©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Studies in Transgenic Mice Reveal Potential Relationships between Secretin-producing Cells and Other Endocrine Cell Types (*)

(Received for publication, August 12, 1994; and in revised form, October 25, 1994)

M. James Lopez (§) Brent H. Upchurch Guido Rindi (¶) Andrew B. Leiter (**)

From the Division of Gastroenterology, New England Medical Center, Tufts University School of Medicine, Boston, Massachusetts 02111

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 beta cells.


INTRODUCTION

The hormone secretin is produced by specific enteroendocrine cells, S cells, in the proximal small intestine of most mammalian species(1) . Labeling with [^3H]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 beta 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) (^1)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.


MATERIALS AND METHODS

Construction of Transgenes

A 1.6-kb rat secretin DNA fragment (EcoRI-NcoI) extending from -1600 of the 5`-flanking sequence to +32 in exon 1 was subcloned into a Bluescript plasmid (Stratagene) at the ApaI/EcoRV sites upstream of the coding sequence of SV40 large T antigen (StuI at 5190 bp to BamHI at 2533 bp)(6, 15) . The KpnI-NcoI fragment of secretin gene (-1600 to +32) was inserted upstream of hGH subcloned into Bluescript. The secretin-Tag and secretin hGH transgenes were separated from plasmid vector sequences by digesting with KpnI and KpnI/NotI, respectively, followed by agarose gel electrophoresis.

Production of Transgenic Animals

Purified transgenes were microinjected into the pronucleus of B(6)D(2)F(1) times B(6)D(2)F(1) mouse embryos and transferred into the oviducts of pseudopregnant CD1 mice(16) . Transgenic mice were identified by DNA blot hybridization with P random primed labeled cDNA probes for either human growth hormone probe or large T antigen. Transgenic pedigrees were maintained on a CD1 background.

Isolation of a Mouse Secretin cDNA

Oligo(dT)-primed first strand cDNA was synthesized from RNA extracted from the murine secretin-producing STC-1 cell line with murine Moloney virus reverse transcriptase(17) . A short mouse secretin cDNA was amplified with mixed primers (GAGTGGATCCACTCGGACGG(C/G)AC(C/G)TT(T/C)ACNAG(T/C); GAGTGCATGCGCTTCCCCACNAGGCCT) encoding the amino acids at the amino and carboxyl termini of secretin and sequenced. A short oligonucleotide (AGCTCAGCCGCTTGCAGGACAGT, sense strand) corresponding to the amplified mouse sequence was used to amplify the 3` end of a mouse secretin mRNA (18) .

Ribonuclease Protection Assay

Total RNA was isolated and quantitated as described previously(6) . Fetal tissues were pooled from several animals (3, 4, 5, 6) to obtain adequate amounts of RNA for analysis. Abundance of secretin and transgene-generated transcripts were determined by ribonuclease protection assays. RNA samples were hybridized overnight at 45 °C with both secretin and hGH cRNA probes or individually with each probe with identical results. Assays using the secretin probe alone revealed that none of the smaller fragments generated were of comparable size or abundance with the major hGH protected fragment. For the analysis of the mouse tissues for secretin, a 409-bp probe consisting of 334 bp of mouse cDNA 3` end, extending from the protein coding sequence to the polyadenylation site, and 75 bp of vector were utilized. The template for the hGH probe consisted of a 335-bp SmaI-NarI fragment of the hGH gene containing 182 bp of exon 5 and most of intron 4 subcloned into pGEM 7. Following linearization with BglII, a 222-nucleotide probe was generated with T7 RNA polymerase. Verification of equal sample RNA content was carried out in parallel experiments using a mouse actin probe. The hybrids were subsequently digested with RNase A (30 mg/ml) and RNase T1 (2 mg/ml) at 25 °C for 30 min; the protected products were separated on 5% sequencing gels and detected by autoradiography.

Immunohistochemical Analysis

Fetal tissues were obtained from timed pregnancies, using noon on the day of the vaginal plug as day 0.5 (e0.5) of gestation. Tissues were fixed by immersion in Bouin's fixative for 4-6 h at room temperature, washed in phosphate-buffered saline, and stored in 70% ethanol. Fixed tissues were embedded in paraffin and sectioned at 4 µm except as noted where tissue was frozen for cryostat sectioning. Primary antisera for immunohistochemistry included: rabbit anti-T antigen (D. Hanahan, University of California, San Francisco, 1:5000 immunoperoxidase (IP), 1:500 immunofluorescence (IF)); guinea pig anti-human growth hormone (Arnel, 1:20,000 IP, 1:2000 IF); rabbit anti-secretin (W. Chey, University of Rochester, 1:4000 IP, 1:400 IF); rabbit anti-gastric inhibitory polypeptide (A. Buchan, University of British Columbia, 1:1000 IP, 1:100 IF); rabbit anti-substance P (R. Kream, 1:2000 IP, 1:200 IF); rabbit anti-serotonin (IncStar, 1:40,000 IP, 1:4000 IF); rabbit anti-neurotensin (IncStar, 1:5000 IP, 1:500 IF); rabbit anti-cholecystokinin (Chemicon, 1:8000 IP, 1:800 IF); rabbit anti-neuron specific enolase (Dako, 1:500 IP); rabbit anti-PGP9.5 (Ultraclone United Kingdom, 1:5000 IP); rabbit anti-glucagon (M. Appel, University of Massachusetts, 1:3000 IP, 1:300 IF); guinea pig anti-insulin (Sorin Biomedica, 1:5000 IP, 1:500 IF); guinea pig anti-peptide YY (G. Aponte, University of California, Berkeley, 1:3000 IP, 1:300 IF); rabbit anti-somatostatin (R. Lechan, Tufts University, 1:3000 IP, 1:300 IF); and rabbit anti-gastrin (S. Brand, Massachusetts General Hospital, 1:500 IF). Primary incubations were done at 4 °C overnight. Controls included nonimmune primary sera, mismatched primary and secondary antisera, known positive sections, and absorption with specific and heterologous antigens. In all cases, absorption of primary antisera with homologous peptides abolished all immunostaining, and absorption with heterologous antigens did not affect immunostaining. More specifically, preadsorption of secretin and glucagon antisera with glucagon and secretin, respectively, indicated no cross-reactivity under the conditions used since immunostaining was unaffected by the heterologous peptide. Serum hGH levels were measured by radioimmunoassay (Nichols Institute).

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.


RESULTS

Tissue Distribution and Developmental Regulation of the Expression of a Secretin-Human Growth Hormone Transgene

Three lines of transgenic mice were established expressing a hybrid gene consisting of 1600 bp of 5`-flanking sequence plus 32 bp of exon 1 of the rat secretin gene cloned upstream to the human growth hormone gene. Mice appeared to be normal in size in all three pedigrees, although hGH was readily detectable by radioimmunoassay in the serum of the one line tested (transgenic range, 22-54 ng/ml, nontransgenic, 0). To determine whether the 1.6 kb of secretin gene 5`-flanking sequence could direct appropriate expression of the hGH reporter, we examined the distribution of hGH mRNA in adult transgenic mice. Expression of the transgene and endogenous secretin transcripts was examined in cellular RNA from different regions of the alimentary tract, pancreas, spleen, liver, thymus, brain, kidney, skeletal muscle, and testis by ribonuclease protection assays using probes for both hGH and secretin. To optimize the sensitivity and specificity for detecting mouse secretin transcripts, a mouse secretin cDNA was amplified from secretin-producing STC-1 cells and used to generate cRNA probes for ribonuclease protection assays. The results indicate that nucleotides -1600 to +32 in the rat secretin gene direct expression of hGH in different regions of the intestine at levels comparable with the endogenous secretin gene (Fig. 1). The distribution of endogenous secretin gene expression seen in transgenic mice was identical to nontransgenic animals. The transgene was expressed in highest abundance in the duodenum, jejunum, and proximal colon, paralleling the abundance of endogenous secretin transcripts in the mouse intestine. Transcripts from both the endogenous secretin gene and the transgene were first detected in the duodenum as early as day 15.5 of gestation (e15.5), indicating that transcription of the transgene initiated at the appropriate time in development.


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.

Expression of the Transgene Is Directed to Secretin-producing Cells

To determine whether the transgene was expressed in the correct cell types, tissues from each pedigree were examined by multiple label immunohistochemistry for secretin and hGH with identical results in each of the three pedigrees. Human growth hormone immunoreactivity was detected only in the small intestine, the colon, and in occasional cells in fetal pancreatic islets. All enteroendocrine cells staining for hGH also stained for secretin immunoreactivity (Fig. 2, a and b, Table 1). This specificity of expression was present throughout development from the time of onset of expression at embryonic day 15. Examination of other duodenal enteroendocrine cells for hGH coexpression revealed that the transgene was coexpressed in subpopulations (<10%) of cholecystokinin (Fig. 2, c and d). In addition, most cells expressing the transgene were positive for serotonin with a smaller percentage staining for substance P immunoreactivity, confirming earlier observations that substance P and serotonin were coexpressed in secretin cells(13, 19) . Coexpression of secretin and cholecystokinin in single cells was confirmed by immunostaining consecutive reverse face sections of duodenum for secretin and cholecystokinin (Fig. 2, e and f). Examination of the other small intestinal endocrine cell types for transgene expression failed to identify hGH in gastric inhibitory polypeptide, gastrin, neurotensin, and somatostatin cells (Table 1).


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.



Tumorigenesis in Secretin/Large T Antigen Transgenic Mice

The transforming oncoprotein large T antigen of SV40 virus has been shown to delay or block differentiation of a number of cell types including endocrine cells. We established nine founder mice expressing large T antigen under control of the same 1.6 kb of 5`-flanking sequence of the secretin gene used earlier to further characterize secretin cell differentiation. Four founders died at an early age as a result of tumorigenesis. Pedigrees were established from the other five founders. Three of the pedigrees that expressed the transgene were examined in detail and exhibited the same phenotype. Mice usually died by approximately 2-4 months of age because of tumor growth.

Intestinal Tract

Developmental expression of Tag in the intestine was examined in two pedigrees at e12.5, e14.5, e15.5, e17.5, and e18.5 as well as at 1 day, 3 days, 2 weeks, 4 weeks, 8 weeks, and 12 weeks of age. A minimum of six animals were examined at each stage of fetal development, and between 6 and 10 animals were examined after birth. Expression of Tag was first observed at e15.5 in single isolated epithelial cells in the villi of the proximal small intestine and persisted throughout life in adult mice. Expression of hormones was observed in a relatively small percentage of cells expressing the transgene, although small numbers of secretin/Tag (Fig. 4a), cholecystokinin/Tag (Fig. 4b), and substance P/Tag were seen. The cytoplasmic staining for each of these hormones in Tag cells was less intense than in normal enteroendocrine cells, possibly indicating reduced synthesis and storage. Tumors of the small intestine were first seen at 4 weeks of age as small intramucosal growths (Fig. 5a), and by 12 weeks they were large and invasive (Fig. 5b), often associated with mucosal ulceration. However, at this stage of tumorigenesis, secretin or other intestinal hormone immunoreactivity was not observed. Tumor cells, nevertheless, stained intensely for PGP 9.5 (Fig. 5b) and neuron specific enolase, cytoplasmic markers of neuroendocrine differentiation that do not depend on the presence of secretory granules(20) . Small clusters of tumor cells were also immunoreactive for chromogranin A, a marker of endocrine granules(20) , indicating the rare presence of more differentiated neuroendocrine cells. The expression of these three markers confirmed the neuroendocrine origin of these neoplasms despite the poorly differentiated phenotype. Nuclear immunostaining for Tag was present in all tumor cells (Fig. 5c). In the proximal colon, adult mice from several pedigrees developed single mucosal tumors distorting the normal glandular architecture (Fig. 5d). All tumor cells showed nuclear Tag immunoreactivity (Fig. 5e). The majority of tumor cells also revealed intense cytoplasmic staining for glucagon (Fig. 5f) with minor subpopulations staining for secretin immunoreactivity, indicating that colonic tumors have retained a more differentiated neuroendocrine phenotype than the small intestinal neoplasms.


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.



Extraintestinal Expression

The onset of pancreatic expression of the secretin Tag transgene was similar to the endogenous secretin gene. Tag expression was first observed in a small subset of single insulin-producing cells e15.5. Throughout development and after birth, Tag expression was restricted to a subset of insulin-producing cells and was never detected in any of the other islet lineages. By 12 weeks of age, at least 80% or more of the islets were substituted by tumors (Fig. 6a) with features similar to those observed in pancreatic neuroendocrine tumors of other transgenic models (21, 22) . Tumor size could reach a few millimeters in maximum diameter and could be seen macroscopically at autopsy. Most tumor cells were positive for insulin immunoreactivity (Fig. 6b) with variable intensity, and all cells showed nuclear Tag staining (Fig. 6c). None of the other islet hormones, including glucagon, somatostatin, pancreatic polypeptide, peptide YY, or secretin, were detected in islet tumors. Large tumors with nuclear Tag immunostaining frequently arose in the liver, spleen, and thymus with cells resembling lymphoid-like elements. The tumor cells showed no immunoreactivity for PGP 9.5, neuron specific enolase, chromogranin A, secretin, or other intestinal hormones, suggesting that they were not of neuroendocrine origin.


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.




DISCUSSION

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 beta 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 beta cells during development. These results indicate that a beta 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.


FOOTNOTES

*
This work was supported in part by a grant from the American Cancer Society and by grants from the National Institutes of Health (DK43673 and DK07471) and the GRASP Digestive Disease Center (DK34928). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Pediatric Gastroenterology, University of Texas Southwestern Medical School, Dallas, TX 75235.

Supported by Public Health Service Fogarty International Research Fellowship TW04781.

**
To whom correspondence should be addressed: Div. of Gastroenterology, #218, New England Medical Center, 750 Washington St., Boston, MA 02111. Tel.: 617-636-8336; Fax: 617-636-4207.

(^1)
The abbreviations used are: hGH, human growth hormone; bp, base pair(s); kb, kilobase pair(s); IP, immunoperoxidase; IF, immunofluorescence; FITC, fluorescein isothiocyanate; e0.5, embryonic day 0.5; PGP, protein gene product; Tag, T antigen.


ACKNOWLEDGEMENTS

We thank Dr. M. B. Wheeler for providing the secretin-Tag fusion gene.


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