Fauser, Jane K.*; Donato, Rino P.‡; Woenig, Joshua A.‡; Proctor, Simon J.§; Trotta, Andrew P.‡; Grover, Phulwinder K.‡; Howarth, Gordon S.*; Penttila, Irmeli A.||; Cummins, Adrian G.‡
*School of Animal and Veterinary Sciences, Roseworthy Campus, University of Adelaide
‡Department of Gastroenterology and Hepatology, The Queen Elizabeth Hospital, Woodville South
§Discipline of Medicine, School of Medicine, University of Adelaide
||Women's and Children's Health Research Institute, North Adelaide, South Australia, Australia.
Address correspondence and reprint requests to Ms Jane K. Fauser, University of Adelaide, School of Animal and Veterinary Sciences, Roseworthy Campus, 5371 South Australia, Australia (e-mail: email@example.com).
Received 5 August, 2011
Accepted 14 December, 2011
The present study was supported by the National Health and Medical Research Council of Australia 508024 project grant to I.A.P. and A.G.C. Parts of this work were submitted by J.K.F. as a thesis in support for a PhD to the University of Adelaide. G.S.H. was supported by the Sally Birch Cancer Council Australia Senior Research Fellowship in Cancer Control.
The authors report no conflicts of interest.
Intestinal crypt fission is a process of longitudinal duplication of crypts in both the small and large intestines that starts in embryonic life in humans (1,2) but postnatally in laboratory rats (3,4). Crypt fission is best characterised in juvenile polyposis and familial adenomatous polyposis syndromes (5–8), in sporadic intestinal adenomas, and in colorectal cancer (9,10), and experimentally as crypt fission during culture of isolated crypt cells as organoids (11,12). Crypt fission peaks from days 11 to 17 in the laboratory rat (4) and from 6 to 12 months in humans (13,14). Mathematical modelling proposes that crypt fission is driven by a 2-fold increase in stem cells (15).
Intestinal stem cells are located not only as crypt base columnar cells (CBCs) in the base of the small intestine but also as +4 stem cells located from cell positions 3 to 7 from the crypt base (16–18). CBCs are dependent on contact with Paneth cells (19,20). They express the Lgr5 stem cell marker (17). The +4 stem cells have lost contact with Paneth cells, are long-lived, and express the Bmi-1 stem cell marker (21,22). Clevers et al advocate that CBCs are primary stem cells (23), whereas Potten et al (18) see the +4 stem cells as the primary stem cell pool.
The Wnt/β-catenin signalling pathway is implicated principally in mediating crypt fission in familial polyposis coli syndrome (7–9). The Wnt/β-catenin pathway subserves several roles in the gastrointestinal tract by directing morphogenesis in early embryonic life, by directing migration of Paneth cells to the base of crypts of the small intestine, by promoting intestinal stem cells, and in maintaining crypt homeostasis throughout postnatal life (23–25). Disruption of Wnt signalling by loss of function of the nuclear Tcf-4 transcription factor or by transgenic overexpression of the canonical Wnt inhibitor, dickkopf, results in disorganised and rudimentary crypts in genetically engineered mice (26–28). Activation of β-catenin by Wnt ligands results in an increase in phosphorylated cytoplasmic β-catenin, translocation to the nucleus, and transcription of Wnt target genes via Tcf-4 transcription factor (23). We have shown that cytoplasmic and nuclear β-catenin peaks in the stem cell region of intestinal crypts in 14-day-old rats (29), which indicates the Wnt/β-catenin signalling is active at this age.
The purpose of the present study was to investigate the effect of Wnt blockade on intestinal crypt fission and on intestinal growth. We also examined changes in expression of Lgr5 and Bmi-1 stem cell markers of CBC and +4 position stem cells, respectively. We used intraperitoneal (IP) administration of recombinant dickkof-1 to postnatal infant rats in which intestinal morphogenesis was essentially complete. The effect on crypt fission and morphometric measures of intestinal growth was examined. Treatment was limited to 5 days and was not intended to completely abrogate Wnt signalling, and a dose-response effect was sought.
Animals and Experimental Design
Members of 3 litters of Hooded Wistar rat pups at 11 days old were randomly allocated by marking the tail into 2 equal experimental and control groups of 14 rat pups for the first study with dickkopf at a dose of 30 ng/day IP. The day of birth was designated day 0. Pups remained with their dams throughout the study. This design was then repeated with 2 groups of 11 rat pups for a second study with dickkopf at a dose of 100 ng/day IP. Experimental rat pups were treated daily IP with recombinant mouse dickkopf (R&D Systems, Minneapolis, MN) in 1% bovine serum albumin (BSA) (Sigma-Aldrich, Castle Hill, Australia) IP daily in phosphate buffer saline (PBS) from days 11 to 15. Control rats were given an equivalent volume of 1% BSA in PBS. Mouse dickkopf has 98% amino acid homology to rat dickkopf (R&D Systems). Rats were killed on day 16 using isoflurane anaesthesia and cervical dislocation. The abdomen was opened and 1-cm segments were obtained from the proximal one-third of the length of the small intestine. One segment was opened longitudinally on cardboard and placed in Clarke fixative (75% v/v absolute ethanol: 25% v/v glacial acetic acid) with brief shaking to remove mucus and to straighten villi. After 16 to 24 hours, they were stored in 70% v/v ethanol. Intestinal segments were used to assess intestinal morphometry and crypt fission. A second segment of intestine was placed in neutral buffered formalin for immunostaining of histological sections. A third segment was placed in RNAlater for RNA extraction (Ambion; Applied Biosystems, Scoresby, Australia).
Assessment of Intestinal Morphometry and Crypt Fission in Crypts
A tissue microdissection technique was performed, as described previously, to assess the intestinal tissue fixed in Clarke fixative overnight and stored in 70% ethanol (30–32). This method provides absolute values of villous area and crypt area that are expressed per appropriate whole villus and crypt anatomical units, as well as crypt fission index, and crypt mitotic count per crypt. The number of the bifid crypts was counted as a percentage of 100 crypts to obtain the percentage of crypt fission index. Means of 15 mitotic figures were recorded.
RNA Isolation and Preparation of cDNA
RNA isolation was undertaken using an RNeasy Lipid Tissue Mini Kit (QAGEN, Doncaster, Australia) with approximately 100-mg samples of tissue. RNA was quantified using a NanoDrop ND-1000 Spectrophotometer (Nanodrop Products, Thermo Fisher Scientific, Wilmington, DE). One microgram RNA was reverse transcribed into cDNA using the QuantiTect reverse transcription kit (QIAGEN, Melbourne, Australia).
Lgr5 and Bmi-1 Polymerase Chain Reaction
Primers for rat Lgr5, Bmi-1, and β-actin cDNA were designed in-house using Primer Design (Premier Biosoft International, Palo Alto, CA) and synthesised commercially (Geneworks, Adelaide, Australia). Quantitative polymerase chain reaction (qPCR) was undertaken using an Opticon real-time thermal cycler (MJ Research, Bio-Rad Laboratories, Minneapolis, MN). All of the reactions were carried out in a 25-μL volume containing 2.4-μL cDNA, forward and reverse primers (Table 1), in 1 × Power SYBR Green PCR Master Mix (Applied Biosytems, Mulgrave, Australia): Taq activated for 10 minutes at 95°C; cDNA denatured at 95°C for 30 seconds, Lgr5 annealed at 64°C, and Bmi-1 at 60°C (β-actin at both temperatures) for 10 seconds and extended at 72°C for 30 seconds, for 35 cycles. A melt curve analysis was performed to exclude nonspecific products. The PCR reaction was optimised for equal amplification efficacy over a range of cDNA concentrations. Gene expression was calculated using the relative ΔΔCt method normalised to β-actin, which we have previously shown is a stable housekeeping gene (4).
Expression of β-Catenin Protein Expression by Immunostaining
Protein expression of β-catenin was assessed by immunostaining. Histological paraffin sections were cut at 4 μm, dewaxed, and rehydrated through an ethanol series to water. Sections on slides were heated in citrate buffer (pH 6.0) at 100°C for 20 minutes for antigen retrieval and incubated with 10% horse serum/1% BSA for 30 minutes to block nonspecific staining. Mouse mononclonal IgG1 anti-β-catenin antibody (Sc-99737; Santa Cruz Biotechnology, Santa Cruz, CA) was applied at 1:1000 in 10% horse serum/1% BSA in PBS and incubated overnight at 4°C. Bound antibody was detected by anti-mouse/rabbit polymer peroxidase and revealed by diaminobenzidine chromogen (Novocastra, Leica Microsystems Pty Ltd, Sydney, Australia). Crypts were blindly scored as either positive or negative for β-catenin.
Means of groups were compared by an unpaired, 2-tailed, Student t test using GraphPad Prism 5.01 for Windows (GraphPad Software, San Diego, CA) software program and significance was determined at P < 0.05.
The study was approved by the animal ethics committees of the North Adelaide Health Service/Institute of Medical and Veterinary Science and of the University of Adelaide. The work was carried out according to the Code of Practice for Use of Animals in Research and Teaching of the National Health and Medical Research Council of Australia.
Effect of 30 ng/day Dickkopf on Intestinal Morphometry, Crypt Fission, and Crypt Cell Proliferation
The effect of 30 ng/day dickkopf on intestinal morphometry is shown in Figure 1. Mean villus and crypt areas were reduced to 71% and 42%, respectively, of control values. The effect of 30 ng/day of dickkopf on percentage of crypt fission and mitotic count per crypts is given in Figure 2. Crypt fission was reduced to 51% of control values, but crypt cell proliferation did not change.
Effect of 100 ng/day of Dickkopf on Intestinal Morphometry, Crypt Fission, and Crypt Cell Proliferation
The effect of 100 ng/day of dickkopf on intestinal morphometry is given in Figure 3. Villous and crypt areas were reduced to 29% and 30%, respectively, of control values. The effect of 100 ng/day dickkopf on the percentage of crypt fission and mitotic count per crypt is given in Figure 4. Crypt fission was reduced to 29% of control values, but crypt cell proliferation did not change.
Effect of Dickkopf on Lgr5 and Bmi-1 RNA Expression
The effect of 100 ng/day of dickkopf on Lgr5 RNA expression is given in Figure 5. Dickkopf reduced the mean of Lgr5 RNA expression to 37% of that of control. Bmi-1 expression was unchanged between treatment and control groups (mean + standard error of the mean, 0.0066 + 0.0020 vs 0.0060 + 0.0013, P = not significant).
Effect of Dickkopf on β-Catenin–positive Crypts
Figure 6 compares representative micrographs of β-catenin staining in the small intestines of control and dickkopf-treated animals. Dickkopf reduced β-catenin staining markedly in a dichotomous manner. Crypts were scored subsequently blindly as showing positive or negative staining and results are given in Figure 7. Dickkopf reduced the mean percentage of positive crypts to 26% of control values.
The present study investigated dickkopf blockade of endogenous canonical Wnt signaling in infant rats at an age when crypt fission has a broad physiological peak, ranging from 11 to 17 days of life (4). Unlike another study using genetically engineered mice that inhibit Wnt signaling from conception (27), dickkopf was given for 5 days at a postnatal age when intestinal morphogenesis is complete. The principal findings were that dickkopf reduced crypt fission and inhibited intestinal mucosal growth as shown by reduced villous and crypt areas, whilst crypt cell proliferation remained unaffected. The efficacy of dickkopf treatment was shown by decreased β-catenin expression in crypts.
There was a dose effect of dickkopf in reducing crypt fission, because the 100 ng/day dose was more effective than the 30-ng/day dose in reducing crypt fission (29% vs 51%, respectively, of control values). The absolute control values for crypt fission of the 30- and 100-ng/day studies differed because the exact ages of each litter could differ up to 12 to 24 hours. This is the reason why we divided members of each litter into equal numbers of experimental and control animals to mitigate any bias. Dickkopf reduced crypt fission, which would reduce the number of crypts supplying each villus with cells. At day 14 of life, about 1.5 crypts normally populate a villus with cells compared with 3.5 at day 72 (A.G. Cummins, unpublished data, 2010) so that, for example, a reduction in up to half of the crypts would be expected to halve villus area. In fact, we found a reduction to 71% and 29% of villous area compared with control animals at doses of 30 and 100 ng/day of dickkopf.
A surprising finding of this study was that crypt cell proliferation was unaffected by dickkopf, and therefore this would not have contributed to the reduction in villous area that was observed. Mitotic activity in crypts is centered on the progeny of stem cells, in other words, the transit-amplifying cells that undergo 4 to 5 cell divisions in the crypts of adult animals. In our study, mitotic activity was low, as we have observed previously and reported on at this age (4), and crypts are small, which indicates that there are few transit-amplifying cells. Cell proliferation increased slowly at first and then increased exponentially after approximately day 21 before plateauing to adult values.
This raises the issue of how the stem cells increase, because crypt cells (including stem cells) have low mitotic activity at this age. The assumption that the Wnt ligands work physiologically at inducing proliferation may not be accurate. This is in spite of the pharmacological action of R-spondin1, an agonist of the Lgr5 receptor in vivo or of studies of intestinal stem cell growth with R-spondin1, jagged 1 of the Notch pathway, and epidermal growth factor (11,12). It is difficult to detect any significant increase in intestinal stem cells in rodents in the first few weeks of life, even though mRNA expression of stem cell markers such as Lgr5 peaks at day 14 (33); however, an increase in stem cells could result from increased proliferation, which was not evident on mitotic counts. Further studies focusing on the potential contribution of apoptosis to intestinal stem cell dynamics are therefore warranted. Thus, crypt fission could be initiated by increased survival of stem cells. The concern of Clevers et al that CBC stem cells could have multiple cell division exceeding the Hayflick limit may not be realised (19).
It remains difficult to assess Lgr5 stem cells because antibodies for Lgr5 only reliably work in humans and not in the rat, and because Lgr5 has a low copy number. Studies of Lgr5 cells always use genetically labelled mice. We used RNA expression of Lgr5 in gut homogenates as a surrogate marker of stem cells; furthermore, we have demonstrated that dickkopf reduced Lgr5 RNA expression, but did not change Bmi-1 expression. We are continuing to attempt to enumerate CBC stem cells in infant rats.
In contrast to the studies using transgenic overexpression of dickkopf (27,28), we did not observe severe disruption of crypt architecture, and in particular, no reduction in crypt mitotic count was seen, which contrasts with data from transgenic dickkopf blockade (27,28). We attribute reduction of crypt cell proliferation in transgenic dickkopf studies to crypt destruction rather than a direct effect of dickkopf. Unlike transgenic studies, our data used quantitative measures rather than subjective assessment.
In summary, our study has shown that short-term Wnt blockade affects crypt fission and intestinal growth. Dickkopf inhibition did not affect crypt cell proliferation, which remained low. We are in the process of identifying which Wnts are present in the small intestine of infant rats and humans. Wnt3 is known to be present in Paneth cells in mice and these are important for maintenance and proliferation of stem cells (27). It is possible to envisage that a Wnt stimulatory factor could be administered luminally or by transfection to augment crypt fission and intestinal growth.
The authors thank Dr Alexandra Keegan and Ms Kerry Lymn for their assistance in animal trials.
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