1 Institute of Zoology, Biozentrum/Pharmazentrum, University of Basel,
Klingelbergstrasse 50, CH-4056 Basel, Switzerland
2 Institute for Molecular Biology, University of Zürich,
Winterthurerstrasse 190, CH-8057 Zürich, Switzerland
3 Department of Anatomy and Cell Biology, University of Saarland, Building 61,
D-66421 Homburg/Saar, Germany
Author for correspondence (e-mail:
frank.hirth{at}unibas.ch)
Accepted 10 February 2003
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SUMMARY |
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Key words: Brain development, Brain evolution, Midbrain/hindbrain boundary, Otx, Hox, Pax, Drosophila melanogaster
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INTRODUCTION |
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In ascidian and vertebrate chordates, a Pax2/5/8 expression domain
is located between the anterior Otx and the posterior Hox expression regions
of the embryonic brain (reviewed by
Holland and Holland, 1999;
Wada and Satoh, 2001
). In
vertebrate brain development, this Pax2/5/8 expression domain is
positioned at the interface of the brain-specific Otx2 and
Gbx2 expression domains; it is an early marker for the isthmic
organizer at the midbrain-hindbrain boundary (MHB), which controls the
development of the midbrain and the anterior hindbrain (reviewed by
Liu and Joyner, 2001
;
Rhinn and Brand, 2001
;
Wurst and Bally-Cuif, 2001
).
The central role of this MHB region in brain development together with the
conserved expression patterns of Pax2/5/8 genes in this region have
led to the proposal that a fundamental characteristic of the ancestral
chordate brain was its tripartite organization characterized by Otx,
Pax2/5/8 and Hox gene expressing regions
(Wada et al., 1998
). To
investigate whether protostomes also possess a tripartite organization of the
brain, and to gain insight into the evolution of the bilaterian brain, we
carried out a comparative analysis of expression and function in the
ecdysozoan Drosophila melanogaster of the orthologues that pattern
the vertebrate MHB region.
Our study reveals striking similarities in the expression and function of genes that pattern the embryonic brains of Drosophila and vertebrates. We find that a Pax2/5/8 expressing domain is located between an anterior otd-expressing region and a posterior Hox-expressing region in the embryonic brain of Drosophila. Moreover, in Drosophila, as in vertebrates, we find that this Pax2/5/8 expressing domain is positioned at the interface between the otd/Otx2 expression domain and a posteriorly abutting unplugged/Gbx2 expression domain. Finally, we demonstrate that inactivation of otd/Otx2 or of unplugged/Gbx2 results in comparable effects on mispositioning or loss of brain-specific expression domains of orthologous genes in both embryonic brain types. These developmental genetic similarities indicate that the tripartite ground plan, which characterizes the developing chordate brain, is also at the basis of the developing insect brain, conferring on all bilaterians a deep similarity in brain development. This suggests that a corresponding tripartite organization already existed in the brain of the last common urbilaterian ancestor of insects and chordates.
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MATERIALS AND METHODS |
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In situ hybridization and immunocytochemistry
For in situ hybridization experiments, digoxigenin-labelled sense and
antisense RNA probes of the spa/Pax2 cDNA clone cpx1
(Fu and Noll, 1997) were
generated in vitro with a DIG-labelling kit (Roche diagnostics) and hybridized
to Drosophila whole-mount embryos by following standard procedures
(Tautz and Pfeifle, 1989
).
Whole-mount immunocytochemistry was performed as previously described
(Hirth et al., 1998
). Rabbit
anti-PAX2 antiserum (Fu and Noll,
1997
) and monoclonal anti-POXN antibodies
(Hassanzadeh et al., 1998
)
were used as primary antibodies at 1:50 and 1:20 dilutions, respectively. To
generate an anti-Otd antiserum, a 835 bp fragment from an otd cDNA
(position 1708-2542) (Finkelstein et al.,
1990
) was amplified by PCR and cloned into the pCR2.1-TOPO vector
(Invitrogen), from which it was removed by digestion at flanking
EcoRI sites and cloned in frame into the pGEX-2T expression vector
(Smith and Johnson, 1988
). The
glutathione-S-transferase/Otd fusion protein was purified according to Smith
and Johnson (Smith and Johnson,
1988
), except that induction occurred at 18°C overnight.
Immunization of rabbits was carried out by Pab Productions (Hebertshausen).
For whole-mount immunocytochemistry, anti-Otd antiserum was used as primary
antiserum at a 1:100 dilution.
Embryos were staged according to Campos-Ortega and Hartenstein
(Campos-Ortega and Hartenstein,
1997).
Laser confocal microscopy
For laser confocal microscopy, a Leica TCS SP microscope was used. Optical
sections ranged from 0.4 to 2 µm, recorded in line average mode with
picture size of 512x512 pixels. Captured images from optical sections
were arranged and processed by the use of IMARIS (Bitplane). Figures were
arranged and labelled by the use of Adobe Photoshop.
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RESULTS |
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The embryonic brain of Drosophila can be subdivided into the
protocerebrum (PC or b1), deutocerebrum (DC or b2) and tritocerebrum (TC or
b3) of the supra-oesophageal ganglion and the mandibular (S1), maxillary (S2)
and labial (S3) neuromeres of the sub-oesophageal ganglion. Expression of
engrailed (en) delimits these subdivisions by marking their
most posterior neurones (reviewed by
Hartmann and Reichert, 1998)
(Fig. 1). Because of
morphogenetic processes, such as the beginning of head involution, the
neuraxis of the embryonic brain curves dorsoposteriorly within the embryo.
Accordingly, henceforth anteroposterior coordinates refer to the neuraxis
rather than the embryonic body axis.
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It is important to note that the DTB is located anterior to the expression
domain of the Drosophila Hox1 orthologue labial
(lab), which is expressed in the posterior tritocerebrum
(Hirth et al., 1998).
Moreover, the DTB is located posterior to the expression domain of the
Drosophila Otx orthologue otd in the protocerebrum and
anterior deutocerebrum (Hirth et al.,
1995
). Thus, in Drosophila as in vertebrates, a
Pax2/Poxn (Pax2/5/8) expression domain is located between
the anterior otd/Otx2 and the posterior Hox-expressing regions. This
raises the question of whether the DTB in the embryonic Drosophila
brain might have developmental genetic features similar to those observed for
the MHB in vertebrate brain development.
Gene expression patterns at the deutocerebral-tritocerebral boundary
region
In the embryonic vertebrate brain, Otx2 is expressed anterior to
and abutting Gbx2. The future MHB as well as the overlapping domains
of Pax2, Pax5 and Pax8 expression are positioned at this
Otx2-Gbx2 interface (Liu
and Joyner, 2001; Rhinn and
Brand, 2001
; Wurst and
Bally-Cuif, 2001
). To investigate if comparable expression
patterns are found in the embryonic fly brain, we determined the
brain-specific expression of the Drosophila Gbx2 orthologue
unplugged (unpg) in relation to that of otd, using
immunolabelling and an unpg-lacZ reporter gene that expresses
ß-galactosidase like endogenous unpg
(Chiang et al., 1995
). The
otd gene is expressed in the protocerebrum and anterior deutocerebrum
of the embryonic brain (Fig.
4A,B) (Hirth et al.,
1995
), as well as in midline cells in more posterior regions of
the CNS (Fig. 4C) (Finkelstein et al., 1990
;
Wieschaus et al., 1992
).
Expression of unpg-lacZ in the embryonic CNS is first
detected at stage 8 in neuroectodermal and mesectodermal cells at the ventral
midline, with an anterior limit of expression at the cephalic furrow
(Chiang et al., 1995
).
Subsequently, the unpg expression domains in the CNS widen and have
their most anterior border in the posterior deutocerebrum
(Fig. 4D-F). Double
immunolabelling of OTD and ß-galactosidase revealed that the posterior
border of the brain-specific otd expression domain coincides with the
anteriormost border of the unpg expression domains along the
anteroposterior neuraxis (Fig.
4G,H, arrows). There is no overlap of otd and
unpg expression in the brain or in more posterior regions of the CNS
(Fig. 4G-I).
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In otd-null mutant embryos, the protocerebrum is absent because
protocerebral neuroblasts are not specified
(Hirth et al., 1995;
Younossi-Hartenstein et al.,
1997
). Analysis of unpg, en and Poxn expression
in otd-null mutant embryos revealed that the anteriormost border of
unpg expression shifts anteriorly into the anterior deutocerebrum
(Fig. 6A,B), while
Poxn fails to be expressed in the deutocerebrum
(Fig. 6C). In contrast to
inactivation of otd, inactivation of unpg does not result in
a loss of cells in the mutant domain of the embryonic brain, as is evident
from the expression of an unpg-lacZ reporter construct in
unpg-null mutant embryos (Chiang
et al., 1995
). Analysis of otd expression in
unpg-null mutants shows that the posterior limit of brain-specific
otd expression shifts posteriorly into the posterior deutocerebrum,
thus extending into the DTB (Fig.
7A,B). This was confirmed by additional immunolabelling studies
examining otd, Poxn and en expression in the
protocerebral/deutocerebral region of the embryonic brain in
unpg-null mutants. Double immunolabelling of OTD and EN in
unpg-null mutants confirms that the posterior border of
brain-specific otd expression extends posteriorly to the
deutocerebral en-b2 stripe into the posterior deutocerebrum
(Fig. 7C, compare with
Fig. 5A). In addition, double
immunolabelling of OTD and POXN in unpg-null mutants confirms that
the posterior border of brain-specific otd expression extends
posteriorly into the Poxn expression domain of the DTB
(Fig. 7D, compare with
Fig. 5B). Moreover, analysis of
lab expression in unpg-null mutants shows that
brain-specific lab expression shifts anteriorly into the anterior
tritocerebrum (Fig. 7E,F).
Thus, in both Drosophila and mammals, mutational inactivation of
otd/Otx2 and unpg/Gbx2 result in the loss or misplacement of
the brain-specific expression domains of orthologous Pax and Hox genes.
Moreover, otd and unpg appear to negatively regulate each
other at the interface of their expression domains.
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DISCUSSION |
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A comparison of the brain-specific topology of gene expression patterns that define this tripartite organization in Drosophila and in mouse suggests that the vertebrate midbrain/hindbrain boundary (MHB) region corresponds to the insect deutocerebral-tritocerebral boundary (DTB) region. If this is the case, one might expect that other patterning genes that characterize the MHB region are also expressed at the insect DTB. Although this expectation is fulfilled for the segment-polarity genes en and wingless (wg) in Drosophila, these two genes are expressed at the borders of all CNS neuromeres, as well as at parasegmental boundaries in the epidermis; hence, their expression may not be indicative of brain-specific requirements.
In addition to remarkable similarities in orthologous gene expression between insects and chordates, our study also shows that several functional interactions among key developmental control genes involved in establishing the Pax2/5/8-expressing MHB region of the vertebrate brain are also conserved in insects. Thus, in the embryonic brains of both fly and mouse, the intermediate boundary regions, DTB and MHB, are positioned at the interface of otd/Otx2 and unpg/Gbx2 expression domains (Fig. 8A,B). These boundary regions are deleted in otd/Otx2-null mutants and mispositioned in unpg/Gbx2-null mutants. Moreover, otd/Otx2 and unpg/Gbx2 genes engage in crossregulatory interactions, and appear to act as mutual repressors at the interface of their brain-specific expression domains. However, not all of the functional interactions among genes involved in MHB formation in the mouse appear to be conserved at the Drosophila DTB. Thus, in the embryonic Drosophila brain, no patterning defects are observed in null mutants of Pax2, Poxn, en or bnl. It remains to be seen if these genes play a role in the postembryonic development of the Drosophila brain.
It is conceivable that the similarities of orthologous gene expression
patterns and functional interactions in brain development evolved
independently in insects and vertebrates. However, a more reasonable
explanation is that an evolutionary conserved genetic program underlies brain
development in all bilaterians. This would imply that the generation of
structural diversity in the embryonic brain is based on positional information
that has been invented only once during evolution and is provided by genes
such as otd/Otx2, unpg/Gbx2, Pax2/5/8 and Hox, conferring on all
bilaterians a common basic principle of brain development. If this is the
case, comparable orthologous gene expression and function should also
characterize embryonic brain development in other invertebrate lineages such
as the lophotrochozoans. This prediction can now be tested in lophotrochozoan
model systems such as Platynereis
(Arendt et al., 2001),
Helobdella (Kourakis et al.,
1997
) or Dugesia
(Pineda et al., 2002
).
Taken together, our results indicate that the tripartite ground plan that characterizes the developing chordate brain is also present in the developing insect brain. This implies that a corresponding tripartite organization already existed in the brain of the last common urbilaterian ancestor of insects and chordates. Therefore, we propose an urbilaterian origin of the tripartite brain.
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ACKNOWLEDGMENTS |
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Footnotes |
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