/. Embryol. exp. Morph. Vol. 62, pp. 229-239, 1981
229
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Analysis of elongating morphogenesis of quail
anterior submaxillary gland: absence of
localized cell proliferation
By HIROYUKI NOGAWA1
From the Zoological Institute, Faculty of Science,
University of Tokyo
SUMMARY
Quail anterior submaxillary glands elongated extensively without branching (more
than sevenfold) from 8 to 10 incubation days. Investigation of mitotic activity of the
rudiments in vivo showed no localized cell proliferation throughout the rudiments, and
recombination experiments in vitro to examine regional differences in mitogenic activity
of the surrounding mesenchyme also showed that no mesenchymal region specifically
stimulates the epithelial cell proliferation.
Histological observation of the rudiments showed that epithelial cells did not lengthen
in a parallel direction to the long axis of the rudiment, and that mesenchymal cells encircled
the epithelial cord perpendicularly to its axis. The basement membrane was obscure in the
distal end of the rudiments, while it was easily detected in the other part of the rudiments.
These results suggest that the elongating morphogenesis of the anterior submaxillary
rudiments is not achieved by localized cell proliferation but by almost uniformly distributed
cell proliferation, and mesenchymal cells surrounding the rudiment or the basement membrane may be involved in the controlling mechanisms of the elongating morphogenesis.
INTRODUCTION
Branching organs, such as mouse salivary gland, lung and mammary gland,
are useful materials to study tissue interactions in morphogenesis. Recombination experiments between these organs have revealed the mesenchymedependent morphogenesis: the mammary epithelium which originally branches
monopodially shows a dichotomous branching pattern when recombined with
the salivary mesenchyme (Kratochwil, 1969; Sakakura, Nishizuka & Dawe,
1976), and the bronchial mesenchyme induces the tracheal epithelium to bud
and branch (Alescio & Cassini, 1962; Wessells, 1970). However, the mechanisms
of inductive action of the mesenchyme on the epithelial morphogenesis have
been poorly understood.
A stimulatory effect of mesenchyme on the growth of epithelium is wellknown in the mouse lung and salivary gland (Alescio & Colombo Piperno,
1
Author's address: Zoological Institute, Faculty of Science, University of Tokyo, Hongo,
Tokyo 113 Japan.
230
H. NOGAWA
1967; Alescio & di Michele, 1968; Lawson, 1974), and a mesenchymal factor
which enhanced the DNA synthesis of the pancreatic epithelium was extracted
from several mesodermal tissues (Ronzio & Rutter, 1973). One possible
explanation of the action of mesenchyme is that the mesenchyme determines
a morphogenetic pattern by stimulating the cell proliferation of a specific part
of the epithelium. Bernfield, Banerjee & Cohn (1972) reported that lobules
of the mouse submaxillary gland were predominantly labelled with [3H]thymidine compared with stalk regions. Wessells (1970) and Goldin&Wessells
(1979) tried to confirm the presence of localized cell proliferation in the supernumerary tracheal bud, but failed to do so.
We have reported in the previous paper that the embryonic quail anterior
submaxillary gland, which is located in the anterior portion of the floor of the
mouth, elongates without branching in vivo (Nogawa, 1978). Since the elongating
morphogenesis of this gland was very simple, this material was thought to be
suitable for studying the relationship between the distribution of cell proliferation and the morphogenetic pattern.
The present paper examines whether a region having a specially high mitotic
activity exists along the long axis of the quail anterior submaxillary rudiments
in vivo, and whether there is a mesenchymal region specifically stimulating
the cell proliferation of the epithelium in vitro. Histological structures of the
normal rudiments were also examined.
MATERIALS AND METHODS
Isolation of anterior submaxillary region
Embryos of Japanese quail (Coturnix coturnix japonica) of the 7th, 8th, 9th
and 10th incubation day were used. A piece of the floor of the mouth including
anterior submaxillary rudiments was isolated from the mandibular bone and
the underlying muscular layer.
Recombination experiments
Explants were cultivated according to the method of Wolff & Haffen (1952).
The medium comprised seven parts of agar (1 % in Gey's solution), three parts
of digestive-tract-free and salivary-gland-free 12-day chick embryo extract
(50 % in Tyrode's solution) and three parts of horse serum (Flow laboratories),
and a trace of penicillin was added as antibiotic.
For recombination experiments, epithelia and mesenchymes were separated
using collagenase (Worthington, CLSPA, 0-3 mg/ml Tyrode's solution at 38 °C
for 60 min). The separated tissue fragments were rinsed twice in a mixture of
Tyrode's solution and horse serum (1:1), and kept in a storing solution consisting of Tyrode's solution, embryo extract and horse serum (7:3:3) at a room
temperature until they were used.
Elongating morphogenesis of quail salivary gland
231
To examine regional differences of mitogenic activity in the mesenchyme,
the surrounding mesenchyme of the 9-day anterior submaxillary rudiment
was isolated from the epithelium using collagenase, and divided into three
equal parts along the long axis of the rudiment; the 9-day mesenchyme was
used since the rudiment was actively elongating at this stage. Since each
isolated mesenchyme was too small, three mesenchymal pieces deriving from
the same position were combined, and the epithelium of a single 8-day anterior
submaxillary rudiment was combined with the three rnesenchymes and cultured;
the 8-day epithelium was used since it was difficult to separate the older epithelium from the surrounding mesenchyme intact.
Measurement of length
Isolated anterior submaxillary regions, being unfixed, were photographed,
and lengths of the rudiments were measured.
Assessment of mitotic activity
Mitotic activity was examined by the aid of colcemid in vivo and in vitro.
In vivo, colcemid (1 jag in 0-05 ml phosphate-buffered saline per egg) was
injected into the amniotic cavity through a hole at the blunt end of the egg.
After 4 h incubation at 38 °C, the anterior submaxillary region was immediately
isolated and fixed in Bouin's fluid. Serial paraffin sections of the rudiment
were cut at 5 /*m transverse to its long axis, and stained with haematoxylin
and eosin. The rudiment was divided into three equal parts (proximal, median
and distal) according to the number of transverse sections of it, and the mitotic
activity of each region was assessed by following procedures: the numbers of
metaphase-arrested and total nuclei were counted in every other section,
summed up separately and expressed in index (%). The mitotic index of each
region was then converted to a ratio to the mitotic index of the median region
to diminish the individual difference in a statistical analysis.
In vitro, after 20 or 40 h cultivation, recombinants were transferred to the
storing solution containing colcemid (2/tg/ml: the concentration accords with
that used by Chopra & Wilkoff, 1977), incubated at 38 °C for 4h, and fixed
in Bouin's fluid. Serial sections of the recombinants were surveyed in the same
manner as in vivo.
Student's /-test was used for a statistical analysis.
The cell number of a whole rudiment was estimated as follows: the sum
of the numbers of total nuclei which were counted in every other section
throughout the whole rudiment was doubled and multiplied by a correction
factor T/(T+D), where T was the thickness of the section and D was the
mean nuclear diameter, according to the method of Abercrombie (1946).
232
H. NOGAWA
100
' 5
Elongating morphogenesis of quail salivary gland
233
Colouration of basement membrane
Isolated anterior submaxillary regions were fixed in Bouin's fluid and
embedded in paraffin. Serial sections of the rudiment were cut at 5 /tm transverse or parallel to its long axis, and stained with Mallory-Heidenhain azan
staining to detect the aniline-blue-positive basement membrane.
RESULTS
Normal development of the anterior submaxillary gland
Anterior submaxillary rudiments appeared as epithelial masses vertically
projecting into the mesenchyme in the anterior floor of the mouth on day 7 of
incubation (Fig. 1). The rudiments elongated laterally on day 8 (Fig. 2), and
thereafter they actively elongated backwards (Figs 3, 4). After day 10, the
elongation rate gradually decreased. The number of the rudiments was usually
six to eight, and the hind two pairs elongated longer than the front pairs.
Lumen structures began to develop within the proximal region of the rudiments
on day 9 or 10 (Fig. 5), and extended distally. Further details were described
in the previous paper (Nogawa, 1978).
The lengths of the hind two pairs of rudiments were measured from day 8
to 10 when the rudiments were actively elongating. The length increased about
fourfold from day 8 (0-23 ±0-09 mm: mean±s.D.) to day 9 (0-88 ±0-22 mm),
and twofold from day 9 to day 10 (1-76 + 0-31 mm). The rudiments elongated
more than sevenfold long during two days from day 8 to 10.
Distribution ofmitotic activity in the anterior submaxillary rudiments
Since the anterior submaxillary rudiments extensively elongated without
branching from day 8 to 10, were mitoses localized in a specific part of these
elongating rudiments ? (Table 1). In the 8-day rudiment, there was no significant
difference in the mitotic activity between the proximal and median or the
Fig. 1. A section of a pair of 7-day anterior submaxillary rudiments. The rudiments
are observed as projections of the oral epithelium into mesenchyme.
Fig. 2. A living 8-day anterior submaxillary region. Three pairs of rudiments are
observed (arrows).
Fig. 3. A living 9-day anterior submaxillary region. The rudiments extensively
elongate backwards (to the lower side of this figure) compared with those in
Fig. 2. Magnification is indicated in Fig. 2.
Fig. 4. A left half of living 10-day anterior submaxillary region. Two long rudiments
run from the upper right to the lower left. Magnification is indicated in Fig. 2.
Fig. 5. A section of the proximal region of 10-day anterior submaxillary rudiment
cut parallel to the long axis. Lumen structures (1) are observed near the opening
(0).
0-82 ±019
106 ± 0 1 8 '
105±0-26 b
1
1
0-94 ±0-30
0-82 ±015
8
9
No. of
observations
12
8
Cell number
of a whole
rudiment
1300-2100
7500-11000
Mitotic index
of a whole
rudiment
(%)
13-2±3-7
1O-6±3O
o>b
* Values express the ratio of the mitotic index to that of the median region (mean ± S.D.).
The distal region of the 9-day rudiment was further halved along the axis, a: proximal and b: distal.
Distal
Median
Proximal
Stage
(days)
Region*
Table 1. Distribution of mitotic activity in the anterior submaxillary rudiments
o
o
X
to
Elongating morphogenesis of quail salivary gland
235
Table 2. Distribution ofmitogenic activity in the 9-day
anterior submaxillary mesenchyme
Region of mesenchyme
Culture
period
Proximal
Median
Distal
20 hr
40 hr
2•6 ±0-7 (12)
2•5 ± 1 1 (10)
2-8 ± 1 0 (12)
2-2 ±0-5 (8)
3 0±l-2 (12)
2•1 ±0-7 (10)
Each value expresses the mitotic index (%) of the 8-day anterior submaxillary epithelium
cultured in recombination with the mesenchyme from each region: mean±s.D. (no. of
recombinants).
proximal and distal regions, but the distal region had lower mitotic activity
than the median region (P < 0-01). In the 9-day rudiment, the proximal region
had lower mitotic activity than the median region (P < 0-01). However, the
median and distal regions of the 9-day rudiment had similar mitotic activity.
These results demonstrate that cell proliferation is not localized in the advancing
distal portion of the rudiment, but is distributed almost uniformly through
the rudiment.
The cell number of the whole rudiment increased comparable to the length
from day 8 to 9 (Table 1), although it was not yet known whether the increase
in cell number was only due to cell proliferation of anterior submaxillary
epithelial cells or partly due to the invagination of neighbouring oral epithelial
cells into the rudiment.
Distribution of mitogenic activity in the anterior submaxillary mesenchyme
To determine whether a regional difference in the mitogenic activity of the
surrounding mesenchyme existed along the long axis of the anterior submaxillary
rudiment, an 8-day anterior submaxillary epithelium was cultured for 20 or
40 h in vitro recombined with the 9-day anterior submaxillary mesenchyme of
the proximal, median or distal regions (Table 2). Mesenchyme from each of
the three regions similarly supported the mitotic activity of the epithelium.
These recombination experiments show that there is no mesenchymal region
specifically stimulating the epithelial cell proliferation along the long axis of
the rudiment.
Histological structures of the anterior submaxillary rudiments
The epithelium of the 8-, 9- and 10-day anterior submaxillary rudiments
consisted of two morphologically different populations of cells (Figs 6, 7).
Cells located in the periphery of the rudiment were lengthening perpendicularly
to the long axis of the rudiment judging from their nuclear orientation, and
those located in the centre were not ordered and had round nuclei. Such
bilayer structure was observed from the proximal region to near the distal
H. NOGAWA
• s •
,.-
50
Fig. 6. A section of the median region of 9-day rudiment cut parallel to the long
axis. Epithelial cells in the periphery are arranged and have an oval nucleus
which lengthens perpendicularly to the axis, while those in the centre are less
arranged and have a round nucleus. The same structure is observed in Fig. 7.
Fig. 7. A transverse section of the median region of 9-day rudiment. Aniline-bluepositive basement membrane is detected in the epithelio-mesenchymal interface.
Mesenchymal cells encircle the rudiment.
Fig. 8. A section of the distal end of 9-day rudiment cut parallel to the long
axis. Orderly arranged mesenchymal cells are not observed in front of the distal
end (d).
Fig. 9. A transverse section of the distal end of 9-day rudiment. Aniline-blue-positive
basement membrane is obscure compared with that in Fig. 7.
end of the rudiments, and mitotic figures were found in both layers. No separate
epithelial cell existed in front of the tip of the rudiments (Fig. 8).
The aniline-blue-positive basement membrane was obscure in the distal end
of the rudiments, while it could be easily detected in the epithelio-mesenchymal
interface of the other part (Fig. 9).
Mesenchymal cells were encircling the epithelial cord perpendicularly to its
axis from the proximal region to near the distal end: this could be observed
particularly in transverse sections of the rudiment (Fig. 7). Orderly arranged
mesenchymal cells were not observed in front of the distal end of the rudiment
where it would elongate (Fig. 8).
Elongating morphogenesis of quail salivary gland
237
DISCUSSION
The quail anterior submaxillary gland extensively elongated without branching, and this characteristic morphogenesis made it a suitable material for the
study of morphogenesis. The present observations on the relationship between
the distribution of cell proliferation and the elongating morphogenesis made
it clear that no specific part along the long axis of the rudiment has specially
high mitotic activity. Only the mitotic activity of the distal region in the 8-day
and of the proximal region in the 9-day rudiment was lower by nearly 20 %
than that of the median region respectively. Although a significant difference
in mitotic activity was statistically detected between them, it seems questionable
to attach importance to this mere 20 % difference instead of the twofold or
threefold difference expected of a controlling mechanism of this elongating
morphogenesis. Further studies with recombination experiments showed that
no specific part of the surrounding mesenchyme significantly stimulates the
cell proliferation of the gland epithelium. From these results, it can be concluded
that the elongating morphogenesis of the anterior submaxillary gland is not
achieved by localized cell proliferation.
The word 'morphogenetically active' is useful in accounting for morphogenetical phenomena. In the case of epithelial morphogenesis proceeding within
mesenchyme, the morphogenetically active site has been thought to be the
one where the epithelium actively goes forward into the mesenchyme, for
example budding points or distal ends of lobules of branching organs. In the
mouse salivary gland, it has been said that cell proliferation is localized in the
morphogenetically active site: Bernfield et al. (1972) reported that lobules
were more predominantly labelled with [3H]thymidine than stalks. Thereafter,
however, results supporting this report have not been presented in studies of
other organs, such as chick thyroid (Smuts, Hilfer & Searls, 1978) and mouse
lung (Goldin & Wessells, 1979). In the quail anterior submaxillary gland, it is
a distal portion of the rudiment that must be morphogenetically active, since,
if the middle portion was morphogenetically active, new buds would be formed
there and the rudiment would take a branching morphology. The present
study clearly demonstrates that cell proliferation is not localized in the distal
portion of the rudiment, suggesting that the morphogenetically active site
does not always correlate with the localized cell proliferation.
What mechanisms do control the elongating morphogenesis of the anterior
submaxillary gland? If the cell mass grows freely, it will expand multi-directionally
and take a spherical shape. In contrast, if the growth of the cell mass is limited
to one direction, it will take an elongating morphology. As for the mechanisms
which limit the direction of growth of the epithelial cell mass, several theories
are possible. The first is the direction of cell division: if all the epithelial cells
divide in a particular direction, the cell mass will take a morphology lengthening
in the direction of mitoses. All the epithelial cells, however, of the anterior
238
H. NOGAWA
submaxillary rudiment never divided in the same direction (data not shown).
The second is the elongation of individual cells: if all the epithelial cells elongate
in a particular direction, the cell mass will consequently take an elongating
morphology. However, the epithelial cells of anterior submaxillary rudiment
did not run parallel to the long axis of the rudiment. On the contrary, the outer
cells near the basement membrane were elongating perpendicularly to the axis.
The third is the contact guidance: if the substratum for epithelial cell locomotion
pre-exists in an elongating form, the epithelial cell mass will elongate along
it. Although the contact guidance of collagen fibres for fibroblasts has been
reported by Dunn & Ebendal (1978), no one has reported the contact guidance
for epithelial cells. We have no data to discuss the possibility of the contact
guidance in the elongating morphogenesis of the anterior submaxillary rudiment.
The fourth is the suppression of swelling of the rudiment by surrounding
structures: if the surrounding structures inhibit swelling of the rudiment, the
increase in volume of the rudiment will generate a motive force for elongation.
In the anterior submaxillary rudiment, mesenchymal cells were encircling the
epithelial cord perpendicular to its axis, and the aniline-blue-positive basement
membrane was detected in the whole part of the rudiment except the distal
end. It seems that the perpendicular arrangement of the surrounding mesenchymal cells is more resistant to the swelling of the long rudiment than the
parallel arrangement, and it has been known that the basement membrane
has a role in maintaining the epithelial morphology (Banerjee, Cohn & Bernfield, 1977). Similar structures were observed in the trachea and the bronchial
stalks of the embryonic mouse lung (Wessells, 1970). In the quail anterior
submaxillary gland, the surrounding structures may have an important role
in controlling the elongating morphogenesis, and the morphogenetic pattern
may be determined by the mesenchymal cells or the basement membrane.
The author wishes to express his deep gratitude to Prof. Takeo Mizuno of the University
of Tokyo for his valuable advice and encouragement during the course of this work.
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(Received 23 June 1980, revised 18 September 1980)