Cellular patterning of the vertebrate embryo

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Cellular patterning of the vertebrate embryo Luc Mathis and Jean-Francois Nicolas Recent studies show that cell dispersal is a widespread phenomenon in the development of early vertebrate embryos. These cell movements coincide with major decisions for the spatial organization of the embryo, and they parallel genetic patterning events. For example, in the central nervous system, cell dispersal is first mainly anterior–posterior and subsequently dorsal–ventral. Thus, genes expressed in signaling centers of the embryo probably control cell movements, tightly linking cellular and genetic patterning. Cell dispersal might be important for the correct positioning of cells and tissues involved in intercellular signaling. The emergence of cell dispersal at the onset of vertebrate evolution indicates a shift from early, lineage-based cellular patterning in small embryos to late, movement-based cellular patterning of polyclones in large embryos. The conservation of the same basic body plan by invertebrate and vertebrate chordates suggests that evolution of the embryonic period preceding the phylotypic stage was by intercalary co-option of basic cell activities present in the ancestral metazoan cell. Published online: 01 November 2002

Early studies using mouse chimeras [1] led to the fascinating finding that any given structure of the embryo, and indeed the embryo itself, derive from pools of progenitor cells termed polyclones (Box 1). Direct visualization of clones in mouse and also in amphibians, fish, birds and mammals demonstrates that this is a general occurrence in

vertebrates (Fig. 1). This observation implies a sustained CELL DISPERSAL (see Glossary) and intermingling of cells (dispersal growth), before their allocation to given structures and fate acquisition. This is in sharp contrast to what happens in most invertebrate species, where the structures of the embryo are derived from a very small number of genealogically related cells and cell specification precedes cell motility, which is absent until late in development [2] (coherent growth: sister cells remain close to each other after cell division) (Fig. 1a, blastomere A7.8). This applies to the two invertebrate groups closest to vertebrates, UROCHORDATES (ascidians) [3] and CEPHALOCHORDATES (amphioxus) [4,5] (Box 2). Therefore, a major operation of development, the allocation of the founder cells to the definitive territories, has evolved from a lineage-based mode in early CHORDATES to a movement-based mode in vertebrates. It is likely that this has involved combination of basic cell activities into complex cellular operations, and intercalation [6] of these operations into preexisting regulatory networks. This ‘intercalary co-option’ could be one of the reasons for the conservation of the phylotypic stage (Box 2) within chordates.

Glossary

Luc Mathis Jean-Francois Nicolas* Unité de Biologie moléculaire du Développement, Institut Pasteur, 25, rue du Docteur Roux, 75724 Paris Cédex 15, France. *e-mail: [email protected]

Cell dispersal: loss of coherent clonal growth; that is, dispersal of the descendants of an individual cell. Cephalochordates (amphioxus): Main character: a small anterior brain vesicle and a notochord extending anteriorly beyond the mouth and dorsal nerve tube and posteriorly to the tip of the tail. Cellular patterning: the process that allocates cells to definitive territories through a temporal and spatial distribution of cell behaviors. Chordates: (sister group hemichordates): groups three phyla urochordates, cephalochordates and vertebrates. Characters common to these phyla are the chord, the neural tube, the longitudinal muscles along the chord, a postanal tail and the ciliated pharyngeal gill slits. Convergence–extension movements: a cellular operation by which cells intercalate to narrow and elongate a tissue. Epiblast: also called primitive ectoderm (mouse), a group of epithelial cells within the blastocyst (mouse) or blastoderm (chick) at the origin of the embryo proper. Epiboly: the movement of the ectoderm to enclose the deeper layers of the embryo. In amphibians and fish it involves radial intercalation. In ascidians it only involves flattening and spreading of ectoderm cells. Gastrulation: a process in which the precursor cells of the endodermal and mesodermal organs move from the surface of the embryo to its interior. Genetic patterning: the assignation of regulatory states to unspecified cells. Germ band: An extending mass of cells along the ventral midline of the early Drosophila embryo from which most of the future embryo will develop. This is the phylotypic stage of insects.

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Hensen’s node: (chick and mouse embryos): a condensation of cells at the most anterior part of the primitive streak. It is the equivalent of the Spemann organizer in amphibians. Intercalary evolution: a mode of evolution in which upstream functions are added from an initial evolutionary state. The structure gains more complexity but the association with the regulatory gene persists from the earliest evolutionary stage [6]. Medio-lateral intercalation: A process by which cells intercalate between their medial and lateral neighbors to narrow the tissue medially and lengthen it antero–posteriorly. Ordered cell mingling: cells intermingle only with their closest neighbors in contrast with dispersive cell mingling. Primitive streak: a thickening of the epiblast cell layer at the posterior region of the avian and mammalian embryo. During gastrulation, cells move through the streak into the interior of the embryo. Radial intercalation: A process by which cells of a multi-layered tissue intercalate in a direction perpendicular to the surface, so thinning and extending the cell sheet. Rhombomeres: a series of periodic swellings due to cell lineage restriction in the hindbrain that divides the rhombencephalon into compartments. Segmentation: a cellular operation that divides the body into morphologically similar units. Spemann organizer: a signaling center of the amphibian embryo that organizes the antero-posterior and dorso-ventral axes. Urochordates (ascidians): main character: a tadpole larvae with a chord only in the tail.

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Box 1. Individualization of progenitor pools: polyclones, founders and mechanisms. Embryogenesis involves the individualization of cell populations into pools of progenitors with shared characteristics. Formally, a given structure acquires a polyclonal origin from a pool of founder cells when the clones derived from these founder cells populate mainly that structure. Founder cells produce progenitors, which are specified uni- or pluripotent cells. The origin of the polyclone (the founder cell population) is defined when mixing of cells of different polyclones no longer occurs, which indicates the clonal individualization of the structure [a]. Different cell behaviors can lead to the individualization of cellular pools of founder cells. A low degree of cell mingling implies that the polyclones will have a low probability of mixing until the structures become individualized. This is a general property of the cell population that has been observed in the forming neural system in frog [b] and in mouse, as a result of a change in the orientation of cell dispersal [c]. Cell movements in opposite directions will also lead to cell populations that have a very low probability of mixing [d]. This particular mechanism involves both population movements and the behavior of individual cells. Finally, the specific arrest at a compartment boundary is another way of separating cell populations [e]. This type of mechanism appears related to cell sorting or cell exclusion [f] and involves the behavior of individual cells rather than cell populations as a whole. Therefore, non-mutually exclusive mechanisms involving different levels of organization (single cells, cell populations, tissues) can ultimately lead to the individualization of cellular populations. References a Eloy-Trinquet, S. et al. (2000) Retrospective tracing of the developmental lineage of the mouse myotome. Curr. Top. Dev. Biol. 47, 33–80 b Wetts, R. and Fraser, S.E. (1989) Slow intermixing of cells during Xenopus embryogenesis contributes to the consistency of the blastomere fate map. Development 105, 9–15 c Mathis, L. et al. (1999) Successive patterns of clonal cell dispersion in relation to neuromeric subdivision in the mouse neuroepithelium. Development 126, 4095–4106 d Mathis, L. and Nicolas, J.F. (2000) Different clonal dispersion in the rostral and caudal mouse central nervous system. Development 127, 1277–1290 e Fraser, S. et al. (1990) Segmentation in the chick embryo hindbrain is defined by cell lineage restrictions. Nature 344, 431–435 f Xu, Q. et al. (1999) In vivo cell sorting in complementary segmental domains mediated by Eph receptors and ephrins. Nature 399, 267–271

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Early gastrulation movements

Clonal analysis, where a cell is marked, enabling its progeny to be followed, permits the description of the expansion of cellular clones from the embryo. This provides an approach to understanding the allocation of the founder cells of the territories and their specialization [7]. Clonal analyses in amphibians [8–11], fish [12–14], chick [15–18] and mouse [19–21] reveal widespread cell dispersal in epithelial sheets of the embryos starting before GASTRULATION. This is in spite of considerable differences in the geometry among the embryos: spherical (amphibians), concave (fish), flat (chicks) or cylindrical (mouse). In all vertebrates, the degree of cell dispersal and the orientation of cell and tissue movements vary greatly depending on their location in the EPIBLAST, with a high degree of cell dispersal along the midline (extension of axial regions), a convergence of ectoderm and presumptive mesoderm towards the midline and the PRIMITIVE STREAK, respectively. In addition, there is a flattening, spreading and RADIAL INTERCALATION of ectoderm cells into the vegetal hemispheres in fish [13] and amphibians (EPIBOLY) [22] (Fig. 2a,b). By contrast, for ascidians, which are generally considered as the prototypes for the ancestral chordate [23] (Box 2), the cleavage pattern up to the http://tig.trends.com

Fig. 1. Cell dispersal in chordates. (a) Photographs of Halocynthia rovetzi (ascidian) embryos in which horseradish peroxidase (HRP) was injected into A7.8 or A7.3 blastomere (in green) at the 64-cell stage. A7.8 blastomere forms part of the spinal cord and some muscle cells. Its division generates a very coherent group of cells; A7.3 blastomere forms the chord by medio-lateral intercalation leading to disruption of clonal coherence. This is the sole structure displaying cell dispersal in ascidians [24]. (b) Map of the clonal progeny of the 64-cell stage blastomere L1211 (injected with a lineage tracer, in green) in the central nervous system and notochord medially, and in all other tissues laterally, of the zebrafish. The cells of the clone are remarkably dispersed with small clusters of labeled cells mixed among unlabeled neighbors [74]. (c) A LacZ clone observed in the central nervous system of a LaacZ transgenic mouse embryo at embryonic day E12.5. The labeling occurred before the extensive rostro-caudal dispersion of the precursors of the central nervous system [21]. Scale bar: (a) 75 µm; (b) 150 µm; (c) 800 µm. SC, spinal cord; rhomb, rhombencephalon.

gastrula stage is invariant and essentially the same in eggs of all species. The allocation of the founder cells of most definitive territories is almost complete by the 64- to 128-cell stage, early gastrulation is executed before the embryonic cells acquire any capability for cell motility [24], and epiboly only involves flattening and spreading of ectodermal cells without any radial intercalation [25]. Similarly, amphioxus (cephalochordates) have a determinate cleavage pattern [4] and their gastrulation involves

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Box 2. The body plan: a major constraint in chordate evolution The chordate body plan is characterized by a specific disposition of distinctive domains (e.g. chord, somites, the neural tube, gill slits and postanal tail). All chordate embryos look alike at a particular stage, the phylotypic stage, despite major morphological differences preceding and succeeding it. The formation of the body plan is the result of a sequence of signals between cellular domains (e.g. induction of the ventral nervous system by the notochord). Most evolutionary constraints act on the phylotypic stage: perturbing this sequence leads to nonviable animals [a,b]. Consequently, the constraints of the phylotypic stage are likely to define homologous traits sharing a common evolutionary history rather than convergent evolution. Earlier events are much less constrained; for instance the mechanisms of determination of the axes in vertebrates appear to be diversified [c]. The identification of not only the molecular but also the cellular events common to the development of all vertebrates should help elucidate the constraints that act on the phylotypic stage. In this article, we argue that the conservation of the body plan of chordates corresponds to the conservation of the genetic coordinates of the axes of the embryo and that the differences in cellular strategy between invertebrates and vertebrate chordates arose, in part, by intercalary evolution from the genetic regulatory architecture of the phylotypic stage.

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Fig. 2. Cell movements in vertebrates. (a) At the onset of gastrulation, the embryonic axis lengthens by (1) expansion (orange arrows) of the axial structures (in red) located in the dorsal part of the blastopore or in the region just anterior to the node, and (2) by the progressive expansion of the ectoderm (green arrows) towards the region of involution (in blue). Simultaneously, the ectoderm covers the whole embryo (epiboly). The homologous organizer structures (node or dorsal lip of the blastopore) are shown in purple. (b) Neurulation movements are characterized by an anterior expansion of forebrain progenitors (restricted movements, red arrows and double arrows) and by the posterior directed movement of spinal cord progenitors (blue arrows). Ant, anterior; Ect, ectoderm; M, presumptive mesoderm; N, presumptive neurectoderm; Post, posterior.

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Fig. I. Metazoan phylogeny. The developmental mode can be by determination in the blastomeres (colored spheres) or after gastrulation (embryo shapes at the phylotypic stage).

References a Raff, R.A. (1996) The Shape of Life: Gene, Development and the Evolution of Animal Form, University of Chicago Press. b Duboule, D. (1994) Temporal colinearity and the phylotypic progression: a basis for the stability of a vertebrate Bauplan and the evolution of morphologies through heterochrony. Development (Suppl.), 135–142 c Beddington, R.S. and Robertson, E.J. (1999) Axis development and early asymmetry in mammals. Cell 96, 195–209

neither exaggerated involution in the region of the dorsal lip of the blastopore nor any marked tendency for epiblast cells to converge towards the dorsal midline [5]. Thus, it appears that the period that precedes gastrulation is characterized in vertebrates by a loss of coherent clonal growth reflected by cell dispersal and, in uro- and cephalochordates, by invariant lineages. As we will see, this difference persists during the following stages of development, in particular during axis elongation. Late gastrulation movements and axis elongation

In vertebrates, following the initial cell movements at gastrula, the elongation of the body axis (trunk, spinal cord) is a very long process, which continues http://tig.trends.com

during primitive streak extension, neurulation and tail development (up to embryonic day [E] 13.5 in the mouse). Elongation movements in Xenopus and zebrafish

In Xenopus [8,26] and zebrafish [27], the cellular basis of the initial movements of axis elongation has been described by time-lapse video-microscopy of labeled cell populations. In these species, the convergence of cells towards the midline and the embryonic shield, and their MEDIO-LATERAL INTERCALATION at the onset of gastrulation lead to the elongation of these cell populations towards the animal pole (extension) (Fig. 3a). The CONVERGENCE–EXTENSION cell movements are maximal in the region of the SPEMANN ORGANIZER (amphibians)/embryonic shield (fish). Starting before the formation of a visible primitive streak (or a shield in fish), the progenitors of the prospective forebrain expand anteriorly [14,28–30]. After the extension of the primitive streak, the progenitors of the spinal cord are displaced towards the posterior in the trunk [8,10,14,15,21]. These cell movements in the epiblast contribute to the initial shaping of the neural tube and to the early segregation of spinal cord and brain progenitor pools, which accompanies the initiation of axis elongation in all vertebrate species (Fig. 2a,b; Box 3). Striking similarities in the initial cell

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Fig. 3. Cell behavior during trunk elongation. (a) Convergence and extension. The convergence of cells (black arrows) and their intercalation result in the elongation of the cell population. Such behavior involves several basic processes, including cell motility, changes in adhesiveness and coordinated polarity of cell movements. (b) Progression of self-renewing precursors. Self-renewing precursors (S, red) resident in the caudal region of the embryo remain posterior and add cells that leave the pool of self-renewing precursors (blue on the left, yellow on the right). These cells mingle with cells located in the neural plate (NP) and eventually form the neural tube where they become more static. The dynamics of cells passing from the caudal neural plate to the neural tube implies that cells adopt each of these types of cell behavior sequentially. (c) For production of a structure by self-renewing permanent precursors there are more cell divisions to generate the posterior regions. Ant, anterior; Post, posterior.

movements are observed among species during early gastrulation (Fig. 2a,b). Later, in the nervous system, movements of convergence–extension are mainly confined to the spinal cord [10,11,14]. Elongation movements in mouse and chick

In mouse and chick embryos, the cellular basis of axis elongation has been described by clonal analysis. Elongation movements appear to result from the proliferation of cells resident in the node region (the equivalent of the dorsal lip of the blastopore in amphibians and of the embryonic shield in fish) and in the primitive streak (Fig. 3b) and later in the tail bud. The analysis of clones, generated using the spontaneous activation of a LacZ gene (termed LaacZ, Box 3) in transgenic mouse embryos, in a derivative of the somite, the myotome [31–35], and in the nervous system [21,36], has provided evidence for this model. The descendants of single cells were frequently distributed along the entire embryonic axis (clonal continuity); however, the clones more frequently give descendants in the posterior part of the embryo. Because the progenitors are labeled at random by a spontaneous recombination event, this suggests that more cell divisions are necessary to generate the posterior regions [21,31] (Fig. 3c). Time-lapse microscopy in the chick following electroporation of green-fluorescent protein (GFP) into neural progenitors in the region of HENSEN’S NODE [18] and clonal analysis in the mouse [21] suggest that dividing http://tig.trends.com

progenitors remain in the node region (see also [37]). Such characteristics are best explained by postulating that clones are produced by self-renewing permanent precursors that progress together with the node (Fig. 3c). The self-renewing precursors could be displaced towards the posterior by the continuous addition of cells in the neural plate. Co-electroporation of a dominant–negative fibroblast growth factor (FGF) receptor with a lineage marker into Hensen’s node in chick embryos markedly alters the elongation of the spinal cord primordium. This indicates that FGFs promote the continuous development of the posterior nervous system by maintaining self-renewing neural progenitors in the region near Hensen’s node [18]. It is not entirely clear whether the differences in mechanisms of elongation described between fish and amphibians, and chick and mouse reflect a radical change from convergence–extension movements to self-renewing progenitors. For instance, in amphibians, the maintenance of cells in the organizer [38] and the continuation of extension movements in the caudal end of the elongating trunk [39] suggest that at least part of the behavior of cells could be conserved between amphibians, mammals and possibly fish [40]. Obviously, methods comparable to the LaacZ approach are required for amphibia and fish to determine whether the mechanism of elongation at neurula and tail-bud stages is also based on self-renewing permanent precursors. It is also clear that more information about the rearrangement of cells during early and late gastrulation is required in mammals and chick. Whatever the case may be, it appears that elongation of the embryo along the antero–posterior axis, by convergence and medio-lateral intercalation, is a complex operation. For instance, cell intercalation must regulate and integrate many basic cell activities, cell polarity, cell motility, orientated protrusive activity of the cells, localized contraction and changes in cell adhesion (reviewed in [41]). The Wnt, Notch and FGF pathways, as well as interactions between these pathways, are involved in elongation movements of the vertebrate axis [18,42–44]. The function of these signals in the control of cellular operations has been resolved only for the noncanonical Wnt pathway in convergence–extension movements in Xenopus and zebrafish. Thus, in Xenopus, inhibition of Dishevelled function, Dishevelled being a component of the Wnt pathway, disrupts convergence–extension movements of cells during gastrulation, because of defects in cell polarity [45–47]. Silberblick, a zebrafish mutant with defects in these movements has a mutation in the Wnt11 gene [42]. Zebrafish knypek mutants that encode a member of the glypican family of heparan sulfate proteoglycans have impaired gastrulation movements of convergence–extension: mutant cells fail to elongate and align medio-laterally [48]. These observations indicate that noncanonical Wnt signaling controls the polarized cell behavior that underlies cell movements leading to convergence–extension.

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Box 3. The LaacZ system Clonal analysis can be used to elucidate the cellular patterning of the embryo. In vertebrate species, the visualization of cellular clones is very challenging owing to the long period of development, the large number of cells and, in mammals, the inaccessibility of the embryo. These difficulties have been partially overcome by using various cell labeling techniques (reviewed in [a]) including tetraparental mouse chimeras, quail grafts in the chick embryo [b], direct cell labeling with dyes, genetic labeling

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The overall picture suggests that cellular operations correspond to amazing combinations of elementary cell properties. Similarly, the process of production and of regionalization of the paraxial mesoderm (somitogenesis) must integrate control of cell dispersal (acquisition of static cell behavior) [34], colinearity of expression of Hox genes [49] and the elements (molecular clock, determination front) of SEGMENTATION [50].

such as infection by replication-deficient retroviruses [c,d] and, more recently, by methods based on the recombination of an inactive reporter gene in the mouse [e,f]. Lines of transgenic mice harboring the LaacZ (β-galactosidase negative, β-gal−) reporter gene have been constructed. Recombinant LacZ (β-gal+) clones are initiated by a homologous recombination event between the duplicated sequences in LaacZ and restoration of a functional LacZ [e]. Precursor cells are labeled at random and at a suitably low frequency (10−6 per cell per generation) to allow the assumption that each labeled embryo contains β-gal+ cells derived from a single recombination event. This method presents several advantageous characteristics for the analysis of successive stages of clonal development. The long-term nature of the labeling allows clones with different dates of birth to be described at the same developmental stage. Clones are initiated at random during development, such that clones representative of all developmental stages are obtained. This is perhaps the most important characteristic of the method because it allows the definition of successive cellular events underlying development [f]. This technique has been used to generate data regarding the mode of cell growth, dispersion and production of the precursors that complement data obtained from fate maps. References a Clarke, J.D. and Tickle, C. (1999) Fate maps old and new. Nat. Cell Biol. 1, E103–E109 b LeDouarin, N.M. (1969) Particularités du noyau interphasique chez la caille japonaise (Coturnix coturnix japonica). Utilisation de ces particularités comme marqueur biologique dans les recherches sur les interactions tissulaires et les migrations cellulaires au cours de l’ontogenèse. Bull. Biol. Fr. Belg. 103, 435–452 c Sanes, J. et al. (1986) Use of a recombinant retrovirus to study post-implantation cell lineage in mouse embryos. EMBO J. 5, 3133–3142 d Price, J. et al. (1987) Lineage analysis inthe vertebrate nervous system by retrovirus mediated gene transfer. Proc. Natl. Acad. Sci. U. S. A. 84, 156–160 e Bonnerot, C. and Nicolas, J.F. (1993) Clonal analysis in the intact mouse embryo by intragenic homologous recombination. C. R. Acad. Sci. III 316, 1207–1217 f Eloy-Trinquet, S. et al. (2000) Retrospective tracing of the developmental lineage of the mouse myotome. Curr. Top. Dev. Biol. 47, 33–80

are already expressing specific chord characters. For the cephalochordates, after gastrulation by simple invagination, the blastopore is incorporated into a tail bud from which the larva elongates posteriorly [5]. Unfortunately, nothing is known about the cell behaviors involved. Clearly, much more must be learnt about the cephalochordates and the sister group of chordates, the hemichordates (Box 2).

Elongation movements in ascidians

A scenario for the evolution of early gastrulation movement and axis elongation in chordates?

For the ascidians, elongation does not disrupt the clonal coherence of the nerve cord (Fig. 1a, blastomere A7.8), the muscles or the epidermis. Clonal coherence, however, is disrupted in the chord by a process that involves cell shape changes, orientated protrusive activity and cell polarity [51,52] (Fig. 1a, blastomere A7.3). These cell activities affect cells that

Is it possible to propose a scenario for the evolution of early gastrulation and axis elongation in chordates? For urochordates, elongation is achieved in part by the pattern of cell divisions (epidermis, nerve cord, muscles) and in part by exploitation of a limited number of cell activities that modify the form of an already differentiated chord. Thus, it is mainly

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lineage-based. Cephalochordates probably represent an intermediate level of complexity. For lower vertebrates, such as fish and amphibians, elongation proceeds by cell rearrangements, coordination of cell movements and orientated cell divisions. Thus, it is mainly movement-based. In each species, these basic cell activities are combined differently in the different regions of the embryo. Finally, for higher vertebrates, to these movements and changes is added the production of cells from pools of cells that renew themselves and function for long periods of time. It is important to note that some non-chordate embryos use several of these basic cell activities. They are not vertebrate innovations. The change of cell shape and fluid-like properties of the gut rudiment during gastrulation in sea urchin is one example [53], and cell intercalation during Drosophila germ-band extension is another [54]. All these basic cell activities are probably present in the ancestral metazoan cell [55]. It is their combination in complex embryonic operations that distinguishes vertebrates from most invertebrates. Therefore, a major operation of development, the allocation of the founder cells to the definitive territories, has possibly evolved from a lineage-based mode in invertebrates to a movement-based mode in vertebrates. Formation of cellular sub-domains after axis elongation, the example of the rhombencephalon

In most invertebrates (including ascidians) during or immediately after the operations described above, cytodifferentiation has occurred. This is not the case in insects and vertebrates that form subdomains before cytodifferentiation, raising the issue of restriction of cell behaviors during this process. Again, the formation of these subdomains involves control of cell movements and coordinated cellular operations. In Drosophila, the segments of the body, termed parasegments, are organized into cell lineage compartments (polyclones), which define developmental units (reviewed in [56]). In vertebrates, the segmental organization in the neural tube is clearer in the rhombencephalon, which is subdivided into distinct domains of gene expression called RHOMBOMERES (r1–r7)[57]. A fundamental observation is that cell movements between adjacent rhombomeres become limited from the time of boundary appearance [58]. The origin of this restriction of cell mixing between adjacent rhombomeres has been the subject of debate, but recent results indicate that it results from both a general arrest of cell movements everywhere in the rhombencephalon and specific lineage restrictions at the boundary between rhombomeres. Following this early CELLULAR PATTERNING, distinct modes of neuronal migration occur throughout the development of central nervous system (they will not be discussed here, see [59]). Two different methods of genetic cell labeling in transgenic mice (Cre–loxP fate mapping and LaacZ cell lineage tracing, Box 3) have provided an overall http://tig.trends.com

description of cell movements in the mouse rhombencephalon. Mice containing a knock-in of Cre recombinase into the Krox-20 locus were crossed with mice ubiquitously expressing a floxed LacZ. Krox20 is expressed before the formation of visible boundaries. The descendants of cells that express Krox-20 even transiently are permanently labeled by LacZ. The resulting fate map of r3/r5 shows that cell movements between adjacent rhombomeres are already limited at the onset of Krox-20 expression; that is slightly before the formation of visible boundaries [60]. The analysis of many LaacZ/LacZ clones born before Krox-20 expression indicates that the extent of cell movements along the antero–posterior axis changes dramatically throughout the rhombencephalon when rhombomeres form (Fig. 3a). Cell dispersal subsequently occurs along the dorso–ventral axis of the neuroepithelium [60]. The organization of these clones persists in the postnatal rhombencephalon [36] showing that cell mixing in the neuroepithelium is very limited during neurogenesis (Fig. 4). These studies on rhombomere formation agree with previous findings in the chick embryo [61], but they add the important notion that the arrest of cell movements occurs in the entire rhombencephalon and not only at the boundaries. Because the antero–posterior order of cells is established at the time of arrest of antero–posterior cell dispersal, this general arrest of cell movements defines the founder cell population at the origin of the rhombomere polyclones (Box 1). In this respect, the clonal clusters generated by dorso–ventral cell movements could be very significant lineage units [36,60]. In the rhombencephalon, the different adhesive properties of odd- and even-numbered rhombomeres have been suggested to cause a specific restriction of cell mixing between rhombomeres [62,63]. Eph receptors and their ephrin ligands are expressed in complementary territories in the rhombencephalon. Eph receptors are expressed in rhombomeres r3/r5, whereas the ligands are expressed in r2/r4/r6. As a result of this complementary expression, interactions of the receptors with their ligands will occur at the interfaces between adjacent rhombomeres. The mosaic expression of a truncated EphA4 receptor leads to the presence of r3/r5 cells in r2/r4/r6 territories in both Xenopus and zebrafish, suggesting that this receptor could regulate cell mixing between rhombomeres [64]. Activation of EphA4 and EphB receptors by the ectopic expression of the ephrin B2 ligand in zebrafish leads to sorting of expressing cells at r3/r5 boundaries, whereas expression of truncated EphA4 receptors, which can activate retrograde signaling through ephrins, leads to cell sorting at the boundaries of r2/r4/r6 [65]. These data provide support for the idea that cell-exclusion mechanisms, mediated at least in part by ephrins, participate in the refinement and stabilization of rhombomeres. There is genetic evidence that EphrinA4 acts downstream from Krox-20 [66] suggesting that the restriction of cell fate within r3 and r5 could result from the combined action

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Fig. 4. The cellular and genetic patternings of the mouse central nervous system. A sequence of movements disperses cells in the embryo. The main signaling centers are indicated by red asterisks to illustrate the temporal and spatial correlation between the cell movements and the patterning mechanisms. Arrows show the movement of self-renewing precursors; double-headed arrows show limited dispersion. The blue double arrow in E6.5 embryo indicates loss of coherent clonal growth in the epiblast. ANR, anterior neural ridge; Ant, anterior; AVE, anterior visceral endoderm; DE, dorsal ectoderm; E, embryonic day; Ecto, ectoderm; N, notochord; Post, posterior; PS, primitive streak; Som, somite.

of the arrest of cell movements and from cell sorting between rhombomeres. This provides possible elements of a genetic pathway that could lead to the progressive refinement of cell distribution. In conclusion, recent results suggest that both a general arrest of cell movements, occurring throughout the rhombencephalon, and cell-sorting at rhombomere boundaries contribute to the limitation of cell mixing between adjacent domains and thus to the definition of segmental cellular domains. The genetic regulation [67] and the sequence of cell movements in the neural tube [68] in fish and mammals are similar, suggesting that this mechanism has been conserved during vertebrate evolution. The use of different levels of cellular organization in lineage restriction (global arrest of cell movements, behaviors of single cells) further suggests that the movement-based strategy accompanies the elaboration of the vertebrate body plan (clearly visible at the phylotypic stage, Box 2) during evolution. Conclusions and challenges: implications for cellular patterning and vertebrate evolution

The progressive formation of the cellular domains of the vertebrate embryo involves a succession of elaborate types of cell behavior [13,21,41,69]: (1) widespread cell dispersal, (2) directional cell dispersal, (3) convergence–extension by cell intercalation, (4) ORDERED CELL MINGLING [21], (5) progression of pools of self renewing precursors, (6) general arrest of cell dispersal, and (7) clonal boundary formation. Most of the genes involved in establishing and controlling the patterns of cell behavior remain to be http://tig.trends.com

discovered, although rapid advances are being made. The temporal and spatial distribution of these cell behaviors produces the final spatial organization of the cells, a process called cellular patterning. Strikingly, the cellular patterning appears to parallel the GENETIC PATTERNING (Fig. 4). For instance, both the cellular and genetic patterning follow the same sequence, first establishing antero–posterior regionalization, then establishing dorso–ventral regionalization. Therefore, the genes expressed in signaling centers of the embryo (the anterior visceral endoderm, the node, the notochord, etc.) that are at the origin of the genetic patterning, might also control cellular patterning. A tantalizing possibility is that cell activities could have been co-opted during evolution by these control genes, thus tightly linking cellular and genetic patterning. Simultaneously, cell movements could be important for the function (and the evolution) of the signaling centers, by correctly positioning cells that will interact (and by promoting novel interactions), by facilitating the withdrawal of cells from the signaling centers or by transferring information over long distances. Studies in vertebrates show that the cellular patterning can diverge between related species [41]. Therefore, any conservation of elements and of the cellular patterning between vertebrate species that diverged over 400 millions of years ago (e.g. fish and mammals) and that have differences in the geometry of their first stages of development could indicate selective pressure on some morphogenetic processes. The conservation of cell movements, which immediately precede gastrulation and accompany the formation of the cellular domains, is striking. Although cell lineage tracing by clonal methods, comparable to that of LaacZ, is now required to study other vertebrate species, the available data suggest that at least part of the cellular patterning might be conserved in otherwise divergent vertebrates. Such conservation must result from strong developmental constraints acting on the movements of cells. These constraints remain to be described and understood; they could be very significant for the construction of the vertebrate body plan. It is possible that cell movements and the shape of the territories are crucial for cell and tissue

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Acknowledgements We thank Margaret Buckingham, Shahragim Tajbakhsh, Sigolène Meilhac and Andrew Ewald for comments on the manuscript. This work has been financially supported by grants from the Pasteur Institute, the CNRS (Centre national pour la Recherche scientifique), the ARC (Association pour la Recherche contre le Cancer) and the AFM, (Association française contre les Myopathies). L.M. is from the Centre National de la Recherche Scientifique, J.F.N. is from the Institut National de la Sante et de la Recherche Medicale. We regret that space constraints preclude us providing a more comprehensive list of primary references.

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interactions in the embryo and that, as a consequence, embryos look similar once the basic vertebrate body plan is in place (at the phylotypic stage, Box 2). This last conclusion raises an apparent paradox: how can the shared body plan of chordates be produced by two apparently different strategies? The solution might reside in the way genomic programs for morphogenesis are designed: the movement-based strategy could have evolved from the lineage-based strategy by a mechanism such as INTERCALARY EVOLUTION [6,70] from the genes which regulate the formation of the body plan of primitive uro- and cephalochordates. This intercalary evolution would presumably have consisted essentially of the recruitment of basic cell activities (or more complex cell behavior) by such regulatory genes. In accordance with this hypothesis, the conservation of the body plan in chordates would result from the conservation of the genetic coordinates of the axes of the embryo. The difference in the strategies used to reach this stage is consistent with the increasing size of the elements of the body plan of the embryo: this increase in size would have permitted more complexity. Also, intercalary recruitment could also explain the rapid evolution of cellular patterning in vertebrates without altering the body plan. Finally, it is interesting to speculate on the evolutionary advantages of cell dispersal (polyclones) that coincides with the emergence of vertebrates. An attractive hypothesis is that keeping structures of a polyclonal origin might have the evolutionary advantage of reducing the impact of mutations [20]. However, the size of the founder cell populations in the embryo (~50–200 cells for most structures [31]) is too low for the frequency of spontaneous somatic mutations (in the range of 10−6 per cell per generation) to be a major issue for the morphogenesis of individuals. By contrast, the size of founder pools is more consistent with the frequency of errors generated by epigenetic controls such as methylation of nucleotides. Indeed, vertebrates are exceptional among animals in that their genomes are extensively methylated. The emergence of vertebrates is associated with an increase in genetic complexity owing to two successive partial duplications of the genome that occurred more than 500 millions years ago, after divergence from the amphioxus

References 1 Russell, L.B. (1978) Genetic Mosaics and Chimeras in Mammals, Plenum Press 2 Davidson, E.H. et al. (1995) Origin of bilaterian body plans: evolution of developmental regulatory mechanisms. Science 270, 1319–1325 3 Nishida, H. and Satoh, N. (1985) Cell lineage analysis in ascidian embryos by intracellular injection of a tracer enzyme. II. The 16- and 32-cell stages. Dev. Biol. 110, 440–454 4 Tung, T.C. et al. (1962) The presumptive areas of the egg of Amphioxus. Sci. Sin. 11, 629–644 5 Zhang, S.C. et al. (1997) Topographic changes in nascent and early mesoderm in Amphioxus embryos studied by Dil labeling and by in situ hybridization for a Brachyury gene. Dev. Genes Evol. 206, 532–535

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lineage [71,72]. Widespread methylation, by lowering the level of transcription, might be a functional adaptation to the higher genetic complexity that resulted from these genome duplications [73]. Epigenetic control by methylation generates a high frequency of genetic errors (variegation and mutability of the methyl cytosine) that could affect morphogenesis. Thus, cell dispersal and a polyclonal origin of the structures of the organism could be a way of reducing the impact of errors generated by epigenetic control and, therefore, could be a relatively direct adaptation to the genome duplications that preceded the emergence of vertebrates. Future

The challenge is to discover the mechanisms underlying the changes occurring in embryos. This will undoubtedly involve a comparative exploration of the regulatory networks of developmental genes during embryogenesis. Such studies could be used to deduce the various forms of the functional architecture of the genomes, and to try to discern the processes that have accompanied evolution [6,70] (intercalary and downstream co-option, or indeed any others). This also absolutely requires a more complete understanding of the cellular operations at work during the embryogenesis of representatives of the major chordates and of vertebrates. The study of amphioxus is essential because it occupies a key position in chordate evolution. Processes such as lineage-based allocation, convergence–extension, production of territories from pools of self-renewing cells and clock-based segmentation are the first examples to be described of the series of cellular operations involved. The other processes implicated should also be studied, and this requires, firstly, looking at embryos by all available methods, from clonal analysis to pattern analyses and micro-cinematography to discover the pathways and cellular behavior associated with cellular patterning of the embryo, and secondly, analyzing the consequences on cellular patterning of mutating genes controlling the genetic patterning. It is likely that only integration of all these elements into a single corpus will allow us to understand the nature of embryos and the contribution of their plasticity to evolution.

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