Proc. Natl. Acad. Sci. USAVol. 91, pp. 12999-13003, December 1994Developmental Biology

Ablation of islet endocrine cells by targeted expression ofhormone-promoter-driven toxigenes

(pancreas/pancreatic polypeptide-fold family/cell lineage/transgenic/diphtheria toxin)

PEDRO-LUIS HERRERA*t, JOAQUIN HUARTE*, ROMAIN ZUFFEREY*, ANTHONY NICHOLS*,BERNADETTE MERMILLOD*, JACQUES PHILIPPE§, PEDRO MUNIESA*¶, FRANCESCA SANVITO*,LELIO ORCI*, AND JEAN-DOMINIQUE VASSALLI**Ddpartement de Morphologie, tCentre d'Informatique Hospitali0re, and §Ddpartement de Gdndtique et Microbiologie, University of Geneva Medical School,CH-1211 Geneva 4, Switzerland

Communicated by Roger H. Unger, September 12, 1994

ABSTRACT Ontogenic relationships between the differenttypes of endocrine cells in the islets of Langerhans wereexplored by generating transgenic mouse embryos in whichcells transcribing the glucagon, insulin, or pancreatic polypep-tide genes were destroyed through the promoter-targeted ex-pression of the diphtheria toxin A chain. Embryos lackingglucagon- or insulin-containing cells did not exhibit alterationsin the development of the nontargeted islet cell types, whereasembryos lacking pancreatic polypeptide gene-expressing cellsalso lacked pancreatic insulin- and somatostatin-containingcells. These results show that neither glucagon nor insulingene-expressing cells are essential for the differentiation of theother islet endocrine-cell types. These results also suggest thatpancreatic polypeptide gene-expressing cells are indispensablefor the differentiation of islet .8 and 6 cells because the formerproduce a necessary paracrine or endocrine factor and/oroperate through a cell-lineage relationship.

The pancreas arises as a double protrusion of the duodenalendoderm, from which both the exocrine (excretory ductsand acini) and the endocrine (islets of Langerhans) portionsof the gland develop (1-3). Hormone-containing cells are firstdetected among the epithelial cells of the primitive ducts (2),and the four types of endocrine cells appear sequentiallyduring development (4-6). On the basis of the reportedcolocalization of different hormones within individual cells inthe mouse embryonic pancreas, a cell-lineage model has beenproposed that suggests cells coexpressing glucagon and in-sulin as precursors to all adult islet endocrine cells (7).However, coexpression patterns do not provide indisputableevidence for the identification of stem cells; additional ap-proaches are thus necessary.The selective ablation of cell lineages through targeted

expression of toxigenes can provide strong evidence for oragainst cell lineage and/or paracrine/endocrine relationshipsduring ontogeny (8, 9). The characterization of regulatorysequences that direct the cell-type-specific expression ofcoding sequences in chimeric transgenes is an indispensablestep for such an experimental approach. For the glucagon-producing a cells and the insulin-producing 13 cells, thisinformation is already available: the regulatory regions of therat glucagon and insulin II genes have been studied previ-ously, and sequences directing transgene expression in theappropriate mouse islet cells have been identified (10-12).Unexpected sites ofexpression have also been observed-forinstance, with the insulin II promoter (7), indicating thatresults obtained in transgenic mice prepared with such pro-moters need to be interpreted carefully. In addition to glu-

cagon and insulin production, another endocrine phenotypeseen early during islet ontogeny is that of the pancreaticpolypeptide (PP)-fold family; the issue of which of thepeptides of this family-i.e., PP, neuropeptide Y, and peptidetyrosine tyrosine-is (are) present in the mouse embryonicpancreas remains controversial because of the cross-reactivity of antibodies raised against these closely relatedpeptides (5, 13, 14). The demonstration that PP mRNA ispresent early during pancreas ontogeny (5, 6) led us to selectthe PP-gene promoter as a third toxigene-targeting sequence.Because a functional characterization of the rodent PP genehas not been reported, we first identified a region of this genecapable of directing cell-type-specific expression in mouseembryonic pancreas.One of the most powerful toxic gene products is the

diphtheria toxin A chain (DT). DT adenoribosylates elonga-tion factor EF2, thus blocking protein synthesis and causingthe selective and cell-autonomous ablation ofcells expressingDT-encoding transgenes (for reviews see refs. 8 and 9). In itsnative form, the diphtheria toxin consists of two chains(subunits), A and B, that are the cleavage products ofa longerprecursor polypeptide. The B subunit is required for endocy-tosis of the A subunit; thus, in the absence of the B subunit,the expression of the A subunit in a given cell causes its deathwithout affecting neighboring cells. Another relevant aspectof the use ofDT for cell ablation is its extreme toxicity (onlya few molecules are sufficient to kill a cell); hence, preciselevels of DT expression may not be critical. For as-yet-unexplained reasons, there is a relatively poor penetrance ofDT-encoding transgenes (15-19), and the generation of mul-tiple transgenic animals is required to obtain individuals orstrains with expression in a majority of targeted cells. An-other inherent drawback of DT-encoding transgenes is thatbecause expression is lethal to the targeted cells, the cell-typespecificity of expression cannot be verified with certainty;that the chimeric (promoter-DT) transgene follows the samepattern of expression as that imposed by the promoter onanother reporter-coding region has to be accepted. Despitethese two limitations, hormone-promoter-DT chimeric trans-genes have been used successfully to assess cell lineages (8,15-18).

In the present work, through the targeted ablation of cellsexpressing the glucagon, insulin, and PP genes, we demon-strate that neither glucagon-producing a cells nor insulin-producing p cells are required for the development of theother endocrine cell types. By contrast, we find that ablationof cells expressing the PP gene results in a marked defect in

Abbreviations: PP, pancreatic polypeptide; GH, growth hormone;hGH, human GIl; DT, diphtheria toxin A chain; Vd, volume density.tTo whom reprint requests should be addressed.VPresent address: Unidad de Anatomia y Embriologia, Facultad deVeterinaria, Universidad de Zaragoza, 50013 Zaragoza, Spain.

12999

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13000 Developmental Biology: Herrera et al.

FIG. 1. PP promoter directs the expression of an hGH-encoding reporter transgene to cells stained with anti-bovine PP antibodies. Threeconsecutive semi-thin sections of the pancreas from a PP-hGH transgenic mouse embryo were stained, respectively, with anti-bovine PP,anti-hGH, and anti-bovine PP antibodies. Arrowheads identify a cell that is stained in all three sections. (Bar = 5 ,um.)

the development of insulin- and somatostatin-producingcells. These results provide additional insights into the on-togeny of the pancreatic islets of Langerhans.

MATERIALS AND METHODSPreparation of the Growth Hormone (GH) and DT Clones.

Human growth hormone (hGH) and DT-encoding transgeneswere prepared as follows:PP-hGH: a 548-bp fragment from the 5' region ofthe rat PP

gene was amplified by PCR, between nt -529 and +19[sequence according to Yonekura et al. (20)] using rat livergenomic DNA as template, inserted in a Bluescript II KSplasmid at the Sma I site (pRPP), and sequenced. A ClaI-Kpn I fragment ofa Bluescript II KS plasmid containing thehGH BamHI/blunted-EcoRI/blunted fragment of pNUT(15) was inserted between the Cla I-Kpn I sites of pRPP.Ins-DT: the rat insulin II gene promoter (660 bp) (10) was

inserted as a BamHI-Xba I fragment (11) in pUC19 (pUC19-RIP); then, a Kpn I-Xba I fragment containing the promoterwas inserted in pDT-A (15) at the corresponding sites (pRIP-DT). A Kpn I-Dra II fragment of simian virus 40 genome wasexcised from a pBluescript II KS containing the KpnI-BamHI early region of simian virus 40 and replaced by aKpn 1-HindIlI fragment from pRIP-DT after suitable endmodifications. This provided a poly(A) signal at the end oftheDT sequence (plns-DT).Glu-DT: two different toxigenes were used to eliminate

glucagon cells: a 1.3-kb Sac I/blunted-Kpn I fragment ofpoCAT plasmid (12), containing the rat glucagon gene pro-moter was inserted into pDT-A at the Kpn I-PstI/bluntedsites (pGlu-DT); the poly(A) signal was added as for theIns-DT gene. In an attempt to improve the penetrance of theDT-encoding transgene, the hGH-coding region (15) wasadded as follows: the metallothionein promoter was excisedfrom pNUT (15) with Kpn I and BamHI digestion andreplaced by the 2.1-kb Kpn I-Bgl II fragment from pGlu-DT.Embryos no. 128 and no. 133 of the Glu-DT group wereobtained by using the Glu-DT-GH toxigene. The twoGlu-DT transgenes yielded a similar proportion of affectedtransgenics.PP-DT: the rat insulin II gene promoter in plns-DT (frag-

ment Sma I-Pst I) was replaced by the rat PP gene promoter(fragment Pvu II-Pst I from pRPP).

Generation and Screening of Transgenic Mice. Transgenicmice were produced by pronuclear injection of B6D2F1 xB6D2F1 zygotes, as described (21), using DNA solutions at5 ng/,ul. The foster mothers were sacrificed at E16.5 (em-bryonic day 16.5, age postcoitum) by cervical dislocation;placental genomic DNAs were extracted, as described (22),for determination of transgenesis using a 32P-labeled cDNAprobe complementary to the 480-bp simian virus 40 cassette,in a DNA dot hybridization assay. A probe complementaryto hGH gene was used for the screening of PP-hGH trans-genic mice. Assessment of transgene expression in DT-transgenic embryos was limited to histological examinations,as toxigene activation destroys targeted cells.

Histological Procedures and Statistical Analysis. Whole pan-creata were fixed in 1% glutaraldehyde and embedded inEpon 812; consecutive semi-thin sections of both portions ofthe gland (dorsal and ventral) were then stained by indirectimmunofluorescence to localize islet endocrine cells withdescribed antisera (5), in addition to rabbit IgG anti-hGH at1:1000 (from D. P. Cameron, Prince Henry's Hospital, Mel-bourne) and rabbit IgG anti-human PP at 1:200 (PeninsulaLaboratories, IHC 7198). As initially noted by others (13), theanti-bovine PP used in our experiments is not specific for PP;rather, it recognizes with comparable affinities all threemembers of the PP-fold family (F. S. and P.-L. H., unpub-lished work).Morphometric analyses were done on the immunostained

semi-thin sections to obtain the volume density (Vd) ofglucagon-, insulin-, PP-fold family-, and somatostatin-containing cells, using a minimum of 14,000 points perpancreas and anti-hormone serum. Vd was calculated by thepoint-counting method (23), according to the formula: Vd =

(P-endocrine/P-pancreas) x 100, where P-endocrine equalspoints of the lattice over endocrine cells, and P-pancreasequals points of the lattice over total pancreas, includingacini, ducts, vessels, and connective tissue. Vd values ofeachislet cell type were compared between groups (control/Ins-DT/PP-DT for glucagon cells, P = 0.02; control/Glu-DT/PP-DT for insulin cells, P < 0.0001; control/Glu-DT/Ins-DTfor PP-fold family cells, not significant; and control/Glu-DT/Ins-DT/PP-DT for somatostatin cells, P < 0.0001) by anested ANOVA on the logarithms of the data; pairwisecomparisons were done using the Bonferroni correction (24),between groups showing statistically significant differences.

Table 1. Generation of Glu-DT, Ins-DT, and PP-DT transgenic miceTransferred E16.5 embryos Transgenic Transgenic embryos

Construct embryos, no. recovered, no. (%) embryos, no. (%) with phenotype, no. (%)Glu-DT 604 60 (10%o) 10 (17%) 3 (30%o)Glu-DT-GH 174 27 (16%) 6 (22%) 2 (33%)Ins-DT 510 67 (13%) 13 (19%o) 5 (38%)PP-DT 617 80 (13%) 13 (16%) 5 (38%)

E16.5, embryonic day 16.5.

Proc. Natl. Acad Sci. USA 91 (1994)

Proc. Natl. Acad. Sci. USA 91 (1994) 13001

GLUCAGON

0 2 _-3

Z A-0 6m

H58 It68

D 69 II-

128 U133 1

(p:0.08)20

a 49 -1m-ch 54 MZ 60 _}

61 I -I

INSULIN

(11)(11)

d_m- (11)IF (10)- (11)

(13).I

(n.s.)(16)(16) -m

(12) -3-

(26) 1{_-(17) * 3.

(10) i(15)(6)

(13)(13)

PP-fold family

(11)s-AM (14)

- (15)-N (7)

-(3- (20)U- (14)

(n.s)*(13)

(11) -m(8) I}

(19) §.(9) 4-

(6) -(n.s)*(7)

(10) 1}(8) a_

(12) _I - .

SOMATOSTATIN

O* (6)a _(8)_ ~~~~~~~~~(a)__} (12)___ (9)

0- (12)(16)

(12) =B- (n.s.) (163)(12) -(13)

(7) l_ (7)(19) * (16)

(13) * (11)I I

(4)(n.s.)

(10)(6) _(7)

(16)fI

(8)(6)(4)(9)

(P:0.03)20.- I 3t-

.; 32 g (16)

aX 54 - (27) 169 M-(13) O

(p < 0.001)(18) 1..

(18) l.(28)(30) l,.(16) .

(p<0.0 01)(19) I(21) l(21) U-(26) 1..(13) .

I I~~~~~~~~

0 2 4 6 8 0 2 4 6 8 0 2 4 6 8 0 0.2 0.4 0.6

Vd

FIG. 2. Box plots (26) representing the Vd of each pancreatic endocrine-cell type in normal (control) and transgenic mouse embryos. Boxessymbolize the interquartile space of each distribution; the median is indicated, when not null, as a vertical line within the box, and dots indicateextreme values. The number of sections scored per embryo for elaboration of the box plots is expressed in parentheses. P values are given ascompared with control embryos by a nested ANOVA: n.s., Not significant; *, no difference. Other significant differences, not indicated, wereas follows: decrease of the Vd of insulin cells in PP-DT embryos vs. Glu-DT embryos (P < 0.001) and a reduction of the Vd of somatostatincells in PP-DT embryos either vs. Glu-DT (P = 0.002) or Ins-DT (P < 0.001) embryos.

RESULTS

The four major adult islet endocrine cell types (glucagon-producing a cells, insulin-producing , cells, somatostatin-producing 8 cells, and PP-producing cells) can be distin-guished on the basis of the selective expression of thehormone genes (25). Previous studies have shown that the660-bp and 1300-bp fragments of the 5' flank of the rat insulinII and glucagon genes, respectively, can be used to direct thecell-type-specific expression of reporter genes-at leastwithin islets of Langerhans (10-12); extrapancreatic expres-sion-e.g., in the developing neural tube for the insulin IIpromoter-has also been described (7). The promoter of thePP gene has not yet been functionally characterized. Wearbitrarily selected a 548-bp sequence of the 5' region of therat PP gene and tested its ability to direct cell-type-specificexpression of a reporter coding sequence in the pancreata oftwo E16.5 transgenic embryos; as a reporter, we used hGH,an antigen that can be revealed immunocytochemically onsemi-thin sections. Cells containing hGH were examined onadjacent semi-thin sections for the presence of the differentislet hormones, using the following experimental strategy.We prepared sets of three consecutive semi-thin sections,stained the middle section with anti-hGH, and stained the twoadjacent sections with antibody against one of the pancreatichormones (see Fig. 1). Ofthe hGH-containing cells that couldbe shown to also contain a pancreatic hormone, 78% had a

peptide of the PP-fold family-i.e., they were stained by theanti-bovine PP antibody (Fig. 1). By contrast, only 12% ofdoubly labeled cells contained insulin, and the remaining 10%contained glucagon. Considering the frequency of coexpres-sion of the latter two hormones with PP-fold family peptidesin the embryonic pancreas (5), it is likely that the cells

containing hGH and insulin or glucagon also contained PP ora related peptide. In addition, no cells containing hGH andsomatostatin were detected. A parallel analysis was alsodone: cells stained by the anti-bovine PP antibody wereexamined on adjacent sections for the presence ofhGH. Only12% of the anti-bovine PP-positive cells-i.e., cells contain-ing one or another member of the PP-fold family-were alsopositive for hGH, indicating that, as expected, the PP pro-moter is not expressed in all PP family-expressing cells but isexpressed only in a subset of these; this agrees with the factthat PP-containing cells are less abundant in the embryonicmouse pancreas than cells containing other members of thePP family (13, 14, and P.-L. H., unpublished observations).Thus, the PP-promoter fragment selected for our experimentsdirected expression of a reporter transgene mostly, andperhaps exclusively, to cells also expressing a PP-fold familygene.The promoters of the three islet hormones were used to

target the expression ofDT encoding toxigenes in transgenicmouse embryos (Table 1). To decrease the influence ofpossible secondary effects of the toxigenes (e.g., embryolethality), we analyzed transgenic embryos at E16.5-i.e., atan early time during development when the four islet endo-crine cell types are already easily detectable. The Vd of cellslabeled by anti-glucagon, anti-insulin, and anti-PP familyantibodies was determined on semi-thin sections of dissectedpancreata. To select embryos with an altered phenotype, weconsidered an 85% or greater reduction in the Vd of thetargeted cell type as evidence of transgene expression; pre-sumably because of the documented poor penetrance of DTtoxigenes (15-19), according to the criterion used, onlyapproximately one-third of transgenic embryos had a clearlyaltered phenotype (Table 1). Additional embryos exhibited a

fetus-#

(10)(15)(16)(25)(12)

Developmental Biology: Herrera et al.

13002 Developmental Biology: Herrera et al.

GLU

-j0Ir-z0

0ELa-

INS PP family

FIG. 3. Expression ofa PP-DT toxigene strongly decreases the number of cells stained by anti-bovine PP and anti-insulin antibodies, whereasglucagon-containing cells remain abundant. Semi-thin sections ofpancreata from control (upper row) and PP-DT transgenic (lower row) embryoswere labeled with anti-glucagon, anti-insulin, and anti-bovine PP antibodies. Erythrocytes are unspecifically stained. (Bar = 10 um.)

less dramatic (e.g., 50-80%) decrease in the Vd of thetargeted cell type.

Five embryos with clear evidence of transgene expressionwere studied for each type of transgene. The Vd of glucagon-,insulin-, PP-fold family- and somatostatin-containing cellswas determined. The quantitative data from these transgenicembryos and from control embryos are presented in Fig. 2.The loss of glucagon-containing cells caused by the expres-sion of Glu-DT transgenes was not accompanied by a sig-nificant decrease in the Vd of the other endocrine cell types.Similarly, expression of the Ins-DT transgene did not affectthe Vd of 8 or PP-family cells, and the Vd of a cells decreasedonly marginally. As expected from the reported expression ofanother insulin-promoter-driven transgene in the developingmouse central nervous system (7), major neural tube malfor-mations were seen in Ins-DT transgenic embryos (data notshown).The loss of PP-containing cells in PP-DT transgenic em-

bryos was accompanied by a parallel and highly significant (P< 0.001) decrease in the Vd of insulin- and somatostatin-containing cells (Figs. 2 and 3); by contrast, the Vd ofglucagon-containing cells was only very slightly reducedcompared with that in controls (P = 0.03) (Figs. 2 and 3). Thedevelopment of PP-DT transgenic embryos was retarded(unfused eyelids, persistence of the umbilical hernia, meanbody length 12.9 mm ± 0.4 vs. 16.3 mm ± 1.4 for nontrans-genic embryos of the same age). PP-DT transgenic embryoswith only a partial effect of the transgene (e.g., a 50-80%decrease in PP-containing cells) experienced a partial de-crease in the Vd of insulin- and somatostatin-containing cellsof similar magnitude (data not shown), confirming the rela-tionship between PP-expressing cells and P and 8 cells.The pancreas of transgenic embryos was also explored for

possible alterations in the development of the exocrine por-tion of the gland. Ablation of a, f3, or PP-fold family cells did

not detectably affect the differentiation of either ducts oracini.

DISCUSSIONThe toxigene-mediated ablation of endocrine-cell popula-tions in developing islets of Langerhans indicates that thepresence of glucagon- or insulin-producing cells is not re-quired for the other cell types to differentiate. By contrast,the ablation ofPP gene-expressing cells is accompanied by asevere defect in the development of insulin- and somatosta-tin-producing cells. The interpretation of these results relies,at least in part, on the specificity of expression of thetoxigenes. The regulatory sequences used have been shownto direct the cell-type-specific expression of reporter se-quences; however, we cannot exclude an influence of theDT-coding region on the spatial or temporal specificity ofexpression of one or multiple transgenes, and because DTkills the transgene-expressing cells, it is inherent in thisapproach that the phenotype ofthe expressing cells cannot beverified.

Despite this limitation, the interpretation ofthe results withat least two of the three toxigenes in unambiguous. Indeed,with the glucagon-promoter- and insulin-promoter-driventoxigenes, only the targeted-cell population was significantlyaffected, suggesting that the specificity of expression wasprecisely dictated by the regulatory promoter sequences. Wecan therefore conclude that cells that express glucagon andcells that express insulin do not appear to produce a paracrineor endocrine factor required for normal islet development,nor do they seem to be the obligatory precursors of either ofthe other islet cell types. The data presented here are thus notcompatible with a model in which cells containing glucagon(either alone or together with insulin) are the precursors of allislet endocrine-cell types (7): such a model predicts thatGlu-DT and/or Ins-DT transgenics should experience a loss

Proc. Natl. Acad. Sci. USA 91 (1994)

Proc. Natl. Acad. Sci. USA 91 (1994) 13003

in nontargeted cell types, which is not seen. That ablation ofinsulin-containing cells does not entail a significant deficit inglucagon-containing cells, and conversely, is in accord withour own observations that failed to reveal a population ofcells containing both hormones in the mouse embryo pan-creas (5) and with the absence of coexpression of insulinpromoter factor 1 and glucagon during development (27).The concomitant ablation of insulin- and somatostatin-

producing cells together with the targeted PP-expressing cellpopulation contrasts sharply with the results obtained withthe other toxigenes. In this case, the interpretation is moredifficult because, for the reason stated above and despite thespecificity of expression of the PP-hGH transgene, it isimpossible to formally exclude that cells that produce no PPmay express the DT-encoding toxigene at one or another timeduring development. Moreover, as PP-DT embryos haveretarded growth, we cannot exclude that some of the ob-served loss of 8 and 8 cells is temporary. Although these areimportant reservations inherent in the experimental strategyused, another interpretation of our results is *that PP-expressing cells are indispensable for the differentiation ofislet p and 8 cells. This relationship could be due to aparacrine or endocrine effect, PP gene-expressing cells pro-ducing a growth factor necessary for the differentiation of ,3and 8 cells. Alternatively, or in addition, a lineage relation-ship may relate , and 8 cells to PP-expressing cells, so thatablation of PP-expressing precursors precludes their differ-entiation to insulin- and somatostatin-producing cells. Thecolocalization of PP (or other members of the PP-fold family)and insulin or somatostatin in a fraction of embryonic pan-creas cells (5) may be the consequence of such a lineagerelationship. In this context, the slight decrease in the Vd ofa cells in PP-DT transgenics is also compatible with thecolocalization of PP family peptides and glucagon in thedeveloping pancreas (5). In this case, however, most a cellsdo not appear to develop from such PP- and glucagon-coexpressing precursors. The near-normal development ofthe a-cell population in PP-DT transgenics also shows thatexpression of the toxigene in the targeted cells and theensuing cell lethality do not indiscriminately affect the dif-ferentiation of all islet endocrine cells.

Further studies will be required to precisely determinewhich members of the PP-fold family are expressed in thepancreas of the mouse embryo; this will depend upon theavailability of antibodies of high specificity. In accord withthe demonstration that PP-family peptides other than PP itselfare more abundant in the embryonic pancreas (13, 14), wehave recently found that only a small fraction of cells stainedwith the anti-bovine PP antibody (which recognizes all threemembers of the family) are also detectably stained by a newlyavailable specific anti-PP antibody. Furthermore, we ob-served that only 12% of cells recognized by the anti-bovinePP antibody express the PP-hGH transgene, suggesting thatthe PP promoter targets a subset of cells expressing PP-foldfamily genes. However, the PP-promoter-driven toxigeneablated most of the anti-bovine PP-stained cells. This resultis compatible with the view that PP-gene-expressing cells arenecessary for the development not only of insulin- andsomatostatin-containing cells but also of cells expressing allmembers of the PP-fold family.

In summary, the use of hormone-promoter-driven toxi-genes allows some firm conclusions to be drawn with respectto cellular relationships in the ontogeny of the endocrinepancreas-in particular, regarding the limited role of gluca-

gon- or insulin-producing cells in this process. In addition,this experimental strategy suggests a role for PP-gene-expressing cells in the development of other endocrine lin-eages in the islets of Langerhans.

We are most grateful to Mrs. I. Condacci for her skillful technicalassistance and to Mr. G. Negro and Mr. G. Andrey for photographicwork. We also thank Dr. R. D. Palmiter for providing the plasmidcontaining the DT cassette. This work was supported by a grant fromthe Swiss National Science Foundation (31-34088.92).

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