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Functional gap junctions in mouse small intestinal crypts.

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THE ANATOMICAL RECORD 212:364-367 (1985)
Functional Gap Junctions in Mouse Small Intestinal
Crypts
MATTHEW BJERKNES, HAZEL CHENG, AND STANLEY ERLANDSEN
Department of Anatomy, University of Toronto, Toronto, Ontario, Canada M5S l A 8 (M.B.,
H. C.) and Department of Anatomy, University of Minnesota, Minneapolis, M N (S.E.)
ABSTRACT
We demonstrate intercellular transfer of Lucifer yellow and the
existence of gap junctions in isolated mouse small intestinal crypts. The pattern of
dye transfer approximates the normal pattern of cell proliferation and differentiation in the intestinal epithelium. These findings indicate that the cells of normal
crypts form a n effective intercellular continuum. This intercellular route may function in the establishment of chemical, ionic, or electrical fields, which in turn may
play a role in the control of cell proliferation, differentiation, and secretion in the
crypt.
It is widely held that establishment of and response to
fields is responsible in large measure for many developmental phenomena (Turing, 1952; Crick, 1970; Meinhardt, 1982). In the embryo, these morphogenic fields
often span many cells. As a result it is thought likely
that many fields (e.g., chemical or ionic gradients) are
established through a direct intercellular route of some
kind. The most likely candidate for the intercellular
channel is the gap junction (Wolpert, 1978; Lo, 1980;
Loewenstein, 1981; Othmer, 1983; Warner et al., 1984;
Guthrie, 1984).
It is plausible that mechanisms similar to embryonic
fields help govern cell proliferation and differentiation
in the adult intestinal epithelium. Crypts do respond to
injury in a manner consistent with the existence of
regulatory fields, although these crypt responses have
usually been ascribed largely to the effects of some form
of feedback from the villus, e.g., through a chalone-like
mechanism (Cairnie, 1967; Rijke et al., 1976; Wright
and Al-Nafussi, 1982). To begin investigating the plausibility of field-like control mechanisms acting in the
intestinal epithelium, we attempted to determine
whether functional gap junctions exist in the normal
adult epithelium. In this paper we report the presence
of functional gap junctions in mouse intestinal crypts,
as demonstrated by fluorescent dye transfer and freezefracture studies.
placed in PBS, and then used immediately to study dye
transfer in the villus epithelial cell population.
Fluorescent Dye Transfer
The ability of mouse duodenal epithelial cells to transfer the dye Lucifer yellow CH (mol. wt. 457.3, Sigma)
was tested by iontophoretic microinjection (micropipettes were pulled from Omega Dot glass and filled with
4% solution of Lucifer yellow in distilled water; 2 rectangular iontophoretic current pulses of 200-msec duration,
and 1- to 2- nA amplitude were applied each second;
Stewart, 1978) into either villus cells in mucosal strips
(20 mice were used to prepare mucosal strips, from each
strip 2-3 villi were studied) or into the cells of isolated
but intact crypts (usually only one or two crypts were
used from any individual mouse; about 100 mice were
used in this study). For any given cell, the injection
period lasted about 30 sec. The injection and transfer of
the dye were monitored through a Dage silicon intensified target video camera mounted on a fluorescence
microscope.
Freeze-Fracture Studies
Five mice were used for freeze-fracture studies. The
crypts were isolated directly into 2.5% glutaraldehyde,
fixed for a n additional 30 min in the same fixative, and
then stored in 0.1 M cacodylate buffer containing 5%
sucrose. The tissue was cryoprotected, fractured, and
MATERIALS AND METHODS
immediately replicated without etching. The replicas
Preparation of Intestinal Crypts and Villi
were collected on Formvar-coated slot grids for examiMale Swiss albino mice weighing 20-25 gm were used nation. The nomenclature for identification of memthroughout the study. Intestinal crypts were isolated as brane surfaces as proposed by Branton et al. (1975) was
described previously (Bjerknes and Cheng, 1981). Briefly, followed.
mice were perfused through the left ventricle with 30
RESULTS
mM EDTA. A segment of duodenum was removed and
Dye
Transfer
Studies
everted, and intestinal crypts were isolated by mechanWe were able to demonstrate efficient dye transfer
ical vibration. The tissue was suspended in PBS and
used immediately following preparation. Only viable between crypt-base columnar cells. When Lucifer yellow
crypts were used for dye injection studies. In other studies, strips of mucosa, about 4-5 villi long and 1 villus
Received November 16,1984; accepted March 14,1985.
wide, were removed from fresh segments of duodenum,
0 1985 ALAN R. LISS, INC
FUNCTIONAL GAP JUNCTIONS IN CRYPTS
was injected into a cell in this region, the dye was
rapidly transferred to many neighboring cells, first to
those immediately adjacent and then to cells more distant (Fig. 1, a-c). At this point, the exact number of cells
involved was difficult to determine because of the threedimensional nature of the tissue. The average time
lapsed (ATL)between the time of injection of a given cell
and the time of dye appearance in a n adjacent cell in
the crypt-base columnar cells was 2.5 f 0.16 sec (X 5
SE, n = 35). Crypt-base columnar cells were not observed to transfer dye to Paneth cells and vice versa. No
dye transfer was observed in most Paneth cells (n = 75),
which were easily recognized from their size, shape, and
granular content. However, a slow transfer of dye to a n
immediately adjacent Paneth cell was observed in a few
cases with a n ATL of 27.5 5 6.94 sec (n = 71, (Fig. 1,d0. (Unlike Paneth cells, mucous and enteroendocrine
cells were not readily identifiable; thus we were not able
to study their ability to transfer Lucifer yellow.) We
observed efficient dye transfer in midcrypt epithelial
cells (Fig. 1,g-i) with a n ATL of 4.8 0.17 sec (n = 28)
and 9.0
0.36 sec (n = 63) in the lower and upper
halves of the midcrypt, respectively. When Lucifer yellow was injected into lower midcrypt cells, the dye was
rapidly transferred to many neighboring cells (Fig. 1, i).
In the case of upper midcrypt cells, the dye appeared to
be transferred to a smaller number of neighboring cells.
Only a low degree of dye transfer was observed in crypttop epithelial cells with a n ATL of 15.2 f 1.15 sec (n =
211, (Fig. 1, j-1). Lucifer yellow transfer was observed
only to immediately adjacent crypt-top cells. We were
not able to demonstrate dye transfer in any region of
the villus (n = 97).
Freezefracture Studies
In freeze-fracture studies we found clusters of intramembranous particles (IMP) resembling gap junctions
in epithelial cells at all levels of the crypt. Fractures
permitting identification of Paneth cell, goblet cell, or
enteroendocrine cell membrane surfaces were rarely observed. Therefore the presence or absence of gap junctions on these cells could not be properly evaluated.
Single gap junctions were routinely seen on the lateral
surface of crypt cells and as many as three gap junctions
were observed in a single cell (Fig. 1,m-0). The size of
the IMP ranged from 6.8 to 10.0 nm in diameter with a
mean size of 8.5 k 0.6 nm (n = 1201, and the number of
IMP in a n individual gap junction varied from a few
dozen to more than several hundred. In individual crypt
cells, gap junctions were observed in fracture faces of
the lateral surface from the level of the nucleus to just
beneath the tight junction. We also found small gap
junctions in villus epithelial cells.
DISCUSSION
Correlation of Dye Transfer With Proliferation
and Differentiation
We found that the pattern of dye transfer approximates the normal pattern of cell proliferation and differentiation in the intestinal epithelium. Specifically, we
found that immature cells with extensive proliferative
abilities, i.e., crypt-base columnar cells and lower midcrypt cells CLeblond and Cheng, 1976), demonstrated
efficient dye transfer. In higher regions of the crypt,
where cells begin to terminally differentiate and hence
365
cease proliferating, cells decreased in dye transfer efficiency until, in the villus (where cells are mature and
do not divide), we were not able to demonstrate dye
transfer. Similarly, no or very low efficiency dye transfer
was observed between Paneth cells, terminally differentiated nonproliferating cells found in the crypt base
(Leblond and Cheng, 1976). In summary, actively proliferating epithelial cells demonstrated a n ability to transfer Lucifer yellow. As these cells stop proliferating and
terminally differentiate they gradually lose their ability
to transfer the dye under our experimental conditions.
Freeze-fracture studies of the intestinal epithelium
have previously reported gap junctions (Staehelin, 1972;
Madara et al., 1981; Curtis et al., 1984); however, the
location of the junctions within the epithelium was not
stated. We found gap junctions in epithelial cells a t all
levels of the crypt. While we also found that villus epithelial cells possess small gap junctions, we could not
demonstrate transfer of Lucifer yellow between villus
epithelial cells. Consistent with the presence of gap junctions is Sheridan’s observation (personal communication) of intercellular communication as demonstrated by
electrotonic coupling in villus epithelial cells in preliminary experiments. Therefore it is plausible that the gap
junctions present in villus cells may be qualitatively
different from those present in crypt cells, e.g., the cellto-cell channels may have been scaled down so that
molecules such as Lucifer yellow can no longer pass but
the channels are still permeable to the smaller ions.
Such regional differences in gap junction permeability
have been observed in other systems (Lo, 1980; Iwatsuki
and Petersen, 1978a).
Gap Junctions and Secretion
Intestinal crypts are also known to be a site of cellular
secretion of secretory IgA, lysozyme, and fluid (Wilson
et al., 1982; Olson and Erlandsen, 1981; Welsh et al.,
1982). It is plausible that intercellular communication
through gap junctions has a role in regulating or possibly synchronizing crypt secretion such as may occur in
pancreas (Petersen and Ueda, 19761, salivary gland
(Roberts et al., 1978; Kater and Galvin, 1978), and lacrimal gland (Iwatsuki and Petersen, 1978b).
In conclusion, the ability of crypt epithelial cells to
transfer Lucifer yellow indicates that direct intercellular exchange of ions and small molecules may be a
normal occurrence in the crypt epithelium. Our observation of gap junctions in crypt cells is consistent with
the conclusion that intercellular transfer may be mediated through gap junctions.
Proliferation and differentiation in the intestinal epithelium is apparently under regulation. The most common view is that the regulation results from a chalonelike feedback from the villus population (Cairnie, 1967;
Rijke et al., 1976; Wright and Al-Nafussi, 1982). We
would like to propose that the present findings suggest
a n additional mechanism that may be important in regulation of epithelial cellular behavior (a detailed mathematical model will be presented elsewhere). The
intercellular continuum formed via the gap junctions
interconnecting most crypt cells could form the physical
substrate within which fields (chemical, electrical, etc.)
are established that in turn influence cellular behavior.
Experimental perturbations that altered the size of the
crypt population (e.g., irradiation with subsequent loss
FUNCTIONAL GAP JUNCTIONS IN CRYPTS
of many crypt cells) would alter the boundary conditions
for the field, resulting in a new field and hence altered
cell behavior (e.g., the pattern of cell proliferation).
ACKNOWLEDGMENTS
This study was supported by grants from the Medical
Research Council of Canada and the Canadian Foundation for Ileitis and Colitis.
LITERATURE CITED
Bjerknes, M., and H. Cheng (1981) Methods for the isolation of intact
epithelium from the mouse intestine. Anat. Rec., 199:565-574.
Branton, D., S. Bullivant, N.B. Gilula, M.J. Karnovsky, H. Moor, K.
Muhlethaler, D.H. Northcote, L. Packer, B. Satir, P. Satir, L.A.
Staehlin, R.L. Steere, and R.S. Weinstein (1975) Freeze-etching
nomenclature. Science, 190:54-56.
Cairnie, A.B. (1967) Cell proliferation studies in the intestinal epithelium of the rat: Response to continuous irradiation. Radiat. Res.,
32240-264.
Crick, F. (1970) Diffusion in embryogenesis. Nature (Lond.), 225:420422.
Curtis, R.L., J.S. Trier, R.A. Frizzell, N.M. Lindem, and J.L. Madara
(1984) Flounder intestinal absorptive cells have abundant gap junctions and may be coupled. Am. J. Physiol., 246:C77-C83.
Fig. 1. a-1. Photomicrographs showing intercellular transfer of Lucifer yellow between crypt epithelial cells. All photomicrographs are
at the same magnification. a x . Dye transfer between crypt-base columnar cells: a) bright-field photomicrograph of a crypt base (intestinal
crypts are roughly test-tube-shaped structures); notice the Paneth cells
with secretory granules below the cell to be injected (which is indicated
by the micropipette in the upper left-hand quadrant); b) fluorescent
photomicrograph of the initial injection into a crypt-base columnar
cell; c) fluorescent photomicrograph of the same cell at a later time
showing transfer of dye to neighboring cells; notice that dye transfer
to cells situated below stopped at the level of the Paneth cells. d-f. Dye
transfer in Paneth cells: d) bright-field photomicrograph of crypt base;
el fluorescent photomicrograph of the Paneth cells shortly after injection; fl fluorescent photomicrograph of the same cell at a later time
showing transfer of dye to a n adjacent Paneth cell; a second injection
into a crypt-base columnar cell can also be seen. g-i. Dye transfer in
midcrypt cells: g) bright-field photomicrograph of midcrypt region; h)
fluorescent photomicrograph of the injected midcrypt cell; i) fluorescent photomicrograph of the same cell at a later time showing transfer
of dye to neighboring cells; two other injections in the same crypt can
also be seen. j-1. Dye transfer in crypt-top cells: j) bright-field photomicrograph of crypt-top region; k) fluorescent photomicrograph of the
injected crypt-top cell; 1)fluorescent photomicrograph of the same cell
at a later time showing transfer of dye to neighboring cells: note that
the orientation of the crypt has been shifted. m-o. Electron micrographs of freeze-fracturereplica of the P-face of mid-crypt cells (bars =
0.1 pm): m) three gap junctions on lateral surface of mid-crypt cell; n)
a small eau iunction consisting of about 25 particles; 0) a large gap
~.
junction consisting of several h<ndred particles.
~
367
Guthrie, S.C. (1984) Patterns of junctional communication in the early
amphibian embryo. Nature (Lond.), 311:149-151.
Iwatsuki, N., and O.H. Petersen (1978a) Electrical coupling and uncoupling of exocrine acinar cells. J. Cell Biol., 79:533-545.
Iwatsuki, N., and O.H. Petersen (197813) Intracellular Ca'+ injection
causes membrane hyperpolarization and conductance increase in
lacrimal acinar cells. Pflugers Arch., 3773185-187.
Kater, S.B., and N.J. Galvin (1978) Physiological and morphological
evidence for coupling in mouse salivary gland acinar cells. J. Cell
Biol., 79:20-26.
Leblond, C.P., and H. Cheng (1976) Identification of stem cells in the
small intestine of the mouse. In: Stem Cells of Renewing Populations. A.B. Cairnie, P.K. Lala, D.G. Osmond, Eds. Academic, New
York, pp. 7-31.
Lo, C.W. (1980) Gap junctions and development. In: Development in
Mammals, Vol. 4. M.H. Johnson, Ed. Elsevier/North-Holland,New
York, pp. 39-80.
Loewenstein, W.R. (1981)Junctional intercellular communication: The
cell-tocell membrane channel. Physiol. Rev, 61:829-913.
Madara, T.L., M.R. Neutra, and J.S. Trier (1981) Junctional complexes
in fetal rat small intestine during morphogenesis. Dev. Biol.,
86:170-178.
Meinhardt, H. (1982) Models of Biological Pattern Formation. Academic, New York.
Olson, R.E., and S.L. Erlandsen (1981) Paneth cell function: The effects
of cholinergic and adrenergic drugs on lysozyme secretion. Anat.
Rec., 1993186A.
Othmer, H.G. (1983) A continuum model for coupled cells. J. Math.
Biol.. 17:351-369.
Petersen, O.H., andN. Ueda (1976) Pancreatic acinar cells: The role of
calcium in stimulus-secretion coupling. J. Physiol., 254:583-606.
Rijke, R.P.C., W.R. Hanson, H.M. Plaisier, and J.W. Osborne (1976)
The effect of ischemic villus cell damage on crypt cell proliferation
in the small intestine: Evidence for a feedback control mechanism.
Gastroenterology, 71:786-792.
Roberts, M.L., N. Iwatsuki, and O.H. Petersen (1978) Parotid acinar
cells: Ionic dependence of acetylcholine-evoked membrane potential changes. Pflugers Arch., 376:159-167.
Staehelin, L.A. (1972) Three types of gap junctions interconnecting
intestinal epithelial cells visualized by freeze-etching. Roc.Natl.
Acad. Sci. USA, 69:1318-1321.
Stewart, W.W. (1978) Functional connections between cells as revealed
by dye-coupling with a highly fluorescent naphthalimide tracer.
Cell, 14:741-759.
Turing, A.M. (1952)The chemical basis of morphogenesis. Phil. Trans.
R. Soc. B, 237337-72.
Warner, A.E., S.C. Guthrie, N.B. Gilula (1984) Antibodies to gapjunctional protein selectively disrupt junctional communication in
early amphibian embryo. Nature (Lond.), 311:127-131.
Welsh, M.J., P.L. Smith, M. Fromm, and R.A. Frizzell (1982) Crypts
are the site of intestinal fluid and electrolyte secretion. Science,
218:1219-1221.
Wolpert, L. (1978) Gap junctions: Channels for communication in development. In: Intercellular Junctions and Synapses (Receptors
and Recognition, Series B, Volume 2). J. Feldman, N.B. Gilula, and
J.D. Pitts, Eds. Chapman and Hall, London, pp. 83-96.
Wright, N.A., and A. Al-Nafussi (1982) The kinetics of villus cell
populations in the mouse small intestine. 11. Studies on erowth
control after death of proliferative cells induced by cytosincarabinoside, with special reference to negative feedback mechanisms.
Cell Tissue Kinet., 15:611-621.
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