenius, 2000), as do modern mono-
tremes. Thus snakes (and other reptiles) have retained a
bone that is typical of primitive amniotes that are either
Synapsids or Sauropsids.
In contrast to the septomaxilla, the pterygoid and
ectopterygoid clearly have a shared condensation as do
the pterygoid and palatine bones. How does this large
condensation give rise to multiple bones? Our data from
the stages 3 and 4 embryos suggests that localized
apoptosis reduces cell numbers in some regions, while
leaving cell density high in others. We also observed
relatively higher intensity of cell proliferation near the
ossification centres but in a more restricted area than
apoptotic cells. Thus, in addition to mechanisms such as
preferential cell adhesion, and increased cell proliferation
proposed by others (Atchley and Hall, 1991; Hall and
Miyake, 1992; Dunlop and Hall, 1995; Hall and Miyake,
2000), intramembranous bone condensations are sculpted
by localized programmed cell death. The early steps of
skeletogenic neural crest migration would not need
adjustment during amniote evolution. Instead, late
reorganization of condensations could occur via local
signals such as bone morphogenetic proteins, which are
known to promote apoptosis (Graham et al., 1996)
Dental development in snakes differs from mammals
In the embryos collected for this study most of the
major steps in tooth morphogenesis could be observed.
Initiation of the dental lamina at stage 1 was followed by
formation of the epithelially derived enamel organ and
mesenchymally derived dental papilla at stages 3 and 4.
The most striking differences as compared to mammals
were much deeper dental laminae that gave rise to 4
generations of teeth (polyphyodont). Thus the very long-
lived dental lamina offers a unique opportunity to study
the signaling required to initiate generational teeth. Not
surprisingly, cell proliferation studies showed that growth
was concentrated in the cervical loop of the teeth and in
the budding generational teeth. In mouse, there are
specific regions of high proliferation in the extending
cervical loop resulting in the formation of the crown
(Shigemura et al., 1999; Tummers and Thesleff, 2003;
Setkova et al., 2006). Programmed cell death was also
observed in python teeth, but unlike mouse where it is
mainly found in the inner enamel epithelium, in the snake
tooth apoptosis is located between the inner and outer
enamel epithelium. The significance of the positional
difference may relate to the simpler conical tooth
morphology of snakes as compared to the multicusped
mammalian teeth (Vaahtokari et al., 1996). The snake
also differs in the way the teeth attach to bone. Rather
than a thecodont attachment, in which a periodontal
ligament forms between the root and bone, the snake
does not form roots and the attachment is directly to
bone. The signals that determine whether a root is made
(Tummers and Thesleff, 2003) have likely been lost in the
snake.
Supralabial gland development in non-venomous
snakes is similar to venomous snakes
In the upper jaw of our python specimens, there were
two types of mucous-secreting salivary glands: (1) the
supra- and infralabial glands; (2) the minor intra-
epithelial glands. The minor salivary glands can be found
in the crypts of the oral cavity and in the midline of the
tongue. They are not associated with teeth and were not
visible prior to stage 10 in our sample. In contrast, the
infralabial and supralabial glands form in a very similar
way to the teeth. Both begin as epithelial thickenings,
followed by invagination into the mesenchyme by
epithelial laminae. These labial glands then develop a
serial, lobular, non-compound structure and mucous
secretion begins at stage 9 or 10. This close relationship of
upper dental lamina to the supralabial salivary gland has
also been described for oviparous, venomous snakes
(Gygax, 1971). It has been hypothesized that the
proximity of developing teeth and glands may have
facilitated the connection of the glands to the fangs of
venomous snakes (Kochva, 1978). Since the python lacks
Duvernoy’s gland or any other venom gland, the
developmental significance of the close relationship of
the teeth and supralabial glands is unclear. It may turn
 ARTICLE IN PRESS
M. Buchtová et al. / Zoology 110 (2007) 231–251 249

out that the secretions of the python salivary glands do

have similar bioactivity to venomous secretions. Such
surprising qualities have already been described for the
secretions of supposedly non-venomous lizards (Fry
et al., 2006).

The position of the tracheal opening relative to the

tongue suggests a different embryonic origin of the
posterior tongue epithelium compared to non-reptilian
amniotes

One of the differences between post-oviposition python

embryos and other amniotes was the position of the
laryngotracheal tube. Normally the trachea is derived
from foregut endoderm caudal to the pharyngeal arches
(Sperber, 2001; Cardoso and Lu, 2006). However, the
orifice of the snake trachea is the centre of the tongue.
Presumably this rostral location is an adaptation to
permit breathing while the oral cavity is obstructed with
large prey. Since the anterior two thirds of the tongue are
derived from the tuberculum impar which is covered with
ectoderm (Sperber, 2001), there are three possible
scenarios: (1) the snake trachea is an island of endoderm
surrounded by tongue ectoderm; (2) the pharyngeal
endoderm extends to encompass trachea and posterior
tongue; (3) the trachea is partially derived from ectoderm.
In any of these situations, one would hypothesize that
genes involved in patterning the snake pharyngeal pouch
endoderm (Graham et al., 2005) would be changed in Fig. 10. Morphology of pharynx and tongue in python as
order to allow this rostral shift in derivation of the compared to chicken. Several key differences are highlighted.
trachea. (A–C) There is an epithelial adhesion in the pharynx seen in
stage 3 python. (D–F) The chicken also has a closed pharynx
but this disappears at much earlier stages (stage 17 Hamburger
The transitory pharyngeal adhesion is not homologous Hamilton, just after neural crest cell migration has ceased).
to the buccopharyngeal membrane There is advanced skeletal development in python (A) compared
to stages when the chicken buccopharyngeal membrane is
In birds and mammals, shortly after neural crest cell present (D). The python adhesion is parallel to the notochord in
migration ends, a bilayered buccopharyngeal membrane sagittal sections whereas in birds, the buccopharyngeal mem-
brane is approximately 901 to the notochord. In chicken, the
is formed from the stomodeal ectoderm and pharyngeal
foregut endoderm and oral ectoderm form a bilayered
endoderm (Figs. 10D–F). In stage 3–4 snakes we noted a membrane (E, F). (B) Instead, the python epithelial adhesion
pharyngeal adhesion, which also separated the pharynx was multilayered. The embryonic origins and rostral position of
from the oral cavity (Figs. 10A–C); however, we do not the tracheal opening in the tongue suggest that the endoder-
think that the pharyngeal adhesion in snakes is homo- m–ectoderm boundary is located near the duct (E). Scale
logous to the buccopharyngeal membrane. There are bar ¼ 200 mm. Abbreviations: ad – adhesion; ah – adenohypo-
several reasons for the lack of homology: (1) the physis; bu – buccopharyngeal membrane; dlt – ductus
pharyngeal adhesion is entirely endodermally derived, laryngotrachealis; md – mandible; nt – notochord; oc – oral
based on the position of the tracheal opening; (2) in birds cavity; prG – preoral gut; t – tongue.
and mammals the buccopharyngeal membrane is present
much earlier and is removed prior to facial prominence
formation (Waterman and Schoenwolf, 1980); and (3) the parallel to the notochord (Figs. 10A–C). For all the
orientation of the snake pharyngeal membrane contrasts above reasons and especially the developmental window
with what is found in other amniotes. In mammals and in which the membrane is normally found, we suspect
birds (Waterman, 1977; Waterman and Schoenwolf, that an ecto-endodermal buccopharyngeal membrane
1980), the membrane is short and vertically oriented with similar to birds and mammals would have been present
regard to the oral cavity (Figs. 10D–F). In python, the in pre-oviposition snake embryos. Since there are very
pharyngeal epithelial adhesion is relatively long and few data published on sectioned early snake embryos it is
 ARTICLE IN PRESS
250 M. Buchtová et al. / Zoology 110 (2007) 231–251
not possible to determine whether such a membrane
exists from available literature. Further studies on
viviparous species or other reptiles that lay their eggs at
much younger stages would help to answer whether a
buccopharyngeal membrane is present in non-avian
reptiles.
The adhesion in the pharynx begins at stage 1 and
completely separates the oral cavity from the pharynx by
stage 3. The pharynx remains closed until at least stage 4
and possibly later (we have no stage 5 data). The reasons
for the short period of time when the pharynx is
separated from the oral cavity in python are not clear
but it may be necessary for gut development. We have
never observed this phenomenon in birds or mammals,
but there may be homologous events in these taxa. The
esophagus in birds becomes occluded at the cranial end
shortly after formation and then reopens (Romanoff,
1960). It is possible that the closure of the pharynx in P.
sebae is analogous to this esophageal obliteration and
luminization in birds. It would be necessary to examine
more caudal python sections to determine whether this is
true.
The importance of snake development to
understanding amniote evolution
In summary, the snake model offers many valuable and
apparently conserved characters that may help to
advance our knowledge of amniote evolution. By
expanding our repertoire to include non-avian reptiles,
biologists can begin to address issues such as the control
of tooth number, tooth generations, salivary gland/dental
lamina decisions and the relationship between condensa-
tions and skeletal patterning, many of which cannot be
studied in other, more rapidly developing model systems.
By undertaking studies on divergent species such as
snakes, it will ultimately be possible to tease apart the
important signals and processes that have been modified
to result in species-specific characteristics.
Acknowledgments
We are very grateful to Paul Springate at the Rain-
forest Reptile Refuge, Surrey, British Columbia, whose
expert reptile husbandry made this research possible. This
work was first funded by CIHR and later by NSERC
grants to JMR. JMR is a Michael Smith Distinguished
Scholar.
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