Download Embryonic development of the Drosophila brain: formation of

Development 121, 3849-3860 (1995)
Printed in Great Britain © The Company of Biologists Limited 1995
DEV2017
3849
Embryonic development of the Drosophila brain: formation of commissural
and descending pathways
Stavros Therianos1, Sandra Leuzinger1, Frank Hirth1, Corey S. Goodman2 and Heinrich Reichert1
1Laboratory of Neurobiology, Institute of Zoology, University of Basel, CH-4051 Basel, Switzerland
2Howard Hughes Medical Institute, Department of Molecular and Cell Biology, University of California,
Berkeley, California 94720,
USA
SUMMARY
The establishment of initial axonal pathways in the
embryonic brain of Drosophila melanogaster was investigated at the cellular and molecular level using antibody
probes, enhancer detector strains and axonal pathfinding
mutants. During embryogenesis, two bilaterally symmetrical cephalic neurogenic regions form, which are initially
separated from each other and from the ventral nerve cord.
The brain commissure that interconnects the two brain
hemispheres is pioneered by axons that project towards the
midline in close association with an interhemispheric
cellular bridge. The descending longitudinal pathways that
interconnect the brain to the ventral nerve cord are prefigured by a chain of longitudinal glial cells and a cellular
bridge between brain and subesophageal ganglion; pioneering descending and ascending neurons grow in close
association with these structures. The formation of the
embryonic commissural and longitudinal pathways is
dependent on cells of the CNS midline. Mutations in the
commissureless gene, which affects growth cone guidance
towards the midline, result in a marked reduction of the
brain commissure. Mutations in the single-minded gene and
in other spitz group genes, which affect the differentiation
of CNS midline cells, result in the absence or aberrant projection of longitudinal pathways. The analysis of axon
pathway formation presented here reveals remarkable similarities as well as distinct differences in the embryonic
development of the brain and the segmental ganglia, and
forms the basis for a comprehensive genetic and molecular
genetic dissection of axonal pathfinding processes in the
developing brain.
INTRODUCTION
hemimetabolous grasshopper, embryogenesis gives rise to adultlike structures (Bate, 1976; Chapman, 1982). In contrast, in the
holometabolous Drosophila, embryonic development produces
a highly derived larval stage, which is refigured postembryonically (Campos-Ortega and Hartenstein, 1985; Truman and Bate,
1988). The degree to which the embryonic brain of Drosophila
is restructured postembryonically has not yet been resolved (see
Truman et al., 1993). (For a review of older literature on insect
brain development see Malzacher, 1968.)
In this report, we analyse the cellular and molecular
processes that give rise to an initial set of axonal projections
in the embryonic brain of Drosophila. We first use histology,
immunocytochemistry and enhancer detection in combination
with light microscopy, laser confocal microscopy and electron
microscopy to determine how the commissural and descending pathways are established. We find that commissural interconnections are formed by axons that project along an interhemispheric cell bridge and that descending interconnections
are prefigured by a chain of glial cells along which pioneering
axons extend. We then use specific mutants to show that CNS
midline cells are involved in the formation of both commissural and descending brain pathways. We find that mutations
in the commissureless gene, which is involved in growth cone
The embryonic CNS of Drosophila has become an important
model system for investigations of neuronal development.
Insight into the mechanisms that control neurogenesis, axonal
pathfinding and target recognition has been gained by exploiting the powerful genetic and molecular genetic techniques
available in Drosophila (for reviews see Campos-Ortega and
Knust, 1990; Grenningloh et al., 1990; Goodman and Doe,
1993). However, most of the investigations on development in
the embryonic CNS of Drosophila have been carried out on
the ventral ganglia. Thus, little is known about the development of the Drosophila brain (see Campos-Ortega and Hartenstein, 1985).
Insight into neurogenesis and axogenesis in an insect brain
has been attained recently in the grasshopper. Embryonic neuroblasts in the grasshopper brain have been identified and some
of their molecular expression patterns characterized (Zacharias
et al., 1993). Moreover, the role of glial cell-bound proliferative
clusters in prefiguring brain pathways and the processes that
generate the axon tracts in the brain have been studied (Boyan
et al., 1995a,b,c). However, it is unclear if the results of these
investigations can be applied to Drosophila. In the
Key words: axonal pathfinding, CNS, glia, embryo, Drosophila,
commissureless, single-minded
3850 S. Therianos and others
guidance towards the midline, result in a marked reduction of
the commissure, and that mutations in the single-minded gene
and in other spitz group genes that are involved in the differentiation of midline cells, result in the absence or ectopic projection of descending pathways.
These findings demonstrate that the embryonic brain of
Drosophila can be characterized at a level of resolution that is
comparable to that attainable in the more simple ventral
ganglia. Moreover, they reveal both distinct differences and
remarkable similarities in the cellular processes and molecular
mechanisms that are involved in early embryonic development
in the brain as compared to the ventral nerve cord. Finally,
these results show that many features of brain development in
the holometabolous Drosophila are strikingly similar to those
that occur in the hemimetabolous grasshopper, thus suggesting
that similar fundamental processes might underlie embryonic
brain development in all insects.
MATERIALS AND METHODS
Fly strains
The wild-type strain was Oregon-R. The following alleles were used:
comm1, logo1 and lola1 (Seeger et al., 1993); simB21.2 (Hilliker et al.,
1980); slitIG107 and spiIIA14 (Nüsslein-Volhard et al., 1984); S1
(Bridges and Morgan, 1919); rho7M43 and pnt8B74 (Jürgens et al.,
1984); fasI/abl2 and fasI/abl3 (Elkins et al., 1990); fasITE (Zinn et al.,
1988). Enhancer detector strains, 2138 P[repo4/lacZ] (Xiong et al.,
1994) and 2035 P[3.7 sim/lacZ] (Nambu et al., 1990) were used.
Embryos were staged according to Campos-Ortega and Hartenstein
(1985) and Klämbt et al. (1991).
Immunocytochemistry
Embryos were dechorionated, fixed and labelled according to Patel
(1994). Primary antibodies were mouse BP102 diluted 1:20; rabbit antiHRP diluted 1:250 (Jackson Immunoresearch); mouse anti-Fasciclin I
diluted 1:5; mouse anti-Fasciclin II diluted 1:5; rat RK2 diluted 1:1250
(Campbell et al., 1994); rabbit anti-prospero diluted 1:1500 (Vaessin et
al., 1991). Secondary antibodies were HRP-conjugated goat antimouse, HRP-conjugated goat anti-rabbit, HRP-conjugated goat anti-rat,
DTAF-conjugated goat anti-mouse, DTAF-conjugated goat anti-rabbit,
DTAF-conjugated goat anti-rat, Cy3-conjugated goat anti-mouse and
Cy3-conjugated goat anti-rabbit (Jackson Immunoresearch) all diluted
1:250, usually after preabsorbtion against fixed embryos. Embryos containing a P[lacZ ]element were incubated with a rabbit antibody against
β-galactosidase (Aurion) which was detected according to Patel (1994).
Fluorescence-labelled embryos were mounted in Vectashield H-1000
(Vector).
Histology and histochemistry
Stained embryos were imbedded in Spurr’s (EMS), oriented
according to neuraxis and cut into 4 µm sections. Sections were
mounted on coated glass slides. Histochemical detection of β-galactosidase was in whole mounts according to Jacobs et al. (1989).
Light and laser confocal microscopy
For light microscopy, a Zeiss compound microscope (Axioskop)
equipped with fluorescence and Nomarksi optics was used. For laser
confocal microscopy, a Leica TCS 4D or a Noran video speed
confocal system were used. Optical sections ranged from 0.8 µm to
3 µm.
Electron microscopy
Embryos were dechorionated, devitellinized and fixed with 6% glutaraldehyde in phosphate buffer (pH 7.2) for 1 hour at room temper-
ature. Six 10-minute washes with phosphate buffer were followed by
postfixation in 1% OsO4 in the same buffer for 90 minutes at 4°C.
Dehydration was in a graded ethanol series and embedding was in
Spurr’s. Ultrathinsections were mounted on Formvar-coated single
slot grids and viewed on a Zeiss EM 10 electron microscope.
RESULTS
The embryonic brain hemispheres derive from the
procephalic neurogenic regions
Development of the Drosophila brain begins with the segregation of neuroblasts from the procephalic neurogenic
ectoderm (see Campos-Ortega and Hartenstein, 1985).
Following segregation, these brain neuroblasts start generating
their neuronal progeny and, during this time, the prospero gene
product (Doe et al., 1991; Vaessin et al., 1991) is expressed in
the brain (Fig. 1A-C). In the early neurogenesis phase, the cells
that express prospero most intensely are these progeny located
interior to superficial layer of brain neuroblasts (Fig. 1D,I).
Longitudinal stripes of these prospero-expressing cells are
observed in both brain hemispheres (Fig. 1G).
The two procephalic neurogenic regions that give rise to the
brain hemispheres are initially separated from each other
(Hartenstein and Campos-Ortega, 1984). This is seen in Fig.
1A where the two regions of prospero-expressing cells in the
two developing brain hemispheres are widely separated from
each other. At the 12/0 stage, the brain hemispheres become
linked to each other at the midline by a bridge-like structure of
prospero-expressing cells (Fig. 1B,C). This is where the brain
commissure will form.
A gap also initially separates the procephalic and the ventral
neurogenic regions (Hartenstein and Campos-Ortega, 1984).
This is seen in Fig. 1D,E,H where the prospero-expressing
regions of the brain hemispheres are clearly separated from the
prospero-expressing ventral neurogenic regions. As neurogenesis proceeds, prospero-expressing cells extend from the brain
to the ventral nerve cord, thus establishing cellular contiguity
around the ingrowing foregut (Fig. 1F,I). This is where the circumesophageal connectives will form.
Formation of the embryonic brain commissure
In the adult insect brain, all of the commissures located within
the brain are preoral structures (see Strausfeld, 1976). Developmental studies in the locust indicate that all of the 73 identified preoral commissural bundles of the adult brain differentiate from a single preoral ‘primary brain commissure’ in the
embryo (Boyan et al., 1995a,b). In Drosophila, a single
embryonic brain commissure is also formed during embryonic
development. We refer to this structure as the (preoral)
embryonic brain commissure. The postoral tritocerebral commissure and the frontal commissure run outside of the brain
(Boyan et al., 1993); in this report we will not refer to these
two structures as brain commissures.
One of the first signs of brain commissure formation is the
generation of two cellular column-like protrusions that extend
from the medial edges of the brain hemispheres towards the
midline. Immunocytochemical analysis with the neuronspecific anti-HRP antibody (Jan and Jan, 1982; Snow et al.,
1987), the neuronal-cell-body-specific anti-elav antibody
Brain development in Drosophila 3851
Fig. 1. Early embryonic brain development. prospero immunoreactivity in cephalic neurogenic regions. (A-C) Two initially separated prosperoexpressing regions (A, arrow) become linked by cellular bridge at midline (C, arrow); dorsal view of wholemounts. (D-F) Initially separated
prospero-expressing regions in hemispheres (E, arrow) and ventral nerve cord are linked by cellular bridges (F, arrow); lateral section (D),
Lateral views of wholemounts (E,F). (G-I) Location of prospero-expressing cells in the hemispheres; dorsal section (G, arrowheads indicate
longitudinal rows of prospero-expressing cells), transverse section (H, note gap between neurogenic regions; arrows), lateral section (I). Stages
11 (A,D,E,G,H), 12/5 (F,I), 12/0 (B,C). Scale bar, 50 µm.
(Campos et al., 1987) and the glia-cell-specific anti-repo (antiRK-2) antibody (Campbell et al., 1994; Xiong et al., 1994;
Halter et al., 1995) indicate that these protrusions are made up
of axons, neuronal cell bodies and a small number of glial cells
(Fig. 2A-C). These protrusions, once they reach the midline,
establish an interhemispheric cell bridge. Both neurons and
glial cells contribute to the completed cell bridge; however,
neuronal and glial cell bodies are in spatially separate layers.
A small group of neurons and glial cells is found at the brain
midline and may also be involved in the formation of this interhemispheric bridge (Fig. 2A-C).
The first axonal projection across the midline in the brain
is established by neurons that differentiate near the medial
edge of each hemisphere and send their axons along the
nascent cellular protrusions towards the midline (Figs 2A,
4D). As they do so, these axons express the cell adhesion
molecule Fasciclin I (Bastiani et al., 1987; Zinn et al., 1988).
At the midline, the axons make contact and fasciculate with
their homologs and, subsequently, grow along their homologs
towards the opposite hemisphere (Figs 2D, 4D). Throughout
its period of formation, this nascent initial brain commissural
fascicle is very closely associated with the interhemispheric
cell bridge (Fig. 2E,F). Indeed, both the developing cellular
bridge and the extending pioneering commissural axons reach
the midline at the same time. As soon as the first commissural axonal pathway in the brain across the midline is established, it is followed by other fasciculating commissural
axons and, during subsequent embryogenesis, this commissural fascicle differentiates further to become the massive
preoral commissure of the embryonic brain (Fig. 2G).
Throughout embryogenesis, a row of glial cells that extends
across the brain midline continues to be associated with this
preoral brain commissure (Fig. 2H,I).
Two minor commissural structures are formed anterior to the
subesophageal ganglion but outside of the embryonic brain;
they will be mentioned briefly here. The postoral tritocerebral
commissure is pioneered before the preoral brain commissure.
This commissure runs outside of the brain and connects the circumesophageal connectives and the brain hemispheres at the
level of the developing tritocerebrum. The frontal commissure
is pioneered by two symmetrical neuronal cell bridges that run
from the developing frontal ganglia to each of the brain hemi-
3852 S. Therianos and others
Fig. 2. Commissure formation. Frontal views through the brain hemispheres (laser confocal microscopy). Neuron-specific anti-HRP
immunoreactivity (A,D,E,F,G), glial cell-specific repo immunoreactivity (H), combined anti-HRP immunoreactivity (red) and repo
immunoreactivity (green/yellow) (B,C,I). (A-C) Cellular protrusions (A, arrows) composed of neurons and glial cells extend from medial edges
of hemispheres towards midline. Small group of neurons and glial cells at midline (open arrow). (A) Reconstruction of brain hemispheres,
(B,C) different optical sections. (D-F) Establishment of initial commissural axonal projection (D,F, arrows) in close association with
interhemispheric cell bridge (E, arrowhead). (D) Reconstruction of brain hemispheres, (E,F) different optical sections. (G-I) Differentiation of
commissural pathway leads to formation of preoral brain commissure (G, arrow); a row of glial cells extending across midline (H, arrowhead)
is closely associated with commissure. Stages 12/0 (A-C), 13 (D-F), 16 (G-I). Scale bar, 10 µm.
spheres. It is established at approximately the same time as the
preoral brain commissure.
Development of circumesophageal axon pathways
Before the first descending longitudinal axonal pathways are
established, two bilaterally symmetrical longitudinal arrays of
repo-immunoreactive glial cells are seen extending from the
brain hemispheres to the subesophageal neuromeres (Fig.
3B,C). In the ventral nerve cord, these longitudinal glial cells
have been described in detail (Jacobs and Goodman, 1989a;
Jacobs et al., 1989). In the head, the longitudinal glial cells are
found at the medial edges of the procephalic and subesophageal neurogenic regions and also extend around the gap
between the brain and ventral ganglia caused by the ingrowing
foregut. The location of these longitudinal glial cells prefigures
the path of the circumesophageal connectives.
The first axonal pathways that interconnect brain and
ventral nerve cord are formed once the longitudinal glial cells
are in place. Initially, a small gap separates the neurons of
each cephalic hemisphere from the neurons of the sube-
sophageal ganglion (Fig. 3D-F). Subsequently, a cellular
bridge composed of anti-HRP immunoreactive neuronal cell
bodies is formed across this gap anterior to the row of longitudinal glial cells (Fig. 3G-I). Then the first longitudinal axon
pathways are pioneered by axons that project across the
neuronal cell bridge in close association with the row of longitudinal glial cells (Fig. 3J-L). Both descending and
ascending axons are involved in pioneering the longitudinal
pathway (Fig. 3A, arrows); they meet and fasciculate approximately half-way around the ingrowing esophagus. This initial
axonal pathway is followed by other fasciculating descending
and ascending axons. Longitudinal glia cells continue to be
closely associated with this longitudinal fascicle as it differentiates further into the circumesophageal connective.
In contrast to the ventral ganglia, in the brain the first longitudinal fascicle is established before the first brain commissural fascicle. Indeed, the first circumesophageal interconnection appears to be formed as the foregut grows through the
embryonic CNS (Fig. 3A). This relatively early time of
formation is advantageous, since the foregut grows and
Brain development in Drosophila 3853
Fig. 3. Formation of circumesophageal axon pathways. (A-C) Sections, (D-L) lateral optical sections in plane of a cirumesophageal connective
(laser confocal microscopy). Neuron-specific anti-HRP immunoreactivity (A,D,G,J), P[repo4/lacZ] revealed with anti-β-galactosidase (B,C),
glial cell-specific repo immunoreactivity (E,H,K), combined anti-HRP immunoreactivity (red) and repo immunoreactivity (green/yellow)
(F,I,L). (A) Ascending and descending axonal processes (arrows) growing around ingrowing foregut (arrowhead); lateral section. (B-C) Rows
of longitudinal glial cells (arrowheads) extend from brain hemispheres around foregut to ventral nerve cord; frontal sections. (D-F) Neurons of
brain and subesophageal ganglion are initially separated by distinct gap (D, arrow); this gap is bridged by row of glial cells (E, arrowhead).
(G-I) Subsequently, a neuronal cell bridge (G, arrow) is formed across gap in close association with row of longitudinal glial cells (H,
arrowhead). (J-L) The first axonal pathways (J, arrow) are established between brain and subesophageal ganglion across neuronal cell bridge
and in close association with longitudinal glial cells (L, arrowhead). Stages 11 (D-F), 12/5 (G,-I), 12/3 (A), 12/0 (J-L) and 13 (B,C). (Note that
stage 12/5 precedes stage 12/3 which precedes stage 12/0; see Klämbt et al., 1991.) Scale bar, 10 µm.
expands rapidly causing a substantial increase in the distance
along which pioneering circumesophageal axons would later
have to project.
A primary axon scaffold in the embryonic brain
After the establishment of the initial brain commissural and circumesophageal fascicles, these structures grow rapidly in size
due to the addition of numerous axons. Longitudinal axonal
pathways extend from the most anterior (according to the
neuraxis) parts of the hemispheres, past the brain commissure
and around the site of foregut ingrowth into the first subesophageal neuromere. (Due to head involution, the brain longitudinal tracts that lie most posteriorly in the embryo correspond to the most anterior part of the brain according to the
neuraxis; see Boyan et al., 1995c.) The preoral embryonic
brain commissure and the postoral embryonic tritocerebral
3854 S. Therianos and others
Fig. 4. Establishment of a primary axon scaffold. (A,C) Sections, (B,D,F) whole mounts, (E) laser confocal microscopy. Axon-specific
monoclonal antibody BP102 immunoreactivity (A,B), Fasciclin II immunoreactivity (C,F), Fasciclin I immunoreactivity (D,E). (A,B) Quasiorthogonal axon scaffold is established in brain. Note paired circumesophageal connectives (arrowheads) around foregut, preoral commissure
(filled arrow) above foregut, postoral tritocerebral commissure (open arrow) below foregut and adjacent three commissures of subesophageal
ganglion in (A). Frontal views in plane of both circumesophageal connectives. (C) Discrete Fasciclin II-expressing axon fascicles in
longitudinal (arrowheads) and commissural (arrows) brain pathways. Frontal section in plane of both circumesophageal connectives.
(D) Pioneer neurons of the brain commissure express Fasciclin I. A pair of neuronal cell bodies on each side of the brain (right pair labelled
with arrowheads) project the first brain commissural axons across the midline (arrow) and fasciculate with their homologs. (E) Many axons in
preoral brain commissure and several fascicles and neurons in two brain hemispheres show Fasciclin I immunostaining. Non-commissural
immunoreactive midline structures (asterisks) belong to stomatogastric system. (F) Two fascicles in primary preoral commissure (arrow) show
Fasciclin II immunostaining. Non-commissural immunoreactive midline structures (asterisks) belong to stomatogastric and the neuroendocrine
systems. Dorsal view of embryo with anterior end downwards. Stages 16 (A,B,C,E,F), 13 (D). Scale bar, 10 µm.
commissure form parallel axonal pathways around the
esophagus. Thus, a quasi-orthogonal primary axon scaffold is
established in the brain at the end of embryogenesis.
Although this axonal scaffold is discernible in whole mount
(Fig. 4B), it is more clearly evident in histological sections
(Fig. 4A), which show that the preoral brain commissure, the
circumesophageal connectives, the tritocerebral commissure
and the commissures of the three subesophageal neuromeres
are all in the same plane at the end of embryogenesis. The
salient features of the postoral axonal structures (tritocerebral
commissure, subesophageal commissures and connectives) are
comparable to those found in the adult. In contrast, the primary
preoral brain commissure in the embryo is still relatively undifferentiated; the numerous individual commissures characteristic of the mature brain are formed postembryonically. Ultrastructural analysis of the developing brain commissure at the
end of embryogenesis confirms this apparently unitary,
compact arrangement of the many commissural axons (Fig.
5A). It also shows that the brain commissure is intimately sur-
rounded by a ring-like array of cells, which may be glial (Fig.
5D). In contrast to the compact nature of the embryonic brain
commissure, there are discrete spaces in the hemispheres (Fig.
5B,C) some of which are associated with ultrastructural indications of cell death (Abrams et al., 1993).
A number of cell adhesion molecules are dynamically
expressed in the ventral nerve cord; their expression patterns
have led to the labelled pathway hypothesis for axonal
guidance (Raper et al., 1984; Goodman et al., 1984). Many of
these molecules are also found in the developing brain indicating that the labelled pathway hypothesis might also apply
to the formation of axon tracts in the brain. For example, many
brain axons express the Fasciclin I cell adhesion molecule
(Bastiani et al., 1987; Zinn et al., 1988). Fig. 4D shows the
expression of Fasciclin I on the axons of the neurons that
pioneer the brain commissure. Fig. 4E shows the intense
Fasciclin I-immunoreactivity in the differentiated brain commissure at a later stage. A more restricted expression pattern
is seen for Fasciclin II (Snow et al., 1988; Harrelson and
Brain development in Drosophila 3855
A
C
D
Fig. 5. Ultrastructure of embryonic brain hemisphere
and preoral commissure. Stage 15. Sections
perpendicular to orientation of commissural axons
near site at which commissure leaves brain
hemisphere. (A) Overview of brain hemisphere
showing peripheral cell body layer (arrows) and
central neuropil (asterisk). (B) Higher magnification
showing individual brain neurons and their axons
(arrow), and remnants of apoptotic cells (stars).
(C) Higher magnification showing individual cortex
glial cells and apoptotic cell (star). (D) Cross-section
through brain commissure. Ring of putative glial cells
(asterisks) surrounds axons of commissure C. Axon
fascicle (arrowhead) projects into commissure. Scale
bar, A, 10 µm; D, 3 µm.
Fig. 6. Commissure differentiation is perturbed in commissureless mutants. (A-E) Frontal views through brain hemispheres (laser confocal
microscopy). (F,G) Sections. Neuron-specific anti-HRP immunoreactivity (A), combined anti-HRP immunoreactivity (red) and glial cellspecific repo immunoreactivity (green/yellow) (B,C), combined axon-specific BP102 immunoreactivity (red) and repo immunoreactivity
(green/yellow) (D), BP102 immunoreactivity (E,G), Fasciclin II immunoreactivity (F). (A-C) An initial commissural axon pathway (A, arrow)
is established along midline cellular bridge (B, arrowheads) composed of neurons and small number of glial cells. (A) reconstruction of
hemispheres, (B,C) different optical sections. (D,E) Subsequent differentiation of commissural pathway is perturbed and only single fascicle of
commissural axons (E, arrows) is formed. Associated glia cells (D, arrowheads) spanning midline appear normal. (F) Commissural fascicle
contains Fasciclin II-expressing cells. Frontal section. (G) Mutant preoral commissure (filled arrow) is highly reduced and all postoral
commissures are missing (open arrow); descending longitudinal tracts (arrow heads) appear normal. Frontal section in plane of both
circumesophageal connectives. Stages 13 (A,B,C), 14 (D,E), 16 (F,G). Scale bar, 10 µm.
3856 S. Therianos and others
Goodman, 1988; Grenningloh et al., 1991). Three major
Fasciclin II-expressing fascicles are found in the circumesophageal connectives and in the preoral brain commissure;
one Fasciclin II-expressing fascicle is found in the tritocerebral commissure (Fig. 4C,F). Fasciclin III (Patel et al., 1987;
Snow et al., 1989) expression was not found.
The embryonic brain commissure is reduced in
commissureless mutants
The characterization of the commissural and longitudinal
pathways represents a first step in the analysis of brain axogenesis. An important second step is the investigation of the
molecular and genetic mechanisms that underlie the formation
of these axon pathways. One way to elucidate these mechanisms is by using mutants to analyse the role of specific genes
in building the axon pathways of the brain.
In the ventral ganglia, mutations in the gene commissureless
have a dramatic and specific phenotype; all commissural
pathways are lacking while other axon pathways appear normal
(Seeger et al., 1993). In the brain, the cellular events involved
in the initial formation of a first brain commissural fascicle
appear normal in commissureless mutants. Thus, cellular protrusions extend from each hemisphere towards the midline, a
cellular bridge spans the gap between the two hemispheres and
pioneering axons establish an interconnection across the
midline (Fig. 6A-C). Subsequently several other commissural
axons project along the pioneered midline pathway, thus,
giving rise to a first brain commissural fascicle (Fig. 6E). This
brain commissural fascicle develops in close association with
a row of repo-expressing glial cells that extends across the
midline (Fig. 6D), and many of the axons in this fascicle
express Fasciclin II as in the wild type (Fig. 6F).
However, in striking contrast to the wild type, subsequent
differentiation of the brain commissure involving the projection of many more axons across the midline and the formation
of additional commissural fascicles does not take place in the
commissureless mutants. For example, the two more anterior
Fasciclin II-expressing fascicles are not formed. Thus, despite
the fact that a cellular bridge has been established across the
midline and that both glial cells and pioneering commissural
axons are in place, many of the follower axons cannot project
across the midline in the absence of the commissureless gene
product. As a result, the brain commissure that is formed
during embryogenesis is markedly reduced; considerably less
than half of the normal commissural axons are formed (Fig.
6G). Thus, although the commissureless gene product is not
necessary for the initial pioneering of a brain commissural
pathway, it is essential for further differentiation of the brain
commissure through the additional projection across the
midline of numerous follower axons.
In commissureless mutants, the tritocerebral commissure
and the commissures of the ventral ganglia do not form. In
contrast, the longitudinal descending connectives appear
normal and the frontal commissure forms in a normal manner.
The circumesophageal connectives fail to form in
single-minded mutants
The single-minded gene encodes an HLH transcription factor
that acts as a master regulator of midline cell development
including the differentiation of midline cells into mature
neurons and glial cells (Crews et al., 1988; Nambu et al., 1990,
1991). Mutations in the gene single-minded have a striking
phenotype in the ventral ganglia; commissures fail to form and
the longitudinal tracts collapse into a fused tract at the midline
(Thomas et al., 1988; Klämbt et al., 1991). In the developing
embryonic brain, mutations in the single-minded gene have an
equally striking yet morphologically completely different
phenotype. In most cases, the longitudinal circumesophageal
connectives fail to form (Fig. 7A,K).
The absence of connectives in single-minded mutants may
be related to a gap in the longitudinal glial cell array. A subset
of the longitudinal glial cells that normally form around the
ingrowing foregut are missing in the single-minded mutants
(Fig. 7B,C). Tenuous neuronal bridges across this gap are
sometimes observed; however, they appear to be unstable and
subsequently disappear. Mutations in the single-minded gene
do not cause a collapse of the longitudinal tracts at the brain
midline, as is the case for the longitudinal tracts in the ventral
nerve cord. A preoral brain commissure is formed in singleminded mutants, and the brain tracts and associated glial cells
that develop anterior to the esophagus appear to differentiate
normally (Fig. 7D-F).
In approximately 20% of the single-minded mutants, ectopic
descending pathways from the brain to the ventral ganglia are
observed. In these cases, the two main longitudinal tracts in the
two brain hemispheres do not project separately around the
esophagus. Rather they fuse medially at the level of the dorsal
esophagus and then project posteriorly and ventrally along the
gut towards the fused segmental ganglia (Fig. 7G,J). Repoimmunoreactive glial cells are not associated with this ectopic
pathway (Fig. 7H,I).
The brain phenotypes observed in single-minded mutants
correlate with the spatial expression of the single-minded gene
in the wild type as assayed by a P[sim/lacZ] transformant line
(Nambu et al., 1991). The P[sim/lacZ] marker indicates that
single-minded is expressed in cells located in the lower part of
the brain (just anterior-dorsal to the subesophageal ganglion),
and some of these cells appear to be glial (Fig. 7L). In the wild
type, the circumesophageal connectives develop in close
proximity to these cells; normal differentiation of these cells
may, therefore, be important for the formation of the paired
circumesophageal connectives.
Based on the P[sim/lacZ] marker, the single-minded gene
does not appear to be expressed in more anterior parts of the
brain. Specifically, there is no evidence for single-minded
expression among the midline neuronal and glial cells at the
site of brain commissure formation. This implies that the functional role of single-minded as a master regulator of midline
cell development does not apply to the major anterior regions
of the embryonic brain.
Other mutations that affect brain pathway formation
A common feature of mutants with defects in either the commissureless gene or the single-minded gene is that the mutant
phenotypes seen in the embryonic brain are strikingly different
from those observed in the embryonic ventral nerve cord. A
difference in mutant phenotype in the brain versus the ventral
ganglia is apparent for several other genes that are involved in
axonal pathway formation.
The single-minded gene is a member of the spitz-group
(Mayer and Nüsslein-Volhard, 1988) that also includes the
genes spitz, Star, pointed and rhomboid. Mutations in these
Brain development in Drosophila 3857
four genes have comparable phenotypes in the ventral nerve
cord, and these phenotypes resemble those seen in singleminded mutants (Klämbt et al. 1991) as well as in slit mutants
(Rothberg et al., 1988, 1990). Given this similarity, we
wondered whether mutations in all of these genes might have
similar effects on the formation brain axon pathways. Our
analysis shows that this is indeed the case, but these defects
are very different from those observed in the ventral nerve
cord. Mutations in the genes spitz and Star typically result in
the absence of circumesophageal connectives. Mutations in
rhomboid and pointed result in the absence or in the formation
of abnormally shortened circumesophageal connectives.
Mutations in slit result in the formation of aberrant ectopic connections between the brain and the ventral ganglia. In all of
these cases, the absence or abnormal formation of connective
pathways correlates with gaps in the set of longitudinal glia
that normally link the brain to the ventral ganglia (Hirth et al.,
unpublished data).
Mutations in the genes longitudinals lacking and longitudinals gone are known to affect the guidance and extension of
longitudinal axons in the ventral ganglia (Seeger et al., 1993).
This does not appear to be the case in the developing brain.
No obvious defects in either longitudinal or commissural axon
pathway formation are observed in the developing brain of
longitudinals gone mutants. Mutations in the longitudinals
lacking gene result in an approximately 50% reduction in the
length and thickness of the embryonic brain commissure.
However, the longitudinal circumesophageal pathways seem
completely normal in longitudinals lacking mutants.
DISCUSSION
Studies of nervous system development in insects have been
significant for understanding the cellular and molecular mechanisms involved in axon outgrowth and pathway formation (for
reviews see Goodman and Doe, 1993; Reichert, 1993). Most
of this work has, however, focused on the segmental ganglia
or the peripheral nervous system; little is known about insect
brain development. In this report, we investigate the processes
that lead to the formation of the major axonal pathways in the
embryonic brain of Drosophila, and we use mutants to analyse
the role of specific genes in building these brain pathways.
The principle problem addressed in this paper stems from
the fact that the insect brain originates as two neural masses
that are separate from each other as well as from the chain of
segmental ganglia, raising the question of how these separate
entities become connected. The manner in which this problem
is solved during embryogenesis reveals both differences and
similarities in the embryonic development of the brain as
compared to the ventral nerve cord. In the following, these differences and similarities are discussed.
Linking the brain hemispheres
In the brains of higher animals, the two hemispheres are
connected by numerous commissural axons. During commissure
formation, axonal pathways must be established across the
midline, which provides a natural boundary in pattern formation.
In insects and in vertebrates, specific cells at the CNS midline
play a prominent role in commissure formation (for reviews see
Dodd and Jessel, 1988; Goodman and Shatz, 1993).
In Drosophila, a small set of pioneering axons establishes
the first brain commissural pathway. These pioneering axons
extend in close association with bridge-like aggregates of
neuronal and glial somata that extend across the brain midline.
Such aggregates of neurons and glial cells extending across the
midline are not observed during commissure formation in the
segmental ganglia. However, surprisingly similar features are
seen in the developing corpus callosum and anterior commissure of the mammalian brain (Silver et al., 1982). There, glia
cells from each hemisphere form an interhemispheric bridge
(‘glial sling’) that is used by the growth cones of the first commissural axons. This suggests that the formation of cellular
bridges across the midline may be a general feature of commissure formation in complex brains, related to the problem of
interconnecting separate brain hemispheres. The mechanisms
that lead to the formation of interhemispheric cellular bridges
in Drosophila are not yet known. Cell migration, local division
or delayed differentiation of neurons and glial cells may be
involved.
Molecules involved in commissure formation in vertebrate
spinal cord have been identified recently (Tessier-Lavigne et
al., 1988; Kennedy et al., 1994; Serafini et al., 1994), but little
is currently known about their involvement in brain development. In Drosophila, molecules involved in brain commissure
development are now known. One of these is the commissureless gene product, which is thought to be secreted from specific
midline cells (Seeger et al., 1993). In commissureless mutants,
the initial commissural fascicle in the brain develops normally,
yet most of the subsequently formed commissural fascicles are
missing. This suggests that molecular signals sufficient for the
initial formation of a brain commissural fascicle are still
present in the commissureless mutant, but that these signals are
not sufficient to allow the majority of commissural axons to
form. The longitudinals lacking gene (Seeger et al., 1993) may
also be involved in differentiation of the embryonic brain commissure. Brain commissural fascicles are established in longitudinals lacking mutations; however, both length and thickness
of these embryonic commissures are markedly reduced.
The specific molecular roles of the genes commissureless,
longitudinals lacking and longitudinals gone in brain commissure and connective formation remain to be determined.
However, in all three cases, our genetic analysis demonstrates
the existence of clear differences in the mechanisms of commissure and connective formation in the brain as compared to
the ventral nerve cord. We, thus, posit that brain-specific mechanisms exist that allow the formation of an initial commissural
fascicle even in the absence of the commissureless gene
product, and that brain-specific mechanisms exist that allow
the formation of longitudinal pathways even in the absence of
the genes longitudinals lacking or longitudinals gone.
Connecting the brain to the ventral nerve cord
The formation of longitudinal pathways that interconnect
brain and ventral nerve cord must deal with two developmental constraints: the initial separation of two neurogenic regions
(see Campos-Ortega and Hartenstein, 1985) and the
ingrowing foregut. The solution to these constraints seems to
involve the early generation of neuronal cell bridges and rows
of longitudinal glial cells, which extend from the brain hemispheres around the ingrowing foregut towards the ventral
ganglia and, thus, link the cephalic and ventral neurogenic
3858 S. Therianos and others
Fig. 7. Paired circumesophageal connectives fail to form in single-minded mutants. (A-C) Lateral optical sections in plane in which
cirumesophageal connective would normally develop (laser confocal microscopy). (D-I) Frontal optical sections in plane in which both
circumesophageal connectives would normally develop (laser confocal microscopy). (J) Midsaggital section. (K) Frontal section. (L) Lateral
view through brain and subesophageal ganglion of P[sim/lacZ] transformed wildtype (laser confocal microscopy). Neuron-specific anti-HRP
immunoreactivity (A,D,G), glial cell-specific repo immunoreactivity (B,E,H), combined anti-HRP immunoreactivity (red) and repo
immunoreactivity (green/yellow) (C,F,I), axon-specific BP102 immunoreactivity (J,K), combined anti-HRP immunoreactivity (red) and anti-βgalactosidase immunoreactivity (green/yellow) (L). (A-C) Longitudinal axon pathways across a gap (A-C, arrows) between brain hemispheres
and subesophageal ganglion do not form and longitudinal glial cells do not bridge gap. (D-F) Commissural fascicles, preoral brain tracts and
associated glial structures are established between brain hemispheres but paired circumesophageal connectives (D, asterisks) do not form
(compare Fig. 2G-I). (G-I) Ectopic midline descending pathways (G, arrow) seen in 1/5 of the mutants. Longitudinal axons fuse medially and
project aberrantly from brain to the ventral ganglia (compare Fig. 2G-I). (J) Aberrant ectopic midline pathway (arrows) projecting along
foregut-associated structures. (K) Preoral commissure (filled arrow) and preoral brain tracts (arrowheads) are present despite absence of paired
circumesophageal connectives (asterisks). (L) P[sim/lacZ] enhancer detector staining indicates that single-minded is expressed in the lower part
of the brain (arrow) located just anterior and dorsal to subesophageal ganglion. Some of the stained cells in this region are also labeled by the
glial cell-specific anti-repo antibody and may, thus, be longitudinal glial cells. Stages 12/3 (A,B,C), 14 (L), 16 (D-K). Scale bar, 10 µm.
Brain development in Drosophila 3859
regions. As in the ventral nerve cord (Jacobs and Goodman,
1989a), the longitudinal glial cells may constitute a preformed
pathway for pioneering axons.
The formation of the longitudinal glial cells linking the
cephalic and ventral CNS depends on the single-minded gene.
In the absence of this gene, a gap in the longitudinal array of
glia appears between the brain and the ventral nerve cord. This
gap is not bridged by longitudinal pioneers and, in consequence, the circumesophageal connectives fail to form
correctly. Since similar events occur in other spitz group genes,
it seems likely that complex molecular genetic interactions
among all of these regulatory genes are involved in controlling
the development of the longitudinal connective pathways.
The single-minded gene product, a transcription factor, is
thought to be a master developmental regulator of CNS midline
lineage (Crews et al., 1988; Thomas et al., 1988; Nambu et al.,
1990, 1991). In the ventral nerve cord of single-minded
mutants, specific midline cells do not differentiate properly. In
consequence, the commissures are lacking and the longitudinal
tracts collapse at the midline (Thomas et al., 1988; Klämbt et
al., 1991). The phenotype of single-minded mutants in the brain
is strikingly different. In single-minded mutants, a brain commissure is established and the longitudinal tracts within the
brain do not collapse. The expression pattern of single-minded
in the brain is also completely different compared to the ventral
nerve cord. We have no evidence for the expression of singleminded anywhere in the brain midline anterior to circumesophageal connectives. These findings suggest that the role of
single-minded as a master developmental regulator may be
limited to the midline of the ventral CNS and to those midline
structures that are involved in circumesophageal connective
formation. Other parts of the brain, including the anterior
midline at the site of brain commissure formation, do not appear
to be under the control of the single-minded gene. The developmental regulator genes that do control midline differentiation
in these major parts of the brain remain to be identified.
Similar features of neuronal development in the
brain and in the segmental ganglia
Although the Drosophila brain derives from a separate neurogenic region, differentiates into an enormously complex
structure and, in doing so, manifests a series of brain-specific
developmental processes, several features of its embryonic
development are reminiscent of the development of the more
simple segmental ganglia. Thus, in both cases: (i) glial cells
are involved in axogenesis; longitudinal glial cells prefigure
descending pathways and glial cells are involved in commissure formation (Jacobs and Goodman, 1989a; Klämbt and
Goodman, 1991); (ii) pioneer neurons establish a simple axon
scaffolding that is used by subsequently outgrowing neuronal
processes for pathfinding (Klämbt et al., 1991; Jacobs and
Goodman, 1989b; Grenningloh et al., 1991); (iii) molecularly
labelled axon pathways are formed during differentiation of the
axon scaffolding (e.g. Raper et al., 1984; Goodman et al.,
1984); Fasciclin II, for example, is expressed in a similar
regionalized manner by specific longitudinal pathways in brain
and ventral ganglia (Grenningloh et al., 1991).
Interestingly, all of the above mentioned features of
embryonic brain development in Drosophila are also found in
grasshopper brain development (Boyan et al., 1995a,b,c), suggesting the generality of these developmental processes in all
advanced insects. Furthermore, many of the developmental
processes that are involved in generating the fly brain are
involved in vertebrate brain development. For example, the
roles of glial cells in prefiguring axon pathways, the function
of pioneer neurons in establishing axon pathways and the
formation of a primary axon scaffolding are also features of
embryonic brain development in vertebrates, including
mammals (Steindler, 1993; Silver, 1993; Chitnis and Kuwada,
1990; Stainier and Gilbert, 1990; Wilson et al., 1990; Easter et
al., 1993).
The authors would like to thank Harald Vaessin for anti-pros
antibody, Daniel Halter for anti-repo antibody, Andrew Tomlinson for
RK2 antibody, Kalpana White for anti-elav antibody; Gerd Technau
for 2138 and Steve Crews for 2035 enhancer trap lines. We also thank
Gabriela Schenker, Markus Dürrenberger, Kurt Ballmer-Hofer and
Jörg Hagmann for help with the confocal systems and Peter Wehrli
for technical assistance. This work was supported by the Swiss NSF.
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(Accepted 27 July 1995)