Expression of Liver Fatty Acid-binding Protein/Human Growth ...

THEJOURNALOF BIOLOGICAL CHEMISTRY 0 1991 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 266, No. 9, Issue of March 25, pp. 5949-5954, 1991 Printed in U.S. A.

Expression of Liver Fatty Acid-binding Protein/Human Growth Hormone Fusion Geneswithin the Enterocyte and Enteroendocrine Cell Populationsof Fetal Transgenic Mice* (Received for publication, August 30, 1990)

Kevin A. Roth$$, Deborah C. Rubinn, Edward H. Birkenmeier 11, and JeffreyI. Gordonll** From the Departments of $Pathology, TMedicine, and **Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 631 10 and )IThe Jackson Laboratory, Bar Harbor, Maine 04609

The intestinal epithelium establishes and maintains gional expressionof genes in the perpetually renewing its continuous intestinalepithelium.Moreover,theyalsoraisethe and rapid renewal.We have used transgenic mice con- possibility of using transgenes to define fundamental temporal changes in the gut’s epithelial cell populataining liver fatty acid-binding proteinlhuman growth hormone (L-FABP/hGH) fusion genes to begin to define tions. the molecular mechanisms which are responsible for appropriate regional and cell-specificexpression of genes inthe gut. Multilabel immunocytochemical methThe spatial organizationof the adult mouse intestinal epiodswere employedto characterize the patterns of thelium is remarkably complex. The epithelium is composed expression of two transgenes in the enteroendocrine and enterocytic populationsof late gestation fetal mice of four major differentiated cell types, enterocytes, Paneth at the time of initial cytodifferentiationof the gastroin- cells, goblet cells, and enteroendocrine cells (the latter contestinal epithelium (fetal days 16-19). Surveys of the sisting of at least 15 different subpopulations based on their enteroendocrine cell population using a panel of anti- principal neuroendocrine products, Ref. 1). These cells arise during a rapid, well organized, bipolar migration. Multiple (or bodies directed against 11 neuroendocrineproducts revealed that these cells are scarce prior to fetal day perhaps a single) multipotent stem cell(s), functionally an17, show a progressive increase in number through chored near the baseof the cryptsof Lieberkuhn, give rise to day 19, and while the relative proportionof subpopu- descendants that undergo commitment to differentiate along lations (defined by their principal peptide product) are the enterocytic, enteroendocrine, and goblet cell lineages as somewhat different than in adults, their geographic they move upward in vertical coherent bands (2-7). Paneth distribution along the duodenal colonic to and intervil- cells arise during migration to the crypt base. Proliferation/ lus(crypt) to villus axes are very similar to that endifferentiation is perpetual and rapid translocationfrom the countered in adult (2-5 month old)mice. Immunoreac- crypt to the apical extrusion zone near the villus tip is comtive L-FABP is first detectable at fetal day 17 and at pleted within 2-3 days in adult mice (8).Regional differences this timeof first appearance shows an adult pattern of in gene expression are not only established and maintained regional enterocytic expression: i.e. it is present cells in along the crypt-to-villus axis of the gut, but also from duooverlying nascent villi but not those in the intervillus denum-to-colon. zone, it is highest in proximal small bowel, declines The molecular signals that orchestrate proper spatial ordistally, andis absent fromcolonocytes. Colocalization ganization of these cell types in the continuously renewing studies indicate that L-FABPis not present in entero- epithelium are just now being defined. We have used transendocrine cells during fetal life. Mapping studies ingenic mice containing rat liver fatty acid-binding protein/ dicate thatnucleotides -596 to +21 of the ratL-FABP human growth hormone(L-FABP’/hGH) fusion genes to gene are sufficient to reproduce an appropriate temporal, cellular, and regional pattern of reporter (hGH) study the geographic differentiation of the adultmouse intesexpression in fetal transgenicmice (with the exception tine (9-11). Expression of the intact endogenous mouse Lthat a subset(s) of enteroendocrine cells, typically con- FABP gene (fubpl) (12) is confined tohepatocytes, small intestinal villus-associated enterocytes, and a small populatainingimmunoreactivegastricinhibitorypeptide, tion of enteroendocrine cells (9, 10). Steady-state levels of support transgene but not L-FABP expression). is This in marked contrast to adult transgenic mice where mRNA and protein fall progressively from the proximal jejunum to the ileum (9). We found that in young adult (2-5 inappropriate hGH accumulation occurs in crypt-asmonth) transgenics, nucleotides -596 to +21 of the L-FABP sociated epithelial cells, in colonocytes, and in many gene “properly” direct reporterexpression to small intestinal enteroendocrine populations.These studiesindicate enterocytes but produce inappropriate hGH accumulationin theimportance of consideringdevelopmentalstage when interpreting the results of any mapping study of crypt epithelial cells, in many enteroendocrine cell subpopucis-acting elements that regulate cell-specific and re- lations, and in colonocytes (9, 10). Addition of nucleotides -4000 to -597 silences colonic expression, and reduces hGH all, enteroendocrine cell * This work was supportedinpart by Grants DK30292 and concentrationsin some, butnot

a precise spatial organization despite

DK37960from the National Institutes of Health and the Lucille Markey Charitable Trust. The costs of publication of this articlewere defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. I To whom correspondence should be addressed.

’ Theabbreviations usedare: L-FABP, liver fattyacid-binding protein;hGH,human growth hormone; IGSS, immunogold-silver intensification staining; GIP, gastric inhibitory peptide; CCK, cholecystokinin;PYY,peptidetyrosinetyrosine;GLP, glucagon-like peptide.

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Transgene Expression inFetal Gut Epithelium

5950 30@ Serotonln

r@JSubstance P

@Secretin

EXPERIMENTALPROCEDURES

Animals-Mice containing nucleotides -4000 to +21 of the rat LFABP gene linked to thehuman growth hormone gene (beginning at its nucleotide +3) were derived from two founders, Gol andG046 (9). Obligate heterozygotes for the L-FABP-596to "'/hGH transgene were obtained from Go13 as described in Ref. 9. Fetal mice were delivered on days 16-19 (15) by Caesarian section. Transgenic micewere distinguished from their normal littermates by Southern blot analysis of carcass DNA. Segments were dissected from the small intestine and colon as described in Ref. 15 and fixed in Bouin's solution for subsequent immunocytochemical studies. Zmmunocytochenistry-The procedures used for single and multiple antigen labeling of single sections are detailed in Refs. 10 and 11 plus the figure legends. Briefly, 5-pm thick,paraffin-embedded tissue sections were deparaffinized and incubated overnight at 4 "C with diluted primary antisera. Immunostaining was subsequently detected by either gold-labeled second antibodies and silver enhancement (Amersham Corp) or with fluorescent labeled second antibodies (Jackson Immunoresearch, West Grove, PA). A panel of antisera were used for these studies. Their sources (and final dilutions for immunogold silver enhancement staining) were: rabbit and goat anti-serotonin (1:4000 and 1:1000, respectively, Incstar, Stillwater, MN), ratanti-serotonin (1:2000, Eugene Tech, Allendale, NJ), rabbit anti-substance P (1:2000, J. Krause, Washington University, Ref. 16), rabbit anti-secretin (1:1000, Peninsula LaboraFetal Day tories, Belmont, CA), rabbit anti-cholecystokinin (CCK, residues 1(1:1000, FIG. 1. Quantitative analysis of enteroendocrine subpopu- 39) (1:1000, PeninsulaLaboratories),rabbitanti-gastrin lations in the fetal mouse small intestine. Cross-sections from Dako Corp., Santa Barbara, CA), rabbit anti-gastric inhibitory pepand )distal (o"--o) small intestine were tide (GIP) (1:1000, Peninsula Laboratories), rabbitanti-glucagon-like the proximal (M taken from late gestation fetal mice and immunostained for neuroen- peptide 1 (GLP-1, residues 1-19)(1:1000, Peninsula Laboratories), docrine products. Experimental animals surveyed included eight 17- rabbit anti-neurotensin (1:1000, Peninsula Laboratories), rabbitantisomatostatin (1:500, Incstar), rabbitanti-pancreatic polypeptide day-old animals (three normal littermates, two L-FABP-40" to +'l/ hGH transgenics (one each from Gol and46), and three L-FABP-596 (1:1000, Dako Corp.), rabbit anti-peptide tyrosine tyrosine (PYY) to +'l/hGH animals (Go13)),five 18-day-old mice (two normals, two (1:1000, a gift from C. J. Evans, UCLA), rabbit anti-L-FABP (1:1000, L-FABP-4W0to +*'/hGH transgenic mice (Go46)),and one L-FABP-596 Ref. 9), goat anti-hGH (1:2000, Ref. 17), andrabbit anti-hGH (1:2000, to "'/hGH transgenic mouse (G013)),and two 19-day-old fetal mice Dako Corp.). Note that the PYY antiserum was raised in rabbits (one normal and one L-FABP-4000to +*l/hGH transgenic mouse against the hexapeptide carboxyl-terminal sequence of PYY. Radioimmunoassay and immunocytochemical studies indicate that it rec(Gal)). To quantitate the expression of neuroendocrine products at least two cross-sections/intestinal segment/animal were counted and ognizes both PYY and neuropeptide tyrosine.' The immunostaining averaged. Since no effect of the transgenes on enteroendocrine cell characteristics and specificities of the remaining antisera have been number or distribution was noted, data for both normal and trans- described by the manufacturers and in our previous reports (9-11, 15). For all tissues examined, nonimmune rabbit, goat, and rat sera genicmicewere combined. The number of immunoreactive cells/ complete cross-section/animal has been averaged and is presented as were used as negative controls. the average -t S.E. RESULTSANDDISCUSSION

subpopulations, but has no effect on transgene expression in the crypts (3, 10). The mouse L-FABP gene starts to be actively transcribed at thesame time that thegut epithelium undergoes conversion from a poorly differentiated stratified epithelium to a monolayer overlying nascent villi (fetal days 16-18, Refs. 13 and 14). Previous developmental studies indicate that nucleotides -4000 to +21 of the L-FABP gene contain cis-acting sequences that produce appropriate regional and temporal patterns of hGH expression in the gastrointestinal tract during late fetal life (15). Immunocytochemical studies indicated that the reporter accumulated in villus associated enterocytes and in some cells that had the histologic appearance of enteroendocrine cells (15). The enteroendocrine cell population represents a very attractive model for assessing the spatial differentiation of the gut given the fact that its numerous subpopulations exhibit distinct regional differences in their distribution in both axes of the gut (1, 10, 11).Therefore, we have now performed a detailed analysis of transgene expression in fetal enteroendocrine subpopulations using several pedigrees of mice, including ones that contain the L-FABP-596to +"/hGH fusion gene. The transgenes have allowed us to define differences between the epithelial cell populations of fetal and adult gut and tofurther map those cis-acting elements that are required for proper developmental stage specific expression of fabpl.

Regional Distribution of Enteroendocrine Cells in the Developing Fetal Mouse Gut-Before comparing the cellular patterns of expression of the L-FABP-4000to "l/hGH and LFABP-596to +*l/hGH transgenes in late gestation fetal intestine, we first surveyed the number and time of appearance of enteroendocrine cell subpopulations in normal fetal C57BL/ 6J X LT/Sv mice and theirtransgenic littermates. At day 16, thereare few cells in the small intestineand colon that produce immunohistochemically detectable amounts of neuroendocrine products. From fetal day 17 to 19, there is a progressive increase in the number of immunoreactive enteroendocrine cells/cross-section of gut (Fig. 1). Serotonin immunoreactive cells are more frequent in proximal than distal fetal small intestine (Fig. lA), similar to adultintestine. While serotonergic cells form a significant proportion of all enteroendocrine cells in thefetal gut, they are not aspredominant in 17-19 day fetal mouse gut as they are in adult mice. Substance P immunoreactive cells exhibit only modest increases in number during late fetal life and in contrast to adult mice, were as frequent in the distal as proximal small intestine (Fig. 1B). Both secretin and CCK immunoreactive cells show a sharp increase in number during this critical period of gut epithelial development. Unlike in adults, by fetal day 19 they are as abundant asserotonin-producing cells and K. A. Roth, D. C. Rubin, E. H. Birkenmeier, and J . I. Gordon, unpublished observations.

Transgene Expressionin Fetal GutEpithelium

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FIG. 2. Immunohistochemical mapping of enteroendocrine cell subpopulations in the fetal small intestine. To quantitate the intervillus-villus distribution of enteroendocrine cell subgroups, cross-sections from the proximal small intestine were immunostained for neuroendocrine products and L-FABP ( A X ) . A section of proximal small intestine from a normal 17-day-old fetal mouse was first incubated with rabbit anti-substance P serum and antigen-antibody complexes were detected by the immunogold-silver intensification staining (IGSS) technique. Sections were subsequently incubated with rabbit anti-L-FABP serum and Texas red-labeled donkey anti-rabbit serum. Three substance P immunoreactive cells, visualized with reflected light polarization microscopy and indicated by arrows, are seen in A . Prominent L-FABP immunoreactivity (visualized as red staining in B ) is limited to villi. Double exposure of the section (C) shows that the three substance P immunoreactive cells are located in the L-FABP negative intervillus zones. The interrelationship of substance P, serotonin, and secretin immunoreactive cells is illustrated in panels D-F. A section from the proximal small intestine of a normal 19-dayold fetal mouse was first stainedfor substance P using the IGSS procedure ( D ) followed by coincubation with goat anti-serotonin ( E ) and rabbit anti-secretin sera ( F ) . The latter two sera were detect.ed with fluorescein-labeled donkey anti-goat and Texas Red-labeled donkey anti-rabbit sera, respectively. The substance P immunoreactive cell labeled1 also contains serotonin. Cell 2 contains substanceP, serotonin, and secretin. Cell 3 contains serotonin and secretin but not substance P. The colocalization of serotonin and gastrin is illustrated inpanels G-I. A section from the proximal small intestine of a normal 19-day-old fetal mouse was coincubated with goat anti-serotonin and rabbit anti-gastrin sera. The serotonin antiserum was detected with fluorescein-labeled donkey anti-goat serum (G) and threeimmunoreactive cells, labeled 1,2, and 3, are visualized. The gastrin antiserum was detected with Texas Red-labeled donkey anti-rabbit serum (H), and three immunoreactive cells are visualized, labeled I , 3, and 4. Double exposure of the section ( I ) shows that cells labeled 1 and 3 contain both serotonin and gastrin, cell 2 only serotonin, and cell 4 only gastrin. (All photomicrographs X 400.)

Transgene Expression

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not only distributed in a characteristic fashion along the duodenal-to-colonic axis but also along the crypt-to-villus axis. However, the crypts of Lieberkuhn do not fully form until the end of the second postnatal week. Studies with mouse aggregation chimeras indicate that thefetal intervillus epithelium is composed of a polyclonal population of cells that undergoes a “purification” to monoclonality (i.e. all cells derived from a common stem cell) by the 14th day after parturition (18).The process of purification may be linked to functional anchorage of stem cells at thecrypt base (19). During late fetal life, L-FABP appears to be confined to ” 5HT SP S CCKGIP G the villus structures of the proximal small intestine, thus Enteroendocrine Subpopulation providing a marker for operationally defining the villus (LFIG. 3. Intervillus-villusdistribution of enteroendocrine FABP’) and intervillus (L-FABP-) cellular populations. We, cell subpopulations in fetal mice. The intervillus-villus distribution of enteroendocrine cell populations was determined in the prox- therefore, used multilabeling techniques to characterize the imal small intestine of four normal (17-19-day old) fetal mice. Sec- location of enteroendocrine cells along the intervillus-villus (i.e. L-FABP- to L-FABP+) axis of the fetal proximal small tions were first stained for neuroendocrine products using primary antisera which were detected with the IGSS technique. They were bowel. The results indicated that at the earliest time of subsequently immunostained for L-FABP using fluorescence tech- cytodifferentiation of the fetal gut, certain enteroendocrine niques. Neuroendocrine positive cells were then quantitated as being cell populations have assumed an “adult” like distribution in either L-FABPpositive villus zones or L-FABPnegative intervillus zones. The percentage of immunoreactive cells located in villi was along its emerging “vertical” axis (Figs. 2, A-C, and 3). For calculated for each subpopulation. Abbreviations: 5HT, serotonin; example, in both 17-19 day fetal and 2-4 month adult mice, substance P immunoreactive cells are located predominantly SP, substance P; S, secretin; G, gastrin. in intervillus zones (crypts) while secretin immunoreactive cells are found almost exclusively on villi. Serotonin, CCK, gastrin, and GIP immunoreactive cells are located primarily on villi in each age group (70-86% are villus associated in normal or transgenic fetal mice and 71-81% in adult normal or transgenic animals). The vast majority of villus-associated enteroendocrine cells in fetal mice do not contain L-FABP (e.g. see Fig. 5 ) . This result is similar to what is seen in adult animals where only a small fraction of enteroendocrine cells produce detectable amounts of L-FABP (10). We previously reported that the distal small bowel of late gestation fetal mice contains L+ + + - + Substance P: FABP immunoreactive cells that histologically resemble en+ - + + - + Serotonin: - + + + + teroendocrine cells (15). Although we noted the same cells in Secretin: this present study, they failed to stain with any of our neuFIG. 4. Substance P/serotonin/secretin interrelationships in the fetal small intestine. Sections from the proximal and distal roendocrine antisera. Thus, the precise classification of these small intestine of three 17-19-day-old fetal mice (one normal and two L-FABP immunoreactive cells awaits further study. L-FABP-4”” to ‘“/hGH transgenic mice (one each from Gol and Enteroendocrine Cell Interrelationships-Although each G,,46)) were analyzed for substance P/serotonin/secretin colocaliza- neuroendocrine product defines a subpopulation of enteroention. Sections were first immunostained for substance P using the docrine cells, these subpopulations may overlap. For example, IGSS technique followed by coincubation with rabbit anti-secretin and goat anti-serotonin antisera which were detected with fluores- in adult mice, most substance P immunoreactive cells coexcein-labeled donkey anti-goat and Texas Red-labeled donkey anti- press serotonin (11).Secretin-producing cells frequently coexrabbit sera (see Fig. 2, D-F). Immunoreactive cells were then sub- press serotonin and occasionally serotonin and substance P classified based on the presence or absence of colocalized products. in the proximal (but not distal) small intestine. These interThe number of cells in each of the possible substance P/serotonin/ relationships between substance P, serotonin, and secretin secretin combinations is presented as a percentage of all combinations cells are also present in the fetus: (i) themajority of substance in that intestinal segment. P immunoreactive cells contain serotonin throughout the are equally distributed between proximal and distal segments length of the small bowel; (ii) 60% of secretin immunoreactive cells in the proximal but less than 5% in the distal small of the small bowel (Fig. 1, C and D).GastrinandGIP immunoreactive cells are limited to theproximal small intes- intestine contain serotonin; and (iii) substance P/serotonin/ tine and show a steady increase in number during late gesta- secretin tripositive cells are found only in the proximal small tion (Fig. 1,E and F ) . PYY, GLP-1, and neurotensin immu- bowel (Figs. 2, B-F, and 4). Other interrelationships are present in fetal but not adult noreactive cells are infrequent before fetal day 19 and are largely confined to the ileum (Fig. 1, G I ) . Somatostatin and gut. Thirty% of gastrin and 5% of CCK immunoreactive cells pancreatic polypeptide immunoreactive cells are very rare at in the proximal intestine contain serotonin. This colocalizaeach time point surveyed and therefore were not analyzed tion was first noted at day 19 and was not encountered in the further (data not shown). It is important to note that the distal small bowel (Fig. 2, G I ) . By contrast, fewer than 3% presence or absence of multiple (up to several hundred) copies of gastrin and CCK immunoreactive cells contain serotonin of either transgene (9) had no discernible qualitative or quan- in the proximal gut epithelium of 6-week-old normal and titative effect on the duodenal-ileal distribution of any of transgenic mice. The interrelationship of gastrin and CCK these enteroendocrine cell subpopulations in late gestation immunoreactive cells with serotonin has previously been noted in the fetal rat duodenum (20). fetal gut. Thus overall, it appears that the enteroendocrine cell popIn adult mice, each enteroendocrine cell subpopulation is ~

Transgene Expression in Fetal Gut Epithelium

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FIG.5. Immunohistocnemlclu stammg 01 L-r-ABP/hGH transgenic mice. The interrelationship of hGH and L-FABP immunoreactivity is illustrated in panels A-C. A section from the proximal small intestine of an 18-day-old fetal transgenic mouse containing the LFABP"WO to +"/hGH transgene (derived from Go46) wascoincubated with goat anti-hGH (A) andrabbit anti-L-FABP ( B ) sera which were subsequently detected with fluorescein-labeleddonkey anti-goat and Texas Red-labeled donkey anti-rabbit sera. Strong Golgi-associatedhGH immunostaining is obtained in enterocytes (A). Prominent diffuse, cytoplasmic L-FABP immunostaining is observed in villus-associated enterocytes ( B ) .Double exposure of the section shows that weak hGH staining (indicated by arrows) is present in the L-FABP negative intervillus zone ( C ) .Golgi-associated enterocytic hGH staining was also observed in transgenic animals containing "only" nucleotides -596 to +21 of the ratL-FABP gene. In panel D,hGH immunoreactivity is observed in a 17-day-old fetal mouse containing the L-FABP-'% to +"/ hGH transgene. Immunostaining in this and other L-FABP-596 to +"/hGH transgenic mice was qualitatively weaker but had a similar distribution as that seen in L-FABP"m to +"/hGH transgenic mice. As in adult L-FABP/hGH transgenic mice, some fetal enteroendocrine cells support transgene expression. Panels E-H illustrate the relationship of L-FABP and hGH to the GIPenteroendocrine subpopulation. A section of proximal small intestine from a 17-day-old transgenic mouse (derived from G046) was first stained for GIP using the IGSS technique followed by coincubation with rabbit anti-L-FABP and goat anti-hGH sera which were subsequently detected with 7-amino-4methylcoumarin-3-acetic acid-(AMCA) labeled donkey anti-rabbit and fluorescein-labeled donkey anti-goat sera, respectively. The GIPlabeled cell ( E ,visualized with reflected light polarization microscopy, and indicated by an arrow) possesses colocalized hGH immunoreactivity (F)but lacks L-FABP immunoreactivity ( G ) . Triple exposure of the section in H illustrates these interrelationships. Although occasional hGH immunoreactive enteroendocrine cells were observed, they are much less frequent in the fetal versus adult intestine. For example, to +"/hGH transgenic mice contain approximately 70% of secretin immunoreactive cells in the adult proximal small intestine of L-FABP" colocalized hGH while in fetal transgenic mice the colocalization is less than 5%. This relationship is illustrated in panels I-M. A section from the proximal small intestine of a 17-day-old fetal mouse containing the L-FABP-4W0to +'*/hGH transgene (Go46)was first stained for secretin using the IGSS technique. The section was subsequently simultaneously incubated with rat anti-serotonin, rabbitanti-L-FABP, and goat anti-hGH sera. Thesesera were detected with &phycoerythrin-labeled donkey anti-rat, AMCA-labeled donkey anti-rabbit, and fluorescein-labeleddonkey anti-goat sera, respectively. Several, but not all, of the secretin immunoreactive cells seen in I contain colocalized serotonin (J, indicated by open arrows). Secretin cells lacking serotonin are also observed (closed arrows in I ) . None of the secretin and/or serotonin immunoreactive cells contain L-FABP ( K ) or hGH immunoreactivity (L)(cells indicated by arrowheads). The interrelationship of all four immunoreactivities is illustratedin thequadruple exposed photomicrograph in panel M. (All photomicrographs X 400.)

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Transgene Expression in Fetal Gut Epithelium

ulation provides a sensitive marker for the early spatial differentiation of the fetal gut epithelium. Moreover, pathways for terminal differentiation of its subpopulations are largely in place coincident with the initial appearance of the monolayer, although some features appear to be unique to thisstage of development. Human Growth Hormone Expression in Fetal Transgenic Mice-Fetalmice containing the L-FABP-4Wuto +*'/hGH fusion gene initially accumulate hGH in their enterocytes between the 15th and 17th days of gestation (15). The reporter is expressed in a distinct duodenal-to-ileal gradient and is more concentrated in villus-associated than intervillus epithelial cells (Fig. 5, A-C and Ref. 15). This geographic pattern of expression is comparable to that seen in adult (2-5-month old) transgenic mice. An identical pattern of cellular (enterocytic) expression was observed in fetal L-FABP-596to +*'/ hGH mice. By contrast, the L-FABP-596to +"/hGH transgene, which displays inappropriate expression in adult colonocytes, is not expressed in the fetal colon. Thus, these 600 nucleotides are sufficient to reproduce the temporal and geographic patterns of expression of theintact, endogenous mouse L-FABP gene (fabpl) in the late gestation fetal intestinal epithelium (Fig. 5 0 ) . The temporal, regional, and cellular patterns of transgene expression were also examined in the fetal enteroendocrine cell population. Adult L-FABP-s96to +*'/hGH transgenic mice exhibit varying degrees of hGH coexpression among their enteroendocrine cell subpopulations (see Ref. 10). Virtually, all GIP, secretin, CCK, and gastrin immunoreactive cells in the duodenum contain colocalized hGH. Addition of nucleotides -597 to -4000 suppresses hGH expression in CCK, gastrin, and secretin subpopulations but has little effect in GIP immunoreactive cells (10). The absolute and relative abundance of hGH immunoreactive enteroendocrine cells is markedly reduced in fetal proximal small intestine of both L-FABP-'"j to and LFABP-4000to +*'/hGHtransgenic mice. Adulttransgenic mice from multiple pedigrees containing either transgenetypically have 80-100 hGH+ enteroendocrine cells/duodenal cross-section while fetal mice from these same pedigrees averaged only 3 & 1cells/cross-section during the 17-19th days of gestation (mean f S.E., n = 7animals). This in part reflects the decreased cross-sectional area in fetal uersus adult intestine. It also represents a "true" reduction in hGH immunoreactive enteroendocrine cells relative to other enteroendocrine cell subpopulations. For example, hGH containing enteroendocrine cells are four times as frequent as secretin cells in the adult mouse duodenum yet represent less than 20% of the total secretin cell population in thefetal proximal small bowel. Similar differences in the ratios of hGH positive enteroendocrine cells to serotonin, substance P, CCK, and GIP immunoreactive cells were noted between fetal and adult animals. Many of the hGH immunoreactive cells present in the proximal intestine of adult L-FABP-400uto +"/hGH and LFABP-"' to '*l/hGH transgenic mice contain colocalized neuroendocrine products. However, those small bowel enteroendocrine cell subpopulations that exhibit a high degree of hGH coexpressionin adulthood, generally lack detectable levels of this reporter in late fetal life (Fig. 5,

,

PYY immunoreactive cells in the proximal colon of adult L-FABP-596to +*l/hGH transgenic mice frequently coexpress hGH, (Ref. 10). hGH is not detectable in the colonic PYY cells of adult mice containing the L-FABP-4u00to +"/hGH fusion gene. Interestingly, none of our pedigrees of fetal transgenic mice (including those containingnucleotides -596 to +21) contain hGH in colonic PYY cells (data not shown). These observations suggest a number of conclusions. While transgenic mice offer a unique and powerful model for mapping cis-acting elements that regulate cell-specificand regionspecific gene expression in the gut epithelium, any interpretation of patterns of transgene expression must consider the developmental stage at which the analysis is performed. For example, our data indicate that although the mouse intestinal epithelium undergoes continuous and rapid renewal throughout life, very different patterns of transgene expression occur in theintervillus (crypt), colonocyte, and enteroendocrine cell populations of fetal and adultanimals. These differences may represent developmental changes that are intrinsic to these cells (e.g. fundamental alterations in stem cell populations, changes in differentiation pathways, or levels of critical transacting factorswhich regulate L-FABP gene expression, etc.). The L-FABP/hGH transgenesappear to be sensitive markers of temporal effects on the gut epithelium, effects that are not operationally definable in these cell populations by noting the transcriptional activity of the intact, endogenous fabpl gene (3). Comparison of the cellular and regional patterns of expression of the intact L-FABP gene and L-FABP/hGH transgenes in fetal intestinal isografts at various times after theirimplantationinto the subcutaneous tissues of nude mouse recipients may, for example, offer a way of evaluating the roles of some of the potentialintrinsicandextrinsic factors listed above. Moreover, our results suggest that itmay be informative to examine the patterns of L-FABP/hGH transgene expression in aging mice. Such studies may also reveal whether fundamental changes occur in the gut epithelial cell population throughout life. Acknowledgment-We thank Cecelia Latham for her help with the immunocytochemical studies. REFERENCES Lechago, J. (1987) Am. J . Surg. Path. 11,63-70 Chang, H., and Leblond, C. P. (1974) Am. J. Anat. 1 4 1 , 537-562 Gordon, J. I. (1989) J. Cell. Biol. 1 0 8 , 1187-1194 Schmidt, G. H., Wilkinson, M. M., and Ponder, B. A. J. (1985) Cell 4 0 , 425-429 5. Winton, D.J., and Ponder, B. A. J. (1990) Proc. R. Soc. Lond. 241, 13-18 6. Winton, D.J., Blount, M. A,, and Ponder, B. A. J. (1988) Nature 333, 463-466 7. Schmidt, G. H., O'Sullivan, J. F., and Paul, D. (1990) Mutation Research 228,149-155 8. Potten, C. S., Schofield, R., and Lajtha, L. G. (1979) Biochim.Biophys. Acta 560,281-299 9. Sweetser, D. A,, Birkenmeier, E. H., Hoppe, P. C., McKeel, D. W., and Gordon, J. I. (1988) Genes Deu. 2,1310-1332 10. Roth, K. A,, Hertz, J. M., and Gordon, J. 1. (1990) J. Cell. Biol. 110,17911801 11. Roth, K. A,, and Gordon, J. I. (1990) Proc. Natl. Acad. Sci. U. S . A. 87, 6408-6412 12. Sweetser, D.A,, Birkenmeier, E. H., Klisak,1. J., Zollman, S., Sparkes, R. S.,Mohandas, T.,Lusis, A. J., and Gordon, J. I. (1987) J . Biol. Chem. 2 6 2 , 16060-16071 13. Trier, J. S., and Moxey, P. C. (1979) Ciba Found. Symp. 70,3-29 14. Colony, P. C., and Neutra, M. R. (1985) Gastroenterology 89,294-306 15. Hauft, S. M., Sweetser, D. A,, Rotwein, P. A,, Lajara, R., Hoppe, P. C., Birkenmeier, E. H., and Gordon, J. I. (1989) J. Bzol. Chem. 2 6 4 , 84191. 2. 3. 4.

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16. MacDonald, M. R., Takeda, J., Rice, C. M., and Krause, J. E. (1989) J . Biol. Chem. 264,15578-15592 'I The presence of hGH immunoreactivity in GIP-producing cells 17. McKeel, D. W., Jr., and Askin, F. B. (1978) Arch. Pathol. Lab. Med. 102, suggests that cis-acting elements not represented between nucleotides 122-128 18. Schmidt, G. H., Winton, D. J., and Ponder, B. A. J. (1988) Deueloprnent -4000 and +21 function during fetal life to repress fabpl expression 103,785-790 in this enteroendocrine subpopulation. L-FABP/hGH transgenic ped19. Potten, C. S., and Hendry, J. H. (1983) in Stern Cells (Potten, C. S., ed) pp. igrees containing additional portions of the (rat) L-FABP gene will 155-199, Churchill Livingstone, Edinburgh, Scotland be needed to further define these regulatory sequences. 20. Larsson, L-I., and Jorgensen, L. M. (1978) Cell Tiss. Res. 1 9 4 , 79-102