/. Embryo/, exp. Morph. Vol. 63, pp. 53-66, 1981
Printed in Great Britain © Company of Biologists Limited 1981
53
,
Active role of embryonic facial epithelium:
New evidence of cellular events
in morphogenesis
By GUILLERMO MILLICOVSKY 1 AND
MALCOLM C. JOHNSTON 2
From the Dental Research Center, University of North Carolina at Chapel fiill
SUMMARY
Epithelial cells of the C57B1/6J mouse embryo participate in a temporal sequence of
events associated with the approximation, fusion and consolidation of components of the
facial primordia into a definitive structure. These cells lose their surface microvilli, and after
a brief period of quiescence they begin to fill the grooves separating facial constituents by
producing a series of surface projections that increase in size and complexity as the process of
fusion nears termination. Cessation of surface activity and the restoration of epithelial
microvilli indicate the end of the temporal sequence. Significantly, the epithelial cells of
primary palates of embryos with genetically- and phenytoin-induced cleft lip remain unchanged
and do not participate in fusion. This epithelial sequence has not been described previously
and we suggest that all of its steps may be critical to the normal development of the mammalian face.
INTRODUCTION
The development of the face, a complex process that begins early in embryonic
life (Johnston, Hassell & Brown, 1975), results from closely timed steps in
growth and fusion of four bilateral tissue outgrowths (Johnston et al. 1975;
Trasler & Fraser, 1979) called facial prominences, processes or elevations
(Slavkin, 1979); the medial nasal, lateral nasal, maxillary and mandibular
prominences (Fig. la). There is abundant evidence that these prominences
remodel and approach each other, but information regarding the mechanisms
involved in these steps is fragmentary and incomplete (Trasler & Fraser, 1979).
Cell differentiation, division, migration and specific interactions between
epithelium, mesenchyme and extracellular matrix are believed to be key elements
in facial morphogenesis (Johnston et al. 1975; Trasler & Fraser, 1979). The
fusion of these prominences shortly after contact consolidates the individual
outgrowths into a more definitive facial structure.
1
Author's address: Dental Research Center, University of North Carolina, Chapel Hill,
North
Carolina 27514, U.S.A.
2
Author's address: Dental Research Center, Departments of Orthodontics and Anatomy,
University of North Carolina, Chapel Hill, North Carolina 27514, U.S.A.
54
G. MILLICOVSKY AND M. C. JOHNSTON
We have studied facial development in C57B1/6J mouse embryos by methods
of scanning and transmission electron microscopy and discovered a sequence of
events exhibited by the epithelial cells located between facial prominences
during periods of consolidation.
Clefts of the upper lip with or without cleft palate are among the most
common developmental defects in humans. Therefore, although we studied all
of the primordia which contribute to the face, this report will emphasize the
epithelial events occurring between the facial prominences of the embryo which
give origin to the upper lip.
MATERIALS AND METHODS
We removed gravid uteri from ten pregnant C57B1/6J mice between 5 p.m.
on gestational day 10 and 8 a.m. on day 11 (day of plug = 0) and made small
uterine incisions over each implantation site before placing the tissues in
refrigerated paraformaldehyde-glutaraldehyde (Waterman, 1974) fixative. One
hour later, we dissected the embryos free from the membranes and staged them
by the number of tail somites posterior to the genital tubercle. At the time the
first tail somite appears, the embryo has approximately 28 body somites. Heads
of staged embryos were immersed in fresh paraformaldehyde-glutaraldehyde
and refrigerated overnight. Specimens were rinsed in 0-1 M cacodylate buffer
and post-fixed in 1 % osmium tetr oxide in Millonig's phosphate buffer for 90
min, followed by rapid dehydration through ethanol and ethanol-l,l,2-trichlorotrifluoroethane. Embryonic heads were critical-point dried from monochlorotrifluoromethane, positioned on aluminium mounts, and sputter coated
with gold-palladium. A total of 72 specimens (from ten litters) were observed
by scanning electron microscopy. In addition, several specimens were studied
by transmission electron microscopy.
RESULTS
Our observations indicate that C57B1/6J embryos possess bilateral facial
depressions (nasal pits) by the stage of two to four tail somites. The primary
palate begins to close at six tail somites and fusion is completed by ten tail
somites. These embryonic events take place over a period of 12 h in utero.
Prior to the initiation of primary palate closure, individual epithelial cells on the
surface of the facial prominences are outlined by rows of microvilli at the cell
margins (Fig. 1 b). Detailed examination of nasal prominences revealed that a
specific group of 10-20 epithelial cells located at the lower portion of the
nasal pit (between the medial and lateral nasal prominences) undergoes transformation beginning with the disappearance of the microvilli. These cellular
alterations coincide with the initiation of primary palate closure at six tail
somites. At the bottom of the nasal pit, there is a triangular area (Fig. 1 a) of
Epithelial activity in facial morphogenesis
55
smooth-surfaced cells (Fig. 2a), but soon this quiescent area will be the site
for a sequence of events that may be critical to the successful closure of the
primary palate. By seven tail somites it is possible to detect the first signs of
surface activity in this initially smooth area. Ridges begin to form on the
smooth triangular surface (Fig. 2b); these ridges (about 0-1 /im across and
about 10-20/^m long) appear in locations previously occupied by rows of
microvilli and course over the epithelial surface. The triangular bottom of the
nasal pit narrows after the appearance of these ridges, and this morphogenetic
rearrangement brings the medial and lateral nasal prominences in closer
apposition. At a slightly later stage epithelial filopodia of about 0-1-0-3/*m
in diameter begin to span from both medial and lateral nasal prominences
establishing the initial bridges (Figs. 3 a and 4) which seem to guide the surface
activity that follows. Our observations with scanning and transmission electron
microscopy suggest that these initial filopodia anchor into the surface by
penetrating between surface cells (Fig. 3a, b). Embryos observed at the stage of
seven to eight tail somites display larger cellular extensions (0-5-0-7 /*m in
diameter) which bridge the area between nasal prominences (Fig. 5 a). Since
these secondary extensions are in close contact with the initial small-diameter
filopodia, it is tempting to speculate that secondary bridges are guided across the
gap by the previously established filopodia. At this stage of primary palate
development, the epithelial connexions between nasal prominences consist of
small and large diameter cellular extensions wrapped around each other (Fig.
5 a). We have observed that secondary extensions often display enlargements
(2-3 /<m) that terminate in filopodia (Fig. 6). We have also observed intercellular
junctions between the enlargements in the secondary bridges and the epithelial
surface of the nasal prominences (Fig. 5b).
In the last 2-3 h of primary palate fusion (about nine tail somites) flattened
cells from the surface of the medial and lateral nasal prominences appear to
move down into the inferior portion of the nasal pit filling the gap between
these prominences (Figs 4, 5a and 6). At this stage of development, we noted
that these migrating cells are in close contact with the initial filopodial and
secondary extensions (Figs 4, 5 a and 6). We found spheroidal particles, 1-3 /im
in diameter (possibly representing cellular debris) on the epithelial surface of
primary palates undergoing active epithelial adhesion as early as six tail somites
and becoming more numerous in the final stages of fusion (Figs 4, 6 and 7).
Finally, by the stage of ten tail somites the surface of the pre-closure epithelium
is re-established. Before the activity described above ceases, rows of epithelial
microvilli reappear forming a pattern different from that observed before the
primary palate began closing. The new rows of microvilli are symmetrically
aligned longitudinally and transversely to the line of primary palate fusion
(Fig. 7). The above developmental steps overlap in time, and fit into the
sequence illustrated in Fig. 8.
56
G. MILLICOVSKY AND M. C. JOHNSTON
For legend see p. 63
57
Epithelial activity in facial morphogenesis
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0-1 urn
For legend see p. 63
58
G. MILLICOVSKY AND M. C. JOHNSTON
FP
0-1 urn
For legend see p. 63
59
Epithelial activity in facial morphogenesis
Q_
O
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6
60
G. MILLICOVSKY AND M. C. JOHNSTON
FC
For legend see p. 63
Epithelial activity in facial morphogenesis
61
LNP
For legend see p. 63
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G. MILLICOVSKY AND M. C. JOHNSTON
62
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Epithelial activity in facial morphogenesis
63
D E S C R I P T I O N OF FIGURES 1-7
FIGURE 1
(a) Overall SEM view of facial primordia of a mouse embryo. Triangle at the
junction of the medial nasal (MNP), lateral nasal (LNP) and maxillary (MxP)
prominences indicates the area of initial fusion of the primary palate. MnP:
mandibular prominence; E: eye primordium.
(b) Higher magnification of surface epithelium demonstrates rows of microvilli
(Mv) at cell margins.
FIGURE 2
(a) Nasal pit of a mouse embryo of six tail somites. A triangular area (dotted lines)
on the floor of the nasal pit has lost the rows of microvilli. The transition between
altered and normal epithelium is best illustrated on the left margin of the triangle.
LNP: Lateral nasal prominence; MNP: medial nasal prominence.
(b) The smooth triangular floor of the primary palate begins to exhibit surface
ridges (SR).
FIGURE 3
(a) Filopodial projections (FP) 01 /«n in diameter frequently establish the initial
bridges between medial (MNP) and lateral nasal prominences (LNP) in embryos of
seven tail somites. These surface projections seem to anchor into the apposing
nasal prominences by penetrating between epithelial cells (asterisk).
(b) TEM view of filopodial projections (FP) penetrating between cells (asterisk) of
the surface epithelium (SE).
FIGURE 4
Sheets of flattened cells (FC) and filopodial projections (FP) actively fuse the area
between the lateral nasal (LNP) and medial nasal prominences (MNP). Spheroidal
particles (S) are very common in areas of intense surface activity.
FIGURE 5
(a) The fusion area of this 8-tail-somite embryo displays filopodial projections (FP)
and secondary projections (SP) wrapped around each other. Note the sheets of
flattened cells (FC) progressing toward the center of the fusion area between the
lateral nasal (LNP) and medial nasal prominences (MNP).
(b) Intercellular junctions (IJ) are present at points of contact between secondary
projections (SP) and surface epithelium (SE).
FIGURE 6
Secondary projections (SP) of this 9 tail somite embryo have 2-3 /im enlargements
(E) that terminate in filopodial projections (FP). Two of these enlargements are
twisted around each other. Note advancing edge of flattened cells (FC) and spheroidal particles (S) present in the fusion area between the lateral nasal (LNP) and
medial nasal prominences (MNP).
FIGURE 7
At the conclusion of the fusion events the rows of epithelial microvilli (Mv) are
reestablished in very symmetrical patterns, and surface activity ceases. Spheroidal
particles (S) remain after fusion is completed.
3-2
64
G. MILLICOVSKY AND M. C. JOHNSTON
Microvilli reappear
|
Flattened cells
Secondary projections
Filopodial projections
Surface ridges
|
Quiescent surface
|
Microvilli disappear
0
1 2
3
4
5
6
7
Time in utero (h)
9
10
11
12
10
Tail somites
Fig. 8. Temporal sequence of events in approximation and fusion of C57B1/6J
mouse embryo primary palate.
DISCUSSION
It is probable that the epithelial events described in this report are critical
for facial morphogenesis since we have recently reported preliminary observations that CL/Fr mouse embryos (genetically predisposed, in our colony, to
spontaneous cleft lip with or without cleft palate) do not exhibit the sequence of
epithelial involvement typical of the C57B1/6J embryos (Millicovsky &
Johnston, 1980 a). Similarly, we observed that the epithelium of embryos from
A/J mice treated with the anticonvulsant phenytoin (producing 90 % cleft lip,
with or without cleft palate) exhibits a depressed ability to participate in
bridging (Millicovsky & Johnston, 19806). Our studies of genetically- and
phenytoin-induced cleft lip and palate indicate that the primary palate epithelium of six tail somite embryos is already different from control, suggesting
that the predisposing changes leading to facial clefts can be detected early in
development. The epithelial cells in these two abnormal models remain relatively
unchanged during the stages of primary palate development.
Our observations suggest that facial consolidation and the filling of grooves
between prominences occurs by a temporal sequence of events which include
the loss of microvilli by the epithelial cells followed by a period of morphologic
quiescence in the depth of the grooves. Next, the presence of filamentous ridges
integrated into the smooth surface of these grooves may be associated with the
reduction in area of these depressions. As the result of morphogenetic rearrangements and differential rates of cell division (Minkoff & Kuntz, 1977),
prominences grow in size and appose the neighbouring prominences. At this
stage of development, filopodial and secondary projections span the distance
between prominences. It is significant that filopodial projections are also
present in the fusion of the free edges of the neural tube of chick (Bancroft &
Bellairs, 1975; Santander & Cuadrado, 1976; Schoenwolf, 1979), hamster
(Waterman, 1975, 1976), and mouse (Waterman, 1975,1976). Short projections
Epithelial activity in facial morphogenesis
65
were also observed in the contact of mouse primary palate at later stages of
development (Gaare & Langman, 1977). In addition, obscured cellular boundaries and appearance of surface filamentous material are characteristic of secondary palate morphogenesis in mouse and man (Waterman, Ross & Meller,
1973; Waterman & Meller, 1974). As the consolidation of facial segments nears
its end, sheets of cells seem to complete the filling of facial grooves, and the
epithelial cells re-establish their microvilli. We have found that steps resembling
those in the epithelium between the medial and lateral nasal prominences occur
in all of the other grooves separating facial prominences during their stage of
consolidation.
In conclusion, we believe that the consolidation of the embryonic face is an
active phenomenon involving a sequence of events dependent on the participation of epithelial cells. We also suggest that the absence of any of the components of this sequence may be associated with conditions leading to facial
clefts.
Based on existing evidence we must speculate that since similar sequences
of cellular activity may exist in other animal models and in other anatomical
areas, it is possible that we are describing a generalized process permitting the
coalescence of embryonic primordia.
This study was supported by N.I.H. grants DE 02668 and RR 05333, and N.I.H. Fellowship DE 05248.
We thank Mrs Dell R. Dorgan for her expert technical assistance, and Ms Renee B.
Williams for typing this manuscript.
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TRASLER,
{Received 11 August 1980, revised 22 October 1980)