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First published online May 18, 2006
Journal of Experimental Biology 209, 2215-2223 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02249
The regenerative nidi of the locust midgut as a model to study epithelial cell differentiation from stem cells
Department of Histology and Pathology, Schools of Sciences and Medicine, University of Navarra, E-31080 Pamplona, Spain
* Author for correspondence (e-mail: lmontuenga{at}unav.es)
Accepted 30 March 2006
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Summary |
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Key words: stem cell, bromodeoxyuridine, BrdU, Locusta migratoria, insect, midgut, proliferation, FMRFa, immunodetection, endoreduplication, epithelial regeneration
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Introduction |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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; Potten et al.,
1997). The production of new differentiated cells usually depends
on a stem cell with the capacity to divide several times, giving rise to one
or more types of differentiated cells
(Potten, 1998;
Potten and Loeffler, 1990;
Slorach et al., 1999). A stem
cell generally divides into two daughter cells, one of which remains a stem
cell while the other goes down the route of differentiation. Amplifying cells
may give rise to an increasing number of differentiation-committed cells. Stem cells, other than haematopoietic stem cells, have thus far been characterized from tissues in a limited number of adult animal organs. New models of pluripotent adult stem cells in animals are required to provide research tools to identify, characterize and purify these cells. These new models for stem cell biology need to be studied both in vitro and in vivo. In vitro studies on isolated cells are simple and very informative but offer less accurate information on the complexity of the fine-tuned regulation of stem cells in vivo.
At present, several animal models are being studied to better understand
the regulation of the proliferative capacity of stem cells in fully developed
organs. Some models, such as haematopoietic stem cell models
(Blau et al., 2001;
Huang and Wang, 2001) or
epidermis and gut epithelium models (Liang
and Bickenbach, 2002; Marshman
et al., 2002; Martin et al.,
1998; Mills and Gordon,
2001; Slorach et al.,
1999), show that some adult stem cells are found in a specific
location of the tissue, known as the stem cell niche. The niche is formed by
stem cells and neighbouring proliferating and differentiating cells.
Neighbouring cells interact with the stem cells, keeping them in the niche,
where a specialized microenvironment preserves their stem cell
characteristics. Niches can be simple or complex. A simple niche contains only
one type of stem cell, generally anchored to a neighbouring cell. Complex
niches house two or more types of stem cells and require more complex
regulation (Ohlstein et al.,
2004). There are six models where stem cell niches have been
described in more detail (Fuchs et al.,
2004; Spradling et al.,
2001). Three of them - the Drosophila ovary
(Xie et al., 2005;
Xie and Spradling, 2000), the
vertebrate bone marrow (Calvi et al.,
2003; Zhang et al.,
2003) and the crypt of the mammalian intestinal villus
(Brittan and Wright, 2002;
Wong, 2004) - are considered
simple niche models. The remaining three - the Drosophila testis
(Xie et al., 2005;
Yamashita et al., 2003), the
subventricular zone of the vertebrate brain
(Song et al., 2002) and the
hair follicle (Tumbar et al.,
2004) - constitute complex niches. In all these models, it has
been proposed that the daughter stem cells start to differentiate when they
detach from the neighbouring cells. Thus, the spatial distribution within the
stem cell niche seems to play an important role as a mechanism of maintaining
stem cell status and regulating cell differentiation
(Ohlstein et al., 2004).
In the present work, we introduce a new stem cell model based on the intestinal epithelium of Locusta migratoria. It is a potentially useful model to study stem cell biology based on a simple and accessible material: the midgut epithelium of the desert locust.
The midgut of Locusta migratoria is a simple columnar epithelium
with three cell types: regenerative, differentiated columnar and endocrine.
Differentiated columnar cells are very tall and have long microvilli at the
luminal side. The columnar cells are metabolically very active, as they are
involved in nutrient absorption, secretion of digestive enzymes and formation
of the peritrophic membrane (Lehane and
Billingsley, 1996).
The endocrine cells are spread along the epithelium and are interspersed
with the columnar cells, just as they are in the mammalian intestinal diffuse
endocrine system (Zudaire et al.,
1998). These cells remain in contact with the basal lamina and
reach the lumen through a long and thin cytoplasmatic prolongation. They are
easily identifiable because they are loaded with cytoplasmic secretory
granules. The granules store active peptides, which can be detected by
specific immunocytochemical techniques. The endocrine cells are particularly
abundant in the posterior region of the midgut and, in particular, in the
ampullae through which the Malpighian tubules drain to the midgut
(Zudaire et al., 1998).
The regenerative cells may give rise to both types of differentiated cells. Each regenerative unit is located basally and is made up by concentrically layered cells that remain in contact with the basal lamina and do not come in contact with the midgut lumen.
Our main hypothesis is that in the midgut epithelium of the locust a stem cell lies in the centre of the niche surrounded by amplification-differentiating cells. The aim of our study was to explore the existence of these two functionally different cell types by means of several experiments designed to determine the proliferative dynamics of the locust midgut epithelial cells and the differentiation activity within the niche.
To test our hypothesis we have used bromodeoxyuridine (BrdU). BrdU is an analogue for thymidine, which is incorporated into DNA during the S phase. Our previous unpublished data showed that, once injected, BrdU bioavailability in the haemolymph lasts for about 30 min. BrdU pulse-chase experiments were designed to follow the fate of the nascent midgut epithelial cells. In addition, data obtained by BrdU immunolocalisation were complemented by simultaneous detection of FMRFamide (FMRFa)-like immunoreactivity as a means of identifying endocrine cells.
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Materials and methods |
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Dissection and processing of the samples
After severing the head and the tip of the abdomen, the entire gut was
pulled out, opened along its length, and cleaned with Mordue's locust saline
(Mordue, 1969). The tissue was
fixed in Bouin's fluid for 24 h. Guts were thoroughly washed in 70% ethanol,
embedded in paraffin and sectioned at 4 µm thickness.
Immunocytochemistry for BrdU
After deparaffination with xylene and rehydration, endogenous peroxidase
was blocked with H2O2. Next, the sections were treated
for one hour at room temperature with a commercial preparation of nuclease
(RPN202; Amersham Pharmacia, Amersham, UK). After blocking non-specific
binding sites with 5% normal goat serum, the sections were incubated overnight
with a primary monoclonal antibody (mouse anti-BrdU immunoglobulins; clone
BU-1; RPN202; Amersham Pharmacia) diluted 1:200 in TBS. A secondary anti-mouse
antibody with polymer-horseradish peroxidase labelling (EnvisionTM;
K4007; Dako, Cambridgeshire, UK) was used. The sections were counterstained
with haematoxylin, dehydrated and mounted in DPX.
Double immunocytochemistry
For the simultaneous immunocytochemical localization of BrdU and FMRFa,
pre-treatment was performed as described above. After blocking non-specific
binding sites with a mix of 5% goat serum and 5% pig serum, sections were
incubated simultaneously with the above-mentioned mouse anti-BrdU monoclonal
antibody and a rabbit polyclonal antibody against FMRFa (IHC8755; Peninsula
laboratories, Weil am Rhein, Germany), diluted at 1:200 and 1:500 in TBS,
respectively. For FMRFa detection, sections were first incubated with a
secondary anti-rabbit biotinylated antibody, followed by a
streptavidin-alkaline phosphatase complex (S-2890; Sigma-Aldrich, UK)
incubation. The samples were developed using a substrate chromogen solution
for alkaline phosphatase, composed of 50 µl Hexazotized New Fucsin [a
mixture of 25 µl of New Fuchsin (N-0638; Sigma) (at 4% in 2 mol
l-1 HCl) and 25 µl of sodium nitrite (6549; Merck, Madrid,
Spain) (at 4% in water)] and 1 ml of a Naftol phosphate solution [1 mg of
Naftol AS-TR-PO4 (N-6000; Sigma) dissolved in 20 µl of
N,N-dimethylformamide (141785, Panreac, Castellar del Vallés,
Spain) and further dissolved in 1 ml Tris-HCl buffer]. The mixture was finally
diluted in 3 ml of Tris-HCl buffer. Detection of BrdU primary antibody and
counterstaining was performed as described above. Finally, sections were
mounted in PBS-glycerol.
Whole mount immunofluorescence
The dissected midgut was fixed for 24 h in 4% formaldehyde. After being
washed with 70% ethanol and saline buffer, the whole-mount samples (
100
µm thickness) were incubated in a 1% Triton solution (T-8532;
Sigma-Aldrich). Next, the midgut portion was simultaneously incubated
overnight at 4°C with both primary antibodies, monoclonal (rat anti-BrdU
immunoglobulins; ab6326; Abcam, Cambridge, UK) and polyclonal (rabbit
anti-FMRFa immunoglobulins; IHC8755; Peninsula Laboratories) diluted at a
ratio of 1:200 and 1:500, respectively. Secondary antibodies, Alexa 488 goat
anti-rat and Alexa 555 donkey anti-rabbit (A-31572 and A-11006, respectively;
Molecular Probes, Breda, The Netherlands), were used for detection. Samples
were incubated in the mixture of secondary antibodies for 30 min at room
temperature. Finally, sections were mounted in an antifading medium (S-2828;
Molecular Probes).
Confocal image acquisition and 3-D reconstruction
Ten images, corresponding to different niches, were acquired from each
sample with a Leica TCS-SP2 AOBS confocal microscope. Confocal stacks were
reconstructed in 3-D with Amira software (Mercury computer systems, Berlin,
Germany). All the confocal and 3-D reconstructions were processed according to
previously published procedures
(Wouterlood, 2006).
Image acquisition and distance measurement in sections from paraffin embedded material
Two images were taken from each immunostained section with an optic Zeiss
800 E microscope at 200x magnification. For each image, two reference
distances were measured using the analySIS software (Soft Imaging System GmbH,
Münster, Germany): the average distance between the longitudinal muscular
layer (LML) and the apical part of the niche ('niche distance') and the
distance between the LML and the apical part of the farthest immunostained
nuclei ('labelled distance'). Both distances were represented graphically
against experimental time.
Experimental procedure
Single BrdU administration
To determine which midgut cells incorporate BrdU and to establish their
fate, we performed a pulse-chase experiment consisting of a single BrdU
administration and tracking of the labelled cells for 8 days.
Twenty insects (two insects per day plus two negative BrdU controls) were collected 24 h after the moult into the sixth instar. Dissection was performed at 1 and 4 h after BrdU administration and then every 24 h from Day 1 to Day 8. Niche distance and labelled distance measurements were taken for all the insects. In addition, we carried out a separate quantitative analysis to distinguish three different types of niche. Niches were characterized by measuring the nuclear diameter of BrdU-labelled cells only in zones where the epithelium was sectioned in a perfectly longitudinal way. Small nuclei were less than or equal to 5-6 µm in diameter, while medium-sized nuclei were between 8 and 12 µm diameters. Niches with one or more small positive nuclei (SN), niches with only medium-sized positive nuclei (MN) and negative niches with no BrdU-stained cells (NN) were quantified separately. Data were analysed with a chi-square test.
Single BrdU administration for confocal microscopy 3-D reconstruction and cell recount
The aim of this experiment was to estimate the total number of
BrdU-labelled cells per niche and to study their spatial distribution pattern.
A total of 15 insects was collected and dissected at different time intervals,
i.e. at 4 h (Day 0) and on Days 1, 2 and 3 after BrdU administration, and were
then prepared for confocal microscopy. Labelled BrdU nuclei and immunostained
FMRFa-like cells were counted in each 3-D reconstructed niche. Data were
analysed with Kruskal-Wallis and Mann-Whitney U statistical
tests.
Continuous BrdU administration
We performed continuous BrdU administration experiments in order to study
the dynamics of cycling cells within the niche and to ascertain whether these
cells are synchronized in their cycle. Two groups of 14 insects were injected
over a period of 8 days. In both groups, BrdU administration was performed 24
h after the moult and then every 24 h for Group 1 and every 48 h for Group 2
until dissection day.
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). This method
extrapolates cell cycle duration from the graphical representation of the BrdU
labelled/unlabelled mitosis ratio against time. The total cell cycle (Tc)
duration is equivalent to the distance between the first and second labelled
mitosis peaks.
Label-retaining cell experiment
According to previous studies (Cairns,
2002; Potten et al.,
2002) label-retaining cells (LRC) are very likely to be stem
cells. To study the presence of LRC, we administered a daily BrdU dose to a
sample of 27 locusts for five days. Locusts were dissected at days 10, 12, 14,
16 and 18 after the last BrdU administration, and the presence of LRCs was
assessed.
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Results |
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In the first 48 h, all the BrdU labelled cells were located in the niche area and no immunoreactive nuclei were observed in the differentiated columnar cells (Fig. 1A). Not all cells within a given niche were immunostained for BrdU. Between 48 and 72 h after a single BrdU administration, a fraction of labelled cells was observed just above the niche area. A group of positive small to medium-sized nuclei remained in the niche. From Day 4 to Day 8, the proportion of BrdU-immunoreactive nuclei in the niche markedly decreased, while an increasing fraction of differentiated columnar cells nuclei were immunostained in the epithelium (Fig. 1B). The distances between the epithelium base and the front wave of labelled cells were measured and represented graphically (Fig. 2). The distance between the center of the regenerative niche and these labelled cells increased progressively with time. The statistical correlation between distance and experimental time is significant (P<0.05), which suggests that cells are progressively moving away from the center of the niche to the taller epithelial region in contact with the lumen.
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50%. Throughout the 8
days, the SN proportion decreased three times, from an initial value of 36% to
22%, then to 12% and finally to 5% on Day 8
(Fig. 3). MN frequency was
approximately 35-40% after 48 h, from Days 2 to 3 it decreased to 15-20% and
was maintained until Day 8 at this level. The frequency of NN increased from
20% to 80% across the 8 days of experimentation, parallel to the decrease of
the other niche types.
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Cell cycle duration
The duration of cell cycle phases was determined by graphical estimation.
Using Willems method (Willems,
1972), we represented the ratio (percentage) of BrdU-labelled
vs total (labelled + unlabelled) mitosis plotted against the time
elapsed from BrdU injection to dissection. The complete cell cycle duration
(Tc) was estimated to be 10 h, This time was calculated from the graph by
measuring the distance between the two peaks of high percentage BrdU-labelled
mitosis (Fig. 7).
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Endocrine cell detection
FMRFa immunostaining was used to detect the endocrine cells that
differentiate within the niche. FMRFa-like immunostaining was found in the
cells of the diffuse endocrine system and was clearly cytoplasmic. The
endocrine cells are in contact with the basal lamina and reach the midgut
lumen through a slender cytoplasmic extension. 3-D reconstruction of the
central midgut region (Fig.
4B,C) showed that 67% of the niches contain a single FMRFa-like
immunoreactive cell, whilst 24% have two cells and 5% have three cells. The
remaining 4% correspond to niches without FMRFa-like positive cells. Double
immunodetection for BrdU and FMRFa was performed to determine the
proliferative status of the endocrine lineage of the niche in several
experimental situations. The nuclei of the endocrine cells are considerably
smaller than the oval-shaped nuclei of the most abundant enterocytes and are
usually located in the periphery of the niches. We performed a thorough search
for double immunostained cells on the ventricle and ampulla midgut regions
using double immunostaining. In the ventricle zone, no BrdU-positive endocrine
cells were found in any of the experiments performed, including the continuous
labelling experiments (Fig.
9A). However, in Group 1, treated during 8 days of continuous BrdU
administration, 10% of endocrine cells of the ampullae showed BrdU and
FMRFa-like colocalisation (Fig.
9B,C).
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Discussion |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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;
Ohlstein et al., 2004). As
expected, the locust midgut niche is similar to the intestinal villus crypt
while nonetheless representing a considerably simpler model. The stem cell of
the mammalian intestinal villus crypt will renew the entire epithelium, giving
rise to four types of differentiated cells, columnar, goblet, endocrine and
Paneth cells (Marshman et al.,
2002), while the stem cell of the locust midgut niche gives rise
only to columnar and endocrine cells needed to renew the intestinal
epithelium. In contrast to the mammalian tissue, in the locust midgut the
regenerative cells are concentrated in a well-defined and easily identifiable
region of the epithelium. They are potentially more accessible for
isolation.
In other described regenerative niches, proliferating cells are also
located in a circumscribed location, but the limits of the niche are not
always evident (Chepko and Dickson,
2003; Ohlstein et al.,
2004; Spradling et al.,
2001). The results of our single BrdU administration experiments
show that, immediately after injection, all the BrdU-labelled cells in the
locust midgut are exclusively located in the niche area. Our previous data
confirm these results as positive cells for PCNA (proliferating cell nuclear
antigen), which are exclusively localized in the niche area and colocalize
with BrdU-labelled cells (Zudaire et al.,
2004).
When the stem cell of the intestinal villus crypt divides asymmetrically
(Bach et al., 2000;
Watt and Hogan, 2000), it
produces a stem cell and a committed daughter cell that enters into a dividing
transient state. These cells are known as transit amplifying cells
(Slorach et al., 1999). In the
mammalian intestine epithelium, transit amplifying cells proliferate and
produce cells that differentiate and migrate towards the villus, where they
perform their function before being shed to the lumen
(Marshman et al., 2002).
According to published reports, the cells produced in the mammalian crypt need
approximately 3-8 days to reach the tip of the villus
(Marshman et al., 2002;
Potten, 1998). In a similar
way, the locust midgut niche stem cell divides and gives rise to transit
amplifying cells. Our data show that BrdU-labelled cells start to leave the
niche between 24 and 48 h after BrdU administration. Differentiated cells
maintain their migration through the gut epithelium towards the lumen for 5-8
days. Columnar cells are shed out of the lumen 8-10 days after they
differentiate. These data are very similar to those observed by Endo in the
cockroach midgut (Endo,
1984).
Transit amplifying cells have a higher probability to be labeled by BrdU
than do stem cells due to their rate of proliferation. When we administered
BrdU every 24 h, all the niches were almost completely labelled. Our results
showing positive and negative cell clusters suggest the existence of
synchronised groups of amplification cells within a given niche. According to
the data obtained by 3-D reconstruction, 5-8 amplification cells could be
found per niche at the BrdU administration time. This number doubled after 24
h of administration and did not change significantly in the following 2 days.
These data suggest that not all the labeled cells that incorporate BrdU (and
thus are synthesizing DNA at Day 0) enter into mitosis. According to previous
studies (Zudaire et al.,
2004), differentiated columnar cells have multiple DNA copies in
their nuclei. To produce polyploid or polytenic cells, the cell must undergo
multiple S phases without entering into mitosis, a process termed
endoreduplication (Edgar and Orr-Weaver,
2001). The non-dividing labeled cells that we have found in this
experiment could be endoreduplicating cells.
The cell cycle duration experiment showed a cell cycle time of 10 h.
This short cell cycle is characteristic of amplification cells, compared with
the longer cell cycle expected for stem cells. The mouse intestinal villus
crypt also presents two different cell populations: transient amplifying cells
and stem cells (Slorach et al.,
1999). The average cell cycle time for amplification cells in the
mammalian intestinal villus is about 12-13 h, while the stem cell cycle lasts
around 24 h. (Bach et al.,
2000; Potten,
1998). Thus, in both models (mammals and locust), amplification
cells divide approximately twice a day.
Lack of specific intestinal stem cell markers is a major problem for
studying the biology of intestinal epithelial differentiation from stem cells
(Leedham et al., 2005;
Marshman et al., 2002;
Potten, 1998). However, the
labelling retaining property is a potential surrogate marker for a stem cell
(Braun et al., 2003;
Braun and Watt, 2004).
Long-term LRCs were round, with a nucleus smaller than that of amplifying or
columnar cells and were localized basally in the niche. Insects dissected on
Days 14-18 showed only 1.5% of the niches having long-term LRC. However,
insects dissected on Days 10 and 12 showed 10% of the niches with short-term
LRC. Results obtained in the quantification of three niche types showed that
the proportion of SN with small short-term LRCs decreases every 48-72 h. From
this, we can estimate that the cell cycle duration of these short-term LRCs is
2-3 days and thus it is slower than that of amplification cells and faster
than that of long-term LRCs.
Studies of intestinal epithelium in mice have shown that endocrine cells
share their clonal origin with other gut cells, suggesting a common stem cell
origin (Thompson et al.,
1990). The locust midgut endocrine cells also seem to be derived
from the common precursor cells present in the niches, at least according to
their spatial localization within the niche and their very close relationship
with the rest of the epithelial cells. We did not observe colocalization among
FMRFa-like immunolabelled cells and BrdU-labelled nuclei in the midgut
ventricle. However, 10% of the endocrine cells of the Malpighian tubules
ampullae draining to the midgut incorporated BrdU after 24 h administration. A
similar lack of tritiated thymidine labelling was observed in the endocrine
cells of Periplaneta americana midgut, suggesting a slower cell cycle
for these cells (Endo, 1984).
Our results suggest that new endocrine cells are indeed being generated in the
ampullar region at a slow rate. Apparently, these cells do not proliferate or
differentiate from stem cell-derived precursors in the ventricle, or, if they
do, the process is extremely slow and not detectable under our experimental
conditions. These regional differences in the generation of new endocrine
cells are very intriguing and point towards a very fine regulation of
endocrine differentiation by the microenvironment or by cell-cell
interactions. The niches located in the ampullar region are bathed by the same
haemolymph as those of the ventricle, suggesting that the regulation of
endocrine cell differentiation within the niches is dependent upon local
rather than systemic stimuli. The ampullar endocrine cells are very abundant
and may be involved in the control of excretory and digestive physiology
(Montuenga et al., 1996;
Zudaire et al., 1998). The
number of endocrine cells per niche did not change throughout the days of
experimentation. Endocrine cells where not shed to the lumen as columnar
enterocytes. Moreover, our unpublished data show that a good percentage of
endocrine cells are already present in the epithelium of all the previous
larval stages, with a particular increase in endocrine cell frequency in the
4th instar insects. All these data suggest that endocrine cells represent a
very stable population with a very low proliferation rate, except in the
ampullar region, where a higher turnover and differentiation of endocrine
cells seems to occur.
In conclusion, we have described here a simple in vivo insect model of epithelial regeneration that may help in understanding the complex regulatory mechanisms of cell proliferation, differentiation and tissue homeostasis.
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Acknowledgments |
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