Growth Factor Superfamilies and Mammalian Embryogenesis

Development 102. 451-460 (1988) Review Article 451 Printed in Great Britain © The Company of Biologists Limited 1988

Growth factor superfamilies and mammalian embryogenesis

MARK MERCOLA and CHARLES D. STILES

Department of Microbiology and Molecular Genetics, Harvard Medical School and the Dana-Farber Cancer Institute, Boston, MA 02115, USA

Summary

With the availability of amino acid and nucleotide unpredicted from the cell biology of most of the sequence information has come the realization that growth factors. Moreover, these actions are reflected growth factors can be clustered into superfamilies. in nonmammalian species where homologues of the Several of these superfamilies contain molecules that mammalian growth factors control crucial steps in the were not initially identified because of growth-promot- choice of developmental fate. This review describes ing activities; rather they were discovered through five growth factor superfamilies and the role these their ability to regulate other processes. Certain molecules may have in controlling proliferation, dif- members of these superfamilies are present during ferentiation, and morphogenesis during mammalian early mammalian embryogenesis. However, until re- development. cently, it has been difficult to manipulate the developing mammalian embryo to observe directly the effects Key words: growth factor, mammal, epidermal growth of inappropriate, excessive, or reduced expression of factor, EGF, insulin-like growth factor, IGF-I, IGF-II, these molecules. Despite this limitation, at least some transforming growth factor-beta, TGF, heparin-binding of these molecules have been implicated in the control growth factor, HBGF, platelet-derived growth factor, of differentiation and morphogenesis, two actions PDGF.

Introduction fibroblast growth factor can induce mesoderm differentiation from ectoderm tissue. Thus, growth factors, Recent analysis of early development of C. elegans, or novel growth-factor-like molecules, seem to guide Drosophila, and Xenopus has emphasized the role morphogenesis and differentiation in these species. -that growtH factors, or related molecules, may play as Can the same be said for mammals? An emerging morphogenetic and differentiation signals. Two well- body of literature suggests that polypeptide growth characterized growth factors have sequence similarity factors which are similar, if not identical, to those to pattern-forming genes. Transforming growth fac- isolated from sera and tissues of adult mammals, tor-beta is homologous to predicted proteins of the influence not only growth but differentiation and decapentaplegic complex of Drosophila and a ma- morphogenesis during mammalian development. ternally encoded mRNA in Xenopus embryos This view is furthered by the realization that several (Padgett et al. 1987; Weeks & Melton, 1987). The molecules originally identified because of their devel- gene encoding epidermal growth factor shares opmental roles are structurally similar to growth domains of homology with the homeotic loci lin-Yl in factors. As shown in Table 1, the expanding number C. elegans and Notch in Drosophila (Greenwald et al. of growth factors can be grouped into superfamilies 1985; Wharton et al. 1985; Knust et al. 1987). More- based on nucleotide and amino acid sequence hom- over, growth-factor-like molecules act as mesoderm- ology as well as similar receptor-binding activity. In inducing signals in developing Xenopus blastocysts Table 2 and the paragraphs below, we summarize the (Smith, J. 1987; Slack et al. 1987; Kimelman & chemistry and biology of these superfamilies as well Kizschner, 1987). Slack etal. (1987) and Kimelman & as the data that link them to mammalian develop- Kirschner (1987) showed that purified bovine basic ment. 452 M. Mercola and C. D. Stiles

The epidermal growth factor family Sundell et al. 1980). However, the analysis of em- Several structurally related peptides exhibit the ac- bryos and embryonal carcinoma cells indicate that tivity first identified with epidermal growth factor TGF-alpha is the primary component of fetal EGF (EGF). EGF was discovered by Stanley Cohen as a activity. A fetal form of EGF was first suggested by contaminant within certain nerve growth factor prep- Nex0 et al. (1980) who noted high levels of EGF arations which triggered premature eyelid opening activity in fetal tissues using a receptor-binding assay and incisor eruption in neonatal mice (for review see but lower levels using a more specific immunoassay. Carpenter & Cohen, 1979). Using this observation as While the mRNA for salivary gland EGF is not a bioassay, Cohen and his associates purified EGF observed during mouse gestation or in F9 embryonal from mouse salivary glands and, by amino acid carcinoma cells, TGF-alpha protein and mRNA are analysis, showed it to be homologous to the human present in mid to late gestation mouse and rat hormone urogastrone. A structurally and functionally embryos (Table 2). In addition to expression of the similar growth factor, transforming growth factor- growth factor, functional TGF-alpha/EGF receptors alpha (TGF-alpha), is produced by certain tumour have been detected in mouse embryos as early as day and retrovirus-transformed cells (Marquardt et al. 11 (Table 2). 1984). Finally, Brown et al. (1985) have described a While studies using embryonic tissue have concen- vaccinia-virus-encoded mitogen which has EGF ac- trated on mid- to late-gestation embryos, exper- tivity. All members of the EGF superfamily compete iments using embryonal carcinoma cells suggest that efficiently with salivary gland EGF for binding TGF-alpha or EGF may function even earlier in 3 to a 170xl0 Mr transmembrane tyrosine-specific development. Retinoic-acid-differentiated F9 and kinase, which is the product of the c-erb B proto- PC13 cells, but not undifferentiated stem cells, se- oncogene (Downward et al. 1984). crete peptides with TGF activity (Rizzino et al. 1983). Early embryological studies have focussed primar- The expression of cell surface TGF-alpha/EGF re- ily on the effects of EGF, administered in utero, on ceptors may reflect the differentiation status of the the developing lung and palate (Catterton et al. 1979; cell. Undifferentiated PC13 and OC15 cells do not express cell surface receptors; however, at least OC15 Table 1. Growth factor families cells contain intracellular receptor molecules (Weller et al. 1987). In contrast, differentiating embryonal carcinoma cells display surface EGF receptors. Thus, Group Members they may be expressed in the embryo as stem cells Epidermal growth factor EGF differentiate. The binding of salivary gland EGF to Transforming growth factor-a\ trophoblast outgrowths of cultured mouse blastocysts TGF-a- supports this view (Adamson & Meek, 1984). Vaccinia growth factor, VGF Whether the cryptic intracellular form of the receptor Insulin-like growth factor IGF-I is expressed in embryonic stem cells, and what IGF-II (Somatomedin-C; function it has, is unknown. multiplication stimulating activity, MSA) EGF-like factors may play a multifunctional role Relaxin during development. Adamson & Meek (1984) demonstrated that, whereas the number of EGF Transforming-growth factor-/? TGF-/8, TGF-ft receptors increases in fetal tissues, the apparent TGF-/5,.2 affinity for EGF declines two- to threefold during Inhibin-A mouse gestation. The authors suggest that these Inhibin B changes correlate with differing roles of the receptor Activin-A as development proceeds from tissue growth to differ- Activin AB Miillerian inhibiting substance entiation. Thus, EGF may stimulate proliferation in stem cells and differentiation or expression of a Heparin-binding growth Acidic HBGF (acidic differentiated phenotype in mature cells. This is factors fibroblast growth factor, aFGF; endothelial cell supported by the range of nonproliferative responses growth factor, ECGF) to EGF seen in differentiated cells (for review see Basic HBGF, bFGF Sporn & Roberts, 1987). The homology between Products of the int-2, hst, and EGF and the homeotic loci Notch and lin-12, men- Kaposi's Sarcoma proto- tioned above, may imply a novel role for EGF and oncogenes TGF-alpha. Like the Notch and lin-12 products, the Platelet-derived growth factor PDGF-A EGF and TGF-alpha precursors are thought to PDGF-B (sis product), encode transmembrane peptides (Rail et al. 1985; PDGF-AB Gentry et al. 1987; Teixido et al. 1987). Thus, these Growth factor superfamilies 453

Table 2. Examples of growth factor expression during embryogenesis Factor Species Material assayed Occurrence Reference TGF-o- Mouse TGF-o- Post-day-7 embryos Proper et al. 1982; Twardzik, 1985 Mouse TGF-o- mRNA Post-day-7 embryos Popliker et al. 1987 Rat TGF-* mRNA Post-day-8 embryos Lee et al. 1985 EGF Receptor Mouse EGF binding Post-day-11 embryos Nex0 et al. 1980; Adamson et al. 1981 Rat Receptor kinase Post-day-10 embryos Hortsch et al. 1983 IGF Rat Serum IGF-II Fetal level > maternal level Moses et al. 1980 Rat Serum IGF-I Maternal level s> fetal level D'Ercole et al. 1980; Sara et al. 1980 Mouse IGF-II Amnion/yolk sac mesoderm Heath & Shi, 1985 Rat IGF-I, II mRNA Post-day-14 embryos/adult Lund et al. 1986 Human IGF-I, II mRNA Predominant expression Han et al. 1987 in connective and mesenchyme -derived tissue IGF Receptor Mouse IGF-I, II receptors Day-9, -12 embryos Smith et al. 1987 Mouse IGF-binding proteins Blastocysts/day-9 embryos Smith et al. 1987 Human IGF-binding Amnion Drop et al. 1984 TGF-jS Mouse TGF-/8 Day-17 embryo Proper et al. 1982 Rat TGF-/3 Day-21 fetal calvariae Pfeilshifter & Mundy, 1987 Rat TGF-/3 Day-12, -13 embryos Twardzik et al. 1982 Human TGF-/3 Placenta Frolick et al. 1983 HBGF Mouse int-2 RNA Day-7-5, -8-5 embryos Jakobovits et al. 1986 precursor molecules may act on the cell surface Midgestation human and rodent tissues express during development to mediate cell—cell interactions IGFs (Table 2). IGF-II mRNA and protein levels by recognizing a complementary receptor on another predominate during gestation and decline within a cell. few weeks after birth. IGF-I, however, exhibits the opposite pattern of expression. From these results, it is generally assumed that IGF-II is primarily a fetal Insulin-like growth factor family mitogen involved in the development of embryonal tissues. In support of this conclusion is the secretion The insulin-like growth factors (IGFs) are a pair of of IGF-II by differentiated embryonal carcinoma growth factors with striking amino acid sequence and lines (Nagarajan et al. 1985; Heath & Shi, 1986). structural similarity to human proinsulin (for review One exception to the pattern of IGF-II expression see Blundell & Humbel, 1980). The IGFs have been in fetal development is noted in neural tissue. High referred to as sulphation factors, nonsuppressable levels of IGF-II protein, mRNA and receptors are insulin-like activity, multiplication stimulatory ac- seen in the adult brain where IGF-II may stimulate tivity (MSA) and somatomedins. IGF-I is the more neural outgrowth (Sara et al. 1982; Recio-Pinto & basic of the IGFs. IGF-II, the more neutral, is Ishii, 1984; Haselbacher etal. 1985). identical to the MSA molecule of rodents which has As shown in Table 2, receptors and binding pro- been well characterized and its gene cloned (Whit- teins for the IGFs have been detected at various field et al. 1984). The analysis of cDNA and genomic stages of mouse embryogenesis. The presence of clones for human IGF-I and -II has shown that the IGF-binding proteins in amniotic fluid and blastocysts IGF-II and insulin genes are contiguous and map to a suggests the early action of IGFs. PC13 embryonal different chromosome than the IGF-I gene (Bell etal. carcinoma cells express IGF receptors and secrete 1985). The genes for the IGFs are homologous to two binding protein into the medium (Heath & Shi, 1986; nonallelic genes which encode human relaxin, a Smith et al. 1987). Upon differentiation of these cells peptide secreted by the ovary and involved in the in response to retinoic acid, IGF-II receptor ex- prenatal maturation of the female reproductive tract pression declines while IGF-II and binding protein (Crawford et al. 1984). IGFs bind to at least two types synthesis increase. Since PCD cells respond to IGF- of IGF receptors, as well as the insulin receptor, with II, Heath and co-workers proposed that these cells different affinities (King & Kahn, 1985). In plasma, and their embryonic analogues are under paracrine circulating IGFs bind specifically to carrier proteins and, following differentiation, autocrine control by which may modulate activity (Zapf et al. 1975). IGF-II. 454 M. Mercola and C. D. Stiles

The transforming growth factor-beta family The heparin-binding growth factor family

Transforming growth factor beta (TGF-beta) is a The heparin-binding growth factors are responsible 3 dimer of two 12-5xlO Mr peptides expressed in a for the activities ascribed to nearly a dozen previously wide array of cell types. TGF-beta was originally identified factors (Lobb et al. 1986). The growth identified by its ability to induce anchorage-indepen- factor activities formerly known as acidic fibroblast dent growth of rat fibroblasts in conjunction with growth factor, endothelial cell growth factor, eye- TGF-alpha or EGF (for review see Sporn et al. 1986; derived growth factor-II, retina-derived growth fac- Massague, 1987). TGF-beta cDNA clones of the tor-I and anionic hypothalamus-derived growth human gene were obtained in 1985 (Derynck et al. factor are either identical to or are enzymatically 1985). More recently, additional TGF-beta-like genes processed derivatives of acidic HBGF. Likewise, and proteins have been described. These include two basic HBGF is identical or related to basic fibroblast new forms of TGF-beta, the Mullerian-inhibiting growth factor, eye-derived growth factor-I, retina- substance (MIS), and the beta subunits of the inhibi- derived growth factor-II, cationic hypothalamus-de- tors and activators of follicle-stimulating hormone rived growth factor-II and macrophage-derived secretion (Cheifetz etal. 1987; Mason etal. 1985; Vale growth factor. The genes encoding the acidic and et al. 1986; Ling et al. 1986; Cate et al. 1986). basic HBGFs have been isolated as cDNA clones TGF-beta best illustrates the multiplicity of re- (Abraham et al. 1986; Jaye et al. 1986). Together, the sponses elicited by growth factor action. Depending HBGFs probably account for much of the activities on the cell type and on other growth factors acting on associated with tumour angiogenesis (see review by the cell, TGF-beta may either promote or inhibit cell Folkman & Klagsbrun, 1987). In addition, three oncogenes (int-2, hst, and a gene isolated from division in culture (Roberts et al. 1985). Differen- Kaposi's sarcoma DNA) encode proteins which are tiation may also be positively or negatively con- members of the HBGF family (Dickson & Gordon, trolled. In cell culture, TGF-beta induces chondro- 1987; Taira et al. 1987; Delli Bovi et al. 1987). genesis and squamous differentiation of bronchial epithelial cells while myogenesis and adipogenesis are Recent work suggests that the HBGFs participate in early mesoderm development. Mouse embryonal inhibited (for review see Sporn et al. 1986). carcinoma cells produce HBGF-like molecules The TGF-beta-related protein, MIS, is produced known as embryonal carcinoma-derived growth fac- by the developing testis and is responsible for the tors (ECDGFs) (Heath & Isacke, 1984; van Veggel et regression of the Miillerian duct. Expression of al. 1987). The ECDGFs are mitogenic for mesoderm- cloned human and bovine MIS in vitro yields a like, but not endoderm- or ectoderm-like, derivatives peptide capable of inducing regression of the rat of PC13 cells. In experiments designed to induce Miillerian duct in organ culture (Cate et al. 1986). mesoderm differentiation from animal pole ectoderm Studies of growth factor expression suggest that TGF- of Xenopus blastocysts, Slack et al. (1987) showed beta itself plays a role during embryogenesis that ECDGF and a purified HBGF, basic fibroblast (Table 2). TGF-beta activity is present in human growth factor, could mimic a component of the placenta and in mid- to late-gestation mouse and rat vegetal mesoderm-inducing signal. These results embryos. Also, conditioned culture media of rat fetal suggest a role for HBGFs in promoting the develop- calvariae explants contains TGF-beta activity. In ment of early mesoderm in mammals as well as in these cultures, osteotrophic hormones that increase Xenopus. In addition, the HGBFs could conceivably bone resorption also increase TGF-beta production. promote angiogenesis during mammalian develop- Since TGF-beta is mitogenic for osteoblasts, these ment. Indirect support for such a role is found in results suggest that TGF-beta links resorption to developing chicks where an angiogenic factor similar subsequent proliferation and differentiation of osteo- to basic HGBF has been isolated from neural tissue blasts. (Risau, 1986). Little is known about the ontogeny of TGF-beta receptors during development. Chiefetz et al. (1987) The platelet-derived growth factor family demonstrated that three types of TGF-beta receptors bind the different forms of TGF-beta with different Platelet-derived growth factor (PDGF) is a potent affinities, thus denning a complex pattern of ligand- mitogen for cells of connective tissue origin and is receptor interactions reminiscent of the IGF system. thought to play a role in wound healing when released TGF-beta receptors are found on essentially all cells. from platelets at the site of injury (for review see Ross If the receptors are also widespread in the early et al. 1986). Purified PDGF from human platelets embryo, it will be important to determine whether yields two distinct polypeptide chains, A and B, upon TGF-beta exerts its influence locally or systemically. reduction. The amino acid sequences of the A and B Growth factor super families 455 chains share greater than 50% positional identity. The initial event in growth factor action is binding The A and B chains are encoded by separate genes to a specific, high-affinity receptor which spans the which map to human chromosomes 7 and 22, respect- outer cell membrane. The receptor is often, but not ively. A and B homodimers are mitogenic for fibro- always, a tyrosine-specific protein kinase (for review blast cells and either homodimer will compete for of kinases, see Hunter, 1987). The receptors for EGF native PDGF isolated from human platelets in recep- (Ullrich et al. 1984; Lin et al. 1984; Xu et al. 1984), tor binding assays. A dimeric structure linked by PDGF (Yarden et al. 1986), insulin (Ullrich et al. disulphide bridges is absolutely essential for PDGF 1985; Ebina et al. 1985), and IGF-I (Ullrich et al. mitogenic activity. PDGF within human platelets may 1986) are tyrosine-specific kinases. The IGF-II recep- consist of A:B heterodimers either in addition to, or tor does not have tyrosine-specific kinase activity in lieu of, A: A and B:B homodimers. Consistent (Morgan et al. 1987). The receptors forTGF-beta and with the mitogenicity of homodimers, the oncogene the HBGFs have not been characterized definitively. of simian sarcoma virus (v-sis) directs the synthesis of The current consensus holds that the binding of a a PDGF B:B homologue. growth factor by its receptor induces soluble, intra- Most developmental work involving PDGF has cellular second messengers which transmit a signal to relied upon embryonal carcinoma cell lines. PDGF the nucleus (see review by Rozengurt, 1986). These activity is detected in conditioned media from undif- second messengers may be phosphoproteins, inositol ferentiated embryonal carcinoma lines (Gudas et al. phosphates, diacylglycerol, cyclic nucleotides, mono- 1983; Rizzino & Bowen-Pope, 1985). Retinoic-acid- valent or divalent ions. Within minutes after forma- induced differentiation greatly reduces the level of tion of the growth factor-receptor complex, changes PDGF activity. While the PDGF secreted by these in gene expression can be detected. Growth-factor- lines is not identical to PDGF from platelets, the inducible genes have been described for PDGF embryonal carcinoma cell data imply the action of a (Cochran et al. 1983; Linzer & Nathans, 1983), EGF PDGF-like factor during early embryogenesis. Con- (Foster et al. 1982) and the IGFs (Zumstein & Stiles, sistent with this idea, transcripts of the A and B (c-sis) 1987), and many of these genes have been isolated as chain genes are seen in mouse embryos at gastru- cDNA clones. lation (M.M. & C.D.S., unpublished observations). Some growth-factor-inducible genes appear to be PDGF receptors are present on embryonal carci- lineage specific. For example, lymphocyte mitogens noma cells. Rizzino & Bowen-Pope (1985) detected stimulate expression of the genes for interleukin-2, PDGF binding to retinoic-acid-differentiated F9 and PC13 cells and to two endoderm-like differentiated the interleukin-2 receptor and gamma interferon in cell lines. In contrast, Gudas et al. (1983) did not see resting T-cells (Kronke et al. 1985; Reed et al. 1986), significant binding to retinoic-acid-treated F9 or PSA- but not in fibroblasts (C.D.S., unpublished obser- 1 cells, although the differentiated line PSA-l-G did vations). These tissue-specific genes dictate, in part, exhibit receptors. No binding is seen in undifferen- the differentiated functions of T-cells. In addition, tiated cells; however, it is plausible that the receptors there appears to be a cohort of genes which are are downregulated by endogenously produced fac- expressed across tissue boundaries in response to tors. numerous growth factors. This set of common genes includes the oncogenes c-myc and c-fos. The protein Intracellular mediators of growth factor action products of both c-myc and c-fos are associated with the cell nucleus and may regulate the expression of Some of the results cited above indicate that growth other genes. It is possible that c-myc and c-fos are factors regulate differentiation and morphogenesis. prototypes of a class of genes whose products behave Thus, it is important to understand how a peptide, as intracellular mediators of the growth factor re- such as HBGF, can function as a mitogen in one sponse (Armelin et al. 1984). context and promote differentiation in another. Re- The influence of the cell's genetic programming in cent data indicate that growth factors, like other determining the cellular response to c-myc and c-fos hormones, regulate gene expression within their tar- expression is seen in the comparison of the different get cells. Two classes of genes are induced: (1) responses of PC12 and fibroblast cells following common genes which seem to be general components growth factor stimulation. The PC12 cell line is of a growth factor response and (2) genes that are derived from a well-differentiated rat pheochromo- characteristic of a cell lineage. The first class of genes cytoma (Greene & Tischler, 1976). These cells pro- may be intracellular mediators of the growth factor liferate rapidly and do not display differentiated response and may themselves influence gene ex- markers until exposure to nerve growth factor pression, while the second class may dictate, in part, (NGF). Contact with NGF causes PC12 cells to the nature of the response. growth arrest and assume a neuronal phenotype. 456 M. Mercola and C. D. Stiles

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