Download Axogenesis in the embryonic brain of the

Development 121, 75-86 (1995)
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Axogenesis in the embryonic brain of the grasshopper Schistocerca gregaria:
an identified cell analysis of early brain development
George Boyan1, Stavros Therianos1, J. Leslie D. Williams2 and Heinrich Reichert1
1Laboratory of Neurobiology, Department of Zoology, University of Basel, CH-4051 Basel, Switzerland
2Max-Planck-Institut für Verhaltensphysiologie, Arbeitsgruppe Kaissling, D-82319 Seewiesen, Germany
SUMMARY
Axogenesis in the embryonic brain was studied at the single
cell level in the grasshopper Schistocerca gregaria. A small
set of individually identifiable pioneer neurons establishes
a primary axon scaffold during early embryogenesis. At the
beginning of scaffold formation, pioneering axons navigate
along and between glial borders that surround clusters of
proliferating neuroblasts. In each brain hemisphere, an
axonal outgrowth cascade involving a series of pioneer
neurons establishes a pathway from the optic ganglia to the
brain midline. At the midline the primary preoral commissural interconnection in the embryonic brain is
pioneered by a pair of midline-derived pioneer neurons. A
second preoral commissural connection is pioneered by two
pairs of pars intercerebralis pioneer neurons. Descending
tracts are pioneered by the progeny of identified neuroblasts in the pars intercerebralis, deutocerebrum and trito-
cerebrum; the postoral tritocerebral commissure is
pioneered by a pair of tritocerebral neurons. All of the pioneering brain neurons express the cell adhesion molecule
fasciclin I during initial axon outgrowth and fasciculation.
Once established, the primary axon scaffold of the brain is
used for fasciculation by subsequently differentiating
neurons and, by the 40% stage of embryogenesis, axonal
projections that characterize the mature brain become
evident. The single cell analysis of grasshopper brain development presented here sets the stage for manipulative cell
biological experiments and provides the basis for comparative molecular genetic studies of embryonic brain development in Drosophila.
INTRODUCTION
In this report, we analyse the cellular processes that give rise
to the initial set of axonal projections in the embryonic brain
of the grasshopper. We use immunocytochemical, intracellular dye injection and electron microscopical techniques to
identify as individuals the neurons that pioneer the initial projections in the embryonic brain and to determine the lineage of
these neurons from identified brain neuroblasts. During early
neurogenesis, the axons from these neurons initially navigate
along glial-bound aggregates of proliferating neuroblasts,
termed proliferative clusters, that are established before axogenesis begins. Through axonal outgrowth and fasciculation,
the identified pioneering neurons rapidly establish a primary
axon scaffold that interconnects all of the proliferative clusters
in the embryonic brain. This scaffold is used for fasciculation
by many of the subsequently differentiating neurons and, by
the 40% stage of embryogenesis tracts, commissures and connectives of the mature brain become apparent.
In the past decade, the embryonic central nervous system (CNS)
of insects has become an important model system for investigations of neuronal development. Many of the cellular mechanisms of neurogenesis and axonal pathfinding have been
analyzed in detail (i.e. Goodman et al., 1984; Bastiani et al.,
1985; Doe et al., 1985). Insight into the molecular mechanisms
that control these developmental processes has also been
gained, both through the use of hybridoma technology (Bastiani
et al., 1987; Harrelson and Goodman, 1988; Snow et al., 1988)
and through molecular genetic analyses of neuronal development in Drosophila (for reviews see Grenningloh et al., 1990;
Campos-Ortega and Knust, 1990; Goodman and Doe, 1994).
Most of the investigations of embryonic development in the
insect CNS have been carried out on the accessible and easily
identifiable cells in the segmental ganglia. The brain, by
contrast, is an enormously complex structure which in large
insects can consist of over a million neurons (Farrel and Kuhlenbeck, 1964). While an initial analysis of neurogenesis in the
grasshopper brain has described neuroblasts from which the
brain develops (Zacharias et al., 1993), virtually nothing is
known about the mechanisms by which the progeny of these
neuroblasts produce the complex axonal projections of the
adult brain (Boyan et al., 1993).
Key words: axogenesis, brain, fasciculation, glia, embryo,
grasshopper, central nervous system.
MATERIALS AND METHODS
Animals
Schistocerca gregaria eggs were kept in moist aerated containers at
30°C. Embryo staging was according to Bentley et al. (1979). This
method stages embryos at time intervals equal to percentage of
embryogenesis completed.
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Whole-mount histology and histological sections
Whole-mount preparations (dorsal side up) were dissected to remove
the stomodium and reveal the interior of brain structures. For serial
sections of osmium-ethyl gallate stained brains, embryos were staged,
dissected and treated according to Zacharias et al. (1993).
Immunocytochemistry
Immunocytochemistry was carried out according to Meier et al.
(1993). Primary antibodies were diluted in preincubation solution as
follows: anti-horseradish peroxidase (HRP), 1:1 (see Jan and Jan,
1982; Snow et al., 1987); anti-fasciclin I, 1:1 (see Bastiani et al., 1987;
Zinn et al., 1988); anti-REGA-1, 1:200 (see Carpenter and Bastiani,
1991); anti-engrailed, 1:1 (see Patel et al., 1989); anti-annulin, 1:1
(see Bastiani et al., 1992).
Intracellular dye filling
Identified cells were impaled with glass microelectrodes, filled electrophoretically with lucifer yellow dye and processed for light
microscopy according to Raper et al. (1983). Alternatively, injected
dye was photoconverted and processed for electron microscopy
according to Sandell and Masland (1988).
Microscopy
For light microscopy, histological material or whole-mount embryos
were viewed in a Zeiss Axioskop microscope equipped for epifluorescence and DIC (differential interference contrast). For documentation, microscopic images were either photographed or, alternatively,
recorded by a CCD camera, digitized and subjected to image processing using the GenIIsys (MTI) and Image 1 systems (Universal
Imaging). For immunoelectron microscopy, embryos were processed
as described by Meier et al. (1993). The primary antibody dilutions
were as above.
Cell lineage analysis
Brain neuroblast identification was according to Zacharias et al.
(1993). Three methods were used to determine the cell lineage of
pioneer neurons. First, an initial identification of the lineage of
pioneer neurons was possible by determining the position of the
neurons in the glia-ensheathed columns relative to their neuroblast of
origin. Second, to confirm this initial analysis, lucifer yellow dye was
injected into the neuroblast of origin of the pioneer neuron. Due to
dye coupling among the neuronal precursors and their early progeny,
it was possible to assign a given pioneer neuron to an identified neuroblast. Third, to analyse the generation of neurons from their neuroblasts of origin in vivo and in situ, 5-bromodeoxyuridine (BrdU) incorporation into proliferating neuroblasts was performed according to
Zacharias et al. (1993). For each pioneer neuron investigated, this and
all of the other experimental analyses reported here were carried out
on one or both contralateral homologs of a minimum of 5 different
preparations.
RESULTS
Formation of glial borders around proliferating
neuroblasts precedes axogenesis
In the embryonic brain of the grasshopper, axogenesis begins
at approximately the 29% stage of embryogenesis. At this
stage, considerable development of the brain has already
occurred, giving rise to a highly structured set of large bilaterally symmetrical neuroblasts (Zacharias et al., 1993), as well
as midline progenitors (see below), developing neurons and
non-neural cells. Since initial axogenesis relies heavily on the
cells in the brain that are in place before the first axon
Fig. 1. Formation of glial-bound proliferative clusters in the
preaxogenesis brain. REGA-1 immunoreactivity. (A) Schematic of
proliferative clusters in one hemisphere of the embryonic brain.
Neuroblast 12 of lateral protocerebrum where the first REGA-1
immunoreactive glial processes appear is highlighted. (B) REGA-1
immunoreactive glial processes (white arrow) surround neuroblast 12
of lateral protocerebrum. No other neuroblasts or neurons in the
brain hemispheres have REGA-1 immunoreactive glial processes at
this stage (26%). (C) At 30% stage REGA-1 immunoreactive glial
processes (black arrows) completely surround individual PI
neuroblast. (D) Glial processes delimit future axonal pathway inside
developing PI proliferative cluster. Dorsal layer of PI neuroblasts has
been removed revealing numerous neuronal cell bodies among which
the labelled glial process (black arrows) is found. 29% stage.
(E) REGA-1-immunoreactive glial processes (white arrows) delimit
borders of PI (upper right) and PI(ALDL) (lower left) proliferative
clusters. Overlying dorsal sheath removed. 30% stage.
(F) Preparation in E at higher magnification. REGA-1
immunoreactive glial processes delimit proliferative cluster border
(white arrows). Glia also extend processes into proliferative clusters
and surround columnar arrays of neuroblasts and progeny (black
arrows). The following abbreviations apply to all figures: GMC,
ganglion mother cell; NB, neuroblast; PI, pars intercerebralis proper;
PI(ALDL), anterior lateral dorsal lobe of pars intercerebralis; PC,
protocerebrum proper; LPC, lateral protocerebrum; DC,
deutocerebrum; TC, tritocerebrum; La, lamina; Me, medulla; Re,
retina; M, midline. In this and subsequent figures, a rectangular box
in the summary diagram indicates the brain region being analysed.
Embryos are viewed dorsally unless otherwise stated. Anterior, A, is
to the top. Scale bar: B, F, 10 µm; C, 6 µm; D, 14 µm; E, 20 µm.
outgrowth is initiated, it is important to characterize the
features of the preaxogenesis brain.
At early embryonic stages (before 25%), the bilaterally symmetrical neuroblasts in the neurogenic region of the future
brain hemispheres are arranged in a sheet-like array, which is
largely homogeneous in appearance. Between 26% and 28%,
this structural homogeneity disappears and the neuroblasts
together with their progeny become arranged into distinct
clustered aggregates. The boundaries of these clusters are
delineated by glial support cells and the differentiation of these
glial borders continues throughout embryonic development.
We refer to the glial-bound aggregates of proliferating neuroblasts, ganglion mother cells and neurons in each of the
embryonic brain hemispheres as proliferative clusters (Fig.
1A). The proliferative clusters prove to be important for axogenesis within the brain hemispheres because the earliest outgrowing axons navigate in the space between these clusters as
well as along their glial borders.
Aspects of glial development can be characterized by the
expression pattern of REGA-1, an antigen present in nonmidline glia in the embryo (Carpenter and Bastiani, 1991;
Seaver et al., 1993). Earliest REGA-1 expression in the brain
hemispheres is seen at the 26% stage and is highly restricted;
immunoreactive glial processes begin to surround a single
brain neuroblast (Fig. 1B). This type of focal initial expression
of an antigen by developing glial support cells is also observed
for annulin (see Bastiani et al. 1992; and below). Subsequently
the spatial expression of the REGA-1 glial marker rapidly
expands to surround the surface of other brain neuroblasts (Fig.
1C) as well as the columnar arrays of their progeny (Fig. 1F).
In addition, the REGA-1 antigen also appears on elongated
glial processes, which extend inside a given proliferative
Brain development in the grasshopper
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B
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cluster and demarcate future pathways along which the neurites
will grow as they project from their neurons of origin outwards
toward the surface of the proliferative cluster (Fig. 1D).
Although REGA-1 immunoreactive glial processes of this type
are observed repeatedly, it has not yet been possible to relate
these processes to identified glial cells or determine the identity
of the neurons whose axons project along the processes. By the
30% stage, all aggregates of proliferating neuroblasts and their
progeny have become bordered by REGA-1 immunoreactive
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glial processes (Fig. 1E,F). These proliferative clusters
subdivide the embryonic brain into the different neurogenic
regions that will give rise to the pars intercerebralis proper, the
anterior lateral dorsal lobe of the pars intercerebralis, the protocerebrum proper, the lateral protocerebrum, the deutocerebrum and the tritocerebrum.
In summary, before axogenesis begins, the bilaterally symmetrical brain neuroblasts are subdivided into the proliferative
clusters, which give rise to the major brain regions. This sub-
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division is accompanied by the formation of glial borders and
by the glial demarcation of future axon pathways. Subsequently both of these glial structures are used as substrata for
axon outgrowth.
Brain pioneer neurons construct an embryonic axon
scaffold
Initial axogenesis in the embryonic brain is carried out by a
small subset of neurons, which are located at discretely spaced
sites in the developing brain. These neurons are found near the
periphery of the proliferative clusters and, in many cases, near
the abutment of two different clusters. Through axonal
outgrowth and fasciculation, this small subset of neurons establishes a primary axon scaffold that interconnects all of the proliferative clusters in the embryonic brain. Given that they are
the first to initiate axogenesis in the insect brain, we refer to
the neurons that establish the initial interconnections among all
of the proliferative clusters in the brain as pioneer neurons in
accordance with the terminology used in the peripheral nervous
system and in the segmental ganglia (Bate, 1976; Bastiani et
al., 1985; Caudy and Bentley, 1986).
We studied axonal pathfinding of the brain pioneer neurons
by direct observation of developing axonal processes using
DIC optics, by intracellular injection of lucifer yellow dye and
by immunocytochemistry with an anti-HRP antibody that
labels the axons and cell bodies of all outgrowing neurons in
the developing insect nervous system (Jan and Jan, 1982; Snow
et al., 1987). A semi-schematic diagram of the brain pioneer
neurons based on a representative anti-HRP stained preparation is shown in Fig. 2A. Many of the brain pioneer neurons
can be identified on the basis of their position, the trajectory
of their axonal projections and their molecular expression
patterns. For several of the brain pioneers, it has also been
possible to identify their neuroblast of origin and determine
their lineage; in all of these cases, the brain pioneer neurons
belong to the initial set of progeny that a given neuroblast
produces.
How do the pioneer neurons establish the initial axonal
pathways in the embryonic brain? To address this question at
the single cell level, we first investigate the formation of the
pioneering axonal pathways that link the peripheral part of the
brain to more medial structures in each hemisphere. We then
analyse the cellular processes through which the commissural
connections between the two hemispheres are established.
Finally we characterize the formation of descending and tritocerebral connections. In each case, we limit our analysis to
initial pioneering neurons and pathfinding processes.
A wave of axonal outgrowth interconnects brain and
optic lobes
At the onset of axogenesis in the brain, a wave of differentiation occurs in which the axons from pioneer neurons located
in lateral and more peripheral parts of the brain extend towards
the axons of more centrally located pioneers, which then
project towards the midline. Throughout this process, the
growth cones of all of the pioneer neurons extend along and
between proliferative clusters and their filopodia make
transient contact with border cells of the proliferative clusters.
The first pioneer neurons that initiate axogenesis in the brain
are found in the developing optic lobes at the interface between
lamina and medulla (Fig. 2A,B). (While the exact mechanisms
are still unclear, at least the distal part of the medulla appears
to form from cells differentiating from the most proximal
regions of the lamina.) There, at the 29% stage, one or a pair
of neurons begin to send their axons towards the protocerebrum. These neurons pioneer a pathway, whose location
suggests that it may be used later by visual neurons of the
medulla (Fig. 2C). The axonal growth cones of these pioneering neurons first grow anteriorly along the border between
lamina and medulla. Then near the anterior edge of the border
they turn medially. At the turn, their growth cones pass
filopodia from a pioneer neuron located at the anterior medullalamina border; subsequently their growth cones encounter
filopodia from a pioneer neuron located in the pars intercerebralis (Fig. 2B). Following reciprocal filopodial contact at the
interface between the developing optic lobes and the lateral
edge of the pars intercerebralis proliferative cluster, the growth
cones of all three sets of pioneer neurons fasciculate with one
another. The axons from the optic lobes then project along the
pars intercerebralis axon into a specific axonal junction zone
in the brain hemisphere.
During the formation of the pioneering connections between
optic lobes and pars intercerebralis, an axonal junction zone is
established in the brain at the interface between the pars intercerebralis, anterior lateral dorsal lobe of the pars intercerebralis
and protocerebrum (Fig. 3A). There, the axon of a pioneer
neuron from the lateral protocerebrum grows out of its proliferative cluster of origin and extends into the intercluster space.
At the interface between the three proliferative clusters, its
growth cone contacts and fasciculates with the growth cone of
the pars intercerebralis neuron which pioneers the pathway
extending peripherally towards the optic lobes (Fig. 3B,C).
(Note that this is the pathway used by the axons from the optic
lobes to grow into the brain hemisphere, see above.) This
pioneer neuron is one of the first two neuronal progeny of pars
intercerebralis neuroblast 14; its putative sibling neuron
pioneers a pathway projecting centrally between proliferative
clusters towards the brain midline (Fig. 3D). In this case, the
lineage analysis of neurons is based on the position of the
neurons in the glial-ensheathed columns relative to their neuroblast of origin; in other cases, intracellular dye-injection experiments and BrdU incorporation were also used (see methods).
After fasciculation, the growth cone of the protocerebral
pioneer neuron first grows centrally along the axon of the
peripherally projecting neuron of the pars intercerebralis. Subsequently (not shown) the axon of the protocerebral pioneer
switches its growth cone to the axon of the other sibling pars
intercerebralis neuron, fasciculates with this cell’s axon and
then continues to grow along the axon of this centrally projecting neuron until it reaches the medial edge of the pars intercerebralis. In this way, a pathway is established from the pars
intercerebralis-protocerebral junction zone to the medial edge
of the brain hemisphere.
Thus, in each hemisphere, the growth cones of brain pioneer
neurons extend along the borders of the proliferative clusters
of the brain and carry out a series of axonal fasciculation
processes at the interfaces between proliferative clusters.
Through these fasciculation processes, initial axon pathways
are established from the optic lobes into the pars intercerebralis
as well as from the lateral protocerebrum via the pars intercerebralis towards the brain midline. With the linkage of these
axon pathways at the pars intercerebralis-protocerebral
Brain development in the grasshopper
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Fig. 2. Pioneer neurons establish an axonal pathway from the optic lobes into the brain. (A) Semi-schematic diagram of neurons that establish a
primary axon scaffolding; summary based on anti-HRP immunocytochemistry of 29-34% stages. (B) Digitized montage of anti-fasciclin I
immunocytochemistry at the 29% stage. The axon (black arrow) of a visual pioneer neuron (white asterisk) from the lamina-medulla interface
is growing along the border of a proliferative cluster into the brain. En route the growth cone (open arrowhead) of the visual pioneer neuron
grows past filopodia (white short arrow) growing into the brain from a neuron (partly visible, white star) located at the edge of the anterior
medulla-lamina border and subsequently encounters filopodia (black arrowheads) directed peripherally into the optic region from another
neuron at the PC/PI(ALDL) interface. All growth cones extend between proliferative clusters; their filopodia (long white arrows) make contact
with border cells within the proliferative cluster. (C) The axon of the visual pioneer neuron establishes a pathway that may later be utilized by
cells of medulla (black arrow). 45% stage. 16 µm horizontal section. Scale bar: B, 15 µm; C, 60 µm.
junction zone, a set of interconnecting neuronal pathways are
established, which extend from the developing optic lobes
towards the midline of the brain. As soon as these initial
pathways in the brain are pioneered, they are used by numerous
other neurons which project their axons along them.
During the construction of the axonal pathways from the
optic lobes to the midline, the growth cones and axons of all
of these reciprocally fasciculating brain pioneer neurons
express the homophilic cell adhesion molecule fasciclin I
(Bastiani et al., 1987; Snow et al., 1988; Zinn et al., 1988).
Fasciclin I expression is dynamic; expression of fasciclin I by
the growth cones and axons of the pioneer neurons is strong
during the formation of the primary axon scaffold early in
embryogenesis; expression of fasciclin I is no longer evident
in these neurons at 40% of embryonic development. Interestingly, the filopodia of the pioneering growth cones often
contact cells located at the borders of the proliferative clusters
that they grow along and some of these contacted cells also
express fasciclin I (see Figs 2B,3B).
Formation of the embryonic brain commissure
By the time the wave of outgrowing axons in each brain hemisphere reaches the medial border of the hemisphere (31%
stage), an initial brain commissural connection has already
been established. How is this embryonic brain commissure
formed? A central role in pioneering the brain commissure is
played by neurons that are generated by a small set of midline
progenitor cells. These midline progenitors do not stain with
G. Boyan and others
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Fig. 3. Contact and fasciculation of pioneer neurons at PI/PI(ALDL)/PC interface. (A) Semi-schematic summary diagram. (B) Digitized
montage, anti-fasciclin I immunocytochemistry of 30% stage. Fasciculation of two outgrowing axons, one from the LPC proliferative cluster
(white asterisk), the other from the PI proliferative cluster (partly visible, white star). Axonal growth cones (black arrows) meet in the space
between the two clusters. (C) Digitized montage showing fasciculating growth cones from PI neuron (white star) and LPC neuron (white
asterisk) in B at higher magnification. Filopodia (black arrows) extend from growth cones to proliferative cluster borders. (D) Schematic
summary data describing lineage relationships of the neuron indicated by white star in B. One of the first born neurons (white star) of
neuroblast 14 projects its axon laterally along a proliferative cluster border to fasciculate with the growth cone of a neuron (white asterisk) from
LPC. The other neuron pioneers a pathway projecting centrally towards brain midline along posterior proliferative cluster border of PI.
Neuroblast 14 and its first born progeny are in solid blue; other progeny outlined in blue, GMC outlined in black. Unlabelled neuroblasts are
protocerebral. Scale bar: B, 20 µm; C, 10 µm.
the anti-HRP antibody. Moreover, they do not express the
engrailed protein. This contrasts with the segmental ganglia,
where large engrailed-positive midline neuroblasts and
progeny are seen (data not shown). However, as is the case for
neuroblasts in the segmental ganglia, the midline progenitors
in the brain do become surrounded by glial sheath cells which
express annulin in correlation with the mitotic activity of the
cells they surround (Bastiani et al., 1992; Singer et al., 1992).
Thus, before the midline progenitors differentiate and become
active, no annulin staining is seen in this region of the brain
Brain development in the grasshopper
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D
(Fig. 4A). When the midline progenitors generate their
progeny, their surrounding glial sheath cells become annulinimmunoreactive and immunoreactive processes surround the
proliferating precursors as well as their progeny (Fig. 4B,C).
We have identified a set of three midline progenitors based on
their position at the midline, on the annulin immunoreactivity
of their glial sheath cells and on the incorporation of BrdU into
the precursors and their progeny (Fig. 4D). Additional, as yet
unidentified midline progenitors may exist.
The first pair of neurons that are generated by the lateralmost progenitors on each side of the midline are fasciclin I
expressing neurons and these pioneer the first commissural
connection in the embryonic brain (Fig. 5A,B). We call these
neurons the primary commissural pioneers (PCP). We carried
out a detailed analysis of the axonal outgrowth process of these
pioneer neurons by intracellular dye filling. At the onset of axogenesis, a single small process extends from each pioneer
neuron towards the midline (Fig. 5B). Shortly thereafter a large
growth cone with numerous filopodia separated by lamellipodial structures is generated (not shown). This growth cone
extends towards the midline and, in doing so, becomes smaller
in size and gives rise to a trailing axonal process (Fig. 5B). The
growth cones of these pioneer neurons extend over the midline
just posterior to the soma of the medial midline precursor. They
then contact and fasciculate with the axons of their contralateral homologs and grow along these axons into the contralateral hemisphere, thus establishing an initial axonal bridge
across the brain midline (Fig. 5A,C).
Once the PCP neurons have established the first axonal
pathway across the midline, another pair of neurons at the
medioposterior edge of the developing pars intercerebralis in
each brain hemisphere, the secondary commissural neurons
(SCN), extend their growth cones medially onto this axon
pathway and fasciculate with it (Fig. 5D,F). At the midline, the
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Fig. 4. Midline progenitors in the brain.
(A-C) Annulin immunoreactivity; (D)
BrdU incorporation. (A) At 26% stage
annulin expression is seen in developing
PI proliferative clusters (asterisks) but
not in preoral brain midline. Annulin
expression is also seen in optic lobes,
deutocerebrum, tritocerebrum and ventral
nerve cord. (B) At 30% stage annulin
expression is seen in preoral midline
progenitor cells (arrow) and in the PI
proliferative clusters (asterisks).
(C) Higher magnification showing
annulin expression surrounding preoral
midline progenitor cells between the PI
proliferative clusters (asterisks). Note
prominent annulin immunoreactive
border (arrowheads) delimiting the
cluster of large progenitor cells from
surrounding epithelial cells. (D) BrdU
labelling of three midline progenitor cells
(arrows). Note progeny (star) of the
central midline progenitor. BrdU
incorporation is also seen in cells of PI
proliferative clusters (upper left and
right). Scale bar: A, 166 µm; B, 120 µm;
C, 14 µm; D, 25µm.
growth cone of each SCN neuron contacts and fasciculates
with its contralateral homolog (Fig. 5F) just like the PCP
neurons did previously (Fig. 5C). Each SCN growth cone then
extends along the axon of its contralateral homolog and grows
into the contralateral brain hemisphere.
Soon after these two initial commissural pathways in the
brain are established (within 1-2% of embryonic development),
numerous other neurons project axons onto them, some
extending growth cones in different directions. The lateral
pioneer (LP), for example, has a cell body at the medial edge
of the developing pars intercerebralis (Fig. 5D). The LP neuron
extends its axon in the opposite direction to the PCP neuron
along the initial midline pathway and establishes a lateral
pathway that extends from the midline commissure peripherally into the brain hemisphere (Fig. 5D,E). Both the secondary
commissural neurons and the lateral pioneer express fasciclin
I during axonal outgrowth. The results of an ultrastructural
analysis of such a nascent commissural fascicle are shown in
Fig. 6. In this experiment, the lateral pioneer and the paired
(contralateral) primary commissural pioneers have been filled
with dye in a 33-34% stage embryo (Fig. 6A). Closer to the
brain midline, the axons of the primary commissure pioneers
are tightly apposed to one another and still at some distance
from the axon of the lateral pioneer (Fig. 6B). Closely associated with these pioneer axons are numerous other unstained
axonal profiles. These unstained axons have projected onto the
initial pioneering pathway and together now form the developing commissural fascicle. Interestingly, a large putative
midline progenitor cell is in close association with the fascicle,
suggesting that this precursor cell might play a role in the commissural pathfinding processes. Further away from the midline,
the primary commissure pioneers have fasciculated with the
lateral pioneer as they run laterally along the posterior margin
of the pars intercerebralis proliferative cluster (Fig. 6C,D).
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Once again, numerous unstained axonal profiles are closely
associated with the commissural pioneer axons. Together these
axons represent the developing commissural fascicle just
before it enters the brain hemisphere. A large glial cell, which
can be identified by the typical involuted membrane structure
surrounding its nucleus, is in close contact with the fascicle.
This glia cell extends processes that envelop all of the axons
in the fascicle. Numerous glial cells of this type are found
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wherever axonal outgrowth occurs in the brain. They are seen
at the midline and in the spaces between the proliferative
aggregates of the brain hemispheres; they can be seen
extending processes along pathways that are subsequently used
by pioneer neurons for axonal outgrowth. These glia are likely
to play important roles in early axogenesis, however, a more
detailed understanding of their function awaits further investigation.
Brain development in the grasshopper
Fig. 5. Formation of the primary brain commissure and of other
protocerebral pathways. (A) A pair of neurons that originate from a
lateral progenitor cell on each side of the midline pioneer the primary
preoral commissure. 30-31% stage. Top: camera lucida drawing of
these primary commissure pioneer (PCP) neurons (arrowheads, cell
bodies). Bottom: anti-HRP immunocytochemistry showing PCP
neurons (white arrows) and axons extending across midline (open
arrows). (B) Details of the primary commissural pioneers following
intracellular lucifer yellow labelling. A PCP neuron initiating process
outgrowth at 30% stage (top). Digitized montage (bottom) shows
axon extension across midline towards contralateral hemisphere at
31% stage of a different preparation. (C) Intracellular lucifer yellow
labelling of a PCP neuron from each brain hemisphere at 32-33%
stage. Arrowhead indicates brain midline. Digitized montage.
(D) Details of the secondary commissural neurons (SCN), digitized
montage of fasciclin I immunoreactivity. The cell body of one SCN
neuron in left PI is shown (asterisk). At midline (white short arrow)
the fascicle bends around midline cells. The lateral pioneer (LP)
(star) located at the medial border of the PI proliferative cluster
directs a process laterally along the axon fascicle. Growth cones
(white arrow) extending into left PI express fasciclin I as they
contact surrounding neurons and proliferative cluster borders. 31%
stage. (E) Intracellular lucifer yellow labelling of lateral pioneer
neuron (star) described in D; different preparation. Arrow indicates
growth cone. (F) Intracellular labelling of SCN neuron (asterisk)
from each brain hemisphere. 31-32% stage. Arrowhead indicates
preoral brain midline. Digitized montage. (G) Summary diagram
showing lineage relationships of neurons involved in early pathway
formation in the brain. 32% stage. Progeny of PI neuroblast 3
contribute to commissural and descending pathways; progeny of
neuroblast 1 contribute to laterally projecting pathway. Progeny from
other unidentified PI neuroblasts contribute to commissural and
descending pathways. Arrowheads indicate direction of initial axon
outgrowth; lineage-related progeny are coloured the same. Scale bar:
A,B, 8 µm; C, 14 µm; D, 10 µm; E, 11 µm; F, 17 µm.
A
C
83
Formation of embryonic descending pathways and
tritocerebral commissure
With the establishment of the primary preoral commissure, the
axon pathways in the two brain hemispheres become interconnected. Concurrently, descending pathways are pioneered
that connect the brain hemispheres with the ganglia of the
ventral nerve cord (Figs 5G,7A). This is a multistage process.
In the protocerebrum, a descending ipsilateral pathway is
pioneered at 31-32% of embryonic development by a pair of
cells derived from neuroblast 3 of the pars intercerebralis (Fig.
5G). Within 1% of embryonic development, the axons of
neurons derived from other pars intercerebralis neuroblasts
project onto this pioneer pathway and through the protocerebrum proper towards the deutocerebrum. Other pioneer
neurons derived from neuroblast 1 of the pars intercerebralis
project axons laterally towards the optic lobes (Fig. 5G). In the
deutocerebrum, neurons derived from neuroblast 5 pioneer a
descending ipsilateral pathway along the medial edges of the
deutocerebrum to the tritocerebrum and from there through the
circumesophageal connectives into the ventral nerve cord (Fig.
7B,C). The axons of all of these descending pioneers express
fasciclin I.
In the tritocerebrum, two very different pathways are
pioneered by the early progeny of tritocerebral neuroblast 12:
a descending ipsilateral pathway and the postoral tritocerebral
commissure (Fig. 7B,C). The neurons responsible for pioneering both pathways are putative sibling cells. Their axons
express fasciclin I and are arranged symmetrically on either
side of the brain. Initially the axons of both siblings follow an
identical route through the tritocerebrum but they soon part and
one sibling directs its axon posteriorly towards the circumesophageal connective. The other directs its growth cone
towards the midline where it meets the growth cone of its
D
Fig. 6. Ultrastructural
analysis of commissural
development.
(A) Schematic summary.
Two primary commissural
pioneer neurons (PCPs)
and the contralateral
lateral pioneer (LP) were
stained in the same
preparation (34% stage) by
B
intracellular injection and
photoconversion of lucifer
yellow. Dashed lines
indicate plane of sections
in B, C. (B) Section shows
the PCP axons (open
arrowhead) separated from
the LP axon (filled
arrowhead). All profiles
are closely associated with
the developing
commissure (arrow) and
with a large midline
precursor (asterisk).
(C) Section more lateral
than (B). Axon of the PCP neurons and the LP neuron are now fasciculated (arrowhead). The labelled axon profiles and other unlabelled axons
of the developing commissure are surrounded by processes of a large glial cell (asterisk). (D) Enlargement of labelled axon profiles (star) seen
in C. Scale bar: B,C, 3 µm; D, 1 µm.
G. Boyan and others
84
A
C
B
D
Fig. 7. Formation of descending pathways and of the tritocerebral commissure. (A) Summary diagram. (B) Formation of the tritocerebral
commissure. Digitized montage based on fasciclin I immunoreactivity. 33-34% stage. Two bilaterally symmetrical tritocerebral neurons (stars),
the tritocerebral commissure pioneers (TCCP), project axons (white arrows) towards postoral midline (open arrow). Axons (arrowheads) from
two pairs of deutocerebral neurons form descending pathways through the tritocerebrum. Inset (white rectangle) is a magnification of midline
region and shows that axons from both TCCP neurons have made contact and fasciculated at the midline (black arrow). (C) Summary
schematic showing lineage relationships of the neurons described in B. TCCP neuron and sibling (white asterisks) derive from tritocerebral
neuroblast 12. TCCP projects across midline; its sibling cell forms a descending axonal projection (black asterisk). Descending pathway from
deutocerebrum into ventral nerve cord (more lateral axons in B) is pioneered by progeny (white triangle) of DC neuroblast 5 (33% stage). Only
one of the sibling progeny contributing axons is shown. Other lineage-related cells are colored alike. (D) Horizontal section (16µm) through
brain and part of subesophageal ganglion showing orthogonal axonal scaffold at 45% stage. Osmium-ethyl gallate staining. P, protocerebrum;
T, tritocerebrum; S1, first subesophageal segment. Scale bar: B, 17 µm; inset, 11 µm; D, 108 µm.
homolog from the other brain hemisphere; these two axons
then fasciculate and grow along their homologs into the contralateral tritocerebrum (Fig. 7B). This initial fascicle of the tritocerebral commissure is pioneered at the 33-34% stage.
Aspects of the early axon scaffold remain evident in
later embryonic stages
Once the axonal scaffolding in the embryonic brain is established, the axonal projections rapidly grow in size. Within 3%
of the initial pioneering of the primary preoral brain commissure, over 100 axons have crossed the midline. Indeed, by the
37% stage, some fascicles of the adult brain, such as the Ushaped commissure PC20 (Boyan et al., 1993), can already be
recognized. By the 40% stage, a massive amount of axonal
outgrowth and fasciculation has occurred in the brain. Tracts,
commissures and descending pathways have increased enormously in size and complexity. Moreover, nerves that extend
from the peripheral nervous system into the brain have formed.
Nevertheless, some aspects of the early orthogonal scaffold of
brain axons are still visible at this stage (Fig. 7D). Lateral axon
tracts extend from the pars intercerebralis and protocerebrum
regions into the optic lobes; descending axon tracts extend
from the pars intercerebralis towards the deutocerebrum and
tritocerebrum and from there through the connectives into the
ventral nerve cord; preoral and postoral brain commissures
formed around the gut link the brain hemispheres. Interest-
Brain development in the grasshopper
ingly, the orthogonal scaffold described above is formed in the
absence of all higher brain structures such as the mushroom
bodies and the central body. These structures only take shape
after 40% of embryonic development.
DISCUSSION
Investigations on insect axogenesis have become increasingly
important for analyzing the cellular and molecular mechanisms
involved in guided outgrowth and pathway formation in the
nervous system (for recent reviews see Reichert, 1993;
Goodman and Doe, 1994). Virtually all of this work has,
however, been carried out on the simple segmental ganglia or
the peripheral nervous system of insects. In consequence, very
little is known about the development of more complex structures such as the insect brain. In this study, we investigate axogenesis in the early embryonic brain of the grasshopper Schistocerca gregaria. Our results show that, despite the enormous
complexity of the adult brain, a set of simple developmental
processes involving individually identifiable cells and axons
underlies early axogenesis in the brain.
Axogenesis in the brain commences in a restricted group of
cells and leads to a simple orthogonal scaffolding of axonal
pathways. The events that occur in the formation of this scaffolding resemble those that are seen in the segmental nervous
system in several ways. First, identifiable pioneering neurons
establish a simple axonal scaffold (Bate, 1976; Bentley and
Keshishian, 1982; Ho and Goodman, 1982). In the brain,
pioneers are involved in the construction both of the commissures that link the two hemispheres and of the tracts and connectives that interconnect the different parts of the brain in
each hemisphere. Second, in the brain and in the segmental
ganglia, all of the initially outgrowing axons derive from the
central nervous system itself (Goodman et al., 1984). Axons
that grow into the brain from structures on the head such as the
antennae, head hairs or the ocelli grow into the axonal scaffolding later and appear to depend on the existence of the
central nervous scaffolding for proper navigation. Third, the
growth cones of neurons pioneering the preoral and postoral
brain commissures meet and then fasciculate with their contralateral homologs in the midline as described for commissure
formation in the segmental ganglia (Myers and Bastiani, 1993).
Fourth, glia play an important role in axonal pathfinding
(Bastiani and Goodman, 1986; Meyer et al., 1987; Jacobs and
Goodman, 1989; Klämbt and Goodman, 1991; Carpenter and
Bastiani, 1991). The pioneering axons of the brain follow glial
borders during their outgrowth and glia are also important in
establishing connections across the midline. Interestingly, glial
bound neuronal aggregates are likely to play important roles in
the postembryonic development of the insect brain (Tolbert
and Oland, 1989) as well as in embryonic brain development
in vertebrates (see Steindler, 1993). Fifth, some cell bodies in
the developing brain may have a function equivalent to that of
‘guidepost cells’ in the peripheral nervous system (Bentley and
Keshishian, 1982; Ho and Goodman, 1982). Such cell bodies
are observed near the borders of proliferative clusters. These
cells express the cell adhesion molecule fasciclin I and are
contacted by outgrowing axons that also express fasciclin I.
These cells, therefore, might play an important role in directing
axon outgrowth.
85
The primary scaffold of axons in the embryonic brain is
established in the absence of higher brain structures such as the
central body, the protocerebral bridge or the mushroom bodies.
These structures are established as part of a second stage of
development that leads to the brain acquiring the anatomical
organization characteristic of the adult. In hemimetabolous
insects like the grasshopper, this second stage of development
occurs in the embryo once the first stage has been achieved
(Boyan et al., unpublished data). Thus, the early embryonic
brain is not constructed as a miniature model of the adult brain.
It initially acquires features that are reminiscent of the developing ventral ganglia. Only later does the embryonic brain
assume the features that are specific for the mature supraesophageal ganglion. In consequence, for a complete understanding of axogenesis in the insect brain, the formation of the
nerve tracts and connections in these later developing, higher
brain structures must also be investigated.
The single cell analysis of early brain axogenesis described
here lays the foundation for further cell biological studies in
which the many experimental advantages of the grasshopper
embryo, such as large size of embryonic neurons, easy accessibility for intracellular labelling and ablation, existence of a
robust embryo culture system and established antibody-block
protocols (Raper et al., 1984; Bastiani et al., 1985; Kolodkin
et al., 1993; Xie et al., 1994) can be exploited. Moreover, this
analysis sets the stage for following molecular genetic investigations
of
embryonic
brain
development
in
Drosophila.(Therianos et al., unpublished data). The merger of
work on the grasshopper with its highly accessible identified
embryonic neurons with work on Drosophila with its powerful
genetics and molecular biology has led to significant advances
in our understanding of neuronal development in general
(Thomas et al., 1984; Goodman et al., 1984; Zinn et al., 1988;
Grenningloh et al., 1990). Indeed, the expectation that mechanisms and molecules, first discovered in insects, have equivalents in the developing vertebrate nervous system has already
been fulfilled in several cases (Thomas and Capecchi, 1990;
McGinnis and Krumlauf, 1992; Goodman and Shatz, 1993;
Kolodkin et al., 1993).
Anti-fasciclin I, anti-annulin and anti-engrailed antibodies were
generous gifts of C. S. Goodman and N. Patel; anti-REGA-1 antibodies were generous gifts of M. J. Bastiani and E. C. Seaver. We
thank T. Meier, F. Xie, D. Sanchez, L. Ganfornina and E. Ball for
technical advice. Supported by the Swiss NSF, the FAG Basel and
the Max-Planck Gesellschaft, Seewiesen.
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(Accepted 20 September 1994)