Download Cell behaviour and cleft palate in the mutant mouse

/ . Embryol. exp. Morph. Vol. 58, pp. 131-142, 1980
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Cell behaviour and cleft palate in the
mutant mouse, amputated
By O. P. FLINT 1
From the Department of Zoology, University of Glasgow
SUMMARY
Cleft palate with a genetic origin normally arises because of a failure of the palatal shelves
to elevate or fuse. Until now attention in studies of palatal development has been focused
on two critical phases, those of elevation and fusion. In the mutant mouse, amputated,
however, cleft palate arises because of a failure of the palatal shelves to make any significant
outgrowth between the 12th day after conception when the palatal shelves are first observed
and the 14th day when elevation and fusion normally occur. When cell proliferation (mitotic
index) was measured in the palatal shelves on days 12, 13 and 14 no difference was found
between mutant and normal. The failure of the mutant palate to grow cannot be accounted
for on grounds of reduced cell proliferation. For this reason the palatal mesenchyme in
12-5-day and 14-5-day normal and amputated mice has been studied with the scanning
electron microscope. This work shows that the mesenchymal cells in the mutant palate are
clumped together and have much greater areas of cell contact than are observed in the normal
palate. The abnormal cell behaviour described in mutant palatal mesenchyme is typical of
amputated embryonic mesenchyme in general, and in other cases has been shown to cause
abnormal morphogenesis. We propose that aberrant cell behaviour causing an aggregation
through increased cell adhesion inhibits palatal outgrowth in the mutant, and for this reason
the palatal shelves subsequently fail to elevate and fuse.
INTRODUCTION
Abnormal cell behaviour causing the cells to clump together and inhibiting cell
movement affects the development of the mouse mutant, amputated, at all
stages involving morphogenetic cell movements. The length of the embryo is
reduced because node and streak regression is retarded (Flint, Ede, Wilby &
Proctor, 1978). Clumping of the somite sclerotome cells may partly contribute
to fusions and distortions later found among the vertebral cartilages (Flint,
\911a). Snout outgrowth is strongly reduced because increased cell adhesion
and cell clumping retards a critical early stage of facial development when the
facial mesenchyme grows by expansion of the extracellular volume, which
normally forces the cells to move apart (Flint, 1977b; Flint & Ede, 1978a). In
in vitro culture, amputated cells clump together and cell movement can be
observed in time-lapse films to have been retarded (Flint, 1977a and Flint, in
preparation).
1
Author's address; ICI Pharmaceuticals Division Mereside Alderley Park Macclesfield
Cheshire SK10 4TG, U.K.
132
O.P.FLINT
Another developmental system involving cell movement is the palate. Cleft
palate is found in all mouse embryos homozygous for the single recessive gene
amputated. Its development is described as part of a programme of research
into the cellular basis of abnormalities in mutant development. We find that
amputated palates fail to rotate and therefore to fuse because palatal shelf
outgrowth is retarded through anomalous cell behaviour similar to that already
described as affecting development in other parts of the embryo. Palate development has been reviewed by Greene & Pratt (1976) who describe two phases of
palate development: (1) elevation (including rotation) of the palatal shelves and
(2) palatal fusion. A failure of elevation or fusion can cause cleft palate. In the
mouse mutant shorthead (Fitch, 1961) elevation of the palatal shelves is arrested,
by a tongue which is too large for the abnormally small buccal cavity. In the
chick, on the other hand, rotation occurs but there is no fusion when the palatal
shelves meet, resulting in the type of cleft palate which is normal in avian
embryos (Greene & Pratt, 1976). Cleft palate arising out of the earliest stage of
palatal shelf outgrowth, as in amputated, has not so far been described, and this
earliest stage of palatal morphogenesis has been neglected.
METHODS
The mice are a CBA/101 hybrid intercross carrying the single recessive gene
amputated. Conception was taken to coincide with the midpoint of the dark
period just prior to noticing a vaginal plug (Snell, Fekete, Hummel & Law,
1940). Matings were designed to produce litters with homozygous amputated
embryos. The techniques of fixation and critical-point drying of mouse embryos
for the scanning electron microscope have been described by Flint & Ede
(19786). After fixation and while in 70 % ethanol the heads were dissected under
the dissecting microscope with micro-surgery scalpels (Moria-France) to
remove the mandibles and tongue, revealing the roof of the buccal cavity. In
some cases the heads were then cut transversely to produce sections of the
palatal shelves. It was found that the effect of cutting like this was not to crush
the tissue but to fracture it in the plane of cutting. The stages of palatal development we describe are not so highly resolved as those described by Walker &
Fraser (1956) or Walker & Crain (1960) since we were not specifically interested
in palatal fusion and also since the earliest stage we describe (12-5 days) is two
days younger than the embryos described by these authors.
Cell proliferation and cell density were measured in normal and amputated
palatal shelves and in the mesenchyme to one side of the base of the palatal shelf
(control area) at 12-5, 13-5 and 14-5 days. Cell proliferation was recorded as
mitotic index; the number of mitotic figures as a percentage of total cell number.
Statistical analysis of the figures was carried out by the standard analysis of
variance procedure.
Cell behaviour and palatal morphogenesis
133
Palatal development
Gross development changes:
Normal. The earliest stage at which the palatal shelves can be seen is at
12-5 days, when they appear as a pair of parallel ridges growing down from
either side of the roof of the buccal cavity (Fig. 1 a). That these are ridges can
be seen at their posterior end where the knife has cut a glancing transverse
section through the tissue. By the 14th day of development (14-5 days) the ridges
have grown well out and away from the palatal roof (Figs. lc,3a, c) and rotation
can be seen to have begun in the anterior half (Fig. 3 a) of at least one shelf. The
palatal rugae (Figs. \c, 3a, c) begin to make their appearance in an anteroposterior sequence at this stage before the palatal shelves meet and fuse. Each
pair of rugae appears at the same level of the palate so that after palatal fusion
(Fig. 3 b) they will form almost continuous ridges across the roof of the mouth.
At the end of the 14th day of development (approximately 15-0 days) fusion has
begun along the meeting central portions of the two palatal shelves (Figs. 2 a, 3 b).
Amputated. As in the normal embryo, mutant palatal shelves begin their
outgrowth during the 12th day of development (Fig. Ib). There are two anatomical features which differ from the normal embryo. First, the width of the
head is greater and the snout shorter (Flint & Ede, 1978 a), so that the palatal
shelves form at a greater distance apart. Secondly, the opening to Rathke's
pouch persists to this late stage in amputated, but not in the normal embryo
(Fig. 1 a, b). By 14-5 days, though, the overall shape of the head in amputated
remains blunter anteriorly and broader than normal, the distance between the
palatal shelves being the same in normal and mutant (Fig. 1 c, d). There is little
or no downgrowth of the palatal shelves away from the roof of the mouth
(Fig. 1 d, 3d), when compared to the normal (Figs. 1 c, 3a, c) at the same stage,
or the amputated embryo at 12-5 days (Fig. \b). As in the normal embryo, the
first palatal rugae make their appearance at 14-5 days (Fig. 1 d) and by the end
of the 14th day of development several more have appeared (Fig. 2b), but there
is no further outgrowth of the mutant palatal shelves. In the normal embryo
growth of the mandibles and the cranial base enlarges the buccal cavity so that
the tongue drops out of the way of the elevating palatal shelves. In amputated,
snout outgrowth is retarded (Flint, 19776 and Flint & Ede, 1978a). But growth
of the tongue is not so strongly inhibited and its impression can be seen in the
palatal shelves in Fig. 2b.
The epithelial surface of the palate
Normal. The aboral epithelial surface of the palate shows some interesting
regional variation at 15-0 days associated with the appearance of the rugae and
the point of fusion. Between the rugae numerous microvilli are seen on the
surfaces of the epithelial cells (Fig. 4 c). These are especially dense at the borders
134
O. P. FLINT
FIGURE 1
Scanning electron micrographs of the heads of normal (o, c) and amputated (b, d)
mice at 12-5 {a, b) and 14-5 (c, d) days of development. The mandibles and tongue
have been dissected away so that a view of the roof of the buccal cavity is obtained
in each case, in, internal nares; pp, primary palate including median process;
pr, palatal ruga; ps, palatal shelf; rp, opening to Rathke's pouch.
Cell behaviour and palatal morphogenesis
135
FIGURE 2
Scanning electron micrographs of normal (a) and amputated (b) mouse heads at
150 days. The preparation is the same as that described in Fig. l.fp, point of fusion
between the palatal shelves; pp, primary palate and median process; /?r, palatal ruga;
ps, palatal shelf; tg, tooth germ (upper incisor) exposed in amputated because the
broadening and foreshortening of the cranium draws the lips apart.
between cells. This is typical of an epithelial surface in contact with a fluid
medium, in this case the amniotic fluid, and has been observed on the chick
limb epidermis (Ede, Bellairs & Bancroft, 1974) and on the naso-frontal region
of the hamster face (Waterman & Meller, 1973), as well as on the mouse
(Waterman, Ross & Meller, 1973) and human (Waterman & Meller, 1974)
palate. There is a transition between inter-rugal epithelium and epithelium
associated with the rugae. Rugal epithelium is comparatively bare of microvilli
and the cell surfaces are much rougher (Fig. 4a). At the point of palatal fusion
all boundary distinction between cells is lost and numerous elongate cell projections are produced. A large quantity of debris also accumulates on the surface.
Amputated. Late in the 14th day of development the inter-rugal epithelium
is very similar in mutant and normal palates (cf. Fig. 4c, d). But the rugae,
far from lacking microvilli, are even more profusely covered, with many more
microvilli on the surface as well as at the junctions between cells (Fig. 4 b).
136
O. P. FLINT
FIGURE 3
Low-power scanning electron micrographs of normal embryos at 145 (a) and 150 (6)
days to show the palatal shelves elevating (a) and fusing (b). Higher powers of sections,
at 14-5 days, of the right palatal shelf in a normal embryo (c) and the left palatal shelf
in an amputated embryo (d) are also shown to compare the outgrowth of the palate in
mutant and normal, in, internal nares; pp, primary palate and median process; pr, palatal
ruga; ps, palatal shelf.
Sectioned palatal shelves
Normal. The section through the raised palatal shelf (Fig. 3 c) is reminiscent
of a section through a developing limb bud, but there is no apical ectodermal
ridge specialization of the distal ectoderm. At 12-5 days cells are spaced well
apart and are connected by numerous fine and very fine filopodia (Fig. 5 a).
By 14-5 days the cells are still well spaced but, in addition to the filopodia, they
are coated with a dense fibrous and globular matrix of intercellular materials
(Fig. 5 c, e). On the basis of the work of Hassell & Orkin (1976), who studied the
production of extracellular materials in the palate, we would identify the long
Cell behaviour and palatal morphogenesis
137
FIGURE 4
The epithelial surface of the palatal shelves at 150 days in normal {a, c, e) and
amputated {b, d) mice. The normal series shows a, the surface of a ruga and the
transition from rugal to inter-rugal surface (at the bottom of the figure); c, the
inter-rugal surface and e, the point of fusion between two palatal shelves. The
mutant series shows b, the rugal surface and d, the inter-rugal surface. A section
through the fusing palatal shelves of a normal mouse at 150 days is shown in/.
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O. P. F L I N T
FIGURE 5
The mesenchyme of the palatal shelves in normal (a, c, e) and amputated (b, dj)
mice at 12-5 days (a, b) and 14-5 days (c, d, e,f) just prior to elevation.
Cell behaviour and palatal morphogenesis
139
Table 1. Mitotic index {mitoses per 100 cells) in the palatal shelves and adjacent
control areas of normal and amputated mouse embryos
Values are given as mean plus or minus the standard deviation.
Tissue
Palate
nbryo
N
am
Control
N
am
12-5 days
13-5 days
14-5 days
Cells
counted
l-94±013
210±014
1-44 ±012
2041015
l-81±014
3-21 ±0-85
l-38±012
l-47±013
O-98±O13
l-86±O13
0-21 ±008
0-72 ±003
9836
4029
10061
5947
fibrous intercellular material as collagen and the globular material as a product
of the basement membrane which can extend far into the palate matrix. At the
point where the two palatal shelves meet a zone of continuity is formed, where
the breakdown of the epithelial interface occurs and the two palatal mesenchymes become one (Fig. 4/). The scanning electron microscope evidence is
consistent with transmission electron microscope studies on the palatal mesenchyme (Babiarz, Allenspach & Zimmerman, 1975 and Innes, 1978).
Amputated. Whereas normal cells at 12-5 days give a well-spaced structure to
the palatal tissue, amputated cells appear to have aggregated together (Fig. 5 b).
The filopodia adhere in a tangled web over all the cell surfaces. This is exactly
similar to preparations of the somite sclerotome in the mutant embryo at
9*5 days (Flint & Ede, 1978&). By 14-5 days there has been no overall change to
this aggregated or clumped appearance (Fig. 5d). But the intercellular spaces,
as in the normal palatal tissue, have become filled with globular and filamentous
extracellular material (Fig. 5d,f).
Cell proliferation in the palatal shelves
Values of mitotic index measured from sections of palatal shelves on 12-5,
13-5 and 14-5 days after conception are given in Table 1.
There is no significant overall difference between normal and amputated
(P > 0-10). But there is a significant overall difference in mitotic index measured
in the palatal shelves and in the adjacent control areas (0-05 > P > 0-025).
This difference largely rests on a drop in mitotic index in the control area between
13-5 and 14-5 days. The only other significant difference occurs at 14-5 days
when in palate and adjacent control areas mitotic indices are higher in amputated than in normal (P < 0-05). No explanation for this difference emerges
from the current work.
Cell density
Values of cell density measured at the same time as mitotic index are given
in Table 2. There is no significant overall difference between normal and
amputated (P > 0-10). But there is a significant overall difference in the cell
IO
EMB 58
140
O. P. FLINT
Table 2. Cell density (cells per 1OZ ju,m2) in the palatal shelves and adjacent control
areas of normal and amputated mouse embryos (same number of cells counted as
in Table 1)
Values are given as mean plus or minus the standard deviation.
Tissue
Embryo
12-5 days
13-5 days
14-5 days
Palate
N
am
Control
N
1408 ±1-52
15-44±l-56
l5-24±3-12
15-24 ±1-72
16-08 ±1-44
15-28 ±1-60
15-40 ±1-96
15-68 ±1-22
ll-92±l-44
1116±l-8O
21-76±l-54
18-68 ±0-98
am
density measured in the palatal shelves and adjacent control areas. This difference rests largely on a drop in cell density in the palatal shelves between
13-5 and 14-5 days.
DISCUSSION
Wherever genetically-caused cleft palate has been described in the mouse the
critical phase at which palate development has been inhibited has been that of
elevation and rotation (i.e. during the 14th day of development).
But there is no significant change in the outgrowth of mutant palatal shelves
from the time when they arefirstobserved at 12-5 days up to early in the 14th
day of development (Figs. 1 b, 3 d). Normal specimens show considerable downgrowth of the palatal shelves on either side of the tongue and away from the
roof of the buccal cavity during this time (Figs. \a, c, 3 a, c).
In spite of abnormal morphogenesis the differentiation of palatal shelf cells
proceeds normally, so that not only is the extracellular matrix material secreted
at the appropriate time accompanied by a parallel reduction in mutant and
normal cell density (see Table 2) but also the palatal rugae make their appearance
on cue (Fig. 1 d, 2b). Between the rugae the normal palatal epithelium is covered
with microvilli (Fig. 4 c), but the rugae have lost their coating of microvilli
(Fig. 4a). Microvilli are most probably a mechanism of conserving cell membrane (O'Neill & Follett, 1970). The development of the elevated ridge-like
rugae in the normal embryo on the expanding surface of the palatal shelves
involves a stretching of the epithelial surfaces and consequently a loss of the
microvilli. In the mutant there is no expansion of the palatal shelves. The
formation of the rugae involves a concentration of the available epithelium,
and consequently an increase in the number of microvilli (Fig. 4 b), rather than
a decrease.
Outgrowth of the naso-frontal region, like that of the palatal shelves, is
inhibited at the earliest stage in the amputated mouse (Flint, 19776; Flint &
Ede, 1978 a). This inhibition cannot be accounted for on the grounds that cell
proliferation is reduced. Similarly mitotic index is the same in mutant and
Cell behaviour and palatal morphogenesis
141
normal palatal shelves throughout the earliest stages from the 12th to the 14th
day of development. In the case of the naso-frontal region we have found that
increased cell adhesion between the cells in the mutant causes clumping similar
to that found elsewhere in the embryo and this retards the tissue expansion
which occurs in the normal embryo by secretion of extracellular material, and
therefore retards morphogenesis from the 10th day of development. This
clumping of cells is typical of the mutant mesenchyme both in vivo (Flint,
1977 a, b; Flint & Ede, 1978/7, b) and in vitro (Flint, 1977 a; Flint, in preparation).
Increased cell contacts and areas of cell-cell adhesion causing an aggregation
of the palatal mesenchyme exactly similar to that described in other parts of the
embryo persist from the 12th (Fig. 5 b) to the 14th (Fig. 5d) day of development. Since it is during this very early stage of palatal morphogenesis prior to
elevation that palatal outgrowth is inhibited, and since there is such a high
correlation in other parts of the embryo between abnormal cell behaviour and
abnormal morphogenesis (see also Introduction), we ascribe the cleft palate in
amputated to an anomaly of cell behaviour causing increased cell adhesion
which inhibits downward growth of the palatal shelves. The increased cell
adhesion does not increase cell density in the mutant mesenchyme. There are
the same number of cells per unit volume as in normal palatal shelves, but
because of increased cell adhesion these cells tend to clump together; they are
not so well dispersed as in the normal palate. Our measurements of the somite
sclerotome in the mutant produced the same result (Flint & Ede, 1978 b).
Matrix secretion causing a drop in cell density in the palatal shelves of both
mutant and normal embryos between the 13th and 14th day of development
comes too late to help the mutant palatal shelf catch up with the growth already
made by normal shelves. Comparing Figs 1 and 2, however, it can be seen that
some expansion of mutant palatal shelves occurs late in the 14th day of
development, probably as a result of the secretion of matrix material. The
development of the palatal shelves between the 12th and the 14th day, when
elevation occurs, has hitherto received little attention, but it appears that this is
a critical phase in palatal development and that disturbance of normal growth
during this stage may produce abnormal cleft palate at a later one.
A comparison of the effect on cell behaviour of amputated with work on
other mutant genes can be found in Flint & Ede (1978 a, b).
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