Embryogenesis: a Question of Pattern

Journal of Experimental Botany, Vol. 44, No. 259, pp. 359-374, February 1993

REVIEW ARTICLE

Embryogenesis: a Question of Pattern KEITH LINDSEY 1 and JENNIFER F. TOPPING Department of Botany, University of Leicester, University Road, Leicester LE1 7RH, UK Received 11 August 1992; Accepted 9 November 1992

Key words: Plant embryogenesis, pattern formation, developmental mutants, gene expression.

INTRODUCTION The development of the plant embryo comprises two processes that are of fundamental interest to developmental biologists: first, the establishment of the precise spatial organization of the component cells derived from a single, fertilized egg cell—pattern formation; and second, the generation of cellular diversity within the developing embryo—cytodifferentiation. Both processes are tightly co-ordinated to create a recognizable morphological structure, many features of which are characteristic of embryogeny within a given species. A central question is, how are these processes regulated? Although most aspects of organogenetic development in plants are carried out in the post-embryonic phase of the life cycle (in contrast to the situation in animals), a study of the embryogenic pathway can nevertheless be expected to address the question of how differentiation and development are co-ordinated. In particular, we can study the role of cell division, the interactions of different cell types and tissues, and the expression patterns of specific genes and sets of genes during a developmental process which is relatively simple and well-defined morphologically, and which can be investigated both in vivo and in vitro. There are three broad questions that we can consider with particular reference to embryogenesis. The first relates to the timing and organization of cell patterning 1

To whom correspondence should be addressed.

© Oxford University Press 1993

and differentiation: at what point do cells become committed to their subsequent fate and how stable is this commitment? The second: what is the number and nature of the genes which determine the characteristic features of embryogenesis? And the third: what are the chemical and other environmental signals that modulate the expression of embryonic genes? While it will become clear to the reader that we are a very long way from assembling a complete picture of the processes that regulate embryogenesis, we aim in this article to illustrate both the principal features of the process and the current experimental approaches that are creating fresh insights, drawing on examples from both monocotyledonous and dicotyledonous systems. P A T T E R N IN E M B R Y O G E N E S I S We will consider patterning in developing embryos at three levels: (1) pattern in the organization of cells; (2) pattern in protein accumulation; and (3) pattern in gene expression. Pattern in cellular organization Embryogenesis in angiosperms is distinct from animal embryo development in several fundamental respects.

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ABSTRACT Embryogenesis in plants appears superficially to be a relatively simple process in terms of morphological development and it is the case that most organogenesis takes place in the post-embryonic phase of the life cycle. This apparent simplicity allows us to exploit embryogenesis as a model experimental system to study the relationship between gene expression and morphogenesis, which is arguably the most important question in developmental biology. However, we are only just beginning to describe changes in gene expression during embryogenesis and to identify regulatory networks. Recent work, exploiting transgenic techniques and the generation and characterization of mutants, points the way ahead in dissecting this developmental process at the molecular level.

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FIG. 1. (A) Generalized structure of an angiosperm ovule, showing the eight haploid nuclei, (B) Schematic representation of the angiosperm double fertilization, to form the diploid zygote (z) and the triploid endosperm (e).

formation in Arabidopsis (Mayer, Torres Ruiz, Berleth, Misera, and Jurgens, 1991, discussed below) and of the latter by the observed effect of mechanical pressure on the plane of cell division in cultured cells (Yeoman and Brown, 1971; Lintilhac and Vesecky, 1984). Cell wall cross-linking, mediated by wall-localized enzymes such as peroxidases, is also expected to modulate the rate and pattern of cell division (Fry, 1988). The reproductive cells of the adult plant derive from the shoot apical meristem, the origins of which are established early in embryo development as a consequence of differential rates and planes of cell division, rather than from a distinct germ line which is characteristic of animals. There is considerable variation in detail of the morphological aspects of embryogenesis between individual species (Steeves and Sussex, 1989), and we have chosen to be selective in this account, which therefore should be considered as illustrative. A model species that has been adopted for the study of the genetics of dicotyledonous plant development, including embryogenesis, is Arabidopsis thaliana. This is a member of the Cruciferae and is closely similar in its pattern of embryogenesis to Capsella bursa-pastoris, itself a model for anatomical studies of embryo development (Mansfield and Briarty, 1991; Schulz and Jensen, 1968). The key features of embryogenesis in these species are illustrated in Figs 2 and 3, and while this is a continuous process, distinguishable stages of embryo development have been described as proembryonic (Fig. 2 A - C ) , octant (Figs 2D, 3A), globular (Figs 2E-F, 3B), heart (Figs 2G, 3C), torpedo (Figs 2H, 3D, E) and cotyledonary (Figs 2i, 3F). Arabidopsis embryogenesis is rapid, being completed typically within 11 -12 d post-fertilization, and the mature, desiccated seed is formed within 14 d. Following fertilization, the zygote undergoes an asymmetric transverse division to form a relatively small apical cell and a larger basal cell. This establishment of polarity must be highly regulated, since it is a consistent feature in embryogenesis in many species and may be of some determinative function: the apical cell will become the embryo proper and the basal cell will form the suspensor, a structure which acts, at least in part, as a conduit for nutrients from the maternal tissues to the developing embryo and may also itself supply essential metabolites. The apical cell divides, first by two vertical divisions at right angles to one another and then by one transverse division, to form a structure of eight isodiametric cells (the 'octant' stage), in which the cells of the embryo proper are organized in two tiers (Figs 2D, 3A). The upper tier is destined to form the cotyledons and shoot apex, while the lower tier will develop into the hypocotyl. The boundary between the two tiers is termed the O' boundary (Tykarska, 1976, 1979). The embryo proper is seated on the hypophysis, the distal cell of the suspensor, which itself will ultimately form the root of the seedling. Even

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Following the transmission of the pollen tube down the style, a double fertilization occurs. The pollen tube contains two sperm cells (following a mitotic division of the generative cell) and enters the multicellular megagametophyte which contains eight haploid nuclei, derived from the megaspore within the ovule. One of the sperm cells fuses with the haploid egg cell to form the zygote while the other fuses with the diploid central cell (containing two polar nuclei) to produce a triploid endosperm (Fig. 1). During the subsequent development of the seed, interactions between the embryo and endosperm comprise an essential pathway of communication that ensures reproductive success and the importance of this interaction is indicated by the observation that mutants affecting endosperm development may result in defective seed, such as in the miniature-1 mutant of maize (Miller and Chourey, 1992). A further characteristic feature of plant embryogenesis is the establishment of structural polarity and of histodifferentiation in the absence of cell movement. This is achieved by a tightly-regulated control of the rate and plane of cell division, resulting in the formation of structurally distinct apical regions, representing precursors of the shoot and root meristems, and of relatively few cell types. This process can be considered to be a consequence of both genetic and biomechanical factors. The importance of the former is illustrated by the identification of single gene mutants of pattern

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Therefore, by the heart stage (3-4 d post-fertilization), the three fundamental tissues of the seedling, namely the 0'-.©J epidermis, ground tissue, and vascular tissue, have been T}"7 oj* oJ o °_y established (Mayer et al., 1991), albeit in a preliminary O o form and which, in the mature plant, comprise many cell o O o , , types which have not yet differentiated in the embryo. o i o O o Furthermore, heart-stage embryos may begin to accumua i \ I \ late chlorophyll and axial elongation continues through o o o / the torpedo stage (3000 cells; 4-5 d post-fertilization) to generate a more clearly-defined hypocotyl and cotyledons which eventually, and in Arabidopsis characteristically, fold back in the cotyledonary stage (Figs 2i, 3F). The mature embryo comprises approximately 20000 cells in a seed which is approximately 500 /*m in length, but the shoot apex is relatively undeveloped and leaf primordia are absent. The development of the endosperm in Arabidopsis has been studied in detail by Marsden and Meinke (1985) and, in particular, Mansfield and Briarty (1990a, b). Following the fusion of sperm cell and central cell, the primary endosperm nucleus undergoes division almost immediately. Commonly in Angiosperms, the central cell is diploid, following fusion of two polar nuclei, and the endosperm nucleus is triploid. The second round of endosperm nuclear divisions are synchronous, but thereafter, asynchronous, and subsequent divisions proceed in the absence of the formation of cell walls, until the embryo proper has developed as far as the early heart stage. One nucleus, a product of an early division, migrates to the chalazal end of the embryo sac, while FIG. 2. Schematic representation of embryogenesis in Arabidopsis another migrates to the micropylar end, adjacent to the thaliana and Capsella bursa-pastoris. ( A - C ) Proembryonic stages; (D) octant stage, showing the position of the so-called 0 ' boundary; zygote. Two other nuclei become located on the longitud(E, F) globular stage; (G) heart stage; (H) torpedo stage; (i) cotyledonary inal walls of the embryo sac, and each divides in situ to stage. The dotted line represents the relative position of the embryo form a layer of endosperm on the wall of the embryo proper. sac. During this period, the coenocytic endosperm is described as 'free-nuclear' (Fig. 4A), and by the time the at this stage, therefore, the relationship between the embryo proper is 100-130 cells in size, the endosperm position of cells in the proembryo and in the seedling is may comprise over 200 nuclei (Marsden and Meinke, established. A further differentiation of cells occurs 1985). By this stage, the embryo is embedded in during the development of the 16-cell embryo, as anticlinal micropylar endosperm tissue. At the chalazal end of the divisions lead to the formation of the protoderm, an embryo sac, in contrast, the free-nuclear endosperm is outer layer of (initially) 8 cells. These cells continue to highly vacuolate and comprises only 10-15 nuclei. During divide in the anticlinal plane as the more central cells this coenocytic phase of endosperm development, the divide both longitudinally and transversely to create, in absence of organized cell divisions is reflected in a Arabidopsis and Capsella, regular tiers. The protoderm random orientation of microtubules and a lack of eventually constitutes the seedling epidermis. During the preprophase bands, in contrast to the organized cytoskeleglobular stage of development, which occurs approxital structure in the embryo proper (Webb and Gunning, mately 2-3 d post-fertilization, cell division proceeds to 1991). Cellularization of the endosperm is initiated first generate an embryo of approximately 30 cells, which is in the micropylar region at about 3-4 d post-fertilization approximately 40 fim in diameter. Subsequent develop(early heart stage; Fig. 4B), but this process is delayed in ment of heart-stage embryos (approximately 250 cells) the chalazal region (Fig. 4c), to approximately 5-6 d involves both the establishment of a bilateral symmetry post-fertilization (early cotyledon stage). At maturity, the to produce distal lobes that constitute the precursors of endosperm comprises only a very small proportion of the the cotyledons and also early stages of the differentiation seed, in contrast to 'endospermic' seeds in graminaceous of cells to generate zones distinguishable as the future species. vascular and ground tissues, respectively. B

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The suspensor in Arabidopsis is a relatively simple structure, comprising at the octant stage a large cell, the hypophysis or basal cell on which is seated the embryo proper, and a distal file of five to seven cells (Figs 2D, 3A). The suspensor begins to degenerate at the torpedo stage, a process which continues as the embryo develops. In other species, the organization of the suspensor is much more complex and may comprise many more cells and form haustoria-like structures, buried in the maternal tissues of the seed (Steeves and Sussex, 1989). The question of the role of the suspensor is an interesting one. The presence of the suspensor during early embryogenesis implies a dependence on it by the young embryo. It has been considered in the past to play a simple structural role in maintaining the embryo in a position which facilitates access to nutrients in the endosperm. However, this is likely to be an over-simplistic interpretation and the presence of haustoria-like projections suggests a role in the active transport of metabolites from maternal to embryonic tissues.

More information on the interaction of embryo and suspensor derives from tissue culture experiments, in which the two structures can be physically separated. For example, Yeung and Sussex (1979) have observed that the capacity for cultured zygotic heart-stage embryos of Phaseolus coccineus to develop into plantlets was much improved if the suspensor was either left attached to, or detached but brought into physical contact with, the embryo. When suspensors were placed 1 cm away from the embryos, the frequency of plantlet regeneration was diminished. However, this effect of the suspensor was itself reduced when the experiment was carried out with more mature embryos. One interpretation of this is that the suspensor may produce one or more metabolites upon which the developing embryo depends and one candidate suggested by Steeves and Sussex (1989) is gibberellic acid. It seems possible that the suspensor, therefore, plays a nutritional or hormonal role, in early embryogenesis and this role is taken over by the endosperm as the suspensor degenerates and the seed

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Fio. 3. Stages in zygotic embryogenesis of Arabidopsis thaliana. (A) Octant stage (x400); (B) globular stage (x400); (c) heart stage (x400); (D) early torpedo stage (x200); (E) later torpedo stage (x200); (F) cotyledonary stage (x 100). (D, E) Stained with methylene blue. In (B) and (c) the suspensors were lost during isolation.

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matures. One question mark over this possibility is raised by the observation that somatic embryos typically do not have suspensors, yet may regenerate into plantlets at high frequency. It may be, however, that the culture media and/or the non-embryonic cultured cells, provide an adequate supply of metabolites essential for embryo maturation, which in vivo are provided by the suspensor and other maternal tissues, but the chemical signals involved remain to be defined. Some progress in this has been made, however, and is discussed later in this review. Some insight into other developmental interactions between the suspensor and embryo have been obtained by studying embryonic-lethal mutants of Arabidopsis which fail to develop beyond the globular stage (Marsden and Meinke, 1985). In these seeds, it was found that, while embryogenesis per se was attenuated, the development of the suspensor was characterized by an aberrant proliferation of component cells. One hypothesis presented by the authors is that, in the wild-type seed, the developing embryo exerts an inhibitory effect on cell division in the suspensor beyond the globular stage, but in at least some mutants blocked at this stage, a presumed diffusible inhibitor is not synthesized and cell division in the suspensor is free to continue. This example illustrates the power of a genetic approach to study not only morphogenesis but also communication systems between tissues, and such an approach will be discussed in more detail below. B Embryogenesis in grasses, as exemplified by maize, has a number of features that are in contrast to those in dicotyledonous species such as Arabidopsis (Sheridan and Clark, 1987). The mature maize embryo is much larger, weighing up to approximately 70 mg in a seed which may be 8 mm long and 200 mg fresh weight. This is reflected in the long time necessary before embryo maturation is reached, namely about 40-50 d post-fertilization. Not only size, but also morphological complexity distinguishes the maize embryo (Fig. 5); the shoot apex is relatively large, several (five or six) true leaf primordia are present before germination and an endosperm is present in the mature seed. During the early stages of embryogenesis, however, there are certain similarities with Arabidopsis. The first division of the zygote is, as for Arabidopsis, asymmetric, producing a small apical cell and a larger basal cell (Fig. 5A). The apical cell divides vertically (Fig. 5B), but subsequent divisions are, unlike Arabidopsis, predominantly asymmetric and the result is a club-shaped embryo FIG. 4. Schematic representation of stages in endosperm development (Fig. 5c, D) comprising an 'integral' suspensor which is in the Arabidopsis thaliana embryo sac (after Mansfield and Briarty, distinguishable from the embryo more by a greater 19906). (A) Mid-globular stage; (B) mid-heart stage; (c) torpedo stage. vacuolation of its component cells than by its general (0) Free-nuclear endosperm; ( • ) cellular peripheral endosperm; (3) second cellular endosperm layer; ( 0 H) maturing cellular morphology: it does not comprise a single file of cells as endosperm; (H) degenerating cellular endosperm. seen in Arabidopsis, but is, like the embryo proper, the product of several asymmetric divisions. A peripheral protoderm is present by about 7 d post-fertilization

364 Lindsey and Topping—Embryogenesis A

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(Fig. 5D), but is not continuous around the embryo and does not, therefore, delineate a globular structure as in Arabidopsis. By about 9 d post-fertilization, the first signs of intra-embryonic differentiation are observable as a region of densely cytoplasmic cells towards one side of the embryo. Within two more days, this zone has differentiated further into two, clearly distinguishable groups of cells which represent the locations of the future shoot and root apices, at lateral and internal sites, respectively (Fig. 5E). Further divisions contribute to organized and, by about 20 d post-fertilization, relatively advanced shoot and root apices, with a relatively large suspensor at the lower side and a shield-like scutellum (Fig. 5F, G), which represent the single cotyledon characteristic of maize and related taxa. As in Arabidopsis, the early phase of endosperm development is coenocytic, but by approximately 4 d post-fertilization cellularization has been initiated. In maize and other cereals, the endosperm becomes differentiated by the formation of a specialized cell layer, the aleurone, in which hydrolytic enzymes are

Carrot (Daucus carota) cell suspension cultures have been exploited widely as a model system to study cellular and molecular aspects of somatic embryogenesis (Steward, Mapes, and Mears, 1958; Reinert, 1959; Krikorian and Smith, 1992; van Engelen and de Vries, 1992). Root explants from seedlings are induced to produce callus on a medium containing 2,4-D and suspension cultures are made from the callus. In such a culture, two types of cell can be identified: relatively large and vacuolate cells and smaller, more densely cytoplasmic cells. Only the latter type are embryogenic. It is not at all clear either where the embryogenic cells originate, or by what mechanism they assume embryogenic competence; but on transferral to a medium lacking growth substances they typically become organized as globular 'proembryonic masses' (PEMs) which, in turn, may develop into bipolar embryonic structures. This somatic embryogenic pathway is morphologically very similar to the analagous zygotic

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FIG. 5. Schematic representation of embryogenesis in maize, (A, B) Proembryonic stages; (c, D) globular stages; (E) initiation of shoot and root apices (hatched areas); (F) approximately 14 d embryo with lateral shoot apex and scutellum apparent; (G) mature embryo, sc, Scutellum; su, suspensor.

synthesized for the mobilization of storage products during germination. Embryogenesis in maize, therefore, contrasts with that in Arabidopsis in two major ways: the predominance of asymmetric cell divisions and the advanced extent to which the embryo, suspensor and endosperm develop in the seed. In both, however, there is a close co-ordination of the development of these tissues, recognizable under the microscope as a tightly organized patterning of cells. A further general feature that is common to both monocots and dicots is the developmental plasticity of somatic cells, such that, under appropriate culture conditions, explanted tissues or indeed protoplasts made from somatic cells may be induced to recapitulate the embryogenic pathway and reorganize into whole and phenotypically normal plants. The efficiency of this process of 'somatic embryogenesis' depends not only on the culture conditions, but also on the particular tissue used as explant material. For example, leaf mesophyll cells and protoplasts of alfalfa (Medicago saliva) can be induced to undergo embryogenesis without an intervening callus phase: such cells will divide in an unorganized fashion in the presence of an auxin such as NAA, but a transient exposure to 2,4-D will induce reorganized cell division and subsequent embryo development in a medium devoid of exogenous growth substances (Dudits, Bogre, and Gyogyey, 1991). As in the zygotic process, the first division is asymmetric. Cereal leaf tissues will not respond to these conditions, however, and successful somatic embryogenesis requires the explantation of immature embryos or immature inflorescences, which then are cultured in medium containing 2,4-D to generate embryogenic callus or derivative suspension cultures. Embryogenesis is induced, as for several dicot species, by transfer to an auxin-free medium (Vasil, Redway, and Vasil, 1990).

Lindsey and Topping—Embryogenesis 365

Pattern in protein accumulation Embryogenesis in both zygotic and somatic situations clearly involves a series of highly organized cell divisions to establish cellular pattern. At the biochemical level, we can detect a different type of patterning, namely comprising the co-ordinated synthesis and accumulation of proteins, lipids and carbohydrates in different parts of the developing seed and at different times during seed development. In this review, we will limit the discussion to proteins, but an excellent account of the other aspects of seed biochemistry may be found in Bewley and Black (1985). Seed proteins may be considered as comprising three broad groups, according to their function (Kreis, Williamson, Forde, Schmutz, Clark, Buxton, Pywell, Marris, Henderson, Harris, Shewry, Forde, and Miflin, 1986): enzymes, such as those involved in the mobilization of food reserves; structural proteins, associated with, for example, membranes and ribosomes; and storage proteins, which may be utilized as food reserves for the germinating seedling or may play a role in protecting the embryo during desiccation (the so-called 'LEA' or lateembryogenesis-abundant proteins). In cereal species, particular attention has been paid to the properties of the seed proteins, to a large extent because of their diverse commercial uses, such as in bread-making and as a source of essential amino acids (Miflin, Field, and Shewry, 1983). In the Gramineae, which yield the most studied seed proteins, the storage proteins (alcoholsoluble prolamins and salt-soluble non-prolamins or globulins) are accumulated predominantly in the endosperm rather than the embryo and in a temporallyregulated manner. For example, the barley hordeins begin to accumulate several days after the initiation of endosperm development and different classes of hordeins accumulate at different rates (Rahman, Kreis, Forde,

Shewry, and Miflin, 1984). Other barley seed proteins, such as the chymotrypsin inhibitors, are synthesized in the embryo as well as the endosperm, albeit under developmental control. In most dicot species studied, the endosperm degenerates during seed development, and the storage proteins accumulate in the embryo proper. In soybean (Goldberg, Barker, and Perez-Grau, 1989), Brassica (Blundy, Blundy, and Crouch, 1991), pea (Wang and Hedley, 1991) and bean (Bustos, Begum, Kalkan, Battraw, and Hall, 1991) for example, storage proteins typically accumulate only from mid-maturation phase onwards, when cell division is completed and the basic form of the embryo has developed. The pattern of protein accumulation within the embryo may also be spatially, as well as developmentally, regulated. In pea, for example, Corke, Hedley, and Wang (1990) used immunofluorescence techniques to demonstrate that the storage protein vicilin was most abundant in the cells on the adaxial surface of the cotyledons. There is also evidence that temporal and spatial patterns of protein accumulation characteristic of the zygotic embryo are recapitulated in somatic embryogenesis. For example Shoemaker, Christofferson, and Galbraith (1987) found that the pattern of protein synthesis, processing and accumulation in zygotic embryos of cotton (Gossypium hirsutum), which was initiated at the globular and early heart stages, was broadly similar in somatic embryos, though the proteins were detected at a slightly earlier stage (early globular) and in lower amounts. Similarly, lower protein levels have been found in somatic, compared with zygotic embryos of Brassica napus (Crouch, 1982). These observations, therefore, indicate that aspects of biochemical, as well as morphological differentiation, are at least qualitatively conserved in the absence of maternal tissues. Pattern in gene expression There is now good evidence that the synthesis of endospermic and embryonic proteins is controlled at the level of gene transcription and the organ- and cell-specific patterns of mRNA accumulation reveal further information on the regulation of the temporal and spatial patterning of seed proteins. In spite of the relatively simple morphology of the developing plant embryo, it is clear that a large number of genes is expressed during embryogenesis, perhaps as many as 20000-30000 (Dure, 1985; Goldberg et at., 1989). Many may comprise multigene families, as described for legumes (Casey, Domoney, and Ellis, 1986) and cereals (Kreis et al., 1986). Diverse patterns of mRNA abundance have been described (Fig. 6), and classes of transcripts can be recognized as being restricted to specific developmental stages of embryogenesis. The stage-specific synthesis of proteins has, in several instances, been demonstrated to shadow the abundance of the encoding transcripts, either by

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pathway, though, as indicated above, the somatic embryo typically lacks a suspensor. These observations are interesting from a number of points of view. They demonstrate that the commitment of cells to a particular pathway of organogenesis and the differentiation of those cells within a given organ system, do not involve the irreversible loss of function of essential genes in those cells, since they can be induced to carry out a complete structural reorganization and regeneration. It is also clear that embryonic cells may develop in a predictable way in isolation from any influence of the embryo sac. Thirdly, the establishment of in vitro embryogenic systems allows us to manipulate the chemical environment to study the embryogenic process and also to investigate the way in which the developing embryo may alter its own chemical environment by secreting specific metabolites. Some of these types of study will be described later.

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FIG. 6. Schematic representation of mRNA sets during seed development (after Goldberg el ah, 1989). Dotted areas indicate the timing of appearance and relative abundance of mRNA populations.

analysing the hybridization of cDNAs (encoding specific proteins) to RNA, or by the in vitro translation of RNAs purified from specific stages of embryogenesis. Transcriptional and post-transcriptional control has been described for the proteinase inhibitors of soybean (Walling, Drews, and Goldberg, 1986) and other legume storage proteins (Gatehouse, Evans, Croy, and Boulter, 1986), Brassica storage proteins (Blundy et al., 1991), and cereal storage proteins (Colot, Robert, Kavanagh, Bevan, and Thompson, 1987; Marris, Gallois, Copley, and Kreis, 1987), enzymes (O'Neill, Kumagai, Majumdar, Huang, Sutliff, and Rodriguez, 1990) and cell wall proteins (Ruiz-Avila, Ludevid, and Puigdomenech, 1991). In situ hybridization studies have been used to localize specific mRNAs to discrete tissues within the developing embryo. As indicated earlier, it is not clear at which stage cells in the embryo may either become committed to a particular fate, or what molecular mechanisms may determine that commitment, but it is possible that such in situ gene expression work may reveal molecular markers that characterize early events of cell determination and commitment to restricted developmental pathways. For example, Perez-Grau and Goldberg (1989) have carried out a detailed analysis of the localization of

A similar type of study has been made to localize mRNAs encoding the napin and cruciferin storage proteins in zygotic embryos of Brassica napus (Fernandez, Turner, and Crouch, 1991), revealing how differences in cell fate are characterized by differences in gene expression. During the late heart stage, napin transcripts accumulate in the cortex of the embryonic axis, in the outer margins of the cotyledons during the torpedo stage, and in the inner cotyledon margins during the cotyledonary stage. This pattern is somewhat reminiscent of that of soybean KTi3 transcripts. The pattern for the Brassica cruciferin mRNAs is similar, but delayed. During embryo maturation, napin mRNAs are localized throughout the embryo except the root apical region, with highest levels in the vascular tissue and shoot apex. An alternative approach to identify patterns of gene expression during embryogenesis is being used in the authors' laboratory. This strategy is one of 'promoter trapping', and relies of the activation of a promoterless gusA reporter gene by native gene promoter sequences following transformation and random integration (Topping, Wei, and Lindsey, 1991; Lindsey, Wei, Clarke, McArdle, Rooke, and Topping, 1993). It is expected that

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GERMINATION SPECIFIC

mRNAs corresponding to two different classes of Kunitz trypsin inhibitor storage proteins of soybean. The two classes, designated KTil/2 and KTi3, are expressed to different levels in the embryo (and also to lesser levels in other organs), with KTi3 the more highly expressed (Jofuku and Goldberg, 1989). During early embryogenesis, the two classes of transcripts exhibited distinct spatial patterns of expression. KTil/2 transcripts were not detectable in globular, heart or cotyledonary stage embryos, whereas KTi3 mRNA was localized at the microplar end of the globular embryo and in the axis of the heart and cotyledonary embryos, though not in the cotyledons themselves. In more mature embryos, the KTi3 transcripts were, in contrast, found in the cotyledons, and were observed to accumulate in a wave-like pattern. At 25 d after flowering (DAF), transcripts were localized to the outer margins of the cotyledons, but as the embryo matured to 70 DAF the transcripts became localized to the inner margins and eventually throughout both cotyledons, being found primarily in the storage parenchyma cells. By 80 DAF the pattern had changed again, with mRNA localized to the cotyledonary margins once more. In older embryos the KTil/2 mRNAs became detectable and, beyond 55 DAF, showed patterns of accumulation similar to the KTi3 transcripts, though at lower levels. When the KTi3 probe was used to localize transcripts in soybean somatic, rather than zygotic, embryos the pattern was found to be the same. This result indicates that, for this experimental system, the pattern of spatial and temporal expression was independent either of events associated with fertilization or of the presence of the embryo sac.

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FIG. 7. Localization of GUS reporter gene expression (blue coloration) in early- (A) and mid- (B) cotyledonary embryos, and the root of the mature plant (C), following activation of a promoter trap vector in a transgenic line of Arabidopsis thaliana. See text for details.

the directed expression of the gusA. gene, which can be localized histochemically in vivo, reflects the promoter properties of the tagged genes. Using this technique in Arabidopsis, it has been possible to identify a range of GUS expression patterns in developing zygotic embryos, each of which is specific to particular transgenic lines, and illustrates embryonic transcriptional control in space and developmental stage (Fig. 7). Using such an approach it is also possible to generate transgenic lines that are defective in specific aspects of the embryogenic process, due to the disruption of embryonically-expressed genes by T-DNA sequences (Errampalli, Patton, Castle, Mickelson, Schnall, Feldmann, and Meinke, 1991). The types of mutants that can be generated by this and by chemical mutagenic techniques will be considered below, in relation to the identification of genes that control specific facets of embryogenesis. GENES ESSENTIAL FOR EMBRYOGENESIS The principal question for developmental geneticists concerns the function of genes expressed during a process such as embryogenesis and, in particular, in the identification of genes that regulate specific aspects of development, rather than which are merely 'symptoms' of a process initiated by more critical events. Biochemical studies tell us that rapidly-dividing cells, such as are found in young embryos, are metabolically active, and that genes encoding enzymes involved in, for example, respiration, cell division and cell wall synthesis are expected to be actively transcribed. In this respect, many of the genes expressed

during embryogenesis would also be expected to be transcriptionally active in other meristematic tissues. For example, we have identified, by gene trapping and in situ localization techniques in Arabidopsis, a gene which is expressed in the basal region of the heart-stage zygotic embryo and also in the root meristem of the adult plant (Fig. 7; Topping and Lindsey, unpublished). Even now, there are still very few embryonically-expressed genes, other than those encoding some enzymes and storage proteins, that have been identified, isolated and characterized. One approach to investigate genes essential for the completion of embryogenesis that has proved to be extremely valuable, and which is expected to be even more fruitful in the future, is the generation and characterization of mutants. This has, to date, been achieved both through the application of chemical mutagens or ionizing radiations, notably with Arabidopsis as a model species (Meinke, 1986; Mayer et ai, 1991), but also in the commercially important pea (Wang and Hedley, 1991), or by exploiting the insertional mutagenic properties of naturally-occurring transposable elements in pea and, to a much greater extent, in maize (Sheridan and Clark, 1987; Clark and Sheridan, 1991). Meinke's laboratory has generated a very large number of embryonic-lethal mutants of Arabidopsis, which are blocked across the full range of developmental stages (Meinke, 1985). In view of the high frequency of lethality, it is expected that many of these mutants would be defective in one or more essential housekeeping functions. For example, embryo rescue experiments identified the

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4k,

368 Lindsey and Topping—Embryogenesis

FIG. 8. Schematic representation of the origin and final form of apical-basal pattern mutants (after Mayer el al., 1991). Genes that affect formation of the solid areas of the prospective seedling (left), mutate to generate the aberrant phenotype (right). Four types of mutant are represented (A-D, see text for details).

Using a chemical mutagenic strategy, Mayer et al. (1991) have identified mutants of Arabidopsis that are seedling lethal, rather than blocked in embryogenesis and which are defective in the establishment of tissue patterning and seedling shape. Since embryogenesis was capable of completion, such mutants were expected not to be due to lesions in housekeeping functions. Genetic complementation studies indicated that the aberrant phenotypes described were determined by nine genes, each represented by an average of eight mutant alleles. The mutant genes affected three distinct aspects of morphological organization, which were (1) apical-basal pattern, determined by four of the nine genes, and characterized by the absence in the seedling of either (a) the cotyledons and shoot apex, (b) the hypocotyl, (c) the root and hypocotyl or (d) the root and shoot regions (Fig. 8); (2) radial pattern, determined by two of the nine genes, characterized by aberrant development and organization of the epidermis, ground tissue and/or vascular tissue; and (3) seedling shape, determined by three of the nine genes identified and characterized by the seedlings retaining the major organs and tissues^but being nevertheless abnormal in shape, such as having large and rounded cotyledons, an altered number of cotyledons or a small and pale morphology. It was estimated that a relatively small number of genes, approximately 40 or so genes in total, may be sufficient to control pattern formation in the Arabidopsis embryo. This number is similar to the estimate for the control of patterning in the Drosophila embryo (St. Johnston and Nusslein-Volhard, 1992). A number of the lines exhibiting aberrant seedling phenotypes were also found to be abnormal in cellular patterning in the heart, globular and even pre-globular stages of embryogenesis, reaffirming the concept that the organization of organs and tissues in the seedling is determined, in part at least, by processes very early in development. These mutant studies demonstrate that it is possible to resolve the effects of genes that control the patterning of tissues, the control of cell division to elaborate morphological structures and the control of basic metabolic processes, each of which is essential for the successful completion of embryogenesis but subject to distinguishable regulatory mechanisms. In plants the nature and mechanism of action of such embryonic 'patterning' genes is unknown, although genes that control the organization of floral organogenesis in Arabidopsis and Antirrhinum, for example, have been identified as being transcription factors (Coen and Meyerowitz, 1991). This is also the case for some genes controlling the expression of animal embryonic pattern (St. Johnston and Nusslein-Volhard, 1992). It is possible that gene products that regulate floral and embryonic patterning may function through broadly similar mechan-

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lesion in one mutant as being in the biosynthetic pathway of biotin, a cofactor involved in CO2 transfer during carboxylation and decarboxylation reactions (Schneider, Dinkins, Robinson, Shellhammer, and Meinke, 1989). Other mutants are defective in embryo and seedling pigmentation (Meinke, 1986). Since these mutants were produced by chemical mutagenesis, it is quite difficult to isolate the respective mutant genes. Although of unknown function, sixteen of the ninety or so mutants isolated in Meinke's laboratory have been mapped in the Arabidopsis genome (Patton, Franzmann, and Meinke, 1991). A number of embryo-lethal mutants have also been identified in maize, either following screens of seed treated with chemical mutagens or following insertional mutagenesis by active transposons such as Robertson's Mutator. Two broad classes of embryonic mutant phenotype have been described in detail: those that comprise both defective embryo and endosperm, the so-called defective kernel (dek) mutants (Sheridan and Clark, 1987), and those that affect only the developing embryo, the embryospecific (emb) mutants (Clark and Sheridan, 1991). Like the Arabidopsis embryo lethals, both dek and emb mutants are arrested across a wide range of developmental stages. From a comparative analysis of different mutant lines, it has been possible to conclude that a large number of loci are responsible for the control of diverse aspects of embryogenesis, and that processes such as cell division and morphogenesis per se, such as the establishment of root versus shoot meristems, or of embryo shape versus endosperm shape, are regulated by sets of genes that can be distinguished readily.

Lindsey and Topping—Embryogenesis

CONTROL OF GENE EXPRESSION DURING EMBRYOGENESIS General considerations

A further fundamental question to be considered is, what determines the cell type-specific expression of embryonic

genes? One possibility proposed by Fernandez et al. (1991) is that the developmental history (lineage) of the cells may influence the gene expression pattern. In this study, they observed that Brassica napin mRNAs exhibited a restricted pattern (boundary) of accumulation that correlated with a boundary of cell divisions apparent in the early globular embryo, the O' boundary. As indicated earlier, this 'boundary' separates the two tiers of cells in the embryo proper (Fig. 2D), which eventually give rise to protoderm and ground tissues. The observed sharp boundary of napin expression in the derivative vascular and marginal cells, but not in the root apex in older embryos, was suggested to coincide, in part at least, with cells derived from the upper, but not lower tier of cells. It was further suggested that specific cells present in the upper part of the embryo, notably the 'epiphysis' (which comprises a group of cells three or four cell layers inside the embryonic shoot apex) contain factors that may determine the expression pattern of the storage proteins. This may represent a relatively stable form of compartmentalization of gene expression, to which specific cell types may be committed and the implication is that the biochemical or gene expression characteristics of particular cells or groups of cells within an organ may be determined at a relatively early developmental stage. With regard to the demarcation of napin gene-expressing cells in epiphysis- and hypophysis-derived cells, this determination may have been established as early as the first, asymmetric division of the zygote. One possible process, still poorly defined in plants, that could allow cell-specific expression patterns through cell division is the 'imprinting' of relatively stable changes to the structure of chromatin domains containing embryonically-active genes. Such an hypothesis has been established for animal embryonic systems and may account, in part at least, for the clonally inherited patterns of expression of homeobox genes in, for example, Drosophila and mouse (Gaunt and Singh, 1990). One possible mechanism for the stable modulation of gene expression in development is DNA methylation and, for example, an inverse correlation between maize seed storage protein gene expression and methylation in a range of tissues has been reported (Bianchi and Viotti, 1988). An alternative, but not necessarily mutually exclusive, view would be that it is the position of the cells within the embryo and so their spatial relationship with chemical or other signals produced by either neighbouring embryonic cells or surrounding tissues such as the endosperm, suspensor or other cells of the developing seed, that determines the pattern of gene expression, rather than the lineage or history of the cell per se. There are arguments in favour of this hypothesis, which has also been discussed widely in animal development: gradients of specific molecules, such as regulatory proteins (St.

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isms, but we must wait for more information before any serious conclusions can be drawn. Recent work on the control of carrot somatic embryogenesis has identified gene products that are essential for that process, but which do not appear to be either regulatory nor involved simply in the maintenance of primary metabolism. These are extracellular glycoproteins that are secreted into the culture medium, and their synthesis is inducible by the culture conditions that promote somatic embryogenesis (de Vries, Booij, Janssens, Vogels, Saris, LoSchiavo, Terzi, and van Kammen, 1988; van Engelen and de Vries, 1992). The functional importance of at least some of these glycoproteins was recognized by their ability to rescue temperature-sensitive mutant embryos that were developmentally arrested. To date, three types of extracellular carrot protein have been characterized, namely cationic peroxidases (Cordewener, Booij, van der Zandt, van Engelen, van Kammen, and de Vries, 1991), lipid transfer proteins (Sterk, Booij, Schellekens, van Kammen, and de Vries, 1991) and chitinases (de Jong, Cordewener, LoSchiavo, Terzi, Vandekerckhove, van Kammen, and de Vries, 1992). What roles might such molecules play in embryogenesis? Expression of a lipid transfer protein, EP2, was detected in the protoderm of both somatic and zygotic globular- and heart-stage embryos, but was found not to be embryo-specific, being found also in the shoot apex, developing inflorescences and, to a lower level, in mature seeds (Sterk et al., 1991). A maize lipid transfer protein mRNA has also been localized to peripheral cell layers in embryos and coleoptiles (Sossountzov, Ruiz-Avila, Vignols, Jolliot, Arondel, Tchang, Grosbois, Guerbette, Miginiac, Delseny, Puigdomenech, and Kader, 1991). It is suggested that the EP2 gene product may be involved in the synthesis of a cutin layer around the developing embryos, perhaps as part of a mechanism to modulate water uptake and hence cell size. However, an essential function for this gene in embryogenesis remains to be demonstrated. Both a carrot peroxidase and chitinase have, however, been demonstrated to have roles that appear to be essential for the maturation of arrested somatic embryos in vitro. It has been speculated by de Jong et al. (1992) that, although chitin is not a component of plant cell walls, the chitinase activity may in fact utilize a cell wall component as substrate and, like peroxidases (Cordewener et al., 1991) act to modify cell wall 'tightening' in order to regulate cell size and perhaps the plane of cell division, presumed to be an essential facet of morphogenesis in embryos.

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370 Lindsey and Topping—Embryogenesis

We have already discussed the evidence pointing to the suspensor as a source of nutrients or growth substances (Yeung and Sussex, 1979), which may act as inductive signals in development and gene expression. Furthermore, it is clear that by changing the chemical environment of embryonic tissues such as by culturing in vitro, the pattern of gene expression and developmental fate can be altered dramatically. Recent experimental work has demonstrated directly that the level of expression of a soybean auxininducible gene is correlated with an endogenous auxin gradient in plant tissues (Li, Hagen, and Guilfoyle, 1991). While this phenomenon was associated with a tropic response, it is possible that such a general regulatory system may operate in a developmental context. Whether such a response would be mediated by the differential availability of receptor molecules to generate differentially competent cells, is unclear. Other types of molecules than growth regulators are likely to be important signals and calcium, for example, is known to be associated, if not with the establishment of polarity, then with the maintenance of polarized growth in the developing Fucus embryo (Kropf and Quatrano, 1987), and to be an essential component of signal transduction/protein phosphorylation pathways in plants. Furthermore, it is becoming clear that the regulation of the phosphorylation of protein kinase complexes is a critical facet of cell division control (Cyert and Thorner, 1989; Kinoshita, Ohkura, and Yanagida, 1990). Whatever the specific mechanisms involved, the main feature of this general hypothesis of patterning rests with an emphasis on local (i.e. intercellular) communication systems to regulate the expression of specific genes, rather than merely a form of 'imprinting' of expression pattern, which may be established earlier in development. We would argue then that the establishment of cellular pattern is a primary determinant in embryogenesis, situated at or near the top of a develop-

mental cascade, but it seems quite likely that the regulation of spatial and temporal patterns of gene expression in development comprises both processes (i.e. imprinting and inductive signals), superimposed one upon the other, to provide on the one hand a stability of gene expression through repeated cell divisions and, on the other, an ability to respond to changing environmental conditions. A great deal of research is required to dissect these control mechanisms. Trans-acting and cis-acting regulators of embryonic gene expression It is apparent, therefore, that there is currently very little information on how embryogenesis and embryonic gene expression are integrated. We have discussed above how, in somatic embryogenesis, the manipulation of auxin in the culture medium is an essential part of the inductive process, but the causal relationship between growth substances and embryogenesis is not understood. It is known that abscisic acid (ABA) is involved in aspects of seed maturation, and the expression of some genes late in embryogenesis appears to be regulated by it (Quatrano, 1986). For example, ABA prevents precocious germination of the embryo in cereals, as demonstrated by the series of viviparous mutants in maize. These plants are deficient either in ABA synthesis or sensitivity to ABA (Neill, Horgan, and Parry, 1986) and, characteristically, produce embryos blocked in development that germinate while still on the parent plant (vivipary). The mutant gene Vp1, responsible for a reduced sensitivity to ABA, appears to encode a transcription factor that may act to potentiate an ABA-mediated developmental pathway in seeds (McCarty, Hattori, Carson, Vasil, Lazar, and Vasil, 1991). This pathway includes the transcription of late embryogenesis abundant genes, such as the Em gene of wheat, the transcription of which has been shown to be ABA-inducible (Marcotte, Bayley, and Quatrano, 1988; Marcotte, Russell, and Quatrano, 1989). The promoters of a number of seed-expressed genes have been studied in order to identify sequences essential for tissue-specific expression (Colot et al., 1987; Marris et al., 1988; Goldberg et al., 1989), but these genes are not of regulatory importance in embryogenesis. Similarly, ABA may act to initiate certain programmes of gene expression, but does not determine embryogenic competence. PROBING CONTROL MECHANISMS We are, therefore, at a point in our understanding of embryogenesis at which we can differentiate, at the genetic level, between mechanisms of determination and downstream mechanisms of growth and metabolism. As we have seen, the processes of embryonic pattern formation, for example, can be distinguished from more 'housekeeping' functions through the study of mutants.

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Johnston and Nusslein-Volhard, 1992) or smaller molecules (Jessell and Melton, 1992) have been correlated with pattern formation and associated gene expression. It may be that the 'patterning' genes revealed by the mutant studies of Mayer et al. (1991) may play an analagous role in plants, by establishing gradients (a) of mRNAs/ proteins encoding either transcription factors that may activate cell-specific gene expression patterns; (b) of receptor molecules sensitive to other regulatory molecules (such as growth substances, which may be provided by various source tissues in the developing seed) that might act to activate specific sets of genes associated with, for example, the control of the correct plane of cell division; or (c) of components of signal transduction pathways involved in downstream processes of differentiation and development and the associated patterns of gene expression. There is currently no concrete information on which to base any firm theories, but there is some circumstantial evidence which may be relevant.

Lindsey and Topping—Embryogenesis 371 or basal structures or in radial symmetry, etc. If the gene had been identified by means other than mutational, such as by library screening, then the generation of a null phenotype by antisense RNA technology may provide information on a broad function. Analysis of the predicted protein sequence of the gene product may provide clues to function, such as the presence of DNAbinding domains or similarities to known gene products. For many embryonic genes, however, it may be extremely difficult to ascertain function, since in most cases there may be no homology to known genes. Significant further progress will require more downstream molecular markers, to assess 'knock-on' effects of null mutants and more mutants of upstream processes, such as signal

A further step in unravelling molecular aspects of embryogenesis is to understand how the expression of other embryonic genes, identified as being 'downstream' of more critical early processes, is affected in mutant plants. To approach this we need a range of molecular markers that characterize specific aspects of embryogenesis—expressed in particular cell types, at particular developmental stages-—such as could be generated with relative ease by promoter trap techniques. Such markers provide a means of visualizing genetic interactions, and a 'secondary mutagenesis' of tagged lines may facilitate the identification of regulatory genes or components of signal transduction pathways, by screening for loss of reporter function. Once a gene of interest has been identified, how can we assess its function? Clearly if its loss of function creates a lesion in normal embryogenesis, we can at least generalize about that area of development in which it first exerts its effect: a role in the specification of apical

5

Some Hierarchies in Embryonic Gene Expression

O