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.

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 mesenchyme-dependent 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 well-known in the mouse lung and salivary gland (Alescio & Colombo Piperno,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 super-numerary 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.

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.

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 mesenchymes 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 μg 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 μm 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 (2μg/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 t-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).

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 5μm transverse or parallel to its long axis, and stained with Mallory-Heidenhain azan staining to detect the aniline-blue-positive basement membrane.

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).

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. 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. 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. 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. 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).

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).

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 of mitotic 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 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.

Table 1

Distribution of mitotic activity in the anterior submaxillary rudiments

Distribution of mitotic activity in the anterior submaxillary rudiments
Distribution of mitotic activity in the anterior submaxillary rudiments

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.

Table 2

Distribution of mitogenic activity in the 9-day anterior submaxillary mesenchyme

Distribution of mitogenic activity in the 9-day anterior submaxillary mesenchyme
Distribution of mitogenic activity in the 9-day anterior submaxillary mesenchyme

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 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).

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. 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. 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. 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).

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).

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.

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.

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).

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 4morphogenetically 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 anteriorsubmaxillary 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.

Abercrombie
,
M.
(
1946
).
Estimation of nuclear population from microtome sections
.
Anat. Rec
.
94
,
239
247
.
Alescio
,
T.
&
Cassini
,
A.
(
1962
).
Induction in vitro of tracheal buds by pulmonary mesenchyme grafted on tracheal epithelium
.
J. exp. Zool
.
150
,
83
94
.
Alescio
,
T.
&
Colombo Piperno
,
E
. (
1967
).
A quantitative assessment of mesenchymal contribution to epithelial growth rate in mouse embryonic lung developing in vitro
.
J. Embryol. exp. Morph
.
17
,
213
227
.
Alescio
,
T.
&
Di Michele
,
M.
(
1968
).
Relationship of epithelial growth to mitotic rate in mouse embryonic lung developing in vitro
.
J. Embryol. exp. Morph
.
19
,
227
237
.
Banerjee
,
S. D.
,
Cohn
,
R. H.
&
Bernfield
,
M. R.
(
1977
).
Basal lamina of embryonic salivary epithelia: production by the epithelium and role in maintaining lobular morphology
.
J. Cell Biol
.
73
,
445
463
.
Bernfield
,
M. R.
,
Banerjee
,
S. D.
&
Cohn
,
R. H.
(
1972
).
Dependence of salivary epithelial morphology and branching morphogenesis upon acid mucopolysaccharide-protein (proteoglycan) at the epithelial surface
.
J. Cell Biol
.
52
,
674
689
.
Chopra
,
D. P.
&
Wilkoff
,
L. J.
(
1977
).
Reversal by vitamin A analogues (retinoids) of hyperplasia induced by N-methyl-N′-nitro-N-nitrosoguanidine in mouse prostate organ cultures
.
J. natn. Cancer Inst
.
58
,
923
930
.
Dunn
,
G. A.
&
Ebendal
,
T.
(
1978
).
Contact guidance on oriented collagen gels
.
Expl Cell Res
.
III
,
475
479
.
Goldin
,
G. V.
&
Wessells
,
N. K.
(
1979
).
Mammalian lung development: the possible role of cell proliferation in the formation of supernumerary tracheal buds and in branching morphogenesis
.
J. exp. Zool
.
208
,
337
346
.
Kratochwil
,
K.
(
1969
).
Organ specificity in mesenchymal induction demonstrated in the embryonic development of the mammary gland of the mouse
.
Devl Biol
.
20
,
46
71
.
Lawson
,
K. A.
(
1974
).
Mesenchyme specificity in rodent salivary gland development: the response of salivary epithelium to lung mesenchyme in vitro
.
J. Embryol. exp. Morph
.
32
,
469
493
.
Nogawa
,
H.
(
1978
).
The development of the salivary glands in the Japanese quail
.
J. Fac. Sci., Univ. Tokyo /JZ
14
,
95
103
.
Ronzio
,
R. A.
&
Rutter
,
W. J
(
1973
).
Effects of partially purified factor from chick embryos on macromolecular synthesis of embryonic pancreatic epithelia
.
Devl Biol
.
30
,
307
320
.
Sakakura
,
T.
,
Nishizuka
,
Y.
&
Dawe
,
C. J.
(
1976
).
Mesenchyme-dependent morphogenesis and epithelium-specific cytodifferentiation in mouse mammary gland
.
Science, N.Y
.
194
,
1439
1441
.
Smuts
,
M. S.
,
Hilfer
,
S. R.
&
Searls
,
R. L.
(
1978
).
Patterns of cellular proliferation during thyroid organogenesis
.
J. Embryol. exp. Morph
.
48
,
269
286
.
Wessells
,
N. K.
(
1970
).
Mammalian lung development: interactions in formation and morphogenesis of tracheal buds
.
J. exp. Zool
.
175
,
455
466
.
Wolff
,
ET.
&
Haffen
,
K.
(
1952
).
Sur une méthode de culture d’organes embryonnaires “in vitro”
.
Tex. Rep. Biol. Med
.
10
,
463
472
.