Cell, Vol. 63, 675-662,

November

30, 1990, Copyright

0 1990 by Cell Press

From Gradients to Axes, from Morphogenesis to Differentiation Laurence Cell

Reid

One of the beauties of developmental biology is the panoply of systems that have been employed to address the question of how a multicellular organism is constructed. The recent EM60 symposium (Heidelberg, September 17-20) carried the ambitious title “The Molecular Biology of Vertebrate Development” and thus focused attention on studies in vertebrate systems and the progress in our understanding at a molecular level. The meeting was remarkably diverse, spanning most of the major fields of vertebrate developmental biology, but had some principal themes. l

l

The putative roles of homeobox-containing genes in vertebrate pattern formation was a recurrent topic. Knowledge of the domains of expression of these likely regulators of pattern has advanced to a stage such that the beginnings of a real molecular understanding of this process are not as remote as they once seemed. Striking advances have been made in understanding the roles of growth factors in differentiation and development. Strong links are now emerging between these factors and the ability of cell adhesion molecules and extracellular matrix molecules to participate in, and potentially regulate, morphogenesis and differentiation.

Pattern

Formation-Of

Mice and Flies

The Paradigm?! The problem of how regions of a developing embryo are specified has until recently been beyond the scope of analysis in mammals. Although there has been a rapid increase in our knowledge of the role of certain growth factors in definition of the primary axes of the developing amphibian, the most advanced understanding of early development and pattern formation exists for the fruit fly, Drosophila melanogaster. An insightful overview of the state of knowledge of early fly development by C. Niisslein-Volhard (MPI, Tiibingen) provided both a reminder of the power of classical genetics in such analysis and an invaluable framework for understanding the mammalian studies. Despite the profound differences between the early fly and vertebrate embryos, and their large evolutionary separation, recent molecular analysis suggests that the mechanisms for regulating pattern formation may actually be more homologous than could ever have been predicted. A major difference between the fly and vertebrate embryos is the fact that early fly development occurs in a syncytium as opposed to a cellular aggregate (blastula or blastocyst) in vertebrates. Four analogous, maternal regulatory systems function, independently, to ultimately position a transcription factor asymmetrically in the develop-

Meeting Review

ing embryo. Three contribute primarily to defining the anterior-posterior axis, while the fourth functions independently to define the dorsal-ventral axis (NiissleinVolhard). This asymmetry is amplified by a zygotic cascade of transcription factors with the result that significant determinative events have subdivided the embryo by the time of cellularization (reviewed in lngham, 1988). Subsequently the fly embryo develops a segmented body pattern, and the diversification of these segments is driven by the products of the homeotic selector genes. These are genes first identified by mutations, such as Bithorax and Antennapedia, that result in homeotic transformations of one segment to ano ther. The homeotic selector genes of Drosophila fall into two linked clusters (the Anrennapedia and Bifhorex complexes) on chromosome 3 (Figure 1). The products of the homeotic selector genes are transcription factors capable of both repression and activation of transcription. They share a region of 81 amino acids of homology, the homeodomain, which includes a helix-turnhelix motif similar to the DNA binding domain of prokaryotic transcriptional regulators. Although a homeodomain initially appeared to be specifically associated with proteins with roles as regulators of development, it now appears to be a generic, DNA binding domain present in many diverse transcription factors (reviewed in Hayashi and Scott, 1990). The targets of the selector genes are poorly defined, but their activation represents a transition from global regulation of body plan by the maternal and early zygotic products to differential specification of tissue types and ultimately body structures. In addition the products of these selector genes clearly interact at this time to define the domains of expression of both other selector genes and themselves (autoregulation) (reviewed in Ingham, 1988). The selector genes are expressed in restricted domains along the anterior-posterior axis. These domains include regions of the embryo that will eventually give rise to those body parts visibly affected by mutations in the genes. An intriguing observation is the fact that the ordering of their sites of action (and indeed domains of expression) along the developing embryo corresponds directly with their position in the cluster. The genes of the cluster are all transcribed in the same direction: those genes at the more 3’ end of the cluster function to define the more anterior regions of the fly (Figure 1; discussed in Lewis, 1978). Looking for the Players The realization that a conserved motif underlay the regulation of many important developmental events prompted the search for homologs of the homeotic genes in higher eukaryotes. Numerous genes containing homeoboxes of various degrees of conservation have been identified, both by homology searches and directly by function (as transcription factors). However, most attention from a developmental viewpoint has focused on the class of vertebrate homeobox (Hex) genes that are homologs of the Drosophila selector genes. The homology of such pro-

Cell 676

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Figure 1. Conservation Clusters of the Murine Homologs in Drosophila

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of Structure of the Hex Genes with their

Four clusters exist in both mice and humans, and each cluster is related to the fly cluster. The fly cluster has been separated into two sections during evolution. All the clusters show a correlation in their organization and the expression pattern of the component genes: genes at the more 5’end of the clusters have more posterior restrictions to their pattern of expression (see text for details). Genes at equivalent positions in different clusters are referred to as paralogs (e.g., Dfd, Hex-2.6, -7.4, -3.5, and -4.2). After a figure kindly supplied by Dr. R. Krumlauf.

49

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teins outside the homeodomain is limited (generally restricted to a small N-terminal region). However, within the extended homeodomain (which includes 6-10 amino acids N-terminal of the homeodomain itself) the homology is highly specific so that counterparts for each gene can be identified. A recent striking series of experiments suggests that the homologies may well be highly significant as the m.ammalian homologs of the Antennapedia and Deformed selector genes are able to perform at least some of the specific functions of the fly genes in the developing fly embryo (McGinnis et al., 1990; Malicki et al., 1990). Mammalian Hox genes fall into four clusters spread on separate chromosomes (see Figure 1). Specific homologs at an equivalent position in the clusters are referred to as paralogs. The clusters contain roughly similar numbers of Hox genes, and strikingly, the order of the Hox genes within the clusters matches that of their counterparts in the fly cluster (reviewed in Akam, 1989). More recently, the colinearity of position and domain of expression has also been shown to be conserved (see below). Expression Patterns-Nothing New under the Sun? The patterns of expression of the members of the mammalian clusters have been analyzed in detail by in situ hybridization in the developing mouse embryo. The principal anterior-posterior axis of the mouse arises at day 6.5, and by 8.5 to 12.5 days clear restricted patterns of expression of the murine Hox genes are apparent. They are expressed primarily in the central nervous system and the somitic mesoderm, and in both tissues the domains of expression of the individual genes essentially extend from a specific anterior limit to the posterior of the embryo. This anterior limit is not at the same point along the axis in the two tissues. In a manner that has clear analogy to the situation in flies, the relative position of the anteriormost margin of expression of a gene corresponds (with few exceptions) directly with its position within the cluster. The genes that lie in the more 5’ regions of the cluster have more posterior restrictions to their pattern of expression. These observations now appear to apply to each of the four defined murine clusters (Krumlauf, NIMR, London;

Duboule, EMBL; Gruss, MPI, G&tingen; reviewed in Kessel and Gruss, 1990). The homologous relationships between the fly and mammalian genes therefore include their clustering, the conservation of gene position within the cluster, and the colinearity of this position and the domain of expression. Although these conservations are highly intriguing, in no organism is their functional implication understood. They do suggest that the mechanisms of specifying regions along a developing axis may have been closely and unexpectedly conserved during evolution. However, the significant differences between the early Drosophila and vertebrate embryos imply that the regulatory hierarchy that precedes activation of the Hox genes must be very different in each organism. The conservation of the cluster structure suggests that there may be some form of regulation occurring over the whole cluster for which the relative gene positions are important. However, initial results with transgenic mice carrying Hox promoter fusions to reporter genes suggest that a large degree of appropriate spatial and temporal regulation may still be conferred upon a transgene that is inserted in the genome away from the cluster (Piischel et al., 1990; Whiting, NIMR, London). The expression patterns of the Hox genes have led to the extension of the idea of a combinatorial code to vertebrates (Gruss). The basic theory of combinatorial code, originally proposed by Ed Lewis, is that the fate of a group of cells is defined by the combination of homeotic selector genes that they are expressing (Lewis, 1978). The idea arose from the observations of the phenotypes of both gain-of-function and loss-of-function mutations of the Drosophila homeotic genes, and the fact that their domain of action was colinear with their position within the Bithorax complex. In fact, the observed patterns of expression in the fly suggest that cell fate is regulated in a hierarchical fashion: the gene with the most 5’ position in the cluster dominates specification of segment identity at any point where it is expressed. The patterns of expression of murine Hox genes actually correspond very closely to those envisaged by Lewis for the fly genes. It should be remembered that the obser-

Meeting a77

Review:

Vertebrate

Development

vations in mice have little functional basis as yet although ectopic expression of Hox-7.7, in regions more anterior than normal, results in partial posterior transformations of both the CNS and the prevertebrae (Kessel et al., 1990). This is analogous to the Drosophila homeotic gain-offunction mutations. In addition, regions of expression do not necessarily directly represent the functional domain of any given protein. Several of the murine paralogs have apparently similar anterior margins of expression in the hindbrain. However, in the prevertebrae there may be significant heterogeneity between the margins of expression of paralogs such that the potential exists to generate a gradation of regulatory complexity. There is no information to date on whether the mechanism of position specification might be combinatorial or hierarchical. Cellular Segregation-Compartments in Vertebrates? The sharply defined domains of expression of putative pattern regulators in mammals begs the question of the degree of likely segmentation or compartmentalization that exists in higher organisms. Compartments were first defined in flies as cell groups of polyclonal origin that were incapable of mixing once determined. They are considered to be a primordial unit of structure in fly development (Garcia-Bellido et al., 1979). The cellular basis for compartmentalization is poorly understood, although candidate mechanisms are emerging (see below). Although vertebrate somites have obvious morphological similarities to segments, the best evidence for true compartmentalization in vertebrates now exists for the chick hindbrain. The pattern of vertebrate neuromeres is superficially metameric, especially in the hindbrain (where the specific neuromeres are known as rhombomeres). That they might really represent vertebrate segments was further suggested by the patterns of neuron differentiation and nerve generation observed there (Lumsden and Keynes, 1989). It was also suggested by the pattern of expression of /@ox-PO (a zinc finger-containing protein) and the Hex genes in hindbrain. In both chick and mouse Krox-20 is expressed in only the third and fifth rhombomeres while Hox-2.9 is expressed in the fourth rhombomere (Wilkinson et al., 1989a, 1989b; Lumsden, Guy’s Hospital, London). With the exception of Hox-2.9, the margins of expression of the Hox genes in the hindbrain of the mouse have a two-segment periodicity to their margins of expression, as do the morphological patterns of the chick hindbrain (Wilkinson et al., 1989b; Lumsden and Keynes, 1989). More recently, experiments using marked cells have shown that, upon formation, rhombomere boundaries serve to restrict clones of cells to their rhombomere of origin. Therefore rhombomeres may actually represent true compartments (Fraser et al., 1990). Calling the Play-Retinolds aa Morphogens? How might the patterns of Hox gene expression arise? Clearly some form of positional information along the axis must underlie the pattern, but its nature is unknown. Indeed, there is still no described system in vertebrate development where the method of specifying positional information is proven.

Figure 2. Patterns of Expression Limb (Duboule, EMIL)

of ffox-4

Genes

in the Developing

(A) The domain of expression of the murine Hox-4.6 gene in the developing limb (Dolle et al., 1989). A frontal section perpendicular to the dorsal-ventral axis is illustrated. Expression extends from a defined margin to the tip of the developing limb bud. A, anterior: Po, posterior; D, distal; Pr, proximal. (6) The colinear progression of the margin of expression of four members of the Hex-4 cluster in the limb, relative to position in the cluster, is shown. All domains extend towards the limb tip as illustrated for Hox4.6 in (A). (C) Pattern of expression of chicken Hex-4.6 in control limb bud (left) and limb treated with bead of retinoic acid for 24 hr. A new domain of expression is induced by the implanted bead. See text for details.

Much attention in vertebrates has concentrated on the role of retinoic acid (RA) in specifying positional information. RA was initially shown to be capable of reproducing the effects of a morphogen in experiments on the chick limb. A graft from the posterior margin of a chick limb bud to the anterior margin of an intact bud can induce mirrorsymmetrical duplication of the bones and digits of the resulting limb. This effect is reproduced faithfully by implantation of a bead soaked in RA. RA therefore either mimics, or is, the chemical responsible for specification of structures along the anterior-posterior axis of the chick limb bud (reviewed by Brickell and Tickle, 1989). G. Eichele (Harvard Medical School) reviewed the evidence for the presence and asymmetric distribution of retinoids in the chick limb bud and presented data suggesting that the asymmetry may result from unequal rates of degradation. The implications of these data for the likely function of RA as a morphogen are presently unclear. The genes of the murine Hox4 cluster are also expressed in restricted, overlapping domains in the developing limb (Dolle et al., 1989; see Figures 2A and 2B). These domains extend from a defined margin to the tip of the limb. The margins of these domains lie along an axis which intersects both the proximal-distal and anteriorposterior axes: the order of these margins is again colinear with gene position in the cluster. Thus the mechanisms underlying both limb development and axis generation in

Cell a78

the trunk may be more similar than initially imagined (Dolle et al., 1989). The chicken homologs of the murine Hox-4 cluster have recently been isolated: their cluster organization is precisely conserved, and the sequences are very highly conserved. They are also expressed in a similar pattern in the developing limb of that organism (Duboule). However, if an RA-soaked bead is implanted in the anterior rim of the developing limb bud, the pattern of Hox gene expression can be altered within 24 hr of the implant. This treatment induces mirror-symmetrical duplications of limb structures and also expression of the 5’ genes of the cluster at the anterior edge of the limb, where they are not normally expressed (Duboule; see Figure 2C). This striking demonstration of coordinate disruption of both body plan and Hox gene expression provides a clear correlation between the asymmetrical patterns of expression of the Hox genes and the asymmetrical development of the limb. In addition to regulation of the pattern of expression with position in the cluster, there also appears to be temporal regulation of the cluster. Sequential activation of the genes along the cluster isobserved as the limb bud grows out (Dolle et al., 1989), and this may provide a mechanism for regulation of the proximal-distal axis of a limb. It has been hypothesized that the age of the dividing cells in the tip of the limb bud (progress zone) might be used as a positional cue (reviewed in Wolpert, 1989) and the observed sequential activation of Hox genes with time and outgrowth is consistent with this theory. Sequential temporal activation of the genes of the Hox-4 cluster is also observed in the mouse trunk (Duboule). Although the mechanism of sequential activation is not yet understood, it is interesting to draw an analogy with the human 8-globin gene cluster, where the order of the genes in the cluster correlates with, and is required for, correct temporal activation (Grosveld, NIMR, London). Of course the globins are not transcription factors, unlike the Hox genes, which have the capability to affect each other’s transcription and may therefore be the effecters of the sequential activation of expression. Evidence for regulation within the homeotic cluster is well documented in flies and is beginning to accumulate in vertebrate systems, although at this stage the studies are all in vitro (Mavilio, Milan; Zappavigna, EMBL). Exposure of the whole mouse embryo to RA is lethal between 5 and 8.5 days of development. However, exposure between 8.5 and 9.5 days causes transformation of posterior body structures toward a more anterior fate, resulting in additional thoracic, lumbar, and sacral vertebrae (Gruss). In contrast, treatment with RA after day 10.5 causes very precise homeotic transformations of several cervical vertebrae towards the form of their immediate posterior neighbor (Gruss). Clearly it will be of great interest to know the effects of these experiments on the pattern of Hox gene expression. If the theory of a combinatorial code and the experiments on regulation of Hox expression have in vivo relevance, then one would predict that the anterior transformations result from down-regulation of the most 5’genes of the cluster in their posterior domains of expression and the posterior transformations

from induction of the more 3’ genes in regions more anterior than their normal boundaries of expression. The first of these predictions is at least partially fulfilled by an observed down-regulation of Hox-4.5 by RA between days 8.5 and 9.5 (Gruss). Retinoic Acid and Hox Genes-In Vitro Models Much attention has focused on the ability of RA to modulate the expression of the Hox genes in embryonal carcinoma and stem cell lines. Preferential activation of genes at the 3’end of the Hox clusters is observed, that is, those genes whose domains of expression extend to the more anterior regions of the developing embryo. The most 5 genes do not respond to RA (and some are in fact downregulated), and there appears to be a gradient of sensitivity along the cluster in human, mouse, and Xenopus (Simeone et al., 1990; Krumlauf). Sequential activation of Hox genes along the cluster is observed with time of exposure to RA. The initial responses are not dependent on protein synthesis, suggesting that they are direct and therefore mediated by the retinoic acid receptors. However, subsequent responses are indirect, implying that some of the early targets of RA are involved in activation of the later ones (Simeone, CNR, Naples; Gruss; Krumlauf). These sequential responses in vitro have clear parallels to those described in limb and trunk in vivo (Duboule). The observed variation in sensitivity along the Hox clusters suggests that a gradient of RA in an embryo in conjunction with sharp thresholds of response could in principle result in the observed stepwise activation of expression of the Hox genes along the anterior-posterior axis. Although this is exactly the type of responses that the original positional information theories suggested (reviewed in Wolpert, 1989), it is worth bearing in mind that proven alternatives to this type of mechanism clearly exist for regional specification in flies. Growth Factors in Inductive Events-Modulating the Pattern In Drosophila a gradient of the transcription factor bicoid encodes positional information relative to the anterior pole in the early embryo (Driever and NQsslein-Volhard, 1988a, 1988b). However, to achieve subdivision of cell type specification, a system of threshold response(s) to the gradient is clearly required. The primary target of bicoid, hunchback, exhibits avery sharp transcriptional response above a specific concentration of bicoid (Driever et al., 1989; Struhl et al., 1989). The importance of the molecular characterization of bicoicf lay in the demonstration that a morphogen with graded positional effects really existed. However, the relevance of a cell-intrinsic signal, functioning in a syncytium, to specification of information in a cellular vertebrate system is clearly moot. Recent studies of the growth factors that appear to regulate axis formation in Xenopus suggest that similar responses can in fact be induced by an extracellular molecule. Early Xenopus development involves a series of inductive events, the first of which involves an interaction between the tissues of the animal and vegetal poles that in-

Meeting 079

Review:

Vertebrate

Development

duces the differentiation of mesoderm at the interface. Growth factors have been identified that will reproduce this induction to a large degree when incubated with an isolated cap (reviewed in Smith, 1989). This is best achieved with a combination of members of the fibroblast growth factor (FGF) and transforming growth factor (3 (TGF-p) families. FGF seems to specify the more ventral mesoderm, while a combination of TGFf3 and FGF specifies the more dorsal forms. However, these inductions do not require localized application of growth factors to the cap in vitro, and therefore some prepattern must exist in the embryo prior to the inductive event. The most potent member of the TGF-f3 family in this assay is actually activin, and activin 6 has recently been shown to be capable of inducing not only the dorsalization of the animal cap, but also a significant degree of anterior-posterior axis formation. The mRNA for the activin l3 chain is present in the blastula-stage embryo at the time when the organizing center of these axes (Spemann organizer) is acting to determine the dorsal-ventral axis. Activin B therefore has many of the properties expected of the Spemann signal (Thornsen et al., 1990). Activin A functions in the mesoderm-inducing assay, although its mRNA is not actually present in vivo until gastrulation. However, it has one remarkable and reassuring property for a putative morphogen. If the cells of the animal cap are dissociated and treated with activin A then the dose-response curve of actin gene expression shows a remarkably sharp response over a 50% change in concentration of activin. Equally intriguing is the precipitous drop in expression observed at slightly higher doses (Smith, NIMR, London; Green and Smith, 1990). This is the first demonstration in a vertebrate system that an extracellular factor (and likely morphogen) may be capable of regulation of gene expression in threshold patterns, analogous to bicoid, despite the requirement for intracellular signal transduction. This type of multiple threshold response is clearly a crucial part of subdividing the fates of adjacent groups of cells. Morphogenesis the Information

and Differentiation-Using

Definitions and Molecules Two important parameters of morphogenesis are the ability of related cells to cluster together (and of unrelated cells to remain apart), and the ability of particular cells to migrate along specific routes at defined times. Interactions between extracellular molecules regulate cell-cell and cell-substratum adhesion, and modulation of these interactions presumably underlies the above phenomena. Certain specific growth factors are capable of interacting with, and regulating expression of, cell adhesion molecules and it is intriguing that these are ones that have been shown to have profound influences on morphogenesis (see below). Two basic systems of adhesion exist in metazoans (reviewed in Edelman, 1988; Takeichi, 1990). One modulates cell-cell interactions and is effected by cell adhesion molecules (CAMS), while the other modulates cell-sub-

stratum interactions and is effected by cell-substratum adhesion molecules (SAMs). Two distinct classes of CAMS exist: those that rely on Ca2+ for adhesion (also known as cadherins) and those that are independent of Ca2+. With a few exceptions, adhesive interactions between the CAMS are homophilic. These interactions can mediate cell sorting in vitro; mixing of populations of fibroblasts that have been transfected with different specific CAMS results in rapid sorting of cells into homogeneous subpopulations on the basis of the CAM that they are expressing. Cell sorting is also achieved on a quantitative basis by cells expressing different levels of the same CAM (Edelman, 1988; Takeichi, 1990). The mechanical basis for cell migration may well ultimately lie in the connections of adhesion molecules with the cytoskeleton. The CAMS are large transmembrane proteins whose function requires both the extracellular domain and an intact cytoplasmic domain. Molecules termed catenins appear to mediate the cytopasmic connections of the cadherins. Three subunits exist (a, 0, Y) and provide a link to the cytoplasm (Ozawa et al., 1989). The complex of the catenins then appears to interact with actin and to be capable of modulating the formation of actin cables in the cytoplasm (Kemler, MPI, Freiburg; Takeichi). The description of a link between molecules regulating cell-cell, and cell-substratum, contacts and actin is at least a first logical finding in beginning to understand the role of extracellular interactions in regulating cell migration. The same intracellular contacts have recently been shown to be capable of inducing cell surface polarity (McNeil1 et al., 1990). The integrins are the cellular receptors for heterophilic interactions with SAMs in the extracellular matrix (ECM). The integrins are transmembrane heterodimers composed of subunits from the a and f3families (each of which has several members; reviewed in Hynes, 1987). They interact with the SAMs in the ECM including fibronectin, collagens, laminin, and proteoglycans. Adhesive Interactions-A Moving Dynamo The patterns of expression of particular CAMS are not tissue specific, although domains of expression can correspond to tissue territories and may then function in the formation of tissue boundaries. Modulation of the function of both CAMS and SAMs is likely to underlie the regulation of cell migration: regulation of either the levels, or the functionality, of these molecules may generate the instability that underlies migration. During migration of chick neural crest cells, substratum adhesion is maintained while cell adhesion is suppressed. Some appropriate CAMS are then reexpressed when the crest cells reach their final position and differentiate (reviewed in Thiery et al., 1990). However, there is still little knowledge of how migration might be controlled. Is there a gradient of substratum molecule along which a cell crawls and that orientates its migration? What are the mechanisms that allow a cell to attach and migrate simultaneously? During development of the neural retina, the pattern of expression alters from predominantly Ncadherin to R-cadherin (Takeichi). The patterns of cells expressing either

Cell 880

kind are complementary, suggesting that alteration of the repertoire of expression of specific cadherins is closely linked to the differentiative events occurring (Takeichi). A similar example comes from study of the neural plate where expression of E-cadherin in the progenitor epithelial sheet is replaced by N-cadherin as the structure begins to fold (Takeibhi). These alterations in expression patterns suggest that specific cell sorting and adhesion events are occurring during these differentiation events. The identification of multiple distinct cadherins in neural tissues suggests that they may contribute to cell sorting during neurogenesis; clearly, if valid, this paradigm is likely to expand to other tissues. J. P Thiery (CNRS, Paris) described recent analysis of a rat bladder carcinoma cell line, NET-II, that displays interesting regulation of CAMS by acidic FGF. Acidic FGF can induce a reversible transition of this cell line from an epithelial to mesenchymal state, causing a rapid and significant activation of motility and concomitant alteration of ceil adhesion properties. This latter is effected by a complete internalization of desmosomes within 5 hr of FGF administration. Clearly, growth factors can affect the cell surface properties and motile properties of cells in a rapid and specific fashion. Short-Range Cellular Repulsion All of the effects described above involve cell-adhesive mechanisms. However, it is clear that short-range cellrepulsive mechanisms could provide an equally potent method of guidance and cell interactions. Recently, attempts to analyze cell-repulsive effects have borne fruit with identification of molecular systems that define prohibited regions of axon outgrowth. These molecules are also cell surface glycoproteins although their primary structure has not yet been characterized. One was identified on the basis of its activity to repel axons of the temporal retina from the posterior tectum (Bonhoeffer, MPI, Tubingen) and a second is expressed in the posterior sclerotome and serves to guide axons from the neural tube through the anterior half of the somite (Keynes, University of Cambridge). Both of these molecules can also cause growth cone collapse in vitro (reviewed in Keynes and Cook, 1990). Therefore both positive and negative interactions occur between cells, and it seems reasonable that these negative interactions will also turn out to be widespread. Although study of negative interactions is in its infancy, its potential importance in contributing to our understanding of developmental events as diverse as axon pathfinding and compartmentalization is already clear. Growth Factors within the ECM-Differentiation Hot Spots A function of the ECM that has been well defined recently is its ability to sequester growth factors such as TGF-8 and basic FGF. This may be a method of generating localized supplies of growth factors and appears to be effected primarily by the proteoglycan components of the ECM. It may then be able to regulate the local supply of these growth

factors in such a way as to influence differentiation. The proteoglycan molecules of the ECM have diverse roles and adhesive properties. They differ from glycoproteins in the type of sugar chains that they carry and the fact that the sugar component is in large excess over the protein content (reviewed in Ruoslahti, 1989). A well-characterized example of modulation of growth factor function by the ECM comes from studies of TGF-81 and its interactions with the small proteoglycan, decorin. Overexpression of decorin in a transformed CHO cell line causes a significant change in morphology and suppression of transformation (Ruoslahti, La Jolla Cancer Research Foundation). This is due to sequestration of TGF81 (which is an autocrine growth factor for the cell line) by the proteoglycan and appears to occur through a specific interaction of the factor with the polypeptide core of the proteoglycan (Yamaguchi et al., 1990). Exogenous addition of decorin has a similar effect. Growth inhibition of the mink MvlLu cell line by TGF-81 can be partially relieved by the addition of decorin. Intriguingly, TGF-81 actually induces expression of decorin (Ruoslahti). The exact meaning of this induction is unclear. It may represent a negative feedback pathway for sequestering and eliminating TGF81. Alternatively, it may create a potential localized reservoir of TGF-81 by incorporation of decorin into the extracellular matrix. Homologs of decorin exist in Drosophila. The best characterized of these is Toll, a transmembrane protein that is likely to be the transmembrane receptor for the dorsal-ventral signal received from the follicle cells surrounding the developing egg (Hashimoto et al., 1988). Its extracellular domain is homologous to decorin, and it is interesting to speculate that a member of the TGF-8 family might therefore be the ligand for Toll, and thus the morphogen for the dorsal-ventral axis, based on the ability of decorin to specifically bind TGF-81. Sequestration of growth factors appears likely to be a general phenomenon. Basic FGF is known to associate specifically with the heparan sulfate components of the ECM. Recent data suggest that a soluble complex of basic FGF with heparan sulfate molecules might actually represent the active form of this molecule in vivo. Binding to free heparan sulfate molecules in solution is proposed to prevent interaction of the growth factor with related proteoglycans bound to the extracellular matrix (Flaumenhaft et al., 1990). Platelet factor 4 is specifically associated with the chondroitin-sulfate side chains of serglycin, the major proteoglycan present in platelets and mast cells, although the functional implication of this is presently unclear (Ruoslahti). These types of interactions may either activate or inactivate a growth factor and may provide a useful reversible attachment mechanism for localization of growth factors in vivo. This may provide a sophisticated method of regulation for cells such as those of the hemopoietic system. The fate of hemopoietic stem cells reflects the regime of growth factors present, although it is not known whether the factors regulate the decision or cause selection for particular cell types after a regulated or stochastic differentiation

Meeting 001

Review:

Vertebrate

Development

event. Progression down a particular lineage is then driven by modulation or loss of activity of the receptors for the growth factors that are inappropriate to that lineage (Dexter, Paterson Institute, Manchester). Hemopoiesis occurs as a result of avery intimate interaction between stem cells and the stromal cells. Heparan sulfate, which is the major glycosaminoglycan of the marrow stroma, can bind both GM-CSF and IL-3 and present them to hemopoietic cells (Roberts et al., 1988). This type of interaction may result in specific stromal microenvironments that dictate particular pathways of differentiation (Dexter). The murine White-spotting (W) and Steel (a) loci are known to play multiple roles in the development of hemopoietic stem cells. These loci encode a tyrosine kinase receptor and its cognate ligand, respectively: the receptor functions in the stem cells while the heavily glycosylated ligand is produced by the stroma and is suggested to bind to the ECM (reviewed in Witte, 1990). A. Bernstein (Mount Sinai Hospital, Toronto) presented data showing that the c-fms receptor may use signal transduction systems similar to Wand that the product of the microphthalmia (mi) locus functions in a signal transduction pathway downstream of both receptors. W, SI, and mi were all discovered by classical mouse genetics and represent a major success for this field. Differentiation of glial cells (oligodendrocytes, type I and type II astrocytes) in the rat optic nerve has been studied by isolation of the primary cells and analysis in vitro of the growth factors that regulate their differentiation. Oligodendrocytes and type II astrocytes arise from a common progenitor at very specific times in development, and these timings are reproduced in vitro (Raff, 1989). The oligodendrocyte pathway seems to be the default mode and the timing of this differentiation is regulated by the ability of the cell to respond to PDGF, which is produced by type I astrocytes. It is unclear why a cell that will become an oligodendrocyte loses its responsiveness to PDGF. A combination of ciliary neurotrophic factor and an unknown factor, which is associated with the ECM and apparently produced by mesenchymal cells, drives the differentiation of the type II astrocyte. Although it is not proven that the regulation is effected by these molecules in vivo, all three of the factors are present in the nerve (Raff, MRC, London). These examples demonstrate the role of growth factors and their interactions with the extracellular matrix in differentiation. It will be interesting to see the degree to which spatial regulation of differentiation by specific sequestrations of growth factors turns out to be a widespread mechanism. Furthermore, the sudden exposure to a different localized regime of growth factors might be a prime mechanism to halt or alter the progress of a migratory cell. Cell adhesion and ECM interactions may provide a link between pattern formation events and cellular differentiation. It is clear that CAMS and SAMs are part of the mechanics of morphogenesis. A very general scheme for consideration of this idea is presented in Figure 3 (Edelman, Rockefeller University). Two of the major questions

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CELL COLLECTION

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ALTER BINDING/ tllGRATlON

Figure 3. Interactions of Developmental Regulatory Growth Factors with the CAM and ECM Systems

Genes

Proven interactions are indicated by solid arrows. Interactions lated and under investigation are indicated by dashed arrows. figure kindly supplied by Dr. G. Edelman.

and postuAfter a

arising in this field now are the following: How are CAM and SAM expression regulated? And how do extracellular interactions between such molecules affect intracellular signal transduction and gene regulation pathways? Overview Diverse approaches to developmental problems are clearly illustrating the strong parallels that underlie fundamental processes in different vertebrate organisms. Advances ii-r gene disruption technology will allow a functional genetic analysis of many of the mouse genes described above. Some, such as the disruption of Wnf-1 (McMahon and Bradley, 1990; Thomas and Cappechi, 1990) have already yielded spectacular and powerful results; others will doubtless follow. Analysis of the likely role of retinoic acid in development continues rapidly and will be aided by disruption of the genes encoding its receptors. The caveat to this is the likely lethal, uninformative phenotypes of mutations in some interesting genes. Progress in the field of vertebrate development is likely to be slow, but the prospect of a real leap in our understanding appears imminent. Acknowledgments I would like to thank Drs. Cori Bargmann, Caroline Donnelly, Denis Duboule, Gerald Edelman, Peter Gruss, Eric Jorgensen, Rolf Kemler, Roger Keynes, Rob Krumlauf, Julian Lewis, Andrew Lumsden, Martin Raff, Erkki Ruoslahti, Jim Smith, Masatoshi Takeichi, Jean Paul Thiery, David Wilkinson, and Lewis Wolpert who read and corrected the original manuscript and offered very helpful criticism. I apologize to those investigators whose work is inadequately cited or cited only via review or collaboration. References Akam, M. (1999). /fox and HOM: homologous and vertebrates. Cell 57; 347-349. Brickell, opment.

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