Download Distribution, classification, and development of Drosophila glial cells

Roux's Arch Dev Biol (1995) 204:284-307
© Springer-Verlag 1995
K e i Ito - J o a c h i m U r b a n • G e r h a r d M a r t i n T e c h n a u
Distribution, classification, and development of Drosophilaglial cells
in the late embryonic and early larval ventral nerve cord
Received: 22 June 1994 / Accepted in revised form: 10 October 1994
Abstract To facilitate the i n v e s t i g a t i o n o f glial d e v e l o p m e n t in Drosophila, w e p r e s e n t a d e t a i l e d d e s c r i p t i o n o f
the Drosophila glial cells in the v e n t r a l n e r v e cord. A
G A L 4 e n h a n c e r - t r a p s c r e e n for g l i a l - s p e c i f i c e x p r e s s i o n
was p e r f o r m e d . U s i n g U A S - l a c Z a n d U A S - k i n e s i n - l a c Z
as reporter c o n s t r u c t s , w e d e s c r i b e the d i s t r i b u t i o n a n d
m o r p h o l o g y o f the i d e n t i f i e d glial cells in the f u l l y diff e r e n t i a t e d v e n t r a l n e r v e cord o f f i r s t - i n s t a r l a r v a e j u s t
after h a t c h i n g . T h e t h r e e - d i m e n s i o n a l s t r u c t u r e o f the
glial n e t w o r k w a s r e c o n s t r u c t e d u s i n g a c o m p u t e r . U s i n g
the strains w i t h c o n s i s t e n t G A L 4 e x p r e s s i o n d u r i n g late
e m b r y o g e n e s i s , w e t r a c e d b a c k the d e v e l o p m e n t o f the
i d e n t i f i e d cells to p r o v i d e a glial m a p at e m b r y o n i c stage
16. W e i d e n t i f y t y p i c a l l y 60 ( 5 4 - 6 4 ) glial cells per abd o m i n a l n e u r o m e r e b o t h i n e m b r y o s a n d early larvae.
T h e y are d i v i d e d into six s u b t y p e s u n d e r three c a t e g o ries: s u r f a c e - a s s o c i a t e d glia ( 1 6 - 1 8 s u b p e r i n e u r i a l glial
cells a n d 6 - 8 c h a n n e l glial cells), c o r t e x - a s s o c i a t e d glia
( 6 - 8 cell b o d y glial cells), a n d n e u r o p i l e - a s s o c i a t e d glia
( 8 - 1 0 n e r v e root glial cells, 1 4 - 1 6 i n t e r f a c e glial cells,
a n d 3 - 4 m i d l i n e glial cells). T h e p r o p o s e d glial classific a t i o n s y s t e m is d i s c u s s e d in c o m p a r i s o n w i t h p r e v i o u s
i n s e c t glial c l a s s i f i c a t i o n s .
K e y w o r d s Drosophila • C e n t r a l n e r v o u s s y s t e m • G l i a
G A L 4 e n h a n c e r trap • C l a s s i f i c a t i o n
Abbreviations %NAP Percent neuromere antero-posterior
%NML Percent neuromere medio-lateral • %NVD Percent
neuromere ventro-dorsal - ac Anterior commissure • act Cell body
fibre tracts to the anterior commissure - aCC anterior corner cell
AEL After egg laying - A-SPG A subperineurial glial cell • B-SPG
B subperineurial glial cell • cbfCell body fibres - CBG Cell body
glia (glial cell) • CG Channel glia (glial cell) - CNS Central
nervous system - D Dorsal • DAB 3,3'-diaminobenzidine • DLSPG Dorsal lateral subperineurial glial cell -dn Dorsal nerve
(transverse nerve) • DV channel Dorsoventral channel • D-CG
K. Ito 1 • J. Urban • G.M. Technau (tS~)
lnstitut ftir Genetik, Universit~t Mainz, Saarstr. 21,
D-55122 Mainz, Germany
Present address:
1 ERATO Project, Mitsubishi Kasei Institute of Life Sciences,
Minami-Ooya 11, Machida, 194 Tokyo, JAPAN
Dorsal channel glial cell • D-IG Dorsal interface glial cell (cluster)
D-MG Dorsal midline glial cell (cluster) - DRG Dorsal roof glia
EG Exit glia (glial cell) - FETi Fast extensor tibiae. GDA
glutardialdehyde • GFAP Glial fibrillary acid protein • G1Gl Glial
glia • IG Interface glia (glial cell) • ISNG Intersegmental nerve root
glia (glial cell) • ISNR Intersegmental nerve root • L Lateral • L1
First instar larvae just after hatching • D-SPG Lateral dorsal
subperineurial glial cell • LG longitudinal glia (glial cell) • LV-SPG
Lateral ventral subperineurial glial cell • L-CBG Lateral cell body
glial cell. L-IG Lateral interface glial cell (cluster) • L-ISNG Lateral intersegmental nerve root glial cell • L-SNG Lateral
segmental nerve root glial cell • M medial • MD-SPG Medial
dorsal subperineurial glial cell - MG midline glia (glial cell)
(cluster) • MM medialmost • MM-CBG medialmost cell body glial
cell • MNB Median neuroblast • MV-SPG Medial ventral
subperineurial glial cell. m-cbfCell body fibre tracts from the
midline neurons • M-CBG Medial cell body glial cell. M-CG
Medial channel glial cell (cluster) • M-ISNG Medial
intersegmental nerve root glial cell • M-SNG Medial segmental
nerve root glial cell • NG Nerve root glia (glial cell) • nho
Neurohemal organ - NPG Neuropilar glia (glial cell) • PBS
phosphate-buffered saline • PBT PBS with Triton-X .pc Posterior
commissure • pct Cell body fibre tracts to the posterior
commissure • PEM PIPES-EGTA-Mg SO 4 buffer. PG Peripheral
glia (glial cell) • PNG Perineurial glia (glial cell). RT room
temperature • SaGl Satellite glia • SBC Segment boundary cell
SNG Segmental nerve root glia (glial cell) • SNR Segmental nerve
root • SPG Subperinenrial glia (glial cell) • TpGl Transport glia
TrGl tracheal glia • V Ventral • VL-CBG Ventral lateral cell body
glial cell. VL-SPG Ventral lateral subperineurial glial cell • VNC
Ventral nerve cord • vt Vertical cell body fibre tracts to the dorsal
neuropile • VUM Ventral unpaired median - V-CG Ventral channel
glial cell • V-IG Ventral interface glial cell • V-MG Ventral
midline glial cell (cluster)
Introduction
N e u r o n s o f the central n e r v o u s s y s t e m ( C N S ) are supp o r t e d b y glial cells in v a r i o u s ways. Vertebrate astrocyte
glia t r a n s p o r t a n d p r o v i d e n u t r i e n t s to n e u r o n s , a n d o l i g o d e n d r o c y t e glia f o r m i n s u l a t i o n layers a r o u n d axons. D u r i n g n e u r o g e n e s i s glial cells also p r o v i d e a scaffold for the
correct m i g r a t i o n o f n e u r o n s a n d g r o w t h c o n e s ( S i n g e r et
al. 1979; H a t t e n 1990; G r a y a n d S a n e s 1992) as w e l l as
g u i d a n c e cues for g r o w i n g a x o n s in insects ( B a s t i a n i a n d
G o o d m a n 1986; J a c o b s a n d G o o d m a n 1989a, b; Kl~imbt
285
et al. 1991; Jacobs 1993; Seeger et al. 1993). It is also
likely that glial cells participate in controlling neuronal
proliferation (Ebens et al. 1993) and in the maintenance
of mature CNS structure (Buchanan and Benzer 1993).
In spite of their importance in neurogenesis and CNS
function, relatively little is known about the mechanisms
of glial determination, development and glia-neuron interactions. Because of the vast array of genetic, molecular and cellular experimental tools available, the fruit fly
Drosophila melanogaster offers an attractive model system to study these mechanisms.
For the interpretation of mutant phenotypes and gene
expression patterns, a detailed knowledge of the types,
distribution and development of glial cells in wild-type
Drosophila is required. Although several glial cells have
been identified in the Drosophila early embryonic CNS
(Jacobs and Goodman 1989a,b; Kl~imbt and Goodman
1991), as well as in the peripheral and postembryonic nervous system (Winberg et al. 1992; Giangrande et al. 1993;
Nelson and Laughon 1993; Choi and Benzer 1994; Giangrande 1994; Prokop and Technau 1994), researchers often have to rely on the analogy with larger insects (mostly
adults) such as the housefly (Sohal et al. 1972; Strausfeld
1976), locust (Hoyle 1986), and moth (Cantera 1993).
In this study, we describe the number, distribution and
morphology of Drosophila glial cells in more detail by
observing GAL4 enhancer-trap strains that label certain
subsets of CNS cells. For the following reasons, we selected the first instar larva just after hatching (termed here
as L1) for identifying glia (see Fig. 1): First, it represents
the end of embryonic development; the state of differentiation reached by each cell makes it easy to clearly distinguish glia and neurons. Second, since there is no cell proliferation in the ventral nervous system of early larvae
(Truman and Bate 1988; Ito and Hotta 1992), the number
of cells should be stable. Third, the morphology and distribution of glial cells in the functional larval CNS provide a better classification of glial cell types; in late embryos, many neurons and glia show immature morphology and there are also some neuroblasts still dividing at
this stage (Prokop and Technau 1991).
We identified 60 glial cells per abdominal neuromere
at L1; the number may vary between segments and individuals from 54 to 64. Using strains with consistent
GAL4 expression during late embryogenesis, we traced
back the identified glia through the late embryonic stages. The resulting glial map of the stage-16 embryonic
ventral nerve cord (VNC, stages according to CamposOrtega and Hartenstein 1985), which can easily be
stained en masse as whole mounts, will be useful for
characterization of gene expression and large-scale
screenings for glial mutants.
Based on our description of identified glial cells in
the Drosophila embryonic and early larval VNC, we propose a glial classification system with six subtypes under
three categories. For the late larval and adult VNC and
brain, the system can accommodate further cell types
without altering its framework. In comparison, previous
glial descriptions for various insects are discussed.
Materials and methods
Screening, fly strains, and markers
Enhancer-trap strains were generated by crossing a pGawB strain
(Brand and Perrimon 1993) with a Pity+; A2-3] strain (Robertson
et al. 1988). To reveal the GAL4 expression pattern, male GAL4
flies were crossed with female flies carrying the UAS-lacZ detector construct (lacZ gene under the control of the GAL4 yeast UAS
(upstream activation sequence); Brand and Perrimon 1993) or with
flies carrying the UAS-kinesin-lacZ construct (courtesy of Ed Giniger; see also Giniger et al. 1993 for the kinesin-lacZ gene construct). The UAS-IacZ construct labelled nuclei and the surrounding cytoplasm; sometimes large protrusions as well as axons were
also labelled. The UAS-kinesin-lacZ construct labelled the cytoplasm of more distal parts of the protrusions, while the nuclei remained unstained (for comparison of staining by the two reporters,
see Fig. 5D, F and Fig. 9A,B).
We generated about 1,600 fly strains carrying single GAL4 enhancer-trap insertions. The first screening was performed for the
embryonic GAL4 expression, and we chose 90 strains that label
various subsets of embryonic CNS cells. The second screening
was carried out by staining the CNS dissected from newly hatched
larvae. Nineteen strains were found to be useful for glial identification. Among these six labelled only glia, while the others also
labelled subsets of neurons (Table 1). To confirm the consistency
of the GAL4 expression pattern during late embryogenesis, we
also stained the CNS dissected from late embryos (mid stage 17;
18-22 h after egg laying: AEL) and compared them with the larval
(24 h AEL) and late stage-16 (15-16 h AEL) CNS.
Most of our strains also showed GAL4 expression in structures
other than the CNS, such as salivary glands, oenocytes, epidermis
and gut. Since we dissected out the larval CNS by cutting the peripheral nerves, glia in the peripheral nervous system (PNS) were
not investigated (exit glia and peripheral glia in Klfimbt and Goodman 1991; dorsal roof glia in Nelson and Laughon 1993).
We also used several markers other than the GAL4 strains. The
P-lacZ enhancer-trap strains AA142, M84, 3-109, and P I O1 were
kindly provided by C. Kl~mbt. The transformant strains sim-lacZ
and slit-lacZ were gifts from S. Crews. The anti-prospero antibody was kindly provided by E. Spana and C. Q. Doe, and anti-repo 4(z3 by D. Halter and A. Travers.
Staining of whole-mount embryos
Collected embryos were devitellinized with Clorox (50%, 4 rain),
fixed for 30 rain with heptane saturated with phosphate-buffered
saline (PBS) containing 10% formaldehyde, and devitellinized
with methanol/heptane mixture. After five washes with PBT (PBS
with 0.5% Triton-X) embryos were pre-incubated for 2 h at room
temperature (RT) with 10% lamb serum in PBT. Incubation with
the anti-fl galactosidase (]3 Gal) rabbit polyclonal primary antibody (Cappel; 1:3000- 1:7000 in 10% lamb serum/PBT) and biotin-conjugated secondary antibody (Dianova; 1:500 in 10% lamb
serum/PBT) were both performed at 4°C over night. Labeling was
revealed using a Vectastain Elite ABC kit with 0.5-1 mg/ml DAB
(3,3'-diaminobenzidine) and 0.05% H202. In some cases 0.06% of
NiC12 was added to enhance the signal contrast. Stained specimens
were dehydrated, cleared, mounted in araldite (Serva; 46% CY
212, 46% HY 964, 5% phtalic acid dibuthyl ester, 3% DY 964),
and sucked into capillaries for observation (Prokop and Technau
1993).
Photographs were taken with an Axiophot microscope (Carl
Zeiss) with x63 Nomarski optics and x2.00ptovar. In some cases,
several photographs with slightly different focal planes were digitally montaged with Adobe Photoshop 2.0 (Adobe) on a Macintosh computer (Apple).
286
Staining of dissected larval and late embryonic CNS
Flies were allowed to lay eggs for 3 h. After 22-24 h at 25°C, larvae
already hatched were removed and, 3 h later, newly hatched larvae
were collected, so they represented a population 0 to 3 h after hatching. The CNS were dissected in PBS with watchmakers' forceps
and transferred with a 200-gl pipette into a 1.5-ml microfuge tube,
which contained about 50 gl of the fixative solution. Mid to late
stage-17 embryonic CNS (stages according to Campos-Ortega and
Hartenstein 1985) were dissected from embryos that were devitellinized and fixed as described. After 30-50 specimens were pooled,
they were further treated with fresh fixative. Either 4% formaldehyde in PEM (0.1 MPIPES, 2 mMEGTA, 1 mM MgSO 4, pH 7.0)
(30-40 min at RT) or 1% glutardialdehyde (GDA) in PBS (10 min
at RT) was used as fixative. For anti-fl GaI polyclonal antibody
staining, using GDA resulted in better conservation of the specimens and lower background. Some monoclonal antibodies, however, showed no staining when fixed with GDA.
The CNS were treated with 0.3% H202 in methanol for 30 min
at RT to inactivate the endogeneous peroxidase, followed by antibody staining as described. Because of the small size and fragile
surface of the dissected CNS, the amount of Triton-X detergent in
PBT was reduced to 0.01-0.005% or PBS was used instead.
The specimens were further incubated in 100% araldite for 6-8 h
at RT, placed in moulds with fresh araldite, and polymerized for
12 h at 45°C and 36 h at 65°C, followed by 1-~tm semi-thin sectioning and counter staining with Richardson's medium (1% toluidine blue and 1% azur II in 1% Borax) for 15 s to 3 min at 50°C.
Three-dimensional reconstruction
For the stereoscopic 3-D reconstruction (Figs. 7, 8), the contours
of the nervous system and the labelled cells were traced from photographs of 1 - g m serial sections onto transparent films, and
scanned into a Macintosh computer. About 30 sections were needed to reconstruct two neuromeres. The scanned data were
three-dimensionally aligned and rendered with StrataVision 2.6
(Strata Inc.) using ray tracing algorithm. Two pictures with six degrees of viewing-angle distance were generated and combined to
achieve the stereoscopic effect.
The schematic 3-D reconstruction (Fig. 10) was drawn by
hand with the aid of the Serial Section Reconstruction System
(SSRS; Eutectics Inc.).
Results
Staining of larval CNS sections
The fixation and antibody staining were done as described earlier;
GDA fixation was preferred. The H202 added to the DAB was reduced to 0.01% in order to avoid Oz bubbles emerging within the
specimens. NiC12 enhancement was avoided, since the resulting
blue-black staining tends to be lost during the subsequent GDA
post-fixation (6% GDA in PBS for 2 h at RT). Specimens were
dehydrated with a series of ethanol (30%, 50%, 70%, 90%, and
two times 100%), cleared with xylene, and incubated in 50% araldite in xylene over night at RT. To allow gradual evaporation of
the xylene, the microfuge tubes were kept open in a draft chamber.
Table 1 Summary of the
staining patterns used in this
study (gray boxes indicate that
the marker labels all or a subset
of cells of the subtype). The
first 23 are enhancer-trap
strains. The expression of the
genes sire, and slit were
revealed with p r o m o t e r - l a c Z
constructs (slit: Rothberg et al.
1988; sire: Nambu et al. 1991;
strains courtesy of S. Crews).
The en, pros, and repo
expression were studied with
antibodies (anti-en 4D9: Patel
et al. 1989; anti-pJvs: courtesy
of E. Spana and C. Q. Doe;
anti-repo 4a3: courtesy of D.
Halter and A. Travers)
The UAS-IacZ and UAS-kinesin-lacZ reporter constructs revealed the cytoplasmic structure of the
GAL4-expressing cells. This enabled us to identify glial
cells by their non-neuronal cell morphology. The enhancer-trap strains labelled various overlapping subsets
of glial cells; we found no single strain that labels all the
glia reported in this study (Table 1). By comparing labelled cells in different strains, we deduced the consensus distribution pattern of 60 glial cells per neuromere
neurons
glia
surface
PNG
M z 97
M z 143
Mz1251
M z 317
M z 512
M z 820
Mz1067
M z 229
M z 376
Mz1127
M z 709
M z 840
M z 770
Mz1457
M z 360
M z 419
M z 520
M z 685
Mz1299
M84
P 101
3-109
A A 142
sire
slit
pros
en
rcpo
~
neuropile
NPG
!--
Table 2 Unique names of
identified glial cells and comparison with previous descriptions (the names of the A and B
glia were changed to A- and
B-SPG, since they were found
to be members of the SPG subtype. The VUM support glia
was found to be the medialmost
member of the CBG. As for the
nerve root glia (NG) subtypes,
we took the names ISNG and
SNG. Characters "M" and "L"
were used to indicate the positions. IG and LG define different sets of cells; IG is defined
by position and LG by origin see text. Although the lateral,
ventral and a part of dorsal IG
are identical to the LG1-6, we
could not establish the
one-to-one relationship. The
origin of IG other than the LG
-unnamed cells in Goodman
and Doe 1993 - is unknown.
We did not find cells equivalent
to the MGP. The MGA and
MGM form one cluster - MG
cluster - at stage 16 and then
randomly separate into two
clusters. It seems there is no
one-to-one relationship between the MGAIMGM and
D-MGIV-MG)
our nomenclature
previous nomenclature
Klambt and
Goodman (1991)
S t a g e 15
S t a g e 16
SPG
CG
A -SPG
B -SPG
MD-SPG
LD -SPG
DL -SPG
VL -SPG
LV -SPG
MV-SPG
A -SPG
B -SPG
MD-SPG
LD -SPG
DL -SPG
VL -SPG
LV -SPG
MV-SPG
D -CG
M -CG cluster (2)
V -CG
D
M
M
V
-CG
-CGl
-CG2
-CG
I
MM-CBG
M -CBG
VL -CBG
L -CBG
-
/
SBC
I : :?:
M -SNG2
L -SNG
M -SNG2
L -SNG
D -IG
D -IG
L -IG
MG
VUM support cell
M 4SNG
1 1h
-----SNG
Goodman and
cluster (3-5)
cluster (2)
cluster (3-5)
LG1-6 +
2-4 unnamed
D -MG cluster (1-2)
V -MG cluster (1-2)
(Fig. 2 and Table 2). The number and position of labelled
glial cells showed some variability between segments
and individuals (54-64 cells per neuromere). To describe
the positions of cells in the ventral nerve cord, we introduced a metameric Cartesian coordinate system (%NAP,
%NML, and%NVD, see Fig. 1).
Unique names were given to single cells that are easily identifiable. Other cells were identified only as groups
of cells (see Fig. 2 for detail). We classified the identified cells in two levels. The individual cells that share
common morphological, positional and molecular features were classified into six "subtypes". These fell into
three "categories" according to their association with the
basic regions of the CNS: the surface, the cortex and the
neuropile.
I: Surface-associated glia
The "surface-associated glia" category represents the
cells that are closely associated with the CNS surface.
The surface is covered by the perineurium, which consists of the acellular neural lamella (Scharrer 1939) and
the surface-associated glial cells underneath (Fig. 3). In
the embryonic and L1 VNC we defined two subtypes of
this glial category. The "subperineurial glia" (SPG) lie
beneath the outer surface of the VNC, and the "channel
glia" (CG) lie along the dorsoventral channels.
Subperineurial glia (SPG)
The distribution pattern of the SPG is metameric but
shows slight variability (Fig. 4A,B at L1, C and D at late
stage 16). The nuclei and the cell bodies of SPG are
round and flat (Fig. 3, also compare Fig. 4D and F). Cytoplasmic extensions of the SPG sometimes protrude a
short distance into the underlying cortex and fill the
space between the outermost neuronal cell bodies (e.g.
Fig. 3C). In a typical case we found eight SPG (sometimes nine) per abdominal hemineuromere (Fig. 2 for
summary).
The ventral surface has two SPG per hemineuromere
(black arrowheads in Fig. 4B and D). The medial one
(medial ventral SPG, MV-SPG) lies at 30-SO%NML
and around SO%NAP. In the thoracic segments it often
lies slightly more medially than in the abdomen. The lateral one (lateral ventral SPG, LV-SPG) lies at
60-90%NML and near the anterior border of the neuromere (0-lO%NAP), hence antero-lateral to the medial
ventral SPG. Both ventral SPG remain at the same position between stage 16 and L1 (compare Fig. 4B and D).
Along the lateral surface, two lateral SPG lie at about
30 and 80%NVD (ventral lateral, VL-SPG and dorsal
lateral, DL-SPG, Fig. 4E and F). Sometimes there are
two ventral lateral SPG instead of one, resulting in a total of three lateral SPG. The exit point of the peripheral
nerve in the abdominal neuromeres is slightly above the
dorsal lateral SPG (see Fig. 4A and C). Anti-prospero
288
A
C
Supraoesophageal Ganglion(brain)
dorsalnerve
~
i
!
i
~
*/.NAP
~ ~ . , / B o ] w i g ' s
~~-2-oesophagus
nerve
0l~ll00
[''[
%NVD
.......
100
Ai
...........
/
]
T1 ¢
I I __
T2
Thorax
A1 A2 A3 A4 A5 A6 A7 A8/9
T3
Abdomen
i
~
Ventral NerveCord (VNC)
SuboesophagealGanglion
E
=
oesophagusforamen
dorsoventralchannel
Fig. 1A-C Schematic drawings of the first-instar larval central
nervous system (CNS). Lateral (A), horizontal (B) and perspective
(C) views of the larval CNS just after hatching. A,B The metameric
Cartesian coordinate system (%NAP percent neuromere antero-posterior - indicates positions along the longitudinal axis,
O%NAP corresponds to the anterior segment border, IO0%NAP
posterior segmental border, %NML percent neuromere medio-lateral - indicates positions along the mediolateral axis, O%NML corresponds to the midline, IO0%NML lateralmost surface of the CNS,
%NVD percent neuromere ventro~torsal - indicates positions
along the vertical axis, O%NVD is the ventral surface, IO0%NVD
dorsal surface). The segment borders are marked by the dorsoventral channel (DV channel), a duct-like structure penetrating the
nervous system vertically on the midline. The inner surface of the
channel is contiguous with the outer ventral nerve cord (VNC) surface (Fig. 3A,H). In early embryos the channels first appear as the
space between a pair of longitudinal connectives and the neighbouring neuromeres (Figs. 6 K and 14E). C Shows the neuropile of
the T3 neuromere and the abdominal ventral nerve cord. One larval
abdominal neuromere (A1-A6) just after hatching is 11-13 gm
thick, 70-80 gm wide and 35-40 gm high. Caudal neuromeres (A7
and A8/9) are narrower and distorted. The thoracic neuromeres are
thicker, narrower and higher than the abdominal ones. With numerous intrinsic axon fibres associated with the commissures and longitudinal tracts, the larval neuromere is much larger and more complex than in early embryos. The midline region between the commissure neuropile is almost devoid of cell bodies (see also Fig. 3).
Each neuromere carries three nerves: a pair of peripheral nerves
and a neurohemal organ. The neurohemal organ connects to the
dorsal VNC at the segment border. The abdominal neurohemal organ forms a V-shaped bifurcation to send a pair of dorsal nerves
(Hertweck 1931; "transverse nerves" in Gorczyca et al. 1994) to
both sides of the body wall, where they are called segment boundary nerves (Bodmer and Jan 1987). The bifurcation occurs just
above the VNC in the embryo. In larvae, especially in late stages,
the stalk between the VNC and the bifurcation point becomes elongated, running above the dorsal midline (see Fig. 1 of White and
Kankel 1978 for late larvae). The thoracic segments lack the lateral
projections from the neurohemal organ (see also Fig. 3 of N~ssel et
al. 1988 for Calliphora). The peripheral nerve consists of two separate fibre bundles until embryonic stage 15-16 (see Fig. l lB,C);
they fuse during stage 16-17. The level where the nerves leave the
VNC is relatively ventral in thoracic segments and more dorsal in
the abdomen (A). The peripheral nerve has two nerve roots. The intersegmental nerve root (ISNR) crosses the segment border and
forms two branches. The anterior branch enters the neuropile of the
anterior segment in the dorsalmost region (90%NVD) at about
50-70%NAP (see also Fig. 3D). The posterior branch enters the
neuropile slightly more ventrally (75%NVD) and posteriorly
(65-80%NML; see also Fig. 3E). Both branches are associated
with the mediolateral fibre tracts in the dorsalmost neuropile. The
segmental nerve root (SNR) forms many small branches that enter
the ventralmost region of the neuropile (50-60% NVD) at various
antero-posterior levels and invade the ventralmost neuropile. In
mid-stage embryos, all the nerve roots run perpendicularly to the
body axis. As the nervous system contracts, most of them are
skewed to pass through the cortex obliquely. This distortion of the
nerve roots does not affect the cortex structure
a n t i b o d y ( c o u r t e s y o f E. Sparta a n d C. Q. D o e ) l a b e l s the
lateral S P G at the p e r i p h e r y o f the lateral c l u s t e r o f the
prospero-positive cells (Fig. 6 o f D o e et al. 1991). T h e
lateral S P G s h o w v e r t i c a l l y e l o n g a t e d b e l t - l i k e m o r p h o l o g y at stage 14. T h i s s u g g e s t s that the lateral S P G are
the e q u i v a l e n t s o f the " b e l t glia" ( D o e et al. 1991). In the
late s t a g e - 1 6 a n d l a r v a l C N S , anti-pros does n o t l a b e l
the lateral S P G (Fig. 16C a n d D).
T h e dorsal surface has t y p i c a l l y f o u r S P G p e r h e m i n e u r o m e r e in the a b d o m e n a n d three in the thorax. T h e
289
Late Stage 16
~,
,j~
,
1st Instar (L1) M.s.,
L-ISNG
~M~SNG
.~"!.:
~
M-SNGI*
I
/L-IS
I
A-SPG
0'~
MV-SpG
0'~:~-M"CG1
%3v2d
LV~PG
PG
1
L-CBG
Neuropile-associated Glia
Nerve Root Gila
Surface-associated Glia
Subperineurial Glia
(SPG)
16-18
A-SPG, B-SPG, MD-SPG, LD-SPG
DL-SPG, VL-SPG*, LV-SPG, MV-SPG
0 Dorsoventral Channel Glia
~
~,
(CG)
7- 8
D-CG, M-CG cluster (M-CG1, M-CG2), V-CG
O Interface Glia
•
(CBG)
(IG)
4
4-6
14-16
(MG)
3-4
D-IG cluster*, L-IG cluster, V-IG
ortex-associated Glia
Cellbody Glia
Intersegmental: M-ISNG,LqSNG
(ISNG)
Segmental:
M-SNGI*, M-SNG2*,L-SNG (SNG)
Midline Gila
D-MG cluster, V-MG cluster (MG cluster)
6- 8
MM-CBG, M-CBG, VL-CBG*, L-CBG
Fig. 2, Summary of glial cells identified in this study. A consensus
distribution of cells in one abdominal neuromere is shown. Left and
right panels show the late stage-16 embryonic and the first-instar
larval CNS, respectively. The top and bottom panels show the horizontal view of the dorsal and ventral half of a neuromere (anterior
to the top). The middle panels show the frontal view (dorsal to the
top). The "unique name" of each identified cell is shown in the figure and in the table below. (The exit glia, EG, are excluded from
the table because they are peripheral nervous system glia.) The
naming scheme is as follows. The characters after the hyphen indicate the subtype the cell belongs to. The characters before the hyphen indicate the position of the cell (D dorsal, V ventral, L lateral,
M medial, MM medialmost). In case of combined characters such
as DL, the second character indicates the region in the neuromere
while the first character shows the precise position within this region. Suffix numbers are added to identify cells in the same area
(M~2G1, M-CG2, M-SNG1 and M-SNG2). Although the A - and
B-SPG should be addressed as anterior and posterior MMD-SPG
according to our scheme, we took the name A- and B- in favour of
compatibility with previous descriptions (see Table 2). In the table,
the word "cluster" is added for cells that are only identified as
groups. The medial CG are singly identifiable at late stage 16 but
not at L1. Cells in the dorsal IG and lateral IG clusters are not identifiable even at stage 16 but can be identified singly until stage 14.
The MG can be identified singly until stage 14, they form one cluster at stage 16 and then randomly separate into dorsal and ventral
clusters. Cells with asterisks showed higher variability than the
others. There are sometimes two ventral lateral SPG instead of one.
The ventrolateral CBG and one of the medial SNG are sometimes
missing. The distribution and number of cells in the dorsal IG cluster are highly variable
Total
54-64
lateral dorsal SPG ( L D - S P G , 8 0 - 9 0 % N M L ) lies near,
but slightly above, the exit point o f the peripheral nerve
(Figs. 4A,C and l lC). The medial dorsal SPG
( M D - S P G , 3 0 - 6 0 % N M L ) lies at the centre between the
intersegmentai nerve roots (Figs. 4 A and 5D,E), slightly
antero-lateral to the medial intersegmental nerve root glial cell ( M - I S N G , see later). Their positions do not
change during late embryogenesis. The medialmost region has two glial cells in the a b d o m e n ( A - S P G and
B - S P G , 2 0 - 3 0 % N M L ) . The A - S P G and B - S P G in the
abdominal segments lie in tandem above the neuropile
(Fig. 4A). They are identical to the "A and B glia" described by Kl~imbt and G o o d m a n (1991, Fig 5A,B). The
thoracic neuromeres have only one glial cell in this area;
they lack the B - S P G . The thoracic A - S P G has the same
position and m o r p h o l o g y as its abdominal counterparts
although it belongs to a different cell lineage ( " A - B - l i k e
glia" in Udolph et al. 1993). The A - S P G lies near the anterior segment border and sends a process to the dorsoventral channel (thin arrows in Fig. 4A). At stage 15 the
A - S P G lies posterolateral to the channel (* in Fig. 5A); it
migrates slightly anteriorly during stage 16 (Fig. 5B). The
cytoplasmic staining at this stage (Fig. 4C) shows a
wing-like structure formed by the A - S P G and the dorsal
channel glial cells ( D - C G ) . The B - S P G lies at about the
centre of the segment ( 5 0 - 6 0 % N A R Fig. 5A,B), sending
Fig. 3 A - H The anatomy of the wild-type Drosophila larval ventral nerve cord (VNC), showing eight of the t7 serial sections
(1 gm) from the first abdominal (A1) neuromere of a mid second-instar larva (30 h after larval hatching) stained with toluidine
blue. Since this stage is slightly before the onset of postembryonic
cell proliferation (Truman and Bate 1988), the number of glial and
neuronal cells should be the same as at L1. The size of the nervous
system, however, has increased by 50-60% during the 30 h of postembryonic development. The acellular neural lamella is lightly
stained along the outer VNC surface and the DV channels; it is thin
at the lateral surface but thick at the dorsal midline and along the
channel. Glial cells are stained darker than the neuronal cells. Unlike the ventral and lateral cortex, the dorsal cortex above the neuropile (15-40% NML) is very thin (about one cell diameter); few
neurons lie in this region. The medialmost region of the dorsal cortex (0-15%NML) is thicker and accommodates two rows (one row
in each hemineuromere) of big neuronal cell bodies, among which
are the aCC (anterior Corner Cell) motoneurons. The cortex/neuropile interface in this region forms a V-shaped "valley". The medial
dorsal IG lie along the slope of this valley, while the dorsal CG and
M G reside in it. Large bundles of cell body fibres (cbf) enter the
neuropile at the ventrolateral region of the cortex/neuropile interface (ac anterior commissure, pc posterior commissure, act cbf
tracts to the ac, pct cbf tracts to the pc, vt vertical cbf tracts to the
dorsal neuropile, m-cbf cbf tracts from the midline neurons, ISNR
intersegmental nerve root, SNR segmental nerve root, see Fig. 2
and Abbreviations list for glial abbreviations)
291
Fig. 4A-F Subperineurial glia (anterior to the left, bar 50 gm).
A-D Strain 347,.97 labelled with UAS-IacZ (Mz97/lacZ). A Dorsal
surface at L1 (montage of 3 pictures). White arrowheads indicate
labelled SPG of one segment (A-, B-, MD- and LD-SPG). Thin
black arrows point to the connections between the A-SPG and the
dorsal CG. The channels are interconnected by thin glial processes
running on the midline. Thick arrows indicate the intersegmental
nerve root glial ceils (ISNG). B Ventral surface of the same specimen (montage of 2 pictures), showing the two ventral SPG per
hemineuromere (LV, MV). C Dorsal surface at late stage 16 (montage of 4 pictures). Note the wing-like structure between the
A-SPG (A) and the dorsal CG (White arrowheads indicate a cluster of IG labelled transiently in some segments). D Ventral surface
of the same specimen (montage of 4 pictures). E Lateral surface of
the L1 VNC of Mz143/kinesin-lacZ, showing lateral SPG and
their fiat processes (DL, VL). F Lateral surface of the stage-16
VNC of M84, showing the flat SPG nuclei
processes in three directions: medially to the midline, anteriorly and posteriorly (Fig. 4C). They form a meshwork
of glial processes that covers the medialmost region of
the dorsal VNC surface (Fig. 5C,D,F).
To see the extent of the thin glial protrusions, which
cannot be observed in whole-mount preparations, we
made a three-dimensional computer reconstruction from
the serial sections of L1 neuromeres of the strain
M z 1 2 5 1 (Fig. 7). The strain labels a large subset of SPG
as well as channel glia and nerve root glia. From the reconstruction it is clear that the larval SPG protrusions
cover most of the VNC outer surface area.
Channel glia (CG)
The CG lie along the dorsoventral channels (DV channels). The cell bodies lie close to and send processes
along the channel surface. Their nuclei are smaller and
more spherical compared to the flat nuclei of the SPG.
We found three groups of channel glial cells: dorsal
(D-CG: 2 cells), medial (M-CG: 3-4 cells), and ventral
(V-CG: 2 cells).
The dorsal CG lie at the dorsal end of the channel
above the neuropile (one per hemineuromere; Fig. 6A, B,
G, H, L). The cells send processes ventrally along the
channel, while receiving processes from the A - S P G
(Fig. 4A,C). The position of the dorsal CG cell body is
292
Fig. 5A-F Subperineurial glia (anterior to the left, bar 50 gm).
A,B Dorsal surface of M84 abdominal VNC at stage 15 (A) and
early stage 17 (B). A and B indicate A- and B-SPG, respectively.
Asterisks indicate the DV channels. Note the more random SPG
distribution and the more anterior position of the A-SPG at stage
17. C Dorsal surface of Mz1251/lacZ at late stage 16, showing the
meshwork structure made by the B-SPG. Note that the structure
exists only in the abdominal neuromeres and disappears at the thorax/abdomen border (T/A). CG, channel glia. D The same strain
(Mz1251/lacZ) at L1. The distorted mesh structure is still visible.
The medial ISNG lies just lateral to the B-SPG. The medial dorsal
SPG (MD) lie anterolateral to the medial ISNG. E Dorsal surface
of Mz143/lacZ VNC at L1, showing flat processes made by the
medial dorsal SPG. F The same area of Mz1251/kinesin-lacZ
(montage of 2 pictures). The thick B-SPG (B) processes cover the
midline region. The thinner processes from the medial dorsal SPG
(MD) are weakly stained. Note that D and F show the same GAL4
strain crossed with different detector strains
restricted between 95 and 100%NVD. The ventral CG
lies at the other end of the channel, but the position may
vary between 0 and 40%NVD (Fig. 6B, C, H, I, L). The
two ventral CG of one segment do not necessarily lie at
the same dorsoventral level; often one lies at the ventral
end and the other more dorsally.
The dorsal and ventral C G send processes to connect
with each other. They form a sheath structure that covers
the inner surface of the DV channels. The structure is established already at late stage 16, when the formation of
the channel is completed (compare Fig. 6B and H). Although the dorsal and ventral CG lie along the midline,
they do not originate from the midline progenitors (see
Bossing and Technau 1994); during stage 14 they migrate from a lateral region towards the midline (Fig. 6J
and K). It is likely that the cells are associated with the
posterior border of segments. They express engrailed
and occupy the posteriormost positions among the
en-positive cells (Fig. 16A,B).
The medial CG lie and send processes only along the
channel at the level of the neuropile (50-95% NVD).
Their cell bodies lie just beneath the ventral side of the
cortex/neuropile interface (Fig. 6D,E, see also Fig. 3A,
H). Unlike the dorsal and ventral CG, sometimes the medial CG cell bodies lie slightly away from the channel
surface (Fig. 6D). Even in these cases, they always send
their processes to and along the channel surface and not
to the neighbouring neuropile or other CNS structures.
The medial CG are not found among the en-positive
cells.
Fig. 6A-L Dorsoventral channel glia (anterior to the left, bar
50 ~tm). A - C L1 VNC of Mz820/lacZ; A Horizontal view of the
dorsal CG. B Lateral view of the dorsal and ventral CG (D and V,
nho neurohemal organ). Montage of 2 pictures. C Horizontal view
of the ventral CG (montage of 2 pictures). D - F Medial channel
glia: D Lateral view of the L1 VNC of Mz709/lacZ showing the
medial CG (M). E Horizontal view of MzTO9/lacZ at the level of
the ventralmost neuropile. The medial CG are identifiable only as
groups of 3-4 cells (arrows). F Stage 17 VNC (18 h AEL) stained
with anti-repo (courtesy of D. Halter and A. Travers). The anterior
M-CG1 (1) and the posterior M-CG2 (2) are identifiable at this
stage. G - I Stage-16 VNC of Mz820/lacZ, showing the views corresponding to the left panels of L1 VNC (A-C). J - K Formation of
the DV channel (Mz820/lacZ). At late stage 14 (J) the dorsal half
of the channel is already formed (asterisks). Since the midline region of the ventral cortex is formed later than the other cortex regions, the ventral half of the channel remains much wider, forming
a downward funnel-Iike opening. At early stage 14, the channel
appears as a wide opening between the immature commissures
and connectives (K). The CG lie at the medial border of this opening. L Frontal optical section of Mz820/lacZ L1 VNC, showing
dorsal (D) and ventral (V) CG (montage of 2 pictures)
294
In mid stage-17 embryos, the medial CG appear to
occupy the four corners of the square flanked by the longitudinal connectives and the posterior and anterior commissures (Fig. 6F). The anterior and posterior medial CG
can be singly identified at this stage (M-CG1 and
M - C G 2 , respectively, see Fig. 2). This distinction is no
longer possible after the VNC condensation; we identified only the medial C G cluster at L1 (Fig, 2 and Table
2). Although we could not trace back the medial CG in
earlier stages, it is very unlikely that they originate from
the midline progenitors. None of the midline-specific
markers label any of the channel glia (see also Bossing
and Technau 1994).
II: Cortex-associated glia
The "cortex-associated glia" are the cells that lie among
the neuronal cell bodies in the cortex. Those that are associated with axonal tracts (e.g. nerve roots) are excluded from this category. Although several subtypes of glia
in this category have been described in the adult nervous
systems of other insects (Hoyle 1986; Cantera 1993), we
found only one subtype in the Drosophila embryonic and
early larval VNC: the "cell body gtia" (CBG).
Cell body glia (CBG)
The C B G are scattered among neuronal cell bodies in the
lateral and ventral cortex between 10 and 75%NVD
(Figs. 8,9,10). There is no CBG in the dorsal cortex. In
the abdominal and thoracic segments there are typically
3 - 4 and 4-5 CBG per hemineuromere, respectively. The
positions of the CBG cell bodies in the cortex vary significantly among individuals as well as between neighbouring segments. The nuclei and cell bodies of the C B G
have quite irregular shapes (Fig. 3D, G, H). Cytoplasmic
protrusions fill the space between neighbouring neuronal
cell bodies. Their distal ends are either sharp or with
blebs (Fig. 10). Some protrusions are up to 30 g m long,
connecting the CBG cell body with the VNC surface and
the cortex/neuropile interface (Figs. 8, 9A and 10). In
some cases CBG processes are associated with bundles
of cell body fibres (Fig. 3A,D).
The medialmost C B G ( M M - C B G ) lies close to the
midline at the centre of the segment ( 1 0 - 2 0 % N M L and
50%NAR Figs. 3C and D, 9, 10). The thoracic hemineuromere has two medialmost C B G instead of one in the
abdomen ( M M - C B G 1 and M M - C B G 2 ; Fig. 9C). The
medialmost CBG flank the V U M (ventral unpaired median) neuron cluster on the midline (Fig. 9B) and, in embryos, they appear to ensheath the V U M neuron cluster
(Fig. 9C). The medialmost CBG, therefore, is identical
to the V U M - s u p p o r t cell (Kl~imbt and Goodman 1991;
Menne and Klfimbt 1994).
The medial CBG ( M - C B G ) lies in the ventral cortex
lateral to the medialmost CBG (Figs. 3F, 9A, 10). The
position of the medial CBG can vary from 20 to
80%NML. The ventrolateral CBG ( V L - C B G ) and lateral
CBG ( L - C B G ) lie in the lateral cortex (Fig. 3F-H, 9A,
10). The ventrolateral CBG is often missing.
The same number of CBG are observed at stage 15,
when we first detect the CBG in the Mz840 strain. The
ventrolateral and lateral CBG lie more close to the ventrolateral VNC surface than at L1 (Fig. 9C).
III: Neuropile-associated glia
The "neuropile-associated glia" category covers the glial
cells that associate with the axonal structures: the nerve
roots and the neuropile that includes the connectives and
commissures. We classified three subtypes in this category: the "nerve root glia" (NG) that is further subdivided
into the "intersegmental nerve root glia" (ISNG) and the
"segmental nerve root glia" (SNG), the "interface glia"
(IG), and the "midline gila" (MG),
Nerve root glia (NG: ISNG and SNG)
The nerve root glial cells lie along the nerve roots. The
dorsally-running intersegmental nerve root has two
ISNG at its medial and lateral ends (Fig. 11A). The medial ISNG, or M - I S N G , (ISG1 of Klfimbt and Goodman
1991; SBC of Goodman and Doe 1993), lies at the mediFig. 7 Stereoscopic views of a computer reconstruction from LI
VNC of Mz1251/kinesin-lacZ, which labels a subset of SPG, CG,
and medial and lateral ISNG (see Fig. 5D,F for the whole-mount
staining pattern). Sections from T3 and AI neuromeres are reconstructed. The two membranes represent the outer surface of the
VNC and the cortex/neuropile interface, respectively. The white
structures show the glial processes labelled by kinesin-lacZ. Marbles represent the positions of glial nuclei, coded by colours according to the glial subtype. The top and middle panels show an
oblique view, with and without the glial processes. The bottom
panels show the frontal view. The fiat processes from the subset of
SPG shown here cover most of the VNC surface. The protrusions
are limited only to the surface region. The borders between the
processes of neighbouring SPG were hardly identifiable. The neurohemal organs (structure above the dorsal midline) are also covered with glial processes, which may derive from either the
B-SPG or the dorsal CG. Due to the relatively low resolution
along the longitudinal axis, the duct-like structure of the channel
is not clear (columns on the midline). Below the SPG processes,
the glial protrusions of ISNG are also visible. The medial ISNG
lie above the neuropile. The lateral ISNG lie just near the exit
points of the peripheral nerves (50%NVD in T3 and 80% in A1)
Fig. 8 Stereoscopic views of a computer reconstruction from L1
VNC of Mz840/kinesin-lacZ, which labels CBG, IG, and MG (see
Fig. 9 for the whole-mount staining pattern). Sections from AI
and A2 neuromeres are reconstructed. The IG and MG cover a significant portion of the cortex/neuropile interface, though their coverage is not as complete as that of SPG along the CNS surface
(Fig. 7). The shape of IG is relatively simple compared to that of
the MG; unlike the MG, the IG do not penetrate deep into the neuropile (see bottom panel). The CBG have extremely irregular
shape and long processes (see also Fig. 10). The flat ends of the
CBG processes contact a small part of the VNC surface
295
t"'-
296
al end of the anterior branch of the intersegmental nerve
root (see Fig. 1C), above the lateral neuropile
(20-40%NML) at about 50-70%NAE In this region the
dorsal cortex is very thin; the medial ISNG contacts both
the perineurium and the cortex/neuropile interface
(Fig. 3C). The position of the medial ISNG is slightly
lateral to the B-SPG (Figs. 4A,C, 5B,C). The medial
ISNG sends a process laterally along the nerve root,
showing a characteristic triangular morphology (Figs. 5C,
11A, 12). In early embryos the cells lie more posteriorly
near the segment border (Fig. 12 A,B), and were hence
referred to as the segment boundary cells (SBC; Bastiani
and Goodman 1986; Jacobs and Goodman 1989a,b;
Goodman and Doe 1993).
The lateral ISNG, or L-ISNG, (ISG2 of Klfimbt and
Goodman 1991) lies near the exit point of the nerve root
(Fig. 11A). In the L1 the cell sometimes lies just outside
of the VNC surface (Fig. 12D, see also arrows in
Fig. 4A).
The ventrally-running segmental nerve root has two
medial SNG, or M-SNG (SG1 and 2 of Kl~imbt and
Goodman 1991), and one lateral SNG, or L-SNG (SG3
of Kl~imbt and Goodman 1991). The two medial SNG lie
in tandem along the longitudinal axis (M-SNG1 and
M-SNG2), covering several branches of the nerve root
(Fig. l lB). Unlike the medial ISNG, which lie at the
junction between the nerve root and the connectives, the
medial SNG are slightly detached from the neuropile
proper.
The lateral SNG lies at the point where the intersegmental and segmental nerve roots meet to form a single
peripheral nerve. As a consequence, the lateral SNG and
the lateral ISNG lie in close proximity; sometimes one of
them appears to be absent (compare nerves A4 and A5 in
Fig. 11C).
Interface glia (IG)
Interface glia are the glial cells that lie at the cortex/neuropile interface and send processes along it (Strausfeld
1976; Meyer et al. 1987; "neuropile cover glia" of Cantera 1993). The embryonic and early larval Drosophila
VNC has 7-8 IG per hemineuromere. The "longitudinal
glia" (Jacobs and Goodman 1989a,b; Klfimbt and Goodman 1991; Goodman and Doe 1993) are a subset of the
IG. In this study we define the longitudinal glia as the
progeny of the longitudinal glioblast (Jacobs et al. 1989;
Doe 1992) and the interface glia as the generic name of
the glial subtype.
The IG nuclei are slightly ellipsoidal. In early embryos
the cell bodies are a bit elongated along the longitudinal
axis (Jacobs and Goodman 1989b); in larvae the cells
have flat cytoplasmic extensions perpendicular to this axis, forming a cage-like structure that surrounds the neuropile (bottom panel of Fig. 8). The processes only occasionally invade the neuropile. The IG cover the ventrolateral region of the interface less densely; many bundles of
cell body fibres enter the neuropile through this region.
Fig. 9A-C Cell body glia (anterior to the left, bar 50 gm). A L1
VNC of Mz840/kinesin-lacZ, showing the ventral half of the cortex that contain 2-3 of the total 3-4 CBG per hemineuromere
(MM, M and VL; montage of 3 pictures). B CBG cell bodies labelled with IacZ at L1 (VUM: ventral unpaired median neurons,
montage of 3 pictures). C Mz840/lacZ at late stage 16. Note that
the abdominal neuromere (At) contains only one medialmost
CBG (MM), while thoracic segments (T3) have two (1 and 2;
montage of 3 pictures)
Fig. 10 Schematic 3-D reconstruction of the cell body glia
(CBG) in an L1 abdominal neuromere. For clarity, three CBG in
the left hemineuromere were omitted
The dorsal interface has up to five dorsal IG (D-IG).
Three or four of them lie at the medialmost area of the
dorsal interface (0-20%NML, arrowheads in Fig. 13)
and separate the neuropile from the pair of rows of big
neuronal cell bodies (Fig. 3B-D, F-H). Another one or
two dorsal IG lie at the more lateral region of the dorsal
interface (204O%NML), which is beneath the thinnest
part of the dorsal cortex (Figs. 3F, G and 13C). Their positions vary significantly in the L1 VNC.
The lateral interface (50% NML) has two lateral IG
(L-IG; Fig. 13A,D). They form two parallel flat processes dorso-ventrally along the lateral interface (Figs. 3A,
B, H and 13D).
The ventral interface has one ventral IG (V-IG) that
lies at the ventralmost region of the interface
of the segment (50% Fig. 11A-C Nerve root glia (anterior to the left, bar 50 pm). A
(15-25%NML) near the
stage-15 CNS of Mz1067/lacZ at 90%NVD. The characterisN ~ P ) ,just lateral to the ventral midline glial cells Late
tic obliquely elongated shape of the medial ISNG (M-ISNG) can
ventra1 IG per be seen (see also Fig. 5C,D). B The same preparation at
(Fig. 13B). In some cases there are
hemisegment (arrowheads in Fig. 13B), lying along the 60%NVD, a pair of medial SNG (M-SNG1 and M-SNG2, 1 and
longitudinal axis. The ventral IG sends processes both 2) can be seen along the SNR. The ISNR and SNR meet, but do
laterally and medially. ~h~ medial process runs towards not fuse, at the exit point from the CNS. A peripheral nerve at this
stage consists of two twisted bundles. C Peripheral nerves at early
the
glial
but
does not connect to stage 17 (M&'/lacZ). The two bundles within peripheral nerves
them (Fig. 3F). It runs along the inner surface of the neu- fuse until this stage. Both the lateral ISNG and lateral SNG are laropile between the anterior and posterior commissures belled along the nerve root A5, while only the lateral ISNG is revealed along A4. The medialmost exit glial cell (EG)lies along the
and seems to contact the medial dorsal IG (Fig. 13E,F).
peripheral nerve. Note the thin dorsal nerves (dn) running from the
The lateral and ventral IG show an interesting migra- neurohemal organ to the lateral body wall
tion during late embryogenesis. At stage 15 all the IG are
aligned dorsally in two rows above the longitudinal connectives (see Fig. 3 of Menne and Klambt 1994, in pile, passing by the cluster of midline glial cells
which the IG are collectively referred to as "longitudinal (Fig. 13E,G). The lateral and ventral IG occupy their figlia"). At early stage 16 two cells in the lateral row begin nal positions at early stage 17 (Fig. 13F). The migrating
to move laterally, and one in the medial row moves ven- cells leave processes behind and thus maintain a connectrally along the medial border of the connectives neuro- tion with the remaining dorsal IG (Fig. 13E,F).
centrue
298
The longitudinal glioblast (Jacobs et al. 1989) and its
progeny (longitudinal glia: LG) express prospero. At
stage 14 anti-pros antibody labels six (occasionally five)
of the seven or eight IG (per hemineuromere) above the
longitudinal connectives. In the late stage-16 and L1
VNC, the two lateral IG, one ventral IG, and two to three
of the five dorsal IG are likely to be the longitudinal glia,
since they are pros positive (Fig. 16C and D).
Midline glia (MG)
The M G associate with the anterior and posterior commissure neuropile. They derive from the mesectodermal
midline progenitors (Kl~imbt and Goodman 1991; Bossing and Technau 1994).
The larval VNC has either three or four M G per neuromere. They are arranged above and below the neuropile at about 50% NAP (Fig. 14B). At embryonic stage
16 the dorsal and ventral M G are still more closely associated (Fig. 14C). The M G cell bodies are relatively
large and round. Their cytoplasmic extensions are restricted to the midline, covering the medial part of the
commissure neuropile. Fine extensions are also observed
within the neuropile (Fig. 14A,C).
Since previous reports described more M G per neuromere than we found (four to six in Fredieu and Mahowald 1989; six in Kl~imbt and Goodman 1991), we carefully studied M G numbers by using various markers
(Fig. 15). They revealed either three or four M G in most
of the abdominal segments both at late stage 16 and L1.
The percentage of segments with more than four M G
was higher in the thorax than in the abdomen.
The AA142 labels up to six cells at stage 13. The posterior cells (MGP according to Kl~imbt and Goodman
1991) then migrate ventrally (arrowheads in Fig. 14D) to
join the median neuroblast (MNB) progeny (see Bossing
and Technau 1994). The staining becomes faint as they
move to the ventral cortex and, at late stage 16, they are
no longer recognizable. Since other strains and markers
labelled no glial cells in this region, it is rather unlikely
that the migrated M G P represent glia.
At stage 14 the M G lie on either side of the midline
(Fig. 14E). As the longitudinal connectives increase their
Fig. 12A-D Late embryonic development of glial cells (SPG,
CG, ISNG, SNG and IG) in the dorsal region of the VNC
(Mz317/IacZ, anterior to the left, bar 50 gm). A Stage 14 (montage of 4 pictures). The dorsal CG lie at the posterolateral side of
the channels, which are still relatively wide at this stage (asterisks). A cluster of exit glia (EG) lies near the exit point of the peripheral nerve; they migrate out to the periphery. B Stage 15 (montage of 5 pictures). The wing-like structure of the A-SPG at the
dorsal channel opening is apparent already at this stage. The transient labelling of about six IG can be seen; two of them lie just
medial to the intersegmental nerve roots with a cluster of four between them. C Late stage 16 (montage of 3 pictures). Note the
wing-like structure of the A-SPG and the meshwork structure of
the B-SPG. D L1 (montage of 2 pictures). Despite the 50% condensation of the VNC, the labelled cells have essentially the same
distribution as in panel C (the SNG are out of focus)
299
Fig. 13A-G Interface glia (A-D and G: anterior to the left, E
and F dorsal to the top, bar 50 ~tm). A L1 dorsal neuropile of
Mzl127/lacZ (montage of 3 pictures). About three medial dorsal
IG (per hemineuromere) lie at the medial region of the dorsal cortex/neuropile interface (D-IG; see also Fig. 3). Some of them lie
adjacent to the dorsal MG. Arrows point to the thin IG processes
perpendicular to the longitudinal axis. The lateral dorsal IG above
the neuropile are out of focus in this composite picture. B Ventral
interface of the same specimen (montage of 2 pictures). The ventral IG lies lateral to the ventral MG, at the centre of the neuromere (50%NAP). Thin IG processes run medio-laterally. In some
neuromeres two IG can be seen (arrowheads 1 and 2). C A lateral
view of L1 Mzl127/lacZ at 25%NML, showing lateral dorsal IG
(D) and the ventral IG (V). D A lateral view of L1 Mz709/lacZ at
width, the M G are a l i g n e d on the m i d l i n e (Fig. 14F). The
M G cell b o d i e s r e m a i n b e t w e e n the l o n g i t u d i n a l c o n n e c tives until stage 16 (Fig. 14C,D). D u r i n g c o n d e n s a t i o n o f
the V N C and the further g r o w t h o f the n e u r o p i l e at stage
17, they m o v e apart f r o m each other to finally o c c u p y
p o s i t i o n s a b o v e and b e l o w the neuropile, w h i l e k e e p i n g
50%NML, showing lateral IG (L). E A frontal view of T1 neuromere of Mz97/lacZ at early stage 16. Because of the 90-degree
bend of the anteriormost VNC (see Fig. 1B), the horizontal view
of the T1 is topologically equivalent to the frontal view of more
posterior neuromeres. The ventral IG (lO lie between the medial
surfaces of the neuropile. The lateral IG (L) are still above the
neuropile. F A frontal view of T1 of Mzl127/lacZ at late stage 16.
The migration of the ventral IG (V) and lateral IG (L) is slightly
advanced compared to panel E. They send processes to contact the
dorsal IG (D). Note the spatial relationship between the ventral IG
and the MG (arrows). G A horizontal view of abdominal neuromeres of stage-16 Mzl127/lacZ, showing the migrating ventral IG
(V) just adjacent to the MG
their c y t o p l a s m i c extensions a r o u n d the c o m m i s s u r e s
(Fig. 14A,B). It seems that cells c h o o s e their final dorsal/ventral p o s i t i o n s rather r a n d o m l y ; m o r e than 60% o f
the investigated n e u r o m e r e s s h o w e d a s y m m e t r i c a l distribution b e t w e e n the two clusters (see Fig. 15).
300
Fig. 14A-F Midline glia (anterior to the left, bar 50 gm). A Lateral view of the L1 VNC of Mz419/kinesin-lacZ, showing the ventral (V) and dorsal (D) clusters of MG. Note the extensive processes in the neuropile. B The same view of Mzl127/lacZ, showing the
spatial relationship between the MG and the CG. C Lateral view
of the stage-16 embryo carrying the sim-lacZ (Nambu et al. 1991)
reporter construct. Clusters of 3-4 MG (arrows) and the ventral
cluster of midline neurons can be seen. D The same view of
stage-16 AA142 VNC. Arrows indicate 3-4 large cells that correspond to the MG shown in panel C. They are identical to the
MGA and MGM (Klfimbt and Goodman 1991) at earlier stages.
Note the smaller nuclei in the ventral cortex near the posterior border of neuromeres (arrowheads), which correspond to the MGP in
earlier stages (Kl~nbt and Goodman 1991). E Horizontal view of
stage-14 Mz419/IacZ, showing a pair of MGA lying beside each
other (arrows). Note the large distance between the two connectives. Asterisks indicate the DV channels. F Horizontal view of
stage-16 Mz419/lacZ The MGM become positive during stage
15, labelling up to four MG in total (arrows). As the two connectives become wider, all the MG shift towards the midline. (Lateral
staining is the transient labelling of a subset of the IG)
Discussion
Effects of reporter gene constructs
in the G A L 4 enhancer-trap system
In this study we took advantage of the G A L 4 enhancer-trap system by using different reporter constructs.
The G A L 4 - r e s p o n s i v e lacZ reporter gene (Brand and
Perrimon 1993) labels nuclei and the cell b o d y cytoplasm, whereas the distal ends of long neuronal fibres
and fine glial protrusions remain unstained. The
UAS-kinesin-lacZ construct (courtesy of E. Giniger)
gives intensive cytoplasmic staining that reveals aspects
of the cellular morphology, thanks to the fused head of
the KINESI~Vmotor protein (Giniger et al. 1993) that actively transports the fl-GaI molecule along microtubules.
Neuronal fibres sometimes show abnormal morphology when labelled with the kinesin-lacZ construct. Several swellings, some of which were as big as the cell b o d y
proper, were observed at the distal ends and branch
points o f axons. This might be due to the accumulation
o f the relatively large KINESIN--flGal fusion molecule upon its unidirectional transport to the distal ends of axons.
At least one strain, Mz376, b e c a m e embryonic lethal
301
Thorax
AA142
,
St. 1 6
(N=18,
n=53)
7
6
6
5
4
5
4
3
01~.......
10
4.0
20
30
40
50
60
Abdomen
L1 ( N = 3 9 ,
67~
n=115)
St. 16(N=lS, n=142)
L1 (N=39,n=312)
6
3
43 ~iiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiSiiiiiiiiiiiililiJliiii
i!ii~iii!i~ii!i!il;!i;i!i!ili;iliJ
2
0'
"0
%
others
2+2 cells
5
4
3
10
20
30
40
56
%
1
0
................... 3.56
10
20
30
40
50
60
%
52
10
20
30
40
50
%
sim-lacZ
N
o' :
St. 16 (N=29, n=87)
5
L1 (,=28,,=8o)
St. 16 (N=29,n=229)
67
5
4
;
4.25
16
20
30
40
50
;-;;-; ;
.............
;I
66
%
Mz 685
,
=
4
3
3.76
3.79
10
20
30
45
56
%
10
20
30
40
50
3.41
;
60
%
10
7
7
6
3
20
30
40
50
%
L1 (N=33,n=263)
6
54 I
4
3
'p
n.d.
=
5
1
o
n.d.
3.84
10
20
30
40
50
1
3.31
o
%
10
25
30
40
50
Mz 419
/
' L
St. 16 (N=21,.=59)
4
L1 (N=38,n=114)
7
6 ~
5
7
6
5
~!i!iii!ii!i!!i;ili;iii;iiii;i!i;iiiiii;iiii!::!il
St. 16 (N=24,n=188)
6
~ili;i:i~it
iiiiiiiiiiiii!jiiiiiiiii!ii:
°4
!iiiii
4
4
L1 (.=3e, .=304)
7
6 J
. . . . . . . . . . . . . . . . .
2
I
3.05
o
10
20
30
40
50
16
66
%
3.57
10
20
30
40
50
%
Fig. 15 Frequency distribution of the number of midline glial
cells (MG) per neuromere. The numbers of MG counted from the
thoracic and abdominal segments were pooled. N and n indicate
the number of specimens and the neuromeres counted, respectively. The number at the bottom-right of each panel indicates the average MG number per neuromere. In the embryonic Mz685 the inspection of MG was not possible because of dense epidermal
staining. AA142 is a P-lacZ enhancer-trap strain (KlSxnbt and
Goodman 1991), and sim-lacZ is a transformant strain with lacZ
under the single minded promoter (Nambu et al. 1991). The GAL4
strains (Mz685 and Mz419) revealed fewer cell numbers more often than the other two markers (see discussion). For L1, cells in
the dorsal and ventral clusters were counted separately; the black
bar indicates the percentage of neuromeres with three or more MG
in either of the two clusters. Less than 40% of the investigated
neuromeres had two cells in both positions ("2+2 cells", gray bar).
The "2+1" combination was as common as the "2+2". The "3+1"
or "4+0" combination occurred in 5-25% of the investigated neuromeres ("others", black bar). The probability of having more
cells in the dorsal and less in the ventral position was essentially
the same as vice versa
when crossed with the U A S - k i n e s i n - l a c Z strain, although it was viable when crossed with the U A S - l a c Z
flies. However, we found no abnormalities in other types
o f cells labelled with the kinesin-IacZ, such as glia, epidermis, muscles, intestine and salivary gland cells. The
aspects o f glial m o r p h o l o g y revealed with the kinesin-lacZ construct were also confirmed with other cyto-
10
20
30
40
50
60
%
10
20
30
40
50
%
plasmic markers as well as with semi-thin sections (compare Fig. 14A, B and C, for example).
Although transient expression was observed in some
cells, the perdurance of ]3 Gal expressed in the G A L 4
lines was generally rather long. In the Mz360 strain, for
example, lacZ was detectable even at L1, which is more
than 12 h after the G A L 4 m R N A expression ends at
stage 14. This feature was vital for our purpose to follow
the fate and migration o f labelled cells during late embryogenesis. The long perdurance m a y be due to the delay in the t w o - s t e p gene activation mechanism; the
G A L 4 protein m a y well continue to activate the UAS
even after the decline o f the enhancer activity. The
U A S - k i n e s i n - l a c Z construct showed shorter perdurance
than the UAS-IacZ; the active transport o f the protein
m a y remove the marker quickly f r o m the cell body.
As is shown in Fig. 15 for the midline glia, G A L 4
strains tend to reveal fewer cells as c o m p a r e d to the conventional P-lacZ enhancer-trap strains and other markers. It seems that the G A L 4 transcription factor - U A S
promoter combination can sometimes fail to activate the
reporter gene; in this case the cells exist but some of
them lack the reporter protein.
Only a few o f our G A L 4 strains show staining patterns in early embryos. This m a y also be due to the delay o f lacZ expression by the t w o - s t e p p r o m o t e r interaction.
302
Fig. 16A-F Glial cells labelled with antibodies (A-C and E: anterior to the left, D and F dorsal to the top, bar 50 gm). A,B
Stage-16 VNC labelled with anti-engrailed, showing dorsal CG
(A) and ventral CG (B; montage of 3 pictures). C,D A subset of
IG labelled with anti-prospero at stage 17: C shows the horizontal
view above the commissure (montage of 3 pictures), and D shows
the frontal view of the T1 neuromere (montage of 2 pictures).
Compare D and F to see that only a subset of D-IG is pros positive. E,F The stage-17 VNC labelled with anti-repo: E shows the
horizontal view (montage of 5 pictures), and F is the frontal view
of the T1 neuromere (montage of 3 pictures). In F, abbreviations
without hyphen indicate the SPG, and abbreviations in italics are
the CBG. Note that some of the glia are out of focus in the picture
Identification of Drosophila embryonic glia
In this study we identified typically 60 glial cells per abdominal neuromere of the ventral nerve cord in Drosophila late embryos and early larvae. The total number varied between segments and individuals from 54 to 64. The
thoracic neuromeres have duplicated medialmost CBG
but lack the B-SPG. They also tend to have more MG.
Among the glial cells we describe here, 32, most of
which are neuropile-associated glia, have previously
been described (Jacobs and Goodman 1989a, b; Kl~imbt
and Goodman 1991; Goodman and Doe 1993; see also
Table 2).
We used 23 different enhancer-trap strains, 2 promote r - l a c Z constructs, and 3 antibodies. All the identified
glial cells were observed using more than one of these
markers (Table 1). Furthermore, they also include all the
cells darkly stained by toluidine-blue on semi-thin sections (Fig. 1). Finally, the number and distribution of
non-neuronal cells labelled by the endoreplicative BrdU
incorporation in the late larval abdominal VNC (Prokop
and Technau 1994) matches the glial population described in this study. These points suggest that we have
identified a significant portion, if not all, of the VNC glial cells made during embryogenesis. They represent
more than 10% of the estimated number of CNS cells per
embryonic neuromere (about 400 neurons per neuromere
plus glia; Goodman and Doe 1993). Among the markers
we examined, the antibody against REPO protein (Xiong
et al. 1994) labelled the largest subset of the glial population. It labels all the identified embryonic CNS glia excluding MG (Campbell et al., 1994; D. Halter, J. Urban
et al., 1995; see also Fig. 16E and F). One previously reported CNS glial type was not described in this study:
the dorsal roof glia (DRG; Nelson and Laughon 1993).
The DRG seems to belong to the neurohemal organ and
thus can be considered as a kind of exit glia of the dorsal/transverse nerve (transverse nerve exit glia in Gorczyca et al. 1994).
We identified cells to the single-cell level when their
identity was clear either positionally, morphologically or
genetically. Some variability, such as dislocation, duplication or absence, was observed. The CG, dorsal IG, lateral IG, and MG were identified only as groups at L1 but
can be identified singly in earlier embryonic stages (see
legend of Fig. 2). Although certain variability always exists, glial distribution is more stereotypic in earlier embryonic VNC, until stage 14, than at L1. The cells that
appear later seem to show more variability than the early
cells (see also Udolph et al. 1993, for the variability in
cell numbers in the neuroblast 1-1 cell lineage). The gli-
303
al distribution in the imaginal wing also shows high variability (Giangrande et al. 1993). On the other hand, certain glial cells show significantly lower variability than
others even in later stages (such as medialmost CBG),
suggesting the importance of the consistent position and
number for their function.
Classification of insect glia
We gave "unique names" to the identified cells according
to their positions and subtypes (Fig. 2). The names are
specific for Drosophila embryonic and early larval VNC;
they are not directly applicable to other CNS regions,
stages, and species because of the different number and
distribution of glial cells in these cases. The "subtypes"
and "categories", on the other hand, should easily be applicable to other CNS regions and stages, as well as to
other insect and arthropod species (Table 3). Without altering the framework, subtypes and categories can accommodate more cell types than proposed in this study,
such as further glia in the late larval and adult CNS.
Among the gtial cells in the Drosophila optic lobe (Winberg et al. 1992), for example, it is likely that the satellite
glia belongs to the cortex-associate glia while the others
are various types of neuropile-associated glia (see Table
1 for the VNC staining pattern of the 3-109 strain, which
was used to reveal the optic lobe glia).
The three different glial categories can clearly be addressed in the lateral and ventral region of the VNC,
where prominent cortex separates the neuropile and the
CNS surface. The difference between the surface-associated and neuropile-associated glia is less obvious in the
dorsal VNC, where the cortex is very thin and glial cells
(A- and B-SPG, medial ISNG, and dorsal IG) contact
both the neuropile and the CNS surface.
There have been debates and confusion about the definition of glia. For example, the parts of the tracheal system that invade the CNS have been assigned to the glial
population ("tracheal glia", Hoyle 1986). The tracheal
branches in the CNS, however, are structurally and functionally contiguous with the outside tracheal system. In
analogy to the capillary cells in the vertebrate brain, we
excluded the tracheal cells from the glial population.
The classification of the outermost cell layer of the
CNS, the perineurial glia (PNG), is also a matter of debate. It has been assumed to be of mesodermal origin
(Bauer 1904; Scharrer 1939) and was reported to be absent in twist mutant embryos that lack the mesodermal
cells ("sheath cells", Edwards et al. 1993). The ultra-structural characteristics of these cells are rather different from the other glial cells (Hoyle 1986). Strausfeld
(1976), Hoyle (1986) and Edwards et al. (1993), therefore, excluded these cells from the glial population. Wigglesworth (1959), Carlson and Saint Marie (1990) and
Cantera (1993), on the other hand, included them in their
glial description. We incorporate the perineurial glia in
the glial population regardless of their origin and feature,
since they form a distinct non-neuronal part of the insect
CNS and probably function with other glial cells to support neurons.
However, none of our enhancer-trap strains labelled
cells that are likely to be the PNG. Using electron microscopy, the Drosophila perineurial layer becomes detectable from stage 17 onwards (Tepass and Hartenstein
1994). In the blowfly Calliphora etythrocephala the
layer is formed even later (Lane and Swales 1978). The
ultrastructural characteristics of glial cells along the
embryonic VNC outer surface resemble the definition
of subperineurial glia by Hoyle (1986) more than that
of perineurium cells (J. Roger Jacobs, personal communication). It is possible that PNG show only immature
morphology in late embryonic stages and at L1, making
it impossible to distinguish them from the SPG at a
light microscopic level. In this case, some of the cells
we classified as the SPG might actually be the PNG.
Another possibility is that we missed the PNG because
we performed the first screening using whole-mount
embryos, which can detect GAL4 expression in the
CNS only until late stage 16. In the third-instar larvae
and imago, markers such as the M84 enhancer-trap
strain (Kl~imbt and Goodman 1991) and the anti-repo
antibody (courtesy of D. Halter and A. Travers) labelled at least two types of surface-associated glia (unpublished observation). The smaller, more abundant
cells, which often lie above the SPG, may correspond
to the PNG.
Comparison of previous classifications
Table 3 summarizes some of the previous glial classifications since the first description of insect glia by Ram6n y
Cajal and S~inchez y Sfinchez (1915). While the glial
classifications in other insects are mainly based on the
observation of larval and/or adult CNS, most of the Drosophila CNS glial cells known so far have been identified in the embryo. The diversity of glial types seems to
be higher in the adult CNS than in larvae (Cantera 1993),
which have similar sets of glia to embryos.
Hoyle (1986) and Cantera (1993) described several
cells we did not find in the Drosophila embryonic VNC.
In the cortex region of large insects, large motoneurons
have more than one glial cell dedicated to their cell
body: satellite glia (SAG1, Hoyle) or cortical type 2 (C2,
Cantera). The lack of this glial subtype in the early larval
Drosophila CNS may be due to its much smaller size.
The FETi (fast extensor tibiae) motoneuron of the adult
locust is 95 gm in diameter, surrounded by 30 gm of glial coat (Hoyle 1986). In comparison, the whole single
neuromere of first-instar larval Drosophila is only
80 gm wide and 13 gm thick. The same dimensional difference may explain the lack of the glial glia (G1G1,
Hoyle 1986), cells that contact only other glial cells, and
the transport glia (TpG1, Hoyle 1986), a subtype of surface-associated glia.
The moth neuropile cover glia type 1 (NC 1) of Cantera (1993) correspond to our interface glia. For the
304
Table 3 Summary of the glial classification and comparison
with previous studies. The left part shows our proposal of the
glial classification. The subtypes written in italics represent the
cells we did not identify in this study. Based on the study of the
adult CNS, one or more subtypes may be added in the cortex-associated glial category (shown here as a dash). The right part
compares some of the previous classifications fitted into our
Our propgsal
(1995, Drosophila embryo and early larva, VNC, enhancer
trap, promoter-lacZ, antibody)
Glial Category GlialSubtype
CNS glia Surfaceassociated Glia
perineurial glia
(PNG)
classification hierarchy. Names written in italics indicate glial
types without corresponding cells found in our study (see discussion). Wigglesworth (1959), Nordlander and Edwards (1969),
Sohal et al. (1972), and Stransfeld (1976) classified glia into a
few large classes. Meyer et al.'s classification (1987) is unique in
that it is based on antibody staining patterns (antibody names
shown in parentheses). Hoyle defined subtypes in more detail.
Ramdn y Cajal and
S~inchez y Sfinchez (1915,
Musca adult, retina and
brain, Golgi / silver nitrate)
Wigglesworth
(1959, Rhodnius
late larva, VNC,
ethyl ganate)
Nordlander and Edwards (1969, Danaus,
larva and adult, brain,
toluidine blue)
--
type i
glial I
glial V
subperineurial glia
Sohal et al.
(1972, Musca,
adult, brain,
EM)
m
(SPG)
Cortexassociated Glia
Neuropileassociated Glia
PNS glia Nerveassociated Glia
channel glia
cell body glia
/CG}
(CBG) epithelial cells (?)
type iii
glial III
nerve root glia
(NG)
type ii
glial II
interface glia
(IG)
type iv
glial IV
midline glia
neuropilar glia
(NG}
(NPG)
neuroglia cells
radiate glia,
displaced epithelial cells
type I
type II
type III
perineurial ~lia
exit glia
(EG)
peripheral glia (PG)
Sensory Organ
Glia
Tracheae
tracheae
adult-specific NC 2, 3 and 4, and NC5 that exists both in
Manduca larvae and imago, we found no corresponding
cells in Drosophila larvae.
In the adult CNS, neuropilar glia (NPG) have been
described that reside within the neuropile (Sohal et al.
1972; Strausfeld 1976; Hoyle 1986; Meyer et al. 1987;
Cantera 1993). We found no strains and markers that label such glia. Semi-thin sections revealed no NPG cell
bodies in early larval VNC (Fig. 3), although we found a
few glial nuclei in the brain neuropile. The existence of
the NPG in the embryonic and early larval Drosophila
VNC, therefore, is rather unlikely, as is also the case in
Manduca larvae (Cantera 1993). Using Manduca pupae,
Tolbert and Oland (1989, 1990) showed that glial cells in
the antennal lobe remain at the cortex/neuropile interface
until early stages of metamorphosis, when they migrate
inward to delineate the antennal lobe glomeruli. This
suggests that the embryonic interface glia later differentiate into further sub-subtypes, some of which represent
neuropilar glia.
The MG have not been described in other insects, although it is unlikely that the VNC of other insects do not
have MG. The CG has not been described, possibly because insects with separate ganglia, as well as Drosophi-
la early embryos (before stage 15), do not have the apparent channel structure. The DV channel is equivalent
to the empty space flanked by the parallel longitudinal
connectives and neighbouring neuromeres in primitive
insects. This relationship suggests that Drosophila channel glia may correspond to the surface-associated glia
along the connectives in these insects.
It is possible that there is essentially no difference between the exit glia (EG) and the peripheral glia (PG)
classified by Kl~imbt and Goodman (1991). During stage
14 and 15 the exit glial cells lie at the exit point of the
peripheral nerve, while the peripheral glial cells lie outside the CNS (Fig. 12A). Before stage 14, however, most
peripheral glial cells lie within the CNS, and all the exit
glial cells migrate out of the CNS after stage 15. The two
terms may only describe a transient situation. The monoclonal antibody 5B12 shows no difference between exit
glia and peripheral glia (Meyer et al. 1987; Klfimbt and
Goodman 1991). The outward migration of PNS glial
cells seems to be rather common; it is also observed for
glial cells along nerves in the developing wing and optic
stalk (Giangrande et al. 1993; Giangrande 1994; Choi
and Benzer 1994). These glial cells however, do not
originate from the CNS. Similarly, some of the embryon-
305
Cantera made a two-level hierarchical system, using descriptive
subtype names followed by a numerical suffix for sub-subtypes.
Edwards et al. deal only with glia along the CNS surface. The
classification of Klfimbt and Goodman is unique in that it is
based on singly identified glial cells. Their nomenclature is
slightly modified by Goodman and Doe (1993, see Table 2). The
names and genus of insects listed here are: Musca domestica:
Common House-fly (Diptera), Rhodnius prolixus: Wheel Bug
(Hemiptera), Danaus plexippus plexippus: Monarch Butterfly
(Lepidoptera), Acheta domesticus: House Cricket (Orthoptera),
Schistocerca americana: Locust (Orthoptera), Manduca sexta:
Hawk Moth (Lepidoptera), and Drosophila melanogaster:
Fruit-fly (Diptera)
Strausfeld
(1976, Musca
adult, brain, Golgi
/ silver nitrate)
Meyer et al.
(1987, Acheta
adult, VNC,
antibody)
' Hoyle
(1986, Schistocerca adult, VNC,
EM)
perineural cells
perineurial glia
(3F6)
perineurium cells
class 1
(perineuralperikaryal glia)
--
subperineurial glia (SuPnGI) perineurial type 2 (P2)
transport glia
('I'pGO
class 2
cortical glia
(1D7, 1E7, 6D7)
cell body glia
(CbGI)
cortical type 1 (Cl)
satellite glia
~lial ~lia
(SAGO
cortical type 2 (C2)
-class 2
(interface glia)
tract glia
(5B12)
interface glia
(5B12)
--
class 3
(neuropilar glia)
class 4
neuropile glia
(3G6,8BI1)
-
-
(Pn)
barrier glia
A glia
B glia
VUM support cell
(GIGII
neuropile cover type 6
nerve root glia
(NC6)
axon-hilloek glia
(AxHGI)
neuropile cover type
1, 2, 3, 4, 5
(ISG, SG)
longitudinal glia
(NC1-5 )
-dark neuropilar glia (DNpGI) neuropile glia (N1, N4)
light neuropilar glia (LNpGI) tract glia
(N2, N3)
(LG)
midline glia
--
(MG)
lemnoblasts
peripheral glia
(5B12)
tracheae
Cantera
Edwards et al.
Kl~mbt and Good(1993, Manduca larva and (1993, Drosophila man (1991, Drosophila
adult, VNC,toluidineblue) embryo,VNC, EM) embryo,VNC,
enhancer trap)
perineurial type 1 (P1) perineurial
-sheath cells (SC)
exit glia
(EG)
peripheral glia (PG)
tracheal glia
(TrGI)
ic PG also seem to emerge outside the CNS, although
their origin is still unknown (D. Halter, J. Urban et al.,
1995).
Structural comparison between insect and vertebrate glia
Four major classes of glia exist in the vertebrate CNS.
The ependymal cells cover the outer surface of the nervous system and are quite similar to the surface-associated glial category in insects.
The astrocyte glia have irregular-shaped cell bodies
with many branched processes, which contact both with
neurons and capillary vessels. They are characterized by
the existence of glial fibrillary acid protein (GFAP, Eng
and DeArmond 1982). Although insect glia lack intermediate filaments like the GFAP (Carlson and SaintMarie
1990), the CBG shares many characteristics with the astrocyte glia.
With regard to the distribution and morphology, the
neuropile-associated giia are comparable to the oligodendrocytes, although insect neurons lack the myelin
sheath. The separation of neuronal cell bodies in the cortex and the dendritic structure in the neuropile may ex-
--
plain the separate distribution o f a s t r o c y t e - l i k e and olig o d e n d r o c y t e - l i k e g l i a in the insect C N S .
M i c r o g l i a are the s m a l l e s t vertebrate glia, w h i c h function as p h a g o c y t e s . T h e y are c o n s i d e r e d to b e o f m e s o d e r m a l origin, and h e n c e are often e x c l u d e d f r o m the
c a t e g o r y o f neuroglia. A l t h o u g h cell death is a ubiquitous p h e n o m e n o n in the e m b r y o n i c D r o s o p h i l a C N S , no
p h a g o c y t e s w e r e o b s e r v e d in the C N S p r o p e r ( A b r a m s et
al. 1993). W h e t h e r cells such as C B G or S P G function as
m i c r o g l i a r e m a i n s to be investigated.
T h e c o m p r e h e n s i v e d o c u m e n t a t i o n o f i d e n t i f i e d glial
cells m a d e in the D r o s o p h i l a e m b r y o n i c C N S p r o v i d e s
us with an i m p o r t a n t basis for s t u d y i n g the o r i g i n and
d e v e l o p m e n t o f glia as well as g i l a - n e u r o n interactions
during e m b r y o n i c n e u r o g e n e s i s b y m e a n s o f mutants,
m o l e c u l a r genetic and cellular e x p e r i m e n t a l tools. It also
serves as a starting p o i n t to investigate the p o s t e m b r y o n ic d e v e l o p m e n t o f the D r o s o p h i l a glia.
Acknowledgements We thank A. Brand (pGawB, UAS-lacZ), S.
Crews (sim-lacZ and slit-lacZ), E. Giniger (UAS-kinesin-lacZ),
D. Halter and A. Travers (anti-repo 4cG), C. Kl~mbt (AA142, M84,
PIO1, 3-109), and E. Spana and C. Q. Doe (anti-pros) for providing us with fly stocks and antibodies. We are grateful to A. Brand
for critically reading the manuscript, and to R. Cantera, V. Harten-
306
stein, A. Prokop, J. R. Jacobs, and the members of our research
group for helpful discussions. This study was supported by post~
doctoral fellowships from the Yamada Foundation and the EMBO
to K.I. and by the DFG grant Te130/4-1 and the EEC grant SCICT92-0790 to J.U. and G.M.T.
Goodman CS, Doe CQ (1993) Embryonic development of the
Drosophila central nervous system. In: Bate M, MartinezArias A (eds) The development of Drosophila melanogaster,
vol II. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, pp 1131-1206
Gorczyca MG, Phillis RW, Budnik V (1994) The role of tinman, a
mesodermal cell fate gene, in axon pathfinding during the development of the transverse nerve in Drosophila. Development
120:2143-2152
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