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Extracellular coat in developing human palatal processesElectron microscopy and ruthenium red binding.

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Extracellular Coat in Developing Human Palatal Processes:
Electron Microscopy and Ruthenium Red Binding'
Department ofAnatomy, Haruard Medical School, Boston, Massachusetts 021 15
The prefusion epithelium of human palatal processes was examined for evidence of specialization which might facilitate epithelial adherence
with the opposing palatal process. A surface coat stained with ruthenium red
(RR) was found on all apical aspects of t h e palatal epithelium. In the prefusion
regions, RR staining was also observed in t h e spaces between t h e superficial cells
of t h e epithelium and in necrotic cells. Adjacent oral and nasal epithelium excluded the RR below t h e level of the apical junctional complex. In t h e absence of
RR, a dense material was observed in t h e most superficial intercellular spaces of
t h e prefusion region. Many superficial cells in the area were in various stages of
necrosis. The combination of degenerating surface cells and a n accumulation of a
poly-anionic substance such as glycoprotein may facilitate epithelial adherence
between opposing human palatal processes.
The adherence of specific regions of epithelial lined structures is a n important morphogenetic event during embryogenesis. The
fusion of t h e neural folds is one such event
t h a t has recently received considerable attention (Waterman, '76). Formation of the secondary palate is a similar event in t h e development of t h e midface in those animals
having a distinct separation of oral and nasal
cavities. The fusion of two distinct epithelial
lined processes in t h e midline results in t h e
formation of a complete secondary palate, a n
event which occurs in t h e human between t h e
seventh to thirteenth weeks after fertilization
(Kraus e t al., '66). Lack of or incomplete fusion would result in a clinical cleft palate.
The ability of t h e palatal epithelia to fuse is
precisely restricted both temporally and spatially. I t has been suggested t h a t rodent palatal shelves in vitro acquire a potential to fuse
t h a t is limited to a period of time, prior to or
after which fusion does not occur (Pourtois,
'66). The age of palates exhibiting t h e ability
to fuse in vitro corresponds to t h e appropriate
time for palatal fusion in vivo (Smiley and
Koch, '71). In addition to precise timing, t h e
palatal epithelium will adhere in vivo only to
another similar palatal process. Ectopic fusions, such as tongue to palatal process, are exANAT. REC. (1978) 190: 223-232.
tremely rare anomalies, yet during the period
of palatogenesis, t h e palatal processes are adjacent to the tongue and cheek epithelium for
prolonged periods of time (Humphrey, '70).
Morphological and biochemical investigations have focused on possible alterations t h a t
might explain the specificity of fusion in the
palatal epithelium. Since t h e initial adherence is a surface event, our interest has focused on t h e surface features of t h e prefusion
Study of palatogenesis in AlJax mice with
t h e scanning electron microscope (SEMI has
revealed a distinctive alteration in t h e palatal epithelium which mimics and precedes
t h e pattern of fusion (Waterman et al., '73).
Morphologically different surface alterations
have been reported in the prefusion areas of
human palatal processes. These areas or zones
of alteration are characterized by a disruption
of t h e usually distinct apical junctions, a general flattening of t h e surface and the appearance of superficial cell blebbing and possible
cellular necrosis (Waterman and Meller, '74).
Histological and transmission electron microscope (TEM) examination of human (Meller
and Waterman, '74) and rodent palatal epithe~~
Received Apr. 5, '77. Accepted Aug. 29. '77.
' This work was supported by NIDR Grant 1 ROL DE04695-01
lium confirms the appearance of degenerating
cells along the surface of the prefusion epithelium (Smiley, '70; Chaudhry and Shah, '73).
The biochemical specificity of t h e fusion has
recently been investigated. Increases i n glycoprotein synthesis in whole palatal shelves at
the time of fusion have been demonstrated in
rabbits (DePaola et al., '75). Attempts to localize the specific sites of glycoprotein synthesis
within the palatal processes have been difficult. Preferential adherence of Concanavillin-A on the surface of cells in the prefusion
region of r a t palatal epithelium has been demonstrated (Pratt and Hassell, '75), indicating
a surface coat rich in carbohydrate.
Ruthenium red (RR) has been used extensively to aid in the visualization of polyanionic
substances such as acid mucopolysaccharides
and glycoprotein in a variety of tissues
(Luft, '71) including mouse palatal processes
(Greene and Kochhar, '74; Souchon, '75). The
present study attempts to identify the possibility of preferential binding of RR in t h e
prefusion zones of human palatal processes.
Embryonic and fetal human palatal tissues
were obtained from specimens acquired one to
two hours after therapeutic interruption of
pregnancy via suction curettage. Thirty-two
specimens were collected for this study and
were graded by degree of palatal development
into one of six developmental stages. This classification was confirmed by heel-toe lengths of
the abortuses, and in 31 of the 32 specimens
collected there was a high degree of correlation. Six separate stages a r e easily recognized
for categorization: I. Early vertical processes;
11. Anterior half of the processes becoming
vertical; 111. Both processes horizontal; IV.
Fusion commencing; V. Anterior four-fifths of
the palate fused; VI. The entire palate fused
with only a small midline groove left in the
uvula region, (Waterman and Meller, '74).
Stages IV and V were most useful in t h e present study since i t is quite obvious'which region of the epithelium will be fusing next. Six
stage IV and five stage V palates were available for this study. Four of t h e stage IV palates
were hemisected and one half of each treated
with ruthenium red in the following manner
(Luft, '71). Palatal processes were dissected
from extraneous tissue in 0.9% saline and
immersed in a 0.2% solution of ruthenium red
( t e t r a a m m i n o r u t h e n i u m hydrochloro chloride) in a modified Ito-Karnovsky ('68) fixa-
tive with cacodylate buffer at pH 7.4. The
solution was mixed and centrifuged just prior
to use, with only the supernatant being utilized. Tissue was left in the fixative at room
temperature (RT) for two hours and then a t
4°C for 16 hours.
A rinse was prepared by centrifuging a 0.1%
solution of RR in cacodylate buffer. The tissues were rinsed in the supernatant for three
10-minute changes. Palates were then osmicated for two hours in the supernatant of a 1%
osmium and 0.1% RR solution in cacodylate
buffer and rinsed with five changes of cacodylate buffer at 5-minute intervals at RT. The
tissues were then subsequently dehydrated
through a graded series of ethanol at RT, then
placed in two changes of propylene oxide for
10-minute intervals, and left for one hour in
a 1:l mixture of propylene oxide and EponAraldite with 1%DMP 30 added. Embeddment
followed in Epon-Araldite with 2% DMP 30.
Contralateral palatal processes and other
specimens were treated for examination in the
SEM and subsequent TEM or directly for the
TEM by fixation in a modified Ito-Karnovsky
('68) primary fixation with cacodylate buffer
at RT for 12 hours, followed by secondary fixation in osmium tetroxide for two hours at RT.
Uranyl acetate en bloc staining was employed
prior to ethanol dehydration (Karnovsky, '67).
Specimens were then either embedded in
Epon-Araldite or prepared for the SEM by
critical point drying from amyl acetate using
liquid CO, in a SAMDRI PVT-3 critical point
apparatus and coated with gold/palladium in
a Technics Hummer I1 coating apparatus.
Embedded specimens were sectioned serially
a t 1 to 2-micrometer intervals with thin sections cut periodically. Specimens were examined in a JEOL 1 0 0 3 TEM, or in a JEOL JSM35 SEM.
SEM examination of a prefusion area of a
stage IV human palate (figs. 1, 3) reveals a n
epithelial lined mesenchymal process with a
distinct flat zone of alteration along the medial edge. Posterior to t h e outlined region of the
processes seen in figure 1, the medial edge
would not yet exhibit the zone of alteration
and would appear no different from the oral
epithelium. The oral and nasal epithelial
areas of the processes a r e uniform in thickness, whereas t h e zone of alteration is variable
in thickness. In the SEM t h e zone of alteration is easily distinguished from adjacent oral
and nasal epithelium (fig. 5 ) . Examination of
the oral and nasal epithelium in t h e TEM
reveals a 2 to 3-layer-thick epithelium with
cuboidal basal and intermediate level cells
and protruding, well-rounded superficial cells
(fig. 4). All cells in this area a r e laden with
large glycogen deposits (Meller and Waterman, '751, which a r e inadequately preserved
because of t h e en bloc staining with maleate
buffered uranyl acetate.
Epithelium in t h e zone of alteration (fig. 6)
is distinguished by a flattening of the superficial cells which become interleafed in a complex manner. A striking feature of this region,
observed in stage IV, and V palates, is t h e
accumulation of a granular, electron-dense
material in the most superficial intercellular
spaces (asterisk: figs. 6,9).Material of similar
density is often seen within t h e condensing
vacuoles of t h e Golgi system t h a t is frequently
found in t h e flattened cells in t h e zone of
alteration (fig. 9). Paradoxically, there a r e
many superficial cells in various stages of apparent degeneration adjacent to cells t h a t appear to be fully viable.
RR binding occurred in all aspects of t h e
palatal epithelium in stage IV. No discernible
difference in t h e thickness of RR stained
material was observed over the epithelial surface of the palatal process anywhere along its
length. These observations are in sharp contrast to those of Souchon ('751, who observed
a n increased thickness of RR staining material a t t h e prefusion sites of mouse palatal epithelium just prior to shelf transposition. A
rather unusual finding in the present study is
the appearance of RR staining in t h e intercellular spaces deep to the flattened surface
cells present on the medial edge (figs. 7, 10).
Regions of RR staining in the intercellular
spaces (fig. 10) correspond to t h e same areas
in t h e thin sections t h a t exhibit the dense intercellular material shown in figure 9. In
these areas the junctional complex apparently
lost its ability to exclude RR. This observation
correlates well with the observations in the
SEM t h a t cells in t h e zone of alteration have
indistinct apical junctions with neighboring
cells. Areas of t h e oral and nasal epithelium
adjacent to t h e medial edge seem to exclude
t h e RR at the level of t h e apical junctional
complex (fig. 8). Degenerating cells stain intensely with t h e RR (fig. 10).Earlier investigations utilizing mouse palates (Greene and
Kochler, '74, Souchon, '75) have not demonstrated any RR staining in intercellular areas,
indicating a n apparent species difference between t h e human and rodent.
I n t h e mesenchymal compartment RR
staining was observed in the areas adjacent to
t h e basal lamina (asterisk: fig. 7 ) . The staining was punctate and may represent visualization of hyaluronic acid, known to appear
in t h e embryonic palatal tissue in humans
(Matthiessen and Andersen, '72).
Epithelial contact and adherence must be
t h e first stage in t h e fusion of t h e midline epithelium during the formation of the secondary
palate. The tight adherence of two joining
palatal processes is evident by the fact t h a t
attempts to separate recently joined processes
results in tearing on either side. Numerous investigators have attempted to ascribe t h e initial adherence of palatal tissues to demonsome formation (DeAngelis and Nalbandian,
'68; Hayward, '69; Morgan '76). While i t is
true t h a t desmosomes offer a focal adhesion,
other membrane specializations a r e known to
provide adhesion a s well. Surface coats are a n
example t h a t have been studied in some detail
(Moscona, '63; Pourtois, '70; Neiders, '72).
In human palatogenesis, cellular degeneration of many superficial cells prior to fusion is
a prominent and consistent finding. Degenerating cells can hardly be expected to be
involved in junctional synthesis. I t is more
plausible to ascribe initial epithelial adherence, of the palatal processes, to the cells
subjacent to t h e superficial layer of degenerating cells, those t h a t contain t h e organelles
for synthesizing t h e cell coat material and
which exhibit a dense coat of polyanionic compounds.
Our investigation shows t h a t RR binding
occurs throughout t h e surface of the palatal
epithelium and in the mesenchyme. The cells
in the prefusion areas permit RR binding to
polyanionic substances within the intercellular spaces. These findings a r e consistent with
the hypothesis t h a t initial epithelial adherence may be related to superficial death
and exposure of cells rich in cell coat material
in a n orderly and carefully localized tissue
Chaudhry, A. P., and R. M. Shah 1973 Palatogenesis in
hamster. 11. Ultrastructural observations on the closure
of palate. J. Morph., 139: 329-350.
DeAngelis, V.,and J. Nalbandian 1968 Ultrastructure of
mouse and rat palatal processes prior to and during secondary palate formation. Arch. Oral Biol., 23: 601-608.
DePaola, D. P., J. F. Drummond, C. Lorente, R . Zarba and S.
A. Miller 1975 Glycoprotein biosynthesis at the time of
palate fusion by rabbit palate and maxilla cultured in
vitro. J. Dent. Res., 54: 1049-1055.
Greene, R. M., and D. M. Kochhar 1974 Surface coat on t h e
epithelium of developing palatine shelves in the mouse a s
revealed by electron microscopy. J. Embryol. exp. Morph.,
31: 683-692.
Hayward, A. F. 1969 Ultrastructural changes in t h e epithelium during fusion of t h e palatal processes in rats.
Arch. Oral Biol., 14: 661-678.
Humphrey, T. 1970 Palatopharyngeal fusion in a human
fetus and its relation to cleft formation. Ala. J. Med. Sci.,
7: 398-429.
Ito, S., and M. J. Karnovsky 1968 Formaldehyde-glutaraldehyde fixatives containing trinitro compounds. J. Cell
Biol., 39: 168a-169a.
Karnovsky, M. 1967 The ultrastructural basis of capillary permeability studied with peroxidase a s a tracer. J.
Cell Biol., 35: (1): 213-236.
Kraus, B. S., H. Kitamura and R. A. Latham 1966 Atlas of
Developmental Anatomy of the Face. Harper and Row,
New York.
Luft, J. H. 1971 Ruthenium red and violet. 11. Fine
structural localization in animal tissues. Anat. Rec., 171:
Mathiessen. M. and J. Andersen 1972 Disintegration of t h e
junctional epithelium of human fetal hard palate. Z.
Anat. Entwicki. Gesch., 137: 153-169.
Meller, S. M., and R. E. Waterman 1974 Correlative SEM/
histological examination of human palatal shelves. J.
Dent. Res., 53: 64a.
1975 Distribution of P A S + material in prefusion human palatal epithelium. J. Dent. Res., 54: 83a.
Morgan, P. R. 1976 The fate of the expected fusion zone
in rat fetuses with experimentally-induced cleft palate.
An ultrastructural study. Dev. Biol. 51 (23: 225-240.
Moscona, A. A. 1963 Inhibition by trypsin-inhibitors of
dissociation of embryonic tissue by trypsin. Nature (London), 199: 379-380.
Neiders, M. E. 1972 Contact phenomena of epithelial
cells. Oral Sci. Rev., 1: 69-101.
Pourtois, M. 1966 Onset of t h e acquired potentiality for
fusion in t h e palatal shelves of t h e rats. J. Embryol. exp.
Morph. 16: 171-182.
- 1970 The fate of r a t palatal shelves cultivated in
t h e presence of periodic acid. Arch. Oral Biol., 16:
P r a t t , R. M., and J. R. Hassell 1975 Appearance and distribution of carbohydrate-rich macromolecules on the
epithelial surface of the developing r a t palatal shelf. Dev.
Biol., 45: 192-198.
Smiley, G. R. 1970 Fine structure of mouse embryonic
palatal epithelium prior to and after midline fusion.
Archs. Oral Biol., 15: 287-296.
Smiley, G. R., and W. E. Koch 1971 Fine structure of mouse
secondary palate development in vitro. J. Dent. Res., 50;
Souchon, R. 1975 Surface coat of the palatal shelf epithelium during palatogenesis in mouse embryos. Anat.
Embryol., 147: 133-142.
Waterman, R. E. 1976 Topographical changes along the
neural fold associated with neurulation in the hamster
and mouse. Am. J. Anat., 246: 151.172.
Waterman, R. E., and S. M. Meller 1974 Alterations in t h e
epithelial surface of human palatal shelves prior to and
during fusion: A scanning electron microscopic study,
Anat. Rec., 180: 11-136.
Waterman, R. E., L. M. Ross and S. M. Meller 1973 Alterations in t h e epithelial surface of A/Jax mouse palatal
shelves prior to and during palatal fusion: A scanning
electron microscopic study. Anat. Rec., Z76: 361-376.
A bbreuiatrons
GC, Golgi complex
Gly, Glycogen
ICS, Intercellular space
J C , Junctional complex
MES, Mesenchyme
OC. Oro-nasal
RR, Ruthenium red
1 A drawing of a stage IV human palate. The upper lip is a t the top of the drawing, the
mandible and tongue are removed; the view is of the oral surface. The dotted lines
indicate the extent of the zone of altered epithelium. The solid line indicates the plane
of section of figure 2.
Light micrograph of 1-micrometer cornonal section of the left-palatal process from a
stage IV palate a t the indicated level. Toluidine blue staining. X 70.
3 Scanning electron micrograph of the left half of a stage IV human palate. The region
that was fused is indicated as the area between the arrows. The oral side is generally
composed of well rounded uniformly shaped superficial cells. X 25.
Transmission electron micrograph of the oral surface cells. Glycogen (Gly) is abundant in all levels of this epithelium. The surface cells are well rounded. Uranyl acetate en bloc, lead stain. X 3,000.
Scanning electron micrograph of the zone of altered cells along the medial edge of the
palate in figure 3. The normal cells of the oral surface are seen a t the uppermost region in sharp contrast to the generally flatter medial edge epithelium. Note the poor
demarcation of cell boundaries and the numerous cellular protrusions, representing
degenerating and the interleafing cells. x 190.
6 Transmission electron micrograph of the epithelium in the flat zone of alteration of a
stage IV palate. The basal cells are cuboidal, but the superficial cells are flat and interleafed. The intercellular space is quite prominent (asterisk) due to a n accumulation of a dense material in these areas. Uranyl acetate en bloc, lead stain. x 5,000.
Samuel M. Meller and Lorraine H. Barton
Transmission electron micrograph of oral epithelium of a stage IV human palatal
process treated with RR. Heavy RR binding is seen along the surface of the epithelium and a punctate staining is observed in the mesenchyme (asterisk). No RR binding is seen within the epithelium. Unstained section. X 9,000.
Transmission electron micrograph of an intercellular junction region between two
surface cells of the oral side of a stage IV human palate treated with RR. The RR is
deposited evenly along the cell surfaces. The apical junctional complex is clearly
seen with no RR binding in the intercellular spaces. Unstained section. X 30,000.
9 Transmission electron micrograph of the zone of alteration. The most superficial intercellular space is seen occupied by a dense material (asterisk). This corresponds to
the density observed in figure 6 (asterisk). Uranyl acetate en bloc, lead stain.
X 40,000.
10 Transmission electron micrograph of the surface of the zone of alteration in a stage
IV RR treated human palate. A superficial cell which is degenerating (outlined by
arrowheads) is heavily stained with RR. The intercellular spaces stain with RR
which is quite intense in areas of dilations and punctate elsewhere. No RR is found
intracellularly. Unstained section. X 30,000.
Samuel M Meller and Lorraine H. Barton
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developing, palatal, extracellular, microscopy, red, ruthenium, processeselectron, human, binding, coat
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