/. Embryo/, exp. Morph. Vol. 36, 2, pp. 225-245, 1976
Printed in Great Britain
225
Developmental aspects of secondary
palate formation
By ROBERT M. GREENE 1 AND ROBERT M. PRATT
Craniofacial Anomalies Section, Laboratory of Developmental Biology and
Anomalies, National Institute of Dental Research, Bethesda, Maryland
SUMMARY
Research on development of the secondary palate has, in the past, dealt primarily with
morphological aspects of shelf elevation and fusion. The many factors thought to be involved
in palatal elevation, such as fetal neuromuscular activity and growth of the cranial base and
mandible, as well as production of extracellular matrix and contractile elements in the palate,
are mostly based on gross, light microscopic, morphometric or histochemical observations.
Recently, more biochemical procedures have been utilized to describe palatal shelf elevation.
Although these studies strongly suggest that palatal extracellular matrix plays a major role in
shelf movement, interpretation of these data remains difficult owing to the complexity of
tissue interactions involved in craniofacial development. Shelf elevation does not appear to
involve a single motive factor, but rather a coordinated interaction of all of the abovementioned developmental events. Further analysis of mechanisms of shelf elevation requires
development of new, and refinement of existing, in vitro procedures. A system that enables
one to examine shelf elevation in vitro would allow more meaningful analysis of the relative
importance of the various components in shelf movement.
Much more is known about fusion of the palatal shelves, owing in large part to in vitro
studies. Fusion of the apposing shelves, both in vivo and in vitro, is dependent upon adhesion
and cell death of the midline epithelial cells. Adhesion between apposing epithelial surfaces
appears to involve epithelial cell surface macromolecules. Further analysis of palatal epithelial adhesion should be directed towards characterization of those cell surface components responsible for this adhesive interaction.
Midline epithelial cells cease DNA synthesis 24-36 h before shelf elevation and contact,
become active in the synthesis of cell surface glycoproteins, and subsequently manifest
morphological signs of necrosis. Death of the midline epithelial cells is thought to involve a
programmed, lysosomal-mediated autolysis. Information regarding the appearance, distribution and quantitation of epithelial hydrolytic enzymes is needed.
The control mechanisms which regulate adhesiveness and cell death in the palatal epithelium are not fully understood. Although palatal epithelial-mesenchymal recombination
experiments have demonstrated a close relationship between the underlying mesenchyme and
the differentiating epithelium, the molecular mechanism of interaction remains unclear.
Recently cyclic nucleotides have been implicated as possible mediators of palatal epithelial
differentiation.
The developing secondary palate therefore offers a system whereby one can probe a
variety of developmental phenomena. Cellular adhesion, programmed cell death and epithelial-mesenchymal interactions are all amenable to both morphological as well as bio1
Author's address: Building 30, Room 414, Laboratory of Developmental Biology &
Anomalies, National Institute of Dental Research, National Institutes of Health, Bethesda,
Maryland 20014, U.S.A.
226
R. M. GREENE AND R. M. P R A T T
chemical analysis. Although research in the field of secondary palate development has been
extensive, there still remain many provocative questions relating to normal development of
this structure.
I. INTRODUCTION
During development, the palatal shelves of most vertebrates undergo a complex reorientation from a vertical position lateral to the tongue to a horizontal
position above the tongue and fuse to form a single, continuous structure, the
secondary palate. This involves four distinct stages including growth of the
individual shelves, reorientation of the shelves and finally epithelial cell adhesion and autolysis.
Past research has emphasized the study of teratogens that induce cleft palate.
This review is not intended to deal with all these studies, but to concentrate on
normal secondary palatal development and to discuss possible areas of future
investigation. Only recently has it been appreciated that a number of developmental events including morphogenetic movements, cellular adhesion, epithelialmesenchymal interaction and programmed epithelial cell death can all be
examined during palatal development. Understanding of these events, which are
accessible to a variety of biochemical analyses, and occur relatively late in
gestation after most organogenic interactions have been completed, may be
relevant to a number of other developing tissues.
II. GROWTH AND ELEVATION OF THE PALATAL SHELVES
After formation of the first pharyngeal arch, the maxillary, frontonasal and
mandibular processes form the boundaries of the oral-nasal cavity. Cells of the
cranial neural crest are known to migrate into and contribute significantly to the
cell population of the face, including the maxillary process (Johnston &
Listgarten, 1972). It is reasonable to assume that much of the original palatal
shelf mesenchyme originates from the neural crest. The palatal shelves initially
appear as small outgrowths at the medial surface of the maxillary processes
which subsequently grow down on either side of the tongue (Fig. 1 A). At this
stage, each shelf consists of mesenchymal cells surrounded by extracellular
matrix and covered by a two- to three-cell layered epithelium (Fig. 2).
The palatal shelves begin movements which will bring them from a vertical
position on either side of the tongue into a horizontal position, superior to the
tongue (Fig. 1B). These movements occur rather late in gestation, well after
most organogenic processes have been completed, both in man (7th week of
gestation (Fulton, 1957)) and in the rat (day 16 of a 21-day gestational period
(Coleman, 1965)). Early investigators envisaged the shelves rotating, as if on a
hinge, from a vertical to a horizontal position (Peter, 1924; Lazzaro, 1940) and
recently, Walker & Ross (1972) have supported this concept.
This interpretation is dependent on the location along the anterior-posterior
length of the shelf, since movement occurs in different fashions in different
Secondary palate development
227
A
Oronasal )[ _ H Nasal
cavin—/V^-v ^^—v\ septum
Palatal
If
Tongue'^
B
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Palatal
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Oral
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Oral
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septum
Tonsue
Fig. 1
Fig. 2
Fig. 1. Schematic illustration of frontal sections taken through the anterior hard
palate of the fetal rat at various times during gestation. (A) Day 15 ( + 8 h) - the
palatal shelves have grown from the maxillary processes and assume a position on
either side of the tongue. (B) Day 16 ( + 8 h) - the shelves have rotated to a position
above the tongue, and medial-edge epithelial surfaces will make contact. (C) Day 17
( + 8 h ) - t h e medial-edge and nasal epithelial surfaces have adhered, and begun
autolysis with some mesenchymal penetration of the epithelial seam (dotted line).
Legend: N, nasal epithelium; O, oral epithelium; M, medial-edge epithelium.
Fig. 2. Photomicrograph of a single, vertically orientated mouse palatal shelf (PS)
just prior to elevation. T, Tongue; M, maxilla, x 250.
regions. Coleman (1965) has observed in the rat fetus that rostrally (anteriorly)
the palatal shelves elevate via rotation, as implied in Fig. 1A and IB, while
caudally (posteriorly) shelf elevation proceeds by means of a 'remodeling' of
the vertical shelf. Remodeling presumably involves a shift of the palatal
mesenchyme resulting in a medially directed protrusion (Fig. 3) which meets the
opposite shelf in the midline above the tongue. Similar observations in other
species have been reported (Walker & Fraser, 1956; Larsson, 1962; Kochhar &
Johnson, 1965; Harris, 1967; Greene & Kochhar, 19736).
(a) Coordinated tissue movements during shelf elevation
After growth from the maxillary processes, the palatal shelves are confronted
with a major obstacle between them, the growing tongue (Fig. 1 A). The tongue
228
R. M. GREENE AND R. M. P R A T T
MP
Fig. 3. Photomicrograph of a single palatal shelf (PS) remodeling from a vertical
position lateral to the tongue (T) to a horizontal position superior to the tongue.
Medial protrusion (MP) of the palatal shelf, x 250.
has long been thought actively to withdraw from between the shelves prior to
palatal shelf elevation. Several of the intrinsic muscles of the tongue have differentiated into fibers, responsive to both direct electrical as well as hypoglossal
nerve nucleus stimulation, at least 4 h prior to palatal shelf elevation (Wragg,
Smith &Boden, 1972). Recently, acetylcholinesterase activity was demonstrated
at developing motor end plates in the tongue of fetal mice prior to shelf elevation
(Holt, 1974). The myoneural apparatus of the tongue is therefore functional at
the time of palatal shelf elevation, suggesting that the tongue has the capacity
for voluntary movement at this time. The close synchrony between the onset
of tongue movements and shelf elevation suggests a relationship between these
two phenomena, but it is unlikely that such movement of the tongue is directly
responsible for shelf elevation. Walker & Patterson (1974«) have demonstrated
that palatal shelves will elevate despite prior removal of the tongue. The obstructive influence of the tongue is further supported by recent studies on shelf movement in vitro (Brinkley Basehoar & Avery, 1974; Brinkley, Basehoar, Branch
& Avery 1975), in which fetal mouse heads cultured prior to in vivo shelf elevation showed the most progress in attaining shelf elevation when the tongue was
removed.
A variety of other fetal movements occur during the time of shelf elevation.
The initiation of fetal mouth-opening reflexes (Humphrey, 1969) as well as
' swallowing' movements (Walker, 1969) is closely followed by palatal elevation.
To test if these were necessary for elevation of the palatal shelves, Jacobs (1971)
Secondary palate development
229
administered muscle relaxants to mice prior to and during the time of palate
elevation. Such treatments failed to produce cleft palate and these studies were
interpreted as indicating that spontaneous or reflexogenic fetal muscular
activity is not essential for normal shelf elevation. It was not, however, demonstrated that these agents actually crossed the placenta and were active in the
fetus. Recently, Walker & Patterson (19746) reported cleft palate in the offspring of pregnant mice treated with barbiturates and tranquillizers, indicating
that the developing fetus is affected, possibly by direct action on the fetal
neuromuscular system, by the action of various central nervous system depressing agents.
It still remains, however, to be clearly demonstrated that fetal movements
play a direct role in palatal development. The evidence available to date supports the concept that a moderate downward growth of the tongue anteriorly
allows elevation to proceed by rotation. In the posterior region, the tongue is
situated firmly in the common oral-nasal cavity so that a moderate movement
downward will not allow for rotation, thereby indirectly forcing the shelf to
remodel around the tongue to a horizontal position.
Growth and straightening of the cranial base has also been proposed as playing a role in palatal elevation. In rodent fetuses, the flexed cartilagenous cranial
base straightens prior to and during shelf elevation and various authors have
suggested that this straightening is necessary for shelf elevation (Harris, 1964,
1967; Verrusio, 1970; Wragg, Klein, Steinvorth & Warpeha, 1970; Smiley,
Hart & Dixon, 1971; Hart, Smiley & Dixon, 1972; Larsson, 1972; Long,
Larsson & Lohmander, 1973; Taylor & Harris, 1973). This movement, combined with growth of the lower jaw (Harris, 1967; Humphrey, 1971) results in
an increased vertical dimension of the anterior one-half of the oral cavity.
Lifting of the anterior portion of the cranial base could facilitate the rotation
of the anterior region of the palatal shelves (Harris, 1967). However, measurements of cranial base changes are usually derived from fixed, embedded
material, subject to shrinkage and other artifacts inherent in tissue preparation.
A study of cranial base changes on frozen sections should prove interesting. The
role of the cranial base in shelf elevation remains controversial, as elevation
in vitro does not require an intact cranial base (Brinkley et al. 1975).
Mandibular growth has also been implicated in palatal elevation (Zeiler,
Weinstein & Gibson, 1964). The forward growth of the mandible presumably
carries the base of the tongue from between the shelves posteriorly, and downward growth of the mandible creates room above the tongue anteriorly for the
shelves to elevate. The evidence, however, for a differential mandibular growth
spurt prior to shelf elevation is presently inconclusive (Zeiler et al. 1964; Hart,
Smiley & Dixon, 1969).
The preceding discussion (II a) points out the fact that the role these external
structures play in palatal elevation is complex and as yet unclear. It seems
reasonable that a highly coordinated interaction of many of these structures
230
R. M. GREENE AND R. M. PRATT
will prove to be necessary for elevation, but at the present state of our knowledge,
it is impossible to say which of these factors is most important.
(b) Role of cellular proliferation and extracellular matrix
An 'internal shelf force' developing from within the shelf was proposed by
Walker & Fraser (1956) to explain elevation of the palatal shelves. While it is
conceivable that rapid proliferation of mesenchymal cells in certain areas of the
palate could induce movement, Walker & Fraser (1956) and Hughes, Furstman
& Berdick (1967) found no evidence of an unusually high mitotic rate in the
shelf mesenchyme at the time of elevation. Jelinik & Dostal (1973, 1974) injected
colchicine into the amniotic fluid of the mouse fetus at various times prior to as
well as during elevation and subsequently counted metaphase figures in the
mesenchymal cells. They found that the peak of proliferation in the mesenchyme
preceded elevation by 24 to 48 h. Similar conclusions have been reached by
measuring the accumulation of DNA with time in the rat palate (Hassell, Pratt
& King, 1974).
It was thought that even after elevation, the shelves had to grow extensively
towards each other to make contact. However, recent studies using freeze-dried
(Wragg, Diewert & Klein, 1972) or freeze-sectioned material (Greene & Kochhar,
1973 a) have shown that the palatal shelves are in contact along at least part of
their medial edges immediately upon elevation. This is most evident in the
anterior region of the shelves where elevation occurs by rotation. Growth does
not appear to play a direct role in elevation of the shelves, although a critical
examination of mesenchymal growth rates in different regions of the shelf both
prior to and during elevation is needed.
Walker & Fraser (1956) postulated that an elastic fiber network in the palatal
mesenchyme provided the necessary force for elevation, although it has since
been demonstrated that no such network is seen in the palate at the time of
elevation (Stark & Ehrman, 1958; Loevy, 1962; Frommer, 1968; Frommer &
Monroe, 1969). Attention thus turned to the role of other extracellular matrix
components in elevation, since the matrix is a prominent constituent of the
shelf mesenchyme and a variety of teratogens alter the synthesis or metabolism
of its components.
Prior to and during elevation the palatal shelves actively synthesize sulfated
proteoglycans (see discussion below) (Larsson, Bostrom & Carlsoo, 1959;
Larsson, 1961; Walker, 1961) and there is also a rapid accumulation of extracellular metachromatic material in the palatal mesenchyme (Larsson, 1961;
Jacobs, 1962). This metachromatic material was degradable by testicular
hyaluronidase (Anderson & Mathiessen, 1967) and based on the known
specificity of this enzyme was presumed to be chondroitin sulfate and/or
hyaluronic acid. Pratt, Goggins, Wilk & King (1973) labeled the glycosaminoglycans synthesized by palatal shelves at the time of elevation in vivo and also in
vitro with[ 3H]-glucosamine and [35S]-sulfate. Subsequently the tissue was treated
Secondary palate development
231
with papain to remove the protein portions and the various polysaccharide
species were resolved by chromatography on DEAE-cellulose. Tissue levels were
found to reflect synthetic activity and showed that hyaluronic acid accounted
for 65% of the total glycosaminoglycans and sulfated glycosaminoglycans
accounted for the remainder.
Hyaluronate is an extremely hydrated high molecular weight unbranched
polysaccharide (Laurent, 1970), and its production and accumulation is often
associated with swelling of embryonic tissues and morphogenetic movements
(Toole, Jackson & Gross, 1972; Pratt, Larsen & Johnston, 1975). Prior to
elevation, the presumptive oral half of the palatal mesenchyme contains larger
extracellular spaces than the presumptive nasal portion of the shelf (Anderson
& Mathiesson, 1967). The accumulation of hyaluronate may be especially high
in this region of the shelf. A number of teratogens, which induce cleft palate by
inhibiting or delaying palatal elevation, alter glycosaminoglycan metabolism in
the palate (cortisone, Larsson, 1962; chlorcyclizine, Wilk, Steffek & King, 1970;
diazo-oxo-nor Leucine, DON, Pratt, Coggins, Wilk & King 1973). These studies
suggest that glycosaminoglycans, especially hyaluronate, play an important role
in shelf elevation.
Another component of the palatal shelf mesenchyme which may play a role
in elevation is collagen. Pratt & King (1971) found a significant increase in the
amount of palatal collagen, estimated to be 4-6% of the total palatal protein,
at the time of shelf elevation (day 15-16 in the rat). /?-aminopropionitrile
(BAPN) appears to inhibit shelf elevation and cause cleft palate by preventing
the crosslinking of collagen in the palate as well as the whole embryo (Steffek,
Verrusio & Watkins, 1972; Wilk, King, Horigan & Steffek, 1972; Pratt & King,
1972#, b). BAPN specifically inactivates lysyl oxidase which ordinarily oxidizes
lysine residues in collagen to aldehydes that react to form the actual crosslinks
(Pinnel & Martin, 1968). The teratogenic action of BAPN implies that collagen
fibers play a structural role in shelf elevation since no change in synthesis or
deposition is noted and presumably only the tensile strength of collagen fibers is
affected.
Collagen fibrils have been observed in the palate of the mouse (Smiley &
Dixon, 1968; Smiley, 1970). In the palate of the rat, collagen fibrils are observed
throughout the mesenchyme, but are particularly abundant in the area adjacent
to the basal lamina of the presumptive oral epithelium. Here, the fibres are
oriented uniformly along the anterior (rostral) to posterior (caudal) axis of the
shelf (Hassell & Orkin, 1974). This location in the palate suggests that these
fibers may serve as attachment or anchorage sites by which a force could be
transmitted to the rest of the mesenchyme during elevation.
Although the components of the palatal matrix have thus far been considered
individually, it is likely that they form functional macromolecular complexes.
In other systems, specific interaction between collagen and sulfated GAG (Toole
& Lowther, 1968; Lowther, Toole & Herrington, 1970; Greenwald, Schwartz &
232
R. M. GREENE AND R. M. PRATT
Cantor, 1975) and also between hyaluronate and proteoglycans (Hardingham &
Muir, 1972) have been observed. The hyaluronate-proteoglycan interaction was
subsequently shown to be essential in the organization of proteoglycan aggregates in cartilage matrices (Hascall & Heinegard, 1974). Additionally, small
molecular weight proteins were shown to be critical for stabilizing the hyaluronate-proteoglycan interaction (Gregory, 1973; Hardingham & Muir, 1974;
Hascall & Heinegard, 1974; Rosenberg, Hellman & Kleinschmidt, 1915a). A
model for these interactions has been discussed recently (Hascall & Heinegard,
1975; Rosenberg et al. 1975 b; Wiebkin, Hardingham & Muir, 1975). Such
complexes have not yet been defined in other tissues but if they occur in palatal
shelves they could be of structural importance during elevation.
Rapid movements in biological systems are usually brought about by contractile elements. The contractile nature of wound granulation tissue has
recently been demonstrated (Gabbiani, Majno & Ryan; 1973), and actin and
myosin have been reported in a variety of tissues and cells other than muscle
(Pollard & Weihing, 1974). Lessard, Wee & Zimmerman (1974) have shown
that actin and myosin are synthesized in the mouse palate at the time of shelf
movement. A calcium-dependent ATPase, presumably myosin, has been observed in the posterior area of the palate and it has been suggested that this
represents smooth muscle which along with striated muscle in the extreme
posterior soft palate, might be involved in shelf elevation (Babiarz, Allenspach
& Zimmerman, 1975).
The relative importance of the extrinsic, as well as various intrinsic, factors
involved in palatal shelf elevation can be further clarified by the development
of new and the refinement of existing in vitro methods. The in vitro procedures of
Brinkley et al. (1975) and of Walker & Patterson (1974a) may offer techniques
by which the process of shelf elevation may be directly observed.
The most widely held concept of palatal elevation supports a direct and
active involvement of the palatal shelves in their own morphogenetic movement.
This view, however, does not rule out the possible involvement of various
external factors previously discussed.
III. FUSION OF THE PALATAL SHELVES
(a) Epithelial cell adhesion
Electron microscopic studies of mouse (Farbman, 1968), rat (Hayward, 1969),
hamster (Chaudhry & Shah, 1973) and human palatal tissue (Matthiessen &
Anderson, 1972; Mato, Smiley & Dixon, 1972) have demonstrated that the fine
structure of the palatal epithelium both before and during epithelial adhesion
and seam formation is remarkably similar in these species. Prior to contact, the
epithelium of the medial edge of the elevating palatal shelves is two to three cell
layers thick with a surface squamous cell layer and underlying cuboidal cell
layer(s). The epithelial-mesenchymal border exhibits an intact basal lamina
Secondary palate development
233
MV
M
Fig. 4. Electron micrograph of a squamous epithelial cell on the surface of a mouse
palatal shelf just prior to contact with the opposite shelf. Note the microvilli (MV)
present at the surface. D, desmosome; G, Golgi apparatus; M, mitochondrion;
RER, rough endoplasmic reticulum. x 36400.
separating the epithelial cells from the underlying mesenchymal cells (DeAngelis
& Nalbandian, 1968; Smiley, 1970; Chaudhry & Shah, 1973). The surface
epithelial cells exhibit prominent microvilli, as cytoplasmic extensions (Fig. 4)
which flatten and subsequently disappear upon contact with the opposite
palatal shelf (DeAngelis & Nalbandian, 1968; Matthiessen & Anderson, 1972;
Waterman & Meller, 1974).
234
R M. GREENE AND R. M. PRATT
Once contact between the two apposing shelves has been made, a tight
adhesion develops, as attempts to separate the shelves after contact produce
tearing of epithelial cell membranes (Zeiler, Weinstein & Gibson, 1964; Farbman, 1968). Although desmosomes may support contiguity between apposing
epithelial surfaces (DeAngelis &Nalbandian, 1968; Brusati, 1969; Chaudhry &
Shah, 1973), Farbman (1968) found no evidence of desmosomes between
adhering palatal epithelial surfaces in the mouse.
Much of the present emphasis on the study of palatal adhesion can be attributed to the earlier work of Pourtois (1966, 1968«, b, 1970) on epithelial cell
adhesion and death during palatal development. Initial adhesion of one shelf
to another may be explained by the presence of an extracellular carbohydraterich surface coat on the developing epithelium of the palatal shelves. Epithelial
cell membranes of apposing shelves are separated by a 10-20 nm. Intercellular
space of uniform width (Hay ward, 1969), presumably containing surface glycoproteins. Initial adhesion mediated by cell-surface carbohydrates may serve to
keep the shelves in contact until more permanent, desmosomal connections can
be made. Farbman (1968) and Matthiessen & Anderson (1972) were unable,
utilizing various histochemical stains, to demonstrate a surface coat, although
Hay ward (1969) using transmission electron microscopy described an 'illdefined fuzzy material' on medial-edge epithelial cell surfaces prior to contact.
A carbohydrate-rich, surface coat has been shown to increase dramatically
on the epithelial surface (medial-edge) prior to contact. This has been shown at
the ultrastructural level by the binding of the plant lectin concanavalin A (CON
A) (Pratt, Gibson & Hassell, 1973a; Pratt & Hassell, 1975) and by staining with
ruthenium red (Greene & Kochhar, 1974). The chemical nature of this surface
coat is probably complex since CON A binds to mannosyl and glucosyl residues
of glycolipids and glycoproteins and ruthenium red binds to glycosaminoglycans
and glycoproteins. Scanning electron microscopic observations of the medialedge epithelial surface prior to contact in several species show the progressive
appearance of a fine filamentous material (Waterman, Ross & Meller, 1973;
Meller, personal communication, 1974), although this material is not observed
in the human (Waterman & Meller, 1974).
Surface glycoproteins and glycosyltransferases have been implicated in cellto-cell adhesion in a variety of systems (Roseman, 1970; McGuire, 1972).
DON (diazo-oxo-norleucine), a glutamine analog, has been shown to prevent
adhesion of mouse teratoma cells (Oppenheimer, 1973) and epithelial adhesion
between homotypic palatal shelves (Pratt, Greene, Hassell & Greenberg,
1975). D-glucosamine reversed the action of DON on both the teratoma cells
and palatal epithelial cells. Shelves cultured in the presence of DON bind much
less labeled CON A at the epithelial surface than shelves cultured without DON
(Greene & Pratt, 1975). These data suggest that amino sugars are incorporated
into macromolecules which are necessary for intercellular adhesion. The observation (Pourtois, 1970, 1972) that adhesion of palatal shelves in vitro was not
Secondary palate development
235
inhibited by proteolytic enzymes could be explained by rapid resynthesis of cellsurface components (Collins, Holland & Sanchez, 1973) or the resistance of
essential surface components to proteolysis.
The presence of a carbohydrate surface coat may explain the phenomenon of
an 'acquired potential' to fuse exhibited by palatal shelves of rats (Pourtois,
1966) and mice (Vargas, 1967) in vitro. It must be pointed out that the term
'fusion' refers only to the merging of mesenchyme of adjacent palatal shelves
after midline epithelial seam breakdown. Epithelial cells from adjacent shelves
adhere to one another, but there is no plasma membrane fusion during formation of the midline seam. Pourtois (1966) and Vargas (1967) have shown that
palatal explants from early mouse and rat fetuses do not fuse in vitro, although
explants from older fetuses show an increasing ability to fuse. Recently, however, Tyler & Koch (1975), using different culture conditions, have shown that
fetal mouse palates, explanted 2-5,- days prior to contact in vivo, w/7/fuse after 72 h
in culture. This suggests that the activating changes necessary for shelf-fusion
can occur in vitro. The acquisition of particular cell-surface properties is not an
uncommon occurrence as cell-surface alterations during development have been
demonstrated in a variety of embryonic systems (Moscona, 1971; Gottlieb,
Merrell & Glaser, 1974; Krach, Green, Nicholson & Oppenheimer, 1974). An
increasing number of observations indicate a functional role for palatal epithelial
cell-surface carbohydrates, and it is likely that their programmed appearance
is necessary for adhesion between adjacent palatal shelves.
B. Epithelial Cell Autolysis
Elimination of certain cells occurs during the development of a variety of
tissues. This process is frequently referred to as programmed cell death, since
the changes occur at precise stages of development (Lockshin & Williams, 1965;
Saunders & Fallon, 1966; Dorgan & Schultz, 1971). Such changes are seen in
palatal medial edge epithelial cells (Mato, Aikawa & Katahira, 1966; Smiley,
1970; Smiley & Koch, 1971; Matthiessen & Anderson, 1972; Chaudhry &
Shah, 1973). Preautolytic alterations include mitochondrial swelling (Sweeny &
Shapiro, 1970; Mato, Smiley & Dixon, 1972) and the appearance of lysosomal
bodies in medial-edge epithelial cells (Mato, Aikawa & Katahira, 1966;
Hayward, 1969).
Studies in the rat in vivo (Hudson & Shapiro, 1973) and in vitro (Pratt &
Martin, 1975) have shown by measuring the incorporation of [3H]-thymidine
that medial-edge epithelial cells cease DNA synthesis as early as 36 h prior
to contact. These cells are not at a local nutritional disadvantage since the
adjacent oral and nasal epithelial cells, as well as the mesenchymal cells,
continue to divide (Fig. 5). Although DNA synthesis ceases, medial-edge
cells incorporate [3H]-uridine into RNA (Pratt & Greene, 1975) and labeled
glucosamine and fucose into macromolecules, some of which appear to be
236
R. M.GREENE AND R. M. PRATT
:
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Fig. 5. Autoradiograms of palatal shelves cultured for 4 h submerged in growth
medium containing [3H]-thymidine at 40 /iCi/ml. (a) Day 14(+12 h) (x 350) and (b)
Day 15(+12 h) (x 350). The various areas of the epithelium are: M, medial edge;
O, oral; N, nasal epithelium; mes, mesenchyme.
transported to the cell surface (Pratt & Hassell, 1974; DePaola, Drummond,
Lorent & Miller, 1974).
Smiley & Koch (1972) demonstrated that selective death occurs in the medialedge epithelial cells of palatal shelves explanted onto Millipore filters. Recently,
Tyler & Koch (1974) demonstrated that isolated palatal epithelium explanted
onto Millipore filters also displays selective cell death in the presumptive medialedge region. Selective death of medial-edge cells was also observed in vivo in
fetuses of the A/Jax mouse that displayed spontaneous cleft lip and palate
(Smiley & Koch, 1972) and in rat fetuses after amniotic sac puncture (Goss,
Bodner & Avery, 1970; Morgan, 1975). In both these cases inhibition of
elevation due to the obstructive position of the tongue prevented epithelial
contact between shelves.
Palatal epithelial-mesenchymal recombination experiments (Pourtois, 1969;
Tyler & Koch, 1975) support the hypothesis that autolysis in medial-edge
epithelial cells is dependent on a prior interaction between these tissues. The
question of the actual time when the medial-edge cells become irreversibly
committed to cell death remains unanswered, although several studies have
Secondary palate development
237
Fig. 6. Photomicrograph of a plastic-embedded secondary palate illustrating early
seam formation between adjacent palatal shelves. S, midline epithelial seam; NS,
nasal septum, x 250.
shown that certain compounds may inhibit fusion in vitro by preventing cell
death in the medial-edge epithelium (/?-thienylalanine, Baird & Verrusio, 1973;
hadacidin, Fairbanks & Kollar, 1974; epidermal growth factor, Hassell, King
& Cohen, 1974; DON, Pratt & Greene, 1975).
The first signs of programmed cell death in the medial-edge cells is a cessation
of DNA synthesis. Numerous studies with various cultured cell lines have shown
that elevated levels of cyclic adenosine 3'-5'monophosphate (CAMP) are
associated with reduced proliferative activity (Otten, Johnston & Pasten, 1972;
Bombik & Burger, 1973). In the rat palatal shelf, levels of cyclic AMP have been
shown to increase dramatically approximately 18 to 24 h prior to epithelial
contact (Pratt & Martin, 1975). Whether or not this increase occurs in the
epithelium or is related to cessation of DNA synthesis in the medial-edge cells
is not known. The addition of dibutyryl cyclic AMP to the immature day-14 rat
shelf in vitro causes a precocious decrease in medial-edge epithelial DNA
synthesis and an increase in glycoprotein synthesis. These induced changes
appear to mimic those that normally occur on days 15 to 16. These results
suggest that activation of medial-edge cells to mature in vivo, involving both
decreased proliferation and increased adhesiveness, may in part be mediated
through cyclic nucleotides. This is further supported by the ability of exogenous
dibutyryl cyclic AMP to prevent EGF-induced inhibition of epithelial cell death
l6
EMB 36
Fig. 7. (Inset) Photomicrograph of a secondary palate illustrating a late midline epithelial seam (S). x 250.
Fig. 8. Electron micrograph of a palatal midline epithelial seam (S) correspondingto the area indicated by the arrow
in Fig. 7, demonstrating an intact basement lamina (BL) separating the cells of the seam (S) from the connective
tissue cells of the palatal mesenchyme (MES). x 14200.
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oo
Secondary palate development
239
(Pratt, Green, Hassel & Greenberg 1975). There are undoubtably other factors
involved in this activation and maturation process, although they remain
unknown at the present time.
The developing palatal shelves in the chick embryo may provide an interesting
system in which to investigate these programmed changes. Chick palatal
shelves do not appear to maintain epithelial contact and therefore formation of
a fused secondary palate never occurs here. A comparison between the programmed changes involved in palatal adhesion and fusion of most vertebrates
and the events which bring about the cleft palate of the chick should prove
interesting. The developing eyelids (Anderson, Ehlers & Matthiessen, 1965;
Anderson et al. 1967) may provide another system to investigate epithelial
adhesion, and control of these developmental events.
After midline epithelial adhesion and the onset of autolysis, the epithelium of
one shelf soon becomes indistinguishable from that derived from the adjacent
shelf, as an epithelial seam is formed in the midline of the future secondary
palate. The midline epithelial seam changes rapidly from the original 4-5 celllayered seam (Fig. 6) to a 1-2 cell-layered strip of cells (Fig. 7) (Smiley & Dixon,
1968; Hayward, 1969). Superiorly, at the nasal surface of the secondary palate,
the seam expands into a triangular area (Smiley & Dixon, 1968), and inferiorly,
at the oral surface, the seam also expands into a triangular area. Both of these
areas are characterized by large intercellular spaces with cells containing
numerous electron-dense granular structures, presumably lysosomal in nature
since they have been shown to be positive for acid phosphatase (Mato, Aikawa
& Katahira, 1967).
Lysosomal bodies appear throughout the palatal epithelial midline seam and
are rare in the epithelium covering the oral and nasal palatal surfaces (Mato,
Aikawa & Katahira, 1967). In a study of the role of acid phosphatase in fusion
of the secondary palate, Angelici & Pourtois (1968) localized all enzyme reactions in the epithelial cytoplasm. Autolysis of the midline seam is accomplished
by lysosomal enzymes produced by the midline epithelial cells, indicating a
primary lysosomal involvement in this type of programmed cell death. This is in
contrast to programmed cell death in the posterior necrotic and interdigital zone
of the developing chick limb, where cells are first fragmented by unknown
mechanisms and these fragments taken up and degraded by macrophages
containing lysosomal enzymes (secondary lysosomal involvement) (Saunders &
Fallon, 1966).
As autolytic breakdown of the midline seam and the superior and inferior
triangular areas proceeds, the epithelial cells become irregular in outline and are
separated by increasingly larger extracellular spaces. The epithelial cells, however, remain connected throughout this process by desmosomes, and initial
epithelial degeneration is not directly associated with apparent breakdown of
the basal lamina (Hayward, 1969) (Fig. 8). The final phases of breakdown of the
midline epithelial seam in both hard and soft palate involve disruption of the
16-2
240
R. M. GREENE AND R. M. PRATT
basal lamina interposed between epithelial and mesenchymal cells, and migration of macrophages into this region with subsequent phagocytosis of degenerating epithelial cells (Koziol & Steffek, 1969; Matthiessen & Anderson, 1972;
Shah & Chaudhry, 1974). Epithelial seam breakdown has been assumed to
allow extensive intermixing of the mesenchymal cells of adjacent palatal shelves.
However, Koch & Smiley (1973) have combined in vitro colchicine-treated
shelves labeled with [3H]thymidine and unlabeled shelves, and have thus shown
that little intermixing takes place between mesenchymal cells derived from
originally separate shelves.
We wish to thank Dr George Martin, Laboratory of Developmental Biology and Anomalies, National Institute of Dental Research, for numerous helpful suggestions during the preparation of this manuscript. We are also indebted to Mrs Barbara Stoops for her excellent
secretarial assistance.
REFERENCES
H., EHLERS, N. & MATTHIESSEN, M. E. (1965). Histochemistry and development
of the human eyelids. Acta Ophthal. 43, 642-668.
ANDERSEN, H., EHLERS, N., MATTHIESSEN, M. E. & CLAESSON, M. H. (1967). Histochemistry
and development of the human eyelids. II. A cytochemical and electron microscopic study.
Acta Ophthal. 45, 288-293.
ANDERSEN, H. & MATHIESSEN, M. E., (1967). Histochemistry of the early development of the
human central face and nasal cavity with special reference to the movements and fusion
of the palatine processes. Acta Anat. 68, 473-508.
ANGELICI, D. & POURTOIS, M. (1968). The role of acid phosphatase in the fusion of the
secondary palate. /. Embryol. exp. Morph. 20, 15-23.
BABIARZ, B., ALLENSPACH, A. L. & ZIMMERMAN, E. F. (1975). Ultrastructural evidence of
contractile systems in mouse palates prior to rotation. Devi Biol. 47, 32-44.
BAIRD, G. & VERRUSIO, A. C. (1973). Inhibition of palatal fusion in vitro by /?-2-thienylalanine. Teratology 7, 37-48.
BOMBIK, B. M. & BURGER, M. M. (1973). Cyclic AMP and the cell cycle: Inhibition of growth
stimulation. Expl Cell Res. 80, 88-94.
BRINKLEY, L. & AVERY, J. K. (1975). Mechanical intervention of the cranial base during
palatal shelf elevation in vitro. J. dent. Res. 54, 80.
BRINKLEY, L., BASEHOAR, G. & AVERY, J. K. (1974). Effects of craniofacial structures on in
vitro palatal shelf elevation. /. dent. Res. 53, 63.
BRINKLEY, L., BASEHOAR, G., BRANCH, A. & AVERY, J. (1975). New in vitro system for studying secondary palatal development. /. Embryol. exp. Morph. 34, 485-495.
BRUSATI, R. (1969). Ultrastructural study of the processes of formation and involution of the
epithelial sheet of the secondary palate in the rat. /. submicrosc. Cytol. 1, 215-234.
CHAUDHRY, A. P. & SHAH, R. M. (1973). Palatogenesis in hamster. II. Ultrastructural
observations on the closure of palate. /. Morph. 139, 329-350.
COLEMAN, R. D. (1965). Development of the rat palate. Anat. Rec. 151, 107-118.
COLLINS, M. F., HOLLAND, K. D. & SANCHEZ, R. (1973). Regeneration of sialic acidcontaining components of embryonic cell surfaces. /. exp. Zool. 183, 217-224.
DEANGELIS, V. & NALBANDIAN, J. (1968). Ultrastructure of mouse and rat palatal processes
prior to and during secondary palate formation. Archs Oral Biol. 13, 601-608.
DEPAOLA, D. P., DRUMMOND, J. F., LORENT, C. A. & MILLER, S. A. (1974). Glycoprotein
biosynthesis associated with fusion of rabbit palates in vitro. J. dent. Res. 53, 134.
DORGAN, W. J. & SCHULTZ, R. L. (1971). An in vitro study of programmed cell death in rat
placental giant cells. /. exp. Zool. 178, 497-512.
FAIRBANKS, M. B. & KOLLAR, E. J. (1974). Inhibition of palatal fusion in vitro by hadacidin.
Teratology 9, 169-178.
ANDERSON,
Secondary palate development
241
A. I. (1968). Electron microscopic study of palate fusion in mouse embryos. Devi
Biol. 18, 93-116.
FROMMER, J. (1968). The lack of evidence for elastic fiber involvement in the closure of the
palatal shelves. Anat. Rec. 60, 471.
FROMMER, J. & MONROE, C. W. (1969). Further evidence for the absence of elastic fibers
during movement of the palatal shelves in mice. /. dent. Res. 48, 155-156.
FULTON, J. T. (1957). Closure of the human palate in embryo. Am. J. Obstet. Gynec. 74,179182.
GABBIANI, G., MAJNO, G. & RYAN, G. B. (1973). The fibroblast as a contractile cell: The
myo-fibroblast. In Biology of Fibroblast (ed. by E. Kulonon & J. Pikkorainon), pp. 139154. London & New York: Academic Press.
Goss, A. N., BODNER, J. W. & AVERY, J. K. (1970). In vitro fusion of cleft palate shelves.
Cleft Palate 7,737-747.
GOTTLIEB, D. I., MERRELL, R. & GLASER, L. (1974). Temporal changes in embryonal cell
surface recognition. Proc. natn. Acad. Sci., U.S.A. 71, 1800-1802.
GREENE, R. M. & KOCHHAR, D. M. (1973 a). Palatal closure in the mouse as demonstrated in
frozen sections. Am. J. Anat. 137, 477-482.
GREENE, R. M. & KOCHHAR, D. M. (19736). Spatial relations in the oral cavity of cortisonetreated mouse fetuses during the time of secondary palate closure. Teratology 8, 153—
162.
GREENE, R. M. & KOCHHAR, D. M. (1974). Surface coat on the epithelium of developing
palatine shelves in the mouse as revealed by electron microscopy. /. Embryol. exp. Morph.
31, 683-692.
GREENE, R. M. & PRATT, R. M. (1975). Inhibition of palatal epithelial cell adhesion in vitro
by diazo-oxo-norleucine. Teratology 11, 19A.
GREENWALD, R. A., SCHWARTZ, C. E. & Cantor, J. O. (1975). Interaction of cartilage proteoglycan with collagen-substituted agarose gels. Biochem. J. 145, 601-605.
GREGORY, J. D. (1973). Multiple aggregation factors in cartilage proteoglycan. Biochem. J.
133, 383-386.
HARDINGHAM, T. E. & MUIR, H. (1972). The specific interaction of hyaluronic acid with
cartilage proteoglycans. Biochim. biophys. Ada 270, 401-405.
HARDINGHAM, T. E. & MUIR, H. (1974). Hyaluronic acid in cartilage and proteoglycan
aggregation. Biochem. J. 139, 565-581.
HARRIS, J. W. S. (1964). Oligohydramnios and cortisone-induced cleft palate. Nature, Lond.
203, 533-534.
HARRIS, J. W. S. (1967). Experimental studies on closure and cleft formation in the secondary
palate. 5c/. Basis Med. Ann. Rev. 356-369.
HART, J. C, SMILEY, G. R. & DIXON, A. D. (1969). Sagittal growth of the craniofacial complex in normal mice. Archs. Oral Biol. 14, 995-997.
HART, J. C, SMILEY, G. R. & DIXON, A. D. (1972). Sagittal growth trends of the craniofacial
complex during formation of the secondary palate in mice. Teratology 6,43-50.
HASCALL, V. C. & HEINEGARD, D. (1974). Aggregation of cartilage proteoglycans. I. The role
of hyaluronic acid. /. biol. Chem. 249, 4232-4241.
HASCALL, V. C. & HEINEGAARD, D. (1975). The structure of cartilage proteoglycans. In
Extracellular Matrix Influences on Gene Expression (ed. H. C. Slavkin & R. C. Greulich),
pp. 423-434. New York & London: Academic Press.
HASSELL, J. R., KING, C. T. G. & COHEN, S. (1974). Inhibited epithelial cell death in cultured
rat palatal shelves. /. dent. Res. 53, 65.
HASSELL, J. R. & ORKIN, R. W. (1974). Synthesis and distribution of collagen in the developing palate. /. Cell Biol. 63, 132 a.
HASSELL, J. R., PRATT, R. M. & KING, C. T. G. (1974). Production of cleft palate in the rat
by growth inhibition. Teratology 9, A-19.
HAYWARD, F. (1969). Ultrastructural changes in the epithelium during fusion of the palatal
processes in rats. Archs Oral Biol. 14, 661-673.
HOLT, M. (1974). A light and electron microscopic cytochemical study of the tongue of
normal and teratogical mice. Anat. Rec. 174, 378.
FARBMAN,
242
R. M. GREENE AND R. M. PRATT
C. D. & SHAPIRO, B. L. (1973). A radioautographic study of deoxyribonucleic acid
synthesis in embryonic rat palatal shelf epithelium with reference to the concept of programmed cell death. Archs Oral Biol. 18, 77-84.
HUGHES, L. V., FURSTMAN, L. & BERDICK, S. (1967). Prenatal development of the rat palate.
/. dent. Res. 46, 373-379.
HUMPHREY, T. (1969). The relation between human foetal mouth opening reflexes and
closure of the palate. Am. J. Anat. 125, 317-344.
HUMPHREY, T. (1971). Development of oral and facial motor mechanism in human fetuses
and their relation to craniofacial growth. /. dent. Res. 50, 1428-1441.
JACOBS, R. M. (1962). Histochemical study of morphogenesis and teratogenesis of the palate
in mouse embryos. Anat. Rec. 149, 691-697.
JACOBS, R. M. (1971). Failure of muscle relaxants to produce cleft palate in mice. Teratology
4, 25-30.
JELINEK, R. & DOSTAL, M. (1973). The role of mitotic activity in the formation of the secondary palate. Ada chir. Plast. 15, 216-222.
JELINEK, R. & DOSTAL, M. (1974). Morphogenesis of cleft palate induced by exogenous
factors. VII. Mitotic activity during formation of the mouse secondary palate. Folia
Morph. (Praha) 22, 94-101.
JOHNSTON, M. C. & LISTGARTEN, M. A. (1972). Observation on the migration, interaction
and early differentiation of orofacial tissues. In Developmental Aspects of Oral Biology (ed.
H. C. Slavkin & L. A. Bavetta), pp. 53-79. New York and London: Academic Press.
KOCH, W. E. & SMILEY, G. R. (1973). An analysis of'fusion' during secondary palate formation. Anat. Rec. 175, 361.
KOCHHAR, D. M. & JOHNSON, E. M. (1965). Morphologic and autoradiographic studies of
cleft palate induced in rat embryos by maternal hypervitaminosis A. /. Embryol. Exp.
Morph. 14, 223-238.
KOZIOL, C. A. & STEFFEK, A. K. (1969). Acid phosphatase activity in palates of developing
normal and chlorcyclizine treated rodents. Archs Oral Biol. 14, 317-321.
KRACH, S. W., GREEN, A., NICHOLSON, G. L. & OPPENHEIMER, S. B. (1974). Cell surface
changes occurring during sea urchin embryonic development monitored by quantitative
agglutination with plant lectins. Expl Cell Res. 84, 191-198.
LARSSON, K. S. (1961). Studies on the closure of the secondary palate. III. Autoradiographic
and histochemical studies in the normal mouse embryo. Acta. morph. neerl.-scan. 4, 349367.
LARSSON, K. S. (1962). Studies on the closure of the secondary palate. IV. Autoradiographic
and histochemical studies of mouse embryos from cortisone-treated mothers. Acta. morph.
neerl.-scan. 4, 369-386.
LARSSON, K. S. (1972). Mechanisms of cleft palate formation. In Congenital Defects. New
Directions in Research (ed. D. T. Janerich, R. G. Skalko & I. H. Porter), pp. 255-273.
New York and London: Academic Press.
LARSSON, K. S., BOSTROM, H. & CARLSOO, S. (1959). Studies on the closure of the secondary
palate. I. Autoradiographic study in the normal mouse embryo. Expl Cell Res. 16, 379-383.
LAURENT, T. C. (1970). Structure of hyaluronic acid. In: Chemistry and Molecular Biology
of the Intercellular Matrix (ed. E. A. Balazs), pp. 703-732. London and New York:
Academic Press.
LAZZARO, C. (1940). Sul meccanismo di chiusura del palato secondario. Monitore zool. ital.
51, 249-273.
LESSARD, J. L., WEE, E. L. & ZIMMERMAN, D. F. (1974). Presence of contractile proteins in
mouse fetal palate prior to shelf elevation. Teratology 9, 113-126.
LOCKSHIN, R. A. & WILLIAMS, C. M. (1965). Programmed cell death. V. Cytolytic enzymes in
relation to the breakdown of the intersegmental muscles of silkmoths. /. Insect Physiol. 11,
831-844.
LOEVY, H. (1962). Developmental changes in the palate of normal and cortisone treated
Strong a mice. Anat. Rec. 142, 375-390.
LONG, S. Y., LARSSON, K. S. & LOHMANDER, S. (1973). Cell proliferation in the cranial base
of A/J mice with 6-AN-induced cleft palate. Teratology 8, 137-138.
HUDSON,
Secondary palate development
243
D. A., TOOLE, B. P. & HERRINGTON, A. C. (1970). Interaction of proteoglycans
with tropocollagen. In Chemistry and Molecular Biology of the Intercellular Matrix (ed.
E. A. Balazs), pp. 1135-1153. London and New York: Academic Press.
MCGUIRE, E. J. (1972). A possible role for carbohydrates in cell-cell adhesion. In Membrane
Research (ed. C. R. Fox), pp. 347-368. New York and London: Academic Press.
MATO, M., AIKAWA, E. & KATAHIRA, M. (1966). Appearance of various types of lysosomes
in the epithelium covering lateral palatine shelves during a secondary palate formation.
Gunma J. med. Sci. 15, 46-56.
MATO, M., AIKAWA, E. & KATAHIRA, M. (1967). Alterations of the fine structure of the epithelium on the lateral palatine shelf during the secondary palate formation. Gunma J. med.
Sci. 16, 79-99.
MATO, M., SMILEY, G. R. & DIXON, A. D. (1972). Epithelial changes in the presumptive
regions of fusion during secondary palate formation. /. dent. Res. 5, 1451-1456.
MATTHIESSEN, M. & ANDERSON, H. (1972). Disintegration of the junctional epithelium of
human fetal hard palate. Z. Anat. EntwGesch. 137, 153-169.
MORGAN, P. (1975). The duration of epithelial degeneration in rat fetuses with cleft palate.
/. dent. Res. 48, LI 3.
MOSCONA, A. A. (1971). Embryonic and neoplastic cell surfaces: Availability of receptors for
concanavalin A and wheat germ agglutinin. Science, N. Y. 171, 905-907.
OPPENHEIMER, S. B. (1973). Utilization of L-glutamine in intercellular adhesion: Ascietes
tumor and embryonic cells. Expl Cell Res. 11, 175-182.
OTTEN, I., JOHNSTON, G. S. & PASTAN, I. (1972). Regulation of cell growth by cyclic AMP.
/. biol. Chem. 247, 7082-7086.
PETER, K. (1924). Die Entwicklung des Saugetiergaumens. Ergebn. Anat. EntwGesch. 25,
448-564.
PINNEL, S. R. & MARTIN, G. R. (1968). The cross-linking of collagen and elastin. Enzymatic
conversion of lysine in peptide linkage to a-aminoadipic a-semialdehyde (allysine) by an
extract from bone. Proc. natn. Acad. Sci., U.S.A. 61, 708-714.
POLLARD, T. D. & WEIHING, R. R. (1974). Actin and myosin in cell movement. Critical
Reviews in Biochem 2, 1-65.
POURTOIS, M. (1966). Onset of acquired potentiality for fusion in the palatal shelves of rats.
/. Embryol. exp. Morph. 16, 171-182.
POURTOIS, M. (1968 a). Amniotic fluid and palatal fusion in the rat. Archs Oral Biol. 13,
87-92.
POURTOIS, M. (19686). La fusion des cretes palatines et son alternative par quelques agents
teratogenes. Archs Biol. 79, 1-74.
POURTOIS, M. (1969). Study on the fusion of palatal processes in vitro and in vivo. J. dent. Res.
48, .1135.
POURTOIS, M. (1970). The fate of rat palatal shelves cultivated in vitro in the presence of
periodic acid. Archs Oral. Biol. 16, 503-508.
POURTOIS, M. (1972). Morphogenesis of the primary and secondary palate. In Developmental Aspects of Oral Biology (ed. H. C. Slavkin & L. A. Bavetta), pp. 81-108. London:
Academic Press.
PRATT, R. M., GIBSON, W. A. & HASSELL, J. R. (1973). Concanavalin A binding to the
secondary palate of the embryonic rat. /. dent. Res. 52, 111.
PRATT, R. M., GOGGINS, J. R., WILK, A. L. & KING, C. T. G. (19736). Acid mucopolysaccharide synthesis in the secondary palate of the developing rat at the time of rotation
and fusion. Devi Biol. 32, 230-237.
PRATT, R. M. & GREENE, R. M. (1975). Inhibition of programmed epithelial cell death in the
developing rat secondary palate by diazo-oxo-norleucine (DON). /. Cell Biol. 67, 344a.
PRATT, R. M. & GREENE, R. M. (1975). Inhibition of palatal epithelial cell death by altered
protein synthesis. Devi Biol. (in press)
PRATT, R. M., GREENE, R. M., HASSELL, J. R. & GREENBERG, J. (1975). Epithelial cell
differentiation during secondary palate development. In Extracellular Matrix Influences on
Gene Expression, (ed. H. C. Slavkin & R. C. Greulich), pp. 561-566. New York: Academic
Press.
LOWTHER,
244
R. M. GREENE AND R. M. PRATT
R. M. & HASSELL, J. R. (1974). Prefusion synthesis of a carbohydrate-rich surface
coat in the rat palatal epithelium. /. dent. Res. 53, 64.
PRATT, R. M. & HASSELL, J. R. (1975). Appearance and distribution of carbohydrate-rich
macromolecules on the epithelial surface of the developing rat palatal shelf. Devi Biol. 45,
192-198.
PRATT, R. M. & KING, C. T. G. (1971). Collagen synthesis in the secondary palate of the
developing rat. Archs Oral Biol. 16, 1181-1185.
PRATT, R. M. & KING, C. T. G. (1972a). Inhibition of collagen cross-linking associated with
/?-aminopropionitrile-induced cleft palate in the rat. Devi Biol. 27, 322-328.
PRATT, R. M. & KING, C. T. G. (19726). Effect of A-aminopropionitrile (BAPN) on collagen
cross-linking in the secondary palate of the rat. Teratology 5, 261.
PRATT, R. M., LARSEN, M. A. & JOHNSTON, M. C. (1975). Migration of cranial neural crest
cells in a cell-free hyaluronate-rich matrix. Devi Biol. 44, 298-305.
PRATT, R. M. & MARTIN, G. R. (1975). Epithelial cell death and cyclic AMP increase during
palatal development. Proc. natn. Acad. Sci., U.S.A. 72, 874-877.
ROSEMAN, S. (1970). Glycosyltransferases and their function in cell-cell adhesion. Chem.
Phys. Lipids 5, 270-297.
ROSENBERG, L., HELLMAN, W. & KLEINSCHMIDT, A. K. (1975). Electron microscopic studies
of proteoglycan aggregates from bovine articular cartilage. /. biol. Chem. 250, 18871883.
ROSENBERG, L., MARGOLIS, R., WOLFENSTEIN-TODEL, C , PAL, S. & STRIDER, W. (1975).
Organization of extracellular matrix in bovine articular cartilages. In Extracellular Matrix
Influences on Gene Expression, (ed. H. C. Slavkin & R. C. Greulich), pp. 415-421. New
York and London: Academic Press.
SAUNDERS, J. W. & FALLON, J. F. (1966). Cell death in morphogenesis. In Major Problems in
Developmental Biology (ed. M. Locke), pp. 289-314. New York: Academic Press.
SHAH, R. M. & CHAUDHRY, A. P. (1974). Ultrastructural observations on closure of the soft
palate in hamsters. Teratology 10, 17-30.
SMILEY, G. R. (1970). Fine structure of mouse embryonal palatal epithelium prior to and
after midline fusion. Archs Oral Biol. 15, 287-296.
SMILEY, G. R. & DIXON, A D. (1968). Fine structure of midline epithelium in the developing
palate of the mouse. Anat. Rec. 161, 293-310.
SMILEY, G. R., HART, J. C. & DIXON, A. D. (1971). Growth of the craniofacial complex
during formation of the secondary palate. /. dent. Res. 50, 1506-1507.
SMILEY, G. R. & KOCH, W. E. (1971). Fine structure of mouse secondary palate development
in vitro. J. dent. Res. 59, 1671-1677.
SMILEY, G. R. & KOCH, W. E. (1972). An in vitro and in vivo study of single palatal processes.
Anat. Rec. 173, 405^16.
STARK, R. B. M. & EHRMAN, N. A. (1958). The development of the center of the face with
particular reference to surgical correction of bilateral cleft lip. Plastic reconstr. Surg. 21,
177-184.
STEFFEK, A. J., VERRUSIO, A. C. & WATKINS, C. A. (1972). Cleft palate in rodents after
maternal treatment with various lathyrogenic agents. Teratology 5, 33-40.
SWEENY, L. R. & SHAPIRO, B. L. (1970). Histogenesis of Swiss white mouse secondary palate
from nine and one half days to fifteen and one half days in utero. I. Epithelial mesenchymal
relationships, light and electron microscopy. /. Morph. 130, 435-450.
TAYLOR, R. G. & HARRIS, J. W. S. (1973). Growth and spatial relationships of the cranial
base and lower jaw during closure of the secondary palate in the hamster. /. Anat. 115,
149-150.
TOOLE, B., JACKSON, G. & GROSS, J. (1972). Hyaluronate in morphogenesis: Inhibition of
chondrogenesis in vitro. Proc. natn. Acad. Sci., U.S.A. 69, 1384-1386.
TOOLE, B. P. & LOWTHER, D. A. (1968). The effect of chondroitin-sulphate protein on the
formation of collagen fibrils in vitro. Biochem. J. 109, 857-859.
TYLER, M. S. & KOCH, W. S. (1974). Epithelial-mesenchymal interactions in the secondary
palate of the mouse. /. dent. Res. 53, 64.
PRATT,
Secondary palate development
245
M. S. & KOCH, W. S. (1975). In vitro development of palatal tissues from embryonic
mice. I. Differentiation of the secondary palate from 12 day mouse embryos. Anat. Rec.
(In press).
VARGAS, V. I. (1967). Palatal fusion in vitro in the mouse. Archs Oral Biol. 12, 1283-1288.
VERRUSIO, A. C. (1970). A mechanism for closure of the secondary palate. Teratology 3,
17-20.
WALKER, B. E. (1961). The association of mucopolysaccharides with morphogenesis of the
palate and other structures in mouse embryos. /. Embryol. exp. Morph. 9, 22-31.
WALKER, B. E. (1969). Correlation of embryonic movement with palate closure in mice.
Teratology 2, 191-198.
WALKER, B. E. & FRASER, F. C. (1956). Closure of the secondary palate in three strains of
mice. /. Embryol. exp. Morph. 4, 176-189.
WALKER, B. E. & PATTERSON, A. (1914a). The mechanism of cortisone-induced cleft palate.
/. dent. Res. 55, 63.
WALKER, B. D. & PATTERSON, A. (19746). Induction of cleft palate in mice by tranquilizers
and barbiturates. Teratology 10, 159-163.
WALKER, B. E. & Ross, L. M. (1972). Observation of palatine shelves in living rabbit embryos.
Teratology 5, 97-102.
WATERMAN, R. E. & MELLER, S. M. (1974). Alterations in the epithelial surface of human
palatal shelves prior to and during fusion: A scanning electron microscopic study. Anat.
Rec. 180, 111-136.
WATERMAN, R. E., ROSS, L. M. & MELLER, S. M. (1973). Alterations in the epithelial surface
of A/Jax mouse palatal shelves prior to and during palatal fusion: A scanning electron
microscopic study. Anat. Rec. 176, 361-376.
WIEBKIN, O. W., HARDINGHAM, T. E. & MUIR, H. (1975). Hyaluronic acid-proteoglycan
interaction on the influence of hyaluronic acid on proteoglycan synthesis by chondrocytes
from adult cartilage. In Extracellular Matrix Influences on Gene Expression (ed. H. C.
Slavkin & R. C. Greulich), pp. 209-224. New York and London: Academic Press.
WILK, A. L., KING, C. T. G., HORIGAN, E. A. & STEFFEK, A. J. (1972). Metabolism of
/?-aminopropionitrile and its teratogenic activity in rats. Teratology 5, 41-48.
WILK, A., STEFFEK, A. J. & KING, C. T. G. (1970). Norchlorcyclizine analogs: relationship
of teratogenic activity to in vitro cartilage binding. /. Pharmac. exp. Ther. Ill, 118-126.
WRAGG, L. E., DIEWERT, V. M. & KLEIN, M. (1972). Spatial relations in the oral cavity and
the mechanisms of secondary palate closure in the rat. Archs Oral Biol. 17, 683-690.
WRAGG, L. E., KLEIN, M., STEINVORTH, G. & WARPEHA, R. (1970). Facial growth accommodation secondary palate closure in rat and man. Archs Oral Biol. 15, 705-719.
WRAGG, L. E., SMITH, J. A. & BODEN, C. S. (1972). Myoneural maturation and function
of the foetal rat tongue at the time of secondary palate closure. Archs Oral Biol. 17,673-682.
ZEILER, K. B., WEINSTEIN, S. & GIBSON, R. D. (1964). A study of the morphology and the
time of closure of the palate in the albino rat. Archs Oral Biol. 9, 545-554.
TYLER,
{Received 12 January 1976)