Proc. Nadl. Acad. Sci. USAVol. 88, pp. 1034-1038, February 1991Cell Biology

Temporal and spatial patterns of transgene expression in agingadult mice provide insights about the origins, organization, anddifferentiation of the intestinal epithelium

(colon/stem cells/progenitor cells/trngnic mice/pattern formation)

STEVEN M. COHN*, KEVIN A. ROTHt, EDWARD H. BIRKENMEIERt, AND JEFFREY 1. GORDON*§Departments of *Medicine, tPathology, and §Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO 63110; andtThe Jackson Laboratory, Bar Harbor, ME 04609

Communicated by Paul Lacy, November 1, 1990

ABSTRACT We have used liver fatty acid-binding pro-tein/human growth hormone (L-FABP/hGH) fusion genes toexplore the temporal and spatial differentiation of intestinalepithelial cells in 1- to 12-month-old transgenic mice. Theintact, endogenous L-FABP gene (Fabpl) was not expressed inthe colon at any time. Young adult transgenic mice containingnucleotides -596 to +21 of the rat L-FABP gene linked to thehGH gene (minus its 5' nontranscribed domain) demonstratedinappropriate expression of hGH in enterocytes and manyenteroendocrine cells of most proximal and mid-colonic crypts(lands). Rare patches of hGH-negative crypts were present.With increasing age, a wave of "extinction" of L-FABP(-596to +21)/hGH expression occurd, first in the distal colon andthen in successively more proximal regios, leaving by 10months ofage only rare hGH-positive multicrypt patches. At notime during this progressive silencing of transgene expressionwere crypts observed that contained a mixture ofhGH-positiveand -negative cells at a particular cell stratum. Young (5-7weeks) mice containing a L-FABP(-4000 to +21)/hGH trans-gene also demonstrated inappropriate expression of the trans-gene in most proximal colonic crypts. However, the additional3.3 kilobases of upstream sequence resulted in much morerapid extinction of reporter expon, leaving by 5 months ofage only scattered single crypts with detectablevels of hGH.This age-related extinction ofL-FABP/hGH expression did notinvolve enterocytes and enteroendocrine cells in the (proximal)small int . These results indicate that cis-acting elementsoutside of nucleotides -4000 to +21 are necessary to fullymodulate suppression ofcolonic L-FABP expression. They alsodefine fndental changes in colonic epithelial cell popula-tions during adult life. Our data suggest that (s) a single stemcell gives rise to all cells that populate a given colonic crypt, (it)stem cells represented in several adjacent crypts may bederived from a common progenitor, and (u) such a progenitorcell may repopulate colonic crypts with stem cells during adultlife. Since each colonic crypt contains the amplii descen-dants of its stem cell, transnes may be powerful tools forcharacterizing the spatial and biological features of gut stemcells and their progenitors during life.

The intestinal epithelium has several unique features thatmake it an attractive model for defining the pathways andmechanisms that regulate epithelial cell differentiation: (A) therapid and continuous renewal of its four principal cell types-all apparently derived from a multipotent stem cell; (ii) itsremarkable spatial organization, with proliferation and dif-ferentiation occurring during migration of the descendants ofthis stem cell from intestinal crypts; and (iiM) the fact that theprogeny of the functionally anchored stem cell undergo an

amplification (involving perhaps four to six divisions) withinthe proliferative zone of each crypt, producing a monoclonalpopulation with a single genotype (reviewed in ref. 1). Thisamplification together with its physical confinement providesan opportunity for disclosing subtle changes in either thestem cell (capable of self-renewal without accompanyingdifferentiation) or its progenitor cell that would not bedefinable in other differentiating epithelia.Mouse aggregation chimeras have been used to describe

several aspects of the organization ofthe gut epithelium (2-6).The intestinal epithelial cell populations ofsome inbred strainsofmice express a receptor for the Dolichos biflorins agglutinin(DBA) lectin (i.e., those having the Dlb-lb rather than theDIb-1a allele). Analysis of adult aggregation chimeras formedfrom Dlb-1b/Dlb_1b and Dlb1a/Dlb-1a strains provided proofthat each small intestinal crypt is composed of a monoclonalpopulation of cells (crypts were either wholly negative orpositive for DBA binding; ref. 3). The number of renewingstem cells within each small intestinal crypt has been examinedbefore or after treatment of mice heterozygous for the Dib-)allele with y radiation or ethylnitrosourea (EtNU). The (am-plified) clonal descendants of a mutated precursor cell can bereadily identified by their lack of staining with peroxidase-labeled lectin in the otherwise positively stained gut epitheliumof these Dlb-1bIDlb-1a mice (7-10). Schmidt et al. (10) ob-served that patches ofmultiple adjacent DBA-negative crypts/villi were produced when 7- to 11-day-old fetal DIb-1b/Dlb_1amice were treated with EtNU (animals were surveyed asadults). These patches were thought to reflect somatic muta-tions in embryonal intestinal stem-cell precursors (i.e., pro-genitor cells). When the same mutagen was given to adultanimals, only isolated, wholly negative, small intestinal cryptswere seen that persisted (9, 10). Their fiequency reached a"steady-state" level of 14 mutations per 101 crypts in the smallintestine 21 weeks after EtNU treatment (9). The frequencywas 0.5 mutation per 10W small intestinal crypts per year inuntreated mice. These data suggest that the epithelial cellpopulation of individual adult small intestinal crypts is re-newed by a single stem cell and that the rate of spontaneousmutation in this cell is extremely low.Comparable studies have not been performed in the large

intestine using the DBA marker system since expression ofthe lectin binding site is variable within its epithelial cellpopulations. However, Griffiths et al. (11) used the X chro-mosome-linked enzyme glucose-6phosphate dehydrogenase(G6PD) to study clonality in the colonic epithelium of adultfemale mice. In mice with wild-type enzyme, all cells in allcolonic crypts (glands) express high levels of activity. Mice

Abbreviations: L-FABP, liver fatty acid-binding protein; hGH, hu-man growth hormone; DBA, Dolichos biflorins agglutinin; G6PD,glucose-6phosphate dehydrogenase; EtNU, ethylnitrosourea; PYY,peptide tyrosine tyrosine; nt, nucleotide(s).

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with G6PD deficiency (GPDX) demonstrate uniformly lowlevels of activity. Adult mice heterozygous for these twoalleles contain patches, encompassing multiple coloniccrypts, of high or low activity: individual crypts do not havemixtures of cells with high and low activity. Dimethylhydra-zine treatment of mice homozygous for the wild-type alleleresults in the production of scattered, individual cryptscomposed of cells with uniformly low enzyme activity. Nomixed crypts were noted, leading Griffiths et al. (11) toconclude that the enterocytes and goblet-cell populations ofeach colonic crypt were also maintained by a single stem cell.No spontaneous G6PD mutations were noted when >105colonic crypts were surveyed in untreated adult animals.We have used transgenic mice containing liver fatty acid-

binding protein/human growth hormone (L-FABP/hGH) fu-sion genes to characterize cis-acting elements that establishand maintain cell-specific, region-specific, and developmen-tal stage-specific patterns of gene expression in the intestinalepithelium (12-15). The mouse "liver" FABP gene (Fabpl) isan attractive model for exploring these aspects of gut entericbiology: it is activated at the time of initial cytodifferentiationof the fetal gut epithelium (13), it is efficiently expressed inenterocytes and in a small subpopulation of enteroendocrinecells (12, 14), and it exhibits distinct regional differences intranscription along both the crypt-to-villus and duodenal-to-ileal axes of the small bowel (L-FABP is not detectable incolonic epithelium; ref. 12). Functional mapping studies intransgenic mice have allowed us to identify cis-acting se-quences within the 5' nontranscribed domain of the L-FABPgene that control its spatial patterns ofexpression (12, 14, 15).These transgenes defined subtle differences in the differen-tiation programs of certain epithelial cell populations thatwere not discernible when these cells were defined only bytheir endogenous gene products (14, 15). Remarkable differ-ences in the spatial and cellular patterns of transgene expres-sion within the continuously renewing intestinal epitheliumhave been noted between late fetal and young adult mice (13),raising the possibility of using transgenes to define funda-mental temporal changes in the gut's epithelial cell popula-tions. We now show that the patterns oftransgene expressionobserved in the colonic epithelium of aging mice provideadditional insights about its clonal organization, mechanismsof cell renewal, and pathways of cellular differentiation.

MATERIALS AND METHODSAnimals. Transgenic mice containing nucleotides (nt)

-4000 to +21 ofthe rat L-FABP gene linked to the hGH genebeginning at its nt +3 [L-FABP(-4000 to +21)/hGH] werederived from founder G046 in ref. 12. Transgenic micecontaining nt -5% to +21 of the L-FABP gene linked to nt+3 ofhGH [L-FABP(-596 to +21)/hGH] were derived fromeither G013 or G019 (12). Mice were fed a standard chow dietad libitum. Animals heterozygous for the transgenes werekilled by cervical dislocation 5-52 weeks after birth. Thecolon was divided into proximal, middle, and distal thirds.The small intestine was rapidly dissected into four segments.All tissues were fixed in Bouin's solution (12).

Immunocytochemistry. The methods and antisera used forthese studies are described elsewhere (12-15). Three to fivemice from each pedigree were studied at each age surveyed.The number of hGH-positive (hGH+) crypts and enteroen-docrine cells were noted for each animal, and the data wereaveraged for animals within each age group. At least twoseparate sections were examined from each small and largeintestinal segment ofeach mouse. A colonic crypt was scoredas positive for transgene expression if there was enterocyticstaining with anti-hGH serum in its uppermost strata of cellsand in its corresponding surface epithelial cuff (cells migrate

up colonic crypts onto the surface epithelium, where theyform hexagonal cuffs around the crypt orifice; ref. 4).

RESULTSTemporal Changes in Transgene Expression in the Colonic

Enterocytic Populations of Adult Mice. Analysis of 5-week- to1-year-old mice indicated that the endogenous gene Fabplwas never expressed in any region ofthe colon. The presenceof multiple (>100) copies of either L-FABP/hGH transgenedid not perturb this quiescent state.

Inappropriate expression of L-FABP(-5% to +21)/hGHwas observed in all colonic segments of 3- to 6-month-oldmice (Fig. LA). In proximal colon, hGH was present in mostcrypts (=90%o, Fig. lA). There were, however, occasionalpatches of several adjacent crypts with no detectable hGH(Fig. 2 A and B). Fewer crypts (418%) expressed hGH in themore distal colonic segments (Fig. LA). Those that did werealso present in patches (Fig. 2C).

Within an individual positive crypt (whether in the proxi-mal or distal colon), little immunoreactive hGH was detect-able in enterocytes located at the lowest cell strata. Intra-cellular levels ofhGH increased as cells migrated towards thesurface epithelium. Within a given stratum, cells had com-parable levels of the reporter. All crypts in each hGH+ patchcontained comparable steady-state levels ofthe reporter. Theboundary between positive and negative crypts was remark-ably sharp (Fig. 2 B and C).The number of crypts expressing hGH declined with in-

creasing age. This extinction of transgene expression pro-gressed from the distal colon to involve progressively more

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FIG. 1. Regional distribution of transgene expression in crypts asa function of age. Sections of proximal colon (PC), mid-colon (MC),and distal colon (DC) were analyzed for enterocytic expression ofhGH in transgenic animals containing either L-FABP(-596 to +21)/hGH (A) or L-FABP(-4000 to +21)/hGH (B).

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proximal segments (Fig. LA). In 10- to 12-month-old mice,transgene expression had been completely extinguished inthe distal colonic epithelium and the number ofhGH' cryptsin the proximal colon had declined to =22% (Fig. lA).Throughout this period (3-12 months) we noted that in

those colonic segments where transgene-expressing crypts

were rare (i.e., <20%/), most hGH' crypts occurred inpatches rather than as isolated individual crypts (Fig. 2C).This was not an insertion-site effect: it was observed in micederived from two different founders. If transgene expressionwere determined independently at the level of the postulatedsingle crypt stem cell, in segments where the probability ofan

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Fig. 2. (Legend appears at the bottom of the opposite page.)

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individual crypt expressing hGH is small, one would expectpositive crypts to occur predominantly in isolation ratherthan in clusters (patches).¶Young transgenic mice containing L-FABP(-4000 to

+21)/hGH also exhibited inappropriate expression ofhGH inmost colonic crypts (Fig. 2D). About 80% of crypts in theproximal colonic epithelium of 1- to 2-month-old mice con-tained immunoreactive hGH (Fig. 1B). While most cryptsshowed uniform expression ofhGH in enterocytes located ata particular cell stratum (Fig. 2 D and E), there were somecrypts that exhibited considerable variability in the steady-state levels of the reporter within comparably positionedcolumnar epithelial cells (Fig. 2F). Addition of nt -4000 to-597 increased the rate of extinction ofcolonic hGH expres-sion, compared with L-FABP(-596 to +21)/hGH mice, butdid not affect the orderly distal to proximal "wave" ofsuppression. By 5 months, the number of hGH+ crypts haddeclined sharply in all colonic segments (Fig. 1B). Cryptsshowing heterogeneous cellular patterns oftransgene expres-sion were no longer apparent. In older L-FABP(-4000 to+21)/hGH mice (5-12 months), isolated, single hGH' cryptswere found in the proximal colon and rarely in the middle anddistal colonic segments (Figs. 1B, 2G, and 2H).Temporal and Spatial Patterns of Exiion of Transgene

Expression in Colonic Enteroendocrine Cell Populations. Wehave described regional differences in the distribution ofenteroendocrine cell populations within the mouse gastroin-testinal tract and correlated their position in the crypt-to-villus and duodenal-to-colonic axes with their ability tosupport L-FABP/hGH expression (14, 15). Serotonin-producing cells represent the most abundant and PYY-immunoreactive cells the next most abundant enteroendo-crine subpopulations in the colonic epithelium. We thereforesought to determine whether the temporal and spatial pat-terns of suppression of transgene expression noted in colonic

IThe predicted distribution of patch sizes for crypts within a tissuecross-section where transgene expression (in each crypt) is inde-pendently determined can be described by F(n) = 2(1 - P)P), whereF(n) equals the fraction of total patches of size n, and P is theprobability that a randomly selected crypt in the tissue section ispositive. The fraction of total crypts expressing the transgene wasused as an approximation of P for this analysis. For colonicsegments of L-FABP (-596 to +21)/hGH transgenic mice wherethe frequency of hGH positive crypts was <20%o, the predictedfrequency distributions of patch sizes were significantly differentfrom the observed distributions, based on %I analysis.

enterocytes also occurred in enteroendocrine cells. The totalnumber of hGH+ colonic enteroendocrine cells closely par-alleled the total number of crypts that supported transgeneexpression in all regions of the large intestine at each agesurveyed (data not shown). hGH+ enteroendocrine cellsappeared to be confined to crypts that demonstrated entero-cytic expression of the reporter.The number of colonic enteroendocrine cells containing

immunoreactive serotonin orPYY did not vary with age (datanot shown). Most PYY-immunoreactive cells present incrypts containing hGH+ enterocytes also contained the re-porter (Fig. 2 I-L). However, none of the PYY cells presentin hGH- crypts/patches supported detectable levels ofL-FABP(-5% to +21)/hGH expression. This suggests thatPYY cells and enterocytes arise from a common progenitorand/or share common regulatory factors that modulate trans-gene expression. By contrast, this transgene was rarelyexpressed in colonic serotonergic cells, even when they werepresent in hGH+ crypts/patches (Fig. 2 M-O).

Age-ReatedSi OfTranagene Expression Does Not Occurin Small Itesial EnteroendocrineCes. Serotonergic cells rep-resent the most abundant enteroendocrine subpopulation in thesmall intestine and exhibit a distinct distribution from duodenumto ileum (14). We detected no changes in their number per crosssection in any ofthe four segments ofsmall bowel surveyed in 3-to 12-month-old normal or L-FABP(-5% to +21)/hGH trans-genic mice (data not shown). The total number of hGH+ ente-roendocrine cells per intestinal cross section also exhibitedcharacteristic differences from duodenum to ileum in adultL-FABP(-5% to +21)/hGH mice (the number decreasing pro-gressively from the proximal to the distal segments; data notshown). In contrast to the colon, the number of enteroendocrinecells that supported transgene expression did not significantlychange with increasing age in the small intestine (data notshown). These results indicate that temporal extinction ofL-FABP(-5% to +21)/hGH expression is limited to enteroen-docrine cell (sub)populations located in the colonic epithelium.Absnce ofAge-Related Differences in Transgene Expression

Within Small Intestinal Enterocytes. Both the L-FABP(-4000to +21)/hGH and L-FABP(-5% to +21)/hGH transgenesare efficiently expressed in small intestinal enterocytes, withhighest steady-state levels of hGH and its mRNA occurringin the proximal small bowel and decreasing distally (12). Wetherefore examined hGH-stained sections prepared from theproximal small intestine of all animals included in our surveyof colonic transgene expression. No comparable age-related

FIG. 2. Immunocytochemical studies on hGH expression in colonic epithelium ofaging transgenic mice. Distribution oftransgene expressionin colonic crypts ofL-FABP(-596 to +21)/hGH (A-C) or L-FABP(-4000 to +21)/hGH (D-G) transgenic mice was analyzed with anti-hGH serumand the immunogold silver intensification staining (IGSS) method. (A) Proximal colon from a 3-month-old L-FABP(-596 to +21)/hGH mousederived from G013, visualized with darkfield microscopy. Note the patches of negatively staining crypts (arrows). (x70.) (B) Proximal colon froma 9-month-old L-FABP(-596 to +21)/hGH transgenic descendent of G019, visualized with brightfield technique. The boundary between patchesof hGH+ and hGH- crypts is sharp. (x 180.) (C) Distal colon from a 3-month-old G013 descendant demonstrating a sharply demarcated patch ofhGH+ crypts (arrow). (Darkfield, x90.) (D) Five-week-old L-FABP(-4000 to +21)/hGH transgenic mouse showing isolated, individualhGH-negative crypts in proximal colon (arrows). (Darkfield, x70.) (E) Tangential section from the proximal colon ofa 5-week-old L-FABP(-4000to +21)/hGH mouse. Enterocytes in cell strata closer to the surface epithelium become uniformly hGH+. (Darkfield, x70.) (F) Tangential sectionof a crypt from a 5-week-old L-FABP(-4000 to +21)/hGH mouse. Note the mixture of hGH+ enterocytes (solid arrows) and hGH- enterocytesat (open arrows) this cell stratum. (x180.) (G) Proximal colon from a 7-month-old L-FABP(-4000 to +21)/hGH mouse demonstrating manyisolated, strongly hGH+ crypts. (Darkfield, x70.) (H) Tangential section through the proximal colon of a 12-month-old L-FABP(-4000 to+21)/hGH mouse. Note the isolated strongly hGH+ crypt. All enterocytes at this cell stratum were hGH+. (Darkfield, x90.) (I-K) Relationshipof L-FABP(-596 to +21)/hGH transgene expression in peptide tyrosine tyrosine (PYY)-containing enteroendocrine cells. A section of proximalcolon from a 7-month-old mouse was immunostained with either rabbit anti-PYY and IGSS (I, visualized with reflected-light polarizationmicroscopy) or goat anti-hGH and fluorescein-labeled donkey anti-goat sera (. Two enteroendocrine cells contained PYY (I, arrows). hGHcolocalized to one of these cells (open arrow) located in an hGH-positive crypt (I and J). The other PYY-containing cell (solid arrow) does notexpress the transgene J). Double exposure ofthe section illustrates these relationships (K). (x 115.) (L-O) Patterns ofL-FABP(-596 to +21)/hGHtransgene expression in PYY and serotonergic enteroendocrine cell populations in the proximal colon of a 3-month-old mouse. PYY-containingcells (solid arrows) were detected with rabbit anti-PYY serum and IGGS (L, visualized with reflected-light polarization microscopy). hGH wasvisualized in the same section by using goat anti-hGH and fluorescein-labeled donkey anti-goat sera (AM). Note that the PYY-containing cells (L,solid arrows) also are heavily stained for hGH (M, solid arrows). Serotonergic cells (open arrows) in the same section were detected using ratanti-serotonin and,-phycoerythrin-labeled donkey anti-rat sera(N). Double exposure ofMandN(O) demonstrates that the serotonergic cells (openarrows) do not contain significant levels of hGH and are distinct from hGH+ PYY-immunoreactive cells. (x180.)

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changes in the cellular or regional patterns of transgeneexpression were observed in small intestinal enterocytes.Together with the enteroendocrine data, these observationssuggest that time-dependent silencing of expression of bothtransgenes appears to be unique to colonic epithelial cells.

DISCUSSIONAnalysis of the expression of L-FABP/hGH fusion genes inadult transgenic mice has revealed remarkable, age-specificdifferences in reporter expression within the colonic epithe-lium. A distinctive, spatially well-organized pattern of ex-tinction ofL-FABP(-5% to +21)/hGH transgene expressionwas observed involving patches of colonic crypts. Theseresults emphasize the importance of considering temporal"gradients" when assessing the appropriateness oftransgeneexpression in the continuously renewing gut epithelium.They suggest that transgenes provide a powerful tool forfurther defining clonal and lineage relationships of colonicepithelial cell populations during life as well as the mecha-nisms that regulate their differentiation in space and time.While the endogenous mouse L-FABP gene (Fabpl) re-

mains silent in the colon during adult life, the L-FABP(-596to +21)/hGH fusion gene appears to be actively and coor-dinately expressed in groups of multiple adjacent crypts withsharply demarcated boundaries. This inappropriate expres-sion initially involves most colonic crypts. "Extinction" oftransgene expression occurs in patches and in a region-specific fashion (from distal colon to proximal colon). Theterm extinction has to be used cautiously because one of theprincipal questions raised by this phenomenon is whether itis due to repopulation ofthe colonic epithelium with cells thathave different regulatory environments for which the trans-gene is a sensitive reporter or whether it reflects the produc-tion of some intra- or extracellular factor that coordinatelymodulates transgene expression within "existing" (differen-tiating) cells. It is difficult to understand how local productionof such a factor(s) could result in sharply demarcated patchboundaries composed of crypts that uniformly express thetransgene at a given cell stratum, unless they affect regula-tory events occurring in stem cells and/or their progenitors.The patches oftransgene expressing crypts are reminiscent ofthe patches observed in the adult colonic epithelium of miceheterozygous for the G6PD alleles (11). Coordinate regula-tion oftransgene expression in multiple adjacent crypts couldarise through determination events occurring in the stem-cellprogenitor. Such a model would require that the progenitorsupply new stem cells to crypts within a colonic patch in adultmice (without this resupplying of colonic crypts, it would benecessary to invoke more complex models).As noted in the Introduction, treatment of adult mice

heterozygous for the G6PD orDlb-1 alleles only resulted in theproduction of isolated crypts with the mutant phenotype: nopatches of multiple crypts were observed. This suggests thatthe target cell for the somatic mutation was the crypt stem cellrather than its progenitor. There are at least two possiblereasons why these marker systems may not be sufficientlysensitive to reveal the postulated active progenitor cell: (t) therates of mutation produced were low and the number of cryptstem-cell progenitors could be very small compared to thenumber of its progeny or (it) their sensitivities to mutagen-induced allelic change may differ. Transgene expression in theamplified products of the progenitor may represent a way ofoperationally defining the biological properties of such a cell.It should be possible to test whether coordinate regulation oftransgene expression in multiple crypts reflects the influenceofthe crypt stem-cell progenitorby examining mice containingboth the L-FABP(-596 to +21)/hGH transgene and a somaticclonal marker expressed in the colon, such as G6PD. If theregulatory event that determines transgene expression occursin the crypt stem-cell progenitor, then patches ofhGH' crypts

may not cross clonal boundaries (as defined by histochemicalstaining for G6PD).Both the "temporal gradients" and spatial patterns of

L-FAPB/hGH expression appeared to be dependent on whatportion of the 5' nontranscribed sequence of the L-FABPgene was represented in the transgene. For instance, the rateof extinction of L-FABP(-4000 to +21)/hGH expressionwas considerably more rapid than that observed with theL-FABP(-5% to +21)/hGH fusion gene. In contrast toL-FABP(-596 to +21)/hGH, the silencing event(s) inL-FABP(-4000 to +21)/hGH appeared to be primarily man-ifested in isolated crypts rather than in patches of contiguouscrypts. All cells in a given crypt appeared to be ultimatelyaffected by the silencing event (compatible with the notionthat extinction involves the single crypt stem cell). However,it is curious that in young L-FABP(-4000 to +21)/hGHtransgenic mice, a striking heterogeneity in cellular hGHlevels occurred within a given stratum of some coloniccrypts. Such heterogeneous crypts were not observed in mice>4 months old and were not detected in L-FABP(-596 to+21)/hGH transgenic mice at any of the ages surveyed. Thissuggests that the additional 3.3 kilobases of upstream se-quence also contains cis-acting elements that detect subtledifferences in the regulatory environments of individualcolonic enterocytes which are not perceived by nt -596 to+21. These data imply that the mechanism of silencing ofFabpl involves regulatory steps that affect multiple levels ofthe differentiation pathway. Each of these regulatory (deter-mination) events can be operationally described on the basisof the different patterns of expression of L-FABP(-596 to+21)/hGH, L-FAPB(-4000 to +21)/hGH, and Fabpl.

In summary, transgenes can be used to characterize subtlechanges that occur in the continuously proliferating/differentiating gut epithelium with time-differences that arenot currently definable with other approaches. Moreover,these molecular probes hold the promise of providing a wayto describe and monitor the properties of stem cells and theirprogenitors from which the intestine's complex cellular pop-ulation continuously arise. Such an inferential approach maybe allowable because of the unique spatial constraints im-posed upon the amplified descendants ofthese stem cells andtheir progenitors, permitting the investigator to "march back-wards" through the spatial and temporal pathways of (gut)epithelial cell differentiation.We thank Douglas Winton and Gunter Schmidt for many helpful

discussions. This work was supported by National Institutes ofHealth Grants DK30292 and DK37690.1. Gordon, J. I. (1989) J. Cell Biol. 1i8, 1187-1194.2. Ponder, B. A. J., Festing, M. F. W. & Wilkinson, M. M. (1985) J.

Embryol. Erp. Morphol. 87, 229-239.3. Ponder, B. A. J., Schmidt, G. H., Wilkinson, M. M., Wood, M. J.,

Monk, M. & Reid, A. (1985) Nature (London) 313, 689-691.4. Schmidt, G. H., Wilkinson, M. M. & Ponder, B. A. J. (1985) Cell 4,

425-429.5. Schmidt, G. H., Wilkinson, M. M. & Ponder, B. A. J. (1986) J. Embryol.

Exp. Morphol. 91, 197-208.6. Schmidt, G. H., Winton, D. J. & Ponder, B. A. J. (1988) Development

1413, 785-790.7. Winton, D. J., Blount, M. A. & Ponder, B. A. J. (1988) Nature (London)

333, 463-466.8. Winton, D. J., Peacock, J. H. & Ponder, B. A. J. (1989) Mutagenesis 4,

404-406.9. Winton, D. J. & Ponder, B. A. J. (1990) Proc. R. Soc. London Ser. B.

241, 13-18.10. Schmidt, G. H., O'Sullivan, J. F. & Paul, D. (1990)Mutat. Res. 228,149-155.11. Griffiths, D. F. R., Davies, S. J., Williams, D., Williams, G. T. &

Williams, E. D. (1988) Nature (London) 333, 461-463.12. Sweetser, D. A., Birkenmeier, E. H., Hoppe, P. C., McKeel, D. W. &

Gordon, J. I. (1988) Genes Dev. 2, 1318-1332.13. Roth, K. A., Rubin, D. C., Birkenmeier, E. H. & Gordon, J. I. (1991) J.

Biol. Chem., in press.14. Roth, K. A., Hertz, J. M. & Gordon, J. I. (1990)J. Cel Biol. 110, 1791-1801.15. Roth, K. A. & Gordon, J. I. (1990) Proc. Nad. Acad. Sci. USA 87,

6408-6412.

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