/ . Embryol. exp. Morph. Vol. 57, pp. 37-49, 1980
Printed in Great Britain © Company of Biologists Limited 1980
37
Regional variation of cell proliferation within
the facial processes of the chick embryo:
a study of the role of ' merging' during
development
By ROBERT MINKOFF 1
From the Department of Orthodontics/School of Dentistry,
Dental Research Center, University of North Carolina
SUMMARY
Variation in rates of cell proliferation along the long axis of the maxillary process, within the
lateral nasal process and in the zone of attachment between these structures was analyzed
employing DNA labeling indices. Chick embryos were labeled with [3H]thymidine for 1 h and
processed for histology and autoradiography. The percentage of labeled mesenchymal cells
was determined in delineated areas.
Analysis of labeling indices indicated that rates of cell proliferation varied within each of
the facial processes. Regions where rates of proliferation were maintained at elevated levels
were the boundary areas of the facial processes (e.g. the anterior tip of the maxillary process)
and the zones of attachment between the facial processes (e.g. between the maxillary process
and the lateral nasal process). Despite the presence of elevated rates of proliferation in
selected regions within the facial processes, however, the percentage of labeled cells in all
areas declined with advancing developmental age.
Thesefindingssupport the hypothesis, proposed by Streeter and Patten, that the ' merging'
of adjacent facial primordia, such as the maxillary and lateral nasal processes, is accomplished
by elevated rates of cell proliferation within the zones of attachment compared to the rates of
proliferation in adjacent regions.
INTRODUCTION
Descriptions of facial morphogenesis have often cited, and contrasted, the
mechanisms of 'fusion' and 'merging' to describe the attachment and joining of
the primordia that form the midface. Fusion - the attachment of the free ends
of outpocketings with subsequent breakdown of the epithelial surfaces followed
by mesenchymal penetration - has been most associated with joining of the
palatal shelves of the maxillary process to form the secondary palate. Merging
has been the mechanism associated with the joining of adjacent outpocketings
such as the maxillary process with the lateral nasal process, the medial nasal
processes with each other, and with the formation of the soft palate (Burdi &
1
Author's address: Dental Research Center 210-H, School of Dentistry, University of
North Carolina, Chapel Hill, NC 27514, U.S.A.
38
R. MINKOFF
Faist, 1967; Patten, 1968; Ross & Johnston, 1972). This was described originally
by Streeter (1948) as the means by which the contours of the developing face are
molded. In Streeter's description of the coalesence of the facial processes, he
stated: 'It is more precise to speak of these structures [the facial processes] as
swellings or ridges which correspond to centers of growth in the underlying
common mesenchyme. The furrows that lie between them on the surface are
smoothed out as the proliferation and fusion of the growth centersfillin beneath.'
Patten later used the term 'merging' to refer to the changes that Streeter
described (1961).
Although these distinctions are presently recognized, the exact role of merging, mesenchymal penetration and epithelial fusion and breakdown in the attachment of certain facial primordia (e.g. the median nasal process with the maxillary
process) remains unclear. Further, the basis for the mechanism of'merging' has
not been established. Streeter (and Patten) both believed that the merging of two
adjacent primordia was accomplished by regional differences in rates of cell
proliferation. In order to test this hypothesis, rates of cell proliferation were
analyzed within the maxillary process, the lateral nasal process and in the zone
of attachment between the two during the formation, enlargement and subsequent
fusion of these structures in the chick embryo.
MATERIALS AND METHODS
Labeling
Chick embryos were incubated, windowed, staged according to Hamburger and
Hamilton (1951), and labeled with [3H]thymidine (specific activity 6-7 Ci/mmol,
New England Nuclear Company) for 1 h by application of label on top of the
embryo through the window in the egg shell. Doses ranged from 15 to 30/tCi,
depending on the age of the embryo; in all cases, the label was diluted to a total
volume of 0 1 ml in a balanced salt solution. The developmental stages studied
range from Stage 21 to Stage 30 and encompass the formation, enlargement
and attachment of the maxillary process with the lateral nasal process.
Histology and autoradiography
For paraffin embedding, embryos were fixed in Boiiin's solution, dissected,
staged, washed in 50 % alcohol, dehydrated through graded alcohols, embedded,
and serially sectioned in the long axis of the maxillary process (Fig. 1) at 4/*m
(Humason, 1972). For plastic embedding, embryos were fixed in glutaraldehyde
and paraformaldehyde (Graham & Karnovsky, 1966), washed in several changes
of cacodylate buffer (0-1 M) for 24 h, dehydrated through graded alcohols to
acetone, and infiltrated with Epon-acetone mixtures of increasing Epon concentration. They were then embedded in Epon, polymerized for 3 days at 6070 °C (Luft, 1961), and sectioned parallel to the long axis of the maxillary
process at 2 /<m. Sections through the center of the maxillary process extending
Variation of cell proliferation in the facial processes
Fig. 1. Scanning electron microscopic photographs (courtesy, K. K. Sulik) of 3-day
(a) and 5-day (b) chick embryos in which the planes of sectioning through the
maxillary process (MP), lateral nasal process (LNP) and the zone of attachment
(ZA) are identified.
39
40
R. MINKOFF
Fig. 2. Representative plastic sections through the maxillary and lateral nasal
processes outlining the areas in which mesenchymal cells were counted to obtain
labeling indices. At early stages (stages 21-23) (a) the reference point is the tip of the
maxillary process from which counting areas were delineated along the maxillary
process (numbered squares) and in the frontonasal region (lettered squares). At later
stages (stages 25-30) (b) the reference point is the epithelial seam between the
maxillary and lateral nasal process at their point of attachment. Counting areas
were delineated from an area at this point (lettered x) along the maxillary process
(numbered squares) and along the lateral nasal process (lettered squares).
through the zone of attachment into the lateral nasal process were analyzed
(Fig. 1).
Slides were washed, coated with liquid autoradiographic emulsion (EastmanKodak NTB3), dried, and stored in light-proof boxes at 4 °C, for varying exposure
times up to 4 months (Rogers, 1973). Slides were then developed and stained
with either Harris's haematoxylin and eosin (paraffin sections) or with Richardson's stain (Epon sections).
Cell counting
Labeled and unlabeled mesenchymal cells were counted in areas delineated by
an ocular grid for the determination of DNA labeling indices. The histological
Variation of cell proliferation in the facial processes
41
Table 1. Comparison of labeling indices in the tip, middle and base of the maxillary
process
Region
.
A
^
Stage
Tip
Middle
Base
24-5
35-9
39-7
28-8
350
41-6
33-6
35-9
390
22-2
27
45-6
39-0
41-4
340
24-6
30-4
37-3
33-0
34-9
Embryos were labeled, embedded in paraffin, serially sectioned in the sagittal plane along
the long axis of the maxillary process and processed for autoradiography as described in the
text. Each horizontal line represents values obtained from one embryo.
sections examined for analysis were separated from each other by a sufficient
amount of tissue to prevent recounting the same population of cells. Labeled
cells were defined as those containing three or more grains above background
over the nucleus and at least four sections from each embryo were examined and
counted. Cell counting was conducted under oil-immersion at a magnification of
1250 x . Labeling indices for each anatomical area were obtained by dividing the
number of labeled cells within a given area by the total number of cells contained
within that area.
Paraffin sections through the center, along the long axis of the maxillary
process, were analyzed. Counting areas were outlined in the tip, at the base (near
the attachment of the maxillary with the mandibular process) and in the middle,
at a point equidistant from the counting areas at the tip and base.
Counting areas on Epon sections were outlined along the length of the
maxillary process, beneath the epithelium, into the zone of attachment between
the maxillary and lateral nasal processes, and in the lateral nasal process (Fig. 2).
The anterior limit of the maxillary process in younger embryos and the epithelial
seam in the zone of attachment between the maxillary and lateral nasal
processes in older embryos were used as reference landmarks. The remaining
counting areas were then marked off from this location in both directions.
Comparable counting areas on each slide were summed and averaged and a
labeling index was obtained for each area with reference to the landmarks noted
above.
RESULTS
A preliminary study, employing serially sectioned paraffin-embedded embryos,
demonstrated regional variation in rates of cell proliferation in selected regions
in the maxillary process. Labeling indices in older embryos (Stage 27) tended to
be lower in the middle of the maxillary process than at the tip or at the base
42
R. MINKOFF
55Stage 21
4535 25 g
55-i
Stage 22
•S
45
"
o
"(3
x>
^3 3 5 ^
45-
Stage 23
35 -
25-
f
g f e d c b
Frontonasal section
a
1
2
3 4 5 6 7 8 9
Maxillary process section
10
Fig. 3. Graphs of DNA labeling indices of mesenchymal cells along the long axis of
the maxillary process and of mesenchymal cells in the fronto-nasal region anterior to
the maxillary process in embryos at Stages 21-23. Embryos were labeled for 1 h and
embedded in Epon. Labeling procedures, delineation and sequencing of counting
areas, determination of labeling indices, etc., are described in the text and illustrated
in Fig. 2. Numbers refer to counting areas in the maxillary process and letters refer to
counting areas in the fronto-nasal region.
(i.e. the junction of the maxillary with the mandibular process). This difference
was not observed in younger embryos (stage 24-5) (Table 1).
A detailed study employing plastic-embedded embryos was then performed in
which labeling indices were obtained in sequence along the entire length of the
maxillary process. This analysis was extended into regions anterior to the
Variation of cell proliferation in the facial processes
43
Table 2. Comparison of the means of labeling indices in the zone of attachment
and the maxillary and lateral nasal processes
Stage
25
Region
Mean
%
Nasal process
17-4
Zone of attachment
23-2
Maxillary process
19-2
Nasal process
281
Zone of attachment
400
Maxillary process
28-7
Nasal process
15-4
Zone of attachment
39-9
Maxillary process
25-3
Nasal process
14-2
Zone of attachment
34-1
Maxillary process
16-7
Difference
5-8
40
26
11-9*
11-3*
28
24-5*
14-6*
30
19 9*
17-4*
The means of labeling indices in the zone of attachment between the maxillary process and
the lateral nasal process (counting areas a, x and 1 of Fig. 4) are compared to those in the
maxillary process (counting areas 3, 4 and 5 of Fig. 4) and the lateral nasal process (counting
areas c,d and e of Fig. 4). An asterisk denotes significant differences between regions.
maxillary process, including, at later stages, the contiguous lateral nasal process
and the zone of attachment (Figs. 3, 4). At early stages (Fig. 3, Stage 21-23)
labeling indices along the maxillary process tended, in general, to be higher than
those anteriorly in the frontonasal region. Beginning at Stage 25 (Fig. 4), however, when the maxillary and lateral nasal processes approximate each other, a
reproducible pattern of variation emerged. Areas of high and low labeling
indices, which corresponded to specific anatomical regions, appeared. For
example, labeling indices in the zone of attachment between the maxillary and
lateral nasal processes and the regions immediately adjacent to the zone of
attachment (sections x, a and 1; Fig. 4 and Table 2) remained high. On either
side of this zone, where labeling indices declined (sections c, d, e and 3, 4, 5;
Fig. 4 and Table 2), the regions coincided, approximately, with the middle of
each process. A rise in cell proliferation was again found as the most proximal
region of the maxillary process and the most superior region of the lateral nasal
process was approached. These regions also correspond to areas of attachment
44
R. MINKOFF
35 25
Stage 25
15
j£
5 -I
45 3525 Stage 26
15i
h g
f e d c b
Nasal process section
aX
1 2
3 4
5 6
7 8910111213
Maxillary process section
Beneath
epithelial seam
Fig. 4. Graphs of DNA labeling indices of mesenchymal cells through the center of
the maxillary process, beneath the epithelial seam in the zone of attachment between
the maxillary and lateral nasal processes and along the long axis of the lateral nasal
process as illustrated in Fig. 1, in embryos at Stages 25 and 26 (a) and at Stages 28 and
30 (b). Embryos were labeled for 1 h and embedded in Epon. Labeling procedures,
delineation and sequencing of counting areas, determination of labeling indices, etc.,
are described in the text and illustrated in Fig. 2. Numbers refer to counting areas in
the maxillary process. Letters refer to counting areas in the lateral nasal process.
X refers to the counting area in the zone of attachment, beneath the epithelial seam.
45
Variation of cell proliferation in the facial processes
453525Stage 28
15-
.8
352515Stage 30
5i
h g
f
e
d
c
b
a
X
l
2
3
Nasal process section
4
5
6
7
8
9
10 11 12 13
Maxillary process section
Beneath
epithelial seam
Fig. 4. For legend see opposite.
of one process with another (e.g. the maxillary process with the mandibular
process, and the superior borders of the lateral nasal with the medial nasal
process).
Data from these and additional embryos were used to calculate labeling
indices within 100 jtim of the tip of the maxillary process from Stages 21 through
30. As seen in Fig. 5, there is a progressive decline in the labeling indices of the
mesenchyme at the tip of the maxillary process throughout this period, despite
the fact that rates of cell proliferation near the tip of the maxillary process are as
high, or higher, than any other region within it.
The differentiation of cartilage and bone in the regions of the chick embryo
examined in this study occurs after 7 days (Murray, 1963). To confirm this,
however, we prepared cleared whole-mount 7-day embryos and stained them
with Alcian blue and Alizarin red S. No evidence of cartilage or bone was found
in any of the regions analyzed in this study.
DISCUSSION
The data presented in this report on regional analysis of cell proliferation
support the view that adjacent facial primordia attach by the mechanism
EMB 57
46
R. MINKOFF
60 i
50 -
40
30
20 -
10 -
21
23
24
25
26
Embryonic stage
27
29
30
Fig. 5. D N A labeling indices of mesenchymal cells within lOO/^m of the tip of the
maxillary process from Stages 21-30. Chick embryos were labeled for 1 h with [ 3 H]thymidine, embedded in Epon and processed for autoradiography as described in the
text. Counting areas were delineated with an ocular grid; each point represents one
embryo in which four or more sections were counted. A regression line was determined by the method of least-squares fit.
described as merging and, further, this mechanism is mediated by regional
differences in rates of cell proliferation. Regions such as the boundary between
the maxillary and lateral nasal processes contain mesenchyme which is proliferating more rapidly than mesenchyme in the center of the maxillary process or the
adjacent region in the lateral nasal process. The data suggest that higher rates of
cell proliferation correspond to other anatomical regions where primordia attach
(e.g. the maxillary and mandibular processes, the medial and lateral nasal
processes superiorly). Differential rates of decline in cell proliferation were
observed as the primordia became joined. Preliminary data indicate that the
decline in cell proliferation as well as the differences in rates of decline can be
attributed to the exit of cells from rapidly proliferating pools and the appearance
of subpopulations of quiescent cells. The size and distribution of these quiescent
subpopulations may account for the differences in rates of cell proliferation that
were found and this is currently being investigated.
The attachment of the facial processes has been extensively discussed in the
literature (Streeter, 1948; Warbrick, 1960; Patten, 1961; Andersen & Matthiessen,
1967; Smith & Monie, 1969; Lejour, 1970; Stark, 1973; Waterman & Meller,
1973; Johnston, Hassell & Brown, 1975; Trasler & Fraser, 1977, and others)
but the details of the sequence of events that occur during attachment remains
unclear. While merging has been considered to be primarily responsible for the
Variation of cell proliferation in the facial processes
47
joining of the lateral nasal process with the maxillary process, the mechanism
for attachment of the medial nasal process with the lateral nasal and maxillary
processes appears to be more complex. The formation of an epithelial 'nasal fin',
formed by adjacent epithelial surfaces, followed by breakdown and mesencyhmal
consolidation is considered by many to be an important early step during attachment (e.g. Streeter, 1948; Warbrick, 1960; Lejour, 1970; Johnston et al. 1975).
Others (Andersen & Matthiessen, 1967) consider the formation of a mesenchymal
'isthmus', produced by the invagination of the nasal placode, to be of primary
importance. After these initial events, merging has been postulated to occur as a
means of consolidating the attachment of the processes. The relative importance
of epithelial seam fusion and breakdown, mesenchymal consolidation, and
merging during the attachment of these structures, however, has not been
determined and a spatial and temporal analysis of regional differences in rates
of cell proliferation of mesenchyme during attachment of the medial nasal
process with the lateral nasal and maxillary processes, correlated with the
accompanying developmental changes in the epithelium, would be warranted.
The findings in this report of higher rates of cell proliferation in areas of
attachment of the facial primordia indicate that developmental patterns have
been established which regulate the cell cycle with reference to boundary
regions. The maintenance of elevated rates of cell proliferation in the zone of
attachment could be due to factors associated with the tip of the maxillary
process prior to attachment or to changes which occur in this region (and the
corresponding one in the lateral nasal process) when the primordia approximate
each other.
Recently theoretical models have been proposed in which cells receive
'positional information' with respect to boundary or reference regions and then
interpret this information by following a specific course of development (Wolpert
& Lewis, 1975). Proximo-distal pattern specification in the chick limb bud has
been attributed to the acquisition of positional information in a 'progress zone'
within 300-400 fim of the tip under the influence of the apical ectodermal ridge
(Summerbell, Lewis & Wolpert, 1973); cells establish their positional values by
a timing mechanism with respect to an internal autonomous 'clock' in which
the successive cell divisions within the 'progress zone' record time (Lewis, 1975).
The diffusion of a substance whose concentration could be detected by cells
from a reference source of fixed concentration has also been proposed as a means
of establishing a positional field (Crick, 1970). Development of the distal parts
of the limb along the antero-posterior axis appears to be regulated by a small
region at the posterior margin of the limb bud (Saunders & Gasseling, 1968)
and recent experiments have implied that a diffusable graded signal emanates
from this region to provide positional information along the antero-posterior
axis of the limb (Tickle, Summerbell & Wolpert, 1975; MacCabe & Parker,
1976; Summerbell, 1979).
Whether these or alternative models can be applied to the development of the
4-2
48
R. MINKOFF
facial primordia remains to be determined. Analogies can be found in the pattern
of change in cell proliferation between the developing limb (Hornbruch &
Wolpert, 1970; Searls & Janners, 1971; Janners & Searls, 1970) and recent work
on cell proliferation in the primary palate (Minkoff & Kuntz, 1977, 1978; Flint
& Ede, 1978). The findings in this report reinforce these comparisons and indicate
that models which have been proposed to explain limb development may be of
value in the analysis of facial morphogenesis.
This investigation was supported by N.I.H. research grants DE-04731 and DE-02668 from
the National Institute of Dental Research.
The author wishes to thank Ross Nash, Rusty Morris and Ms. Diane Marbach for their
skillful technical assistance.
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