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The dependent and independent relationships between cytodifferentiation and morphogenesis in developing salivary gland secretory cells.

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THE ANATOMICAL RECORD 196:341-347 (1980)
The Dependent and Independent Relationships
Between Cytod iff erent iat i on and Mo r phogenesis
in Developing Salivary Gland Secretory Cells
Department of Oral Dingnosis, University of Connecticut, School of Dental
Medicine, Farmington, Connecticut 06032
The mesenchymal capsule was removed from the epithelial anlage of 15 day (unbranched) and 16 day (initially branched) fetal rat submandibular gland (SMG) rudiments. The undifferentiated epithelial portion of the SMG
rudiments was placed in tissue culture and examined by light and electron microscopy and histochemistry for secretory peroxidase. The 15 day fetal SMG epithelial
rudiments failed to attach, and spread over the culture dish and degenerated by 3
days in culture. The 16 day epithelial rudiments attached to the dish and the cells
spread radially from the explant. Mitotic activity was minimal. Cells spreading
from the 16 day rudiments underwent cytodifferentiation, giving rise to two secretory cell types: 1) peroxidase containing “proacinar cells,” and 2) secretory
“terminal tubule” cells. The results suggest that in the developing SMG, morphogenesis and cytodifferentiation are partially coupled but independently regulated
processes. The earliest phases of morphogenesis (rudiment down growth and primary branching) seem to be required to initiate cytodifferentiation. Once initiated,
cytodifferentiation can proceed in the absence of continued morphogenesis (tissue
organization) or significant amounts of connective tissue elements.
The fact that one developing tissue can influence the development of another developing
tissue was demonstrated by Spemann in 1901,
introducing the concept of “inductive tissue interactions.” This idea of two or more tissues of
different histogenetic origin, and function becoming closely associated and subsequently effecting their final developmental pathways,
has become generally accepted as a major regulatory force in embryonic development (Grobstein, ’56; Lehtonen, ’76). Epithelial-mesenchymal interactions of this nature are required
for the development of a wide variety of tissues,
including the salivary glands, skin, teeth,
mammary glands, ureteric bud, and several
others. The mechanism(s)by which these tissue
interactions mediate their inductive influences
are not fully understood.
The organization of chemically unique cells
into a functional structural configuration
is required for the normal development of a
particular tissue. In most cases, cells are first
organized into specialized structural units
(morphogenesis) and then these cells develop
their architectural and biochemical (production of cell-specific molecules)identity (cytodifferentiation) (Rutter et al., ’67).During the de-
000-3276x180/1963-0341$01.400 1980 ALAN R. LISS, INC.
velopment of rodent salivary glands and the
development of virtually all exocrine organs,
the initial phases of morphogenesisprecede the
onset of low levels of cell-specific protein production. In most of these systems, morphogenesis is relatively advanced before production of
cell-specific protein reaches the level a t which
the proteins are packaged into demonstrable
secretory granules. It is not known, for salivary
glands or for most exocrine glands, to what
degree the processes of morphogenesis and subsequent cytodifferentiation are linked with regard to temporal sequencing or with regard to
maintenance of cytodifferentiation in the absence of morphogenesis. Numerous studies
have shown that a finite (relatively large)
quantity of mesenchyme is necessary for the
morphogenesis of rodent salivary gland tissue
(Borghese, ’50; Grobstein, ’53a, b; Lawson, ’72,
’74). However, no evidence is available with
regard to the necessity of the presence of mesenchyme for the cytodifferentiation of rodent
salivary gland exocrine cells. The current report describes studies which suggest that
morphogenesis and cytodifferentiation are
Received May 3, 1979 accepted July 26, 1979.
separately controlled processes, which are only
partially coupled in the development of the rat
submandibular gland (SMG). Further, the
results suggest that different types of epithelial-mesenchymal interactions may be
required for morphogenesis and cytodifferentiation of the rat SMG.
Isolation and culture of the SMG Anlage
Sprague-Dawley rats were bred under rigidly controlled conditions as previously reported (Cutler and Chaudhry, '73a). On the
15th and 16th days of gestation, fetuses were
aseptically delivered by sterile laparotomy.
The SMG rudiments, which show no morphologic signs of cytodifferentiation (Cutler and
Chaudhry, '74; Jacoby and Leeson, '591, were
excised, washed twice in calcium-magnesium
free Tyrode solution, and then incubated for 40
minutes a t room temperature in 0.25% trypsin
(Difco 1/250) in calcium-magnesium free
Tyrode solution in the well of a glass depression
slide. The incubation was terminated by removing the trypsin solution and washing the
rudiments with McCoy 5A medium, containing
10%fetal calf serum. The mesenchymal capsule was removed by carefully passing the rudiments in and out of a sterile pasteur pipette
and then gently dissecting (under a dissecting
microscope with sterile microdissecting instruments) and removing the capsule from the
epithelial anlage.
The isolated epithelia were cultured in the
center well of a plastic organ culture dish (Falcon plastics) directly on the plastic substratum.
The cultures were incubated in McCoy 5A medium or Fitton-Jackson modified BGJ medium
(GIBCO, Grand Island, NY); both media were
supplemented with 10%fetal calf serum, gentamycin, and neomycin. The culture incubations were carried out at 37°C in a humidified
95% air-5% CO, atmosphere. The medium was
changed daily.
Light and electron microscopy
Cultures were monitored daily by phase contrast microscopy. At 3 and 5 days of culture,
cells were fixed by removing the medium and
flooding the culture dish with cold (4°C) 1.25%
glutaraldehyde in 0.1 M cacodylate buffer (pH
7.3) for 5 minutes. The fixative was removed
and the cultures were washed 4 times (10 minutes each) with cold 0.1 M cacodylate buffer (ph
7.3). Some of the dishes were then incubated in
0.05 M tris-HC1 buffer (pH 7.0) containing 10
mM DAB (3,3'-diaminobenzidine tetrahydrochloride, Sigma Chemicals, St. Louis, MO), 5
mM KCN and 1 mM (0.003%)Hz02to demonstrate secretory peroxidase (Graham and Karnovsky, '66). The incubation was carried out for
1hour a t 37°C in the dark. Following the incubation, the cultures were washed twice in 0.1 M
cacodylate buffer (pH 7.3) and then postfixed in
1%cacodylate buffered osmium tetroxide. Control experiments included dishes incubated in
medium depleted of H,O, or DAB and dishes
fixed for 3 hours with 3% cacodylate buffered
After postfixation, the cultures were stained
for 1hour in 0.25% aqueous uranyl acetate. A t
this point, some cultures were lightly stained in
1%methylene blue in 1%borax and examined
by light microscopy. Other cells were lifted
from the culture dish with a rubber policeman,
suspended in 25% acetone, and concentrated by
centrifugation (2500 g for 10 minutes). The resulting pellet was dehydrated through a graded
series of acetones and embedded in Spurr low
viscosity resin (Spurr, '69).
Thick (0.5 p ) and thin (6OCk900 A) sections
were cut on a n LKB-ultrotome 111. Thick sections were checked by light microscopy for orientation, and thin sections were then examined
to confirm the specificity of the histochemical
reaction. The thin sections were subsequently
counterstained with lead citrate (Venable and
Coggeshall, '65) to enhance contrast and examined on a Zeiss EM10 electron microscope.
Forty-three of 48 explanted 15 day fetal SMG
epithelial analgen failed to spread over the culture dish. After 24 hours in culture, these rudiments had rounded into small spheres of cells
and by 48 hours in culture, all had detached
from the substratum and were floating in the
medium. Most of these epithelial spheres had
degenerated by 72 hours in culture. The five
explants that did attach and spread over the
culture dish behaved like the 16 day explants.
Thirty-one of 46 explanted 16 day fetal SMG
epithelial anlagen attached to the plastic substratum and cells spread radially from the
main explant. Cell division was minimal and
although there were 7 or 8 rudiments per culture dish, confluence was not reached during
the 5 days of culture. By 2-3 days of culture,
many cells could be seen with small granules in
their cytoplasm and many of these granules
contained peroxidase (Fig. 1).The cells did not
show any polarity of granule disposition in the
cytoplasm and the cells did not show any tendency t~ form acinar or ductal structures (Figs.
1, 2).
A t the ultrastructural level, cells which were
Fig. 1. Light micrograph of cells from a 16 day fetal SMG epithelial anlage explant after 3 days in culture. The cells were
fixed in glutaraldehyde and histochemically stained to demonstrate peroxidase activity. Two epithelial cell types can be
identified by the type ofgranule in their cytoplasm (A, B). The granules are generally small in both cases, but the peroxidase
containing granules appear much darker because of the oxidized DAB-osmium reaction product (B). Cells without definable
granules can also be identified ( X 750).
Fig. 2. Light micrograph of cells from a 16 day fetal SMG epithelial anlage explant a f k r 5 days in culture. These cells were
fixed and stained to demonstrate peroxidase activity. Two epithelial cell types can be readily identified by the granule type in
their cytoplasm (A, B). The majority of cells in this field (B) contain peroxidase positive granules in their cytoplasm. The
granules are randomly distributed in the cytoplasm and no polarity of organelle distribution can be seen. The cells do not
group into organized acinar or ductal structures ( x 750).
Fig. 3. Electron micrograph of a cell from a 16day fetal SMG epithelial anlage explant after 3 days in culture. The cell has
been stained to demonstrate peroxidase activity. Weak DAB reaction product (peroxidaseactivity) can be seen in the cisternae
of the endoplasmic reticulum (ex.) while more intense reaction product can be seen in the nuclear envelope (arrows). No
secretory granules are evident ( X 10,000).
apparently free of granules but showed peroxidase reactivity in the cisternae of the rough
endoplasmic reticulum (e.r.1and nuclear envelope, were seen in the 16 day explants after 3
days of culture. Peroxidase activity was more
pronounced in the nuclear envelope than in the
e.r. (Fig. 3). Several cells were also seen with
marked peroxidase reactivity in the cisternae
of the e.r. and with several small peroxidasecontaining granules in their cytoplasm (Fig. 4).
At 5 days in culture, two cell types were clearly
definable on the basis of secretory granule type.
One cell type had peroxidase-positivegranules
and the other had secretory granules which did
not contain peroxidase (Fig. 5). Those 16 day
epithelial rudiments which failed to attach and
spread on the culture dish behaved similarly to
the 15 day fetal rudiment explants.
The cytochemical controls were generally
nonreactive in all cases. Faint reaction product
was seen in the HzO,-depleted control and in
the dishes fixed for 3 hours. This sparse reaction product was due, in part, to low levels of
endogenous H,O, and to the residual peroxidase activity after prolonged fixation. No reaction product was seen in the DAB-depleted controls.
The results of this study indicate that exocrine cells in the rat SMG can undergo cytodifferentiation in the absence of continued or advanced morphogenesis or substantial amounts
of mesenchymal tissue. These observations
confirm and extend those of Spooner and coworkers ('77) on the exocrine pancreas and suggest that a large degree of independence exists
between cytodifferentiation and morphogenesis. Further, this independencemay be common
to all exocrine tissues. As has been pointed out
by Spmner et al. ('771, this separation of cytodifferentiation and morphogenesis refers
uniquely to the transition of protodifferentiated cells (cellsproducing low levels of cell specific proteins) into more highly differentiated
cells. It must be stressed that the initial phases
of morphogenesis (up to and including the
initiation of rudiment branching) seem to be a
prerequisite to cytodifferentiation. Thus, initial morphogenetic events seem to be obligatorily linked to cytodifferentiation.
The results of the current study are also in
agreement with a recent report by Sakakura
and coworkers ('761, which showed that cytodifferentiation of mammary epithelium was unaffected by the morphogenetic organization of the
epithelium in heterotypic cultures with sali-
vary mesenchyme, and with a report by Lawson ('74) on the regulation of parotid gland cytodifferentiation. These investigators stressed
the importance of mesenchymal factors in
the regulation of morphogenesis. The specific
biosynthetic function of developing epithelium
is apparently determined genetically during
the developmental history of the epithelium,
and once initiated, seems to proceed independently of morphogenesis. It has been shown
that a few cells in the end-buds of the initial
branches of the 16 day SMG rudiment contain
secretory peroxidase within the cisternae of
their endoplasmic reticulum. Thus, low levels
of specific secretory proteins are present at
least 2 days prior to the formation of peroxidase
containing secretory granules in the developing proacinar cells (Yamashina and Barka, '72;
Cutler and Mooradian, unpublished) indicating that these cells are indeed protodifferentiated. No cells of this type have been seen in 15
day SMG rudiments. The timing of granule appearance in the cultured 16 day SMG epithelial
cells is in close agreement with the appearance
of secretory granules both in vivo (Yamashina
and Barka, '72, '73; Cutler and Chaudhry, '73b,
'74) and in organ culture (Rufo and Barka, '76;
Cutler, '77) of the developing rat SMG. The
observation of two varieties of cells, based on
secretory granule type, is also consistent with
the developmental sequence seen in vivo and in
organ culture.
The observation that 15 day fetal SMG epithelium failed to survive in culture in the absence of significant amounts of mesenchyme is
consistent with the early classical studies on
salivary gland development which showed that
at this stage of development the epithelial anlage of the SMG required mesenchyme in order
to survive in culture (Borghese,'50; Grobstein,
'53a, b). On the other hand, 16 day fetal SMG
epithelium will attach, spread, and differentiate into definitive proacinar cells and secretory
terminal tubule cells in the absence of significant amounts of mesenchyme.This observation
suggests that between days 15 and 16 of gestation, changes occur in the developing SMG epithelia which permit cytodifferentiation to proceed in the absence of mesenchyme. Some of the
changes in the SMG cells, which occur during
this period may be at the cell surface and may
be reflected in the increased ability of cells from
the 16 day rudiments to attach to the culture
dish. Attachment to substrata is known to be
required for differentiation of some cell types.
However, a wide variety of cytoplasmic and/or
nuclear changes may also be involved in the
Fig. 4. Electron micrograph of portions of three cells from the outgrowth of a 16 day fetal SMG epithelial anlage which was
in culture for 3 days. The cells have been stained to demonstrate peroxidase activity. Two ofthe cells (A, B) show DAB reaction
product indicative of peroxidase activity in their endoplasmic reticulum (e.r.1and nuclear envelope (arrowheads). These cells
also contain several small DAB positive secretory granules (small arrows). The third cell (C) contains several large granules
(g)which do not show peroxidase activity. Strands of endoplasmic reticulum that do not contain peroxidase activity can also be
seen (large arrows) ( x 4,000).
Fig. 5. Electron micrograph of portions of two cells (A, B) from the outgrowth of a 16 day fetal SMG epithelial anlage which
was in culture for 5 days. Cell A contains numerous, large pale granules (g). Cell B contains many electron dense granules
(arrows) ( x 4,000). Inset: Higher magnification electron micrograph showing the different types of granules ( x 10,000).
developmental changes which take place i n the
SMG cells between the 15th and 16th days of
gestation. Independent of the nature of these
changes, it appears that the epithelial cells
must be in close association with the mesenchyme during this brief but important time
period for the changes required for cytodifferentiation to occur.
These observations suggest that epithelialmesenchymal interactions of some type are required for cytodifferentiationto proceed. There
appear to be at least two types of epithelialmesenchymal interactions involved i n SMG
development. There are epithelial-mesenchyma1 interactions involved in initiating and
maintaining morphogenesis of the SMG. These
interactions require that relatively large quantities of mesenchyme remain in close association with the developing epithelium for relatively prolonged periods in order to maintain
morphogenesis (Borghese, '50; Grobstein, '53a,
b). The epithelial-mesenchymal interactions
which appear to be involved in the cytodifferention of the SMG are apparently of a more transient nature and do not require continued association of epithelium and mesenchyme once
they have occurred. The transient, direct heterotypic, cell-cell (epithelial-mesenchymal)
interactions known to occur in the developing
SMG rudiment prior to cytodifferentiation in
vivo and in vitro could represent this second
type of interaction (Cutler and Chaudhry, '73;
Cutler, '77). Transient epithelial-mesenchymal
contacts of this type have been described in a
variety of developing tissues other than salivary glands including teeth, duodenum, skin,
and lung (see Lehtonen, '76; and Cutler, '77 for
review). The exact mechanism(s) of transmission of developmental signals by contact-mediated tissue interactions is not known, but current theories have recently been reviewed by
Lehtonen ('76).
The current report represents only the second experimental study dealing with the relationship between morphogenesis and cytodifferentiation in developing exocrine systems.
The results suggest that in the development of
the rat SMG, morphogenesis and cytodifferentiation are partially coupled, independently
regulated processes and support the data of
Spooner et al. ('77) in the developing exocrine
pancreas. This confirmation suggests that the
observation may represent a general biologic
phenomenon in the development of secretory
A preliminary report of this study has been
presented Journal of Dental Research58A:288,
The author wishes to thank Ms. M. Reid and
Mrs. C. Christian for their technical assistance
and Mrs. V. Every for her secretarial help.
This study was supported in part by N.I.H.
grant DE-03933.
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developing, secretory, gland, salivary, cytodifferentiation, dependence, independence, relationships, morphogenesis, cells
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