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Development 117, 59-74 (1993)
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59
Origins, migration and differentiation of glial cells in the insect enteric
nervous system from a discrete set of glial precursors
Philip F. Copenhaver
Department of Cell Biology and Anatomy L215, Oregon Health Sciences University, 3181 SW Sam Jackson Park Road,
Portland, Oregon 97201, USA
SUMMARY
The enteric nervous system (ENS) of the moth, Mand uca sexta, consists of two primary cellular domains and
their associated nerves. The neurons of the anterior
domain occupy two small peripheral ganglia (the frontal
and hypocerebral ganglia), while a second population of
neurons occupies a branching nerve plexus (the enteric
plexus) that spans the foregut-midgut boundary. Previously, we have shown these two regions arise by separate programs of neurogenesis: cells that form the anterior enteric ganglia are generated from three discrete
proliferative zones that differentiate within the foregut
epithelium. In contrast, the cells of the enteric plexus
(the EP cells) emerge from a neurogenic placode within
the posterior lip of the foregut. Both sets of neurons subsequently undergo an extended period of migration and
reorganization to achieve their mature distributions. We
now show that prior to the completion of neurogenesis,
an additional class of precursor cells is generated from
the three proliferative zones of the foregut. Coincident
with the onset of neuronal migration, this precursor
class enters a phase of enhanced mitotic activity, giving
rise to a population of cells that continue to divide as
the ENS matures. Using clonal analyses of individual
precursors, we demonstrate that the progeny of these
cells become distributed along the same pathways taken
by the migratory neurons; subsequently, they contribute
to an ensheathing layer around the branches of the
enteric plexus and the enteric ganglia. We conclude that
this additional precursor class, which shares a common
developmental origin with the enteric neurons, gives rise
to a distinct population of peripheral glial cells. Moreover, the distribution of enteric glial cells is achieved by
their migration and differentiation along the same pathways that are formed during the preceding phases of
neuronal migration.
INTRODUCTION
brates, as well, where glial cells may provide directional
cues for axonal outgrowth during normal development
(Katz et al., 1983; Maggs and Scholes, 1986; Norlander and
Singer, 1982; Silver, 1984) and can have significant modulatory effects on axon regrowth following injury (Bunge,
1983; Rudge and Silver, 1990; So and Aguayo, 1985). Nonneural cells expressing glial-related markers have been
detected in advance of peripheral nerve formation, as well,
(Carney and Silver, 1983; Noakes and Bennett, 1987),
although a role for glia in guiding peripheral nerve outgrowth remains controversial (Noakes et al., 1988; Poston
et al., 1988; Rickman et al., 1985).
Besides their functional interactions during development,
neurons and glia are often linked by their developmental
origins. In the vertebrate brain, all of the cell types of a
particular region can be generated by a common stem cell
within the neuroepithelium (Turner and Cepko, 1987; Wetts
and Fraser, 1988; Galileo et al., 1990) while, in the peripheral nervous system (PNS), ‘bipotential precursors’ from
the neural crest apparently give rise to both the sensory neurons and their glial support cells (Rohrer, 1985; Frank and
A key feature in the formation of the nervous system is the
coordinated generation of both neurons and supportive,
non-neuronal classes of glial cells. As a group, glial cells
have been shown to serve both structural and metabolic
roles in the mature nervous system (e.g. Roitbak, 1983; Vernadakis, 1988), but they also subserve a number of important developmental functions, as well. During the formation
of the central nervous system (CNS) of insects, for example, discrete sets of glial cells have been shown to prefigure the major longitudinal tracts of the segmental ganglia
that are subsequently ‘pioneered’ by the outgrowing
processes of central neurons (Poulson, 1950; Jacobs and
Goodman, 1989). Other glial subsets help delineate the
major commissural tracts that form within each ganglion
(Fredieu and Mahowald, 1989; Klambt et al., 1991) and
form the pathways that are followed by motor and sensory
axons as they leave the CNS (Bastiani and Goodman, 1986;
Carr and Taghert, 1988; Jacobs and Goodman, 1989). Similar interactions have been indicated in the CNS of verte-
Key words: enteric nervous system, glial progenitor, Manduca
sexta, glial differentiation, cell lineage
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P. F. Copenhaver
Sanes, 1991; Hall and Landis, 1991). Similarly, in the insect
CNS, transplantation studies have suggested that single
progenitors can produce both neurons and glia, at least in
some cases (Technau and Campos-Ortega, 1986). However,
while both neuroblasts and glioblasts arise from the same
neuroectoderm, most of the neural and glial lineages normally diverge before the cells differentiate (Campos-Ortega
and Hartenstein, 1985; Jacobs et al., 1989; Klambt et al.,
1991). At least some peripheral neurons and glia (those
associated with sensory hairs and bristles) do share a
common precursor (Lawrence, 1966; Bate, 1978; Bodmer
et al., 1989; Hartenstein and Posakony, 1990), although
how other classes of peripheral glia arise in insects and
what functions they perform during the formation of peripheral nerves have not been defined.
Recently, we have characterized the developmental origins of the enteric nervous system (ENS) of the moth, Man duca sexta. The ENS is organized into two distinct domains
(Fig. 1): anteriorly, the enteric neurons are clustered into a
pair of ganglia (that resemble miniature ganglia of the
CNS), while posteriorly, a second group of about 300 neurons are distributed throughout the enteric plexus, a branching nerve plexus that spans the foregut-midgut boundary
(Copenhaver and Taghert, 1989a). While all of the enteric
neurons arise locally from the foregut ectoderm, these two
populations are distinguished by the manner in which they
are generated. Neurons of the anterior enteric ganglia originate from three neurogenic zones that form in the middorsal epithelium of the embryonic foregut (Copenhaver
and Taghert, 1991). Each of the zones gives rise to a progression of mitotically active precursor cells, most of which
undergo one or two equal divisions and produce neurons
that subsequently populate the enteric ganglia. In this
regard, these precursors resemble the midline precursor
(MP) class of progenitor cells in the insect CNS, which
divide only once and give rise to pairs of neurons in the
insect CNS (Bate and Grunewald, 1981). In contrast, the
neurons of the enteric plexus (the EP cells) are generated
from a neurogenic placode that invaginates from the posterior lip of the foregut (Copenhaver and Taghert, 1990);
invagination refers to the inpocketing of epithelial cells
from the basal surface of an epithelium into the body cavity
(Fristrom, 1988). The EP cells become postmitotic as they
emerge from the epithelial layer and subsequently achieve
their mature distributions by active migration along a set
of preformed pathways (Copenhaver and Taghert, 1989b).
An unresolved issue has been the origins and differentiation of the glial cells that populate the ENS. In this report,
we present evidence that most of the glial cells ensheathing the enteric plexus and ganglia share a common developmental origin with the enteric neurons, but they are generated from a distinct class of progenitors. Specifically,
towards the end of their neurogenic phase, each of the three
neurogenic zones of the foregut produces an additional set
of precursor cells; these precursors remain closely associated with the neuronal populations of the ENS but do not
themselves become neurons. Rather, they give rise to a
group of mitotically active cells that ultimately form an
ensheathing layer around the enteric ganglia and nerves.
Thus in this simple system, neurogenesis and gliogenesis
are closely linked, involving the sequential generation of
Fig. 1. Cellular domains within the ENS of Manduca sexta. Two
distinct populations of enteric neurons can be distinguished by
their position and organization. One group of about 70 neurons
forms a pair of small ganglia on the anterior surface of the
foregut: the frontal ganglion (FG), which is connected to the brain
lobes (BR) via the frontal ganglion connectives (FGC), and the
hypocerebral ganglion (HG), which is continuous with the
recurrent nerve (RN) that lies mid-dorsally on the foregut surface.
Paired nerves also connect the recurrent nerve to the neurohemal
organs of the brain, the corpora cardiaca-corpora allata (CC-CA).
A second group of about 300 neurons (the EP cells) occupies the
enteric plexus, a branching set of nerves that extend along specific
radial muscle fibers on the foregut and along eight longitudinal
muscle bands on the midgut (only the dorsal muscle bands are
shown: L1-L2 and R1-R2). Scale, 0.1 mm.
Gliogenesis in the insect ENS
distinct classes of precursors from a common position
within the neuroectoderm. In addition, we show that glial
migration follows neuronal migration during the formation
of the enteric plexus, in that the mitotically active glial precursor cells become gradually dispersed along the nerve
pathways that were previously formed by the migratory EP
cells. A preliminary account of these results has appeared
previously (Copenhaver, 1991).
MATERIALS AND METHODS
Staging and visualization of the embryonic ENS
Rearing and staging of Manduca embryos was performed as
already described (Copenhaver and Taghert, 1989a, 1990) and by
reference to external and internal markers (Broadie et al., 1991;
Copenhaver and Taghert, 1989a; Dorn et al., 1987). Embryos were
dissected in a culture medium of the following composition
(vol/vol): 50% Schneider’s Drosophila medium, 40% Eagle’s
basic salts, 9.9% heat-inactivated fetal calf serum, 0.09% penicillin-streptomycin, 0.01% insulin; supplemented with Manduca
hemolymph (Copenhaver and Taghert, 1990; after Chen and LeviMontalcini, 1969 and Seecof et al., 1971). The developing ENS
could then be viewed using a compound microscope equipped
with Nomarski optics or by immunohistochemical staining with
the monoclonal antibody, TN-1 (ascites fluid diluted 1:20,0001:40,000). TN-1 recognizes a membrane-associated molecule that
is expressed by a variety of neural and non-neural cell types
(Taghert et al., 1986; Carr and Taghert, 1988) and that may be
related to fasciclin II (Nardi, 1990). As in previous reports (Copenhaver and Taghert, 1989b, 1990), it was used in the present study
to identify the components of the ENS throughout embryogenesis. Whole-mount immunohistochemistry was performed using 2%
paraformaldehyde or a modified Zamboni’s fixative (4%
paraformaldehyde, 15% saturated picric acid, in sodium phosphate
buffer, pH 7.2), followed by the ABC-HRP reaction protocol of
Vector Laboratories (Copenhaver and Taghert, 1989a). Antisera
against the neuronal protein ELAV from Drosophila (generously
provided by Drs Kalpana White and Steven Robinow) were also
used for whole-mount staining of the developing ENS at dilutions
of 1:500.
Scanning electron microscopy was performed on staged
embryos as previously described (Copenhaver and Taghert,
1989b). Animals were dissected in culture medium, fixed in 2%
glutaraldehyde plus 2% paraformaldehyde in PBS for 24 hours,
then dehydrated in ethanol and dried using a Tousimi critical point
dryer. Samples were gold-coated (200 A) and viewed on a Phillips
SEM501 microscope.
Glial proliferation and differentiation in the ENS
Mitotic patterns within the developing ENS were examined using
the thymidine analogue, 5-bromo-2′-deoxyuridine (BrdU; Sigma)
to label cells undergoing active DNA synthesis (Truman and Bate,
1988; Copenhaver and Taghert, 1990). Staged embryos were dissected at various times after the completion of neurogenesis (4070% of development; 1% of development equals ~1 hour of real
time) and incubated in culture medium containing BrdU (50
µg/ml) for 2 hours. The preparations were then fixed and stained
with an antibody to BrdU (Becton-Dickinson) at concentrations
of 1:30 to 1:50 (Gratzner, 1982); in some cases, the preparations
were also counterstained with TN-1 to show the overall morphology of the ENS.
Clonal analyses of the glial precursor cells within the developing ENS were performed using intracellular injections of fluorochrome-coupled dextran amines (Copenhaver and Taghert,
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1990; after Gimlich and Braun, 1985 and Wetts and Fraser, 1988).
Lysinated tetramethylrhodamine dextran amine (LRD) or lysinated fluorescein dextran amine (LFD; from Molecular Probes,
Inc.) was injected into individual precursor cells in staged, dissected embryos at about 40% of development; each preparation
was then briefly examined with a heavily filtered UV light source
to verify that only a single cell was labelled. Embryos were then
placed in culture for an additional 24 hours of development and
subsequently analyzed to determine the numbers and distributions
of labelled progeny. The reliability of this technique for performing clonal analyses of embryonic cells has been previously
described (Wetts and Fraser, 1988; Copenhaver and Taghert,
1990). TN-1 counterstaining was often performed using the appropriate secondary antibodies to complement the injected fluorochrome (e.g. rhodamine-conjugated antibodies were used in conjunction with LFD). Labelled cells were then photographed in
whole-mount preparations using a modified Nikon UM-2 epifluorescent microscope equipped with the appropriate filter sets and
drawn by camera-lucida techniques.
RESULTS
Cellular distributions in the larval and embryonic
ENS
All of the components of the ENS in postembryonic stages
of Manduca lie superficially along the alimentary tract (Fig.
1) and supply innervation to the visceral musculature and
possibly the midgut epithelium. Anteriorly, approximately
70 neurons are organized into two peripheral ganglia: the
frontal ganglion, which communicates with the overlying
brain lobes via the frontal ganglion connectives, and the
hypocerebral ganglion, which is joined with the recurrent
nerve. Posteriorly, the recurrent nerve is continuous with
the enteric plexus, a branched set of nerves that project
along specific groups of muscle fibers on both the foregut
and midgut and that contain the second major class of
enteric neurons, the EP cells (Copenhaver and Taghert,
1989a).
As in other regions of the insect PNS (c.f. Radojcic and
Pentreath, 1979), both the nerves and ganglia of the ENS
are ensheathed by glial cells that contribute to their structural integrity and provide an effective barrier against the
circulating hemolymph (Treherne et al., 1984; Ziller et al.,
1987). During the initial formation of the ENS, however,
this glial layer was not yet apparent. By 40% of development, the neurons of both the anterior and posterior domains
of the ENS could be distinguished on the foregut surface
(Fig. 2A), but had not yet achieved their mature distributions. At this time, cells derived from the neurogenic zones
of the foregut had begun to coalesce into the anterior enteric
ganglia and recurrent nerve, while the EP cells (derived separately from a neurogenic placode) formed an enlarged, premigratory packet at the posterior end of the foregut.
In addition to the two populations of neurons (ranging
from 8 to 15 µm in diameter), an additional class of somewhat larger cells (~20-25 µm) could be distinguished within
the developing ENS, interspersed among the neurons on the
foregut surface. In particular, a cluster of 8-10 of these
larger cells was invariably found at the apex of the EP cell
packet, juxtaposed between the EP cells and the developing recurrent nerve (Fig. 2B). As previously described,
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P. F. Copenhaver
Fig. 2. Distribution of cell groups
within the developing ENS following
the completion of neurogenesis (40%
of development). (A) Scanning
electron micrograph of the dorsal
surface of the foregut (anterior towards
top of page) reveals both anterior and
posterior groups of enteric neurons
prior to their differentiation. Cells
derived from the neurogenic zones of
the foregut have begun to coalesce into
the recurrent nerve (rn) and anterior
enteric ganglia, including the
hypocerebral ganglion (hg) and the
frontal ganglion (see Fig. 3); br,
developing brain lobes. In addition,
cells derived from the neurogenic
placode of the foregut have formed a
distinct packet of cells (the EP cells;
ep) that are now in contact with the
posterior end of the recurrent nerve.
Adjacent to the EP cell group, a cluster
of somewhat larger cells (derived from
the neurogenic zones) can be
distinguished by their size and
organization (demarcated by the white
arrowheads). (B) Whole-mount
preparation of a similarly staged
embryo stained with the monoclonal
antibody, TN-1, which recognizes epitopes on both the zone-derived and placode-derived cell groups. Black arrowheads denote the same
relative position as white arrowheads in A, indicating the boundary between the EP cells and the zone-derived precursors; arrow points to
one of the precursors that is in the plane of view (see also figure 4A). (C) Immunohistochemical staining with an anti-BrdU antibody
following a 2 hour incubation with BrdU (preparation was also lightly counterstained with TN-1). Several nuclei of cells within the
cluster of zone-derived precursors shows positive staining, indicating active DNA synthesis, whereas none of the EP cells are mitotically
active at this time. Scale, 30 µm.
these apical cells are among the last to emerge from the
third neurogenic zone of the foregut, whereupon they establish contact with the neurogenic placode of the enteric
plexus (Copenhaver and Taghert, 1991). Moreover, this
continuity is maintained throughout subsequent phases of
morphogenesis (40-70%), as the neuronal populations of
the ENS become reorganized into their mature configurations.
Identification of glial precursor cells by patterns
of mitotic activity
In past reports (Copenhaver and Taghert, 1990, 1991), we
have shown that neurogenesis in the ENS is essentially
complete by 40% of development, at which time all of the
neurons of both the enteric ganglia and the plexus are
present and undergo no further rounds of division. During
the period of neurogenesis, we observed little or no mitotic
activity within the apical cell group. Coincident with the
onset of neuronal migration, however, these cells commenced a new phase of proliferation. When BrdU labelling
was used to map the distribution of cells undergoing active
DNA synthesis at this time, many of the cells in the apical
cluster began to show evidence of mitotic activity, in contrast to the unlabelled EP cells (Fig. 2C). During the subsequent phases of development, progeny associated with
this apical cluster continued to proliferate and gradually
spread throughout the enteric plexus (Figs 3, 4).
From 40-55%, the EP cells underwent a circumferential
phase of migration, gradually spreading down both sides of
the foregut next to the foregut-midgut boundary (Fig. 4AC; Copenhaver and Taghert, 1989b). As this first phase of
neuronal migration progressed, the number of labelled
nuclei within the precursor population increased steadily
(Figs 3, 4E-G). At the same time, labelled nuclei also began
to spread laterally across the surface of the EP cell packet.
Then during the second phase of neuronal migration (55-
Fig. 3. Camera-lucida drawings to show the distribution of
mitotically active cells within the developing ENS following
neurogenesis. Staged embryos were incubated with BrdU and
stained in whole-mount with an anti-BrdU antiserum to label
nuclei that were undergoing active DNA synthesis. Labelled cells
in the surrounding epithelial layers were not drawn. Arrowheads
at 40% indicate the boundary between the EP cell group and the
zone-derived cluster of precursor cells (see Fig. 2). During
subsequent phases of development, arrowheads mark the position
of labelled nuclei along the migratory pathways taken by the
postmitotic EP cells. FG, frontal ganglion; HG, hypocerebral
ganglion; RN, recurrent nerve; L1-L2 and R1-R2, dorsal sets of
muscle bands followed by the EP cells during migration. Same
scale as Fig. 1.
Gliogenesis in the insect ENS
Fig. 3
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P. F. Copenhaver
Fig. 4. Paired photomicrographs of identically staged embryos stained either with TN-1 (A-D) or anti-BrdU following BrdU incubation
(E-H). Small arrows indicate the directions taken by the EP cells during their migration; arrowheads mark the positions of labelled nuclei
generated by the zone-derived precursor cells. (A, E) 45%. The EP cells have begun to spread bilaterally down both sides of the foregut,
adjacent to the foregut-midgut boundary (black bars). The cluster of larger zone-derived precursors is clearly distinguishable in A and
includes a number of synthetically active nuclei. (B, F) 50%. The EP cells have continued to spread circumferentially around the foregut;
the mitotically active precursor group has increased in number and begun to spread bilaterally, as well. (C, G) 55%. The EP cells are just
beginning to migrate onto the muscle bands of the midgut, while the mitotically active cells have become dispersed bilaterally across the
EP cell population. (D, H) 60%. The EP cells have migrated posteriorly onto the longitudinal bands of the midgut (including bands L1 and
R1) as well as onto radial muscle fibers of the foregut (out of the plane of view); labelled nuclei can now be seen along the major branches
of the developing enteric plexus as far posteriorly as the foregut-midgut boundary. Same scale as Fig. 2.
60%; Fig. 4C, D), when the EP cells moved rapidly out
along the muscle bands of the foregut and midgut, mitotically active progeny could also be found along the pathways formed by the migratory neurons (Figs 3, 4G-H).
However, while the neuronal population had completed its
dispersal by 65% of development (Copenhaver and Taghert,
1989b), mitotically active cells continued to be found along
the nerve pathways during subsequent periods of embryogenesis. By 65%, labelled nuclei were detected across the
foregut-midgut boundary (Fig. 5A,B) and, by 70%, they
could be found in the vicinity of the postmigratory clusters
of EP cells along the midgut muscle bands (Figs 3, 5E).
Throughout this developmental period, a similar increase in
mitotic activity accompanied the differentiation of the
enteric ganglia. While most of the neurons in the anterior
ENS were generated by 40% of embryogenesis (Copenhaver and Taghert, 1991), BrdU labelling during subsequent
stages of development revealed a steadily increasing
number of synthetically active nuclei that were associated
with the ganglia and nerves of the foregut (Fig. 3). Most
of the labelled nuclei were associated with the superficial
layers of the frontal and hypocerebral ganglia and the recur-
rent nerve (Fig. 5C, D), and later with their peripheral nerve
roots (Fig. 3, 70%). Detectable levels of mitotic activity in
the ENS continued throughout the remainder of embryogenesis, well after the enteric neurons had commenced the
expression of their mature phenotypes (Copenhaver and
Taghert, 1989a, 1991). These results indicated that, in
addition to the apical cluster of precursors next to the EP
cell packet, an additional population of precursors with similar mitotic behavior was distributed throughout the anterior regions of the ENS.
Identification of glial precursor cells by clonal
analysis
To gain insight into the number and fate of progeny arising from this precursor class, we injected individual cells
with either LFD or LRD (e.g. Fig. 6A) and examined the
distribution of labelled progeny after 24 hours in culture.
Representative examples chosen from over 70 such preparations are included in Figs 6 and 7. When an individual
EP cell (a presumptive neuron) was injected at 40% of
development, we subsequently observed only a single
labelled neuron that had migrated along one of the path-
Gliogenesis in the insect ENS
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Fig. 5. Continued dispersal of mitotically
active cells after the completion of
neuronal migration. Large arrows indicate
the orientation of the EP cell clusters that
have migrated onto the dorsal pair of
midgut muscle bands (L1 and R1).
Arrowheads in B and E mark the position
of labelled nuclei along the pathways
formed during EP cell migration. (A) TN-1
stained preparation at 65% of development
to show the dispersed subsets of EP cells
on the mid-dorsal muscle bands. (B)
Embryo at the same stage that was stained
with anti-BrdU, revealing that mitotically
active cells can now be detected posterior
to the foregut-midgut boundary (black
bars). The EP cells remain unlabeled,
although heavy labelling of epithelial cells
in the midgut can be seen on either side of
the muscle bands. (C-E) Mitotic activity at
70% of development in different regions of
the ENS. (C) Labelled nuclei in the
peripheral layer of the frontal ganglion
(fg); labelled glioblasts can also be seen in
the brain lobes (br). (D) Labelled nuclei
associated with the peripheral layer of the
recurrent nerve (rn). (E) Labelled nuclei
along the midgut branches of the enteric
plexus, indicating the continued dispersal
of progeny from the zone-derived
precursors (compare with Fig. 4). Same
scale as Fig. 2.
ways of the enteric plexus (Fig. 6B; in this preparation, the
enteric plexus was lightly counterstained with a fluorescent
antiserum). In contrast, when individual cells within the
apical group of large precursors were injected at 40%, clusters of labelled progeny were subsequently detected along
one or more of the branches of the plexus (Figs 6C-E, 7IL). In general, labelled progeny were found in the approximate vicinity of the original injected cell, although the
extent of their dispersal varied from animal to animal. In
optimal preparations, the dye remained uniformly dispersed
throughout the cytoplasm of the labelled cells, revealing
that the progeny of the injected cell had formed an
ensheathing layer around one or more nerves of the enteric
plexus (Figs 6C, 7K-L). In other preparations, the dye was
localized primarily to the nuclei of labelled cells (Figs 6E,
7I) or was concentrated into punctate intracellular blebs that
were distributed superficially along particular branches of
the plexus (Fig. 6D).
While only a single precursor cell was labelled in each
preparation (see Methods), the number of progeny derived
from a single precursor cell varied significantly in these
experiments, ranging from a minimum of 4-6 cells to more
than 20. In preparations where the dye had become compartmentalized in a punctate manner (e.g. Fig. 6D), it was
not possible to determine the number of cells that had been
labelled but only the approximate extent of their dispersal.
Similar variability in the intracellular distribution of lysinated dextrans following injection has also been reported in
other systems (e.g. Wetts and Fraser, 1988). A small number
of preparations (<5%) contained no labelled cells, presumably due to the death of the injected precursor cell. In such
cases, we could detect no evidence of the dye tracer in either
the neurons or glial cells of the ENS, as might result from
the phagocytosis of lysed fluorescent material. The preparations in Figs 6 and 7 were chosen to show the range in
the number of labelled cells and the dispersal of dye that
we observed. Despite this variability, these results corroborated our BrdU-labelling experiments, indicating that this
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P. F. Copenhaver
additional class of precursors continued to proliferate well
after the completion of neurogenesis and gave rise to a population of cells that assumed a glial morphology.
Similar patterns of labelled cells were seen following the
injection of mitotically active precursors in more anterior
regions of the developing recurrent nerve, as well. Injec-
Fig. 6. Clonal analysis of the glial precursor cells by intracellular injection of lineage-tracing dyes. LRD ( A, B, D, F) or LFD (C, E) was
injected into a single precursor within the developing ENS at 40% of embryogenesis, and the distribution of labelled progeny was
examined after 24 hours of subsequent development in embryo culture. In A-B and E-F, the preparations were also counterstained with
TN-1 (using a complementary fluorochrome) to show the outline of the developing ENS. (A) Example of an injected glial precursor cell
at 40% of embryogenesis (adjacent to the EP cell packet; ep), prior to the onset of gliogenesis. This preparation was fixed immediately
and counterstained with TN-1; rn, recurrent nerve. (B) Preparation in which one of the EP cells was injected at the same stage as A (40%)
and allowed to develop in culture. Only a single neuron within the enteric plexus was labelled after 24 hours, illustrating the postmitotic
status of the EP cell group. (C-F) Labelled progeny of individual glial precursor cells that were injected at 40% (similar to A) and allowed
to develop for 24 hours. In C, the progeny of a glial precursor had spread down one of the lateral branches of the plexus and had begun to
form an ensheathing layer around the neurons and processes of the nerve branch. In D, progenitors from the injected cell had spread down
several branches of the developing plexus, but the dye has become compartmentalized within the cytoplasm of the cells in a punctate
manner. In E, 10-12 labelled progeny had spread down both sides of the developing enteric plexus (compare with Fig. 4H) but the dye
was localized in the vicinity of the nuclei; not all of the cells are in the same plane of focus. In F, injection of a glial precursor that
occupied a more anterior position on the foregut resulted in the labelling of a cluster of progeny that ensheathed a region of the recurrent
nerve (rn; lightly counterstained with TN-1). Scale, 50 µm.
Gliogenesis in the insect ENS
tions of precursor cells at intermediate positions along the
foregut resulted in labelled patches of an ensheathing layer
around the recurrent nerve (Figs 6F, 7E-H), again with
varying degrees of dispersal from the site of the original
injection. More anteriorly, individual precursors often gave
rise to progeny that invested the nerve roots associated with
the enteric ganglia as well as the recurrent nerve (Fig. 7AD). Identifying glial precursor cells within the developing
ganglia was more difficult, due to the complexity of the
aggregating cell groups (Copenhaver and Taghert, 1991).
Nevertheless, occasional examples of clones were found
that ensheathed portions of one of the enteric ganglia (not
shown), in which the morphology of the labelled cells
resembled the perineurial glial cells of the CNS (Swales
and Lane, 1985). In general, precursors from the more anterior zones ensheathed more anterior regions of the ENS,
although we did not detect any clear boundaries between
the domains occupied by glial cells of adjacent zones.
When the levels of mitotic activity within the zonederived cell populations were examined throughout the
developing ENS, two distinct periods of proliferation could
be distinguished (Fig. 8). From 25% to 40% of development, a period of relatively low mitotic activity coincided
with the generation of neurons that eventually formed the
anterior enteric ganglia (Copenhaver and Taghert, 1991).
Subsequently, from 40% to 75%, a period of enhanced divisional rates was clearly evident, corresponding to a new
wave of proliferation stemming primarily (if not exclusively) from the larger precursor cells. These observations
indicated that cells derived from the neurogenic zones of
the foregut expressed one of two distinct fates: most of the
zone-derived precursors underwent a limited number of
divisions and produced cells that followed a neuronal program of differentiation, while the late-emerging population
of precursors subsequently commenced an extended phase
of proliferation and gave rise to comparatively large clones
of glial cells.
A neuronal-specific antigen distinguishes
neurons from glia in the ENS
To corroborate the morphological analysis of the presumptive glial cell populations within the ENS, we also
attempted to label the developing ENS with markers for
glial cells from other systems. A number of antibodies
against glial subtypes in cricket (Meyer et al., 1987) and
antisera against glial cells in the adult moth brain (C. E.
Krull and L. P. Tolbert, personal communication) failed to
differentiate any of the components of the developing ENS.
However, using a recently generated antiserum against the
ELAV protein, an RNA-binding protein that is expressed
by neurons but not glia in the fly nervous system (Robinow and White, 1991), we were able to distinguish the neuronal populations of the ENS from the putative glial lineages. When embryos were stained with anti-ELAV
antisera at 65% of development, the EP cells that had
migrated onto both the foregut and midgut pathways stained
positively (Fig. 9), as did the neurons of the anterior enteric
ganglia (not shown). In contrast, none of the presumed glial
cells within the branches of the enteric plexus or along the
recurrent nerve showed positive staining at any time that
we examined during development (Fig. 9, arrowheads).
67
These results further support our hypothesis that the residual class of precursors in the ENS gives rise to a distinct
population of cells that follow a non-neuronal sequence of
differentiation, ultimately assuming a glial fate.
In preliminary work, we also found that antibodies
against the Drosophila form of neuroglian (Bieber et al.,
1989) stained a number of processes in the developing ENS
of Manduca during the initial formation of the enteric ganglia. Specifically, in embryos between 25 and 30% of development, anti-neuroglian antibodies transiently labelled a
small number of processes that spanned the distance
between the newly formed neurogenic zones and extended
into regions of the foregut where the enteric ganglia would
subsequently form (unpublished results). In Drosophila,
neuroglian is expressed by large subsets of neuronal and
glial cells in the developing CNS and peripheral nerve roots
and is believed to play a role in neuronal and glial cell
adhesion (Bieber et al., 1989). Thus it is possible that an
additional class of non-neuronal cells arises early in the formation of the ENS and participates in the formation of the
enteric ganglia, as has been shown to occur in the insect
CNS (Jacobs and Goodman, 1989). In the present study,
we focussed only on the late-maturing glial populations that
contribute an ensheathing layer to the enteric nerves and
ganglia.
DISCUSSION
A model for neurogenesis and gliogenesis in the
ENS
The experiments described in this paper have shown that
the enteric glial cells of Manduca share an ectodermal
origin with the enteric neurons. Together with our previous
observations (Copenhaver and Taghert, 1990, 1991), these
results now permit the construction of a unified model for
the developmental origins of the ENS (Fig. 10). Neurogenesis in this system occurs via two distinct but concurrent
processes during which epithelial cells of the foregut
become committed to a neuronal fate. (1) Starting at 25%
of embryogenesis, three neurogenic zones (Z 1, Z 2, Z3) form
at the dorsal midline of the foregut, which has recently
extended into the body cavity from the surface ectoderm.
Each of these neurogenic zones gives rise to a series of
mitotically active precursor cells (Fig. 10, yellow cells) that
subsequently divide once or twice, producing neurons (blue
cells) that will populate the anterior enteric ganglia and the
recurrent nerve (Copenhaver and Taghert, 1991). (2)
Shortly thereafter, zone 3 disappears and the surrounding
epithelium commences a second proliferative sequence,
giving rise to a neurogenic placode (green cells; arrowhead
in Fig. 10) that invaginates onto the foregut surface. All of
the placode-derived cells stop dividing as they leave the
epithelium and subsequently differentiate into the neurons
of the enteric plexus (the EP cells; Copenhaver and Taghert,
1990). By 40% of development, both of these neurogenic
sequences are complete: all three proliferative zones have
been obliterated as the last of their cells emerge onto the
foregut and the neurogenic placode has completed its
invagination to form the packet of EP cells. Virtually all of
68
P. F. Copenhaver
the neurons of the ENS appear to have been generated by
this stage (Copenhaver and Taghert, 1990, 1991) and
undergo no additional rounds of mitosis.
Prior to their obliteration, however, the three neurogenic
zones produce an additional population of precursor cells
that do not lose their mitotic activity (red cells). These
include a cluster of 8-10 precursors (arrow in Fig. 10) that
are among the last to emerge from zones 2 and 3 and that
remain apposed to the invaginating packet of EP cells
(Copenhaver and Taghert, 1991). In contrast to the enteric
neurons, these precursors remain mitotically active and,
during the subsequent phases of neuronal migration, they
commence a new phase of proliferation (40-65% of development). While the zone-derived (blue) neurons migrate
anteriorly into the enteric ganglia and the placode-derived
(green) EP cells migrate around the foregut and then out
across the gut musculature, the progeny of the glial precursor cells (red) continue to proliferate and gradually
spread along the pathways taken by the migrating neurons.
Even after neuronal migration is complete and the neurons
have begun to express their mature phenotypes, this mitotically active cell class continues to disperse along the
lengthening nerves of the foregut and midgut. By the time
of hatching (100% of development), these cells have differentiated to form an ensheathing layer (red layer) that surrounds the neuronal components of the ENS.
The strongest evidence supporting this model was found
during the development of the enteric plexus: the apical
cluster of glial precursors could be distinguished from the
EP cells at all stages of development (e.g. Figs 2, 4) and
the delayed proliferation of these precursors was readily
documented both with BrdU labelling and the lineage tracing techniques that we employed. More anteriorly, identification of individual precursors was somewhat problematic, due to the intermingling of the zone-derived cells as
the enteric ganglia formed. For example, some of the glial
precursor cells might be generated simultaneously with the
neuronal precursors, although, in a previous study, we
found that the three proliferative zones gave rise to neurons
prior to 40% of development (Copenhaver and Taghert,
1991). In general, our observations were consistent with a
similar pattern of glial differentiation in the anterior ENS
as well as in the enteric plexus. Large cells similar to the
apical precursors were among the last of the cells to emerge
from each of the neurogenic zones, but remained relatively
quiescent until neurogenesis was complete. From 40% of
development on, however, BrdU labelling showed a greatly
enhanced level of mitotic activity throughout the anterior
portions of the ENS (Figs 3, 5), while clonal analysis
showed that the progeny of these larger cells assumed a
glial-like phenotype, ensheathing the enteric nerves and
ganglia of the foregut in a manner that resembled the differentiation of glial progeny in the enteric plexus.
Thus we propose that throughout the ENS, separate
classes of precursors for neurons and glia arise from a
common region of epithelium in a sequential manner: the
three neurogenic zones of the foregut first give rise to a
series of neuronal precursors (that will form the enteric ganglia) and then produce a smaller subset of glial precursors
(whose progeny will populate both the enteric ganglia and
the enteric plexus). As a result, the ensheathing glial pop-
ulations are generated only after neurogenesis is complete.
Both the neuronal and glial precursors are created by segregation from the non-neuralized epithelium, but the neuronal precursors undergo a limited number of divisions
around the time that they delaminate, whereas the glial precursors enter a delayed phase of proliferation that accompanies the morphogenesis of the ENS. Finally, both neurons and glial cells participate in an extended period of
reorganization, during which the late-arising glial populations follow the pathways established during the preceding
waves of neuronal migration (Copenhaver and Taghert,
1989b, 1991), whereby the glial progeny invest the enteric
nerves and ganglia.
Characteristic features of the enteric glial cells
Our conclusion that the progenitor cells that we have
described in this paper are glial precursors is supported by
a number of criteria used to identify glial cells in previous
studies (Radojcic and Pentreath, 1979; Roitbak, 1983;
Hoyle, 1986): (1) they have an ectodermal origin and (as
in most other systems) are derived from a region of ectoderm that is also neurogenic (Le Douarin et al., 1984;
Thomas et al., 1988; Bodmer et al., 1989; Galileo et al.,
1990); (2) their progeny remain closely associated with the
neuronal populations of the ENS but assume a distinctly
non-neural morphology and (3) as in the peripheral nervous
systems of other animals, this glial population forms a barrier between the neurons (which are ectodermal) and adjacent tissues that are mesodermally derived (Bray et al.,
1981; Gabella, 1981; Roitbak, 1983; Vernadakis, 1988).
Besides these morphological criteria, a number of biochemical markers have been described that can distinguish
neuronal cell types from glial cells in other systems. For
Fig. 7. Camera-lucida images redrawn to show the distribution of
dye-labelled progeny following the injection of individual glial
precursor cells at 40% of development. The small figures to the
left of A, E and J indicate the approximate positions of precursor
cells that were injected in the preparations of each row. (A-D)
Examples of labelled progeny in the vicinity of the frontal and
hypocerebral ganglia (fg and hg) and associated with the lateral
branches of the recurrent nerve (rn). (E-H) The progeny of glial
precursors that were located in the middle and posterior regions of
the developing recurrent nerve at time of their injection; in H,
some of the labelled progeny had dispersed across the foregutmidgut boundary and onto the anterior portion of the enteric
plexus. (J-L) The progeny of precursor cells that were originally
adjacent to the EP cell packet (see Fig. 6A). Labelled cells were
typically found along one or more of the branches of the enteric
plexus that had been formed during the migratory dispersal of the
EP cells, prior to glial differentiation. The variable patterns of dye
dispersal after 24 hours in culture are indicated by the manner in
which labelled cells are represented: in A, E and I, dye was
confined primarily to the nuclei, with only weak labelling of the
cytoplasm (stippled regions). In B, F and J, clusters of labelled
cells could be distinguished within a particular region of the ENS,
but the extent of their processes was not always apparent. In all
remaining preparations, the dye was dispersed throughout the
cytoplasm of labelled progeny, revealing that they had contributed
to the formation of an ensheathing layer around local regions of
the ENS, although the number of labelled cells could not easily be
discerned (compare with Fig. 6D-F). Same scale as in Fig. 1.
Gliogenesis in the insect ENS
example, monoclonal antibodies have been generated that
recognize glial-specific epitopes in a number of insect
preparations (e.g. Meyer et al., 1987; Fredieu and
69
Mahowald, 1989; Carpenter and Bastiani, 1990); while in
Drosophila, specific genes have been identified that are
expressed in developing glial cells (or their precursors) and
Fig. 7
70
P. F. Copenhaver
neurons but not glial cells in the embryonic nervous system
of the fly (Robinow and White, 1991). Similarly, in Man duca, we found that antisera against ELAV stained all of
the neurons in both the anterior enteric ganglia and the
enteric plexus but none of the putative glial lineages (Fig.
9), further supporting our identification of the glial precursor class in the developing ENS. A definitive marker for
glial cells in the ENS (such as an antiserum that could be
applied in conjunction with immunoelectron microscopy)
has yet to be obtained, however.
Fig. 8. Patterns of mitotic activity within the cell groups that arise
from the neurogenic zones of the foregut. Synthetically active
nuclei were counted at each developmental stage after labelling
with BrdU for 2 hours in embryonic culture. Each circle
represents the analysis of a single preparation. Only the nuclei of
cells that were derived from the neurogenic zones (enteric
ganglion neurons and glial precursor cells) were included in this
analysis. The mean values for each developmental stage are
connected by lines. Dotted segments of the lines indicate the
temporal overlap of neurogenesis and gliogenesis (as indicated by
lineage-tracing techniques; see discussion).
that regulate particular aspects of gliogenesis or neuronalglial interactions (Thomas et al., 1988; Bieber et al., 1989;
Uemura et al., 1989; Rothberg et al., 1990). While we have
not identified probes that selectively label the enteric glia
in Manduca, we were able to differentiate neuronal from
non-neuronal cell types on the basis of anti-ELAV staining. The ELAV protein, a molecule that is expressed
throughout the developing nervous system in Drosophila
and that may be involved in post-transcriptional processing
of mRNA (Robinow et al., 1988; Robinow and White,
1988), has recently been found exclusively in postmitotic
The relationship between neuronal and glial
lineages
Several comparisons may be drawn between the results of
our lineage-tracing experiments in the ENS and previous
data that has been obtained from the insect CNS. During
the formation of the segmental ganglia, separate lineages
arise for neurons and glia during the initial delamination of
precursor cells (neuroblasts and glioblasts) from the neurectoderm (Bate and Grunewald, 1981; Taghert and Goodman,
1984; Rothberg et al., 1988; Thomas et al., 1988). As has
been shown for the precursors of longitudinal glia in the
CNS (Jacobs et al., 1989), we found that glial precursors
in the ENS arose from specific locations within the neurogenic epithelium of the foregut and subsequently gave rise
to mitotically active progeny that migrated to their mature
positions. Jacobs et al. (1989) suggested that glioblasts of
the CNS became committed to their specific fates by virtue
of their original positions, a mechanism that is similar to
that proposed for neuroblast determination (Doe and Goodman, 1986). If such ‘position-specific’ mechanisms of cell
determination regulate precursor formation in the ENS, the
nature of these mechanisms must be changing with time:
all three of the neurogenic zones of the foregut produce
neuronal precursor cells prior to the emergence of the glial
precursor classes that we have described. Moreover, the
epithelial region in which zone 3 forms undergoes an additional transformation once that zone has been obliterated,
Fig. 9. Distribution of ELAV-like
immunoreactivity in the developing ENS of
Manduca. (A) Photomicrograph of an
embryonic gut (dorsal view) that was
dissected at 65% of development and stained
in whole-mount with an antiserum against the
neuron-specific protein ELAV. Black bars
indicate the foregut-midgut boundary
(anterior is to the top of the page). Positive
staining can be seen in the post-migratory EP
cells that have migrated along the muscle
bands of the midgut (straight arrows) and
foregut (curved arrows; some of the cells are
out of focus), but none of the presumptive
glial cells along the nerve branches of the
enteric plexus are immunoreactive
(arrowheads; see Fig. 4, 5A). (B) Cameralucida drawing of the same preparation to
show the complete distribution of labelled
(filled) and unlabelled (clear) cells; shaded
cells indicate a ventral position below the
more superficial branches of the enteric
plexus. RN, recurrent nerve. Scale, 30 µm.
Gliogenesis in the insect ENS
71
Fig. 10. A unified model for the origins
of the ENS in Manduca. Two programs
of neurogenesis and a related program
of gliogenesis give rise to distinct cell
populations during development. 25%
of embryogenesis: three neurogenic
zones (Z1, Z2, Z3) appear in the foregut
epithelium and produce neuronal
precursor cells (yellow cells). Each
neuronal precursor divides once or
twice, generating neurons that will
populate the anterior enteric ganglia
and recurrent nerve (blue cells). 35%:
invagination of a neurogenic placode
from the posterior lip of the foregut
epithelium (arrowhead) gives rise to the
packet of EP cells (green cells) that
emerge onto the foregut surface. At the
same time, prior to their obliteration,
each of the neurogenic zones of the
foregut produces an additional group of
precursors (red cells) that will form
glial precursors. 40%: all of the neurons
of both the anterior enteric ganglia and
enteric plexus have been generated and
are postmitotic. In contrast, the glial
precursor population (including a
cluster of precursors near the EP cell
packet; arrow) enters a new phase of
proliferation as the structures of the
ENS begin to form. 50%: the anterior
neurons continue to aggregate into the
enteric ganglia, while the EP cells
commence the first phase of their
migratory dispersal down both sides of
the foregut. The glial precursors
continue to divide and spread behind
the migrating neurons. 60%: the EP
cells enter their second phase of
migration along the muscle bands of
the foregut and midgut, while the glial
precursors begin to spread along the
pathways formed during neuronal
migration. 70%: neurons of the anterior
enteric ganglia and the enteric plexus
have achieved their mature positions
and have commenced the expression of
differentiated phenotypes. The glial cell
populations however, continue
to be mitotically active and to disperse along the nerves of the ENS. 100% (time of hatching): by the completion of embryogenesis, the
glial cell populations have established an ensheathing layer that completely surrounds the enteric ganglia and the peripheral branches of
the enteric plexus, providing a protective layer that separates the ENS from the circulating hemolymph. For more discussion, see text.
as it is subsequently incorporated into the neurogenic placode that gives rise to the EP cells (Copenhaver and
Taghert, 1990, 1991; see Fig. 9). As each of the neurogenic
zones is approximately the diameter of three to four epithelial cells, the degree to which multiple positional values
may be expressed within these structures is not known.
In general, we found that, once the precursor cells of the
ENS were segregated from the epithelium of the foregut,
they gave rise to neurons or glia but not both. In a small
number of preparations, however, when we dye-injected
individual cells while they were still within the epithelial
layer of the foregut, we subsequently found a mixed population of both neurons and glial cells that were labelled
within the developing ENS (unpublished observations).
Thus it is possible that some of the zone-derived precursors may go through a limited number of neurogenic divisions before becoming respecified to produce glia. Alternatively, an early division of a zone cell might give rise to
a pair of progenitors that are themselves committed to one
or another lineage type. The possibility that neuroblasts in
72
P. F. Copenhaver
the insect CNS may subsequently produce glial cells has
been suggested but not proven (e.g. Norlander and Edwards,
1968; Vanhems, 1985; Meyer et al., 1987; Fredieu and
Mahowald, 1989). A more extensive analysis of the developmental potentials of zone-derived cells in vitro may clarify the relationship between the precursor populations of
the ENS.
Neuronal-glial interactions during morphogenesis
Several aspects of glial development described in this paper
coincided with particular morphogenetic events in the ENS,
suggesting that regulatory interactions between the enteric
neurons and glial cells may contribute to the differentiation
of this system. In particular, we found that the main period
of glial proliferation commenced only after neurogenesis
was complete (Figs 6-8) and occurred during a period of
extensive migration and reorganization on the part of the
enteric neurons. We also found that the dispersal of the
mitotically active glial cells proceeded along the same pathways that had been established during neuronal migration,
so that the glial progeny remained in intimate association
with the enteric neurons and their processes. Finally, while
an enhanced level of glial proliferation was observed in
both the anterior and posterior domains of the ENS (Figs
3-5), the manner in which the glial cells distributed themselves reflected the organization of the local neuronal populations. Thus the glia of the anterior domain became incorporated into an ensheathing layer around the enteric ganglia,
while the glia that followed the migratory populations of
EP cells invested the diffuse sets of nerve branches that
constitute the enteric plexus.
These observations suggest that glial proliferation and
migration may be regulated in part by neuronally derived
cues, providing coordination between the two cell types so
that the nerves and ganglia are ensheathed and in a timely
manner. For example, the marked increase in glial proliferation at around 40% of development might be induced by
the same signal(s) that triggers the onset of EP cell migration. Alternatively, glial proliferation might occur in
response to altered characteristics of the neurons themselves, such as a change in their adhesive properties associated with the onset of migration (c.f. Daniloff et al., 1986;
Rutishauser, 1986; Antonicek et al., 1987). In a similar
manner, the migratory dispersal of the glial cells during
later stages of development might be in response to the
same directional cues that the EP cells follow (compare Fig.
5A and E) or might simply reflect an adhesive preference
of the glial cells for neuronal membranes versus mesodermal components on the adjacent gut surface.
Ample precedent for these types of regulatory interactions have been documented in other systems: membraneassociated components of both developing and regenerating axons can stimulate the proliferation and dispersal of
Schwann cells or their precursors (Salzer et al., 1980; Pleasure et al., 1985; Ratner et al., 1988), while mature peripheral neurons may exert an inhibitory influence on the
mitotic activity of Schwann cell populations (Wood, 1976;
Salzer et al., 1980). Similarly, enteric neurons from guinea
pig have been found to inhibit glial cell proliferation in a
variety of experimental contexts (Bannerman et al., 1987;
Eccleston et al., 1987, 1989). With the establishment of a
primary culture preparation for embryonic cells from Man duca, in which the migratory neurons and glial precursors
of the ENS can be selectively isolated and identified in vitro
(Copenhaver, 1991; and unpublished observations), we can
now investigate whether the proliferation of the glial precursors can be modulated by the presence of postmitotic
enteric neurons and whether the glial progeny will migrate
along isolated muscle band pathways independent of the
neurons that normally precede them.
I wish to thank Dr Paul Taghert (in whose laboratory this work
began) for his extensive support and insightful criticisms of this
manuscript. I am indebted to Ms Marisa LaGrange for her excellent technical assistance and Drs Steven Matsumoto and Mark
Meyer for their helpful comments. I also wish to thank Drs
Kalpana White and Steven Robinow for permission to use their
antiserum against the ELAV protein, and Drs Mark Meyer, John
Edwards, Cathy Krull, Leslie Tolbert, Cory Goodman and Allan
Bieber for the use of their glial-specific antisera. This work was
supported by NSF grant no. F32NS07957 and a research initiation
grant from the Medical Research Foundation of Oregon.
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(Accepted 7 October 1992)