Roundabout signalling, cell contact and trophic support confine longitudinal glia and axons in the Drosophila CNS

Abstract

Contrary to our knowledge of the genetic control of midline crossing, the mechanisms that generate and maintain the longitudinal axon pathways of the Drosophila CNS are largely unknown. The longitudinal pathways are formed by ipsilateral pioneer axons and the longitudinal glia. The longitudinal glia dictate these axonal trajectories and provide trophic support to later projecting follower neurons. Follower interneuron axons cross the midline once and join these pathways to form the longitudinal connectives. Once on the contralateral side, longitudinal axons are repelled from recrossing the midline by the midline repulsive signal Slit and its axonal receptor Roundabout. We show that longitudinal glia also transiently express roundabout, which halts their ventral migration short of the midline. Once in contact with axons, glia cease to express roundabout and become dependent on neurons for their survival. Trophic support and cell-cell contact restrict glial movement and axonal trajectories. The significance of this relationship is revealed when neuron-glia interactions are disrupted by neuronal ablation or mutation in the glial cells missing gene, which eliminates glia, when axons and glia cross the midline despite continued midline repellent signalling.

Full text

INTRODUCTION
Flies, like vertebrates, are bilateral symmetrical organisms, in
which the two sides are separated by the midline. The fly ventral
nerve cord (VNC) of the central nervous system (CNS) extends
along the main body axis, like the spinal cord of vertebrates,
linking the brain with the sensory and motor systems.
Interneurons project axons that cross the midline once and then
extend along the longitudinal pathways (Goodman and Doe,
1992). The insect ventral midline and its vertebrate equivalent,
the floorplate, are sources of antagonistic repulsive (Slit) and
attractive (Netrins) signals that regulate the crossing of axons
(Tear, 1999; Tessier-Lavigne and Goodman, 1996; Thomas,
1998). The combination of these molecules instructs axons to
cross the midline, to leave the midline once they have reached
it and never to cross it again. However, despite our
understanding of the control of midline crossing, not enough is
known about how longitudinal axons are maintained laterally.
Repulsion from the midline is a key mechanism to keep
axons along longitudinal pathways (Tear, 1999; Tessier-Lavigne
and Goodman, 1996; Thomas, 1998). The repulsive signal Slit
(Sli) is produced by the midline glia (Kidd et al., 1999).
Interneuron axons express the Sli receptor Roundabout (Robo)
and thus remain parallel to the midline from a certain distance
(Kidd et al., 1998a). To allow robo-expressing axons to reach
the midline prior to extending along the longitudinal pathways,
the midline glia also express the Commissureless protein, which
is responsible for downregulating robo expression as axons
approach the midline (Kidd et al., 1998b; Tear et al., 1996). At
the end of axonogenesis, all longitudinal axons express robo
and remain contralateral, away from the midline.
The longitudinal pathways are pioneered by four neurons per
hemisegment, pCC, MP1, dMP2 and vMP2, whose axons
never cross the midline (Bastiani et al., 1986; Bate and
Grunewald, 1981; Hidalgo and Brand, 1997; Jacobs and
Goodman, 1989; Lin et al., 1994). These ipsilateral pioneer
axons form a scaffold for the later selective fasciculation of
follower axons. During the formation of the first longitudinal
fascicle, pioneer growth cones also express the Robo receptor,
which prevents them from crossing the midline (Kidd et al.,
1998a). During their pathfinding, the pioneer axons interact
with a class of glial cells, the interface glia (Ito et al., 1995),
which at the end of embryogenesis overlie the longitudinal
axons (Hidalgo and Booth, 2000). The longitudinal glia are the
interface glia derived from the segmentally repeated lateral
glioblasts, located at the edge of the neuroectoderm.
Longitudinal glia, like the midline glia, are reminiscent of
vertebrate oligodendrocytes since they originate from highly
migratory and proliferative precursors and enwrap CNS axons
(Halter et al., 1995; Jacobs et al., 1989; Schmidt et al., 1997).
The longitudinal glioblasts divide and migrate ventrally until
they contact the cell bodies of the pioneer neurons, where they
halt at a certain distance from the midline. The first
longitudinal fascicle is formed as the descending axons of
207
Development 128, 207-216 (2001)
Printed in Great Britain © The Company of Biologists Limited 2001
DEV9748
Contrary to our knowledge of the genetic control of midline
crossing, the mechanisms that generate and maintain the
longitudinal axon pathways of the Drosophila CNS are
largely unknown. The longitudinal pathways are formed by
ipsilateral pioneer axons and the longitudinal glia. The
longitudinal glia dictate these axonal trajectories and
provide trophic support to later projecting follower
neurons. Follower interneuron axons cross the midline once
and join these pathways to form the longitudinal
connectives. Once on the contralateral side, longitudinal
axons are repelled from recrossing the midline by the
midline repulsive signal Slit and its axonal receptor
Roundabout. We show that longitudinal glia also
transiently express roundabout, which halts their ventral
migration short of the midline. Once in contact with axons,
glia cease to express roundabout and become dependent on
neurons for their survival. Trophic support and cell-cell
contact restrict glial movement and axonal trajectories.
The significance of this relationship is revealed when
neuron-glia interactions are disrupted by neuronal ablation
or mutation in the glial cells missing gene, which eliminates
glia, when axons and glia cross the midline despite
continued midline repellent signalling.
Key words: robo, Connectives, Glia, Cell survival, Drosphila
melanogaster, CNS
SUMMARY
Roundabout signalling, cell contact and trophic support confine longitudinal
glia and axons in the
Drosophila
CNS
Edward F. V. Kinrade
1
, Tamar Brates
1
, Guy Tear
2
and Alicia Hidalgo
1,
*
1
NeuroDevelopment Group, Department of Genetics, University of Cambridge, Downing Street, Cambridge CB2 3EH, UK
2
Molecular Neurobiology Group, New Hunt’s House, Guy’s Hospital Campus, King’s College, London SE1 1UL, UK
*Author for correspondence (e-mail: a.hidalgo@gen.cam.ac.uk)
Accepted 3 November; published on WWW 21 December 2000

208
dMP2 and MP1 meet the ascending axons of pCC and vMP2
(Bate and Grunewald, 1981; Hidalgo and Brand, 1997).
Longitudinal glia migrate anteroposteriorly slightly ahead, but
in close contact with, the extending pioneer growth cones
and stall at choice points relevant for axon guidance and
fasciculation (Hidalgo and Booth, 2000). Following the
formation of the first longitudinal fascicle, glia continue
migrating, occupying choice points to instruct axonal
defasciculation and refasciculation. The final pattern of pioneer
axon trajectories is dictated by glia (Hidalgo and Booth, 2000).
Two mechanisms are likely to promote the cell interactions
that generate the longitudinal pathways, although the
molecules involved are largely unknown. The first one is cell
contact, which is manifested in axon-axon and axon-glia
adhesion. Axon-axon contact is responsible for selective
fasciculation, such as fasciculation of follower with pioneer
axons (Bastiani et al., 1984; Hidalgo and Brand, 1997; Raper
et al., 1983; Raper et al., 1984). Axon-glia contact enables
the glia to guide pioneer growth cones and provoke their
fasciculation or defasciculation at choice points (Hidalgo and
Booth, 2000). The second mechanism is trophic support.
Survival of follower neurons depends on glia, thus contributing
to the maintenance of contralateral follower axons (Booth et
al., 2000). However, it is not known whether these interactions
can prevent axons and glia from re-crossing the midline.
Here we address the question of what are the mechanisms that
confine longitudinal axons and glia away from the midline. We
show that robo is transiently required to stop longitudinal glia
migration short of the midline. We also show, however, that from
the time that the glia contact axons, robo is no longer responsible
for restricting glial movement. When glia are in contact with
axons, trophic support and cell-cell contact maintain glia and
axons in lateral positions. These later mechanisms operate on
axonal patterns contemporarily with, but independently of,
midline-derived repulsion, since upon interference with neuron-
glia interactions glia and axons can cross the midline despite
expressing the repulsion receptor Robo.
MATERIALS AND METHODS
Fly stocks
Wild-type: Canton-S. Mutants: gcm
∆
P1
/CyOlacZ (Jones et al., 1995)
(null). gcm
∆
P1
/CyOlacZ; Df (3L) H99/TM6B. Both robo alleles are
nulls that do not produce any protein: robo
Z14
/CyOlacZ; robo
Z1772
/
CyOlacZ (Kidd et al., 1998a); Df (3L) H99/TM6B (White et al., 1994).
Ablations: w; ftzNGAL4 (Lin et al., 1995). w; ftzNGAL4 Df (3L)
H99/TM6B. w; UAS ricin (UFR1.1)/CyOlacZ (Hidalgo et al., 1995).
w; UAS rpr/TM3lacZ. w; UFR1.1; Df (3L) H99/ SM6a-TM6B and
w; UAS rpr Df (3L)H99/TM6B (Booth et al., 2000). Other: w; 158
(Booth et al., 2000). w; UAS sli (Kidd et al., 1999). Robo rescue:
sgcmGAL4151; robo
Z14
/CyO. w; robo
Z14
/CyO; UAS robo.
sgcmGAL4151; robo
Z1772
/CyO. w; robo
Z1772
/CyO; UAS robo.
Cell ablations
Ablation of neurons was carried out with the GAL4 system driving
either the toxin, ricin (Hidalgo et al., 1995) or reaper (rpr) expression
(Booth et al., 2000). The line ftzNGAL4 drives expression in all four
pioneer neurons, pCC, MP1, dMP2 and vMP2 from their initial
specification and in many other neurons (Hidalgo and Brand, 1997; Lin
et al., 1995). It also drives expression transiently in some of the glioblast
progeny. Expression does not affect most glia derived from the glioblast,
since ricin or rpr expression with this line leaves many longitudinal glia
intact at stage 13 and since longitudinal glia survival can be rescued in
ablated embryos in a rpr mutant background (Fig.7D). This line is not
expressed in midline glia. Only embryos in which ablation had taken
place were analysed. Furthermore, non-ablated embryos were also
identified by the expression of lacZ from the reporter balancer
chromosomes, which was visualised with anti-β-gal antibodies.
Rescue of
robo
mutant phenotype
Homozygous robo
Z14
mutants do not express any Robo protein. robo
expression was provided to glia in a mosaic fashion with
sgcmGAL151 (Booth et al., 2000). Only embryos with mosaic robo
expression were analysed.
Immunohistochemistry
Antibody stainings were carried out following standard procedures.
Rabbit anti-Repo (reversed polarity) was used at 1:300; rabbit anti-
Htl (Heartless) at 1:1000; mouse Fasciclin 2 (FasII/Fas2/mAb1D4) at
1:5; mouse anti-Robo at 1:5; mouse anti-Sli at 1:10; rabbit anti-β-gal
at 1:5000. Antibody concentrations were doubled in fluorescent
stainings and fluorescence signal was amplified with Streptavidin-
Alexa 488 (Molecular Probes). Fluorescent stainings were analysed
in an MRC 600 confocal microscope.
RESULTS
Longitudinal glia migrate over the midline in
robo
mutants
Interface glia normally overlie the longitudinal connectives of
the CNS and are not found over the midline (Ito et al., 1995).
Mutations in robo cause interface glia to migrate over the
midline (n=12/12 embryos, Fig. 1C,D). However, not all glia
migrate over the midline: some remain along the longitudinal
tracts (Fig. 1D), whose position is nevertheless closer to the
midline than in normal embryos (compare Fig. 1C,D with
A,B). This differential effect of robo mutations on glia
correlates with similar differences in the axonal phenotype. For
instance, only the pCC/MP2 (central) fasII fascicle, but not the
outer two fascicles, is affected in robo mutants (Fig. 1C; Kidd
et al., 1998a). The longitudinal glia are responsible for the
formation of the three fasII fascicles (Hidalgo and Booth,
2000). Hence, these data suggest that either there is a
differential requirement for robo amongst the longitudinal glia,
or that other members of the robo family may also play a role
in these glia or that further factors, other than robo function,
may determine glial positions along the longitudinal fascicles.
robo
is transiently expressed in longitudinal glia
Since in robo mutants longitudinal glia migrate over the
midline, we wondered if robo may be expressed in glia as well
as in axons. If so, robo would keep glia away from the midline
in normal embryos and would render them insensitive to
midline repulsion in robo mutants. Prior to axonogenesis, robo
is expressed in two broad longitudinal bands at either side of
the sli-expressing midline (Fig. 2A, stage 12.3). Sli protein is
also found within these bands at stages 12.2 and 12.1 (Fig. 2B).
By stage 13 robo expression resolves into segmental clusters
(Fig. 2C), preceding the overt expression in axons.
robo is also expressed in one transverse stripe per
hemisegment at stage 11 (Fig. 2D). These lateral stripes
include the tracheal pits. The longitudinal glioblasts originate
just ventrally of the tracheal pits, and they divide as they
migrate medially within these lateral bands of robo expression
E. Kinrade and others

209Robo, cell contact and trophic support
(Fig. 2D). Glia stop migrating medially as they enter the
longitudinal bands of robo expression (Fig. 2E,F). Within these
robo expression domains glia express robo themselves. This
was confirmed by double labelling experiments. Firstly, glial
nuclei were visualised with the glial marker anti-Repo (Halter
et al., 1995) and found to be totally surrounded by robo-
expressing cytoplasm and membranes (stages 12.4-12.1, Fig.
3A,C-E). Secondly, glia were found to coexpress the
longitudinal glia cell membrane marker Heartless (Shishido et
al., 1997) and Robo (stages 12.4-12.2, Fig. 3B,F-H). Thirdly,
longitudinal glia, expressing lacZ under GAL4 control by the
glial line 158 (Booth et al., 2000), were found to coexpress β-
gal and Robo (stage 12.3, Fig. 3I-K, note view of glial
projections in a 0.5 µm section).
In glial cells missing (gcm) mutants, in which glial fate is
transformed to neuronal fate (Hosoya et al., 1995; Jones et al.,
1995; Vincent et al., 1996), there is a reduction of robo
expression between stages 12.2 and 13 (Fig. 4C,D).
Furthermore, the remaining signal is often concentrated on
what appears to be individual cells, which could correspond to
pCC (stage 13, Fig. 4D). robo signal increases to normal levels
in gcm mutants from stage 14. If robo expression were limited
to neurons, in gcm mutants an increase (not a reduction) in robo
expression at stages 12.2-13 would have been expected.
These data show that longitudinal glia express robo and
respond to midline-derived repulsion within a narrow time
window.
Expression of robo in normal embryos disappears from glia
from stage 13, as glia occupy more dorsal positions over the
longitudinal tracts. By stage 14, Robo is clearly present
only in axons (see Kidd et al., 1998a). Since glia maintain
lateral positions in the longitudinal pathways throughout
embryogenesis despite ceasing to express robo, further
mechanisms must restrict glial movement.
robo
is required cell-autonomously in the
longitudinal glia
To test whether robo function is required cell-autonomously in
the longitudinal glia, we analysed whether ectopic expression
of the repulsive signal Sli affects glial migration.
In normal embryos, longitudinal glia migrate towards the
pioneer neurons. We expressed Sli ectopically in all postmitotic
neurons with elavGAL4. This has been shown to cause axonal
misrouting across the midline late in embryogenesis, as
visualised with BP102 antibodies (Kidd et al., 1999). Upon
ectopic Sli expression, in old embryos we observed
defasciculation defects and rather subtle misroutings across the
midline with fasII (not shown). For the analysis of glial patterns
we focused on early embryogenesis, at the time when the
interface glia migrate and establish contact with the pioneer
neurons. We observed a range of phenotypes between stages
12.5 and 13 (altogether with 60% penetrance, n=46 embryos),
comprising delayed glial migration (Fig. 5D), defective
migration of glia along axons (Fig. 5E), increased glial
numbers (Fig. 5F) and increased distance from the midline
between the connectives (not shown). After stage 13, we
observed more glia clustered at the exit of the nerves from the
Fig. 1. Glial phenotypes of
robo mutants. Axons
visualised with fasII
antibodies (brown) and glia
nuclei with anti-Repo
antibodies (black).
(A,B) Wild-type and (C,D)
robo mutant stage 16
embryos focusing on the
axons (A,C) and on the glia
(B,D). Note in C that only
the first (pCC/MP2) fascicle
crosses the midline (arrow;
arrowheads indicate outer
fascicles), and that the
distance between the two
connectives is shorter than
in wild type (compare C,D
with A,B). Note in D that
not all glia are located over
the midline (arrowheads;
arrows indicates glia over
midline). Anterior is up.
Fig. 2. Glia migrate within robo expression domains. (A-C) Detection
of anti-Robo (brown, arrows) and anti-Sli (blue, arrowheads) at stages
12.4 (A), 12.1 (B) and 13 (C). Note in B there is Sli protein in the
longitudinal pathways (arrowheads). (D-F) Detection of anti-Robo
(brown, arrows) and the glial nuclear marker Repo (black arrowheads)
at stages 11 (D) and 12.4 (E,F). The embryo in E is slightly younger
than that in F. (D) The glioblast starts migrating along the transverse
Robo domains. (E) The glioblast progeny enter the longitudinal
domains of Robo. (F) The glioblast progeny have divided, and are still
located within the Robo domain, which is narrowing down. Anterior is
up. Tp, tracheal pits (white arrowhead).

210
CNS, and missing interface glia along the connectives (not
shown). However, older embryos look to have recovered
somewhat compared to the earlier defects and can look normal.
These data show that glial migration is affected upon ectopic
expression of Sli. The increase in glial numbers suggests that
whereas some glia do not migrate, the glia that reach the
pioneer neurons divide further, thus increasing the overall
numbers of glia. This increase in glial numbers would explain
the remarkable recovery of glial presence along the connectives
in late embryogenesis. The presence of glia in the connectives
despite the ectopic expression of sli in axons is consistent with
the fact that glia do not express robo after stage 13 (see above).
Robo is insufficient to restrict all glial movement
Interface glia express robo only transiently, and they can overlie
longitudinal axons despite their ectopic expression of Sli. We
therefore determined whether robo expression is sufficient to
restrict glia to lateral positions in the longitudinal pathways.
We targeted robo expression to the glioblast in robo mutant
embryos. Glia (marked with anti-Repo antibodies) expressing
robo are most often found in lateral positions, indicating that
robo expression can keep glia away from the midline compared
to their usual position in robo mutants (n=16/18 clones) (Fig.
E. Kinrade and others
Fig. 3. The repulsion receptor Robo is expressed in the longitudinal
glia. Confocal images, representing projections of 0.5 µm thick
sections. (A,C-E) Robo protein (green) surrounds glial nuclei
visualised with anti-Repo antibodies (red) at stage 12.2. (A) Robo
protein is distributed in broad longitudinal bands at this stage. Total
thickness: 1.5 µm. (C) Robo, green; (D) merged images of C and E;
(E) Repo, red. Robo protein is located in the cytoplasm and
membranes of individual glia. Higher magnification details. Total
thickness: 1.5 µm. (B,F-H) Robo (red) protein is colocalised with Htl
(green) in longitudinal glia (colocalisation in yellow) at stage 12.4.
(B) Total thickness: 4.5 µm. Note the black nuclei, which do not
express Robo or Htl, surrounded by both signals. (F) Htl, green; (G)
merged images of F and H (colocalisation in yellow); (H) Robo, red.
Higher magnification detail of one cell. Total thickness: 2 µm. Note
the black nucleus in the centre of the cell surrounded by both signals.
(I) Robo, green; (J) merged images of I and K (colocalisation in
yellow); (K) β-gal, red. Glia visualised with anti-β-gal antibodies
(red) in embryos expressing lacZ in glia (genotype: 158 GAL4/UAS
tau lacZ) and Robo (green), at stage 12.2. Total thickness: 0.5 µm.
Note colocalisation in cytoplasmic projections (arrows). Note also
black nuclei (arrowheads), surrounded by both signals. White bar
represents position of midline. Anterior is up, except for C-E, in
which anterior is to the left.
Fig. 4. Decrease in Robo protein in gcm mutant embryos.
(A,B) Wild-type embryos stained with anti-Robo (brown) and anti-
Sli (blue) at stages 12.2 (A) and 13 (B). Note the clusters of Robo
protein (arrowheads), more pronounced at stage 13. (C,D) gcm
mutant embryos stained with anti-Robo antibodies (brown). Note the
reduced extent of the Robo clusters, and the concentration of Robo
signal in individual round spots, which appear to be individual cells.
Anterior is up.

211Robo, cell contact and trophic support
6B). However, these robo-expressing glia remain in contact
with axons and thus are still closer to the midline than in
normal embryos (see also Fig. 6E,F). In some cases glia were
found over the midline despite expressing robo (n=2/18 clones,
Fig. 6C). These data suggest that robo is not sufficient to
restrict glial migration and that glial positions also depend on
contact with axons.
Since glia are required for formation of normal axonal
trajectories, we wondered if targeting robo expression to glia
alone in robo mutants would restore the mutant axonal
phenotypes. Targeting robo expression to the longitudinal
glioblast, in mutants lacking robo expression, does not rescue
the robo axonal phenotype. The pCC fascicle misroutes across
the midline despite the expression of robo in adjacent glia
(n=40 clones, Fig. 6E,F). These data confirm the notions that
the pCC fascicle requires robo autonomously to
remain ipsilateral (Kidd et al., 1998a) and that
pathfinding by the pCC growth cone does not require
glia (Hidalgo and Booth, 2000; Hosoya et al., 1995;
Jones et al., 1995).
Axon-glia contact contribute to keep glia
laterally
Longitudinal glia require contact with axons to
maintain their survival. In normal embryos some glia
die from the time when they reach the longitudinal
axons (A. H., E. K. and M. Georgiou, unpublished
data). Consistently, ablation of neurons leads to loss of
glia (genotype: ftzNG4/UAS ricin; Fig. 7C; also A. H.
and A. Brand, unpublished data). In rpr mutant
embryos in which apoptosis is blocked, the distribution
of interface glia along the connectives is normal (Fig.
7B). If neuronal ablation takes place in rpr embryos
(genotype: rpr ftzNG4 / UAS ricin rpr; n=10/10
embryos), interface glia are rescued, as monitored with
anti-Repo (Fig. 7D). These results show that interface
glia apoptosis is induced in the absence of neurons.
Although the rescued glia in the experiment above
no longer die, they do not migrate normally (Fig. 7D).
Glia do not acquire their normal lateral positions and
are found over the remaining axons, or clustered around
their original location, or scattered throughout, or along
the edges of the ventral nerve cord, or clustered over
the midline (Fig. 7D). These observations imply that
interface glia require contact with longitudinal axons to
survive. Furthermore, interface glia require contact
with axons to acquire their normal positions along the
longitudinal pathways.
Interface glia are also in contact across the midline.
We have observed that cytoplasmic projections of the
longitudinal glia reach the midline and contact glia
from the contralateral side (Fig. 8). Glial projections
were visualised with reporter lacZ expression driven
by the glial-specific GAL4 line 158 (Booth et al.,
2000; Fig. 8A) and with anti-Heartless (Htl) antibodies
(Fig. 8B). These midline-reaching longitudinal glia
projections were observed between stages 13-14 (in all
segments of all embryos stained with anti-Htl), after
robo expression has disappeared from the longitudinal
glia. Since glia dictate axonal patterns (Hidalgo and
Booth, 2000), these glial projections may provide a
pathway across the midline when axons are deprived of their
normal lateral fasciculation cues.
Interference with axon-glia interactions causes
axons to cross the midline despite expressing
robo
To test whether longitudinal axon-glia interactions play a role in
maintaining longitudinal axons away from the midline, we
disrupted the normal axon-glia interactions to answer several
questions: (1) does elimination of glia provoke midline crossing
by axons?; (2) does elimination of pioneer neurons provoke
midline crossing by axons or glia?; (3) are the potential effects
dependent or independent of Robo-mediated repulsion?
To analyse the consequences of absence of glia in
longitudinal pathways, we analysed gcm mutant embryos
lacking functional glia, since they are transformed to neurons
Fig. 5. robo is required cell-autonomously in longitudinal glia. Effects in glia
of ectopic panneural expression of sli (genotype: elavGAL4/UAS sli) in
embryos stained with fasII (brown, pioneer neurons and axons) and anti-Repo
(black, glia). (A-C) Wild-type embryos at stage 12.3 (A) and 12.1 (B,C)
focusing on a dorsal (B) or ventral (C) plane; (D-F) elavGAL4/UAS sli
embryos at stage 12.3 (D) and 12.1 (E,F) focusing on a dorsal (E) or ventral
(F) plane. Note in A that glia are in contact with pioneer neurons at this stage
(arrowheads), but if neurons express sli, glia do not migrate as far
(D, arrowheads; severely affected specimen). Note in B that at stage 12.1 in
normal embryos the longitudinal glia migrate along the pioneer axons. Upon
neuronal sli expression (E) glia are repelled from establishing contact with
axons (arrowheads; arrows indicate glia that migrate normally in the same
specimen). Interface glia remain clustered around the neuronal cell bodies.
Note in C the ventral glia also labelled with anti-Repo (arrowheads). Upon
neuronal sli expression there are higher numbers of ventral glia, which are also
disorganised (F, arrowheads). These glia could correspond to interface glia
which failed to migrate. Anterior is up.

212
(Hosoya et al., 1995; Jones et al., 1995; Vincent et al., 1996).
We find that in gcm mutant embryos, axonal fascicles, which
normally extend along the longitudinal pathways, may cross
the midline (in at least one segment – and most frequently in
several – in 17 out of 18 embryos examined; Figs 8D and 9B).
To analyse the consequences of absence of pioneer neurons
on the positions of axons and glia, we ablated all pioneer neurons
and other neurons with ftzNGAL4 (Lin et al., 1995) driving ricin
expression (Hidalgo et al., 1995). Such ablation causes fasII
positive fascicles, which would normally project along the
longitudinal pathways, to cross the midline within the
commissures (most segments in 16/16 embryos, Fig. 9C; see
also Hidalgo and Brand, 1997). Longitudinal glia also migrate
over the midline in these embryos (n=4/7 embryos; Fig. 7C).
Given that the midline is the source of the repulsive signal
Sli and longitudinal axons express the Sli receptor Robo (Kidd
et al., 1999; Kidd et al., 1998a), midline crossing by axons was
unexpected. Misrouting across the midline could occur if the
midline glia were damaged, leading to loss of the Sli repulsive
signalling molecule. However, neither the gcm gene nor the
ftzNGAL4 line are expressed in midline glia, (Hidalgo and
Brand, 1997; Hosoya et al., 1995; Jones et al., 1995; Vincent
et al., 1996) and sli expression is normal in the midline of both
gcm mutants (Fig. 9G) and ablated embryos (Fig. 9H). In both
gcm mutants (n=13/17 embryos) and ablated embryos (n=8/8
embryos) the misrouted axons cross the midline despite
expressing normal levels of the Sli receptor Robo (Fig.
9G,H).These data show that, unexpectedly, interference with
axon-glia interactions results in midline crossing despite
midline derived repulsion.
These data suggest that longitudinal axon-glia interactions
are involved in keeping both axons and glia away from the
midline independently of midline-derived repulsion.
Trophic support confines axonal and glial lateral
positions
Trophic support between neurons and glia is a means of
restricting glial movement and axonal trajectories. There are
reciprocal (although asymmetric) trophic interactions between
neurons and glia during pathfinding. Pioneer neurons maintain
the survival of longitudinal glia (see above and Fig. 7C), and
glia maintain the survival of follower neurons (Booth et al.,
2000). When axon-glia interactions are intact, axonal CNS
patterning is virtually normal in the absence of cell death (rpr
mutants, Fig. 9F), although subtle axonal defects are found,
such as longitudinal growth cones projecting towards the
midline (Fig. 8C). However, when neuron-glia interactions are
perturbed in rpr mutant embryos, that are unable to undergo
programmed cell death, the misrouting of axons across
the midline is dramatically enhanced, compared either
with rpr mutants alone or with perturbing neuron-glia
interactions in embryos in which apoptosis can occur
normally. In embryos double mutant for gcm and rpr
E. Kinrade and others
Fig. 6. robo expression in glia partially confines glia laterally in robo
mutants. Longitudinal fascicles visualised with fasII (brown) at
stages 16 (A) and 14 (D-F). (A,D) Wild-type embryos.
(B,C,E,F) Rescue experiments: mosaic expression of robo in glia in
robo mutants (genotype: sgcmG4 151; robo
Z14
/robo
Z14
; UAS robo).
(B) Glia (anti-Repo, black) expressing robo (brown) occupy lateral
positions (black arrowheads) whereas glia that do not express robo
are still found over the midline (white arrowheads). (C) Glia (anti-
Repo, black) span the midline despite expressing robo (brown,
arrowheads). (E,F) Expression of robo (blue, arrowheads) in the
longitudinal glia (E, plane of glia) can rescue lateral positions of glia
but it does not rescue axonal misrouting across the midline (fasII,
brown, plane of axons F). Anterior is up.
Fig. 7. Glia do not acquire their lateral positions if deprived
of neuronal cues. Axons visualised with fasII antibodies
(brown) and glia with anti-Repo antibodies (black) (stage
16). (A) Wild-type embryo; (B) rpr mutant; (C) neuronal
ablation (ftzNG4/UAS ricin) causes glial depletion (arrows)
and remaining axons and glia cross the midline
(arrowheads); (D) ablation of neurons in rpr mutant
embryos (ftzN rpr/UAS ricin; rpr). Glial positions are not
restored. Arrows point to segments with particularly high
clustering of glia over the midline, which resembles the
robo glial mutant phenotype. Anterior is up.

213Robo, cell contact and trophic support
(gcm rpr, n=9/9 embryos, Fig. 9D) or upon neuronal ablation
in a rpr mutant background (ftzNGAL4 rpr/UAS ricin rpr,
n=10/10 embryos, Fig. 9E), axonal extension along the
longitudinal pathways is severely affected, as visualized with
fasII. Longitudinal connectives are virtually missing and fasII-
positive axons cross the midline in every segment. In the case
of ablated rpr mutant embryos the phenotype often resembles
(albeit more severely) the robo mutant phenotype (Fig. 9E).
These midline crossing axons express robo. In embryos double
mutant for gcm and rpr (n=6/11 embryos, Fig. 9I) or upon
neuronal ablation in a rpr mutant background (n=9/9 embryos
Fig. 9J) there is no robo expression along the longitudinal
connectives, but instead all axons expressing robo cross the
midline. These observations imply that attraction towards the
midline is the default pathway taken by axons and glia despite
midline repulsion in the absence of normal axon-glia
interactions in the longitudinal pathways. They also mean that
the pressure for survival normally keeps cells in contact with
their normal neighbours, in this case in lateral positions.
Taken together, these data show that in normal embryos
control of cell survival and cell-cell contact are means of
confining glia and axons laterally.
DISCUSSION
Pioneer axons, interface glia and interactions between them are
necessary for the formation and maintenance of longitudinal
Fig. 8. Projections from longitudinal
glia reach the midline.
(A,B) Longitudinal glia projections
(high magnification) span the midline
(arrowheads) in normal embryos
visualised with: (A) anti-βgal
antibodies (green) in glia driving tau
lacZ expression (genotype: 158
GAL4/ UAS tau lacZ), stage 14. Glial
nuclei are visualised with anti-Repo
antibodies (red). (B) anti-Htl (blue).
This embryo has also been stained
with the neuronal marker 22c10
(brown), stage 13. (C,D) Axon
misrouting towards midline
(arrowheads; visualised with fasII
antibodies) in rpr (C) or gcm (D)
mutant embryos. Anterior is up.
Fig. 9. Midline crossing despite robo
expression upon interference with axon-glia
interactions. (A-E) Longitudinal axons
visualised with fasII antibodies (brown) at
stage 16. In B-E arrowheads indicate
misroutings across the midline and arrows
indicate lack of longitudinal connectives.
(A) Wild-type embryo. Arrowheads indicate
the three longitudinal fascicles at either side
of the midline, which do not cross the
midline. (B) gcm mutant (this embryo has
also been stained with anti-Repo, in black).
(C) Embryo in which all pioneer neurons
and other neurons have been ablated
(genotype: ftzNGAL4/UAS ricin).
(D) Embryo double mutant for gcm and rpr,
lacking programmed cell death. Most axons
cross the midline, compare with A and B.
(E) Neuronal ablation in the absence of
programmed cell death (genotype:
ftzNGAL4 rpr/UAS ricin rpr). The
longitudinal connectives are missing or
incomplete and fasII-positive axons cross
freely over the midline, reminiscent of the
robo phenotype (compare with Fig. 1C). (F-
J) Detection of Sli (blue) and Robo (brown)
at stage 16. (F) rpr mutants, which have a
morphologically normal ventral nerve cord,
except that more midline cells express sli.
(G) gcm mutant. Longitudinal axon tracts
are thinner (arrows) and Robo is detected in
the commissures (arrowheads). (H) Ablated
embryo (genotype: ftzNG4/UAS ricin). Longitudinal axons misroute across the midline expressing robo (arrowheads). (I,J) In gcm rpr double
mutants (I) and in embryos ablated in a rpr mutant background (ftzNG4 rpr/UAS rpr, rpr) (J), Robo-expressing axons do not form longitudinal
pathways (arrows), but they all misroute dramatically across the midline (arrowheads). Anterior is up.

214
pathways of the VNC. Here, we show that the longitudinal glia
also respond transiently to the Sli/Robo repulsive mechanism,
which enables them to stop migrating ventrally, short of the
midline. In fact, we have shown that longitudinal glia migrate
within broad dorsoventral and subsequently longitudinal bands
of robo expression. Within these domains, glial nuclei are
surrounded by robo-expressing cytoplasm and membranes.
This was confirmed by demonstrating that glial cell cytoplasm
and membranes coexpress robo and the longitudinal glia
membrane marker htl as well as cytoplasmic lacZ driven by
GAL4 control in glia. Moreover, in gcm mutants, in which glial
fate is transformed to a neuronal one (Hosoya et al., 1995;
Jones et al., 1995; Vincent et al., 1996), there is a reduction in
robo expression during the time window in which robo is
normally expressed in glia. Furthermore, ectopic expression of
sli can affect glial migration. And finally, expression of robo
in glia in robo mutants favours the lateral distribution of glia.
Our data have also shown that the sensitivity of glia to
repulsion is transient, consistent with the fact that the
expression of robo in glia disappears as it is switched on in
axons. From this time on, glia associate with longitudinal
axons to maintain their lateral positions. From the moment
when glia contact axons, trophic support and cell-cell contact
prevail to restrict the position of glia. In fact, interface glia can
overlie longitudinal connectives that express sli ectopically,
particularly at stages when glia do not normally express robo.
Furthermore, some glia with targeted robo expression in robo
mutants can still overlie the midline despite expressing robo.
Our data show that these additional cellular mechanisms also
participate in confining axons to the longitudinal pathways.
The involvement of trophic support and cell contact in
maintaining the lateral positions of axons and glia is revealed
upon interference with neuron-glia interactions. Firstly, in the
absence of pioneer neurons, glia die, but if their survival is not
compromised, they do not acquire their normal positions.
Instead, they become dispersed within the VNC migrating over
the midline and to other positions. This means that glia need
contact with axons to acquire their normal positions. Secondly,
in the absence of either glia or pioneer neurons, longitudinal
axons that would not normally cross the midline, now cross the
midline despite expressing the repulsion receptor Robo. This
midline crossing by follower axons is likely to be due to a
combined loss of axonal fasciculation cues, glial contact and
trophic support by glia. Thirdly, since midline crossing is
enhanced in embryos also lacking programmed cell death, the
dependence on neuron-glia contact for survival forces these
two cell types to remain associated along the lateral pathways.
Misrouting of axons across the midline despite the
expression of robo has also been observed in calmodulin and
Son of sevenless mutants (Fritz and VanBerkum, 2000). In this
case, calmodulin and Son of Sevenless are required to transduce
the Sli signal by Robo. It is conceivable that interfering with
neuron-glia interactions similarly alters the response of cells to
Robo signalling. However, this is unlikely to explain our cases
of midline misrouting, since we eliminated cells, and thus
interfered with cell-cell communication, but molecular
functions were not directly altered.
Temporal sequence of
robo
expression
robo is initially expressed in the growth cones of pCC and in
other pioneer axons, and subsequently in all contralateral
longitudinal axons (Kidd et al., 1998a). We found that prior to
its expression in axons, robo expression is dynamic. robo is
initially expressed in broad transverse and longitudinal domains
(these longitudinal domains demarcate the future positions of the
longitudinal pathways); subsequently the transverse domains
vanish and the longitudinal domains become more restricted and
include glia. robo is then further restricted to one cell cluster per
hemisegment, and finally robo expression disappears from these
clusters and becomes apparent in axons. Thus, robo expression
is switched on in a strict temporal and spatial manner prior to its
expression in axons.
Presumably robo expressing glia receive the repulsive signal
Sli emanating from the midline, thus halting their migration at
E. Kinrade and others
Fig. 10. Sequential model of lateral confinement. (A) robo is expressed in longitudinal and transverse stripes (grey, shaded), along which the
glioblast (GB) migrates. Dotted line indicates midline (ml) which secretes Sli. (B) Robo expression in the glia along these longitudinal bands
confines glial migration. The pCC growth cone expresses robo and extends parallel to the midline. (C) When the pioneer axons extend
anteroposteriorly to form the first longitudinal fascicle, glia no longer express robo. Glial movement is restricted by cell contact and the
dependence of glia on pioneer neurons for survival. (D) The pCC/MP2 fascicle is the most sensitive to loss of robo. Glia provide trophic
support to follower neurons, thus contributing to maintain their longitudinal trajectories. If interactions between longitudinal glia and axons are
disturbed, longitudinal axons and glia cross the midline. Anterior is up.

215Robo, cell contact and trophic support
a certain distance from the midline. We can also detect Sli
expression in the longitudinal domains of robo expression from
the time when glia reach these positions and pioneer axons
project longitudinally. Presumably this detection of Sli is
protein diffused from the midline and bound by robo-
expressing cells within the lateral domains (Kidd et al., 1999).
Axons and glia seek to establish normal or
alternative contacts
Several pieces of evidence suggest that cell-cell contact –
presumably in the form of adhesion – plays a major role in the
formation of longitudinal pathways. There is evidence for
contact between axons, in the form of fasciculation; contact
between axons and glia, and glia-glia contact.
Axons fasciculate selectively with different pioneer fascicles
(Bastiani et al., 1984; Hidalgo and Brand, 1997; Raper et al.,
1983; Raper et al., 1984). Therefore, the need to maintain
fasciculation is likely to contribute to abnormal midline crossing
under our experimental conditions. In fact, since follower axons
normally fasciculate with pioneer axons, when the pioneer
neurons are eliminated, follower axons are likely to cross the
midline in search of alternative axonal as well as glial contact.
Interface glia at either side of the midline are in physical
contact through their cytoplasmic projections, which could
facilitate midline crossing of both axons and longitudinal glia.
For instance, in the absence of pioneer axons, follower axons
may follow these glial projections across the midline. When
lateral neuron-glia interactions are disturbed both axons and
glia migrate to locations where they can re-establish axon-glia
contact. For instance, glial ablation causes both axons and
remaining glia to associate over the commissures (Hidalgo et
al., 1995). In the case of gcm mutants, it is conceivable that
some of the misroutings we have found in fact correspond to the
normal trajectories of the transformed neurons. In fact, in gcm
mutants, the transformed neurons have unique and stereotypic
projections, which cross the midline (Jones et al., 1995).
However, since in gcm mutants there are no functional glia, we
do not know if the projections of the transformed neurons
would have still crossed the midline in the presence of interface
glia. We have provided two pieces of evidence indicating that in
gcm mutants there is ectopic misrouting across the midline.
Firstly, midline misroutings, visualised with fasII and anti-
Robo, were found primarily in later embryogenesis (from stage
15), indicating problems relating to the midline in the
maintenance rather than the establishment of axonal
trajectories. Secondly, in gcm rpr double mutant embryos there
are virtually no longitudinal axons expressing either fasII or
Robo, and axons project mostly across the midline.
Embryos lacking programmed cell death have an almost
normally patterned VNC, but when axon-glia interactions are
disturbed in these embryos axons project by default towards the
midline. This implies that axon-glia interactions are necessary
to maintain axons longitudinally, and they are sufficient if cell
survival is not compromised. Several molecules are known to be
involved in cell adhesion and are expressed in the longitudinal
pathways of the CNS, for instance Neuroglian (Bieber et al.,
1989), Neurotactin (Speicher et al., 1998), FasII (Lin et al.,
1994) and Connectin (Gould and White, 1992). Mutations in
these molecules cause fasciculation defects. However, it is not
known whether these molecules are involved only in axonal
fasciculation or also in axon-glia interactions.
Trophic support is a means of restricting cell
movement
Cells in animals are programmed to die unless they receive
input from their neighbours (Raff et al., 1993). In the nervous
system, target cells provide trophic factors to extending axons,
thus ensuring correct innervation. In the Drosophila CNS,
trophic support between neurons and glia plays an instructive
role during the formation of longitudinal pathways. We have
provided further evidence by showing that glia numbers are
depleted upon neuronal ablation, and that they can be rescued
by blocking programmed cell death. Longitudinal glia
normally undergo apoptosis at the time when they first come
into axonal contact (A. H., E. K. and M. Georgiou, unpublished
data). Pioneer neurons do not require longitudinal glia for
survival, but they require glia for pathfinding (Booth et al.,
2000; Hidalgo and Booth, 2000). Thus by regulating glia
survival, the pioneer neurons anchor longitudinal glia to their
axons to enable their pathfinding. Subsequently, longitudinal
glia maintain the survival of follower neurons, thus aiding the
maintenance of the axonal fascicles in lateral positions (Booth
et al., 2000). Altogether these data show that survival pressure
is instructive in determining the positions of glia and axons
during pathfinding.
We have provided further evidence here in support of this
notion. Firstly, in embryos lacking programmed cell death, some
axons project across the midline. Secondly, when neuron-glia
interactions are disturbed in embryos lacking programmed cell
death, axons and glia dramatically cross over the midline. These
are more severe misroutings than if only neuron-glia interactions
are disturbed. This reveals the roles of axon-glia interactions in
keeping both axons and glia laterally, and it also shows that
combining lack of programmed cell death with other genotypes
does not lead to additive but synergistic phenotypes. This means
that cells respond differently if their survival needs are removed.
Interestingly, blocking programmed cell death has been used as
a means of unravelling functions of neurotrophins other than in
survival (Patel et al., 2000). Our observations, however, imply
that preventing cell death does not recreate a normal although
death-free environment, but instead generates a novel one in
which cells are subject to different kinds of pressures. In the
normal Drosophila embryo, the pressure for cell contact to
survive keeps axons and glia away from the default midline
pathway, and along lateral positions.
Sequential model of lateral confinement of axons
and glia
We present a model for longitudinal pathway formation that
integrates the response to repulsive signalling and interactions
between axons and glia at lateral positions (Fig. 10). There are
two key features. (1) Temporal sequence: robo is expressed in
glia transiently to confine their migration, and it disappears
from glia when they become dependent on axons for survival.
(2) Balance of forces: subsequently robo is expressed in axons
confining them to extend parallel to the midline (Kidd et al.,
1998a; Kidd et al., 1998b), whereas axon-glia interactions
drive the need to establish cell contact and maintain survival
of both cell types within lateral positions (Booth et al., 2000)
(A. H., E. K. and M. Georgiou, unpiblished data). If pioneer
neurons or glia are eliminated, follower axons and glia will
cross the midline despite midline repellent signalling.
Initially, robo is expressed in longitudinal glia (this work)

216
and subsequently in pioneer axons (Kidd et al., 1998a),
confining both glia and pioneer axons to a fixed distance from
the midline. This may depend solely on midline-derived Sli
(Kidd et al., 1999). Expression of robo in the pCC growth cone
(Kidd et al., 1998a) alone, independent of interactions with
glia, confines its extension parallel to the midline. In fact,
whereas other pioneer axons are affected by the absence of
glia, pCC can extend normally in the absence of glia (Hidalgo
and Booth, 2000) but it cannot in the absence of robo (Kidd et
al., 1998a). Hence, pCC defines the initial trajectory of
longitudinal fascicles. As robo is switched on in the pioneer
axons, it is switched off in glia. From this time on, glia depend
on pioneer axons for their survival (see also A. H., E. K. and
M. Georgiou, unpublished data). Hence, trophic dependence
on neurons anchors the longitudinal glia to the pioneer axons
during growth cone guidance. Subsequently, axon-glia contact
and the mutual dependence of follower neurons and glia for
trophic support (Booth et al., 2000) keeps both axons and glia
along the lateral pathways. If the normal interactions between
axons and glia are disrupted, the need for cell-cell contact
forces axons and glia to establish alternative contacts. The
requirement to establish contacts that maintain survival
predominates and axons can cross the midline despite normal
repellent signalling. Thus, trophic support is instructive to keep
both axons and glia in their lateral positions.
We thank G. Booth and M. Landgraf for discussions and comments
on the manuscript; P. Badenhurst, A. Brand, J. Castelli-Gair, C.
Goodman, K. Hosono, M. Landgraf, C. Mirth, J. Roote, N. Sanchez-
Soriano and A. Travers for antibodies and flies. This work was
supported by a Wellcome Trust Fellowship to A. H. and a MRC
Fellowship to G. T. T. B. held a BBSRC studentship.
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