Development 126, 4149-4156 (1999) 4149 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 the enteroendocrine cells repopulated the intestine in cells, enterocytes, Paneth cells and goblet cells, arise from normal numbers, suggesting that a common early a common totipotent stem cell located in the mid portion endocrine progenitor was spared. Expression of BETA2, a of the intestinal gland. The secretin-producing (S) cell is basic helix-loop-helix protein essential for differentiation one of at least ten cell types belonging to the diffuse of secretin and cholecystokinin cells was examined in the neuroendocrine system of the gut. We have examined the proximal small intestine. BETA2 expression was seen in developmental relationship between secretin cells and all enteroendocrine cells and not seen in nonendocrine other enteroendocrine cell types by conditional ablation cells. These results suggest that most small intestinal of secretin cells in transgenic mice expressing herpes endocrine cells are developmentally related and that a simplex virus 1 thymidine kinase (HSVTK). Ganciclovir- close developmental relationship exists between secretin- treated mice showed markedly increased numbers of producing S cells and cholecystokinin-producing and L apoptotic cells at the crypt-villus junction. Unexpectedly, type enteroendocrine cells. In addition, our work shows ganciclovir treatment induced nearly complete ablation of the existence of a multipotent endocrine-committed cell enteroendocrine cells expressing cholecystokinin and type and locates this hybrid multipotent cell type to a peptide YY/glucagon (L cells) as well as secretin cells, region of the intestine populated by relatively immature suggesting a close developmental relationship between cells. these three cell types. In addition, ganciclovir reduced the number of enteroendocrine cells producing gastric inhibitory polypeptide, substance-P, somatostatin and Key words: Enteroendocrine cells, Secretin, Intestine, serotonin. During recovery from ganciclovir treatment, Cholecystokinin, Cell ablation, Thymidine kinase

INTRODUCTION in the small intestine of most mammalian species (Chey and Escoffery, 1976; Larsson et al., 1977; Straus and Yalow, 1978) At least 10 different enteroendocrine cell types have been as well as in the colon (Lopez et al., 1995). In addition, secretin described in the small intestine of mammals (Solcia et al., is transiently expressed in insulin-producing β cells of the 1998). Subtype classification relies on the identification of the developing rat endocrine pancreas (Wheeler et al., 1992). main hormonal cell content by immunohistochemical methods Labeling studies with [3H]thymidine indicate that all four as well as on the ultrastructure of their secretory large dense epithelial cell types of the mouse intestine, namely enterocytes, core endocrine granules. Several cell types may coexpress Paneth cells, goblet cells and enteroendocrine cells, originate more than one hormone. The heterogeneity of hormone from a common totipotent stem cell and turnover every 3-4 expression in enteroendocrine cells is believed to reflect their days (Cheng, 1974; Cheng and Leblond, 1974a,b). In rats, peculiar multipotency and is observed in gut neuroendocrine labeling with [3H]thymidine showed that secretin-producing tumors and derived cell lines (Klöppel and Heitz, 1988; cells turnover once every 4-5 days like the other intestinal cell Madsen et al., 1986; Oie et al., 1983; Pilato et al., 1988; types (Inokuchi et al., 1986). The absence of labeled secretin Tischler, 1983). Secretin-producing S cells are located mainly cells earlier than 24 hours after the first [3H]thymidine 4150 G. Rindi and others injection suggested that differentiation towards secretin cells MATERIALS AND METHODS may occur within the crypt in precursor cells expressing secretin at levels below the detection limit of Construction of transgene immunohistochemistry. 1.2 kb of HSVTK coding sequences (−53 to +1438) attached to about Coexpression of more than one hormone by individual 1.2 kb of Simian Virus polyadenylation signal sequences were enteroendocrine cells is consistent with the idea that obtained after BglII digest of the pGK-TK plasmid (a gift from M. enteroendocrine cells arise from multipotential endocrine Low, Portland, Oregon) and subcloned in pBluescriptKS at the BamHI site (pBlueKS-HSVTK). Not-Xho digest from pBlueKS-TK was cells. A fraction of intestinal secretin cells coexpress ′ serotonin and substance P, suggesting a developmental 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 relationship between these two lineages (Cetin, 1990). fragment orientation was confirmed by DNA sequencing before Transgenic mice expressing easily detected reporter genes microinjection. The secretin-TK transgene was separated from like human growth hormone (hGH) in intestinal epithelial plasmid vector sequences by digesting with KpnI-XhoI followed by cells have provided further insight into relationships between agarose gel electrophoresis. different enteroendocrine cell types. The detection of reporter genes such as hGH has proved in some cases to be much more Production of transgenic mice sensitive than directly staining enteroendocrine cells with Purified transgene was microinjected into the pronucleus of B6D2F1 × antibodies directed against specific hormones. Sequences in B6D2F1 mouse embryos and transferred into the oviducts of the first 500 bp of 5′ flanking sequence of the liver fatty acid pseudopregnant CD1 mice (Hogan et al., 1994). Transgenic mice were identified by DNA blot hybridization with 32P random primed labeled binding protein (L-FABP) gene directed expression of a hGH cDNA probes for HSVTK. Transgenic pedigrees were maintained on reporter gene to several enteroendocrine cell types including a CD1 background. secretin cells as well as enterocytes, in which L-FABP is normally expressed (Roth and Gordon, 1990; Roth et al., Ablation procedure 1990). The results suggested complex lineage differentiation Sec-HSVTK mice and controls of about 35 g weight (mean 35.5 ±5.8 pathways. g) were treated with 4.8 mg/day of the nucleotide analog ganciclovir We previously showed that 1.6 kb of 5′ flanking sequence (Citovene, Sintex, Ca, USA) for 5 days (Borrelli et al., 1989). Ablation of the rat secretin gene directed developmentally regulated, by ganciclovir was accomplished by continuous infusion with cell-specific transgene expression in major gastrointestinal subcutaneously implanted osmotic minipumps loaded with either ganciclovir or saline solution according to the manufacturer’s sites of secretin gene expression in transgenic mice. The instructions (Alzet, model 2002, Alza). Controls consisted either of colocalization of this sensitive reporter gene in multiple transgenic mice treated with saline solution or age-matched non- enteroendocrine cell types provided the initial evidence that transgenic CD-1 mice treated with ganciclovir. Some transgenic mice secretin is coexpressed in CCK in the small intestine, and were allowed to recover for 5 weeks after treatment to assess glucagon, peptide-YY and neurotensin cells in the colon. endocrine cell repopulation. At the end of the treatment and recovery These data were consistent with the existence of a period, transgenic mice and controls were killed for histochemical multipotential endocrine-committed cell type from which studies. secretin cells terminally differentiate. However, coexpression Histology and immunohistochemistry of multiple hormones in subpopulations of specific Three samples of about 2 cm each in length from different parts of enteroendocrine cell types cannot establish that a particular the small intestine (duodenum, ileum and terminal ileum) were lineage arises from a common progenitor expressing another removed, fixed by immersion in Bouin’s solution or 10% formalin for hormone. 6-8 hours at room temperature and processed into paraffin wax. Serial We have generated transgenic mice expressing the herpes sections (3-5 µm) were stained with haematoxylin and eosin for simplex 1 virus thymidine kinase (HSVTK) under control of conventional histology and PAS/Alcian blue for identification of 1.6 kb of 5′ rat secretin gene. HSVTK expression in mucins. Immunohistochemical tests were performed using the avidin- transgenic mice is nontoxic. However, the viral enzyme biotinylated peroxidase complex (ABC, Vector, Burlington, USA) renders tissues sensitive to the nucleoside analog ganciclovir, method. The following specific sera were used: anti-HSVTK (W. C. allowing conditional cell ablation. This strategy has been Summers, Yale University, New Haven, CT, USA; rabbit polyclonal, used with success to target cell types for cell-lineage studies 1:2000), secretin (D. Gossens, Free University of Brussels, Belgium; rabbit polyclonal, 1:15,000). Other primary antibodies have been in transgenic mice (Borrelli et al., 1989; Canfield et al., 1996; described previously and include rabbit anti-glucagon (1:3,000), Heyman et al., 1989). The objective of the present study was rabbit anti-cholecystokinin (1:8,000), rabbit anti-gastrin (1:2,000), to target an inducible toxic phenotype to secretin-expressing guinea pig anti-peptide-YY (PYY) (1:15,000), rabbit anti- cells by specific cell ablation in order to (i) investigate the somatostatin (1:3,000), rabbit anti-gastric inhibitory polypeptide (GIP, existence of an endocrine-committed multipotent progenitor 1:1,000), 5-HT (1:5000), rabbit anti-substance-P (1: 2,000), rabbit cells; (ii) identify the developmental lineage relationship anti-neurotensin (1:5,000) (Lopez et al., 1995; Upchurch et al., 1996). between secretin cells and the other enteroendocrine cell Reverse-face sequential immunoperoxidase staining was performed types and (iii) determine the relationship between the putative for HSVTK and secretin immunolocalization. Specificity tests for the endocrine-committed progenitor cells and the nonendocrine immunostaining consisted of absorption of each antiserum with its cell types of the intestinal mucosa. The data reported here homologous antigen (10 nmol/ml of diluted antiserum), omission of either the primary or secondary antibody, and use of control tissue indicate that the secretin cell lineage is most closely related with or without the pertinent antigen. to enteroendocrine cells that express CCK and PYY/glucagon Intestinal tissue from BETA2+/− mice was fixed and stained for β- which appear to arise from a secretin-producing, multipotent, galactosidase activity, embedded, and sectioned at 4 µm as described endocrine-committed cell type. Secretin cells appear to be previously (Mutoh et al., 1998). Following microwave antigen less closely related to 4 additional endocrine cell types. retrieval, sections were immunostained as described previously with Secretin cell ablation 4151 antibodies against secretin, cholecystokinin, GIP, serotonin, magnification revealed small numbers of cells in the mid to substance-P, somatostatin, glucagon, and peptide YY. Primary lower regions of the crypt compartment with pyknotic, antibodies were detected by immunoperoxidase using DAB as a hyperchromatic nuclei and shrunken cytoplasm, features substrate. typical of cells undergoing apoptosis (Fig. 2B, arrows). In DNA fragmentation analysis contrast, we failed to identify apoptotic cells in the crypts of control animals. Some of the dying cell stained for secretin Sections of formalin-fixed, paraffin wax-embedded samples of small intestine from ganciclovir-treated and control mice were analyzed for immunoreactivity (Fig. 3B). DNA fragmentation with a modified TUNEL method (Gavrieli et al., Staining for DNA fragmentation by the TUNEL method in 1993). In brief, after treatment with proteinase K, sections were samples of the small intestine of ganciclovir-treated transgenic incubated with 3′ terminal transferase in the presence of biotin-labeled mice confirmed the histologic observations (Fig. 2B). In dUTP at 37¡C for 2 hours. DNA labeling was revealed using avidin- ganciclovir-treated transgenic mice, numerous cells showed peroxidase-biotin complexes as above. nuclear staining for DNA fragmentation, indicative of apoptosis, in the lower to mid portion of the crypts (Fig. 2C,E). Morphometry and statistical analysis Apoptotic cells were not seen in the crypts of either saline- Only tissue samples correctly oriented along the longitudinal axis treated transgenic mice or in ganciclovir-treated CD1 mice. In were used for quantitative analysis. Immunoreactive cells for 9 addition, relatively rare cells with labeled nuclei were observed gastrointestinal hormones were counted per linear millimeter of mucosal length as previously described (Langhans et al., 1997). Data at the tip of the villus in both transgenic and control mice (Fig. were collected from three different groups of mice: controls, 2C,D). The occasional dying cells at the villus tip probably ganciclovir-treated transgenic mice and ganciclovir-treated transgenic represent apoptosis that occurs during normal epithelial cell mice after recovery. A total of 6 mice, two for each group, were turnover in the intestine. These features indicate that the cell studied. Six to ten observations (mean 7.7) for each cell type were death in the neck zone of the crypt of ganciclovir-treated performed for each mouse in all groups. The length of the mucosa transgenic mice was a specific effect of transgene expression available for counting in each mouse, for each specific hormone and ganciclovir treatment. antiserum ranged from 13.17 mm to 75.27 mm (mean value 37.27 We examined the small intestine from control mice and mm). The mean length of mucosa evaluated was 27.84 mm for ganciclovir-treated transgenic mice with a panel of ten specific controls (range 13.17-38.88 mm, 75 observations), 52.29 mm for antisera directed against secretin and nine gastrointestinal treated transgenic mice (range 29.59-75.27 mm, 70 observations) and 31.34 mm for treated transgenic mice after recovery (range 18.03- hormones. In ganciclovir-treated transgenic mice, secretin 37.69, 63 observations). immunoreactive cells were reduced in number compared to Data were first analyzed for equality of population with the controls (Fig. 3A,B). A significant fraction of the few Kruskal-Wallis test (analysis of variance) to elicit difference between remaining secretin cells appeared abnormal with pyknotic the three groups. P<0.05 was considered statistically significant. nuclei with cytoplasmic shrinkage (Fig. 3D) when compared Parameters statistically different at the previous analysis were then to secretin cells in control animals (Fig. 3C), suggesting that compared by the two sample Wilcoxon rank-sum, Mann-Whitney, test they were undergoing programmed cell death. The numbers of to compare data between two groups of observations. A P value of most other enteroendocrine cells appeared to be reduced in × <0.05 was considered statistically significant after Bonferroni ( 3) addition. correction. A detailed morphometric analysis for 9 intestinal endocrine cell types enabled us to quantitate the effect of ganciclovir RESULTS treatment on different enteroendocrine populations of transgenic mice (Fig. 4). The mean number of secretin cells Thirteen transgenic lineages were established after transgene was reduced by 86% in transgenic mice as compared to control microinjection. Two transgenic pedigrees with the highest transgenic animals treated with saline (Fig. 4). Unexpectedly, enzymatic activity were selected for further studies with cells expressing CCK (89% reduction), glucagon (85%) and ganciclovir (nos. 933 and 995). Both pedigrees showed a peptide YY (88%) were similarly reduced in number in similar phenotype following treatment and one (no. 995) was ganciclovir-treated transgenic mice, suggesting a close used for subsequent detailed studies. Transgenic mice were developmental relationship between these cell lineages. normal in size and reproduced normally. To assess the correct Several populations were partially depleted by ganciclovir tissue-specific expression of the transgene, samples of the treatment, including GIP cells (59%), substance-P (58%), small intestine of adult transgenic mice were investigated by somatostatin (55%) and 5HT (45%) cells. In contrast, gastrin- immunohistochemistry with an antiserum specific for HSVTK. immunoreactive cells decreased only by 13% suggesting that HSVTK immunoreactivity was detected in discrete mucosal they were not significantly affected by ganciclovir treatment. cells of the small intestine (Fig. 1A) which coexpressed The number of endocrine cells of transgenic mice returned to secretin in the adjacent section (Fig. 1B), thus confirming that values similar to controls after 5 weeks recovery, indicating transgene expression was specifically directed to secretin cells. that the stem cell population was spared (Fig. 4). The extremely The small intestine from ganciclovir-treated transgenic mice low number of neurotensin-expressing cells in the small showed relatively normal crypt-villus cytoarchitecture intestine precluded meaningful quantitative analysis of this indicating that most cells in the intestine were spared (Fig. 2A). lineage. The equality of population test (variance analysis) Paneth cells, enterocytes and goblet cells appeared relatively revealed that observed differences between ganciclovir-treated normal (not shown). Thus the drug did not induce relatively transgenic mice and either controls or transgenic animals nonspecific, generalized ablation that could potentially occur postrecovery were statistically significant (P≤0.006) for all due to reuptake of phosphorylated ganciclovir released enteroendocrine cell types examined except gastrin- from correctly targeted dying cells. Examination at higher immunoreactive (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.

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 secretin- and 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 Fig. 2. Ganciclovir targets cells in the crypt region of the small described, containing a targeted deletion in a single BETA2 intestine of SEC-HSVTK transgenic mice. Note the preserved general architecture of the small intestine, with normal villi (A). At allele. Most of the region coding for BETA2 was replaced with higher magnification (B) apoptotic cell bodies are visible in the neck an in frame fusion of the lacZ reporter gene, allowing us to area (arrowheads). The analysis of DNA fragmentation by the identify cells expressing β-galactosidase activity by X-gal TUNEL method identified several nuclei labeled in cells at the tip of histochemistry (Naya et al., 1997). The sensitivity of this the villi (C and enlarged in d) and cell bodies in the same neck zone method is far greater than immunostaining with BETA2 as in b (C and enlarged in E). Hematoxylin and eosin staining (A,B), antibodies described earlier (Mutoh et al., 1997). Nuclear β- scale bar (A) 164 µm, (B) 66 µm; TUNEL method, peroxidase (C- galactosidase activity was seen in each of the small intestinal E), scale bar (C) 164 µm, (D,E) 41 µm. 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 other than gastrin cells (Mann-Whitney test) showed that found. However, the majority of the peptide YY/glucagon cells results from treated transgenic mice were statistically different showed β-galactosidase activity. To determine if BETA2 is when compared to those of controls or transgenic mice after expressed only in enteroendocrine cells, we stained sections of Secretin cell ablation 4153 small intestine for both β-galactosidase activity and 8 6 7 chromogranin A, a marker for endocrine secretory granules. 7 5 6 All chromogranin A immunoreactive cells showed staining for 6 β 5 -galactosidase activity and virtually all X-gal-stained cells 5 4 4 showed colocalized staining for chromogranin A, indicating 4 3 that most if not all small intestinal enteroendocrine cells 3 3 express BETA2 and that BETA2 expression is restricted to 2 cells/mm length 2 endocrine cells in the intestine. 2 * 1 1 1 * * 0 0 0 secretin CCK peptide YY(PYY) DISCUSSION 7 8 4 6 7 This paper describes the effect of inducible selective ablation 5 6 of secretin cells in the small intestine of transgenic mice. The 3 5 1.6 kb of 5′ sequence used to drive expression of herpes virus 4 4 * 1 thymidine kinase (HSVTK) is sufficient to direct 2 3 * 3 developmentally regulated tissue and cell-specific expression 2 of reporter genes in transgenic mice (Lopez et al., 1995). The cells/mm length 1 * 2 sensitivity of secretin-producing enteroendocrine cells to 1 1 0 0 0 ablation after 5 days treatment with ganciclovir indicates that glucagon GIP substance P the secretin gene is expressed in proliferating cells. Prior to the present work, it was believed that expression of secretin was 4 25 12 10 restricted to terminally differentiated, non-proliferating cells 20 based on the localization of secretin cells exclusively to the 3 8 nondividing villus compartment of the small intestine as 15 opposed to the proliferating crypt compartment. The failure of 2 6 * 10 secretin cells to incorporate radiolabel from a single injection * 4 3 of [ H]thymidine into rats provided additional evidence that 1 secretin cells were nonreplicating. cells/mm length 5 2 3 Continuous [ H]thymidine labeling showed increased 0 0 0 incorporation of the isotope into secretin cells with all cells somatostatin serotonin gastrin labeled after 5 days, the renewal time for small intestinal Controls Recovery enteroendocrine cells. Thus secretin cells were believed to Treatment arise from a less differentiated, continuously self-renewing progenitor cell. The present work supports the existence of a Fig. 4. Effect of ganciclovir treatment on enteroendocrine cell proliferating progenitor cell and indicates that a subpopulation populations of the small intestine in control mice, transgenic mice, of cells destined to differentiate into secretin cells continue to and transgenic mice after recovery. Open bars show results from divide and express the secretin gene. This subpopulation of control mice, black bars from transgenic mice treated with ganciclovir, and striped bars from ganciclovir-treated transgenic mice cells appears to express secretin at levels below the limits of allowed to recover after treatment. Results shown as mean ± s.d. of detection by immunohistochemistry. the number of immunoreactive cells observed per linear mm of Histology and DNA fragmentation analyses indicate that the mucosa. Mann-Whitney test (see Material and Methods); cells targeted by the nucleoside analog in transgenic mice are *statistically significant difference observed between treated located in the mid portion of the crypt in contrast to mature transgenic mice and the other two groups (observed P ranging from secretin cells found mainly in the villus compartment. The ≤0.0008 to 0.03). 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 secretin- expressing 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

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). 4154 G. Rindi and others enteroendocrine progenitor cells segregate from nonendocrine small intestine of transgenic mice. However, the transgene was lineages and stem cells. This population of immature cells not expressed in small intestine endocrine cells expressing expresses relatively low levels of secretin and thymidine kinase glucagon, peptide YY, somatostatin, GIP or gastrin. The yet is ablated by ganciclovir due to the sensitivity of cells to ablation of a significant fraction of glucagon/peptide YY, the drug. Continuous infusion of ganciclovir for 5 days, the somatostatin and GIP cells reported here was not anticipated approximate turnover time for secretin cells, resulted in in light of our earlier observations that the secretin gene or a depletion of greater than 90% of secretin cells. Thus, it is likely secretin promoter driven reporter did not appear to be that the nucleoside is not directly targeting mature villus coexpressed in these cells (Lopez et al., 1995). Secretin secretin cells but rather, an immature, proliferating progenitor expression may fall below the limits of detection by population. immunostaining in the latter 3 cell lineages. Only 10% of CCK A small fraction of secretin cells (about 10%) survived after cells coexpress secretin, yet treatment with ganciclovir killed 5 days of ganciclovir treatment. These cells may represent a over 90% of this cell type. We interpret these results to indicate small subpopulation of terminally differentiated secretin cells that relatively low levels of thymidine kinase expression driven that turnover more slowly. It is also possible that these cells by the secretin gene are sufficient to confer sensitivity to were targeted by the nucleoside but are only just entering the ganciclovir. The results suggest that cell lineage ablation is a apoptotic pathway and have yet to show morphologic changes much more sensitive approach to study enteroendocrine cell consistent with programmed cell death. Although unlikely, we lineage than hormone coexpression. We have previously shown cannot exclude the possibility that the transgene may not be that the same 1.6 kb of secretin gene 5′ flanking sequence expressed in the few secretin cells surviving ablation. directs tissue specific, cell-specific and developmentally It is well established that enteroendocrine cells frequently regulated expression of reporter genes in transgenic mice, express more than one hormone. Coexpression of serotonin making it unlikely that the observed ablation of (5HT) and substance-P in a fraction of secretin cells has been glucagon/peptide YY, somatostatin and GIP cells resulted from well documented (Cetin, 1990; Roth and Gordon, 1990; Roth promiscuous expression of thymidine kinase (Lopez et al., et al., 1990). Subpopulations of small intestinal CCK- 1995). expressing (Lopez et al., 1995) and glucagon- (GLP-1) Transgenic mouse studies examining expression of human expressing cells (Aiken et al., 1994) coexpress secretin, further growth hormone (hGH) under control of the L-FABP gene in suggesting that S type enteroendocrine cells are related to enteroendocrine cells showed hGH in small intestinal several other enteroendocrine lineages. Ganciclovir treatment endocrine cells expressing secretin, CCK, GIP, serotonin, of SEC-HSVTK mice resulted in depletion of CCK-expressing substance P, and glucagon (GLP-1), suggesting that each of and PYY/glucagon-expressing (type L cells) enteroendocrine these cell lineages may share a common developmental cells in addition to secretin cells. This finding suggests a much program. In the present work we show expression of the basic closer lineage relationship between secretin cells, CCK and helix-loop-helix transcription factor, BETA2, also known as PYY/glucagon cells than was previously appreciated from neuro D, in cell types targeted by ganciclovir. The expression coexpression studies. The data presented here confirms that at of BETA2 in all small intestine enteroendocrine cells provides least CCK and PYY/glucagon cells share a common further evidence that multiple enteroendocrine cell lineages are differentiation pathway with secretin cells until almost developmentally related. terminally differentiated. The close relationship between secretin and CCK- Ganciclovir treatment of Sec-HSVTK transgenic mice expressing enteroendocrine cells is further highlighted by the depleted cell lineages producing GIP, substance-P, serotonin failure of mice containing a targeted deletion of BETA2 to and somatostatin to a lesser extent than cells producing develop either secretin or CCK cells (Naya et al., 1997). This secretin, CCK and PYY/glucagon. This finding may suggest protein has a major role in the terminal differentiation of that such cell types are also related to secretin cells in their secretin-expressing enteroendocrine cells by coordinating lineage differentiation pathway. Alternatively, selected transcription of the secretin gene with cell cycle arrest and cell subpopulations of these four cell types may be able to express turnover (Mutoh et al., 1998). The requirement for a functional secretin before terminal differentiation. Although unlikely, we BETA2 protein for the development of secretin and CCK cells cannot exclude that loss of secretin after ablation may reduce is an example of a common differentiation pathway shared by expression of other hormones leading to an apparent depletion these two cell lineages. We previously noted the presence of of its cell type. In contrast to the other small intestinal other enteroendocrine cell lineages in BETA2−/− mice in endocrine cell lineages, gastrin-producing cells were contrast to secretin and CCK cells. Thus, this protein may be unaffected by treatment with ganciclovir, indicating a separate a marker of enteroendocrine cells, but is not required for differentiation pathway with respect to secretin cells. After transcription of genes that lead to expression of GIP, substance- cessation of ganciclovir treatment, each of the depleted cell P, glucagon, peptide YY, serotonin and somatostatin. lineages repopulates the intestine to pretreatment numbers. The In summary, enteroendocrine cells appear to differentiate ability of each enteroendocrine lineage to recover suggests that from progenitor cells expressing more than one hormone each lineage arises from an earlier progenitor/stem cell that through a multistep process (Fig. 6). Cell lineage ablation does not express the secretin gene and is therefore not targeted experiments imply the existence of at least two types of by ganciclovir. multipotent enteroendocrine progenitor cells. Secretin- We previously showed that the same region of the secretin expressing enteroendocrine cells differentiate from a gene 5′ flanking sequence directed the expression of a human endocrine-committed progenitor cell type that may give rise to growth hormone reporter gene to secretin cells as well as other cell lineages. This earliest progenitor does not express subpopulations of CCK, substance-P, and serotonin cells in the the secretin gene and in our transgenic mice was not sensitive Secretin cell ablation 4155

B. L., HD 17379 and DK 55325 to M. J. T., and IRCC. Policlinico Sec L CCK S P 5HT Som GIP San Matteo grant 030RCR96/04 to G. R. G. R. was supported by a Gas United States Public Health Service Fogarty International Fellowship TW04781 and M. E. C. by a IRCCS. Policlinico San Matteo fellowship.

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At least three terminally obliteration: creation of transgenic mice with controlled immune dificiency. differentiated enteroendocrine lineages including secretin Proc. Natl. Acad. Sci. USA 86, 2698-2702. cells, CCK cells and glucagon/peptide YY cells segregate from Hogan, B., Costantini, F. and Lacy, E. (1994). Manipulating the Mouse Embryo. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory. these late progenitor cells as an obligate intermediate step. Cell Inokuchi, H., Fujimoto, S., Hattori, T. and Kawai, K. (1986). Tritiated lineage ablation experiments also indicate that some thymidine radioautographic study on the origin and renewal of secretin cells enteroendocrine cells do not arise from the secretin-expressing in the rat duodenum. Gastroenterology 89, 1014-1020. intermediate cells as is likely for gastrin-expressing cells. Other Klöppel, G. and Heitz, P. U. (1988). Pancreatic endocrine tumors. Pathol. cell lineages expressing GIP, substance-P, somatostatin and Res. Pract. 183, 155-168. Langhans, N., Rindi, G., Chiu, M., Rehfeld, J. F., Ardman, B., Beinborn, serotonin may terminally differentiate through two distinct M. and Kopin, A. S. (1997). Abnormal gastric histology and decreased acid pathways. Approximately half of enteroendocrine cells production in cholecystokinin-B/gastrin receptor-deficient mice. expressing substance-P, 5HT, GIP and somatostatin arise from Gastroenterology 112, 280-286. the secretin-expressing progenitor cell type whereas the other Larsson, L. I., Sundler, F., Alumets, J., Håkanson, R., Schaffalinsky De Muckadell, D. B. and Fahrenkrug, J. (1977). Distribution, ontogeny and half differentiate from an earlier committed endocrine cell ultrastucture of the mammalian secretin cell. Cell Tissue Res. 181, 361-368. progenitor without ever expressing the secretin gene. Lopez, M. J., Upchurch, B. H., Rindi, G. and Leiter, A. B. (1995). Studies Our data reveal a much closer developmental relationship intransgenic mice reveal potential relatioships between secretin-producing between different enteroendocrine cell types than was cells and other endocrine cell types. J. Biol. Chem. 270, 885-981. previously appreciated from hormone coexpression studies, Madsen, O. D., Larsson, L. I., Rehfeld, J. F., Schwartz, T. W., Lernamark, A., Labrecque, A. D. and Steiner, D. F. (1986). Cloned cell lines from a attesting to the sensitivity of cell lineage ablation as a method transplantable islet cell tumor are heterogeneous and express for studying cell lineage relationships. This work raises several cholecystokinin in addition to islet hormones. J. Cell Biol. 103, 2025-2034. interesting questions about how intestinal endocrine cells Mutoh, H., Fung, B., Naya, F., Tsai, M.-J., Nishitani, J. and Leiter, A. B. differentiate. First, does each cell type differentiate from the (1997). The basic helix-loop-helix transcription factor BETA2/NeuroD is expressed in mammalian enteroendocrine cells and activates secretin gene proliferating multipotent progenitor on the basis of a expression. Proc. Natl. Acad. Sci. USA 94, 3560-3564. predetermined differentiation cascade or (and) in response to Mutoh, H., Naya, F. J., Tsai, M. J. and Leiter, A. B. (1998). The basic helix- specific physiological stimuli? Secondly, since a number of loop-helix protein BETA2 interacts with p300 to coordinate differentiation enteroendocrine cells share a significant part of their of secretin-expressing enteroendocrine cells. Genes Dev. 12, 820-830. differentiation pathways, can the phenotype change so that one Naya, F. J., Huang, H. P., Qui, Y., Mutoh, H., DeMayo, F. J., Leiter, A. B. and Tsai, M. J. (1997). Diabetes defective pancreatic morphogenesis, and cell type may differentiate into another? abnormal enteroendocrine differentiation in BETA2/neuroD-deficient mice. Genes Dev. 11, 2323-2334. This work was supported in part by NIH grants DK43673, Oie, H. K., Gazdar, A. F., Minna, J. D., Weir, G. C. and Baylin, S. B. (1983). DK52870, the GRASP Digestive Disease Center P30-DK34928 to A. Clonal analysis of insulin and somatostatin secretion and L-DOPA 4156 G. Rindi and others

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