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157 Development Supplement 2, 1991, 157-168 Printed in Great Britain © The Company of Biologists Limited 1991 Cell movements driving neuruiation in avian embryos GARY C. SCHOENWOLF Department of Anatomy, University of Utah, School of Medicine, Salt Lake City, Utah 84132, USA Summary Neuruiation, formation of the neural tube, a crucial event of early embryogenesis, is believed to be driven by the coordination of a number of diverse morphogenetic cell behaviors. Such behaviors include changes in cell number (division, death), cell shape and size (wedging, palisading and spreading), cell position (rearrangement or intercalation) and cell-cell and cell-matrix associations (including inductive interactions). The focus of this essay is on epiblast cell movements and their role in shaping and bending of the neural plate. Neuruiation is a multifactorial process requiring both intrinsic (within the neural plate) and extrinsic (outside the neural plate) forces. The origin and movements of three populations of epiblast cells have been studied in avian embryos by constructing quail/chick transplantation chimeras and by labeling cells in situ with identifiable, heritable markers. MHP (median hinge-point neurepithelial) cells originate principally from a midline epiblast area rostral to and overlapping Hensen's node. In addition, a few caudal MHP cells originate from paranodal epiblast areas. MHP cells stream down the length of the midline neuraxis in the wake of the regressing Hensen's node. This streaming occurs as a result of cell division (presumably oriented so that daughter cells are placed into the longitudinal plane rather than into the transverse plane) and rearrangement (intercalation), resulting in a narrowing of the width of the MHP region with a concomitant increase in its length. L (lateral neurepithelial) cells originate from paired epiblast areas flanking the rostral portion of the primitive streak, and they stream down the length of the lateral neuraxis concomitant with regression of Hensen's node. They do so both by oriented cell division and by intercalation. SE (surface epithelial) cells originate from the epiblast of the area pellucida, as far lateral as near the area pellucidaarea opaca border. From this area they stream medially, toward the forming lateral margins of the neural plate. Collectively, movements of the three populations of epiblast cells generate the convergent-extension movements characteristic of the epiblast during neuruiation. Heterotopic grafting has been used to assess the relationship between cell position and cell fate and to determine whether transplanted heterotopic cells can adopt the behaviors typical of the new site. For example, SE cells can replace L cells, changing their fate and adopting L-cell behavior. Similarly, prospective MHP and L cells both can change their fate and adopt the behavior of SE cells. L cells, when placed into prospective MHP-cell territory, move out of this territory by intermingling with adjacent host L cells. Likewise, prospective MHP cells placed into L-cell territory, move out of this territory by intermingling with host MHP cells. Collectively, these results suggest that cell fate is determined principally by the ultimate position of cells, and that adjacent, different cell populations are restricted from intermingling with one another. How positional information is specified, the nature of restriction of intermingling and the guidance cues used for cell navigation during streaming remain to be elucidated. Introduction tail bud, into an epithelial, longitudinal cord, the medullary cord, which subsequently cavitates, thereby establishing a lumen and forming the caudal neural tube. Why the embryo builds its neural tube in two strikingly different manners is unknown, but the fact that it does so underscores the importance of using restraint when generalizing among species and among developing systems. The only way to determine with certainty how one species constructs its rudiments, is to carefully catalog and analyze the events underlying the formation of each rudiment in that organism. Generalization does provide insight and allows one to make Formation of the neural tube, the process of neuruiation, occurs in two phases in avian and mammalian embryos. During the first phase or primary neuruiation, the flat, ectodermal neural plate rolls up into a cylindrical neural tube. This phase of neuruiation is the one that has received the most attention and, consequently, it is the one that is best understood. Secondary neuruiation, the second of the two phases, begins when primary neuruiation is nearing completion. It involves the aggregation of mesenchymal cells, derived from the Key words: cell division, cell rearrangement, cell shape, ectoderm, epiblast, neural plate, neural tube, neurepithelium, surface epithelium. 158 G. C. Schoenwolf predictions, but insight and predictions do not necessarily provide facts. I will restrict my discussion in this essay to primary neurulation, a process that can be subdivided into four distinct but spatially and temporally overlapping stages (Fig. 1): (1) formation of the neural plate, (2) shaping of the neural plate, (3) bending of the neural plate and (4) closure of the neural groove. My discussion will be further restricted to just two of these stages, shaping and bending of the neural plate, and will be based principally on information obtained, largely during the last five years in my laboratory, from chick and quail embryos. Recent studies have shown that shaping and bending of the neural plate are driven by certain morphogenetic cell behaviors, which occur both within the neural plate as well as within the surrounding tissues. A major task, which is not yet complete, has' been to catalog the relevant behaviors and to assess their respective roles in neurulation. This catalog reveals that neurulation is not an all-or-none event, but rather requires an interacting series of diverse cell behaviors, which are precisely coordinated in both time and space. Such behaviors include changes in cell number (division and death), cell shape and size (wedging, palisading and spreading), cell position (rearrangement or intercalation) and cell-cell and cell-matrix associations (including inductive interactions). The mechanisms underlying these obligatory cell behaviors, and the manner in which they are coordinated, are largely unknown and remain as challenges for future studies. Rather than discussing all cell behaviors involved in neurulation, my focus in this brief essay will be on one particular behavior, cell movement, which results in a change in the cell's position within the embryo. Neurulation is discussed more broadly in several recent reviews (Gordon, 1985; Jacobson et al. 1986; Martins-Green, 1988; Copp et al. 1990; Schoenwolf and Smith, 1990a,6; Schoenwolf, 1991). Shaping and bending of the neural plate are multifactorial processes Shaping and bending of the neural plate are multifactorial processes involving both intrinsic (i.e. within the neural plate) and extrinsic (i.e. outside the neural plate) forces acting in concert. Although the initiation of shaping precedes bending, with the onset of bending, these two processes become contemporaneous (e.g. Schoenwolf, 1982, 1983). Shaping involves intrinsic forces Shaping of the neural plate includes three characteristic events (Fig. 1A,B; Burnside and Jacobson, 1968; Jacobson and Gordon, 1976; Morriss-Kay, 1981; Jacobson and Tarn, 1982; Schoenwolf, 1985; Schoenwolf and Powers, 1987; Schoenwolf and Alvarez, 1989): (1) apicobasal thickening as most neurepithelial cells composing the pseudostratified, columnar epithelium of the flat neural plate increase their height; (2) transverse narrowing: (a) as neurepithelial cells increase in height, consequently decreasing their diameter; and (b) as neurepithelial cells rearrange, intercalating such that the number of cells spanning the transverse dimension of the neural plate decreases, while the number spanning the longitudinal dimension increases; and (3) longitudinal lengthening: (a) as neurepithelial cells intercalate, moving to decrease the width of the neural plate and to increase its length; and (b) as neurepithelial cells divide, with most daughter cells positioned so as to increase the length of the neural plate rather than its width. These processes still occur when the neural plate is isolated from lateral tissues (Schoenwolf, 1988), caudal tissues (Schoenwolf et al. 1989a) and underlying tissues (Alvarez and Schoenwolf, 1991a), suggesting that requisite forces for shaping of the neural plate originate exclusively (or at least principally) within the neural plate; that is, these forces are intrinsic to the neural plate. Bending involves intrinsic and extrinsic forces Bending of the neural plate includes two characteristic events (Figs 1B-D, 2; Schroeder, 1970; Morriss-Kay, 1981; Jacobson and Tarn, 1982; Schoenwolf, 1982, 1983; Schoenwolf and Desmond, 1984): (1) furrowing; and (2) folding. Furrowing of the neural plate, the formation of shallow, gutterlike depressions, which extend down the length of the neural plate, is driven (at least for the median hinge-point region; see below) by intrinsic forces and it is associated with the formation of localized regions termed hinge points. Three hinge points form during neurulation, a single median and paired dorsolateral; the paired regions of neurepithelium between the median and dorsolateral hinge points on each side are called the L (lateral) regions. The median hinge point (MHP) forms at the midbrain through spinal cord levels and consists of the midline neurepithelium and underlying notochord (actually, it could be argued that the MHP also forms at the forebrain level, where it consists of the midline neurepithelium and underlying prechordal plate mesoderm; my main reason for excluding the forebrain level is that this region, unlike the rest of the neuraxis, does not form a floor plate, a structure seemingly derived from MHP cells; Schoenwolf et al. 1989/?; Fraser et al. 1990). It is defined as the midline area where the neural plate becomes anchored to the notochord and longitudinal furrowing of the neurepithelium occurs. This furrowing occurs autonomously within the MHP (i.e. it does not require the presence of forces from more lateral tissues; Schoenwolf, 1988), and it occurs as a result of a change in neurepithelial cell shape from columnlike to wedgelike (i.e. cell wedging) with an accompanying decrease in neurepithelial cell height (Schroeder, 1970; Brun and Garson, 1983; Schoenwolf and Franks, 1984; Schoenwolf, 1985). Presumably, such changes in cell shape and size generate intrinsic forces, which deform the neural plate locally and establish the longitudinal furrow. Much less is known about the paired dorsolateral hinge points (DLHPs). These form throughout most brain levels, as well as the most caudal Cell movements driving neundation 159 A (A* flbr 1 1 H» • i Fig. 1. Light micrographs of living chick blastoderms on the surface of the yolk viewed dorsally through the vitelline membranes. Formation of the neural plate occurred earlier in development, so that a flat neural plate (diagonal lines) is already present in A. (A) Flat neural plate stage. (B) Initial neural groove stage; note that considerable shaping of the neural plate has occurred and that bending of the neural plate (arrow) has been initiated. (C) Incipient neural tube stage: note that the neural groove has closed at the future midbrain level (arrow). (D) Definitive neural tube stage: note that the neural tube has formed throughout most of the length of the neuraxis. ps. primitive streak: nt. neural tube. Adapted (with permission) from Schoenwolf and Watterson (1989). Bar=200/im (A); 400/im (B-D). 160 G. C. Schoenwolf Fig. 2. Scanning electron micrographs of transverse slices of chick blastoderms during shaping and bending of the neural plate. Neurepithelial cells of the MHP and DLHPs have been outlined. (A-C) Low power overviews; (D,E) enlargements of the MHP region showing an early and a late stage (E is an enlargement of C); (F) enlargement of a DLHP region, n, notochord; nf, neural fold; ng, neural groove; np, neural plate; ps, primitive streak; se, surface epithelium; arrow (in F), initial furrowing of the DLHP. Bars=100,i<m (A-C); lOjum (D-F). Cell movements driving neurulation primary neural tube level (i.e. the region of the caudal neuropore, a level derived from the sinus rhomboidalis portion of the ectodermal neural plate) and are composed, on each side, of the dorsolateral neurepithelium and the adjacent surface epithelium; that is, the two layers constituting the paired bilaminar neural folds. Whether furrowing of the DLHPs occurs autonomously within these regions and whether the associated changes that occur in the shape of its neurepithelial cells (i.e. wedging and increase in neurepithelial cell height) are induced by surface epithelium have not been studied. Folding of the neural plate, the uplifting of the neural folds with the consequent formation of the neural groove, is centered around the three hinge points and requires (at least for neural fold elevation; see below) extrinsic forces (i.e. forces generated outside the neural plate). As extrinsic forces act upon the neural plate, furrowing of the neurepithelium within the hinge points facilitates subsequent neural plate folding, and anchorage of the neurepithelium to the underlying tissues (as well as apicobasal polarization of the neurepithelium) assists in preventing the neural plate from being everted; thus, 'invagination' of the neural plate occurs rather than evagination, establishing the neural groove. Extrinsic forces cause the neural folds first to rotate dorsally (i.e. to undergo elevation), with their axis oi rotation centered on the MHP, and then medially (i.e. to undergo convergence), with the axis of rotation on each side centered on the corresponding DLHP. Thus, in the hinge-point model (reviewed by Schoenwolf and Smith, 1990b), there are 'hinge pins' (formed by localized anchorage and furrowing at the hinge points) and 'hinge plates' (formed by the paired L regions of the neural plate for the MHP and by each neural fold for the DLHPs), which are acted upon by forces arising outside the neural plate. Historically, bending of the neural plate has been viewed as being driven exclusively by intrinsic forces (reviewed by Schoenwolf and Smith, 1990a). This idea arose with the classical experiments of Wilhelm Roux (in 1885 and 1895; translated by Weiss, 1939), who reported that the neural plate 'when isolated could bend into a tube without external assistance.' However, experiments in my laboratory (unpublished) have shown that although pieces of avian epiblast do rapidly roll up when isolated completely in a fluid medium (Fig. 3A-C), they consistently roll up inside out (i.e. the epithelium curls so that its original apical side, as indicated by the presence of mitotic figures, lines the outside of the vesicle, whereas its original basal side, lines the inside of the vesicle; Fig. 3D). Hence, the fact that isolated neural plates roll up in a direction directly opposite to that occurring during normal neurulation (also see experiments by Burnside, 1972; Jacobson, 1981; Vanroelen et al. 1982; Stern et al. 1985), suggests not that neurulation is driven exclusively by intrinsic forces (as originally concluded from Roux's experiment), but that such rolling up is an artifact of the isolation and culturing of epithelial sheets. The role of extrinsic forces in folding of the neural 161 Fig. 3. Light micrographs of isolated plugs of epiblast. (A-C) Living plugs viewed from their basal (ventral) side over a 2 h period (lettered in order of increasing time) following their isolation; note the direction of curling. (D) Section through an isolated plug; note the direction of curling. Arrow, mitotic figure at the apical side of the plug. Bar=120f<m (A-C); 30jxm (D). 162 G. C. Schoenwolf plate has been assessed in two general ways: by ablation experiments, and by the analysis of the behaviors of cells in non-neurepithelial tissues. Ablation experiments involving enzymatic (or otherwise) depletion of various components of the extracellular matrix, or microsurgical removal of tissues lateral to the neural plate, resulted in a delay, severe inhibition or complete failure of bending of the neural plate (Morriss-Kay and Crutch, 1982; Morriss-Kay etal. 1986; Morriss-Kay and Tuckett, 1989; Anderson and Meier, 1982; Schoenwolf and Fisher, 1983; Schoenwolf, 1988; Smits-van Prooije etal. 1986; Tuckett and Morriss-Kay, 1989). In contrast, removal of tissue caudal to or beneath the neural plate had little or no effect on this process (Schoenwolf et al. 1989«; Alvarez and Schoenwolf, 1991a). Collectively, these results suggest that tissues (i.e. cells and associated extracellular matrix) lateral to the neural plate cause folding of the neural plate. What might the relevant tissues be? Three tissues form lateral to the neuraxis: (1) the surface epithelium, continuous medially with the neural plate; (2) the mesoderm, consisting principally of lateral plate; and (3) the endoderm, forming the fore, mid- and hindgut regions. Removal of the endoderm and mesoderm, but not the ectoderm, lateral to the neural plate does not prevent folding of the neural plate, implicating the only remaining tissue, the surface epithelium and its associated extracellular matrix, in this process (Alvarez and Schoenwolf, 1991a). What are the morphogenetic cell behaviors that occur within the surface epithelium to generate extrinsic neurulation forces? Much work remains to be done. Nevertheless, our initial study of the surface epithelium indicates that at least three cell behaviors are involved (Schoenwolf and Alvarez, 1991): cell division, cell rearrangement and change in cell shape from columnar to squamous (i.e. cell spreading). Spreading of the epidermis (i.e. surface epithelium) toward the dorsal midline also occurs 4A _ during neurulation in amphibians (Jacobson, 1962; Jacobson and Jacobson, 1973; Brun and Garson, 1983). Cell movements within the epiblast during neurulation The movements of three types of epiblast cells, MHP cells (i.e. neurepithelial cells within the MHP), L cells (i.e. lateral neurepithelium cells between the MHP and DLHPs) and surface epithelial (SE) cells (i.e. ectodermal cells lateral to the neural plate) have been studied during shaping and bending of the neural plate. Two experimental paradigms have been used to analyze the direction and amount of such movement in each cell population (Figs 4, 5): (1) construction of quail/chick transplantation chimeras (Schoenwolf et al. 19896; Schoenwolf and Alvarez, 1989, 1991; Alvarez and Schoenwolf, 1991b); and (2) microinjection of a fluorescent-histochemical marker (rhodamine-conjugated horseradish peroxidase) into the epiblast, through which single epiblast cells or small groups of epiblast cells are labeled (Schoenwolf and Sheard, 1989, 1990). In essence, the two approaches provided the same information, so their results will be combined in the following discussion. MHP cells Shortly after formation of the neural plate, prospective MHP cells are located in three areas of the epiblast. The first two of these areas contribute almost all the MHP cells; the last area's contribution is relatively minor. Essentially all the MHP cells of the future brain levels are situated in a single, midline area located just rostral to Hensen's node. From this area cells stream caudally, in synchrony with the regression of Hensen's node, to form the MHP cells of the midbrain and hindbrain levels, as well as contributing some of the MHP cells of the spinal cord level (see below). Cells from the B Fig. 4. Sections of a quail/chick chimera 24 h after transplantation (A; arrows indicate quail cells in the surface epithelium of the neural fold) and of a chick embryo (B), whose flat neural plate was injected 24h earlier with rhodamine-conjugated horseradish peroxidase (arrows indicate labeled cells in the neural tube). Bar=20,um (A); 15jum (B). Cell movements driving neurulation 163 Fig. 5. Combined fluorescent and bright-field micrographs of the dorsal surface of chick blastoderms injected with rhodamine-conjugated horseradish peroxidase. Two cases are shown (A,B> one case; C,D, another case). Note that injected areas of the epiblast (encircled) move medially and caudally during shaping and bending of the neural plate. A,B, Oh and 6.5h after injection, respectively, n, notochord; ps, primitive streak; hn, Hensen's node. C,D, Oh and 6.5h after injection, respectively. Bar=200,um. prenodal area also move rostrally to contribute to the floor of the forebrain and the ventrolateral aspects of the optic vesicles. Hensen's node is a second area that contributes MHP cells. Although it is unclear exactly where such prospective MHP cells arise in the node, because the node is capped by epiblast, which is directly continuous with the surrounding perinodal epiblast, it seems likely that the cap is the source of these cells. Hensen's node contributes MHP cells to the spinal cord level, supplementing those derived from the prenodal area. Recent experiments suggest that the paranodal areas immediately flanking Hensen's node collectively constitute a third area normally providing MHP cells; however, the paranodal areas provide only a few MHP cells and they do so only to the caudal levels of the primary neural tube (Schoenwolf and Alvarez, 1991). Interference with the contribution made by any one of the three sources of MHP cells, results in a compensa- 164 G. C. Schoenwolf tory increase in the number of cells contributed by the remaining sources (Smith and Schoenwolf, 1991; Schoenwolf et al. unpublished data). Hence, each of the three sources of MHP cells apparently gauges the number of such cells required at each rostrocaudal level throughout the process of MHP formation; it then inserts the appropriate number of cells into the forming MHP region to fill in any gaps that would arise otherwise. Seemingly, such a system would work only if prospective MHP cells are induced rather than preprogrammed (which they are; see below), and it would increase the assurance that a normal MHP region, an important region for organizing neurons within the developing neural tube (Placzek et al. 1990a; Hirano et al. 1991), would form. Prospective MHP cells exhibit at least four types of morphogenetic behavior during shaping and bending of the neural plate: cell division, change in cell shape and size, cell-cell inductive interactions and cell rearrangement. The cell-cycle time of MHP cells is about 12 h (e.g. at brain levels), approximately 65% longer than that of cells of the flat neural plate prior to formation of MHP and L regions (Smith and Schoenwolf, 1987). Furthermore, most (i.e. approximately 70%) MHP cells are wedgeshaped, in contrast to cells of the fiat neural plate, most (i.e. approximately 70%) of which are spindleshaped, and MHP cells have a decreased height that is about 25 % less than that of the cells of the flat neural plate (Schoenwolf and Franks, 1984; Schoenwolf, 1985). These changes in cell shape and size, relative to that of neurepithelial cells of the flat neural plate, arise as a result of cell-cell inductive interactions with the notochord (van Straaten et al. 1988; Smith and Schoenwolf, 1989; Placzek et al. 1990fo). Finally, MHP cells undergo extensive cell rearrangement during neurulation, namely, lateromedial intercalation (Schoenwolf and Alvarez, 1989). During such intercalation, cells move medially within the width of the neural plate, and as a consequence of this rearrangement, the number of cells arrayed within the longitudinal plane of the neural plate is increased, while the number of cells spanning its transverse dimension is decreased. Such intercalation results in convergent-extension of the neural plate, a movement defined as a narrowing of the width of the neural plate with a concomitant increase in its length. Convergentextension has perhaps been best documented in echinoderm, amphibian and fish embryos, where it is believed to be one of the principal motors driving gastrulation (Keller, 1980; Keller et al. 1985; Keller and Trinkhaus, 1987; Ettensohn, 1985; Hardin, 1989; Warga and Kimmel, 1990). L cells L cells apparently have a simpler origin than do MHP cells. Shortly after formation of the neural plate, prospective L cells are located in paired, bandlike epiblast areas flanking Hensen's node and the rostral portion of the primitive streak (hence, they overlap the areas that contribute a few MHP cells to the caudal primary neural tube). From these bilateral areas cells stream caudally, in synchrony with the regression of Hensen's node, to form the paired L regions of the midbrain through spinal cord levels. Cells from the bilateral areas also move rostrally to contribute to the roof of the forebrain and the dorsolateral aspects of the optic vesicles. The exact longitudinal extent of the areas alongside the primitive streak that contribute to the L regions of the neural plate is a matter of controversy; estimates of the area's length range from about 500 ,um (Spratt, 1952) to something over 1 mm (Schoenwolf and Sheard, 1990). Prospective L cells exhibit at least three types of morphogenetic behavior during shaping and bending of the neural plate: cell division, change in cell shape and size and cell rearrangement. The cell-cycle time of L cells is about 8h (e.g. at brain levels), identical to that of cells of the flat neural plate prior to formation of MHP and L regions (Smith and Schoenwolf, 1987). Furthermore, most (i.e. approximately 70%) L cells are spindleshaped, like the cells of the flat neural plate, but L cells have an increased height that is about 30 % greater than that of the cells of the flat neural plate (Schoenwolf and Franks, 1984; Schoenwolf, 1985). Finally, L cells undergo extensive rearrangement during neurulation as a result of lateromedial intercalation, and through this process they generate a convergent-extension movement (Schoenwolf and Alvarez, 1989). SE cells Shortly after formation of the neural plate, prospective SE cells are located in an ovalshaped ring (with its long axis oriented rostrocaudally) whose outer edge lies near the area pellucida-area opaca border of the epiblast. Cells within this ring form both extraembryonic and intraembryonic SE cells. Extraembryonic SE cells remain within the peripheral portion of the area pellucida, and with formation of the body folds (i.e. the head, lateral and tail body folds), these cells become distinguishable from the intraembryonic SE cells. They remain peripheral to the body folds and contribute to the extraembryonic membranes (i.e. amnion and chorion). Extraembryonic SE cells exhibit at least two types of morphogenetic behavior during shaping and bending of the neural plate (Smith and Schoenwolf, 1987; Schoenwolf and Alvarez, 1991): cell division, and change in cell shape and size. Extraembryonic SE cells undergo about two divisions (i.e. the cell-cycle time of SE cells is about 10 h, or in other words, it is shorter than that of MHP cells but longer than that of L cells). In addition, extraembryonic SE cells change their shape and size, converting from cuboidal to squamous and thereby decreasing their height by about 60%. As a consequence of cell division and change in cell shape and size, grafts of extraembryonic SE cells exhibit radial expansion (as considered en face in surface view), increasing their surface area extensively. Intraembryonic SE cells become incorporated into the embryo proper with formation of the body folds, and they contribute to the outer epithelial 'tube' (i.e. the 'skin') of the typical vertebrate tube-within-a-tube Cell movements driving neurulation body plan. Intraembryonic SE cells, like extraembryonic SE cells, exhibit at least two types of morphogenetic behavior (Smith and Schoenwolf, 1987; Schoenwolf and Alvarez, 1991). These cells undergo about two divisions during shaping and bending of the neural plate, but they essentially maintain their original shape and size. Additionally, intraembryonic SE cells undergo extensive rearrangement, namely, lateromedial intercalation, which generates a convergentextension movement. How are cell fates determined? There are two basic scenarios to account for the specification of cell fate during neurulation. Either cells could be predetermined for a particular developmental fate prior to initiating their movement (i.e. cells already could be specified to be: neurepithelium or surface epithelium; MHP neurepithelial cells or L neurepithelial cells; or extraembryonic or intraembryonic surface epithelial cells), or they could be undetermined for a particular development fate until after their movement is completed (i.e. cell fate could be specified by local cell-cell inductive interactions near the final area of cell residence). Recent experiments, based either on (1) addition or subtraction of notochords or notochordal precursor cells, or on (2) heterotopic grafts of quail epiblast plugs to host chick embryos, provide strong evidence that the latter scenario is the principal one used. In the first type of experiment, it was shown that in the absence of the notochord, MHP cells fail to form, and in the presence of ectopic notochords, supernumerary MHPs develop (van Straaten etal. 1988; Smith and Schoenwolf, 1989; Placzek et al. 1990ft). Thus, instructive, inductive interactions are required for MHP formation. In the second type of experiment, it was shown that prospective L cells, isolated in MHPcell territory, form MHP cells when they come to overlie the notochord, and prospective MHP cells, isolated in L-cell territory, form L cells when they are removed from the influence of the notochord (Alvarez and Schoenwolf, 19916). This experiment provides further evidence of the induction of MHP cells by the notochord and also provides evidence of the induction of L cells (namely, because L cells differ from cells of the flat neural plate, and when the latter are transplanted, they form L cells; therefore, this difference between cells of the flat neural plate and L cells depends upon the cell's location, not its area of origin). Whether this induction occurs 'vertically' (i.e. a signal originating from mesodermal and/or endodermal tissues underlying L cells) or 'horizontally' (i.e. an 'edgewise' signal originating from adjacent L cells) remains to be elucidated. Further experiments of the second type have shown that SE and neurepithelial cells also arise as a result of local inductive interactions (Schoenwolf and Alvarez, 1991). For example, prospective SE cells placed into L-cell territory form typical L cells and, conversely, prospective L cells placed into surface epithelium form surface epithelium (alternatively, in this latter case the surface epithelium could be merely a default pathway). Collectively, this series of exper- 165 iments demonstrates that cells of avian embryos possess a tremendous ability to adapt to changes in their local environment, presumably accounting (at least in large part) for their capability to regulate and reconstitute regions of the neurepithelium deleted experimentally (e.g. Alvarez and Schoenwolf, 1991ft). How are patterns of cell movement established? The patterns of cell movement during neurulation are complex and the question of how they are established is far from answered. Here, I will consider two points. First, individual cell movement is highly influenced by movement of like cells in a surrounding cell stream. Thus, for example, when plugs of prospective L cells are transplanted to L-cell territory with the correct apical-basal orientation, but with incorrect rostralcaudal and medial-lateral orientations, their pattern of rearrangement still corresponds to that of the host (Alvarez and Schoenwolf, 1991ft). This result shows that cells do not go 'upstream' (i.e. against a 'current' of streaming cells) even when 'disoriented' in their normal territory and surrounded by like cells. Second, patterns of cell movement for some types of cells are shown to be labile and for other types of cells are shown to be fixed, when cells are placed in atypical locations (Alvarez and Schoenwolf, 1991ft; Schoenwolf and Alvarez, 1991). For example, prospective L cells (or prospective MHP cells) placed into surface epithelium adopt the rearrangement pattern of surrounding host SE cells, and, conversely, prospective SE cells placed into L-cell territory adopt the rearrangement pattern of surrounding host L cells. By contrast, prospective MHP cells placed into L-cell territory, prospective L cells placed into MHP-cell territory and prospective SE cells placed into MHP-cell territory cannot adopt the rearrangement pattern typical of their new site. Instead, these cells intermix (when the opportunity exists) with adjacent host cells of the like type, preferentially intercalating with them. How and when such cell rearrangement preference is established is unknown. Hypothesis: inhibition of cell rearrangement results in neural tube defects According to the hinge-point model, discussed above, bending of the neural plate is driven by both intrinsic forces (i.e. generated by cell wedging, which leads to furrowing of the neural plate) and extrinsic forces (generated by lateral tissues, presumably surface epithelium and its associated matrix, which leads to folding of the neural plate; that is, to the elevation and convergence of the neural folds). Cell rearrangement during shaping of the neural plate facilitates subsequent folding of the neural plate by decreasing the width of the neural plate and by increasing its length, thereby allowing extrinsic forces to lift the neural folds toward the dorsal midline where they fuse to establish a closed neural tube. An hypothesis that emerges from the work discussed above is that inhibition of neurepithelial cell rearrangement results in neural tube defects; namely, that such inhibition would result in a neural plate with a wider (mediolateral) and shorter (rostrocaudal) con- 166 G. C. Schoenwolf figuration than is normal, and consequently, that such an ungainly neural plate would serve as an impediment, opposing extrinsic forces in their endeavor to elevate the neural folds. Unpublished evidence from my laboratory suggests that in many cases, embryos with anencephaly have apparently wider and shorter neural tubes than do embryos undergoing normal neurulation (Fig. 6). Moreover, recall that SE cells, like neurepithelial cells, also undergo rearrangement, and that this process is implicated in providing driving forces for bending of the neural plate. Consequently, inhibition of cell rearrangement within the surface epithelium might decrease the overall magnitude of the extrinsic forces, thereby resulting in neural tube defects. The possibility that some neural tube defects are generated by decreased cell rearrangement is an important consideration worthy of future studies, and it emphasizes the value of careful analysis of the cell behaviors underlying normal morphogenesis. Undoubtedly, neural tube defects arise in a multiplicity of ways, and it is not unreasonable to suspect that inhibition of any of the fundamental morphogenetic cell behaviors would cause neurulation to go awry. Thus, we must remember the complexity of the system when we consider the highly relevant but difficult-to-answer question underlying birth defects: What went wrong? Concluding remarks Analysis of cell behaviors during morphogenesis reveals both the native beauty of the embryo as well as its immense complexity. In view of such complexity, how do we proceed in our analysis? The approach we have chosen in my laboratory is based on the assumption that the behaviors of individual cells and groups of cells drive morphogenesis. Consequently, my laboratory has focused on cataloging normal cell behaviors and exploring how such behaviors are regulated. Many questions about mechanisms of regulation still remain. The embryo is 'a difficult nut to crack," but with the proper "nutcrackers" the job can be done. I wish to thank Jennifer Parsons and Fahima Rahman for excellent assistance, and Dr I. Santiago Alvarez for providing Figs 3D and 4A, unpublished micrographs from his studies in my laboratory. The original research described herein from my laboratory was funded by grants principally from the National Institutes of Health. References ALVAREZ. I. S. 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