100

GROWTH FACTOR GENES ARE EXPRESSED DURING MORPHOGENESIS OF THE MOUSE FIRST BRANCHIAL ARCH

H.C. Slavkin, M.L. Snead, W. Luo, P. Bringas, Jr., Y. Sasano, S. Kikunaga, LB. Rail*, D.A. Rappolee*, and Z. Werb*. Lab. Develop. Biol., Univ. Southern Calif., Los Angeles, and *Laboratory of Radiobiology and Environmental Health, Univ. Calif., San Francisco, CA 94143-0750.

Morphogenesis of first branchial arch (FBA) from embryonic (day 10) mice grown in vitro parallels development in vivo. Cartilage, bone, teeth and tongue form by 9 days in culture in serumless, chemically-defined medium, implying that FBA produces endogenous growth factors. Messenger RNA phenotyping by reverse transcription coupled with primed polymerase chain reactions (RT-PCR) demonstrated that TGF-alpha, TGF-beta , EGF, IGF- I and IGF-II mRNA transcripts were expressed in developing FBA both in vivo and in culture. Growth factors were also detected in developing mandil~Jes by immunocytoehemistry. FBA labeled with °°S- cysteine produced proteins that specifically bind to heparin. Spatial distribution of growth factor mRNA transcripts was analyzed by computer- assisted, three-dimensional reconstruction of serial in situ hybridizations. EGF transcripts were localized to regions of maximal growth and differentiation in Meckel's cartilage and adjacent alveolar bone. These data suggest that sequential time- and position-restricted expression of growth factors may provide epigenetic signals for allocation, determination and differentiation of specific phenotypes associated with mandibular morphogenesis. These studies were supported by research grants DE-00133, DE-06425, HD 23539, a NRSA ES 07106 from NIH-USPHS, and a contract DE-AC03-76-SF01012 from US DOE-OHER.

101

IMMUNOCYTOCHEMICAL ANALYSIS OF EGF RECEPTORS LOCALIZATION IN A431 CELLS

A. Sorokin, V. Romanov, G. Reshetnikova, N.Vinogradova, A. Nesterov, N. Nikolsky Institute of Cytology, Leningrad, USSR

Surface replication, label-fracture and freeze- etching methods were used for the detection of EGF receptors in A431 cells. About 20% of unoc- cupied receptors were in microaggregates. The similarity of distribution of immunogold label (monoclonal antibodies to ECF receptor) on the surface of cells incubated with EGF (i hour, 4°C) with distribution of immunogold label in control cells indicated that EGF didn't induce aggregation of factor-receptor complexes at 4°C). In such conditions (EGF 1 hour, 400C) antiphosphotyrosine antibodies stained the bor- ders of cells (shown by immunofluorescence). If the temperature was increased up to 37°C aggre- gation was dramatically increased. Quantity analysis of induction of microaggregation and internalization of EGF receptors using immune- gold labelling was performed. At 37°C phospho- tyrosinylated EGF receptors were observed in ruffles and then within endosomes.

102

EFF~CI~ OF DETI~G~qT ON EG'F-R~'IL~ ~ E AL'IXVlTY. M. Spaargaren I, L.H.K. Defize 2, J. Boonstra s and S.W. de Last 2. llnstltute of Molecular Biology and Medical Biotechnology, University of Utrecht, Pndualann 8, 3586 CH Utrecht; 2|[ubrecht I.aboratory, Nether|ands Institute Of Developmental Biology. Utrecht: SDepartment of Molecular Cell Biology, University of Utrecht, The Netherlands. The anti-reeeptor monoclonal antibody 2E9, which blocks F~F binding to the low affinity EGF-receptor subclass, has been used to show that the hlgh affinity EGF-receptor subclass plays a major role in ECF- induced proteln-klnase activity and early signal transductlon (Deflze at al., submitted). Honoclonal antibody 2E9 does not stimulate EGF- receptor protein tyroslne klnase actlvlty in intact cells, however, 2E9 as well as several other anti- receptor antibodies, strongly stimulated phosphoryl- atlon of EGF-receptor on tyrosine residues in an in vitro assay using A431 membrane vesicles. This discrepancy turned out to be due to the presence of detergent in the in vitro phosphorylatlon experiments. Antl-receptor antibody treatment of intact cells, in detergent containing buffer, also leads to enhat~ed tyrosIne phoaphorylation. Furthermore, monovalent Fab fragments of these antibodies where not able to activate receptor phosphorylation. As detergent treatment of intact cells also caused enhanced receptor dimerizatlon, these results suggest the existance of a detergent sensitive factor that precludes spontaneous or antibody induced receptor- receptor interactions, subsequently leading to receptor klnase avtlvatlon. Since EGF itself efficiently activates the kinase in membranes and intact cells, it is tempting to speculate that EGF causes receptor dimerization by overcoming or abolishing this factor.

103

INSULIN AND INSULIN-LIKE GROWTH FACTOR I (IGF I) IN EARLY MOUSE EMBRYOGENESIS

R. Spaventi and K. Paveli6

Growth factors have an important role in the regulation of cell growth,division and dif- ferentiation. These substances induce proces- ses which are necessary for transmission of cells from resting to the cell division, namely the DNA, RNA and protein synthesis. Growth factors are also involved in the regu- lation of embryonic growth and differentiation. It seems that insulin and insulin-like growth factors play an important role in these events. It was interesting to find if these peptides are secreted by embryonic cells in early sta- ges of embryogenesis. Our results show that mouse embryos 7 to 20 days old contain measu- rable amounts of insulin and IGF I activity. Concentrations of these peptides are higher in the first than in the second half of the investigated period. We also found that embryo- nic~cells from 8, 9and 10 days old mouse embryos secret insulin, IGF I and/or related molecules in serum-free cultures. Furthermore, same growth factors, when added to the culture of 9 days old mouse embryonic cells, stimulate their proliferation. These results suggest that insulin and IGF I play a role in growth regulation during early stages of mouse embryo- genesis.

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