38
Growth factors in development
Random 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 by
the animal is random, or organised as a succession of clones. Our approach has been to study genetically
marked 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 vitro
assay for granulocyte-macrophage precursors. We assume that different growth factors act on colonyforming cells at different developmental stages.
We confirmed that such progenitors formed clones in culture and, by analysing the proportions of genetic
markers of their colonies, studied whether progenitors at different developmental stages respond to
different haemopoietic growth factors. If haemopoiesis is random there should be no significant difference
in the proportions of genetically marked cells responding to different growth factors. Conversely, if clones
of haemopoietic stem cells are utilised successively then we might expect to see proportional differences
between 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 were
found which possessed both Pgk allelic forms. Distinct populations of progenitors can respond to different
stimulants, so our findings are consistent with clonal succession. Significant differences can be found in the
proportions of alloenzymes in similar sets of these progenitors cultured from similar and neighbouring
bones. This suggests that the system is locally organised. We have found no evidence of any single
population 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 have
been investigated and will be discussed.
Cell cycle control and growth factor action in differentiating embryonal
carcinoma 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 by
retinoic 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 growing
EC and END cells (PC13) and of synchronized EC cells in the presence and absence of RA, and cell cycle
parameters were determined according to a modified transition probability model. EC and END cells have
generation times (Tg) of ~11 h and ~20 h, respectively. RA had no effect on cell cycle kinetics during the
first two generations following addition to synchronized EC cells, but caused an increased Tg from 11 h to
16 h in the third, and a further increase to 20 h in the fourth and fifth generation. This increase in Tg was
principally due to an increase in the length of the S phase, and was associated with cell death occurring
frequently in certain sister pairs, as if susceptibility to RA toxicity was programmed and genetically
determined. Morphological differentiation was shown to begin already in the second generation, thus
preceding the altered growth characteristics. END cells, in contrast to EC cells, respond mitogenically to
exogenous growth factors, such as EGF. This property related to the acquisition of functional EGF
receptor/signal
transduction systems upon differentiation. Not only had EC cells no binding capacity for
125
I-EGF (as found by others), but EGF was also unable to induce a number of normal early responses. In
contrast END cells showed
~80,000 EGF binding sites, and EGF induced a variety of early responses, such
as activation of Na + /H + exchange and Na+,K+-ATPase in EC cells and tyrosine phosphorylation of the
EGF receptor and of exogenous peptide substrates by EC cell membranes. We conclude that RA-induced
differentiation of EC cells results in programmed cell cycle-dependent alterations in cell cycle kinetics, and a
transition from EGF-independent to EGF-dependent growth control which relates to the expression of
functional EGF receptor/signal transduction systems upon differentiation.
Growth factors in development
39
Clathrin and transferrin-receptor distributions during embryonic and
neonatal rat skin development
Martin R. Green and John R. Couchman, Biosciences Division, Unilever Research, Colworth House,
Sharnbrook, Bedfordshire MK441LQ
Specific polyclonal rabbit antibodies to bovine brain clathrin (1) and the human placental transferrinreceptor (Tf-R) (2) have been used on frozen sections to map the distribution of these antigens during rat
skin development. At 16 days embryonic development (day one is taken as the day following plug
formation) Tf-R staining is almost undetectable while clathrin localisation is restricted to a ribbon like
network in the region of the epidermal basal cells. By 17 days both antibodies weakly stain the membrane
region of all the epidermal cells and the ribbon staining for clathrin is lost. As the epidermis thickens and
stratifies with increasing age the intensity of staining with both antibodies increases until all the viable
epidermal cells are clearly delineated. In addition both antigens at birth have an intracellular punctate
localisation 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 the
epidermis. The Tf-R on the epidermal basal cells of the neonatal animal is partially polarised to the lower
cell 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 cells
and by the cells in the upper epidermis furthest away from the supply of nutrients from the dermis. Both
antibodies stain the hair follicle outer root sheath cells but the intensity is much reduced on dermal papilla
fibroblasts 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, Hills
Road, Cambridge, U.K.
Intracellular 125I-epidermal growth factor processing in rat skin
detected by electron microscope autoradiography
Martin R. Green, Claire Mycock, Colin G. Smith and John R. Couchman, Biosciences Division, Colworth
Laboratory, Sharnbrook, Bedfordshire MK441LQ
The distribution of EGF-receptors in embryonic and neonatal skin has previously been characterised
using 125I-EGF, viable skin explants and light microscope autoradiography (1, 2). Heavy labelling of the
basal cells of the epidermis, hair shaft and hair bulb was seen. In addition,
the epidermal EGF-receptor
number during neonatal development was found125to be related to the 3H-thymidine labelling index (1). We
report here on the intracellular processing of I-EGF in the basal epidermal cells and hair bulb cells
determined by incubating viable 3 day rat skin with 125I-EGF at 25
°C and analysis by electron microscope
autoradiography. Silver grains, indicating the localisation of 125I-EGF and presumably its associated
receptor, were associated with coated membrane imaginations. In some areas label appeared to collect near
hemidesmosomes. Internalised label was seen in the Golgi apparatus and in multivesicular, lysosomal
bodies 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 endothelial
cells confirming light microscope observations (1). Inclusion of sodium azide (2mM) in the incubation
medium restricted label to the plasma membrane region. Thus, intracellular processing of EGF in the
epithelial 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 growth
factor in cultured cells. Exp. Cell Res. 146, 163-175.
40
Growth factors in development
The effect of insulin and epidermal growth factor on rat embryonic
development in vitro
A. 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 is
suboptimal compared to growth in 100 % rat serum (Priscott, 1983). However, supplementation of, for
example, human serum with rat serum to a concentration of 10 % can restore the ability of human serum to
support 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 been
exhausted by repeated culture (Al-Alousi, 1983) with dialysed rat serum to a concentration of 10 % will not
support adequate growth and differentiation, it is likely that a number of dialysable factors present in rat
serum 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 are
likely to contribute to the growth promoting qualities of rat serum.
This communication presents evidence for the probable involvement of insulin and EGF in the
development of explanted early head-fold rat embryos in vitro. Rat serum dialysed for 6 days against four
changes of 400 ml of a balanced salt solution (Cockroft, 1979) using 18/32 dialysis tubing (Scientific
Instruments) was used for culture of 9-5 day rat embryos for 48 h. Culture in dialysed rat serum
supplemented 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 either
insulin (4 ng/ml) or EGF (4 ng/ml) to the culture medium marginally improved embryonic growth. A
significant improvement of embryonic growth was observed when both insulin and EGF were added to the
culture medium. Significantly smaller values for the morphological score (Brown & Fabro, 1980) and total
embryonic protein were observed for all experimental embryos compared to those values for embryos
cultured in 100 % undialysed rat serum. These results suggest that insulin and EGF can synergistically
improve the growth of rat embryos in culture. However, since the addition of both insulin and EGF resulted
in suboptimal embryonic growth compared to growth in undialysed rat serum, it is likely that dialysable
factors present in rat serum in addition to insulin and EGF could also be involved in the development of
early 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 for
the 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 their
expression in the extra-embryonic tissues of the early post-implantation embryo. The tissue distribution of
these growth regulatory molecules has lead to the proposal that proliferation in the early embryo may be
co-ordinated by reciprocal interactions between primitive ectoderm cells and their differentiated
derivatives.
The availability of serum-free culture conditions has facilitated the purification and structural
characterisation of an apparently novel growth factor species secreted by EC cells. Embryonal carcinoma
derived growth factor (ECDGF) will stimulate the proliferation of specific normal embryonic tissues and the
immediate differentiated derivatives of EC cells in vitro, suggesting a possible growth regulatory function in
vivo. ECDGF will also inhibit the spontaneous differentiation of certain EC cell lines in vitro. This action is
inhibited in the presence of retinoic acid. These findings argue for a linkage between cell proliferation and
differentiation in vitro and suggest that specific growth factors may have an important function in the
coordinated development of the early post-implantation embryo.
Growth factors in development
Mechanism of action of platelet-derived growth factor and its
relation to oncogenes
41
C.-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. Our
working hypothesis is that a constitutive expression of any of the regulatory components along the mitogenic
pathway (the growth factor, the membrane receptor, or the postreceptor signal system which leads to
initiation of DNA synthesis) may leadsis to transformation. Amino acid sequence analysis of PDGF has
revealed a striking homology with p28 , the transforming protein of simian sarcoma virus (SSV). The B
chain of PDGF is almost identical to part of p28sis, whereas the A chain is about 60 % homologous to the B
chain. Apparently SSV acquired transforming activity via transduction of the gene for the B chain of
PDGF. In normal cells, PDGF exerts its mitogenic activity via interaction with a 185 kDa membrane
receptor. The mitogenic signal is probably transduced via activation of a tyrosine kinase associated with the
receptor. By use of an antiserum against phosphotyrosine, we have identified phosphorylated components
in PDGF-stimulated human fibroblasts. The major substrate is the PDGF receptor itself, which becomes
autophosphorylated after binding of PDGF. In addition, other components, e.g. one of Mr 115,000, was
found to be phosphorylated. Although the function of these substrates is still not known, they are interesting
candidates for being involved in the intracellular transmission of the mitogenic signal. An involvement of
the sis-proto-oncogene in human malignancy is suggested by the finding that certain osteosarcoma and
glioma cells in culture synthesize and release a 31 kDa factor with close resemblance to PDGF. In order to
investigate whether production of a PDGF-like growth factor leads to autocrine growth stimulation of the
producer cell, a clone of human osteosarcoma cells was studied, which showed a lowered production of
growth factor and, in contrast to the maternal cell line, retained a limited number of PDGF receptors. This
clone responded to stimulation by the endogeneously produced growth factor in a manner similar to how
human fibroblasts respond to PDGF. Furthermore, immunoprecipitation with phosphotyrosine antibodies
led to identification of a 115 kDA component. Since a similar protein has been found also in normal
fibroblasts, but only after exposure to PDGF, this may indicate that the PDGF-stimulated mitogenic
pathway is activated in this clone of U-2 OS cells.
Proliferative properties of stromal fibroblasts from human embryonic
corneas in vitro
Louise Hyldahlx and Anthony R. Rees 2. 1Department of Ophtalmology, Karolinska Hospital, S-104 01
Stockholm, 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 8-12
week old human foetuses. By the use of different techniques, it has been possible to establish pure corneal
endothelial cell cultures as well as pure stromal fibroblast cultures from this material.
Furthermore, substantial multiplication of human embryonic corneal stromal fibroblasts has been
obtained 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 mg
ovalbumin/ml,
50 /xg High Density Lipoprotein (HDL)/ml, 50 /LCE LOW Density Lipoprotein (LDL)/ml and
10~6 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 system
contains 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 of
EGF-receptors. In contrast no displaceable binding of * -I-insulin could be detected above the background
level. This finding suggests that this cell type does not exhibit specific receptors for insulin.
42
Growth factors in development
Laminar 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 the
enhanced 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 Petriperm
permeable membranes. Cultures are fixed and prepared for autoradiography using stripping film. Labelled
nuclei from processed cultures are identified using digital image analysis and replotted on a compressed
scale to show the whole wound.
A circular plate was fixed in the culture medium at a small distance above the cell sheet. This suppressed
the convection currents that normally occur in culture dishes. Under these conditions, higher levels of serum
are required to show edge-enhanced tritiated thymidine incorporation. By rotating the plate slowly, we were
able to induce a steady laminar flow of low shear rate between the plate and the cell sheet. When the flow
was arranged to cross the wound perpendicularly, we found that tritiated thymidine labelling was
significantly enhanced down-stream of the wound.
These regimes for controlling medium flow allow the testing of different hypotheses of growth control at
wound edges. The results so far are consistent with diffusion limitation hypotheses but contradict those
based solely on growth inhibition mediated by cell contact.
The multipotential stem cells of the hemopoietic system: the factor
approach to their detection and expression in culture
N. N. Iscove*, Basel Institute for Immunology, CH-4005 Basel, Switzerland
Cells of both the hemopoietic and the immune systems derive continuously throughout adult life from
self-renewing multipotential stem cells. These earliest cells have yet to be identified by a satisfactory clonal
assay. However, subsequent pluripotential and committed stages in their progression down the various
hemopoietic lineages have been charted in vitro in this way. In addition to giving clear operational definition
to these differentiative stages, the in vitro clonal methods have also permitted identification, purification and
cDNA cloning of stage- and lineage-specific hemopoietic growth factors. The progress achieved in defining
these factors is now expected to support an effort to identify the additional conditions necessary for clonal
detection of the earliest multipotential stem cells.
Growth factors in development
Differentiation in vitro represses oncogene expression
43
M. Jaye \ W. Drohan 1, B. Tong 2, T. Deuel2and T. Maciag*3.1Department of Molecular Biology, Meloy
Laboratories, 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 an
environment which limits cell division (Maciag, et al., Journal of Cell Biology, 94: 511-520, 1982). The
modification of fibronectin (FN) by plasmin also promotes HEC organization by the stimulation of NEC
migration. These tubular structures represent the non-terminal differentiated form of the endothelial cell in
vitro. Since (i) bovine EC synthesize a platelet-derived growth factor (PDGF)-like mitogen (DiCorleto and
Bowen-Pope, Proc. Natl. Acad. Sci. U.S.A., 80, 1919-1923,1983) and (ii) PDGF possesses a homology to
the sis oncogene of the simian sarcoma virus (Waterfield, et al., Nature, 304: 35-39, 1983), we studied the
effect of HEC differentiation upon the expression of RNA for FN and the sis oncogene. Preparations of
RNA were derived from non-differentiated, monolayer and organized/differentiated in vitro populations of
HEC (umbilical vein). These RNA preparations were resolved by agarose gel electrophoresis and
hybridized on a Northern blot with a synthetic cDNA probe for the v-sis oncogene and a cDNA probe for
human FN (F. Ramerez, Rutgers University School of Medicine, New Brunswick, N.J.). We observed that
the RNA derived from the HEC monolayer hybridized with the v-sis probe (4-2 Kb) whereas the RNA from
the differentiated HEC cultures did not. Although RNA preparations from either HEC monolayer or
tubular structures hybridize with the cDNA probe for FN (6-7 Kb), the differentiated population of HEC
contains considerably more RNA for FN than the non-differentiated HEC monolayer system. These results
demonstrate: (i) HEC express at least one RNA for a PDGF-like moiety, (ii) HEC differentiation represses
the expression of the sis oncogene perhaps by regulation of transcription and (iii) HEC differentiation
results in elevated levels of RNA for FN. Together these data emphasize the reciprocity between HEC
growth and differentiation at the molecular level and demonstrate the uniqueness of the HEC system as a
model for the study of the molecular developmental biology of the human vasculature.
Early effects of unilateral nephrectomy on cellular autophagy in kidney
tubular 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 between
10.30 h and 11.30 h. The animals were killed 3 h 40 min to 8 h after the operations, i.e. between 14.00 h
and 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 the
physiological diurnal shift of cellular autophagy to low values was already present, the volume fraction of
the different types of AV containing endoplasmic reticulum and ground substance, mitochondria, and
peroxisomes was reduced in the experimental animals to 60 %, 41 %, and 52 %, respectively, of the values
found in the sham-operated controls. The reduction by 49 % (p < 0-005) of the total AV-volume fraction is
more 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 that
inhibition of intracellular catabolism plays an important role already shortly after a growth stimulus. Such a
reaction has been found also in liver and heart muscle (Pfeifer, 1982). Biochemical data from literature are
consistent 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 development
Seasonal variation of the regenerative regulatory mechanisms in
Triturus
S. 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 the
extent and the rate of appendage regeneration in vertebrates. These variations have been attributed to
internal factors, mainly endocrine fluctuations, (Schauble, K. M., Tyler, B. D., 1972. The effect of prolactin
on the seasonal cyclicity of newt forelimb regeneration. J. Exp. Zool., 182, 41-46), but also cosmic
radiation, inherent biologic rhythms, etc. There is however little information on the control mechanisms
operating during regeneration and appropriately ensuring the completion of an appendage.
During a study on 100 regenerating forelimbs of the crested newt, (Triturus cristatus), under controled
environmental conditions, (20 ± 1 °C and 12 h light cycle), a seasonal difference was observed. Single
puncture with an hypodermic needle, either into the stump or inside the 20 days blastema, always resulted in
abnormal regenerates, as hypo-, ectro-, polydactyly, etc., during Spring months, (March to May). Injection
of cultured cells from various strains at different concentrations, induces the development of a great variety
of abnormal appendages. Although the end effects of plain puncture and/or cell administration seem to be
macroscopically 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 control
mechanisms, 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 of
normal limbs. From these data we deduce that the presence of the added low molecular weight nutrients in
the regenerating area, renders the morphogenetic field capable of expressing itself without defect. Duplicate
experiments carried out in the Winter, (December to February), brought unexpected results. All 100
animals, 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 skeletal
muscles in the rabbit
L. Lefaucheur and P. Vigneron, INRA Station de Recherches sur I'Elevage des Pores, 35590, St-Gilles, and
Station de Physiologie Animale, ENS A, 34060 Montpellier, Cedex, France
Published studies dealing with muscular insulin receptors have been carried on isolated membranes or on
small whole muscles. These two methods cannot be used for comparing simultaneously postnatal changes of
insulin binding in several muscles since muscle weight and extractability of membranes vary with muscles
and age. We have developed a technique that enabled us to carry out such a study in the rabbit. The
semi-membranosus proprius and psoas major muscles which are respectively slow twitch oxidative and fast
twitch 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 containing
3 10~ n M of 125I-insulin (130-150 /ici//ig). In the competition
assays the total binding was measured
at various concentrations of unlabeled insulin (5-5 10~ n M to 5-5 10~9 M). All data have been corrected for
nonspecific binding. The precipitability by trichloracetic acid showed that less than 5 % of the hormone was
degraded. Results were analysed by using the Scatchard
method, taking exclusively into account the high
affinity 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) and
then they changed differently. In the psoas major muscle it decreased immediately after birth and reached a
minimum value of 0*2 fmoles at about 1«2 kg body weight (B.W.). It then increased slightly to reach the
adult level of 1-3 fmoles from 2 kg B.W. onwards. In the semi-membranosus proprius muscle the number of
receptors remained unchanged until 0-6 kg B.W. and then dramatically decreased to a minimum value of
0-9 fmoles at about 1-2 kg B.W. It subsequently increased to 3-7 fmoles at about 2 kg B.W. and reached a
plateau, approximately 5-6 fmoles, in the adult animal. Physiological significance of these results is
discussed.
Growth factors in development
Attempts to extract and characterize the neural- and lens-inducing
activities from chicken embryonic tissues
45
A. T. Mikhailov and N. A. Gorgolyuk, Koltzov Institute of Developmental Biology USSRAcad. ScL,
Vavilovst. 26, Moscow 117334, USSR
An attempt has been made to extract from chicken embryonic retina and brain the factors having neuraland lens-inducing activities upon amphibian early gastrula ectoderm (EGE). Explantation techmque was
used; extracts and fractions were added into culture medium. EGE of Rana temporaria L. was used as a
reacting tissue. When cultivated in media without proteins this EGE cannot spontaneously form neural or
lens 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 did
not change epidermal pattern of differentiation; only with NGF some explants (about 10 %) became
neuralized. These agents also did not induce formation of 'free' lentoids. No signs of crystallin-specific
fluorescence were observed in the untreated EGE. Inducers: Supernatants after 100 000 g and 20 000 g
centrifugation of water extracts from 7 to 8-day-old embryos were used. These showed pronounced
neuralizmg and lens-inducing activities which were Proteinase K-sensitive but did not change after RNA-ase
treatment. Neuralizing activity was exerted by soluble proteins (100 000 g supernatant) but not by
microsomal fractions of the extracts. Lens-inducing activity was associated with either supernatant or pellet
after 100 000 g centrifugation. 80S ribosomal proteins from 10-day-old chick embryos showed no
neuralizing activity but they induced 'free' lentoids in EGE explants (up to 30 %).
Influence of adult female urine on the weights of growing male rats
O. 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 the
males. Likewise, at 60 days of age, male mice exposed to an adult female weighed more than when reared in
its absence (Vandenberg, 1971). On the other hand, it has been described several pheromonal effects from
male rodents on the females and vice versa. It was intended to investigate the influence of the adult female
urine on the body weight of male rats. 66 Wistar male rats were used and divided in two groups of 33 in two
different acconditioned rooms at 22 ± 2 °C and a light/dark period of 12/12 h. Both groups were isolated
from 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 industrial
standard laboratory diet and water ad libitum. 5 ml of female urine collected from rats in the estrus phase
were 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 of
destillated water, in both cases from the day of birth until 60 days of age. The body weight at birth was (in
grams; mean ± standard error of the mean): Group I, 6-29 ± 0*111; Group II, 6-26 ± 0-018. The mean of
the 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 the
estrus females has some factor that, in our conditions, inhibits the normal rate of weight increase in the male
rats. These results conflict with those from Vandenbergh (1971) in mice. This may be due to: i), in the
Vandenbergh's conditions the adult female was in the cage of the males, with visual and tactile stimuli, in
addition 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.
J. G. (1971). The influence of the social environment on sexual maturation in male mice. J.
Reprod. Fert. 24, 383-290.
VANDENBERGH,
46
Growth factors in development
Isolation 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 adult
male 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 molecule
from embryonic chicks using a variety of biochemical and cell culture techniques. As EGF is a low molecular
weight molecule (< 10,000 Da), a low molecular weight fraction of a chick embryo homogenate was
obtained by acid precipitation of all the large molecules and the ultrafiltration of the supernatant through a
PM10 membrane. This low molecular weight fraction was found to consist of a number of small proteins by
SDS gel electrophoresis. That this low molecular weight fraction contains an EGF-like molecule was
demonstrated by 1) its ability to bind to EGF receptor sites on125
embryonic chick lung systems (as shown by
competitive binding of the low molecular weight fraction with I-EGF) and 2) its mitogenic effect on the
epithelium of chick lung systems grown in organ culture (as shown by autoradiographic analysis of
thymidine incorporation into the epithelium of cultured chick lungs). Characterization and purification of
the active components by RRA and by SDS-PAGE has, been initiated. The conclusion we draw is that
embryonic chicks contain EGF or an EGF-like molecule and that this molecule has an important role to play
in 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 from
PC13 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 to
endoderm-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 growth
factors (reviewed by Heath, 1983). We have analysed the change in growth phenotype for a PC13 EC cells
using video recordings and autoradiography. We have shown that the endoderm-like cells have a longer cell
cycle than their undifferentiated counterparts (1800 min compared to 800 min, 5 cell divisions after
exposure to medium ± retinoic acid). The endoderm-like cells also have a progressively decreasing
probability of dividing again and this indicates that the differentiation of PC13 EC cells is accompanied by
the acquisition of a limited lifespan. The characteristics of mortal cells are well documented and the
endoderm-like cells demonstrate the properties of such cells. In addition we have confirmed previous
observations 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 express
EGF receptors. Using video recordings, we have demonstrated that the effect of EGF is to shorten the cell
cycle of the differentiating cells. However, we have also shown that EGF can enhance the survival and
thereby prolong the lifespan of the endoderm-like cells. It is known that EGF and other growth factors can
prolong the lifespan of mortal cells derived from normal tissue, but we have demonstrated that EGF can
have this effect on the differentiated derivatives of a tumour cell.
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 during
embryonal carcinoma cell differentiation. /. Cell Sci. 58, 331-344.
REES, A. R., ADAMSON, E. D. & GRAHAM, C. F. (1979). Epidermal growth factor receptors increase during
the differentiation of embryonal carcinoma cells. Nature, Lond. 281, 309-311.
HEATH,
Growth factors in development
Epidermal growth factor and insulin-like growth factor precursors
47
/ . 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 protein
of 133 kd. EGF resides towards the COOH terminus. The amino terminal of the precursor contains nine
EGF-like peptides. The precursor has a membrane spanning domain adjacent to the COOH terminus of
EGF andfivepotential glycosylation sites. In the adult mouse EGFP mRNA is abundant in the submaxillary
gland, tooth buds and distal convoluted tubule of the kidney: lower levels are found in the proximal
intestine, pancreas and lactating breast. The biosynthesis of EGFP has been examined in organ cultures of
the submaxillary gland and kidney. In the submaxillary gland EGFP is processed to release 6 kd
immunoreactive 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 and
20,100 kd respectively. IGF I and II have NH2 terminal signal sequences linked directly to the mature
protein and COOH terminal extensions of 35 and 89 amirio acids residues.
Epidermal growth factor inhibits morphogenesis and differentiation in
the developing tooth
/. Thesleff *, A.-M. Partanen and P. Ekblom, Institute of Dentistry and Department of Pathology, University
of Helsinki, SF-00280 Helsinki 28, Finland
Epidermal growth factor (EGF) stimulates the proliferation of various cell types in culture, and in some
systems this is accompanied by an inhibition of cell differentiation. We have studied the effect of EGF on
the development of embryonic mouse teeth in organ culture. The chemically defined culture medium was
supplemented with 50 /xg/ml transferrin previously shown to support early tooth morphogenesis (Partanen
et at., Differentiation, in press). EGF (20 ng/ml) prevented morphogenesis of bud and cap-staged teeth (first
molars of 13 and 14-day embryos), and this was followed by inhibition of odontoblast and ameloblast cell
differentiation. However, when the teeth were explanted at the early bell stage (15-day embryos) their
morphogenesis and differentiation continued also in the presence of EGF. Thus the EGF-sensitive period of
tooth development corresponded closely to the transferrin-dependent period shown earlier. Autoradiographic localization of thymidine incorporation showed that the most significant increase of cell proliferation
was caused by EGF in the dental epithelium.
The differentiation of odontoblasts and ameloblasts in the developing tooth is preceded by active cell
proliferation. Furthermore, it has been well established that the proliferation as well as differentiation of
these cells is regulated by epithelial-mesenchymal interactions. Our results suggest that by stimulating cell
proliferation during early tooth development EGF interferes with the interactions of the differentiating cells
and disturbs the tightly regulated program of cell differentiation.
48
Growth factors in development
Diadenosine 5', 5'"-P 1 , P4-tetraphosphate pyrophosphohydrolase in
Drosophila embryos: isolation and characterisation
Carmen 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 found
with short doubling times and changes are observed during the cell cycle. Ap4A has been found to induce
DNA replication in permeabilised Gi-arrested cells. This inducing effect might be exerted through its found
binding to one of the subunits of DNA polymerase a. The apparent necessary regulation of Ap4A levels
during the cell cycle can be attained by a balance between the synthetic and the degradative activities. This
can in turn be achieved by raising or lowering the levels of both enzymatic activities and/or by the metabolic
regulation of these enzymes. The Ap4A-synthetic activity of some tRNA synthetases is stimulated by Zn 2+ .
Ap4A is degraded specifically to ATP and AMP by the enzyme dinucleosidetetraphosphatase
in eucaryotes
and to ADP in procaryotes. The Ap4A-splitting specific enzyme is also regulated. Zn 2+ and Ca2+ have
been found to inhibit it in eucaryotes as well as tetra, tri, di and mononucleotides. However, the effect of
Ap4A on DNA replication and cell division has been studied in cultured cells. An embryonic system like
Drosophila, 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 and
characterising 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 the
corresponding tri and mononucleotides. It does not split dinucleoside triphosphates or the synthetic
substrate for alkaline phosphatase, bis-p-nitrophenylphosphate. It has an absolute requirement for divalent
cations. The optimum pH is 7-0-7-5. The Michaehs constant 2+
for Ap^A is Km = 2 /uM. Mononucleotides
Ap4, ATP, ADP, AMP inhibit in a competitive
manner. Zn inhibits in a competitive
fashion with an
2+
inhibition constant Ki = 1-4 /xM. Ca , inhibits is an uncompetitive manner. Co 2+ , a strong stimulator
of the procaryotic enzyme, has no special effect on the Drosophila enzyme. The described properties of the
dinucleoside 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 of
Ap4A-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 vitro
only in the presence of the appropriate stimulatory molecules; in the absence of these stimulating factors the
cells are lost from culture within hours. These factors have two roles, firstly to act as a survival signal and
secondly to facilitate proliferation and development. One such growth factor (HCGF), which has been
purified and characterised, can promote the survival and self-renewal of stem cells and the survival and
development of committed progenitor cells. This growth factor can also maintain haemopoietic precursor
cell lines (FDC-P cells) which are absolutely dependent on HCGF for their survival and proliferation; in its
absence they die within 12-48 h. Using these cells the nature of this factor dependence has been investigated.
In the presence of HCGF, ATP levels are maintained in the FDC-P cells; in the absence intracellular ATP
levels undergo a steady depletion. Also removal of the HCGF leads to a rapid fall in the glycolytic flux of
these cells. However, the addition of agents which increase the rate of glycolysis such as high extracellular
levels of glucose or glycolytic intermediates can maintain FDC-P cell viability to some extent in the absence
of HCGF. Furthermore, HCGF can stimulate the uptake of 2-deoxyglucose into the FDC-P cells in a dose
dependent fashion. This uptake is inhibited by the addition of cytochalasin B, an inhibitor of the glucose
transport protein. L-glucose, which is not a substrate for the glucose transport protein is not taken up to any
great extent by FDC-P cells in the absence or presence of HCGF. These results suggest that HCGF can
activate the glucose transport protein. This activation then leads to an enhanced rate of glycolysis which
maintains the ATP levels of the cells, allowing them to survive and proliferate. Thus the control of primitive
haemopoietic cell survival and growth may befinelyregulated by the provision of growth factors. A possible
first step in leukaemic transformation could then be a loosening of this control of hexose uptake by specific
growth factors either because the cells have bypassed the need for, or are themselves constitutive producers
of, these regulatory molecules which allow survival and proliferation.