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First published online 13 August 2008
doi: 10.1242/dev.022343
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Department of Biochemistry and Biophysics and Department of Biology, Program in Molecular Biology and Biotechnology, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-3280, USA.
* Author for correspondence (e-mail: steve_crews{at}unc.edu)
Accepted 10 July 2008
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: CNS midline, Drosophila, Glia, Neuroblast, Neuron, Notch
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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22 cells, including midline
glia (MG), local interneurons, projection neurons, peptidergic motoneurons and
neuromodulatory motoneurons (Wheeler et
al., 2006
). The embryonic expression patterns of nearly 300
midline-expressed genes have been identified
(Kearney et al., 2004), and
transcriptional maps permit detailed genetic analysis of the entire process of
midline cell development (Bossing and
Brand, 2006; Wheeler et al.,
2006). Thus, the Drosophila midline cells combine
cellular diversity with extensive molecular genetic characterization for the
study of CNS development.
The Drosophila midline cells originate from about eight precursor
cells/segment that undergo synchronous cell division (
1414)
at stage 8 (Foe, 1989) to give
rise to 16 cells (Bossing and Technau,
1994). These cells are characterized by expression of the
single-minded (sim) gene
(Crews, 2003;
Thomas et al., 1988). By late
stage 11, the midline cells consist of about ten MG, comprising two
populations, the anterior midline glia (AMG) and posterior midline glia (PMG),
two midline precursor 1 (MP1) neurons, two MP3 neurons, six ventral unpaired
median (VUM) neurons (two VUM4s, two VUM5s and two VUM6s) and the median
neuroblast (MNB) (Wheeler et al.,
2006). The PMG die during embryogenesis along with about half of
the AMG. The remaining three AMG ensheathe the axon commissures. Whereas the
two MP1 neurons appear to be identical, the MP3 neurons differentiate into the
dopaminergic H-cell and glutamatergic H-cell sib. Each VUM precursor (MP4-6)
divides once, giving rise to a GABAergic VUM interneuron (iVUM4-6) and a
glutamatergic/octopaminergic VUM motoneuron (mVUM4-6). Thus, MPs can give rise
to either two identical neurons (MP1) or two non-identical neurons (MP3-6).
The MNB stem cell divides asymmetrically to generate about eight GABAergic
neurons during embryogenesis, and a much larger number postembryonically
(Truman et al., 2004). Despite
the small number of embryonic midline cells, the origins of midline neurons
and glia remain largely unknown. In this study, for the first time, we
identified each MP and described their patterns of cell division. This
information was then utilized to reveal multiple roles of Notch
signaling in midline neuronal and glial cell development.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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),
numb4 (Skeath and Doe,
1998), spdoG104 and
spdoZ143 (Skeath and
Doe, 1998), N55e11, Nts1,
P[12xSu(H)bs-lacZ] (Go et al.,
1998) and Gbe-lacZ
(Furriols and Bray, 2001).
Gal4 and UAS lines used were: sim-Gal4
(Xiao et al., 1996),
UAS-numb (Wang et al.,
1997), UAS-spdo
(O'Connor-Giles and Skeath,
2003), UAS-Su(H).VP16
(Kidd et al., 1998) and
UAS-tau-GFP (Brand,
1995). For N temperature-shift experiments,
N55e11/Nts1 embryos were collected for
2 hours at 18°C, further incubated for 2 hours at 18°, then shifted to
the restrictive temperature (30°C) for 6 hours, followed by fixation
(approximately stage 14).
In situ hybridization and immunostaining
Embryo collection, in situ hybridization and immunostaining were performed
as previously described (Kearney et al.,
2004). Primary antibodies used were: mouse (Promega) and rabbit
(Cappel) anti-β-galactosidase, rabbit anti-Cas
(Kambadur et al., 1998), mouse
and rat anti-Elav [Developmental Studies Hybridoma Bank (DSHB)], mouse anti-En
MAb 4D9 (Patel et al., 1989),
anti-Futsch MAb 22C10 (DSHB), guinea pig anti-Hb [East Asian Distribution
Center (EADC)] (Kosman et al.,
1998), chicken anti-GFP (Upstate), rabbit anti-GFP (Abcam), guinea
pig anti-Lim1 (Broihier and Skeath,
2002), guinea pig anti-Numb
(O'Connor-Giles and Skeath,
2003), rabbit anti-Odd (Ward
and Skeath, 2000), rabbit anti-Period (Per)
(Liu et al., 1992), rabbit
anti-phosphohistone H3 (Millipore), guinea pig anti-Runt (EADC), guinea pig
and rat anti-Sim (Ward et al.,
1998), rabbit anti-Spdo
(O'Connor-Giles and Skeath,
2003), mouse anti-Tau (Sigma) and rat anti-Tup
(Broihier and Skeath, 2002).
Alexa Fluor-conjugated secondary antibodies were used (Molecular Probes). The
Tyramide Signal Amplification System (Perkin Elmer) was employed for some
immunostaining.
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; Wheeler et al.,
2006). Midline cells were examined in abdominal segments A1-8.
Owing to the three-dimensional structure of the midline cells, it was
difficult to represent all relevant cells in a single focal plane, so, for
clarity, irrelevant portions of single images within a stack of confocal
images were subtracted and projections were generated. Thus, a single
composite image is made from different focal planes that each contained
relevant data.
Live imaging of midline cells
Time-lapse imaging of midline cell development was carried out in
sim-Gal4 UAS-tau-GFP and sim-Gal4 UAS-tau-GFP;
Dl3/Dl3 embryos by visualizing GFP.
Embryos were collected for 1 hour, aged for an additional 4 hours,
dechorionated, mounted on a glass coverslip, and immersed in halocarbon oil
700 on slides containing an oxygen-permeable membrane. GFP-fluorescent images
were captured using a Nikon Eclipse TE300 equipped with a Perkin Elmer
Ultraview confocal scanner and 40x or 60x oil-immersion
objectives. Embryos were visualized for 4 hours with an image captured
every 30 seconds. Movies were assembled from images of single focal planes
using MetaMorph software (Molecular Devices). Ten movies of wild-type embryos
viewing 29 segments, and five movies of Dl mutant embryos viewing 14
distinct groups of cells, were analyzed.
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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), which allowed identification of individual midline cells.
In this paper, we mapped the midline cell expression of 16 genes
(Fig. 1 and see Fig. S1 in the
supplementary material) at multiple periods during stages 10-11 of
embryogenesis. Each of the MPs, the MNB, and their progeny, were defined and
distinguished from each other by gene expression differences, position, size
and visualization of cell division. These data provided strong evidence that
MP divisions occur during stage 11, as confirmed by time-lapse imaging of
midline cells in sim-Gal4 UAS-tau-GFP embryos
(Fig. 2 and see Movie 1 in the
supplementary material).
At early stage 10, the midline cells constitute a monolayer along the
anterior-posterior axis. However, beginning at late stage 10, MPs began to
delaminate, and migrated basally (internally). As the cells migrated, they
retracted a cytoplasmic process from the apical surface. The MP1,3,4
precursors acquired a flattened shape, resided internal to the MG, and were
separated from other MPs by MG. The five MPs were arranged in a defined order,
MP1
MP3MP4MP5MP6 (anterior to posterior), within the
segment. However, they delaminated and divided in the order
MP4MP3MP5MP1MP6. The MP divisions were characterized
by loss of an apical projection, retraction of the MG that separate the MPs,
and the subsequent juxtaposition of neuronal progeny. The MP3-6 divisions were
along the apical-basal axis, whereas the MP1 division was perpendicular to the
apical-basal axis. After the MPs divided, the MNB delaminated posterior to the
MP6 progeny and began dividing to generate ganglion mother cells (GMCs).
Notch signaling promotes midline glia, MNB and MP5,6 formation and inhibits MP1,3,4 formation
Based on the important roles of Notch signaling in CNS
development, Delta (Dl) and Notch (N)
mutants were screened for midline phenotypes, including alterations in
expression of midline-expressed genes. In both Dl3
homozygotes and Dl3/Dl7
transheterozygotes, an increase was observed in the number of midline neurons
at the expense of MG (Fig. 3).
At stage 14, the number of MP1 neurons increased from two cells/segment to
9.3±1.6 (n=14 segments) cells
(Fig. 3A,F). The number of
H-cells increased from one cell/segment to 9.6±1.1 (n=17)
(Fig. 3B,G), and the number of
mVUMs increased from three cells/segment to 11.5±1.7 (n=51)
(Fig. 3C,H). H-cell sib- and
iVUM-specific gene expression was absent in Dl mutants (not shown).
As described below, in the absence of Notch signaling, all MP3
neurons are H-cells and all VUMs are mVUMs owing to cell fate defects. Both
MP1 and MP3 neurons increased 5-fold in Dl mutant embryos. The
VUM neurons, by contrast, increased only 2-fold.
This disparity led us to investigate the identity of the mVUM neurons
observed in Dl mutants. All mVUMs can be uniquely identified in the
midline by Tyramine β hydroxylase (Tbh)
expression, and mVUM4-6 can be distinguished from each other based on
Tyrosine kinase-related protein (Tkr) and Castor (Cas)
levels. The wild-type mVUM4 and mVUM5 neurons are Tkr-,
whereas mVUM6 is Tkr+
(Fig. 4A). The expanded
Tbh+ mVUMs in Dl mutants were
Tkr- (Fig.
4C), indicating that none was mVUM6. The one significant
difference between wild-type mVUM4 and mVUM5 is that mVUM4 has low levels of
Cas (Caslo) and mVUM5 has high levels of Cas (Cashi)
(Fig. 4B). Quantitation of Cas
staining intensity was measured using the Mean Gray Value (MGV) function of
ImageJ (Abramoff et al., 2004).
In wild type (n=4 segments), mVUM4, mVUM5 and mVUM6 showed MGVs of
67, 155 and 22, respectively. In Dl mutants (n=6 segments),
all Tbh+ cells showed similar MGVs with an average of 67,
identifying them as mVUM4s. These results, together with the observation that
the 11.5 mVUMs/segment observed in Dl mutants was close in number to
the approximately nine MP1s and ten H-cells observed, suggested an expansion
of a single VUM precursor, probably the MP4.
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30) could be due to either: (1) a transformation of all of the 16
midline cells to MPs1,3,4, followed by a single division of each MP; or (2) an
overproliferation of one or a few MP1,3,4 cells, accompanied by the death or
unrecognizable fate change of the other midline cells. This was tested by
assaying stage 10-11 Dl mutant embryos for gene expression and
positions and timing of cell division. Late stage 10 mutant embryos had an
increased number of Odd-skipped (Odd)+ MP1s (4.1±1.2;
n=17) (Fig. 4E,F).
Live imaging of Dl mutant embryos during stage 11 indicated that the
observable MP divisions occurred within a relatively short time interval
(88±16 minutes) (see Movie 2 in the supplementary material). Divisions
of closely juxtaposed cells were frequently observed to occur in close
temporal sequence in both live imaging and fixed embryos stained for
phosphohistone H3 (Fig. 4G).
There was no evidence of cell death. Confocal imaging of stage 11 Dl
embryos, after division, revealed 7.9±2.1 (n=19)
Odd+ Cas+ MP1 neurons, 6.9±1.4 (n=12)
Odd- Cas- MP3 neurons, and 10.0±2.2
(n=7) Odd- Cas+ MP4s
(Fig. 4H). These data are most
consistent with a model in which there is a transformation of 16 midline
cells into approximately five MP1s, five MP3s and six MP4s, followed by a
single division of each MP. In contrast to the expansion of MP1,3,4-derived neurons in Dl mutants, there was an absence of MG and of the MNB. MG gene expression was reduced from 10.0±1.3 (n=15) cells/segment in the wild type to 0.1±0.2 (n=176) cells/segment in Dl mutants (Fig. 3E,J). The wild-type MNB has prominent expression of three genes: worniu (wor) (Fig. 3D), miranda (mira) (not shown), and sanpodo (spdo) (not shown), which are specific to the MNB after stage 11. In stage 14 Dl mutant embryos, wor (n=84) (Fig. 3I), mira (n=56) and spdo (n=47) expression was absent from the midline. Involvement of the Notch receptor was confirmed by analysis of a N mutant combination, Nts1/N55e11, that showed similar phenotypes to Dl mutants, although at a reduced frequency (not shown).
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) in all
midline cells. Stage 14 sim-Gal4 UAS-Su(H).VP16 embryos were examined
because MG undergo apoptosis beginning at stage 15. At stage 14, these embryos
showed a 3-fold increase in the number of MG (30±5.5 cells/segment,
n=19) compared with wild type (10.0±1.3, n=15)
(Fig. 3E,O, and see Fig. S2A-H
in the supplementary material). The expanded MG had wild-type properties: they
underwent apoptosis, both AMG and PMG were present, and they wrapped
commissural axons. In addition, there was a near complete absence of midline
axons (see Fig. S3A,B in the supplementary material) and fewer than one
MP-derived neuron/segment was present (Fig.
3K-M). The larval and adult phenotypes of these midline
neuron-less animals were assessed: 62% of embryos survived to adulthood, but
were female sterile (see Fig. S3C in the supplementary material), and larvae
had reduced motility (see Fig. S3D in the supplementary material). When
sim-Gal4 UAS-Su(H).VP16 embryos were stained for the MNB markers
wor (Fig. 3N),
mira and spdo, there was an increase in cell number from one
cell/segment in the wild type to 4.9±1.8 (n=12). These
wor+ cells also had MG gene expression. The expansion of
MNB gene expression was consistent with the Dl mutant data indicating
that Notch signaling was required for MNB formation. By contrast,
there was no evidence that Su(H).VP16 misexpression resulted in
additional MP5,6 progeny.
To further understand the spatial and temporal dynamics of midline
Notch signaling, the expression of two reporters of Su(H)
activity was examined: P[12xSu(H)bs-lacZ]
(Go et al., 1998) and
Gbe-lacZ (Furriols and Bray,
2001). Reporter expression was observed in AMG and PMG during
stage 10, and was maintained through to the end of embryogenesis, although
levels were low by stage 17 (see Fig. S2I-L in the supplementary material).
Expression was dependent on Notch signaling, as it was absent in the
CNS midline cells in Dl mutant embryos (see Fig. S2M,N in the
supplementary material). In addition to MG, expression of
P[12xSu(H)bs-lacZ] was present in MP5,6 and in the MNB (see Fig.
S2I-K in the supplementary material) during stage 11, prior to their division.
MP5 expresses a low level of P[12xSu(H)bs-lacZ], MP6 an intermediate
level, and the MNB higher levels. After division, the MP5,6 and MNB progeny
express P[12xSu(H)bs-lacZ] at the same relative levels as the
precursors (see Fig. S2L in the supplementary material). The neuronal
expression is maintained throughout embryogenesis. No expression of the
reporter was observed in MP1,3,4 or their progeny. The expression pattern of
Gbe-lacZ was similar, although levels of lacZ expression
were reduced compared with P[12xSu(H)bs-lacZ]. These data indicate
that Notch signaling is occurring in MG, MP5, MP6 and the MNB during
stages 10-11, consistent with genetic requirements for Notch
signaling in these cells.
numb and spdo direct sibling neuronal fates in MP asymmetric divisions
MPs either divide symmetrically (MP1) or asymmetrically (MP3-6). A possible
mechanism for generating MP asymmetric cell fates is asymmetric localization
of Numb in conjunction with Notch signaling. To assess cell fate in
Dl, numb and spdo mutant and overexpression embryos, the
MP1, MP3 and VUM neurons were analyzed for changes in the expression of 37
genes, which encode transcription factors, signaling molecules,
neurotransmitter biosynthetic enzymes, neurotransmitter receptors and
neuropeptide receptors. Additionally, axonal trajectories were analyzed based
on sim-Gal4 UAS-tauGFP visualization.
MP3 neurons
Analysis of 19 genes expressed in the H-cell and H-cell sib neurons showed
that H-cell-specific gene expression was absent in numb mutant
embryos (Fig. 5A,B,F,G), but
was present in both neurons in spdo mutants
(Fig. 5K,L). The opposite
results were observed for H-cell sib-specific gene expression
(Fig. 5C,D,H,I,M,N). Another
indicator of neuronal cell fate is axonal trajectory. Consistent with the gene
expression results, numb mutants showed an absence of H-cell axons
and the presence of H-cell sib axons, whereas spdo mutants showed the
opposite phenotype (see Fig. S4A-C in the supplementary material). These
results were confirmed by analysis of H-cell gene expression in
numb-overexpression and Dl mutant embryos. When
numb was overexpressed in all midline cells, there were two
pale (ple)+ cells (H-cells), an absence of
CG13565+ H-cell sib, and a duplication of H-cell axons
(Fig. 5P,Q; see Fig. S4D in the
supplementary material). Overexpression of spdo did not result in
cell fate defects (Fig. 5R).
Analysis of Dl mutant embryos revealed an expansion of neurons
derived from the MP3. Only ple+ H-cells
(Fig. 3G), and not
CG13565+ H-cell sibs (data not shown), were present. Four
genes, including POU domain protein 2 (pdm2)
(Fig. 5E,J,O), that are
expressed in both cell types had no alterations in expression in either
numb or spdo mutant embryos, indicating that numb
and spdo affect cell type-specific gene expression, but not
expression present in both cells. Thus, assays of both neuronal morphology and
gene expression indicated that Notch controls all of the divergent
aspects of H-cell versus H-cell sib cell fate.
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MP1 neurons
The MP1 neurons are unique among MP progeny in that they appear identical.
Consequently, their development might be independent of numb and
spdo regulation. This was addressed by examining mutant and
overexpression embryos for ten MP1-expressed genes (see Table S1 and Fig. S5
in the supplementary material). There were no alterations in MP1 neuronal gene
expression in numb, spdo or sim-Gal4 UAS-numb embryos (see
Fig. S5 in the supplementary material), nor were there alterations in MP1
neuronal axonal trajectories (see Fig. S5A-E,G in the supplementary material).
These data indicate that numb and spdo do not play a role in
the cell fate specification of MP1 neurons. In Dl mutant embryos, we
observed an expanded set of neurons that are Hunchback+ and
Odd+ (Fig. 3F);
within the midline, these genes are specific for the MP1 neurons. Taken
together, the Dl, numb and spdo mutant results suggest that
Notch signaling is not important for MP1 cell fate determination.
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;
O'Connor-Giles and Skeath,
2003). In summary, MPs asymmetrically generate a Numb+
intracellular Spdo+ neuron (H-cell, mVUM) and a Numb-
cortical Spdo+ neuron (H-cell sib, iVUM). What happens in the MP1, which generates two identical neurons? In this case, Numb was uniformly localized to the membrane prior to, during and after MP1 cell division (Fig. 7E,F). Spdo was found at the membrane and in cytoplasmic puncta prior to and during division, and in both progeny after division (Fig. 7K,L). Although Numb is present in both MP1 neurons, other mechanisms must cause these cells to be refractory to Notch signaling because numb mutants do not exhibit changes in MP1 gene expression.
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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; Goodman et
al., 1981; Jia and Siegler,
2002). We propose that the Drosophila cells described
here are homologous, and that MP4 gives rise to the anterior pair of VUMs
(VUM4s), MP5 to the medial VUM pair (VUM5s), and MP6 to the posterior VUM pair
(VUM6s). This picture of Drosophila stage 11 MP divisions runs
counter to the prevailing Drosophila models, which propose that the
MP divisions occur at stage 8 during the 1414 synchronous
cell division (Bossing and Technau,
1994; Jacobs,
2000; Klambt et al.,
1991). Instead, we propose that the precursors dividing at stage 8
give rise to glial-glial, neuronal-neuronal and mixed glial-neuronal lineages
(Fig. 8). In general, this new
model fits DiI-labeling data from previous reports in which mixed clones were
noted (Bossing and Technau,
1994; Schmid et al.,
1999), but no compelling arguments put forward for how they
arose.
Notch signaling directs the formation of midline glia and inhibits neurogenesis
Dl mutant and Su(H) misexpression experiments indicated
that: (1) Notch signaling is required for the formation of both AMG
and PMG; (2) Dl is a ligand for N; and (3) transcriptional output involves
Su(H) beginning at stage 10. Consistent with these results, analysis of a
Nts mutant showed changes in expression in MG and neuronal
enhancer-trap lines, but lacked specific markers to fully characterize the
phenotype (Menne and Klambt,
1994). Genes of the Enhancer of split-Complex
[E(spl)-C] are commonly activated by Notch signaling and
repress transcription. We note that the HLHm5 E(spl)-C gene is
expressed specifically in MG at stages 10-11
(Fig. 1), and other
E(spl)-C members are also expressed in midline cells
(Kearney et al., 2004;
Wech et al., 1999). While
E(spl)-C genes could be direct targets of Su(H) and repress midline
neuronal gene expression in MG, what activates MG gene expression? The
sim gene was previously shown to activate MG gene transcription
(Ma et al., 2000;
Wharton et al., 1994), and
could be a direct target of Su(H).
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). Do the
expanded Dl mutant MP1,3,4 neurons result from transformation of MG
precursors to MPs, or from excessive division of a small number of MPs?
Analysis of Dl mutants at stages 10-11 suggests that the midline
cells at these stages consist of approximately five MP1s, five MP3s and six
MP4s. If each divided once, this would equal the ten MP1 neurons, ten H-cells
and 12 mVUM4s observed in Dl mutant embryos at later stages. In this
model (Fig. 8), Notch
signaling promotes MG development, while MP1, MP3 and MP4 are selected from
their respective MP fields. This midline role for Notch parallels
known functions of Notch in both Drosophila and vertebrates,
in which it promotes gliogenesis and inhibits neurogenesis
(Gaiano et al., 2000;
Morrison et al., 2000;
Udolph et al., 2001).
Notch signaling promotes MNB and MP5,6 formation
The progeny of MP5,6 and the MNB were absent from Dl mutants,
indicating that Notch signaling is required for the formation of the
MNB as well as for VUM5,6. This was a surprising result for the MNB because in
the ventral nerve cord, Notch signaling inhibits NB formation early
in development (Campos-Ortega,
1993) and plays no apparent role in the asymmetric division of
postembryonic nerve cord NBs (Almeida and
Bray, 2005). However, Notch signaling controls central
brain NB number (Lee et al.,
2006; Wang et al.,
2007), indicating a parallel between the MNB and brain NBs. Thus,
the MNB has a number of properties distinct from other nerve cord NBs in that
it is not part of a neural/epidermal equivalence group and does not utilize
the Hunchback>Krüppel>Pdm>Cas cascade
(Isshiki et al., 2001).
Similarly, it is unusual that VUM5,6 require Notch function, as
Notch signaling inhibited MP1,3 and MP4 neurogenesis. Consistent with
the genetic data, P[12xSu(H)bs-lacZ] expression is restricted to
MP5,6 and the MNB. This suggests that the different responses to
Notch signaling might reflect anterior-posterior location. However,
there might also be differences with respect to cell type, because
sim-Gal4 UAS-Su(H).VP16 embryos have expanded MNB-like cells, but the
MP5,6 cells were not expanded. One potential model involves successive waves
of signaling, by Notch or other signaling molecules, to generate the
MNB, MP5,6 and MG, similar to what happens during development of the
Drosophila retina (e.g. Doroquez
and Rebay, 2006). Bossing and Brand have proposed an equivalence
group in which Notch signaling would inhibit cells from becoming a
MNB, and instead promote the VUM cell fate
(Bossing and Brand, 2006).
However, our Dl mutant and Su(H).VP16 misexpression data
indicate that Notch signaling promotes, not inhibits, MNB formation.
Another view is that the presence of PMG is required for MP5,6 and MNB
formation, and that the absence of PMG in Dl mutants also results in
the loss of the neural precursors. In summary, alterations in Notch
signaling have revealed its requirement in the formation of MP5,6 and the MNB,
but additional work will be required for mechanistic insight.
Notch signaling and numb generate asymmetric midline neuronal cell fates
Asymmetric neuronal cell fates of MP3-6 progeny are determined by Numb and
Spdo asymmetric localization in one of the two daughter cells
(Fig. 8), similar to asymmetric
cell fate determination of the non-midline MP2 cell and GMCs
(O'Connor-Giles and Skeath,
2003; Spana and Doe,
1996; Spana et al.,
1995). In the H-cell sib and iVUMs, Numb is absent, and
Notch signaling, in combination with cortical Spdo, activates H-cell
sib- and iVUM-specific gene expression and represses H-cell and mVUM gene
expression. Genes that are expressed in both siblings are not dependent on
Notch signaling. The MP1 progeny are identical by gene expression and
morphological criteria. Numb is present in both MP1 neurons, but the
significance of this is unclear because MP1 gene expression and morphology
were unaffected in numb mutants; nor were defects observed in
Dl mutants. This suggests that Notch signaling does not
influence MP1 development.
Another difference between MP1 and the other MPs is that MP1 divides perpendicular to the apical-basal axis, whereas MP3-6 rotate their spindles during cell division along the apical-basal axis. The basal cell is always the Numb+ cell, which is the Notch-independent H-cell or mVUM. The orientations of the divisions might aid in positioning the cells towards their final locations in the CNS. In the mature CNS, the iVUMs are apical to the mVUMs, and during MP divisions the iVUM is the more apical sibling. In the case of the MP1s, it might be important that both cells are in the same position along the basal/apical axis.
Kuwada and Goodman examined the development of grasshopper MP3
(Kuwada and Goodman, 1985).
Their data suggested a model in which the two MP3 neurons are born equivalent
with an H-cell sib dominant fate, and, within 5 hours, signaling between the
two cells generates different fates. These data appear inconsistent with the
Drosophila results, as the Drosophila MP3 neurons
asymmetrically localize Numb and are inherently different at birth. However,
it is important to recognize that the grasshopper and Drosophila
results are based on different types of experiments (genetic versus
experimental ablation), and the grasshopper data might be revealing additional
levels of regulation or different mechanisms for generating cell fates.
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Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/18/3071/DC1
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Abramoff, M. D., Magelhaes, P. J. and Ram, S. J.
(2004). Image processing with ImageJ. Biophotonics
Int. 11,36
-42.
Almeida, M. S. and Bray, S. J. (2005).
Regulation of post-embryonic neuroblasts by Drosophila Grainyhead.
Mech. Dev. 122,1282
-1293.[CrossRef][Medline]
Bate, C. M. and Grunewald, E. B. (1981).
Embryogenesis of an insect nervous system II: a second class of neuron
precursor cells and the origin of the intersegmental connectives.
J. Embryol. Exp. Morphol.
61,317
-330.[Medline]
Bossing, T. and Technau, G. M. (1994). The fate
of the CNS midline progenitors in Drosophila as revealed by a new method for
single cell labelling. Development
120,1895
-1906.[Abstract]
Bossing, T. and Brand, A. H. (2006).
Determination of cell fate along the anteroposterior axis of the Drosophila
ventral midline. Development
133,1001
-1012.
Brand, A. (1995). GFP in Drosophila.
Trends Genet. 11,324
-325.[CrossRef][Medline]
Broihier, H. T. and Skeath, J. B. (2002).
Drosophila homeodomain protein dHb9 directs neuronal fate via crossrepressive
and cell-nonautonomous mechanisms. Neuron
35, 39-50.[CrossRef][Medline]
Campos-Ortega, J. A. (1993). Early neurogenesis
in Drosophila melanogaster. In The Development of
Drosophila melanogaster, vol. 2 (ed. M.
Bate and A. M. Arias), pp. 1091-1129. Cold Spring
Harbor, NY: Cold Spring Harbor Laboratory Press.
Crews, S. T. (2003). Drosophila bHLH-PAS
developmental regulatory proteins. In PAS Proteins: Regulators and
Sensors of Development and Physiology (ed. S. T. Crews), pp.69
-108. New York: Kluwer Academic
Publishers.
Doroquez, D. B. and Rebay, I. (2006). Signal
integration during development: mechanisms of EGFR and Notch pathway function
and cross-talk. Crit. Rev. Biochem. Mol. Biol.
41,339
-385.[CrossRef][Medline]
Foe, V. E. (1989). Mitotic domains reveal early
commitment of cells in Drosophila embryos. Development
107, 1-22.[Abstract]
Furriols, M. and Bray, S. (2001). A model Notch
response element detects Suppressor of Hairless-dependent molecular switch.
Curr. Biol. 11,60
-64.[CrossRef][Medline]
Gaiano, N., Nye, J. S. and Fishell, G. (2000).
Radial glial identity is promoted by Notch1 signaling in the murine forebrain.
Neuron 26,395
-404.[CrossRef][Medline]
Go, M. J., Eastman, D. S. and Artavanis-Tsakonas, S.
(1998). Cell proliferation control by Notch signaling in
Drosophila development. Development
125,2031
-2040.[Abstract]
Goodman, C. S., Bate, M. and Spitzer, N. C.
(1981). Embryonic development of identified neurons: origin and
transformation of the H cell. J. Neurosci.
1, 94-102.[Abstract]
Hutterer, A. and Knoblich, J. A. (2005). Numb
and alpha-Adaptin regulate Sanpodo endocytosis to specify cell fate in
Drosophila external sensory organs. EMBO Rep.
6, 836-842.[CrossRef][Medline]
Isshiki, T., Pearson, B., Holbrook, S. and Doe, C. Q.
(2001). Drosophila neuroblasts sequentially express transcription
factors which specify the temporal identity of their neuronal progeny.
Cell 106,511
-521.[CrossRef][Medline]
Jacobs, J. R. (2000). The midline glia of
Drosophila: a molecular genetic model for the developmental functions of glia.
Prog. Neurobiol. 62,475
-508.[CrossRef][Medline]
Jia, X. X. and Siegler, M. V. (2002). Midline
lineages in grasshopper produce neuronal siblings with asymmetric expression
of Engrailed. Development
129,5181
-5193.[Medline]
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.
Kearney, J. B., Wheeler, S. R., Estes, P., Parente, B. and
Crews, S. T. (2004). Gene expression profiling of the
developing Drosophila CNS midline cells. Dev. Biol.
275,473
-492.[CrossRef][Medline]
Kidd, S., Lieber, T. and Young, M. W. (1998).
Ligand-induced cleavage and regulation of nuclear entry of Notch in Drosophila
melanogaster embryos. Genes Dev.
12,3728
-3740.
Klambt, C., Jacobs, J. R. and Goodman, C. S.
(1991). The midline of the Drosophila central nervous system: a
model for the genetic analysis of cell fate, cell migration, and growth cone
guidance. Cell 64,801
-815.[CrossRef][Medline]
Kosman, D., Small, S. and Reinitz, J. (1998).
Rapid preparation of a panel of polyclonal antibodies to Drosophila
segmentation proteins. Dev. Genes Evol.
208,290
-294.[CrossRef][Medline]
Kuwada, J. Y. and Goodman, C. S. (1985).
Neuronal determination during embryonic development of the grasshopper nervous
system. Dev. Biol. 110,114
-126.[CrossRef][Medline]
Lee, C. Y., Andersen, R. O., Cabernard, C., Manning, L., Tran,
K. D., Lanskey, M. J., Bashirullah, A. and Doe, C. Q. (2006).
Drosophila Aurora-A kinase inhibits neuroblast self-renewal by regulating
aPKC/Numb cortical polarity and spindle orientation. Genes
Dev. 20,3464
-3474.
Liu, X., Zwiebel, L. J., Hinton, D., Benzer, S., Hall, J. C. and
Rosbash, M. (1992). The period gene encodes a predominantly
nuclear protein in adult Drosophila. J. Neurosci.
12,2735
-2744.[Abstract]
Ma, Y., Certel, K., Gao, Y., Niemitz, E., Mosher, J., Mukherjee,
A., Mutsuddi, M., Huseinovic, N., Crews, S. T., Johnson, W. A. et al.
(2000). Functional interactions between Drosophila bHLH/PAS, Sox,
and POU transcription factors regulate CNS midline expression of the slit
gene. J. Neurosci. 20,4596
-4605.
Menne, T. V. and Klambt, C. (1994). The
formation of commissures in the Drosophila CNS depends on the midline cells
and on the Notch gene. Development
120,123
-133.[Abstract]
Morrison, S. J., Perez, S. E., Qiao, Z., Verdi, J. M., Hicks,
C., Weinmaster, G. and Anderson, D. J. (2000). Transient
Notch activation initiates an irreversible switch from neurogenesis to
gliogenesis by neural crest stem cells. Cell
101,499
-510.[CrossRef][Medline]
O'Connor-Giles, K. M. and Skeath, J. B. (2003).
Numb inhibits membrane localization of Sanpodo, a four-pass transmembrane
protein, to promote asymmetric divisions in Drosophila. Dev.
Cell 5,231
-243.[CrossRef][Medline]
Patel, N. H., Martin-Blanco, E., Coleman, K. G., Poole, S. J.,
Ellis, M. C., Kornberg, T. B. and Goodman, C. S. (1989).
Expression of engrailed proteins in arthropods, annelids, and chordates.
Cell 58,955
-968.[CrossRef][Medline]
Schmid, A., Chiba, A. and Doe, C. Q. (1999).
Clonal analysis of Drosophila embryonic neuroblasts: neural cell types, axon
projections and muscle targets. Development
126,4653
-4689.[Abstract]
Skeath, J. B. and Doe, C. Q. (1998). Sanpodo
and Notch act in opposition to Numb to distinguish sibling neuron fates in the
Drosophila CNS. Development
125,1857
-1865.[Abstract]
Spana, E. P. and Doe, C. Q. (1996). Numb
antagonizes Notch signaling to specify sibling neuron cell fates.
Neuron 17,21
-26.[CrossRef][Medline]
Spana, E. P., Kopczynski, C., Goodman, C. S. and Doe, C. Q.
(1995). Asymmetric localization of numb autonomously determines
sibling neuron identity in the Drosophila CNS.
Development 121,3489
-3494.[Abstract]
Thomas, J. B., Crews, S. T. and Goodman, C. S.
(1988). Molecular genetics of the single-minded locus: a
gene involved in the development of the Drosophila nervous system.
Cell 52,133
-141.[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.
Udolph, G., Rath, P. and Chia, W. (2001). A
requirement for Notch in the genesis of a subset of glial cells in the
Drosophila embryonic central nervous system which arise through asymmetric
divisions. Development
128,1457
-1466.[Abstract]
Uemura, T., Shepherd, S., Ackerman, L., Jan, L. Y. and Jan, Y.
N. (1989). numb, a gene required in determination of cell
fate during sensory organ formation in Drosophila embryos.
Cell 58,349
-360.[CrossRef][Medline]
Wang, H., Ouyang, Y., Somers, W. G., Chia, W. and Lu, B.
(2007). Polo inhibits progenitor self-renewal and regulates Numb
asymmetry by phosphorylating Pon. Nature
449,96
-100.[CrossRef][Medline]
Wang, S., Younger-Shepherd, S., Jan, L. Y. and Jan, Y. N.
(1997). Only a subset of the binary cell fate decisions mediated
by Numb/Notch signaling in Drosophila sensory organ lineage requires
Suppressor of Hairless. Development
124,4435
-4446.[Abstract]
Ward, E. J. and Skeath, J. B. (2000).
Characterization of a novel subset of cardiac cells and their progenitors in
the Drosophila embryo. Development
127,4959
-4969.[Abstract]
Ward, M. P., Mosher, J. T. and Crews, S. T.
(1998). Regulation of bHLH-PAS protein subcellular localization
during Drosophila embryogenesis. Development
125,1599
-1608.[Abstract]
Wech, I., Bray, S., Delidakis, C. and Preiss, A.
(1999). Distinct expression patterns of different enhancer of
split bHLH genes during embryogenesis of Drosophila melanogaster.
Dev. Genes Evol. 209,370
-375.[CrossRef][Medline]
Wharton, K. A., Jr, Franks, R. G., Kasai, Y. and Crews, S.
T. (1994). Control of CNS midline transcription by asymmetric
E-box-like elements: similarity to xenobiotic responsive regulation.
Development 120,3563
-3569.[Abstract]
Wheeler, S. R., Kearney, J. B., Guardiola, A. R. and Crews, S.
T. (2006). Single-cell mapping of neural and glial gene
expression in the developing Drosophila CNS midline cells. Dev.
Biol. 294,509
-524.[CrossRef][Medline]
Xiao, H., Hrdlicka, L. A. and Nambu, J. R.
(1996). Alternate functions of the single-minded and rhomboid
genes in development of the Drosophila ventral neuroectoderm. Mech.
Dev. 58,65
-74.[CrossRef][Medline]
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