Embryonic
growth
factors
John K. Heath and Vicky Valancius-Mange1 University
of Oxford,
Oxford,
UK
The role of growth factors in development is under analysis on three main fronts: examination of patterns of growth factor expression in embryogenesis, studies of biological activity in vitro, and mutational analysis in viva. Recent findings indicate that growth factors control developmental decisions, are strictly controlled in their delivery to responding cells, and act in conjunction to create tissue-specific regulatory networks.
Current
Opinion
in Cell Biology 1991, 3:935-938
Introduction
knowing the identity of individual growth factor components.
Growth factors appear to be pivotal intercellular mediators in embryonic development. The evidence for this assertion is largely of three types. Firstfy, significant progress has been made in defining the spatial and temporal patterns of growth factor expression in developing systems, revealing remarkably complex patterns of expression, as well as the fact that the embryonic stages of development represent the major (or in some cases, sole) arena of activity of many growth factors. The second type of evidence has come from in vitro studies in which the action of exogenousty applied growth factors can be shown to reproduce, partially or completely, normal embryonic regulatory processes. The third category of evidence comes from the genetic analysis of growth factor expression and function through an examination of both existing developmental mutants and de nova mutations in genes encoding growth factors and their receptors, created by sequence-based genetic techniques such as gene targeting. Consideration of the current evidence also reveals some important features of the action of growth factors in a developmental setting. Firstly, growth factors are not necessarily mediators of cell multiplication; in an embryonic context they often (if not predominantly) act to control development decisions such as the control of altemarive pathways of differentiation. Secondly, the presentation and dispersal of control signals is a key issue in development; growth factors exhibit sophisticated mechanisms that control their state of activity and their delivery to responding target cells. Finally, growth factors exist in regulatory control circuits, whereby their expression, presentation and resulting cellular responses are controlled by the prior or contemporary action of other growth factors. In the end, unravelling the design of these regulatory circuits will probably prove to be more important for understanding developmental control processes than
Anatomical
studies of growth factor expression
The availability of cloned growth factor cDW and corresponding antibodies has permitted a detailed analysis of the spatial and temporal patterns of expression of a rapidly increasing number of growth factors in the developing embryo in vim Taken as a whole, these data reveal that many growth factors exhibit complex, but characteristic anatomical patterns of expression and that most are expressed in multiple cell types and at a variety of developmental stages. An individual growth factor may therefore be active in a number of different contexts. This has been illustrated strikingly by comparison of the expression of members of the transforming growth factor (TGF)-P superfamily of growth factors. TGF-@s 1, 2 and 3 are encoded by separate genes but share significant structural homology and in many respects appear to have qualitatively similar biological activities in most in vitro assays. Nevertheless, in situ hybridization studies [ 1*,2*,3*] show that they have distinctive patterns of expression in vim These include overlapping expression at sites such as the whisker follicles, and complementary patterns of expression in, for example, the lung (where TGF-Pl is expressed in the mesenchyme, and TGF-fJ2 and -3 have mutually distinct patterns of expression in epithelia). Situations also exist in which only one form of TGF-P appears to be expressed in a developing tissue, an example being the transient expression of TGFp2 observed in the developing nervous system. At first sight the most obvious conclusion to draw from these findings (which is not self-evident from in vitro studies) is that each TGF-P family member has distinct biological functions that can be correlated with different types of developmental activity. Nevertheless, it is
Abbreviations BMP-bone morphogenetic protein; FGF-fibroblast growth factor; bFC&basic FCF; MC&mast cell growth factor; OZA-oligodendrocyte-type 2 astrocyte progenitor; PDCF-platelet-derived growth factor; H-Patch; S/-Steel; SP-Steel-Dickie; TGF-transforming growth factor; W-Dominant White Spotting.
@ Current
Biology Ltd ISSN 0955a74
935
936
Cell differentiation
worth bearing in mind that cellular responses may be programmed in unique ways, as much by specific combinations of different growth factors as by unique properties of individual growth factors. For example, parallel studies of the expression of bone morphogenetic protein (BMP)-2 and BMP-4 [4*,5*], which are more distant relatives of the TGF-P family, reveal significant domains of co-expression with specific TGF-P family members in locations such as the heart. This suggests that interactions between TGF-fl superfamily members may occur in rkm and that the precise combination of factors expressed in a tissue may be more biologically significant than the expression of any individual member. Thus, different TGF-j3 family members may indeed be functionally redundant and their association with different types of developmental events might depend upon the co-expression of other factors.
Growth
factors
and development
in vitro
The diversity of biological effects that growth factors can exert on the differentiation of many cell types in vitro occupies an expanding area of the embryological literature. Such studies can be misleading, as interpretation of the biological behavior of cloned cell lines depends on the available means of recreating in culture a faithful representation of the cellular context in z~it~o. On the other hand, studies of tissue fragments pose the contrary problem, which is an inability to accurately control the endogenous expression and presentation of other growth factors in the material under study. In vitro studies can be particularly revealing in exposing novel aspects of growth factor action with relevance to normal development. One example of a moderately well characterized in vitro growth factor-mediated differentiation system is seen in the ability of exogenously added members of either the fibroblast growth factor (FGF) family or the activin family to reproduce the effects of normal vegetal hemisphere cells in the induction of mesodermal differentiation and axial patterning in the animal hemisphere of the pre-gas trulation Xenopus fuevB embryo. One of the unanswered questions in this area, however, is the mechanism by which a complex set of cell types forming a spatially organized structure can be generated by the action of a seemingly restricted number of signals (reviewed in [6] ). Even if FGF and activin-like factors proved to be expressed in a spatially heterogeneous manner (which is not clear), it still remains to be determined how such spatial heterogeneity in a signal could be interpreted into multiple and discrete domains of differentiation. Theoretical models of developmental processes often emphasize the need for threshold responses to external signals as a means of generating spatial heterogeneity in patterns of gene expression. This supposes that cells can somehow ‘interpret’ a continuous quantitative distribution of growth factor concentration into qualitatively discrete biological responses. That responses to growth fac-
tor signals can apparently exhibit ‘threshold-like effects’ has been shown in the Xenopusmesoderm induction system by Green and Smith [7*]. Exposing reaggregated animal pole cells to varying concentrations of activin A and examining the expression of a set of genes that exhibit regionalized expression in the normal embryo revealed a striking threshold effect on the induction of marker gene expression. Changes in activin concentration as small as 1.5-fold were observed to induce discrete changes in the combinations of marker genes expressed. Two further noteworthy features were revealed by this study. Firstly, the threshold responses of animal pole cells to activin A operate over a much narrower concentration range than that observed for another mesodenn inducer, Xenopus basic FGF (bFGF) (J Smith, personal communication), suggesting that the values’ of the thresholds are defined by the identity of the ligand and its cognate receptor(s). Secondly, whereas only two classes of signals have so far been found to act in the mesoderm induction system, at least three distinct combinations of gene expression were generated. These considerations may point to the existence of multiple and mutually interactive activin receptor-signal transduction systems with differing affiities for the l&and. A limited number of signals can, in principle, generate multiple responses by a ‘combinatorial’ mechanism whereby growth factors acting in combination elicit responses that are distinct from each factor acting alone. An interesting example of such a mechanism comes from analysis of the bipotential oligodendrocyte-type 2 astrocyte progenitor (02A) cells from the rat optic nerve. Previous in vitro studies had established that, in the absence of growth factors, these cells prematurely execute a terminal differentiation programme, but the normal in zko timing of differentiation could be restored in vitro in the presence of platelet-derived growth factor (PDGF). Bogler et al. [8=] have now shown that simultaneous exposure of 02A cells to both PDGF and bFGF induces a programme of continuous self-renewal in the absence of differentiation, whereas exposure to bFGF alone results in premature differentiation of 02A cells, which continue to proliferate. The effects of specific combinations of growth factors in this system are therefore not readily predictable from the effects of each factor in isolation. This type of study indicates clearly that the integration of separate signals is a viable mechanism for generating heterogeneity of response, and cautions against the assumption that any individual growth factor plays a dominant determining function in a given tissue.
Growth
factor
genetics
Despite the evident importance of growth factors in controlling developmental processes in vitro and the abundant evidence for their expression in the developing embryo, to date there has been little direct evidence for the function of growth factors in controlling developmental processes in viva. In other words, in most cases we do not really know what growth factors actually do. This pic-
Embryonic
ture is beginning to change as genetic methods for directly analysing the phenotypic consequences of mutation in growth factor genes and their receptors becomes available. The genetic evidence arises from two sources. The first comes from analysing the molecular basis of naturally occurring mutations. Two ‘spotting’ mutations in the mouse, Steel (SO and Dominant W%iteSpotting ( L@, which have analogous phenotypic effects on the maturation of erythroid cells, melanocytes and germ cells, have arisen from mutations in the mast cell growth factor (MCF) gene and its receptor (c-kit), respectively. These are reviewed in detail by Besmer (this issue, pp 939-946). It is important to note from these studies that the identification of multiple alleles of both these mutations has proved to be particularly informative regarding the normal biological function of MCF and its receptor. A good example of this is the Iinding that one mutation in the Sl (MCF) gene, SteeCDickie(Sp), arises from a 4.0-kb deletion within the MCF gene, resulting in the inability of the gene to produce a membrane-associated form of the MCF protein. Despite this, the diffusible, non-membraneassociated form of the protein appears to be produced in equivalent amounts, and with equivalent biological activity, to the wildtype counterpart [9~“,10~~]. In other words the St” mutation seems to a&t the dissemination of the ligand rather than its biological activity. This, combined with heterozygous effects of Sf mutations, provides strik ing genetic evidence that the presentation of a growth factor signal can be of considerable importance for its biological function in uivo, as has been suggested from in vitro studies of other growth factors such as bFGF 1111 and leukemia inhibitory factor [ 121. The MCF receptor, c-kit, is located within 630 kb of the PDGF-a receptor (which binds all three forms of the PDGF dimer). Molecular analysis of another spotting mutation, Patch (Ph) (which is linked to the Wlocus), and large deletions in the vicinity of the Wgene, reveals that Ph mutations are associated with deletions in the gene encoding the PDGF-a receptor [ 13*,14*]. Although the current data do not prove beyond doubt that Ph and the PDGF-a receptor are allelic, it is of considerable interest that Ph m ice with homozygous deletions of the PDGF-a receptor die in midgestation with multiple and complex morphological defects, including abnormalities in the neural tube, fluid retention and crania-facial abnormalities. These findings may accordingly provide the first genetic evidence for the biological function in vivo of PDGF, and detailed molecular analysis of further Pb mutations is likely to be of considerable importance. The availability of naturally occurring and informative mutations of growth factors and their receptors is likely to be limited in mammals. The advent of reliable techniques for creating specified mutations in genes in situ by genetargeting approaches, and the determination of the phenotypes of these mutations by embryonic stem-cell technology, will consequently have a significant impact on the dissection of growth factor function in Ljivo. The wnt family of growth factors are an evolutionary conserved set of related genes encoding secreted polypep-
growth
factors Heath and Valancius-Mange1
tides that physically associate with the extracellular matrix; the prototype gene writ- 2 was Iirst identi6ed by genetic methods in Drosophila as being invoked in the specification of segment polarity. In situ hybridization studies of wnt-1 in the mouse revealed that expression was principally confined to the developing nervous system. In addition, over-expression of mouse wnt-1 in Xenopus embryos was found to be associated with duplication of the embryonic axis. Two groups [ 15*=,16**] have now initiated an investigation into the biological function of writ- 2 in the mouse by creating loss-of-function mutations of the wnt-1 gene. Homozygous mutant individuals exhibit severe malformations of the developing central nervous system, including an absence of the majority of the midbrain and parts of the mesencephalon. This observation is consistent with the writ-1 growth factor acting in the (direct or indirect) determination of the developing midbrain. Furthermore, the effects of wnt-1 deletion clearly extend into tissues which do not express wnt-1 itself, arguing for the participation of wnt- 2 in a regulatory system that extends beyond the physical limits of the wnt-1 signal. Moreover, there are no discernible phenotypic effects in a number of tissues in which wnt-1 is expressed, such as the hindbrain and spinal cord. These findings have been used to argue for a functional redundancy of wnt-1 in these tissues,which is perhaps compensated by functionally equivalent members of the writ- 1 gene family. Nevertheless, these studies clearly demonstrate that an individual factor may participate in quite different control circuits in different tissues.
Conclusion Models and ideas on growth factor action derived from in oivo expression patterns and in vitro functional assays are beginning to inIluence the design of genetic studies. In particular, ‘threshold’ effects of growth factor concentration, the existence of regulatory networks and the biological significance of ligand presentation each have distinctive and testable features amenable to biologically relevant genetic analyses. It is worth remembering, nevertheless, that loss-of-function mutations are not necessarily the most informative class of genetic modiIications for many purposes, and the future application of more sophisticated methods for site-directed mutagenesis of growth factor genes in situ will prove to be a key step in evaluating the significance of the concepts described above.
References
and recommended
reading
Papers of special interest, published within the annual period of review, have been highlighted as: . of interest .. of outstanding interest 1. .
PELTON RW. DICKINSXN ME, MOSES HL, HOGAN BLM: In
SITU
Hybridization Analysis of TGF-Beta-3 RNA Expression Dur-
937
938
Cell diirentiation ing Mouse Development: Comparative Stud& with TGFBeta-l and Beta-2. Dew@ment 1990, 110(2):6op620. see [PI.
see [lo**]. 10. BRANNANC, Lw
2. .
Dickie Mutation Encodes a c-Kit Ligand Lacking Transmembrane and Cytoplasmic Domains. Proc Nat1 Acud Sci USA 1991, 88:4671-4674. Two SNdieS [9**,10**] which provide genetic evidence for the significance of growth factor presentation in the MCF/c-kit system. 11. YAYON A, KLVXBRIJN M, ESKO JD, LEDER P, Ow?z DM: Cell Surface, Heparin-Like Molecules are Required for Binding of Basic Fibroblast Growth Factor to its High Af?inity Receptor. Ceu 1991, 64:641+i8. 12. RATHENPD, TOTH S, WIUS A, HEA’rwJK. S~ml AG: Difkrentiation Inhibiting Activity is Produced in Matrix-Associated and Diffusible Forms that are Generated by Alternate Usage. Cell 1990, 62:110>1114. STEPHENSON DA, MERCOLA M, ANDERSON E, WANG C, STL~ESC, 13. . BOWEN-POPE D, CHAPMAN V: Platelet-Derived Growth Factor Receptor a Subunit Gene (Pdgfra) is Deleted in the Mouse Patch (Ph) Mutation. Proc Natl Acud Sci USA 1991, 886-10. see 114*]. L, WATSON MI CHOUDHURY 14. Sm EA, SELLXN MF, m
S~HMIDP, Cox D, BILEE G, hlrUE~ R, McM~STERGK: Differential Expression of TGF Beta-l Beta-2 and Beta-3 Genes During Mouse Embryogenesis. Develc@ment 1991, 111(1):117-130.
see I5.1. Muw F4 DENHEZ F, KOND~AH P, ,&HURST E Embryonic Gene Expression Pattern of TGF Beta-l Beta-2 and Beta-3 Suggest DifTerent Developmental Function In Vfva Development 1991, 111(1):131-144. see (5.1.
3. .
4. .
KM, PELTONRW, HOCANBL Organogenesis and Pattern Formation in the Mouse: RNA Distribution Patterns Suggest a Role for Bone Morphogenetic Protein-U (BMPU). Devekpment 1990, 109(4):833-&N see I5.1. LYONS
CM, LYONS KM, HOCAN BLM: lnvohrement of Bone Morphogenetic Protein-4 bmp4 and vgr-1 in Morphogenesis and Neurogenesis in the Mouse. Development 1991, 111(2):531-542. Five detailed papers [ l*-5*] describing the expression of TGF-b superfamily members by in siru hybridization techniques. 5. .
JONES
6.
MELTON Science
DA: Pattern Formation During Animal Development. 1991, 252:234-241.
7. .
GREEN JBA, Sm
JC: Graded Changes in Dose of a Xenopus Activin A Homologue Elicit Stepwise Transitions in Embry onic CeU Fate. Nature 1991, 347391-394. Direct experimental evidence for threshold mechanisms in the inter. pretation of growth factor signals.
8.
BOGLER0, WREN D, BARNETTSC, Lum H, NOBLE M: Ccqeration Between Two Growth Factors Promotes Extended Selfrenewal and lnhibits Differentiation of Oligodendrocytem 2 Astrocyte (0-U) Progenitor Cells. Proc Nat1 Acad Sci USA 1990. 87:63&372. A clear example of the combinatorial effects of growth factors on cell dilferentiation and multiplication. .
9. ..
JG, CHAN DC, IEDER P: Transmembrane Form of the Kit Ligand Growth Factor is Determined by Altemative Splicing and is Missing in the Sld Mutant. Cell 1991, 64:102F1035.
..
.
SD, WILLIAMSDE,
DM, COSMAN D, BEDELL m
E~SENMAN J, ANDERSON JENKINS N, COPELAM) NG: Steel-
GG, IAUEY PA, PIERCE J, AARONSON S, BARKER J, NAYLOR SL SMGUCHI AY: Mouse Platelet-Derived Growth Factor Re-
ceptor a Gene is Deleted in ~19” and Patch Mutations on Chromosome 5. Proc NatlAcud Sci USA 1991, 88:4811-4815. Two papers [ 13*,14*] revealing that deletions of the PDGF-a receptor are associatedwith the Ph developmental mutation. 15. MCMAHON AP, BRADY A: The Wnt-1 (int-1) Proto-oncogene .. is Required for Development of a Large Region of the Mouse Brain. Cefl 1’999,62:1073-1085. see [16**]. 16. THohw KR, CAPECCHI MR: Targeted Disruption of the .. Murine int-1 Proto-oncogene Resulting in Severe Abnormalities in Midbrain and Cerebellar Development. Nature 1990, 346:847-850. Creation and analysis of homozygous loss-of-function mutations of the wnl- 1 gene, revealing a role for writ-I in central nervous system devel-
opment (see also [ 15**] ).
WG~
JK Heath and V Valancius-Mangel,CRC Growth Factor Group, Depart. merit of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QLI, UK.
growth
factors
John K. Heath and Vicky Valancius-Mange1 University
of Oxford,
Oxford,
UK
The role of growth factors in development is under analysis on three main fronts: examination of patterns of growth factor expression in embryogenesis, studies of biological activity in vitro, and mutational analysis in viva. Recent findings indicate that growth factors control developmental decisions, are strictly controlled in their delivery to responding cells, and act in conjunction to create tissue-specific regulatory networks.
Current
Opinion
in Cell Biology 1991, 3:935-938
Introduction
knowing the identity of individual growth factor components.
Growth factors appear to be pivotal intercellular mediators in embryonic development. The evidence for this assertion is largely of three types. Firstfy, significant progress has been made in defining the spatial and temporal patterns of growth factor expression in developing systems, revealing remarkably complex patterns of expression, as well as the fact that the embryonic stages of development represent the major (or in some cases, sole) arena of activity of many growth factors. The second type of evidence has come from in vitro studies in which the action of exogenousty applied growth factors can be shown to reproduce, partially or completely, normal embryonic regulatory processes. The third category of evidence comes from the genetic analysis of growth factor expression and function through an examination of both existing developmental mutants and de nova mutations in genes encoding growth factors and their receptors, created by sequence-based genetic techniques such as gene targeting. Consideration of the current evidence also reveals some important features of the action of growth factors in a developmental setting. Firstly, growth factors are not necessarily mediators of cell multiplication; in an embryonic context they often (if not predominantly) act to control development decisions such as the control of altemarive pathways of differentiation. Secondly, the presentation and dispersal of control signals is a key issue in development; growth factors exhibit sophisticated mechanisms that control their state of activity and their delivery to responding target cells. Finally, growth factors exist in regulatory control circuits, whereby their expression, presentation and resulting cellular responses are controlled by the prior or contemporary action of other growth factors. In the end, unravelling the design of these regulatory circuits will probably prove to be more important for understanding developmental control processes than
Anatomical
studies of growth factor expression
The availability of cloned growth factor cDW and corresponding antibodies has permitted a detailed analysis of the spatial and temporal patterns of expression of a rapidly increasing number of growth factors in the developing embryo in vim Taken as a whole, these data reveal that many growth factors exhibit complex, but characteristic anatomical patterns of expression and that most are expressed in multiple cell types and at a variety of developmental stages. An individual growth factor may therefore be active in a number of different contexts. This has been illustrated strikingly by comparison of the expression of members of the transforming growth factor (TGF)-P superfamily of growth factors. TGF-@s 1, 2 and 3 are encoded by separate genes but share significant structural homology and in many respects appear to have qualitatively similar biological activities in most in vitro assays. Nevertheless, in situ hybridization studies [ 1*,2*,3*] show that they have distinctive patterns of expression in vim These include overlapping expression at sites such as the whisker follicles, and complementary patterns of expression in, for example, the lung (where TGF-Pl is expressed in the mesenchyme, and TGF-fJ2 and -3 have mutually distinct patterns of expression in epithelia). Situations also exist in which only one form of TGF-P appears to be expressed in a developing tissue, an example being the transient expression of TGFp2 observed in the developing nervous system. At first sight the most obvious conclusion to draw from these findings (which is not self-evident from in vitro studies) is that each TGF-P family member has distinct biological functions that can be correlated with different types of developmental activity. Nevertheless, it is
Abbreviations BMP-bone morphogenetic protein; FGF-fibroblast growth factor; bFC&basic FCF; MC&mast cell growth factor; OZA-oligodendrocyte-type 2 astrocyte progenitor; PDCF-platelet-derived growth factor; H-Patch; S/-Steel; SP-Steel-Dickie; TGF-transforming growth factor; W-Dominant White Spotting.
@ Current
Biology Ltd ISSN 0955a74
935
936
Cell differentiation
worth bearing in mind that cellular responses may be programmed in unique ways, as much by specific combinations of different growth factors as by unique properties of individual growth factors. For example, parallel studies of the expression of bone morphogenetic protein (BMP)-2 and BMP-4 [4*,5*], which are more distant relatives of the TGF-P family, reveal significant domains of co-expression with specific TGF-P family members in locations such as the heart. This suggests that interactions between TGF-fl superfamily members may occur in rkm and that the precise combination of factors expressed in a tissue may be more biologically significant than the expression of any individual member. Thus, different TGF-j3 family members may indeed be functionally redundant and their association with different types of developmental events might depend upon the co-expression of other factors.
Growth
factors
and development
in vitro
The diversity of biological effects that growth factors can exert on the differentiation of many cell types in vitro occupies an expanding area of the embryological literature. Such studies can be misleading, as interpretation of the biological behavior of cloned cell lines depends on the available means of recreating in culture a faithful representation of the cellular context in z~it~o. On the other hand, studies of tissue fragments pose the contrary problem, which is an inability to accurately control the endogenous expression and presentation of other growth factors in the material under study. In vitro studies can be particularly revealing in exposing novel aspects of growth factor action with relevance to normal development. One example of a moderately well characterized in vitro growth factor-mediated differentiation system is seen in the ability of exogenously added members of either the fibroblast growth factor (FGF) family or the activin family to reproduce the effects of normal vegetal hemisphere cells in the induction of mesodermal differentiation and axial patterning in the animal hemisphere of the pre-gas trulation Xenopus fuevB embryo. One of the unanswered questions in this area, however, is the mechanism by which a complex set of cell types forming a spatially organized structure can be generated by the action of a seemingly restricted number of signals (reviewed in [6] ). Even if FGF and activin-like factors proved to be expressed in a spatially heterogeneous manner (which is not clear), it still remains to be determined how such spatial heterogeneity in a signal could be interpreted into multiple and discrete domains of differentiation. Theoretical models of developmental processes often emphasize the need for threshold responses to external signals as a means of generating spatial heterogeneity in patterns of gene expression. This supposes that cells can somehow ‘interpret’ a continuous quantitative distribution of growth factor concentration into qualitatively discrete biological responses. That responses to growth fac-
tor signals can apparently exhibit ‘threshold-like effects’ has been shown in the Xenopusmesoderm induction system by Green and Smith [7*]. Exposing reaggregated animal pole cells to varying concentrations of activin A and examining the expression of a set of genes that exhibit regionalized expression in the normal embryo revealed a striking threshold effect on the induction of marker gene expression. Changes in activin concentration as small as 1.5-fold were observed to induce discrete changes in the combinations of marker genes expressed. Two further noteworthy features were revealed by this study. Firstly, the threshold responses of animal pole cells to activin A operate over a much narrower concentration range than that observed for another mesodenn inducer, Xenopus basic FGF (bFGF) (J Smith, personal communication), suggesting that the values’ of the thresholds are defined by the identity of the ligand and its cognate receptor(s). Secondly, whereas only two classes of signals have so far been found to act in the mesoderm induction system, at least three distinct combinations of gene expression were generated. These considerations may point to the existence of multiple and mutually interactive activin receptor-signal transduction systems with differing affiities for the l&and. A limited number of signals can, in principle, generate multiple responses by a ‘combinatorial’ mechanism whereby growth factors acting in combination elicit responses that are distinct from each factor acting alone. An interesting example of such a mechanism comes from analysis of the bipotential oligodendrocyte-type 2 astrocyte progenitor (02A) cells from the rat optic nerve. Previous in vitro studies had established that, in the absence of growth factors, these cells prematurely execute a terminal differentiation programme, but the normal in zko timing of differentiation could be restored in vitro in the presence of platelet-derived growth factor (PDGF). Bogler et al. [8=] have now shown that simultaneous exposure of 02A cells to both PDGF and bFGF induces a programme of continuous self-renewal in the absence of differentiation, whereas exposure to bFGF alone results in premature differentiation of 02A cells, which continue to proliferate. The effects of specific combinations of growth factors in this system are therefore not readily predictable from the effects of each factor in isolation. This type of study indicates clearly that the integration of separate signals is a viable mechanism for generating heterogeneity of response, and cautions against the assumption that any individual growth factor plays a dominant determining function in a given tissue.
Growth
factor
genetics
Despite the evident importance of growth factors in controlling developmental processes in vitro and the abundant evidence for their expression in the developing embryo, to date there has been little direct evidence for the function of growth factors in controlling developmental processes in viva. In other words, in most cases we do not really know what growth factors actually do. This pic-
Embryonic
ture is beginning to change as genetic methods for directly analysing the phenotypic consequences of mutation in growth factor genes and their receptors becomes available. The genetic evidence arises from two sources. The first comes from analysing the molecular basis of naturally occurring mutations. Two ‘spotting’ mutations in the mouse, Steel (SO and Dominant W%iteSpotting ( L@, which have analogous phenotypic effects on the maturation of erythroid cells, melanocytes and germ cells, have arisen from mutations in the mast cell growth factor (MCF) gene and its receptor (c-kit), respectively. These are reviewed in detail by Besmer (this issue, pp 939-946). It is important to note from these studies that the identification of multiple alleles of both these mutations has proved to be particularly informative regarding the normal biological function of MCF and its receptor. A good example of this is the Iinding that one mutation in the Sl (MCF) gene, SteeCDickie(Sp), arises from a 4.0-kb deletion within the MCF gene, resulting in the inability of the gene to produce a membrane-associated form of the MCF protein. Despite this, the diffusible, non-membraneassociated form of the protein appears to be produced in equivalent amounts, and with equivalent biological activity, to the wildtype counterpart [9~“,10~~]. In other words the St” mutation seems to a&t the dissemination of the ligand rather than its biological activity. This, combined with heterozygous effects of Sf mutations, provides strik ing genetic evidence that the presentation of a growth factor signal can be of considerable importance for its biological function in uivo, as has been suggested from in vitro studies of other growth factors such as bFGF 1111 and leukemia inhibitory factor [ 121. The MCF receptor, c-kit, is located within 630 kb of the PDGF-a receptor (which binds all three forms of the PDGF dimer). Molecular analysis of another spotting mutation, Patch (Ph) (which is linked to the Wlocus), and large deletions in the vicinity of the Wgene, reveals that Ph mutations are associated with deletions in the gene encoding the PDGF-a receptor [ 13*,14*]. Although the current data do not prove beyond doubt that Ph and the PDGF-a receptor are allelic, it is of considerable interest that Ph m ice with homozygous deletions of the PDGF-a receptor die in midgestation with multiple and complex morphological defects, including abnormalities in the neural tube, fluid retention and crania-facial abnormalities. These findings may accordingly provide the first genetic evidence for the biological function in vivo of PDGF, and detailed molecular analysis of further Pb mutations is likely to be of considerable importance. The availability of naturally occurring and informative mutations of growth factors and their receptors is likely to be limited in mammals. The advent of reliable techniques for creating specified mutations in genes in situ by genetargeting approaches, and the determination of the phenotypes of these mutations by embryonic stem-cell technology, will consequently have a significant impact on the dissection of growth factor function in Ljivo. The wnt family of growth factors are an evolutionary conserved set of related genes encoding secreted polypep-
growth
factors Heath and Valancius-Mange1
tides that physically associate with the extracellular matrix; the prototype gene writ- 2 was Iirst identi6ed by genetic methods in Drosophila as being invoked in the specification of segment polarity. In situ hybridization studies of wnt-1 in the mouse revealed that expression was principally confined to the developing nervous system. In addition, over-expression of mouse wnt-1 in Xenopus embryos was found to be associated with duplication of the embryonic axis. Two groups [ 15*=,16**] have now initiated an investigation into the biological function of writ- 2 in the mouse by creating loss-of-function mutations of the wnt-1 gene. Homozygous mutant individuals exhibit severe malformations of the developing central nervous system, including an absence of the majority of the midbrain and parts of the mesencephalon. This observation is consistent with the writ-1 growth factor acting in the (direct or indirect) determination of the developing midbrain. Furthermore, the effects of wnt-1 deletion clearly extend into tissues which do not express wnt-1 itself, arguing for the participation of wnt- 2 in a regulatory system that extends beyond the physical limits of the wnt-1 signal. Moreover, there are no discernible phenotypic effects in a number of tissues in which wnt-1 is expressed, such as the hindbrain and spinal cord. These findings have been used to argue for a functional redundancy of wnt-1 in these tissues,which is perhaps compensated by functionally equivalent members of the writ- 1 gene family. Nevertheless, these studies clearly demonstrate that an individual factor may participate in quite different control circuits in different tissues.
Conclusion Models and ideas on growth factor action derived from in oivo expression patterns and in vitro functional assays are beginning to inIluence the design of genetic studies. In particular, ‘threshold’ effects of growth factor concentration, the existence of regulatory networks and the biological significance of ligand presentation each have distinctive and testable features amenable to biologically relevant genetic analyses. It is worth remembering, nevertheless, that loss-of-function mutations are not necessarily the most informative class of genetic modiIications for many purposes, and the future application of more sophisticated methods for site-directed mutagenesis of growth factor genes in situ will prove to be a key step in evaluating the significance of the concepts described above.
References
and recommended
reading
Papers of special interest, published within the annual period of review, have been highlighted as: . of interest .. of outstanding interest 1. .
PELTON RW. DICKINSXN ME, MOSES HL, HOGAN BLM: In
SITU
Hybridization Analysis of TGF-Beta-3 RNA Expression Dur-
937
938
Cell diirentiation ing Mouse Development: Comparative Stud& with TGFBeta-l and Beta-2. Dew@ment 1990, 110(2):6op620. see [PI.
see [lo**]. 10. BRANNANC, Lw
2. .
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JK Heath and V Valancius-Mangel,CRC Growth Factor Group, Depart. merit of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QLI, UK.
