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/. Embryol. exp. Morph. Vol. 70, pp. 45-60, 1982
Printed in Great Britain © Company of Biologists Limited 1982
45
Localization of H-2K k in developing mouse palates
using monoclonal antibody
By MICHAEL MELNICK,1 TINA JASKOLL1
AND MARY MARAZITA1
From the Laboratory for Developmental Biology,
University of Southern California
SUMMARY
Using monoclonal antibodies to H-2Kk antigen, we sought to develop a reproduceable
method of in situ localization in embryonic tissue and to determine whether there are
specific patterns of H-2 localization in time and space in the developing palatal tissues of
B10.A(H-2a) embryonic mice, with and without corticosteroid pretreatment at 12 days
gestation. Our procedure employs ethanol-glacial acetic acid fixation, paraplast embedding,
and enzymatic predigestion with purified hyaluronidase and neuraminidase. H-2 antigens
were detected in palatal mesenchyme as well as basement membranes but not in oral or
nasal epithelium. The pattern of distribution in mesenchyme of untreated embryos changed
with progressive shelf development: vertical -> horizontal ->• epithelial fusion -»• epithelial
seam degeneration -> mesenchymal confluence. Although the palatal shelves of treated
embryos remained vertical, corticosteroid treatment does not appear to alter the detectable
spatiotemporal distribution of H-2 antigens in developing palates of embryonic BIO.A mice.
INTRODUCTION
There is an explicit and implicit theoretical expectation among many developmental biologists that differentiating cells of vertebrate embryos will be found
to have specific cell surface antigens at different stages of development and
that cataloguing the localization and potential functions of these antigens will
provide a more clear understanding of morphogenesis (Hay, 1980). Major
histocompatibility complex (MHC) cell surface antigens of the mouse (H-2)
are integral plasma membrane constituents which are associated with allograft
rejection and immune response (Klein, 1979). Over the past decade a number
of workers have speculated that the MHC codes for cell surface antigens that
control critical cell-cell interactions during embryonic development (Bennett,
Boyse & Old, 1971; Ohno, 1977; Bonner, 1979). However, prior to any
investigation of the function of H-2 antigens in progressive embryonic development one must initially determine (1) when H-2 antigens are first expressed
1
Author's address: Laboratory for Developmental Biology, University of Southern
California, Andrus Gerontology Center, P.O. Box 77912, Los Angeles, California 90007,
U.S.A.
46
M. MELNICK, T. JASKOLL AND M. MARAZITA
in developing embryonic tissues, (2) whether the expression of H-2 antigens
is restricted to certain cell types, and (3) whether there are specific patterns of
H-2 localization in time and space that can be elucidated in situ in embryonic
tissues.
Virtually all of the work reported has been confined to addressing the first
two questions. The onset of H-2 antigen expression appears to occur in
cleavage to blastocyst embryos (see reviews by Ostrand-Rosenberg, 1980; and
Johnson & Calarco, 1980). In older embryos H-2 antigens have been detected
on 15-day embryonic liver cells in suspension (McClay & Gooding, 1978), on
cultured 14-day and 15-day embryonic cells derived from thymus, salivary
gland, head and neck skin, thyroid, and lung rudiments (Jenkinson, Owen &
Aspinall, 1980), on cultured 18-to 20-day foetal mouse skin cells (Worst &Fusenig,
1973), and on cultured cells of mid-gestation mouse embryonic skin, gut, lung,
limb bud and heart (Kirkwood & Billington, 1981). Investigators have failed
to detect H-2 antigens on cultured mid-gestation embryonic cells from gonads
and kidneys (Kirkwood & Billington, 1981), as well as on suspensions of
15-day embryonic brain cells (McClay & Gooding, 1978). In fact, H-2 in brain
has not been detected until 8 days postnatal (Schachner & Sidman, 1973). Even
though one interesting study of 12-day otocysts has successfully used immunoperoxidase techniques and Normaski optics (Prystowsky, Khan & Marovitz,
1978), to date, the in situ localization of H-2 antigens in embryonic tissues has
been difficult for a variety of reasons, mostly technical (see Discussion below).
Our objectives in this set of experiments were twofold. First, we sought to
develop a practical and reproducible method for the in situ localization of
H-2 antigens in embryonic tissues; second, using monoclonal antibodies to
H-2K k antigen, we sought to determine whether there are specific in situ
patterns of H-2 localization in time and space in the developing palatal tissues
of B10.A(H-2Kk) embryonic mice. Our procedure employs ethanol-glacial
acetic acid fixation, paraplast embedding, and enzymatic predigestion with
purified hyaluronidase and neuraminidase. H-2 antigens were detected in palatal
mesenchyme as well as basement membranes but not in oral or nasal epithelium.
The pattern of indirect immunofluorescent staining for H-2K k antigens in
mesenchyme changed with progressive palatal shelf development: vertical shelf
position -> horizontal shelf position -> epithelial fusion -> epithelial seam degeneration -> mesenchymal confluence. Since H-2K k is significantly associated
with embryonic susceptibility to corticosteroid-induced cleft palate (Melnick,
Jaskoll & Slavkin, 1981), we repeated the study in mice treated at 12 days
gestation with triamcinolone hexacetonide, a synthetic analogue of cortisol.
Although the palatal shelves of treated embryos remained vertical, the patterns
of distribution changed with time in a manner similar to untreated embryos.
We believe this is the first time specific in situ patterns of H-2 antigen localization
have been demonstrated in the progressive differentiation of embryonic tissues.
Fig. 1. Early stage of palate development in which both shelves are vertical: (A) view of the palate as seen with removal of
lower jaw, line is the plane of section for B; (B) H & E stained section through the embryonic mouse head showing the relationship
between the vertical shelves (v) and the tongue (/)(x 103); (C) immunofluorescent localization of H-2Kk in one of the vertical shelves,
see text for details ( x 200),
1
s-
I
Fig. 2. Stage of palate development in which one shelf is horizontal and one shelf is vertical: (A) View of the palate as seen with
removal of lower jaw, line is the plane of section for B; B. H & E stained section through embryonic mouse head showing
relationship of horizontal (h) and vertical (v) shelves to the tongue (/) (x 104); (C) Immunofiuorescent localization of H-2Kk in
the horizontal shelf, see text for details (x 200).
H
N
r
r
o
C/3
z
r
4
00
Fig. 3. Stage of palate development in which both shelves are horizontal: (A) View of the palate as seen with removal of
lower jaw, line is the plane of section for B; (B) H & E stained section through embryonic mouse head showing the relationship
between the horizontal shelves (h) and the tongue (/) (x95); ( O Immunofluorescent localization of H-2Kk in one of the
horizontal shelves, see text for details (x 200).
I"
a
Fig. 4. Stage of palate development in which both horizontal shelves contact one another: (A) View of palate as seen with removal
position of the tongue (/) (x 100); (C) Immunofluorescent localization of H-2Kk in the fusing palatal shelves, see text for details
of lower jaw, line is the plane of section for B; (B) H & E stained section through embryonic mouse head showing the formation
of an epithelial seam (arrow) between the contacting horizontal shelves and the (x210).
3
r
Z
o
H
N
2
O
r
r
O3
a
H-2Kk and palate development
51
MATERIALS AND METHODS
Mice
Virgin female B10.A/SnSg(H-2Kk) mice, obtained from Jackson Laboratory
(Bar Harbor, Maine), were housed in groups of five and were acclimatized to the
vivarium environment in our institution during a quarantine period of 2 weeks.
When the animals were 12-13 weeks old, groups of three females were placed
in a cage with a male overnight. Females were examined daily in the morning
for the presence of vaginal plugs. The date of plug detection was designated
as day 0 of gestation. Pregnant dams were housed individually in solid-bottom,
plastic cages. The vivarium facility is temperature controlled with an average
daily temperature of 24 °C. Alternate 12 h periods of light and darkness were
maintained daily. All animals were maintained on Wayne's Mouse Breeder
Blox containing 20 % crude fat and a maximum of 2 % crude fibre, and were
supplied with drinking water ad libitum.
One group of experimental animals received a single intramuscular injection
(in the proximal portion of the lower limb) of 2 mg/kg body weight triamcinolone hexacetonide, a long-acting, synthetic analogue of cortisol, at 9.00 a.m.
on day 12 of gestation. The remaining pregnant animals were untreated.
Tissue preparation
On the morning of days 14-16 gestation, dams were killed by cervical dislocation. By laparotomy, the uterus and all its contents were exposed. Each
live embryo was dissected out of its extraembryonic membranes, placed in
phosphate-buffered saline (PBS), and staged according to the external morphologic developmental criteria established by Theiler (1972). After staging,
embryonic heads were removed with a scalpel and forceps and fixed overnight
at 4°C in absolute ethanol-glacial acetic acid (99:1). The heads were then
dehydrated for 1 h in absolute ethanol (4 °C), cleared in xylene for 1 h (4 °C),
and embedded in paraplast (56-59 °C). Specimens were serially sectioned at
5/tm in a plane perpendicular to the anteroposterior axis. Sections were
placed on clean glass slides and stored at - 2 0 °C for short periods until stained.
Anti-H-2 McAb
Monoclonal anti-mouse H-2K k was purchased from Becton Dickenson
(Sunnyvale, CA). Their clone 11-4-1 was derived from hybridization of mouse
NS-1 myeloma cells with spleen cells from BALB/C(H-2 d ) mice immunized with
CKB(H-2Kk) spleen cells. The monoclonal antibody (McAb) was obtained from
serum and/or ascites fluid of tumour-bearing mice and purified by protein A
affinity column chromatography. The specificity and purity of each lot of McAb
is tested at Becton Dickenson by two-dimensional gel electrophoresis, cytotoxicity, and indirect immunofluorescence staining of spleen cells followed by
fluorescence-activated cell-sorter analysis. The McAb was supplied as 0-5 mg
Fig. 5. Stage of palate development in which the horizontal palatal shelves have almost completely fused: (A) View of palate as seen
with removal of lower jaw, line is the plane of section for B; (B) H & E stained section through embryonic mouse head showing the
degeneration of the epithelial seam (arrow) and the initiation of mesenchymal confluence (xlOO); (C) Immunofluorescent
localization of H-2Kk in the later stages of palatal fusion, see text for details (x 220).
8/
H
N
r
r
O
o
w
r
to
H-2Kk and palate development
53
Fig. 6. Immunofluorescent localization of H-2Kk in the palatal shelf of a corticosteroid-treated early 16-day mouse embryo; note that the palatal shelf has
remained vertical and that the tongue (seen on the far right) has not changed
position, as is the case during normal palatal development; see text for details
( x 260).
purified immunoglobulin in 0-5 ml of PBS containing 0-1% sodium azide
and was stored at 2-4 °C. McAb was additionally checked in our laboratory
for specificity and radioactivity by indirect immunofluorescent labelling of
B10.A(H-2Kk) and B10.D2(H-2Kd) adult spleen cell suspensions using the
methods previously described by Eskinazi et al. (1979). B10.D2(H-2Kd) cells
were routinely negative for fluorescence.
Section staining
Deparaffinized sections were incubated with a 1:5 dilution of rabbit antimouse IgG [heavy and light chains, F ' (ab) fragment] (Cappel Laboratories,
Cochranville, PA) at room temperature for 30 min and washed 3 times (10 min
each) in PBS (pH 7-1) containing 0-025 % Triton x 100 (PBS/TRX). Following
this, the sections were incubated with 20 /d/section of essentially protease-free
streptomyces hyaluronidase (100 TRU/ml) (Calbiochem, San Diego, CA) in
0-2 M sodium acetate buffer (pH4-8), washed twice with PBS/TRX (pH7-l)
for 10 min each, and incubated with 20 /^I/section of vibro cholerae neuraminidase (1 IU/ml) (Calbiochem, San Diego, CA) in 0-05 M sodium acetate
buffer (pH5-5). Each enzymatic digestion was performed for 2 h at room
temperature. Following enzymatic treatment, the sections were washed 6 times
(10 min each) with PBS/TRX (pH 7-1) and then incubated at 4 °C with monoclonal anti-mouse H-2K k antibody in PBS (pH 7-1) containingO-4 % Triton x 100
(10 /ig protein/20 ^I/section) for 22-24 h. The sections were then washed for
54
M. MELNICK, T. JASKOLL AND M. MARAZITA
3 h (9 changes) at room temperature in PBS/TRX (pH7-l) and incubated
with 1:40 dilution of FITC-labelled rabbit anti-mouse IgG [heavy and light
chains, F ' (ab) fragment] (Cappel Laboratories, Cochranville, PA) for 30 min
at room temperature. Finally the slides were washed 5 times (10 min each) in
PBS/TRX (pH 7-1), washed once for 10 min in PBS (pH 7-1), air dried, and
mounted with a coverslip in aquamount. The slides were examined with a Zeiss
Epifluorescence Photomicroscope equipped with a high-pressure mercury
burner (HBO-50) and tungsten lamp (12 V, 60 W). Photomicrographs were
recorded using Kodak Ektachrome 400 film at constant exposure.
In addition to the positive and negative controls described above the adult
spleen cell suspensions, a number of other controls were undertaken: (1) unlabelled rabbit anti-mouse IgG alone; (2) unlabelled rabbit anti-mouse IgG
followed by FITC-labelled rabbit anti-mouse IgG; (3) monoclonal H-2Kk antibody alone; (4) monoclonal H-2Kk antibody followed by unlabelled rabbit
anti-mouse IgG (1:40 dilution); (5) FITC-labelled rabbit anti-mouse IgG
alone; (6) hyaluronidase and/or neuraminidase alone; (7) rabbit anti-mouse
unlabelled IgG followed by hyaluronidase and neuraminidase followed by
FITC-labelled rabbit anti-mouse IgG. Controls were routinely negative for
fluorescence.
RESULTS
Progressive development of the normal secondary palate of the mouse is
pictured in the drawings and histologic sections of Figs. 1-5. A number of
excellent reviews of normal secondary palate development are extant (e.g.
Pourtois, 1972; Trasler & Fraser, 1977). In short, the palatal shelves are initially
in a vertical position on either side of the tongue and subsequently reorient
themselves to a horizontal position above the dorsum of the tongue. The
principal forces causing the 'horizontalization' of the shelves are thought to
be hydration of glycosaminoglycans in the mesenchymal extracellular matrix
and perhaps contractile elements in the mesenchymal cells themselves. Upon
becoming horizontal the shelves contact and strongly adhere to one another.
The apposed epithelia fuse to form an epithelial seam which soon breaks
down allowing complete fusion and mesenchymal confluence within the palate.
The palatal mesenchyme then differentiates into osseous, muscular, and fibrous
elements; the palatal epithelium differentiates into pseudostratified ciliated
columnar on the nasal surface and keratinized and stratified squamous on the
oral surface (Tyler & Koch, 1975).
H-2 and normal palate development
Using monoclonal antibodies and indirect immunofluorescence, H-2K k antigen was detected in palatal mesenchyme as well as basement membrane regions,
but not in oral or nasal epithelium (Figs. 1-6). The fluorescent-staining pattern
in the mesenchyme changed with progressive palatal shelf development. Shelves
H-2Kk and palate development
55
in the vertical position generally showed a diffuse mesenchymal staining
(Fig. 1); as the shelves elevated to a horizontal position this staining became
more intense but remained somewhat uniform throughout the mesenchyme
(Figs. 2, 3). Upon fusion of the apposed epithelia the fluorescent staining
began to localize in the direction of the nasal cavity (Fig. 4). As the epithelial
seam began to break down the localization beneath the nasal epithelium
became more prominent and the staining appeared to intensify (Fig. 5). This
staining pattern persisted through the stage of complete mesenchymal confluence.
H-2 abnormal palate development
Although the palatal shelves of embryos exposed previously to triamcinolone
hexacetonide remained vertical, the patterns of fluorescent staining in the
mesenchyme changed with time in a manner similar to untreated embryos.
Specifically, mesenchymal staining in early day-16 embryos was found to be
more localized in an area beneath what would normally become nasal epithelium (Fig. 6).
DISCUSSION
H-2 antigens and palatogenesis
Using monoclonal antibodies to H-2K k antigen, we were able to demonstrate
specific in situ patterns of H-2 localization in time and space in the developing
palatal tissues of B10.A(H-2Kk) embryonic mice. In addition the fluorescent
staining appeared to be cell specific, being associated with mesenchymal but
not with epithelial cells. It remains obscure as to why the H-2 staining finally
localized in the mesenchyme immediately subjacent to the nasal epithelium and
not the oral epithelium. Perhaps it is related to the distinctly separate fates of
the two epithelia during subsequent differentiation.
Of course, demonstrating changing patterns of specific antigens in embryonic
tissues does not necessarily reveal the potential or actual functions of these
antigens. In the case of H-2 antigens the function remains an enigma, even
though a number of hypothetical models have been proposed which view
H-2 antigens as playing a role in cell-cell recognition (Edelman, 1976; Bonner,
1979). There is mounting evidence that embryonic pattern formation depends
on strictly local cell-cell interactions (Bryant, French & Bryant, 1981). Although
our experiments do not in any way allow us to conclude that this is the function
of H-2 antigens during development, it is not unreasonable to think that the
changing patterns of distribution of this immunoglobulin-like molecule is
more than a fortuitous occurrence in a dynamic mesenchyme.
Although palatal shelves of corticosteroid-treated embryos remained vertical
through the beginning of gestation day-16, the patterns of detectable H-2Kk
distribution changed with time in a manner similar to untreated embryos. This
is not altogether surprising, since corticosteroid-induced cleft palate in mice is
thought primarily to be due to a reduction in shelf force and a resulting delay
56
M. MELNICK, T. JASKOLL AND M. MARAZITA
in shelf movement both in relation to chronological and developmental age
(Walker & Fraser, 1957). Relatively normal elevation and fusion can be achieved
in treated foetuses by excising the tongue (Walker & Quarles, 1976). In any
event, although there is known to be a strong embryonic effect vis-a-vis the
relationship of H-2K k (or closely linked genes) and high susceptibility to
corticosteroid-induced cleft palate (Melnick et al. 1981a), corticosteroid treatment does not appear to alter the detectable spatiotemporal distribution of
H-2 antigens in developing palates of embryonic BIO.A mice. These data
suggest that we should look elsewhere for the mechanism of steroid-induced
clefting, perhaps in the transmembrane relationship between H-2 antigen and
the microfilament protein actin (Kock & Smith, 1978). Even though it is
admittedly dangerous to argue from single instances of association, the investigation of effects on transmembrane relationships may be of particular importance
if the effect of the drug is to act on some process subsequent to H-2 expression.
Methodological considerations
Previous investigators have reported the indirect immunofluorescent localization of H-2 in tissue sections from placenta (Voisin & Chaouat, 1974) and
various adult organs including liver, spleen, pancreas, kidney, and brain
(Gervais, 1968; Gervais, 1970; Saunders, Beals & Schultz, 1979). It has been
noted by others that these studies are not, however, completely satisfactory
for several reasons: (1) there is considerable tissue distortion in the unfixed
cryostat sections (Gervais, 1972a; Parr, 1979); (2) there is often high nonspecific background staining (Gervais, 1972&; Saunders et al. 1979); (3) it is
almost impossible to make alloantisera specific for individual antigenie components in sufficient quantity with great reproducibility (Swaab, Pool & Van
Leeuwen, 1977; Milstein & Lennox, 1980). We believe our protocol has allowed
in situ detection of H-2 antigens in embryonic tissue while avoiding the
difficulties just enumerated.
We are aware of the extensive study by Gervais (1972 a) on the effects of
various fixatives and the use of tissue preparations other than frozen sections.
It is difficult to compare our results in embryonic tissues with those of Gervais
(1972a) in adult tissue. It should be noted, though, that paraffin embedding
has proved successful in the immunofluorescent localization of other antigens
in embryos, e.g. fibronectin and collagen (Melnick et al. 1981 b; Silver, Foidart
& Pratt, 1981). Further, although Gervais (1972a) concluded that H-2 antigens
are destroyed with various fixatives, Parr & Oei (1973 a, b) published results
suggesting that paraformaldehyde fixation immobilizes mouse H-2 antigens on
cell surface membranes, and thus enhances detection. In addition, Voisin &
Chaouat (1974) have also successfully utilized ethanol fixation for indirect
immunofluorescent studies of H-2 antigens in tissues.
The primary questions raised in studies of this sort revolve around the
specificity of the antigen-antibody reaction. Swaab et al. (1977) have concluded
H-2Kk and palate development
57
that if antisera are tested properly, few, if any, of them will be found to be
monospecific. Akeson (1979) has noted that although it is generally inferred
that the observed pattern of cell and tissue reactivity is due to the presence of
a unique macromolecule(s) this is not necessarily the case, particularly when
values for positive cells are not greatly above control values (e.g. Saunders
et al. 1979). To be sure, there is no substitute for analyzing the suspected or
putative macromolecule by biochemical means. Nevertheless, the use of monoclonal antibodies obviously eliminates the problem of multiple antigenic recognition by a mixed antibody population, even though it does not completely
eliminate the possibility of cross reactions resulting from antigenic similarities
(Milstein & Lennox, 1980). In the view of Milstein & Lennox (1980), this
latter possibility, if it occurs at all, is not common enough to blur the recognition of individual 'differentiation' antigens.
The interpretation of results obtained with monoclonal antibodies must be
made with caution. For example, the fine recognition of McAb may introduce
a serious bias when a given antigenic determinant is exposed in one environment
but hidden in others (Milstein & Lennox, 1980). This problem may be particularly acute in embryonic tissues which contain large amounts of extracellular matrix macromolecules such as glycosaminoglycans and collagens.
These macromolecules are closely associated with cell surfaces and may serve
to mask them. It thus seems both reasonable and prudent to 'unmask' craniofacial tissues such as palatal mesenchyme, which are known to have large
extracellular quantities of hyaluronate associated with their cell surfaces
(Toole, 1976). In our laboratory hyaluronidase pretreatment is found to
enhance the fluorescent labelling. Similarly, we have pretreated with collagenases but this seems to have no effect. In another context, it is worth noting
that Saunders et al. (1979) suggested that the high background staining they
observed might actually be due to nonspecific accumulation of FITC-labelled
secondary antibody in extracellular matrix. Thus enzymatic pretreatment may
serve a dual function.
It has been shown that when over 95 % of the sialic acid contained in H-2
glycoprotein is removed the sialic acid-free H-2 siloantigens still retain their
activity for the tested specificities (Shimada & Nathenson, 1971). It has also
been shown that removal of the sialic acid moiety by Vibrio cholerae neuraminidase enhances the immunogenicity of the cells so treated (Currie et al.
1968; Simmons et al. 1971). It has been postulated that perhaps the sialic acid
moiety acts by steric hindrance to prevent access to underlying antigenic
determinants (Currie, van Doorninck & Bagsharve, 1968; Simmons, Rios &
Ray, 1971). This would be particularly so if the antigenic determinants are
closely spaced on the surface of the cells (Kent, Ryan & Siegel, 1978). We
found that pretreatment of sections with Vibrio cholerae neuraminidase also
enhanced fluorescent labelling of BIO.A embryonic tissue. This result was not
altogether unexpected since neuraminidase treatment was necessary to demon-
58
M. MELNICK, T. JASKOLL AND M. MARAZITA
strate H-2 associated variations in adhesion rates when cells with a BIO genetic
background were used, but not with A or C3H genetic backgrounds (Bartlett
& Edidin, 1978). In addition it has been reported that BIO erythrocytes contain
more sialic acid per cell than do erythrocytes of other strains, and that this
interferes with assays of haemaglutinating anti-H-2 antibodies (Rubenstein,
Liu, Streun & Decary, 1974).
In conclusion, then, the results of our experiments provide a manageable
and a reproducable method for the immunofluorescent labelling of H-2 antigens
in embryonic tissue. Initial results with this method seem promising and
appear to have the potential for opening up new avenues of investigation
relative to specific spatiotemporal patterns of H-2 localization during progressive
embryonic morphogenesis.
This study was supported by grant DE-05440 from the National Institute of Dental
Research, National Institutes of Health. Dr M. Melnick is a recipient of public Health
Research Career Development Award DE-OOO83.
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(Received 17 July 1981, revised 16 December 1981)