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SU M M ARY

Palatogenesis is a com plex developm ental


process that requires tw o m ain events: elevation
and then fusion of the palatal shelves. There
rem ains controversy concerning the m echa-
nism (s) responsible for palatal shelf elevation, it
being proposed that an intrinsic shelf elevation
force m ight be produced either by the genera-
tion of a turgor pressure follow ing hydration of
the extracellular m atrix via its glycoconjugate
m olecules or by proliferation, m igration and/or
contraction of the palatal shelf m esenchym al
cells. Recent evidence indicates that the shelf
elevation force is related to the presence of
hyaluronan in the extracellular m atrix, to an as
yet unknow n m olecule that is packaged in the
m esenchym al cellsG olgi com plex, and to CD 44
receptor functioning. For fusion of the palatal
shelves to occur, the breakdow n of the m idline
epithelial seam relates to apoptosis and rediffer-
entiation of the epithelial cells and this appears
to be signalled by the synthesis of type IX colla-
gen just prior to the breakdow n of the basem ent
m em brane around the m idline epithelial seam .
The events associated w ith palatogenesis are
controlled by the palatal shelf m esenchym e,
under the influence of a variety of hom eobox
genes and transcription factors and and of sev-
eral grow th factors (particularly TG F-s).
Key words: Palatogenesis Palatal shelf eleva-
tion H yaluronan CD 44 TG F-
IN TRO D U CTIO N
Palatogenesis is a com plex event and is often
disturbed to produce the congenital defect
know n as cleft palate. Consequently, the events
and m echanism s responsible for the develop-
m ent of the palate have been m uch studied,
although som e controversy rem ains.
The definitive palate (or secondary palate)
develops in the hum an fetus betw een the sixth
and eighth w eek of intra-uterine life (e.g. Fergu-
son, 1978a; Johnston and Sulik, 1990; Sadler,
2000; Berkovitz et al., 2002). By the sixth w eek
of developm ent (Fig. 1), the prim itive nasal cav-
ities are separated by a prim ary nasal septum
53
The devel opment of t he pal at e a br i ef r evi ew
B.J. Moxham
Cardiff School of Biosciences, Cardiff University, United Kingdom and
The School of Medicine, St. Georges University, Grenada
Correspondence to:
Prof. Bernard J . Moxham. Cardiff School of Biosciences, Cardiff University, Museum
Avenue, Cardiff, CF10 3US, United Kingdom. Telephone: +44 (0)29 20874031; Fax: +44
(0)29 20875964. E-mail: moxham@cardiff.ac.uk
Web URL: www.cardiff.ac.uk/ biosi/
Submitted: March 10, 2003
Accepted: J une 12, 2003
Figure 1. D iagram show ing the state of developm ent of the palate
by the sixth w eek of intra-uterine life. A = prim itive nasal
cavities; B = prim ary nasal septum ; C = prim ary palate.
REVI EW
Eur J Anat, 7 Suppl. 1: 53-74 (2003)
and are partitioned from the prim itive oral cavi-
ty by a prim ary palate. Both the prim ary nasal
septum and prim ary palate are derived from the
frontonasal process of the developing face. The
stom odeal cham ber is divided at this stage into
the sm all prim itive oral cavity beneath the pri-
m ary palate, and the relatively large oronasal
cavity behind the prim ary palate. As show n in
Figure 2, during the sixth w eek of developm ent,
tw o palatal shelves develop laterally behind the
prim ary palate from the m axillary facial process-
es. A secondary nasal septum grow s dow n from
the roof of the stom odeum behind the prim ary
nasal septum , thus dividing the nasal part of the
oronasal cavity into tw o.
Figure 3 show s the developing head during
the seventh w eek of developm ent. At this stage,
the oral part of the oronasal cavity becom es
com pletely filled by the developing tongue.
G row th of the palatal shelves continues such
that they com e to lie vertically. Tw o peaks of
D N A synthesis occur as the palatal shelves are
form ed: during initial shelf outgrow th and during
vertical shelf elongation (Burdett et al., 1988).
The reason for m am m alian shelves form ing w ith
a vertical orientation is unknow n. It has been
suggested that the potential space in the
oronasal cavity is insufficient because of the evo-
lution of a large tongue in m am m als (H ayw ard
and Avery, 1957). H ow ever, Young et al. (1990)
have show n that there is no spatio-tem poral rela-
tionship betw een the developm ent of the tongue
and the palatal shelves.
D uring the eighth w eek of developm ent (Fig.
4), the stom odeum enlarges, the tongue drops
B. J . Moxham
54
Figure 2. D iagram show ing the developm ent of the palate during
the sixth w eek of intra-uterine life. A = lateral palatal
shelves; B = prim ary palate; C = secondary nasal septum .
Figure 3. Coronal section through the developing head during the seventh w eek of developm ent show ing the palatal shelves (A). B = devel-
oping tongue. (M assons trichrom e). x 55.
A A
B
and the vertically-inclined palatal shelves becom e
horizontal. It has been suggested that the descent
of the tongue is related to m andibular grow th
and/or a change in the shape of the tongue (e.g.
H um phrey, 1971; D iew ert, 1974). O n becom ing
horizontal, the palatal shelves contact each other
(and the secondary nasal septum ) in the m idline
to form the definitive or secondary palate. The
shelves contact the prim ary palate anteriorly so
that the oronasal cavity becom es subdivided into
its constituent oral and nasal cavities. Figure 5
show s a coronal section through the developing
oronasal regions follow ing contact of the palatal
shelves and secondary nasal septum . After con-
tact, the m edial edge epithelia of the tw o shelves
fuse to form a m idline epithelial seam (M ES).
Subsequently, this degenerates so that m es-
enchym al continuity is established across the
now intact, and horizontal, secondary palate.
Fusion of the palatal processes is com plete by the
tw elfth w eek of developm ent. Behind the sec-
ondary nasal septum , the palatal shelves fuse to
form the soft palate and uvula.
Concerning the origin of the m esenchym e
w ithin the fetal processes contributing to the
developm ent of the palate, all of the skeletal and
connective tissues that form the face are derived
from neural crest (N C) cells that originate along
the dorsal m argins of the m idbrain and rostral
hindbrain (N oden, 1978; Couly et al., 1992; Knt-
ges and Lum sden, 1996; Le D ouarin and Kalcheim ,
1999). Indeed, Been and Song (1978) have show n
that localized destruction of m idbrain N C inter-
The development of the palate a brief review
55
Figure 5. Coronal section through developing oronasal regions follow ing contact of the palatal shelves (A) and secondary nasal septum (B);
C = m idline epithelial seam ; D = developing bone of m axilla. (M assons trichrom e). x 55.
B
A
D
A
C
Figure 4. D iagram show ing the state of developm ent of the palate
during the eighth w eek of intra-uterine life. A = palatal
shelves; B = prim ary palate.
feres w ith palatal closure. In m am m alian fetuses,
cranial N C cells do not alw ays m igrate before the
neural tube closes (e.g. Tan and M orriss-Kay,
1986). Furtherm ore, recent w ork suggests that
craniofacial developm ent does not depend on N C
pre-program m ing but is controlled by a com plex
com bination of cell and tissue interactions involv-
ing N C plasticity (Trainor and Krum lauf, 2001).
Evidence is also available that suggests that N C
cells can be reprogram m ed and that their fate and
identity depends upon the cellular signals they
receive as they m igrate to their target tissues (e.g.
Schilling et al., 2001). This seem s to occur as a
result of alteration in Hox gene identity. H ow ever,
there is som e w ork indicating that N C cells have
som e identity at their place of origin near the neur-
al tube; although elaboration of their developm ent
is reached via integration and interaction w ith sig-
nals from surrounding tissue environm ents
through w hich they m igrate (G ram m atopoulos et
al., 2000; Pasqualetti et al., 2000).
Recent research on palatogenesis has concen-
trated on tw o m ain events: palatal shelf elevation
and the initial stage of fusion of the shelves.
PALATAL SH ELF ELEVATIO N
Several m echanism s have been proposed to
account for the rapid m ovem ent (Ferguson, 1978;
Brinkley, 1980) of the palatal shelves from the ver-
tical to the horizontal position and the source of
the force(s) responsible for palatal shelf reorienta-
tion/elevation is a m atter of controversy. Tw o cat-
egories of explanations have been provided: either
the forces are extrinsic to the shelves or they are
generated intrinsically by the shelf m esenchym e.
Those extrinsic forces that have been proposed
often relate to m ovem ent of the tongue. For
exam ple, there have been hypotheses that include
dow nw ard m ovem ent of the tongue due to a
m andibular grow th spurt clearing a path for
palatal shelf elevation (e.g. Asling et al., 1960;
D iew ert, 1974), a dow nw ard displacem ent of the
tongue by the nasal septum again clearing a path
for shelf elevation (e.g. Zeiler et al., 1964), and a
low ering of the tongue due to a fetal m outh open-
ing reflex (e.g. H um phrey, 1969, 1971). It has also
been suggested that the tongue physically pushes
the palatal shelves upw ards (e.g. W alker, 1971).
H ow ever, it is now generally thought that the
palatal shelf elevation force is not extrinsic in ori-
gin. Ferguson (1978a) review ed the literature relat-
ing to extrinsic forces and concluded that the
chronology of events extrinsically did not neces-
sarily synchronise w ith shelf elevation. Further-
m ore, follow ing tongue excision during palatoge-
nesis, no spatio-tem poral relationship exists and
aglossia and m icroglossia in hum ans does not pre-
vent palatal closure (Young et al., 1990). In addi-
tion, palatal shelves are seen to elevate in organ
culture in the absence of a tongue or a low er jaw .
It has been proposed that the intrinsic shelf
elevation force m ight develop as a result of
hydration of extracellular m atrix (ECM ) com po-
nents (principally hyaluronan) in the shelf m es-
enchym e (e.g. Larsson et al., 1959; Pratt et al.,
1973; Ferguson, 1978a; Brinkley and M orris-
W im an, 1984, 1987; Singh et al., 1994, 1997), or
as a result of m esenchym al cell activity (e.g.
Shah, 1979, 1980; Innes, 1978; W ee et al., 1979;
Zim m erm an, 1979; Babiarz et al., 1979; Luke,
1984; Bulliet and Zim m erm an, 1985; Brinkley and
Bookstein, 1986; Shah et al., 1989). O f course, the
intrinsic shelf elevating force m ight be m ultifac-
torial, although there is as yet no experim ental
evidence to support w hat otherw ise m ight be
considered a com m onsenseview .
M uch recent w ork has focussed on the
changes occurring in the ECM of the palatal
shelfs m esenchym e during shelf elevation. The
changing am ounts of glycosam inoglycans (G AG )
during developm ent of the anterior (presum ptive
hard) and posterior (presum ptive soft) palates
have been reported by Singh et al. (1994) and
are illustrated in Fig. 6. The findings show that
the m ost significant changes occur after eleva-
B. J . Moxham
56
Figure 6. G raphs illustrating the changing am ounts of glycosam ino-
glycans (G AG ) during developm ent of the anterior (pre-
sum ptive hard) and posterior (presum ptive soft) palates.
Stage A prior to shelf elevation; Stage B after shelf ele-
vation; Stage C during shelf fusion and early histogene-
sis; Stage D a stage of m arked histogenesis after fusion.
Courtesy of D r G .D . Singh and B.J. M oxham and the edi-
tor of Archives of Oral Biology.
tion and that, during the tim e of elevation, there
are no differences betw een the anterior and pos-
terior regions of the shelves even though, in the
species studied here (the rat), the posterior
region of the shelf does not elevate but grow s
initially w ith a horizontal disposition (Colem an,
1965; Cleaton-Jones, 1976a; Singh et al., 1994).
Singh et al. (1997) have also reported that, w hen
palatal clefts are induced in the rat by 5-fluoro-
2-deoxyuridine (FU D R), G AG biosynthesis is
suppressed.
Three types of G AG are found in the devel-
oping palatal shelves in vivo (Singh et al., 1994,
1997): hyaluronan, heparan sulphate and chon-
droitin-4-sulphate (Fig. 7). If palatal shelves are
cultured in vitro, derm atan sulphate is also pre-
sent (e.g. Burkitt, 1990), highlighting the prob-
lem of extrapolating from the findings of tissue
culture to the in vivo situation. Furtherm ore,
there m ay be species differences since, using
early histochem ical techniques, it has been
claim ed that chondroitin-6-sulphate m ay be pre-
sent in m ouse palatal shelves (Larsson, 1962).
M uch attention has been paid to the role of
hyaluronan in shelf elevation. It has been pro-
posed that hyaluronan is a G AG involved in
shelf elevation because it is highly electrostati-
cally charged, it displays non-ideal osm olarity,
and its open coil m olecule is capable of binding
up to 10 tim es its ow n w eight in w ater (e.g. Pratt
et al., 1973; Brinkley and M orris-W im an, 1987).
This view has the support of the w ork of Fore-
m an et al. (1991), w here an increase in w ater
content of the palatal shelves w as observed up
until shelf fusion.
Figure 8 show s a section through a vertical
(pre-elevation) palatal shelf stained using the
hyaluronectin/anti-hyaluronectin technique
(G irard et al., 1986) and show s intense staining
for hyaluronan w ithin the palatal shelf m es-
enchym e. Singh et al. (1994) have investigated
the changing concentrations of hyaluronan w ith-
in the anterior and posterior regions of palatal
shelves (Fig. 9). Statistically, there is significantly
m ore hyaluronan in the shelves im m ediately
before elevation than im m ediately after eleva-
tion. H ow ever, the data do not agree w ith som e
reports that there is less hyaluronan posteriorly
than anteriorly (K nudsen et al., 1985), the pat-
tern of change in hyaluronan again being sim ilar
both anteriorly and posteriorly even though the
posterior region does not undergo elevation to
reach the horizontal (Colem an, 1965; Cleaton-
Jones, 1976a; Singh et al., 1994).
Singh et al. (1994) also reported that,
although heparan sulphate and chondroitin-4-
sulphate are present w ithin the palatal shelves
throughout palatogenesis, these G AG s do not
show detectable changes at the tim e of palatal
shelf elevation.
The development of the palate a brief review
57
Figure 7. D ensiom etric scan of electrophoretogram s show ing the
G AG s w ithin the palatal shelves. H A hyaluronan; H S
heparan sulphate and C
4
S chondroitin 4-sulphate.
Courtesy of D r G .D . Singh and B.J. M oxham and the edi-
tor of Archives of Oral Biology.
Figure 8. Section through a vertical (pre-elevation) palatal shelf (A)
stained using the hyaluronectin/anti-hyaluronectin tech-
nique to dem onstrate the presence of hyaluronan. x 40.
A A
That FU D R is associated w ith the production
of cleft palates (e.g. Singh et al., 1997) has also
been im plicated in supporting the notion that
hyaluronan is im portant in palatogenesis.
Accordingly, Ferguson (1978b) has reported that
FU D Rs interference in D N A synthesis fits w ith its
reported effects on glycoconjugate production in
vitro (e.g. D orfm an et al., 1975).
M ore recent studies (Thom as, 1999; Thom as,
H all and M oxham , unpublished data) have
revealed the presence during palatogenesis of
enzym es associated w ith hyaluronan synthesis, of
a cell surface receptor associated w ith hyaluro-
nan, of the hyaluronan binding ECM com ponents
versican and hyaluronectin, and of hyaluronan
binding sites. Furtherm ore, using an organ culture
system (Figs. 10-13), agents that alter hyaluronan
content or size, that disrupt G AG substitution on
proteoglycans, or that alter the balance of m atrix
m olecules secreted via the G olgi com plex and
hyaluronan produced at the cell surface all affect
palatogenesis. Figure 10 illustrates the effects of
streptom yces hyaluronidase, an enzym e that
specifically degrades hyaluronan. Streptom yces
hyaluronidase treated shelves produced clefts. In
addition, link protein is absent w hilst versican
w as evident in the m esenchym e and CD 44 in the
ectoderm . The results suggest that palate devel-
opm ent is disrupted in the absence of hyaluro-
nan. Figure 11 show s the effects of chlorcyclixine,
a substance that enhances degradation of hyaluro-
nan and chondroitin sulphate to low er the m ole-
cular w eight products, w ith little effect on their
synthesis and no appreciable effect on D N A syn-
thesis. Chlorcyclixine treated shelves exhibit a
cleft. CD 44 and link protein are absent from the
shelves but versican is evident throughout the
m esenchym e. Therefore, the size of the G AG
chain m ay influence palatogenesis. The effects of
U D P-xylose are illustrated in Fig. 12. U D P-xylose
is a natural inhibitor of U D PG D , the enzym e
responsible for the conversion of U D P-glucose to
U D P-glucuronic acid. U D P-xylose treated shelves
exhibited norm al palate developm ent. Link pro-
tein w as again absent, but versican and CD 44
exhibited the sam e distribution as seen in control
cultures. Therefore, inhibition of U D PG D has no
effect on palate developm ent (assum ing that it
w as able to enter the tissue). Figure 13 show s the
effects of Brefeldin A. This inhibits vesicular trans-
port through the G olgi com plex. H yaluronan syn-
thesis is not affected as this G AG undergoes a dif-
ferent synthetic pathw ay to the other G AG s, being
form ed at, or near, the plasm a m em brane by the
hyaluronan synthase/enzym e com plex. Brefeldin
A produced a cleft. W hile versican w as evident
throughout the m esenchym e, link protein and
CD 44 w ere absent. Exposure to BFA at sequential
10h w indow periods suggests that BFA only caus-
es a cleft in the initial 30h. The results indicates
B. J . Moxham
58
Figure 9. G raphs show ing the changing concentrations of hyaluronan w ithin the anterior and posterior regions of palatal shelves. A-D stages
of palatogenesis described in Figure 6. Courtesy of D r G .D . Singh and B.J. M oxham and the editor of Archives of Oral Biology.
The development of the palate a brief review
59
Figure 10. The production of clefts produced during organ culture of rat developing palates follow ing the introduction of streptom yces
hyaluronidase to the culture m edium and show n by scanning electronm icroscopy (A) and light m icroscopy (B). N S = nasal sep-
tum ; PS = unfused palatal shelves. Scale bars: 10A = 500 m ; 10B = 400 m . Courtesy of S.Thom as, R. H all and B.J. M oxham .
A
B
PS
B. J . Moxham
60
Figure 11. The production of clefts produced during organ culture of rat developing palates follow ing the introduction of chlorcyclixine to
the culture m edium and show n by scanning electronm icroscopy (A) and light m icroscopy (B). CB = cranial base; PS = unfused
palatal shelves. (11A = x 50; 11B = x 100). Courtesy of S.Thom as, R. H all and B.J. M oxham .
A
B
The development of the palate a brief review
61
Figure 12. N orm al palatogenesis during organ culture of rat palates follow ing the introduction of U PD -xylose to the culture m edium and
show n by scanning electronm icroscopy (A) and light m icroscopy (B). P = fused palatal shelves. (12A = x 60; 12B = x 95). Cour-
tesy of S.Thom as, R. H all and B.J. M oxham .
A
B
P
B. J . Moxham
62
Figure 13. The production of clefts produced during organ culture of rat developing palates follow ing the introduction of brefeldin A to the
culture m edium and show n by scanning electronm icroscopy (A) and light m icroscopy (B). CB = cranial base; PS = unfused palatal
shelves. (13A = x 50; 13B = x 100). Courtesy of S.Thom as, R. H all and B.J. M oxham .
A
B
PS
that a set of m acrom olecules other than hyaluro-
nan, and synthesised in the G olgi com plex, plays
an im portant role in norm al palate developm ent.
O ther recent studies at our laboratories at
Cardiff (H udson and H all, unpublished data)
have been concerned w ith the expression of
hyaluronan binding protein splice variants of
CD 44, versican and RH AM M and isoform s of
hyaluronan synthases (Has) and hyaluronidases
(Hyal) in the developing rat palate. Expression
of CD 44 (the m ajor hyaluronan binding protein)
w as found to be both transient and dynam ic dur-
ing shelf elevation w ith differential expression of
CD 44 transcripts containing variant exons v1, v2,
v8 and v9. It w as also noted that larger tran-
scripts (containing m ore variant exons) w ere
present after shelf elevation. It can be argued
that expression of distinct Has and Hyal splice
variants is necessary during palatogenesis in
order for correct tissue form ation to occur
because both enzym es produce different sizes of
hyaluronan, thus prom oting distinct cellular
responses depending on cell type (Itano et al.,
1999). Sm all hyaluronan chains can induce gene
expression (M cK ee et al., 1996), cell signalling
responses and cell differentiation (Term eer et al.,
2000), and cell proliferation and grow th (M oha-
patra et al., 1996, Bourguignon et al., 1997),
w hereas large hyaluronan chains at high con-
centrations inhibit cell grow th and induce cell
adhesion and m igration (N oble et al., 1998). Ver-
sican splice variants differ in size and G AG chain
num ber and are thought to form bridges, help-
ing to stabilize the ECM and create the necessary
turgor pressure to enable shelf elevation.
O ther ECM com ponents, including proteogly-
cans, are probably of im portance to shelf eleva-
tion. Versican and decorin (but not biglycan)
have been identified at a range of m olecular
w eights corresponding to various processed
form s. The extent to w hich aggregation and dis-
aggregation of proteoglycans occurs at different
locations of the palatal shelf and at different
stages of palatogenesis is unknow n; although
this could have significant functional im plica-
tions associated w ith shelf elevation. The role of
collagen w ithin the palatal shelves is disputed.
Pratt and K ing (1972) show ed that cleft palates
can result from the adm inistration of lathyrogens
that have specific effects on collagen crosslink
form ation. H assell and O rkin (1976) described
collagen bundles w ith defined orientation next
to the basem ent m em brane of the palatal shelves
and reported that the rate of collagen synthesis
w as greatest just prior to shelf elevation. Indeed,
it has been suggested that these collagen fibres
directthe shelf elevation force (e.g. Bulleit and
Zim m erm an, 1985) and/or contribute to a critical
volum e of the shelves necessary for their re-ori-
entation (Ben-K haial and Sha, 1994). Im m uno-
histochem ically, type 1 collagen can easily be
identified (Fig. 14) (Ferguson, 1988). Indeed,
stout bundles of collagen can be seen running
dow n the centre of the palatal shelf and these
are orientated from the base tow ards the tip of
the shelf.
The development of the palate a brief review
63
Figure 14. Section of a palatal shelf labelled im m unocytochem ically w ith antibodies against type I collagen. A = collagen bundles; B = base
of palatal shelf; C = tip of palatal shelf. x 300. Courtesy of Professor M .W .J. Ferguson.
C
A
B
The role of the m esenchym al cells w ithin the
palatal shelves has also been controversial.
There is evidence that a critical num ber of cells
are required for palatal shelf elevation to occur
(e.g. Shah et al., 1989) but there is no reliable
evidence as yet that these cells, by their rapid
division and proliferation or by their m igration
or contraction, can generate a palatal shelf ele-
vation force (particularly in view of the rapidity
of shelf elevation). The density of palatal shelf
m esenchym al cells appears to change during
palatogenesis (e.g. Brinkley and Bookstein,
1986). This could be the result of variations in
cell num ber and/or of cell redistribution. It w as
once believed that differential rates of cell
m itoses/proliferation m ight produce the shelf
elevation force (Luke, 1984; Bulliet and Zim -
m erm ann, 1985).
3
H -thym idine studies have
show n that there are differential rates of m es-
enchym al cell proliferation (e.g. Cleaton-Jones,
1976b). H ow ever, the differential rates are prob-
ably related to histogenic changes and do not
necessarily account for the generation of the
shelf elevation force. Brinkley and Bookstein
(1986) show ed that shelf re-orientation is
accom panied by changes in m esenchym al cell
density and distribution. They suggested that
high local cell densities w ere enhanced by cell
division but that decreased cell density (w hich
cannot be accounted for by an increase in cell
size) w as probably related to displacem ent of
cells by expansion of the ECM . Ferguson
(1978a) also noted the closely packed nature of
m esenchym al cells before elevation and com -
m ented upon the greater cell density w ithin the
posterior region of the developing palate (a
region w hich is the last to fuse).
In addition to m esenchym al cell proliferation,
the production of a shelf elevation force m ight
also be related to changes, at the critical tim e, in
cellular m orphology (e.g. Brinkley and Book-
stein, 1986) and in particular to changes in the
intracellular m icrofilam entous and m icrofibrillar
system s (e.g. K uhn et al., 1980). Babiarz et al.
(1979) reported that palatal shelf m esenchym al
cells before elevation w ere elongated and polar-
ized, the cells nearest the basem ent m em brane
being perpendicularly aligned to the m em brane.
After shelf elevation, the cells becam e m ore
rounded w ith short cellular projections. Babiarz
et al. (1979) considered that these changes w ere
indicative of cell contraction and that this could
be the m eans of generating the shelf elevation
force. Innes (1978) and Shah (1979, 1980)
reported that shelf m esenchym al cells possess
contractile, m icrofilam ents. In addition, contrac-
tile proteins have been isolated from palatal
shelf m esenchym al cells, leading to the claim
that actin- and m yosin-like system sm ay be
involved in shelf elevation (Babiarz, Allenspach
and Zim m erm an, 1975). Indeed, Babiarz et al.
(1979) reported on the presence of m icrofila-
m ents containing actinom yosin and suggested
that these w ere associated w ith cell m igration
that could be responsible for shelf elevation.
W ee and Zim m erm ann (1980) reported that
cytochalasin B inhibits palate shelf elevation by
disrupting actin crosslinking in the cytoskeleton.
H ow ever, they also found that curare (a m icro-
filam ent antagonist) enhanced shelf elevation in
vitro, thus providing evidence against the notion
that m icrofilam entous system s are prim arily
responsible for shelf re-orientation. Further-
m ore, it is not clear w hether changes in the
palatal shelf m esenchym al cells are prim arily
associated w ith the re-orientation m echanism or
w hether they are the effect of cell displace-
m ents/cell activities caused by changes in the
ECM during the period of shelf re-orientation
(e.g. Pratt et al., 1973).
There have been m any qualitative electron-
m icroscopic investigations of the palatal shelf
m esenchym al cells (e.g. D e Angelis and N alban-
dian, 1968; Babiarz et al., 1975; Innes, 1978,
1981, 1985; Ferguson, 1981a). Essentially, these
studies show that the m esenchym al cells appear
to be very active, possessing m any m itochon-
dria, abundant cisternae of endoplasm ic reticu-
lum , a w ell-developed G olgi com plex, and large
num bers of glycogen particles (organelles appro-
priate for cells actively synthesising and secret-
ing ECM proteins and entirely consistent w ith the
view that the gradual accum ulation of G AG is
correlated w ith the synthesizing organelles of the
m esenchym al cells). Shah (1979) described ultra-
structural changes occurring during norm al
palatogenesis, noting that the cells elongated
after shelf elevation. Lieb and D e Paola (1981)
found that the m esenchym e w as tightly packed
w ith polygonal cells possessing centrally placed
ovoid nuclei w ith prom inent nucleoli. They also
reported that there w as a large com plem ent of
free ribosom es and polysom es and very little
intercellular space. Recently, it has been report-
ed that filopodia-like structures appear on the
surface of palatal shelf cells at the tim e of fusion
(Taya et al., 1999). Sim ilar events occur during
developm ent of the interm axillary segm ent
w hen the facial processes fuse (Sym ons and
M oxham , 2002). D espite these m any studies, to
date there have been rem arkably few quantita-
tive electronm icroscopic studies. Brinkley and
Bookstein (1986) have undertaken som e quanti-
tative studies on the developm ent of the m ouse
secondary palate. The purpose of their investi-
gation w as to determ ine differences in cell den-
sity at various stages of palatogenesis in vitro
and consequently, w ith the exception of the
nuclei, they did not m easure the organelles w ith-
in the m esenchym al cells.
It is obvious that, w hether or not the palatal
shelf m esenchym al cells are involved in the
B. J . Moxham
64
generation of the shelf elevation force, the cells
have to m aintain (and control) events taking
place in the shelf ECM . Indeed, using special
silver staining techniques to highlight nucleolar
organiser regions (N O Rs), the degree of protein
synthesising activity of the m esenchym al cells
in the palatal shelf at different stages of palato-
genesis has been assessed (Singh and M oxham ,
1993) (Fig. 15). The num ber and configuration
of grainsw ithin the N O Rs reflect the overall
degree of protein synthesis by the cells. This
staining procedure confirm ed that the rate of
protein synthesis during palatogenesis is high,
is higher before elevation than after elevation,
and is higher still during later stages of histoge-
nesis. These results accord w ith the changes
occurring in G AG synthesis at various stages of
palatogenesis. The AG -N O R staining technique
further show s that protein synthesis is severely
depressed during cleft form ation, but the tech-
nique is unable to dem onstrate m ajor differ-
ences betw een anterior and posterior regions.
A lthough hyaluronan in the palatal shelves
is m ost often associated w ith the developm ent
of a turgor pressure for shelf elevation via
attraction of w ater m olecules, this G A G also
influences cellular activity. For exam ple,
hyaluronan produces large intercellular spaces
during early palatogenesis to prevent cell-cell
and cell-m atrix interactions, allow ing assem bly
of ECM constituents and presentation of grow th
factors that in turn influence cell grow th and
differentiation by altering the local concentra-
tion of intercellular signals (Toole, 2000). Fol-
low ing shelf elevation, there is a decline in
hyaluronan shelf content (Singh et al., 1994),
probably via CD 44 receptor-m ediated endocy-
tosis of hyaluronan and hyaluronidases that
produce shorter hyaluronan chains. This
enables the onset of palatal tissue differentia-
tion. H yaluronan that is taken up into cells can
bind to intracellular hyaluronan binding pro-
teins, including som e RH A M M splice variants.
Such binding induces cell signalling pathw ays
that can, in turn, induce changes in the
cytoskeleton. D uring differentiation, the inter-
cellular m atrix becom es m ore dense w here
hyaluronan is replaced by proteoglycans, but
the rem aining hyaluronan binds to such pro-
teoglycans (including hyaluronan binding pro-
teins such as versican, cell surface RH A M M and
CD 44) to form a stable ECM .
Finally, although the production of cleft
palates follow ing the adm inistration of FU D R is
thought to be related to interference in ECM gly-
coconjugate production (e.g. D orfm an et al.,
1975; Ferguson, 1978b; Singh et al., 1997), alter-
native explanations are possible in term s of cell
activity w ithin the palatal shelves. Indeed,
Am w ayi and Luke (1990) reported that FU D R
produces a decrease in m esenchym al cell prolif-
eration.
The development of the palate a brief review
65
Figure 15. Silver staining of the palatal shelves (A) to assess the degree of activity of the m esenchym al cells. The black silver grains in the m es-
enchym al cell nuclei highlight N ucleolar O rganiser Regions (N O Rs). Silver stain. x 500. Courtesy of D r. G .D . Singh and B.J. M oxham .
A
FU SIO N O F TH E PALATAL SH ELVES
O nce the palatal shelves have elevated, they
contact each other (initially in the m iddle third of
the palate; Ferguson 1988) and adhere by m eans
of an adhesiveglycoprotein that coats the sur-
face of the m edial edge epithelia of the shelves
(G reene and K ochhar, 1974; Pratt and H assell,
1975; Souchon, 1975; G reene and Pratt, 1977).
Additionally, the epithelial cells develop desm o-
som es (D e Angelis and N albandian, 1968; M or-
gan and Pratt, 1977) and consequently an epithe-
lial seam is form ed (M organ and Pratt, 1977;
Ferguson, 1988) (see Fig. 5). The adherence of
the m edial edge epithelia is specific as palatal
epithelia w ill not fuse w ith epithelia from other
sites (e.g. the tongue) (Ferguson et al., 1984).
This m ay be related to the fact that the proteins
associated w ith the form ation of desm osom es
(i.e. desm oplakin) appear specifically on the cell
m em branes of the m edial edge epithelia just
prior to shelf contact (Ferguson, 1988). An intact
basal lam ina lies on either side of the epithelial
seam .
The signals that are responsible for the break-
dow n of the m idline epithelial seam (M ES) are not
yet fully understood. N evertheless, the breakdow n
of the basal lam ina is likely to be a significant
event (e.g. Ferguson, 1988). Figure 16 dem on-
strates the fusing palatal shelves im m unocyto-
chem ically stained w ith antibodies against type IV
collagen found in basal lam ina (Ferguson, 1988;
Fyfe and Ferguson, 1988). At this early stage of
fusion, the basal lam ina rem ains intact. At a later
stage of fusion (Fig. 17), w ith m igration of the
epithelial cells into the m esenchym e, the M ES is
disrupted and the m igrating cells initially carry
w ith them fragm ents of the disrupted basal lam i-
na (Fyfe and Ferguson, 1988). Fibrils com prising
tenascin and type III collagen have been show n to
run at right angles to the basal lam ina and m ay
provide guiding pathw ays for the m igrating
epithelial cells (Fyfe and Ferguson, 1988; Fyfe et
al., 1988). Evidence indicates that the events lead-
ing to the breakdow n of the M ES occur in single
isolated palatal shelves and therefore do not
depend upon shelf contact (Ferguson et al., 1984;
Ferguson, 1988).
Alm ost as soon as the M ES is form ed, it thins
to a layer tw o or three cells thick (M ato et al.,
1966; Ferguson, 1988). This thinning m ay be the
result of three processes. First, the M ES is
thinned by grow th of the palate (in term s of
oronasal height) and by epithelial cell m igration
from the region of the M ES onto the oral and
nasal aspects of the palate (e.g. Fyfe and Fergu-
son, 1988). Second, there is program m ed cell
death (apoptosis) in the M ES. For exam ple, by
using the TU N EL technique, and by assessing the
presence of m acrophages, M artinez-Alvarez et al.
(2000) have show n that M ES cells die in the
developing m ouse palate at the tim e of fusion.
Program m ed cell death is also suggested by the
finding that D N A synthesis ceases in the m edial
B. J . Moxham
66
Figure 16. Fusing palatal shelves (A) im m unocytochem ically labelled w ith antibodies against type IV collagen found in basal lam ina
(arrow ed). x 120. Courtesy of Professor M .W .J. Ferguson.
A
A
edge epithelial cells one day prior to shelf con-
tact (H udson and Shapiro, 1973). Furtherm ore,
cyclic AM P increases just before shelf fusion (e.g.
Ferguson, 1987) and exogenous cyclic AM P is
associated w ith precocious cell death in the
m edial edge epithelia (Pratt and M artin, 1975). It
has also been show n that epiderm al grow th fac-
tor (EG F) inhibits m edial edge cell death (H as-
sell, 1975; Pratt et al., 1984; Pratt, 1984) and that
this inhibition is blocked by exogenous cyclic
AM P (H assell and Pratt, 1977). Care m ust be
taken, how ever, w hen interpreting the effects of
cyclic AM P since physiologically it is an intracel-
lular m essenger and m ay therefore be m ediating
differential gene expression triggered by other
events occurring at the cell surface. M artinez-
Alvarez et al. (2000) also suggested that TG F-
3
is an inducer of apoptosis during palatal fusion.
Third, there is good evidence that som e of the
epithelial cells m igrate from the M ES into the
palatal shelf m esenchym e and differentiate into
cells indistinguishable from the m esenchym al
cells (e.g. Ferguson, 1988). Indeed, it is w ell
know n that epithelial cells can m igrate and dif-
ferentiate into m esenchym al-like cells in other
circum stances during developm ent. Although
labelling of M ES cells w ith vital lipophilic m ark-
ers has not clarified w hether such cells m igrate
and/or transform into m esenchym e, in vitro
studies that involve infecting the cells w ith the
replication-defective helper-free retroviral vector
CXL carrying the Escherichia coli lacZ gene (thus
enabling analysis of -galactosidase activity in
the cells and the determ ination of cell fate) indi-
cate that the cells of the M ES transform into m es-
enchym e during palatal fusion (M artinez-Alvarez
et al., 2000).
There have been m any experim ents to help
clarify the nature of the epithelial-m esenchym al
interactions during fusion of the palatal shelves.
In the m ain, these experim ents have involved
the separation and then the recom bination in
culture of the epithelial and m esenchym al com -
ponents of the shelves. O verall, these experi-
m ents have show n that, as w ith epithelial-m es-
enchym al interactions for tooth developm ent, it
is the m esenchym e that signals epithelial differ-
entiation and behaviour (e.g. Ferguson and
H onig, 1984). The nature of this signal is con-
troversial. Figure 18 show s the m edial edge
The development of the palate a brief review
67
Figure 17. Late stage of fusion of the palatal shelves im m unocy-
tochem ically labelled for type IV collagen and show -
ing disruption of the m idline epithelial seam . x 250.
Courtesy of Professor M .W .J. Ferguson.
Figure 18. The m edial edge epithelia of palatal shelves failing to
label im m unocytochem ically for type IX collagen
before shelf elevation. A = palatal shelves; B = epithe-
lium covering floor of the m outh. x 560. Courtesy of
Professor M .W .J. Ferguson.
A
B
epithelia of palatal shelves labelled im m unocy-
tochem ically for type IX collagen before shelf
elevation (Fyfe and Ferguson, 1988). Although it
w as once proposed that the palatal m esenchym e
could signal epithelial differentiation directly by
cell-to-cell contact, m esenchym al-epithelial cell
contacts are very rare during palatogenesis (Fer-
guson, 1988). It seem s that ECM m olecules m ay
provide the signal and w ork has been undertak-
en to assess the role of type IX collagen (Fergu-
son, 1988). Figure 18 show s that, at the earliest
stages before shelf elevation, the m edial edges
of the palatal shelves label poorly for type IX
collagen com pared w ith floor of the m outh
epithelia. Figure 19 show s the m edial edge
epithelia of palatal shelves labelled im m unocy-
tochem ically for type IX collagen at a tim e w hen
m edial edge epithelial differentiation occurs as
determ ined by recom bination experim ents. At
this stage, type IX collagen appears around the
surfaces of the m edial edge epithelial cells. It is
believed that the control of the synthesis of type
IX collagen is influenced by grow th factors (Fer-
guson, 1988).
Ferguson (1988), using im m unocytochem ical
labelling w ith antibodies against epiderm al
grow th factor receptors, has dem onstrated the
presence of such receptors on the m esenchym al
cells adjacent to the M ES of fusing palatal shelves.
Epiderm al grow th factor (EG F), or its em bryonic
hom ologue know n as transform ing grow th factor
(TG F- ), is know n to inhibit palatal m edial
edge epithelial cell death in the presence of m es-
enchym e (Tyler and Pratt, 1980). Furtherm ore, it
has been show n that the synthesis of ECM m ole-
cules (including type IX collagen) is stim ulated by
factors such as TG F and TG F and is inhibit-
ed by fibroblast grow th factors (FG F) (Sharpe and
Ferguson, 1988; Ferguson, 1988; Sharpe et al.,
1993). W hen palatal shelves are organ-cultured
w ith EG F, the m edial edge of the palatal shelf
show s a nipple-like bulge, m edial edge epithelial
cell death is absent, and the m esenchym e pos-
sesses increased quantities of ECM m olecules (Jel-
nick and D ostal, 1974; N anda and Rom eo, 1975;
Cleaton-Jones, 1976a; Ferguson, 1988). It has
been proposed, therefore, that the palatal shelf
m esenchym e produces grow th factors that either
directly signal epithelial differentiation or, by
stim ulating ECM production, indirectly influence
differentiation through this m atrix. Ferguson
(1988) has suggested that EG F receptors show
regional heterogeneity and that the receptors only
appear beneath the m edial edge of the shelves
w hen the epithelial seam is degenerating.
H yaluronan is also critical during breakdow n
of the M ES, providing a suitable m atrix for som e
of these cells to undergo epithelial-m esenchym al
transform ation in order to subsequently m igrate
to the oral and nasal aspects of the palate.
H yaluronan produced by Has 2 is vital for
epithelial-m esenchym al transform ation during
heart m orphogenesis (Cam enisch et al., 2000)
and is proposed to function in a sim ilar m anner
in the developing palate.
Recent studies have highlighted the im por-
tance of TG F during fusion of the palatal
shelves. TG F-
1
,
2
and
3
expression during m ouse
palatogenesis has been extensively studied (both
tem porally and spatially) and results suggest that
TG F-s act as regulators at palatal shelf fusion.
Im m ediately before palatal fusion, TG F-
3
expres-
sion is localised in the m edial edge epithelium
(Pelton et al., 1990: Fitzpatrick et al., 1990). Short-
ly afterw ards, TG F-
1
expression is also detected
in the m edial edge epithelium but TG F-
2
expres-
sion can only be seen in the m esenchym al cells.
Furtherm ore, for TG F-
3
knockout m ice, the TG F-

3
-null m utant fetuses develop cleft palate so that
all TG F-
3
-null pups die shortly after birth (Proetzel
et al., 1995). The TG F-
3
knockout m ouse is char-
acterised by appearing to have no other m orpho-
logical anom alies (excepting the lung). TG F-
1
knockout m ice, how ever, do not develop cleft
palate (Shull et al., 1992; Kulkarni et al., 1993) and
B. J . Moxham
68
Figure 19. The m edial edge epithelia of palatal shelves labelled
im m unocytochem ically for type IX collagen at a tim e
w hen m edial edge epithelial differentiation occurs as
determ ined by recom bination experim ents. x 500.
Courtesy of Professor M .W .J. Ferguson.
cleft palate, along w ith m any other types of
abnorm alities, is observed (but at low er incidence
rates) in TG F-
2
knockout m ice (Sanford et al.,
1997). That TG F-
3
plays an im portant role in
palatal shelf fusion is also show n by the fact that
palate fusion fails to occur in vitrow hen the activ-
ity of TG F-
3
is inhibited by antisense oligonu-
cleotide or by neutralising antibody (Brunet et al.,
1995). M ore recently, Taya et al. (1999) reported
that m utation of the TG F-
3
gene results in cleft
palate form ation and that, w hen palates from
transgenic m ice w ith TG F-
3
deletions are grow n
in organ culture such that shelves w ere placed in
hom ologous (+/+ vs +/+, -/- vs -/-, +/- vs +/-) or
heterologous (+/+ vs -/-, +/- vs -/-, +/+ vs +/-)
paired com binations, pairs of -/- and -/- shelves
failed to fuse w hile pairs of +/+ and =/+ shelves
show ed com plete disappearance of the M ES
w hereas -/- and +/+ shelves retained som e rem -
nants of the M ES. They also studied the ability of
TG F-
3
fam ily m em bers to rescue the fusion
betw een -/- and -/- palatal shelves in vitro by
adding to the culture m edium recom binant hum an
TG F-
1
, porcine TG F-
2
, recom binant hum an
TG F-
3
, recom binant hum an activin, or porcine
inhibin. It w as reported that, for untreated organ
culture -/- palate pairs that w ould be expected to
show com plete failure to fuse, TG F-
3
treatm ent
induced com plete palatal fusion w hereas TG F-
1
or TG F-
2
produced near norm al fusion and
activin and inhibin had no effect. The m echanism
w hereby TG F-
3
rescued the fusion w as claim ed
to be related to the appearance of filopodia-like
process on the surface of the M ES cells that are
coated w ith m aterial resem bling proteoglycan.
O nce fusion is com plete, the hard palate ossi-
fies intram em branously from four centres of ossi-
fication, one in each developing m axilla and one
in each developing palatine bone (Sperber, 2001;
Berkovitz et al., 2002; M eikle, 2002). The m axil-
lary ossification centre lies above the developing
deciduous canine tooth germ and appears in the
eighth w eek of developm ent. The palatine centres
of ossification are situated in the region form ing
the future perpendicular plate and appear in the
eighth w eek of developm ent. Incom plete ossifica-
tion of the palate from these centres defines the
m edian and transverse palatine sutures. There
does not appear to be a separate centre of ossifi-
cation for the prim ary palate in M an (in other
species there being a separate prem axilla). Fig-
ure 20 provides a coronal section through the
developing hard palate to show early ossification.
CLIN ICAL CO N SID ERATIO N S
M alform ations of palatogenesis m ay result in the
appearance of clefts (Sperber, 2001; Berkovitz et
The development of the palate a brief review
69
Figure 20. Coronal section through the developing hard palate show ing early ossification. A = developing body of m axilla; B = bone extend-
ing from body of m axilla into palate; C = nasal cavity. (M assons trichrom e). x 160.
C
A
B
al., 2002; M eikle, 2002). Clefts of the palate, like
those of the lip, are m ultifactorial m alform ations,
involving both genetic (polygenic) and environ-
m ental factors. Clefts m ay result from distur-
bances of any of the processes involved during
palatogenesis, i.e. from defective palatal shelf
grow th (e.g. Abbott et al., 1990); delayed shelf
elevation or failure of elevation (e.g. Ferguson,
1981a); defective shelf fusion or lack of degen-
eration of the M ES; or failure of m esenchym al
consolidation and/or differentiation (e.g. Abbott
and Birnbaum , 1989).
The m ildest form of cleft is that affecting the
uvula, such a disturbance occurring relatively late
in the process of palatal m alfusion. D isturbances
occurring during the early phases of palatal
fusion can result in a m ore extensive cleft involv-
ing m ost of the secondary palate. Should the cleft
involve the prim ary palate, it m ay extend to the
right and/or left of the incisive foram en to
include the alveolus, passing betw een the lateral
incisor and canine teeth. Cleft palate m ay be
associated w ith cleft lip, though the tw o con-
ditions are independently determ ined. D ental
m alform ations are com m only associated w ith a
cleft involving the alveolus. A subm ucous cleft
describes a condition w here the palatal m ucosa is
intact, but the bone/m usculature of the palate is
deficient beneath the m ucosa. Less problem atic
than clefts (but m ore com m on) is the retention of
epithelial rem nants in the m idline that eventually
becom e cystic.
H ypotheses to explain the m echanism s
responsible for cleft palate form ation range from
genetic predisposition (e.g. Bonner and Slavkin,
1975) to the adm inistration of teratogens (e.g.
Fraser and Fainstat, 1951). Ferguson (1981b) has
also proposed that the expression of a cleft
palate is a m anifestation of phylogeny - birds
develop a physiological cleft and the oral and
nasal cavities are not separated (e.g. Shah and
Craw ford, 1980). Recent research indicates that
retinoids in excess have a teratogenic effect, pro-
ducing clefts of the palate and the abnorm al
appearance of islandsof cartilage in the m es-
enchym e (Em m anouil-N ikoloussi et al., 1999;
Em m anouil-N ikoloussi et al., 2000). M ore recent-
ly, w ork by G unston, M oxham and Em m anouil-
N ikoloussi (unpublished data) show s that all-
trans retinoic acid (RA) is the m ost teratogenic
isom er of RA in term s of rat palatal abnorm alities,
that the tim e of adm inistration of RA is m ore crit-
ical than dose, but that im m unohistochem ical
labelling for cartilage ECM m olecules fails to
detect ectopic cartilage w ithin the palates.
Ectopic localization of Sonic hedgehog protein
(Shh) in the developing rostral neural tube is also
associated w ith craniofacial defects (N asrallah
and G olden, 2001). This is thought to be due to
disruption in norm al genes expression patterns
(e.g. wnt-3a, wnt-4, Pax-6, HNF-3( and Ptc).
Studies using transgenic m ice suggest that
m any hom eobox genes and transcription factors
are involved in palatogenesis. For exam ple,
Satokata and M aas (1994) have highlighted the
possible significance of Msx1 and W inograd et
al. (1997) of Msx2. Tissier-Seta et al. (1995) have
suggested a role for Barx 1. G endron-M aguire et
al. (1993) and Rijli et al. (1993) suggest that
H oxa2 is im portant and M artin et al. (1995) have
im plicated Mhox. Peters et al. (1997) have deter-
m ined a role for Pax9 and M o et al. (1997) have
reported on the significance of Dli and Dli3.
Finally, Takihara et al. (1997) suggest that there
is expression of rae28 during palatogenesis and
Takagi et al. (1998) deltaEF1. Additionally, m any
cytokines (and their receptors) are also involved
in palate developm ent. For exam ple, TG F-
alpha/EG F receptors, TG F-
2
, and TG F-
3
have
im portant functions (M iettinen et al., 1999; San-
ford et al., 1997; Proetzel et al., 1995; K aartinen
et al., 1995; see also above). Furtherm ore, im por-
tance has also been claim ed for activin-A,
activin-receptor type II and follistatin (M atzuk et
al., 1995a, b, c). Lohnes et al. (1993, 1994) have
show n a role for retinoic acid receptor gam m a
during palate developm ent and K urihara et al.
(1994) have reported on endothelin. O rioli et al.
(1996) have suggested an involvem ent of
sek4/nuk1.
ACK N O W LED G EM EN TS
I w ould like to thank all m y colleagues w ho, at
the U niversities of Bristol, Cardiff and Thessa-
loniki, have collaborated w ith m e in the study of
palatogenesis, nam ely: D r. G .D . Singh, D r. W .
M cLean, D r. R. H all, D r. S. Thom as, M s L. H ud-
son, M s E. G unston, Prof. G . Em bery, D r. R.J.
W addington, D r. M .S. Langley, D r. E-N .
Em m anouil-N ikoloussi. M any thanks for your
hard w ork and inspiration. I w ould also like to
thank St. G eorges U niversity (G renada), and
especially Prof. R. Jordan, for providing m e w ith
facilities necessary to com plete this paper.
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