Acta histochem. 89, 213-235 (1990) Gustav Fischer Verlag Jena
*Department of Oral Pathology, College of Dentistry, and **Department of Pathology, College of Medicine, Seoul National University, Seoul, Korea, and ***Department of Oral and Maxillofacial Surgery, Asahi University School of Dentistry, Gifu, Japan
Prenatal development of human major salivary glands and immunohistochemical detection of keratins using monoclonal antibodies By SUK-KEUN LEE*, CHANG YUN LIM*, JE GEUN CHI**, KAZUTO YAMADA***, KOUJI HASHIMURA***, MAYUKO KUNIKATA***, and MASAHIKO MORI*** With 9 Figures (Received April 4, 1990)
Summary The major salivary glands were examined from 69 human fetuses ranging from 10 to 40 weeks of gestation. Prenatal growth curves of developing salivary glands could be established by histological scoring, and development was divided into the early developmental stage (EDS) from 10 to 18 weeks, early intermediate developmental stage (EIDS) from 19 to 24 weeks, late intermediate developmental stage (LIDS) from 15 to 32 weeks, late developmental stage (LDS) from 33 to 40 weeks. Characteristic morphogenesis and cytodifferentiation occurred in glandular duct cells during the period of EIDS and LIDS. In the LDS, acini and ducts of the salivary glands histologically developed into a mature state similar to adult glands. Immunohistochemical staining with monoclonal antibodies (MaAhs) PKKI, KLl, K8.12, K8.l3, K4.62, RPN 1160, 1162, 1163, 1164, and 1165 was performed. During the fetal period, keratin expression as revealed by MoAbs PKKI, KLl, K8.12 was well established, and the staining pattern for each of these antibodies was comparable. Other antibodies showed rare or negative staining except K8.l3 which had a diffuse, non-specific staining pattern. Accordingly, the proliferation and cytodifferentiation of fetal stage keratin staining in ductal cells as revealed by MoAbs PKKI, KLl, and K8.12 showed a heterogenic distribution in both luminal and basal cells. It is a characteristic finding that the cytodifferentiation of ductal luminal cells precedes ductal basal cells. Ductal basal cells stained with MoAb K8. 12 and show heterogeneity of keratin distribution continuously until the full term of gestation. The keratin staining of oral epithelium was also examined to compare with distribution of salivary gland ductal cells and oral epithelial cells. In the present study, the developmental sequence of salivary gland cells and the immunohistochemical properties of keratin proteins in these cells were described in relation to the histogenesis of salivary gland tumours.
1. Introduction
In order to investigate the histogenesis of salivary glands and their tumours, exploration of stem cells may be regarded as a fundamental problem in the developmental biology. There is little doubt that the parotid gland arises from the stomodeum and is therefore of ectodermal derivation. The sublingual and submandibular glands remain somewhat obscure as to their derivation, but probably they are ectodermally based, contrary to lingual glands (including von Ebner's glands) which are undoubtedly endodermal derivatives (AVERY et al. 1987, BATSAKIS 1980).
214
S.-K. LEE et al.
The existence of intennediate sized keratin proteins associated with cell organization and integration has been reported in ductal epithelia of salivary glands (CASELITZ et al. 1986, DARDICK et al. 1987, 1988, MARSHAK and LEITER 1987, MORI et al. 1987, 1989 TAKAI et al. 1988). The salivary glands of several mammals including rodents, domestic animals, and humans have been reported to contain keratins in excretory, striated or grandular convoluted duct, and intercalated ducts (TAKAI et al. 1985). The frequentcy of occurrence of salivary gland tumours has been shown to greatly vary among major salivary glands and histopathological type of tumours. Those differences were probably related to the histologic varieties of prenatal developing salivary glands. Compared to the development of rat or mouse salivary glands, which are differentiated predominatly postnatally, the development of human salivary glands has not elucidated in detail. Recently, Gustavsson et al. (1988) demonstrated the expression of monoclonal antibodies of 3 keratins and 2 vimentins in developing salivary glands in man. In the present study, we attempt to investigate histological details during prenatal development of human salivary glands, and immunohistochemical detections of keratins using monoclonal antibodies.
2. Materials and methods A total of 70 aborted human fetuses, confinned as nonnal by histopathological examination, was obtained from the files of the RCM (Registration of Congenital Malfonnation, Department of Pathology, Seoul National University Children's Hospital, Seoul, Korea). Their gestational ages ranged from 10 weeks to 40 weeks. The tissue bank includes 57 parotid, 60 submandibular and 51 sublingual glands, fixed in 10% neutral fonnalin. Serial paraffin sections at 4 11m were used for immunohistochemical detection of keratins and to identify histologic features using HE and PAS stain.
Table l. Immunohistochemical detection of monoclonal antibodies for keratins Immunogens
KLl
human keratinized squamous epithelium
PKKI
pig kidney epithelium
K8.12 K8.13
}
bovine epidermal kreatin
Kreatin type no. of relative molecular masses
55,000 '" ",000
RPN1160
PtKl cells
no. 18
RPN1162
PTl12 bladder Ca. cell line
no. 7
RPNl163
PtKl cells
J
Immunoteck (Marsille) Labsystems (Helsinki)
]
no. I, 5, 6, 7, 8 (basic) no. 10, 11, 18 (acidic) no. 19
RPN1l65
1I80
no. 13, 16
Cultured human breast carcinoma
SV 40 transformed human keratinocyte
Products
40,000, 45,000, 52,000, 5,000 no. 19, 18, 8
K4.62
RPNl164
)
Dilution
no. 8 (type II)
no. 19
Bio-Yeda (Israel)
1I20
Amersham (Buckinghamshire)
215
Prenatal development of human major salivary glands
In order to estimate the developmental stage of fetal salivary glands, incremental scores for glandular structures from each salivary gland were made. In the present study, excretory ducts, striated ducts, intercalated ducts, and acinar cells were observed in different developmental stages, each exhibiting characteristic histology. 2 early stages in fetal salivary gland development were added for the purpose of descriptive representation. From incidental findings of oral epithelium usually associated with sublingual salivary glands, immunohistochemistry of epithelium in oral mucose was used to compare keratin staining of all major salivary glands. Fetal salivary glands were removed from 43 fetuses of estimated gestational ages: 10, 15, 17, 18, 19,20 (2),21 (3),22,23 (3), 24 (3),25 (2), 26 (5),27 (2), 28 (2), 29 (2), 30, 31, 32, 33 (2), 34, 36, 38 (3), 39, and 40 (2) weeks. For immunohistochemical detection of keratins, MoAbs PKKI (nos. 8, 18, 19), KLI (56KD), and K8. 12 (nos. 13, 16) were routinely examined, and additionally MoAbs K8.13 (nos. 1,5,6,7,8, 10, 11), K4.62 (no. 19), RPN 1160 (no. 18), 1162 (no. 7), 1163, 1164 (no. 8), 1165 (no. 19) were used to further elucidate different types of keratin (Table I). Details of irrunnnohistochemical staining methods have been described (TAKA! et al. 1988).
3. Results 3.1. Development of salivary glands and histological scoring The development stages of salivary glands were estimated using the criteria for developmental scoring of tissue elements of salivary glands listed in Table 2. 3.1.1. Excretory ducts: In the excretory duct, score 1 is for the initial stage of growth of cordlike strands in salivary gland mesenchyme. Score 2 is for the stage of luminalization with the appearance of inner ductal cell arrangement in the main stalk of the salivary epithelium. Score 3 is for the stage of ductal development in the extra-lobular space. Score 4 is for the thickened excretory ductal epithelium composed of 3 to 4 cell layers (stratification) and shows abundant vascular proliferation around the excretory duct. Score 5 is for the stage of the differentiation of inner duct cells into columnar cells with abundant apical cytoplasm. Score 6 is for the stage of maturation of the excretory duct including the presence of a conspicuous basement membrane. 3.1.2. Striated ducts: In the striated duct, score 1 is for the initial stage of cord-like epithelial strands. Score 2 is for the stage of luminalization with the appearance of an inner ductal cell arrangement, usually in the branched ductal epithelium. Score 3 is for the stage of a 2-celllayer ductal structure, usually in the intralobular space. Score 4 is for the stage of elongation and Score
25 20 15 10 5
• 5
10
15
o : Sublingual gland • : Submandibular gland * : Parotid gland
20
25
30
35
40
Gestational Age (Weeks)
Fig. I. Growth curve by the sum of histological scoring. The growth stage of developing major salivary gland could be assessed by the total score of 4 tissue elements (excretory ducts, striated ducts, intercalated ducts, and acinar cells).
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Table 2. Histological scores for the observation of developing major salivary glands A. Excretory duct I Cord-like epithelial strands grow deeply into the salivary gland mesenchyme making a several branches. 2 Luminalization begins from the distal tubules, and there appears the arrangement of an inner ductal cell layer. 3 A 2-cell layer ductal structure composed of inner columnar cell layer and outer cuboidal cell layer usually prominent in the extra-lobular space. 4 The duct thickened and became a 3-to 4-cell layer ductal structure in the interlobular space. Abundant vascularity appeared in the vicinity of the duct. 5 Inner ductal cells show columnar nuclei and abundant apical cytoplasm, and the outer ductal cell is cuboidal in shape. 6 Mature excretory ducts show a well aligned columnar inner cell layer and a slightly columnar basal cell layer with a conspicuous basement membrane. The distal side of the excretory duct discloses and increased multi-cell layer of ductal epithelium compared to the proximal side excretory duct which is usually located in the interlobular space. B. Striated duct I Cord-like epithelial strands grow deeply into salivary gland mesenchyme making a several branches. 2 Luminalization begins from the distal tubules and there appears the arrangement of an inner ductal cell layer. 3 A 2-celllayer ductal structure composed of inner and outer cuboidal cell was seen in the intralobular space. 4 The duct became elongated and convoluted. Abundant vascularity appeared in the vincitiy of the duct. 5 Inner ductal cells show columnar nuclei and abundant apical cytoplasm, and the outer cuboidal ductal cells are decreased in number. 6 Mature striated ducts consist of a layer of tall columnar cells with definite basement membrane, occasionally polyhedral cells were found in the basal area. C. Intercalated duct I 2 3 4 5 6
Club-like epithelial strands grow deeply into salivary gland mesenchyme making several terminal epithelial bulbs. Luminalization begins from the distal tubules, and there appears the arrangement of an inner ductal cell layer. A I-cell layer ductal structure in direct conncetion with acini. Single layered inner ductal cells are cuboidal in shape and infrequently surrounded by flattended basal cells. A 2-cell layer ductal structure composed of inner cuboidal cells and outer flattened cells. Mature intercalated ducts consist of a definite cuboidal cell layer and frequent outer polyhedral cells. The basement membrane become conspicuous.
D. Acinar cells I 2 3 4 5 6
Knob-like bulging of the proximal epithelium. Several terminal blub-like structure form. The primitive acini obtain an alveolar pattern. Initial acinic cell differentiation. Moderate acinic cell differentiation. Complete acinic cell differentiation.
convolution of the striated duct and abundant capillary proliferation. Score 5 is for the stage of differentiation of inner ductal cells into tall columnar cells, resulting in the outer basal cells becoming reduced in number. Score 6 is for the maturation of the striated duct which consists of almost I-cell layer with a conspicuous basement membrane. There was infrequently found polyhedral cells in the basal area of the duct. 3.1.3. Intercalated ducts: In the intercalated duct, score 1 is for the initial stage of club-like epithelial proliferation in the terminal branches. Score 2 is for the stage of luminalization with the appearance of an inner ductal cell arrangement. Score 3 is for the stage of I-cell layer ductal structure connected with the terminal bulging of the acini. Score 4 is for the stage of proliferation of the ductal cells, resulting in the inner ductal cells being surrounded by flattened outer cells. Scroe 5 is for the stage of differentiation of duct cells into inner luminal cells of cuboidal shape
Prenatal development of human major salivary glands
217
Fig.2. A Developing sublingual gland from 15 weeks old fetus. After luminalization in the branching salivary gland epithelium produced 2- cells layer structure of excretory and striated ducts were seen. HE, X 32. B Intercalated duct from 21 weeks old fetus consists of monolayer of cuboidal cells at the terminal branches. HE, X 160. C Intercalated duct from 23 weeks old fetus showed proliferation of outer cell layer surrounding the inner ductal cells. HE, X 160. D Excretory duct from 23 weeks old fetus showed well aligned luminal cells and basal cells. HE, X 160. E Striated duct from 38 weeks old fetus consisted of almost monolayer of tall columnar cells showing the abundant apical cytoplasm. HE, X 160, 15
Acta histochem., Bd. 89, 2
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and outer basal cell of a flattended shape. Score 6 is for the stage of maturation of the intercalated duct which is composed of cuboidal luminal cells and flattened basal cells with a conspicuous basement membrane. 3.1.4. Acinar cells: In the acinar compartment, score 1 is for the stage of initial knob-like bulging of the proximal epithelium. Score 2 is for the stage of multiple bulb-like formations in terminal epithelium. Score 3 is for the stage of occurrence of primitive acini having an alveolar arrangement. Score 4 is for the stage of initial acinar cell differentiation in the glandular lobules. Score 5 is for the stage of moderate acinar cell differentiation in the glandular lobules. Score 6 is for the stage of almost complete differentiation of acinar cells. In brief, the excretory ducts were characterized by the most rapid maturation compared with others. Excretory duct maturation was identified predominately by the stratification of ductal epithelium and by the cytodifferentiation of luminal epithelium. Excretory duct maturation occured from about 24 to 25 weeks of gestation in all major salivary glands (Fig. 2D). The striated ducts were principally composed of 2-celllayers in the early fetal period: inner luminal cells and outer basal cells. A characteristic of striated duct maturation was that the luminal ductal cells gradually developed into tall luminal cells occupying the whole ductal thickness and forming a basement membrane at their basal border. With maturation, the number of ductal basal cells Table 3. Histological scoring of tbe developing major salivary glands in human fetuses G. A. [weeks]
Cases (No.)
Types of salivary glands Submaxillary gland
Parotid gland A
18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 36 38 39 40
1 1 2 2 1 3 3 2 4 5 5 8 2 3 5 1 3 1 3 2 1 5 2 5
Total
70
10 15 17
B
C
Sublingual gland C
D
3.0
3.0
2.0
4.0 4.0 4.5 4.0 4.0 4.0 4.8 5.0 5.1 6.0 6.0 6.0
4.0 4.0 3.5 4.0 4.0 4.0 4.0 4.0 4.9 6.0 5.7 6.0
4.0 3.0 3.5 3.5 4.0 4.0 4.5 5.0 5.1 5.5 5.0 5.5
4.0 4.0 3.5 4.0 4.0 4.0 4.8 4.8 5.1 5.5 5.0 5.5
6.0 6.0 6.0 6.0 6.0 6.0
(6.0) (6.0) (6.0) (6.0) (6.0) (6.0)
5.5 6.0 6.0 6.0 6.0 6.0
6.0 6.0 6.0 6.0 6.0 6.0
6.0
(6.0)
6.0
6.0
D
A
B
C
D
A
2.0 3.0 3.0 3.0 4.0 4.0 4.0 4.0 4.5 5.0 5.0 5.3 5.5 6.0 6.0 6.0 6.0
1.0 3.0 3.0 3.0 3.0 3.0 4.0 4.0 4.0 4.0 4.0 4.1 4.0 5.0 5.0 6.0 6.0
1.0 3.0 3.0 3.0 3.0 3.0 3.5 4.0 4.0 4.3 4.7 5.0. 5.0 5.0 5.0 5.0 5.0
2.0 2.0 3.0 3.0 3.0 3.0 3.0 3.8 4.0 4.3 5.0 5.0 5.0 5.0 5.0 5.0
3.0
6.0 6.0 6.0 6.0 6.0 6.0
6.0 6.0 6.0 6.0 6.0 6.0
5.0 5.0 5.0 5.1 6.0 6.0
5.0 5.5 6.0 6.0 6.0 6.0
3.0 3.0 4.0 4.0 4.0 4.0 4.3 4.5 5.0 5.1 5.0 5.7 6.0
3.0 3.0 3.0 3.0 4.0 4.0 4.0 4.0 4.0 4.1 4.0 4.0 5.0
3.0 3.0 3.0 3.0 3.7 4.0 4.0 4.0 4.0 4.6 5.0 5.0 5.0
3.0 3.0 3.0 3.0 3.0 3.0 3.7 4.0 4.3 5.0 5.0 5.0 5.0
6.0 6.0 6.0 6.0 6.0 6.0 6.0 6.0
5.0 6.0 5.7 6.0 6.0 6.0 6.0 6.0
5.0 5.0 5.0 5.0 5.0 5.0 5.0 6.0
5.0 5.0 5.0 5.0 5.0 5.0 6.0 6.0
B
Abbreviation; GA gestation age, A excretory duct, B striated duct, C intercalated duct, D acinus, ( ) illdefined observation of striated duct of sublingual gland
Prenatal development of human major salivary glands
219
Table 4. Sum of the histological scoring of the developing major salivary glands in human fetuses
G.A. [weeks]
Cases (No.)
Types of salivary glands Parotid gland
Submaxillary gland
12.0 12.0 13.0 13.0 14.7 15.0 16.0 16.5 17.3 18.0 19.0 19.7 21.0
4.0 11.0 11.0 12.0 13.0 13.0 14.5 15.0 16.3 17.3 18.0 19.4 19.5 21.0 21.0 22.0 22.0
I I
10 15 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 36 38 39 40
3 2 1 5 2 5
Total
70
2 2 I
3 3 2 4 5 5 8 2 3 5 I
3 I
21.0 22.0 21.7 22.0 22.0 22.0 23.0 24.0
22.0 22.5 23.0 23.5 24.0 24.0
Sublingual gland
11.0 11.0 15.0 15.0 15.0 15.5 16.0 16.0 18.0 18.8 20.1 23.0 21.7 23.0 23.5 24.0 24.0 24.0 24.0 24.0 24.0
gradually decreased. The striated ducts of sublingual glands began to mature from 26 weeks of gestation. From 31 weeks of gestation, the striated duct was dilated similar to the excretory duct and became indistinguishable in histologic preparations. The striated ducts in submandibular and parotid glands also began to mature from 28 to 29 weeks of gestation and were characterized by a tall columnar single cell layer (Fig. 2E). The intercalated ducts continued to proliferate until 31 weeks of gestation in the sublingual gland, 38 weeks in submandibular gland, and 39 weeks in the parotid gland (Fig. 2B, 2C). The acinar development was remarkably comparable with the PAS stain. The sublingual gland, composed of mucous acini, showed a maximum reaction with PAS staining from 27 weeks of gestation forth. The sublingual gland consisted of short striated ducts, intercalated ducts, and relatively dilated excretory ducts. The submandibular gland partially consisted of mucous acini, and contained abundant PAS positive materials from 31 weeks of gestation. The parotid gland was an entire serous gland showing abundant PAS reaction from 36 weeks of gestation. Using the histological scoring system described above for the 4 tissue elements, the growth state of developing major salivary glands could be assesed by their growth curve (Table 3, 4, Fig. 1). The present study disclosed that the development of the sublingual gland was the most rapid among the 3 major salivary glands (Fig. 2A), and the submandibular and parotid glands had a similar rate of development. However, the submandibular gland tended to develop slightly faster using histological scores than the parotid gland. The growth curve of major salivary glands
220
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-
LEE
et al.
-- -
- _8
Fig. 3. A Strong PAS positive reaction in the terminal branches of developing salivary gland epithelium of 10 weeks gestation. Abundant PAS positive materials in the center of the epithelium. PAS, X 200. B PAS staining of excretory duct of 15 weeks gestation. Noted the abundant PAS positive materials in the intercellular space between the basal and luminal cell layer (white arrow). Luminal surface also showed granular PAS positive materials (small arrow). PAS, X 100.
indicates that their growth rate was characteristically accelerated from the 15th week to 32nd week of gestation, and at about 35 weeks of gestation complete maturation of major salivary glands was accomplished.
221
Prenatal development of human major salivary glands
A
~
.....
-- ..
Fig. 4. A Immunodeposition of PKK 1 in the developing glandular epithelium of 17 weeks gestation, EDS, noted the slight distribution of PKKI in the luminal border of suets. X 150. B Immunodeposition of K8.12 in same area of A, noted the condensed demonstration in the basal border of ducts. X 75. C Immunodeposition of KL 1 in the excretory duct of 25 weeks gestation, increased deposition both in the luminal cells and in the basal cells. X 150. D Immunodeposition ofK8,12 in the same area ofC showed confined distribution in the basal cells of the duct. x 150. E lmmunodeposition of PKK 1 in the same salivary gland of 27 weeks gestation, disclosing strong positive staining in the striated duct and luminal border of intercalated duct and acini. x 75. F Immunodeposition of KL I in the same salivary gland of E (LIDS), disclosing strong positive staining in the striated duct and luminal border of intercalated duct and acini. x 75.
16 Acta histochem., Bd. 89,2
222
S.-K. LEE et al.
3.2. Developmental stages of fetal salivary glands As illustrated on the growth curve, fetal salivary glands were divided into 4 stages. The 1st is the early development stage (EDS) from 10 weeks to 18 weeks of gestation, the 2nd is the early intermediate development stage (EIDS) from 19 weeks to 24 weeks. The 3rd is the late intermediate development stage (LIDS) from 25 weeks to 32 weeks, and the 4th is the late development stage (LDS) or mature stage from 33 weeks to full term. During the EDS, glandular epithelial proliferated rapidly producing luminalization and forming several branches in epithelial cords. Generally, the intermediate development stage (IDS) offetal salivary glands coincided with the period of most rapid proliferation and .:ytodifferentiation using histological scoring; i. e., the score continuously increased during this period. In the EIDS, glandular ducts proliferated to from a typical ductal structure, that is, the excretory ducts became multiple layered, and the striated and intercalated ducts became elongated, consisting of 2-celllayers. This was contrary to acinar cell development which developed into lobular structures and began to differentiate into mature acinar cells. In the LIDS, ductal cells gradually differentiated into mature ductal cells, and the multi-cell layer of the excretory duct differentiated into columnar luminal cells for inner layer and cuboidal basal cells for outer layer. Luminal cells of striated duct became tall columnar cells with abundant apical cytoplasm, and intercalated duct cell became a 2-celllayer structure composed of cuboidal luminal cells and polyhedral basal cells. The acinar cells showed marked maturation with abundant PAS positive material in the lumen. In the LDS, salivary gland elements gradually matured until full term of gestation, and PAS positive material was present in the acini and occasionally in the ductal lumen.
3.3. PAS staining in fetal salivary glands Before the onset of luminalization, terminal branches showed abundant accumulations of PAS positive material in the center of the glandular cords (Fig. 3A). At approximately 10 weeks, the terminal branches showed signs of luminalization. The PAS positive material continuously increased in cells from 10 weeks to 17 weeks of gestation, especially in the luminal border of inner duct cell and in the intercellular space between the luminal duct cells and basal cells (Fig. 3B). After luminalization was completed, PAS positive material gradually decreased in the ductal epithelium and ductal lumen. From 19 weeks of gestation, a moderate PAS positive reaction was found in the lumen of acinar cells of major salivary glands; on the contrary, the ductal lumen had only a small of PAS positive material (Table 5). Mucous acinar cells represent more abundant PAS positive material than serous cells. The sublingual gland from 21 week of gestation showed a moderate positive reaction; thereafter, PAS staining gradually increased until full term. The submandibular gland was slightly admixed with mucous acini so that the PAS positive reaction was relatively prominent in comparison with the parotid gland. Submandibular glands showed moderate PAS staining from 27 weeks of gestation, parotid glands showed moderate staining from 33 weeks of gestation, and at full term, almost all of the acinar cells from major salivary glands showed an abundant PAS positive reaction irrespective of the different type of histology, serous or mucous.
Fig. S. A Immunodeposition of PKKI in the salivary gland of 38 weeks gestation, showing most strong in the luminal border of excretory duct (ED) and striated duct (SD), only slight staining in the limited luminal cells of intercalated duct (ID) and almost negative in the acinar (A) cells. x 90, B Immunodeposition of KLi in the salivary gland of A (LDS). Strong positive staining showed in the whole ED cells and SD cells (SD), and slight staining in the glandular acinar cells. x 180, C Immunodeposition of K8.12 in the salivary glands of 24 weeks gestation (EIDS). Discriminative positive staining is noted in the basal cells of SD, also in limited basal cells of ID. and almost negative in the acinar (A) cells. x 90.
223
Prenatal development of human major salivary glands
A
10
.
,
.I
, 10
SD .'
*Department of Oral Pathology, College of Dentistry, and **Department of Pathology, College of Medicine, Seoul National University, Seoul, Korea, and ***Department of Oral and Maxillofacial Surgery, Asahi University School of Dentistry, Gifu, Japan
Prenatal development of human major salivary glands and immunohistochemical detection of keratins using monoclonal antibodies By SUK-KEUN LEE*, CHANG YUN LIM*, JE GEUN CHI**, KAZUTO YAMADA***, KOUJI HASHIMURA***, MAYUKO KUNIKATA***, and MASAHIKO MORI*** With 9 Figures (Received April 4, 1990)
Summary The major salivary glands were examined from 69 human fetuses ranging from 10 to 40 weeks of gestation. Prenatal growth curves of developing salivary glands could be established by histological scoring, and development was divided into the early developmental stage (EDS) from 10 to 18 weeks, early intermediate developmental stage (EIDS) from 19 to 24 weeks, late intermediate developmental stage (LIDS) from 15 to 32 weeks, late developmental stage (LDS) from 33 to 40 weeks. Characteristic morphogenesis and cytodifferentiation occurred in glandular duct cells during the period of EIDS and LIDS. In the LDS, acini and ducts of the salivary glands histologically developed into a mature state similar to adult glands. Immunohistochemical staining with monoclonal antibodies (MaAhs) PKKI, KLl, K8.12, K8.l3, K4.62, RPN 1160, 1162, 1163, 1164, and 1165 was performed. During the fetal period, keratin expression as revealed by MoAbs PKKI, KLl, K8.12 was well established, and the staining pattern for each of these antibodies was comparable. Other antibodies showed rare or negative staining except K8.l3 which had a diffuse, non-specific staining pattern. Accordingly, the proliferation and cytodifferentiation of fetal stage keratin staining in ductal cells as revealed by MoAbs PKKI, KLl, and K8.12 showed a heterogenic distribution in both luminal and basal cells. It is a characteristic finding that the cytodifferentiation of ductal luminal cells precedes ductal basal cells. Ductal basal cells stained with MoAb K8. 12 and show heterogeneity of keratin distribution continuously until the full term of gestation. The keratin staining of oral epithelium was also examined to compare with distribution of salivary gland ductal cells and oral epithelial cells. In the present study, the developmental sequence of salivary gland cells and the immunohistochemical properties of keratin proteins in these cells were described in relation to the histogenesis of salivary gland tumours.
1. Introduction
In order to investigate the histogenesis of salivary glands and their tumours, exploration of stem cells may be regarded as a fundamental problem in the developmental biology. There is little doubt that the parotid gland arises from the stomodeum and is therefore of ectodermal derivation. The sublingual and submandibular glands remain somewhat obscure as to their derivation, but probably they are ectodermally based, contrary to lingual glands (including von Ebner's glands) which are undoubtedly endodermal derivatives (AVERY et al. 1987, BATSAKIS 1980).
214
S.-K. LEE et al.
The existence of intennediate sized keratin proteins associated with cell organization and integration has been reported in ductal epithelia of salivary glands (CASELITZ et al. 1986, DARDICK et al. 1987, 1988, MARSHAK and LEITER 1987, MORI et al. 1987, 1989 TAKAI et al. 1988). The salivary glands of several mammals including rodents, domestic animals, and humans have been reported to contain keratins in excretory, striated or grandular convoluted duct, and intercalated ducts (TAKAI et al. 1985). The frequentcy of occurrence of salivary gland tumours has been shown to greatly vary among major salivary glands and histopathological type of tumours. Those differences were probably related to the histologic varieties of prenatal developing salivary glands. Compared to the development of rat or mouse salivary glands, which are differentiated predominatly postnatally, the development of human salivary glands has not elucidated in detail. Recently, Gustavsson et al. (1988) demonstrated the expression of monoclonal antibodies of 3 keratins and 2 vimentins in developing salivary glands in man. In the present study, we attempt to investigate histological details during prenatal development of human salivary glands, and immunohistochemical detections of keratins using monoclonal antibodies.
2. Materials and methods A total of 70 aborted human fetuses, confinned as nonnal by histopathological examination, was obtained from the files of the RCM (Registration of Congenital Malfonnation, Department of Pathology, Seoul National University Children's Hospital, Seoul, Korea). Their gestational ages ranged from 10 weeks to 40 weeks. The tissue bank includes 57 parotid, 60 submandibular and 51 sublingual glands, fixed in 10% neutral fonnalin. Serial paraffin sections at 4 11m were used for immunohistochemical detection of keratins and to identify histologic features using HE and PAS stain.
Table l. Immunohistochemical detection of monoclonal antibodies for keratins Immunogens
KLl
human keratinized squamous epithelium
PKKI
pig kidney epithelium
K8.12 K8.13
}
bovine epidermal kreatin
Kreatin type no. of relative molecular masses
55,000 '" ",000
RPN1160
PtKl cells
no. 18
RPN1162
PTl12 bladder Ca. cell line
no. 7
RPNl163
PtKl cells
J
Immunoteck (Marsille) Labsystems (Helsinki)
]
no. I, 5, 6, 7, 8 (basic) no. 10, 11, 18 (acidic) no. 19
RPN1l65
1I80
no. 13, 16
Cultured human breast carcinoma
SV 40 transformed human keratinocyte
Products
40,000, 45,000, 52,000, 5,000 no. 19, 18, 8
K4.62
RPNl164
)
Dilution
no. 8 (type II)
no. 19
Bio-Yeda (Israel)
1I20
Amersham (Buckinghamshire)
215
Prenatal development of human major salivary glands
In order to estimate the developmental stage of fetal salivary glands, incremental scores for glandular structures from each salivary gland were made. In the present study, excretory ducts, striated ducts, intercalated ducts, and acinar cells were observed in different developmental stages, each exhibiting characteristic histology. 2 early stages in fetal salivary gland development were added for the purpose of descriptive representation. From incidental findings of oral epithelium usually associated with sublingual salivary glands, immunohistochemistry of epithelium in oral mucose was used to compare keratin staining of all major salivary glands. Fetal salivary glands were removed from 43 fetuses of estimated gestational ages: 10, 15, 17, 18, 19,20 (2),21 (3),22,23 (3), 24 (3),25 (2), 26 (5),27 (2), 28 (2), 29 (2), 30, 31, 32, 33 (2), 34, 36, 38 (3), 39, and 40 (2) weeks. For immunohistochemical detection of keratins, MoAbs PKKI (nos. 8, 18, 19), KLI (56KD), and K8. 12 (nos. 13, 16) were routinely examined, and additionally MoAbs K8.13 (nos. 1,5,6,7,8, 10, 11), K4.62 (no. 19), RPN 1160 (no. 18), 1162 (no. 7), 1163, 1164 (no. 8), 1165 (no. 19) were used to further elucidate different types of keratin (Table I). Details of irrunnnohistochemical staining methods have been described (TAKA! et al. 1988).
3. Results 3.1. Development of salivary glands and histological scoring The development stages of salivary glands were estimated using the criteria for developmental scoring of tissue elements of salivary glands listed in Table 2. 3.1.1. Excretory ducts: In the excretory duct, score 1 is for the initial stage of growth of cordlike strands in salivary gland mesenchyme. Score 2 is for the stage of luminalization with the appearance of inner ductal cell arrangement in the main stalk of the salivary epithelium. Score 3 is for the stage of ductal development in the extra-lobular space. Score 4 is for the thickened excretory ductal epithelium composed of 3 to 4 cell layers (stratification) and shows abundant vascular proliferation around the excretory duct. Score 5 is for the stage of the differentiation of inner duct cells into columnar cells with abundant apical cytoplasm. Score 6 is for the stage of maturation of the excretory duct including the presence of a conspicuous basement membrane. 3.1.2. Striated ducts: In the striated duct, score 1 is for the initial stage of cord-like epithelial strands. Score 2 is for the stage of luminalization with the appearance of an inner ductal cell arrangement, usually in the branched ductal epithelium. Score 3 is for the stage of a 2-celllayer ductal structure, usually in the intralobular space. Score 4 is for the stage of elongation and Score
25 20 15 10 5
• 5
10
15
o : Sublingual gland • : Submandibular gland * : Parotid gland
20
25
30
35
40
Gestational Age (Weeks)
Fig. I. Growth curve by the sum of histological scoring. The growth stage of developing major salivary gland could be assessed by the total score of 4 tissue elements (excretory ducts, striated ducts, intercalated ducts, and acinar cells).
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Table 2. Histological scores for the observation of developing major salivary glands A. Excretory duct I Cord-like epithelial strands grow deeply into the salivary gland mesenchyme making a several branches. 2 Luminalization begins from the distal tubules, and there appears the arrangement of an inner ductal cell layer. 3 A 2-cell layer ductal structure composed of inner columnar cell layer and outer cuboidal cell layer usually prominent in the extra-lobular space. 4 The duct thickened and became a 3-to 4-cell layer ductal structure in the interlobular space. Abundant vascularity appeared in the vicinity of the duct. 5 Inner ductal cells show columnar nuclei and abundant apical cytoplasm, and the outer ductal cell is cuboidal in shape. 6 Mature excretory ducts show a well aligned columnar inner cell layer and a slightly columnar basal cell layer with a conspicuous basement membrane. The distal side of the excretory duct discloses and increased multi-cell layer of ductal epithelium compared to the proximal side excretory duct which is usually located in the interlobular space. B. Striated duct I Cord-like epithelial strands grow deeply into salivary gland mesenchyme making a several branches. 2 Luminalization begins from the distal tubules and there appears the arrangement of an inner ductal cell layer. 3 A 2-celllayer ductal structure composed of inner and outer cuboidal cell was seen in the intralobular space. 4 The duct became elongated and convoluted. Abundant vascularity appeared in the vincitiy of the duct. 5 Inner ductal cells show columnar nuclei and abundant apical cytoplasm, and the outer cuboidal ductal cells are decreased in number. 6 Mature striated ducts consist of a layer of tall columnar cells with definite basement membrane, occasionally polyhedral cells were found in the basal area. C. Intercalated duct I 2 3 4 5 6
Club-like epithelial strands grow deeply into salivary gland mesenchyme making several terminal epithelial bulbs. Luminalization begins from the distal tubules, and there appears the arrangement of an inner ductal cell layer. A I-cell layer ductal structure in direct conncetion with acini. Single layered inner ductal cells are cuboidal in shape and infrequently surrounded by flattended basal cells. A 2-cell layer ductal structure composed of inner cuboidal cells and outer flattened cells. Mature intercalated ducts consist of a definite cuboidal cell layer and frequent outer polyhedral cells. The basement membrane become conspicuous.
D. Acinar cells I 2 3 4 5 6
Knob-like bulging of the proximal epithelium. Several terminal blub-like structure form. The primitive acini obtain an alveolar pattern. Initial acinic cell differentiation. Moderate acinic cell differentiation. Complete acinic cell differentiation.
convolution of the striated duct and abundant capillary proliferation. Score 5 is for the stage of differentiation of inner ductal cells into tall columnar cells, resulting in the outer basal cells becoming reduced in number. Score 6 is for the maturation of the striated duct which consists of almost I-cell layer with a conspicuous basement membrane. There was infrequently found polyhedral cells in the basal area of the duct. 3.1.3. Intercalated ducts: In the intercalated duct, score 1 is for the initial stage of club-like epithelial proliferation in the terminal branches. Score 2 is for the stage of luminalization with the appearance of an inner ductal cell arrangement. Score 3 is for the stage of I-cell layer ductal structure connected with the terminal bulging of the acini. Score 4 is for the stage of proliferation of the ductal cells, resulting in the inner ductal cells being surrounded by flattened outer cells. Scroe 5 is for the stage of differentiation of duct cells into inner luminal cells of cuboidal shape
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Fig.2. A Developing sublingual gland from 15 weeks old fetus. After luminalization in the branching salivary gland epithelium produced 2- cells layer structure of excretory and striated ducts were seen. HE, X 32. B Intercalated duct from 21 weeks old fetus consists of monolayer of cuboidal cells at the terminal branches. HE, X 160. C Intercalated duct from 23 weeks old fetus showed proliferation of outer cell layer surrounding the inner ductal cells. HE, X 160. D Excretory duct from 23 weeks old fetus showed well aligned luminal cells and basal cells. HE, X 160. E Striated duct from 38 weeks old fetus consisted of almost monolayer of tall columnar cells showing the abundant apical cytoplasm. HE, X 160, 15
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and outer basal cell of a flattended shape. Score 6 is for the stage of maturation of the intercalated duct which is composed of cuboidal luminal cells and flattened basal cells with a conspicuous basement membrane. 3.1.4. Acinar cells: In the acinar compartment, score 1 is for the stage of initial knob-like bulging of the proximal epithelium. Score 2 is for the stage of multiple bulb-like formations in terminal epithelium. Score 3 is for the stage of occurrence of primitive acini having an alveolar arrangement. Score 4 is for the stage of initial acinar cell differentiation in the glandular lobules. Score 5 is for the stage of moderate acinar cell differentiation in the glandular lobules. Score 6 is for the stage of almost complete differentiation of acinar cells. In brief, the excretory ducts were characterized by the most rapid maturation compared with others. Excretory duct maturation was identified predominately by the stratification of ductal epithelium and by the cytodifferentiation of luminal epithelium. Excretory duct maturation occured from about 24 to 25 weeks of gestation in all major salivary glands (Fig. 2D). The striated ducts were principally composed of 2-celllayers in the early fetal period: inner luminal cells and outer basal cells. A characteristic of striated duct maturation was that the luminal ductal cells gradually developed into tall luminal cells occupying the whole ductal thickness and forming a basement membrane at their basal border. With maturation, the number of ductal basal cells Table 3. Histological scoring of tbe developing major salivary glands in human fetuses G. A. [weeks]
Cases (No.)
Types of salivary glands Submaxillary gland
Parotid gland A
18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 36 38 39 40
1 1 2 2 1 3 3 2 4 5 5 8 2 3 5 1 3 1 3 2 1 5 2 5
Total
70
10 15 17
B
C
Sublingual gland C
D
3.0
3.0
2.0
4.0 4.0 4.5 4.0 4.0 4.0 4.8 5.0 5.1 6.0 6.0 6.0
4.0 4.0 3.5 4.0 4.0 4.0 4.0 4.0 4.9 6.0 5.7 6.0
4.0 3.0 3.5 3.5 4.0 4.0 4.5 5.0 5.1 5.5 5.0 5.5
4.0 4.0 3.5 4.0 4.0 4.0 4.8 4.8 5.1 5.5 5.0 5.5
6.0 6.0 6.0 6.0 6.0 6.0
(6.0) (6.0) (6.0) (6.0) (6.0) (6.0)
5.5 6.0 6.0 6.0 6.0 6.0
6.0 6.0 6.0 6.0 6.0 6.0
6.0
(6.0)
6.0
6.0
D
A
B
C
D
A
2.0 3.0 3.0 3.0 4.0 4.0 4.0 4.0 4.5 5.0 5.0 5.3 5.5 6.0 6.0 6.0 6.0
1.0 3.0 3.0 3.0 3.0 3.0 4.0 4.0 4.0 4.0 4.0 4.1 4.0 5.0 5.0 6.0 6.0
1.0 3.0 3.0 3.0 3.0 3.0 3.5 4.0 4.0 4.3 4.7 5.0. 5.0 5.0 5.0 5.0 5.0
2.0 2.0 3.0 3.0 3.0 3.0 3.0 3.8 4.0 4.3 5.0 5.0 5.0 5.0 5.0 5.0
3.0
6.0 6.0 6.0 6.0 6.0 6.0
6.0 6.0 6.0 6.0 6.0 6.0
5.0 5.0 5.0 5.1 6.0 6.0
5.0 5.5 6.0 6.0 6.0 6.0
3.0 3.0 4.0 4.0 4.0 4.0 4.3 4.5 5.0 5.1 5.0 5.7 6.0
3.0 3.0 3.0 3.0 4.0 4.0 4.0 4.0 4.0 4.1 4.0 4.0 5.0
3.0 3.0 3.0 3.0 3.7 4.0 4.0 4.0 4.0 4.6 5.0 5.0 5.0
3.0 3.0 3.0 3.0 3.0 3.0 3.7 4.0 4.3 5.0 5.0 5.0 5.0
6.0 6.0 6.0 6.0 6.0 6.0 6.0 6.0
5.0 6.0 5.7 6.0 6.0 6.0 6.0 6.0
5.0 5.0 5.0 5.0 5.0 5.0 5.0 6.0
5.0 5.0 5.0 5.0 5.0 5.0 6.0 6.0
B
Abbreviation; GA gestation age, A excretory duct, B striated duct, C intercalated duct, D acinus, ( ) illdefined observation of striated duct of sublingual gland
Prenatal development of human major salivary glands
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Table 4. Sum of the histological scoring of the developing major salivary glands in human fetuses
G.A. [weeks]
Cases (No.)
Types of salivary glands Parotid gland
Submaxillary gland
12.0 12.0 13.0 13.0 14.7 15.0 16.0 16.5 17.3 18.0 19.0 19.7 21.0
4.0 11.0 11.0 12.0 13.0 13.0 14.5 15.0 16.3 17.3 18.0 19.4 19.5 21.0 21.0 22.0 22.0
I I
10 15 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 36 38 39 40
3 2 1 5 2 5
Total
70
2 2 I
3 3 2 4 5 5 8 2 3 5 I
3 I
21.0 22.0 21.7 22.0 22.0 22.0 23.0 24.0
22.0 22.5 23.0 23.5 24.0 24.0
Sublingual gland
11.0 11.0 15.0 15.0 15.0 15.5 16.0 16.0 18.0 18.8 20.1 23.0 21.7 23.0 23.5 24.0 24.0 24.0 24.0 24.0 24.0
gradually decreased. The striated ducts of sublingual glands began to mature from 26 weeks of gestation. From 31 weeks of gestation, the striated duct was dilated similar to the excretory duct and became indistinguishable in histologic preparations. The striated ducts in submandibular and parotid glands also began to mature from 28 to 29 weeks of gestation and were characterized by a tall columnar single cell layer (Fig. 2E). The intercalated ducts continued to proliferate until 31 weeks of gestation in the sublingual gland, 38 weeks in submandibular gland, and 39 weeks in the parotid gland (Fig. 2B, 2C). The acinar development was remarkably comparable with the PAS stain. The sublingual gland, composed of mucous acini, showed a maximum reaction with PAS staining from 27 weeks of gestation forth. The sublingual gland consisted of short striated ducts, intercalated ducts, and relatively dilated excretory ducts. The submandibular gland partially consisted of mucous acini, and contained abundant PAS positive materials from 31 weeks of gestation. The parotid gland was an entire serous gland showing abundant PAS reaction from 36 weeks of gestation. Using the histological scoring system described above for the 4 tissue elements, the growth state of developing major salivary glands could be assesed by their growth curve (Table 3, 4, Fig. 1). The present study disclosed that the development of the sublingual gland was the most rapid among the 3 major salivary glands (Fig. 2A), and the submandibular and parotid glands had a similar rate of development. However, the submandibular gland tended to develop slightly faster using histological scores than the parotid gland. The growth curve of major salivary glands
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- _8
Fig. 3. A Strong PAS positive reaction in the terminal branches of developing salivary gland epithelium of 10 weeks gestation. Abundant PAS positive materials in the center of the epithelium. PAS, X 200. B PAS staining of excretory duct of 15 weeks gestation. Noted the abundant PAS positive materials in the intercellular space between the basal and luminal cell layer (white arrow). Luminal surface also showed granular PAS positive materials (small arrow). PAS, X 100.
indicates that their growth rate was characteristically accelerated from the 15th week to 32nd week of gestation, and at about 35 weeks of gestation complete maturation of major salivary glands was accomplished.
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Prenatal development of human major salivary glands
A
~
.....
-- ..
Fig. 4. A Immunodeposition of PKK 1 in the developing glandular epithelium of 17 weeks gestation, EDS, noted the slight distribution of PKKI in the luminal border of suets. X 150. B Immunodeposition of K8.12 in same area of A, noted the condensed demonstration in the basal border of ducts. X 75. C Immunodeposition of KL 1 in the excretory duct of 25 weeks gestation, increased deposition both in the luminal cells and in the basal cells. X 150. D Immunodeposition ofK8,12 in the same area ofC showed confined distribution in the basal cells of the duct. x 150. E lmmunodeposition of PKK 1 in the same salivary gland of 27 weeks gestation, disclosing strong positive staining in the striated duct and luminal border of intercalated duct and acini. x 75. F Immunodeposition of KL I in the same salivary gland of E (LIDS), disclosing strong positive staining in the striated duct and luminal border of intercalated duct and acini. x 75.
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3.2. Developmental stages of fetal salivary glands As illustrated on the growth curve, fetal salivary glands were divided into 4 stages. The 1st is the early development stage (EDS) from 10 weeks to 18 weeks of gestation, the 2nd is the early intermediate development stage (EIDS) from 19 weeks to 24 weeks. The 3rd is the late intermediate development stage (LIDS) from 25 weeks to 32 weeks, and the 4th is the late development stage (LDS) or mature stage from 33 weeks to full term. During the EDS, glandular epithelial proliferated rapidly producing luminalization and forming several branches in epithelial cords. Generally, the intermediate development stage (IDS) offetal salivary glands coincided with the period of most rapid proliferation and .:ytodifferentiation using histological scoring; i. e., the score continuously increased during this period. In the EIDS, glandular ducts proliferated to from a typical ductal structure, that is, the excretory ducts became multiple layered, and the striated and intercalated ducts became elongated, consisting of 2-celllayers. This was contrary to acinar cell development which developed into lobular structures and began to differentiate into mature acinar cells. In the LIDS, ductal cells gradually differentiated into mature ductal cells, and the multi-cell layer of the excretory duct differentiated into columnar luminal cells for inner layer and cuboidal basal cells for outer layer. Luminal cells of striated duct became tall columnar cells with abundant apical cytoplasm, and intercalated duct cell became a 2-celllayer structure composed of cuboidal luminal cells and polyhedral basal cells. The acinar cells showed marked maturation with abundant PAS positive material in the lumen. In the LDS, salivary gland elements gradually matured until full term of gestation, and PAS positive material was present in the acini and occasionally in the ductal lumen.
3.3. PAS staining in fetal salivary glands Before the onset of luminalization, terminal branches showed abundant accumulations of PAS positive material in the center of the glandular cords (Fig. 3A). At approximately 10 weeks, the terminal branches showed signs of luminalization. The PAS positive material continuously increased in cells from 10 weeks to 17 weeks of gestation, especially in the luminal border of inner duct cell and in the intercellular space between the luminal duct cells and basal cells (Fig. 3B). After luminalization was completed, PAS positive material gradually decreased in the ductal epithelium and ductal lumen. From 19 weeks of gestation, a moderate PAS positive reaction was found in the lumen of acinar cells of major salivary glands; on the contrary, the ductal lumen had only a small of PAS positive material (Table 5). Mucous acinar cells represent more abundant PAS positive material than serous cells. The sublingual gland from 21 week of gestation showed a moderate positive reaction; thereafter, PAS staining gradually increased until full term. The submandibular gland was slightly admixed with mucous acini so that the PAS positive reaction was relatively prominent in comparison with the parotid gland. Submandibular glands showed moderate PAS staining from 27 weeks of gestation, parotid glands showed moderate staining from 33 weeks of gestation, and at full term, almost all of the acinar cells from major salivary glands showed an abundant PAS positive reaction irrespective of the different type of histology, serous or mucous.
Fig. S. A Immunodeposition of PKKI in the salivary gland of 38 weeks gestation, showing most strong in the luminal border of excretory duct (ED) and striated duct (SD), only slight staining in the limited luminal cells of intercalated duct (ID) and almost negative in the acinar (A) cells. x 90, B Immunodeposition of KLi in the salivary gland of A (LDS). Strong positive staining showed in the whole ED cells and SD cells (SD), and slight staining in the glandular acinar cells. x 180, C Immunodeposition of K8.12 in the salivary glands of 24 weeks gestation (EIDS). Discriminative positive staining is noted in the basal cells of SD, also in limited basal cells of ID. and almost negative in the acinar (A) cells. x 90.
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Prenatal development of human major salivary glands
A
10
.
,
.I
, 10
SD .'
