|
|
|
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
First published online 15 December 2008
doi: 10.1242/dev.027854
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Department of Neuroscience and Cell Biology, University of Texas Medical Branch School of Medicine, Galveston, TX 77555, USA.
* Author for correspondence (e-mail: kmbhat{at}utmb.edu)
Accepted 13 November 2008
 |
SUMMARY |
|---|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
GMC-1RP2/sib, suggest that at least part of the process
operates in GMCs. That is, a GMC or a neuronal identity need not be determined
at the NB or NE level. This is demonstrated by showing that Mid is expressed
in a row 5 GMC (M-GMC), but not in its parent NB or NE cell. In mid
mutants, M-GMC changes into GMC-1 and generates an RP2 and a sib without
affecting the expression of key genes at the NE/NB levels. Expression of Mid
in the M-GMC in mid mutants rescues the fate change, indicating that
Mid specifies neurons at the GMC level. Moreover, we found a significant
plasticity in the temporal window in which a neuronal lineage can develop.
Although the extra GMC-1 in mid mutants is born 
2 hours later
than the bona fide GMC-1, it follows the same developmental pattern as the
bona fide GMC-1. Thus, a GMC identity can be independent of parental identity
and GMC formation and elaboration need not be strictly time-bound.
Key words: Neurogenesis, Drosophila, Midline, Transcription, Ancestry
|
INTRODUCTION |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
30 NBs in
a given hemisegment, 320 distinct and highly specialized neurons and 30
glial cells are generated (reviewed by
Gaziova and Bhat, 2006
).
For several years, we have been focusing on a few typical NB lineages with
the aim of understanding neuronal lineage elaborations in the ventral nerve
cord of the Drosophila embryo. One such lineage is
NB4-2GMC-1RP2/sib, a well-studied neuronal lineage
(Chu-LaGraff and Doe, 1993;
Bhat and Schedl, 1994;
Bhat et al., 1995;
Bhat, 1996;
Bhat and Schedl, 1997;
Duman-Scheel et al., 1997;
Bhat, 1998;
Buescher et al., 1998;
Wai et al., 1999;
Lear et al., 1999;
Bhat et al., 2000;
Mehta and Bhat, 2001;
Yedvobnick et al., 2004;
Bhat and Apsel, 2004;
Bhat, 1999;
Gaziova and Bhat, 2006). NB4-2
is a row 4, column 2 cell, and is formed as one of 30 NBs in a
hemisegment; it is an S2 NB formed during the second wave of NB delamination.
The identity of this NB is determined at the neuroectodermal (NE) level: the
segmentation protein Patched (Ptc) represses the expression of Gooseberry
(Gsb) in the precursor NE cells, allowing Wingless (Wg) signal reception and
the specification of NB4-2 identity in this cell. This NB4-2 specification
allows the cell to generate its first GMC, GMC-1 (also known as GMC4-2a);
GMC-1 then divides asymmetrically into a motoneuron called RP2 and a sibling
cell of as yet undetermined fate. Thus, a sequence of events governed by
combinatorial and interdependent genetic programs set in motion prior to the
formation of a NB appears to guide the identity of progeny neurons. This
theory has been further extended by the observation that once a NB divides,
its gene expression program changes, and this then dictates the identity of
the next GMC, as well as the identity of the daughter cells generated from
this GMC (Isshiki et al.,
2001). Thus, an ancestry-dependent mechanism, i.e. gene expression
programs in precursor cells, appears to strictly guide lineage development in
the Drosophila CNS. However, it is unclear if the integration of the
positional and temporal cues occurs in a linear fashion (i.e. one sets the
other) or whether cells at the level of GMCs and neurons can respond to
temporal cues independently of positional cues. A strict linear model seems
unlikely because temporal factors are not present in parent cells. Could it
be, then, that the positional cues are priming cells so that they can receive
signals later in development?
In a screen for mutants that affect the RP2/sib lineage, we identified
extra, a mutation characterized by an RP2-like extra neuron in each
hemisegment at the periphery of the nerve cord (K.M.B. and P. Wai,
unpublished). Our genetic analysis of extra indicated that it is
allelic to the previously identified mutation midline (mid)
(Nusslein-Volhard et al.,
1984) or lost in space (los)
(Kolodziej et al., 1995).
mid mutants exhibit cuticle defects, suggesting that Mid protein is
required for ectodermal patterning
(Nusslein-Volhard et al.,
1984). mid mutants also exhibit defects in the lateral
chordotonal axons, with shorter and defasciculated dorsally routed axons in
the peripheral nervous system (Kolodziej
et al., 1995). Given these results, we sought to examine
mid mutants in detail.
Cloning of the gene by us and others revealed that it encodes a T-box (Tbx)
protein. The Tbx proteins, which are highly conserved among metazoans, are
defined by the presence of a T-box domain, a 180-230 amino acid DNA-binding
domain. One of the most striking attributes of Tbx proteins is their dosage
sensitivity, i.e. developmental processes appear to be sensitive to the levels
of some Tbx proteins. For example, upper limb malformation and congenital
heart disease in Holt-Oram syndrome are caused by a haploinsufficiency of
TBX5 (Li et al.,
1997; Basson et al.,
1999). Haploinsufficiency of the Tbx genes mouse brachyury and
human TBX3 and TBX1, also produces dominant phenotypes:
short tails/tailless, Ulnar-Mammary syndrome and DiGeorge syndrome,
respectively (Bamshad et al.,
1997; Merscher et al.,
2001).
Whereas a previous study, based on limited analysis, suggested that the
extra neuron present at the periphery of the nerve cord in mid
mutants is not an RP2 neuron (Buescher et
al., 2006), our analyses indicate that it is indeed an RP2 neuron.
We found that loss of Mid activity causes a GMC from a row 5 NB (M-GMC) to
adopt the GMC fate of the RP2/sib lineage (GMC-1) without affecting the
expression pattern of key genes at the parent NB or NE level. Expression of
Mid in the M-GMC in mid mutants is sufficient to suppress the fate
change, indicating that Mid specifies neurons at the GMC level (or just as
post-mitotic cells are produced). We also found that there is significant
plasticity in the temporal window in which a neuronal lineage can develop.
Although this extra GMC-1 in mid mutants is born 2 hours later
than the bona fide GMC-1, it follows the same developmental pattern as the
bona fide GMC-1. Within the CNS, there appears, in general, to be a temporally
progressive restriction on the ability of a NB to generate earlier-born
neurons. Our results indicate that at the organismal level, an earlier lineage
can be generated at a later point in development (although in a different NB
lineage). In summary, our results show that a GMC or a neuronal identity can
be independent of parental identity and their formation and elaboration need
not be developmentally time-bound.
|
MATERIALS AND METHODS |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
Molecular characterization of mid alleles
Based on intron-exon prediction, four sets of primers were designed to
amplify all four exons and surrounding parts of the introns of the
mid gene. Genomic DNA from embryos homozygous for each of the
mid alleles was amplified and PCR products from three independent
reactions were sequenced.
Rescue experiments with mid
A full-length mid cDNA (RE27439; Berkeley Drosophila
Genome Project) was subcloned into the pUAST P-element vector.
Transgenic flies carrying one or two copies of a UAS-mid construct in
the DfH15,mid mutant background were crossed with
DfH15,mid or los1 carrying a specific
transgenic GAL4 driver. Several drivers were tested for their ability
to rescue/suppress the mid phenotype: sca-GAL4, ptc-GAL4,
wg-GAL4, ftz-GAL4 and eve-GAL4. The rescue efficiency was
analyzed at 22°C and 26°C.
Immunohistochemistry
Embryo collection, fixation and immunostaining were performed according to
standard procedures. The following antibodies were used: anti-Eve (gift of
Manfred Frasch, 1:2000), anti-Zfh1 (gift of Eric Lai, 1:400), 22C10
[Developmental Studies Hybridoma Bank (DSHB), University of Iowa, 1:1],
anti-Wg (DSHB, 1:5), anti-En (DSHB, 1:1), anti-Gsb (gift of Bob Holmgren,
1:3), anti-Gsb-n (gift of Bob Holmgren, 1:1), anti-Slp (gift of Ken Cadigan,
1:400), anti-β-galactosidase (Cappel, 1:100) and anti-Mid (this study,
1:100). For color visualization, AP-conjugated and HRP-conjugated secondary
antibodies were used. For double staining, secondary antibodies conjugated
with Alexa Fluor 488 and Alexa Fluor 635 were used.
Generation and purification of anti-Mid antibodies and western blot analysis
A DNA fragment corresponding to 125 amino acids (aa 49-173) of the
N-terminal portion of the Mid protein, which has the least homology to H15,
was cloned into pMal-KK-1 to create a fusion with the maltose-binding protein
(MBP). The MBP-Mid fusion protein was expressed in E. coli
BL21-CodonPlus (Stratagene) and purified using amylose columns. About 0.6 ml
of the MBP-Mid fusion protein (0.5 mg/ml) was used for rat immunization
according to a standard protocol (Covance). To remove any antibodies specific
to MBP from the polyclonal serum, we applied the serum to AminoLink Plus
coupling gel (Pierce) immobilized with MBP2 protein and then affinity purified
Mid-specific antibodies using AminoLink Plus coupling gel immobilized with
MBP-Mid fusion protein. For western analysis, extracts from 30
Drosophila embryos per lane were used (mutants were identified by the
absence of GFP balancer) using the anti-Mid antibody at 1:100. As a loading
control, we used anti-Tubulin antibody (Abcam, 1:2000).
Transactivation assay
Two reporter vectors were prepared to analyze Mid and H15 activation
potential in vitro. The luciferase gene in pGL3-Basic vector was fused with
the Hsp70Bc minimal promoter. We inserted either the double-stranded
oligonucleotide 5'-TTAATTTCACACCTAGGTGTGAAATT-3', to create a
T-site consensus reporter (see Kispert and
Herrmann, 1993), or 367 bp of the gsb-n promoter (-1279
to -912) containing the TBE putative T-site upstream of the hsp70
promoter. Full-length mid cDNA (RE27439) and H15 cDNA
(IP01538) were fused with the 6xHis epitope sequence of the
pAc5.1/V5-HisA vector. The predicted full-length cDNA of org-1 was
amplified by RT-PCR of RNA from Drosophila embryos and fused with the
6xHis epitope sequence of pAc5.1. As a control, an expression vector
containing the EGFP gene fused with the 6xHis epitope sequence of pAc5.1
was used. Renilla luciferase (Rluc) under the control of the HSV
thymidine kinase (HSV-TK) promoter in pRL-TK vector showed weak activation; an
NheI-XbaI fragment containing the entire Rluc gene was fused
with the actin gene promoter in the pAc5.1 vector via subcloning in
pBluescript to create the normalization vector Ac-Rluc (see
Porsch at al., 2005).
Drosophila S2 cells were grown in Shields and Sang Medium (Sigma)
with 10% FBS at 25°C. For transient transfection experiments, S2 cells
were seeded in 24-well plates at a dilution of 1.2-1.5x106
and transfected 24 hours later using calcium phosphate precipitation. Each
well containing 0.5 ml of S2 cells was treated with 25 µl of transfection
mixture. For each transfection, 20 ng of Ac-Rluc and 40 ng of T-site
reporter, or 10 ng of Ac-Rluc and 50 ng of gsb-n T-site
reporter, were used per 500 ml of transfection mixture. The expression vectors
were used at three different concentrations: 100 ng, 500 ng and 2 µg per
500 ml of transfection mixture. Total DNA concentration was adjusted with
pBluescript SK+ to 10 µg per 500 ml. The transfected S2 cells of each well
were lysed 48 hours later in 120 µl of Passive Lysis Buffer (Promega) and
luciferase assays performed using the Dual Luciferase Reporter Assay System
(Promega) according to the manufacturer's protocol and analyzed on a Veritas
Microplate Luminometer. The transfections were set up in triplicate. The
relative luciferase activity values shown are the mean of three independent
experiments after normalization.
|
RESULTS |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
GMC-1RP2/sib
lineage is affected in embryos mutant for mid using an antibody
against Even skipped (Eve), a GMC-1RP2/sib lineage marker. This lineage
is generated by NB4-2, which is formed as an S2 NB at 4.5 hours of
development. It generates its first GMC (GMC-1) by a self-renewing asymmetric
division at 6-6.5 hours of development. This GMC-1 divides into an RP2
and a sib at 7.45 hours of development. The RP2 begins to project its
axon ipsilaterally towards the intersegmental nerve bundle (ISN) by 10
hours of development and innervates muscle numbers 2, 9 and 11.
|
;
Bhat and Schedl., 1994;
Gaziova and Bhat, 2006). Both
the nuclear division and cytokinesis of GMC-1 are asymmetric, and thus there
is a size difference between GMC-1 (7.5 µm), an RP2 (5 µm) and a
sib (3 µm). Similarly, the nucleus of GMC-1 is 6.5 µm, whereas
that of an RP2 is 4 µm and that of a sib 2.5 µm. There is also a
difference in the level of marker gene expression between an RP2 and a sib, as
well as a difference in the temporal dynamics of expression of these markers;
the future RP2 cell expresses markers such as Eve more strongly than does a
future sib. The cell that assumes a sib identity undergoes a size reduction
and further downregulation of RP2-specific marker genes. By 14 hours of
development, expression of all RP2-specific markers is lost from the sib.
Finally, there is a subset of markers that only a mature RP2 expresses but not
the sib or GMC-1. These include Futsch (MAP1B) as detected by Mab 22C10, which
stains the membrane and the axonal projection of only a subset of neurons in
each hemisegment (Fujita et al.,
1982), allowing us to visualize the axon morphology of these
neurons (see Fig. 1G), and Cut,
a transcription factor.
When 14-hour-old embryos mutant for mid were examined with an
antibody against Eve, a strongly Eve-positive cell was observed close to the
periphery of the nerve cord (Fig.
1B). This cell is located between two clusters of EL neurons of
the adjacent hemisegments; EL neurons are a group of 8-10 Eve-positive neurons
(Fig. 1B). This phenotype was
observed in all the mid mutants, the strongest phenotype observed
being in a deficiency that removes mid and an adjacent gene that
encodes another T-box protein, H15 (Table
1). Among the mid alleles, mid1 was
genetically the strongest. We also observed variation in the penetrance of the
defect in some of the alleles depending on whether the embryos were collected
from balanced (CyO balancer) parents or parents in a wild-type
background (Table 1),
indicating a parental balancer-induced influence on the phenotype (see
Bhat et al., 2007).
|
|
9 hours of development
(Fig. 2C) and an RP2 (eRP2) and
a smaller sib-like cell (esib) by 9.5 hours
(Fig. 2D). We note that an
Eve-positive esib was observed in 50% of the hemisegments in this extra
lineage in middf embryos. It appears that in the remaining
50% of the hemisegments, Eve expression only became apparent following
eGMC division; because the eve gene is not transcribed in a sib, that
esib cells are not detected in such hemisegments is not surprising.
We further examined the lineage development using Zfh1 staining. We found
that the eGMC in a 9-hour-old embryo is weakly positive for Zfh1
(Fig. 2G), and that the Zfh1
expression becomes stronger in the eRP2
(Fig. 2H) but not in its
smaller esib cell (Fig. 2H).
The above result led us to carefully examine Zfh1 expression in the bona fide
GMC-1RP2/sib cells. We found that the bona fide GMC-1 has no Zfh1
expression as an early GMC-1 (Fig.
2I-K), but a late GMC-1 does have low levels of Zfh1
(Fig. 2L-N). Most of the Zfh1
appeared to segregate into an RP2; however, rarely, we also observed low
levels of Zfh1 in a sib that are likely to be inherited from a GMC-1 (data not
shown). These results refine our knowledge of the temporal expression of Zfh1
in this lineage and, more importantly, suggest that the expression of Zfh1 in
the eGMC is not unusual or is perhaps even a distinct property of that cell.
Thus, the Zfh1 results indicate that the extra lineage in mid mutants
behaves as an RP2/sib lineage (see below) and that it is formed 2-2.5 hours
after the development of the bona fide RP2 lineage
(Fig. 2O).
inscuteable, numb, pdm1/2 and wingless RP2 phenotypes are epistatic to the mid RP2 phenotype
It has been shown that the asymmetric division of a GMC is guided by the
asymmetric localization of proteins such as Numb
(Uemura et al., 1989). In the
RP2 and sib lineages, we and others have shown that the asymmetric division of
GMC-1 involves several proteins, including Notch, Delta, Inscuteable (Insc)
and Numb (Buescher et al.,
1998; Wai et al.,
1999; Lear et al.,
1999) (reviewed by Gaziova and
Bhat, 2006). Asymmetric localization of Insc to the apical end of
the cell causes a basal localization of Numb, resulting in one of the two
cells inheriting Numb. This Numb prevents the reception of Notch signaling in
this cell by inhibiting the proteolytic cleavage of the intracellular domain
of Notch. As a result, this cell becomes an RP2. The cell that does not
inherit Numb is able to respond to Notch signaling and becomes a sib.
Thus, in insc mutant embryos, the GMC-1 of the RP2/sib lineage
symmetrically divides into two RP2s (Fig.
3A,C), whereas in numb mutants it symmetrically divides
into two sibs (Fig. 3E). To
obtain additional lines of evidence that the extra lineage is indeed an
additional RP2/sib lineage, we generated double-mutant embryos between
insc and mid as well as between numb and
mid. If the extra lineage is an eRP2 lineage, our expectation is that
the eGMC should yield two eRP2s in insc, mid double mutants and two
esibs in numb, mid double mutants. Indeed, we observed two eRP2
neurons in insc, mid double mutants
(Fig. 3B,D). In numb,
mid double mutants we observed a numb phenotype with two esib
cells (Fig. 3F). Note that
numb is maternally deposited, and that in this numb mutant
allele the penetrance of the defect is restricted to 50% of the
hemisegments. We observed full penetrance of the numb phenotype in
the eRP2/sib lineage. This is likely to be a consequence of the late formation
of this extra lineage: by the time this eGMC-1 is dividing, the maternally
inherited Numb must have been exhausted, resulting in complete penetrance of
the numb phenotype in the extra lineage.
|
;
Bhat et al., 1995;
Yeo et al., 1995). Thus, a
mutant for both genes causes a fully penetrant loss of the Eve-positive
GMC-1RP2/sib lineage (Fig.
3G). We constructed triple mutants between pdm1, pdm2 and
mid and examined these embryos with Eve staining. As shown in
Fig. 3H, the eRP2 neurons are
completely missing from the triple mutant, indicating that, just as with the
bona fide RP2 lineage, this extra lineage is dependent on
pdm1/2 for the specification of its identity.
It has been shown that Wg signaling is necessary for the specification of
the RP2/sib lineage (Chu-LaGraff and Doe,
1993; Bhat, 1996;
Bhat, 1998). In wg
mutants, the Eve-positive RP2 lineage is missing owing to an NB4-2 formation
and specification defect (Fig.
3I). We constructed double mutants between wg and
mid and examined these embryos with Eve staining for the extra RP2
lineage. These double-mutant embryos were missing the eRP2 lineage in 75-96%
of the hemisegments (Fig. 3J;
n=12 embryos). It has been reported that in wg mutants, the
formation and/or specification of row 4 and 6 NBs are affected, but row 5 NBs
are normal (Chu-LaGraff and Doe,
1993). Accordingly, our mid, wg double-mutant result
suggests that the eRP2 lineage originates from either a row 4 or a row 6 NB; a
failure in the formation or specification of the parent NB will cause the
wg phenotype to be epistatic to the mid phenotype.
Alternatively, as loss-of-function of wg also affects row 5 NBs, the
eRP2 lineage might originate from one of the NBs in row 5 (see below).
Expression of Mid in the CNS: defining the M-lineage and its transformation into an extra RP2 lineage
A fine mapping of mid mutations using deletions led us to a gene
that encodes a Tbx transcription factor of 580 amino acids that is located in
close proximity to another T-box gene, H15. We sequenced the gene in
three different mid alleles and found mutations in all of these
alleles in this gene (Fig. 4A).
The mid1 allele had a CT change that produced a stop
codon at amino acid position 128 (this was the strongest allele),
mid2 had a CT change at 361, whereas in
los1 a 22 bp deletion caused a deletion of seven amino
acids at position 321 and a frame shift leading to a stop codon at amino acid
position 350 (thus, the protein in this allele had 28 amino acids that were
different from wild type, in addition to the truncation). In the meantime, a
report was published showing that this gene corresponds to mid
(Buescher et al., 2006),
although they reported a different mutation in the gene for the
mid2 allele.
Because we repeated the sequencing several times, we are confident that the
mutational change in mid2 is as reported in this study. We
further confirmed the identity of the gene by performing western analysis
(Fig. 4B) using a polyclonal
antibody generated against Mid. In the wild type, we observed a band of
75 kDa, which is larger than that expected for a protein of 580 amino
acids. This band was absent in embryos that were homozygous for a deficiency
that removes both mid and H15. In mid1
we did not observe any Mid protein, whereas in los1 and
mid2 we observed a truncated protein of expected size from
the sequence data. There was no maternal deposition of mid RNA
(Fig. 4C) or Mid protein
(Fig. 4D) to developing
embryos.
|
We next examined the expression of Mid in NBs in developing embryos
(Fig. 5). We found that the
expression of Mid in NBs is restricted to midline NBs (MNBs), three NBs in row
7 (NB7-1, 7-2 and 7-4) and two NBs in row 1/2 (NB 2-5 and 3-2) at 4.5
hours of development (Fig.
5A,B). At 6.5 hours of development, NB3-2, NB7-1 and NB7-2
became negative for Mid, but NB1-2, NB6-4 and NB6-2 in row 6 started to
express Mid (Fig. 5D,E). In a
10-hour-old embryo, we observed a Mid-positive cell in the same location as
the eRP2 (Fig. 5G,H) and named
this cell an M-neuron (M for Mid). This cell is located in the row of cells
expressing Wg (Fig. 5I) and
thus the cell appears to originate in row 5. No NBs in this location expressed
Mid, indicating that the parent NB of this M-GMC does not express Mid.
Since the Mid antibody detects Mid protein in los1 embryos (Fig. 4B), we examined whether the M-neuron is the same as that which gets transformed into the eRP2 by double staining los1 mutant embryos with Mid and Eve antibodies. We found that this is indeed the case (Fig. 5J-L). These results argue that Mid functions in this M-lineage to prevent it from developing into a second RP2/sib lineage.
We next sought to examine the development of the M-lineage in detail.
First, we stained wild-type embryos with Mid and Engrailed (En) antibodies.
The results indicate that the M-GMC is located adjacent to the anterior row of
En-expressing cells and appears at 7.45 hours of development
(Fig. 6B); no such cell was
observed in or near that location in a 7-hour-old embryo
(Fig. 6A). Thus, the GMC must
have formed between 7 and 7.45 hours of development. This cell divides into a
larger M-neuron and a smaller M-sib cell at 9-9.5 hours of development
(Fig. 6C-F). Interestingly, the
smaller M-sib cell appears to become Mid-negative soon after its birth as it
is no longer visible by Mid staining (Fig.
6G,H). The same conclusions were also reached from the results
obtained with Mid and Eve staining (Fig.
6I-L). Note that in the wild-type embryo, the M-neuron is smaller
than an RP2 (Fig. 6L). In
embryos older than 15 hours, the level of Mid in the M-neuron seemed to fade
(Fig. 6M). A similar pattern
was also observed in at least one other (and possibly more) Mid-positive
lineage in which the GMC divides to generate two asymmetric cells: the smaller
cell inherited a reduced amount of Mid
(Fig. 6N).
|
|
The extra RP2 lineage in mid mutants originates from a row 5 NB
In order to determine from which row of NBs this GMC is generated, we
performed rescue (this is also a suppression of formation of the eRP2 lineage)
experiments by spatially expressing mid in the mid mutant
using a UAS-mid transgene. We generated UAS-mid transgenic
lines and sought to determine whether the mutant eRP2 lineage phenotype could
be rescued by expressing the gene in different rows of NBs. We crossed the
UAS-mid to the following GAL4 lines: wg-GAL4 (in row 5),
en-GAL4 (rows 6 and 7 and one NB in row 1), ptc-GAL4 (rows
2-5) and sca-GAL4 (all NBs). As shown in
Table 2, we found that
expression of the mid transgene in row 5 significantly suppresses the
formation of the eRP2 lineage (the M-GMC and its neuronal pairs now express
Mid; data not shown). This was also the case with ptc-GAL4, although
the suppression with ptc-GAL4 was only observed at 26.5°C. The
formation of the eRP2 lineage was also suppressed upon expression of
mid using sca-GAL4. However, with en-GAL4, no
suppression was observed (Table
2). These results argue that the extra lineage in mid
mutants arises from a row 5 NB. Since staining of mid mutant embryos
for Huckebein (Hkb) expression, a NB4-2 marker, did not reveal any additional
Hkb-positive NBs in row 5 (or in rows 4 and 6; data not shown), it was
possible that NB5-4 or NB5-5 is changed into NB4-2 in mid mutants;
both these NBs express Hkb and therefore have the potential to change into
NB4-2. Moreover, NB5-4 is formed as a S4 NB at 6.5 hours of development
and NB5-5 is formed as a S5 NB at 7 hours of development, and the timing
of their formation is consistent with a first GMC from either of these NBs
changing to eGMC-1, of which NB5-4 seems the more likely by the timing
(Fig. 6T; see also below).
|
).
The only way to reconcile the los, wg double-mutant results and the
suppression data is to conclude that row 5 NBs are indeed affected in
wg mutants. Therefore, we re-examined the formation of row 5 NBs in
wg-null mutants by staining the mutant embryos with an antibody
against Gsb (also referred to as Gsb-distal). In the wild type, Gsb is
expressed in NBs from rows 5 and 6 and in NB7-1 in row 7
(Fig. 7A). In embryos mutant
for wg, we found that row 5 NBs were indeed affected. Thus, 70%
of the hemisegments were missing one or more NBs, such as NB5-4 (which was
affected the most), NB5-5 and NB5-2 (Fig.
7B). These results suggest that Wg function is necessary for the
formation of some of the row 5 NBs, and it is therefore probable that the eRP2
lineage originates from a row 5 NB. Since we did observe NB5-4 (and NB5-5) in
wg mutant embryos in 30% of the hemisegments, the presence of
the eRP2 lineage in mid, wg double mutants in some of the
hemisegments is consistent with a row 5 NB, most likely NB5-4, giving rise to
a GMC that transforms into eGMC-1 in the absence of Mid activity.
Fate transformation in mid mutants occurs at the GMC level and not at the NB level
The presence of Gsb in the precursor NE cells that give rise to NB4-2 will
block Wg from specifying NB4-2 identity to a NB
(Bhat, 1996) (reviewed by
Bhat, 1999). The finding that
the GMC that transforms into GMC-1 of the RP2/sib lineage (M-GMC) is
Mid-positive, whereas none of the row 5 NBs is Mid-positive, suggests that the
fate transformation is likely to be at the GMC level (Mid being a
transcription factor). If this were true, row 5 NBs and the precursor NE cells
should all have Gsb expression intact. As shown in
Fig. 7C, all these NBs
expressed Gsb. The precursor NE cells of the row 5 NBs also expressed Gsb
(data not shown). These NBs also had normal Wg
(Fig. 7E), Sloppy paired (Slp)
and Hkb expression (data not shown).
There is a second gsb gene, gsb-neuro (gsb-n)
(also known as gsb-proximal). This gene is expressed in GMCs and
neurons that originate from the same parent NBs that express Gsb
(Gutjahr et al., 1993). In
mid mutants, among the cluster of Gsb-n-positive neurons, only the
M-neuron was lacking Gsb-n expression (Fig.
7F-H; Eve-positive, Gsb-n-negative cell in the right hemisphere at
the top), and double staining for Eve indicated that this neuron is indeed the
eRP2. These results also confirm that eRP2 is generated from a row 5 NB.
We further tested the possibility raised above that the fate transformation occurs at the GMC level by suppression experiments. The UAS-mid transgene was induced with ftz-GAL4 and eve-GAL4. These two GAL4 drivers are expressed in GMC-1 of the RP2 lineage. As shown in Table 2, the UAS-mid transgene expressed using ftz-GAL4 and eve-GAL4 suppressed the transformation defect, indicating that the fate transformation in mid mutants is at the GMC level.
|
|
;
Lee et al., 2003). As shown in
Fig. 8C, in control experiments
with the consensus TBE, Org-1 activated the reporter gene strongly, whereas
Mid and H15 activated transcription 2- to 3-fold as compared with the
control protein GFP. With the gsb-n promoter
(Fig. 8D), H15 showed the
highest activation: by 6-fold, at the highest DNA concentration tested.
This was followed by Mid, which was 1.5-fold that of the control, and
then Org-1 (see Discussion). |
DISCUSSION |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
).
Similarly, identical neurons can be specified by different lineages. For
instance, for bilateral neurons among the six sensory neurons involved in
mechanosensation, although derived from the same founder cell, their lineages
diverge four cell divisions prior to the terminal division (WormBase;
www.wormbase.org).
However, Drosophila is not driven by lineages, except for NBs, but
they produce distinct progeny lineages specific to a given NB. Therefore, the
above conclusion as drawn from studies in C. elegans was not an
obvious, or expected, one in Drosophila and this makes our findings
with mid significant. The fate of a cell is not specified simply by a
single transcription factor, but instead by a complex combination of
cell-autonomous and cell-non-autonomous genetic circuitry. Our results
indicate that Mid plays a central role in this process in M-GMC, preventing it
from becoming GMC-1 of the RP2/sib lineage. Absence of Mid activity initiates
a cascade of events in M-GMC that ultimately transforms M-GMC into GMC-1.
A NB undergoes multiple self-renewing asymmetric divisions, each time
producing a GMC of specific identity, which then generates two neurons of
distinct identities. The identity of the first GMC from a NB is dependent upon
the gene expression program in the NE cells from which the parent NB is
delaminated, and this identity is thought to be invariant
(Bhat, 1996;
Bhat and Schedl, 1997;
Duman-Scheel et al., 1997)
(see also Chu-LaGraff and Doe,
1993) (reviewed by Bhat,
1999; Isshiki et al.,
2001). Following division to generate a GMC, the gene expression
program in the NB changes so that it produces a second GMC of different and
distinct identity from the first GMC
(Isshiki et al., 2001). Based
on these and several other similar studies, it is currently believed that the
identity of a GMC and its neuronal pairs is already determined in the NE and
NB levels, i.e. it is ancestry-dependent. However, our results with
mid show that this ancestry-dependent fate specification is not as
stringent as once thought, and that the identity of a GMC can be altered
without altering the gene expression program in the NB or NE level. Thus, a
specific set of neurons (in this case RP2/sib) can be derived or specified
from a GMC other than the bona fide GMC by altering the activity of a single
gene, in this case mid. One should keep in mind that the ultimate
specification of the identity of a GMC (in this case M-GMC/eGMC-1) certainly
depends on a complex interplay of many gene products. Our study, however,
identifies Mid as a key player in preventing M-GMC from becoming GMC-1 of the
RP2 lineage. One should also point out that some GMCs, although being
generated by different NBs, may have similar potentials and that there might
be only one gene responsible for their differences; we believe that we have
identified mid as one such gene.
Our results also show that duplication of the RP2/sib lineage, an
extensively studied neuronal lineage, can occur by a mechanism or route that
is different from those previously described. There are several ways the RP2
lineage can be duplicated. The most common way is through a second NB changing
its identity into NB4-2, the parent of the RP2/sib lineage
(Bhat and Schedl, 1997;
Duman-Scheel et al., 1997).
RP2 lineage duplication can also occur when a GMC-1 divides symmetrically to
produce two GMC-1s (Yang et al.,
1993; Bhat and Schedl,
1994; Bhat et al.,
1995), each producing an RP2. A GMC-1 can also divide
asymmetrically to self-renew and generate an RP2, and the self-renewed GMC-1
divides again to generate another (or more) RP2 or sib
(Bhat and Schedl, 1994;
Bhat and Apsel, 2004). A GMC-1
can also divide symmetrically to generate two RP2s
(Mehta and Bhat, 2001;
Bhat and Apsel, 2004). All
these scenarios are different from the one we have described in mid
mutants, in which an unrelated GMC (M-GMC) in a relatively distant location
changes its identity to GMC-1 and generates a second set of RP2/sib cells at
this distant site. This occurs without changing the expression of any of the
genes known to be crucial for fate determination in the precursor NB or NE
cells. This has not been observed before and as such adds to the novelty of
our results.
A third set of our results that we think are novel comes from the fact that
a second GMC-1RP2/sib lineage can be formed 2-2.5 hours after the
formation of the bona fide GMC-1RP2/sib lineage. This type of plasticity
in the timing of formation of a lineage has, to our knowledge, never been
shown before for this or any other lineage in the CNS. There is a certain
degree of plasticity in the timing of formation and elaboration of a lineage
in the CNS between hemisegments. For example, formation of NB4-2 and its
division can be delayed by 15 minutes between hemisegments. In the case
of gsb or en/invected mutants, for example, NB5-3 (which is
located close to NB4-2) transforms into NB4-2, thus duplicating the RP2
lineage. NB5-3 (whether transformed into NB4-2 as in these mutants, or not) is
formed 30 minutes prior to the formation of NB4-2. Thus, the sequential
production of the duplication can be delayed by as much as 45 minutes in an
embryo. A similar interval in the sequential production of the RP2 lineage is
also observed in embryos mutant for lottchen
(Buescher and Chia, 1997), in
which a second NB (possibly NB3-2, located adjacent to NB4-2) changes into
NB4-2. Our results with mid indicate that an additional
GMC-1RP2/sib lineage can be formed as much as 2 hours later than
normal for this lineage, and at a site relatively distant from the original
location of this lineage. This indicates considerable plasticity in terms of
the developmental timing of a neuronal lineage, and that the nerve cord is
capable of generating an early forming neuronal lineage also at a later point
in time. Moreover, in all previous cases in which a second RP2/sib lineage was
formed, it was always formed close to the bona fide RP2/sib lineage. The
duplication of the RP2/sib lineage in mid mutants is the first case
in which the second lineage is formed at an ectopic site.
These results are also interesting from another angle. A NB loses it
ability, later in development, to produce earlier neurons. In other words,
there is a temporally guided progressive restriction on the ability of a NB to
generate earlier-born neurons. Indeed, a previous study showed that NBs indeed
gradually lose competence to generate earlier-born cells
(Pearson and Doe, 2003).
Although it is not clear whether this is true for all lineages, our results
show that at an organismal level, an earlier lineage can be generated at a
later point in development. Thus, whereas the same NB, later in its life, may
lose its ability to generate an earlier-born neuron, an earlier-born neuron
can still be generated in the CNS at a later point in development, albeit in a
different NB or GMC lineage.
Our results show that Mid plays a unique role in preventing M-GMC from
becoming GMC-1, 2 hours after the formation of the bona fide RP2 lineage.
It is possible that in the wild type, during evolution a combination of gene
expression patterns converged at this 2-hour time point with the
potential to push the M-GMC into GMC-1, but because a nerve cord does not need
two RP2s, evolution found a way to prevent this from occurring via expression
of Mid in this M-GMC. We suspect that a similar mechanism might exist in many
more lineages than just the M-lineage.
The extra neuron in mid is an RP2 neuron
It has been suggested previously that the extra cell is not an RP2 neuron
(Buescher et al., 2006).
However, this conclusion was based on the observation that this cell does not
have an axon projection similar to that of RP2. Since the location of this
eRP2 is at the periphery of the nerve cord, one would not expect to observe an
ipsilateral projection from this neuron. We found that the growth cone from
this neuron projects anterior and towards the midline
(Fig. 1), where a choice point
for an RP2 projection might exist. This growth cone often fasciculates with
the ISN along with the projection from the bona fide RP2. We have employed a
number of experiments involving different markers, mutant combinations and a
very detailed and thorough analysis of this extra lineage. Our analysis
reveals that it is indeed an RP2: the GMC divides into a larger and a smaller
cell akin to the division of the GMC-1 into an RP2 and a sib. One of the two
cells, similar to a sib, loses the expression of Eve and does not express
RP2-specific markers such as Zfh1. Furthermore, in a mid, insc
double-mutant embryo, the esib adopts an RP2 fate, with both cells being the
same size and expressing the same RP2 markers as the bona fide RP2 lineage in
insc mutants. Similarly, in mid, numb double mutants, both
cells become sibs. The two POU genes, pdm1 and pdm2, are
required for the specification of GMC-1 of the RP2/sib lineage. In mid,
pdm1, pdm2 triple mutants, the eGMC-1 fails to adopt a GMC-1 identity
just as the bona fide GMC-1 also fails to adopt a GMC-1 identity.
However, there are temporal differences in the gene expression pattern between the bona fide RP2/sib lineage and the eRP2/sib lineage. For example, Eve expression begins later in the eRP2 lineage in at least 50% of the hemisegments, as late as subsequent to the eGMC-1 division. Thus, we often find hemisegments with no Eve-positive esib. Since loss-of-function for Eve has no drastic effect on the RP2/sib lineage, this late expression of Eve is likely to be non-consequential to the development of the lineage.
Plasticity in the developmental timing of the GMC-1RP2/sib lineage
The bona fide GMC-1RP2/sib lineage originates from NB4-2, an S2 NB
formed at 4.5 hours of development (at 22°C). The GMC-1 is formed at
6-6.5 hours of development, although it becomes Eve-positive at 7 hours
of development; it then divides at 7.45 hours into an RP2 and a sib. The
cells undergo a complex migration and then settle within the anterior
commissure (Bhat, 2007). An RP2
begins to project its axon growth cone at 10 hours of development. The
eGMC-1 appears to be formed at 8 hours, becoming Eve-positive at 9
hours of development (Fig. 6).
It then divides at 9.5 hours and begins to project its axon at 12
hours of development. This indicates that there is significant plasticity in
terms of developmental timing as far as the ability of the embryo to generate
an RP2 lineage is concerned. All the requisite genetic pathways must still be
operational even after 2 hours of development of the bona fide RP2/sib
lineage.
Transformation of a row 5 GMC into GMC-1 and not the transformation of a NB into NB4-2 is responsible for the extra RP2 lineage
Our results indicate that the M-GMC from a row 5 NB (most likely NB5-4) is
transformed into GMC-1, as opposed to a NB being transformed into NB4-2. It
has previously been shown that in order to specify a NB as NB4-2, that cell
should be Gsb-negative. First, our `suppression' results indicate that the
eRP2 is generated by a row 5 NB and not a row 4 or 6 NB. However, none of the
NBs in row 5 is Gsb-negative in mid embryos
(Fig. 7); row 5 NBs also had
normal expression of three other markers: Wg, Slp and Hkb. This indicates that
the identity of these NBs is unlikely to be affected in mid mutants.
Second, whereas none of the NBs in row 5 expresses Mid, a row 5 GMC that
generates the neuron that transforms into an RP2 in the mutant expresses Mid
(Fig. 6). It is possible that
Mid is expressed in the parent NB of M-GMC but at an undetectable level.
However, our conclusion was based on three sets of results: (1) by RNA in situ
hybridization using a mid probe, we did not observe any
mid-positive NBs at this location (data not shown); (2) we generated
mid-promoter-lacZ transgenic lines and the expression of
lacZ was basically the same as expression observed with the Mid
antibody; and (3) we were able to rescue/suppress the mid phenotype
(i.e. the formation of an extra RP2 lineage) by expressing Mid in M-GMC in
mid mutants. Finally, the timing of NB versus GMC specification is
also consistent with the conclusion that the transformation occurs at the NB
level. We conclude that a row 5 GMC becomes GMC-1 of the RP2/sib lineage in
the absence of wild-type Mid function.
One issue that we were not able to resolve conclusively is the identity of the parent NB for the M-lineage. Our current results indicate that it is NB5-4; the first GMC of this NB gives rise to the M-lineage. Alternatively, it might be NB5-5, in which case the NB has to generate the M-GMC within 1 hour, or it could be a later-born GMC of NB5-3, although based on the position of the M-GMC this latter possibility is unlikely. We have not been able to address the ultimate fate of the M-neuron or its sibling, as to whether they are motoneurons, interneurons or some other cell type (it is unlikely to be glial as they do not express Repo, a glial cell marker), or the function of these cells.
Our results indicate that row 5 NBs are affected in wg mutants
(Fig. 6B), not just rows 4 and
6 as was previously thought (Chu-LaGraff
and Doe, 1993). In the previous work, the authors used a
temperature-sensitive (ts) mutant and an allele of wg,
wgCX4. Whereas the ts mutation is likely to be a hypomorph and
retains some Wg activity, wgCX4 is considered a null.
However, we have noticed that this allele carries a background mutation(s)
that suppresses the wg loss-of-function effect; a partial
recombination did eliminate the background suppressor mutation(s) and this
`cleaned up' wgCX4 mutation in trans to another allele of
wg, wgIG22, did have the missing row 5 NB defect. We
believe that because of the effect of wg mutation on row 5 NBs, the
wg phenotype is mostly epistatic to the mid phenotype in
wg, mid double mutants in terms of the extra RP2 lineage defect.
Mid and H15 as transcriptional activators
The T-box-binding element (TBE) was first defined as a 20-bp degenerate
palindromic sequence with the highest affinity for the Brachyury protein
(Kispert and Herrman, 1993).
However, analysis of the actual target genes reveals that the TBE is highly
variable in sequence, number and distribution within their promoters. In our
experiments, with the consensus TBE only the Org-1 protein showed strong
activation of the reporter gene, whereas Mid or H15 showed only an 2-fold
increase in transcriptional activation over the GFP control. However, with the
gsb-n promoter, which contains a degenerate palindromic TBE sequence,
activation by Org-1 was only slightly greater than that by the control
protein. By contrast, there was a significant level of activation (4-fold
that of the control) by H15 from the same promoter element (H15 shares 62%
identity with Mid), and the level of activation by Mid was 1.5-fold that
of the control, which is slightly more than the stimulation by Org-1. That
Org-1 behaves differently to Mid and H15 is consistent with the fact that Mid
and H15 belong to the Tbx20 subfamily, whereas Org-1 belongs to the Tbx1
subfamily. This result also shows that although these proteins are all in the
Tbx family, they diverge significantly in their sequence preference with
regard to the activation of transcription. The Tbx family of proteins is also
known to repress transcription (Porsch et
al., 2005). Whereas the Tbx domain binds to DNA, albeit with
different specificities according to variations in DNA sequence in the binding
site, the rest of the protein is likely to be responsible for either
activation or repression.
|
Footnotes |
|---|
|
REFERENCES |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
Bamshad, M., Lin, R. C., Law, D. J., Watkins, W. C., Krakowiak,
P. A., Moore, M. E., Franceschini, P., Lala, R., Holmes, L. B., Gebuhr, T. C.
et al. (1997). Mutations in human TBX3 alter limb, apocrine
and genital development in ulnar-mammary syndrome. Nat.
Genet. 16,311
-315.[CrossRef][Medline]
Basson, C. T., Huang, T., Lin, R. C., Bachinsky, D. R.,
Weremowicz, S., Vaglio, A., Bruzzone, R., Quadrelli, R., Lerone, M., Romeo, G.
et al. (1999). Different TBX5 interactions in heart and limb
defined by Holt-Oram syndrome mutations. Proc. Natl. Acad. Sci.
USA 96,2919
-2924.
Bhat, K. M. (1996). The patched signaling
pathway mediates repression of gooseberry allowing neuroblast specification by
wingless during Drosophila neurogenesis. Development
122,2921
-2932.[Abstract]
Bhat, K. M. (1998). frizzled and frizzled 2
play a partially redundant role in wingless signaling and have similar
requirements to wingless in neurogenesis. Cell
95,1027
-1036.[CrossRef][Medline]
Bhat, K. M. (1999). Segment polarity genes in
neuroblast formation and identity specification during Drosophila
neurogenesis. BioEssays
21,472
-485.[CrossRef][Medline]
Bhat, K. M. (2007). Wingless activity in the
precursor cells specifies neuronal migratory behavior in the Drosophila nerve
cord. Dev. Biol. 311,613
-622.[Medline]
Bhat, K. M. and Schedl, P. (1994). The
Drosophila miti-mere gene, a member of the POU family, is required for the
specification of the RP2/sibling lineage during neurogenesis.
Development 120,1483
-1501.[Abstract]
Bhat, K. M. and Schedl, P. (1997). Requirement
for engrailed and invected genes reveals novel regulatory interactions between
engrailed/invected, patched, gooseberry and wingless during Drosophila
neurogenesis. Development
124,1675
-1688.[Abstract]
Bhat, K. M. and Apsel, N. (2004). A mechanism
for the self-renewing asymmetric division of neural precursor cells in the
Drosophila CNS. Development
131,1123
-1134.
Bhat, K. M., Poole, S. J. and Schedl, P.
(1995). The miti-mere and pdm1 genes collaborate during
specification of the RP2/sib lineage in Drosophila neurogenesis.
Mol. Cell. Biol. 15,4052
-4063.[Abstract]
Bhat, K. M., van Beers, E. H. and Bhat, P.
(2000). Sloppy paired acts as the downstream target of wingless
in the Drosophila CNS and interaction between sloppy paired and gooseberry
inhibits sloppy paired during neurogenesis.
Development 127,655
-665.[Abstract]
Bhat, K. M., Gaziova, I. and Krishnan, S.
(2007). Regulation of axon guidance by Slit and Netrin signaling
in the Drosophila ventral nerve cord. Genetics
176,2235
-2246.
Buescher, M. and Chia, W. (1997). Mutations in
lottchen cause cell fate transformations in both neuroblast and glioblast
lineages in the Drosophila embryonic central nervous system.
Development 124,673
-681.[Abstract]
Buescher, M., Yeo, S. L., Udolph, G., Zavortink, M., Yang, X.,
Tear, G. and Chia, W. (1998). Binary sibling neuronal cell
fate decisions in the Drosophila embryonic central nervous system are
nonstochastic and require inscuteable-mediated asymmetry of ganglion mother
cells. Genes Dev. 12,1858
-1870.
Buescher, M., Tio, M., Tear, G., Overton, P. M., Brook, W. J.
and Chia, W. (2006). Functions of the segment polarity genes
midline and H15 in Drosophila melanogaster neurogenesis. Dev.
Biol. 292,418
-429.[CrossRef][Medline]
Chu-LaGraff, Q. and Doe, C. Q. (1993).
Neuroblast specification and formation regulated by wingless in the Drosophila
CNS. Science 261,1594
-1597.
Doe, C. Q. (1992). Molecular markers for
identified neuroblasts and ganglion mother cells in the Drosophila central
nervous system. Development
116,855
-863.[Abstract]
Duman-Scheel, M., Li, X., Orlov, I., Noll, M. and Patel, N.
H. (1997). Genetic separation of the neural and cuticular
patterning functions of gooseberry. Development
124,2855
-2865.[Abstract]
Fujita, S. C., Zipursky, S. L., Benzer, S., Ferrus, A. and
Shotwell, S. L. (1982). Monoclonal antibodies against the
Drosophila nervous system. Proc. Natl. Acad. Sci. USA
79,7929
-7933.
Gaziova, I. and Bhat, K. M. (2006). Generating
asymmetry with and without self-renewal. In Recent Advances in
Molecular Biology (ed. A. M. Coelho), pp.143
-178. Berlin, Germany:
Springer-Verlag.
Gaziova, I. and Bhat, K. M. (2007). Generating
asymmetry: with and without self-renewal. Prog. Mol. Subcell.
Biol. 45,143
-178.[CrossRef][Medline]
Gutjahr, T., Patel, N. H., Li, X., Goodman, C. S. and Noll,
M. (1993). Analysis of the gooseberry locus in Drosophila
embryos: gooseberry determines the cuticular pattern and activates gooseberry
neuro. Development 118,21
-31.[Abstract]
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]
Kispert, A. and Herrmann, B. G. (1993). The
Brachyury gene encodes a novel DNA binding protein. EMBO
J. 12,3211
-3220.[Medline]
Kolodziej, P. A., Jan, L. Y. and Jan, Y. N.
(1995). Mutations that affect the length, fasciculation, or
ventral orientation of specific sensory axons in the Drosophila embryo.
Neuron 15,273
-286.[CrossRef][Medline]
Lear, B. C., Skeath, J. B. and Patel, N. H.
(1999). Neural cell fate in rca1 and cycA mutants: the roles of
intrinsic and extrinsic factors in asymmetric division in the Drosophila
central nervous system. Mech. Dev.
88,207
-219.[CrossRef][Medline]
Lee, H. H., Norris, A., Weiss, J. B. and Frasch, M.
(2003). Jelly belly protein activates the receptor tyrosine
kinase Alk to specify visceral muscle pioneers. Nature
425,507
-512.[CrossRef][Medline]
Li, Q. Y., Newbury-Ecob, R. A., Terrett, J. A., Wilson, D. I.,
Curtis, A. R., Yi, C. H., Gebuhr, T., Bullen, P. J., Robson, S. C., Strachan,
T. et al. (1997). Holt-Oram syndrome is caused by mutations
in TBX5, a member of the Brachyury (T) gene family. Nat.
Genet. 15,21
-29.[CrossRef][Medline]
Mehta, B. and Bhat, K. M. (2001). Slit
signaling promotes the terminal asymmetric division of neural precursor cells
in the Drosophila CNS. Development
128,3161
-3168.
Merscher, S., Funke, B., Epstein, J. A., Heyer, J., Puech, A.,
Lu, M. M., Xavier, R. J., Demay, M. B., Russell, R. G., Factor, S. et al.
(2001). TBX1 is responsible for cardiovascular defects in
velo-cardio-facial/DiGeorge syndrome. Cell
104,619
-629.[CrossRef][Medline]
Moerman, D. G. and Fire, A. (1997). Muscle:
structure, function and development. In C. elegans
II (ed. D. L. Riddle, T. Blumenthal, B. J. Meyer and J. R.
Priess), pp. 417-470. Cold Spring Harbor, NY: Cold
Spring Harbor Laboratory Press.
Nusslein-Volhard, C., Wieschaus, E. and Kluding, H.
(1984). Mutations affecting the pattern of the larval cuticle in
Drosophila melanogaster. I. Zygotic loci on the second chromosome.
Roux Arch. Dev. Biol.
193,267
-282.[CrossRef]
Pearson, B. and Doe, C. Q. (2003). Regulation
of neuroblast competence and temporal identity in Drosophila.
Nature 425,624
-628.[CrossRef][Medline]
Porsch, M., Hofmeyer, K., Bausenwein, B. S., Grimm, S., Weber,
B. H., Miassod, R. and Pflugfelder, G. O. (1998). Isolation
of a Drosophila T-box gene closely related to human TBX1.
Gene 212,237
-248.[CrossRef][Medline]
Porsch, M., Sauer, M., Schulze, S., Bahlo, A., Roth, M. and
Pflugfelder, G. O. (2005). The relative role of the T-domain
and flanking sequences for developmental control and transcriptional
regulation in protein chimeras of Drosophila OMB and ORG-1. Mech.
Dev. 122,81
-96.[CrossRef][Medline]
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]
Wai, P. B., Truong, B. and Bhat, K. M. (1999).
Cell division genes promote asymmetric interaction between Numb and Notch in
the Drosophila CNS. Development
126,2759
-2770.[Abstract]
Yang, X., Yeo, S., Dick, T. and Chia, W.
(1993). The role of a Drosophila POU-homeodomain gene in the
specification of neural precursor cell identity in the developing embryonic
CNS. Genes Dev. 7,504
-516.
Yedvobnick, B., Kumar, A., Choudhury, P., Opraseuth, J.,
Mortimer, N. and Bhat, K. M. (2004). The asymmetric division
function of Mastermind is separable and distinct from its neurogenic function
during Drosophila neurogenesis. Genetics
166,1281
-1289.
Yeo, S. L., Lloyd, A., Kozak, K., Dinh, A., Dick, T., Yang, X.,
Sakonju, S. and Chia, W. (1995). On the functional overlap
between two Drosophila POU homeo domain genes and the cell fate specification
of a CNS neural precursor. Genes Dev.
9,1223
-1236.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati 
Twitter What's this?
This article has been cited by other articles:
|
|
 |
|
  Q.-X. Liu, M. Hiramoto, H. Ueda, T. Gojobori, Y. Hiromi, and S. Hirose Midline governs axon pathfinding by coordinating expression of two major guidance systems Genes & Dev., May 15, 2009; 23(10): 1165 - 1170. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
