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2121
Development 129, 2121-2128 (2002)
Printed in Great Britain © The Company of Biologists Limited 2002
DEV2815
Inductive patterning of the embryonic brain in Drosophila
Damon T. Page
MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK
Accepted 11 February 2002
SUMMARY
In vertebrates (deuterostomes), brain patterning
depends on signals from adjacent tissues. For example,
holoprosencephaly, the most common brain anomaly in
humans, results from defects in signaling between the
embryonic prechordal plate (consisting of the dorsal
foregut endoderm and mesoderm) and the brain. I have
examined whether a similar mechanism of brain
development occurs in the protostome Drosophila, and
find that the foregut and mesoderm act to pattern the fly
embryonic brain. When the foregut and mesoderm of
Drosophila are ablated, brain patterning is disrupted. The
loss of Hedgehog expressed in the foregut appears to
mediate this effect, as it does in vertebrates. One
INTRODUCTION
Induction of neural tissue across tissue layers is a mechanism
of brain development that has been extensively studied in
deuterostomes (vertebrates). Tissues adjacent to the brain, such
as the foregut endoderm and mesoderm (which constitute the
prechordal plate) send developmental instructions to the brain
(Ang and Rossant, 1993; Camus et al., 2000; Dale et al., 1997;
Pera and Kessel, 1997; Shimamura and Rubenstein, 1997).
Disruption of signaling between the prechordal plate and brain
results in holoprosencephaly (HPE), a brain birth defect that
includes a loss of ventral brain structures and incomplete
separation of the forebrain into right and left hemispheres, and
has a prevalence in humans of 1:250 in embryogenesis and
1:10,000 to 1:20,000 among live births (Chiang et al., 1996;
Muenke and Beachy, 2000). Several of the molecular cues that
are involved in the induction of pattern in the vertebrate brain
have been identified and include members of the Hedgehog
signaling pathway (Dale et al., 1997; Pera and Kessel, 1997;
Shimamura and Rubenstein, 1997).
In contrast to the situation in deuterostomes, the induction
of brain patterning in protostomes has been little studied. With
the fruit fly Drosophila, there are a variety of genetic tools now
available that allow us to address the question of whether the
protostome brain is patterned by induction. By systematically
removing tissues adjacent to the embryonic Drosophila brain,
I have found that the foregut and the mesoderm are involved
in establishing and refining brain pattern by influencing brain
size and apoptosis. Furthermore, I have investigated the role
of the Hedgehog signaling pathway in Drosophila brain
mechanism whereby these defects occur is a disruption
of normal apoptosis in the brain. These data argue
that the last common ancestor of protostomes and
deuterostomes had a prototype of the brains present in
modern animals, and also suggest that the foregut and
mesoderm contributed to the patterning of this ‘protobrain’. They also argue that the foreguts of protostomes
and deuterostomes, which have traditionally been
assigned to different germ layers, are actually
homologous.
Key words: Drosophila, Brain, Patterning, Foregut, Mesoderm
patterning. The results argue that the protostome embryonic
brain pattern is influenced by induction as it is in
deuterostomes, and that Hedgehog signaling has a conserved
role in this effect among these groups.
MATERIALS AND METHODS
Drosophila stocks
As a wild-type stock, Oregon-R flies were used. The following mutant
strains were also used: forkhead6, hedgehogE23, hedgehog-lacZ
(hedgehogP30), hedgehog-GAL4 (unpublished line kindly provided by
M. Calleja and G. Morata), Scer/GAL4how–24B (mesodermal GAL4
driver), UAS-ricin (Rcom\RAScer\UAS.cBa), patched-GAL4 (Scer\
GAL4patched–559.1), UAS-tau-GFP (Avic\GFPScer\UAS.T:Btau\MAPT),
1407-GAL4 (Sweeney et al., 1995), UAS-patched∆loop2 (Briscoe et al.,
2001) and tinEC40. For analyzing gene expression, GAL4 lines were
crossed with UAS-tau-GFP lines, thereby forming a microtubulebound GFP reporter. For identifying mutant embryos, balancers
expressing GFP or β-galactosidase were used.
Laser ablations
Dechorionated cellularized-blastula stage embryos were lined up on
glass slides after staging on a dissecting microscope in Voltalef 3S oil.
All staging of embryos was carried out using standard guidelines
(Campos-Ortega and Hartenstein, 1997). The cells of the foregut
anlage were ablated using a Laser Sciences VSL-337ND-S nitrogen
laser. Cells were exposed to 1-second bursts at 10 Hz, and the area
around where the laser was focused was checked for blebbing, thereby
indicating cell death. I destroyed what appeared to be most of the cells
within the foregut anlage (see Hartenstein and Campos-Ortega, 1985).
Subsequent to laser treatment, embryos were placed at 25°C until they
2122 D. T. Page
reached embryonic stage (ES) 15, as determined using a dissecting
microscope. Approximately 60% of the laser-treated embryos clearly
lacked a foregut; these were the embryos that were used for
subsequent experiments.
Immunohistochemistry and microscopy
Embryos were collected, fixed and immunostained according to
standard procedures (Patel, 1994). The following primary antibodies
were used: BP102 [mouse, 1:200, Developmental Studies Hybridoma
Bank (DSHB)], anti-Fasciclin II (Bastiani et al., 1987) (mouse, 1:20),
anti-Elav (rat, 1:100, DSHB), anti-Repo (Halter et al., 1995) (rabbit,
1:750), anti-GFP (rabbit, 1:1000, Molecular Probes), anti-βgalactosidase (rabbit, 1:1500, Cappel), anti-Hunchback (Struhl et al.,
1989) (rat, 1:2000), anti-Prospero (mouse, 1:100, DSHB), antiFasciclin III (mouse, 1:100, DSHB) and anti-Hedgehog (Tabata and
Kornberg, 1994) (rabbit, 1:1500). For light microscopy, biotinconjugated secondary antibodies were used (1:200, Jackson).
Embryos were viewed and photographed on a Zeiss Axiophot
microscope. Secondary antibodies used for laser confocal microscopy
were Texas Red-conjugated anti-mouse (1:200, Jackson), FITCconjugated anti-rabbit (1:200, Jackson), and Cy5-conjugated anti-Rat
(1:200, Jackson). Images were collected on BioRad MRC 1024 or
Radiance confocal microscopes, and were processed using Adobe
PhotoShop and Illustrator.
Acridine Orange staining
Staining for apoptosis using Acridine Orange was done according to
standard procedure (Abrams et al., 1993). Briefly, embryos were
dechorionated, then shaken for 5 minutes in a 1:1 mixture of 5 µg/ml
Acridine Orange in PBS and heptane, and then mounted in Voltalev
10S oil. Embryos at late ES13 were examined on a BioRad Radiance
confocal microscope for staining at the level of the pre-oral brain
commissure at the dorsal midline of b1.
Cell counting
To quantify brain phenotypes, stained embryos were examined in the
following ways:
(1) To estimate the area occupied by neuronal nuclei and the
number of glia in b1, confocal sections were collected from at the
dorsal most region of the ES15 brain, progressing ventrally through
the brain at 2.5 µm increments for 35 µm (which is a level slightly
ventral to the preoral brain commissure in the wild-type brain), using
a 60× oil immersion objective on a BioRad Radiance confocal
microscope. These sections were then projected into one image using
BioRad software. The number of glia present was counted directly
and the area occupied by neurons was measured by tracing the outline
of the anti-Elav marked brain using Adobe PhotoShop and obtaining
the total pixel area within the outlined image. The pixel area was then
multiplied by the conversion factor 0.154 µm2/pixel (this value was
obtained by dividing the area of a scanning box provided by the
BioRad confocal software by the number of pixels in such a box as
measured in Adobe PhotoShop) to obtain an estimate of the area
occupied by neuronal nuclei.
(2) To estimate the number of glia and the area occupied by neurons
in b2-S3, a procedure similar to that used in b1 was used except that
optical sectioning began at the lateral edge of the foregut (or 5µm
from the midline when the foregut was ablated) and progressed
medially at 2.5 µm increments for 25 µm. Images were then projected
and analyzed as described above. The boundaries of b2-S3 were
estimated based on the positions of commissures.
(3) For statistical analysis of the difference in cell counts between
wild-type and mutant brains, Student’s t-test was used (Sokal and
Rohlf, 1997).
It is important to note that the data provided are not intended to
represent the absolute area or number of cells in the brains of embryos,
but rather a relative comparison of brain size and glia number between
groups of embryos as measured using consistent techniques.
RESULTS
Ablation of the foregut results in brain patterning
defects
The embryonic brain of Drosophila develops in close
association with the foregut ectoderm, as well as the visceral
and procephalic mesoderm (Fig. 1A,B). Thus, these tissues are
candidates for being involved in inductive patterning of the
brain. The strategy employed was to remove these tissues
systematically and see what effect this had on brain
morphogenesis. The wild-type embryonic brain of Drosophila
has a stereotyped arrangement of cells and axon tracts (Nassif
et al., 1998a) (Fig. 1C,D), defects that can be readily detected
using molecular markers. The structure consists of six
segments: b1-b3 lie dorsal and lateral to the foregut and S1-S3
are ventrolateral and ventral to the foregut. All segments
contain paired longitudinal connectives, which are called
circumesophageal connectives lateral to the foregut. The
preoral brain commissure is in b1, and connects the right and
left hemispheres of the brain, which are otherwise separated at
the midline, and the frontal commissure originates in b2 and
extends into b1, where it joins with neuronal soma and glia to
form the frontal ganglion. b3 and S1 have one commissure
each, and S2 and S3 have two commissures each.
Does the removal of the foregut affect the development of
the brain? To answer this question, I ablated the anlage of the
embryonic Drosophila foregut using a laser. When the foregut
did not invaginate, the following defects were seen in 90% of
embryos (n=20): the pattern of neurons was disrupted so that
the right and left hemispheres of the brain were either fused at
the midline or separated by an abnormally small space (≤4µm),
because of excess cells in this region; the area occupied by
neuronal nuclei was decreased; the preoral brain commissure
did not defasciculate normally, so that it appeared thickened;
and the frontal commissure and ganglion were missing (Fig.
2A). In b2-S3, there was an apparent reduction in the area
occupied by neuronal nuclei and a decrease in the number of
glia (Fig. 2B). In addition, in 50% of these embryos, the
longitudinal connectives in b2-S3 were disrupted.
Genetic ablation experiments agreed with these
observations. Foregut formation is defective in embryos
lacking function of the transcription factor Forkhead (Jürgens
and Weigel, 1988). Seventy percent of Forkhead loss-offunction mutant embryos examined (n=20) exhibited defects in
b1 that resembled the phenotype seen in embryos with laser
ablated foreguts. This included an unusually small space
between the brain hemispheres, abnormal defasciculation of
the preoral brain commissure, and a decrease in the area
occupied by neuronal nuclei in b1 and b2-S3, as well as a
reduction in the number of glia in b2-S3 (Fig. 2C,D; Table 1).
Examining the expression pattern of forkhead using an antiForkhead antibody showed that, while Forkhead is expressed
in the central and lateral parts of the b1 hemispheres, it is not
expressed at the dorsomedial edges of b1 up to ES13 (Fig. 2E).
This suggested that the phenotype seen in the dorsal midline
in Forkhead null embryos was not due to a loss of Forkhead
function in this region. Interestingly, the vertebrate homolog of
forkhead, Hnf3b, is expressed in the prechordal plate and brain,
and embryos lacking function of this gene have severe defects
in foregut morphogenesis and brain patterning (Ang and
Rossant, 1994).
Brain patterning 2123
Fig. 1. Drosophila embryonic brain anatomy. (A) DIC lateral view of
an ES15 whole wild-type embryo stained with BP102, an antibody
that recognizes CNS axons. The brain, which forms around the
foregut, is outlined by the box. (B) Schematic representations of the
brain and adjacent tissues at ES12 and late ES13. Mesoderm is in
green, foregut ectoderm is in red, neuroblasts are in blue and neurons
are in purple. (C) Dorsal view of the embryonic brain. b1 is the only
segment visible in this view, and shows distinct separation into left
and right hemispheres. (D) Lateral view of the embryonic brain, with
b1-S3 visible. In this and the following figure panel, the outline of
the foregut is shown by broken lines. Schemes represent a simplified
arrangement of brain structures. CC, corpora cardiaca; CEC,
circumesophageal connective; D/T, deutocerebral/tritocerebral
neuron cluster; FC, frontal commissure; FG, frontal ganglion; PBC,
preoral brain commissure; VNC, ventral nerve cord.
do form foregut ectoderm (D. T. P., unpublished) (Bodmer et
al., 1990; Azpiazu and Frasch, 1993). In 65% of Tinman lossof-function embryos (n=20) there were excess cells at the
dorsal midline of b1, the area occupied by neuronal nuclei was
increased when compared with wild type in this region of the
brain, and the preoral brain commissure was abnormally thin
(Fig. 3A,B; Table 1).
To verify that the removal of the mesoderm had an effect on
brain development, a pan-mesodermal driver (Brand and
Perrimon, 1993) was used to express the toxin Ricin (Hidalgo
et al., 1995), thereby specifically ablating mesodermal cells.
The expression of this line in the anlage of the foregut
mesoderm was verified using a GFP reporter (data not shown).
In 45% of embryos expressing Ricin in the mesoderm (n=20),
the brain phenotype resembled that of the tinman loss-offunction embryos in that there were excess cells at the dorsal
midline and there was thinning of the preoral brain commissure
(Fig. 3C,D); however, the frontal commissure and ganglion
were also absent.
Disruption of mesoderm formation results in brain
patterning defects
As the Drosophila foregut invaginates, it normally becomes
ensheathed by visceral mesoderm. Thus, when the foregut is
ablated, visceral mesoderm is displaced from its normal
position adjacent to the brain. How much does the loss of
mesoderm contribute to the brain phenotype seen in foregut
ablated animals? I examined embryos lacking function of the
NK-2 class transcription factor Tinman, which have defects in
forming mesoderm around the foregut, as examined using
mesodermal markers for such as Fasciclin III expression, but
Ablation of the foregut and mesoderm results in
changes in the pattern of brain apoptosis at the
dorsal midline
Why were there excess cells at the dorsal midline in foregut- and
mesoderm-ablated embryos? During normal brain development,
more neurons are born than will be present in the adult brain and
apoptosis eliminates the excess cells (for a review, see Hutchins
and Barger, 1998). Defects in apoptosis could contribute to the
observed defects in brain patterning by failing to remove excess
cells. To see if apoptosis was perturbed when the foregut and
mesoderm were ablated, Acridine Orange staining, which labels
apoptotic cells (Abrams et al., 1993), was carried out. In
forkhead loss-of-function embryos, the pattern of apoptosis in
the brain at the level of the preoral brain commissure was clearly
different from wild type at late ES13. In the wild-type b1
neuromere, there were groups of apoptotic cells at the
dorsomedial edges of the hemispheres (Fig. 4A). This correlates
with previous observations regarding the expression of the
apoptosis regulatory protein Reaper (Nassif et al., 1998b). In
forkhead loss-of function embryos, there was a clear reduction
in the number of these cells (Fig. 4B). Examination of tinman
loss-of-function embryos showed that removal of mesoderm
results in a similar reduction in the number of apoptotic cells at
the dorsal midline (Fig. 4C), thus suggesting that the mesoderm
and possibly the foregut have an influence on the normal pattern
of apoptosis in brain development.
2124 D. T. Page
Fig. 2. Ablation of the foregut results in brain patterning defects. For
fluorescent images, staining is as follows: α-Fasciclin II (which
labels a subset of axons) in red; α-Elav (which labels neuronal
nuclei) in blue; and α-Repo (which labels peripheral glial nuclei) in
green. All embryos at ES15. (A) Dorsal view of embryo with laserablated foregut, showing a missing frontal commissure and ganglion,
a fusion of brain hemispheres at the midline (arrowhead), and
abnormal defasciculation in the preoral brain commissure in b1
(arrow). (B) Lateral view of an embryo with a laser-ablated foregut.
(C,D) Dorsal and lateral views, respectively, of forkhead (fkh) lossof-function mutant embryos. Brain defects resemble those seen in
laser-ablated embryos (arrows and arrowhead as in A,B).
(E) Expression pattern of Forkhead in b1 at ES13, as visualized using
anti-Forkhead antibody staining (brown). Forkhead appears to be
expressed in the central and lateral regions of the b1 hemispheres
(arrowhead), but not in the dorsomedial edges of b1 up to ES13. The
staining at the midline seen in this image is from Forkhead
expression in the foregut (arrow), which lies in a focal plane
immediately adjacent to the b1 midline.
development. Null mutations in Drosophila Hedgehog resulted
in a phenotype that strongly resembled the one seen in the
foregut ablation experiments in 70% of embryos (n=20). In b1,
the right and left hemispheres of the brain were joined at the
midline or separated by an abnormally small space because of
excess cells in this region, and the preoral brain commissure
showed abnormal defasciculation. In addition, in b1 the frontal
commissure was missing, and there was a significant decrease
in the area occupied by neuronal nuclei and the number of glia
(Fig. 5A; Table 1). In b2-S3, the longitudinal connectives were
disrupted, and the area occupied by neuronal nuclei and the
number of glia was significantly reduced (Fig. 5B; Table 1),
and the number of Fasciclin II-expressing neurons was
reduced.
Loss of Hedgehog function results in brain
patterning defects
The results of foregut and mesoderm ablation experiments
strongly suggest that the brain is patterned by induction from
these tissues. Did ablation of these tissues remove inductive
signals required for normal brain development? What
molecular signals could be mediating this effect? In
vertebrates, Hedgehog signaling originating from the
prechordal plate functions in forebrain patterning. Thus, the
Hedgehog pathway in Drosophila seemed a good place to
begin to look for inductive signals involved in brain
hedgehog and patched are expressed in the foregut
and the brain
To see where the Hedgehog signal originated from, I analyzed
hedgehog expression in the brain. Using a GFP reporter of
hedgehog expression [hedgehog-GAL4 line kindly provided
by M. Calleja and G. Morata, generated by the method
outlined by Calleja et al. (Calleja et al., 1996)], I found that
there was a source of Hedgehog in the foregut, i.e.
immediately adjacent to the brain (Fig. 5C,D). This
expression was present as the stomodeaum invaginated
posterior through the region of brain neuroblasts, and
continued through ES13, at which time the foregut expression
Table 1. Penetrance and expressivity of phenotypes
Average cell counts in +++ embryos (s.d.)
Severity of defects
(% penetrance)
b1
Genotype
+
++
+++
Neurons*
(ES 15)
Wild type
forkhead6
tinmanEC40
hedgehogE23
−
5
15
0
−
25
20
30
−
70
65
70
7142 (328)
5453 (842)
8810 (595)
6323 (422)
b2-S3
Glia
(ES 15)
Neurons
(ES 15)
Glia
(ES 15)
73 (5)
76 (5)
71 (3)
44 (7)
6185 (811)
4333 (591)
5987 (305)
4143 (698)
47 (4)
39 (2)
46 (5)
35 (6)
*The value given for neurons is the average area (µm2) occupied by Elav-expressing nuclei. Statistically significant deviations from wild type (as determined
using a t-test) are indicated by italicized numbers (n=20 in all cases). Severity of defects was rated as follows: +++, embryos showed the complete phenotype
described for the mutation in the text and in Table 2; ++, embryos showed some of this phenotype; +, embryos did not show this phenotype.
Brain patterning 2125
Loss of Hedgehog function results in changes in
apoptosis
To see if changes in apoptosis underlie the b1 dorsal midline
defects seen in Hedgehog null embryos, Acridine Orange
staining was carried out. This showed that there was a decrease
in the number of apoptotic cells at the dorsal midline of the
brain at late ES13 (Fig. 5H), suggesting that the defects in this
region in Hedgehog null embryos were due to a disruption in
the normal pattern of apoptosis.
Blocking Hedgehog signaling in neural cells
influences brain size, but not apoptosis at the dorsal
midline
To see if Hedgehog signaling is directly required in brain cells,
I expressed a form of Patched that is insensitive to Hedgehog
(Briscoe et al., 2001) specifically in neural tissues using the
1407-GAL4 line (Sweeney et al., 1995). In these embryos, the
size of the brain was decreased; however, excess cells did not
appear to be present at the dorsal midline (Fig. 5I). This
suggests that Hedgehog signaling has a direct function in
influencing brain size; however, its function in inducing
apoptosis in the dorsal midline of b1 may be indirect.
Fig. 3. Brain defects resulting from removal of mesoderm. Staining
as described for Fig. 2. All embryos at ES15. (A,B) Dorsal and
lateral views, respectively, of tinman (tin) mutant embryos. b1 shows
a thinning of the preoral brain commissure (arrow) and abnormal
closeness of the brain hemispheres (arrowhead). (C,D) Dorsal and
lateral views, respectively, of embryos in which the mesoderm was
ablated using a pan-mesodermal GAL4 driver to express Ricin toxin.
These embryos also show a thinning of the preoral brain commissure
(arrow) and a fusion of the brain hemispheres at the dorsal midline
(arrowhead). In addition, these embryos lack the frontal commissure
and ganglion.
was lost immediately adjacent to the brain. I also observed
that hedgehog was expressed in a segmental pattern in the
brain itself; this expression pattern is in agreement with
previous reports (Lee et al., 1992; Taylor et al., 1993). A
similar expression pattern was observed in a hedgehog-lacZ
line and by staining with anti-Hedgehog antibody (data not
shown). Importantly, patched, a gene encoding a putative
receptor for Hedgehog (for a review, see Ingham, 1998), was
found to be upregulated in brain cells surrounding the foregut
Hedgehog source, including neurons, glia and precursors of
these cells (Fig. 5E-G). In addition, patched was upregulated
in the visceral mesoderm after it ensheathed the foregut at
ES13.
Influence of the foregut and mesoderm on the
formation of neural precursors
When do the inductive influences of the foregut and mesoderm
exert their effect on brain development? To answer this
question, I stained embryos lacking foregut (Forkhead null) or
mesoderm (Tinman null) with anti-Hunchback antibody, which
stains neuroblasts in the brain (Kambadur et al., 1998). At
ES10, after the brain neuroblasts have started to delaminate,
the pattern of Hunchback-expressing neuroblasts appeared to
be the same as wild type in all these embryos (Fig. 6A-C). This
suggested that the initial formation of brain neuroblasts was
normal in the absence of a foregut or mesoderm. Next, I
examined the pattern of ganglionic mother cells (GMCs) using
the marker anti-Prospero (Doe et al., 1991). In this case, the
number of brain GMCs in embryos lacking a foregut was
clearly decreased at ES11 when compared with wild type (Fig.
6D,E). By contrast, in embryos lacking mesoderm, the pattern
of GMCs did not appear to be affected (Fig. 6F). This
suggested that there are two inductive events: an earlier event
that is mediated by signals originating from the foregut, in
which neural precursor cells of the brain receive a signal that
influences the formation of GMCs. That Hedgehog may be
involved in this signaling event is suggested by the fact that in
a Hedgehog null background, the number of Prosperoexpressing GMCs is reduced (Fig. 6G). The later event appears
Fig. 4. Changes in apoptosis associated with brain
patterning defects. All embryos are at late ES13, and are
view dorsally at the level of the preoral brain
commissure. Acridine Orange staining for apoptotic cells
(green) is superimposed onto a DIC image taken
simultaneously on a confocal microscope. The outline of
the brain is indicated by white broken lines for reference.
(A) The wild-type brain shows groups of apoptotic cells
at the dorsomedial edges of the brain hemispheres
(arrows). (B) In fkh loss-of-function embryos, there is a
reduction in the number of apoptotic cells at the dorsal
midline (arrow). (C) A similar reduction in the number of apoptotic cells is seen in tin loss-of-function embryos.
2126 D. T. Page
Fig. 5. Involvement of Hedgehog signaling in patterning the brain. All embryos at stage 15
unless otherwise noted. (A,B,I) α-Fasciclin II is in red, α-Elav is in blue and α-Repo is in
green. (C-G) α-GFP is in green and BP102 is in red. (A,B) Dorsal and lateral views,
respectively, of a hedgehog (hh) null mutant embryo. (A) b1 with the phenotype of a missing
frontal commissure, fused hemispheres (arrowhead), and a abnormally defasciculated preoral
brain commissure (arrow). (B) The axon tracts in b2-S3 are thinned or broken, and there is a
decrease in the number of glia and a disruption in the pattern of neurons in this region.
(C,D) Lateral and frontal views, respectively, of ES13 hedgehog-GAL4, UAS-tau-GFP
embryos, showing expression of hedgehog in a segmentally repeated pattern in b1-S3
(arrowhead), and in the foregut adjacent to b1 (arrow). (E-G) Lateral, frontal and dorsal views,
respectively, of ES13 patched-GAL4, UAS-tau-GFP embryos. patched (ptc) appears to be
upregulated in a segmental pattern complimentary to that of hedgehog in the brain (arrowhead), and in a group of brain cells surrounding the
foregut (arrow). (H) Late ES13 hedgehog null embryo strained with Acridine Orange for apoptosis. There is a reduction in the number of
apoptotic cells at the dorsal midline in these embryos (arrow) when compared with wild type. (I) Dorsal view of embryo in which expression of
UAS-patched∆loop2 was driven using the nervous system specific line 1407-GAL4. The size of the brain in these embryos is decreased when
compared with wild type, but there does not appear to be excess cells at the dorsal midline of b1. Note that the frontal commissure is present.
to be mediated by the visceral mesoderm and seems to
influence the survival of late GMCs or postmitotic cells.
DISCUSSION
Evidence that the foregut and mesoderm induce
pattern in the brain
When the foregut and mesoderm are ablated, specific sets of
defects occur in the brain (summarized in Table 2), thus
suggesting that these tissues induce patterning of the brain.
Ablation of the foregut results in a smaller brain, and disruption
of mesoderm formation enlarges b1. These results may be
explained as follows: the foregut influences the size of the brain
and the mesoderm influences apoptosis at the dorsal midline of
b1. Thus, ablation of the foregut, which also removes the
visceral mesoderm, results in a smaller brain size, in spite of
excess cells resulting from apoptosis defects. By contrast,
ablation of the mesoderm alone results in a slightly larger brain
because of excess cells are present at the dorsal midline, but
brain size is not reduced because the foregut is present.
Considering that the preoral brain commissure did not
appear to defasciculate normally in foregut-ablated embryos,
and appeared thinned in mesoderm-ablated embryos, the
foregut and mesoderm may have additional distinct roles in
influencing preoral brain commissure development. The cause
for the differential effects of removing these tissues on the
formation of this commissure is not entirely clear based on
these experiments. However, these phenotypes may be related
to the fact that removal of either tissue affects the arrangement
of glia associated with this commissure; rearrangement of glia
has been shown to influence commissure formation elsewhere
in the Drosophila nervous system.
Table 2. Effects on brain development of ablation of
adjacent tissues
Tissue
Effects of ablation on gross brain morphology
Foregut
Fusion or abnormal closeness of brain hemispheres at
midline in b1
Decreased area occupied by neuronal nuclei in b1
Abnormal defasciculation of the pre-oral brain
commissure
Loss of frontal commissure
Decreased area occupied by neuronal nuclei in b2-S3
Reduction in number of glia in b2-S3
Mesoderm
Fusion or abnormal closeness of brain hemispheres at
midline in b1
Increase in the area occupied by neuronal nuclei in b1
Thinning of pre-oral brain commissure
Brain patterning 2127
Fig. 6. Influence of the foregut and
mesoderm on the formation of neural
precursors. All embryos viewed laterally.
(A-C) ES10 wild type, forkhead loss-offunction and tinman loss-of-function
embryos, respectively. The pattern of
neuroblasts in b1 was visualized using
anti-Hunchback antibody staining (brown)
and did not show obvious differences
between these groups. (D-F) ES11 wild-type, forkhead loss-offunction, and tinman loss-of-function embryos, respectively. GMCs
in the brain were stained using anti-Prospero antibody (brown).
(E) In the absence of Forkhead function, the number of GMCs in
b1 appeared to be reduced (arrow). (F) A similar reduction in
GMCs was not seen in Tinman null embryos. (H) ES11 Hedgehog
null embryo, showing a reduction in the number of GMCs in b1
similar to that seen in Forkhead null embryos (arrow).
Involvement of Hedgehog in the induction of brain
pattern
When function of Hedgehog is lost, brain patterning defects
occur that resemble those seen in foregut ablated embryos,
including a fusion of brain hemispheres in b1, a reduction in
the size of the brain and abnormal defasciculation of
the preoral brain commissure. Importantly, Hedgehog is
expressed in the foregut adjacent to the brain, and patched,
which encodes a putative receptor for Hedgehog, is expressed
in brain cells surrounding the foregut. Loss of Hedgehog
function causes a reduction in brain size that resembles that
seen when the foregut is ablated, thus suggesting that the
foregut source of Hedgehog influences brain size. Hedgehog
loss of function also results in a disruption of apoptosis that
resembles what is seen when the mesoderm is removed;
however, hedgehog does not appear to be expressed in the
visceral mesoderm surrounding the foregut. This suggests that
Hedgehog from the foregut may be received by the mesoderm
(where patched is upregulated), which then responds by
producing another signal that influences apoptosis at the
dorsal midline. This hypothesis is supported by the
observation that inhibiting Hedgehog signaling specifically in
neural tissue using a mutant form of Patched results in a
decrease in b1 size, but not in an excess of cells, at the dorsal
midline.
Significance for understanding disease and
evolution
Considering the reduction in the number of brain cells and the
joining of the brain hemispheres, the foregut ablation brain
phenotype resembled aspects of human HPE. Importantly, in
the vertebrate nervous system, Sonic hedgehog is involved in
the formation of oligodendrocytes and motoneurons, and
in regulating apoptosis (Ericson et al., 1995; Nery et al.,
2001). Disruption of Hedgehog signaling causes HPE;
correspondingly, in Drosophila Hedgehog mutants, a HPE-like
phenotype occurs, including a reduction in the number of glia
and Fasciclin II expressing motoneurons. The recapitulation of
aspects of the human HPE phenotype in Drosophila – i.e. the
loss of brain cells and the defects in hemispheric separation –
means that fly embryos might have use for understanding some
mechanisms of this disease.
Furthermore, this work demonstrates that brain patterning
via induction by the foregut and mesoderm appears to be
a mechanism that is shared between protostomes and
deuterostomes. This finding supports the hypothesis that the
ground plan for the brain was established in the last common
ancestor of bilaterally symmetric animals (Arendt and NublerJung, 1996; Arendt and Nubler-Jung, 1999; Dohrn, 1875;
Leydig, 1864). This also suggests how the brain of the muchdebated last common ancestor of Bilateria may have
developed. I hypothesize that this animal had a brain that
formed in close association with the foregut, and molecules
such as Hedgehog expressed in the foregut patterned this brain.
As regards the origins of the brain in evolution, if the foregut
is assumed to be more ancient than the brain, then the
possibility arises that the ventral neural mass of a bilaterian
ancestor that lacked a brain could have expanded dorsally,
using the foregut (which would probably have already been
expressing patterning molecules such as Hedgehog) as a
scaffolding, thus forming a brain.
The apparent homology between the foreguts of protostomes
and deuterostomes raises a problem in nomenclature: the
foregut of protostomes is considered ectodermal in origin,
while the foregut of deuterostomes is considered endodermal.
However, there appears to be homology in function,
blastodermal fate map position (Arendt and Nubler-Jung,
1997), gene expression (Arendt et al., 2001) and induction
between tissues derived from what have been traditionally
regarded as distinct germ layers. Perhaps the assignment of the
protostome foregut to the ectoderm and the deuterostome
foregut to the endoderm should be reconsidered, as there are
no absolute criteria for these assignments.
Many thanks to Peter Lawrence for providing the laboratory space
as well as the support that made this research possible. For critical
readings of this manuscript at various stages of completion, I thank
Peter Lawrence, Katharina Nübler-Jung, John L. R. Rubenstein and
Klaus Sander. To José Casal and Marta Llimargas, I am grateful for
stocks and helpful discussions and suggestions. My gratitude goes to
Maximiliano Suster-Rathjens, Gary Struhl, Manfred Frasch, Alicia
Hidalgo, Acaimo González-Reyes and the Umeå and Bloomington
Stock Centers for stocks, and to Andrew Travers, Corey Goodman,
Steve Crews, Paul Macdonald, Tetsuya Tabata, Jorge Bolivar and the
DSHB for antibodies. I am also very grateful to Manuel Calleja and
Gines Morata for letting me use the unpublished hedgehog-GAL4 line
that they generated.
2128 D. T. Page
REFERENCES
Abrams, J. M., White, K., Fessler, L. I. and Steller, H. (1993). Programmed
cell death during Drosophila embryogenesis. Development 117, 29-43.
Ang, S. L. and Rossant, J. (1993). Anterior mesendoderm induces mouse
Engrailed genes in explant cultures. Development 118, 139-149.
Ang, S. L. and Rossant, J. (1994). HNF-3 beta is essential for node and
notochord formation in mouse development. Cell 78, 561-574.
Arendt, D. and Nubler-Jung, K. (1996). Common ground plans in early brain
development in mice and flies. BioEssays 18, 255-259.
Arendt, D. and Nubler-Jung, K. (1997). Dorsal or ventral: similarities in fate
maps and gastrulation patterns in annelids, arthropods and chordates. Mech.
Dev. 61, 7-21.
Arendt, D. and Nubler-Jung, K. (1999). Comparison of early nerve cord
development in insects and vertebrates. Development 126, 2309-2325.
Arendt, D., Technau, U. and Wittbrodt, J. (2001). Evolution of the bilaterian
foregut. Nature 409, 81-85.
Azpiazu, N. and Frasch, M. (1993). tinman and bagpipe: two homeo box
genes that determine cell fates in the dorsal mesoderm of Drosophila. Genes
Dev. 7, 1325-1340.
Bastiani, M. J., Harrelson, A. L., Snow, P. M. and Goodman, C. S. (1987).
Expression of fasciclin I and II glycoproteins on subsets of axon pathways
during neuronal development in the grasshopper. Cell 48, 745-755.
Bodmer, R., Jan, L. Y. and Jan, Y. N. (1990). A new homeobox-containing
gene, msh-2, is transiently expressed early during mesoderm formation of
Drosophila. Development 110, 661-669.
Brand, A. H. and Perrimon, N. (1993). Targeted gene expression as a means
of altering cell fates and generating dominant phenotypes. Development 118,
401-415.
Briscoe, J., Chen, Y., Jessell, T. M. and Struhl, G. (2001). A hedgehoginsensitive form of patched provides evidence for direct long-range
morphogen activity of sonic hedgehog in the neural tube. Mol. Cell 7, 12791291.
Calleja, M., Moreno, E., Pelaz, S. and Morata, G. (1996). Visualization of
gene expression in living adult Drosophila. Science 274, 252-255.
Campos-Ortega, J. and Hartenstein, V. (1997). The Embryonic Development
of Drosophila melanogaster. Berlin, Heidelberg: Springer.
Camus, A., Davidson, B. P., Billiards, S., Khoo, P., Rivera-Perez, J. A.,
Wakamiya, M., Behringer, R. R. and Tam, P. P. (2000). The
morphogenetic role of midline mesendoderm and ectoderm in the
development of the forebrain and the midbrain of the mouse embryo.
Development 127, 1799-1813.
Chiang, C., Litingtung, Y., Lee, E., Young, K. E., Corden, J. L., Westphal,
H. and Beachy, P. A. (1996). Cyclopia and defective axial patterning in
mice lacking Sonic hedgehog gene function. Nature 383, 407-413.
Dale, J. K., Vesque, C., Lints, T. J., Sampath, T. K., Furley, A., Dodd, J.
and Placzek, M. (1997). Cooperation of BMP7 and SHH in the induction
of forebrain ventral midline cells by prechordal mesoderm. Cell 90, 257269.
Doe, C. Q., Chu-LaGraff, Q., Wright, D. M. and Scott, M. P. (1991). The
prospero gene specifies cell fates in the Drosophila central nervous system.
Cell 65, 451-464.
Dohrn, A. (1875). Der Ursprung der Wirbelthiere und das Princip des
Functionswechsels. Leipzig: Verlag von Wilhelm Engelmann.
Ericson, J., Muhr, J., Placzek, M., Lints, T., Jessell, T. M. and Edlund, T.
(1995). Sonic hedgehog induces the differentiation of ventral forebrain
neurons: a common signal for ventral patterning within the neural tube. Cell
81, 747-756.
Halter, D. A., Urban, J., Rickert, C., Ner, S. S., Ito, K., Travers, A. A. and
Technau, G. M. (1995). The homeobox gene repo is required for the
differentiation and maintenance of glia function in the embryonic nervous
system of Drosophila melanogaster. Development 121, 317-332.
Hartenstein, V. and Campos-Ortega, J. A. (1985). Fate-mapping in wildtype Drosophila melanogaster. I. The spatio-temporal pattern of embryonic
cell divisions. Roux’s Arch. Dev. Biol. 194, 181-195.
Hidalgo, A., Urban, J. and Brand, A. H. (1995). Targeted ablation of glia
disrupts axon tract formation in the Drosophila CNS. Development 121,
3703-3712.
Hutchins, J. B. and Barger, S. W. (1998). Why neurons die: cell death in the
nervous system. Anat. Rec. 253, 79-90.
Ingham, P. W. (1998). Transducing Hedgehog: the story so far. EMBO J. 17,
3505-3511.
Jürgens, W. and Weigel, D. (1988). Terminal versus segmental development
in the Drosophila embryo: the role of the homeotic gene fork head. Roux’s
Arch. Dev. Biol. 197, 345-354.
Kambadur, R., Koizumi, K., Stivers, C., Nagle, J., Poole, S. J. and
Odenwald, W. F. (1998). Regulation of POU genes by castor and
hunchback establishes layered compartments in the Drosophila CNS. Genes
Dev. 12, 246-260.
Lee, J. J., von Kessler, D. P., Parks, S. and Beachy, P. A. (1992). Secretion
and localized transcription suggest a role in positional signaling for products
of the segmentation gene hedgehog. Cell 71, 33-50.
Leydig, F. (1864). Vom Bau des thierischen Körpers. Tübingen: Laupp and
Siebeck.
Muenke, M. and Beachy, P. A. (2000). Genetics of ventral forebrain
development and holoprosencephaly. Curr. Opin. Genet. Dev. 10, 262-269.
Nassif, C., Noveen, A. and Hartenstein, V. (1998a). Embryonic development
of the Drosophila brain. I. Pattern of pioneer tracts. J. Comp. Neurol. 402,
10-31.
Nassif, C., Daniel, A., Lengyel, J. A. and Hartenstein, V. (1998b). The role
of morphogenetic cell death during Drosophila embryonic head
development. Dev. Biol. 197, 170-186.
Nery, S., Wichterle, H. and Fishell, G. (2001). Sonic hedgehog contributes
to oligodendrocyte specification in the mammalian forebrain. Development
128, 527-540.
Patel, N. (1994). Imaging neuronal subsets and other cell types in wholemount Drosophila embryos and larvae using antibody probes. In
Drosophila melanogaster: Practical Uses in Cell and Molecular Biology
(ed. L. Goldenstein and E. Fryberg), pp. 445-487. San Diego: Academic
Press.
Pera, E. M. and Kessel, M. (1997). Patterning of the chick forebrain anlage
by the prechordal plate. Development 124, 4153-4162.
Shimamura, K. and Rubenstein, J. L. (1997). Inductive interactions direct
early regionalization of the mouse forebrain. Development 124, 2709-2718.
Sokal, R. and Rohlf, F. (1997). Biometry. New York: W.H. Freeman and
Company.
Struhl, G., Struhl, K. and Macdonald, P. M. (1989). The gradient
morphogen bicoid is a concentration-dependent transcriptional activator.
Cell 57, 1259-1273.
Sweeney, S. T., Broadie, K., Keane, J., Niemann, H. and O’Kane, C. J.
(1995). Targeted expression of tetanus toxin light chain in Drosophila
specifically eliminates synaptic transmission and causes behavioral defects.
Neuron 14, 341-351.
Tabata, T. and Kornberg, T. B. (1994). Hedgehog is a signaling protein with
a key role in patterning Drosophila imaginal discs. Cell 76, 89-102.
Taylor, A. M., Nakano, Y., Mohler, J. and Ingham, P. W. (1993). Contrasting
distributions of patched and hedgehog proteins in the Drosophila embryo.
Mech. Dev. 42, 89-96.