* Department of Microbiology and Department of Histology and Cell Biology, Umeå University, S-901 87 Umeå, Sweden; § Research Institute of Molecular Pathology, A-1030 Vienna, Austria; and
Developmental Biology Institute of Marseille,
University de Mediterranee, CNRS/INSERM, Campus de Luminy, 13288 Marseille cedex 09, France
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Abstract |
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Classical cell dissociation/reaggregation experiments with embryonic tissue and cultured cells
have established that cellular cohesiveness, mediated
by cell adhesion molecules, is important in determining
the organization of cells within tissue and organs. We
have employed N-CAM-deficient mice to determine
whether N-CAM plays a functional role in the proper
segregation of cells during the development of islets of
Langerhans. In N-CAM-deficient mice the normal localization of glucagon-producing cells in the periphery of pancreatic islets is lost, resulting in a more randomized cell distribution. In contrast to the expected
reduction of cell-cell adhesion in N-CAM-deficient
mice, a significant increase in the clustering of cadherins, F-actin, and cell-cell junctions is observed suggesting enhanced cadherin-mediated adhesion in the
absence of proper N-CAM function. These data together with the polarized distribution of islet cell nuclei
and Na+/K+-ATPase indicate that islet cell polarity is
also affected. Finally, degranulation of
cells suggests
that N-CAM is required for normal turnover of insulin-containing secretory granules. Taken together, our results confirm in vivo the hypothesis that a cell adhesion molecule, in this case N-CAM, is required for cell type
segregation during organogenesis. Possible mechanisms
underlying this phenomenon may include changes in
cadherin-mediated adhesion and cell polarity.
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Introduction |
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CELL-CELL interactions mediated by cell-cell adhesion molecules (CAMs)1 are thought to be crucial
for tissue formation and organogenesis by regulating the spatial and temporal dissociation, migration, aggregation, and sorting of cells. Neural cell adhesion molecule (N-CAM), a member of the immunoglobulin super-gene
family of CAMs mediating homophilic and heterophilic
cell-cell interactions, has been implicated in some of these
processes during development of the nervous system
(Schachner, 1989; Rutishauser, 1991
, 1993
; Walsh and
Doherty, 1991
, 1997
; Thiery, 1996
). N-CAM is encoded by
a single gene whose primary transcript exhibits a complex
pattern of alternative splicing (Gennarini et al., 1986
; Cunningham et al., 1987
; Owens et al., 1987
; Santoni et al.,
1989
; Walsh and Dickson, 1989
). The various isoforms of
N-CAM can be categorized into three main groups according to the size of their cytoplasmic tails and cell surface membrane association: N-CAM-120, -140, and -180 (Cunningham et al., 1983
; Chuong and Edelman, 1984
;
Gennarini et al., 1984
). During early phases of embryogenesis these proteins are expressed in cells from all three
germ layers. However, as development proceeds its expression pattern becomes more restricted to neuronal tissues (Goridis and Brunet, 1992
). Recently, N-CAM mutant mice, lacking either all N-CAM forms (Cremer et
al., 1994
) or only the 180-kD isoform (N-CAM-180; Tomasiewicz et al., 1993
) were generated. Although these
mice appear healthy and are fertile, a reduction of the olfactory bulb and deficits in spatial learning are observed in
adult mice, phenotypes attributed to defects in cell migration. In adult animals N-CAM is also expressed in various nonneuronal cells, for example in pancreatic endocrine
cells (Langley et al., 1989
; Rouiller et al., 1990
; Moller et al.,
1992
). However, N-CAM's functional role in these cell
types, in particular during organogenesis, remains elusive.
Pancreatic development involves both cytodifferentiation and morphogenesis, in that order (Gittes and Rutter,
1992). Initially, pancreatic progenitor cells form a dorsal
and ventral evagination of the foregut endoderm. During
the outgrowth of these buds a branched ductular tree containing pancreatic progenitors is formed. It is thought that
the organization of endocrine cells in islets of Langerhans
is established through a series of morphogenetic events involving cell sorting, cell migration, and cell reaggregation, processes for which cell adhesion is thought to be important (Pictet and Rutter, 1972
; Slack, 1995
). Initially, the endocrine cells are present in the primitive pancreatic duct
epithelium. These cells eventually sort out of the duct epithelium and begin to aggregate into islets of Langerhans
with a distinct cell architecture; non-
cells (
, D, and PP
cells) in the periphery and
cells in the center. The fact
that islet cell organization is perturbed in humans with diabetes and in animal models for the disease, suggest that
the ability to organize endocrine cells properly could be
crucial for islet function (Gepts and Lecompte, 1981
; Gomez Dumm et al., 1990; Tokuyama et al., 1995
). Moreover,
it was demonstrated that the molecular mechanisms that
regulate insulin secretion depend on
cell contacts, further emphasizing the need for intact cell-cell interactions
within islets of Langerhans (Lernmark, 1974
; Halban et al.,
1982
; Bosco et al., 1989
; Salomon and Meda, 1986
; Philippe et al., 1992
).
Using a transgenic approach, we recently demonstrated
that members of the cadherin family of CAMs are required for the aggregation of endocrine cells into pancreatic islets (Dahl et al., 1996). However, with these experiments we could not explore whether cadherins are also
necessary for sorting of cell types into the typical islet cell
architecture. Based on a series of experiments that involved the reaggregation of dissociated rat pancreatic islet cells in vitro, it was recently proposed that N-CAM may
regulate cell type segregation of pancreatic islet cells (Cirulli et al., 1994
). We have employed N-CAM knockout
mice (Cremer et al., 1994
) to test this hypothesis in vivo.
Our data reveal that N-CAM is essential for islet cell type segregation, thus providing in vivo evidence for the requirement of a CAM in cell sorting during organogenesis. Alterations in the subcellular distribution of cell polarity markers and cell-cell junctional components and structures suggest that cell polarity is affected and that cadherin-mediated adhesion may be enhanced in N-CAM mutant mice. Furthermore, our data indicate that N-CAM-mediated cell- cell interactions are important for the turnover and activity of intracellular organelles within islets.
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Materials and Methods |
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Animals
Control (+/+), N-CAM heterozygous (+/), and N-CAM homozygous
(
/
) mutant mice in a C57Bl/6J background were used (Cremer et al.,
1994
). Control experiments demonstrated that no N-CAM protein is expressed in the pancreas of N-CAM
/
mice (see Fig. 2).
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Islet Isolation
Mice were killed and pancreata were perfused with HBSS () with 3 U/ml collagenase (Worthington Biochemical Corporation), and 10 µg/ml DNase (), incubated at 37°C for 30 min, and washed once with HBSS () with 3% goat serum and three times in RPMI () supplemented with 10% FBS (). Islets were handpicked under microscope and incubated overnight in RPMI () supplemented with 10% FBS ().
Cell Transfections
L cells and L cells transfected with cDNAs for murine E-cadherin (LE;
Nose et al., 1988) and N-cadherin (LN; Miyatani et al., 1989
) were generously supplied by Dr. M. Takeichi (Kyoto University, Kyoto, Japan).
These cell lines were transiently transfected with expression-vectors containing cDNAs for enhanced green fluorescence protein (eGFP; ClonTech), murine N-cadherin (pRc/CMV; Invitrogen), and murine N-CAM-120, N-CAM-140, or N-CAM-180 (pRc/CMV; Invitrogen), by using
Lipofectamine Reagent according to the manufacturer's instructions
(). Except for eGFP, which was regulated by the MSV-LTR
promoter (pMexNeo), all cDNAs were under the influence of the CMV
promoter. Cells were either fixed in 4% paraformaldehyde for 10 min or
in methanol at
20°C for 5 min, washed, and processed for immunofluorescence staining as described under immunohistochemistry, with the exception that the incubation-time for primary antibodies were 3 h (see below). Samples were analyzed both by standard fluorescence and confocal
laser scanning microscopy.
Histological Analysis
Pancreata were removed and fixed in 4% paraformaldehyde overnight, washed in PBS overnight, dehydrated in graded alcohols, and embedded in paraffin. 6-µm sections were stained with hematoxylin-eosin and photographed using a Axioplan light microscope.
Immunoblotting
Islets were solubilized by boiling in sample buffer (63 mM Tris, pH 6.8, 1% SDS, 10% glycerol, 5% -mercaptoethanol, and 10 µg/ml of bromphenol blue) for 5 min, separated by SDS-polyacrylamide gels, and electrophoretically transferred onto nitrocellulose filters (Bio-Rad) in 192 mM glycine, 20% methanol, and 25 mM Tris-HCl. Blocking (overnight)
and all antibody incubations were in HBST-Ca2+ (10 mM Hepes, pH 7.4, 150 mM NaCl, 1 mM CaCl2, and 0.1% Tween 20). The first and secondary
antibodies were applied for 3 h and 60 min, respectively. To visualize the
antigen-antibody complexes, filters were incubated with a conjugated secondary antibody, which was visualized by chemiluminiscence using the ECL-detection kit () according to the manufacturer's specifications.
Immunohistochemistry
Tissues (pancreata) were collected and fixed in HBS (10 mM Hepes, pH
7.4, 150 mM NaCl) supplemented with 4% paraformaldehyde for 2 hours
at room temperature. For cryostat protection, tissues were incubated in
serial sucrose solutions (12, 15, and 18%) in HBS supplemented with 1 mM
CaCl2 for 2-3 h each at 4°C. Tissues were embedded in Tissue Tek compound and frozen in liquid nitrogen. 8-µm-thick sections on polylysine
()-coated glass slides were washed in HBS, heated in a microwave
oven (only for N-cadherin antibody), postfixed in 20°C methanol for 20 min, and blocked in TBS-Ca2+ (10 mM Tris, pH 7.6, 150 mM NaCl, and 1 mM
CaCl2) supplemented with 5% skim milk for 30 min at room temperature.
The first antibody was added in TBS-Ca2+ supplemented with 5% skim
milk overnight at 4°C. Secondary antibodies, FITC- or Cy3-streptavidin
were added for 60 min each. When HRP staining was used (Vectastain
ABC kit) endogenous peroxidase was blocked with 3% H2O2 in methanol
during postfixation (see above).
Insulin and Glucagon Measurements
Pancreatic insulin and glucagon were measured in total pancreatic extracts from five fed animals (4-5 mo of age) of each genotype using a commercially available radioimmunoassay for rat insulin and glucagon (Linco Research, Inc.). Total pancreatic protein concentration was determined using Bio-Rad protein assay (Bio-Rad). Values of pancreatic insulin and glucagon were within the normal range according to the manufacturer (Linco Research, Inc.). Statistical analysis was performed using the Chi-square test.
Immunoreagents
The following antibodies were used at the indicated dilutions for immunoblotting and immunohistochemistry experiments; rat mAb against E-cadherin (ECCD-2; Shirayoshi et al., 1986; 1:40); rat mAb against N-cadherin
(MNCD-2; Matsunami and Takeichi, 1995
; 1:200); affinity-purified rabbit
anti-Na+/K+-ATPase (Nelson and Hammerton, 1989
; 1:100); rabbit anti-N-CAM (Rasmussen et al., 1982
; 1:1,000); rat mAb against ZO-1 (Chemicon; 1:100); rabbit anti-rat amylase (Przybyla et al., 1979
; 1:1,000); rabbit
anti-carboxypeptidase (Biogenesis; 1:1,000); rabbit anti-PDX1 (Ohlsson
et al., 1993
; 1:400); guinea pig anti-insulin (Linco Research, Inc.; 1:1,000);
rabbit anti-glucagon (Linco Research, Inc.; 1:500); FITC-conjugated anti-guinea pig and anti-rabbit (Molecular Probes; 1:500); indocarbocyanine
(Cy3)-conjugated anti-rabbit (Molecular Probes; 1:300); biotin-conjugated
anti-rat and anti-rabbit (Molecular Probes; 1:500). Cy3-conjugated streptavidin were purchased from Molecular Probes and used at 1:1,000 dilution.
Rhodamine-phalloidin was purchased from Molecular Probes and used
according to the manufacturer's instructions. The Vectastain ABC kit was
from Vector Laboratories, Inc.
Cell Distribution Measurements within Islets
To estimate the distribution and the number of cells in islets, whole pancreata were sectioned and sections separated by 200 µm were stained with
anti-glucagon polyclonal antibodies. Data were obtained by analyzing
more than 80 individual islets per mouse in four animals (4-8 mo old) of
each genotype. Normal islets were defined as islets in which all
cells
were found within the three most peripheral cell layers. Islets with a radius of five or less cell layers were not included. Analysis of variance (ANOVA) and the Scheffe multiple comparison test were used to compare the values in Table I. Statistics were performed by using SPSS 7.5 for
Windows (SPSS Inc.).
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Transmission Electronmicroscopy
Specimens were fixed overnight in 2.5% glutaraldehyde in 0.1 M phosphate buffer at 4°C, rinsed in phosphate buffer, postfixed in 1% OsO4 in 0.1 M phosphate buffer for 2 h, dehydrated in graded alcohol solutions, and embedded in Poly/Bed (Polyscience Inc.). Ultrathin sections were cut on a LKB ultratome. The sections were placed on gold grids, stained with uranyl acetate and lead citrate, and examined in a Jeol 100 CX electron microscope.
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Results |
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N-CAM Expression during Pancreatic Ontogeny
N-CAM's expression pattern during pancreatic development is reminiscent of its general expression pattern in the
embryo, i.e., initially it is expressed more or less ubiquitously, whereas at later stages of development and in adult
tissue its expression becomes more restricted. During the
early stages of pancreatic ontogeny, N-CAM is expressed
both in the pancreatic mesenchyme and endoderm (Fig. 1,
a and b). Gradually, N-CAM becomes confined to aggregating endocrine cells (Fig. 1, c-f), which in addition to peripheral nerve endings and ganglia are the only pancreatic cells that express N-CAM in adult mice (Fig. 1 g). In contrast to pancreatic islets in the rat, which express higher
levels of N-CAM in non- cells than in
cells (Rouiller et al.,
1990
; Moller et al., 1992
), the mouse appears to express similar levels in all endocrine cells, at least when judged by immunohistochemistry (Fig. 1 g). The predominant N-CAM
polypeptide expressed in adult islets is the glycosylphosphatidylinositol-linked 120-kD isoform (Fig 1 h). In conclusion, the pattern of N-CAM expression during pancreatic organogenesis suggests an involvement in islet morphogenesis.
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N-CAM Is Required for Islet Cell Type Segregation
N-CAM does not appear to be required for the differentiation of pancreatic cells or for major pancreatic morphogenetic events, since a normal pancreas, consisting of all
pancreatic cell types and islets of normal size and number
scattered within the exocrine tissue, formed in N-CAM-deficient mice (Fig. 2, a-d). However, close examination
of the mutant animals revealed alterations in the organization and morphology of islet cells. Notably, these changes
are apparent in both heterozygous (N-CAM +/) and homozygous (N-CAM
/
) animals. Control experiments
demonstrated that N-CAM protein is completely absent in
N-CAM
/
islets and that N-CAM +/
islets express
~50% of the protein levels detected in wild-type mice
(Fig. 2, e-h). The subcellular distribution of N-CAM in
N-CAM +/
islets was unaltered as compared with wild-type islets, i.e., N-CAM is predominantly expressed in
cell-cell contacts (Fig. 2 f).
In the mouse, islets begin to form around 17.5-18 days
postcoitum (dpc; Herrera et al., 1991). Already at this
stage the islets begin to adopt their final cell organization,
i.e.,
cells in the center and non-
cells in the periphery.
However, the segregation of these cell types is not yet
complete and in many aggregates the cells are more or less
intermixed. In fact, by using the distribution of glucagon-producing
cells as the criterion of cell type segregation after birth, it was noted that it is not until 4-5 wk of age
that the majority of mouse islets adopt their final cell configuration (data not shown). This cell architecture is maintained at least up to 11 mo of age, which is the oldest age
that we have analyzed. To investigate whether N-CAM
has any influence on islet cell type segregation we compared the ratio of normal and mixed islets between control
and N-CAM-deficient mice. We defined normal islets as islets with all
cells distributed within the three most peripheral cell layers and mixed islets as islets, which contain
one or more
cells positioned centrally to the three most
peripheral cell layers. Although mixed islets were found in
control mice (22%), the majority of the islets was defined
as normal (78%; Table I, Fig. 3). However, in N-CAM-
deficient mice the ratio of normal and mixed islets completely changed. In these mice the number of normal islets
were diminished, whereas the majority of the islets was defined as mixed (71% +/
, 67%
/
vs. 22% +/+; Table I,
Fig. 3). This effect becomes apparent only when islet cell type segregation is usually complete, i.e., around 4-5 wk of
age. In further support of N-CAM's involvement in islet
cell type segregation, the extent of inter-mixing of cells
within mixed islets of N-CAM-deficient mice was markedly
higher as compared with the few mixed islets of control
mice. Thus, the percentage of
cells positioned centrally to
the three most peripheral cell layers was significantly higher in N-CAM-deficient animals than in control mice (20 +/
,
21
/
vs. 14 +/+; Table I). Taken together these data
suggest that N-CAM regulate cell type segregation during
the development of islet of Langerhans. There are, however, several potential explanations for these observations.
Either the number of islet cells has changed, or the segregation of islet cells is affected in N-CAM-deficient mice.
However, the fact that the number of
cells and pancreatic insulin and glucagon levels exhibits no marked difference between the different genotype mice (Table I and II),
suggests that N-CAM is, indeed, a key regulator of islet
cell sorting. Besides
cells and
cells, islets consist of two
other endocrine cell types, somatostatin-producing D cells
and pancreatic polypeptide-producing PP cells. Similarly to
, D, and PP cells are also preferentially confined to the
outer rim of islets in control mice, however, to a much lower
degree. Their distribution was not detectably affected in
N-CAM mutant mice (data not shown).
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Islet Cell Polarity Is Affected in N-CAM Mutant Mice
To elucidate the molecular mechanisms behind the altered
cell type segregation within islets of Langerhans in N-CAM-deficient mice, the subcellular distribution of molecules
that are known to affect morphogenetic behaviors of cells
or act as molecular markers for cell polarity were examined. Thus far, three members of the classic cadherins have
been found to be expressed in islets, E-, N-, and R-cadherin
(Begemann et al., 1990; Moller et al., 1992
; Hutton et al.,
1993
; Dahl et al., 1996
; Esni, F., and H. Semb, unpublished
observations). Only N-cadherin and E-cadherin localize to
cell-cell contacts (Begemann et al., 1990
; Dahl et al., 1996
),
whereas R-cadherin distribute mainly in the cytoplasm (Dahl, U., F. Esni, and H. Semb, unpublished observations). Analysis of N-CAM-deficient mice revealed striking changes in the subcellular distribution of N-cadherin
and E-cadherin, whereas R-cadherin was unaffected. As
mentioned previously, these cadherins normally localize to
all regions engaged in cell-cell adhesion without any apparent clustering (Begemann et al., 1990
; Dahl et al., 1996
; Fig. 4 a). In N-CAM-deficient mice, N-cadherin (Fig. 4, a-d)
but also E-cadherin (data not shown) accumulate in discrete regions of cell-cell contacts, suggesting that N-CAM
prevents clustering of cadherins within islets of Langerhans in control mice. Because cadherins are capable of affecting cortical F-actin organization and cell polarity, the
subcellular distribution of F-actin and Na+/K+-ATPase
(Hammerton et al., 1991
; Drubin and Nelson, 1996
), apical and basolateral epithelial cell polarity markers, respectively, were investigated in N-CAM-deficient mice. In control islets F-actin's preferential distribution is adjacent to
cell-cell contacts (Fig. 4 e), whereas in N-CAM-deficient
mice it accumulates together with the cadherins (Fig. 4, f-h).
The visualization of the redistribution of N-cadherin,
E-cadherin, and F-actin appears as multicellular rosette
structures, which are composed of all endocrine cell types, and are reminiscent of the apical enrichment of E-cadherin and F-actin in exocrine acinar cells (insets in Fig. 4, d
and h). We propose to refer to these structures as endocrine
acini. The striking changes in the organization of cadherins
and actin filaments in islets of N-CAM-deficient mice suggest that cell polarity may also be affected. In further support for alterations in islet cell polarity, the subcellular localization of the basolateral epithelial cell polarity marker
Na+/K+-ATPase and the nuclei within islets of N-CAM-deficient is reminiscent of their distribution within fully
polarized exocrine acinar cells (Fig. 5, compare a with c
and d, and e with g and h). In normal control islets, Na+/
K+-ATPase colocalizes with N-cadherin and E-cadherin in
cell contact regions (Fig. 5 b, and data not shown), whereas
in islets of N-CAM-deficient mice Na+/K+-ATPase does
not accumulate in regions with increased cadherin and
F-actin clustering (Fig. 5, c and d). Furthermore, while islet cell nuclei are randomly distributed in control islets
(Fig. 5 f), cell nuclei are preferentially localized to the basal
regions of those islet cells that are organized in endocrine
acini in N-CAM-deficient mice (Fig. 5, g and h). Similar to
the effect on cell type segregation, changes in the subcellular localization of cadherins and F-actin in N-CAM-deficient mice were first observed at 4-5 wk of age. To examine whether transdifferentiation of endocrine cells into
exocrine acinar cells could explain the appearance of the
endocrine acinar structures, expression of acinar markers, such as amylase or carboxypeptidase, was investigated.
However, none of these exocrine markers is expressed in islets of N-CAM +/
and N-CAM
/
mice, indicating that
the appearance of acinar structures in islets of Langerhans
does not resemble a transdifferentiation process (data not
shown).
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Taken together, our data suggest that N-CAM is required for islet cell type segregation. Moreover, N-CAM may play a functional role in the regulation of islet cell polarity, probably by modulating the organization of cadherins and F-actin within islet cells.
Ultrastructural Changes in Islet Cells of N-CAM Mutant Mice
Changes in cell-cell interactions and in cellular sorting
may also affect intracellular organization and possibly
physiological functions of a cell. To investigate this possibility we analyzed islets of Langerhans of control mice and
N-CAM-deficient mice by transmission electron microscopy. Although in both N-CAM +/ and N-CAM
/
animals the majority of endocrine cells appeared morphologically normal, a significant fraction of
cells and
cells
appeared ultrastructurally perturbed. The reorganization of cadherins and F-actin as seen by light microscopy (Fig.
4) appears to correlate with the accumulation of cell-cell
junctions in multicellular endocrine structures (Fig. 6, b
and c). Although each of these junctions, including desmosomes and adherens type junctions, are found scattered in
islet cell contacts of control mice, they appear to accumulate towards the center of groups of cells in islets of
N-CAM-deficient mice (Fig. 6, b and c). Moreover, a significant fraction of
cells contained a diminished number of secretory granules (Fig. 6, compare a to b) together with
an increased number of residual bodies that are frequently
associated with the plasma membrane (Fig. 6, b-d). Secretory granules were occasionally observed within residual
bodies (Fig. 6 d), suggesting increased autophagy. Dilation
of the rough endoplasmic reticulum was seen in both
cells and
cells (Fig. 6 c, and data not shown). As the corresponding changes were not observed in control mice, it
appears that N-CAM-mediated cell-cell interactions are
required for organizing islet cell-cell contacts, as well as
for maintaining normal activity and turnover of organelles
within islet cells.
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N-CAM Affects the Epithelial Cell Morphology of Cadherin-expressing L Cells
To examine whether N-CAM's effects on pancreatic endocrine cell morphology also applies to other cell types, and
to assess more directly whether N-CAM can influence
cadherin function, we performed a series of cell transfection experiments. It has previously been demonstrated
that expression of cadherins in fibroblast cell lines results
in the transition from a mesenchymal to an epithelial cell
morphology (Nose et al., 1988). To determine whether
N-CAM affects the epithelial phenotype of cadherin-
expressing L cells, we transiently transfected parental L
cells and L cells that expressed either E-cadherin (LE; Nose
et al., 1988
) or N-cadherin (LN; Miyatani et al., 1989
) with
cDNA constructs encoding N-CAM-120, N-CAM-140, and
N-CAM-180. Both N-CAM-140 and N-CAM-180, but not
N-CAM-120, induced extensive neurite-like extensions or
filopodia on the cells, regardless whether the L cells expressed cadherins or not (Fig. 7, e-g). Notably, the majority of the N-CAM-expressing cells left the monolayer and
migrated on top of neighboring cells (Fig. 7, e and f). Furthermore, thick protrusions were observed on the dorsal
side of N-CAM-expressing cells (data not shown).
|
Next, we examined N-CAM's effect on the subcellular localization of E-cadherin and N-cadherin. In the majority of N-CAM-expressing cells cadherins were diffusely distributed on the cell surface, in particular in cells with neurite-like extensions (Fig. 7, g and h). However, in N-CAM-expressing cells that remained within the cell monolayer, the distribution of E-cadherin and N-cadherin was not significantly changed (Fig. 7, i and j). Expression of either eGFP or another cell adhesion molecule, N-cadherin, did not result in morphological changes in any of the cell lines (Fig. 7, c and d, and data not shown). The failure of N-CAM-120 to induce morphological changes is probably due to the fact that, although expressed at significant levels, N-CAM-120 was not localized to the cell membrane. Thus, the results with L cells in vitro are consistent with our findings in N-CAM-deficient mice in vivo. Together, the results suggest that N-CAM-mediated changes in cell morphology are dominant over cadherin function.
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Discussion |
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Previous analyses of N-CAM-deficient mice showed that
mutant mice appear healthy and are fertile, demonstrating that N-CAM is not essential for embryogenesis (Tomasiewicz et al., 1993; Cremer et al., 1994
). However, close
examination of the development and function of the nervous system in these mice have revealed interesting new
insights into the role of N-CAM in the nervous system
(Cremer et al., 1997
; Shen et al., 1997
; Krushel et al., 1998
;
Moscoso et al., 1998
).
Based on a series of reaggregation experiments of dissociated rat islets in vitro, it was proposed that N-CAM is
also involved in pancreatic islet morphogenesis by regulating islet cell type segregation (Cirulli et al., 1994). We have
now employed N-CAM knockout mice to investigate
N-CAM's functional role in pancreas organogenesis in
vivo. Normally, glucagon-producing
cells are preferentially distributed in the periphery, while insulin-producing
cells are found in the center. Both in N-CAM +/
and
N-CAM
/
mice, islet cell type segregation was affected,
resulting in a more random distribution of
cells within islets. To our knowledge this is the first demonstration of
the involvement of N-CAM in developmental cell sorting
in vivo. Concomitant with these changes, the subcellular
distribution of epithelial cell polarity markers, cadherins,
F-actin, and cell-cell junctions were altered, suggesting that
N-CAM may influence cell polarity and cadherin-mediated adhesion in islet cells. Finally, the diminished number
of secretory granules in
cells of N-CAM mutant mice
suggests that N-CAM-mediated cell-cell interactions are
crucial for the normal turnover of insulin.
Interestingly, all our findings in the N-CAM-deficient
mice were seen to the same extent in heterozygous and homozygous animals, suggesting that N-CAM protein levels
are critical for the processes described above. This is in accordance with the recent demonstration that one functional N-CAM allele is not sufficient to compensate for
the effects on aggressive behaviors and tumor cell dissemination in N-CAM-deficient mice (Stork et al., 1997; Perl,
A.-K., U. Dahl, P. Wilgenbus, H. Cremer, H. Semb, and
G. Christofori, manuscript submitted for publication), indicating that a distinct dosage of N-CAM expression is
crucial for several cellular processes.
How could N-CAM's involvement in islet cell type segregation be explained in molecular terms? Initially, the
mechanisms for cell rearrangement during development
such as tissue spreading movement and segregation of unlike cells were explained by Holfreter as differences in tissue affinities (Townes and Holfreter, 1955). Steinberg and
Takeichi (1994)
showed that two motile cell types differing
only in the levels of expression of a single adhesion system,
will not only segregate from one another but also arrange themselves in the form of an envelope of less cohesive
cells surrounding a core of more cohesive cells. The differential adhesion hypothesis could thereby be attributed to
forces generated by intercellular adhesions within and between migrating cell populations (Steinberg, 1996
). Steinberg and coworkers then went on to explain in physical terms that tissue surface tensions determine the arrangement of cells that are free to rearrange within tissues (Foty
et al., 1996
; Davis et al., 1997
). According to the differential adhesion hypothesis,
cells, forming the core of islets
of Langerhans, should be more cohesive than peripheral
non-
cells. Is there any evidence that islet cell type segregation could be explained by differences in adhesion properties? In rat, Ca2+-dependent adhesion appears to be similar in
cells and non-
cells, whereas non-Ca2+-dependent
adhesion, including that mediated by N-CAM, is more pronounced in non-
cells (Rouiller et al., 1990
, 1991
). In
mouse, the picture is not as clear, since similar immunohistochemical methods failed to reveal any differential expression of N-CAM within mouse islets (Fig. 1 g). The
only CAM that has been shown to be differentially expressed in mouse islets is R-cadherin. However, this cadherin appears to be differentially expressed in
cells at
very low levels. Most importantly, it is predominantly localized to the cytoplasm (Dahl, U., F. Esni, and H. Semb,
unpublished observations). In addition to R-cadherin,
E-cadherin and N-cadherin are also expressed in islets
(Begemann et al., 1990
; Moller et al., 1992
; Hutton et al.,
1993
; Dahl et al., 1996
). However, in the mouse neither
E-cadherin nor N-cadherin appears to be differentially expressed by the various cell types within islets (Begemann
et al., 1990
; Rouiller et al., 1991
; Esni, F., and H. Semb, unpublished observations). Thus, the existence of a CAM
that by virtue of its differential expression would make
cells more adhesive than non-
cells remains elusive.
Nonetheless, it is important to consider the fact that the
cell surface expression level of a CAM may not be a reliable indicator of a cell's adhesiveness.
Alternatively, islet cells may express molecules, which
negatively influence the basic adhesion machinery, which
in islets is primarily mediated by cadherins. Our results
suggest that N-CAM might fulfil the criteria for a molecule that negatively influences the basic adhesion mechanisms. Differential expression of N-CAM in non- cells, or
at least in
cells, would make them less cohesive than
cells, resulting in their peripheral distribution within islets.
Concomitant with the cell sorting defect, the subcellular
distribution of cadherins and F-actin were altered in
N-CAM /
and N-CAM +/
mice. Recent data suggest
that one prerequisite for strong cadherin adhesion is the
clustering of cadherin molecules (Adams et al., 1996
; Yap
et al., 1997
). During the development of cell-cell contacts
in epithelial cells it is thought that E-cadherin and actin filaments form localized clusters, designated "puncta" by
Adams et al. (1996)
, which gradually merge in order to
strengthen adhesion in cell-cell junctions. This is presumably the mechanism by which E-cadherin concentrates to
the cell-cell junctional region of acinar cells in the exocrine pancreas. However, in normal pancreatic islets no
apparent clustering of N-cadherin and E-cadherin is apparent. In contrast, in islets of N-CAM
/
and N-CAM
+/
animals, cadherins appear to cluster more efficiently,
even forming acinar structures, suggesting that cadherin-mediated adhesion becomes stronger. This notion is further
supported by ultrastructural studies showing an accumulation of cell-cell junctions in areas reminiscent of areas with
increased clustering of cadherins and F-actin. These observations, together with the polarized localization of cell nuclei
and the basolateral cell polarity marker, Na+/K+-ATPase,
within rosette-like structures, provide rather compelling evidence for changes in islet cell polarity.
To explain a possible attenuating influence of N-CAM on
cadherin-mediated adhesion, we speculate that N-CAM, either directly or indirectly, interferes with the clustering of
cadherins through changes in cell morphology. It is known
that N-CAM isoforms, which contain polymers of -2,8-linked polysialic acid (PSA-N-CAM), have strong anti-adhe-sive properties (Rutishauser et al., 1988
; Acheson et al.,
1991
; Rougon, 1993
). Although conflicting evidence exists regarding the expression of PSA-N-CAM in rat islets (Moller et al., 1992
; Kiss et al., 1994
), the molecule was not detected in mouse islets by the use of a PSA-N-CAM-specific mono-clonal antibody (5A5; Dodd et al., 1988
; data not shown).
To further elucidate the mechanism for a possible connection or cross-talk between N-CAM and cadherins, and
to examine if it applies to other cell types, we performed a
series of L cell transfection experiments. These in vitro experiments reveal that N-CAM exhibits a dominant effect
over cadherins on cell morphology. They also indicate
that our observations in pancreatic endocrine cells may
apply to other cell types as well. However, expression of
N-CAM-120 and N-CAM-140 in fully polarized epithelial
MDCK cells did not result into changes of cell morphology, raising the possibility that different cell types may exhibit varying responses to N-CAM expression (Powell et al.,
1991). Future experiments will have to identify the mechanisms by which N-CAM affects the subcellular organization of cadherins.
Regarding N-CAM's involvement in F-actin organization, it is unlikely that this involves a direct physical interaction with the actin cytoskeleton, since the major N-CAM
isoform expressed in the islets is the glycosylphosphatidylinositol-linked N-CAM-120. Alternatively, N-CAM
could indirectly regulate actin filament organization either
through the observed rearrangement of cadherins or through
the activation of intracellular signaling cascades. There is
now considerable evidence that N-CAM can activate the FGF receptor through a direct physical interaction (Doherty
and Walsh, 1994; Saffell et al., 1997
). The possible involvement of N-CAM in actin filament rearrangement through
the FGF receptor signaling pathway is further supported
by the fact that N-CAM-mediated activation of the FGF
receptor leads to activation of GAP-43 (Walsh et al.,
1997
), an intracellular protein that is involved in the organization of the actin cytoskeleton.
Finally, the question remains whether cell-cell contacts
and correct islet cell organization are required for normal
islet function. One line of evidence suggests that this
could, indeed, be the case. When cells are separated, insulin biosynthesis and secretion decrease, especially in response to glucose concentrations that physiologically stimulate pancreatic islets. However, these changes are rapidly
corrected after cell reaggregation (Lernmark, 1974
; Halban et al., 1982
; Salomon and Meda, 1986
; Bosco et al., 1989
; Philippe et al., 1992
). It is interesting to note that
perturbation of islet cell organization in diabetic patients,
as well as in animal models of the disease, suggests that the
inability to organize endocrine cells may explain the hyperglycemic phenotype (Gepts and Lecompte, 1981
; Gomez Dumm et al., 1990; Tokuyama et al., 1995
). Therefore,
the altered cell type segregation in islets of N-CAM
/
and N-CAM +/
animals seemed appropriate for testing this hypothesis. However, even though occasional glucose-intolerant individuals were found among N-CAM mutant
mice, no statistically significant alterations in glucose-tolerance of N-CAM
/
and N-CAM +/
mice were observed (data not shown). This was rather surprising in light
of the ultrastructural changes observed in islet cells of
N-CAM-deficient mice, which indicated that function could be impaired in at least a fraction of the endocrine
cells. Maybe the absence of a grossly impaired islet function is due to the fact that the reported effects are only
found in a fraction of the islet cells. However, these data
warrant further studies to elucidate whether N-CAM or
other CAMs are directly involved in glucose-mediated insulin secretion and, if so, whether the expression of any of
these CAMs is affected in diabetic patients and in animal
models of the disease.
|
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Footnotes |
---|
Received for publication 21 July 1998 and in revised form 30 November 1998.
Address correspondence to Henrik Semb at Institute of Medical Biochemistry, Göteborg University, Box 440, S-405 30 Göteborg, Sweden,
Tel.: 46 31 773 3779. Fax: 46 31 41 61 08. E-mail: henrik.semb{at}medkem.gu.se
We wish to thank Drs. E. Bock, H. Edlund, R.J. MacDonald, J. Nelson, and M. Takeichi for immunoreagents and cDNAs and Åsa Gylfe for help with statistical evaluation. Kristina Forsgren is gratefully acknowledged for the EM work.
This work was supported by research grants from the Swedish Cancer Society (3157-B96-06XAB), Kempestiftelserna, and M. Bergvalls Stiftelse (F. Esni and H. Semb), the Swedish Medical Research Council (12x-2288; I.-B. Täljedal), and the Austrian Industrial Research Promotion Fund (A.-K. Perl and G. Christofori).
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Abbreviations used in this paper |
---|
CAMs, cell adhesion molecules; Cy3, indocarbocyanine; dpc, days postcoitum; eGFP, enhanced green fluorescence protein; N-CAM, neural cell adhesion molecule.
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References |
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