4149
Development 126, 4149-4156 (1999)
Printed in Great Britain © The Company of Biologists Limited 1999
DEV4179
Targeted ablation of secretin-producing cells in transgenic mice reveals a
common differentiation pathway with multiple enteroendocrine cell lineages
in the small intestine
Guido Rindi1,*, Christelle Ratineau2, Anne Ronco2, Maria Elena Candusso1, Ming-Jer Tsai3
and Andrew B. Leiter2,‡
1Department of Human Pathology, University of Pavia, Italy
2Division of Gastroenterology, GRASP Digestive Disease Center,
and Tupper Research Institute, New England Medical Center,
Boston, MA 02111, USA
3Department of Cell Biology, Baylor College of Medicine, Houston, TX 77030, USA
*Present address: Department of Anatomic Pathology, University of Brescia, Brescia, Italy
‡Author for correspondence (e-mail: [email protected])
Accepted 18 June; published on WWW 23 August 1999
SUMMARY
The four cell types of gut epithelium, enteroendocrine
cells, enterocytes, Paneth cells and goblet cells, arise from
a common totipotent stem cell located in the mid portion
of the intestinal gland. The secretin-producing (S) cell is
one of at least ten cell types belonging to the diffuse
neuroendocrine system of the gut. We have examined the
developmental relationship between secretin cells and
other enteroendocrine cell types by conditional ablation
of secretin cells in transgenic mice expressing herpes
simplex virus 1 thymidine kinase (HSVTK). Ganciclovirtreated mice showed markedly increased numbers of
apoptotic cells at the crypt-villus junction. Unexpectedly,
ganciclovir treatment induced nearly complete ablation of
enteroendocrine cells expressing cholecystokinin and
peptide YY/glucagon (L cells) as well as secretin cells,
suggesting a close developmental relationship between
these three cell types. In addition, ganciclovir reduced the
number of enteroendocrine cells producing gastric
inhibitory polypeptide, substance-P, somatostatin and
serotonin. During recovery from ganciclovir treatment,
the enteroendocrine cells repopulated the intestine in
normal numbers, suggesting that a common early
endocrine progenitor was spared. Expression of BETA2, a
basic helix-loop-helix protein essential for differentiation
of secretin and cholecystokinin cells was examined in the
proximal small intestine. BETA2 expression was seen in
all enteroendocrine cells and not seen in nonendocrine
cells. These results suggest that most small intestinal
endocrine cells are developmentally related and that a
close developmental relationship exists between secretinproducing S cells and cholecystokinin-producing and L
type enteroendocrine cells. In addition, our work shows
the existence of a multipotent endocrine-committed cell
type and locates this hybrid multipotent cell type to a
region of the intestine populated by relatively immature
cells.
INTRODUCTION
in the small intestine of most mammalian species (Chey and
Escoffery, 1976; Larsson et al., 1977; Straus and Yalow, 1978)
as well as in the colon (Lopez et al., 1995). In addition, secretin
is transiently expressed in insulin-producing β cells of the
developing rat endocrine pancreas (Wheeler et al., 1992).
Labeling studies with [3H]thymidine indicate that all four
epithelial cell types of the mouse intestine, namely enterocytes,
Paneth cells, goblet cells and enteroendocrine cells, originate
from a common totipotent stem cell and turnover every 3-4
days (Cheng, 1974; Cheng and Leblond, 1974a,b). In rats,
labeling with [3H]thymidine showed that secretin-producing
cells turnover once every 4-5 days like the other intestinal cell
types (Inokuchi et al., 1986). The absence of labeled secretin
cells earlier than 24 hours after the first [3H]thymidine
At least 10 different enteroendocrine cell types have been
described in the small intestine of mammals (Solcia et al.,
1998). Subtype classification relies on the identification of the
main hormonal cell content by immunohistochemical methods
as well as on the ultrastructure of their secretory large dense
core endocrine granules. Several cell types may coexpress
more than one hormone. The heterogeneity of hormone
expression in enteroendocrine cells is believed to reflect their
peculiar multipotency and is observed in gut neuroendocrine
tumors and derived cell lines (Klöppel and Heitz, 1988;
Madsen et al., 1986; Oie et al., 1983; Pilato et al., 1988;
Tischler, 1983). Secretin-producing S cells are located mainly
Key words: Enteroendocrine cells, Secretin, Intestine,
Cholecystokinin, Cell ablation, Thymidine kinase
4150 G. Rindi and others
injection suggested that differentiation towards secretin cells
may occur within the crypt in precursor cells expressing
secretin at levels below the detection limit of
immunohistochemistry.
Coexpression of more than one hormone by individual
enteroendocrine cells is consistent with the idea that
enteroendocrine cells arise from multipotential endocrine
cells. A fraction of intestinal secretin cells coexpress
serotonin and substance P, suggesting a developmental
relationship between these two lineages (Cetin, 1990).
Transgenic mice expressing easily detected reporter genes
like human growth hormone (hGH) in intestinal epithelial
cells have provided further insight into relationships between
different enteroendocrine cell types. The detection of reporter
genes such as hGH has proved in some cases to be much more
sensitive than directly staining enteroendocrine cells with
antibodies directed against specific hormones. Sequences in
the first 500 bp of 5′ flanking sequence of the liver fatty acid
binding protein (L-FABP) gene directed expression of a hGH
reporter gene to several enteroendocrine cell types including
secretin cells as well as enterocytes, in which L-FABP is
normally expressed (Roth and Gordon, 1990; Roth et al.,
1990). The results suggested complex lineage differentiation
pathways.
We previously showed that 1.6 kb of 5′ flanking sequence
of the rat secretin gene directed developmentally regulated,
cell-specific transgene expression in major gastrointestinal
sites of secretin gene expression in transgenic mice. The
colocalization of this sensitive reporter gene in multiple
enteroendocrine cell types provided the initial evidence that
secretin is coexpressed in CCK in the small intestine, and
glucagon, peptide-YY and neurotensin cells in the colon.
These data were consistent with the existence of a
multipotential endocrine-committed cell type from which
secretin cells terminally differentiate. However, coexpression
of multiple hormones in subpopulations of specific
enteroendocrine cell types cannot establish that a particular
lineage arises from a common progenitor expressing another
hormone.
We have generated transgenic mice expressing the herpes
simplex 1 virus thymidine kinase (HSVTK) under control of
1.6 kb of 5′ rat secretin gene. HSVTK expression in
transgenic mice is nontoxic. However, the viral enzyme
renders tissues sensitive to the nucleoside analog ganciclovir,
allowing conditional cell ablation. This strategy has been
used with success to target cell types for cell-lineage studies
in transgenic mice (Borrelli et al., 1989; Canfield et al., 1996;
Heyman et al., 1989). The objective of the present study was
to target an inducible toxic phenotype to secretin-expressing
cells by specific cell ablation in order to (i) investigate the
existence of an endocrine-committed multipotent progenitor
cells; (ii) identify the developmental lineage relationship
between secretin cells and the other enteroendocrine cell
types and (iii) determine the relationship between the putative
endocrine-committed progenitor cells and the nonendocrine
cell types of the intestinal mucosa. The data reported here
indicate that the secretin cell lineage is most closely related
to enteroendocrine cells that express CCK and PYY/glucagon
which appear to arise from a secretin-producing, multipotent,
endocrine-committed cell type. Secretin cells appear to be
less closely related to 4 additional endocrine cell types.
MATERIALS AND METHODS
Construction of transgene
1.2 kb of HSVTK coding sequences (−53 to +1438) attached to about
1.2 kb of Simian Virus polyadenylation signal sequences were
obtained after BglII digest of the pGK-TK plasmid (a gift from M.
Low, Portland, Oregon) and subcloned in pBluescriptKS at the BamHI
site (pBlueKS-HSVTK). Not-Xho digest from pBlueKS-TK was
subcloned in a pIBI31-Sec vector containing 1.6 kb of 5′ flanking
sequences of rat secretin gene cloned in the BamHI-SacI site. Correct
fragment orientation was confirmed by DNA sequencing before
microinjection. The secretin-TK transgene was separated from
plasmid vector sequences by digesting with KpnI-XhoI followed by
agarose gel electrophoresis.
Production of transgenic mice
Purified transgene was microinjected into the pronucleus of B6D2F1
× B6D2F1 mouse embryos and transferred into the oviducts of
pseudopregnant CD1 mice (Hogan et al., 1994). Transgenic mice were
identified by DNA blot hybridization with 32P random primed labeled
cDNA probes for HSVTK. Transgenic pedigrees were maintained on
a CD1 background.
Ablation procedure
Sec-HSVTK mice and controls of about 35 g weight (mean 35.5 ±5.8
g) were treated with 4.8 mg/day of the nucleotide analog ganciclovir
(Citovene, Sintex, Ca, USA) for 5 days (Borrelli et al., 1989). Ablation
by ganciclovir was accomplished by continuous infusion with
subcutaneously implanted osmotic minipumps loaded with either
ganciclovir or saline solution according to the manufacturer’s
instructions (Alzet, model 2002, Alza). Controls consisted either of
transgenic mice treated with saline solution or age-matched nontransgenic CD-1 mice treated with ganciclovir. Some transgenic mice
were allowed to recover for 5 weeks after treatment to assess
endocrine cell repopulation. At the end of the treatment and recovery
period, transgenic mice and controls were killed for histochemical
studies.
Histology and immunohistochemistry
Three samples of about 2 cm each in length from different parts of
the small intestine (duodenum, ileum and terminal ileum) were
removed, fixed by immersion in Bouin’s solution or 10% formalin for
6-8 hours at room temperature and processed into paraffin wax. Serial
sections (3-5 µm) were stained with haematoxylin and eosin for
conventional histology and PAS/Alcian blue for identification of
mucins. Immunohistochemical tests were performed using the avidinbiotinylated peroxidase complex (ABC, Vector, Burlington, USA)
method. The following specific sera were used: anti-HSVTK (W. C.
Summers, Yale University, New Haven, CT, USA; rabbit polyclonal,
1:2000), secretin (D. Gossens, Free University of Brussels, Belgium;
rabbit polyclonal, 1:15,000). Other primary antibodies have been
described previously and include rabbit anti-glucagon (1:3,000),
rabbit anti-cholecystokinin (1:8,000), rabbit anti-gastrin (1:2,000),
guinea pig anti-peptide-YY (PYY) (1:15,000), rabbit antisomatostatin (1:3,000), rabbit anti-gastric inhibitory polypeptide (GIP,
1:1,000), 5-HT (1:5000), rabbit anti-substance-P (1: 2,000), rabbit
anti-neurotensin (1:5,000) (Lopez et al., 1995; Upchurch et al., 1996).
Reverse-face sequential immunoperoxidase staining was performed
for HSVTK and secretin immunolocalization. Specificity tests for the
immunostaining consisted of absorption of each antiserum with its
homologous antigen (10 nmol/ml of diluted antiserum), omission of
either the primary or secondary antibody, and use of control tissue
with or without the pertinent antigen.
Intestinal tissue from BETA2+/− mice was fixed and stained for βgalactosidase activity, embedded, and sectioned at 4 µm as described
previously (Mutoh et al., 1998). Following microwave antigen
retrieval, sections were immunostained as described previously with
Secretin cell ablation 4151
antibodies against secretin, cholecystokinin, GIP, serotonin,
substance-P, somatostatin, glucagon, and peptide YY. Primary
antibodies were detected by immunoperoxidase using DAB as a
substrate.
DNA fragmentation analysis
Sections of formalin-fixed, paraffin wax-embedded samples of small
intestine from ganciclovir-treated and control mice were analyzed for
DNA fragmentation with a modified TUNEL method (Gavrieli et al.,
1993). In brief, after treatment with proteinase K, sections were
incubated with 3′ terminal transferase in the presence of biotin-labeled
dUTP at 37°C for 2 hours. DNA labeling was revealed using avidinperoxidase-biotin complexes as above.
Morphometry and statistical analysis
Only tissue samples correctly oriented along the longitudinal axis
were used for quantitative analysis. Immunoreactive cells for 9
gastrointestinal hormones were counted per linear millimeter of
mucosal length as previously described (Langhans et al., 1997). Data
were collected from three different groups of mice: controls,
ganciclovir-treated transgenic mice and ganciclovir-treated transgenic
mice after recovery. A total of 6 mice, two for each group, were
studied. Six to ten observations (mean 7.7) for each cell type were
performed for each mouse in all groups. The length of the mucosa
available for counting in each mouse, for each specific hormone
antiserum ranged from 13.17 mm to 75.27 mm (mean value 37.27
mm). The mean length of mucosa evaluated was 27.84 mm for
controls (range 13.17-38.88 mm, 75 observations), 52.29 mm for
treated transgenic mice (range 29.59-75.27 mm, 70 observations) and
31.34 mm for treated transgenic mice after recovery (range 18.0337.69, 63 observations).
Data were first analyzed for equality of population with the
Kruskal-Wallis test (analysis of variance) to elicit difference between
the three groups. P<0.05 was considered statistically significant.
Parameters statistically different at the previous analysis were then
compared by the two sample Wilcoxon rank-sum, Mann-Whitney, test
to compare data between two groups of observations. A P value of
<0.05 was considered statistically significant after Bonferroni (×3)
correction.
RESULTS
Thirteen transgenic lineages were established after transgene
microinjection. Two transgenic pedigrees with the highest
enzymatic activity were selected for further studies with
ganciclovir (nos. 933 and 995). Both pedigrees showed a
similar phenotype following treatment and one (no. 995) was
used for subsequent detailed studies. Transgenic mice were
normal in size and reproduced normally. To assess the correct
tissue-specific expression of the transgene, samples of the
small intestine of adult transgenic mice were investigated by
immunohistochemistry with an antiserum specific for HSVTK.
HSVTK immunoreactivity was detected in discrete mucosal
cells of the small intestine (Fig. 1A) which coexpressed
secretin in the adjacent section (Fig. 1B), thus confirming that
transgene expression was specifically directed to secretin cells.
The small intestine from ganciclovir-treated transgenic mice
showed relatively normal crypt-villus cytoarchitecture
indicating that most cells in the intestine were spared (Fig. 2A).
Paneth cells, enterocytes and goblet cells appeared relatively
normal (not shown). Thus the drug did not induce relatively
nonspecific, generalized ablation that could potentially occur
due to reuptake of phosphorylated ganciclovir released
from correctly targeted dying cells. Examination at higher
magnification revealed small numbers of cells in the mid to
lower regions of the crypt compartment with pyknotic,
hyperchromatic nuclei and shrunken cytoplasm, features
typical of cells undergoing apoptosis (Fig. 2B, arrows). In
contrast, we failed to identify apoptotic cells in the crypts of
control animals. Some of the dying cell stained for secretin
immunoreactivity (Fig. 3B).
Staining for DNA fragmentation by the TUNEL method in
samples of the small intestine of ganciclovir-treated transgenic
mice confirmed the histologic observations (Fig. 2B). In
ganciclovir-treated transgenic mice, numerous cells showed
nuclear staining for DNA fragmentation, indicative of
apoptosis, in the lower to mid portion of the crypts (Fig. 2C,E).
Apoptotic cells were not seen in the crypts of either salinetreated transgenic mice or in ganciclovir-treated CD1 mice. In
addition, relatively rare cells with labeled nuclei were observed
at the tip of the villus in both transgenic and control mice (Fig.
2C,D). The occasional dying cells at the villus tip probably
represent apoptosis that occurs during normal epithelial cell
turnover in the intestine. These features indicate that the cell
death in the neck zone of the crypt of ganciclovir-treated
transgenic mice was a specific effect of transgene expression
and ganciclovir treatment.
We examined the small intestine from control mice and
ganciclovir-treated transgenic mice with a panel of ten specific
antisera directed against secretin and nine gastrointestinal
hormones. In ganciclovir-treated transgenic mice, secretin
immunoreactive cells were reduced in number compared to
controls (Fig. 3A,B). A significant fraction of the few
remaining secretin cells appeared abnormal with pyknotic
nuclei with cytoplasmic shrinkage (Fig. 3D) when compared
to secretin cells in control animals (Fig. 3C), suggesting that
they were undergoing programmed cell death. The numbers of
most other enteroendocrine cells appeared to be reduced in
addition.
A detailed morphometric analysis for 9 intestinal endocrine
cell types enabled us to quantitate the effect of ganciclovir
treatment on different enteroendocrine populations of
transgenic mice (Fig. 4). The mean number of secretin cells
was reduced by 86% in transgenic mice as compared to control
transgenic animals treated with saline (Fig. 4). Unexpectedly,
cells expressing CCK (89% reduction), glucagon (85%) and
peptide YY (88%) were similarly reduced in number in
ganciclovir-treated transgenic mice, suggesting a close
developmental relationship between these cell lineages.
Several populations were partially depleted by ganciclovir
treatment, including GIP cells (59%), substance-P (58%),
somatostatin (55%) and 5HT (45%) cells. In contrast, gastrinimmunoreactive cells decreased only by 13% suggesting that
they were not significantly affected by ganciclovir treatment.
The number of endocrine cells of transgenic mice returned to
values similar to controls after 5 weeks recovery, indicating
that the stem cell population was spared (Fig. 4). The extremely
low number of neurotensin-expressing cells in the small
intestine precluded meaningful quantitative analysis of this
lineage. The equality of population test (variance analysis)
revealed that observed differences between ganciclovir-treated
transgenic mice and either controls or transgenic animals
postrecovery were statistically significant (P≤0.006) for all
enteroendocrine cell types examined except gastrinimmunoreactive (G) cells P=0.08). Comparison of the groups
4152 G. Rindi and others
Fig. 1. Herpes thymidine kinase transgene expression is directed to
secretin cells of secretin-HSVTK transgenic mice. Transgene
expression in the small intestine of SEC-HSVTK transgenic mice.
The arrowhead denotes a single secretin (S) cell coexpressing
secretin (A) and HSVTK (B). Reverse face, consecutive sections,
immunoperoxidase, hematoxylin counterstain. Scale bar, 28 µm.
Fig. 3. Enteroendocrine cells in the small intestine of controls and
SEC-HSVTK transgenic mice after ganciclovir treatment.
Distribution of secretin (S) cells in ganciclovir-treated CD-1 (A,
arrowheads). In ganciclovir-treated transgenic mice (B), S cells
(arrowheads) are markedly reduced in number. In contrast to normal
appearing S cells in control mice (C), the few remaining secretin
cells have shrunken, pyknotic nuclei with abnormal cytoplasmic
shape (D). Immunoperoxidase, hematoxylin counterstain, scale bar,
(A,B) 164 µm, (C,D) 10 µm.
Fig. 2. Ganciclovir targets cells in the crypt region of the small
intestine of SEC-HSVTK transgenic mice. Note the preserved
general architecture of the small intestine, with normal villi (A). At
higher magnification (B) apoptotic cell bodies are visible in the neck
area (arrowheads). The analysis of DNA fragmentation by the
TUNEL method identified several nuclei labeled in cells at the tip of
the villi (C and enlarged in d) and cell bodies in the same neck zone
as in b (C and enlarged in E). Hematoxylin and eosin staining (A,B),
scale bar (A) 164 µm, (B) 66 µm; TUNEL method, peroxidase (CE), scale bar (C) 164 µm, (D,E) 41 µm.
other than gastrin cells (Mann-Whitney test) showed that
results from treated transgenic mice were statistically different
when compared to those of controls or transgenic mice after
recovery (observed P≤0.03). Conversely, no statistically
significant difference was observed between controls and
transgenic mice after recovery.
We previously showed that mice containing a targeted
deletion in the gene encoding the basic helix-loop-helix
transcription factor, BETA2, failed to develop both secretinand CCK-expressing enteroendocrine cells, leading us to
suggest a close relationship between these two lineages (Naya
et al., 1997). To determine whether expression of BETA2 was
related to the observed results in cell lineage ablation
experiments, we examined small intestine endocrine cell types
for expression of BETA2 using mice that we have previously
described, containing a targeted deletion in a single BETA2
allele. Most of the region coding for BETA2 was replaced with
an in frame fusion of the lacZ reporter gene, allowing us to
identify cells expressing β-galactosidase activity by X-gal
histochemistry (Naya et al., 1997). The sensitivity of this
method is far greater than immunostaining with BETA2
antibodies described earlier (Mutoh et al., 1997). Nuclear βgalactosidase activity was seen in each of the small intestinal
endocrine cell types examined in the proximal small intestine
(Fig. 5). β-galactosidase staining was less intense in the distal
small intestine where most peptide YY/glucagon cells are
found. However, the majority of the peptide YY/glucagon cells
showed β-galactosidase activity. To determine if BETA2 is
expressed only in enteroendocrine cells, we stained sections of
Secretin cell ablation 4153
8
cells/mm length
small intestine for both β-galactosidase activity and
chromogranin A, a marker for endocrine secretory granules.
All chromogranin A immunoreactive cells showed staining for
β-galactosidase activity and virtually all X-gal-stained cells
showed colocalized staining for chromogranin A, indicating
that most if not all small intestinal enteroendocrine cells
express BETA2 and that BETA2 expression is restricted to
endocrine cells in the intestine.
7
cells/mm length
6
4
4
3
5
4
3
3
2
2
2
1
*
secretin
4
3
1
*
0
CCK
2
1
glucagon
6
7
5
6
2
1
GIP
0
substance P
12
10
8
15
2
6
*
1
0
*
3
1
20
3
peptide YY(PYY)
4
*
2
0
*
5
25
4
0
8
3
*
1
7
4
0
cells/mm length
5
5
DISCUSSION
Fig. 5. BETA2 is expressed in multiple enteroendocrine
lineages in the small intestine. Sections from a 1-month old
BETA2+/− mouse showing nuclear β-galactosidase activity
(blue) colocalized with (large arrows) chromogranin A (A),
cholecystokinin (B), serotonin (C), GIP (D), somatostatin
(E), and peptide YY (F). Small arrows in B indicate cells
expressing β-galactosidase activity that do not stain for
cholecystokinin. Hormones stained by immunoperoxidase,
hematoxylin counterstain; scale bar, 20 µm (A-C); 10 µm
(D-F).
7
6
0
This paper describes the effect of inducible selective ablation
of secretin cells in the small intestine of transgenic mice. The
1.6 kb of 5′ sequence used to drive expression of herpes virus
1 thymidine kinase (HSVTK) is sufficient to direct
developmentally regulated tissue and cell-specific expression
of reporter genes in transgenic mice (Lopez et al., 1995). The
sensitivity of secretin-producing enteroendocrine cells to
ablation after 5 days treatment with ganciclovir indicates that
the secretin gene is expressed in proliferating cells. Prior to the
present work, it was believed that expression of secretin was
restricted to terminally differentiated, non-proliferating cells
based on the localization of secretin cells exclusively to the
nondividing villus compartment of the small intestine as
opposed to the proliferating crypt compartment. The failure of
secretin cells to incorporate radiolabel from a single injection
of [3H]thymidine into rats provided additional evidence that
secretin cells were nonreplicating.
Continuous [3H]thymidine labeling showed increased
incorporation of the isotope into secretin cells with all cells
labeled after 5 days, the renewal time for small intestinal
enteroendocrine cells. Thus secretin cells were believed to
arise from a less differentiated, continuously self-renewing
progenitor cell. The present work supports the existence of a
proliferating progenitor cell and indicates that a subpopulation
of cells destined to differentiate into secretin cells continue to
divide and express the secretin gene. This subpopulation of
cells appears to express secretin at levels below the limits of
detection by immunohistochemistry.
Histology and DNA fragmentation analyses indicate that the
cells targeted by the nucleoside analog in transgenic mice are
located in the mid portion of the crypt in contrast to mature
secretin cells found mainly in the villus compartment. The
totipotent intestinal stem cell as well as other relatively
immature cells are believed to reside in this part of the
intestinal gland. Therefore, the ablated secretinexpressing cells in Sec-HSVTK mice appear to be
located physically close to relatively undifferentiated
cells, suggesting that cells were targeted at an
early stage of differentiation, after committed
6
10
*
5
somatostatin
0
Controls
4
2
serotonin
Treatment
0
gastrin
Recovery
Fig. 4. Effect of ganciclovir treatment on enteroendocrine cell
populations of the small intestine in control mice, transgenic mice,
and transgenic mice after recovery. Open bars show results from
control mice, black bars from transgenic mice treated with
ganciclovir, and striped bars from ganciclovir-treated transgenic mice
allowed to recover after treatment. Results shown as mean ± s.d. of
the number of immunoreactive cells observed per linear mm of
mucosa. Mann-Whitney test (see Material and Methods);
*statistically significant difference observed between treated
transgenic mice and the other two groups (observed P ranging from
≤0.0008 to 0.03).
4154 G. Rindi and others
enteroendocrine progenitor cells segregate from nonendocrine
lineages and stem cells. This population of immature cells
expresses relatively low levels of secretin and thymidine kinase
yet is ablated by ganciclovir due to the sensitivity of cells to
the drug. Continuous infusion of ganciclovir for 5 days, the
approximate turnover time for secretin cells, resulted in
depletion of greater than 90% of secretin cells. Thus, it is likely
that the nucleoside is not directly targeting mature villus
secretin cells but rather, an immature, proliferating progenitor
population.
A small fraction of secretin cells (about 10%) survived after
5 days of ganciclovir treatment. These cells may represent a
small subpopulation of terminally differentiated secretin cells
that turnover more slowly. It is also possible that these cells
were targeted by the nucleoside but are only just entering the
apoptotic pathway and have yet to show morphologic changes
consistent with programmed cell death. Although unlikely, we
cannot exclude the possibility that the transgene may not be
expressed in the few secretin cells surviving ablation.
It is well established that enteroendocrine cells frequently
express more than one hormone. Coexpression of serotonin
(5HT) and substance-P in a fraction of secretin cells has been
well documented (Cetin, 1990; Roth and Gordon, 1990; Roth
et al., 1990). Subpopulations of small intestinal CCKexpressing (Lopez et al., 1995) and glucagon- (GLP-1)
expressing cells (Aiken et al., 1994) coexpress secretin, further
suggesting that S type enteroendocrine cells are related to
several other enteroendocrine lineages. Ganciclovir treatment
of SEC-HSVTK mice resulted in depletion of CCK-expressing
and PYY/glucagon-expressing (type L cells) enteroendocrine
cells in addition to secretin cells. This finding suggests a much
closer lineage relationship between secretin cells, CCK and
PYY/glucagon cells than was previously appreciated from
coexpression studies. The data presented here confirms that at
least CCK and PYY/glucagon cells share a common
differentiation pathway with secretin cells until almost
terminally differentiated.
Ganciclovir treatment of Sec-HSVTK transgenic mice
depleted cell lineages producing GIP, substance-P, serotonin
and somatostatin to a lesser extent than cells producing
secretin, CCK and PYY/glucagon. This finding may suggest
that such cell types are also related to secretin cells in their
lineage differentiation pathway. Alternatively, selected
subpopulations of these four cell types may be able to express
secretin before terminal differentiation. Although unlikely, we
cannot exclude that loss of secretin after ablation may reduce
expression of other hormones leading to an apparent depletion
of its cell type. In contrast to the other small intestinal
endocrine cell lineages, gastrin-producing cells were
unaffected by treatment with ganciclovir, indicating a separate
differentiation pathway with respect to secretin cells. After
cessation of ganciclovir treatment, each of the depleted cell
lineages repopulates the intestine to pretreatment numbers. The
ability of each enteroendocrine lineage to recover suggests that
each lineage arises from an earlier progenitor/stem cell that
does not express the secretin gene and is therefore not targeted
by ganciclovir.
We previously showed that the same region of the secretin
gene 5′ flanking sequence directed the expression of a human
growth hormone reporter gene to secretin cells as well as
subpopulations of CCK, substance-P, and serotonin cells in the
small intestine of transgenic mice. However, the transgene was
not expressed in small intestine endocrine cells expressing
glucagon, peptide YY, somatostatin, GIP or gastrin. The
ablation of a significant fraction of glucagon/peptide YY,
somatostatin and GIP cells reported here was not anticipated
in light of our earlier observations that the secretin gene or a
secretin promoter driven reporter did not appear to be
coexpressed in these cells (Lopez et al., 1995). Secretin
expression may fall below the limits of detection by
immunostaining in the latter 3 cell lineages. Only 10% of CCK
cells coexpress secretin, yet treatment with ganciclovir killed
over 90% of this cell type. We interpret these results to indicate
that relatively low levels of thymidine kinase expression driven
by the secretin gene are sufficient to confer sensitivity to
ganciclovir. The results suggest that cell lineage ablation is a
much more sensitive approach to study enteroendocrine cell
lineage than hormone coexpression. We have previously shown
that the same 1.6 kb of secretin gene 5′ flanking sequence
directs tissue specific, cell-specific and developmentally
regulated expression of reporter genes in transgenic mice,
making it unlikely that the observed ablation of
glucagon/peptide YY, somatostatin and GIP cells resulted from
promiscuous expression of thymidine kinase (Lopez et al.,
1995).
Transgenic mouse studies examining expression of human
growth hormone (hGH) under control of the L-FABP gene in
enteroendocrine cells showed hGH in small intestinal
endocrine cells expressing secretin, CCK, GIP, serotonin,
substance P, and glucagon (GLP-1), suggesting that each of
these cell lineages may share a common developmental
program. In the present work we show expression of the basic
helix-loop-helix transcription factor, BETA2, also known as
neuro D, in cell types targeted by ganciclovir. The expression
of BETA2 in all small intestine enteroendocrine cells provides
further evidence that multiple enteroendocrine cell lineages are
developmentally related.
The close relationship between secretin and CCKexpressing enteroendocrine cells is further highlighted by the
failure of mice containing a targeted deletion of BETA2 to
develop either secretin or CCK cells (Naya et al., 1997). This
protein has a major role in the terminal differentiation of
secretin-expressing enteroendocrine cells by coordinating
transcription of the secretin gene with cell cycle arrest and cell
turnover (Mutoh et al., 1998). The requirement for a functional
BETA2 protein for the development of secretin and CCK cells
is an example of a common differentiation pathway shared by
these two cell lineages. We previously noted the presence of
other enteroendocrine cell lineages in BETA2−/− mice in
contrast to secretin and CCK cells. Thus, this protein may be
a marker of enteroendocrine cells, but is not required for
transcription of genes that lead to expression of GIP, substanceP, glucagon, peptide YY, serotonin and somatostatin.
In summary, enteroendocrine cells appear to differentiate
from progenitor cells expressing more than one hormone
through a multistep process (Fig. 6). Cell lineage ablation
experiments imply the existence of at least two types of
multipotent enteroendocrine progenitor cells. Secretinexpressing enteroendocrine cells differentiate from a
endocrine-committed progenitor cell type that may give rise to
other cell lineages. This earliest progenitor does not express
the secretin gene and in our transgenic mice was not sensitive
Secretin cell ablation 4155
Sec
L
Late
Progenitor
S P 5HT Som GIP
CCK
Gas
X
B. L., HD 17379 and DK 55325 to M. J. T., and IRCC. Policlinico
San Matteo grant 030RCR96/04 to G. R. G. R. was supported by a
United States Public Health Service Fogarty International Fellowship
TW04781 and M. E. C. by a IRCCS. Policlinico San Matteo
fellowship.
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Ablation
Early Committed
Multipotential
Endocrine Cell
Stem Cell
Fig. 6. Model for enteroendocrine cell differentiation in the small
intestine of mouse. The totipotent stem cell generates an early
endocrine-committed multipotent cell type that does not express the
secretin gene. This early progenitor cell can differentiate into a
secretin-expressing progenitor that is sensitive to ablation with
ganciclovir in secretin-HSVTK mice. Most if not all secretin-,
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