Printed in Sweden Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved 0014-4827/79/070121-C6WZ,~/O Experimental Cell Research 121 (1979) 121-126 INHIBITION OF MORPHOGENETIC CELL INTERACTIONS BY 6-DIAZO-5-0X0-NORLEUCINE (DON) PETER EKBLOM,’ JAMES W. LASH,2 EERO LEHTONEN,’ STIG NORDLING’ and LAURI SAXEN’ ‘Department of Pathology, University of Helsinki, SF-00290 Helsinki 29, Finland, and =Department of Anatomy G3, School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA SUMMARY The glutamine analogue 6-diazo-5-oxo-norleucine (DON), known to interfere with the synthesis of glycosaminoglycans and glycoproteins, inhibited kidney tubule induction., while the subsequent stages of metabolic activation and overt morphogenesis were unaffected wtth the same concentra- tions. Moreover, close cell contacts, previously shown to be of importance in kidney tubule induction, were retained. Thus, carbohydrates, most likely located at the inductor-target in- terface, seem to be involved in this form of intercellular communication. The induction of kidney tubules in meta- nephric mesenchyme seems to require a close apposition of the interacting tissues [16, 211. Two mechanisms that may be in- volved are (1) an interaction through junc- tional complexes, such as gap junctions, permitting transfer of molecules [4, 133or (2) interaction between cell surface compo- nents, such as surface-associated glycopro- teins or proteoglycans. Such surface-asso- ciated molecules have been proposed to have developmental significance [3, 6, 121. In the present study we have investigated the role of proteoglycans and glycoproteins in kidney tubule induction by interfering with the glycosylation of their precursors, utilizing the glutamine analogue 6-d&o-5- oxonorleucine (DON) [ 181. MATERIAL AND METHODS Tissues Metanephric kidney rudiments were removed from 1l- day-old BALBlc XCBA mouse embryos (0 being the day of appearance of the vaginal plug). After a short incubation in 0.02% EDTA, the ureter bud was sen- arated from the surrounding mesenchyme. Do&l spinal cord used as kidney tubule inductor [7j was dis- sected from the same embryos. Cultures In one set of experiments the kidney rudiments were cultivated intact on top of a Nucbporea filter (Nucle- pore, Pleasanton, Calif.). Alternatively, the separated metanephric mesenchvme were placed on one side of a filter and a piece of spinal cordon the opposite side, as previously described [15]. The culture medium con- sisted of Eagle’s minimum essential medium (MEM) supplemented with 10% fetal calf serum (FCS) and 2 mM glutamine. Unless specified, glutamine was omitted when DON was used. The total culture time was 72 h, and the spinal cord was removed after 24 h. In some experiments, the spinal cord was left in place for the whole culture period, as indicated in Results. Estimation of the tub&e formation response The tubule formation in intact whole kidneys was estimated from histological sections stained with hematoxylin and eosin. In recombination experiments, the response of the mesenchyme was evaluated from whole-mount preparations. After cultivation, the spinal cord was removed (if still present), the mesen- thyme fixed in 4% formaldehyde, and stained with hematoxylin and eosin. Whole mounts were then made Exp Cell Res I21 (1979)