/. Embryo/, exp. Morph. Vol. 53, pp. 1-13, 1979
Printed in Great Britain © Company of Biologists Limited 1979
A cellular mechanism for the palatal shelf
reorientation from a vertical to a horizontal plane
in hamster: light and electron microscopic study
By RAVINDRAM. SHAH1
From the Department of Oral Biology, Faculty of Dentistry, The University
of British Columbia
SUMMARY
Cellular and subcellular events during reorientation of the palatal shelf in hamster fetuses
are described. An alteration in the morphology of the epithelial and the mesenchymal cells is
observed during shelf realignment from a vertical to a horizontal plane. The mesenchymal
cells elongate and subsequently appear to protrude in the medially bulging palatal shelf.
Microtubules, microfilaments and close contacts are associated with elongation of the mesenchymal cells. The cells of the thickened epithelium may play a mechanical role in providing
direction to the mesenchymal cells during palatal shelf reorientation. The altered morphology
of the mesenchymal cells may be associated with the intrinsic shelf force implicit in Walker
and Fraser's theory of palatal shelf reorientation.
INTRODUCTION
Following the publication of Dursy's study in 1869 on the development of
the secondary palate, one of the much discussed questions was how the vertical
palatine processes, at first separated by the tongue, are able to move up to
a horizontal position. Several possibilities were suggested and disputed regarding the mechanism underlying reorientation of the palatal shelf (see Ferguson,
1978 for details). However, the mechanism still remains unclear.
A literature review of the electron microscopic observations of palatal development in rodents and human fetuses indicates that most studies were concerned
with differentiation and fate of midline epithelial cells after the palatal shelves
were realigned from a vertical to a horizontal plane. However, few studies
describing ultrastructural aspects of both the epithelial and the mesenchymal
cells during palatal shelf reorientation have been made (Walker, 1961; Babiarz,
Allenspach & Zimmerman, 1975; Innes, 1978; Ferguson, 1978). These were
carried out in mice and rats and the results were conflicting.
Shah & Travill (1976 a) observed that the reorientation of palatal shelves
from a vertical to a horizontal plane in hamster fetuses occurred between days
1
Author's address: Department of Oral Biology, Faculty of Dentistry, The University of
British Columbia, Vancouver, B.C. V6T 1W5, Canada.
2
R. M. SHAH
12:00 and 12:04 (12 days 4 h) of gestation. They further noted that the mode
of palatal shelf reorientation in hamster fetuses resembled that in humans
(Anderson & Matthiessen, 1967; Aronov, 1970) and mice (Walker & Fraser,
1956; Greene & Kochhar, 1973). The purpose of the present investigation is to
study cellular and subcellular events taking place at the time of change in position
of the palatal shelf from a vertical to a horizontal plane in the hamster fetus.
Further, an effort will be made to relate the observations of the present study
to those reported in the literature to propose a hypothetical mechanism of shelf
reorientation.
MATERIALS AND METHODS
Maintenance and breeding of Golden Syrian hamster have been described
elsewhere (Shah & Travill, 1976a). The pregnant hamsters, in a group of five,
were killed at 1 h intervals between days 12:00 and 12:04 of gestation. Half the
fetuses from each litter were fixed in Bouin's solution for light microscopy and
the remainder in 3 % phosphate-buffered cold glutaraldehyde for electron
microscopy. Fetuses for light microscopy were processed for parafin embedding.
Frontal serial sections, 7/tm thick, were stained with hematoxylin and eosin. The
method for processing glutaraldehyde fixed palates for electron microscopy was
similar to one described by Shah & Travill (19766). Prior to embedding, the
palates were divided into anterior, middle and posterior thirds and carefully
oriented to procure 1 jam frontal sections. Thin sections were obtained from the
middle third of the secondary palate and stained with methanolic uranyl
acetate (Stempak & Ward, 1964) followed by lead citrate (Reynolds, 1963). The
sections were observed in a Philips 300 or Hitachi HU-11E-1 electron
microscope.
RESULTS
Light microscopy
The vertical palatal shelf (Fig. 1) was composed of mesenchymal tissue
covered by one to three cell layers of epithelium (Fig. 2). Examination of serial
sections revealed a focal epithelial thickening on the medial aspect of the vertical
shelf. The epithelial thickening was localized and was seen only in the middle
third of the secondary palate. The thickening was composed of three to five
layers of cells. The palatal mesenchyme was composed of stellate cells scattered
in an amorphous ground substance. An occasional cell undergoing mitosis was
seen in the mesenchyme. The epithelium on the medial aspect of the vertical
shelf, i.e. the~prospective midline epithelium that is destined to fuse and disintegrate, showed no mitotic figures.
Reorientation of the shelf from a vertical to a horizontal plane started in the
middle third of the secondary palate (Fig. 3). In the frontal section, a medial
bulge was observed above the area of epithelial thickening (Fig. 4). The prospective midline fusion epithelium showed no histological change from that of the
Palatal shelf reorientation in hamster
Fig. I. Ventral view of hamster palate at day 12:00 of gestation. Both the lower jaw
and tongue are removed. The palatal shelves are in vertical position, x 16.
Fig. 2. A vertical palatal shelf at day 12:00 of gestation showing thickening of the
epithelium on its medial aspect (arrow). Epon thick section, x 76.
Fig. 3. Ventral view of hamster palate at day 12:02 of gestation. Both the lower jaw
and tongue are removed. The palatal shelves are reorienting in the middle third but
are still vertical in the posterior third, x 16.
Fig. 4. A reorienting palatal shelf at day 12:02 of gestation. Some of the mesenchymal cells are elongated (arrows). Note the epithelial thickening (E). Epon thick
section. x52.
R. M. SHAH
Figs 5-7. For legends see facing page.
Palatal shelf reorientation in hamster
5
vertical shelf. Some of the mesenchymal cells were, however, elongated and
appeared to be polarized along the transverse axis of the medially bulging palatal
shelf. Other mesenchymal cells were stellate or appeared to be undergoing
elongation.
Subsequently, when the palatal shelf became horizontal (Fig. 5), the focal
thickening of the epithelium was on its medial edge (Fig. 6). The mesenchymal
cells were either stellate or elongated and resembled those of the vertical or
reorienting shelf. However, as the opposing horizontal shelves approached
one another toward the midline all mesenchymal cells were stellate.
Electron microscopy
The epithelium covering the medial aspect of the vertical palatal shelf
(Fig. 7) was separated from the underlying mesenchyme by a continuous basal
lamina. Numerous spaces were present between the epithelial cells. The superficial epithelial cells were flat. They were attached to one another and to the
subjacent epithelial cells by desmosomes. They contained a flattened nucleus
surrounded by polyribosomes, mitochondria, a few cisternae of rough endoplasmic reticula and bundles of tonofilaments. The basal cells were roughly
cuboidal. Each contained a relatively large, oval or irregularly shaped nucleus,
polyribosomes, a few cisternae of rough endoplasmic reticula, mitochondria,
coated vesicles and a small Golgi complex.
The cells of focal epithelial thickening (Fig. 8) varied in their shapes. The cells
near the basal lamina were generally cuboidal and those away were flat. A few
spaces intervened between the cells of thickened epithelia which were otherwise
attached to one another by desmosomes. The cells in thickened epithelium contained a spherical or oval nucleus surrounded by numerous polyribosomes, a small
Golgi complex, few strands of rough endoplasmic reticula and mitochondria.
The mesenchymal cells were stellate and contained a large nucleus surrounded
by few cytoplasmic organelles and a well developed Golgi complex.
During reorientation of the palatal shelf (Fig. 9), the basal lamina remained
F I G U R E S 5-7
Fig. 5. Ventral view of hamster palate at day 12:04 of gestation. Both the lower jaw
and tongue are removed. The palatal shelves are horizontal, x 16.
Fig. 6. The horizontal palatal shelves at day 12:04 of gestation. The thickened epithelium is present on the medial edge of the shelves (arrows). Epon thick section.
x42.
Fig. 7. Electron micrograph of a vertical palatal shelf at day 12:00 of gestation.
A continuous basal lamina (BL) separates the epithelium from the mesenchyme.
Numerous spaces (JCS) are present between the superficial and the basal cells and
between the latter. The superficial cell is characterized by a flat nucleus (N) surrounded by polyribosomes, mitochondria (M) and tonofilaments (Tf). The basal
cell, in addition, contains a small Golgi complex (GC) and a few cisternae of rough
endoplasmic reticula (RER). The nucleus (N) in the basal cell is irregular. The
superficial cell is attached to the basal cell by desmosomes (D). x 13200.
R. M. SHAH
Fig. 8. Electron micrograph of a vertical palatal shelf at day 12:00 of gestation.
Thickened epithelium. The cells near the basal lamina (BL) are cuboidal and those
away are flat. Intercellular spaces (ICS) are few and the cells are attached to one
another by desmosomes (D). The cells of thickened epithelium contain a spherical
or oval nucleus (N), numerous polyribosomes, rough endoplasmic reticulum (RER)
and mitochondria (M). x 8800.
continuous. The spaces between the epithelial cells were increased both in
number and size from that of the earlier stage. The basal cells appeared to have
flattened as compared to that of the vertical shelf. Lysosomes appeared for the
first time in both the superficial and the basal cells. Other cytoplasmic features
remained unchanged.
The thickened epithelium on the reorienting palatal shelf (Fig. 10), on the
other hand, showed a reduction in number and size of the spaces between its
cells. Also the cells near the basal lamina were roughly columnar rather than
cuboidal as seen on the vertical shelf. Other cells were flat. The Golgi complex
was well developed. As in the other epithelial cells, lysosomes were recognized
for the first time in the cells of the thickened epithelium.
Palatal shelf reorientation in hamster
Fig. 9. Electron micrograph of a reorienting palatal shelf at day 12:02 of gestation
showing numerous intercellular spaces (ICS) in the epithelium. The basal lamina
(BL) is intact. Lysosomes (Ly) are present in both the superficial and the basal cells,
x 8800.
The mesenchymal cells of reorienting palatal shelves were elongated (Fig. 11).
The nuclei of the elongated cells were flattened. In addition, microtubules
(Fig. 12) and condensation of microfilaments (13) were also present. Often two
neighbouring elongated cells were attached to one another by close junctions
(Fig. 14).
The epithelium covering the horizontal palatal shelf was separated from the
subjacent mesenchyme by a continuous basal lamina. The spaces between the
epithelial cells were reduced in number and size. The basal cells were roughly
cuboidal. The cytoplasm showed an increase in organelles. The morphology and
content of the thickened epithelium remained unchanged.
DISCUSSION
The mechanism by which the palatal shelf changes its position from a vertical
to a horizontal plane is not understood. External factors such as muscular
pressure by the tongue (Humphrey, 1969, 1971; Walker, 1969, 1971) and an
alteration at the cranial base (Harris, 1967; Taylor, 1978) have been considered
to be responsible for the shelf reorientation. However, palatal closure has been
R. M. SHAH
Fig. 10. Electron micrograph of a reorienting palatal shelf at day 12:03 of gestation
showing few intercellular spaces (ICS) (compare with Fig. 9) in the thickened
epithelium. The basal cells (Bas) are roughly columnar (compare with Fig. 8).
Mitochondria (M), Golgi complex (GC), rough endoplasmic reticulum (RER),
nucleus (N), lysosome (Ly). x 8800.
FIGURES
11-14
Fig. 11. Electron micrograph of a reorienting palatal shelf at day 12:02 of gestation.
The elongated mesenchymal cells contain a flattened nucleus (N), polyribosomes
and numerous cisternae of rough endoplasmic reticula (RER). x 8800.
Fig. 12. Electron micrograph of a reorienting palatal shelf at day 12:03 of gestation.
The elongated mesenchymal cell contains microtubules (arrows) which are oriented
along the long axis of the cell, x 31000.
Fig. 13. Electron micrograph of a reorienting palatal shelf at day 12:03 of gestation.
The elongated mesenchymal cell contains microfilaments (arrow) which are oriented
along the long axis of the cell, x 32300.
Fig. 14. Electron micrograph of a reorienting palatal shelf at day 12:03 of gestation.
The two neighbouring mesenchymal cells are attached to one another by close
junctions (arrows). Microfilaments (mf). x 28600.
Palatal shelf reorientation in hamster
Figs 11-14. For legends see facing page.
10
R. M. SHAH
noted in human cases of aglossia and microglossia (see review by Shah, 1977)
and no substantial alterations at the cranial base have been noted at the time of
shelf reorientation (Hart, Smiley & Dixon, 1972; Diewert, 1974). Further,
in vitro elevation of shelves has also been observed in the absence of the tongue
and cranial base (Brinkley, Basehoar, Branch & Avery, 1975; Wee, Wolfson &
Zimmerman, 1976). Thus both clinical and experimental observations seem to
indicate that external factors may not be necessary for shelf elevations.
Walker & Fraser (1956) have suggested that reorientation of the palatal
shelves by the mode of medial bulging may be due to an 'intrinsic shelf force'.
The nature of such a force, if any, is still undetermined. The contention that
an increased acid mucopolysaccharide concentration in the palatal shelf at the
time of elevation may provide the necessary internal shelf force (Walker, 1961;
Larsson, 1962; Jacobs, 1964; Pratt, Goggins, Wilk & King, 1973; Ferguson,
1978) has been disputed (Nanda, 1971; Andrew & Zimmerman, 1971).
Suggestions that the intrinsic shelf force may reside in the elastic fibers (Walker
& Fraser, 1956; Clark, 1956) have been discarded by subsequent histochemical
and ultrastructural observations in which no evidence of elastic fibers in the shelf
tissue was found (Walker, 1961; Isaacson & Chaudhry, 1962; Frommer &
Monroe, 1969). Suggestions that an increase in cellular proliferation within the
palatal shelf tissue at the time of elevation may be the source of the shelf force
(Schorr, 1908; Mott, Toto & Hilgers, 1969; Jelinek & Dostal, 1974; Nanda &
Romeo, 1975) have also been disputed (Walker & Fraser, 1956; Cleaton-Jones,
1976). Indeed it has been recently indicated that such growth does not play
a direct role in the shelf reorientation (Greene & Pratt, 1976).
The most recent hypothesis to explain the shelf reorientation through intrinsic
mechanism is the molecular interactions of the contractile proteins, actin and
myosin. These proteins are synthesized in the mesenchymal cells just prior to
reorientation (Lessard, Wee & Zimmerman, 1974) and are represented by
a cytoplasmic filament system (Babiarz et ah 1975; Innes, 1978). The results of
the present study are in accordance with this hypothesis. In addition, our
findings showed that there is an alteration in the morphology of both the mesenchymal and the epithelial cells during palatal shelf reorientation. The
mesenchymal cells undergo elongation and appear to protrude into the medially
bulging palatal shelf. The epithelial cells also flatten and appear to adapt
themselves to the subjacent growth forces. These morphological alterations may
be associated with the necessary power implicit in Walker & Fraser's intrinsic
force theory.
The alterations in the morphology of mesenchymal cells coincide with the
appearance of microtubules and microfilaments. Microtubules play an important
role in the determination of cell shape during organogenesis (Byers & Porter,
1964; Handel & Roth, 1971), and thus may have a similar role in elongation
of mesenchymal cells. Presence of microfilaments in non-muscle cells is considered to be associated with cellular movement during morphogenesis (Wessels
Palatal shelf reorientation
in hamster
11
et al. 1971; Pichichero & Avers, 1973) since they contain contractile proteins
(Goldman, Lazarides, Pollak & Weber, 1975). Ultrastructural identification of
microfilaments in the elongated mesenchymal cells of the palate, as observed in
the present study, may correspond to the contractile proteins identified by
Lessard et al. (1974) and Krawczky & Gillon (1976) in mouse palate. Morphologically, however, they do not resemble the myofibrils described by Babiarz
et al. (1975) and Innes (1978). The lack of resemblance may be due to differences
in the areas of observation. In the present study observations were made in the
middle third of the hard palate whereas the other authors observed the muscle
cells in the posterior part of the palate. In any case, the presence of cytoplasmic
filaments in palate mesenchyme may permit marshalling of internal stresses
for movement of mesenchymal cells, and thus in general our results support
the hypothesis that internal shelf force may reside in the mesenchymal cells of
the palatal shelf.
Close contacts observed between the elongated mesenchymal cells during
reorientation of the palatal shelves may also be important in generating internal
shelf force since Dehaan (1963), Hay (1968) and Sanders (1973) have shown that
such contacts play an important role in cellular movement during morphogenesis.
An interesting finding during the present study was occurrence of focal
epithelial thickening on the medial aspect of the vertical palatal shelf. The cells
in the thickening were closely packed. The reorientation of the palatal shelf
started immediately above the thickened epithelium. A similar thickening of
epithelium, however, has not been reported during mouse or human palatal
development and its significance is, therefore, entirely speculative. The epithelial
thickening may provide necessary guidance for the elongated mesenchymal cells
to move into the medially bulging palatal shelf during the reorientation stage.
The focal epithelial thickening could then act as a 'mechanotactic' pivot around
which the mesenchymal cells can glide. Similar mechanotactic guidance has also
been suggested during corneal development (Nelson & Revel, 1975).
In brief, the foregoing Results and Discussion indicate that reorientation of
the hamster palatal shelf occurs through the mode of medial bulging and thus
resembles that of humans and mice. A transient elongation of the mesenchymal
cells, which subsequently appears to protrude in the medially bulging shelf, may
be associated with generation of the intrinsic force for the palatal shelf realignment. Microtubules, microfilaments and close contacts are all associated
with the morphological changes of the mesenchymal cells. The epithelial changes
may be adaptive and guiding in nature.
The work was supported by a grant from the Medical Research Council of Canada. The
author extends his gratitude to Mrs V. Koulouris and Miss Ruth Scheuing for their
assistance.
12
R. M. SHAH
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EMB 53