Proc. Natl. Acad. Sci. USAVol. 87, pp. 6408-6412, August 1990Cell Biology

Spatial differentiation of the intestinal epithelium: Analysis ofenteroendocrine cells containing immunoreactive serotonin,secretin, and substance P in normal and transgenic mice

(pattern formation/epithelial differentiation/lineage relationships)

KEVIN A. ROTH*t AND JEFFREY I. GORDONH§Departments of *Pathology, *Medicine, and §Biochemistry and Molecular Biophysics, Washington University School of Medicine, Saint Louis, MO 63110

Communicated by William H. Daughaday, May 18, 1990 (received for review February 19, 1990)

ABSTRACT The mammalian intestinal epithelium under-goes continuous and rapid renewal of its four principal termi-nally differentiated cell types. These cells arise from multipo-tent stem cells located at or near the base of the crypts ofLieberkuhn. The differentiation process is precisely organizedalong two spatial dimensions (axes)-from the crypt to thevillus tip and from the duodenum to the colon. The enteroen-docrine cell population provides a sensitive marker of theintestine's topologic differentiation. At least 15 different re-gionally distributed subsets have been described based on theirprincipal neuroendocrine products. We have used immuno-cytochemical methods to characterize the spatial relationshipsof the serotonin-, secretin-, and substance P-containing ente-roendocrine cell subsets in normal adult C57BL/6J X LT/Svmice as well as in transgenic littermates that contain rat liverfatty acid-binding protein-human growth hormone fusiongenes. Our results reveal precise spatial interrelationshipsbetween these populations and suggest a differentiation path-way that may involve the sequential expression of substance P,serotonin, and secretin.

The mouse intestine is lined with a monolayer of epithelialcells that undergoes rapid and continuous renewal (reviewedin ref. 1). Functionally anchored multipotent stem cellslocated near the base of the crypts of Lieberkuhn give rise todaughter cells that differentiate into several different lineages(2, 3). Differentiation takes place during a well-organizedbipolar migration along the crypt-to-villus axis of the gut.Polarized absorptive enterocytes, mucous-producing gobletcells, and enteroendocrine cells arise as descendants of thesedaughter cells and migrate in vertical coherent bands up thevillus (4) towards the apical extrusion zone.

Enteroendocrine cells represent <1% of the terminallydifferentiated cells in the intestinal mucosa (5). However, thiscell population provides a sensitive marker of the gut'stopologic differentiation. At least 15 different subpopulationshave been defined based on their principal neuroendocrineproducts (6, 7). Each maintains a distinctive geographicdistribution (7, 8).We have used (8) transgenic mice to further characterize

these cells. These animals contained nucleotides -4000 to+21 or -596 to +21 of the rat gene encoding liver fattyacid-binding protein (L-FABP) linked to the gene encodinghuman growth hormone (hGH) (beginning at its nucleotide+3). The intact endogenous mouse L-FABP gene (fabpl) isnormally transcribed in hepatocytes, enterocytes, and someenteroendocrine cells (8, 9). Immunocytochemical studiesrevealed differences in transgene expression between andwithin several enteroendocrine populations that had beenpreviously grouped only on the basis of their principal

neuroendocrine products. These differences in several casescould be correlated with the positions cells occupied alongthe crypt-to-villus and duodenal-to-colonic axes. We pro-posed that transgenes represented a potentially sensitive andpowerful tool for (i) identifying subtle differences in theregulatory environments of enteroendocrine cell subpopula-tions, (it) examining and classifying the relationships betweenenteroendocrine cell subsets, and (iii) defining the molecularmechanisms that establish and maintain cellular and regionaldifferentiation of this class of intestinal epithelial cells.

In the study reported here, we characterized serotonin-,secretin-, and substance P/tachykinin-containing enteroen-docrine cells in normal and transgenic mouse gut. Serotonin-containing cells represent the largest enteroendocrine cellpopulation in the mammalian gastrointestinal tract (7, 8).Many intestinal enteroendocrine tumors (carcinoids) pro-duce large amounts of serotonin and the polypeptide productsof the preprotachykinin gene-substance P and related ta-chykinins (10-13). The tachykinin peptides may producesome of the clinical symptoms associated with carcinoidneoplasms (14). Some have proposed that substance P-immunoreactive cells represent an "independent" cell typeunrelated to serotonergic cells (15), while others have pos-tulated that cells containing immunoreactive substance Prepresent a subpopulation of serotonergic cells (16). Duringthe course of an earlier mapping study of mouse enteroen-docrine populations (8), we observed that some serotonin-producing cells also contained secretin. Therefore, in thiscurrent study we focused on defining the interrelationships,if any, between enteroendocrine cells producing these threeneuroendocrine products. The data "reveal" a model forstudying spatial differentiation of the gut.

MATERIALS AND METHODSAnimals. Mice from several transgenic pedigrees and their

normal littermates (C57BL/6J x LT/Sv) were analyzed. Micecontaining nucleotides -4000 to +21 of the rat L-FABP genelinked to the hGH gene (L-FABP-0 to '21-hGH) werederived from two founders-Gol and G046 (see ref. 9). Obli-gate heterozygotes for the L-FABP-596 to +21-hGH transgenewere obtained from G013 and G,19 (9). Mice were fed astandard chow diet ad libitum and maintained under a 12-hrlight/12-hr dark cycle (lights on between 0600 and 1800). Theywere sacrificed at 4-6 months of age. Protocols for dissectionof their gastrointestinal tracts are provided in ref. 9.Immunocytochemical Methods. A detailed description of

the immunofluorescence and immunogold/silver-enhance-

Abbreviations: L-FABP, liver fatty acid-binding protein; hGH, hu-man growth hormone.tTo whom reprint requests should be addressed at: Department ofPathology, Washington University School of Medicine, 660 SouthEuclid Avenue, Box 8118, Saint Louis, MO 63110.

6408

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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ment detection procedures that we use is available (8).Briefly, sections of paraffin-embedded intestine are deparaf-finized and incubated overnight at 40C with diluted primaryantisera. After several wash steps, immunostaining is de-tected with either gold-labeled second antibodies and silverenhancement (Amersham) or with fluorescent-labeled sec-ond antibodies (Jackson ImmunoResearch).A panel of seven antisera were used for these studies. Their

sources and final dilutions were: rabbit and goat anti-serotonin (1:4000 and 1:800, Incstar, Stillwater, MN), ratanti-serotonin (1:2000, Eugene Tech, Allendale, NJ), rabbitanti-secretin (1:1000, Peninsula Laboratories), rabbit anti-substance P (1:2000, from J. Krause, Washington University;ref. 17), rabbit anti-hGH (1:2000, Dako, Santa Barbara, CA),and goat anti-hGH (1:2000; ref. 18). The specificity of theanti-secretin and anti-substance P sera was verified whenpreincubation of the diluted primary antisera with an excess(1 ,tM) of the synthetic peptide against which they wereraised abolished specific immunostaining. Both secretin andsubstance P antisera were produced by using the respectivecomplete polypeptide sequences. RIAs have revealed thatthe substance P antiserum is highly specific for the COOH-terminal amidated substance P sequence. However, it haslow (<1%) cross-reactivity with other tachykinins (neuroki-nin A, neuropeptide K, neuropeptide y, and neurokinin B),which have similar COOH-terminal sequences (17). Forbrevity we refer to the immunoreactivity of this antiserum assubstance P-like. We cannot exclude the possibility that someof this immunoreactivity is due to other tachykinins. Sub-stance P immunoreactivity was not affected by preincubationwith secretin or serotonin. The specificity of the goat andrabbit hGH antisera are well documented (8, 18).Methods for double-, triple-, and quadruple-label immu-

nocytochemistry are described in ref. 8 and in the legend toFig. 2.

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RESULTS

Spatial Relationships of Serotonin-, Substance P-, and Se-cretin-Containing Enteroendocrine Cells in the Adult MouseIntestine. Fig. 1A summarizes the distribution of cells con-taining immunoreactive serotonin, substance P, and secretinalong the proximal-to-distal (duodenal-to-colonic) axis ofadult C57BL/6J x LT/Sv mouse intestine. Serotonin-producing cells were distributed throughout the length of theintestine. The distribution of substance P- and serotonin-producing cells was qualitatively but not quantitatively sim-ilar throughout the small intestine and proximal colon. How-ever, substance P-immunoreactive cells were not repre-sented in the distal colon. The secretin population was theleast abundant and most restricted in its distribution-confined to the duodenum and jejunum.

Fig. 1B shows that each cell type has a unique distributionalong the crypt-to-villus axis. Secretin-producing cells werefound exclusively on villi-typically in their upper half. Cellswith immunoreactive substance P were primarily located inthe crypts and lower half of the villus (Fig. 2 A and B).Serotonin-containing cells showed an intermediate pattern.

Coexpression of Serotonin, Secretin, and Substance P inSubpopulations of Enteroendocrine Cells. Serotonin/sub-stance P. Serotonin and substance P were frequently coex-pressed in individual enteroendocrine cells (Fig. 2 C and D).Coexpression was related to the location of these cells in boththe crypt-to-villus and duodenal-to-colonic axes. Fig. 1Cshows that -60% of duodenal crypt-associated substanceP-immunoreactive cells contained serotonin. This numberincreased progressively from jejunum to proximal colon (tonearly 100%). Fig. 1D shows that virtually all substanceP-producing cells located on villi produced serotonin and thatthis was true throughout the length of the intestine. Thus, in

FIG. 1. Quantitative analysis of serotonin-, secretin-, and sub-stance P-containing enteroendocrine cells in adult normal and trans-genic C57BL/6J x LT/Sv mice. (A and B) Regional distribution ofserotonin-, secretin-, and substance P-immunoreactive enteroendo-crine cell subpopulations in the mouse intestinal tract. To quantitatethe expression of serotonin (o), secretin (A), and substance P (a) inenteroendocrine cells, at least 10 complete cross-sections of theintestine for each of the regions indicated on the abscissa wereexamined (n = six animals; D, duodenum; PJ, proximaljejunum; DJ,distal jejunum; I, ileum; PC, proximal colon; and DC, distal colon).The number (#) of immunoreactive cells per complete cross-sectionwas counted, and the results were averaged. These data are pre-sented as the average ± SEM in A. The percentage of immuno-reactive cells found on villi in each segment is presented in B. (C-E)Interrelationships of serotonin-, secretin-, and substance P-immu-noreactive enteroendocrine cell subpopulations. For substance P-immunoreactive enteroendocrine cells, we determined the percent-age of coexpressed serotonin or secretin immunoreactivity in eachintestinal segment. The substance P-immunoreactive cells weredivided into crypt (C)- and villus (D)-associated groups. Secretin-immunoreactive cells (found only in villi) were also assessed forcoexpression of serotonin and substance P (E). (F and G) Expressionof hGH in the substance P-immunoreactive enteroendocrine cellsubpopulation of transgenic mice. The percentage of substanceP-immunoreactive cells containing colocalized hGH immunoreactiv-ity was determined for crypt (F)- and villus (G)-associated cells.Segments harvested from two animals from each of the transgenicmice pedigrees containing nucleotides -4000 to +21 (A) and nucle-otides -596 to +21 (e) of the rat L-FABP gene were examined (2-12sections per segment).

the proximal intestine, the percentage of coexpression ofserotonin and substance P was different in crypt and villus(60% versus -100%). This difference disappeared in theileum and proximal colon (Fig. 1 C and D). It is important tonote that the percentage of serotonin-immunoreactive cellsthat contain substance P per cross-section varied markedly

D PJ DJ PCIntestinal Segment

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FIG. 2. Immunohistochemical mapping of enteroendocrine cell subpopulations. (A and B) Tissue from the ileum of a normal mouse stainedwith rabbit anti-substance P serum. Substance P immunoreactivity is found in both the myenteric plexus (open arrows) and in enteroendocrinecells (closed arrows) (A). Preincubation of the substance P antiserum with excess substance P (1 ,uM) completely abolished the immunostainingof both nerves and enteroendocrine cells (B). (C and D) Tissue from a section of normal mouse duodenum. The section was first incubated withrabbit anti-substance P serum, and antigen-antibody complexes were detected by the immunogold/silver-intensification staining (IGSS)procedure. Sections were subsequently incubated with rat anti-serotonin serum, which in turn was detected with f3-phycoerythrin-labeled donkeyanti-rat serum. In C the substance P immunoreactivity was visualized with reflected light polarization microscopy. Strong substance Pimmunostaining is observed in the myenteric plexus in several crypt-associated enteroendocrine cells and in a few low villus-associated cells.In D double exposure of the section shows substance P immunoreactivity as turquoise, serotonin immunostaining as red, and colocalizedsubstance P/serotonin immunoreactivity as yellow. Serotonin immunoreactivity is not present in the substance P-labeled myenteric plexus(arrow heads) but is present in a subpopulation of crypt-associated substance P-immunoreactive enteroendocrine cells and in all thevillus-associated substance P-immunoreactive cells (substance P+/serotonin+ cells are indicated by closed arrows and substance P+/serotonin-cells are indicated by open arrows). We used a combination of IGSS and double- or triple-label immunofluorescence to localize three or fourantigens in a single section. We found that deposition of silver around the complex of primary antiserum and gold-labeled secondary antiserumduring the IGSS procedure effectively abolished its immunoreactivity without interfering with subsequent immunofluorescence staining foradditional antigens (8). Thus, four antigens can be localized in a single section by using two antisera raised in rabbits, a third antiserum raisedin a goat, and a fourth raised in a rat. (E-I) Tissue from the duodenum of a transgenic mouse containing the L-FABP-4 to +21-hGH fusiongene. Multilabeling of this section was performed by using rabbit anti-substance P serum detected by IGSS (E), followed by rat anti-serotonin(F), rabbit anti-secretin (G), and goat anti-hGH (H) sera, which were detected by ,B-phycoerythrin, 7-Amino-4-methylcoumarin-3-acetic acid(AMCA), and fluorescein-labeled donkey anti-rat, anti-rabbit, and anti-goat sera, respectively. The substance P immunoreactivity in E wasvisualized by combined reflected-light polarization and transmitted-light bright-field microscopy after light counterstaining of the section with

Proc. Natl. Acad Sci. USA 87 (1990)

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along the duodenal-to-colonic axis: >75% of the, serotonincells in the ileum contained substance P immunoreactivity,whereas <5% of the serotonergic cells in the distal coloncontained this peptide (see Fig. 1A).

Serotonin/secretin. Fig. 1E shows that the coexpression ofserotonin and secretin was also affected by the position ofcells along the proximal-to-distal axis. About 80% of thesecretin-immunoreactive cells located in the duodenum con-tained serotonin. This percent colocalization decreasedsteeply from the duodenum to the distal jejunum, where only20% of secretin-producing cells produced serotonin. Allsecretin-containing cells were located on the villus (Fig. 1B).Therefore, the subpopulation that coexpressed serotonin alsowas villus-associated whether they were located in the duo-denum or the proximal or distal jejunum (Fig. 2 E-H).Substance Pisecretin. Since all secretin-containing cells

were associated with the villus, it follows that none of thecrypt-associated substance P-producing cells produced de-tectable levels of secretin. Approximately 40% of villus-associated substance P-containing cells in the duodenumcontained secretin (Fig. 1D). These cells typically were foundin the lower villus. Substance P-positive secretin-negative(substance P+/secretin-) cells were commonly seen in thecrypt below these double-positive (substance P+/secretin+)cells, whereas substance P-negative secretin-positive (sub-stance P-/secretin+) cells were frequently encounteredabove them.The percentage of substance P-containing cells with colo-

calized secretin decreased steadily from duodenum (40%) toproximal colon (<10%; see Figs. 1D and 2 E-H). Nonethe-less, the crypt-to-villus sequence of substance P+/secretin--* substance P+/secretin+ -* substance P-/secretin+ cellswas encountered throughout the proximal-to-distal axis ofthe adult mouse intestine.

Serotonin/secretin/substance P. The multilabeling im-munocytochemical method described in the legend to Fig. 2indicated that serotonin was frequently produced in thesubstance P+/secretin+ cells described above.Mapping Serotonin/Secretin/Substance P-Containing En-

teroendocrine Cell Populations in Transgenic Mice ContainingL-FABP-hGH Fusion Genes. We previously noted (8) thattransgenic mice containing different portions of the 5' non-transcribed domain of the rat L-FABP gene linked to thehuman growth hormone (hGH) gene express the hGH re-porter in subpopulations of enteroendocrine cells. Expres-sion could be related in part to the position ofthese cells alongthe proximal-to-distal and crypt-to-villus axes of the gut.Approximately 30% of serotonin-immunoreactive cells in theduodenum of L-FABP-596 to +21-hGH transgenics containedhGH. Coexpression decreased sharply (to <5%) in the distalsmall intestine and colon. Serotonin+/hGH+ cells werefound primarily on villi, although occasional crypt-associatedserotonergic cells also supported transgene expression. Add-ing nucleotides -597 to -4000 of the L-FABP gene had onlya minor (inhibitory) effect on the percentage of serotonin-producing cells containing hGH no matter which portion ofthe two axes were surveyed. In contrast, the addition ofthese3.3 kilobases (kb) of upstream sequences had a demonstrableeffect on hGH expression in secretin cells: in mice containingthe L-FABP-596 to +21-hGH transgene, virtually all (>90%)secretin-immunoreactive cells were positive for hGH, whilethe percent colocalization of hGH and secretin decreased to60% in duodenal and proximal jejunal villi of mice with theL-FABP-4" to +21-hGH transgene (8). Transgene expres-

sion in substance P-immunoreactive enteroendocrine cellswas not analyzed in this earlier study.

Neither transgene had an effect on the absolute numbers ordistribution of substance P (or serotonin or secretin)-producing cells in the intestinal epithelium. hGH was pro-duced in a subpopulation of substance P-immunoreactivecells, but transgene expression was modified by three factors:cellular location along the crypt-to-villus and proximal-to-distal axes and by nucleotides -597 to -4000 of the ratL-FABP gene. Analysis of eight mice from four differenttransgenic pedigrees revealed that in all animals <10% of theduodenal crypt-associated substance P-containing cells ex-pressed hGH (Fig. 1F), whereas -50% of duodenal villus-associated substance P-containing cells contained this re-porter (Figs. 1G and 2 I-L). The percent colocalization ofsubstance P and hGH in villi decreased progressively fromduodenum (-50%) to ileum (<10%; see Fig. 1G). Addition ofnucleotides -597 to -4000 produced a modest (up to 20%)reduction in the percentage of villus-associated substanceP-producing cells that coexpress hGH in animals derivedfrom two different founders. This effect was evident through-out the small intestine (Fig. 1G).

Multilabeling studies indicated that nucleotides -596 to+21 of the L-FABP gene directed expression of hGH invirtually all villus-associated serotonin+/secretin+ and sub-stance P+/serotonin+/secretin+ cells throughout the smallintestine. Some substance P+/serotonin+ cells also ex-pressed the transgene. hGH expression was least frequent incells that only produced serotonin (Fig. 2 D-I). The upstreamcis-acting elements had little effect on hGH expression inthese cells (data not shown).

DISCUSSIONWe have examined the interrelationships of the serotonin-,secretin-, and substance P-immunoreactive enteroendocrinecell populations in normal adult mice and transgenic animalscontaining L-FABP-hGH fusion genes. The results revealthat the spatial relationships between these populations arecomplex but highly structured.

Substance P is a member of a family of peptides known asthe tachykinins that possess the same COOH-terminal aminoacid sequence and overlapping biological activities (reviewedin ref. 10). The cellular distribution of substance P and othertachykinins has been examined previously in the mammaliannervous system, including the small intestinal myentericplexus (19, 20). Our studies show that antibodies that recog-nize the common COOH-terminal region of tachykinins reactwith a subpopulation of serotonergic enteroendocrine cells.Some cells, located primarily in duodenal and jejunal crypts,were identified that reacted with substance P antiserum butnot with our anti-serotonin sera. However, the majority ofsmall intestinal epithelial cells with immunoreactive sub-stance P also contain serotonin. This is particularly strikingin the ileum where virtually all of the serotonergic populationwas substance P+/serotonin+. Coexpression was negligiblein serotonergic cells located in the distal colon. This findingcan be correlated with the production of these neuroendo-crine products in human intestinal carcinoids. Nearly alltumors arising in the ileum contain both serotonin- andsubstance P-immunoreactive cells (12, 21, 22). In contrast,carcinoid tumors that arise in the distal colon and rectumfrequently produce serotonin but only rarely contain immu-noreactive substance P (22).

hematoxylin. Quadruple exposure of the section (I) shows the interrelationships of the four antigens. Six nucleated immunolabeledenteroendocrine cells and portions of two others are seen in this section in addition to the Golgi-associated hGH immunostaining of enterocytes.Cells labeled 1 and 6 contain serotonin, substance P, and hGH; cells labeled 2 and 5 contain serotonin, secretin, and hGH; cell 7 contains serotoninand hGH; and cell 4 contains only secretin. The visualized portions of cells labeled 3 and 8 appear to contain only serotonin. (A and B, x 190;C-I, x120.)

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Cheng and Leblond used [3H]thymidine labeling methodsto show that enteroendocrine cells undergo bidirectionalmigration, with the majority of cells moving upward from thecrypt to villus (2, 5). Inokuchi et al. combined [3H]thymidinelabeling and immunocytochemical methods to examine themigration of rat serotonin, gastrin, and secretin cells (23).Although some crypt-associated serotonin- and gastrin-immunoreactive cells contain label shortly after initiation of[3H]thymidine injection, no secretin-containing cells werefound in crypts, and 60% of villus-associated secretin-producing cells were labeled 2 days after the initiation of anevery-6-hr injection schedule. These labeled secretin (S) cellswere replaced in 2 days by nonlabeled cells, leading theseworkers to conclude that functional differentiation of secretincells does not occur until they "reach" the villus (23).

Multiple labeling immunocytochemical techniques and theability to define subpopulations of enteroendocrine cellsbased on their capacity to support transgene expression mayprovide important clues about their potential lineage rela-tionships. Although the distribution of serotonin-, substanceP-, and secretin-immunoreactive cells along the crypt-to-villus axis is well organized, additional methods are requiredto determine whether these subsets of enteroendocrine cellsrepresent points on a continuum of cellular differentiation(possibly migration dependent) with associated sequentialregulation of peptide gene expression or represent distinctsubsets of cells that do not arise from a common lineage (butsynthesize common products during the course of theirterminal differentiation).Transgenes may be useful tools to further dissect potential

differentiation pathways. More than 90% of secretin-immunoreactive cells coexpress the L-FABP-596%to 21-hGHtransgene. Addition of nucleotides -4000 to -597 reducedcoexpression to -70% throughout the proximal-to-distalaxis. In contrast, coexpression of hGH in serotonergic cellswas markedly "sensitive" to cellular position in the duode-nal-to-colonic axis, decreasing from 35% of cells in theduodenum of L-FABP-596 to 21-hGH transgenics to <5% intheir ileum. The upstream regulatory elements had only aminor inhibitory effect on this pattern of hGH expression inserotonergic cells. Expression of hGH in enteroendocrinecells containing immunoreactive substance P was affected bytheir location in both axes of the intestine and by theadditional upstream nucleotide sequences.Our studies allow us to operationally define several subsets

of serotonin-immunoreactive cells based on their coexpres-sion of secretin and/or substance P and their ability tosupport transgene expression. Villus-associated serotonergiccells that express hGH are in large part composed ofcells thatalso contain secretin and/or substance P. The hGH transgeneis less frequently expressed in cells that contain only sero-tonin. Expression of the transgene may require serotonergicprecursor cells to traverse the proposed substance P -*serotonin -+ secretin pathway of differentiation. Transgenescan be used as molecular markers for such events and alsooffer the opportunity to functionally map cisacting elementsthat respond to differentiation-dependent changes in theregulatory environments of these cells.The factors that are responsible for initiating and main-

taining these regional differences have not been defined. One

potential source of these "spatial signals" is the lumen. Therelative contribution of luminal versus intracellular factorscan be surveyed by using intestinal isografts-i.e., implan-tation of fetal intestinal segments under the subcutaneoustissue or renal capsules of syngenic adult animals. Thesegrafts grow and are able to establish regional differences inthe expression of some enterocytic markers (e.g., brushborder hydrolases; ref. 24).

In conclusion, we believe that the serotonin-, substance P-,and secretin-containing enteroendocrine populations of de-veloping and adult, normal, and transgenic mice, represent anideal model system for defining mechanisms that establishand maintain spatial differentiation of the gut epithelium inthe face of its continuous renewal.

We thank Edward Birkenmeier for his help in maintaining thetransgenic mouse pedigrees and Cecelia Latham for expert technicalassistance. This work was supported by National Institutes of HealthGrant DK30292 and the Markey Foundation.

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M. J., Monk, M. & Reid, A. (1985) Nature (London) 313,689-691.

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5. Cheng, H. & Leblond, C. P. (1974) Am. J. Anat. 141, 503-520.6. Lewin, K. J. (1986) Pathol. Annu. 1, 1-27.7. Sjolund, K., Sanden, G., Hakanson, R. & Sundler, F. (1983)

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