DEVELOPMENTAL

BIOLOGY

153,44-58 (19%)

A Conceptual Framework for Maize Leaf Development MICHAELFREELING Depurtment

of Plant Biology,

University

of California,

Accepted June 5,

Berkeley,

California

947.20

1992

What is and is not known about the maize leaf is reviewed. Analysis of genetic mosaics and direct observation with the SEM have broken leaf development into three distinct phases: recruitment of cells within the meristem, cell division into the 0.6-mm tall primordium, and postprimordial division and differentiation into the mature leaf. New data are presented that imply that cell division rates in the leaf are coordinated by inductive signals from the internal cells. Leaf cells that tend to divide more are held in check by slower growing neighbors; this complicates the search for developmental compartments. Experiments with recessive mutants that remove the ligule and auricle have been important in identifying an inducer signal with the specific meaning “make ligule-auricle.” We have studied many dominant mutant alleles at seven different genes. Each mutant alters the position of the ligule boundary. We conclude the following. First, the mutants act in particular domains of the primordium. Second, the dominant mutants all move the ligule boundary in the same direction. Third, the mutants all retard developmental stage transitions. Fourth, three and probably four of the seven genes for which dominant mutants have been studied specify homeodomain proteins in the wrong place. The concept of “maturation schedule” is used to explain these data. All of the dominant mutant phenotypes are seen as consequences of immature cells being in the wrong place when inductive signals pass through the leaf. Several specific questions of leaf development and especially questions as to source of inductive signals or homologies among juvenile o 1992 Academic PRSS, IX and adult organ parts are recast in light of this “maturation schedule” hypothesis.

relatively undifferentiated cells-presumably naive for a time-that, subsequently, generate embryos or meristerns de novo, and then whole plants again. The origin and maintenance of polarities may be readily studied in plant systems. Third, typical plants have the potential to grow forever by virtue of having meristems, (Lord and Hill, 1987; Steeves and Sussex, 1989). Because of this essential indeterminism, there is a huge amount of genetic variability in developmental time-keeping mechanisms and rates of developmental stage transitions within any one plant species (McDaniel et al, 1992). For example, (dayneutral) maize cultivars exist that differ in time-to-flowering from 6 to 14 weeks, but are cross-fertile. Animal species generally maintain a more fixed generation time. Theory pertaining to the evolution of developmental processes has generally emphasized the importance of the relative timing of developmental events (Alberch et al., 1979). Specifically, dissociation of inductive signal from the cells competent to properly receive the signal leads to novelty (Raff and Wray, 1989). Such examples fall into a field of theoretical biology called heterochronism, a field which is presently lacking a firm foundation in molecular biology. Our recent work has shown that some maize mutants altered in rate of developmental stage transitions express radical phenotypes in different time-to-flowering backgrounds (Bertrand-Garcia and Freeling, 1991; Freeling et al., 1992). It was possible to predict the position within a plant that organ

INTRODUCTION

Plant systems present distinct experimental advantages over animal systems when it comes to delineating mechanisms measuring position (in space) from those measuring developmental history (over time). Three specific advantages of using plant systems are discussed. First, cell positions in a plant primordium are fixed and readily measurable, which facilitates experiments where position is to be held constant. Plant cells are cemented into place as they come to exist, and do not migrate nor rotate thereafter. Adjacent plant cells may establish cytoplasmic bridges, but otherwise, membranes do not touch each other. The expected consequence is that the relative positions of cells when primary determinative events take place, such as in progenitor cells where positional or temporal information is acquired, correspond directly to relative positions in the adult organ. Second, experiments on truly naive cells, cells devoid of retained developmental history, may be possible in some plant systems. Wolpert’s positional information hypothesis predicts that graded morphogens exist (Wolpert 1969, 1989). This hypothesis also implies that cells about to acquire positional identity either be naive or at least have equal developmental potentials. There may be no point in a higher animal’s development where such a naive state should exist. There are now many cases where individual plant cells generate clumps of 0012.1606/92 $5.00 Copyright 411 rights

ca 1992 by Academic Press, Inc. of reproduction in any form reserved.

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younger but adult. Thus, the position of a leaf on the plant is correlated with the absolute age or youth of the leaf. I will use the term younger to indicate that there are relatively many cell divisions or maturation stages to go before terminal differentiation; juvenile is not a synonym for young. The confusion of position within a shoot apical meristem and age of a cell at that position is a natural consequence of meristematic growth, where leaves are recruited from meristematic cells sequentially in a timedependent process. Not surprisingly, it has proven exceedingly difficult to distinguish timing (developmental history) mechanisms from positional mechanisms at any stage of plant development, but it should be possible as discussed in the Introduction. A large leaf of the corn plant is founded by a ring of about 250 cells, actually a ring with overlapping ends, like a key ring, consisting of at least two layers of the apical meristem (Poethig, 1984; McDaniel and Poethig, 1988). This ring of founder cells may look somewhat like the sketch in Fig. 2. This ring of cells is defined as plasTHE MAIZE LEAF tochron 0 (PO), or the leaf at the point just before rapid primordial cell divisions begin. It is not yet known how The strap-like maize leaf (reviewed by Freeling and the founder cells originate from the leaf initial cells, Lane, 1992) is the most visible organ of the repeating, those cells that first acquire leaf identity. Although the vegetative shoot segment (the “phytomer,” composed of word “plastochron” is usually defined as the unit of time leaf, prophyll, axillary bud, and internode; Galinat, 1959; revised by Johri and Coe, 1983). Although each of it takes the meristem between generating one leaf and the 20 leaves of a typical commercial corn line is unique, the next, a developing leaf is often given a plastochron all leaves are basically similar to the largest, fully ma- designation indicating plastochrons elapsed. A plasture leaf diagrammed in Fig. 1. The longitudinal dimen- tochron 3 leaf (P3) is three plastochrons old; it is three sion of the leaf is divided into basal sheath and distal nodes below the ring of founder cells. It is important to blade, with the wedge-like auricles separating the two, give a maize leaf from any inbred line both a leaf (L) and and the ligule, a fringe of epidermis, rising from the a plastochron (P) designation. [L15,P4] is an immature, adult leaf 15th from the base of the plant and it is only adaxial (upper) surface at the sheath-auricle boundary. The diagnostic cell shapes and surface structures of the about 0.6 cm tall. [L4,P15], on the other hand, is a fully mature blade (Fig. 1A) and sheath (Fig. 1B) are shown mature, juvenile leaf, fourth up from the base. Once a in the scanning electron microscopic (SEM) micro- leaf matures, the P designation is meaningless. graphs. In cases where a mutant allele transforms one The use of replica surfaces for SEM studies (Williams part of the leaf to another, such as the common mutant et al., 1987) has been useful. Descriptions using replica phenotype of a blade region transforming to sheath, ob- SEM methods have shown that leaf development hapservations of epidermal cell surfaces usually suffice to pens in three phases: recruitment of meristematic establish identity (Beer-aft and Freeling, 1989; Ber- founder cells, primordial development, and postprimortrand-Garcia and Freeling, 1991; Sylvester et ah, 1990). dial cell division and differentiation (Sylvester et al., The transverse dimension of the leaf is subdivided into 1990). The following plastochron designations apply to leaves near L7. The first phase is the recruitment of a the distinctive tissue layers, and the lateral dimension of the leaf is subdivided into domains of gene expres- few hundred cells within the meristem, the founder sion, as will be discussed. cells. This recruitment process has not yet been obThe leaf that is closest to the ground, leaf 1, is the served directly and it is not clear that PO is really the oldest leaf since it is the first elaborated from the meri- earliest plastochron. That is, leaf initial cells may exist stem. The lower approximately five leaves, the fetal in rings higher up within the meristem that might be leaves of the dry kernel, are often called juvenile be- called P - 1 (P minus l), for example. Further, the word cause they share some of a constellation of organ and “recruitment” is meant to be vague, as it is not clear cellular traits (reviewed by Poethig, 1990). Older leaves whether or not just any cell could volunteer. Sharman are more juvenile leaves while the upper leaves are (1942) observed that the position of divisions spread

transformations would occur by a concept of “slipping time frames,” one frame measuring time-to-flowering and one measuring rate of developmental stage transitions. Plants in general may afford the biological systems where heterochronic theory submits to experimental tests. Especially for the three reasons detailed above, plant systems may be used to answer developmental questions of interest to all developmental biologists. We chose the maize leaf because it is large and many mutants affecting leaf morphology and cell identity were described, and because an important developmental boundary on the leaf, the ligule, was known to be dispensable. Genetic dissections are most effective when the structure is dispensable. The use of maize ensures that any Mendelian mutant that expresses reliably will eventually lead to the appropriate cloned DNA sequences, and permits crucial experiments requiring the addition or subtraction of chromosomal segments.

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from a major procambial strand (the dot on sketch of Fig. 2) around the dome in both directions. The second phase is where each founder cell continues to divide about the same number of times to generate the approximately 6 mm long, P3-4 leaf primordium. The finished primordium is characterized by a complete set of lateral veins, and the beginnings of the periclinal epidermal divisions that generate the ligule (Fig. 2E). The postprimordial, or third, phase of leaf development begins at the tip of the leaf, where growth and differentiation begin and end first. Differentiation works its way down the leaf (basipetally), with the basal cells always being last to divide and differentiate. Although the midvein and laterals differentiated acropetally during primordial development, the remaining majority of the veins of the adult leaf arise as branches from primordial veins (diagrammed in Fig. 1) and differentiate basipetally along with the other cells of the leaf (Sharman, 1942; Esau, 1943) during the third phase. These intermediate (basipetal) veins anastomose near the ligule (also shown in Fig. l), where usually one basipetal vein differentiates into the sheath (Sharman, 1942) and progresses to join basipetal vascular network of the stem, a network separate from the primordial vein network (Kumazawa, 1961). All of the mutants with which we have chosen to work affect the leaf before the primordium (P3-4) has been completed. The SEMs of replicas shown in Fig. 2 are of primordial leaf surfaces at [L7,P2, and P3] (Sylvester et al., 1990). The ligule is a ridge of closely packed, hair-like cells that rise out from the plane of the adaxial epidermis at P3-4. At P2 (in adaxial view, Fig. 2B), the epiderma1 cells that will be ligule have not yet undergone any extra mitoses, but the arrows bracket a region of cells, cells displaying a distinctive “square” shape. This young primordial region we call the ligular region, and it may even be present at Pl, but we have not yet been able to observe primordia this early. Thus, presheath, ligular region, and preblade exist early. Some of the cells in the ligular region will divide anticlinally, within the plane of the epidermis, at P3 (Fig. 2D) to generate columnar cells that will then go on to divide periclinally at the primordial stage (Becraft et ah, 1990). The anticlinal divisions happen uniformly in time across the leaf, but the periclinal divisions begin at two foci at the midrib boundaries (Fig. 1 shows midrib region in mature blade) and progress both to the margins and to the midvein. Sylvester and co-workers (1990) described epidermal cells, and their division frequencies and directions, in about every other leaf from P2 until terminal differentiation. The progression of ligular region [P2] to ligular band [P3] to young ligule [P3-41 is of particular interest because we have studied many mutant alleles at several genes that interrupt or alter this process.

VOLUME153,1992 ARE

THERE

DEVELOPMENTAL COMPARTMENTS IN THE MAIZE LEAF?

Garcia-Bellido and Merriam (1971, reviewed by Brower, 1985) defined a compartment boundary in the fruit fly as a line through an organ over which cells do not divide. Lines of mitotic restriction were identified by constructing genetic mosaics where a clone of genetically fast-growing somatic cells would take over regions of an organ, but would have a straight side, straight because cells could only pile up but not cross an anatomically invisible barrier. These compartment boundaries turned out to delineate parasegments, and the action domains of segment-identity genes. Following the experimental plan used in Drosophila, I used the dominant dwarf allele, 08-O (0 for original; Harberd and Freeling, 1989) as a way to slow down cell division in a cellautonomous fashion. A plant carrying one or more Ds-0 alleles is slow to develop and the leaf only has 40% of the wild-type number of cells in the longitudinal dimension (Phinney, 1947; this laboratory, unpublished data). Plants doubly heterozygous for closely linked alleles D80 and lw, a recessive white leaf allele, are green and dwarf. The genetic configuration (repulsion, diagrammed in Fig. 3) is such that, if the DS-0 chromosome within a young meristematic cell is detached from its centromere by x rays, a mosaic meristem occurs, and leaves including these cells as founders will carry a white ((/Zw) somatic sector that is also not genetically dwarf. Figure 3 diagrams the chromosomes, the shoot apex as seen from the tip of the plant, and some of the resulting sectors of white, fast-growing tissue. What is particularly informative about this experiment is that one sector within the meristem generates a sequence of leaves, each with genetically identical sectors which are positioned differently within the leaf. Three of the leaves of a single plant show the growth consequences of a sector that is totally within the blade (Fig. 3B, bottommost), one where the sector includes the margin of the blade (middle), and one leaf where a large sector has clearly overgrown the green blade at the tip (uppermost). Before discussing observations of these sectors of white, potentially fast-growing cells, the cell autonomy of the dwarf phenotype conferred by mutant D8-0 must be addressed. It is entirely expected for dominant phenotypes to be cell autonomous for one component of phenotype and noncell autonomous for another, and that is certainly true for 08. A hand-cut transverse section of blade was cut across a boundary between dwarf green tissue and a particularly large white sector; its position is indicated on the uppermost leaf of Fig. 3B. This transverse section was examined under fluorescent light where chlorophyll autoemits red; the red color of Fig. 3A

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longitudinal transverse

-k I’ IT IV

-

lateral

intermediate veins - lateral vein midvein

blade surface * i midrib auricle ,igu,e 3 adaxial surface

::,:;;

L

intermediate vein anastomosis I)

6 sheath surface

FIG. 1. An adaxial view of an adult, mature maize leaf with the parts and veins labeled. The inset photograph of the region of the ligule is labeled: s. sheath; Ig, ligule; a, auricle; mr, midrib; t), blade. The transverse dimension is not visible. The SEMs are of replicas of wild-type, adaxial epidermal surfaces. (A) Blade surface showing long and short epidcrmal cells with their interlocking wall crenations. The macrohair is

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DEVELOPMENTALBIOLOGY

marks the green part of the sector. Note the differential size of the two major xylem elements of the vein bisected by the sector (Fig. 3A), and note the wider intermediate vein placement within the white, nondwarf blade. Cell counts and measurements in this and similar sections indicate that cells are between 10 and 20% smaller if 08-O is present, which is consistent with our previous observations in homozygotes (Harberd and Freeling, 1989). The cell-size component of the D&Ophenotype, a small reduction in cell size, is consistent with cell autonomy (excluding the epidermis, where D&O does not act, Harberd and Freeling, 1989). Figure 3A and its legend show this, although the exact border of a dwarf and a wild-type cell cannot be seen in this photograph. That one component of phenotype is cell autonomous does not predict that the cell division component of 08 phenotype will also be cell autonomous, tissue autonomous, or domain autonomous. My observations are preliminary in the sense of accurate quantification and allometry, but the major result of the experiment was apparent without need for rigor. Of the about two dozen leaves with sectors examined, some cells within most white sectors showed a distinct tendency to grow too much, especially in sectors that occupied over 30% of the lateral distance from midvein to margin. The sectors bubbled out in their central regions. In other words, the 08 cell division component of phenotype displays a tendency for organ-region autonomy. Sectors overgrew more at the tips of leaves. However, most of the white cells were “held in check” by their surrounding slower-growing cells. When overgrowth occurred, marginal sectors did not tend to grow more than sectors bordered by dwarf tissue on both sides.

This rule is illustrated in the three leaves of Fig. 3B. In plant 11136-13,the white, non-D8 sector occurred in the developing meristem such that five sequential leaves had a white sector somewhere in the leaf; three of these leaves are shown (Fig. 3B). Arrows in Fig. 3B indicate positions where nondwarf cells are clearly “trying” to overgrow. No ripping was apparent. Clearly, non-D8 cells, although they have a tendency to divide relatively rapidly, do not presumably because the 08 cells nearby send signals to divide more slowly. Perhaps it will prove different for the meristem, but the Drosoph,ilu method for establishing compartment boundaries simply will not work for leaves because of cell-cell coordination of mitotic rates. Fortunately, we can observe cell division patterns directly and can dis-

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cover the domains of leaf cell-identity genes; compartments will probably emerge using alternative methods. I believe a more important conclusion is strongly suggested by these results: the cell-cell communication mechanism that coordinates cell division within the leaf is more likely to utilize a chemical as opposed to cell-cell waves of stress (a force). It has been exceedingly difficult to test any of the various hypotheses that invoke (biophysical) force as a signal important for communication, and strain as the morphological response to the force. If stress were the signal that is being used to (almost) keep non-D8 cells in check, then non-D8 cells that find themselves at the margin of the leaf should be able to overgrow without restraint. In fact, the position of genetically identical sectors within leaves, be they totally internal or marginal, made no difference to the tendency to overgrow. For the simple reason that cells restrained from both sides are under more stress than cells held from but one, I suggest that cell division is coordinated within the leaf via chemical rather than stress signals. TISSUE-TISSUE

INDUCTIONS

IN LEAF

DEVELOPMENT

The three tissues of the leaf are defined histologically: the outer layer or epidermis, the vascular tissue (xylem, phloem), and everything else, called “ground tissue.” The mature blade cross section of Fig. 3A shows these layers. The developmental reality of the primordial leaf in cross section is that there are five cell layers in three positions: the two outer epidermal cell layers, the central layer, and the two in-between layers. Cells of the central layer have been seen to divide periclinally from the adaxial inner tissue layer (Sharman, 1942). Using genetic mosaics where the lineage of primordial cells was genetically marked, Langdale and co-workers (1989) showed that the central layer of the leaf primordium generates provascular cells that include bundle sheath photosynthetic cells in their lineage, and the middle mesophyll photosynthetic cells as well. If plant tissues were defined developmentally rather than physiologically, the leaf would be composed of three tissues: outer, middle, and ground. Two of these “tissues” display obvious patterns, the rows of specific cells characterizing the epidermis (outer) and the venation pattern (middle). Figure 4 (from Freeling and Lane, 1992) shows an epidermal pattern and the underlying venation pattern between two lateral veins in a large maize leaf.

subtended by basal cells, and marks a row of bulliform cells. An arrow marks a row of stomata1 complexes which are elongated in the longitudinal dimension. (B) Sheath surface composed of cells with straight walls and stomata1 complexes elongated in the lateral dimension. Ectopic regions of sheath within blade retain the sheath identity reflected in B. Micrographs reproduced, by permission of the publisher, from Sylvester and co-workers (1990).

REVIEWS

Founder

margin

blade

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cells

midrib

blade

margin

FIG. 2. Early development of the leaf primordium. The sketch of the founder cells within the meristem is a reconstruction based on Pocsthig (1984) and McDaniel and Poethig (1988); the dot is the position of the immanent midvein. The lower sketch of the unrolled founders shows lateral regions illustrating the hypothesis that the founder cells already possess identity of the three major regions of the blade. The SEMs ar(’ of replicas of P2 and P3 primordial approximately L7 leaves. (A) An apex with the P2 leaf still attached with its abaxial surface visible. (B) A later P2 leaf spread out to expose the adaxial surface. The arrows point to the boundaries of a region of “squarish” cells: these comprise the preligular region. (C) An apex with a P3 leaf attached. (D) The adaxial preblade region of a P3 leaf. (E) The adaxial ligular region of a P3 leaf with a distinctive band of anticlinal divisions showing both possible division planes. This ligular band will later divide periclinally to generate a young ligule. (F) Adaxial view of P3 presheath cells. Micrographs reproduced, by permission of the publisher, from Sylvester and co-workers (1990).

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Clearly, the patterns are related; either the middle layer induces the outer or vice versa, or some whole-organ signal coordinates all tissues at once. As a geneticist, I am accustomed to visualizing form as controlled from the inside out. In this case, it seems natural that the middle layer of the primordium, the procambial-middle mesophyll layer, induces the patterns of cell differentiation in the epidermis. This “central control” hypothesis is supported only by circumstantial evidence. First, veins differentiate before the epidermal cells. Second, two mutants that affect growth and age identity of all three tissues of the leaf, Knl-0 (Knottedl, Sinha and Hake, 1990 and references therein) and the previously mentioned 08-O (Harberd and Freeling, 1989) have been tested for function independently in the different tissues using organs that are mutant in one tissue but wild-type in adjacent tissue, and vice versa. The conclusion is that mutant function in the epidermis was not necessary or sufficient for some components of the phenotype. In other words, epidermal phenotypes were shown to be induced from tissues deeper within the primordial leaf. Although these two arguments do not constitute proof that provascular cellsmiddle mesophyll are inducers, they do detract from any hypothesis that places primary morphological control in the epidermis. Specifically, the coordination of cell division phenotype of 08 and the retarded organ maturation phenotypes of Knl affect the epidermis through cell-cell induction only.

SOMATIC INHERITANCE OF PATTERN PRIMORDIUM TO ADULT LEAF

FROM

Plant cells, similar to the cells of other higher organisms, differentiate according to position, not lineage (reviewed by Dawe and Freeling, 1991). This conclusion derives from numerous examples where, during postprimordial growth (e.g., phase 3 of leaf development), cells from one lineage find themselves caught up with cells generally from another lineage. It is customary for plant biologists to discount any developmental function for special cell lineage rules, similar to stem cell division

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rules, because of the many cases of position-dependent differentiation without regard to lineage. I believe that cell lineage rules may play an important role in primordial divisions (e.g., phase 2 of leaf development), but not during the postprimordial phase of massive leaf growth. Consider the problem of initiating a basipetal vein as a branch from a lateral vein at the tip of a P4 leaf, a primordium only about 6 mm long. This vein may differentiate to be 4 feet long in the adult leaf and stem. As expected from preceding studies, it was shown that this differentiation process sometimes recruits cells from the noncentral lineage, and these differentiate strictly according to position irrespective of lineage (Langdale et al., 1989). However, Langdale and co-workers (1989) found that a typical middle layer leaf primordial cell divided unequally, giving rise to a precursor to a halfvein and to another middle mesophyll cell. This sort of division may indicate a classical stem cell division. Stem cell divisions in the middle tissue layer of the primordial leaf, being unequal, naturally generate a periodic pattern that could explain the typical venation pattern of the blade in the transverse dimension (Dawe and Freeling, 1991): vein-middle mesophyll cell-middle mesophyll cell-vein-etc. (Russell and Evert, 1985). Once this venation pattern is established, Dawe and Freeling (1991) marshalled evidence in support of the notion that the pattern could be perpetuated by largely agenic mechmechanisms: anisms (i.e., structure-begets-structure Sonneborn, 1969; Green, 1980; Newman and Comper, 1990), where existing veins serve as sfrucfurd templutes for the organization of cells recruited in a lineage-independent way. This structural inheritance scheme is simply a new application of an ancient and robust idea.

RECESSIVE MUTANTS THAT REMOVE THE LIGULE AND AURICLE

Two genes, 1~1 and 1~2, are defined by recessive mutants that, when homozygous, remove the ligule and auricle from at least the basal leaves of the plant. Becraft and co-workers (1990) showed that 1~1 blocks ligule development before the anticlinal divisions of P3. This

dwarf mutant, DX-0, is lost in a sector of meristem, generating FIG. 3. X-ray-induced genetic mosaics where a dominant, “cell-autonomous” plants with more than one leaf carrying a sector of nondwarf tissue marked with 1~, a cell-autonomous allele that specifies white cells. The sectors shown here included all three tissue layers. The upper part of the figure shows the chromosomal constitution of 2-day seedlings when submitted to 1000-2000 rad of x rays; upper leaves are not yet founded at this stage. The diagram shows how a single meristematic sector could engender a succession of leaves with a white, nondwarf sector. (A) The red color in this sector is green chlorophyll autofluorescence. This examination of a hand-cut, fresh transverse section over a white-green boundary, as indicated, demonstrates that a small reduction of cellular diameter is a cell autonomous property conferred by the L&-O allele. Note that the dwarf (green) major xylem tube within a mosaic lateral vein is smaller, the blade is thinner, the intermediate veins are closer together, and the like. The smaller cell size component of the dwarf phenotype is autonomous. (8) Cannon laser copies of the second, third, and fourth leaf from the top of plant 11136-13, a plant in which the upper five leaves reflected the mosaic meristem. Note that these leaves express the same genetic sector of white, nondwarf tissue; an arrow marks positions where this tissue tends to overgrow. Especially note that the marginal sector of the middle leaf does not overgrow.

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observation puts LgP action at P2 (Figs. 2A and 2B) or before. No other effects of lgl homozygosity on cell division rate or direction were found (Sylvester et al., 1990). Using precise tissue-layer somatic sectors, Becraft and co-workers also found that Lg+ product is required autonomously in the epidermis to produce ligule and required in the inner leaf to produce auricle. When the wild-type allele is removed in a sector including all three tissues of the leaf, both ligule and auricle disappear within the sector, and sheath blends into blade over a larger region we guess to be the cellular descendants of the ligular region of P2 (Sylvester et ab, 1990, and see Fig. 2B). So, Lgl+ product is not needed to separate sheath and blade, but only to do so abruptly. Recently, Becraft and Freeling (1991) showed that Lgl’ product was also necessary to propagate a “make ligule-make auricle” signal from the midrib boundary out to the margin. We hypothesized that this gene’s function was necessary to help direct differentiation from among preligular cells already competent to respond and to transmit the signal that carries this instruction. Homozygotes for unlinked ZgZhave a similar phenotype to lgl mutant homozygotes, but preliminary genetic mosaic analyses suggest that cells expressing wild-type Lg2+ product readily signal nearby cells to develop auricle, and ligule itself may be induced from nearby wild-type cells in the transverse dimension only (L. Harper, unpublished data). In other words, the LgZ’ function is less autonomous (more regulative) than the LgP function. We have additional liguleless mutants that have not yet been tested for allelism, and we probably have not saturated this phenotype with all possible recessive mutants. DOMINANT MUTANTS THAT ALTER THE POSITION OF THE LIGULE: LIGULE POLARITY MUTANTS

Seven genes are defined by dominant mutant alleles that specify a similarity of phenotype: each mutant allele changes the shape of the sheath-blade boundary such that some sheath “pushes into” blade. An eighth gene is defined by the single rs2 mutant allele, an allele that may be recessive or may be such a mild dominant that two doses are required to express; it specifies an Rsl-O-like phenotype when it does. Figure 5 (Freeling et ah, 1992) diagrams the essential phenotypes of seven representative dominant mutant alleles, one for each of the seven genes. Note how the adult morphology of each is that blade transforms to sheath, but not vice versa. This unidirectionality of the transformation phenotype is why these mutant alleles are named “ligule polarity mutants.” These seven genes fall into groups. The Knotted1 group contains the four genes that encode proteins with Knl-like homeodomains, including Knl (Voll-

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brecht et ah, 1991), Rsl (P. Becraft, S. Hake, and M. Freeling, unpublished data), Lg3 (J. Fowler and M. Freeling, unpublished data), and Lg.4 (linked tightly to a homeobox, but not yet proved to carry one, J. Fowler, and M. Freeling, unpublished data). Proof that a mutant allele is of a gene with a homeobox requires that the phenotype can be knocked out by transposon insertion and that the insertion concomitantly alters the DNA fragment containing the homeobox. The phenotypes of mutant alleles at these four genes are confined primarily but not exclusively to the ligular region and/or blade, not the sheath, and mutants at these genes do not respond in a regular fashion to genetic backgrounds that differ in time-to-flowering. A second group of dominant mutant alleles define the pleiotropic, heterochronic genes Hsfl and Lxml. These mutant alleles affect many organs on the plant in addition to the leaf. Hsfl-0, the original of two mutants at this gene, expresses very different phenotypes in different time-to-flowering backgrounds (Bertrand-Garcia and Freeling, 1991); this has important evolutionary implications (Freeling et ah, 1992). The single mutant allele defining Lxml displays similar background effects on expressivity (D. Porter, unpublished data), and neither Hsfl nor Lxml appear to be linked to a Knl-like homeobox. The last group of mutants, defining one or more Rld (Rolled) genes, are unique in inverting adaxial and abaxial epidermal identities in regions of the leaf, leading to ligule on the abaxial epidermis, and push sheath up into blade in this region as well. Three generalizations can be drawn from observations of the phenotypes of these mutants. First, the ligule boundary is always changed in the direction that the sheath projects into blade; this aspect of phenotype is strictly polar. Further, there are often developmentally immature cells associated with the regions of transformation, as was first observed around the whitish lateral veins of Knotted mutants (Freeling and Hake, 1985). Second, the mutants differ in exactly which lateral region of the blade is altered. The founder cells sketched in Fig. 2 depict three such lateral regions although it is not certain that these domains exist as early as the founder cells. In any case, these mutants identify domains along the lateral dimension of the blade. These lateral regions of blade are discussed. Third, with the possible exception of rs2, these ligule polarity mutant alleles are dominant, and are certainly not losses of function. Consequently, obtaining information on the nonmutant function of these genes is not straightforward. These nonmutant genes may have much to do with development, but little to do with leaf development specifically. Referring back to Fig. 1, note the lateral dimension of blade. Here, the midvein is the line of symmetry, pro-

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l macrohair 0 bulliform cell veins I \

intermediate (basipetal) transverse primary intermediate (basipetal)

I

lateral

FIG. 4. The adaxial and abaxial patterns of epidermal cells aligned with the venation pattern that lies within the leaf. The patterns are related. Especially note how rows of stomata1 complexes are never over veins and how bulliform cell rows sometimes rcplacc stomata1 complexes at regular intervals predicted by vein placement. Reprinted, by permission of the publisher, from Freeling and Lane (1992) from original data by Ms. Deverie Bongard-Pierce when she worked in this laboratory.

ceeding to the midrib boundary, the expanse of blade, and finally the margin. Six of the seven genes are defined by mutant alleles that do not express uniformly throughout this bilateral dimension. Lg3 and Lxml mutants alter the midrib and central region only. Hsfl mutants affects the margin, but not the blade proper. Lgh and Rid mutant alleles affect an internal region of the blade, and Knl mutants alter strips of cells around lateral veins preferentially in this internal region (Freeling and Hake, 1985). Only Rsl mutant alleles seem to affect cells in the entire lateral dimension, but Rsl is region specific in the longitudinal dimension. Rsl is very specific to the ligular region, as if ligular region cells of P2 were its domain (P. Becraft, unpublished data, and Fig. 2B). Judging from the regional phenotypes of these mutant alleles, the leaf seems to be pieced together. Almost all ligule polarity mutants are dominant. One explanation for this involves the special flower morphology of the corn plant, where pollen derives from cells at

the tip of the shoot apical meristem and eggs derive from cells at the tips of certain axillary bud meristems in the axes of the largest vegetative leaves. This separation of male and female lineage affects the ease with which recessive mutants may be screened. A somatic sector carrying a recessive mutant would never be able to homozygose in the first generation and would go unnoticed. Similar sectors in, for example, barley, Arabido~s,sis,or tomato would generate ovules with naturally self-pollinated seeds segregating 25% for a recessive mutant homozygote. This is because these species have perfect flowers with compatible male and female parts. Thus, the excess of dominants reflects the huge numbers of corn plants grown each year, and the relative paucity of recessives is explained anatomically. A more satisfying explanation for the abundance of dominant ligule-shape mutant alleles derives from what we know of the flagship gene of the ligule polarity mutants, Krrl. No dominant mutants in maize are haploin-

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Knl-0

Rsl-0

La4-0

Lxml-0

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Hsfl-0

Rld 1-O FIG. 5. Ligule and blade phenotypes of seven representative ligule-polarity mutants. These phenotypes are relatively mild, with no complexities involving growth consequences of blade transforming to sheath. In reality, these mutants have been known to prolong sheath in lateral regions other than those depicted here. Notations: s, sheath; Ig, ligule; a, auricle; h, blade. Redrawn from Freeling and co-workers (1992).

REVIEWS

sufficient (knockouts) since all possible monosomes and segmental monosomes have been observed to be without specific phenotypes. Aneuploid studies (Freeling and Hake, 1985) indicated that mutants at Knl behave as neomorphs. That is, the KNOTTED phenotype is unaffected by added copies of the normal region carrying the wild-type knotted gene. In addition, the knotted phenotype-foci of blade overgrowth called “knots,” whitish veins, ectopic ligule, and ligule displacement-appeared to be focused in cells around lateral veins in the central region of the primordium. These cells, including bundle sheath, mesophyll, and sclerenchyma, did not differentiate beyond simple parenchymatous cells in the Knl-0 mutant. Later, it was shown that the epidermis over these immature laterals differentiated as sheath, not blade (Becraft and Freeling, 1989), as if sheath identity might be the property of retarded or immature blade identity. Thus, the KNOTTED1 phenotype was seen as developmental retardation leading to cells around lateral veins acting in an immature fashion in response to normal developmental signals. Ectopic expression of normal Knl product around lateral veins was predicted (Freeling and Hake, 1985). Since Knl has been cloned by transposon-tagging (Hake et al., 1989) and the cDNA has been sequenced, it is now clear that Knl encodes a protein with a homeodomain (Vollbrecht et al., 1991) and is likely to be a transcriptional factor. Antibodies prepared against KNl protein localize to the wild-type shoot apical meristem, but were not detected in the leaf founder cells or leaf; in Knl mutants, there is KNl product ectopically expressed around lateral veins (L. Smith, B. Greene, B. Viet, and S. Hake, 1992,unpublished manuscript) as predicted. Thus, Knl mutants confer developmentally retarded phenotypes because a homeodomain protein is ectopically expressed around lateral veins. Rsl, Lg3, and Lg4, those genes encoding Knl-type homeodomain proteins, are still being characterized genetically and molecularly; ectopic leaf expression has been shown at the message level for both Rsl and Lg3 (P. Becraft and J. Fowler, respectively, unpublished data). Perhaps homozygous knockouts of these genes encoding transcriptional factors leads to dead embryos or kernels, or that the encoded homeodomain proteins function necessarily in the gametophytes. Dominant mutants may be the only sort of mutants that are viable. As a final alternative, dominant mutant alleles may define genes that encode duplicate functions, However, in Drosoph,ila, important developmental genes are unique sequences. TIME

VERSUS LIGIJLE

POSITION IN UNDERSTANDING REGION SPECIFICATION

There are two general ways by which these dominant mutant alleles might alter the shape of the ligule: First,

55

early without changing relative cell division rates or, second, postprimordially via differential growth. Using methods developed previously (Sylvester et ah, 1990), Sylvester and co-workers (unpublished) observed the surfaces of very early primordia of the Lg3-0 mutant heterozygote genotype. The photographs of Fig. 2 depict the wild-type surfaces at these early stages. We showed that P2 primordia have altered ligular regions and anticlinal division bands. Additionally, the lateral domain of Lg3-0 mutant action, the area between presumptive blade-midrib borders, was clearly visible in primordia. The lateral domains of the blade might be determined in the meristem itself because the founder cells for these domains are at least present there. The sketch of Fig. 2 includes these hypothetical lateral domains. Nothing is known as to how the meristem cells are recruited as leaf founders. If the midvein is specified first, as suggested by Sharman (1942), and this midpoint on the surface of the meristem then sends recruitment signals that are propagated around the dome, then the domains could be specified in a time-dependent manner. However, the involvement of true positional information-where graded information is interpreted qualitatively in naive cells or at least equipotent cells-has not been excluded for within the meristem itself. Similarly, the ligular region, between sheath and blade, could be specified positionally in a very early primordium composed of naive or equipotent cells. Certainly, whatever mechanism is altered by the dominant mutants happens very early. However, there is a far more compelling way to explain how the ligule boundary is established and altered by the dominant mutants (introduced first in Freeling et al., 1992). This hypothesis simultaneously explains the ligule-polarity phenotypes (Fig. 5), developmental retardation of transformed regions, and predicts that dominant neomorphic mutants would cause these phenotypes. This explanation involves the idea that meristem leaf founder cells start their developmental clocks at time zero and, from that point, each lateral domain of the leaf matures semi-independently according to a strict schedule. I now explain this maturation schedule hypothesis in greater detail. A CONCEPTUAL FRAMEWORK FOR LEAF DEVELOPMENT INVOKING A MATURATION SCHEDlJLE

Assume that the about 250 leaf founder cells, a keyring-like group of cells circling within the meristem (sketch, Fig. 2), acquire their regional identities by virtue of the action of genes that we have not yet studied directly. The lateral regions already implicated are margin, blade, and midrib, but there are likely to be additional regions at well. The ectopic expression of ho-

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meodomain proteins (for Knl, Rsl, Lg3, and probably Lg4) or the product of Lxml, Hsfl, or Rld is proposed to interfere with this identity acquisition process. If one sees this identity process as “starting the developmental clock” in the leaf, then “interference” comprises retarding the maturation schedule, thus leaf developmental stage transitions simply happen more slowly in the dominant mutants. The ectopic expression of a transcriptional factor might certainly “gum up the works” and interfere with the regulation of this schedule. The similarity of all of the phenotypes drawn in Fig. 5 is thus explained. Following this line of reasoning, the leaf is a mosaic of lateral regions, each maturing semiautonomously according to its own schedule. The notion that embryonic cells count time in oscillations and operate via morphogenetic schedules is not new. Cooke and Smith (1990) recently reviewed a body of experimental evidence, largely involving the timing of amphibian gastrulation after various chemical treatments of the egg or early embryo, and concluded that competence to respond to a developmental signal is dependent on progress along a schedule, and a schedule that does not count cell divisions nor require new protein synthesis. Gurdon (1992) suggested that a simple mechanism that might keep time in this fashion is “the progressive degradation of proteins or of an mRNA that encodes a very short-lived protein.” Thus, a mechanism for the internal clock that times gastrulation is at least imaginable. Following this maturation schedule reasoning, the smooth ligule-auricle line that bisects the wild-type leaf develops smoothly only because each separate region matured normally, such that all cells at the ligule-competent maturation stage were in their wild-type positions. The dominant mutants, presumably by ectopitally expressing product that slows maturation, expose the ligular region and blade as a developmental mosaic. Thus, this conceptualization of leaf development envisions the ligular boundary being drawn as a consequence of coincidental developmental histories and not positional information. The maturation schedule hypothesis for leaf development may be summarized in four steps. Step 1. The founder cells acquire regional identities within the meristem. This sets the developmental clocks at time zero independently in each region. Positional information might operate at this step. Step 2. Each regional group of founder cells begins a strictly determined sequence of maturation events reflected as a sequential movement through windows of competencies. Cell divisions occur during this time, but are independent of the timekeeping mechanism. This entire process may be termed a maturation schedule, and I hypothesize that the dominant mutants of Fig. 5

VOLUME 153, 1992

retard such a maturation process. It is clear from visual observation that the presheath, preligule, and preblade parts of the leaf are apparent at P2, thus the sheath, ligule-auricle, and blade “stops” along the maturation schedule must happen very soon after the regional groups of founder cells begin dividing. Step 3. Specific regional identities, like sheath, ligular region, or blade, depend on a cell’s competency, dictated by its progress along the schedule, and not its position, at the time when a developmental induction signal occurs. The “make ligule-make auricle” signal identified in our ligulelessl mutant studies exemplifies such a specific signal, as discussed previously, but several additional signals must happen. Perhaps inductive signals emanate primarily from the primordial midvein, secondarily from the primordial lateral veins, and finally from the young intermediate veins in an induction cascade, but the idea that provascular cells are inducers is a prediction, not a fact. Step 4. Commitment to a particular differentiation outcome happens early in primordial development, but must then propagate itself through the many cell divisions between the 6-mm-long primordium and the lOOOmm-long leaf. During this propagation process, cells from various lineages get recruited to various fates, and these differentiate according to position only. Positional information is not involved in this sort of position-dependent development. Structural templates might operate during this postprimordial phase of massive growth, as has been discussed. INDEPENDENT SHEATH

EVIDENCE THAT THE IS A JUVENILE BLADE

LEAF

The first, basal segments elaborated by the shoot apical meristem generate leaves that have a constellation of characteristics that are, in total, called juvenile. Poethig (1990) has reviewed recently the general area of juvenility and the change of an apex from juvenile stages to adult stages. The notion is that the shoot apex passes through maturation stages (phases), and these are reflected by the morphologies of the corresponding leaves. In the conceptualization of leaf development presented in the preceding section, sheath identity was seen as a maturation stage closer to “time zero” than the blade. In other words, the sheath was seen as a juvenile blade in the same sense that organs at the base of a plant are more juvenile than younger organs at the top. In both cases, meristematic or progenitor cells are envisioned as passing through developmental maturation stages. Studies on one of the ligule-polarity mutants (Fig. 5F) the dominant mutant Hairy-sheath-frayedl-0 (Hsfl-0; Bertrand-Garcia and Freeling, 1991) showed that dif-

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ferent time-to-flowering genetic backgrounds moved the phenotype, slow developmental stage transitions, to predictable regions of the plant. Observations of this phenotype demonstrated that there was a tendency to simultaneously prolong (a specific heterochronic term defined by Takhtajan, 1972) characteristics of a juvenile (basal) apex up the shoot, and to push the sheath into the blade in the leaf (blade transformed to sheath in the marginal region, as in Fig. 5F). This double, leaf and shoot, prolongation phenotype supports the idea that sheath moving into blade is a prolongation of a juvenile identity. This notion is especially appealing since the ligule polarity mutants would simply need to slow down the maturation process to result in a prolongation of more juvenile maturation stages. It is not customary to discuss juvenility as a property of organs. To reverse custom entirely, when the meristem switches to being determined to flower, perhaps that becomes equivalent to time zero in the leaf founder cells. That is, the adult meristem might be seen as beginning a determined series of maturation stages in a similar fashion to the leaf founder cells of the meristem. DOUBLE

MUTANTS

AND

CURIOUS

PHENOTYPES

Many of the double mutants among the dominant ligule-polarity mutants have been made. Most double mutant combinations act additively, but there are curious exceptions (this laboratory, unpublished data). The interpretation of double dominant mutant phenotypes is less direct than are conclusions drawn from double recessive or knockout mutants. For example, Lxml-0 in one dose eliminates the lateral vein phenotype of Knl-0: Lxml-0 suppresses Knotted1 (Freeling, 1991). The maturation schedule concept suggested a biological explanation. Perhaps retarded development of the midrib and midvein region retarded the primary signaling mechanism of the leaf. Since Knl-0 phenotype has been explained as retarded maturation around lateral veins, perhaps the retarded signal and retarded target find themselves back in synchrony. More conventional explanations, like some sort of transcriptional cascade, are not ruled out. We need to characterize the products of all of the ligule-polarity genes before any particular model can be tested unequivocally. Many of the phenotypes of the mutants simplified in Fig. 5 have expansive new boundaries where ectopic sheath or auricle meet blade. While ligule always occurs at a blade-sheath union, such a juxtaposition is clearly not sufficient to induce ligule. Figure 5A, depicting the ectopic ligule fringes around knotted lateral veins, is an excellent example. Ligule is induced at very specific regions where sheath meets blade, but other blade-sheath junctions are barren. This specificity for certain blade-

sheath boundaries, which also applies to the phenotypes of the other ligule-polarity mutants as well, may reflect the continuity of leaf maturation phases, only some of which are competent to induce ligule. Specifically, cells may pass through not one but many different sheath identities and blade identities, but only the most mature sheath stage in contact with blade may be competent to respond to the “make ligule-make auricle” signal. Once again, the maturation schedule hypothesis helps to place biologically complex results into a conceptual framework. Continued recovery, cloning, and careful characterization of genes necessary to the leaf, in situ localizations of gene products in double and triple mutants, characterization of knockout derivatives of dominant mutants, and a reemphasis on experimental embryology should ensure a test of the maturation schedule concept. THE

LEAF

There is excellent evidence in Arabidopsis and Arlti(Coen and Meyerowitz, 1991) that the cell types of the dicot leaf comprise a developmental ground state. Sepals, petals, stamens, and carpels (or their whorls, depending on how one looks at it) are all converted into almost perfect leaves when three functions are simultaneously lacking. In Arabidopsis, a triple homozygote for mutant alleles a@?-1, a@-1, and ag-1 accomplishes this transformation. Although there are insufficient mutants in maize or other grasses to immediately test a similar hypothesis for the monocots, it seems likely that the leaf is a ground-state organ for all flowering plants. Unlike flower organs, the leaf is not dispensable and may not be reducible to any simpler structure. The lack of any gene product important for leaf identity may be lethal. We who work with higher plants will need to be particularly clever in our attempts to cover lethal mutations in the haploid gametophytes and to assay lethal mutations in somatic sectors if we are to use the genetic approach to its fullest advantage.

rrhinum

I thank members of my laboratory, past and present, for patience, insight, and data from which this paper derives. Barbara Lane and Assunta Chytry helped technically with the data of Fig. 3. I am particularly indebted to Anne Sylvester for permission to cite important unpublished results, and to Julie Vogel and my graduate students John Fowler, Phil Becraft, Lisa Harper, and Denise Porter for data, discussion, and criticism. This research is supported by NIH. REFERENCES Alberch, P., Gould, S. J., Oster, G. F., and Wake, D. B. (1979). Size and shape in ontogeny and phylogeny. Puleobiology 5, 296-317. Becraft, P., and Freeling, M. (1989). Use of the scanning electron microscope to ascribe leaf regional identities even when normal anatomy is disrupted. Maize Genet. Coop. News Lett. 63, 37. Becraft, P., and Freeling, M. (1991). Sectors of liqcluleless-1 tissue in-

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terrupt an inductive signal during maize leaf development. Plant Cell 3, 801-807. Becraft, P., Bongard-Pierce, D. K., Sylvester, A. W., Poethig, R. S., and Freeling, M. (1990). The li&eless-1 gene acts tissue-specifically in maize leaf development. Dev. Biol. 141,220-232. Bertrand-Garcia, R., and Freeling, M. (1991). Hairy-she&h fruyedl-0: A systemic, heterochronic mutant of maize that specifies slow developmental stage transitions. Am. J Bof. 78, ‘747-765. Bowman, J. L., Smyth, D. R., and Meyerowitz, E. M. (1991). Genetic interactions among floral homeotic genes of Arubidopsis. Develop merit 112, l-12. Brower, D. L. (1985). The sequential compartmentalization of Drosophila segments revisited. Cell 41, 361-364. Coen, E. S., and Meyerowitz, E. M. (1991). The war of the whorls-Genetic interactions controlling flower development. Nuture 353, 3137. Cooke, J., and Smith, J. (1990). Measurement of developmental time by cells of early embryos. Cell 60, 891-894. Dawe, R. K., and Freeling, M. (1991). Cell lineage and its consequences in higher plants. Plant J. l(l), 3-8. Esau, K. (1943). Ontogeny of the vascular bundle in Zea mays. Hi&rdin 15, 327-368. Freeling, M. (1991). Lzml-0 suppresses the Knl-0 lateral vein phenotype. Muize Genet. Coop. Newslett. 65, 33. Freeling, M., and Hake, S. (1985). Developmental genetics of mutants that specify knotted leaves in maize. Genetics 111, 617-634. Freeling, M., Bertrand-Garcia, R., and Sinha, N. (1992). Maize mutants and variants altering developmental time and their heterochronic interactions. BioEssays 14,227-236. Freeling, M., and Lane, B. (In press). The maize leaf. In “The Maize Handbook” (M. Freeling and V. Walbot, Eds.). Springer-Verlag, New York. Galinat, W. C. (1959). The phytomer in relation to floral homologies in the American Maydeace. Bot. MUS. Lea& Hurt. Univ. 19,1-32. Garcia-Bellido, A., and Merriam, J. (1971). Parameters of the wing imaginal disc development in Drosoph,ilu melunoguster. Dev. Biol. 24,61-87. Green, P. B. (1980). Organogenesis-A biophysical view. Annu. Rev. Pltrnt Physiol. 31, 51-82. Gurdon, J. B. (1992). The generation of diversity and pattern in animal development. Cell 68, 185-199. Hake, S., and Freeling, M. (1986). Analysis of genetic mosaics shows that the extra epidermal cell divisions in Knotted mutant maize plants are induced by adjacent mesophyll cells. Nature 320,21-623. Hake, S., Vollbrecht, E., and Freeling, M. (1989). Cloning Knotted, the dominant morphological mutant of maize using DsZ as a transposon tag. EMBO J. 8, 15-22. Harberd, N. P., and Freeling, M. (1989). Genetics of dominant gibberellin-insensitive dwarfism in maize. Genetics 121, 827-838. Johri, M. M., and Coe, E. H., Jr. (1983). Clonal analysis of corn plant development. I. The development of tassel and ear shoot. Dev. Biol. 131, 154-172. Kumazawa, M. (1961). Studies on the vascular course in maize plant. July 1961, 128-139. Ph ytomorphology

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Langdale, J. A., Lane, B., Freeling, M., and Nelson, T. (1989). Cell lineage analysis of maize bundle sheath and mesophyll cells. Dev. Biol. 133,128-139. Lord, E. M., and Hill, J. P. (1987). Evidence for heterochrony in the evolution of plant form. In “Development as an Evolutionary Process” (R. A. Raff and E. C. Raff (Eds.), pp. 47-70. A. R. Liss, New York. McDaniel, C. N., and Poethig, R. S. (1988). Cell lineage patterns of the shoot apical meristem in the germinating corn embryo. Planfu 175, 13-22. McDaniel, C. N., Singer, S. R., and Smith, S. M. E. (1992). Developmental states associated with the floral transition. Dev. Biol. 153,59-69. Newman, S. A., and Comper, W. D. (1990). “Generic” physical mechanisms of morphogenesis and pattern formation. Development 110, 1-18. Phinny, B. 0. (1946). Gene action in the development of the leaf in Zeu rnn!~s L. Ph.D. thesis, University of Minnesota, Minneapolis, MN. Poethig, S. (1984). Cellular parameters of leaf morphogenesis in maize and tobacco. 1n “Contemporary Problems in Plant Anatomy” (R. A. White and W. C. Dickison, Eds.), pp. 235-259. Academic Press, New York. Poethig, R. S. (1990). Phase change and the regulation of shoot morphogenesis in plants. Science 250, 923-930. Raff, R. A., and Wray, G. A. (1989). Heterochrony: Developmental mechanisms and evolutionary results. J. Evol. Biol. 2, 409-434. Russell, S. H., and Evert, R. F. (1985). Leaf vasculature in Zeu rnal4.sL. Planta 164,448-458. Sharman, B. C. (1942). Developmental anatomy of the shoot of Zeu muys L. Ann. Bot. 6,245-284. Shaver, D. L. (1976). Conversion for earliness in maize inbreds. Maize Genet. Coop. Newslett. 50, 20-23. (Cited by permission). Sinha, N., and Hake, S. (1990). Mutant characters of Knotted maize leaves are determined in the innermost tissue layers. De?!. Biol. 141, 203-210. Steeves, T. A., and Sussex, I. M. (1989). “Patterns in Plant Development” 2nd ed. London, Cambridge University Press. Sonneborn, T. M. (1970). Gene action in development. Proc. R. Sot. London Ser. B 19,347-366. Sylvester, A. W., Cande, W. Z., and Freeling, M. (1990). Division and differentiation during normal and l@uleless-1 maize leaf development. Development 110,985-1000. Takhtajan, A. (1972). Patterns of ontogenetic alterations in the evolution of higher plants. Phytomorphology 22, 164-171. Vollbrecht, E., Veit, B., Sinha, N., and Hake, S. (1991). The developmental gene Knotted-l is a member of a maize homeobox gene family. Nature 350,241-243. Williams, M. H., Vesk, M., and Mullins, M. G. (1987). Tissue preparation for scanning electron microscopy of fruit surfaces: comparison of fresh and cryopreserved specimens and replicas of banana peel. Micron. Microsc. Acta 18, 27-31. Wolpert, L. (1969). Positional information and the spatial pattern of cellular differentiation. J. Theor. Biol. 25, l-47. Wolpert, L. (1989). Positional information revisited. Development (Suppl.) 3-12.