Growth and pattern formation occur simultaneously in many epimorphic fields and it has been suggested that specification of positional information is somehow linked to cell division. It is possible, therefore, that boundary regions responsible for the specification of positional information produce cell growth factors. In this paper I review the properties of some known growth factors, describe their effects on the cell cycle and discuss how they might act. In developing a convenient in vitro assay for morphogenetic factors it will be much easier to measure incorporation of [3H]thymidine into responding cells than to estimate changes in positional value.

The development of a multicellular organism from fertilized egg to adult does not proceed by an orderly series of cell doublings. It is by differential growth and cell division (together with cytodifferentiation and pattern formation) that the features of the adult body are established (Wolpert, 1969, 1971). This is well illustrated by the development of the wrist and forearm of the chick wing. Initially, the rudiments of these two elements are very similar in length, but while the wrist remains more or less the same size the forearm grows considerably to gain its final prominence (Summerbell, 1976; Lewis, 1977). In the terminology of Lewis & Wolpert (1976) cartilage cells in the wrist and cartilage cells in the forearm are ‘non-equivalent’, and their different growth properties reflect their different ‘positional values’.

But cell division during development should not be viewed only as a response to positional value once positional value has been specified; it is becoming clear that cell division may be an important parameter in the specification process itself. This is most obvious in the ‘progress zone’ model for the specification of positional information along the proximodistal axes of the chick limb bud and the regenerating amphibian limb (Summerbell, Lewis & Wolpert, 1973). In this model positional information is specified by the time a cell spends in the ‘progress zone’ at the tip of the limb, and this time may be measured in terms of cell division cycles (Lewis, 1975; Wolpert, Tickle & Sampford, 1979).

In many systems growth and specification of positional information occur simultaneously. Along the anteroposterior axis of the chick limb bud growth and cell division are enhanced by a graft of an additional zone of polarizing activity (Tickle, Summerbell & Wolpert, 1975; Fallon & Crosby, 1977; Summerbell & Tickle, 1977; Smith & Wolpert, 1981; Summerbell, 1981), which causes mirror-image duplication of the limb about its long axis (Saunders & Gasseling, 1968). Indeed, in any epimorphic field, including the appendages and imaginai discs of developing insects and the regenerating amphibian limb (French, Bryant & Bryant, 1976) pattern formation or regulation may be causally linked to cell division (Summerbell et al. 1973). One is reminded of Holtzer’s concept of the ‘quantal mitosis’ (Hotzler, Weintraub, Mayne & Mochan, 1972).

The question of how cell division is controlled during embryogenesis is thus of great importance to the study of pattern formation. Although Summerbell has obtained some evidence for density-dependent inhibition of growth in the chick limb bud (Summerbell & Wolpert, 1972; Summerbell, 1977; Cooke & Summerbell, 1980), most information regarding the control of cell division has been obtained from cells grown in culture. Here it has been found that a variety of factors can regulate the cell cycle: nutrients (Eagle, 1955; Ley & Tobey, 1970; Rubin, 1972), cell contact (Todaro, Lazar & Green, 1965; Dulbecco, 1970), cell shape (Folkman & Moscona, 1978), cell substrate (Gospodarowicz & Ill, 1980; Gospodarowicz, Delgado & Vlodavsky, 1980), growth factors (Sato & Ross, 1979) and inhibitors of cell growth (Balazs, 1979). However, it is not clear which, if any, of these might control cell division in developing embryos. In this review I shall describe the effects of some growth factors on cells in culture and in vivo, discuss how these growth factors might act and consider whether these, or similar, molecules might be active during development and perhaps even represent the elusive morphogens.

Most cells in culture require serum (the liquid portion of clotted blood) in order to proliferate (Carrel, 1912; Eagle, 1955; Todaro et al. 1965). Cells derived from tumours or exposed in vitro to oncogenic viruses or chemicals have a reduced serum requirement (reviewed by Brooks, 1975). Serum contains many components which function in different ways to promote growth (see Stiles, Cochran & Scher, 1981). Some of these, such as trace elements, amino acids and lipids, fulfil a nutritional requirement; others may transport such low molecular weight nutrients (transferrin, for example, is an iron carrier); some, like fibronectin, mediate cell attachment. In addition, serum contains the substances we now know as growth factors - hormones which stimulate growth but are not required as nutrients; that is, they are not molecules that may be used within the cell as metabolic substrates or co-factors (Gospodarowicz & Moran, 1976). (While this definition of a growth factor is adequate for the purposes of this review, it should be pointed out that until the molecular mechanism of action of a growth factor is elucidated its metabolic function is unknown.) Of all its functions, the most important role of serum does appear to be to provide hormones or growth factors. Hayashi & Sato (1976) have succeeded in growing an established rat pituitary cell line in a defined serum-free medium containing physiological concentrations of four hormones and transferrin.

Serum is a complex mixture of a variety of substances and the concentration of mitogens is low; thus, growth factors have been isolated from serum only with the greatest difficulty (see Gospodarowicz & Moran, 1976). More success has been obtained in isolating growth factors from tissue or from cultured cells. A fibroblast growth factor (FGF) has been isolated from bovine pituitary and brain (Gospodarowicz, 1975; Gospodarowicz, Bialecki & Greenburg, 1978 a); an epidermal growth factor (EGF) from the male mouse submaxillary gland (Cohen, 1962); and nerve growth factor (NGF) from the same source (Cohen, 1960). Cultured adipocytes produce a potent growth factor for endothelial cells (Castellot, Karnovsky & Spiegelman, 1980).

Below, I shall describe briefly the properties of six growth factors which may be involved in development. Although none of these has been implicated in any way in the interactions occurring during pattern formation they could give an idea of the kind of molecule to look for.

Endothelial cell growth factors

There is much interest in factors that control endothelial cell growth and migration because these are the processes involved in vascularization of tissues during both development and tumour growth (Ausprunk & Folkman, 1977). Growth factors for endothelial cells cultured in vitro have been isolated from a variety of sources; from tumour-cell-conditioned medium (Folkman, Hauden-schild & Zetter, 1979; Zetter, 1980); bovine hypothalamus (Maciag et al. 1979); and cultured 3T3 cells (Birdwell, Gospodarowicz & Nicolson, 1977; McAuslan, Hannan & Reilly, 1980). For developmental biologists perhaps the most interesting is a growth factor produced by differentiated 3T3 adipocytes, disvered by Castellot et al. (1980).

These authors have partially characterized a growth factor produced by 3T3-F442A adipocytes (Green & Kehinde, 1976). The factor is non-dialysable and it is not inactivated by heat or proteases. It is only produced by differentiated adipocytes, and of all the cell types examined only vascular endothelial cells respond to it. Adipose tissue is very highly vascularized and it is possible that the factor identified by Castellot and colleagues stimulates vascularization in vivo. Whether the factor also stimulates migration of endothelial cells remains an important question.

Epidermal growth factor

Epidermal growth factor was first isolated by Cohen (1962) from the submaxillary gland of the adult male mouse (mEGF). Extracts of the submaxillary gland injected into newborn mice induced precocious eyelid opening and incisor eruption, later shown to be due to a stimulation of epidermal growth and keratinization (Cohen, 1965). This mEGF was found to be a heat-stable singlechain polypeptide with 53 amino-acid residues. It has a pl of 4·5, three disulphide bonds (which are required for biological activity) and a molecular weight of 6045 (see Carpenter & Cohen, 1979).

A similar molecule (hEGF) has been isolated from human urine (Cohen & Carpenter, 1975; Savage & Harper, 1981). Of its 53 amino acids 37 are common to mEGF and its three disulphide bonds are in the same relative positions. Human EGF appears to be identical to urogastrone, a gastric antisecretory hormone isolated from urine (Gregory, 1975).

The biological activities of mEGF and hEGF are very similar and they possess some common antigenic sites. Both, as mentioned above, will stimulate epidermal growth and keratinization on injection into newborn mice. They will also stimulate epithelial cell proliferation in several organ culture systems, including the chick embryo cornea (Savage & Cohen, 1973). In tissue culture mEGF (hEGF was not tested) has little effect on the growth of small colonies of early passage human keratinocytes but does enhance growth of larger colonies. It also prolongs the culture lifetime of these cells (Rheinwald & Green, 1975).

EGF is mitogenic (at concentrations around 4 ng/ml; 0·7 nM) for a variety of fibroblast cells, both established lines, like 3T3 (Hollenberg & Cuatrecasas, 1973) and early passage human diploid fibroblasts (Cohen & Carpenter, 1975). Malignant transformation is sometimes associated with a loss of the growth requirement for EGF (Cherington, Smith & Pardee, 1979).

EGF is a powerful tool for studying the interactions of hormones with their target cells. It can be tagged with radioactive iodine (Carpenter & Cohen, 1976), fluorescein (Haigler, Ash, Singer & Cohen, 1978), rhodamine (Shechter et al. 1978 b) and ferritin (McKanna, Haigler & Cohen, 1979) while remaining active and able to bind to its receptor. These studies have demonstrated that on binding EGF the receptors aggregate and are taken into the cell by endocytosis. It is not clear whether internalization is required for the mitogenic action of EGF; this is discussed below.

Erythropoietin

The production of erythrocytes in both normal and anaemic mammals is regulated by erythropoietin, a blood-borne hormone whose level in the circulation is under the control of the kidney. The effects of erythropoietin have been studied on expiants of bone marrow and foetal liver. The hormone stimulates mitosis of proerythroblasts and promotes the differentiation of this enlarged population of erythroid precursors to erythroblasts, with the concomitant onset of globin synthesis (Chui, Djaldetti, Marks & Rifkind, 1971; Stephenson & Axelrad, 1971). Enrichment of cell populations for proerythroblasts has confirmed that these are the target cells for erythropoietin (Cantor, Morris, Marks & Rifkind, 1972) and enabled study of the expression of the globin gene (reviewed by Browder, 1980). The first detectable effect of erythropoietin is a stimulation of 4S, 5S and ribosomal RNA synthesis, but synthesis of globin mRNA does not occur until the proerythroblasts have undergone a round of DNA synthesis and mitosis. DNA synthesis is essential for the onset of globin mRNA synthesis, for if it is inhibited by hydroxyurea globin mRNA is not produced. Erythropoietin may, therefore, control a ‘quantal’ cell cycle (Holtzer et al. 1972).

Analysis of the nature of erythropoietin has been hampered because there is little starting material (plasma or urine from anaemic animals or man) available and the purification procedure is inefficient (Adamson & Brown, 1978). Isolated from sheep, erythropoietin appears to be a glycoprotein of molecular weight 46000 (Goldwasser & Kung, 1972) whereas the human form may have a molecular weight half of this (Dorado, Espada, Langton & Brandon, 1974). About 11% of the molecule consists of sialic acid, which is necessary for its in vivo activity; desialated erythropoietin is removed from circulation by the liver (Morell, Irvine, Sternlieb & Scheinberg, 1968). Desialated erythropoietin is, however, active in vitro (Goldwasser, Kung & Eliason, 1974).

Fibroblast growth factor

Fibroblast growth factors (FGFs) have been isolated from bovine brain and pituitary by Gospodarowicz and his colleagues (Gospodarowicz, 1974, 1975; Gospodarowicz, Moran & Bialecki, 1976). There is presently some confusion about the nature of these factors. Pituitary FGF appears to be a basic molecule with a molecular weight of 13000 on SDS-polyacrylamide gels under reducing conditions. It is active at 1 ng/ml (0·1 nM) (Gospodarowicz, 1975). Purification of brain FGF led to the isolation of two biologically active polypeptides, FGF-1 and FGF-2, which seemed to originate from a common precursor (Gospodarowicz et al. 1978 a). These were later shown to be produced by limited proteolysis of myelin basic protein (Westall, Lennon & Gospodarowicz, 1978). However, Bradshaw and his colleagues (Thomas et al. 1980) have now shown clearly that the mitogenic activity of brain FGF preparations resides in a small acidic (pl between 4·8 and 5·8) protein that represents only 1% of the protein in preparations of brain FGF made according to the procedure of Gospodarowicz. This protein is not related to myelin basic protein.

Preparations of pituitary FGF and the ‘crude’ brain FGF are mitogenic for many cell types. These include 3T3 cells, foreskin fibroblasts, glial cells, kidney fibroblasts, amniotic cells, chondrocytes, myoblasts, endothelial cells, smooth muscle cells, adrenal cortex cells, granulosa cells and blastemal cells (reviewed by Gospodarowicz, Moran & Mescher, 1978 b).

Fibroblast growth factors have been implicated in the control of vertebrate limb regeneration, although the rationale for these experiments (the assumption that FGF is a breakdown product of myelin basic protein) now appears invalid. The regeneration of the amphibian limb is dependent upon innervation (Todd, 1823; Singer, 1952, 1974), and the suggestion (see Gospodarowicz & Mescher, 1980) was that peptides with FGF activity would be released from injured peripheral myelin. These FGFs might direct regeneration by acting directly on blastemal cells, by-promoting endothelial cell growth and thereby vascularization (see Smith & Wolpert, 1975), and by stimulating Schwann cell proliferation and remyelination of regenerating nerves (Gospodarowicz & Mescher, 1980). To test the hypothesis that FGF regulates amphibian limb regeneration Gospodarowicz and his colleagues injected FGF into newt forelimb blastemas which had been denervated 3 days previously. The results indicated that FGF could indeed stimulate mitotic activity in the blastemas. Furthermore, repeated injections of pituitary FGF into the amputated limbs of adult frogs (which do not normally regenerate) resulted in the initial phases of blastema formation (Gospodarowicz & Mescher, 1980).

These results are consistent with the notion that a growth factor is active during vertebrate limb regeneration, but the finding of Thomas et al. (1980) that FGF is not a breakdown product of myelin basic protein makes it less likely that the growth factor is FGF.

Nerve growth factor

The first evidence for a nerve growth factor (NGF) was obtained in 1948 by Bueker. Implants of mouse sarcoma 180 to the body wall of chick embryos caused a 20 –40% enlargement of the dorsal root ganglions that innervated the tumour. Motor neurons were not stimulated to grow. Levi-Montalcini & Hamburger (1951, 1953) then showed that sympathetic ganglia were also enlarged and contributed nerves to the tumour. Furthermore, by transplanting sarcoma 37 or 180 to the chorioallantoic membrane, they demonstrated that the effects of the tumours were mediated by a diffusible agent. This agent became known as nerve growth factor.

The first attempts to purify NGF used sarcoma 180 as a starting material but it was then found that NGF is present in snake venom (Cohen & Levi-Montalcini, 1956) and in adult male mouse submaxillary glands (Cohen, 1960). NGF purified from the submaxillary glands of adult male mice (now the usual source) is a dimer of two identical peptide chains (each consisting of 118 amino acids, 3 disulphide bonds and with a molecular weight of 13259) linked by non-covalent forces.

NGF is not mitogenic in vivo or in vitro. The hyperplasia of sensory ganglia exposed to NGF, first observed by Bueker (1948), is probably due to enhanced neuronal survival (see Mobley et al. 1977); a variety of experiments have demonstrated that NGF plays a role in maintaining the survival of sympathetic and some embryonic sensory neurons (Levi-Montalcini & Booker, 1960; Levi-Montalcini & Angeleti, 1963). In addition, NGF causes an increase in cell body size and in neurite outgrowth in responsive cells; it may be chemotactic for axon growth; and it may play a role in the maintenance of the differentiated state of some neurons (reviewed by Greene & Shooter, 1980).

There appear to be three cellular receptors for NGF: on the plasma membrane, on the nucleus and at the synaptic ending (see review by Vinores & Guroff, 1980). The membrane and nuclear receptors were demonstrated by [125I]NGF binding studies on a variety of cells (including dorsal root ganglia cells and the NGF-responsive clone PC 12). Synaptic receptors were first proposed as a result of experiments in which [125I]NGF was injected into the anterior chamber of the eye of a mouse. It was found that there was a preferential accumulation of radioactivity in the superior cervical ganglion of the injected side, and this was interpreted to be due to retrograde axonal transport following uptake of NGF at the synaptic ending (Hendry, Stockel, Thoenen & Iverson, 1974). This retrograde transport seems to be of biological significance; if it is interrupted by axotomy neuronal degradation results, and this effect can be reversed by administration of exogenous NGF (Hendry & Campbell, 1976).

Somatomedins

Growth hormone (GH) promotes skeletal growth by stimulating epiphyseal cartilage cells to proliferate and increase matrix secretion (see Williams & Hughes, 1977). However, GH does not act directly, but through substances now known as somatomedins (Daughaday et al. 1972), whose levels in vivo are under the control of GH. This was first proposed by Salmon & Daughaday (1957) who observed that GH had no effect on expiants of cartilage cultured in vitro but incorporation of [35S]sulphate into proteoglycans was enhanced by addition of normal serum or serum from hypophysectomized rats which had been treated with GH.

Purification and characterization of substances capable of stimulating incorporation of [35S]sulphate into cartilage expiants demonstrated that the effects of these agents are not restricted to cartilage. Thus, substances classified as somatomedins should meet the following criteria: their concentration in serum should be regulated by GH; they should stimulate incorporation of sulphate into proteoglycans of cartilage; they should be mitogenic for fibroblasts; and they should have insulin-like effects on adipose and muscle cells (Daughaday et al. 1972).

Four factors or groups of factors which meet these criteria have been identified. These are: somatomedin A (Hall, 1972); somatomedin C (Van Wyk et al. 1974); insulin-like growth factors (IGF) (Rinderknecht & Humbel, 1976); and multiplication-stimulating activity (MSA) (Pierson & Temin, 1972; Smith & Temin, 1974). A peptide isolated by following stimulation of [3H]thymidine uptake into human glial cells was termed somatomedin B (Uthne, 1973; Westermark, Wasteson & Uthne, 1975) but it cannot be classed as a true somatomedin because it does not stimulate incorporation of [35S]sulphate into cartilage expiants.

Characterization of the somatomedins has been slow because of the difficulties in obtaining pure material. The liver is widely assumed to be the site of synthesis but the somatomedins are not concentrated there nor in any other organ (Chochinov & Daughaday, 1976; Daughaday, 1977; Rechler et al. 1979). Consequently, large amounts of blood are required. It has been found that all four factors are single-chain acid-soluble polypeptides of molecular weights 5000 to 10000. All are structurally related to insulin and compete for binding to the insulin receptor (reviewed by Blundell & Humbel, 1980).

That the somatomedins bind to the insulin receptor explains their weak insulin-like effects, but there is a second set of receptors with a high affinity for the somatomedins and a low affinity for insulin. It is through these receptors that the growth-promoting effects of the somatomedins and of insulin are exerted (see Blundell & Humbel, 1980).

Although the somatomedins are presumed to mediate the effect of GH during somatic growth there has been no direct confirmation of this because large quantities of somatomedins are difficult to obtain (Van Wyk & Underwood, 1978). However, it has recently been demonstrated directly that somatomedin C, at least, is active in vivo. DNA synthesis and mitosis are abolished in the lens epithelium of the frog after hypophysectomy but may be restored by injection of purified somatomedin C or human GH (Rothstein et al. 1980).

Many growth factors, like the endothelial cell growth factor, erythropoietin and NGF, have specific target cells. Others, like EGF, FGF and the somatomedins have little target specificity but may be highly specific for the portion of the cell cycle in which they act. This was first suggested by experiments in which growth factors were observed to function synergistically, suggesting that they controlled different events in the cell cycle. More sophisticated experiments have been able to dissect the sequence in which these growth factors act.

Synergism between growth factors

Synergism between growth factors was first observed by Armelin (1973), who found that hydrocortisone would enhance the growth-promoting effects of pituitary extracts. Subsequently, Gospodarowicz & Moran (1974) demonstrated that pituitary FGF would only induce optimal DNA synthesis in the presence of a corticosteroid and insulin.

More recently, Jimenez de Asua and colleagues (1977 a, b, 1979, 1981) have studied the initiation of DNA synthesis in quiescent, confluent Swiss 3T3 cells. They found that prostaglandin F2 α (PGF2 α) and insulin act synergistically to initiate DNA synthesis 15 h after addition of the hormones. By adding the hormones at different times they established that PGF2 α alone could initiate DNA synthesis, and addition of insulin to such PGF2 α-treated cultures did not alter the lag phase but did increase the rate at which the cells entered the S phase. Hydrocortisone would inhibit growth stimulation by PGF2 α, but only if it was added within 3 h. These results suggested that there are two steps in G0/G1 which control the entry of cells into S phase. The first is mediated by PGF2 α and inhibited by hydrocortisone, the second is regulated by insulin and controls the rate of entry of cells into S phase. Recently Jimenez de Asua et al. (1981) have demonstrated that EGF, like PGF2 α, will stimulate the initiation of DNA synthesis but that these two factors act through different cellular or biochemical pathways.

Competence and progression

Pledger, Stiles, Antoniades & Scher (1977, 1978) have attempted to characterize the factors in serum that are required for the growth of BALB/c-3T3 cells. Serum can be separated into two sets of components that function synergistically to regulate growth. One component is the platelet-derived growth factor (PDGF), a heat-stable cationic polypeptide contained in the α-granules of platelets and released when blood clots (Kohler & Lipton, 1974; Ross, Glomset, Kariya & Harker, 1974; Antoniades, Scher & Stiles, 1979; Kaplan et al. 1979). The other set of components is contained within platelet-poor plasma, which is prepared from unclotted blood and therefore lacks PDGF.

Brief treatment of quiescent, density-arrested BALB/c-3T3 cells with PDGF renders the cells ‘competent’ to replicate their DNA. Competence persists for many hours after removal of PDGF from the medium but PDGF-treated cells make no ‘progress’ through G0/G1 towards S phase until they are exposed to the complementary set of growth factors contained in platelet-poor plasma; 12 h later the cells begin to enter S phase. Two growth-arrest points have been identified in this 12 h period. One, the V point, is located 6 h before S phase; the other, the W point, is at the G1/S boundary. The rate of entry of cells into S phase from the W point depends upon the concentration of plasma.

The ‘progression activity’ of platelet-poor plasma can be replaced by EGF, somatomedin C and transferrin (Stiles et al. 1979 a, b; Leof, Wharton, Van Wyk & Pledger, 1980). Somatomedin C is required in the latter half of the progression sequence, at the V and W points (Stiles et al. 1979b; Wharton, Van Wyk & Pledger, 1981). By this time there has already occurred in the cells an increase in receptors for somatomedin C, in response to PDGF and factors in somatomedin C-deficient plasma (Clemmons, Van Wyk & Pledger, 1980).

Different growth factors act on different cells and at different points in the cell cycle. It is very unlikely, therefore, that they will work by similar mechanisms, and to make generalizations about the ways in which growth factors function is difficult. Below, I describe the early responses of cells to growth stimulation, the interaction of 125I-Iabelled EGF with cells and recent evidence that at least one growth factor, PDGF, acts via a cytoplasmic ‘second message’. Finally, I suggest that growth factors in vivo need not act while in solution; they could be active while adherent to the extracellular matrix.

Cellular responses to growth factors

Stimulation of growth in quiescent cells by serum or by purified growth factors rapidly (within a few minutes) induces a complex set of changes in the responding cells. These include stimulation of the Na-K pump (probably initiated by H+/Na+ exchange) (Rozengurt & Heppel, 1975; Rozengurt & Mendoza, 1980; Moolenaar, Mummery, van der Saag & de Laat, 1981); increase in 2-deoxy-D-glucose uptake (Sefton & Rubin, 1971); phosphorylation, and therefore trapping, of intracellular uridine (Rozengurt, Stein & Wiggles-worth, 1977); changes in the uptake of different amino acids (Hochstadt, Quinlan, Owen & Cooper, 1979); increase in phosphate uptake (Cunningham & Pardee, 1969); and changes in intracellular cyclic nucleotide concentrations (Seifert & Rudland, 1974). These responses can occur in the absence of protein synthesis but later changes include increases in the rates of protein and RNA synthesis (see Hershko, Mamont, Shields & Tomkins, 1971; Pardee, Dubrow, Hamlin & Kletzein, 1978). These reactions have been termed as a whole the ‘pleiotypic response’ (Hershko et al. 1971).

It is not clear how these events are directed towards the onset of DNA synthesis and cell division. One suggestion is that changes in nutrient transport directly trigger and sustain DNA synthesis and cell proliferation by providing the materials needed for growth (Holley, 1972). Rubin and his colleagues (Rubin & Sanui, 1979) have suggested that Mg2+ ions mediate the response to extracellular stimuli by increasing rates of protein synthesis. It is certainly true that inhibition of protein or RNA synthesis after serum stimulation of quiescent cells will inhibit or delay entry into S phase (Brooks, 1977; Chadwick, Ignotz, Ignotz & Lieberman, 1980) and that inhibition of protein synthesis in cycling cells will greatly extend the G1 period (Rossow, Riddle & Pardee, 1977). Furthermore, a variant line of Chinese Hamster cells which lacks a measurable G1 period has been shown to have an elevated rate of protein synthesis during mitosis (Rao & Sunkara, 1980); a G1 period could be induced by slowing their rate of protein synthesis with cycloheximide (Liskay, Kornfeld, Fullerton & Evans, 1980).

However, at least for quiescent 3T3 fibroblasts the elevated rate of protein synthesis that is induced by growth factors is not sufficient to induce DNA synthesis. Quiescent BALB/c-3T3 cells treated briefly with PDGF and then incubated in medium containing platelet-poor plasma experience an increase in protein synthesis and enter S phase after a minimum lag of 12 h. Brief treatment of cells with PDGF without subsequent incubation in plasma-containing medium or continuous incubation in medium containing plasma produce a similar increase in protein synthesis, but the treated cells do not enter S phase (Cochran, Lillquist, Scher & Stiles, 1981).

Interactions of[125I]EGF with responding cells

Epidermal growth factor is frequently used to study the interactions of growth factors with their target cells because it is available in large quantities, it may be tagged in a variety of ways while remaining active and as an acidic peptide it is easy to handle. Human and mouse EGF have been radio-iodinated. Both compete for the same receptor and these receptors are present on a variety of cultured cells (see Carpenter & Cohen, 1979). Different human fibroblasts were reported to have 40000 and 100000 binding sites per cell (Hollenberg & Cuatre-casas, 1975; Carpenter, Lembach, Morrison & Cohen, 1975).

The EGF receptors are normally randomly distributed, but on binding the growth factor they aggregate and are taken into the cell by endocytosis (see review by Hopkins, 1980). Once inside the cell the EGF-receptor complexes fuse with lysosomes where both EGF and the receptor are degraded (Carpenter & Cohen, 1976; King, Hernaez & Cuatrecasas, 1980). Carpenter & Cohen (1976) demonstrated that the degradation, but not the internalization, of EGF was prevented by chloroquine, an inhibitor of lysosomal protease activity. They also observed that internalization of EGF was associated with a loss of EGF binding sites from the cell surface. This phenomenon has been termed ‘down regulation’ and has also been observed with insulin (Gavin et al. 1974) and human GH (Lesniak & Roth, 1976).

The roles of EGF-receptor internalization and degradation in the mitogenic response to EGF are unclear. It is known that EGF must be present in the medium for several hours in order to induce DNA synthesis (Carpenter & Cohen, 1975; Shechter, Hernaez & Cuatrecasas, 1978a; Aharonov, Pruss & Herschman, 1978) and that only a small fraction of the EGF receptors need be occupied in order to induce the maximal mitogenic response (Das & Fox, 1978; Shechter et al. 1978 a). Furthermore, King et al. (1980) have demonstrated that degraded receptors are continually replaced by newly synthesized receptors so that the process seems to be geared towards a constant transport of EGF and its receptor into the cell (see King, Hernaez-Davis & Cuatrecasas, 1981).

The importance of the internalization and subsequent degradation of the EGF-receptor complex in the induction of the mitogenic response has been studied with inhibitors of the breakdown process. Savion, Vlodavsky & Gospodarowicz (1980) attempted to inhibit degradation of [125I]EGF with leupeptin, and found that the mitogenic response to EGF was unaffected. This suggested that breakdown of the EGF-receptor complex was not required for mitogenesis. However, King et al. (1981) showed that primary alkylamines, which also inhibit intracellular processing of ligand-receptor complexes (but not internalization, as had previously been thought), will inhibit mitogenesis in response to EGF. Hence, the role of the degradation of the EGF-receptor complex remains unknown. Hopkins (1980) suggests that there is no compelling reason why internalization and degradation should play a part in the mitogenic response and prefers the view that signalling by the EGF-receptor complex begins on the cell surface.

PDGF induces a cytoplasmic ‘second message’

The cytoplasm of cells undergoing DNA synthesis contains agents which will initiate DNA synthesis in non-replicating nuclei. This has been demonstrated by cell fusion experiments in which fusion with an S phase cell initiates DNA replication in the nuclei of G0/G1 cells (Rao & Johnson, 1970; Graves, 1972; Mercer & Schlegel, 1980) or of chick erythrocytes (Harris, 1965; Lipsich, Lucas & Kates, 1978) and by in vitro studies in which cytoplasmic extracts from growing cells promote DNA synthesis in isolated frog cell nuclei (Jazwinski, Wang & Edelman, 1976; Floros, Chang & Baserga, 1978; Das, 1980). The nature and mode of action of these cytoplasmic agents are unknown, but broadly there are two possibilities. Some may be precursors or enzymes which are produced as cells approach and enter S phase and which are directly involved in DNA replication (see Kornberg, 1980). Others may be cell-cycle regulatory molecules produced, perhaps, in response to extracellular growth factors. Such regulatory molecules would be of greater interest in the study of the control of cell growth because the critical events controlling cell division occur before the onset of DNA synthesis (see above).

Until recently, it had not been possible to distinguish between these two possibilities because exposure to growth factors could not be uncoupled from growth itself. However, the discovery that PDGF can render cells ‘competent’, but that competent cells will not ‘progress’ towards S phase unless incubated in medium containing platelet-poor plasma enabled Smith & Stiles (1981) to do just that. Cells treated briefly with PDGF, or cytoplasts derived from them, were fused to ‘recipient’ cells; these recipient cells were rendered competent and when incubated in medium containing platelet-poor plasma entered S phase after the typical lag of 10 –12 h. The ability of PDGF-treated cells to transfer competence in this way was abolished by inhibitors of RNA synthesis but not by inhibitors of protein synthesis. These results suggest that PDGF induces the formation of a second message, perhaps an mRNA, which is responsible for the competent state. Possible translation products of this mRNA have been identified by Pledger, Hart, Locatell & Scher (1981); PDGF rapidly induces the formation of five polypeptides (molecular weights 29000, 35000, 45000, 60000 and 70000) within treated cells, the production of which is inhibited by inhibitors of RNA synthesis.

Growth factors may bind to cell substrata

The substrate on which cells are maintained can alter cellular requirements for growth factors. For example, smooth-muscle cells maintained on the extracellular matrix produced by corneal endothelial cells are able to proliferate in medium containing platelet-poor plasma (Gospodarowicz & Ill, 1980; Gospodarowicz et al. 1980); the same cells maintained on tissue-culture plastic require further additions to the medium, such as PDGF or FGF (see above references and Ross et al. 1974). Recently, Smith et al. (1981) have investigated the possibility that the ability of the extracellular matrix to enhance cell proliferation is due to adherent growth factors.

Tissue-culture plates, either coated with collagen (a major component of the extracellular matrix) or uncoated, were treated briefly with PDGF or FGF and then washed thoroughly. The growth of BALB/c-3T3 cells (which, like smoothmuscle cells, require PDGF or FGF for optimum growth in vitro) on these treated substrates or on untreated plates was compared. Cells maintained on PDGF- or FGF-treated plates grew rapidly compared with cells on untreated plates. This stimulation of growth was due to adherent growth factor, because when half a plate was treated and quiescent cells were seeded in medium containing platelet-poor plasma and [3H]thymidine, only those cells on the treated portion of the dish became labelled after autoradiography.

This observation suggests a means by which a localized and persistent in vivo stimulation of cell growth might be obtained, even in the absence of soluble growth factor. If morphogenetic factors are able to adhere to extracellular matrix this would provide a basis for a ‘positional memory’ (Smith, 1979).

I have described the properties of six growth factors thought to be active during development and attempted to give a general idea of how they act. It has been suggested (see Introduction) that growth and pattern formation are causally linked, so it is possible that molecules resembling growth factors are involved in the specification of positional information. How could this be investigated?

Two broad approaches might be adopted. While there is no reason to believe that any one of the growth factors discussed here, or indeed any known growth factor, is a ‘morphogen’, one should not rule out its involvement in pattern formation simply because the organ which produces it in the adult animal has not yet developed or because the field under study is remote from that organ. If a growth factor is produced in the adult the potential for making it is present in every embryonic cell. Thus, one approach would be to examine the effects of known growth factors (say, EGF or somatomedin C) in developing systems. The presence of the factors could be studied by radioimmunoassay; receptors might be identified using 125I-labelled growth factors (see D’Ercole & Underwood, 1980). The presence of a growth factor and its receptor does not prove that the factor is active in stimulating growth. To demonstrate this, in vitro experiments might be required or exogenous growth factors could be administered in vivo. This could be achieved by implants of growth-factor-impregnated Silastic (Heaton, 1977) or by the use of osmotic minipumps (Stiles & Smith, work in progress).

A second approach would be to study signalling regions to see if they produce growth factors - extracts could be assayed in vitro or in vivo as above - and to search for pleiotypic responses in the responding cells. This approach might prove fruitful in the chick limb where it has been demonstrated that a graft of the zone of polarizing activity (ZPA) rapidly stimulates the growth of responding cells (Cooke & Summerbell, 1980). However, it is unlikely that any novel morphogenetic growth factor could be purified from this source. Purification of 18 μg of PDGF required 500 units of human platelets (about 78 g protein) (Antoniades et al. 1979). Assuming the same efficiency and recovery, purification of 18 μg of ZPA growth factor would require one to ten million dozen eggs.

I am supported by a NATO postdoctoral fellowship at the Sidney Farber Cancer Institute. I thank Chuck Stiles and Fiona Watt for their helpful comments and Lynne Dillon for preparing the manuscript.

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