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,