Department of Cell Biology, Emory University School of Medicine,
Atlanta, GA 30322, USA
*
Author for correspondence (e-mail:
Kbhat{at}cellbio.emory.edu
)
Accepted 4 June 2001
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SUMMARY |
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Key words: Neurogenesis, Slit, Roundabout, Cell signaling, Ganglion mother cells, Asymmetric division, Drosophila melanogaster
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INTRODUCTION |
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In the Drosophila ventral nervous system, hundreds of different
cell types are generated outside of the midline from a relatively few primary
precursor cells called neuroblasts (Bate,
1976; Goodman and Doe,
1993
). During neurogenesis,
about 30 neuroblast (NB) cells in each hemisegment delaminate in a stereotyped
and spatiotemporal pattern. Following formation, each neuroblast functions as
a stem cell and divides asymmetrically renewing itself and producing a chain
of secondary neuronal precursor cells called ganglion mother cells (GMCs). A
GMC is always committed to differentiate and does not self-renew; it divides
asymmetrically to generate two distinct neurons (Bate,
1976
; Thomas et al.,
1984
; Skeath and Doe,
1998
; Dye et al.,
1998
; Buescher et al.,
1998
; Wai et al.,
1999
).
Recently, several genes such as inscuteable (insc),
miranda (mira), numb (nb) and
Notch (N) have been shown to be required for the asymmetric
division of neural precursor cells (Buescher et al.,
1998; Wai et al.,
1999
; Schober et al.,
1999
; Lu et al.,
1999
). The asymmetric
divisions mediated by these proteins appear to be tied to their asymmetric
segregation into one of the two daughter cells during division. For instance,
during the division of GMC-1 of the RP2/sib lineage, Insc localizes to the
apical end of GMC-1 while Nb segregates to the basal end. The cell that
inherits Nb is specified as RP2 owing to the ability of Nb to block Notch
signaling from specifying sib fate, whereas the cell that inherits Insc is
specified as sib by Notch. Thus, in insc mutants, Nb is distributed
to both cells and both the progeny of GMC-1 adopt RP2 fate; whereas in
nb mutants, they assume sib fate (Buescher et al.,
1998
; Wai et al.,
1999
; Lear et al.,
1999
).
In a screen for mutations that affect the development of
NB4-2GMC-1
RP2/sib, a typical neuroblast lineage, we discovered
that loss of function of sli and its upstream activator
single-minded (sim) affects the elaboration of GMC-1 and
GMC-1-1a of the aCC/pCC lineage in a partially penetrant manner. In this
study, we show that Sli signaling promotes the terminal asymmetric division of
GMCs of the RP2/sib and aCC/pCC lineages. In the RP2/sib lineage, in embryos
mutant for sli, robo or sim, the GMC-1 symmetrically divides
to generate two RP2 neurons. Analysis of the expression of Insc in
sli mutant embryos and analysis of double mutants between sli,
nb and sim; nb indicates that Sli signaling promotes the
asymmetric division of GMCs by regulating the asymmetric localization of Insc.
Our results also indicate that the disruption of the asymmetric localization
of Insc by loss of sli in at least GMC-1 of the RP2/sib lineage is
due to the up-regulation of the two POU proteins, Nubbin (Nub; also known as
Pdm1) and Mitimere (Miti; also known as Pdm2). Consistent with this, up
regulation of miti or nub in a late GMC-1 by over-expression
leads to mis-localization of Insc and symmetric division of GMC-1 to generate
2 RP2s. Moreover, while the penetrance of the symmetrical division phenotype
is not high in both sim and sli mutant embryos, doubling the
dosage of miti and nub in sim mutant background,
for instance, significantly enhances the penetrance; similarly, halving the
dosage of these POU genes suppresses the symmetrical division phenotype.
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MATERIALS AND METHODS |
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Miti and Nub over-expression experiments
To determine the effect of over-expression of miti and
nub, transgenic lines carrying these genes under the control of
hsp70 promoter were used. To determine if the GMC-1 undergoes a
symmetrical mitosis at elevated levels of Miti, Hs-miti or
Hs-nub transgenic embryos were collected for 2 hours, aged for 7
hours and subjected to a heat shock at 37°C for 20 minutes. These embryos
were allowed to develop until they reached stage 12 and 14 before being fixed.
To determine if the localization of Insc is affected in embryos ectopically
expressing Miti or Nub at high levels, embryos collected and aged as above
were heat shocked for 25 minutes. These embryos were allowed to recover for 20
minutes before being fixed and double stained for Eve and Insc. As control,
non-heat shocked transgenic embryos and heat shocked wild-type embryos were
used.
Antibodies and immunostaining
Embryos were fixed and stained with the following antibodies: Eve
(polyclonal, 1: 2000 dilution; monoclonal, 1:5), Zfh-1 (1:400), 22C10 (1:4),
Insc (1:500), Nub (1:50), Miti (1:10), Sim (1; 500), Robo (1:10), ß-gal
(1:3000 or 1:400), Ftz (1:50), Sli (1:200) and alpha-spectrin II (1:10). For
confocal microscopy, cy5 and FITC-conjugated secondary antibodies
were used. For light microscopy, alkaline phosphatase or DAB-conjugated
secondary antibodies, were used. Mutant embryos were identified using blue
balancers, marker phenotypes or immunostaining (lack of positive staining). To
examine Huckebein expression, a lacZ enhancer-trap line,
hkb5953 in the hkb locus was used.
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RESULTS |
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Initially, embryos mutant for sli were examined with
anti-Even-skipped (Eve) antibody. Eve is first expressed in GMC-1 of the
RP2/sib lineage (Fig. 1A); it
is also expressed in a newly formed RP2 and sib
(Fig. 1B). The sib eventually
loses Eve expression whereas RP2 maintains Eve
(Fig. 1C). Eve staining of
sli mutant embryos (sli2 or
sliGA20) revealed that the GMC-1 in sli mutants
frequently divides symmetrically to generate two RP2s instead of an RP2 and a
sib. Thus, while approx. 7-hour old sli embryos had only one GMC-1 as
in wild type, in 10% of the hemisegments (number of hemisegments examined,
n=1092; the maximum penetrance that we observed in a sli
mutant embryo was approx. 20%), approx. 8.5-hour old mutant embryos had two
cells of equal sizes, both expressing Eve
(Fig. 1D and G). This is in
contrast to the wild type where a larger RP2 and a smaller sib are faithfully
observed by 7.5-8 hours of age (Fig.
1A). It must be pointed out that there is no de novo synthesis of
Eve in sib; thus the entire stock of Eve protein in a sib is inherited from
GMC-1 and thus, the immunoreactivity for Eve in sib is ancestry
dependent/indicator. Similarly, in wild type the difference in the size of the
nuclei between RP2 and sib is generated prior to cytokinesis, and thus
inherent to the lineage (compare Buescher et al.,
1998). When approx. 10-hour old
sli embryos were examined with Eve, 7% of the hemisegments
(n=780) had two RP2s (Fig.
1E,H). In 13-hour old sli embryos, two RP2s of equal
sizes were observed (Fig. 1F,I)
in 11% of the hemisegments (n=776). Moreover, as shown in
Fig. 1G-I, symmetric division
of GMC-1 to generate two RP2s was also observed in single-minded
(sim) embryos in 9% of the hemisegments (n=210). Sim is a
transcription factor and is the upstream activator of sli (Thomas et
al., 1988
; Crews et al.,
1988
; Nambu et al.,
1990
; Klambt et al.,
1991
). We note that RP2 and
aCC neurons are occasionally misplaced within a hemisegment in the CNS of
14-hour or older sli mutant embryos, however, in embryos that are
less than 10 hours old the CNS is not severely affected and these cells do not
cross the midline or segmental boundary. Thus, the RP2 or aCC duplications
observed here are not due to migration of RP2s across the midline or segmental
boundaries (see also below).
|
We sought to obtain more direct evidence for the symmetric mitosis of
GMC-1. If the GMC-1 in a sli mutant embryo divides symmetrically to
generate two RP2s, the cytokinesis and nuclear division of GMC-1 must also be
symmetrical as opposed to the non-symmetrical nuclear and cytokinesis of GMC-1
in wild type. Thus, it must be possible to observe symmetrical versus
non-symmetrical division by examining GMC-1s that are undergoing cytokinesis
with cell cortex markers in combination with nuclear markers. Therefore,
sli mutant embryos were stained with the cell cortex marker spectrin
(Byers et al., 1987; Prokopenko
et al., 1999
) and the lineage
specific nuclear marker Eve. As shown in
Fig. 2A and C, the asymmetric
cytokinesis of GMC-1 (and the unequal nuclear sizes of daughter nuclei) to
generate two unequal cells in wild type can be faithfully observed using these
markers. However, in sli mutant embryos the GMC-1 undergoes a
symmetric cytokinesis with two nuclei of equal sizes to generate two equal
sized cells was observed (Fig. 2B and
D). (The method of visualization of GMC-1 division in live embryos
using green fluorescent protein was not possible since it takes >3 hours
for this protein to become fluorescent after it is made and this window of
time is too long as the GMC-1 completes its division by then.) Finally,
transformation of some other neuroblast into NB4-2 was not observed in
sli mutants as judged by the expression of Huckebein, a NB4-2
specific marker (Chu-LaGraff et al.,
1995
; Bhat,
1996
). Thus, these results
indicate that GMC-1 in sli or sim mutants undergoes a
symmetrical division to generate two RP2s.
|
The generation of an RP2 at the expense of the sib was further confirmed using additional cell-type specific markers. First, mutant embryos were double stained for Eve and Zfh-1. In wild type, Zfh-1 is never expressed in GMC-1, GMC1-1a, sib, pCC, or newly formed RP2 and aCC. Zfh-1 begins to be expressed in RP2 and aCC at approx. 9 hours of development (at 22°C) and continues to be expressed thereafter (Fig. 3A and B). In approx. 10-hour old sli embryos, both the progeny of GMC-1 of the RP2/sib lineage co-express high levels of Eve and Zfh-1 (Fig. 3C) and they continue to co-express high levels of Eve and Zfh-1 in approx. 13-hour or older mutant embryos (Fig. 3D). In the aCC/pCC lineage, both the progeny of GMC1-1a co-express Eve and Zfh-1 (Fig. 3E and F) in 8% of the hemisegments (n=1100), indicating that the two daughter cells have adopted an aCC identity in these hemisegments. Consistent with the possibility that the duplicated Eve- and Zfh-1-positive cells are RP2 and aCC neurons in sli embryos, they express 22C10 and have axon trajectory that of an RP2 (Fig. 3H) and aCC (Fig. 3I), respectively.
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The relationship between Slit, Insc and Nb during the asymmetric
division of GMC-1
The symmetric division of GMCs in sli mutants was similar to that
observed in insc, Notch or rapsynoid (raps; also
known as pins) mutants and opposite to that of nb (compare
Buescher et al., 1998; Wai et
al., 1999
; Lear et al.,
1999
; Yu et al.,
2000
). Previous results show
that the cytoplasmic adaptor protein Insc is required for the asymmetric
division of GMC-1 into RP2 and sib (Buescher et al.,
1998
). During GMC-1 division,
Insc protein localizes to the apical side (see
Fig. 4A) and Nb to the basal
side. The Nb-negative daughter cell becomes specified as sib by Notch
signaling whereas the cell that inherits Nb becomes an RP2 owing to the
blocking of Notch signaling by Nb. Thus, in insc mutants, both cells
inherit Nb and are specified as RP2 while in nb mutants both progeny
becomes sib. Given the similarity of sli, sim and insc
mutant phenotypes, we examined the relationship between Sli and Insc. First,
in sli mutants the localization of Insc in GMC-1, when examined, was
not asymmetric (Fig. 4B). About
7% of the hemisegments (n=440) showed this phenotype. A similar
non-localization of Insc was also observed in GMC1-1a of the aCC/pCC lineage
(Fig. 4D). In raps
mutant embryos, Insc is also not localized and as in sli the GMC-1
divides symmetrically to generate two RP2s (Yu et al.,
2000
). Thus, failure to
localize Insc in these GMCs in sli mutants is responsible for their
symmetric mitosis.
|
In insc, nb double mutants both the daughters of GMC-1 are
specified as sib by Notch signaling. In sli, nb (or sim, nb)
double mutant embryos also, both the progeny of GMC-1 adopt a sib fate
(Fig. 4F). Thus, Sli is
required upstream of Nb during the asymmetric division of GMC-1. Since the
GMC-1 symmetrically divides to yield two RP2s in Notch; nb double
mutants (Wai et al., 1999) and
two sibs in sli, nb double mutants, Sli is also upstream of Notch
signaling during the asymmetric division of GMC-1. These results also indicate
that when the GMC-1 in sli mutants symmetrically divides, both
daughters inherit Nb.
Slit signaling regulates the asymmetric division of GMC-1 by down
regulating Nubbin and Miti
Since previous results tie the two POU genes, miti and
nub, to the normal elaboration of the GMC-1 RP2/sib lineage
(Yang et al., 1993
; Bhat and
Schedl, 1994
; Bhat et al.,
1995
; Yeo et al.,
1995
), we examined the
expression of these genes in sli mutant embryos. In wild type, the
levels of Nub (or Miti), which are normally high in a newly formed GMC-1
(Fig. 5A,B), are down regulated
prior to the asymmetric division of GMC-1
(Fig. 5E,F). In sli
mutants the expression of Nub (or Miti) in a newly formed GMC-1 was comparable
to that of wild type, but, in a late GMC-1 the level remained high compared to
wild type (Fig. 5G and H). Previous results showed that a brief ectopic expression of these POU genes
from the hsp70 promoter prior to GMC-1 division induces GMC-1 to divide
symmetrically to generate two GMC-1s; each then divides asymmetrically to
generate an RP2 and a sib (Yang et al.,
1993
; Bhat et al.,
1995
). If the symmetric
division of GMC-1 in these mutants has anything to do with the lack of down
regulation of Nub and Miti in GMC-1, ectopic expression of miti or
nub should also induce GMC-1 to divide symmetrically to generate two
RP2 neurons. Indeed, a brief over-expression of miti (or
nub) in a late GMC-1 causes this GMC to divide symmetrically into two
RP2 neurons (Fig. 5I,J,L) in
27% of the hemisegments (n=770).
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Insc is non-localized in GMC-1 expressing high levels of Miti
The loss-of-function effects of sli on the distribution of Insc in
GMC-1 (and thus the symmetrical division of GMC-1) could be due to this lack
of down regulation of Miti and Nub in GMC-1. To test this possibility, the
miti transgene was ectopically expressed from the hsp70 promoter. A
25-minute induction of miti was sufficient to alter the localization
of Insc and the distribution of Insc in these embryos resembled the
distribution of Insc in sli embryos
(Fig. 5M).
The penetrance of the symmetrical division phenotype in sim
mutant is sensitive to the dosage of nub and miti genes
The penetrance of the symmetric division of GMC-1 phenotype in sli
and sim mutants was approx. 10%, indicating a partial genetic
redundancy for this pathway. Since the loss of asymmetric division of GMC-1 in
sli or sim appears to be due to a failure in the down
regulation of Nub and Miti, we reasoned that the penetrance of the phenotype
might be enhanced by increasing the copy numbers of these POU genes in
sli or sim background. Since a duplication for nub
and miti exists but the duplication is on the second chromosome {Dp
(2; 2) GYL)}, we examined sim; GYL embryos for the GMC-1 division
phenotype. As shown in Fig. 6C and
D, the penetrance of the phenotype in these embryos was enhanced
to 42% (n=70; symmetrical division of GMC-1 in GYL is approx. 2%,
n=840). Similarly, halving the copy numbers of the two POU genes in
sim background using a small deficiency [sim/sim;
Df (2L) GR4/+] that eliminates these two genes (Yeo et al.,
1995) suppressed the phenotype
to 1.4% (n=74; see also Fig.
6E).
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The above results indicate that the symmetrical division of GMC-1 in sli mutants is due to the up regulation of the two POU genes and that these two POU genes are the targets of Sli signaling in GMC-1; however, the partial penetrance of these phenotypes in sli mutants indicate that additional pathways also mediate this very same process and regulate the levels of the two POU proteins in GMC-1. Since the penetrance in insc mutants is also partial, additional pathways must exist to mediate the asymmetric division of GMC-1 to partially complement the loss of the Insc/Sli pathway.
Robo, a key receptor for Slit, partially mediates GMC-1 asymmetric
division
How is the Sli signal transmitted from outside to inside? Previous results
show that one of the receptors for Sli is the transmembrane protein encoded by
the robo locus (Kidd et al.,
1999; Wang et al.,
1999
; Brose et al.,
1999
; Li et al.,
1999
; Ba-Charvet et al.,
1999
). To determine if the
effect of Sli signaling on GMC-1 is mediated via Robo, the expression of Robo
in the GMC-1
RP2/sib and GMC-1-1a
aCC/pCC lineages was examined.
Double staining of wild-type embryos with anti-Eve and anti-Robo shows that
both these GMCs express Robo (Fig.
7A,B). Consistent with this, in robo null mutants (or
robonull/robodeficiency), GMC-1 and
GMC1-1a were found to divide symmetrically to generate two RP2s and two aCCs
at the expense of sib and pCC (Fig.
7C,D). Although the penetrance of the RP2 lineage phenotype was
low in robo mutants (
2.3%, n=1300), the fact that Robo
is expressed in GMC-1 and that the phenotype was observed only in
robo null mutants, argue that Robo at least partially transmits the
Sli signal and promotes the asymmetric division of GMC-1 into RP2 and sib.
Since three additional robo genes, robo2, robo3 (Rajagopalan
et al., 2000
; Simpson et al.,
2000
) and robo4 (our
unpublished results) exist in Drosophila, the weak penetrance is
likely to be due to genetic redundancy between these robo genes.
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DISCUSSION |
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The results presented here show that in sli mutants the GMC-1
divides symmetrically to produce two RP2s. Several lines of evidence support
this possibility. For instance, initially only one large cell with the
characteristics of a GMC-1 (in terms of its location and marker gene
expression pattern: Eve, Miti and Nub positive but Zfh-1 negative) appears in
a hemisegment. This GMC-1 undergoes a symmetrical nuclear division and
cytokinesis to yield two identical cells as revealed by spectrin and Eve
staining (Fig. 2). Unlike
previous studies on the problem of asymmetric mitosis of secondary neuronal
precursor cells (compare Buescher et al.,
1998; Wai et al.,
1999
; Yu et al.,
2000
), this particular
experiment provides more direct evidence for the symmetric division of GMC-1.
That both these cells adopt an RP2 identity is indicated by the expression of
RP2-specific genes and axon trajectory
(Fig. 3). Similarly, in the
aCC/pCC lineage, the GMC1-1a generates two aCC neurons at the expense of a pCC
cell.
The GMC-1 and GMC1-1a phenotypes in sli mutants mimicked those
phenotypes in insc mutants indicating that these genes function in
the same pathway. Consistent with this possibility is the finding that
localization of Insc in sli mutants was affected in both GMC-1 and
GMC1-1a (Fig. 4). A similar
non-asymmetric localization of Insc and duplication of RP2 neurons was also
observed in embryos mutant for raps, which encodes a protein required
for the proper localization of Insc (Yu et al.,
2000). Thus, these results tie
Sli signaling to insc, a gene known to regulate GMC-1 and GMC1-1a
asymmetric division. Our results show that the phenotypes in sli or
sim null mutants are partially penetrant. Since the penetrance in
sim mutants is also partial, additional pathways must also regulate
asymmetric division of GMCs. Moreover, it should be pointed out that in
insc mutants the GMC-1 division is normal in approx. 30% of the
hemisegments (n=280) despite having no insc. Similarly, the
penetrance of the symmetrical division of GMC-1 in raps (where Insc
localization is affected as in sli mutant embryos or embryos over
expressing miti or nub genes) or nb is also
partial, indicating the presence of additional (partially redundant) pathways
mediating the asymmetric division of GMC-1 independent of Insc or Nb. These
very same additional pathways must also contribute to the partial penetrance
of the phenotypes in sli mutants. Nonetheless, we emphasize that
while the penetrance of the phenotype is weak in sim, sli, and
robo mutants, it is significantly enhanced by doubling the copy
numbers of the two downstream target genes, miti and nub,
nearly to the same extent as in insc mutants.
In sli mutants, the distribution of Insc in GMC-1 is not asymmetric and this is likely to be the reason for the symmetric division of GMC-1 or GMC1-1a. While the regulation of Insc localization appears to require down-regulation of the two POU genes, it is not known how elevated levels of these POU genes alter the localization of Insc. Since Miti and Nub have the structural motif of DNA-binding proteins, elevated levels of Miti or Nub might repress genes that are required for the localization of Insc.
Our results indicate that the loss of Sli signaling affects the expression
and localization of Insc in GMC1-1a as it does in GMC-1 of the RP2/sib
lineage. Interestingly, nub and miti appear not to be the
targets of Sli signaling in GMC1-1a. This is based on the finding that ectopic
expression of miti or nub which alters the division pattern
of GMC-1, has no effect on the division pattern of GMC1-1a (Yang et al.,
1993; Bhat and Schedl,
1994
; Bhat et al.,
1995
). Similarly, while the
loss of miti and nub genes leads to a mis-specification of
GMC-1 identity, this has no effect on GMC1-1a (Bhat and Schedl,
1994
; Bhat et al.,
1995
; Yeo et al.,
1995
). Moreover, altering the
dosage of miti and nub in sim mutant background had
no effect on the division of GMC1-a. However, Sli signaling appears to
regulate the terminal asymmetric division of GMC1-1a by regulating Insc
localization. This is consistent with the fact that in insc mutants
GMC1-1a often generates two aCC neurons at the expense of a pCC.
The results described here indicate that GMC-1 also divides symmetrically
into 2 RP2s in embryos mutant for robo. Furthermore, we have shown
that Robo is expressed on the surface of GMC-1. Thus, Sli secreted from the
midline acts as a long-range signal and interacts with Robo on GMC-1 in row 4,
column 2 of the ventral nerve cord to regulate its division. The effect of
loss of robo on GMC-1 division is weakly penetrant. One possibility
is that there is a genetic redundancy between robo, robo2, robo3 (see
Simpson et al., 2000;
Rajagopalan et al., 2000
) and
robo4 (our unpublished results).
In summary, the following picture emerges from this study. The Sli-Robo signaling down regulates the levels of Nub and Miti in late GMC-1, allowing the asymmetric localization of Insc and the asymmetric division of GMC-1. We entertain the possibility that loss of sibling cells in sli mutants would mean that some projections will be duplicated, while others are eliminated. Depending upon the extent, this might have an overall bearing on the pathfinding defects in sli mutants. Since Sli signaling is conserved in vertebrates, it is possible that this signaling may regulate generation of asymmetry during vertebrate neurogenesis as well.
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ACKNOWLEDGMENTS |
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