@ Masson,Paris, 1990
J. Physiol.,Paris,1990,84,27-32
Gif Lecturesin Neurobiology
"Expressionand assemblyof a functionalneuron"
Segmentation and the development
of the vertebrate nervous system
Roger KEYNES (r), Geoffrey COOK (r), Jamie DAVIES 1r;, Andrew LUMSDEN
(2), Wendie NORRIS (3) and Claudio STERN (3)
(t) Department of Anatomy, Downing Street, Cambridge C82 3DY, U.K. (2) Department of Anatomy, tlnited Medical
Schools, Guy's Hospital, London SRl 9RT. U.K.t)
Department of Human Anatomy, South Parks Road, Oxford OXi
Suvruenv:
l' Recent experiments on the development of neural
segmentation in chick embryos are reviewed.
2' Segmentationofthe spinal peripheral nervesis governed
by a subdivision of the somite-derivedsclerotome into anterior
and posterior halves. Migrating neural crest cells and outgrowing motor axons are conflned to the anterior sclerotome as a
result, in part, of inhibitory interactions with posterior sclerotome cells.
3' The sclerotomal distribution of certain molecules
known to influence growing nerve cells in vitro, namely laminin,
flbronectin, N-CAM, N-Cadherin and Jl/tenascin/cytotactin,
suggest that these molecules play no critical role in determining
the preference of nerve cells for anterior sclerotome.
4' Peanut agglutinin (PNA) recognises cell surfaceassociatedcomponents on posterior cells which, when incorporated into liposomes, cause the abrupt collapse of sensory
growth cones ln vitro.The PNA receptor(s) may be inhibitory
for nerve cells in vivo.
5" The chick hindbrain epithelium is segmented early in its
development. Each branchiomotor nucleus in the series of
cranial nervesV, VII and IX derives from a pair of segments
lying in register with an adjacent branchial arch. Neurogenesis
of motor and reticular axons begins in alternate segments,
suggestingparallels with insect pattern formation.
Key-words.' Segmentation.Somites.Axon growth. Neural crest
cells. Rhombomeres. Pattern formation.
INTRODUCTION
Segmentation is an important patterning process
during the development of many higher organisms. In
this paper we review its role during the development of
the vertebrate nervous system,and focus on two separable aspectsof neural segmentation; first, its contribution
to axon guidance in the developing peripheral nervous
Received March 30th, 1989.
Reprint requests: R. KEYNES,above mentioned address
and Dental
3QX, U.K.
system; and second, its significanceas a mechanism for
patterning neuronal populations in the central nervous
system.
A - Axon guidance during segmentation in the peripheral
nerYoussystem.
The original studies of Lnnrr,r,l.lrN (1927) and
DETwILER (1934) establishedthat the segmented,ladderlike arrangement of the spinal peripheral nerves arisesas
a result of segmentation of the somitic mesoderm.
DETwILER showed, for example, that grafting an additional somite alongside the neural tube in axolotl
embryos generates an additional spinal nerve. More
recently, we have found that spinal nerve segmentation in
higher vertebrate embryos is a direct consequenceof the
subdivision of the somitic mesoderm into anterior
(A, cranial) and posterior (P, caudal) parts. Specifically,
migrating neural crest cells and outgrowing motor and
sensory axons are confined to the anterior half of each
somite-derived sclerotome as they course laterally from
the neural tube region (KEvNES and SrpRN, 1984;
RICKMANNet a|.,1985; BRoNNnn-FRASER,
1986; TprlLET et al.,1987 ; LonINc and EnrcrsoN, 1987).In chick
embryos, reversal of a strip of paraxial mesoderm along
the A-P axis, before somite segmentation,results in axons
traversing the posterior (original anterior) parts of the
reversed sclerotomes (KEYNES and STERN, 1984).
Segmentationof the peripheral nerves is therefore governed by differencesbetween A- and P-sclerotome,which
can be detected by neural crest cells and by the growth
cones of motor and sensory nerves.
We have reviewed recently the implications of the
A/P subdivision of the sclerotome for the mechanismsof
segmentation (KEYNES and STBRN, 1988). Another
feature of the phenomenon is that it provides an apparently simple experimental system for the study of the
molecular mechanismsof axon and crest cell guidance.In
principle, both stimulatory (anterior half) and inhibitory
28
R. KEYNES et al.
(posterior half) interactions could be involved in the
preference of nerve cells for A-sclerotome. Since motor
axons are able to grow from the neural tube after removal
of the somitic mesoderm (LEwts et al.,l98l), any positive
(for example, adhesiveor chemotropic) influence is unlikely to be an absolute requirement for nerve growth. This
suggests,in turn, that inhibitory interactions may be
more critical in directins axons and crest throueh the
sclerotome.
Journal de Physiologie
begin their migration at around the I I somite stage
(Tnrrnv et al., 1982). Second, crest cells have been found
to round up when cultured on a substrate of cytotactin/tenascin, in sharp contrast to their behaviour when
cultured on fibronectin. another substrateadhesionmolecule. Third, our own immunohistochemical studies of the
distribution of tenascin and the immunologically related
Jl (FAISSNERet a|.,1988) in chick somites have shown a
complicated, evolving pattern of localisation in the sclerotome (SrrnN, NoRRIS, BRoNNBn-FRASER,CARLSON,
FArssNER, KEyNEs, SHACHNER, submitted; Fig. l).
BETWEENANTERIORAND Before the sclerotome develops, immunoreactivity to
I) MOLECULARDIFFERENCES
J1/tenascin appears in association with the extracellular
POSTERIOR
CELLS
matrix surrounding the epithelial somite, and with the
A number of approacheshave been adopted to iden- few mesenchymalcells that lie in the central core of the
tify molecular differences between A- and P-sclerotome epithelial somite. When the sclerotome first appears,
cells. The simplest is to look for A/P differencesusing Jlitenascin is concentratedin the posterior half at a stage
probes, such as monoclonal antibodies, recognisingmole- when crest cells migrate selectively into the anterior half.
cules known to influence growing nerve cells in vitro. Only later does immunoreactivity localise to the anterior
Immunohistochemical studies using antibodies to lami- half-sclerotome. Moreover, unlike T.q.Ner al. (1987), we
nin, fibronectin, N-CAM and N-cadherin have failed to find that after neural crest ablation, immunoreactivity
reveal any differential distribution of these molecules remains predominantly in posterior half-sclerotome,
et al., 1985; rather than becoming localised to the A-half ; and, after
within the sclerotome (RtcrueNN
KRorosKI et al.,1986; DunnNo et a|.,1987 ;HATTA et crest ablation, the molecular weights of moieties carrying
al..1987: Meczun et al.,1988). While theseresultsrule epitopes for J1/tenascinantibodies are found, by affinityout the possibility that there are major quantitative diffe- purification of somite samples, to be different from those
rences in the A/P distribution of these molecules, it is seen in samples from unoperated embryos. The most
important to note that more subtle, qualitative differences parsimpnious interpretation of these findings is that cytohave yet to be excluded. It is conceivable, for example, tactin/tenascin cannot be the critical factor in determithat certain regions of the fibronectin molecule may be ning the pathway of nerve cells through the somite. It
present in A-sclerotome but modified or absent in P- may be involved, however, in later crest-related events,
sclerotome. The production of antibodies which specifi- such as the arrest of migration and the aggregation of
cally recognisesuch sites is neededin order to assessthis crest cells into dorsal root ganglia.
possibility further.
T.q.Ner al. (1987) have also described the sclerotomal
Recent immunohistochemical studies of the distribudistribution of a cytotactin-binding proteoglycan (CTB
tion of the < substrate adhesion molecule > cytotactin in proteoglycan). Like cytotactin, CTB proteoglycan provichick embryos (TaN et al.,1987), and the (probably iden- des a poor substratefor crest cell migration in vitro. CTBtical) glycoprotein tenascin in quail embryos (MecrIn et proteoglycan is evenly distributed in both A- and Pa\.,1988), have revealed that cytotactin/tenascin is sclerotome halves when the sclerotome first appears, and
concentrated in anterior sclerotome. Cytotactin is repor- so cannot be solely responsiblefor directing the crest into
ted to be restricted to the anterior half sclerotome after A-sclerotome. At later stagesit becomesconcentrated in
ablation of the neural crest, and both cytotactin and the posterior half-sclerotome. It could be argued that,
tenascinhave been shown to be produced by somitic cells because CTB proteoglycan binds peanut lectin (PNA),
when cultured in vitro. It has therefore been proposed and becausethis lectin has been shown to stain the caudal
that cytotactin/tenascin is involved in some way in deter- halves of the sclerotomes of the chick embryo (Srr,RN er
mining the preference of nerve cells for A-sclerotome, for
al.,1986; seebelow), CTB proteoglycan may be responexample by providing an adhesive matrix for crest cells sible for the pattern of staining seenwith PNA. However,
and growth cones. A number of further observations PNA binding is never seen in both halves of the scleroshow, however, that such an interpretation is likely to be tome.
an over-simplification.
First. there is no correlation in time between the
2) PEANUT AGGLUTININ RECEPTORS AND AxoN
earliest expressionof cytotaetin in A-sclerotome and the GROWTH.
earliest migration of the neural crest ; in chick embryos,
Other screening procedures in the search for molecytotactin is detectable in A-sclerotome only after the
30 somite stage; trunk crest cells, on the other hand, cular differences between A- and P-sclerotome have used
Vol.84,n" 1, 1990
VERTEBRATE NEU RAL SEGMENTATION
29
Distrib uti o n of J 1/tenasci n-related m olecu les i n un operated somites
Distribution of J1/tenascin-related molecules after crest ablation
Ftc. I . - Schematic diagram showing immunohistochemical localisaiion of J 1/tenascin in unoperated (above) and neural crest-ablated (below)
chick embryo somites. Anterior is to the right. Seven sclerotomes are shown, each divided (dashed line) into anterior (A) and posterior (P) halves.
Jl/tenascin immunoreactivity is indicated by the irregular 1ines.Three epithelial somites are also shown to the left, each surrounded by Jl/tenascin
immunoreactivity.
A-cells (SrnnN et al., 1986). Examination of isolated Pcells stained with fluorescein-conjugated PNA shows that
the receptor is localised to the cell surface (Fig. 2). By
means of affinity chromatography using immobilised
lectin, we have demonstrated that the principal PNAbinding activity is associatedwith polypeptides of apparent Mr 49 and 55 K. Moreover, when chick A- and Phalf sclerotomes are dissected and analysed by SDSPAGE, only thesetwo molecular weight speciesare found
to be missing from the anterior halves by silver staining.
Recently, we have performed experiments using a strategy for assaying growth cone-inhibitory activity devised
by Dr J.A. RRpnn (University of Pennsylvania).
Detergent-extracted somite material, incorporated into
Ftc. 2. - fung reaction, with patching and capping, of FITClabelledPNA-receptor(s)on posteriorsclerotomecells.A suspensionof
liposomes, causes the rapid collapse of sensory growth
P cellswas labelledwith FlTC-conjugatedPNA for I hour at 4'C.
cones rn vitro, whlle prior incubation of the solubilised
material with immobilised PNA, before incorporation
into liposomes, removes the collapsing activity. These
observations (DAvIEs, CooK, KevNns and Stnnx, in
preparation)
support the notion that inhibition of nerve
enzymehistochemistry(showingbutyrylcholinesterase
to
growth
by
P
cells,
in association with PNA-binding matebe localisedin A-sclerotome;LAYSRet al., 1988)and
major
rial,
may
be
a
influence responsible for segmentaplant lectins.In a study using a variety of peroxidaseperipheral
tion
of
the
nervous system.
conjugatedlectins to stain sectionsof somites,it was
found that PNA recoenisesP-sclerotomecells and not
In addition to the molecular differences between A-
30
R. KEYNES et al.
Journal de Physiologie
and P-cells detected by one-dimensional gel electrophoresis, several other A/P differencesare present on twodimensional gels of sclerotome cells. These are stage- and
region-specific, and may reflect a more complex temporal
and spatial organisation beyond the simple subdivision of
each sclerotome into A- and P-halves (NoRRls el c/.,
1989).
-------l
I
\\ I
B - Segmentation in the central nervous system.
During the late lgth and early 20thcenturies,a substantial literature appeared on the subject of neuromeres,
periodic swellings visible in the epithelium of the vertebrate neural tube soon after its formation. It is interesting
to note that the prevailing perspective of these studies was
from the standpoint of evolution rather than development, and in particular the search for homologies in relation to segmentation of the vertebrate head. In a review
of the subject in 1918,NEAL argued that neuromeresare
of doubtful importance ; and the dependenceof peripheral nerve segmentation on mesodermal segmentation led
DETwILER (1934) to conclude that the spinal cord is not
intrinsically segmented. More recent descriptions of
neuromereshave continued to emphasisemore their existence than their significance (for review, see VAAGE,
1969). It is possible, nevertheless,that neuromeres do
represent important units of construction of the CNS.
The most obvious way to establishthis would be to correlate the development of individual neuromereswith any
underlying segmental patterns of neuronal development
within the neural epithelium. Previous attempts at this,
examining the relation between the neuromeres of the
hindbrain and the development of the cranial nerve
motor nuclei, produced conflicting results, probably
becauseof the low resolution of the anatomical techniques then available (SrReernR, 1908; Nnel, l9l8). The
advent of higher resolution methods for identifying and
tracing developing neuroneshas allowed us to re-examine
this question, and to show that the hindbrain of higher
vertebrates is constructed segmentally (LurusoEN and
KnvNns, 1989).
1) RHOMBoMERES.
In the chick embryo the hindbrain swellings, or
< rhombomeres )), are present between days 2 and 4 of
development. They are conspicuous as periodic undulations of the neural epithelium, lying symmetrically on
either side of the midline floorplate ; their boundaries are
marked by concentrations of axons growing transversely
in the marginal zone of the epithelium. The relation
between the pattern of neurogenesisof the cranial branchiomotor nerves and that of the rhombomeres has been
xll
I
Ftc. 3. - Schematicdiagramof the 3 day chick embryohindbrain,
basedon neurofilament-stained
and Dil-injectedpreparations.The relationship of cranial sensoryganglia (gV-gX), branchial motor nuclei,
somatic motor nuclei (IV. VI. XII) and the combined roots of the
sensoryand branchial motor nerves(mV-mXI) to the rhombomeres
(rl-r8) and branchialarches(b1-b3)is shown.ov, otic vesicle;fp, floor
plate; i, isthmus/midbrain-hindbrain boundary. (Reproduced by
permissionfrom Nature, Vol. 337, p.428. Copyright (c) 1989,Mac
Millan MagazinesLtd.)
assessedby the local application of lipid-soluble carbocyanine dyes (DiI and"DiO) to individual cranial nerve
roots ; the fluorescent dye diffuses in the axon membranes
to the parent cell bodies, allowing the early motor nuclei
to be identified. The results are illustrated schematically
in Fig. 3 (LutvtsoEN and KpyuEs, 1989). Each consecutive motor nucleusin the seriesof cranial branchiomotor
nerves V (trigeminaD, VII (facial) and IX (glossopharyngeal) derives from a specific, consecutive pairing of rhombomeres, each pair lying in register with an adjacent branchial arch. Thus, the nucleus of nerve V derives from
rhombomeres2 and 3 (r2,3), oppo_site
the l't arch; VII
derives from r4,5 opposite the 2n" arch; and IX from
Vol. 84, n' 1, 1990
16,7 opposite the 3'd arch. Neurogenesis begins in an
alternating pattern : the anterior member of each rhombomere pair produces motor axons before the posterior
member, and contains the motor nerve root. An alternating pattern is also seen in the differentiation of reticular
neurones within the hindbrain epithelium : as for the
branchiomotor nerves, reticular axons first appear in 12,
14 and 16.
These observations show that the gross neuromeric
pattern visible in the hindbrain is reflected at the cellular
level. Neurogenesis begins in alternating segments, and
follows a two-segment repeat. The alternating sequenceis
matched, moreover, by the expression pattern of a mouse
zinc finger gene, Krox-20, which is transcribed exclusively
in 13 and 15 during early embryonic development
(Wu-rINsoN et a|.,1989). Such phenomena, being reminiscent of the spatial expression patterns of the < pairrule > genesduring epidermal segmentation in Drosophila
(NUsslnm-VoLHARD and WrBscnAUS, 1980; AKArra,
1987), raise the possibility that the process of vertebrate
hindbrain segmentationmay be similar to segmentation
mechanisms operative during fly development. With this
in mind, it may also be significant that many mouse
homeobox geneshave A/P boundaries of expressionthat
lie within the embryonic hindbrain (HolraNo
and
HocAN, 1988).
2) SpcrvlpNrATroN oF THE NEURAXTS.
If segmentation can now be seen as an important
feature of hindbrain development in higher vertebrate
embryos, is this also the casefor other CNS regions, and
for lower vertebrates ? Segmentally arranged neurones
have been described in the nerve cord of Amphioxus
(BoNn, 1960),the spinal cord of the larval brook-lamprey
(WHrrrNc, 1948), and the spinal cord and hindbrain of
embryonic zebrafish (Mvnns, 1985; MnTcALFE et ql.,
1986; HaNNEMAN et al., 1988). Such arrangements are
not present, however, during the development of the
chick spinal cord : the neuromeres(< myelomeres>) visible in this region of the neural tube are likely to arise as
a result of mechanical interactions between the neural
epithelium and the adjacent somites(Lrru, 1987),as originally suggestedby NEAL (1918). It seemsprobable that
during the course ofvertebrate evolution, as brain centres
for movement control became progressivelymore dominant over local spinal reflex circuits, intrinsic spinal cord
segmentationdisappeared.Simultaneously,segmentation
in the neighbouring mesoderm appears to have assumed
control as the major influence over tissue pattern in the
trunk. Whether CNS segmentation extends beyond the
hindbrain into the mid- and forebrain remains to be established. The recent description of a monoclonal antibody
with specificity for neurons in the telencephalon, but not
VERTEBRATE NEURAL SEGM ENTATIO N
3l
other CNS regions(MonI et al.,1987),raisesthe possibility that position-specific,possibly segment-related,
antigensmay exist in the developingvertebrateCNS. If
more suchantigensare identified,it will be important to
assesstheir boundariesof expressionin relation to the
neuromericboundaries.
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