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41
Development 103 Supplement, 41-60 (1988)
Printed in Great Britain © The Company of Biologists Limited 1988
Palate development
MARK W. J. FERGUSON
Animal & Human Reproduction, Development and Growth Research Group, Department of Cell & Structural Biology, University of
Manchester, Coupland 3 Building, Manchester M13 9PL, UK
Summary
In all vertebrates, the secondary palate arises as
bilateral outgrowths from the maxillary processes. In
birds and most reptiles, these palatal shelves grow
initially horizontally, but do not fuse with each other
resulting in physiological cleft palate. In crocodilians,
shelf fusion occurs resulting in an intact secondary
palate. Mammalian palatal shelves initially grow vertically down the side of the tongue, but elevate at a
precise time to a horizontal position above the dorsum
of the tongue and fuse with each other to form an
intact palate. Palatal shelf-elevation is the result of an
intrinsic shelf elevating force, chiefly generated by the
progressive accumulation and hydration of hyaluronic
acid. In all vertebrates the nasal epithelium differentiates into pseudostratified ciliated columnar cells and
the oral epithelia differentiates into stratified
squamous cells, but the medial edge epithelial (MEE)
phenotype differs in different groups. In mammals,
the MEE of opposing shelves adhere to each other to
form an epithelial seam which then disrupts by cell
death and cell migration into the mesenchyme accompanied by an epitheliomesenchymal transformation. In birds, the MEE keratinize resulting in cleft
palate whereas, in alligators, the MEE migrate onto
the nasal aspect of the palate. In all vertebrates, this
regional, temporal and species-specific epithelial dif-
ferentiation is specified by the underlying mesenchyme. Signalling of this interaction is complex but
involves both extracellular matrix and soluble factors
e.g. minor collagen types, tenascin, EGF, TGFa-,
TGF£, PDGF, FGF. These soluble growth factors
have a biphasic effect: directly on the epithelia and on
the mesenchyme where they stimulate or inhibit cell
division and synthesis of specific extracellular matrix
molecules. The extracellular matrix molecules (and
bound growth factors) synthesized by the mesenchymal cells may then directly affect the epithelium.
These signals cause differential gene expression via
second messenger systems e.g. cAMP, cGMP, Ca 2 + ,
pH, pi etc. Molecular markers for nasal, medial and
oral epithelial cell differentiation include the types of
cytokeratin intermediate filaments and specific cell
surface molecules recognized by monoclonal antibodies: the genes for such molecules are probably
expressed in response to mesenchymal signals. Using
such an approach, it is possible to go from a morphological description of palate development to a cellular
analysis of the mechanisms involved and then to
identification of candidate genes that may be important for screening and diagnosis of cleft palate.
Introduction
investigating fundamental mechanisms common to
the embryogenesis of many structures as it appears
relatively late in embryogenesis, can be easily excised
and cultured under chemically defined, serum-free
conditions (Ferguson, Honig & Slavkin, 1984) and
exhibits morphogenetic movements (Ferguson,
1978), extracellular matrix synthesis (Brinkley &
Morris Wiman, 1984, 1987; Pratt & King, 1971;
Silver, Foidart & Pratt, 1981), neurotransmitter synthesis (Zimmerman & Wee, 1984), cell adhesion
Development of the mammalian secondary palate is a
complex and critical event, which, in man, is frequently disturbed resulting in the common and distressing birth defect of cleft palate. Thus palate
embryogenesis has been the target of much research
with the long-term aim of rational prevention or
improved treatment for cleft palate. Moreover, the
developing palate is often used as a model system for
Key words: palate, extracellular matrix,
epitheliomesenchyme interaction, growth factor.
42
M. W. J. Ferguson
(Greene & Pratt, 1977), epithelial-mesenchymal interactions and regional patterning (Ferguson &
Honig, 1984).
Several reviews summarize the literature on palate
development (Greene & Pratt, 1976; Ferguson, 1978;
Shah, 1979; Pratt & Christiansen, 1980; Melnick,
Bixler & Shields, 1980; Zimmerman, 1984; Pratt,
1984; Pisano & Greene, 1986; Ferguson, 1987).
Rather than re-review the extensive literature on
palate development in this paper, I first provide an
overview of secondary palate development in vertebrates and then focus on important developmental
questions, particularly in relationship to recent work
from our laboratory on the mechanism of regional
specification of palatal epithelial differentiation by
the underlying mesenchyme.
Overview
Mesenchymal cells from the neural crest migrate to
the primitive oral cavity where in association with
craniopharyngeal ectoderm they form the bilateral
maxillary processes. Bilateral palatal shelves arise
from these maxillary processes at embryonic day 12 in
mice, day 6 in chickens, day 17 in alligators and day 45
in man. In mammals (both eutherian and protherian),
these bilateral palatal shelves at first grow vertically
down the sides of the tongue (Fig. 1), but at a precise
developmental stage they rapidly elevate to a horizontal position above the dorsum of the tongue
(Fig. 2). The medial edge epithelia of the approximating palatal shelves then fuse with each other to
form a midline epithelial seam (Fig. 2), which rapidly
degenerates, so establishing mesenchyme continuity
across the intact horizontal palate. At approximately
the same time as the midline epithelial cells die, the
epithelia on the nasal aspect of the palate differentiate into pseudostratified ciliated columnar cells,
whilst those on the oral aspect of the palate become
stratified squamous, nonkeratinizing cells. Osteogenic blastemata for the palatal processes of the
maxillary and palatine bones differentiate in the
mesenchyme of the hard palate (and include secondary cartilages in the midline region of the palatal
suture) whilst several patterned myogenic blastemata
develop in the soft palate. Cleft palate may result
from disturbances at any stage of palate development: defective palatal shelf growth, delayed or failed
shelf elevation, defective shelf fusion, failure of
medial edge cell death, postfusion rupture and failure
of mesenchymal consolidation and differentiation
(Ferguson, 1987).
In birds, the bilateral palatal shelves arise from the
maxillary processes, but instead of growing vertically
(as in mammals) they initially develop and grow
horizontally above the dorsum of the tongue (Shah &
Crawford, 1980; Koch & Smiley, 1981; Ferguson etal.
1984; Ferguson & Honig, 1985; Shah, Cheng, Suen &
Wong, 1985; Shah, Cheng, MacKay & Wong, 1987).
The horizontal avian shelves approximate and contact each other, but their medial edge epithelia
neither adhere, fuse nor die, but rather keratinize so
that birds have a naturally cleft palate (Koch &
Smiley, 1981; Ferguson el al. 1984; Ferguson &
Honig, 1984, 1985; Shah etal. 1985, 1987).
In amphibians and some reptiles e.g. certain
species of turtles and snakes, the roof of the mouth is
largely formed by a posterior growth of the primary
palate which is of frontonasal process origin (Voeltzkow, 1903; Fuchs, 1907; Fleischmann, 1910). In other
reptiles e.g. lizards, secondary palatal shelves arise
from the maxillary processes, grow horizontally
above the dorsum of the tongue, either never contact
each other, resulting in a large choanal groove in the
palate, or else contact each other but their medial
edge epithelia do not adhere, fuse or die but keratinize resulting in natural cleft palate (Goppert, 1903;
Hofmann, 1905; Sippel, 1907; Fuchs, 1908; Fleischmann, 1910). The general developmental scenario is
analogous to that in birds.
One group of reptiles; the crocodilians, however,
have an intact, fused mammal-like secondary palate
(Ferguson, \9%\a,b, 1984, 1985). In alligators and
crocodiles, bilateral palatal shelves arise from the
maxillary processes and in the anterior four fifths of
the palate, grow horizontally above the dorsum of the
tongue. In the posterior one fifth of the palate, the
shelves grow vertically and remodel into a horizontal
position later in development, except in their posterior extremities where they fuse with the floor of the
mouth to form the basihyal valve (Ferguson, 1981a,
1984, 1985). Upon contact, however, the medial edge
epithelia of alligator palatal shelves show a very
restricted region (near the oral edge) of adherence,
fusion and cell death. Instead the shelves establish
mesenchymal continuity largely by a merging process
with mesenchymal infilling and migration of the
medial edge epithelia onto the nasal aspect of the
palate (Ferguson, 1981a, 1984, 1985). A fused epithelial seam with medial edge cell death is therefore
not a feature of alligator secondary palate development.
Secondary palate development appears to be absent in lower vertebrates including fish. Phylogenetically, therefore, cleft palate is ancestoral to intact
palate, initial vertical growth of the palatal shelves is
peculiar to mammals, keratinization of the medial
edge palatal epithelia (and consequently cleft palate)
occurs in birds and some reptiles, limited medial edge
cell adhesion, fusion and death, but massive cell
migration (and intact palate) occurs in alligators and
Palate development
crocodiles, whilst medial edge epithelial cell adherence, fusion to form a seam and death (and intact
palate) occurs in mammals. These species-specific
morphogenetic and differentiative events also occur
under identical chemically defined, serum-free culture conditions (Ferguson et al. 1984) and can be
experimentally exploited to investigate fundamental
mechanisms in palate development (Ferguson &
Honig, 1984, 1985).
Why do mammalian palatal shelves develop
initially in a vertical direction?
There has been almost no research on the initial
phases of palatal shelf outgrowth from the maxillary
processes, despite the fact that this is the developmental time at which most drugs are administered to
experimentally induce cleft palate (Salomon & Pratt,
1979). Recently it has been shown that there are two
peaks of DNA synthesis around the time of initial
palate development, one corresponding with initial
shelf outgrowth and the other with elongation of the
shelf in a vertical direction (Burdett, Waterfield &
Shah, 1988). Why mammalian palatal shelves initially
grow vertically and subsequently elevate, as opposed
to growing horizontally ab initio, as in birds and
reptiles, is unresolved. Arguments relating to the
volume of potential space in the primitive oronasal
cavity with the evolution of the large muscular
mammalian tongue and cheeks have been advanced
(Shah, 1977; Ferguson, 1981) but remain intellectually unsatisfying. To date, no fossil or extinct
animal with vertical palatal shelves in the adult state
has been described so that phylogenetic arguments
relating to the function of vertical palatal shelves in
respiration, feeding or hearing are highly speculative.
What is the mechanism of mammalian palatal
shelf elevation?
Numerous theories have been advanced to account
for palatal shelf elevation (see Ferguson, 1978 for a
review). In principle, an intrinsic force is progressively generated within the palatal shelves; once this
reaches a threshold level which exceeds the force of
resistance factors (e.g. frictional tongue resistance)
shelf elevation occurs. Shelf elevation is a rapid
event, probably occurring over a matter of minutes or
hours in vivo (Ferguson, 1978; Brinkley, 1980). It
involves a swinging 'flip up' mechanism in the anterior one third of the palate but an oozing remodelling 'flow' mechanism in the posterior two thirds of
the palate (Figs 1, 2) (Brinkley & Morris-Wiman,
1984, 1987a). The intrinsic shelf elevating force is
43
multifactorial, but the chief component appears to be
a regionally specific accumulation (Figs 3, 4) of glycosaminoglycans, predominantly hyaluronic acid, as
development progresses (Pratt, Goggins, Wilk &
King, 1973; Ferguson, 1978; Brinkley & MorrisWiman, 1984, 1987a,b). There is more hyaluronic
acid in the anterior palate than posteriorly and more
in the future oral aspect than the future nasal aspect
(Knudsen, Bulleit & Zimmerman, 1985; Brinkley &
Morris-Wiman, 1987a). Hyaluronic acid is a highly
electrostatically charged, open coil molecule, capable
of binding up to ten times its own weight in water.
Small changes in the concentration of hyaluronic acid
result in large changes in osmotic concentrations.
Regional accumulation of hyaluronic acid results in
swelling of the extracellular matrix and a corresponding decrease in mesenchyme cell density (Brinkley &
Bookstein, 1986). This separation of cells may also be
important in preventing contact and cell-cell, cellmatrix interactions at this stage of development: in
later palatal development such interactions are critical and their onset corresponds with a decrease in
hyaluronic acid content and an increase in cell density. Synthesis of hyaluronic acid by palatal mesenchyme cells is stimulated by epidermal growth factor
(EGF) (Turley, Hollenberg & Pratt, 1985; Dixon,
Foreman, Schor & Ferguson, 1988) and transforming
growth factor beta (TGF/?) (Sharpe, Foreman, Carette, Schor & Ferguson, 1988; Sharpe & Ferguson,
1988). Vertical palatal shelves increase in size as much
by extracellular matrix swelling as by mesenchymal
cell division (Brinkley & Bookstein, 1986). The latter
is greatest at the shelf tip (Jelinek & Dostal, 1974;
Nanda & Romeo, 1975; Cleaton-Jones, 1976).
The erectile shelf elevating force is partly directed
by stout bundles of type I collagen which run down
the centre of the vertical shelf from its base (where
they are associated with the bony blastemata) to its
tip (Fig. 5). Moreover the epithelial covering and
associated basement membrane of the palatal shelf
exhibit differential traction, which serve to constrain
and direct the swelling osmotic force, in much the
same way that the skin constrains an inflating balloon
(Bulleit & Zimmerman, 1985; Brinkley, 1984). The
alignment of mesenchymal cells within the core of the
palatal shelf may further serve to direct the elevating
force (Zimmerman & Wee, 1984). On the basis of in
situ cell morphology, palatal mesenchyme has been
divided into three principal regions which undergo
different changes in cell shape and orientation during
elevation (Babriarz, Wee & Zimmerman, 1979; Zimmerman & Wee, 1984). Palatal mesenchymal cells are
themselves contractile (Zimmerman, Clark, Ganguli
& Venkatasubramanian, 1983) and secrete various
neurotransmitters e.g. serotonin, acetylcholine (Zimmerman & Wee, 1984). These neurotransmitters
44
M. W. J. Ferguson
Fig. 1. Histological section through the anterior region of the palate in a day-13 embryonic mouse head. Note the
vertical palatal shelves (p) firmly wedged against the tongue (t) in preparation for shelf elevation. x21.
Fig. 2. Transverse histological section through the mid-palate region of an early day-14 and late day-14 mouse embryo.
The palatal shelves first approximate each other, their medial edge epithelia (arrowed), then contact each other and fuse
to form the midline epithelial seam. x21.
Fig. 3. Transverse cryosection through the vertical palatal shelf of a day-13 mouse embryo in the anterior region. The
section has been immunocytochemically stained for hyaluronic acid using the hyaluronectin/anti-hyaluronectin
technique (Girard, Delpech & Delpech, 1986) and immunoperoxidase. Note the intense staining for hyaluronic acid
within the palatal shelf mesenchyme. x84.
Fig. 4. Transverse cryosection through the anterior palatal region of a day-14 mouse embryo immunocytochemically
stained for hyaluronic acid using the hyaluronectin/anti-hyaluronectin technique. Note the concentration of hyaluronic
acid along the medial edge mesenchyme adjacent to the midline epithelial seam. x84.
affect both mesenchymal cell contractility and glycosaminoglycan degradation, and so may play a modulatory role in palate morphogenesis (Zimmerman &
Wee, 1984).
Palatal shelf elevation occurs in a conducive orofacial environment. During the period of shelf elevation, there is almost no growth in head width, but
constant growth in head height (Diewert, 1978). This
means that the position of least resistance for the
expanding palatal shelves to occupy is above the
dorsum of the tongue. Moreover, the tongue muscles
become functional around the time of shelf elevation
(Wragg, Smith & Borden, 1972) and fetal reflexes are
present (Humphrey, 1968,1969). Recently it has been
demonstrated that human embryos start to hiccup
around the time of palatal shelf elevation, but that
jaw opening, sucking and swallowing behaviour are
not observed until after the time of palatal closure
(De Vries, Visser & Prechtl, 1982, 1985). It is
tempting to speculate that the profound pressure
Palate development
45
Fig. 5. Transverse cryosection of a day-13 vertical anterior embryonic mouse palatal shelf immunocytochemically
stained with antibodies against collagen type I. Note the stout bundles of collagen type I down the centre of the palatal
shelf orientated from the base towards the tip. xl79.
Fig. 6. Transverse cryosection through a day-14 fusing mouse palatal shelf. The medial edge epithelia are just
contacting each other. The section has been stained immunocytochemically with antibodies against desmoplakin. Note
the localization of these molecules on the surfaces of the medial edge epithelia (antibodies kindly supplied by Dr D.
Garrod, Southampton). x450.
changes produced in the oronasal cavity by hiccuping
may be a trigger for rapid palatal shelf elevation.
How does the epithelial seam form?
After elevation, the palatal shelves approximate and
contact each other, first in the region of the second
ruga (middle third of the palate) from which point
fusion spreads in posterior and anterior directions.
The medial edge epithelia of opposing palatal shelves
adhere to each other by means of a sticky cell surface
glycoprotein coat (Greene & Kochhar, 1974; Pratt &
Hassell, 1975; Souchon, 1975; Greene & Pratt, 1977)
and desmosomes (De Angelis & Nalbandian, 1968;
Morgan & Pratt, 1977) to form an epithelial seam.
This is an interesting developmental phenomenon:
(1) medial edge epithelial cells, which were originally
superficial, develop cell adhesion molecules and
desmosomes and become the central cells of the seam
and (2) medial edge epithelial cell adherence is
specific: the medial edge epithelia will not normally
fuse with other epithelia e.g. floor of mouth, tongue,
cheek etc. (Ferguson et al. 1984). Recently, we have
demonstrated that mouse medial edge epithelial cells
rapidly form desmosomes and accumulate desmoplakin - one of the desmosomal plaque proteins - on
their superficial cell membranes just prior to shelf
contact (Fig. 6). This suggests that desmosomal components are rapidly assembled, specifically in medial
edge epithelial cells, just prior to and upon medial
edge epithelial cell contact: this may be one of the
mechanisms conferring specificity on medial edge
epithelial cell adherence. The turnover rates of various desmosomal components prior to, during and
after medial edge epithelial cell adherence may be an
important controlling mechanism in palatal fusion
and require investigation. Moreover, we have also
produced monoclonal antibodies that recognize cell
surface molecules on palatal epithelial cells (Dixon,
White & Ferguson, 1988). These molecules vary in
distribution by palatal region, by layers of the epithelium and with developmental time. One molecule
is distributed between the layers of epithelial cells but
is absent from their superficial surfaces in vertical
palatal shelves. It appears on the superficial surface of
the medial edge epithelia prior to and during shelf
contact. It is tempting to speculate that these are
epithelial cell adhesion molecules which might again
confer specificity on medial edge epithelial cell adher-
48
M. W. J. Ferguson
Fig. 7. Transmission electron micrograph through the midline epithelial seam (e) of a pair of fusing mouse palatal
shelves. The specimen was prepared in tannic acid containing fixative. Note the intact basement membrane on both
sides of the epithelial seam and the accumulation of numerous lysosomes within the medial edge epithelial cells. X39OO.
Fig. 8. Transmission electron micrograph through a disrupted midline epithelial seam. Note the mesenchymal
penetration and the resynthesis of the basement membrane along the epithelial seam cells (e). X3900.
ence. None of the known cell adhesion molecules
(Obrink, 1986) exhibit a similar staining pattern when
localized immunocytochemically. Superficial medial
edge epithelial cells also stain markedly with certain
lectins e.g. ConA and Ulex europaeus agglutinin
suggesting the presence of surface molecules rich in
carbohydrate. Chick medial edge epithelia show none
of these specializations of surface coat, do not form
desmosomes and normally do not adhere.
How does the epithelial seam disrupt?
Almost as soon as the epithelial seam has formed
(Fig. 7), it starts to thin ultimately becoming two or
three cells thick. Thinning is achieved by expansion in
palatal height (oronasally) and epithelial cell migration onto the oral and nasal aspects of the palate.
The seam cells also rapidly accumulate lysosomal
enzymes (Mato, Aikawa & Katahiva, 1966) and
undergo apototic cell death (Figs 7,8). Palatal mesenchyme becomes continuous in areas where the
seam has disrupted (Fig. 8). The basement membrane on each side of the epithelial seam remains
intact even when it has completely thinned (Fig. 9)
and a basal lamina is rapidly reconstituted around the
ends of the seam or isolated epithelial islands, in
areas of mesenchymal penetration (Fig. 8) (K. Hall &
M. W. J. Ferguson, unpublished data).
Medial edge epithelial cells cease DNA synthesis
24-36h prior to shelf contact (Hudson & Shapiro,
1973) and this has been referred to as programmed
cell death. Cyclic AMP increases transiently just prior
to shelf fusion, and much has been made of this
observation in relation to competitive inhibitor
studies of the prostaglandin, cyclic AMP, metabolic
pathways (for reviews see Greene & Garbarino, 1984;
Goldman, 1984; Pratt, 1984; Pisano & Greene, 1986;
Ferguson, 1987). Exogenous cyclic AMP causes precocious medial edge epithelial cell death in mammals
(Pratt & Martin, 1975) but does not induce it in birds
(Tyler, 1986). Epidermal growth factor inhibits medial edge epithelial cell death (Hassell, 1975; Pratt,
Kim & Groove, 1984; Pratt, 1984) but only in the
presence of palatal mesenchyme (Tyler & Pratt,
1980): exogenous cyclic AMP blocks this EGFinduced inhibition (Hassell & Pratt, 1977). Despite a
wealth of data on the biosynthesis and effects of cyclic
AMP on palatal mesenchymal and epithelial cells
(Greene & Garbarino, 1984; Goldman, 1984; Pratt,
1984; Pisano & Greene, 1986), it must be remembered that cyclic AMP, like intracellular calcium, pH
and phosphatidylinositol lipids, is physiologically an
intracellular second messenger. Its peak prior to shelf
fusion suggests that it may be mediating differential
gene expression, as a result of events at the cell
surface e.g. binding of various molecules.
Recently, we have demonstrated that epithelial cell
death is not the only method of seam disruption. A
large number (perhaps up to 50 %) of epithelial seam
cells migrates into the palatal mesenchyme initially
carrying with them fragments of their disrupted
basement membranes (Fig. 10) (Fyfe & Ferguson,
1988; K. Hall & M. W. J. Ferguson, unpublished
Palate development
47
Fig. 9. Cryosection through a pair of fusing palatal shelves in an embryonic day-14 mouse immunocytochemically
stained with antibodies against type IV collagen. Note that the basement membrane is intact even though the seam is
only one to two cells thick. Note also the staining of blood vessel basement membranes. X112.
Fig. 10. Cryosection through an embryonic day-14 mouse head illustrating recently fused palatal shelves
immunocytochemically stained with antibodies against type IV collagen. Note the disruption of the midline epithelial
seam and the dispersion of clumps of basement membrane material with the epithelial cells which migrate into the
mesenchyme. X179.
Fig. 11. Cryosection through a horizontal day-14 embryonic mouse palatal shelf immunocytochemically stained with
antibodies against tenascin. Note the fibrils of tenascin emerging from the basement membrane region and running
perpendicularly into the mesenchyme particularly around the medial edge of the palatal shelf. X179.
Fig. 12. Cryosection through an embryonic day-13 mouse palatal shelf immunocytochemically stained with a monoclonal
antibody recognizing heparan sulphate proteoglycan. This specimen has been prepared using the immunogold silver
enhancement technique. Note the distribution of heparan sulphate proteoglycan in the basement membrane on the stout
collagen I bundles (Fig. 5), and on the surfaces of the palate mesenchymal cells. X179.
48
M. W. J. Ferguson
data). These fragments disappear after a few hours,
the cells lose their staining for cytokeratins and
express vimentin intermediate filament staining and
so quickly become indistinguishable from other palatal mesenchyme cells. For this reason, the ultimate
developmental fate of these cells is unknown. It is
unclear whether these migratory cells constitute a
specific epithelial subpopulation e.g. basal stem cells
which do not die. During seam disruption, the seam
epithelial cells are less adherent to their basement
membranes and display blebbing of their basal surfaces. They also develop well-organized bundles of
microfilaments and microtubules in their cytoplasms.
Interestingly in other cell systems increased cyclic
AMP levels cause marked decreases in the numbers
of cell surface fibronectin receptors (Allen-Hoffmann
& Mosher, 1987), and it would be interesting to
determine if this is true for palatal medial edge
epithelial cells. Moreover, the basement membranes
of the epithelial seam show a progressive loss of
laminin, whilst fibrils of tenascin and type III collagen
accumulate in the sub-basement membrane zone at
right angles to the basement membrane (Fig. 11)
(Fyfe & Ferguson, 1988; Fyfe, Ferguson & ChiquetEhrisman, 1988). The medial edge epithelial cells
appear to migrate into the underlying mesenchyme
along these fibrils. Chick medial edge epithelia do not
cease DNA synthesis, show no peak of cyclic AMP
(Greene et al. 1983) and no vertical fibrils of tenascin
or type III collagen (Fyfe & Ferguson, 1988; Fyfe et
al. 1988).
Recently a mesenchymally produced soluble factor
(scatter factor) has been isolated which causes the
rapid disruption of epithelial junctions (Stoker, Gherardi, Perryman & Gray, 1987). Such a factor could be
important in epithelial seam disruption. We therefore
investigated the production of scatter factor by regionally derived clones of mouse embryonic palate
mesenchyme cells from the oral, nasal and medial
surfaces grown in vitro. The prediction was that
medial edge mesenchymal cells would synthesize
more scatter factor. Table 1 shows the results of
scatter factor activity in the conditioned media of
such cells. Although palate mesenchymal cells synthesize scatter factor, regional differences in production are insignificant, so that the role of scatter
factor in palatal epithelial seam disruption is currently
unclear and awaits experimental and immunological
investigations.
Does medial edge epithelial cell differentiation
(death) depend upon shelf contact?
Experiments in which mouse, alligator or chick palatal shelves were organ cultured either in isolation or
Table 1. The scatter factor activity (units ml ') of
palate mesenchymal cells derived from different
regions of the palate (nasal, medial or oral) when
cultured in vitro for various passages
Palate mesenchyme
Cell type
Passage number
3
4
5
6
Nasal
24
48
24
24
Medial
24
48
12
24
Oral
48
48
24
48
These values are not statistically significant. Analysis by Sir
Michael Stoker, FRS, IJniversity of Cambridge.
in homologous pairs, revealed that medial edge
epithelial cell differentiation (death in mice, migration in alligators, keratinization in chicks) occurred in single palatal shelves and so was independent of shelf contact (Ferguson et al. 1984). Regional
differentiation of the nasal epithelia into pseudostratified ciliated columnar cells and the oral epithelia
into stratified squamous cells also occurred in single
palatal shelves of all three species. Each palatal shelf
is therefore a developmental field with three defined
regions of epithelial differentiation (nasal, medial,
oral), one of which (medial) varies between vertebrate species.
Is mouse palatal medial edge epithelial cell
death a suicide or a murder?
An extensive series of epithelial-mesenchymal recombination experiments has been performed including homologous, heterologous, heterochronic and
isochronic combinations of mandibular, limb and
palatal tissues both within and between mouse, chick
and alligator embryos (Ferguson & Honig, 1984). In
alligator and chick, the situation is simple. The
mesenchyme signals nasal, medial and oral epithelial
differentiation in a species-specific fashion, even to
heterologous epithelia. The palatal epithelium appears to play a passive role, receiving its instructions
from the underlying mesenchyme (Ferguson &
Honig, 1984). The results in mice are similar, except
that mouse palatal epithelia are biased to differentiate into nasal, medial and oral phenotypes from the
onset of palatogenesis and will do so if placed on a
'neutral' e.g. mandibular, mesenchyme. Mouse palatal mesenchyme, however, signals regionally specific
epithelial differentiation to heterologous epithelia or
to palatal epithelia from other species e.g. chick,
alligator at all stages of palatogenesis (Ferguson &
Honig, 1984). If mouse palatal epithelial sheets are
Palate development
recombined with mouse mesenchyme with the original medial edge of the epithelial sheet at right angles
to the medial edge of the mesenchyme (crossed
recombination), then there is complete respecification of palatal epithelial differentiation into the
regions defined by the orientation of the new mesenchyme. If, however, mouse palatal epithelia are
recombined with alligator mesenchyme in a crossed
recombination, then the alligator mesenchyme completely respecifies the nasal and oral fields and a new
medial edge with a cobblestoned migratory phenotype typical of the alligator; but a line of cell death
dividing the new oral and nasal fields still persists
along the original medial edge of the mouse epithelial
sheet (Ferguson & Honig, 1984). This suggests that
there is some kind of signalling hierarchy between
vertebrate mesenchymes and epithelia. In summary,
epithelial-mesenchymal recombination experiments
have demonstrated that nasal, medial and oral palatal
epithelial differentiation is specified by the mesenchyme, and that signalling of medial edge epithelial
differentiation goes across different vertebrates in a
species-specific fashion (Ferguson & Honig, 1984).
Medial edge epithelial cell death in mammals is
therefore a murder by the underlying mesenchyme
rather than an intrinsic epithelial suicide.
What are the physlcochemlcal properties of the
mesenchymal signal?
In theory, there are four principle ways in which
mesenchyme could signal epithelial differentiation;
(1) extracellular matrix molecules, (2) soluble factors, (3) direct cell-cell contact, (4) combinations of
1-3. Extensive TEM surveys of the epithelial-mesenchymal interface at all stages of mouse palate development reveal that direct mesenchymal-epithelial
cell contacts are very rare, even in the degenerating
epithelial seam! (K. Hall & M. W. J. Ferguson,
unpublished data). Direct cell-cell contact is therefore an unlikely signalling mechanism. There are,
however, differences in the number and density of
mesenchymal pseudopodial processes adjacent to the
basement membrane at different developmental
times and in different regions of the palate: these may
be important in extracellular matrix synthesis/remodelling and soluble factor secretion.
We have conducted extensive immunocytochemical surveys of all stages of the developing mouse and
chick palate using affinity-purified polyclonal or
monoclonal antibodies against: collagen types I-X,
laminin, fibronectin, tenascin, heparan sulphate proteoglycan, chondroitin 0,4,6, sulphates; (Fyfe & Ferguson, 1988; Fyfe et al. 1988). Collagen types II, VIII
and X are absent from the developing mouse palate.
49
Collagen types I, III, chondroitin sulphates and
fibronectin are fairly ubiquitously distributed in the
palate at all stages of development (Fig. 5) (Silver et
al. 1981). These molecules are rapidly synthesized in
the fusion zone following epithelial seam degeneration. Collagen type IV and laminin are present in the
basement membranes of the palatal epithelia and
blood vessels at all stages of development (Figs 9,10).
Their distribution during seam disruption was described earlier. Collagen types V and VI and heparan
sulphate proteoglycan (Fig. 12) are present in basement membranes and in certain regions of the mesenchyme e.g. type VI collagen is present around the
nasal angle and in the midoral regions of an individual
palatal shelf. Types V and VII collagen are found
around epithelial cells particularly of the midline
epithelial seam. Tenascin accumulates preferentially
beneath the medial edge basement membranes of
horizontal mouse palatal shelves (Fig. 11). Heparan
sulphate proteoglycan is present in the basement
membranes and on the mesenchyme cell surfaces,
particularly in vertical tracts of collagen down the
centre of the shelf (Figs 5 and 12).
Type IX collagen is the most interesting extracellular matrix molecule in terms of signalling epithelialmesenchymal interactions in the mouse palate (Fyfe
& Ferguson, 1988). Type IX collagen is absent from
the basement membranes, mesenchymal and epithelial cell surfaces of the medial edge epithelia of
day-12 to -13 embryonic mouse palates (Fig. 13). It is
present on the cell surfaces of floor of the mouth
epithelia at these stages (Fig. 13). However, just prior
to shelf elevation type IX collagen appears on the cell
surfaces of palatal medial edge epithelial cells (Fig.
14). This corresponds precisely with the time of
signalling of medial edge epithelial differentiation as
determined by recombination experiments (Ferguson
& Honig, 1984), so the molecule appears in the right
place at the right time. It is absent from developing
chick palates. Cryoimmunoelectron microscopy reveals that type IX collagen is present at intersections
of other collagen molecules (e.g. types III and I) with
either themselves or the basal lamina (Fig. 15). It is
also present on the medial edge epithelial cell surfaces (Fig. 16). We believe that type IX collagen acts
as a linker molecule, physically and functionally
connecting components of the extracellular matrix
both with themselves and with the epithelial cell
surface (Fyfe & Ferguson, 1988). If this is true, then
its appearance at the time of signalling of epithelialmesenchymal interactions could represent a significant developmental control mechanism. Within such
a hypothesis all of the elements of the signalling
mechanism (different collagen types, basal lamina,
cell surface) could be developed and in place, awaiting only the synthesis of a linker molecule (type IX
50
M. W. J. Ferguson
collagen) to activate the mechanism. Type IX collagen has been described as linking together type II
collagen fibrils at their intersections in cartilage
(Muller-Glauser et al. 1986). Recently, a new collagen molecule, type XII, has been described, and it
is suggested that this molecule acts as a minor
collagen linking fibrils of types I and III collagen
(Olsen, personal communication). As the types IX
and XII molecules allegedly exhibit homology in at
least one of the regions where our antibodies bind, it
is possible that what we have described as type IX
collagen in the palate, is in fact type XII. This
possibility will be tested once details of type XII
collagen are elucidated and published, and typespecific antibodies generated against both types IX
and XII collagens. It may be that minor collagen
types (including species yet to be described) play a
crucial role in embryogenesis and that such a role
need not be structural but, rather, predominantly one
of directing morphogenesis and differentiation. Indeed, it could be intuitively argued that if the chief
role of collagen was structural then it is unlikely that
there would be so many different types, particularly
of a minor variety.
Knowledge that type IX collagen appears in the
right place at the right time to be a candidate
signalling molecule for mouse palatal medial edge
epithelial cell differentiation, merely begs the question as to what controls the regional synthesis of type
IX collagen. We have investigated this coincident
with investigating the role of certain soluble factors as
mesenchymal-signalling agents of epithelial differentiation.
It is known that palate mesenchymal cells have
large numbers of receptors for epidermal growth
factor (EGF) (Yoneda & Pratt, 1981a,/>; Pratt et al.
1984) and that EGF (1) inhibits palatal medial edge
epithelial cell death, but only in the presence of
mesenchyme (Tyler & Pratt, 1980), and (2) stimulates
oral epithelial cell division, hypertrophy and keratinization (Grove & Pratt, 1984) and mesenchymal
synthesis of type V collagen and hyaluronic acid
(Silver, Murray & Pratt, 1984; Turley et al. 1985).
Transforming growth factor alpha (TCFcr) is likely
the embryonic homologue of EGF. Transforming
growth factor beta (TGF/3) is known to kill cells,
particularly of epithelial lineage (Sporn, Roberts,
Wakefield, de Crombrugghe, 1987; Cheifetz et al.
1987; Massague, 1987) and to stimulate fibronectin
synthesis (Ignotz & Massague, 1986). It has been
localized in the developing mouse palate (Heine et al.
Fig. 13. Cryosection through the medial edge (m) and floor of the mouth epithelia (/) of an embryonic day-13 mouse
head immunocytochemically stained with antibodies against type IX collagen. Note the absence of type IX collagen
staining on the medial edge epithelia of the tip of the palatal shelf. Such staining is present in the floor of the mouth
epithelia. X280.
Fig. 14 Cryosection through an early day-14 embryonic mouse head illustrating the approximating medial edge epithelia
(m) of the two palatal shelves immunocytochemically stained with an antibody against type IX collagen. Note the
localization of type IX collagen around the surfaces of these medial edge epithelial cells. x280.
Palate development
51
Fig. 15. Cryoelectron microscopy of the medial edge epithelia (w), basement membrane (b) and underlying
mesenchyme of an embryonic day-14 mouse palatal shelf. The specimen has been prepared with 15 nm gold probes
linked to a secondary antibody detecting the primary antibody against laminin which distributes in the basement
membrane. The specimen is double labelled with 5nm immunogold probes identifying a type IX collagen antibody.
Note the type IX collagen appears to be linking the basement membrane with an underlying type I collagen fibril
(arrow), x 16500.
Fig. 16. Cryotransmission electron micrograph of a medial edge epithelial cell (m) on a day-14 embryonic mouse palatal
shelf. The specimen has been prepared with 5 nm immunogold probes detecting a type IX collagen antibody. Note that
the type IX collagen is localizing on the surface of the epithelial cells. X30250.
Fig. 17. Scanning electron micrograph of regionally derived mouse embryonic palatal mesenchymal cells (m) cultured to
confluence on the surface of a type I collagen gel (c). X137.
Fig. 18. Transmission electron micrograph illustrating an embryonic mouse palatal mesenchymal cell (m) embedded
within a type I collagen gel. Note the collagen fibrils, the loose nature of the collagen gel and the reorganization of
extracellular matrix molecules around the mesenchymal cell, x 11000.
1987; Sharpe & Ferguson, 1988) and is therefore a
potentially interesting molecule. Acidic and basic
fibroblast growth factors (FGF) or related molecules
are known to be important in signalling epithelial
mesenchymal interactions during amphibian gastrulation (Slack, Darlington, Heath & Godsave, 1987)
and so are of potential interest in the palate. Plateletderived growth factor (PDGF) is important in wound
healing (Lynch, Nixon, Colvin & Antoniades, 1987)
and so of potential interest in terms of epithelial seam
formation, degeneration and mesenchymal consolidation.
We have therefore investigated the individual ef-
fects of EGF, TGFar, TGF/3, PDGF, FGF acidic and
basic (FGFa/FGFb), on mouse palatal development
in organ culture, mesenchyme cell culture and epithelial cell culture (Sharpe et al. 1988; Sharpe &
Ferguson, 1988; Dixon, Foreman, Schor & Ferguson,
1988). For mesenchyme cell culture the general
strategy has been to determine the dose-response
curves for the growth factor and then to study its
effects on cell division and extracellular matrix biosynthesis when mouse palatal mesenchyme cells are
cultured on plastic, on collagen films and within
three-dimensional hydrated collagen gels (Figs 17,
18) i.e. physiologically relevant substrata. Sum-
52
M. W. J. Ferguson
Fig. 19. Cryosection of a type I collagen gel with mouse embryonic palatal mesenchymal cells cultured on its surface
(see Fig. 17). The section has been stained with antibodies against type IX collagen and virtually none is present. The
cultures were performed in the presence of 2-5 % donor calf serum in the DMEM/F12 media, x 132.
Fig. 20. Cryosection of a type I collagen gel with embryonic mouse palatal mesenchymal cells cultured on its surface.
These cells were cultured in the presence of DMEM/F12 media supplemented with 2-5 % donor calf serum and
lOngml"' EGF. The section is immunocytochemically stained for type IX collagen. Note the appearance of type IX
collagen in a basement-membrane-like structure on the surface of the collagen gel. Type IX collagen synthesis has been
stimulated by epidermal growth factor (compare with Fig. 19). X132.
Fig. 21. Immunocytochemical staining of the matrix deposited by mouse embryonic palatal mesenchymal cells on a
plastic glass slide immunocytochemically stained with antibodies against fibronectin. The embryonic palatal
mesenchymal cells were cultured in the presence of transforming growth factor /3 which stimulated the synthesis of
fibronectin. xl68.
Fig. 22. Scanning electron micrograph of a mouse palatal epithelial sheet cultured on the surface of a type I collagen gel
which had previously been conditioned (see Figs 19-21) by the prior culture of mouse embryonic palatal mesenchymal
cells on the surface of the gel followed by lysis of these cells using ammonia. The epithelial sheet was derived from a
late day-13 mouse embryo and was therefore already determined. Note differentiation of the palatal epithelia into nasal
pseudostratified ciliated columnar cells (n), medial edge cell death (m) and oral stratified squamous cells (O). X180.
marized, these so called growth factors have major
specific effects on extracellular matrix biosynthesis
which are much more important than their mitogenic
effects when cells are cultured in a physiologically
relevant environment. TGF/3 increases markedly the
synthesis of fibronectin and also of collagen types III,
IV and V and possibly IX (Fig. 21). It inhibits the
synthesis of type I collagen and only increases the
synthesis of hyaluronic acid in sparse cultures but not
in dense ones (Sharpe etal. 1988; Sharpe & Ferguson,
1988). TGF/3 inhibits palatal mesenchymal cell
growth and kills some cells. EGF (or TGFa) stimu-
ates the synthesis of all collagen types and increases
the synthesis of glycosaminoglycans and proteoglycans (Figs 19, 20). Interestingly EGF stimulates the
synthesis of collagen which is underhydroxylated
(Dixon et al. 1988). EGF (or TGFo-) increases the
rate of palatal mesenchymal cell division, but less so
when cells are cultured in or on collagen gels. PDGF
stimulates cell division in a similar way (but to a lesser
extent) to EGF, but has no effect on extracellular
matrix biosynthesis. FGFa or FGFb inhibits extracellular matrix biosynthesis, which is interesting
in view of its previously documented effect of
Palate development
stimulating cell migration during gastrulation (Slack
et al. 1987). Therefore, the synthesis of extracellular
matrix molecules including type IX collagen is stimulated by certain soluble factors e.g. TG¥a, TGF0
known to be present in the palate and inhibited by
others e.g. FGF.
We have investigated the effects of these various
combinations of extracellular matrix molecules on
palatal epithelial differentiation by lysing the palatal
mesenchymal cells off the conditioned gel surface
using ammonia and plating on sheets of isolated
palatal epithelia (Fig. 22). To date, we have been able
to maintain the differentiation of already determined
palatal epithelial sheets in these mesenchyme-free
culture conditions (Fig. 22): experiments are in progress to investigate the effects of these conditioned
matrices in directing epithelial differentiation. It
should also be noted that many soluble factors bind to
extracellular matrix molecules such as heparan or
fibronectin (Smith, Singh, Lillguist, Goon & Stiles,
1982; Fava & McClure, 1987) and this may be
important both in the embryo and in interpreting
these conditioned gel experiments.
Thus one population of mesenchymal cells could
secrete extracellular matrix molecules and bound
growth factors. These may remain either inactive or
locally active (Smith et al. 1982; Fava & McClure,
1987) until at a subsequent developmental time the
ECM is remodelled by another population of cells,
the bound growth factors released and exert their
effects on this new population of cells. Cause and
effect may therefore be separated by developmental
time.
Mouse palatal shelves have been organ cultured in
the presence of these various growth factors (M. J.
Dixon, D. M. Fyfe & M. W. J. Ferguson, unpublished data). No effects were noted in cultures at the
air-gas interface, presumably due to the failure of the
growth factor to penetrate the tissue despite the
omission of Millipore filters as a culture substratum.
By contrast in submerged organ cultures effects were
noted. In EGF-supplemented cultures, the medial
edge of the palatal shelf develops a nipple-like bulge;
interestingly, this is the area in vivo where cells have
the highest mitotic indices (Jelnick & Dostal, 1974;
Nanda & Romeo, 1975; Cleaton-Jones, 1976). The
mesenchyme had increased quantities of extracellular
matrix molecules, medial edge epithelial cell death
was absent and the medial edge plus oral epithelia
were heavily keratinized. In TGF/3-supplemented
cultures, the epithelia were very thin and poorly
differentiated, MEE cell death was marked, mesenchyme cells were clumped and the fibronectin content
of the mesenchyme was increased. These results
indicate that exogenous growth factors exert effects
on organ-cultured mouse palatal shelves in a fashion
53
similar to their effects in cell culture and that controlled physiological levels of such factors may be
important in mouse palate development.
How Is mesenchymal signalling of epithelial
differentiation regionally specified?
Our model of mesenchymal signalling of terminal
palatal epithelial differentiation is complex and involves a dynamic interaction between soluble growth
factors and the extracellular matrix. The model
supposes that the mesenchyme produces a growth
factor, or factors, which may (1) act directly on the
epithelia to induce a phenotype, (2) act by autocrine
stimulation of extracellular matrix production by
mesenchyme cells and these extracellular matrix molecules may then directly influence epithelial differentiation. Additionally, the extracellular matrix may
modulate the response of epithelia to growth factors
or vice versa. The system may selfregulate via a
biphasic response of the epithelia and mesenchyme to
the same growth factor i.e. the factor may directly
affect the epithelia and the mesenchyme, the extracellular matrix molecules produced by the mesenchyme may then modulate (e.g. turn off) the direct
effects of the factor on the epithelia.
Regional specification of epithelial differentiation
may depend upon the clonal heterogeneity of palatal
epithelial and mesenchymal cells. Not all the palatal
mesenchymal cells produce all the extracellular
matrix molecules known to be present (Sharpe et al.
1988; Dixon et al. 1988). Heterogeneity in extracellular matrix production therefore exists. Not all the
palatal mesenchymal cells respond to soluble growth
factors (Sharpe et al. 1988; Dixon et al. 1988) either in
relationship to mitosis or extracellular matrix production. This may reflect the presence or absence of
receptors for these various growth factors on the
mesenchyme cells. Preliminary immunocytochemical
studies of the regional localization of the EGF receptor during different stages of mouse palate development suggest that there is regional heterogeneity (M.
W. J. Ferguson, unpublished data). When the palatal
shelves are vertical, the mesenchyme cells beneath
the medial edge appear to lack EGF receptors, but
the latter appear when the shelves are horizontal and
the epithelial seam is degenerating (Fig. 23). Growth
factors and extracellular matrix molecules may cause
the clonal expansion of subpopulations of mesenchyme cells which may be important in the regional
signalling of epithelial mesenchymal interactions
(Schor & Schor, 1987).
Palatal mesenchyme cells are heterogeneous not
only in terms of production of extracellular matrix
molecules and response to soluble factors but also in
5*
M. W. J. Ferguson
Fig. 23. (A) Histological section through a day-13 vertical mouse palatal shelf which had been stained en bloc with
antibodies against the epidermal growth factor receptor. These antibodies were then detected via secondary antibodies
linked to colloidal gold particles which were enhanced using the silver enhancement technique. The specimen is viewed
in epipolarizing light and the EGF receptor appears as a white spot. Note the distribution of this receptor in the nasal
and oral regions particularly in the core of the palatal shelf, but its absence from the mesenchymal cells at the tip of the
palate. This absence is a real result and not due to a penetration-of-antibody technical problem as we have deliberately
disrupted the epithelium at the tip of the palatal shelf. (B) A similar preparation of a pair of horizontal fusing palatal
shelves in a day-14 mouse embryo. Note the appearance of mesenchymal cells expressing the EGF receptor adjacent to
the midline epithelial seam (e). xl69.
growth factor production (Heine et al. 1987). Synthesis of growth factors at particular locations within
the palate may give rise to diffusion gradients and
thus to differing regional responses by both epithelial
and mesenchymal cells. In this context, it should be
noted that the same growth factor can stimulate cell
division at one concentration and inhibit it at another;
populations of cells can respond differently to the
same concentration of a growth factor depending
upon their density and the nature of their substratum
(Sporn et al. 1987; Massague, 1987). Gradients of
growth factors within the developing palate may also
be generated by (1) the binding and sequestration of
growth factors in specific locations by binding proteins or extracellular matrix molecules, (2) their
presence in amniotic fluid and local diffusion into
palatal tissues via the oronasal cavity. Gradients
of growth factors may be superimposed upon gradients of receptors on responding cells and the density
and substrata of the latter. The situation is therefore
potentially highly complex and interactive. Moreover
different growth factors interact with each other to
produce either synergistic or antagonistic effects, thus
layering on another complicating tier (Sharpe et al.
1988; Dixon et al. 1988; Sporn et al. 1987).
Palatal epithelial tissues may also be heterogeneous
in terms of their response (e.g. possession of receptors) to either growth factors or extracellular matrix
molecules. Investigation of such a complex model is
not as daunting as it might appear. First, we have
recently demonstrated that regionally derived,
uncloned populations of mesenchyme cells from the
nasal, medial, and oral regions of the palate, exhibit
differences in response (extracellular matrix pro-
Palate development
duction and mitosis) to various growth factors even
after several passages in vitro (M. J. M. Carette & M.
W. J. Ferguson, unpublished data). It is therefore
possible to conduct mesenchyme conditioning/epithelial target experiments on collagen gels (as described
in the previous section) using either regionally derived or cloned palatal mesenchyme cells. Second, we
have produced monoclonal antibodies against specific
cell surface determinants on mouse palatal nasal,
medial and oral epithelial cells, before, during and
after epithelial differentiation (Fig. 25) (Dixon,
White & Ferguson. 1988). These antibodies can be
used to regionally sort (using FACS) isolated palatal
epithelial populations into regional types which can
then be used in targeting experiments using cloned
mesenchyme cells.
Such a dynamic interactive model of mesenchyme
signalling is even more intriguing when it is realized
(1) that specific extracellular matrix molecules can
bind and sequester growth factors (Smith et al. 1982;
Fava & McClure, 1987) and (2) that certain growth
factors (e.g. TGF#) can stimulate the expression of
cell adhesion protein receptor sites in mouse epithelial cell lines (Ignotz & Massague, 1987). Thus,
presence of a certain growth factor could stimulate
extracellular matrix production by mesenchyme cells,
bind locally to this newly synthesized matrix and
simultaneously stimulate the expression of receptors
for such matrix and soluble factors in adjacent epithelial cells: epithelia and mesenchyme would then
interact. In this context, soluble factors such as EGF,
TGFa-, TGF£, PDGF, FGF are somewhat misnamed
as 'growth factors'. They act more like 'differentiation factors' during embryonic development and
their principal effect is not on mitogenesis when cells
are investigated in a physiologically relevant situation
(Sharpe etal. 1988; Dixon etal. 1988). Interestingly, it
has recently been shown that proteins encoded by
pattern-forming genes in Drosophilia are homologous with EGF and TGF/3 (Padgett, Johnston &
Gelbart, 1987).
How do the responding epithelial cells process
the epigenetlc signals?
It is hypothesized that palatal epithelial cells possess
receptors for extracellular matrix and soluble factors.
Apart from some data for the EGF receptor (Yoneda
& Pratt, 1981a,b) nothing is known about the nature
or affinity of such receptors, or how they vary by
region or with developmental time. Changes in the
extracellular matrix substrata underlying the epithelia
may affect the polarity and cytoskeletal organization
of the latter. Such shape changes may directly induce
differential gene expression and/or affect the dispo-
55
sition, numbers and affinity of other receptors on the
cell surface e.g. for soluble factors.
Most events at the cell surface are transduced by
second messenger systems, intracellular: cyclic
nucleotides, calcium, pH and phosphatidylinositol
lipids. There are extensive data on the regulation of
palatal cyclic AMP by prostaglandins, some data on
cyclic GMP, few data on phosphatidylinositol lipids
and almost no data on intracellular pH and calcium
(reviewed in Greene & Garbarino, 1984; Goldman,
1984; Pisano & Greene, 1986; Ferguson, 1987). Surprisingly, there have been few investigations of
second messenger systems using defined palatal cell
populations - even epithelia or mesenchyme! There
have been equally few studies of the chain of intracellular events following stimulation of the cell surface with one particular factor either extracellular
matrix or soluble.
How do these signals result In the terminal
differentiation of the epithelial cells?
Ultimately, this question asks what genes and their
encoded proteins make a nasal cell pseudostratified
ciliated columnar, an oral cell stratified squamous and
a medial edge cell in the mouse die? We have
addressed this issue by trying to develop better
molecular markers for nasal, medial and oral epithelial cells. First, we have mapped immunocytochemically the distribution of cytokeratin and vimentin
intermediate filaments in epithelial and mesenchymal
cells during mouse and and chick palate development
(Ferguson, Fyfe & Lane, 1988). In mouse, cytokeratin 18 is present only in nasal epithelia, not in medial
edge nor oral epithelia (Fig. 24A). It appears progressively from the palatal shelf nasal base (day 12) to
the shelf tip (day 14-5) corresponding with the progressive differentiation of the nasal epithelia into
pseudostratified ciliated columnar cells. Cytokeratins
1 and 19 are present in all epithelia at all stages of
development. Cytokeratin 8 is present in the future
nasal and oral regions of vertical palatal shelves but
absent from the medial edge epithelia (Fig. 24B).
Cytokeratin 10 appears in the oral epithelia at day 14
when the shelves are fused. Vimentin is present in
shelf mesenchyme cells and appears in medial edge
epithelia at the time of midline epithelial seam
disruption.
Second, we have produced monoclonal antibodies
that recognize cell surface determinants on mouse
nasal, medial and oral epithelia at different stages of
development (Dixon, White & Ferguson, 1988). Of
the 104 antibody secreting hybridomas produced,
four (designated 2H6, 3H2, 6A11, 6D3) are of
considerable interest (Fig. 25). 2H6 and 3H2 recog-
56
M. W. J. Ferguson
Fig. 24. (A) Histological section through the superior region of a vertical palatal shelf (p) in a day-13 embryonic mouse
head. The specimen has been immunocytochemically stained with antibodies against cytokeratin 18. Note the presence
of this cytokeratin in the developing nasal epithelia at the superior aspect of the palatal shelf and their absence from the
adjacent tongue (r). x375. (B) A similar preparation stained with an antibody against cytokeratin 8. Note this
cytokeratin is present in the future nasal and oral epithelia but is absent from the tip of the palatal shelf and the future
medial edge epithelia. xl69.
Fig. 25. Cryosection through a day-13 embryonic mouse palatal shelf in the anterior region. The specimen has been
stained with monoclonal antibody 6D3 which recognizes future nasal and some basal medial edge epithelial cells but not
future oral epithelia. Note also the staining in the tongue (r). x210. (B) A similar preparation stained with monoclonal
antibody 6A11 which recognizes predominantly cells in the medial edge of the palatal shelf and also on the future nasal
aspect as well as the tongue (/). p, palatal shelf. x84.
Palate development
nize an epitope which is present on palatal epithelia
from the onset of palatogenesis but which progressively disappears as regional differentiation occurs.
6D3 recognizes an epitope which is expressed in
differentiating epithelia in a specific temporal and
spatial pattern. 6A11 recognizes an epitope which is
expressed on the tips of the palatal shelves just prior
to fusion i.e. in determined and differentiating medial
edge epithelia. It should also be noted that these cells
exhibit a specific lectin-staining pattern (see earlier
section entitled 'How does the epithelial seam
form?').
Thus the molecular message that makes a nasal cell
pseudostratified ciliated columnar or an oral cell
stratified squamous may involve the synthesis of a
specific repertoire of cytokeratins and cell surface (?
adhesion) molecules, not to mention receptors for
various matrix and soluble factors. Use may be made
of these molecular markers of regional palatal epithelial differentiation for experimental analysis of the
nature of mesenchyme signalling of epithelial differentiation.
What are the Important candidate genes for
normal and cleft palate?
At a phenomenological level the different developmental mechanisms (e.g. failure of shelf elevation,
inhibition of cell division, excessive head width etc.)
causing cleft palate have been summarized previously
(Ferguson, 1987). It follows from the previous
account that the genes coding for the following
molecules could be important in normal and cleft
palate development: extracellular matrix molecules
particularly hyaluronic acid, minor collagen types
e.g. type IX, tenascin; soluble factors e.g. EGF,
PDGF, TGFo-, TGF/3, FGF, cytokeratins, cell surface molecules. Moreover, as many aspects of the
intracellular signalling pathway utilized by growth
factors can be subverted by oncogene products, it is
likely that certain proto-oncogenes e.g. erb b, c cis
etc, may be particularly important in palate development. Adamson (1987) has recently summarized the
literature on proto-oncogenes in developmental systems and the differential expression of a panel of
proto-oncogenes during mouse palate development is
currently being investigated in my laboratory.
There are also data implicating the H-2 locus in
cleft palate development (Bonner & Slavkin, 1975;
Goldman, 1984) whilst a gene involved in X-linked
cleft palate has recently been mapped to the
Xql3-Xq21 regions of the human X chromosome
(Moore et al. 1987). However, in general genes
controlling pattern formation in the palate or indeed
the head are virtually unknown. To date few homeo-
57
tic genes have been localized in the cranial region.
However, there exists the tantalizing possibility that
mechanisms of pattern formation in the head (and
palate) may be similar to that elsewhere in the body,
for instance the limbs. The investigation of such
possibilities awaits the isolation of homeotic genes
localizing in the head and palate.
Work reported in this review has been and is supported,
by grants from: The Medical Research Council, Wellcome
Trust, Action Research for the Crippled Child and Birthright. Numerous collaborators and students have made
important contributions especially Larry Honig, Seth
Schor, David Garrod, Shirley Ayad, Anne White, David
Fyfe, Mike Dixon, Paul Sharpe, David Foreman, Martin
Carette, Katy Hall and Bill Moser.
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