Segmentation and the origin of regional diversity in the vertebrate central nervous system

Neuron,

Vol. 2. I-9,

lanuary,

1990, Copyright

0

1990 hy Cell Pres\

Segmentation and the Origin of Regional Diversity in the Vertebrate Central Nervous System Roger Keynes and Andrew Lumsden Department of Anatomy Cambridge University Cambridge CT32 3DY England Department of Anatomy United Medical and Dental Schools Guy’s Hospital London SE1 9RT England

The primitive segmentation of the vertebrate brain is a problem which has probably attracted as much of the attention of morphologists as any one of the great, unsettled questrons of the day, and many views have been advanced which have, it is true, reached one important point of agreement; namely, that the primitive brain was undoubtedly a segmented structure. But beyond this, in regard to the character of these segments and the number of segments of which the brain originally consisted, I think it can be said with perfect freedom that nothing whatever has been definitely proved. -Charles F. W. McClure, 1890

To understand the development of the vertebrate central nervous system (CNS) it is necessary to understand how regional variation in the connectivity and arrangement of nerve cells originates. How does a simple tube formed from an epithelial sheet generate such different structures as, for example, the cerebral cortex and the spinal cord? Classical studies in amphibian embryos by Mangold (1933) and others have demonstrated that the overall regional differentiation of the neural tube into forebrain, midbrain, hindbrain, and spinal cord results from signals transmitted to the neural ectoderm from the underlying axial mesoderm. Less is known about how the subsequent differentiation of local regions of the neural tube is achieved. Subdivision of the epithelium into a number of independent units is one possible strategy for producing local variations in pattern. Early segmentation of the neuraxis could confer upon the individual segmental units a degree of independence during development; it would also convey the potential for generating self-contained anatomical variation and thus the flexibility necessary for evolutionary change. Segmentation is found in many invertebrate phyla, and recent genetic and molecular analyses of Drosophila development have advanced significantly our understanding of the mechanisms of segmentation in the insect integument (Lewis, 1978; Nusslein-Volhard and Wieschaus, 1980; Akam, 1987; fngham, 1988). In vertebrate embryos, on the other hand, it is unclear whether segmentation even exists in many body regions, let alone how the segmental patterns may arise. There has been a tendency to regard only the somitic mesoderm as truly segmented (Figure I), while

Review

the status of other mesodermal tissues (Jacob et al., 1986; Meier, 1979; Jacobson, 1988) and the neural epithelium remains controversial. What is the evidence for segmentation in the nervous system? When assessing neural segmentation in vertebrates, it is important to distinguish its manifestations in the peripheral and central nervous systems. We now know that the periodic arrangement of the spinal nerves in higher vertebrates does not represent an inherent property of the nervous system; rather, this arrangement is a direct consequence of segmentation in the somitic mesoderm alongside the neural tube (Lehmann, 1927; Detwiler, 1934; Keynes and Stern, 1984, 1988; Keynes et al., 1989). It remains possible, however, that segmentation does exist within the epithelium of the neural tube itself. von Baer (1828) first observed repeated swellings along the neural tube, which later became known as “neuromeres” (Orr, 1887). Since then, an extensive literature has arisen charting the appearance and disappearance of neuromeres in embryos of many different species (reviewed by McClure, 1890; Grgper, 1913; Neal, 1918; Streeter, 1933; Bergquist and Kgllen, 1954; KZllen, 1956; Vaage, 1969; Kuhlenbeck, 1973). These accounts culminated in two opposing views: first, that neuromeres are either fixation artifacts or the products of mechanical interactions between the neural tube and adjacent mesoderm; and second, that neuromeres, particularly in the hindbrain region (rhombomeres, Figure I), are evidence in favor of intrinsic neural segmentation, having perhaps arisen in adaptation to the adjacent branchial arch segmentation (Figure 1). Neal (1918) nevertheless argued that there is no consistent relationship between specific rhombomeres and specific cranial nerves. For neuromeres to be of developmental significance, at least some of the following criteria should be met. First, the overt neuromeric pattern should correspond to an underlying segmental pattern of cellular or molecular differentiation. Second, the neuromere pattern should be matched by an equivalent pattern of cell proliferation. Third, the boundaries between neuromeres should represent barriers to cell movement, thereby comprising lineage restriction boundaries. Finally, genes with possible regulatory roles during development should be expressed in patterns that relate in some way to the neuromeric pattern. These criteria are met to differing degrees in different regions of the CNS. The first, for example, is met by the lower vertebrate spinal cord, in which repeating patterns are imposed by segmentation of the somites. However, all of these criteria are met by the higher vertebrate hindbrain. In the hindbrain, we can now ask: Is such segmentation analogous to the segmentation and compartmentation in invertebrates? If so, what role does it play in the specification of neuronal fates?

Neuron 2

1986) and probably also in other teleosts (Fetcho, 1987). In the zebra fish, there are three primary motor neurons per segment that are distinguished on the basis of their position within the cord and their axon trajectories outside the cord. Thus far, there is no published account of segmentally arranged interneurons in teleosts. The same is true for any neuronal type in the amphibian spinal cord (Coghill, 1913; Youngstrom, 1940; Silver, 1942; Blight, 1978; Forehand and Farel, 1982). The possibility remains that in amphibians a segmental arrangement of motor neurons does exist very early in development but bet-omes rapidly obscured through the relative displacement of the myotomes (Westerfield and Eisen, 1985; Nordlander, 1986). The

Higher

As for periodic

Figure

1. Segmentation

in the

Chick

Embryo

Diagram of a stage 18 (Hamburger and Hamilton, 1951) chtck embryo showing the hindbrain and its rhombomeres (rl-r8, coarse stipple), the cranial motor nerves (Ill-XII), and branchial arches (bl-b4). The mesodermal somites (fine stipple) and spinal motor nerves are also shown; somites alongside r7 and r8 are dispersed (broken lines). Sensory ganglia have been omitted. Note that whereas the Vth, Vllth, and IXth nuclei occupy serially adjacent positions along the A-P axis, later forming a continuous column of branchial and visceral efferent cell bodies, the nuclei of the somatic motor system origlnate and remain in a discontinuous cell column. The generation of these cells contorms, nevertheless, to a segmental pattern. The neurons of the IVth nerve lie in rl; those of the Vlth nerve arise en bloc between the r4-r5 and the r6-r7 boundaries, occupying two rhombomeres that are out of phase with the adjacent branchial motor nuclei (VII and IX). Finally, the Xllth nerve nucleus lies in the region of the medulla adjacent to the occipital somites 02-04 (r8). ~6, somite number six; SC, spinal cord; m, mesencephalon ImIdbrain); d, dtencephalon; t, telencephalon.

The

Lower

Vertebrate

Spinal

Cord

There are several descriptions of segmentally arranged neurons in the spinal cords of fishes and protochordates. Bone (1960) found that both interneurons and motor neurons are segmented with a period equal to the myotomes in amphioxus. Whiting (1948) described a class of segmentally arranged interneuron in the larval lamprey, again matching the periodicity of segmentation in the adjacent mesoderm. Finally, primary motor neurons are segmentally arranged in the spinal cord of zebra fish embryos (Myers, 1985; Eisen et al., 1986; Westerfield et al., 1986; Myers et al.,

the

Vertebrate developing swelling of

Spinal

the

Cord

brain, the existence ot overt spinal neural tube, myelo-

meres, has long been recognized. Neal (1918) argued persuasively that these are likely to represent the outcome of mechanical molding by the contours of the adjacent somites rather than evidence of any segmental cellular arrangement. Although there have been no clear descriptions of segmental proliferation patterns in the spinal cords of normal chick embryos (Hamburger, 1948; Corliss and Robertson, 1963), till& (1962) nevertheless described periodic mitotic maxima in colchicine-arrested animals. A recent reexamination of the effects of colchicine arrest (Lim, Jaques, Fraser, Stern, and Keynes, submitted), however, has been interpreted entirely on the basis of mechanical interaction with the somites. Other studies of the chick have found no segmental arrangement of motor neurons or interneurons (Lim, 1987) or relay neurons (Schlosser and Tosney, 1988). Although there are no primarily segmental arrangements of neurons, the clustering of motor axons in the white matter, which follows their preferential outgrowth into the anterior halves of somites (Keynes and Stern, 1984), leads also to a secondary reciprocal enlargement of ventral white matter columns (e.g., Hoffman‘s nucleus in birds and reptiles, the nucleus marginalis in mammals: Anderson et al., 1964). The absence ot interneuromeric boundaries and segmental arrangements of neurons in the chick spinal cord distinguish this region of the neuraxis from the brain. This difference is matched by the absence of lineage restrictions at any predictable periodic positions along the A-P axis (Lim et al., submitted). Such regional organization as exists along the A-P axis of the spinal cord extends over a distance equivalent to a large multiple of mesodermal segments; for example, the limb motor columns, preganglionic motor columns, and Clarke’s column are all plurisegmental. It is thus likely that regulatory genes involved in spinal cord regionalization would be expressed in a suprasegmental pattern. The expression patterns of some homeobox genes appear to conform to such expectations (Holland and Hogan, 1988; Bogarad et al., 1989).

Rev~cw: 3

Neural

Segmrn~at~on

I” Vcrtrbratr\

Segmentation

in the

Higher

Vertebrate

Hindbrain

Early in its formation, the rhombencephalon displays distinct periodic undulations along its length (Figure 2); each rhombomere comprises an external (pial) ballooning of the epithelium and is continuous with its neighboring rhombomere at an internal (ventricular) ridge. To what extent does this overt pattern reflect any underlying anatomical, cellular, or molecular organization? A recent study of the chick hindbrain (Lumsden and Keynes, 1989) revealed that from the time the first neurons differentiate, the boundaries between rhombomeres become delineated by transversely oriented axons (Figure 3A), which grow through laminin-rich intercellular spaces on the pial side of the epithelium, directly beneath the ridges on the ventricular surface (Figure 3B). The nuclei of the somatic motor system are segmentally arranged (Figure I), and neurons of the reticular formation differentiate in the same alternating pattern as the branchiomotor neurons. Neurogenesis of the branchial motor neurons conforms to the rhombomeric pattern; each consecutive nerve (V, VII, IX) derives from a specific, consecutive pairing of rhombomeres, each pair lying in register with an adjacent branchial arch. Thus, the neurons of the fifth cranial nerve derive from rhombomeres 2 and 3 (r2 and r3), opposite the first arch; the seventh from r4 and r5, opposite the second arch; and the ninth from r6 and r7, opposite the third arch (Figures 4A and 48). Neurogenesis involves, moreover, a superimposed periodicity of a higher order: the anterior (A, rostral) member of each rhombomere pair produces motor axons before the posterior (P, caudal) member and contains the motor nerve root. Is there any indication that cells are already organized in units corresponding to the future segments before neuronal differentiation? In a study of mitotic patterning in the chick embryo CNS using colchicine, Kallen (1962) showed that mitoses accumulate maximally around the centers of rhombomeres and that the boundaries are mitotic minima. A later study (Tuckett et al., 1985) found no such segmental patterns. We recently confirmed Kallen’s findings in un-

Figure

2. Rhombomeres

in Higher

Vertebrate

Hindbrains

(A) Scanning electron micrograph of 3.day-old chick embryo hindbrain displaying the ventricular surface of basal and alar plates on either side of the midline floor plate. Neuromeres are visible in the hindbrain but not the spinal cord. Scale bar, 300 pm. Reprinted by permission from Nature, Vol. 337, pp. 425. Copyright 0 1989 Macmillan Magazines Ltd. (B) Photomicrograph of the hindbrain of a living Xenopus tadpole displaying prominent rhombomeres. Unstained.

treated embryos (Guthrie, Butcher, Keynes, and Lumsden, unpublished data); the centers of rhombomeres have a higher mitotic density and shorter cell cycle time than the boundaries. The pattern of bromodeoxyuridine uptake by hindbrain cells indicates that theventricularlpial interkinetic nuclear migration typical of the neural epithelium (Sauer, 1935) is normal within rhombomeres but reduced or absent at their boundaries: here the cells divide at the ventricular surface, but their nuclei remain close to this surface throughout the cell cycle. The relative stasis of dividing cells, together with a local increase in cell-cell adhesion (Lumsden and Keynes 1989), suggests that the boundaries could form barrtcrs to the translocation of cells along the A-P plane, from one rhombomere to the next. In parallel, neuronal fates could be speci-

, z,:: rl

fF

gv

_,

r2

-

Figure 3. Rhombomere Hindbrain

Boundaries

in the

Higher

Vertebrate

r3

(A) Wholemount 2.day-old chick embryo hindbrain stained by indirect immunoperoxidase using anti-68 kd neurofilament antibody. The boundaries between rhombomeres contain clusters of axons. On the left side, the trigeminal (V) and facial (VII) nerve roots and the otic vesicle (OV) remain attached and shown by arrows. Scale bar, 300 pm. (B) Parasagittal section of 3-day-old chick embryo hindbrain stained by indirect immunofluorescence using anti-68 kd neurofilament antibody. At the rhombomere boundaries, embrasures containing axons extend toward the ventricular ridges. Scale bar, 100 pm. Reprinted by permission from Nature, Vol. 337, pp. 426. Copyright 0 1989 Macmillan Magazines Ltd.

fied early in development, in segmental groups whose boundaries would prevent mixing between cells of different segmental origins. These possibilities were tested recently (Fraser, Keynes, and Lumsden, submitted) by a clonal analysis in the chick hindbrain using intracellular fluorescent marking to follow the descendants of single labeled

Figure meres

4. Relatlonshtp

(A) Wholemount

between

&day-old

chic-k

Cranial embryo

Nerves hlndbraln

and

Rhomboatter

Inlet-

cells. From the time of their first appearance rhombomere boundaries are not crossed by expanding clones, despite the potential for widespread movement by individual cells in the plane of the neural epithelium. When marked before the appearance of boundaries, however, the progeny of a single cell sometimes extend into more than one rhombomere. This result implies that rhombomere boundaries do act as lineage restriction boundaries. The majority of clones produce, moreover, a single neuronal phenotype. Such early determination indicates that in the hindbrain, unlike other CNS regions analyzed to date, cell lineage ancestry may play an important role in establishing cell fates. In the mammalian cerebral cortex (Walsh and Cepko, 1988; Price and Thurlow, 1988; Luskin et al., 1988) and retina (Turner and Cepko, 1987), chick mesencephalic tectum (Gray et al., 1988), and frog retina (Wetts and Fraser, 1988; Holt et al., 1988), a single parent cell produces a variety of neuronal phenotypes. Cell fates are determined, presumably by cell-cell interactions, shortly before terminal differentiation. Given the morphological evidence for organization of the hindbrain into segmental units, is there any evidence for an underlying segmental pattern of gene expression? A number of candidate genes have now been identified that encode DNA binding proteins and that may have a role in the regulation of vertebrate development. These include many homeobox genes and a zinc finger gene, whose patterns of expression have been mapped in mouse embryos (Holland and Hogan, 1988; Wilkinson, 1989). A striking feature of these patterns is the frequency with which they involve the developing hindbrain, where a sharp anterior limit often terminates a continuous domain of expression extending from the spinal cord (Graham et al., 1989). Moreover, for the Hox-2 genes these anterior limits were recently found to coincide with rhombomere boundaries (Figure 5); for example, Hox2.1, Hox-2.6, Hox-2.7 and Hox-2.8 are expressed up to the spinal cord-r8 boundary, the r6-r7 boundary, the r4-r5 boundary, and the r2-r3 boundary, respectively (Wilkinson et al., 198913). At the same stage, in contrast, Hox-2.9 is expressed within and is coextensive with one rhombomere, r4 (Wilkinson et al., 1989b;

tion of the trigeminal (V) and facial (VII) motor nerve roots with Dil. The motor nucleus of each nerve occupies two adjacent rhombomeres with the exit point in the anterior member of each pair. The rhombomere boundaries (dashed lines) are visible under the combined fluorescence/bright field optics. Reprinted by permission from Nature, Vol. 337, pp. 42% Copyright 0 1989 Macmillan Magazines Ltd. (6) Schematic diagram of chick embryo hindbrain showing the relationship of cranial sensory ganglia fgV-gX), branchial motor nuclei and exit points (mV-mXI), and somatic motor nuclei and exit points (IV-XII) to the rhombomeres (rl-r8) and branchial arches (bl-b3). ov, otic vesicle; fp, floor plate; i, isthmus/midbrain-hindbrain junction. Reprinted by permission from Nature, Vol. 337, pp. 428. Copyright 0 1989 Macmillan Magazines Ltd.

ChickEn En2 ,r--.

/-

5. Gene

< :

: ,+,-:

I’

--fl

-.

m ‘.

‘;’ .--

’

.I’

\

Hox

Figure

,r*:

rl

2.1

Expression

Patterns

and

Rhombomeres

Data on Hox genes from Wilkinson et al. (1989b); data on Hex-2.9 also from Murphy et al. (1989); data on &ox-20 from Wilkinson et al. (1989a); and data on ChickEn (antibody staining of protein product) from Gardner et al. 11988).

Murphy et al., 1989). The mouse and chick homologs of the Drosophila segment polarity gene enpiled, En-2 (Davis et al., 1988), and ChickEn (Gardner et al., 1988) are expressed more anteriorly, in a domain that includes the posterior midbrain and anteriormost hindbrain, probably terminating at the rl-r2 boundary (see also Hemmati Brivanlou, and Harland, 1989). At present, it is not clear whether these homeobox genes might be involved in generating the segmental pattern itself or whether they could impart individual identities to particular segments at a later stage. The fact that the expression patterns described above are not seen until after the appearance of rhombomeres argues against a role in producing the primary periodic pattern. On the other hand, the zinc finger gene Krox-20, which is expressed as the pattern is established, could play such a role. Krox-20 is expressed in two alternate rhombomeres, r3 and r5 (Wilkinson et

al., 1989a), domains that coincide with the posterior halves of the trigeminal and facial motor nuclei (Lumsden and Keynes, 1989). Krox-20 could therefore function in a manner analogous to the pair rule genes of Drosophila, which are expressed in alternate epidermal segments and are proposed to define the segmental boundaries (Ntisslein-Volhard and Wieschaus, 1980). Alternatively, Krox-20 could be involved in establishing a two-segment repeat with respect to segment identity, as necessitated by the paired-rhombomere construction of the branchial motor nuclei. The spatial correspondence of homeobox gene expression boundaries and segment boundaries in the ectoderm-derived hindbrain is reminiscent of the tight coupling of these boundaries in Drosophila epidermal development (Akam, 1989) and contrasts with the looser association between segment position and fate found in the mesoderm of the vertebrate trunk (Bateson, 1894; Lankester, 1904). This raises the interesting possibility that, like the fly, the vertebrate hindbrain uses metamerism to generate level-specific anatomical structures with great and reliable precision. In the hindbrain, as for many invertebrates, lineage ancestry may be important in the determination of cell fate; the segmentation seen in this body region could therefore reflect a similar condition once present in the ancestor common to vertebrates and invertebrates. Segmentation

in the

lower

Vertebrate

Hindbrain

The hindbrain of zebra fish embryos displays a segmental series of rhombomeres equivalent to those found in higher vertebrates. There are seven clearly identifiable segments posterior to the midbrain, and an uncertain number (two or three) of less distinct segments in the posterior hindbrain/occipital somite region (Hanneman et al., 1988) (for convenience these posterior hindbrain segments are referred to collectively as “r8” in the chick [Lumsden and Keynes, 19891; the first seven rhombomeres are likely to be homologous in all vertebrates; see also Figure 2B and Graper [1913]). Segmentation

of the

Head

Mesoderm

A long standing question is whether the segmental pattern of branchial arches is related to segmentation in the cranial paraxial mesoderm. This idea has been supported by some (Balfour, 1877; van Wijhe, 1882; Goodrich, 1918) and rejected by others (Koltzoff, 1901; Stockard, 1906; Kingsbury, 1926). These early studies focused predominantly on fish embryos, in which segmentation of the preotic paraxial mesoderm is conspicuous. In amniote embryos the problem is further compounded by the lack of overt segmentation in this tissue. However, the more recent studies by Meier and colleagues, using scanning electron microscopy, have revealed that the apparently continuous column of cranial paraxial mesoderm may be pat-

terned into a longitudinal series of varicosities and constrictions known as “somitomeres” (Meier, 1979; Anderson and Meier, 1981; Jacobson and Meier, 1984; Jacobson, 1988). This mesoderm contributes the myoblasts that form the voluntary muscles of the branchial apparatus (Noden, 1983a). Somitomeres are claimed to have a one-to-one relationship with rhombomeres (Jacobson and Meier, 1984). This may hold for the early stages of chick development, when there are first three and then four rhombomeres that can be matched with the four somitomeres lying at the hindbrain level. Later, however, this simple numerical relationship breaks down as the number of rhombomeres increases. Eventually, segments of the three principal components (brain, mesoderm, and branchial arch) are registered approximately in the ratio of 2:l:l. This view of head segmentation is appealing in its simplicity but may be misleading. Somitomeres could be passively molded by the contours of the brain, in which case there would be as many somitomeres as neuromeres at the time the molding is effected and the registration would be trivial. Recent work on the interactions and fates of craniofacial mesenchyme cells in the chick has demonstrated that it is the neural crest, and not the mesoderm, that is responsible for patterning this region (Noden, 1986,1988). The paraxial mesoderm is positionally unspecified; somitomeres, if they exist at all, are passive elements in the patterning process. The early search for a segmental relationship between the branchial arches and the cranial paraxial mesoderm can thus be seen as misdirected. If there is an overall plan of head segmentation in amniotes, it would involve a matching between branchial arches and segmental levels of the neural crest. Noden’s (1983b) grafting experiments have suggested that each axial level of the presumptive branchial neural crest contains the morphogenetic identity of its arch before migration. We have presented morphological evidence that there is a simple match between rhombomeres and anterior branchial arches, the motor nerve of each arch being derived from two rhombomeres (Lumsden and Keynes, 1989). It remains to be seen whether the neural crest of a particular rhombomere, or rhombomere pair, carries axial positional information that enables the matching of nerve and target. The

Rest

of the

Brain

The segmental status of mid- and forebrains and their subdivisions is uncertain. Bergquist and Kallen (1954), Vaage (1969), and Keyser (1972) have provided detailed accounts of the appearance and disappearance of successive bulges and furrows in the anterior neural tube. Further studies indicate that these neuromeres may correspond to centers of epithelial cell proliferation (Kallen, 1956; Puelles and Martinez de la Torre, 1987), and several interneuromeric boundaries are colonized by axons (Coggeshall, 1964; Keyser, 1972).

Rrvlew:

Neural

Sermentatlon

III Vertebrates

In two cases, molecular markers have been identified that may correspond to segmental forebrain territories. Puelles et al. (1987) describe the early appearance of acetylcholinesterase in cells at the centers of the diencephalic neuromeres, and Mori et al. (1987) have described the staining pattern of a monoclonal antibody specific for cells in the telencephalon. Taken together, these phenomena appear to satisfy some of the segmentation criteria outlined above; it remains to be seen, however, whether the boundaries described represent lineage restriction boundaries and/or boundaries of gene expression. Finally, the cerebellum may also have a specific segmental origin, from rl (von Kupffer, 1906). Conclusions It is well known that the neural epithelium originates from an inductive interaction between the midline chordamesoderm and the overlying ectoderm, and there are suggestions that such interactions may influence the polarity of the neuraxis (e.g., Spemann, 1912; Detwiler, 1943; Jacobson, 1964). A question therefore raised by the existence of segmental neuronal patterns is whether the mechanism that produces them is contained within the epithelium itself or derives from the mesoderm. We do not know the answer, but can speculate that at trunk levels the mesoderm could directly imprint its periodic character on the spinal cord to produce, for example, segmental groups of primary motor neurons in lower vertebrates. In the higher vertebrate head, however, the paraxial mesoderm never becomes overtly segmented, and an intrinsic patterning mechanism for rhombomeres is indicated. It should also be pointed out that neither the mesoderm nor the neural plate is segmented at the ventral midline at any level of the axis; the notochord is coextensive with a continuous midline floor plate (epichordal strip) that shares none of the cellular or lineage restriction patterns seen laterally at the hindbrain level (Lumsden and Keynes, 1989; Fraser et al., submitted). The lack of intrinsic segmentation in the chick spinal cord stands in striking contrast to the hindbrain, and it is necessary to ask why this should be so. Assuming that the fish spinal cord represents a primitive evolutionary stage, it is reasonable to suppose that the differential state of the higher vertebrate neuraxis is the result of a loss of spinal segmentation rather than the acquisition of hindbrain segmentation. In the higher vertebrate trunk, perhaps because evolutionary constraints involved a transition from a tightly localized neuromuscular control system for swimming to one more dependent upon long range integration and control by higher centers, the segmental arrangements of neurons are no longer required. The mesoderm has assumed total dominance and sole control in axial segmentation-of skeletal, muscular, and peripheral nervous systems. Segmentation in the spinal cord may thus have been lost in subservience to par-

axial segmentation. Furthermore, while axial musculature and vertebrae retain a segmental origin, the limb musculature and innervation have plurisegmental origins requiring a different, perhaps novel, patterning mechanism capable of transforming a series of segments into individual harmonious structures. Thus, the loss of spinal cord segmentation also parallels the rise to prominence of the (unsegmented) lateral plate mesoderm as a patterning influence. In contrast, the heads of higher vertebrates retain units of construction, the branchial arches, whose developmental derivatives are distinct both anatomically and functionally. Because such units require independent as well as integrated control, segmental motor and sensory units would be highly conserved. In the head also, paraxial segmentation is at best feeble, and the lateral plate is unimportant in the patterning process. Instead it is the neural crest, with its segmental rhombomeric origins, that is responsible for patterning the flexible interlinked components of each branchial arch. Segmentation of the head, therefore, may depend fundamentally upon the early subdivision of the neural tube and its derivatives. A number of important questions are raised by recent studies of hindbrain segmentation. We need to know, for example, how the pattern is established and the precise role that genes such as those of the Hox-2 cluster and Krox-20 play in the process. To this end, experiments involving the manipulation of such genes and their products are now feasible. The phenomenon of compartmentation, first identified in insect embryos, has now been extended to vertebrates. Why is this process confined to the hindbrain? How do cell movements become restricted at the boundaries? The chick system promises to be particularly suitable for addressing these questions and for analyzing neural segmentation at both cellular and molecular levels. References Akam, M. (1987). The molecular basis the Drosophila embryo. Development Akam, Insects

M. (1989). and

Hex

and

vertebrates.

HOM:

Cell

for metameric 707, l-22.

homologous

gene

pattern

in

clusters

I”

57, 347-349.

Anderson, C. B., and Meier, 5. (1981). The lnfluente of the metamerrc pattern I” the mesoderm on migration of cranial neural crest cells in the chick embryo. Dev. Biol. 85, 385-402. Anderson, The nucleus Neural.

C. B., Meadwows, marginalis of the

M. M. (1964). cord. J. Comp.

723, 97-100.

Balfour,

F. M.

J. Anat.

Physiol.

(1877).

The

(London)

Bateson, W. A. (1894). don: Mac millan).

J. Comp.

development

Neural.

of ela5mobranch

fishes.

77, 406-490.

Materials

Bergqulst, H., and KBll&, sis and morphogenesis tebrates.

I., and Chambers, mammalian spinal

tar

the

Study

of Variation.

(Lon-

B. (1954). Notes on the early histogeneof the central nervous system in ver100, 627-660.

Blight, A. R. (1978). Golgi-staining motoneurons in the developing Comp. Neural. 180, 679-690.

of “primary” and “secondary” spinal cord of an amphibian.

1.

Bogarad, L. D., U&et, M. F., Awgulewitsch, A., Hart, C. P., and Ruddle, F. H. (1989). The developmental expresslon pattern of a new murine homeobox gene: Hex-2.5. Dev. Biol. 733, 537-549.

Neuron 8

Bone, Comp.

Q. (1960). The central Neural. 775, 241-270.

in Amphloxu\.

I.

Study of dientephallc Neural. 722, 241-270.

development

in

Coghill, C. E. (1913). The primary ventral root\ tor column of Amblystoma. J. Comp. Neural.

and somatic 21, 121-143.

Coggeshall, the albino

R. E. (1964). rat. J. Camp.

nervous

system

mo-

Corliss, C. E., and Robertson, G. C. (1963). The pattern ot mltotlc density in the early chick neural epitheltum. J. Exp. Zool. 153, 125-140. Davis, C. A., Noble-Topham, (1988). Expression of the delineates a specific region Genes Dev. 2, 361-371.

S. E., Rossant, J., and Joync’r, A 1. homeobox-containing gene tn.2 of the developing mouse brain.

Detwiler, S. R. (1934). An experimental study of spinal nerve segmentation in Amblystoma with reference t” the plurisegmental contrlhution to the brarhial plexus. J. Exp. Zool. 67. 395-441. Detwiler, S. R. (1943). Reversal of the medulla bryos. J. Exp. Zool. 94, 169-179. Eisen, J. S., Myers, P. A., and Westerfield, lection by growth cone< of identified zebra fish embryos. Nature 320, 269-271.

111Ambly$tomd

em-

M. (1986). Pathway \emot”neurone\ in live

Fetch”, J. R. (1987). A review of the organlratlon and of motoneulons innervating the axial musculature tebrates. Brain Res. Rev. 12, 243-280. Forehand, C. J., and Farel, P. B. (1982). Spinal in anuran larvae: 1. Primary and secondary Neural. 209, 386-394.

evolution of ver-

cord development neuron\. I. Comp.

Jacobson, vertebrate

A. (,. 11988). Somltomeres: embryos. Development

mesodermdl 704. 209-220.

segment\

or

Jac obzon, A. C;., and Meier, S. (1984). Morphogrne\l\ of the hedcf of a newt: me\odcrmal segments, neuromerr<. and dlstnhutlon of neural ( rr\t. Dev. Biol. 106, 181-193. Jatohzon, C.-c ). 11964). Motor nut IPI, (ramal nerve roots, and tI ber pattern in the medulla “hlongata atter reversal experiment\ on the neural plate of axolotl larvae. 7001. Bidrag. Uppsala 36, 73-160. Kall@n, B. (IY5b) (.ontrihutlon to the knowledge tion of the prolileratlon proc eases in the vertebrate ontogene\i<. Ac ta Anat. 22 351-360.

ot the brain

quidduring

Kalltn, B. (1962). Mitotic pdtternlng I” the< t,ntTal nervous system of chtck embryo\; studied bv a c olrhlc ~nr method. Z. Anat. Lnt wickI.-Gesc h. 121. 309-319. Keynes, tebrate

R. J dnd Stern. ( i). 11984). \egmentation ncrw,u\ svstem. Ndturc 170, 786-789.

111the vt’r

Keynq R. I., .rnd stt,rn, C I). 11988). Met hdnl>m\ segmentatton I)evelopmt~nt 103, 41 1.-429

ot vertchratt,

Keynes, R. I., ( ook, (;. M. W.. Davlrs. J., Lumsden, 4.. N”rr~\, W and Stern, ( 11989). Segmentation and the development of thrx vertebrate nt’rvr~u\ system. I. Physlol. (Paris). in pre\\ Keysrr, A. I I (I 721 The development ot thr dlentephalon of tht> Chinrsr ham\tclT An Invr\tlgatlon ot the validity of the ( rltt’rla ot suhdlvlslon of the braln. Ac ta Anat. 81 (SuppI. 59), l-178. KIngsbury, B. f 11926). Branc hiomeri\m segmentation. 1. Morphol. 42, 83-109.

and the’ theory

of hedtl

Gardner, C. A., Darnell. D. K., Poole, 5. J., Ordahl, (.. P.. dnd Baraid, K. F. (1988). Expression of an engralled-like gene during development of the early embryonic chlrk nervous system. I. Neurosci. Res. 21, 426-437

Koltzoff, N K 0901). Fntwlc klungcge\< hlc hte des Kopte\ VOII Petromyzon planrri. Ein Beltrag zur Lehre van der Metamerlr, des Wirbrlthlrrkoptes. Bull. Sot. Imp. Natural. Moscou li 259-589.

Goodrich, the head

Kuhlenbec k, ti tehrate\, Volumcl

E. S. (1918). in Scyllium.

On the development of the segments Q. J. Microsc. Sci. 63, l-30.

ot

Graham, A., Papalopulu, N., and Krumlauf, R. (1989). 7 he munnc and Drosophila homeobox gene complexes have common tedtures of organization and expression. Cell 57, 367-378. Graper, L. (1913). Die Rhombomeren gen. Arch. mike. Anat. 83, 380-429.

and

ihrr

Nervenheriehun-

Gray, G. E., Clover, j. C., Majors, J., and Same\, J. R. (1988). Radial arrangement of clonally related cells in the chicken optic teeturn: lineage analysis with a recombinant TetToviTus. Pro<. Natl. Acad. Sci. USA 85, 7356-7360. Hamburger, V. (1948). The mitotic patterns in the spInal cord of the chick embryo and their relation to histogenetic processes. J. Camp. Neural. 88, 221-284.

Lankester, thropoda.

(lY77). Ihc* (.rntral Nc*rvou\ 3, Part 2 tBd\el: S. Kargtsr).

system

1. R. (1904). The structure and clas\~tledtlon 0. J. MI< ro~c. Sti. 4Z 523-582

01 Vt,r ot th(l a,

Lehmann, F. (19271. Further studies on the morphogenetit of the somite\ I” the development ol th(n nervou< system phibian,. 1. Fxp. Zool. 49, 93-l 11. Lewis, I. B. (1978) A gent’ complex Drosoph,/a. Nature 276, 565570. Llm. T. M. (1987). Segment&Ion embryo. Ph.11 thesis, Unlverzity gland. Lumsden, A., dnd ronal development

controlling

rol(, of am

segmentation

in the neural of Cambridge,

I[,

tube ot the thl( k Camhndge, tn-

Keynes, R. (1989). Segmental patterns ot neu in the (hick hindbraln. Nature 337, 424-428.

Hamburger, V., and Hamilton, H. L. (1951). A serif\ 01 normal stages in the development of the chick embryo. J. Exp. Lool. 771. 457-502.

Luskin, M. R., Pcarlman,A. I ., and Sanrs, 1. R. (1988). Cell line++, nn the cerebral cortex of the mouse qtudled in viva and in vitro with a recomhlndnt retrovlru\. Neuron 7. 635-647.

Hanneman, E., Trevarrow, B., Metcalfe, W. Ii., Kimmel, c‘. B., dnd Westerfield, M. (1988). Segmental pattern of development of the hindbrain and spinal cord of the zebrafish embryo. Development 103, 49-58.

Mangold, 0. (19171. irber eren Bezlrkedrr Neurule 761-766.

die Induktions van Vrodelrn.

McClure, (. F. M. (1890). tebrate hrdin. I. Morphol.

The segmcntatlon 4, 35-56.

Hemmati Brivanlou, A., and Harland, R. M. (1989). Expression of an engrailed-related protein is induced in the anterior neural ectoderm of early Xenopus embryos. Development, 706, 611-617. Holland, P. W. H., and homeobox genes during Dev. 2, 773-782.

Hogan, mouse

B. I.. M. (1988). Expression of development: a review. Genes

Holt, C. E., Bertsch, T. W., Ellis, H. M., and Harris, W. A. (1988). Cellular determination in the Xenopus retina is independent of lineage and birth date. Neuron 7, 15-26. Ingham, P. W. (1988). The molecular genetlrs of embryonic tern formation in Drosophila. Nature 335, 25-34. Jacob, H. J., ment of the Developing (New York:

pat-

Jacob, M., and Christ, B. (1986). The early developintermediate mesoderm in the c-hick. In Somitcs in Embryos, R. Bellairs, D. A. Fde, and J. W. Lash. eds. Plenum Press;), pp. 61-68.

tahigkelt der verF< hled NaturwissenschafterL’1, ot thr

prlmitlvr

Vera

Meier, S. (1979). Development of the c hick mesohlast. Formdtmrr of the embryonic axls and establishment of the metamerlc pat tern. Dev. B~ol. 71, 25-45. Metcalfe, W. K., Mendelson, B., and Ktmmel, (.. B. (1986). Segmental homologies among rettculosplnal neurons In the handbrain of the zebrafish larva. J. Comp. Neural. 157, 147-159. Mori, K., Fullta, 5 C., Watanabe, Y., Ohdta, K., dnd Hayaishl, 0. (1987). Telencephalon-spec-ific antigen identified by monoclonal antibody. Pro<. Natl. Acad. Sci. USA 84, 3921-3925. Murphy, spectfic mouse

P., Ddvldson, D. R., and Hill, R. t. expression ot a homoeohox-containing hindbrdtn. Nature 347. 156-159

(1989). Segmentgene in the

Krwew 0

Neural

Segmentatw!

I” Vertebrate

Myers, P. Z., Ersen, J. S., and Westerfield, M. (1986). and axonal outgrowth of identified motoneurons fish. J. Neurosci. 6, 2278-2289. Neal,

H. V. (1918).

Neuromeres

and

Development In the rebra-

metameres.

J. Morphol.

Walsh, C., and Cepko, show several migration

37,

293-315.

C. L. (1988). Clonally patterns. Science

related cortrcal 247, 1342-1345.

Westerfield, M., and Eisen, J. S. 11985). The growth axons in the spinal cord of Xenopus embryos. Drv.

of motor Btol. 709,

96-101.

Noden, D. M. (1983a). The embryonic and cervical muscles and associated Anat. 168,257m276.

origins of avran cephalic connectrve trssues. Am. 1.

Westerfield, M., MrMurray, J. V.. and fied motoneurons and their innervation zebrafish. J. Neurosci. 6, 2267-2277.

Noden, D. M. (1983b). The role of the of avian cranial skeletal, c-onnective, Brol. 96, 144-165.

neural crest and muscle

Wetts, R., and Fraser, S. E. (1988). Multrpotent precursors give rise to all major cell types ot the frog retina. Science

Noden, D. M. (1986). Patterning Dev. Biol. 776, 347-356. Noden,

D. M.

mrsenchyme.

(1988).

In pattermng tissues. Dev.

and

c raniofatial

fates

703 (SuppI.),

of avian

musc.les.

Whrtrng, H. P. (1948). Nervous young larval Brook-lamprey.

craniofacial

Wilkinson,

121-140.

of the

R. H. (1986). Motoneurons of the tail of young J. Comp. Neural. 253, 403-413.

Nusslein-Volhard, C., and Wieschaus, rng segment number and polarrty

Xeno-

E. (1980). Mutations affectin Drosophila. Nature 287

to the

embryology

Price, J., and Thurlow, L. (1988). Cell cortex: a study using retroviral-mediated ment 104, 473-482.

of the

IiTard.

I.

lineage in the rat cerebral gene transfer. Develop-

Puelles, L., and Martinez-de-la-Terre, M. (1987). Autoradiographrc and Colgi study of the early development of n. Isthmi1 principalrs and adjacent grisea viewpiont. Anat. Embryol. Puelles,

L.,

Amat,

rn the chick 176, 19-34.

J. A.,

and

embryo:

a todimensional

Martinez-de-la-Torte.

M.

(1987).

Segment-related, mosaic neurogenetic pattern in the forebrain and mesencephalon of early chick embryos: I, Topography of AChE-positive neuroblasts up to HH18. J. Comp. Neural. 266,

247-268. F. C. (1935).

Mitosis

in the

neural

tube.

J. Comp.

Neural.

62, 377-405. Schlosser, C., and send axons through

Tosney, K. W. (1988). Projection-neurons the lumbar spinal cord of the chick

are not obviously distributed J. Neurosci. Res. 27, 410-419.

in a segmentally

Srlver, M. L. (1942). The motoneurons frog. J. Comp. Neural. 77, I-40. Spemann, H. (1912). ijber teile bei Amphibienembryonen. Stockard, C. R. (1906). rn Bdellostoma stouti. Streeter, nervous

die

of the

that embryo

reprtitrve spinal

manner. cord

of the

Entwicklung umgcdrehter Zool. Jahrb. 753 (SuppI.),

HirnI-48.

The development of the Am. J. Anat. 5, 481-475.

C. L. (1933). The status system of chick embryos.

mouth

and

gills

of metamerism in the central J. Comp. Neural. 57 455-475.

.Tuc kett, F., Lim, L., and Morriss-Kay, G. M. (1985). The ontogenesis of cranial neuromeres rn the rat embryo. I. A scanning elertron microscope and kinetic study. J. Embryol. Exp. Morph. 87, 131-136. Turner, neurons ture

D. L., and Cepko, and glia persists

C. L. (1987). A common progenitor in rat retina late in development.

The segmentatron of the prrmrtive (Callus domesticus). Adv. Anat.

neural Embryol.

van Wijhe, J. W. (1882). tntwicklung der Nerven Akad. Wiss., Amsterdam.

iiber des

die Mesodermsegmente Selachierkopfes. Naturk.

von

ijber

die

Thiere.

for Na-

328, 131-136.

Vaage, S. (1969). tn chick embryos Biol. 47, l-88.

Baer,

K. E. (1828).

Entwicklungsgeschichte

tube Cell

and dre Verh. K. der

Konigsberg.

von Kupffer, K. (1906). Die Morphogenie des Zentralnrrvensystems, 0. Hertwig, ed., Handbuch der vergleichenden und expermentellen Entwic klungslehre der Wtrbelthiere. Volume 2 (Jena:

FIscherI.

D. C. (1989).

vertebrate

CNS.

Wilkrnson, D. C., Bhatt, P. (1989a). Segment-specific the developing nervous 461-464.

795-801. Orr, H. (1887). Contribution Morphol. 7, 311-372.

Eisen, J. \. (1986). ot axral muscles

IdentrIn the tan 239.

1142-1145.

of avran

Interactions

Development

Nordlander, pus tadpoles.

Sauer,

cells

structure of the spnal cord ot the Q. I. Mic-rose. SCI. 89, 359-384.

Homeobox Bioessays

genes

and

the development

10, 82-85.

S., Chavrier, P., Bravo, R.. and Charnay, expression of a zinc finger gene rn system of the mouscs. Nature 13;:

Wilkinson, D. C., Bhatt, S., Cook, M., Boncrnrlli. F.. and Krum Iauf, R. (1989b). Segmental expressron of Hex-2 homeobox genes in the developing mouse hrndbrain. Nature 347, 405-409. Youngstrom, K. A. (1940). A primary and a secondary somatic motor innervation in Amblystoma. J. Comp. Neural. 73, 139-151.