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  • 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 selectedCD44, 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 resembledthat 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.

    Intioduction

    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 observedas 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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    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 jg 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 inject