/ . Embryo/, exp. Morph. Vol. 46, pp. 65-74, 1978
65
Printed in Great Britain (© Company of Biologists Limited 1978
Cell proliferation and cell density
of mesenchyme in the maxillary process and
adjacent regions during facial
development in the chick embryo
By ROBERT MINKOFF 1 AND AMY J. KUNTZ 2
From the Department of Orthodontics and Department of Biostatistics,
University of North Carolina
SUMMARY
Cell proliferation, as measured by DNA labeling indices was analyzed during the early
development of the maxillary process. Chick embryos were labeled with [3H]thymidine for
.1 h and processed for autoradiography. The percentage of labeled mesenchymal cells was
determined within delineated areas in the maxillary processes and in adjacent regions.
Analysis of labeling indices in each of the areas at successive stages of development demonstrated a pattern of declining rates of cell proliferation with advancing developmental age.
Cell proliferation in adjacent regions declined earlier and, in some instances, faster than it
did in the maxillary process.
Cell density was measured in the maxillary process and the roof of the stomodeum and
was found to be higher in the maxillary process throughout the period studied. Cell density
and cell proliferation data were analyzed with reference to the operation of' density-dependent
inhibition' of growth as a regulatory mechanism for the observed changes.' Density-dependent
inhibition' of growth was not a satisfactory explanation for the observed differences between
the maxillary process and adjacent regions.
INTRODUCTION
The maxillary processes in avian and mammalian embryos are derived
embryologically from the first pharyngeal arch (Romanoff, 1960; Hamilton,
Boyd & Mossman, 1972), and contribute to the structures of the midface,
including the upper jaw, the palate, the upper lip, or beak, etc. The early
development of the maxillary processes are similar in avian and mammalian
embryos and includes the formation of outgrowths from the cranial ends of the
first pharyngeal arch which contain mesenchyme derived largely from cells of
the cranial neural crest that have migrated into this region (Johnston, 1966;
Noden, 1975; Le Lievre & Le Douarin, 1975). These outgrowths enlarge and
1
Author's address (for reprints): Department of Orthodontics, School of Dentistry 209H,
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, U.S.A.
66
R. MINKOFF AND A. J. KUNTZ
Fig. 1. A representative section through the maxillary process and the roof of the
stomodeum outlining the areas in which mesenchymal cells were counted to obtain
data for labeling indices. Mx, Maxillary process; R.S., roof of stomodeum.
extend below the eye and form the lateral parts of the primitive stomodeum.
During the course of their enlargement, they impinge and finally fuse with the
lateral and medial nasal elevations, thus contributing to the formation of the
primary palate. Later in development, shelves arise from the maxillary processes
and grow medially to form the secondary palate.
Descriptions of early development have referred to the presence of ' growth
centers' within each of the processes (Streeter, 1948; Warbrick, 1960; Andersen
& Matthiessen, 1967). It was stated, or implied, that proliferation of mesenchymal cells increased during the progressive enlargment and development of
both the nasal processes and the maxillary processes.
A recent study (Minkoff& Kuntz, 1977), however, reported that evidence for
increases in rates of cell proliferation of mesenchyme during development of
the nasal processes was not found. It was observed that rates of proliferation
were approximately unchanged during early stages and then declined during
later stages of development. More rapid rates of decline in cell proliferation
were observed in the regions adjacent to the nasal processes.
Because of these findings, we investigated the role of cell proliferation during
maxillary process formation in order to confirm our previous conclusions
regarding the nature of the 'growth centers' that are present during facial
morphogenesis. In addition, preliminary investigations were begun to examine
the mechanisms that underlie these alterations in cell proliferation.
Cell proliferation in the maxillary process
67
MATERIAL AND METHODS
Labeling. Chick embryos were incubated, windowed, staged, according to
Hamburger & Hamilton (1951), and labeled with [3H]thymidine (sp. act. 6-7 Ci/
m-mole, New England Nuclear Co.) for a 1 h pulse, either by injection directly
into the yolk sac or, at comparable stages, by application on top of the embryo
through a window in the egg shell. Doses ranging from 10 /^Ci to 30 /*Ci were
used and in all cases the label was diluted to a total volume of 0-1 ml in balanced
salt solution. Labeling was terminated by rapid fixation in Bouin's solution and
embryos were then washed, staged, dissected, dehydrated through graded
alcohols, embedded in paraffin and serially sectioned in the frontal plane at
4/trn. The developmental stages studied were from stage 20 to stage 32
(Hamburger & Hamilton, 1951), a period encompassing the early development
and subsequent fusion of the maxillary process with the nasal processes.
Autoradiography and cell counting. Slides containing cross-sections through
the middle, anteroposteriorly, of the maxillary process were used for analysis
(Fig. 1) and a delineated area of the maxillary process was examined. Slides
containing sections from appropriate areas were washed, coated with liquid
autoradiographic emulsion (Eastman-Kodak-NTB-2) (Rogers, 1973), dried,
stored in light-proof boxes at 4 °C for exposure times ranging from 1 to 6 weeks,
developed, and then stained with Harris' hematoxylin and eosin. The area to be
examined in the maxillary process was delineated by means of an ocular grid
and both labeled and unlabeled cells were counted for determination of DNA
labeling indices. Three to ten sections separated those sections used to obtain
data. Areas between the maxillary processes, in the roof of the stomodeum, were
delineated by means of an ocular grid on the same sections and comparable
methods were used to obtain labeling indices in these areas (Fig. 1).
In all cases, labeled cells were defined as those containing three grains above
background over the nucleus (background was low, less than one grain per
nucleus). At least three or more sections from each embryo were examined and
counted. Approximately 1000 cells were counted in each area and examinations
were conducted using an oil immersion objective at a magnification of 1000 x .
Cell counts were made primarily by one investigator but control counts were
done by a second and sometimes a third investigator. 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.
Cell density measurements were performed on the same slides used for determining labeling indices. An ocular grid was used to delineate the counting area
and all cells contained within a predetermined number of grid squares were
counted. Cell density measurements were converted and plotted as the number
of cells per 1000 /.im2.
R. MINKOFF AND A. J. K U N T Z
Mx
Fig. 2. Photographs of embryos in which the maxillary process is shown during initial
outgrowth (stage 21) (2 A), enlargement (stage 23-24) (2B), approximation of
maxillary process with nasal processes (stage 26) (2C), and fusion of maxillary
process with nasal processes (stage 28) (2D). Mx, Maxillary process; N.P., nasal pit.
RESULTS
The appearance of the maxillary processes during the developmental period
studied are shown in Fig. 2.
In Fig. 3, the percentage of labeled cells of the mesenchyme of the maxillary
process is plotted against the corresponding developmental age of the embryo.
The values for the labeling index do not increase during successive developmental stages; at later stages (from stage 24 to stage 30), the indices decline
sharply from previous levels.
An area on the roof of the stomodeum, between the maxillary processes, was
also delineated by means of an ocular grid (Fig. 1). When the labeling indices of
this region were compared to those of the maxillary processes at comparable
stages of development (Fig. 3), a rapid drop in cell proliferation was observed
in the roof of the stomodeum, from stages 20 to 25, followed by a leveling of
this decline until, at stage 30, the rates of cell proliferation in the maxillary
processes and in the roof of the stomodeum approached each other.
Additional regions were examined and compared to the maxillary processes
Cell proliferation in the maxillary process
69
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302010-
20
21
22
23
24
25
26
27
28
29
30
31
32
Developmental stages
Fig. 3. DNA labeling indices of mesenchyme of the maxillary process ( # ) and of
the roof of the stomodeum (O). Each data point represents counts of approximately
1000 cells. A total of 45 embryos were analyzed. Embryos were labeled with [3H]thymidine for 1 h, fixed, and processed for autoradiography as described in text.
in a limited series of embryos at selected stages of development. Areas below
the eye, lateral to the maxillary processes, and regions of mesenchyme between
the brain and the eye were delineated and counted on sections from the same
slides that were used to obtain data for the maxillary processes. When the labeling
indices of the mesenchyme of these regions were compared to the labeling
indices within the maxillary processes (Fig. 4), a rapid drop in cell proliferation
was found in the adjacent regions from stages 22 to 24 followed by a leveling
of this decline until at stage 30 the rates of cell proliferation in the maxillary
processes and those adjacent regions (below and medial to the eye) were approximately the same.
Cell density measurements were obtained employing ocular grids to delineate
areas on the same slides from which labeling indices were determined in the
maxillary process and the roof of the stomodeum.
Examination of cell density at successive stages of development (Fig. 5)
revealed a higher level in the maxillary process than in the roof of the stomodeum
at all stages studied. When individual sections are examined, the difference in
cell density is readily apparent (Fig. 1).
Examination of Fig. 5 indicates that cell density in the maxillary process is
approximately 80-100% higher than in the roof of the stomodeum in the early
stages studied (stages 20-22) and 30-50% higher at later stages (stages 27-30).
When the data was analyzed on an embryo by embryo basis, the cell density
70
R. MINKOFF AND A. J. K U N T Z
SO70605040302010-
24
27
30
Developmental stages
Fig. 4. Comparison of labeling indices of the maxillary process with additional
adjacent regions in a limited series of embryos at stages 22, 24,27 and 30. Areas below
the eye and between the brain and eye were delineated and counted employing the
same techniques that were used for the maxillary process. Each point represents the
mean of pooled data of approximately 4000 cells obtained from three tofiveembryos.
One standard error above and below the means is indicated by a bar. # , Maxillary
process; D, below the eye; A, between the brain and eye.
in the maxillary process exceeded that in the roof of the stomodeum in every
case. The values for the difference ranged from 15 to 134%.
DISCUSSION
A labeling index represents a composite of many parameters of cell proliferation (Thrasher, 1966; Cleaver, 1967) and although a change in the labeling
index does not indicate which aspect of cell proliferation has been altered
(e.g. cell cycle time v. growth fraction), it is still a useful tool to use for comparisons of proliferative activity.
The findings in this report of decelerating rates of proliferation during
development, based upon labeling indices, are consistent with what had been
found in a previous study of fronto-nasal morphogenesis (Minkoflf & Kuntz,
1977), and are consistent with the pattern of cell proliferation found during
limb development in the avian embryo (Hornbruch & Wolpert, 1970; Searls &
Janners, 1971; Ede, Flint & Teague, 1975) and with an analysis of cell proliferation during development of the secondary palate in the mammalian embryo
(Nanda & Romeo, 1975).
Cell proliferation in the maxillary process
71
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20
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23
24
I
25
26
I
27
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28
I
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29
30
Developmental stages
Fig. 5. Comparison of cell density in the maxillary process ( # ) with that in the roof
of the stomodeum (O)- The same slides were used, and regions counted, from which
labeling index data was obtained. Counting procedures, histological preparation, etc.
are described in the text. Least square lines were fitted to the data. Cell density is
expressed as number of cells/1000/tm2.
Cell density in the mesenchyme of the maxillary process was greater than
that in the roof of the stomodeum throughout the period of development studied.
This is consistent with the findings of Warbrick (1960) in his study of the upper
lip and nasal cavity in the human embryo. Warbrick noted that the mesenchyme
of the maxillary process was condensed relative to that of the roof of the stomodeum. There was, in fact, a clear demarcation between the two regions in cell
density. Warbrick's observations were concerned with the question of a 'septal
process'-mesenchyme derived from the maxillary process (and, therefore, the
first pharyngeal arch) which appeared to migrate into the roof of the stomodeum
to form the nasal septum. The differences in cell density between the two regions
led him to believe that there was little 'mixing' of the mesenchyme of the two
regions. Our observations on the differences in cell density between these
regions are in agreement with Warbrick's findings although the question of a
72
R. MINKOFF AND A. J. KUNTZ
'septal process' and the origin of the mesenchyme of the nasal septum is still
unresolved.
Cell density measurements were also analyzed in order to test the hypothesis
that the difference in the timing and rate of decline in cell proliferation between
the maxillary process and the roof of the stomodeum may have been due to
a mechanism involving 'contact inhibition' or 'density-dependent inhibition'
of growth.
Many examples of in vitro regulation of cell division have been described in
which rates of cell division decline at 'saturation' density. Cells in those in vitro
systems eventually become quiescent either because of postulated effects of
direct contact between cells (Todaro, Green & Goldberg, 1964) or because of
limitation of any of a variety of macromolecular or low molecular weight
factors present in the serum or synthetic medium (Holley & Kiernan, 1968;
Holley, 1975).
Recently, in vivo 'density-dependent inhibition' of cell division has been
reported. Inverse correlations between mitotic rate and cell density have been
found by Summerbell & Wolpert (1972) and Summerbell (1977) in the development of the chick limb-bud. The authors believed that this represented the first
demonstration of density-dependent growth control in vivo (Summerbell &
Wolpert, 1972).
During the early development of the maxillary process, the labeling index of
the mesenchyme in the roof of the stomodeum drops sharply from stage 20 to
25 (Fig. 3) from 44 to 13 %, in contrast to a relatively moderate decline in the
labeling index of the mesenchyme in the maxillary process (from 54 to 40%).
The level of cell density in the maxillary process, however, is higher than the
roof of the stomodeum at all stages studied both in the aggregate and on an
individual embryo basis.
It is, therefore, difficult to apply the concept of density-dependent regulation
of growth to the differentially greater rate of decline observed in the roof of the
stomodeum. If density-dependent regulation is operative, then it must be
operating in a region specific manner.
The declines in the labeling indices observed in the latter stages of development of the maxillary process are also associated with increasing cell density
and the concept of density-dependent regulation may be operative; however,
this is the period when differentiation of tissues is occurring and correlations
between proliferation indices and cell density may be a reflection of regional
differences in the pattern of cell differentiation (Ede et ah 1975; Thorogood &
Hinchliffe, 1975).
Correlation and regression analyses were performed on the data of cell
density and labeling index for stages 20-25, 26-30, and 20-30 for both the
maxillary process and the roof of the stomodeum. Significant inverse correlations were found in some cases but not in others. In the maxillary process
r = - 0-45 (P < 0-05) for stages 20-25, but r = -0-09 at stages 26-30. Similarly,
Cell proliferation in the maxillary process
73
in the roof of the stomodeum r = - 0-53 (P < 0-05) for stages 20-25 but
r = +0-43 for stages 26-30.
In summary, the development of the maxillary process is accompanied by
declining rates of cell proliferation in the maxillary process and adjacent regions,
and differences in rate and timing of the declines exist between these regions.
Cell densities are greater in the maxillary process throughout the period studied.
Although some inverse correlations between proliferation indices and cell
density were found, density-dependent regulation of cell division was not
found to be a satisfactory explanation of the early larger drop in cell proliferation in the roof of the stomodeum than in the maxillary process.
This investigation was supported by N.T.H. research grants RRO5333 from the Division
of Research Facilities and Resources, DE-04731 from the National Institute of Dental
Research and GM05021 from the National Institute of General Medical Science.
The authors wish to thank Dr Malcolm Johnston for his constructive comments and are
most grateful to Mrs Janice Davis and Mr Ross Nash for their skillful technical assistance.
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{Received 22 November 1977, revised 5 March 1978)