|
|
|
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
First published online 21 February 2007
doi: 10.1242/dev.02814
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Biozentrum, University of Basel, Klingelbergstrasse 50, CH-4056 Basel, Switzerland.
* Author for correspondence (e-mail: Robert.Lichtneckert{at}stud.unibas.ch)
Accepted 18 January 2007
 |
SUMMARY |
|---|
 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Key words: empty spiracles, ems, Brain development, Neuroblast lineage, MARCM, Drosophila, Neurogenesis
|
INTRODUCTION |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
;
Skeath and Thor, 2003). In
insects such as Drosophila, which has complete metamorphosis,
neuroblasts generate the primary neurons of the larval brain during embryonic
development. Following a period of quiescence, most neuroblasts resume their
asymmetric mode of proliferation during postembryonic larval development and
generate the adult-specific secondary neurons that make up the bulk of the
adult central nervous system (Prokop and
Technau, 1991; Truman and
Bate, 1988). The adult-specific neurons that are generated during
larval life from each persistent neuroblast form a lineage-related cluster of
immature neurons that extend fasciculated primary neurites into the neuropile
but wait until metamorphosis to complete their extension to synaptic targets
(Dumstrei et al., 2003;
Pereanu and Hartenstein, 2006;
Truman et al., 2004;
Zheng et al., 2006). During
metamorphosis, development of the adult brain is completed through neuronal
remodelling of larval functional neurons and final morphogenesis of
adult-specific neurons (Lee et al.,
2000; Marin et al.,
2005; Zheng et al.,
2006).
Recent analyses of Drosophila neurogenesis have identified
developmental control genes that are involved in generating the larval brain.
Expression analysis for over 30 of these genes has shown that specific
combinations of gene expression characterize each of the approximately 100
embryonic brain neuroblasts (Urbach and
Technau, 2003). For a number of these genes, loss-of-function
analyses have revealed severe defects in neurogenesis, patterning and circuit
formation during embryonic brain development
(Hirth et al., 1998;
Hirth et al., 2003;
Hirth et al., 1995;
Kammermeier et al., 2001;
Noveen et al., 2000;
Urbach and Technau, 2003). By
contrast, only a few of these developmental control genes have been studied in
postembryonic development of the adult brain
(Callaerts et al., 2001;
Hassan et al., 2000;
Hitier et al., 2001;
Kurusu et al., 2000;
Pereanu and Hartenstein,
2006).
The empty spiracles (ems) gene plays a central role in
embryonic development of the brain
(Lichtneckert and Reichert,
2005). The ems gene encodes a homeodomain transcription
factor that acts as a cephalic gap gene during early embryogenesis
(Cohen and Jurgens, 1990;
Dalton et al., 1989;
Walldorf and Gehring, 1992).
During embryonic neurogenesis, ems is expressed in 11 bilaterally
symmetrical neuroblasts and later is found in the deutocerebral and
tritocerebral embryonic brain neuromeres
(Hirth et al., 1995;
Urbach and Technau, 2003).
Mutation of the ems gene results in the absence of cells in the
deutocerebral and tritocerebral anlagen; this is due to the failure of
neuroblasts to form in the mutant domain
(Younossi-Hartenstein et al.,
1997). By contrast to the insight into the role of ems in
embryonic brain development, virtually nothing is known about expression or
function of ems during postembryonic development of the adult
brain.
This lack of information on ems action in postembryonic brain
development in Drosophila contrasts with the wealth of information on
the role of the ems orthologues, Emx1 and Emx2, in
mammalian brain development. Both of these mammalian genes are expressed in
the early neuroectoderm and the embryonic progenitor cells that give rise to
telencephalic brain regions (Simeone et
al., 1992a; Simeone et al.,
1992b). Mutant analysis indicates that these genes play important
roles in early patterning and proliferation of anterior brain regions
(Bishop et al., 2003;
Cecchi and Boncinelli, 2000;
Shinozaki et al., 2002).
Mammalian Emx genes also act in later phases of brain development and
are expressed in cells of the adult brain
(Briata et al., 1996;
Cecchi, 2002;
Gulisano et al., 1996).
Moreover, mutant analysis suggests a role of Emx genes in
differentiation and maintenance of cortical neurons and in pathfinding of
cortical efferents (Bishop et al.,
2003; Shinozaki et al.,
2002).
The finding that mammalian Emx genes play multiple roles in
different stages of brain development underscores an emerging theme in
vertebrate neuronal development. Thus, many developmental control genes
implicated in early neurogenesis and patterning, are re-expressed and have
different roles in later embryogenesis and postembryonic brain development
(Salie et al., 2005;
Zapala et al., 2005). Given
the conservation of expression and function of fly ems and mammalian
Emx genes in embryonic brain development, we set out to determine if
the Drosophila ems gene might also play important roles in
postembryonic development of the adult brain.
Here we show that ems is expressed in the adult brain in the clonal progeny of a single neuroblast in each brain hemisphere. All adult-specific secondary neurons in this lineage already express ems during larval development and continue to do so throughout metamorphosis and into the adult. To investigate the role of the ems gene in the persistently expressing, adult-specific brain lineage, we used mosaic analysis with a repressible cell marker (MARCM). Our findings demonstrate that ems function is cell-autonomously required for the correct number of cells in this lineage. Moreover, they indicate that ems is also required cell autonomously for the formation of the correct neuritic projections in this specific lineage. This analysis of ems function reveals novel and unexpected roles of a cephalic gap gene in determining the anatomical features of an individual lineage-based unit in the adult brain of Drosophila.
|
MATERIALS AND METHODS |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
) on
chromosomal arm 3R and tubP-GAL4, UAS-mCD8::GFPLL5 on
chromosome 2. For MARCM analysis (Lee and
Luo, 1999) +; UAS-mCD8::GFPLL5,
UAS-nlslacZ20b; FRT82B, ems9Q64 or
UAS-mCD8::GFPLL5, UAS-nlslacZ20b;
FRT82B males were crossed to hs-FLP; tubP-GAL4; FRT82B
tubP-GAL80LL3 females
(Bello et al., 2003), resulting
in ems mutant or wild-type clones. Ems or P35 rescue experiments were
performed by combining UAS-ems (H.R., unpublished) or
UAS-P35BH1 on chromosome 2 with FRT82B, ems9Q64
and crossing them to the stock hs-FLP; tubP-GAL4,
UAS-mCD8::GFPLL5; FRT82B tubP-GAL80LL3 respectively
to generate MARCM clones. For MARCM experiments, embryos of appropriate genotype were collected on standard medium over a 4 hour time window and raised at 25°C for 21 to 25 hours before heat-shock treatment (37°C for 60 minutes).
Immunolabelling
Larval and adult brains were fixed and immunostained as previously
described (Bello et al., 2003).
The following antibodies were used: rat anti-Ems (1:200; gift of U. Walldorf,
University of Saarland, Homburg, Germany), rabbit anti-Ems (1:500; gift of U.
Walldorf), rabbit anti-Grh (1:200), rabbit anti-H3P (1:400; Upstate
Biotechnology), rabbit anti-cleaved caspase 3 (1:75; Cell Signalling
Technologies) rat anti-Elav Mab7E8A10 (1:30; DSHB), mouse anti-cycE (1:50;
gift of H. Richardson, Peter MacCallum Cancer Centre, East Melbourne,
Victoria, Australia), mouse anti-Pros MaMR1A (1:10; DSHB), mouse
anti-ßGAL (1:20; DSHB), mouse anti-Nrt BP106 (1:10; DSHB), mouse
anti-BrdU (1:100; DSHB), mouse monoclonal nc82 (1:20; gift of A. Hofbauer,
University of Regensburg, Regensburg, Germany), rabbit anti-Castor
(Kambadur et al., 1998).
Secondary antibodies were Alexa 488, Alexa 568 and Alexa 647 antibodies
generated in goat (1:300; Molecular Probes).
To estimate the number of dividing cells in wild-type or ems
mutant clones induced at early first instar stage, larvae were transferred to
BrdU-containing standard medium (final concentration: 1 mg/ml) at 60 hours
after hatching and raised for 12 hours before dissection
(Truman and Bate, 1988).
Brains were fixed immediately in 4% paraformaldehyde for 15 minutes at RT and
incubated with the anti-Ems and anti-ßGAL primary antibodies.
Subsequently, an additional fixation step in 2% paraformaldehyde was applied
and the brains were incubated in 2N HCl for 30 minutes to denature
BrdU-labelled DNA before incubation with the anti-BrdU antibody.
Microscopy and image processing
Fluorescent images were recorded using a Leica TCS SP scanning confocal
microscope. Optical sections were taken at 1 µm intervals in line average
mode with a picture size of 512x512 pixels. Digital image stacks were
processed using ImageJ
(http://rsb.info.nih.gov/ij/).
For visualizing particular MARCM clones, image stacks with few non-interfering
clones were selected and stained processes and cell bodies from other clones
were removed using the lasso tool in every single optical section. Digital
3D-models were generated using the AMIRA software by manually labelling
structures of interest such as cell bodies, processes, whole clones or
neuropile and subsequent automated 3D surface rendering.
|
RESULTS |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
The compact aspect of the ems-expressing cell clusters suggests
that they might represent clonally restricted neuroblast lineages. To
investigate this, we carried out a MARCM-based analysis
(Lee and Luo, 1999;
Lee and Luo, 2001). In these
experiments, GFP-labelled wild-type clones were induced at random in early
first instar larvae (21-25 hours after egg-laying) to specifically label the
secondary, adult-specific lineage of individual larval neuroblasts. The brains
of adult flies that contained GFP-labelled MARCM clones were then co-labelled
with anti-Ems and anti-Nc82. Brains, in which GFP-labelled clones were
anti-Ems-immunoreactive, were analysed further by confocal microscopy.
Co-labelling of cell bodies with GFP and anti-Ems was restricted to one clone per brain hemisphere (Fig. 1D). All the GFP-labelled cells of this particular clone coexpressed ems. In addition, a few ems-expressing cells that were not GFP-labelled were closely associated with the cell cluster that coexpressed GFP and ems (Fig. 1F). These findings indicate that the majority of the cells in the ems-expressing clusters of the adult brain are secondary adult-specific neurons that derive in a clonal manner from a single larval neuroblast. (The non-GFP-labelled cells in the ems-expressing clusters may represent primary neurons generated by the same persistent neuroblast during embryogenesis.)
The ems-expressing cells in the GFP-labelled neuroblast clones were associated with a GFP-labelled fascicle that extended to more dorsal brain regions. To facilitate the analysis of this fascicle, a digital 3D model of the projection and the major neuropile compartments along which the fascicle projected was generated (Fig. 1E). This showed that the fascicle projected from the GFP-labelled cell bodies medially along the antennal lobe to the ipsilateral superior medial protocerebrum. Hereafter this will be referred to as the `protocerebral fascicle' of the ems-labelled clonal cells. Close to the cell bodies, a dense arbor of labelled, dendrite-like processes was observed; these short processes extended ventrally into the suboesophageal neuropile (Fig. 1G, asterisk).
|
; Pereanu
and Hartenstein, 2006).
A total of eight cell clusters with ems expression were found in
each brain hemisphere. These cell clusters could be unambiguously identified
based on their relative positions and on the projection pattern of their
primary neurite bundles (Pereanu and
Hartenstein, 2006; Truman et
al., 2004). Each cell cluster contained a large
ems-expressing cell near the cortex surface associated with a
columnar-like aggregate of smaller cells, which co-expressed ems and
Neurotactin (Fig.
2C,D). In each cell cluster, a Neurotactin-expressing
fascicle emerged from the smaller ems-expressing cells
(Fig. 2D). This fascicle
extended towards the brain neuropile (Fig.
2E). A digital 3D model of all eight ems-expressing cell
clusters and their primary neurites is shown in
Fig. 2F. The medial cluster
(asterisk) with its dorsally projecting fascicle (arrow) is clearly
identifiable in this model.
These findings suggest that each ems-expressing cell cluster in the late third instar brain is composed of a persistent neuroblast and its progeny, which project fasciculated primary neurites into the neuropile. To determine if ems expression in the late third instar brain is restricted to adult-specific lineages, a MARCM-based clonal analysis was carried out. GFP-labelled wild-type MARCM clones, induced in early first instar larvae, were recovered in the late third instar brain for all eight Ems-positive cell clusters. This indicates that each cell cluster represents a neuroblast clone. A clear difference in ems expression was observed between the medial ems-expressing lineage (hereafter referred to as the EM lineage) and the remaining seven ems-expressing lineages. In these seven lineages, ems expression was present in the neuroblast and in a small subset of the adult-specific neurons located adjacent to the neuroblast; these represent the late born cells in the neuroblast clone (Fig. 2G). By contrast, early born neurons located further away from the neuroblast did not express ems (Fig. 2H). This spatially restricted ems-expression pattern within neuroblast clones is illustrated in a digital 3D model of one representative of the seven lineages (Fig. 2I; arrowhead in 2F).
A markedly different ems-expression pattern was observed in the EM lineage. In this lineage, ems-expression was present throughout the secondary lineage, including the neuroblast and the adult-specific neurons, irrespective of their position within the clone (Fig. 2J,K). A digital 3D model of the EM lineage (Fig. 2L) illustrates the fact that ems is expressed throughout the GFP-labelled clone. In addition, GFP-negative cells located close to the early born, adult-specific neurons of the GFP-labelled clone also expressed ems, albeit at a lower level (open arrowheads in Fig. 2K; lightly coloured cells in Fig. 2L). These could be progeny generated by the EM neuroblast during embryonic development before MARCM clone induction.
|
To investigate this, GFP-labelled wild-type MARCM clones induced in early first instar larvae were examined at late third instar stage and at 24, 48 and 72 hours after puparium formation (APF). In all cases, only one GFP-labelled clone, which coexpressed ems in all labelled cells, was observed per brain hemisphere (Fig. 3). These clones were comparable in size and location in the brain cortex and had similar fascicle projections. Moreover, their overall morphology at 72 hours APF was very similar to that of the single ems-expressing clone in the adult brain (compare Fig. 3D with Fig. 1D). By contrast, ems expression in the other lineages was strongly reduced in the early pupa and completely disappeared at later pupal stages. This indicates that the larval EM lineage persists through metamorphosis, maintaining both its ems expression features and its neurite fascicle projection pattern. One morphological change that did occur during metamorphosis in the cells of the EM lineage was the emergence of dense dendrite-like arborizations (Fig. 3 insets). In the early pupa, these short arborizations extended ventrally into the neuropile of the suboesophageal ganglia. They were retained through metamorphosis and remained present in the adult brain (Fig. 1G).
Based on its overall morphology, we tentatively assign the EM lineage to
the BAmas2 secondary lineage defined by Pereanu and Hartenstein
(Pereanu and Hartenstein,
2006). Accordingly, this lineage is a member the basoanterior
group of the ventral deutocerebrum, which surrounds the antennal compartment
and projects its secondary lineage axon tract (SAT) upwards along the medial
edge of the brain along the median bundle.
Neuronal precursors and postmitotic neurons are present in ems mutant EM lineages
To determine the role of the ems gene in the development of the EM
lineage, ems mutant and wild-type MARCM clones were induced randomly
in early first instar larvae and analysed in late third instar brains. Mutant
lineages were homozygous for ems9Q64, an embryonic lethal
loss-of-function allele of ems. This allele encodes a truncated
non-functional protein that is detected by the anti-Ems antibody in the
cytoplasm.
All labelled wild-type and ems mutant EM lineages contained one
large cell, the neuroblast, which consistently expressed the transcription
factor Grainyhead (Grh) (Fig.
4A,B). Smaller Grh-expressing ganglion mother cells (GMCs) were
found directly adjacent to the neuroblast in both wild-type and mutant EM
lineages. Moreover, expression of the mitotic markers Cyclin E (CycE) and
anti-phosphorylated histone-H3 (H3p) was seen in neuroblasts and GMCs of both
wild-type and mutant clones (Fig.
4C-F). These findings indicate that neuroblasts and GMCs are
present and mitotically active in wild-type and ems mutant EM
lineages at late third instar stage
(Almeida and Bray, 2005;
Bello et al., 2003;
Cenci and Gould, 2005).
|
), which
were expressed in postmitotic neurons of the wild-type EM lineage, were also
seen in the neural progeny of the ems mutant EM lineage
(Fig. 4I-L). Taken together,
these results indicate that mitotically active progenitor cells and
differentiating neuronal progeny are present in the wild-type and ems
mutant EM lineage in the late third instar brain.
The ems gene is required for correct neuronal cell number in the EM lineage
Although ems mutant EM lineages did contain postmitotic neurons,
the number of neurons per clone seemed to be reduced
(Fig. 4). Reduction in clone
size was clearly manifest in mutant clones of late third instar brains
(Fig. 5B-G). Quantification of
GFP-labelled cells revealed that the wild-type EM lineage contains on average
79 adult-specific cells (s.d.=3.4; n=7), whereas ems mutant
clones had an average of only 36 labelled cells (s.d.=12.8; n=20)
(Fig. 5A). To confirm that this
reduction was due to ems loss of function, we carried out a clonal
rescue experiment. For this, Ems expression was targeted in ems
homozygous mutant clones using a UAS-ems transgene under the control
of the MARCM tub-GAL4 driver. When examined in late third instar, the
size of these rescued clones was restored to an average of 77 cells (s.d.=4.5;
n=10), which was almost wild type
(Fig. 5A, also compare
Fig. 5H-J with
Fig. 5B-D). These findings
indicate that the ems gene is required cell autonomously for the
correct number of adult-specific neurons in the EM lineage.
In order to obtain insight into the mechanisms of clone size reduction in ems mutants, a more detailed characterization of the EM lineage during postembryonic development was carried out for both wild-type and mutant clones. First, the number of cells in EM clones (induced at early first instar) was determined at different larval and pupal stages (Fig. 6). At 48 hours after larval hatching (ALH), mutant and wild-type clones contained a similar number of cells, suggesting that initially postembryonic proliferative activity in the EM clones was not affected by ems loss of function. Marked differences between mutant and wild-type clones became apparent at 72 hours ALH in that the ems mutant clones contained fewer cells than the wild-type clones. This difference had increased at 96 hours ALH and remained large through pupal development and in the adult.
To determine if this difference in clonal cell number might be due to reduced proliferative activity in the ems mutants, we next studied the incorporation of BrdU into wild-type and mutant clones at the mid-third instar stage (see Materials and methods). In wild-type clones, an average of 16.2 (s.d.=2.2; n=13) and in ems mutant clones an average of 15.9 (s.d.=1.9; n=13) labelled cells were observed, indicating that mitotic activity was similar in the two cases. Furthermore, the percentage of EM neuroblasts expressing the mitotic marker H3p at mid-third instar stages was comparable in both cases; 43% of wild-type neuroblasts (n=74) and 50% of ems mutant neuroblasts (n=14) expressed the marker. (Comparable findings were obtained for late third instar larva; data not shown.) Taken together, these findings imply that the proliferative activity was not significantly reduced in ems mutant EM clones.
To investigate if the reduction in clonal cell number might involve the death of postmitotic cells, we initially stained ems mutant clones in late third instar brains with the apoptosis marker cleaved Caspase 3. All the ems mutant EM clones studied contained one to four cleaved Caspase 3-positive cells (average=2.4, s.d.=1.0, n=10), indicating the presence of apoptosis in the mutant lineages. To determine if apoptosis can account for the reduction in clonal cell number observed in the ems mutant lineage, we next blocked cell death in ems mutant clones through misexpression of the pancaspase inhibitor P35. For this, clones were induced in early first instar larva and cell numbers determined at the late third larval stage. Blocking cell death resulted in mutant clones containing an average of 70 (s.d.=15; n=9) cells. This is comparable to an average of 79 cells in wild-type EM clones and an average of 77 cells in ems mutant clones misexpressing an ems transgene, and it is significantly higher than the average of 36 cells in ems mutant clones (see above). These findings imply that the reduction in clonal cell number in ems mutant EM lineages is due to apoptosis.
The ems gene is required for correct projections in the EM lineage
When examined in the adult brain, ems mutant MARCM clones in the
EM lineage showed a second marked ems mutant phenotype. In many
cases, mutant clones lacked the prominent protocerebral fascicle that
projected to the superior medial protocerebrum in the wild-type control
(Fig. 7A-D). In other mutant
clones, a somewhat reduced protocerebral fascicle was formed
(Fig. 7E,F). Moreover, in all
ems mutant clones examined (n=8), aberrant projections
extended without obvious pattern towards adjacent neuropiles
(Fig. 7C-F, arrowheads).
Misdirected projections of this type were never observed in the wild-type
control. These projection defects were fully restored in rescue experiments in
which the ems transgene was misexpressed in the ems mutant
EM clone (Fig. 7G,H). Rescued
clones had a normal protocerebral fascicle and never showed short aberrant or
misdirected process extensions. These findings indicate that the ems
gene is required cell autonomously for the correct projection pattern of
adult-specific neurons in the EM lineage.
|
|
To determine if the cell-autonomous requirement of the ems gene for correct projection of the EM lineage occurs at the level of postmitotic cells, we analysed single cell MARCM clones in third instar larval brains. Single labelled wild-type cells had neuronal morphologies that were expected for the EM lineage (Fig. 8A,B). Thus, labelled cells had a cell body located in the appropriate region of the brain cortex as well as a projection pattern in the brain neuropile consisting of a single process that extended towards the midline, turned rostrally, projected to the anterior protocerebrum, and there formed arborizations. By contrast, most of the single labelled ems mutant cells showed dramatic projection defects. In some cases, labelled cells extended processes posteriorly that arborized in the suboesophageal ganglion (Fig. 8C,D). In other cases, labelled cells failed to extend any process (Fig. 8E,F). These observations indicate that individual postmitotic EM cells manifest a cell-autonomous requirement for the ems gene in order to establish their appropriate neuronal morphology.
|
|
DISCUSSION |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
By contrast, in the eighth neuroblast lineage, ems expression is persistent. During larval development the neuroblast and all its adult-specific progeny express ems; this expression continues throughout metamorphosis and into the adult in all postmitotic cells of the EM lineage. The mechanisms responsible for the maintenance of ems expression in the adult-specific cells of the EM lineage are currently unknown. However, there is some evidence that ems is also expressed and maintained in the primary neurons of the EM lineage generated during embryogenesis. In all postembryonic stages and in the adult, approximately 30 ems-expressing neurons are closely associated with the early born, adult-specific neurons of the EM clone. These neurons are not generated postembryonically, and their number does not change significantly during postembryonic development. This suggests that the mechanisms responsible for the persistence of ems expression in the EM lineage may operate in all cells of the lineage, embryonic and postembryonic.
During early embryogenesis, ems is expressed in a total of eleven
neuroblasts per embryonic brain hemisphere
(Urbach and Technau, 2003). An
unambiguous link between these embryonic brain neuroblasts and the eight
postembryonic ems-expressing neuroblasts has not yet been
established. If the persistent expression of ems is a unique feature
of the EM lineage, it should be possible to trace this lineage back into
embryonic stages and identify its embryonic neuroblast of origin. For the
remaining seven postembryonic ems-expressing neuroblasts, this may be
more difficult and require a combination of molecular markers and
neuroanatomical lineage mapping (Pereanu
and Hartenstein, 2006;
Younossi-Hartenstein et al.,
2006).
The postembryonic expression of ems in the fly brain has
interesting parallels to the expression of the Emx1 and Emx2
genes in the mammalian brain. In addition to early expression in the neural
plate, the Emx1 gene is expressed in many differentiating and mature
neurons of the murine cortex (Briata et
al., 1996; Gulisano et al.,
1996). Brain-specific expression of Emx2 appears to be
more transient in later stages and in the adult brain seems to be restricted
to neural stem cells (Gangemi et al.,
2001; Mallamaci et al.,
1998; Mallamaci et al.,
2000). Thus, spatially restricted persistent and transient
expression patterns are observed for the ems/Emx genes in neural
progenitors and in neurons during brain development and maturation in flies
and mice.
Functional roles of ems in the EM lineage
For mutant analysis of ems function we focused on the EM lineage
and used clonal techniques to ensure that the secondary adult-specific neurons
are mutant from the time of their birth onwards. Two lineage-specific mutant
phenotypes are apparent in these loss-of-function experiments. The number of
adult-specific neurons is reduced and projection defects occur in mutant
clones. Both phenotypes are cell autonomous, and both can be fully restored in
genetic rescue experiments. Moreover, both mutant phenotypes are seen in
larval stages and persist in the adult brain. These findings implicate the
ems transcription factor in translating lineage information into
neuronal cell number control and neurite projection specificity.
There are several possible explanations for the 50% reduction in cell number observed in ems mutant EM clones. First, proliferation of the mutant neuroblast might cease due to cell cycle arrest or to premature neuroblast death. This seems unlikely, because proliferating neuroblasts can be identified in larval ems mutant clones based on expression of specific markers. Second, cell division of ganglion mother cells might be suppressed in favour of a direct differentiation of each neuroblast progeny into a single neuron, resulting in a total clone size reduction of 50%. This also appears unlikely, as GMCs expressing a cell proliferation marker can be identified repeatedly in mutant clones, indicating that they divide normally to produce two daughter cells. Third, the time window of proliferative activity or the proliferation rate of the persistent neuroblast is shortened in ems mutants. While we cannot rule out this possibility, it also appears unlikely for the following three reasons: first, mutant and wild-type clones contain a similar number of cells at 48 hours ALH, suggesting that the proliferation rate is not affected at this stage; second, BrdU-incorporation studies reveal no difference in mitotic activity at late larval stage brains of wild-type versus ems mutant clones; third, the percentage of neuroblasts expressing the mitotic marker H3p at late larval stages was comparable for wild-type and ems mutant clones. The final explanation for the marked reduction in cell number seen in mutant clones is that postmitotic cells die due to apoptosis. This possibility is supported by two observations: (1) late larval ems mutant EM clones contain apoptotic cells, as assayed by the apoptosis marker cleaved Caspase 3; (2) blockage of cell death in the ems mutant lineage through a pancaspase inhibitor results in significant restoration of the clonal cell number to a value comparable to that observed in the wild type. Based on these findings, we posit that ems is required in the adult-specific EM lineage for survival of clonal postmitotic progeny.
|
A second projection defect is observed in the adult brain in approximately half the ems mutant EM lineages. It consists in the complete absence of the fascicle projecting to the superior medial protocerebrum. This projection phenotype in the adult has a corresponding projection phenotype in the larva, in that the primary neurite bundle is missing in approximately half the mutant lineages. These observations suggest that the formation of the primary neurite bundle during larval development might be a prerequisite for the process extension to adult-specific targets during metamorphosis; this would indicate a larval requirement of ems for neurite fascicle formation.
Both projection phenotypes seen in mutant neuroblast clones, short ectopic neurite projections and the absence of the fascicle to the protocerebrum, are also apparent in ems mutant single cell clones of the larval brain. Given that all other cells in the lineage, including the EM neuroblast, are wild-type-like in these experiments, this finding indicates that individual postmitotic neurons of the EM lineage have a cell-autonomous requirement for the ems gene in order to form correct projections in larval brain development.
Are features of ems function in brain development general?
Our analysis of ems function in the EM lineage demonstrates that
homeobox transcription factors can influence adult brain architecture in a
cell-autonomous and lineage-specific way. A lineage-specific, cell-autonomous
requirement of other transcription factors in brain development has been shown
for the olfactory projection neurons and for mushroom body neurons in
Drosophila (Callaerts et al.,
2001; Komiyama et al.,
2003; Kurusu et al.,
2000; Martini and Davis,
2005; Martini et al.,
2000; Noveen et al.,
2000). Thus, increasing evidence indicates that key developmental
control genes, which operate early in embryogenesis, also act later in a
lineage-specific manner in controlling anatomical features of the adult
Drosophila brain. It may be a general feature of brain development
that developmental control genes implicated in early neurogenesis and
patterning are re-expressed and have different roles in later embryogenesis
and postembryonic brain development (Salie
et al., 2005; Zapala et al.,
2005).
|
;
Shinozaki et al., 2002). The
cortical surface area of Emx1/Emx2 double mutants is about half that
of wild type, and the thickness of the preplate and cortical plate is reduced,
suggesting that Emx genes regulate the numbers of cortical neurons.
Moreover, Emx1/Emx2 double mutants have major defects in the
pathfinding of most cortical axons, implying an important role for
Emx genes in axonal pathfinding in the brain. Thus, mutant analyses
in Drosophila and mouse suggest that loss of function of
ems/Emx genes may result in comparable brain phenotypes, namely in
reduction of neuronal cell number and in neurite projection defects. This in
turn suggests that the morphological differentiation of brain architecture in
both flies and mammals may involve conserved functions of orthologous
ems/Emx homeobox genes, not only in the early embryo but also during
later stages of brain development. |
ACKNOWLEDGMENTS |
|---|
|
REFERENCES |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Almeida, M. S. and Bray, S. J. (2005). Regulation of post-embryonic neuroblasts by Drosophila Grainyhead. Mech. Dev. 122,1282 -1293.[CrossRef][Medline]
Bello, B. C., Hirth, F. and Gould, A. P. (2003). A pulse of the Drosophila Hox protein Abdominal-A schedules the end of neural proliferation via neuroblast apoptosis. Neuron 37,209 -219.[CrossRef][Medline]
Bishop, K. M., Garel, S., Nakagawa, Y., Rubenstein, J. L. and O'Leary, D. D. (2003). Emx1 and Emx2 cooperate to regulate cortical size, lamination, neuronal differentiation, development of cortical efferents, and thalamocortical pathfinding. J. Comp. Neurol. 457,345 -360.[CrossRef][Medline]
Briata, P., Di Blas, E., Gulisano, M., Mallamaci, A., Iannone, R., Boncinelli, E. and Corte, G. (1996). EMX1 homeoprotein is expressed in cell nuclei of the developing cerebral cortex and in the axons of the olfactory sensory neurons. Mech. Dev. 57,169 -180.[CrossRef][Medline]
Callaerts, P., Leng, S., Clements, J., Benassayag, C., Cribbs, D., Kang, Y. Y., Walldorf, U., Fischbach, K. F. and Strauss, R. (2001). Drosophila Pax-6/eyeless is essential for normal adult brain structure and function. J. Neurobiol. 46, 73-88.[CrossRef][Medline]
Cecchi, C. (2002). Emx2: a gene responsible for cortical development, regionalization and area specification. Gene 291,1 -9.[CrossRef][Medline]
Cecchi, C. and Boncinelli, E. (2000). Emx homeogenes and mouse brain development. Trends Neurosci. 23,347 -352.[CrossRef][Medline]
Cenci, C. and Gould, A. P. (2005). Drosophila
Grainyhead specifies late programmes of neural proliferation by regulating the
mitotic activity and Hox-dependent apoptosis of neuroblasts.
Development 132,3835
-3845.
Cohen, S. M. and Jurgens, G. (1990). Mediation of Drosophila head development by gap-like segmentation genes. Nature 346,482 -485.[CrossRef][Medline]
Dalton, D., Chadwick, R. and McGinnis, W.
(1989). Expression and embryonic function of empty spiracles: a
Drosophila homeo box gene with two patterning functions on the
anterior-posterior axis of the embryo. Genes Dev.
3,1940
-1956.
de la Escalera, S., Bockamp, E. O., Moya, F., Piovant, M. and Jimenez, F. (1990). Characterization and gene cloning of neurotactin, a Drosophila transmembrane protein related to cholinesterases. EMBO J. 9,3593 -3601.[Medline]
Dumstrei, K., Wang, F., Nassif, C. and Hartenstein, V. (2003). Early development of the Drosophila brain: V. Pattern of postembryonic neuronal lineages expressing DE-cadherin. J. Comp. Neurol. 455,451 -462.[CrossRef][Medline]
Gangemi, R. M., Daga, A., Marubbi, D., Rosatto, N., Capra, M. C. and Corte, G. (2001). Emx2 in adult neural precursor cells. Mech. Dev. 109,323 -329.[CrossRef][Medline]
Gulisano, M., Broccoli, V., Pardini, C. and Boncinelli, E. (1996). Emx1 and Emx2 show different patterns of expression during proliferation and differentiation of the developing cerebral cortex in the mouse. Eur. J. Neurosci. 8,1037 -1050.[CrossRef][Medline]
Hassan, B. A., Bermingham, N. A., He, Y., Sun, Y., Jan, Y. N., Zoghbi, H. Y. and Bellen, H. J. (2000). atonal regulates neurite arborization but does not act as a proneural gene in the Drosophila brain. Neuron 25,549 -561.[CrossRef][Medline]
Hirth, F., Therianos, S., Loop, T., Gehring, W. J., Reichert, H. and Furukubo-Tokunaga, K. (1995). Developmental defects in brain segmentation caused by mutations of the homeobox genes orthodenticle and empty spiracles in Drosophila. Neuron 15,769 -778.[CrossRef][Medline]
Hirth, F., Hartmann, B. and Reichert, H. (1998). Homeotic gene action in embryonic brain development of Drosophila. Development 125,1579 -1589.[Abstract]
Hirth, F., Kammermeier, L., Frei, E., Walldorf, U., Noll, M. and
Reichert, H. (2003). An urbilaterian origin of the tripartite
brain: developmental genetic insights from Drosophila.
Development 130,2365
-2373.
Hitier, R., Chaminade, M. and Preat, T. (2001). The Drosophila castor gene is involved in postembryonic brain development. Mech. Dev. 103,3 -11.[CrossRef][Medline]
Jurgens, G., Wieschaus, E., Nusslein-Volhard, C. and Kluding, H. (1984). Mutations affecting the pattern of the larval cuticle in Drosophila melanogaster. Rouxs Arch. Dev. Biol. 193,283 -295.
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.
Kammermeier, L., Leemans, R., Hirth, F., Flister, S., Wenger, U., Walldorf, U., Gehring, W. J. and Reichert, H. (2001). Differential expression and function of the Drosophila Pax6 genes eyeless and twin of eyeless in embryonic central nervous system development. Mech. Dev. 103,71 -78.[CrossRef][Medline]
Komiyama, T., Johnson, W. A., Luo, L. and Jefferis, G. S. (2003). From lineage to wiring specificity. POU domain transcription factors control precise connections of Drosophila olfactory projection neurons. Cell 112,157 -167.[CrossRef][Medline]
Kurusu, M., Nagao, T., Walldorf, U., Flister, S., Gehring, W. J.
and Furukubo-Tokunaga, K. (2000). Genetic control of
development of the mushroom bodies, the associative learning centers in the
Drosophila brain, by the eyeless, twin of eyeless, and Dachshund genes.
Proc. Natl. Acad. Sci. USA
97,2140
-2144.
Lee, T. and Luo, L. (1999). Mosaic analysis with a repressible cell marker for studies of gene function in neuronal morphogenesis. Neuron 22,451 -461.[CrossRef][Medline]
Lee, T. and Luo, L. (2001). Mosaic analysis with a repressible cell marker (MARCM) for Drosophila neural development. Trends Neurosci. 24,251 -254.[CrossRef][Medline]
Lee, T., Marticke, S., Sung, C., Robinow, S. and Luo, L. (2000). Cell-autonomous requirement of the USP/EcR-B ecdysone receptor for mushroom body neuronal remodeling in Drosophila. Neuron 28,807 -818.[CrossRef][Medline]
Lichtneckert, R. and Reichert, H. (2005). Insights into the urbilaterian brain: conserved genetic patterning mechanisms in insect and vertebrate brain development. Heredity 94,465 -477.[CrossRef][Medline]
Mallamaci, A., Iannone, R., Briata, P., Pintonello, L., Mercurio, S., Boncinelli, E. and Corte, G. (1998). EMX2 protein in the developing mouse brain and olfactory area. Mech. Dev. 77,165 -172.[CrossRef][Medline]
Mallamaci, A., Mercurio, S., Muzio, L., Cecchi, C., Pardini, C.
L., Gruss, P. and Boncinelli, E. (2000). The lack of Emx2
causes impairment of Reelin signaling and defects of neuronal migration in the
developing cerebral cortex. J. Neurosci.
20,1109
-1118.
Marin, E. C., Watts, R. J., Tanaka, N. K., Ito, K. and Luo,
L. (2005). Developmentally programmed remodeling of the
Drosophila olfactory circuit. Development
132,725
-737.
Martini, S. R. and Davis, R. L. (2005). The dachshund gene is required for the proper guidance and branching of mushroom body axons in Drosophila melanogaster. J. Neurobiol. 64,133 -144.[CrossRef][Medline]
Martini, S. R., Roman, G., Meuser, S., Mardon, G. and Davis, R. L. (2000). The retinal determination gene, dachshund, is required for mushroom body cell differentiation. Development 127,2663 -2672.[Abstract]
Noveen, A., Daniel, A. and Hartenstein, V. (2000). Early development of the Drosophila mushroom body: the roles of eyeless and dachshund. Development 127,3475 -3488.[Abstract]
Pearson, B. J. and Doe, C. Q. (2004). Specification of temporal identity in the developing nervous system. Annu. Rev. Cell Dev. Biol. 20,619 -647.[CrossRef][Medline]
Pereanu, W. and Hartenstein, V. (2006). Neural
lineages of the Drosophila brain: a three-dimensional digital atlas of the
pattern of lineage location and projection at the late larval stage.
J. Neurosci. 26,5534
-5553.
Prokop, A. and Technau, G. M. (1991). The origin of postembryonic neuroblasts in the ventral nerve cord of Drosophila melanogaster. Development 111, 79-88.[Abstract]
Salie, R., Niederkofler, V. and Arber, S. (2005). Patterning molecules; multitasking in the nervous system. Neuron 45,189 -192.[Medline]
Shinozaki, K., Miyagi, T., Yoshida, M., Miyata, T., Ogawa, M., Aizawa, S. and Suda, Y. (2002). Absence of Cajal-Retzius cells and subplate neurons associated with defects of tangential cell migration from ganglionic eminence in Emx1/2 double mutant cerebral cortex. Development 129,3479 -3492.
Simeone, A., Acampora, D., Gulisano, M., Stornaiuolo, A. and Boncinelli, E. (1992a). Nested expression domains of four homeobox genes in developing rostral brain. Nature 358,687 -690.[CrossRef][Medline]
Simeone, A., Gulisano, M., Acampora, D., Stornaiuolo, A., Rambaldi, M. and Boncinelli, E. (1992b). Two vertebrate homeobox genes related to the Drosophila empty spiracles gene are expressed in the embryonic cerebral cortex. EMBO J. 11,2541 -2550.[Medline]
Skeath, J. B. and Thor, S. (2003). Genetic control of Drosophila nerve cord development. Curr. Opin. Neurobiol. 13,8 -15.[CrossRef][Medline]
Truman, J. W. and Bate, M. (1988). Spatial and temporal patterns of neurogenesis in the central nervous system of Drosophila melanogaster. Dev. Biol. 125,145 -157.[CrossRef][Medline]
Truman, J. W., Schuppe, H., Shepherd, D. and Williams, D. W.
(2004). Developmental architecture of adult-specific lineages in
the ventral CNS of Drosophila. Development
131,5167
-5184.
Urbach, R. and Technau, G. M. (2003). Molecular
markers for identified neuroblasts in the developing brain of Drosophila.
Development 130,3621
-3637.
Walldorf, U. and Gehring, W. J. (1992). Empty spiracles, a gap gene containing a homeobox involved in Drosophila head development. EMBO J. 11,2247 -2259.[Medline]
Younossi-Hartenstein, A., Green, P., Liaw, G. J., Rudolph, K., Lengyel, J. and Hartenstein, V. (1997). Control of early neurogenesis of the Drosophila brain by the head gap genes tll, otd, ems, and btd. Dev. Biol. 182,270 -283.[CrossRef][Medline]
Younossi-Hartenstein, A., Nguyen, B., Shy, D. and Hartenstein, V. (2006). Embryonic origin of the Drosophila brain neuropile. J. Comp. Neurol. 497,981 -998.[CrossRef][Medline]
Zapala, M. A., Hovatta, I., Ellison, J. A., Wodicka, L., Del
Rio, J. A., Tennant, R., Tynan, W., Broide, R. S., Helton, R., Stoveken, B. S.
et al. (2005). Adult mouse brain gene expression patterns
bear an embryologic imprint. Proc. Natl. Acad. Sci.
USA 102,10357
-10362.
Zheng, X., Zugates, C. T., Lu, Z., Shi, L., Bai, J. M. and Lee, T. (2006). Baboon/dSmad2 TGF-beta signaling is required during late larval stage for development of adult-specific neurons. EMBO J. 25,615 -627.[CrossRef][Medline]
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati 
Twitter What's this?
Related articles in Development:
This article has been cited by other articles:
|
|
 |
|
  R. Lichtneckert, L. Nobs, and H. Reichert empty spiracles is required for the development of olfactory projection neuron circuitry in Drosophila Development, July 15, 2008; 135(14): 2415 - 2424. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
