(Received for publication, August 12, 1994; and in revised form, October 25, 1994)
From the
We have produced transgenic mice expressing fusion genes
consisting of 1.6 kilobase pairs of the secretin gene 5` flanking
region to direct the expression of human growth hormone (hGH) or simian
virus 40 large T antigen to secretin-producing cells. Analysis of
different mouse tissues for hGH transcripts revealed expression in each
of the major secretin-producing tissues, namely the intestine and
endocrine pancreas. Multiple label immunohistochemistry demonstrated
that the transgene was correctly directed to secretin cells in the
intestinal tract, including a previously unrecognized population of
secretin cells in the colon of adult and developing mice. In the small
intestine, subpopulations of hGH-containing cells frequently
coexpressed substance P, serotonin, and cholecystokinin, whereas in the
colon, cells expressing hGH frequently coexpressed glucagon, peptide
YY, or neurotensin. Transgenic mice expressing large T antigen in
secretin cells developed poorly differentiated neuroendocrine tumors of
the small intestine, well differentiated colonic tumors containing
glucagon-expressing cells, and insulin-producing tumors in pancreas.
These studies indicate that the major cis-regulatory sequences
necessary for secretin expression in enteroendocrine cells and fetal
islets are localized with 1.6 kilobase pairs of the transcriptional
start site. Coexpression of reporter transgenes with several
gastrointestinal hormones suggests a potential relationships between
secretin cells and other enteroendocrine cell types, as well as
pancreatic cells.
The hormone secretin is produced by specific enteroendocrine
cells, S cells, in the proximal small intestine of most mammalian
species(1) . Labeling with [H]thymidine
in rats indicated that secretin-producing cells turnover approximately
once every 4-5 days, similar to other small intestinal endocrine
cells(2) . Although the duodenum and jejunum are the major
sites of secretin production, secretin gene expression also occurs in
the ileum and colon in some species(3, 4) . In the rat
intestine, secretin cells first appear on approximately day 16 of
gestation(5) . In addition, secretin is transiently expressed
in a subpopulation of
cells in rat developing pancreatic
islets(6) . In the central nervous system, secretin
immunoreactivity was reported by several groups (7, 8) but not by others(3) . With the
availability of a cloned secretin cDNA, sensitive and specific RNA
blot-hybridization assays indicated that the central nervous system was
not a major secretin-producing tissue(4) .
Little is known
about how intestinal stem cells become committed to the enteroendocrine
pathway of differentiation, nor is it clear how each of the
approximately 15 enteroendocrine cell types differentiate. Transgenic
mice represent a powerful system for gaining insight about
cell-specific and developmentally regulated gene expression in
enteroendocrine cells and provide a sensitive genetic approach to
label-related neuroendocrine cells. Expression of reporter genes in
specific cell types of the pancreatic islets of transgenic mice as well
as developmental studies in normal mice have demonstrated that each of
the four lineages arises from a multipotential endocrine
cell(9, 10, 11) . Most recent evidence
suggests that the hormone peptide YY may be one of the earliest markers
of endocrine commitment in the developing islet cells(11) .
Similarly, transgenic mice expressing human growth hormone (hGH) ()under control of the liver fatty acid binding protein gene
5`-flanking sequence have been used to infer potential lineage
relationships among enteroendocrine cells. Liver fatty acid binding
protein is normally expressed in enterocytes, hepatocytes, and a
subpopulation of serotonin-producing enteroendocrine cells. In the
small intestine, the liver fatty acid binding protein-hGH transgene was
expressed in most cholecystokinin, gastric inhibitory polypeptide,
secretin, and glucagon cells (12) and in 10-40% of
substance P- and serotonin-producing cells(13) . In the colon,
cells producing glucagon, peptide YY, neurotensin, and cholecystokinin
coexpressed the transgene, whereas substance P- and serotonin-producing
cells did not. These findings suggested that cells producing peptide
YY, glucagon, neurotensin, and cholecystokinin represent one branch of
the colonic enteroendocrine lineage, whereas cells expressing serotonin
and/or substance P represent the other(14) .
We have previously described a positive cis-regulatory domain of the rat secretin gene localized within 200 bp of the transcriptional start site, which is capable of enhancing its transcription in secretin-producing cell lines(6) . The aim of the present work is to characterize secretin cell differentiation in transgenic mice. Here we have expressed two reporter genes, human growth hormone and simian virus 40 large T antigen (Tag) under control of a 1.6-kb fragment of the rat secretin gene 5`-flanking region in transgenic mice. The hybrid genes contain the sequence information necessary for developmentally regulated reporter gene expression in all major secretin-producing tissues. The results suggest that enhancers necessary for secretin gene transcription in normal secretin cells are also contained within 1.6 kb of the transcriptional start site. Furthermore, the expression of reporter genes in transgenic mice identified several additional extraintestinal tissues where the secretin gene is normally transcribed at low levels. Detailed examination of transgene expression in the enteroendocrine population revealed potential relationships between S cells and several other endocrine cell types.
Immunoperoxidase labeling was performed with a Vectastain ABC kit (Vector Labs) using 3,3`-diaminobenzidine or aminoethylcarbazole precipitation for detection. Nuclear Tag detection was enhanced with subtilisin digestion and 3,3`-diaminobenzidine-nickel chloride precipitation(17) . Cytoplasmic hormone immunoreactivity was detected with conventional 3,3`-diaminobenzidine precipitate (brown), in Tag-stained cells. Hormone co-localization was determined by double immunofluorescent labeling with FITC-, and Texas red-conjugated, donkey anti-guinea pig, anti-rabbit, and anti-mouse IgG secondary antibodies that were immunoabsorbed for multiple labeling (Jackson ImmunoResearch Laboratories). Single labeled sections incubated with mismatched secondary antibodies showed no immunostaining, confirming the specificity of the secondary antisera. Immunofluorescence was observed on an Olympus BH-2 microscope fitted with appropriate barrier filters to achieve complete color separation for the different fluorophores. When primary antisera were from the same species, hormones were colocalized in reverse-face serial 3 µm sections by single immunoperoxidase staining.
Figure 1: Tissue distribution of secretin and human growth hormone mRNA in secretin-hGH transgenic mouse. Forty µg of total cellular RNA from different transgenic mouse tissues was analyzed by ribonuclease protection assays using hGH exon 5 and mouse secretin cDNA sequences to generate antisense RNA probes. The mobilities of the secretin and hGH probes and their predicted protected fragments of 334 and 182 bases, respectively, are indicated in the margin. The panels under B are longer exposures of the same gel as under A. Autoradiographs were exposed with two intensifying screens for 36 h (A), 10 days (B), and 7 days (C).
Developmental regulation of secretin gene expression in the mouse appeared to be similar to the rat(6) . Secretin transcripts were readily detected in the fetal pancreas at e15.5 but not in the adult pancreas (Fig. 1). However, expression of the transgene in the pancreas continued after birth at low levels (Fig. 1). The transgene was not expressed in several nonsecretin-producing tissues including stomach (corpus and antrum), brain, skeletal muscle, and kidney. hGH transcripts were unexpectedly expressed in relatively high abundance in the spleen, liver, testis, and thymus. With prolonged autoradiographic exposure, secretin transcripts were detected at low levels in (Fig. 1, B and C) colon, testis, spleen, and thymus but not in the liver. Although RNase protection assays could not detect secretin transcripts in mouse liver, an appropriately sized fragment could be amplified by reverse transcription polymerase chain reaction. The unexpected extraintestinal expression of the transgene was identical in all three pedigrees, indicating that expression in these tissues did not result from host chromosomal DNA sequences near the integration site.
Figure 2: Expression of the secretin-hGH transgene in small intestine and fetal pancreas. a-f, tissues from an adult transgenic mouse stained for different hormones. Scalebar = 14 µm. a and b, double immunofluorescent staining for secretin (a) and hGH (b) in the same cell localized with Texas red and FITC-conjugated secondary antibodies, respectively. c and d, cholecystokinin-containing cells (c, Texas red), one of which coexpresses hGH (d, FITC). e and f, reverse face consecutive sections showing a single intestinal mucosal cell with immunoperoxidase staining for secretin (e) and cholecystokinin (f). g, immunoperoxidase staining for hGH in a single pancreatic islet cell of a transgenic fetus at day 18 of gestation. Scalebar = 25 µm.
In the
colon of late fetal (e17, e18) and newborn mice, single cells
expressing both secretin and hGH were visualized by double
immunofluorescence (Fig. 3, a and b). All hGH
containing cells in the colon stained for secretin. Most
secretin/hGH
cells appeared to
coexpress glucagon (Fig. 3, c and d) and
peptide YY consistent with the known coexpression of these latter two
hormones (14) in enteroglucagon cells. In addition,
hGH
cells in the colon often revealed immunostaining
for neurotensin (Fig. 3, e and f). The
transgene was not expressed in either serotonin or substance P
immunoreactive cells in the colon. Occasional human growth hormone
positive cells were also seen in pancreatic islets late in fetal
development (Fig. 2g).
Figure 3: Secretin expression in enteroendocrine cells of the developing colon. Sections from a newborn (p 0.5) transgenic mouse. a and b, double immunofluorescent staining of a single colonic mucosal cell stained for secretin (a, Texas red) and hGH (b, FITC). c and d, single cell staining for secretin (c, Texas red) and glucagon (d, FITC) immunoreactivity. e and f, single cell with immunofluorescent staining for hGH (e, Texas red) and neurotensin (f, FITC). Bar = 22 µm.
Figure 4: Large T antigen expression in single duodenal enteroendocrine cells. a, double immunoperoxidase staining showing black nuclear Tag staining that co-localized in a single cell with brown (lighter) cytoplasmic staining for secretin in a 12-week-old adult transgenic mouse. b, colocalization of nuclear Tag staining in a cell with brown cytoplasmic staining for cholecystokinin. Scalebar = 14 µm.
Figure 5: Development of intestinal tumors in secretin-Tag transgenic mice. a-c, small intestine. a, small intramucosal tumor swelling and distorting the normal structure of a single villus with ulceration in a 4-week-old transgenic mouse. Hematoxylin and eosin, scalebar = 100 µm; b, large tumor showing showing strong PGP 9.5 immunoperoxidase staining in a 12-week-old mouse. Note a strongly positive neuronal cell body in the myenteric plexus (lowerright). Scalebar = 100 µm. c, strong nuclear Tag-immunoreactivity in spindle-shaped cells of the tumor shown in b. Note the lack of any immunoreactivity in the nuclei of the epithelial cell of the glands at the lowerright and those in the upperleft portions of the micrograph. Immunoperoxidase. bar = 50 µm. d-f, large intestine, small intramucosal tumor developing in the proximal colon of an adult transgenic mouse. The tumor effaces the normal gland structure of the colonic mucosa (d) and is composed by cells showing strong nuclear Tag immunoreactivity (e) and diffuse glucagon immunoreactivity in the majority of cells (f). Consecutive frozen sections; hematoxylin and eosin (d); immunoperoxidase (e and f); scalebar = 45 µm.
Figure 6: Pancreatic islet cell tumors in secretin-Tag transgenic mice. a, low power photomicrograph of an adult transgenic mouse pancreas showing two large tumors, several smaller tumors, and dysplastic islets (small arrows). Compare with one single islet showing normal size and structure (large arrow). Hemotoxylin and eosin; scalebar = 200 µm. b, cytoplasmic immunostaining for insulin in a large pancreatic islet tumor. Immunoperoxidase, ABC method, bar = 50 µm. c, nuclear large T antigen immunostaining from the same tumor shown in b. Compare with negative nuclei of the exocrine parenchyma (upperright part of the micrograph). Immunoperoxidase, ABC method, bar = 50 µm.
This work describes the expression of reporter proteins in secretin-producing cells of transgenic mice. The results provide new information regarding the tissue and cell-specific regulation of secretin gene expression, potential relationships between secretin-producing cells and other endocrine cell types, and neuroendocrine tumorigenesis in the intestinal tract and pancreas.
Our data indicate that 1.6 kb of rat secretin gene 5`-flanking sequence contained sufficient information to direct expression of reporter genes to all established secretin-producing tissues. The reporter genes, hGH and Tag, were expressed in secretin-producing enteroendocrine cells in parallel with the endogenous gene, indicating that the transgene contained the necessary cis-active regulatory sequences to enhance transcription in the major secretin-producing cell type at the appropriate times in development. The distribution of secretin mRNA in the mouse intestine, with highest abundance in duodenum and jejunum, paralleled the distribution of secretin immunoreactivity in most mammalian species(4) . The expression of both transgenes as well as endogenous secretin gene mRNA in the colon and the developing endocrine pancreas extends and confirms earlier observations reporting secretin gene expression in these rat tissues(4, 15) . The absence of both hGH transgene and secretin expression in the central nervous system reported here suggests that the central nervous system in the mouse, like the rat, does not represent a major site of secretin gene expression.
The relatively high abundance of hGH transcripts expressed under control of the rat secretin gene in the spleen, testis, thymus, and liver was unanticipated prompting a reexamination of these tissues for expression of the endogenous secretin gene. Sensitive ribonuclease protection assays and cDNA amplification demonstrated the presence of secretin transcripts at low abundance in the spleen, testis, thymus, and possibly in the liver of the mouse. The high level of transgene expression in these four tissues suggests that the 1.6 kb of sequence from the secretin gene included in the transgene does not contain elements that normally restrict secretin gene transcription. The identification of neuroendocrine gene transcripts in spleen and thymus is not without precedent since other gut hormones, including somatostatin, have been described in cells of the immune system(23, 24) .
Expression of the viral oncoprotein, SV40 large T antigen in transgenic mice under the control of the secretin gene produced tumors in the small intestine, colon, pancreatic islets, liver, spleen, and thymus. In the small intestine, the transgene was frequently expressed in single enteroendocrine cells eventually giving rise to discrete tumors. Other investigators have attributed the difficulty in generating small intestinal tumors by expressing large T antigen to the rapid turnover of the small intestinal epithelium(25) . The 4-day turnover rate of enterocytes, goblet cells, and enteroendocrine cells may not allow sufficient time for transformation to occur. Small intestinal tumors generated in the present study were aggressive, invading the lamina propria and lymphatics very early. In addition, the tumors were also notable for the absence of hormone immunoreactivity, although they continued to express several neuroendocrine marker proteins. Removal of cell proliferation controls induced by Tag may have interfered with the ability of these cells to terminally differentiate. Blocked differentiation has been frequently described in epithelial tissues of transgenic mice expressing Tag, including endocrine cells of the gastrointestinal tract. This phenomenon has resulted in the loss of expression differentiation markers, including the endogenous copy of the gene used to drive expression of the viral oncoprotein(11, 17, 26, 27, 28) . Alternatively, expression of Tag may have interfered with hormone storage and/or release.
Tumors developed less frequently in the proximal large intestine. In contrast to the tumors of the small intestine, the colonic tumors retain a well differentiated endocrine phenotype. Colonic enteroendocrine cells turnover once every 23 days, much more slowly than small intestinal endocrine cells(29) . The likelihood of transformation may be greater with the increased exposure to Tag and may not require the degree of aggressiveness and loss of differentiation seen in the small intestine.
The endocrine
pancreas developed cell tumors involving most islets. The
frequency of islet transformation observed with the secretin-T antigen
transgene was far higher than that observed either with insulin-Tag or
glucagon-Tag transgenes(21, 22) . The principal
hormonal product of these tumors was insulin, thus reinforcing our
earlier observation that secretin is expressed in
cells during
development. These results indicate that a
cell-specific
enhancer, active in vivo, must be localized within 1.6 kb of
the transcriptional start site and are consistent with in vitro DNA transfection studies in insulinoma cells identifying a
cis-active regulatory domain between -174 and
-53(6) . Neoplasms frequently arose in liver, spleen, and
thymus, consistent with the level of transgene expression in each
tissue and may reflect their underlying susceptibility to
transformation. However, the tumors in the latter tissues failed to
stain for the markers PGP 9.5, neuron specific enolase, chromogranin A,
or any gastrointestinal hormone, suggesting that they were not of
neuroendocrine origin.
In the small intestine we report the novel finding of coexpression of secretin and reporter genes in cholecystokinin-producing cells. In addition, the secretin gene 5` region directed reporter gene expression to substance P and serotonin-producing cells in the small intestine, confirming a potential relationship between these enteroendocrine cell types(13) . It has been previously suggested that secretin cells in the small intestine arose from sequential differentiation of substance P-producing enteroendocrine cells originating in the crypt compartment. As substance P cells migrated up the crypt-villus axis, increasing numbers of cells coexpressed serotonin. A subpopulation of the substance P-serotonin cells coexpressed secretin in the villus but not the crypt compartment, suggesting that expression of secretin was a relatively late event in enteroendocrine differentiation.
Secretin cells have not been previously identified in the colon. Here we show that these cells are much more abundant in developing mice than in adults. Colonic secretin cells frequently coexpress glucagon, peptide YY, and neurotensin but not substance P or serotonin. Previously, two distinct lineage branches have been proposed for colonic enteroendocrine cells(14) . One branch, consists of cells producing glucagon, peptide YY, cholecystokinin, and neurotensin. The other branch includes cells producing substance P and serotonin(14) . The data presented here suggest that colonic secretin cells are more closely related to the glucagon/peptide YY/neurotensin/cholecystokinin lineage branch rather than colonic substance P and serotonin cells. Our findings suggest that secretin-producing enteroendocrine cells share a previously unappreciated relationship with cholecystokinin-containing cells (I cells) in the small intestine and with neurotensin and glucagon/peptide YY cells (L cells) in the large intestine.
Tritiated thymidine labeling studies have shown that the four intestinal epithelial cell types, enteroendocrine cells, enterocytes, goblet cells, and Paneth cells, arise from a common stem cell(30) . However, relatively little is known regarding how cells become committed to the endocrine differentiation pathway. The present work suggests a potential relationship among several cell types including secretin, glucagon/peptide YY, cholecystokinin, neurotensin and substance P cells and may indicate the existence of a multipotential endocrine-committted cell type from which secretin cells terminally differentiate. Unequivocal evidence supporting a common progenitor for these enteroendocrine cell types will eventually require genetic ablation studies in transgenic mice.