/ . Embryol. exp. Morph. Vol. 40, pp. 101-113, 1977
Printed in Great Britain © Company of Biologists Limited 1977
\Q\
Cell proliferation during morphogenetic
change; analysis of frontonasal morphogenesis in
the chick embryo employing DNA labeling indices
By ROBERT MINKOFF1 AND AMY J. KUNTZ2
From the Departments of Orthodontics and Biostatistics,
University of North Carolina
SUMMARY
The role of cell proliferation was analyzed in the chick embryo system employing DNA
labeling indices during the invagination of the olfactory placode and the development of the
lateral and medial nasal processes. Chick embryos were labeled for 1 h with [3H]thymidine
and processed histologically and autoradiographically. The percentage of labeled mesenchymal cells in delineated areas within and adjacent to the nasal processes was determined.
From analysis of labeling indices of each area at successive developmental stages, it was concluded that cell proliferation of mesenchyme, as measured by DNA labeling indices,
did not appear to increase during the formation of the nasal processes, and that cell proliferation actually declined during the later stages of nasal process formation.
Differences were also found between the labeling indices of the mesenchyme of the nasal
processes as compared to that of adjacent areas. These differences tended to become greater
as development progressed. In all of the areas studied, cell proliferation declined during the
Jater stages of development but the magnitude of the decline was greater in the areas adjacent
to the nasal processes. Differential rates of decline, rather than acceleration of cell proliferation, therefore, appears to be operative as a morphogenetic mechanism during early primary
palate formation.
INTRODUCTION
Facial development in avian and mammalian vertebrates begins with a
thickening of the epithelium overlying the embryonic forebrain. This thickening,
the nasal placode, which represents the initial event in the formation of the
nasal chambers and in the development of the midface, is soon followed by the
invagination of the placode and the formation of a nasal groove. The areas
medial and lateral to the nasal groove enlarge during this time and these tissue
outpocketings have been termed the lateral and medial nasal processes. The
enlargement and fusion of these structures with each other and with the maxillary process, a third outpocketing which develops at the same time from the
first branchial arch, represent the primordia from which the structures of the
1
Author's address: Department of Orthodontics, School of Dentistry, University of
North Carolina, Chapel Hill, North Carolina 27514, U.S.A.
2
Author's address: Department of Biostatistics, School of Public Health, University of
North Carolina, Chapel Hill, North Carolina 27514, U.S.A.
102
R. MINKOFF AND A. J. KUNTZ
midface develop; the nose, upper lip, maxilla, etc. (Romanoff, 1960; Patten,
1968; Hamilton & Mossman, 1972; Waterman & Meller, 1973).
Classical descriptions of the development of this region in embryological
literature have ascribed the formation of the medial and lateral nasal processes
to the presence of 'growth centers' within each structure. Either explicitly
stated, or implied, descriptions of normal development refer to localized increases in the rate of cell proliferation of the underlying mesenchyme as the
mechanism by which these growth centers arise (Streeter, 1948; Warbrick,
1960; Patten, 1964; Andersen & Matthiesen, 1967; Hamilton & Mossman,
1972). For example, Warbrick (1960) in his study of the early development of
the nasal cavity, stated: 'The nasal groove is probably formed and deepened
by the proliferation of mesoderm round its edges'. Patten (1964) described the
development of this area in similar terms. Hamilton & Mossman (1972) also
considered the development of this region due to the proliferation of mesenchyme which brought about the elevation of the surface epithelium surrounding
the nasal placodes. Andersen & Matthiessen (1967), in their study of the early
development of the human central face, also state that the processes result from
localized mesenchymal proliferation.
In the present investigation, an analysis of cell proliferation during the
development of this region was performed. Labeling indices, derived from
[3H]thymidine incorporation into DNA, were obtained in defined anatomical
areas through successive developmental stages in the chick embryo. The conclusions drawn from analysis of these indices do not support the explanations
that have previously appeared in the embryological literature. Evidence for
increases in rates of cell proliferation in mesenchyme during successive developmental stages in the formation of the nasal processes was not found.
METHODS AND MATERIALS
Chick embryos (White Leghorn) were incubated, windowed, staged according
to Hamburger & Hamilton (1951) and reincubated. [3H]thymidine (sp. act.
6-7 Ci/mmole, New England Nuclear Co.) was diluted with chick Ringer's
solution. 0-1 ml (20 jnCi) was injected directly into the yolk-sac of each egg
through a hole in the shell at the blunt end. The developmental stages studied
were from stage 15 to stage 28 (Hamburger & Hamilton, 1951), the period
encompassing the invagination of the nasal placode and the formation and
fusion of the nasal processes. Labeling was terminated by rapid fixation of
embryos. Following fixation in Bouin's solution, embryos were dissected,
staged, washed and dehydrated in graded alcohols. The embryos were then
double embedded in 2 % nitrocellulose and paraffin (Humason, 1972) in order
to preserve morphologic relationships within the regions containing the invaginating nasal placodes. Embryos were embedded and oriented so that the
plane of sectioning was approximately at right angles to the nasal placode or
Cell proliferation during primary palate formation
103
OP
Fig. 1. Schematic diagram (oblique frontal plane) illustrating the method used to
delineate the boundaries of the areas in which cell counts were done. The areas
utilized to obtain labeling indices were (1) the lateral nasal process, (2) the base of
the olfactory placode and (3) the medial nasal process. OP, Olfactory placode;
FP, forebrain.
groove and sections were cut at 5 fivcv. Serial sections were obtained on all
embryos, and areas containing the nasal placodes and processes, and other
areas of interest, were identified. A minimum of three to ten sections separated
those sections used to obtain data for labeling indices.
A utoradiography
Slides containing sections through appropriate areas were thoroughly washed,
coated with liquid autoradiographic emulsion (Eastman-Kodak-NTB-2), dried,
stored in light tight boxes at 4 °C, and exposed for lengths of time ranging from
2 days to 4 weeks (Rogers, 1973). Exposure times for each embryo were determined individually by examination of test slides until satisfactory grain density
with minimal background was obtained. Slides were developed and stained
with either Harris' haematoxylin and eosin or Mayer's haematoxylin. The
areas in the region of the nasal placode to be studied were defined by means of
an ocular grid; the latter was also used when counting labeled and unlabeled
cells. After establishing a background count, labeled cells were defined as those
containing three or more grains above background. (Background counts were
low - less than 0-5 grains/nucleus.) Examination was done using an oil immersion objective at a magnification of 1000 x .
At least three or more sections from each embryo were examined and counted
and both left and right sides of bilateral structures, such as the lateral nasal
process, were counted. Left and right sides were pooled. In most cases, at least
1000 cells were counted for each labeling index. The areas within the facial
processes and beneath the olfactory placode that were counted were delineated
by the dimensions of the placode itself. Determinations were made at the level
104
R. MINKOFF AND A. J. KUNTZ
corresponding to the greatest depth of the placode or groove, and the counting
area was outlined with the aid of an ocular grid, as indicated in Fig. 1, utilizing
the dimensions of the placode and the outline of the forebrain as boundaries.
In addition, serial sections of the region above and below the olfactory placode
and nasal processes were examined, processed and counted in a limited series
of embryos.
Labeling indices for each anatomical area at each developmental stage were
determined as the percentage of labeled cells to the total number of cells within
the area studied. In the data shown in Figs. 3-6, duplicate determinations of
several, and in some cases all, labeling indices were made by a second investigator and in several cases were confirmed by a third investigator.
Statistical methods
Both parametric and non-parametric statistical techniques were used to
analyze the data (Siegal, 1956; Snedecor & Cochran, 1973). T-tests were used to
determine differences in labeling index between different stages of development.
Self-pairing of embryos with reference to anatomical location was employed for
paired ?-tests used to determine differences in labeling index among different
anatomical locations. A multivariate model was constructed to determine if the
decline in the labeling index from early and middle to late stages was different
in the nasal processes from that in adjacent areas.
These parametric tests were supported by non-parametric tests, which resulted
in the same conclusions. The Mann-Whitney Wilcoxon test was used to examine
the differences in labeling index at different stages as well as to evaluate declines
in the nasal processes and the adjacent areas. A sign test was used on a selected
series of embryos to support the conclusions of the paired t-ttsi.
RESULTS
Lateral and medial nasal processes
Representative sections of the areas studied are shown in Fig. 2. In Fig. 3 the
percentage of labeled cells of the lateral nasal process is plotted against the
corresponding developmental age of the embryo. The values remain within a
narrow range throughout the period from stage 15 to stage 25 - a period characterized by marked changes in the morphology of the region. During this time
the nasal placode invaginates, the nasal groove forms, and the lateral nasal
and medial nasal processes develop into distinct morphological entities. The
Fig. 2. Representative sections through the invaginating olfactory placode and the
medial and lateral nasal processes during the developmental time period studied.
Invagination of the placode (Fig. 2A), formation of the nasal groove (Fig. 2B),
deepening and enlargement of the nasal groove and nasal processes (Fig. 2C). Contact and fusion of the nasal processes (Fig. 2D). LNP, Lateral nasal process; MNP,
medial nasal process; OP, olfactory placode; FP, forebrain.
Cell proliferation during primary palate formation
pp
100 nm
FP
MNP
MNP
LNP
LNP
105
106
R. MINKOFF AND A. J. K U N T Z
IV)
60 50
-°
•1
30
t . .
•
U •
40
o
20 10 i
i
i
i
i
i
i
i
i
i
i
i
i
i
15 16 17 18 19 20 21 22 23 24 25 26 27 28
Developmental stages
Fig. 3. Labeling indices of lateral nasal process. Each data point represents one
embryo. Embryos were labeled with [3H]thymidine for 1 h, fixed, and processed for
histology and autoradiography as described in the text. Combined cell counts of a
minimum of three sections were used in determining labeling indices. Both left and
right sides were counted and pooled. Counting areas were delineated as described in
Fig. 1. • , Labeling index of mesenchyme of the lateral nasal process; O, Labeling
index of head mesenchyme prior to placode invagination.
IV)
ibeled c:ells
60
•
50 - o
40
•
•
! t•
o
•
•
•
30
*
20 10 •
i
i
i
i
,
i
i
i
i
i
15 16 17 18 19 20 21 22 23 24 25 26 27 28
Developmental stages
Fig. 4. Labeling indices of medial nasal process. Counting procedures, histological
and autoradiographic techniques, etc. are the same as those in Fig. 3. • , Labeling
index of mesenchyme of the medial nasal process; O, labeling index of head mesenchyme prior to placode invagination.
labeling indices of the mesenchyme of the lateral nasal process, however, vary
between 40 % and 48 % throughout this period of development. There is no
indication from the present data that the change in morphology is accompanied
by an increase in cell proliferation, as measured by a DNA synthesis index. In
addition, the data indicate that at later stages (stages 26-28) the rate of cell
proliferation decreases during development of the lateral nasal process.
The medial nasal process (Fig. 4) was also studied. When labeling indices of
the medial nasal process were plotted against developmental age, findings
similar to those for the lateral nasal process were obtained. The labeling index
of the medial nasal process varied within a narrow range throughout the period
from stages 18-25, and then declined during stages 26-28. In addition, it did not
Cell proliferation during primary palate formation
107
70 r
60
=
50
o
TJ 40
—
I
30
^
20
10
15 16
17 18
19 20 21 22 23 24 25
Developmental stages
26 27 28
Fig. 5. Labeling indices of areas adjacent to the nasal processes. Counting procedures, histological and autoradiographic techniques, etc. are the same as those employed for determining labeling indices of the medial and lateral nasal processes.
The adjacent areas analyzed were: • , the mesenchyme at the base of the olfactory
placode (as delineated in Fig. 1); O, the mesenchyme superior to the nasal processes;
A, the mesenchyme inferior to the nasal processes. ©, The labeling index of head
mesenchyme prior to placode invagination.
exceed the level found for 'head mesenchyme' at stage 15 prior to placode invagination. Increased cell proliferation, therefore, did not appear to account
for the marked changes in morphology that occurred in either the lateral or the
medial nasal process.
Areas adjacent to the nasal processes
In addition to the areas previously prepared and examined, an area was
defined directly beneath the invaginating olfactory placode at early stages of
development, or directly beneath the olfactory groove at later developmental
stages. The boundaries of this area were determined by the olfactory placode
epithelium and the forebrain as illustrated in Fig. 1. In a limited series of
embryos, comparable areas both above and below the nasal processes were
identified, defined and counted by the same procedure and criteria that was
used for obtaining data within the nasal processes. When labeling indices from
these areas adjacent to the nasal processes were collated and graphed (Fig. 5),
the results indicated that the labeling index within these adjacent areas approximated that found in the lateral and medial nasal processes. This level also
declined with advancing developmental age; the decline, however, was more
pronounced in the adjacent areas than it was within the nasal processes.
A comparison of the data obtained from areas within the nasal processes
with data obtained from adjacent areas (i.e. above or below the nasal processes
and beneath the olfactory placode) demonstrated comparable labeling indices
during early stages of development (Fig. 6). A divergence appeared as development progressed, and the values for the labeling indices became markedly
divergent during later stages (stages 25-28).
The observations just described are summarized in Table 1 in which the
108
R. MINKOFF AND A. J. KUNTZ
60 r
50
40
-o
20
10
18
19
20
21
22
23
24
Developmental stages
25
26
27
28
Fig. 6. Comparison of labeling indices of mesenchyme within the nasal processes
with labeling indices of mesenchyme from adjacent areas. Data points which are in a
vertical array are from the same embryo. Each cluster of data points is from the
same developmental stage. • , Labeling indices of mesenchyme of either the medial
or the lateral nasal process. • , Labeling indices of mesenchyme of adjacent areas,
including the mesenchyme at the base of the olfactory placode, and superior and
inferior to the nasal processes.
Table 1. Means of labeling indices of anatomical areas during early,
middle, and late stages of development
Location
Developmental
time period
(stages)
Lateral
nasal
process
Medial
nasal
process
18-19-20
22-23-24
25-26-28
44-6
45-2
37-9
42-2
43-7
35-8
Base of
placode
Lateral and
medial nasal
processes
combined
Adjacent areas
including base
of placode
42-2
410
26-4
43-4
44-4
36-8
40-5
39-9
26-7
The developmental time period analysed (stage 18—28) was divided into three segments:
early (stages 18-20), middle (stages 22-24) and late (stages 25-28). Data presented for areas
individually are on the left. On the right, data are combined for areas within the processes
(the average of the values for the lateral and medial nasal processes) and adjacent to the
processes (the average of the values for the base of the placode, and above and below the
nasal processes).
developmental time period of nasal process formation is divided into early,
middle and late stages. In all anatomical areas, the mean of the labeling index
during the late developmental time period was lower than that during both the
middle and early time periods. These differences were statistically significant
(P < 0-05). The means of the labeling index of the early and middle periods
Cell proliferation during primary palate formation
109
Table 2. Comparison of labeling index of nasal processes and adjacent areas
Anatomical region
Labeling index
of nasal
Stage
processes
40-7
42-2
41 1
46-7
45-6
47-0
40-3
51-5
42-2
39-4
460
42-8
330
48-8
32-2
36-2
38-2
300
33-3
18
18
18
20
20
22
22
22
23
23
24
25
26
26
26
26
28
28
28
Difference between labeling index of processes
minus labeling index of adjacent areas
Labeling index
of adjacent
areas
By embryo
37-8
36-6
41-7
44-4
41-7
38-5
35-7
48-5
35-7
49-3
49-6
316
27-5
34-8
22-6
341
26-5
170
17-8
+ 2-9
+ 5-6
-0-6
+ 2-3
+ 3-9
+ 8-5
+ 4-6
+ 3-0
+ 6-5
-0-9
+ 5-4
+ 11-2
+ 5-5
+ 140
+ 9-6
+ 2-1
+ 11-7
+ 130
+ 15-5
By developmental time
period (early, middle,
late)
By stage
)
+ 2-6
+ 2-9
J
J
+ 3-1J
TJ
1. + 5-4
i
I
j
1
'
A.e
+4 J
+ 2-8
+ 5-4
+ 11-2 >
i
T
/
7.0
0
+ 10-8
1
1
+ 13-4
J
The first column is the average of the values for the lateral and medial nasal processes, the
second column is the average of the values for adjacent areas (beneath the olfactory placode,
below or above the nasal processes), and the third column is the difference between the two,
within each embryo, as well as by stage and by stage grouping into early, middle and late
developmental time periods.
were essentially the same and no statistically significant differences were obtained between these periods.
When the means of the labeling index of either of the nasal processes and the
means of the labeling index of adjacent areas were compared, significant
differences were also found (Table 1) at the late developmental time period
(stages 25-26-28). The means for both the lateral and medial nasal process
differ significantly from the mean for the base of the placode; and the combined
means for the nasal processes differ significantly from combined data obtained
from adjacent areas (P < 0-05). Differences were also found between the
labeling index of the nasal processes and adjacent areas at the early and middle
developmental time periods although, of lesser magnitude. A self-paired t-test,
used to analyze the results from a small series of embryos in stages 18-24 in
which all areas were included in the analysis, indicated significant (P < 0-05)
differences between the labeling index of the nasal processes and the adjacent
areas. This result indicates that there may be differences between the nasal
8
EMB 40
110
R. M I N K O F F
AND
A . J.
KUNTZ
Table 3. Decline in the labeling index between stages 18-24 and
stage 25-28 in the nasal processes and adjacent areas
Anatomical area
Decline in % of
labeled cells between
stages 18-24 and
25-28
Lateral
nasal
process
Medial
nasal
process
Base of
placode
7-0
7-1
15-1
Lateral and
medial nasal
processes
combined
Adjacent areas
including base
of placode
7-1
13-5
On the left, individual regions are compared; on the right, the values for the lateral and
medial nasal process are combined and compared to the combined value of adjacent areas.
processes and the adjacent areas at the early and middle developmental period
as well as at the later period.
When the difference between means of the labeling index of the nasal pro­
cesses and that of the adjacent areas was calculated for only those embryos in
which data were obtained from both regions (Table 2), the array displayed a
consistently higher value for the labeling index of the nasal processes in all
stages and developmental time periods studied. Further, in 17 embryos out of a
total of 19, the labeling index is higher in the nasal processes. By application of a
sign test, the consistency of higher values for the labeling index in the nasal
process was found to be statistically significant (P < 0-001). This array of
values again lends support to the possibility that significant differences exist
between the labeling index of the nasal processes and those of adjacent areas,
not only at later stages, but at all of the developmental time periods studied.
In addition to the decline in labeling index between early and late stages, a
difference in the magnitude of this decline was observed in the mesenchyme of
the nasal processes in contrast to that of adjacent areas. In Table 3, the magni­
tude of decline in the labeling index from the early and middle period into the
late period is displayed for the areas within and adjacent to the nasal processes.
When subjected to multivariate analysis, the decline was found to be signifi­
cantly greater in the adjacent areas than in the nasal processes (P < 0-05).
DISCUSSION
Cell proliferation as a morphogenetic mechanism in the facial
processes
Previous examples have been cited which imply that the invagination of the
nasal placode and the development of the adjacent facial elevations are due to
increased rates of cell proliferation in selected anatomical regions. The data
provided in this paper do not support this assumption. Labeling indices indicate
that rates of cell proliferation do not increase during this time period. Not only
Cell proliferation during primary palate formation
111
are increases in cell proliferation not apparent, but at later stages of develop­
ment, cell proliferation actually declines within these surface elevations as they
become larger.
Labeling indices of areas adjacent to the nasal processes also decline at later
stages and the decline is much greater in these adjacent areas than in the nasal
processes. Differential rates of decline in cell proliferation, rather than accelera­
tion, may be responsible for the alterations of form in this region during de­
velopment.
Evidence is present that differential rates of cell proliferation also exist at
earlier stages. The data in Table 2 display a consistent pattern of differences in
labeling indices from stage 18 to stage 28 and although the magnitude of the
differences at early stages is smaller than at later stages, the probability that this
pattern of differences is due to chance is extremely small (P < 0-001). The
progressive increase in the magnitude of these differences with advancing
developmental age indicates that differential rates of proliferation may be
operative as a developmental mechanism at early as well as late stages.
Comparisons with limb outgrowth
The role of cell proliferation during the development of the limb in the chick
embryo has been extensively studied (Amprino, 1965; Cairns, 1966; Hornbruch
& Wolpert, 1970; Janners & Searls, 1970; Searls & Janners, 1971; Ede, Flint &
Teague, 1975; and others).
Searls & Janners (1971) observed differential rates of decline in cell prolifera­
tion during limb morphogenesis. The labeling indices in the limb-bud declined
at a slow rate from the initiation of limb outgrowth, at stage 17, to approxi­
mately stage 20, after which the decline in the labeling index of the wing was
rapid. The labeling index of the adjacent flank region, however, exhibited a
dramatic decline during stages 17 and 18 and then continued to decline at a
slower rate. The pattern of cell proliferation that was found by Searls & Janners
to accompany limb outgrowth was one characterized by differential rates of
decline during morphogenetic change, contrary to their expectation of enhanced
rates of proliferation within the mesenchyme of the limb outgrowth.
In a similar fashion, the determination of labeling indices in this study also
demonstrated a pattern of decline with advancing developmental age during
the formation of the nasal processes and the invagination of the olfactory
placode; and when adjacent areas (e.g. beneath the olfactory placode) were
examined, differential rates of decline were also found. This pattern, however,
is most evident at later stages in the development of the nasal processes, in
contrast to the limb-bud in which the pattern appears during the initial events
of limb morphogenesis. The events involved in the development of the limb,
however, are quite different from those observed during primary palate forma­
tion. Instead of a mesenchymal condensation followed by an outpocketing
of the surface epithelium, the initial events of primary palate formation include
8-2
112
R. MINKOFF AND A. J. KUNTZ
the thickening of surface epithelium to form the olfactory placodes which is
then followed by the invagination of these placodes to form depressions, and
then grooves which become the primitive choanae. The initiating events may,
therefore, be accompanied by different developmental mechanisms.
While a labeling index represents a composite of many parameters of cell
replication (Thrasher, 1966; Cleaver, 1967), a change in the labeling index does
not indicate which aspect of cell proliferation is being modified (e.g. cell cycle
time versus proportion of dividing cells). Nevertheless the labeling index is a
useful tool with which to measure and compare differences in proliferative
activity of different cell populations. Analysis of the cell cycle, and other aspects
of cell replication of the mesenchyme of the facial processes, are currently under
investigation. Initial conclusions, however, drawn from this study indicate that
the modulation of one or more aspects of cell replication, resulting in differential
rates of decline is, in all probability, a significant morphogenetic mechanism
operative during primary palate formation.
This investigation was supported by N.I.H. research grant no. RRO5333 from the Division
of Research Facilities and Resources and N.I.H. National Research Service Award GM05021
from the National Institute of General Medical Science.
We wish to thank Professors Phillip Hirsch, William Proffit, and Malcolm Johnston for
their constructive comments. We are most grateful to Ms Joyce Schumann and Janice Davis
and Mr Ross Nash for their skillful technical assistance.
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{Received 6 October 1976)