Transcript
Page 1: Transient Absence ofCD44Expression andDelayin Development ...cgd.aacrjournals.org/cgi/reprint/8/11/1211.pdf · Transient Absence ofCD44Expression andDelayin Development byAnti-CD44

Vol. 8, 1211-1223, November 1997 Cell Growth & Differentiation 1211

Transient Absence of CD44 Expression and Delay inDevelopment by Anti-CD44 Treatment duringOntogeny: A Surrogate of anInducible Knockout?1

Margot Z#{246}ller,2Karin Herrmann, Sumy B#{252}chner,Simone Seiter, Christoph Claas,Charles B. Underhill, and Peter M#{246}ller

Department of Tumor Progression and Immune Defense, GermanCancer Research Center, 69120 Heidelberg, Germany [M. Z., K. H.,S. B., S. S., C. C.]; Department of Applied Genetics, University ofKarlsruhe, 7900 Karlsruhe, Germany [M. Z.]; University Hospital ofDermatology, Heidelberg, Germany [S. S.]; Department of Anatomy andCell Biology, Georgetown Medical Center, Washington, D. C. [C. B. U.];and University Institute of Pathology, 59081 Ulm, Germany [P. M.]

AbstractBecause the lack of some adhesion molecules inducedby site-directed mutagenesis has been described to belethal, whereas the lack of others apparently has noeffect, we were interested in seeing whether thedeveloping organism might gradually adapt to theabsence of adhesion molecules. Therefore, we chose aform of transient interference by i.v. injection ofantibody into pregnant rats. As a model, we selected

CD44, which has been reported to play a key roleduring embryogenesis. Rats received either anantibody recognizing an epitope on the CD44 standardisoform (CD44s) or on exon v6 (CD44v6).

In the presence of anti-CD44s, delivery wasfrequently delayed, and intrauterine abortions wereoften observed. The fetuses were smaller, particularlythe anlage of the hair follicle of the whisker, and theformation of alveoli in the lung, of the tubular systemof the kidney, and of villi in the gut was delayed.The development of fetuses receiving antl-CD44v6 washampered until days 16-18 of gestation.lmmunodetection revealed a weaker expression of thetarget molecules at the implantation site and thecomplete absence of CD44s and CD44v6 expression infetal tissue until day 12. During the late stages ofgestation, the expression pattern of CD44 resembled

that of 2-3-day-younger fetuses of untreated rats.Interestingly, degradation of hyaluronate was alsodelayed, particularly in the kidney. Thus, the

Received 5/15/97; revised 7/10/97; accepted 9/2/97.The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to mdi-cate this fact.1 Supported by Deutsche Forschungsgememnschaft Grant Zo40-5/2 (toM. 1).2 To whom requests for reprints should be addressed, at Department ofTumor Progression and Tumor Defense, German Cancer Research Cen-ter, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany. Phone: 6221-422454; Fax: 6221-424760; E-mail: M.ZoellerODKFZ-Heidelberg.DE.

diaplacental antibody passage was very efficient andshould make it possible to obtain a clearly defined anddifferentiated concept of the requirements for theCD44 molecule during ontogeny and also for fail-safemechanisms. Both experiences may be missed in theknockout proper.

Inti’oduction

The generation of homologous knockout mutants providesone of the most elegant proofs of function (1-3). However,there may also be drawbacks to the knockout approach.Deletion of a gene may be lethal at early stages of gestation,or the knockout mouse may be in perfect health despitecontrary expectations. Whereas the former outcome doubt-lessly proves the vital necessity of a gene product, the latteris regarded as evidence of the plasticity of developmentalregulation of gene expression. Respective examples of ad-hesion molecules are the knockout of vascular cell adhesionmolecule 1 and E-cadherin, which have been described to belethal (4-6), whereas minor alterations have been observed

as a result of gene disruption of contact site A protein orintercellular adhesion molecule 1 (2, 7, 8). Unfortunately, bothan unaltered phenotype and lethality at early stages of ges-tation may not allow for a differential analysis. In thesesituations, the reversible modulation of a gene product byantibody treatment during embryogenesis may be advanta-geous. The approach, although restricted to surface mole-cules expressed during ontogeny, has several advantages:(a) it may offer the possibility of defining the critical stage of

expression of the gene product in question (9) and analyzingthe alternate pathway of development; (b) it may allow anestimate of the outcome of an inducible knockout proper;and (c) it is technically easy, inexpensive, and quickly estab-lished. As an example, we chose the CD44 family of adhe-sion molecules.

CD44 is a rather ubiquitous family of adhesion molecules(1 0) that can differ in the form of glycosylation (1 1 , 1 2) and inthe insertion of variant exons through alternative splicing ofpre-mRNA (13-16). There exists a multitude of variant iso-

forms (1 7, 18). The hematopoietic isoform of CD44, CD44s,3is expressed on many cells and tissues in the adult organism,whereas CD44v isoforms are only detected on some spe-cialized epithelial cells and on activated lymphocytes (19-24). Different from the adult organism, CD44v isoforms are

3 The abbreviations used are: CD44s, CD44 standard isoform; b-PG.biotinylated mixture of proteoglycan and link protein; CD44v, CD44 van-ant isoferm; CD44V6, CD44 isoform containing oxen v6; HA, hyaluronicacid; mAb, monoclonal antibody.

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1212 Delay in Development by Anti-CD44

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Fig. 1 . Diaplacental passage of immunoglobulin and enrichment in the fetal tissue. A, control and pregnant (day 8 of gestation) BDX rats (5 rats/group)received a single iv. injection of 200 �g of 125l-labeled mouse lgGl of irrelevant specificity (3-9) or anti-CD44s (Ox50; mouse lgGl) in which the amountof 1251 was adjusted to 20 x 106 cpm/rat. Rats were sacrificed after 24 h, and the amount of antibody in serum, skin, muscle, liver, lung, kidney, gut, lymphnode, uterus, fetus within the extraembryonic coelom, and the amniotic cavity was determined in a gamma counter. The mean cpm ± SD/g tissue is shown.B, control and pregnant (day 10 ofgestation) BDX rats (5 rats/group) received a single iv. injection of200 �g of 125l-labeled anti-CD44s (Ox50; mouse lgGl)or IgGi of irrelevant specificity in which the amount of 1251 was adjusted to 20 x 1 06 cpm/rat. Rats were sacrificed after 24 and 48 h, and the amount ofantibody in serum, skin, muscle, liver, lung, kidney, lymph node, uterus, fetus, and placenta was determined in a gamma counter. The ratio of cpm/gtissue:cpm/g serum is presented.

abundantly expressed during implantation, gastrulation, and

embryogenesis (23-27). By the transfer of CD44v cDNA, butalso by antibody inhibition studies, we have provided evi-

dence on the functional activity of CD44v in tumor progres-

sion and lymphocyte activation (28-31). Now we will de-

scribe the transient inhibition of embryonic development by

as simple an approach as two weekly injections of anti-CD44

into the tail vein of a pregnant rat.

ResultsInfluence of Anti-CD44 on Implantation, Placentation,Embryogenesis, and Fetal Development. In a pilot exper-iment, the distribution of IgGi in the maternal organism, thepassage through the maternal placenta, and the retention of

antibody in the fetal tissue were evaluated (Fig. 1A). A group

of nonpregnant female rats and a group of pregnant rats at 8

days of gestation received an iv. injection of 1251-labeled

mouse IgGi of irrelevant specificity or of 1251-labeled anti-

CD44s (mouse IgG1). Rats were bled by heart puncture and

killed 24 h thereafter. Organs, including the uterus and, in

pregnant rats, the fetus within the extraembryonic coelom

and the amniotic cavity, were excised and weighed, and

radioactivity was determined in a gamma counter. Although

there was a clear accumulation of IgGi in the fetal tissue as

compared, for example, to well-vascularized organs of the

mother such as the heart and muscle, the total amount of

IgG1 reaching the fetus was only in the range of �2.5 �g/g

fetal tissue, which corresponded at day 9 of gestation (n =

1 7; mean weight, 29.1 �g) to 0.071 jtg/fetus/200 j�g injectedantibody. These values accounted for a nonbinding anti-

body. In rats receiving the CD44s-specific antibody, a higher

amount of antibody was retained in CD44s-positive maternal

tissue such as the lymph nodes. Nonetheless, the total

amount of IgG1 in the fetal tissue was in the same range as

that after injection of an antibody with irrelevant specificity

(n = 21 ; mean value, 0.067 j.tg/fetus). Comparable amounts

of anti-CD44s, which binds heavily to many tissues and cells

in the maternal organism, and of an antibody of the IgGi

isotype, which neither binds to maternal nor to fetal tissue,were recovered in the fetus, which pointed toward a selective

passage of IgG1 through the placental tissue. To seewhether, in addition, anti-CD44s would be specifically re-

tamed, antibody was injected into pregnant rats (1 0 days of

gestation), and the relative retention of a CD44s-binding

versus a nonbinding antibody in comparison to the peripheralblood was evaluated at 24 and 48 h after antibody applica-

tion (Fig. 1B). Accumulation of anti-CD44s was noted par-

ticularly in the lymph nodes and also in the lung and the fetus,but not in placental tissue. Taking into account that at day 12

of gestation (48 h after antibody injection), only a minority offetal cells express CD44, fetal retention seems to be very

efficient. No such retention in the fetus was seen with the

antibody of irrelevant specificity, which was slightly enriched

only in organs involved in degradation and excretion such as

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Fig. 2. Delay in fetal development in anti-CD44s-treated rats. Sagittal sections through embryos atdays 1 6 and 21 of gestation are shown, demon-strating the size differences of fetuses from con-trol lgG- and anti-CD44s-treated rats. Bar, 0.4 cm.

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Cell Growth & Differentiation 1213

Table 1 Crown-rum p length of anti-CD4 4s- and anti-CD44v6-tre ated fetuses

Day ofgestation

Crown-rump len gth (mm)a

Averageb No lgG Control lgGl Anti-CD44s (�)C Anti-CD44v6 (�)C

141618

8.5

13.520.5

8.2 ± 0.34

13.0 ± 0.5119.9 ± 0.53

8.4 ± 0.41

13.1 ± 0.2520.2 ± 0.40

5.8 ± 0.32 (0.008)

10.5 ± 0.59 (0.012)

14.3 ± 0.47 (0.008)

5.7 ± 0.28 (0.008)10.2 ± 0.50 (0.012)

15.2 � 1.70 (0.012)

a mean ± SD of 10-15 fetuses.b Ref. 79.C p values in parentheses referring to fetuses of BDX rats treated with control IgGi.

antiCD44s control IgGI

the lung, liver, and kidney. At 72 h after antibody injection,

around 99% of the irrelevant IgGi had been excreted,

whereas retention of the anti-CD44s antibody in lymph

nodes and the fetus was still in the range of 5-1 0% (data not

shown). Thus, although only low amounts of antibody

reached the fetus, the relative retention of anti-CD44s was

high and persisted for a prolonged period of time.

Depending on the time of application, the amount of anti-

body reaching the fetus was sufficient to influence embryo-

genesis. When female rats received anti-CD44s or anti-

CD44v6 starting at the time of mating, conception was

infrequent, even when the potential mating period was ex-

tended up to 4 days. When, on the other hand, antibodies

were injected starting at 24 h after mating, a comparison ofparaffin sections of the implantation site at 8 and 1 0 days of

gestation revealed no marked differences between tissues

from rats treated with a control IgG1 or anti-CD44s or anti-

CD44v6 (data not shown). This observation, together with the

finding that anti-CD44s was not particularly retained in the

placental tissue (Fig. 1B), was interpreted in the sense that

anti-CD44s and anti-CD44v6 did not induce abortion. How-

ever, antibody application at 24 h after mating influenced

fetal development (Table 1 and Fig. 2). The 1 0-21 -day-old

anti-CD44s-treated embryos were smaller than control em-

bryos, and delivery was frequently delayed by 2-3 days. In

about 50% of pregnant rats, the prolonged gestation re-

suited in intrauterine abortion of the fetuses. At 10-1 8 daysof gestation, development of the fetus was also stunted by

treatment with anti-CD44v6. However, normal-sized fetuses

were delivered at term, i.e. , after 22-23 days of gestation,

and no intrauterine abortions were noted. Organ develop-

ment, demonstrated for the lung (Fig. 3), was also delayed by

1-2 days. Besides the lung, the heart muscle was concor-

dantly hypoplastic in anti-CD44s-and anti-CD44v6-treated

fetuses; the anlage of the hair follicles of the whisker and the

development of the villi of the gut and of the tubular system

of the kidney were delayed by about 2 days in anti-CD44s-

treated rats and by about 1 day in anti-CD44v6-treated rats

(data not shown). With the exception of the lung, which was

still hypoplastic at birth (Fig. 3, G-I), the influence of anti-

CD44s treatment had vanished at term, and the influence of

anti-CD44v6 had vanished at day 21 of gestation. These

changes were only seen when antibody application was

started early after fertilization, i.e. , no gross changes in de-

velopment were noted when pregnant rats received anti-

CD44 starting at days 14 or 1 8 of gestation (data not shown).

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Fig. 3. Delay of lung development by anti-CD44s and anti-CD44v6 treatment. Whole fetuses and newborns of control IgGi -, anti-CD44s-, andanti-CD44v6-treated rats were fixed in formalin and embedded in paraffin. Sections of 2 j.tm through the lung of 1 8- and 21 -day-old fetuses and of newbornsare shown. Besides differences in size (A-C), the delay in the formation of alveoli (0-I), which is still visible at birth in the anti-CD44s-treated fetus (H), isparticularly noteworthy.

Anti-CD44 Interferes with Expression of CD44 in theDeveloping Organism. We next asked whether the anti-

CD44s- and anti-CD44v6-mediated growth inhibition of the

developing fetus may have been related to the expression of

CD44s and CD44v6, thus providing direct evidence for func-

tional activity of the molecules. This was the case (Table 2).

We have recently observed that expression of CD44s and

CD44v6 is induced at the implantation site (27). When preg-

nant rats were treated with anti-CD44s or anti-CD44v6, ex-

pression of CD44s at the implantation site was much weaker,

and expression of CD44v6 was completely absent (Fig. 4).

Expression of CD44s and CD44v6 on the epithelium of the

uterus, on the other hand, was unaltered (data not shown).

Expression of CD44s on fetal tissue (Table 2) was first

noted on day 12 (limb bud, neural tube, heart, and hemato-

poietic cells in the liver). Additional staining of the epidermis

was seen at day 14, additional staining of connective tissue

as well as that of lung and gut epithelia was seen at day 16,

and additional staining of the thymus and the tubular system

of the kidney was seen at day 1 8. In anti-CD44s-treated

embryos, CD44s was not detected at all in 12-day-old em-bryos; in 14- and 1 6-day-old embryos, only hematopoietic

cells in the liver were faintly stained. Even in 18-day-old

embryos, CD44s was not expressed in lung and kidney tis-sue, whereas weak expression was noted in the other or-

gans. At 21 days of gestation, expression of CD44s in the

lung was stronger in anti-CD44s-treated rats than it was in

control rats. When rats received anti-CD44v6, expression of

CD44s was not completely suppressed but was much

weaker until at least day 1 8 of gestation (exemplified by the

staining of the nasal epithelium of a 16-day-old fetus in

Fig. 5, A-C).

During embryogenesis, the expression pattern of CD44v6

is quite similar to the expression pattern of CD44s, with the

exception of connective tissue. The pericardia and the sub-

cutis, for example, do not express CD44v6 (Table 2). Expres-

sion of CD44v6 was completely eliminated by anti-CD44s in

the developing embryo up to day 1 8 of gestation and was

absent or very weak in anti-CD44v6-treated embryos, includ-

ing day 1 6 of gestation (exemplified by the lung and the gut

of a 16-day-old fetus in Fig. 5, D-I). On the other hand,

hematopoietic cells in the liver, thymocytes, and the lung

stained brightly with anti-CD44s and anti-CD44v6 in 21 -day-

old antibody-treated embryos but not in controls (Table 2).

Down-modulation of antigen expression by anti-CD44 was

not exclusively restricted to CD44s and CD44v. While stain-

ing with a panepithelial-specific antibody, D5.7 (32), the rat

homologue of EGP314,4 was completely unaltered in the

anti-CD44s- and anti-CD44v6-treated embryos, the expres-

sion of MHC class I molecules on hematopoietic cells of the

embryo was clearly delayed by treatment with anti-CD44s.

Anti-CD44v6 had no effect on MHC class I expression (data

not shown). Whether anti-CD44 directly influenced MHC an-

tigen expression or whether, more likely, expression was

4 J. W#{252}rfel,M. Rbsel, and M. Zbller, The rat homologue ofthe panepithelialantigen EGP31 4 supports metastasis formation of non-epithelial tumors,manuscript in preparation.

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4-.,.1

Fig. 4. Down-modulation of expression of CD44s and CD44v6 at the implantation site by diaplacental antibody treatment. Whole fetuses of control IgGi(A and D)-, anti-CD44s (B and E)-, and anti-CD44v6 (C and F)-treated rats were excised at day 8 of gestation. Fetuses were shock-frozen in liquid nitrogen.Sections of 5 �m were stained with anti-CD44s (A-C) and anti-CD44v6 (0-F). The uterine tissue at the implantation site is shown. In contrast to controlIgGi -treated fetuses, the ectoplacental cone of anti-CD44s- and anti-CD44v6-treated fetuses stained weakly, and the decidua did not stain at all;trophoblast giant cells did not express CD44s or CD44v6. A-F: 1 , embryo; 2, embryonic ectoderm cells; 3, ectoplacental cone; 4, yolk sac cavity; 5,trophoblast giant cells; 6, decidua.

Cell Growth & Differentiation 1215

CD

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CD44S staining

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delayed according to the developmental stage of maturation

remains to be explored.

Influence of Anti-CD44 on HA Distribution. CD44, asone of the receptors for HA (33, 34), is known to be involved

in assembly of the pericellular matrix (35) as well as in HA

degradation (36, 37). It therefore was of special interest to

evaluate whether in the absence of CD44 expression, grosschanges in the distribution of HA would be noted. This wasevaluated by staining with a biotinylated proteoglycan prep-

aration. Appearance of HA was delayed according to the

retardation in fetal development, but it was detected before

expression of CD44. For example, a similar distribution pat-tern was seen in a 1 0-day-old fetus treated with control IgGl

and a 12-day-old fetus treated with anti-CD44s (Fig. 6, A and

B). Thus, the production of HA seemed to be independent of

CD44v6 staining

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CD44 expression. Whether this independence also ac-

counted for HA degradation could not be unambiguously

elucidated. At 18 days of gestation, abundant amounts of HA

were detected in the kidneys of control and anti-CD44-

treated fetuses (Fig. 6, C-E); at 21 days of gestation, when

CD44 was expressed even in the kidneys of anti-CD44-

treated fetuses (Table 2), bright staining with b-PG was onlyseen in the kidney of anti-CD44s-treated fetuses and not in

the kidneys of control lgGl or anti-CD44v6-treated fetuses

(Fig. 6, F-H). Thus, the lack of HA degradation could just as

well be due to the delayed appearance of CD44 as to theanti-CD44-mediated delay in organ development.

Shedding and Internalization of Anti-CD44 on FetalCells. By the coincidence of the lack of CD44 expression

and the delay in development, the question arose as to the

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1216 Delay in Development by Anti-CD44

Fig. 5 CD44s staining CD44v6 staining CD44v6 staining

day 18 day 21

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Cell Growth & Differentiation 1217

mechanism of the antibody-mediated down-regulation of

CD44 expression. As revealed by flow cytometry, expression

of CD44 in adult fibroblasts remained unaltered when cul-tured in the presence of anti-CD44 (data not shown). Instead,when 14-day-old fetuses were meshed and cultured in thepresence of anti-CD44, surface expression of CD44 wasstrongly reduced after 4 days of culture, and intermediatelevels of CD44 expression were seen after 2 days of culture(Table 3). The difference in modulation of CD44 expression in

adult versus fetal cells and the gradual decline in CD44expression prompted us to examine whether shedding and

internalization may be different in fetal as compared to adultcells. Cell suspensions obtained from 14-day-old fetuseswere cultured for 2 days in the presence of anti-CD44s (toremain at an intermediate level of CD44 expression; see

Table 3) and rested for an additional 2 days. As revealed byindirect surface staining, the resting period of 48 h was

required and sufficient to avoid competition of bound anti-

. CD44 with the 1251-Iabeled anti-CD44. Thereafter, controlcells and transiently anti-CD44-treated cells were incubatedfor 30 mm at 4#{176}Cwith 125l-Iabeled anti-CD44s or 1251-labeledanti-CD44v6. After washing, shedding was followed for anincubation period of 2 h at 37#{176}C,and internalization wasmonitored by treatment with proteinase K (Fig. 7). Interest-ingly, shedding from fetal cells was strongly reduced ascompared to that from adult fibroblasts, and the lower rate ofshedding from fetal as compared to adult cells correlatedinversely with a higher rate of internalization. The internalizedantibody, as revealed by the uptake of FITC-labeled anti-CD44s, did not recirculate; i.e. , even after 2 days, the cyto-plasm contained high levels of anti-CD44s. On the otherhand, fetal cells transiently cultured with anti-CD44s re-

leased close to 90% of the bound antibody within 15 mm ofculture at 37#{176}C.This accounted for 125I-labeled anti-CD44s

as well as 1251-Iabeled anti-CD44v6 (data not shown). Uptakeof 125l-Iabeled anti-CD44 in anti-CD44-pretreated fetal cellswas negligible.

Thus, fetal cells internalized and retained anti-CD44 morestably and at a higher rate than did adult cells. These featurescould argue for a lack of outside-in signals as the responsiblemechanism of delayed development. However, once the fe-tal cells were pretreated with anti-CD44, shedding wasstrongly augmented, which would support the interpretationthat a missing interaction between CD44-positive cells andtheir surroundings hampered development. Thus, the ques-tion of the cause and effect relationship cannot be answeredthus far, although a lack of outside-in signaling by CD44

internalization on the first contact with antibody may be themore likely mechanism.

DiscussionCD44, one of the principle surface receptors for HA (33, 34)with further binding domains for fibronectin, glucosaminogly-

cans, and possibly other elements of the extracellular matrix(reviewed in Ref. 1 1), has been shown repeatedly to beimportant in morphogenesis (36-43). It has been observedthat CD44 influences neural development (42, 44). It is

strongly expressed at the leading edge of the limb bud,

thereby suggesting an involvement in the development oflimbs (40, 43). Maturation of hematopoietic cells can also be

followed by differential expression of CD44 (45-48), andanti-CD44 is known to block hematopoiesis (49). Here wedescribed the systemic influence of anti-CD44s and anti-CD44v6 on fetal development in the rat. We want to discussthe possible modes of action, the consequences of down-regulation of CD44 expression, and the advantage of study-ing the function of the adhesion molecule CD44 in ontogenyby antibody blocking studies.

Depending on the time of antibody application, both anti-CD44s and anti-CD44v6 interfered with the development ofthe growing fetus. When female rats received anti-CD44s oranti-CD44v6 starting at the time of mating, conception wasinfrequent, even when the potential mating period was ex-

tended up to 4 days. Thus, anti-CD44 might have interferedwith the process of fertilization. Because we were primarily

interested in the process of embryogenesis, this questionwas not pursued systematically in the present study. Anti-body injections starting at day 14 of gestation or thereafterdisplayed no measurable effect on either CD44 expression orfetal development. When rats received the first injection ofanti-CD44 at 24, 72, or 120 h after observation of the vaginalplug, growth of the developing fetuses was clearly impaired

(data not shown). On the other hand, even when antibodyapplication had been started at 24 h after mating, we did notobserve differences between control and anti-CD44-treatedrats at the implantation site up to day 10 of gestation. Thisimplies that neither anti-CD44s nor anti-CD44v6 interferedstrongly with the process of implantation itself. Because it

had become obvious by the inefficiency of antibody appli-cation starting at day 14 of gestation that there was only awindow of susceptibility to antibody treatment, we intendedto optimally use this window and thereafter started all of the

experiments described with the anti-CD44 applications at

Fig. 5. Down-modulation of expression of CD44s and CD44V6 in the developing fetus by diaplacental antibody treatment. Whole fetuses of control lgGl�4, D, and G)-, anti-CD445(B, E, and H)-, and anti-CD44v6(C, F, and 1)-treated rats were excised at 16 days ofgestation and shock-frozen in liquid nitrogen.Sections (5 �&m) through the nasal cavity (A-C) were stained with anti-CD44s; sections through the lung (0-F) and the gut (G-!) were stained withanti-CD44v6. A-C: 1, nasal epithelium; 2, dental primordium. 0-F: 3, bronchus; 4, bronchiolus. G-I: 5, gut epithelium; 6, mucosa; 7, mucesa muscularis;8, pancreas.

Fig. 6. HA distribution in control lgGl-, anti-CD44s-, and anti-CD44v6-treated fetuses. Whole fetuses of control IgGl (A, C, and F)-, anti-CD44s(B, D, andG)-, and anti-CD44v6 (E and H)-treated rats were excised at 10 (A), 12 (B), 18 (C-E), and 21 (F-H) days of gestation. Fetuses were fixed in formalin andembedded in paraffin. Section of 2 �.un were stained with a biotinylated proteoglycan. The pattem of HA distribution was similar in 10-day-old controllgGl-treated fetuses (A) and 12-day-old anti-CD44s-treated fetuses (B). In the latter group, the general hypoplasia, besides the delay in development,should be noted. Whereas an abundance of HA in 18-day-old kidneys (C-E) was independent of antibody treatment, at 21 days of gestation (F-H), HA wasdegraded except in the anti-CD44s-treated fetus. A and B: 1, neural tube; 2, paraxial mesoderm; 3, forelimb bud; 4, heart; 5, foregut; 6, amnion; 7, yolksac. C-H: 8, glomeruli; 9, proximal tubuli; 10, distal tubuli.

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1218 Delay in Development by Anti-CD44

Table 2 M odulation of CD44s and CD44v6 expressi on by anti-CD44s and anti-CD44v6

Day ofgestation

Or ang

Staining with anti-CD44s (Ox49) Staining with anti-C D44v6 (A2.4)

Control Anti-CD44s-treated Anti-CD44v6-treated Control Anti-CD44s-treated Anti-CD44v6-treated

8 Implantation site + + + ± ± + - -

Uterine glands +++ +++ ++ +++ + +

1 0 Implantation site + + + - - + + + - -

Uterine glands + + + + + + + + + + + +

Trophoblast +++ ± - - - -

12 Ljmbbud +++ - + +++ - +

Neuraltube + - ± + - ±

Heart + - ± + - ±

Uver (hematopoietic cells) + + + - + + + + - +

14 Heart ++ - + + - -

Liver (hematopoietic cells) + + + ± + + + - -

Epidermis/squamous epithelia + + - + + + + +a + +

16 Pericardia + + - + - - -

Liver (hematopoietic cells) + + +b + + + + - +b

Epidermis/squamous epithelia + + + - ± + + + - ±

Lung epithelia + + - ± + + + - -

Gut +++ - ± ++ - ±

Connective tissue + + + + - - -

1 8 Liver (hematopoietic cells) - + +b + +b+ +b

+b

Thymus +++ + + +++ + ++

- Epidermis/squamous epithelia + + + + + + + + + ± + +

Lung ++ - ± +++ - ±

Stomach +++ ± + +++ ± +

Gut +++ ±c + +++ ± +

Kidney ++ - - - - -

21 Liver (hematopoietic cells) - + , + - + +

Thymic epithelium ++ ++ ++ + + +

Thymocytes ± + + ± + + + + +

Epidermis/squamous epithelia + + + + + + + + + + + + + + + +

Subcutis +++ +++ +++ - - -

Lung +++ +++ +++ ± + +

Esophagus +++ +++ +++ +++ + +

Stomach +++ +++ +++ +++ + +

Gut +++ +++ +++ +++ + +

Kidney +++ +++ +++ - - -

a Only epithelia of the oral cavity were stained.b Megacaryocytes only.C Smooth muscles only.

24 h after mating. However, it should be mentioned that in

selected groups of fetuses in which antibody was given onlyafter implantation, similar, albeit somewhat weaker, effects

were observed throughout the gestation period.

Considering the above-mentioned window of susceptibil-ity and the vanishing efficiency of anti-CD44 treatment at lategestational stages, there are two possible, not mutually ex-clusive explanations: (a) given a certain size of the fetus, theamount of antibody is no longer saturating; and (b) alterna-tively, there exists a real window of susceptibility, i.e. , cells ata defined stage of maturation do not respond to anti-CD44with loss of CD44. For the following reasons, we argue that

both suggestions are relevant. The amount of IgG recovered

in the fetus was found to be not more than roughly 0.05% of

the injected antibody, i.e. , about 4 x 1 01 1 molecules. Takingan estimate of 1 O� CD44 molecules per cell, the bivalent

antibody molecules would be sufficient for covering roughly

8 x 1 O� cells. Thus, until midgestation, the quantity of anti-CD44 reaching the fetus should be saturating. In the last

trimester, however, this can no longer be expected to be thecase. Nonetheless, loss of susceptibility toward antibodytreatment may be an additive important factor, because, as

discussed below, we did not observe anti-CD44-mediatedmodulation of CD44 expression in tissue culture lines of adultrats.

Both anti-CD44s and anti-CD44v6 harmoniously retarded

the growth and differentiation of the developing fetus. Be-cause it is known that the expression of CD44s begins at anearlier stage and is generally stronger than that of CD44v6, itwas to be expected that the effect of anti-CD44s would bemuch more pronounced than the effect of anti-CD44v6. Fur-

thermore, because the anti-CD44s antibody recognizes at

least the vast majority of CD44v isoforms (24), it is likely that

anti-CD44s affected cells expressing CD44s as well as cellsexpressing CD44v isoforms. Therefore, it is not possible todifferentiate in the anti-CD44s-treated fetus between alter-ations brought about by a blockade of CD44s or CD44v.Instead, treatment with anti-CD44v6 clearly demonstratedthat exon v6 influenced development, particularly that of the

gut, the lung, and skin appendages. In the last stages ofgestation, the effects of anti-CD44s and anti-CD44v6 van-ished. Only anti-CD44s treatment delayed delivery.

An overall growth retardation of the fetus was clearly thedominating feature. Besides, the differentiation of organs,

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Cell Growth & Differentiation 1219

5 U. GUnthert and C. Schwarzler, T cell maturation in CD44v6 knockoutmice, manuscript in preparation.

Table 3 CD44 expression on cultured fetal cells

Anti-CD44 treatment

Percentage of stained cells� (relative intensity of staining)

Small cells (68%)

FS/logSS” 10.8/8.8

Medium-sized cells (22%) Large cells (7%)

51.5/45.831.3/17.0

NoneAnti-CD44s, 2 daysAnti-CD44s, 4 days

Anti-CD44v6, 2 daysAnti-CD44v6, 4 days

13.9 (44.0)7.6 (40.5)1 .0 (40.4)

6.5 (38.3)4.3 (33.1)

42.0 (54.5)17.7 (29.9)

2.9 (31 .1)

27.4 (48.4)12.3 (41.3)

63.1 (148.2)17.1 (55.9)

4.1 (45.9)

31.3(80.5)3.1 (111.6)

a Cells were derived from 14-day-old fetuses and cultured for 4 days; for the staining procedure, see “Materials and Methods;” relative intensity refers to

the population of CD44� cells.b FS/logSS, forward scatter/logarithmic sideward scatter.

which strongly express CD44 early during development, has

been particularly hampered. This could be demonstratedmost convincingly for branching of the lung, villi formation inthe gut, and the development of skin appendages. Interest-ingly, these are the same tissues that express CD44v6 at ahigh level. It is therefore tempting to speculate that we areactually dealing with two overlapping effects of antibody

treatment, malnutrition and interference with the differentia-tion of selected organs. This assumption is supported by thefollowing two observations: (a) because not all developingtissues express CD44, the overall growth retardation will bebased on an antibody-mediated down-regulation of CD44 onextraembryonic tissue, such as the visceral yolk sac, with theconsequence of malnutrition of the fetus proper. These ad-jacent tissues brightly express CD44s but only partially ex-press CD44v6. In fact, growth retardation was more pro-nounced, and intrauterine absorption was exclusivelyobserved in anti-CD44s-treated fetuses; and (b) delayed dif-ferentiation, instead, was restricted to selected organs,which, at the time of differentiation, express CD44v6.Whether impaired differentiation relies exclusively on thedown-modulation of CD44v expression is currently beingexplored using CD44v6 knockout mice, kindly provided by U.GOnthert (Basel Institute for Immunology, Basel, Switzer-land).5

The changes in development correlated with down-mod-ulation of the corresponding antigens. Anti-CD44s treatmentduring ontogeny led temporarily to the complete absence ofexpression of CD44s and CD44v6; anti-CD44v6 affectedexpression of CD44v6, but not as strongly as anti-CD44s.The observation in antibody-treated rats that the CD44 mol-ecule was undetectable in fetal tissues and development wasdelayed was suggestive of a linkage between both phenom-enons. The phenomenon of down-modulation of CD44 ex-pression by antibody treatment was restricted to embryo-genesis and was noted in neither the adult organism nor oncultured lines of mature cells (data not shown). The differentresponse of immature versus mature cells to ligation of sur-face receptors via antibody is not unique to ligation of CD44molecules by anti-CD44. For example, anti-lgM treatmentleads to activation of mature B cells but to loss of receptor

expression in the developing B cell (50). It should be pointedout that down-modulation in itself did not provide a clue asto the underlying mechanism. Given the absence of surfaceexpression, antibody binding must have led either to inter-nalization and subsequent degradation or to shedding. Thelatter phenomenon has been repeatedly described to be ofrelevance for the level of CD44 expression (51-54). Indeed, in

vitro studies with cultured fetal cells pointed toward abun-dant shedding from anti-CD44-pretreated fetal cells. Al-though further experimental verification is required, we none-

theless favor internalization as the underlying mechanism,

because on first contact with anti-CD44, fetal cells internal-ized the antibody much more readily than did adult cells andretained it for a prolonged period.

Besides the mode of down-regulation, there remains an-other question. Have the observed effects been a conse-quence of the absence of CD44 or have they been initiatedby the supposed antibody-receptor interaction? By the cur-rent state of knowledge, neither hypothesis can be experi-mentally proven or discarded. Because of the following func-tions of CD44, we consider a direct linkage between theabsence of CD44 and the observed phenomena as morelikely: (a) there are several examples for induction of cytokineproduction by CD44-ligand interactions (55-58); (b) particu-lar CD44v isoforms are known to bind cytokines/chemokines(59-62); (c) CD44 is involved in the assembly of the pericel-lular matrix (35); and (d) CD44 mediates degradation of HA(36, 37). A defect in any one of these functions could welldelay embryonic development. Because the involvement ofCD44 in HA distribution in the developing organism is par-ticularly well described (36, 37, 40, 41 , 63, 64), we consid-ered the evaluation of the distribution of HA in the anti-CD44-treated fetus as potentially informative in support of ourworking hypothesis. In general, the distribution of HA inanti-CD44-treated fetuses was similar to previously de-

scribed patterns, but the appearance and degradation of HAwere delayed. However, it should be noted that the assemblytook place before the appearance of CD44; thus, the delayedappearance has likely been a consequence of the general

retardation in development. The delayed degradation couldhave been due to either the delay in development or thedelay in the appearance of CD44. Thus, for intrinsic reasons,this observation did not allow us to differentiate between

cause and effect. The same argument accounts for the highexpression of CD44 at late stages of gestation, which be-

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adult flbroblastsfetal cells

lii

fetal cells fetal cells#{224}CD44streatedA 30 adult flbroblasts

25

:20�150E0.0 10

fetal cellsacD44s treated

� Osupernatant

I Ucells

I�1��T

B 100

90

80

70

� 6000

.z’� 50

0

� 40

30

20

10

015 30 60 120 15 30 60 120 15 30 60 120

minutes of incubation at 37#{176}C

15 30 60 120 15 30 60 120 15 30 60 120

minutes of incubation at 37#{176}C

1220 Delay in Development by Anti-CD44

Fig. 7. Influence of anti-CD44 on shedding and intemalization of CD44 in fetal cells. Whole fetuses at 1 4 days of gestation were meshed and cultured for2 days in the presence or absence of anti-CD44s (1 0 �g/ml). Thereafter, all cultures were kept for an additional 2 days in the absence of anti-CD44.SV4O-transformed fibroblasts of adult rats and cultured fetal cells were incubated for 30 mm at 4�C with 125l-labeled anti-CD44s. After washing the cellsto remove unbound antibody, cells were incubated for 1 5-1 20 mm at 37CC, and the amount of shed versus cell-bound antibody was evaluated (A). Todifferentiate between surface-bound and intemalized antibody, cells were washed again and treated for 5 mm with proteinase K (1 00 �zg/ml). The relativeamount of 1251 in the supernatant, of 1251 released by proteinase K treatment (cell membrane), and of 1251 in the proteinase K-treated cell pellet (internalized)is shown (B). A and B, mean ± SD of triplicate cultures.

came especially apparent in the lung and the thymus, where

expression of CD44v6 is only noted during certain stages of

gestation (27). High expression of CD44 at late stages of

gestation was no artifact, as indicated by the fact that sec-tions incubated with only the second dye-labeled detection

antibody were not stained at all. Thus, it has been ruled out

that iv. injected antibodies interfered with the immunohisto-

logical detection of surface expression. According to thedelayed degradation of HA, the most likely explanation will

be that at a stage of development in which the diapiacental

route of antibody transfer became inefficient, the cells

reached with delay a stage of differentiation where they

normally express high levels of CD44. Finally, considering the

delay in development as a possible consequence of a defect

in cytokine production and binding by CD44, it should be

mentioned that different isoforms apparently proceed via

independent signaling pathways and that signaling via CD44

is still poorly understood. Unless ligands and signaling func-

tions of CD44 isoforms are clearly delineated, the cause and

effect relationship of the absence of CD44 in embryogenesiswill be difficult to dissect. Irrespective of these remaining

open questions, diaplacental application of antibody proved

to be very efficient for a transient knockout of CD44 at the

protein level and offers the possibility to explore in more

detail the functions of CD44 isoforms in ontogeny.

Adhesion molecules are essential for the development of

multicellular organisms (65-69). They are known for theirplasticity and interchangeability (66). They mostly belong to

large families, the members of which can show variable

glycosylation, modulation of the protein structure, or a dif-

fering composition of subunits. Interchangeability and diver-

sity make the proof of function by the knockout approach

rather difficult. Can antibody treatment during developmentsupplement for an inducible homologous recombination ap-

proach or even provide additional information? As outlined

above, antibodies can function in multiple ways, and the

diverse modes of function may impede conclusions on thefunctional activity of adhesion molecules by antibody inter-

ference (70). Even taking this caveat into account, the par-

ticularly frequent reaction of the developing organism to

respond to contact with an antibody by down-modulation, as

shown for the stem cell factor (9), surface 1gM (50), and here

for CD44, allows us to define which cells and at which stage

of maturation functional activity of a particular cell surface

molecule is required. Taking into account that this informa-tion may even be obtained independent of the mode of

antibody interference, i.e. , induction of shedding, internaliza-

tion, or blockade, it is to be expected that intrauterine anti-body treatment may allow us in many instances to gain

insight into the functional activities of developmentally reg-

ulated cell surface molecules.

Materials and MethodsRats and Treatment. Adult BDX rats were obtained from Charles River

(Sulzfeld, Germany). Fetuses and newborn rats were obtained by breedingof BDX rats in the animal facilities of the German Cancer Research Center.

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Cell Growth & Differentiation 1221

Rats were kept under specific pathogen-free conditions and receivedwater and standardized food ad libitum. Male and female rats were mated

overnight. Female rats were then separated, and conception was verifiedby the vaginal plug. Pregnant rats received an iv. injection of 200 �.tg ofpurified lgGl antibody twice per week. If not stated otherwise, antibodyinjections were started at 24 h after observation of the vaginal plug. Thecontrol group was injected with a gallium chelate-specific antibody (3-9,71), whereas two other groups received either anti-CD44s (0x50; Ref. 72)

or anti-CD44v6 (1 .1ASML; Refs. 29 and 73). Pregnant rats were sacrificedat 8, 10, 12, 14, 16, 18, and 21 days of gestation. Newborn rats were

sacrificed within 24 h of birth. The significance of differences in the

crown-rump length was determined using Wilcoxon’s rank-sum test.Antibodies. The mAbs 1 .1ASML and A2.4, antirat CD44v6 (mouse

lgGl ; Refs. 29 and 73); Ox49 and 0x50, antirat CD44s (mouse IgG2A and

mouse lgGl , respectively; Ref. 72); D5.7, a panepithelial-specific antibody

(mouse lgGl ; Ref. 32); F16.4.4.1 1 , antirat MHC class I (IgGl ; Ref. 74); and

3-9, anti-gallium chelate (mouse lgGl ; Ref. 71) were purified by thepassage of culture supematants over protein G-Sepharose 4B (75). The

eluted fractions were dialyzed against PBS, concentrated to 1 mg/mI, andfiRer-sterilized. Polyclonal, biotinylated, or FITC-labeled antimouse lgGantibody was obtained from Southern Biotechnology (Birmingham, AL).

Antibody Distribution Profiles in Vitro. Purified mAbs were labeled

with 1251 according to the procedure described by Fraker and Speck (76).Briefly, 100 iLg of purified mAb in 100 �d of sodium phosphate buffer (pH

7.4) were incubated for 5 mm with 100 ACi of Na1251 in iodogen-coated

tubes. After blocking the reaction with 400 �l of 0.05 M sodium phosphate,the labeled mAb was separated from the free 1251 by passage over aNAP-5 column (Pharmacia), according to the manufacturer’s instructions.

The amount of immunoreactive 125l-labeled mAb ranged between 75 and

80%. Control rats and pregnant rats at days 8-1 0 of gestation received ani.v. injection of a mixture of 1251-labeled and unlabeled antibody (200 �g;

20 x 1 0e cpm). Rats were sacrificed 24 and 48 h thereafter. The organswere excised and weighed, and antibody distribution was determined by

measuring radioactivity in a gamma counter. Antibody distribution is pre-

sented as mean cpm ± SD/g wet tissue.Antibody Binding and Shedding. Short-term culture-derived fetal

cells and adultflbroblasts(104 cells/100 �d) were incubated for30 mm with125l-labeled antibody. Cells were washed three times and resuspended in

100 �l of RPMI 1640 supplemented with 2% FCS. After an incubation

period of 15-1 20 mm at 37#{176}C,supernatant was collected. After washing,cells were treated at 37#{176}Cwith proteinase K (100 �g/ml) for 5 mm, whichreleased CD44 from the cell surface, whereas internalized CD44-anti-

body complexes were not affected. Surface expression was confirmed by

fluorescence-activated cell-sorting analysis, which was performed ac-cording to routine procedures, incubating 5 x 1O� cells with the first

unlabeled antibody after washing with a FITC-labeled antimouse lgGantibody. Fluorescence staining was determined with an Epics XL(Coulter, Hialeah, FL).

Histological Examination. Whole fetuses were either fixed in 5%

formalin and embedded in paraffin according to routine procedures or

shock-frozen in liquid nitrogen. Paraffin-embedded and frozen fetuseswere cut in 5-nm-thick sections. Paraffin-embedded fetuses were stainedwith H&E according to routine procedures. For immunohistochemistry(77), frozen sections were mounted on gelatin-coated glass slides andair-dried overnight. They were fixed in acetone for 10 mm at -20#{176}Candimmunostained immediately. Sections were incubated for 20 mm at roomtemperature with the first antibody. We used two different antibodies for

staining CD44s (Ox49 and 0x50) and CD44v6 (A2.4 and A2.6), solely toprovide an internal safety control. The results were identical throughout.Antibody-treated slides were washed intensively and incubated at roomtemperature for 30 mm with the second, biotinylated antimouse lgGantibody. For the detection of HA by b-PG from cartilage (78), paraffinsections were dewaxed and rehydrated in a graded series of alcoholsolutions. The slides were treated with 10% H202 for 5 mm to eliminateendogenous peroxidase activity and then incubated for 1 h with 20 �g/mlb-PG. After washing, all sections were incubated with a horseradishperoxidase-conjugated streptavidin complex; 3,3’-diaminobenzidine wasused as substrate for the enzyme. For positive control purposes, each

slide contained a section of the small intestine of adult rats, which isknown to stain brightly with anti-CD44s, anti-CD44v6, the anti-panepi-thelial antibody, and the anti-MHC class I antibody. As a negative control,the staining of each sample was performed without applying the primary

antibody or by using an antibody (3-9) of irrelevant specificity. The resultswere evaluated according to the following scoring system: + + + , veryhigh intensity of staining; + +, high intensity of staining; +, distinctstain-

ing; ±, weak staining; and - , no staining.

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