Center for Regenerative Medicine, Department of Biology and Biochemistry,
University of Bath, Bath BA2 7AY, UK
* Present address: UK Centre for Tissue Engineering, Room 3.446, Stopford
Building, Biological Sciences, University of Manchester, Manchester M13 9PT,
UK
Author for correspondence (e-mail:
a.ward{at}bath.ac.uk)
Accepted 18 November 2002
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SUMMARY |
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For both skin and colon, the histological organisation of structural-proliferative units was unaltered with increasing IGF2 dose, although there was a higher fraction of dividing cells in the proliferative compartment. In the colon an increase in IGF2 dose increases the overall area of the epithelium. This is due to an increase in the number of crypts with no change of cell size or of crypt area. Growth stimulation appears to be due to a reduction in the duration of crypt fission. The conclusion is that the IGF2 pathway can stimulate the multiplication of colonic crypts independently of stimulating increased cell proliferation.
The results for the skin are consistent with this. An increase of IGF2 dose increases the proportion of dividing cells in the basal layer, the thickness of the epidermis and the total area of the epidermis.
By comparison with Drosophila, these results show no effects on cell size, but do show the possibility of inducing disproportionate growth. These differences may represent properties of the SPU organisation that is characteristic of vertebrate tissues.
Key words: Insulin-like growth factor 2 (IGF2), Growth, Size, Proportion, Intestinal crypts, Epidermis, Structural-proliferative units (SPUs), Proliferating cell nuclear antigen (PCNA)
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INTRODUCTION |
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Growth can be considered at various levels: subcellular, cellular and organismal. At the subcellular level, there is the molecular biology of the various component processes: the cell division cycle, the action of growth factors or the mechanism of apoptosis. These are all key processes that have been the subject of much recent research. But most of the information comes from studies on tissue culture cells. Although very convenient for molecular biology, cells in culture are not a good model for the growth of tissues in the body, as they are typically homogeneous monocultures showing exponential growth. In vivo, exponential growth is rarely found and most cell division occurs at particular sites within a defined three-dimensional tissue architecture. Furthermore, in vivo, most tissues consist of multiple cell types, the production and turnover of which needs to be coordinated.
Organs are not homogeneous masses of similar cells. They normally consist
of microanatomical structural units and these are often also units of cell
proliferation control (Potten,
1978; Potten,
1998
). This may be demonstrated by examples in which the clonal
organisation of structural units can be visualised, such as mouse aggregation
chimeras between different strains, X-chromosome inactivation mosaics or
mutagenesis studies (Gordon et al.,
1992
). If a structural unit is fed by a single stem cell, then it
will necessarily appear monoclonal under conditions where individual clones
can be experimentally distinguished. Monoclonality is seen, for example, in
the intestinal crypts (Ponder et al.,
1985
; Winton et al.,
1988
), cell patches in the epidermis
(Potten, 1974
;
Jones et al., 1995
),
endometrial glands (Jiang et al.,
1996
), terminal ductal-lobular units in the breast
(Tsai et al., 1996
) and cell
columns in the adrenal cortex (Weinberg et
al., 1985
). In fact, such observations do not preclude structural
units from containing more than one stem cell, because random elimination of
individual cellular lines of descent can also lead to monoclonality
(Schmidt et al., 1988
).
Histological structural units that are also units of cell proliferation will
be referred to here as `structural-proliferative units' or SPUs.
Significant growth of an organ cannot occur without an increase in the
number or size of SPUs, so the regulation of the multiplication of SPUs is a
central and crucial issue when considering the control of growth generally. In
general, the number of SPUs increases during the growth of the animal but
achieves a steady state with minimal turnover once full size is attained. For
example, the number of nephrons in the kidney stops increasing about 40 days
after birth in rats (Canter and Goss,
1975), while in the colon the fission of crypts declines to a
steady state of 1-2% by 8-10 weeks
(Maskens, 1978
;
Cheng and Bjerknes, 1985
). The
ability to repair damage to tissues and organs is dependent on whether the
ability to produce new SPUs is retained, and this is associated with, although
not identical to, the pattern of normal multiplication. For example new
nephrons cannot be produced in the rat kidney beyond about 50 days, not much
longer than the normal growth period
(Canter and Goss, 1975
), while
new intestinal crypts can arise at any stage in response to irradiation or
other damage (Cairnie and Millen,
1975
). So not only is the control of the number of SPUs during
growth central to normal growth control, but it is also crucial for the
prospects of repair to damaged tissues and organs.
There are very few ways known in which it is possible experimentally to
vary the relative growth of parts during embryogenesis. The majority of cases
where this has been achieved have involved the insulin/insulin-like growth
factor (IGF) signalling system in Drosophila, where insulin-like
ligands stimulate their tyrosine kinase receptor, and thereby activate PI3
kinase, protein kinase B and S6 ribosomal protein kinase
(Oldham et al., 2000;
Leevers, 2001
). In
vertebrates, the homologous pathway involves three ligands (insulin, IGF1 and
IGF2) interacting with at least two signalling receptors (IR and IGF1R), with
effects on growth mediated by IGF1R
(Efstratiadis, 1998
). In
humans, IGF2 overexpression is associated with disproportionate overgrowth in
Beckwith-Weidemann syndrome (Ward,
1997
). Although rare, by analogy with the discovery of tumour
suppressor genes in rare familial cancers, a better understanding of such
growth conditions is likely to have an importance much greater than is
suggested by the relatively small proportion of affected individuals.
In the present work, we have studied the effects of the IGF2 pathway in a
mammalian system. We have used three strains of mice differing in their IGF2
status to investigate the relationship between the multiplication of the SPUs
within particular organs and the growth of the organ as a whole. We have
examined two contrasting types of SPU: in the colon and in the epidermis of
the skin. In the colon, each crypt represents a single SPU. The stem cells are
located at the base, the transit-amplifying cells in the lower part of the
crypt and the differentiating cells in the upper part. Cells are produced in
the basal region, progress up the crypt and become shed into the lumen of the
colon (Wright and Alison,
1984). In the epidermis, an SPU consists of a stem cell together
with its progeny (Potten,
1974
). The stem cells and the transit cells lie in the basal
layer, while cells in suprabasal layers are postmitotic and differentiating.
They progress upwards and eventually lose their nuclei and become shed from
the exterior surface of the skin (Watt,
1998
).
When the IGF2 dose to either of these systems is altered, the number of cell divisions is altered. We were interested to know whether an increase in cell division affects the growth of the organ or simply drives cells faster through their differentiation pathway, speeding up the cell turnover. Furthermore, if growth is affected, does this arise from a change in size of SPUs or in the total number of SPUs, or by some combination of both effects? Our results show that IGF2 dose does affect growth and it does so by controlling the number rather than the size of SPUs. The IGF2 pathway must therefore, in some way, control the multiplication of the SPUs, as well as affecting the cell division cycle itself.
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MATERIALS AND METHODS |
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For the present study two strains of mouse were used, with over- and
under-expression of IGF2, and were compared with the wild type.
K:Igf2 is a transgenic containing an Igf2 gene driven by a
keratin promoter (Ward et al.,
1994). The full designation is Tg(Igf2)B1Ward, and it has
previously been referred to as `Blast'. There is overexpression of IGF2 in
several tissues, including the skin, colon and endometrium. The mice show
overgrowth of the affected organs and the DNA content of the listed organs is
increased, showing that the cell number is increased and that the enlargement
is not simply due to an increased content of water or extracellular materials.
The Igf2 knockout shows a considerable growth deficit during
embryonic life. Newborns are about 60% of wild-type size and remain small
throughout life. Although small, the mice are otherwise normal and the
relative proportions of body parts are conserved
(De Chiara et al., 1990
).
Control animals used in the study were wild-type littermates of the
Igf2 knockout and K:Igf2 mice.
Igf2 knockout and K:Igf2 hemizygous transgenic male mice were crossed to non-transgenic F1 (C57BL6xCBA) females. The imprinted nature of the Igf2 locus means that paternal transmission of the disrupted knockout allele results in heterozygous Igf2 knockout offspring with a growth retardation phenotype that is essentially indistinguishable from homozygotes. The K:Igf2 transgene acts in a dominant manner, but cannot be transmitted maternally because of phenotypic effects on the uterus rendering transgenic females subfertile. The results on the number, size and labelling indices of the SPUs are presented in terms of `IGF2 dose', which we know increases in the series: Igf2 knockout<wild type<K:Igf2.
In addition, we have used H253 mice, a strain that carries several
lacZ genes on the X chromosome in the region subject to
X-inactivation (Tan et al.,
1993), to visualise the clonal makeup of tissues. H253 transgenic
males were crossed to non-transgenic F1 (C57BL6 x CBA)
females, and female offspring were tested for the presence of the X-linked
transgene by ß-galactosidase staining of earclips. Transgenic females
from these crosses were obligate hemizygotes, giving mosaic expression of the
transgene. Expression of the X-linked transgene was ubiquitous in epidermis
and colon crypts from homozygous females or males (data not shown).
Genotyping
Igf2 knockout samples were distinguished from wild type by
multiplex PCR using primers Igf2-91
(5'-CTGTGAGAACCTTCCAGCCTTTTC-3'), Igf2-92
(5'-GTGAGAGACCAGTGCGGAATAATC-3'), Neo-80
(5'-CATCGCCTTCTATCGCCTTC-3') and Igf2-95
(5'-CATGCCAGCAAGGATAGTCA-3'). These primers generate a 400 bp
product from the Igf2 knockout disrupted gene and a 640 bp product
from the intact wild-type gene. Ear clips from animals to be tested were
boiled for 10 minutes in 1 ml freshly prepared 50 mM NaOH, then neutralised by
the addition of 50 µl 1 M Tris HCl (pH 8.0). This mixture (1 µl) was
added to 24 µl reaction mix to give a 25 µl reaction containing 75 mM
Tris HCl (pH 9.0), 20 mM (NH4)2SO4, 0.01%
Tween-20, 3 mM MgCl2, 250 µM dNTPs, 1U Taq polymerase
and 150 nM each primer. Thirty-six reaction cycles were carried out, each
consisting of 60 seconds denaturation at 95°C, 60 seconds annealing at
60°C and 60 seconds extension at 72°C.
K:Igf2 samples were distinguished from wild type by slot blotting.
Genomic DNA was prepared from tail biopsies as previously described
(Ward et al., 1997) and
approximately 10 µg was denatured by addition of an equal volume of 1 M
NaOH and incubation for 10 minutes at room temperature. Denatured DNA was
loaded onto nylon membrane (Hybond-N+, Amersham) pre-incubated in 0.5 M NaOH
and placed in the slot-blot apparatus (Flowgen). The filter was neutralised,
air-dried and UV crosslinked, then probed using a radiolabelled fragment
corresponding to exons 4-6 of the Igf2 gene
(Ward et al., 1994
). The
K:Igf2 transgene has more than 25 copies of this region of the
Igf2 gene; hence, the probe readily distinguishes between endogenous
and transgene-related signal.
Histological analysis
The histological analysis was carried out at three developmental stages:
neonatal, when SPUs are newly established, about 2 weeks postnatal, when whole
organism growth is still rapid, and about 3 months, when whole organism growth
is slowing. Some studies focused more closely on the crucial 2-4 week
postnatal period.
Sample collection
Samples were collected at 0.5, 13.5 and 83.5 days post-partum (neonate,
2-week- and 12-week-old mice). Mice were killed by cervical dislocation and
samples dissected from the mid-portion of the colon and from the dorsal skin
roughly in the centre of the back. Samples were fixed for 24 hours in 10%
formaldehyde in PBS, or in Zamboni's fixative [85 mM sodium phosphate buffer
(pH 7.3), 0.85% paraformaldehyde, 15% saturated picric acid], then embedded in
paraffin wax, and sections cut at 7 µm. Conventional Haematoxylin and Eosin
(H&E) staining was performed for nuclear counting.
Total crypt number analysis
For analysis of colon crypt density, crypt fission and water content,
samples were collected at 13.5, 20.5 and 27.5 days post-partum (2-, 3- and
4-week-old mice). The entire colon was dissected open in PBS, the contents
washed out and the colon squashed flat between glass slides. The lumenal
surface was examined under a dissecting microscope and photographed using a
digital SPOT-RT camera (Diagnostic Instruments) at 8x magnification.
Randomly chosen areas of the distal, mid and proximal colon were also
photographed at 100x magnification. SPOT V3.3 software was used to
measure the surface area of the entire colon at low power and to count the
number of crypts across randomly selected fields (though note that it was
necessary to reject fields containing folded tissue, where it was impossible
to accurately count crypts across the entire field) of the distal, mid and
proximal regions sampled at high magnification.
Crypt fission analysis
Regions of dissected mid-colon were fixed and processed through to H&E
stained sections as above, then examined for fissioning crypts. Crypts were
only counted if they could clearly be seen to extend from the base to the
lumenal surface, as evidenced by a lumenal brush border. Fissioning crypts
were so designated if a region of bifurcation was visible in the lower 75% of
the crypts, eliminating false positives caused by two mature adjacent crypts
overlapping slightly near their tops in the plane of section. To reduce
sampling bias, one person scored all of the samples in a blind fashion. It
should be noted that this method is relatively simple and allowed for a robust
comparison between samples from animals of different genotypes. A more
definitive fission index might be derived using microdissected colon tissue
(Goodlad, 1994).
Water content analysis
Part of the dissected colon was accurately weighed and then dessicated for
6 days in a 65°C oven to remove water content. It was then re-weighed and
the water content determined.
ß-galactosidase staining
H253 colon samples were fixed in 100 mM phosphate buffer (pH 7.4), 4%
paraformaldehyde, 2 mM MgSO4 and 5 mM EGTA for 30 minutes at
4°C. Tissues were then rinsed and incubated for 30 minutes at room
temperature in the same solution minus the paraformaldehyde, then rinsed and
incubated for 5 minutes at room temperature in 100 mM phosphate buffer (pH
7.4), 2 mM MgCl2, 5 mM K3Fe(CN)6, 5 mM
K4Fe(CN)6, 0.01% sodiumdeoxycholate and 0.02% NP-40.
Tissues were then stained overnight at 28°C in the same solution
containing 1 mg/ml X-Gal. Stained colon samples were refixed overnight in 10%
formaldehyde in PBS, wax embedded and sectioned. Sections stained with X-Gal
were counterstained with nuclear Fast Red.
Epidermal wholemounts
Dorsal mouse skin was removed from the carcass and pinned out. Fur was
removed using Immac hair removal cream (Reckitt Benckiser), followed by gentle
scraping with a blunt scalpel. The denuded skin was cut into 1.5 cm pieces,
washed in PBS and incubated overnight in DMEM containing 2.5 mg/ml Dispase II
(Boeringher) at 4°C. The epidermis was then carefully separated from the
underlying dermis using a scalpel blade, washed in PBS and fixed for 5 minutes
in 10% formaldehyde in PBS and stained for ß-galactosidase as above.
Epidermal wholemounts were mounted under coverslips and photographed using
brightfield illumination on a Leica DMRB equipped with a digital SPOT-RT
camera.
Antibody staining
Anti-proliferative cell nuclear antigen (PCNA) antibody was used to mark
proliferating cells. Slides from samples fixed in formaldehyde were dewaxed
and rehydrated, then placed in a domestic pressure cooker containing boiling
10 mM sodium citrate buffer pH 6.0 and pressure cooked for 60 seconds to
retrieve the antigen. Slides were washed for 10 minutes in PBS with 0.1%
Tween-20 (PBST), then endogenous peroxidase activity was quenched by
incubation in 3% H2O2 (wt/vol) in tap water for 5
minutes, followed by 0.5% H2O2 in methanol for 30
minutes. Slides were washed three times for 10 minutes each in PBST, then
blocked and incubated with primary and secondary antibodies using the Vector
Mouse-on-Mouse (MOM) kit (Vector), according to the manufacturers
instructions. The primary antibody (anti-PCNA, monoclonal PC10, Dako) was used
at a concentration of 1:500. After application of secondary antibody,
streptavidin-horseradish peroxidase complex (ABC kit, Vector) and
diaminobenzidine (DAB kit, Vector) were used for detection. Slides were then
counterstained with nuclear Fast Red, dehydrated and mounted.
Cell counts and proliferative index measurement
In the skin, an unfurrowed region of epidermis containing no hair follicles
was aligned horizontally along a 300 µm frame at x400 magnification
and all epidermal nuclei were counted. In the colon, crypt heights were
counted only if the crypts could clearly be seen to extend from the base to
the brush border at the lumenal surface. Crypts in cross-section were counted
only if they appeared circular to reduce counting errors due to oblique
sections.
As proliferating cells were not found above the basal layer, the proliferative index (PI) in transverse skin sections was taken to be the ratio of PCNA-positive cells to the total number of cells in the basal layer only. In the crypts, the PCNA index was taken to be the ratio of PCNA positive cells to the total number of cells in the crypt.
The use of cell-cycle-related antigens as a measure of proliferative index
has well-documented pitfalls (Ezaki,
2000). In this study, we have attempted to overcome these
variables by using a standardised immunohistochemical regime throughout, and
by all sections being scored by one person. This has allowed us to measure
relative differences in PCNA staining between genotypes with confidence, but
the data should not be taken as an accurate measure of the actual number of
proliferating cells in the samples.
Apoptotic index measurement
Wax sections (7 µm) from 3-week-old mid-colon samples were processed for
TUNEL staining using the ApopTag Red in situ apoptosis detection kit
(Intergen), according to the manufacturer's instructions. Apoptotic nuclei
were stained with rhodamine, and nuclei were counter-stained with DAPI. As for
crypt height measurement, crypts were only selected for scoring if they
extended longitudinally from base to lumen, with the brush border visible. At
least 50 such crypts were counted from each animal, and the number of
apoptotic nuclei in these crypts was counted. Apoptotic rhodamine signal was
checked for veracity by comparison with the blue (DAPI) channel (to ensure
that a cell nucleus was present) and by comparison with the green channel (to
eliminate false positives due to auto-fluorescence). The apoptotic index was
derived by placing the number of apoptotic nuclei over the total number of
cells scored (number of crypts counted x height x 2; where height
was the number of cells on one side of the crypt, counted from base to top,
and a factor of 2 has been used to estimate the number of cells visible in the
whole crypt).
As with PCNA staining, the limitations of TUNEL staining are well
documented, but largely centre around false positives rather than lack of
sensitivity. The use of sections, rather than examining whole crypts has also
been carefully compared and the errors introduced are fairly consistent
(Potten and Grant, 1998). The
figures given for apoptotic indices should therefore not be considered as true
measures of the levels of cell death, but rather as useful metrics to make
comparisons between genotypes.
Statistical analysis
The Kruskal-Wallis test was applied to data to test for differences between
the three genotypes at each timepoint. This one-way analysis of variance test
does not assume a normal distribution in the data. The level of significance
was set at P<0.05.
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RESULTS |
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Colon
It is already known that IGF2 is ectopically expressed throughout the gut
epithelium of the K:Igf2 mouse, and that the colon becomes larger
than normal (Smith et al.,
2000). It has a higher DNA content, indicating that the difference
is based on the presence of more cells rather than just water or extracellular
material (Ward et al., 1994
).
But it was not known to what extent the increase in cell number was in the
epithelium and to what extent in the connective tissues. Nor was it known how
the increased overall size related to the SPU structure of the epithelium. We
have made a detailed analysis to examine this question.
We measured water content in the present study and found that that the ratio of wet to dry weight was, as expected, constant in the three strains (results not shown). In order to gain a comparative estimate of overall epithelial area, we measured the total lumenal area of the colon of knockout, wild-type and K:Igf2 strains during the rapid growth phase of 2-4 weeks postnatal. The results show a substantial effect of IGF2 dose. By 4 weeks of age, the K:Igf2 colon area is almost double that of the knockout (Fig. 2A). In the case of the knockout to wild type difference, this is consistent with the overall size of the animals. In the case of the wild type to K:Igf2 difference, it arises from a disproportionate growth of the colon relative to the whole body, with the proximal colon showing a relatively greater increase in surface area.
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Does this difference arise from larger crypts, more crypts, or a mixture of both effects? To answer this, we examined the size of crypts. In this context it is the size in the plane of the epithelium that is important and, as we did not see any indication of a change in cell size, this was measured by counting the number of cells around the circumference of the crypts. The results showed rather little difference: the K:Igf2 crypt circumference was a little larger than the others at 2 weeks but this difference was lost by 12 weeks (data not shown). This shows that the increase of area with IGF2 dose is not due to an increase in crypt size. It might, in principle, be due to an increase in crypt number, or to a larger amount of space between crypts. In order to resolve this, the total number of crypts was counted as described in the Materials and Methods. Comparison of these results with the area measurement shows that indeed virtually all of the area increase is attributable to an increase in crypt number (Fig. 2B).
Of course, crypt size may change in the other axis, perpendicular to the surface. To examine this, the crypt heights were measured for the three strains and the results are shown in Fig. 2C. There is an increase in crypt height with time for all strains, and also an effect of IGF2 dose which is much stronger at 2 weeks than at 12 weeks. At 2 weeks, the K:Igf2 mice show crypt heights almost double the knockouts, while by 12 weeks all the crypts have increased in height and the difference between K:Igf2 and the others is only about 15%.
So driving extra cell production with IGF2 does increase the crypt size,
but only temporarily, and not in the axis that will affect the total area of
the epithelium. It is known that the crypt number increases during normal
development by binary fission, with the division commencing at the crypt base
(Maskens, 1978;
Cheng and Bjerknes, 1985
;
Bjerknes, 1986
)
(Fig. 3). We measured the
fission index during the critical growth phase and found the overall mean
index to be 8.7%, with no significant difference between the three strains at
any of the time points examined (Fig.
4A). There may be a reduction in the index after 2 weeks, but as
this is a difficult parameter to measure accurately we cannot be confident
about this. If the log of crypt number is plotted against time
(Fig. 2B), only the
Igf2 knockout data produce a straight line, suggesting uniform
exponential growth. In this case the doubling time is 12 days, so the 8.7%
fission index would lead to an approximate estimate of fission duration of
1.04 days or 25 hours. For the other two strains there is no straight line
relationship: as may be seen in Fig.
2B, the crypt numbers increase at a faster rate than exponential,
indicating that the presence of IGF2 accelerates the crypt fission rate during
the rapid growth phase. For the 3-4 week interval the doubling time is about 7
days for both strains, and so the 8.7% mean fission index would predict a
duration of fission of approximately 0.61 days or 15 hours. These estimates
are not very accurate because of the variation in crypt fission index between
individuals and indeed ANCOVA analysis of the slopes of the semi-log plots do
not show significant differences (P>0.05). However the indication
is that the presence of IGF2 drives faster crypt fission by reducing the time
taken for each crypt to divide.
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Crypt fission must be driven by an increase in cell numbers, although cell
production feeds the turnover of the epithelium, as well as increases in size.
Fig. 5A-D shows the PCNA
labelling pattern of the three strains, and in
Fig. 4B a graphical
representation of the PCNA index at 2 weeks and 12 weeks. It is exceedingly
difficult to calculate absolute net cell production rates for epithelia in
situ (Wright and Alison, 1984)
and we have not attempted to do this. But it is likely from the direct
comparison of the PCNA indices that K:Igf2 is producing more cells
than the other two strains, and that the difference is present both at 2
weeks, during rapid growth of the organism, and at 12 weeks, when growth is
slowing down. So the rate of crypt number growth increases from knockout to
wild type, while the proliferative index increases from wild type to
K:Igf2. This suggests that the main effect of the normal IGF2 level
may be to suppress cell death, while an increase above normal increases
overall cell turnover.
|
Apoptosis in colonic crypts was measured by TUNEL staining and compared
between K:Igf2 and wild-type mice at three weeks of age
(Fig. 5E,F). Igf2
knockout crypts were excluded from this analysis because knockout crypts were
indistinguishable from those of wild-type animals, both in terms of height
(Fig. 2C) and proliferation
indices (Fig. 5A-D). For
K:Igf2 mice (n=5), the mean apoptotic index was 0.24%
(range, 0.06-0.47%), and for wild type (n=3), the mean apoptotic
index was 0.34% (range, 0.17-0.46%). Thus, there is no significant difference
in the steady-state level of apoptosis between the two genotypes. This should
be considered in light of the fact that the baseline level of apoptosis in the
large intestine is very low, and that our figures fall within the range given
for previously published data (Potten,
1998; Potten and Grant,
1998
). It should be further borne in mind that apoptotic cell
clearance may occur in less than 2 hours, and that very small (and hence
difficult to measure) shifts in the apoptotic index could result in large
changes in cell flux.
In conclusion, it is clear that IGF2 is not necessary to maintain the colonic crypt structure because the organisation is normal in the Igf2 knockout mouse. But an increasing IGF2 dose does drive more cell division in the crypts. Some of the new cells end up contributing to new growth, as opposed to cell turnover, and this takes place by reducing the duration of crypt fission such that the number of crypts, and thus the total area of the epithelium increases.
Skin
The K:Igf2 mouse has the IGF2 gene driven by a bovine keratin
10 promoter that is active in the suprabasal layers of keratinocytes
(Smith et al., 2000). Although
the excess IGF2 is not expressed in the basal layer itself, the protein
evidently reaches the basal layer as in all three strains of mice this is the
only layer containing dividing cells. There is clearly an increase in total
skin area with IGF2 dose. For knockout and wild type the increase is
proportional to the surface area of the animal. For the K:Igf2, the
mice are normal size but the skin is distinctly wrinkled
(Ward et al., 1994
). This
means that the skin is definitely increased in area but it is not possible to
make an accurate quantitative estimate.
The main difference between the three strains is that the total skin thickness is greatly increased in the K:Igf2s. This is apparent both from the histological sections (Fig. 6) and from the total epidermal cell counts (Fig. 7A). The PCNA labelling shows that dividing cells are confined to the basal layer in all three strains (Fig. 6). There is some tendency for the labelling index to be higher with IGF2 dose (Fig. 7B), although clear statistical significance is only reached by the 2 week data, corresponding to the maximum growth rate of the organism. So, as in the colon, the cell division is confined to the normal location, and there is some increase in height of the SPUs. Because it is not possible to visualise the SPUs, we cannot be sure that the number has increased, although this would be consistent with the increase of overall skin area.
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The number of cells in the basal layer were counted along a fixed distance for all three genotypes as the denominator for the PCNA index and showed no significant difference (data not shown). This supports a lack of change in cell size with IGF2 dosage.
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DISCUSSION |
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We were interested to know whether these changes in cell division affected
the growth of the organ or simply drove cells through their differentiation
pathway at a different rate. The results show that in both colon and skin
there is an effect on overall growth. In this context, it is appropriate to
consider what fraction of cell production is feeding growth compared with that
feeding renewal. In the case of the colon this may be estimated from published
data (Wright and Alison,
1984). The figures show some variation with position, but, as the
crypts are smaller in regions of lower cell production, the estimated cell
number doubling (i.e. turnover) times do not vary greatly and are in the range
1.5-3.3 days, with a mean of 2.4 days. There is little effect of age of the
mouse up to 70 days postnatal. For our wild-type mice, the minimum time for
crypt number to double is about 7 days at 3 weeks postnatal, indicating that
the normal situation involves about one third of cell production being devoted
to growth and two thirds to turnover. On the basis of the PCNA index, the cell
division rate appears similar in Igf2 knockout and wild type but the
crypt number doubling time falls from 12 days to a minimum of 7 days,
indicating a shift from about 20% to 30% of cell production devoted to growth.
This shift could be due to a suppression of cell death as this is a function
previously attributed to IGF2 in both normal and hyperplastic growth states
(Christofori et al., 1995
;
Burns and Hassan, 2001
). In our
analysis of wild-type and K:Igf2 mice, no significant change in
apoptotic index was detected, but it should be borne in mind that small
changes in the rate of apoptosis/cell survival could contribute significantly
to tissue growth. From wild type to K:Igf2, the crypt number growth
rate is only slightly increased, while the PCNA index increases significantly,
suggesting that most of the additional cell division goes to turnover. The
small increment of net production devoted to growth does, however, produce a
considerable cumulative effect over the entire growth phase of the mouse.
Next, we were interested in whether a growth effect arises from a change in size of SPUs, or in the total number of SPUs, or some combination of both. Our results on the colon clearly show that the effect of increased IGF2 is to increase the number of SPUs. There is an increase in crypt length during the maximal growth period, but it is not permanent and does not affect the packing density of crypts in the plane of the epithelium. The number of crypts depends on the rate of crypt fission, and we found that this is accelerated by IGF2 with the main effect being to reduce the duration of crypt fission. The supra-exponential rate of crypt fission is easy to explain under conditions where the majority of the cell production is feeding renewal rather than growth. A small increase in the net proportion devoted to growth will increase the rate of crypt division above the exponential and also shorten the fission time, as observed.
There have been previous studies on the effects of increased cell
production on crypt fission
(Berlanga-Acosta et al., 2001;
Park et al., 1997
). Treatment
with epidermal growth factor (EGF) increases the labelling index in a similar
way to IGF2, but this does not lead to a sustained increase in crypt numbers.
Instead there is an early rise and a later fall in crypt fission. Treatment
with the mutagen dimethylhydrazine has marginal effects on measured rate of
cell production but does increase the fission index, possibly as a
regenerative response to tissue damage. These studies concur in identifying
the control of crypt division as being separate from the simple production of
cells. We also note that a recent study on human colorectal adenomas found an
increase in crypt fission, as well as increased crypt height and an expansion
of the proliferative zone, precisely the features found in K:Igf2
colon (Wong et al., 2002
).
This is of interest, as many colorectal cancers frequently show loss of
imprinting (and concomitant overexpression) of IGF2, and IGF2 is the most
abundant mRNA overexpressed in colorectal cell lines and tumours
(Zhang et al., 1997
). The
ApcMin/+ mutant has a genetic tendency to
develop multiple intestinal adenomas. When the K:Igf2 transgenic line
used in this study was crossed to the ApcMin/+
mutant, the frequency of colon adenomas was increased 10-fold
(Hassan and Howell, 2000
).
Furthermore, Bjerknes and Cheng have noted the presence of elongated crypts in
the `transitional epithelium' adjacent to adenomas
(Bjerknes and Cheng, 1999
).
This is of interest, as the authors suggest that the adenomas may secrete
epithelial trophic factors acting in a paracrine manner to trigger adjacent
wild-type crypt enlargement. It is tempting to speculate that IGF2 might be
one such trophic factor.
Another aspect of the results is the recognised regional difference in cell
kinetics in the colon (Wright and Alison,
1984). We did observe the greatest increase in surface area in the
proximal colon, and this is consistent with the effect observed in rat colon
when ectopic EGF was administered, suggesting a proximodistal gradient of
sensitivity of crypt fission to growth factor stimulation
(Ribbons et al., 1994
;
Park et al., 1997
).
It is often not realised that the epidermis of the skin is organised into
SPUs. This concept was first advanced by Potten, who studied the cell renewal
patterns of epidermis on the dorsal surface of the mouse and proposed that a
single stem cell fed each proliferative unit
(Potten, 1974). This
conception was developed by work of Jones and Watt, who found that stem cells
could be identified by integrin immunostaining
(Jones et al., 1995
).
Subsequently, it was supported by cell labelling studies. Mackenzie
(Mackenzie, 1997
) showed that
single lacZ-labelled epidermal stem cells would feed a single
columnar unit within the epidermis. Likewise, Zhang et al.
(Zhang et al., 2001
) showed
that UV-induced p53 mutant cells will rapidly fill up individual SPUs.
Although all these studies support the idea of SPUs fed by a single stem cell,
the structure is not in general as regular as that originally described by
Potten (see also the irregularity of patches in our
Fig. 1B). This means that in
the skin we have no simple way of measuring the size of SPUs. However, our
data are still consistent with the idea that the mechanism is the same as in
the colon, and that excess IGF2 drives a slight increase in the number of
cells contributing to growth, which leads to an increase in the number of SPUs
and thereby increases the overall area of the skin.
Comparison with Drosophila
The most detailed studies of the effects of the insulin/IGF signalling
pathway on growth have previously been made in Drosophila.
Hypomorphic mutation of components of the pathway will decrease overall size,
while overexpression will increase overall size. However there is an important
difference between the situation in Drosophila and in mice, which is
that in Drosophila a significant component of the effect is on cell
size as well as cell number (Stocker and
Hafen, 2000). In our case, the difference between the knockout and
the wild type is one of SPU number, and therefore also cell number, with
conservation of normal cell size. Cell size has also been shown to be normal
in the small mouse embryos having Igf2-paternal null genotype
(Burns and Hassan, 2001
). A
further difference concerns the issue of disproportion arising from regional
differences in signalling activity. In Drosophila, a local increase
of signalling in imaginal discs, induced by mitotic recombination, yields
clones that themselves may be large and have large cells, but do not affect
the overall proportions of the imaginal structure or of the fly as a whole. In
our K:Igf2 mice, a local increase in signalling does cause a local
and disproportionate increase in size. The reasons for these differences will
require further investigation, but one possibility is that they arise from an
absence of an SPU-type of organisation in the imaginal discs.
Because of the general lack of histological analysis and cell kinetic
studies of invertebrates, it is hard to say at present whether SPUs are
widespread in the animal kingdom or are a specific vertebrate characteristic.
Interestingly, vertebrate SPUs are somewhat comparable with the polyps of
colonial invertebrates such as hydroids, bryozoa or ascidians
(Brusca and Brusca, 1990). Like
colonial invertebrates, SPUs can produce new SPUs by budding. It is clear from
the present study, as well as others, that budding is not simply a consequence
of increased cell production. There must be a level of control that determines
whether new cells will feed SPU multiplication or increased cell turnover. The
nature of this control remains entirely mysterious. One possibility that has
been suggested for small intestinal crypts is that the crucial determinant is
the number of stem cells, rather than the total number of dividing cells
(Loeffler et al., 1997
). If
so, this once again points to the factors controlling stem cell identity as a
key issue in the control of growth, development and size.
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ACKNOWLEDGMENTS |
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REFERENCES |
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---|
Berlanga-Acosta, J., Playford, R. J., Mandir, N. and Goodlad, R.
A. (2001). Gastrointestinal cell proliferation and crypt
fission are separate but complementary means of increasing tissue mass
following infusion of epidermal growth factor in rats.
Gut 48,803
-807.
Bjerknes, M. (1986). A test of the stochastic theory of stem cell differerentiation. Biophys. J. 49,1223 -1227.[Abstract]
Bjerknes, M. and Cheng, H. (1999). Colossal
crypts bordering colon adenomas in Apc(Min) mice express full-length Apc.
Am. J. Pathol. 154,1831
-1834.
Brusca, R. C. and Brusca, G. J. (1990). Invertebrates. Sunderland, MA: Sinauer Associates Inc.
Burns, J. L. and Hassan, A. B. (2001). Cell
survival and proliferation are modified by insulin-like growth factor 2
between days 9 and 10 of mouse gestation. Development
128,3819
-3830.
Cairnie, A. B. and Millen, B. H. (1975). Fission of crypts in the small intestine of the irradiated mouse. Cell Tiss. Kinet. 8,189 -196.[Medline]
Canter, C. E. and Goss, R. J. (1975). Induction of extra nephrons in unilaterally nephrectomized immature rats. Proc. Soc. Exp. Biol. Med. 148,294 -296.[Abstract]
Cheng, H. and Bjerknes, M. (1985). Whole population cell kinetics and postnatal development of the mouse intestinal epithelium. Anat. Rec. 211,420 -426.[Medline]
Christofori, G., Naik, P. and Hanahan, D. (1995). Deregulation of both imprinted and expressed alleles of the insulin-like growth factor 2 gene during ß-cell tumorigenesis. Nat. Genet, 10:196 -201.[Medline]
De Chiara, T., Efstratiadis, A. and Robertson, E. (1990). A growth-deficiency phenotype in heterozygous mice carrying an insulin-like growth factor-ii gene disrupted by targeting. Nature 345,78 -80.[CrossRef][Medline]
Efstratiadis, A. (1998). Genetics of mouse growth. Int. J. Dev. Biol. 42,955 -976.[Medline]
Ezaki, T. (2000). Antigen retrieval on formaldehyde-fixed paraffin sections: its potential drawbacks and optimization for double immunostaining. Micron 31,639 -649.[CrossRef][Medline]
Goodlad, R. A. (1994). Microdissection-based techniques for the determination of cell proliferation in gastrointestinal epithelium: application to animal and human studies. In Cell Biology: A Laboratory Handbook. (ed. J. E. Celis), pp.205 -216. London: Academic Press.
Gordon, J. I., Schmidt, G. H. and Roth, K. A.
(1992). Studies of intestinal stem cells using normal, chimeric
and transgenic mice. FASEB J.
6,3039
-3050.
Goss, R. J. (1972). Regulation of Organ and Tissue Growth. New York, London: Academic Press.
Hassan, A. B. and Howell, J. A. (2000).
Insulin-like growth factor II supply modifies growth of intestinal adenoma in
Apc(Min/+) mice. Cancer Res.
60,1070
-1076.
Jiang, X., Hitchcock, A., Bryan, E. J., Watson, R. H., Englefield, P., Thomas, E. J. and Campbell, I. G. (1996). Microsatellite analysis of endometriosis reveals loss of heterozygosity at candidate ovarian tumor suppressor gene loci. Cancer Res. 56,3534 -3539.[Abstract]
Jones, P. H., Harper, S. and Watt, F. M. (1995). Stem cell patterning and fate in human epidermis. Cell 80,83 -93.[Medline]
Leevers, S. J. (2001). Growth control: invertebrate surprises. Curr. Biol. 11,R209 -R212.[CrossRef][Medline]
Loeffler, M., Bratke, T., Paulus, U., Li, Y. Q. and Potten, C. S. (1997). Clonality and life cycles of intestinal crypts explained by a state dependent model of epithelial stem cell organization. J. Theor. Biol. 186,41 -54.[CrossRef][Medline]
Lyon, M. F. (1961). Gene action in the X chromosome of the mouse (Mus Musculus L.). Nature 190,370 -373.
Mackenzie, I. C. (1997). Retroviral transduction of murine epidermal stem cells demonstrates clonal units of epidermal structure. J. Invest. Dermatol. 109,377 -383.[Abstract]
Maskens, A. P. (1978). Histogenesis of colon glands during postnatal growth. Acta Anatomica 100, 17-26.[Medline]
Oldham, S., Bohni, R., Stocker, H., Brogiolo, W. and Hafen, E. (2000). Genetic control of size in Drosophila. Philos. Trans. R. Soc. 355,945 -952.[CrossRef]
Park, H. S., Goodlad, R. A., Ahnen, D. J., Winnett, A., Sasieni, P., Lee, C. Y. and Wright, N. A. (1997). Effects of epidermal growth factor and dimethylhydrazine on crypt size, cell proliferation, and crypt fission in the rat colon. Am. J. Pathol. 151,843 -852.[Abstract]
Petrik, J., Pell, J. M., Arany, E., McDonald, T. J., Dean, W.
L., Reik, W. and Hill, D. J. (1999). Overexpression of
insulin-like growth factor-II in transgenic mice is associated with pancreatic
islet cell hyperplasia. Endocrinology
140,2353
-2363.
Ponder, B. A. J., Schmidt, G. H., Wilkinson, M. M., Wood, M. J., Monk, M. and Reid, A. (1985). Derivation of mouse intestinal crypts from single progenitor cells. Nature 313,689 -691.[Medline]
Potten, C. S. (1974). The epidermal proliferative unit: the possible role of the central basal cell. Cell Tiss. Kinet. 1,77 -88.
Potten, C. S. (1978). Epithelial proliferative subpopulations. In Stem Cells and Tissue Homeostasis (ed. B. I. Lord, C. S. Potten and R. J. Cole), pp.317 -334. Cambridge: Cambridge University Press.
Potten, C. S. (1998). Stem cells in gastrointestinal epithelium: numbers, characteristics and death. Philos. Trans. R. Soc. Lond. Ser. B Biol. Sci. 353,821 -830.[CrossRef][Medline]
Potten, C. S. and Grant, H. K. (1998). The relationship between ionizing radiation-induced apoptosis and stem cells in the small and large intestine. Br. J. Cancer 78,993 -1003.[Medline]
Raff, M. C. (1996). Size control: the regulation of cell numbers in animal development. Cell 86,173 -175.[Medline]
Ribbons, K. A., Howarth, G. S., Davey, K. B., Georgenascimento, C. and Read, L. C. (1994). Subcutaneous but not intraluminal epidermal growth-factor stimulates colonic growth in normal adult-rats. Growth Factors 10,153 -162.[Medline]
Schmidt, G. H., Winton, D. J. and Ponder, B. A. J. (1988). Development of the pattern of cell renewal in the crypt-villus unit of chimaeric mouse small intestine. Development 103,785 -790.[Abstract]
Slack, J. M. W. (1996). The mysterious mechanism of growth. Curr. Biol. 6, 348.[Medline]
Smith, J., Goldsmith, C., Ward, A. and LeDieu, R. (2000). IGF-II ameliorates the dystrophic phenotype and coordinately down-regulates programmed cell death. Cell Death Diff. 7,1109 -1118.[CrossRef][Medline]
Stocker, H. and Hafen, E. (2000). Genetic control of cell size. Curr. Opin. Genet. Dev. 10,529 -535.[CrossRef][Medline]
Tan, S. S., Williams, E. A. and Tam, P. P. L. (1993). X-chromosome inactivation occurs at different times in different tissues of the post implantation mouse embryo. Nat. Genet. 3,170 -174.[Medline]
Tsai, Y. C., Lu, Y., W, N. P., Zlotnikov, G., Jones, P. A. and Smith, H. S. (1996). Contiguous patches of normal human mammary epithelium derived from a single stem cell: Implications for breast carcinogenesis. Cancer Res. 56,402 -404.[Abstract]
Ward, A. (1997). Beckwith-Wiedemann syndrome and Wilms' tumour. Mol. Hum. Reprod. 3, 157-168.[Abstract]
Ward, A., Bates, P., Fisher, R., Richardson, L. and Graham, C.
F. (1994). Disproportionate growth in mice with Igf-2
transgenes. Proc. Natl. Acad. Sci. USA
91,10365
-10369.
Ward, A., Fisher, R., Richardson, L., Pooler, J.-A., Squire, S., Bates, P., Shaposhnikov, R., Hayward, N., Thurston, M. and Graham, C. F. (1997). Genomic regions regulating imprinting and insulin-like growth factor-II promoter 3 activity in transgenics: novel enhancer and silencer elements. Genes Funct. 1, 25-36.[Medline]
Watt, F. M. (1998). Epidermal stem cells: markers, patterning and the control of stem cell fate. Phil. Trans. R. Soc. B 353,831 -837.[CrossRef]
Weinberg, W. C., Howard, J. C. and Iannaccone, P. (1985). Histological demonstration of mosaicism in a series of chimeric rats produced between congenic strains. Science 227,524 -527.[Medline]
Winton, D. J., Blount, M. A. and Ponder, B. A. J. (1988). A clonal marker induced by mutation in mouse intestinal epithelium. Nature 333,463 -466.[CrossRef][Medline]
Wong, W. M., Mandir, N., Goodlad, R. A., Wong, B. C. Y., Garcia,
S. B., Lam, S. K. and Wright, N. A. (2002). Histogenesis of
human colorectal adenomas and hyperplastic polyps: the role of cell
proliferation and crypt fission. Gut
50,212
-217.
Wright, N. A. and Alison, M. (1984). The Biology of Epithelial Cell Populations, Vols1 and 2 . Oxford: Clarendon Press.
Zhang, L., Zhou, W., Velculescu, V. E., Kern, S. E., Hruban, R.
H., Hamilton, S. R., Vogelstein, B. and Kinzler, K. W.
(1997). Gene expression profiles in normal and cancer cells.
Science 276,1268
-1272.
Zhang, W. G., Remenyik, E., Zelterman, D., Brash, D. E. and
Wikonkal, N. M. (2001). Escaping the stem cell compartment:
Sustained UVB exposure allows p53-mutant keratinocytes to colonize adjacent
epidermal proliferating units without incurring additional mutations.
Proc. Natl. Acad. Sci. USA
98,13948
-13953.