38 Growth factors in developmentRandom haemopoiesis or clonal succession?

/. Adam and M. Rosendaal, Department of Anatomy and Embryology, University College London,Gower Street, London WC1E6BT

Work in this laboratory is addressing the question of whether the utilisation of haemopoietic stem cells bythe animal is random, or organised as a succession of clones. Our approach has been to study geneticallymarked precursor cells at various developmental stages from distinct haemopoietic sites.

We use the X-linked enzyme phosphoglycerate kinase (Pgk-1) with a and b alleles coupled to an in vitroassay for granulocyte-macrophage precursors. We assume that different growth factors act on colony-forming cells at different developmental stages.

We confirmed that such progenitors formed clones in culture and, by analysing the proportions of geneticmarkers of their colonies, studied whether progenitors at different developmental stages respond todifferent haemopoietic growth factors. If haemopoiesis is random there should be no significant differencein the proportions of genetically marked cells responding to different growth factors. Conversely, if clonesof haemopoietic stem cells are utilised successively then we might expect to see proportional differencesbetween the set of progenitors responding to one growth factor and another.

Our results show that the culture system employed is indeed a clonal assay. No discrete colonies werefound which possessed both Pgk allelic forms. Distinct populations of progenitors can respond to differentstimulants, so our findings are consistent with clonal succession. Significant differences can be found in theproportions of alloenzymes in similar sets of these progenitors cultured from similar and neighbouringbones. This suggests that the system is locally organised. We have found no evidence of any singlepopulation which is permanently derived from a limited set of precursor cells.

Other related questions such as whether there is any mixing between different haemopoietic sites havebeen investigated and will be discussed.

Cell cycle control and growth factor action in differentiating embryonalcarcinoma cells

S. W. de Laat*, C. L. Mummery, P. T. van der Saag, A. Feijen and C.E. van den Brink, Hubrecht Laboratory, International Embryological Institute,

Uppsalalaan 8,3584 CT Utrecht, The Netherlands

Differentiation of embryonal carcinoma (EC) cells to endoderm-like (END) cells can be induced byretinoic acid (RA). In this study we have characterized the effects of RA treatment on (1) cell cycle kinetics,(2) the acquirement of functional membrane receptor for epidermal growth factor (EGF), and (3)susceptibility for exogenous growth control by EGF. Time-lapse films were made of exponentially growingEC and END cells (PC13) and of synchronized EC cells in the presence and absence of RA, and cell cycleparameters were determined according to a modified transition probability model. EC and END cells havegeneration times (Tg) of ~11 h and ~20 h, respectively. RA had no effect on cell cycle kinetics during thefirst two generations following addition to synchronized EC cells, but caused an increased Tg from 11 h to16 h in the third, and a further increase to 20 h in the fourth and fifth generation. This increase in Tg wasprincipally due to an increase in the length of the S phase, and was associated with cell death occurringfrequently in certain sister pairs, as if susceptibility to RA toxicity was programmed and geneticallydetermined. Morphological differentiation was shown to begin already in the second generation, thuspreceding the altered growth characteristics. END cells, in contrast to EC cells, respond mitogenically toexogenous growth factors, such as EGF. This property related to the acquisition of functional EGFreceptor/signal transduction systems upon differentiation. Not only had EC cells no binding capacity for125I-EGF (as found by others), but EGF was also unable to induce a number of normal early responses. Incontrast END cells showed ~80,000 EGF binding sites, and EGF induced a variety of early responses, suchas activation of Na+/H+ exchange and Na+,K+-ATPase in EC cells and tyrosine phosphorylation of theEGF receptor and of exogenous peptide substrates by EC cell membranes. We conclude that RA-induceddifferentiation of EC cells results in programmed cell cycle-dependent alterations in cell cycle kinetics, and atransition from EGF-independent to EGF-dependent growth control which relates to the expression offunctional EGF receptor/signal transduction systems upon differentiation.

Growth factors in development 39Clathrin and transferrin-receptor distributions during embryonic and

neonatal rat skin developmentMartin R. Green and John R. Couchman, Biosciences Division, Unilever Research, Colworth House,

Sharnbrook, Bedfordshire MK441LQSpecific polyclonal rabbit antibodies to bovine brain clathrin (1) and the human placental transferrin-

receptor (Tf-R) (2) have been used on frozen sections to map the distribution of these antigens during ratskin development. At 16 days embryonic development (day one is taken as the day following plugformation) Tf-R staining is almost undetectable while clathrin localisation is restricted to a ribbon likenetwork in the region of the epidermal basal cells. By 17 days both antibodies weakly stain the membraneregion of all the epidermal cells and the ribbon staining for clathrin is lost. As the epidermis thickens andstratifies with increasing age the intensity of staining with both antibodies increases until all the viableepidermal cells are clearly delineated. In addition both antigens at birth have an intracellular punctatelocalisation which is particularly prominent for clathrin, suggesting active receptor mediated endocytosis.Clathrin levels are also particularly intense around the necks of hair follicles where they merge with theepidermis. The Tf-R on the epidermal basal cells of the neonatal animal is partially polarised to the lowercell membrane and, in addition, the level of receptor noticeably increases in the upper epidermal cell layers.These observations imply that orientation and density of Tf-R facilitate efficient Tf uptake by the basal cellsand by the cells in the upper epidermis furthest away from the supply of nutrients from the dermis. Bothantibodies stain the hair follicle outer root sheath cells but the intensity is much reduced on dermal papillafibroblasts and hair bulb cells lying below the tip of the dermal papilla.

(1) BLOOM, W. S., FIELDS, K. L., YEN, S. H., HAVER, K., SCHOOK, W. & PUSKIN, S. (1980). Brain Clathrin:Immunofluorescent patterns in cultured cells and tissues. Proc. Natl. Acad. Sci. U.S.A. 77, 5520-5524.

(2) The kind gift of Dr J. Bleil, Laboratory of Molecular Biology, Medical Research Council Centre, HillsRoad, Cambridge, U.K.

Intracellular 125I-epidermal growth factor processing in rat skindetected by electron microscope autoradiography

Martin R. Green, Claire Mycock, Colin G. Smith and John R. Couchman, Biosciences Division, ColworthLaboratory, Sharnbrook, Bedfordshire MK441LQ

The distribution of EGF-receptors in embryonic and neonatal skin has previously been characterisedusing 125I-EGF, viable skin explants and light microscope autoradiography (1, 2). Heavy labelling of thebasal cells of the epidermis, hair shaft and hair bulb was seen. In addition, the epidermal EGF-receptornumber during neonatal development was found to be related to the 3H-thymidine labelling index (1). Wereport here on the intracellular processing of 125I-EGF in the basal epidermal cells and hair bulb cellsdetermined by incubating viable 3 day rat skin with 125I-EGF at 25 °C and analysis by electron microscopeautoradiography. Silver grains, indicating the localisation of 125I-EGF and presumably its associatedreceptor, were associated with coated membrane imaginations. In some areas label appeared to collect nearhemidesmosomes. Internalised label was seen in the Golgi apparatus and in multivesicular, lysosomalbodies indicating that degradation of 125I-EGF and possibly associated EGF-receptor was in progress.Interestingly, label was seen over some cell nuclei. Grains were also associated with vascular endothelialcells confirming light microscope observations (1). Inclusion of sodium azide (2mM) in the incubationmedium restricted label to the plasma membrane region. Thus, intracellular processing of EGF in theepithelial cells of skin appears to follow the conventional pattern described for cells in culture (3).(1) GREEN, M. R., BASKETTER, D. A., COUCHMAN, J. R. & REES, D. A. (1983). Distribution and number of

epidermal growth factor receptors in skin is related to epithelial cell growth. Develop. Biol. 100,506-512.(2) GREEN, M. R. & COUCHMAN, J. R. (1984). Distribution of epidermal growth factor receptors in rat

tissues during embryonic skin development, hair formation and the adult hair growth cycle. /. Invest.Dermatol. (In press).

(3) WILLINGHAM, M. C , HAIGLER, H. T., FITZGERALD, D. J. P., GALLO, M. G., RUTHERFORD, A. V. &PASTAN, I. H. (1983). The morphological pathway of binding and internalisation of epidermal growthfactor in cultured cells. Exp. Cell Res. 146, 163-175.

40 Growth factors in developmentThe effect of insulin and epidermal growth factor on rat embryonic

development in vitroA. P. Gulamhusein, I. M. Huxham and N. Faisal, Department of Anatomy, The Medical School, University

of Leicester, University Road, Leicester LEI 7RH

Growth of post-implantation rat embryos in culture (New, 1978) using 100 % heterologous serum issuboptimal compared to growth in 100 % rat serum (Priscott, 1983). However, supplementation of, forexample, human serum with rat serum to a concentration of 10 % can restore the ability of human serum tosupport embryonic growth and differentiation (Reti, Beck & Bulman, 1982; Gupta & Beck, 1983; Lear et al.1983). Since supplementation of human serum (Faisal, unpublished data) or rat serum which has beenexhausted by repeated culture (Al-Alousi, 1983) with dialysed rat serum to a concentration of 10 % will notsupport adequate growth and differentiation, it is likely that a number of dialysable factors present in ratserum are presumably required for embryonic growth. Many growth factors like epidermal growth factor(EGF), nerve growth factor, somatomedins, insulin and many others, are of small molecular weight and arelikely to contribute to the growth promoting qualities of rat serum.

This communication presents evidence for the probable involvement of insulin and EGF in thedevelopment of explanted early head-fold rat embryos in vitro. Rat serum dialysed for 6 days against fourchanges of 400 ml of a balanced salt solution (Cockroft, 1979) using 18/32 dialysis tubing (ScientificInstruments) was used for culture of 9-5 day rat embryos for 48 h. Culture in dialysed rat serumsupplemented with undialysed rat serum (to a concentration of 10 %), 2 mg/ml D-glucose and vitamins(MEM, Flow Laboratories, U.K.) resulted in severe embryonic growth retardation. The addition of eitherinsulin (4 ng/ml) or EGF (4 ng/ml) to the culture medium marginally improved embryonic growth. Asignificant improvement of embryonic growth was observed when both insulin and EGF were added to theculture medium. Significantly smaller values for the morphological score (Brown & Fabro, 1980) and totalembryonic protein were observed for all experimental embryos compared to those values for embryoscultured in 100 % undialysed rat serum. These results suggest that insulin and EGF can synergisticallyimprove the growth of rat embryos in culture. However, since the addition of both insulin and EGF resultedin suboptimal embryonic growth compared to growth in undialysed rat serum, it is likely that dialysablefactors present in rat serum in addition to insulin and EGF could also be involved in the development ofearly post-implantation rat embryos.

The identification and expression of early embryonic growth factors

JohnK. Heath*, Hilary A. Oakley andW.-K. Shi, Department of Zoology, University of Oxford,South Parks Road, Oxford 0X13PS

The development of serum-free defined media has allowed the identification of molecules necessary forthe proliferation of murine embryonal carcinoma (EC) cells and their differentiated derivatives in vitro.That these molecules may serve a similar function in early mammalian embryogenesis is suggested by theirexpression in the extra-embryonic tissues of the early post-implantation embryo. The tissue distribution ofthese growth regulatory molecules has lead to the proposal that proliferation in the early embryo may beco-ordinated by reciprocal interactions between primitive ectoderm cells and their differentiatedderivatives.

The availability of serum-free culture conditions has facilitated the purification and structuralcharacterisation of an apparently novel growth factor species secreted by EC cells. Embryonal carcinomaderived growth factor (ECDGF) will stimulate the proliferation of specific normal embryonic tissues and theimmediate differentiated derivatives of EC cells in vitro, suggesting a possible growth regulatory function invivo. ECDGF will also inhibit the spontaneous differentiation of certain EC cell lines in vitro. This action isinhibited in the presence of retinoic acid. These findings argue for a linkage between cell proliferation anddifferentiation in vitro and suggest that specific growth factors may have an important function in thecoordinated development of the early post-implantation embryo.

Growth factors in development 41Mechanism of action of platelet-derived growth factor and its

relation to oncogenesC.-H. Heldin*, B. Ek, C. Betsholtz, A. Johnsson, M. Nistir, L. Ronnstrand, A. Wasteson andB.

Westermark, Dept. ofMedical and Physiological Chemistry and Dept. of Pathology, University of Uppsala,S-75123 Uppsala, Sweden

Recent findings link platelet-derived growth factor and its mechanism of action to oncogenes. Ourworking hypothesis is that a constitutive expression of any of the regulatory components along the mitogenicpathway (the growth factor, the membrane receptor, or the postreceptor signal system which leads toinitiation of DNA synthesis) may lead to transformation. Amino acid sequence analysis of PDGF hasrevealed a striking homology with p28sis, the transforming protein of simian sarcoma virus (SSV). The Bchain of PDGF is almost identical to part of p28sis, whereas the A chain is about 60 % homologous to the Bchain. Apparently SSV acquired transforming activity via transduction of the gene for the B chain ofPDGF. In normal cells, PDGF exerts its mitogenic activity via interaction with a 185 kDa membranereceptor. The mitogenic signal is probably transduced via activation of a tyrosine kinase associated with thereceptor. By use of an antiserum against phosphotyrosine, we have identified phosphorylated componentsin PDGF-stimulated human fibroblasts. The major substrate is the PDGF receptor itself, which becomesautophosphorylated after binding of PDGF. In addition, other components, e.g. one of Mr 115,000, wasfound to be phosphorylated. Although the function of these substrates is still not known, they are interestingcandidates for being involved in the intracellular transmission of the mitogenic signal. An involvement ofthe sis-proto-oncogene in human malignancy is suggested by the finding that certain osteosarcoma andglioma cells in culture synthesize and release a 31 kDa factor with close resemblance to PDGF. In order toinvestigate whether production of a PDGF-like growth factor leads to autocrine growth stimulation of theproducer cell, a clone of human osteosarcoma cells was studied, which showed a lowered production ofgrowth factor and, in contrast to the maternal cell line, retained a limited number of PDGF receptors. Thisclone responded to stimulation by the endogeneously produced growth factor in a manner similar to howhuman fibroblasts respond to PDGF. Furthermore, immunoprecipitation with phosphotyrosine antibodiesled to identification of a 115 kDA component. Since a similar protein has been found also in normalfibroblasts, but only after exposure to PDGF, this may indicate that the PDGF-stimulated mitogenicpathway is activated in this clone of U-2 OS cells.

Proliferative properties of stromal fibroblasts from human embryoniccorneas in vitro

Louise Hyldahlx and Anthony R. Rees 2. 1Department of Ophtalmology, Karolinska Hospital, S-104 01Stockholm, Sweden. 2Laboratory of Molecular Biophysics, University of Oxford, South Parks Road, Oxford

We have recently shown that a variety of primary cell cultures can be established from eye globes of 8-12week old human foetuses. By the use of different techniques, it has been possible to establish pure cornealendothelial cell cultures as well as pure stromal fibroblast cultures from this material.

Furthermore, substantial multiplication of human embryonic corneal stromal fibroblasts has beenobtained in basal medium MCDB 104 supplemented with 25 ng Epidermal Growth Factor (EGF)/ml,10 fig insulin/ml, 20 fig transferrin/ml, 25 ng Multiplication Stimulating Activity (MSA)/ml, 0-5 mgovalbumin/ml, 50 /xg High Density Lipoprotein (HDL)/ml, 50 /LCE LOW Density Lipoprotein (LDL)/ml and10~6 M hydrocortisone. Even though the growth rate appears to be similar to that obtained in 10 % serum,the cells maintained in such serum-free medium cease proliferating at a lower density. The culture systemcontains no deliberately added undefined substances or components and is chemically defined.

Human embryonic corneal stromal fibroblasts exhibit a significant displaceable binding of 125I-EGF.Scatchard analysis revealed that this binding is biphasic, indicating the existence of two sets ofEGF-receptors. In contrast no displaceable binding of * -I-insulin could be detected above the backgroundlevel. This finding suggests that this cell type does not exhibit specific receptors for insulin.

42 Growth factors in developmentLaminar flow alters cell growth at wound edges in monolayers

G. W. Ireland and G. A. Dunn, MRC Cell Biophysics Unit, 26-29 Drury Lane, London WC2B5RL

Using techniques for controlling the flow pattern of medium in 3T3 cultures, we have re-examined theenhanced tritiated thymidine incorporation found at the edges of wounds made in confluent cell sheets.Precise parallel-sided wounds are made in confluent monolayers of 3T3 cells growing on Petripermpermeable membranes. Cultures are fixed and prepared for autoradiography using stripping film. Labellednuclei from processed cultures are identified using digital image analysis and replotted on a compressedscale to show the whole wound.

A circular plate was fixed in the culture medium at a small distance above the cell sheet. This suppressedthe convection currents that normally occur in culture dishes. Under these conditions, higher levels of serumare required to show edge-enhanced tritiated thymidine incorporation. By rotating the plate slowly, we wereable to induce a steady laminar flow of low shear rate between the plate and the cell sheet. When the flowwas arranged to cross the wound perpendicularly, we found that tritiated thymidine labelling wassignificantly enhanced down-stream of the wound.

These regimes for controlling medium flow allow the testing of different hypotheses of growth control atwound edges. The results so far are consistent with diffusion limitation hypotheses but contradict thosebased solely on growth inhibition mediated by cell contact.

The multipotential stem cells of the hemopoietic system: the factorapproach to their detection and expression in cultureN. N. Iscove*, Basel Institute for Immunology, CH-4005 Basel, Switzerland

Cells of both the hemopoietic and the immune systems derive continuously throughout adult life fromself-renewing multipotential stem cells. These earliest cells have yet to be identified by a satisfactory clonalassay. However, subsequent pluripotential and committed stages in their progression down the varioushemopoietic lineages have been charted in vitro in this way. In addition to giving clear operational definitionto these differentiative stages, the in vitro clonal methods have also permitted identification, purification andcDNA cloning of stage- and lineage-specific hemopoietic growth factors. The progress achieved in definingthese factors is now expected to support an effort to identify the additional conditions necessary for clonaldetection of the earliest multipotential stem cells.

Growth factors in development 43Differentiation in vitro represses oncogene expression

M. Jaye \ W. Drohan 1, B. Tong 2, T. Deuel2and T. Maciag*3.1Department of Molecular Biology, MeloyLaboratories, Springfield, Viriginia 22151.2Departments of Medicine and Biochemistry, Washington

University, St. Louis, Missouri 63110.3Department of Cell Biology, Revlon Biotechnology Research Center,Rockville, Maryland 20850

Human endothelial cells (HEC) differentiate into three dimensional tubular structures in vitro in anenvironment which limits cell division (Maciag, et al., Journal of Cell Biology, 94: 511-520, 1982). Themodification of fibronectin (FN) by plasmin also promotes HEC organization by the stimulation of NECmigration. These tubular structures represent the non-terminal differentiated form of the endothelial cell invitro. Since (i) bovine EC synthesize a platelet-derived growth factor (PDGF)-like mitogen (DiCorleto andBowen-Pope, Proc. Natl. Acad. Sci. U.S.A., 80, 1919-1923,1983) and (ii) PDGF possesses a homology tothe sis oncogene of the simian sarcoma virus (Waterfield, et al., Nature, 304: 35-39, 1983), we studied theeffect of HEC differentiation upon the expression of RNA for FN and the sis oncogene. Preparations ofRNA were derived from non-differentiated, monolayer and organized/differentiated in vitro populations ofHEC (umbilical vein). These RNA preparations were resolved by agarose gel electrophoresis andhybridized on a Northern blot with a synthetic cDNA probe for the v-sis oncogene and a cDNA probe forhuman FN (F. Ramerez, Rutgers University School of Medicine, New Brunswick, N.J.). We observed thatthe RNA derived from the HEC monolayer hybridized with the v-sis probe (4-2 Kb) whereas the RNA fromthe differentiated HEC cultures did not. Although RNA preparations from either HEC monolayer ortubular structures hybridize with the cDNA probe for FN (6-7 Kb), the differentiated population of HECcontains considerably more RNA for FN than the non-differentiated HEC monolayer system. These resultsdemonstrate: (i) HEC express at least one RNA for a PDGF-like moiety, (ii) HEC differentiation repressesthe expression of the sis oncogene perhaps by regulation of transcription and (iii) HEC differentiationresults in elevated levels of RNA for FN. Together these data emphasize the reciprocity between HECgrowth and differentiation at the molecular level and demonstrate the uniqueness of the HEC system as amodel for the study of the molecular developmental biology of the human vasculature.

Early effects of unilateral nephrectomy on cellular autophagy in kidneytubular cells

N. Jurilj 1 and U. Pfeifer 2. 1 Department of Biology, Stomatological Faculty, University of Zagreb,Yugoslavia. ^Department of Pathology, University ofWurzburg, West Germany

Unilateral nephrectomy or a sham-operation was performed in 24 male Sprague-Dawley rats between10.30 h and 11.30 h. The animals were killed 3 h 40 min to 8 h after the operations, i.e. between 14.00 hand 19.00 h. By means of a special morphometric technique, the volume fraction of autophagic vacuoles(AV) in cortical tubular cells was determined. At all time intervals investigated, except at 19.00 h where thephysiological diurnal shift of cellular autophagy to low values was already present, the volume fraction ofthe different types of AV containing endoplasmic reticulum and ground substance, mitochondria, andperoxisomes was reduced in the experimental animals to 60 %, 41 %, and 52 %, respectively, of the valuesfound in the sham-operated controls. The reduction by 49 % (p < 0-005) of the total AV-volume fraction ismore extensive in this early phase of compensatory kidney growth than it has been found at the first day(42 %) and second day (25 %) after unilateral nephrectomy in an earlier study. These results indicate thatinhibition of intracellular catabolism plays an important role already shortly after a growth stimulus. Such areaction has been found also in liver and heart muscle (Pfeifer, 1982). Biochemical data from literature areconsistent with this assumption.

PFEIFER, U. (1982). Kinetic and subcellular aspects of hypertrophy and atrophy. Int. Rev. Exp. Path. 23,1-45.

44 Growth factors in developmentSeasonal variation of the regenerative regulatory mechanisms in

TriturusS. Koussoulakos, D. Polydorou, N. Zilakos and V. Kiortsis, University of Athens, Zoological Laboratory,

Panepistimiopolis, Gr. 15701, Athens, Greece

A number of factors, including temperature, light cycle and season, have been shown to affect both theextent and the rate of appendage regeneration in vertebrates. These variations have been attributed tointernal factors, mainly endocrine fluctuations, (Schauble, K. M., Tyler, B. D., 1972. The effect of prolactinon the seasonal cyclicity of newt forelimb regeneration. J. Exp. Zool., 182, 41-46), but also cosmicradiation, inherent biologic rhythms, etc. There is however little information on the control mechanismsoperating during regeneration and appropriately ensuring the completion of an appendage.

During a study on 100 regenerating forelimbs of the crested newt, (Triturus cristatus), under controledenvironmental conditions, (20 ± 1 °C and 12 h light cycle), a seasonal difference was observed. Singlepuncture with an hypodermic needle, either into the stump or inside the 20 days blastema, always resulted inabnormal regenerates, as hypo-, ectro-, polydactyly, etc., during Spring months, (March to May). Injectionof cultured cells from various strains at different concentrations, induces the development of a great varietyof abnormal appendages. Although the end effects of plain puncture and/or cell administration seem to bemacroscopically similar, histological examination reveals great differences, (Koussoulakos, S., Zilakos, N.,Kiortsis, V., 1983. Effect of normal and malignant cells injected into the regenerating forelimb field. Proc.Hell. Biol. Soc, 5, 35-36). Abnormal regenerates reflect disturbances in morphogenetic controlmechanisms, and can be used as tools for identifying the nature of those mechanisms. On the other hand,injection of a convenient volume of culture media, (DMEM or Leibovitz), results in the production ofnormal limbs. From these data we deduce that the presence of the added low molecular weight nutrients inthe regenerating area, renders the morphogenetic field capable of expressing itself without defect. Duplicateexperiments carried out in the Winter, (December to February), brought unexpected results. All 100animals, tests and controls, developed normal appendages irrespectively of the treatment, which in turn,and in contrast with the Spring experiments, significately alters the rate of regeneration.

Postnatal changes of insulin binding in slow and fast twitch skeletalmuscles in the rabbit

L. Lefaucheur and P. Vigneron, INRA Station de Recherches sur I'Elevage des Pores, 35590, St-Gilles, andStation de Physiologie Animale, ENS A, 34060 Montpellier, Cedex, France

Published studies dealing with muscular insulin receptors have been carried on isolated membranes or onsmall whole muscles. These two methods cannot be used for comparing simultaneously postnatal changes ofinsulin binding in several muscles since muscle weight and extractability of membranes vary with musclesand age. We have developed a technique that enabled us to carry out such a study in the rabbit. Thesemi-membranosus proprius and psoas major muscles which are respectively slow twitch oxidative and fasttwitch glycolytic in the mature animal were chosen in this work. 40 fim cryostat microtome muscle slices(approximately 3 mg of fresh muscle per tube) were incubated for 22 h at 4 °C in a buffer containing3 10~n M of 125I-insulin (130-150 /ici//ig). In the competition assays the total binding was measuredat various concentrations of unlabeled insulin (5-5 10~n M to 5-5 10~9 M). All data have been corrected fornonspecific binding. The precipitability by trichloracetic acid showed that less than 5 % of the hormone wasdegraded. Results were analysed by using the Scatchard method, taking exclusively into account the highaffinity binding sites. The Kd was about 0*7 10~9 M in both muscles and did not change with age. At birth,the numbers of receptors were nearly identical in both muscles (5-3 femto-moles per mg fresh muscle) andthen they changed differently. In the psoas major muscle it decreased immediately after birth and reached aminimum value of 0*2 fmoles at about 1«2 kg body weight (B.W.). It then increased slightly to reach theadult level of 1-3 fmoles from 2 kg B.W. onwards. In the semi-membranosus proprius muscle the number ofreceptors remained unchanged until 0-6 kg B.W. and then dramatically decreased to a minimum value of0-9 fmoles at about 1-2 kg B.W. It subsequently increased to 3-7 fmoles at about 2 kg B.W. and reached aplateau, approximately 5-6 fmoles, in the adult animal. Physiological significance of these results isdiscussed.

Growth factors in development 45Attempts to extract and characterize the neural- and lens-inducing

activities from chicken embryonic tissuesA. T. Mikhailov and N. A. Gorgolyuk, Koltzov Institute of Developmental Biology USSRAcad. ScL,

Vavilovst. 26, Moscow 117334, USSRAn attempt has been made to extract from chicken embryonic retina and brain the factors having neural-

and lens-inducing activities upon amphibian early gastrula ectoderm (EGE). Explantation techmque wasused; extracts and fractions were added into culture medium. EGE of Rana temporaria L. was used as areacting tissue. When cultivated in media without proteins this EGE cannot spontaneously form neural orlens tissues and differentiates only to atypical epidermal or sucker cells. In the presence of cyclic nucleotides(or their derivatives), fetal calf serum, rabbit globulin and nerve growth factor (NGF) EGE generally didnot change epidermal pattern of differentiation; only with NGF some explants (about 10 %) becameneuralized. These agents also did not induce formation of 'free' lentoids. No signs of crystallin-specificfluorescence were observed in the untreated EGE. Inducers: Supernatants after 100 000 g and 20 000 gcentrifugation of water extracts from 7 to 8-day-old embryos were used. These showed pronouncedneuralizmg and lens-inducing activities which were Proteinase K-sensitive but did not change after RNA-asetreatment. Neuralizing activity was exerted by soluble proteins (100 000 g supernatant) but not bymicrosomal fractions of the extracts. Lens-inducing activity was associated with either supernatant or pelletafter 100 000 g centrifugation. 80S ribosomal proteins from 10-day-old chick embryos showed noneuralizing activity but they induced 'free' lentoids in EGE explants (up to 30 %).

Influence of adult female urine on the weights of growing male ratsO. A. Mora, 5. Guisado and L. Prieto, Departamento de Fisiologia, Facultad de Medicina, Universidades

Complutense v de Alcald de Henares, Madrid, Spain

It is a known fact that the presence of the female mice induces changes in the sexual development of themales. Likewise, at 60 days of age, male mice exposed to an adult female weighed more than when reared inits absence (Vandenberg, 1971). On the other hand, it has been described several pheromonal effects frommale rodents on the females and vice versa. It was intended to investigate the influence of the adult femaleurine on the body weight of male rats. 66 Wistar male rats were used and divided in two groups of 33 in twodifferent acconditioned rooms at 22 ± 2 °C and a light/dark period of 12/12 h. Both groups were isolatedfrom females of the same litter and other adult males or females from the day of birth, except their mothers.The weaning was at 21 days of age and there were 3 or 4 rats in each cage. The foodstuff was an industrialstandard laboratory diet and water ad libitum. 5 ml of female urine collected from rats in the estrus phasewere put daily in the bed of the cage of the group I, while in the cages of the group II were put daily 5 ml ofdestillated water, in both cases from the day of birth until 60 days of age. The body weight at birth was (ingrams; mean ± standard error of the mean): Group I, 6-29 ± 0*111; Group II, 6-26 ± 0-018. The mean ofthe body weight at 54, 56, 58 and at 60 days old was: Group I, 233-71 ± 3-58; Group II, 245-38 ± 4-095.The statistical difference is highly significant (p < 0-01, Student't' Test). We think that the urine of theestrus females has some factor that, in our conditions, inhibits the normal rate of weight increase in the malerats. These results conflict with those from Vandenbergh (1971) in mice. This may be due to: i), in theVandenbergh's conditions the adult female was in the cage of the males, with visual and tactile stimuli, inaddition to gustatory and olfactory ones, or ii) the events, in the rat, may be different to those in the mouse.The last possibility is, we believe, less likely.

VANDENBERGH, J. G. (1971). The influence of the social environment on sexual maturation in male mice. J.Reprod. Fert. 24, 383-290.

46 Growth factors in developmentIsolation of an EGF-like molecule from embryonic chicks

L. A. Opperman, S. H. Kidson and G. V. Goldin, Zoology Department, University of the Witwatersrand,Johannesburg 2001, South Africa

Epidermal growth factor is a potent mitogen first isolated from the submaxillary salivary glands of adultmale mice by Cohen (1962) and subsequently isolated from a variety of tissues including human urine,guinea pig prostate gland and embryonic mice. In this paper we report the isolation of an EGF-like moleculefrom embryonic chicks using a variety of biochemical and cell culture techniques. As EGF is a low molecularweight molecule (< 10,000 Da), a low molecular weight fraction of a chick embryo homogenate wasobtained by acid precipitation of all the large molecules and the ultrafiltration of the supernatant through aPM10 membrane. This low molecular weight fraction was found to consist of a number of small proteins bySDS gel electrophoresis. That this low molecular weight fraction contains an EGF-like molecule wasdemonstrated by 1) its ability to bind to EGF receptor sites on embryonic chick lung systems (as shown bycompetitive binding of the low molecular weight fraction with 125I-EGF) and 2) its mitogenic effect on theepithelium of chick lung systems grown in organ culture (as shown by autoradiographic analysis ofthymidine incorporation into the epithelium of cultured chick lungs). Characterization and purification ofthe active components by RRA and by SDS-PAGE has, been initiated. The conclusion we draw is thatembryonic chicks contain EGF or an EGF-like molecule and that this molecule has an important role to playin the development of the embryonic chick lung system.

COHEN, S. (1962). / . Biol. Chem. 237, (1555-1562).

Acquisition of a limited lifespan by differentiating cells derived fromPC13 embryonal carcinoma cells

Michael J. Rayner* and James A. J. Pulsford, Department of Zoology, Oxford University,South Parks Road, Oxford 0X13PS

Retinoic acid has been shown to induce the differentiation of mouse embryonal carcinoma (EC) cells toendoderm-like cells which have a slower rate of proliferation and are non-tumourigenic (see for example,Rayner & Graham, 1982). These cells also acquire the ability to respond to a range of exogenous growthfactors (reviewed by Heath, 1983). We have analysed the change in growth phenotype for a PC13 EC cellsusing video recordings and autoradiography. We have shown that the endoderm-like cells have a longer cellcycle than their undifferentiated counterparts (1800 min compared to 800 min, 5 cell divisions afterexposure to medium ± retinoic acid). The endoderm-like cells also have a progressively decreasingprobability of dividing again and this indicates that the differentiation of PC13 EC cells is accompanied bythe acquisition of a limited lifespan. The characteristics of mortal cells are well documented and theendoderm-like cells demonstrate the properties of such cells. In addition we have confirmed previousobservations that epidermal growth factor (EGF) can stimulate the proliferation of the endoderm-like cells(Rees, Adamson & Graham, 1979) and have shown, using autoradiography, that 92 % of these cells expressEGF receptors. Using video recordings, we have demonstrated that the effect of EGF is to shorten the cellcycle of the differentiating cells. However, we have also shown that EGF can enhance the survival andthereby prolong the lifespan of the endoderm-like cells. It is known that EGF and other growth factors canprolong the lifespan of mortal cells derived from normal tissue, but we have demonstrated that EGF canhave this effect on the differentiated derivatives of a tumour cell.

HEATH, J. K. (1983). Regulation of murine embryonal carcinoma cell proliferation and differentiation.Cancer Surveys 2, 141-164.

RAYNER, M. J. & GRAHAM, C. F. (1982). Clonal analysis of the change in growth phenotype duringembryonal carcinoma cell differentiation. / . Cell Sci. 58, 331-344.

REES, A. R., ADAMSON, E. D. & GRAHAM, C. F. (1979). Epidermal growth factor receptors increase duringthe differentiation of embryonal carcinoma cells. Nature, Lond. 281, 309-311.

Growth factors in development 47Epidermal growth factor and insulin-like growth factor precursors

/ . Scott*, L. Rail, R. Crawford and G. Bell, Molecular Medicine Group, M.R.C. Clinical Research Centre,Harrow, Middlesex

The nucleotide sequence of mouse epidermal growth factor precursor (EGFP) cDNAs predicts a proteinof 133 kd. EGF resides towards the COOH terminus. The amino terminal of the precursor contains nineEGF-like peptides. The precursor has a membrane spanning domain adjacent to the COOH terminus ofEGF and five potential glycosylation sites. In the adult mouse EGFP mRNA is abundant in the submaxillarygland, tooth buds and distal convoluted tubule of the kidney: lower levels are found in the proximalintestine, pancreas and lactating breast. The biosynthesis of EGFP has been examined in organ cultures ofthe submaxillary gland and kidney. In the submaxillary gland EGFP is processed to release 6 kdimmunoreactive EGF, whereas in the kidney EGFP remains as a single, high molecular weight species.

cDNAs coding for human insulin-like growth factors (IGF) I and II predict precursors of 14,600 and20,100 kd respectively. IGF I and II have NH2 terminal signal sequences linked directly to the matureprotein and COOH terminal extensions of 35 and 89 amirio acids residues.

Epidermal growth factor inhibits morphogenesis and differentiation inthe developing tooth

/. Thesleff *, A.-M. Partanen and P. Ekblom, Institute of Dentistry and Department of Pathology, Universityof Helsinki, SF-00280 Helsinki 28, Finland

Epidermal growth factor (EGF) stimulates the proliferation of various cell types in culture, and in somesystems this is accompanied by an inhibition of cell differentiation. We have studied the effect of EGF onthe development of embryonic mouse teeth in organ culture. The chemically defined culture medium wassupplemented with 50 /xg/ml transferrin previously shown to support early tooth morphogenesis (Partanenet at., Differentiation, in press). EGF (20 ng/ml) prevented morphogenesis of bud and cap-staged teeth (firstmolars of 13 and 14-day embryos), and this was followed by inhibition of odontoblast and ameloblast celldifferentiation. However, when the teeth were explanted at the early bell stage (15-day embryos) theirmorphogenesis and differentiation continued also in the presence of EGF. Thus the EGF-sensitive period oftooth development corresponded closely to the transferrin-dependent period shown earlier. Autoradio-graphic localization of thymidine incorporation showed that the most significant increase of cell proliferationwas caused by EGF in the dental epithelium.

The differentiation of odontoblasts and ameloblasts in the developing tooth is preceded by active cellproliferation. Furthermore, it has been well established that the proliferation as well as differentiation ofthese cells is regulated by epithelial-mesenchymal interactions. Our results suggest that by stimulating cellproliferation during early tooth development EGF interferes with the interactions of the differentiating cellsand disturbs the tightly regulated program of cell differentiation.

48 Growth factors in developmentDiadenosine 5', 5'"-P1, P4-tetraphosphate pyrophosphohydrolase in

Drosophila embryos: isolation and characterisationCarmen G. Vallejo, Departamento de Enzimologia, Institute de Investigations Biomtdicas del CSIC.

Facultad de Medicina, UAM, Madrid-34, Spain

Diadenosine 5', 5'"-?1, P4 tetraphosphate (Ap^\) is a nucleotide detected in eucaryotic and, recently,procaryotic species at concentrations of 10~8-10~° M. An increase in the cellular levels of Ap4A is foundwith short doubling times and changes are observed during the cell cycle. Ap4A has been found to induceDNA replication in permeabilised Gi-arrested cells. This inducing effect might be exerted through its foundbinding to one of the subunits of DNA polymerase a. The apparent necessary regulation of Ap4A levelsduring the cell cycle can be attained by a balance between the synthetic and the degradative activities. Thiscan in turn be achieved by raising or lowering the levels of both enzymatic activities and/or by the metabolicregulation of these enzymes. The Ap4A-synthetic activity of some tRNA synthetases is stimulated by Zn2+.Ap4A is degraded specifically to ATP and AMP by the enzyme dinucleosidetetraphosphatase in eucaryotesand to ADP in procaryotes. The Ap4A-splitting specific enzyme is also regulated. Zn2+ and Ca2+ havebeen found to inhibit it in eucaryotes as well as tetra, tri, di and mononucleotides. However, the effect ofAp4A on DNA replication and cell division has been studied in cultured cells. An embryonic system likeDrosophila, where nuclear division occurs in a rapid and synchronised way up to 2 h after fertilization,seems a more physiological system where to study the proposed role of A p ^ . I have started by isolating andcharacterising the splitting enzyme from late embryos (18-20 h).

The Ap4A-splitting enzyme is a thiol-protein, MT 26 K. It splits dinucleoside tetraphosphates to thecorresponding tri and mononucleotides. It does not split dinucleoside triphosphates or the syntheticsubstrate for alkaline phosphatase, bis-p-nitrophenylphosphate. It has an absolute requirement for divalentcations. The optimum pH is 7-0-7-5. The Michaehs constant for Ap^A is Km = 2 /uM. MononucleotidesAp4, ATP, ADP, AMP inhibit in a competitive manner. Zn2+ inhibits in a competitive fashion with aninhibition constant Ki = 1-4 /xM. Ca2+, inhibits is an uncompetitive manner. Co2+, a strong stimulatorof the procaryotic enzyme, has no special effect on the Drosophila enzyme. The described properties of thedinucleoside tetraphosphatase of Drosophila allow to characterise this enzyme as a typical eucaryotic one.Changes in the levels of the enzyme as well as in its negative effectors can allow the regulation ofAp4A-degradative activity and, therefore, modulate the velocity of nuclear division.

The mode of action of haemopoietic cell growth factor (HCGF-IL-3)A. D. Whetton*, G. W. Bazilland T. M. Dexter, Paterson Laboratories, Christie Hospital & Holt Radium

Institute, Manchester M20 9BX

Haemopoietic stem cells and progenitor cell populations undergo proliferation and development in vitroonly in the presence of the appropriate stimulatory molecules; in the absence of these stimulating factors thecells are lost from culture within hours. These factors have two roles, firstly to act as a survival signal andsecondly to facilitate proliferation and development. One such growth factor (HCGF), which has beenpurified and characterised, can promote the survival and self-renewal of stem cells and the survival anddevelopment of committed progenitor cells. This growth factor can also maintain haemopoietic precursorcell lines (FDC-P cells) which are absolutely dependent on HCGF for their survival and proliferation; in itsabsence they die within 12-48 h. Using these cells the nature of this factor dependence has been inves-tigated.

In the presence of HCGF, ATP levels are maintained in the FDC-P cells; in the absence intracellular ATPlevels undergo a steady depletion. Also removal of the HCGF leads to a rapid fall in the glycolytic flux ofthese cells. However, the addition of agents which increase the rate of glycolysis such as high extracellularlevels of glucose or glycolytic intermediates can maintain FDC-P cell viability to some extent in the absenceof HCGF. Furthermore, HCGF can stimulate the uptake of 2-deoxyglucose into the FDC-P cells in a dosedependent fashion. This uptake is inhibited by the addition of cytochalasin B, an inhibitor of the glucosetransport protein. L-glucose, which is not a substrate for the glucose transport protein is not taken up to anygreat extent by FDC-P cells in the absence or presence of HCGF. These results suggest that HCGF canactivate the glucose transport protein. This activation then leads to an enhanced rate of glycolysis whichmaintains the ATP levels of the cells, allowing them to survive and proliferate. Thus the control of primitivehaemopoietic cell survival and growth may be finely regulated by the provision of growth factors. A possiblefirst step in leukaemic transformation could then be a loosening of this control of hexose uptake by specificgrowth factors either because the cells have bypassed the need for, or are themselves constitutive producersof, these regulatory molecules which allow survival and proliferation.