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First published online 22 February 2006
doi: 10.1242/dev.02299
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1 Department of Entomology, University of Maryland, College Park, MD 20742,
USA.
2 Division of Hematology Oncology, Department of Medicine, University of
Massachusetts Memorial Medical Center, Worcester, MA 01655, USA.
3 Department of Neurobiology, University of Massachusetts Medical School,
Worcester, MA 01605, USA.
Author for correspondence (e-mail:
tzumin.lee{at}umassmed.edu)
Accepted 24 January 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Polyhomeotic, Polycomb group, Neuronal cell fate maintenance, Ecdysone, Metamorphosis
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Cells
imprint histories of development in their genomes; and differential imprinting
of genomes is fundamental to cell differentiation (reviewed by
Orlando, 2003). The Polycomb
group (PcG) of nuclear proteins, including Polyhomeotic (Ph), is known to
modulate gene expression globally in the genomes according to their patterns
of imprinting (reviewed by Pirrotta,
1998; Beuchle et al.,
2001). However, little progress has been made to elucidate roles
of PcG proteins in neuronal development since the initial characterization of
the neural phenotypes of ph mutants 17 years ago
(Smouse et al., 1988).
Neural tissues differentiate step by step. First, distinct neuronal
precursors, characterized with different transcriptional codes, are specified
during patterning of neuroectoderm (Briscoe
et al., 2000; Urbach et al.,
2003). Second, distinct precursors further give rise to different
characteristic sets of multiple subtypes of neurons during neurogenesis
(Lee et al., 1999;
Schmid et al., 1999;
Jefferis et al., 2001). Both
processes of cell diversification involve spatial and/or temporal patterning
of tissues (reviewed by Jacob and Briscoe,
2003; Zhong,
2003). Tissue patterning permits acquisition of different gene
expression profiles in originally equivalent cells, while maintenance of cell
type-specific transcription programs depends on various mechanisms of
epigenetic functions (Orlando,
2003).
Much of our knowledge about roles of epigenetic imprint in cell
differentiation comes from characterization of several PcG genes that were
originally identified through mutant screens based on derepression of homeotic
(Hox) genes in Drosophila embryos (e.g.
Lewis, 1978;
Duncan, 1982;
Jurgens, 1985). Proper
patterning of Drosophila embryos requires expression of distinct Hox
genes in different spatially restricted regions along the anteroposterior axis
(e.g. McGinnis and Krumlauf,
1992). Expression of Hox genes is initially controlled by the Gap
proteins, such as Hunchback and Kruppel, which set the limits of Hox gene
expression by repressing transcription during early embryogenesis (reviewed by
Bienz and Muller, 1995).
Interestingly, such repression of Hox genes lasts through cell divisions and
in the absence of the Gap repressors. Heritable silencing of Hox genes at the
later developmental stages requires PcG proteins (e.g.
Lewis, 1978;
Duncan, 1982;
Jurgens, 1985;
Bienz and Muller, 1995). Both
the Hox genetic system and its late epigenetic maintenance exist in higher
organisms (e.g. Gould, 1997;
Forlani et al., 2003). PcG
proteins, thus, constitute a widely conserved cell memory system that prevents
changes in cell identity by maintaining transcriptional repression of
previously suppressed genes throughout development and in adulthood.
PcG proteins are thought to maintain gene silencing by controlling
chromatin accessibility (e.g. Boivin and
Dura, 1998; Zink and Paro,
1995; Fitzgerald and Bender,
2001). In vivo, different PcG proteins form at least two distinct
multimeric chromatin silencing complexes
(Ng et al., 2000). The PRC1
complex, containing Polycomb (Pc) and Polyhomeotic (Ph) among others, appears
physically associated with the chromatin of specific cis-regulatory sequences
in Hox genes, called Polycomb response elements (PREs) (e.g.
Shao et al., 1999;
Saurin et al., 2001;
Horard et al., 2000;
Bloyer et al., 2003;
Ringrose et al., 2003).
Meanwhile, experiments with DNA-tethered PcG proteins, such as
GAL4-Pc, have provided evidence that PcG proteins function as potent
transcriptional repressors (Muller,
1995). But detailed molecular links remain missing from binding of
PcG complexes with PREs to chromatin remodeling and transcriptional silencing.
It is also unclear how transiently expressed factors help chromatin silencing
complexes find their way onto targets and how these complexes retain
characteristic chromatin-binding patterns from one cell generation to the
next. In addition, evidence accumulates to challenge our conventional views on
the biological functions of PcG. First, several PcG proteins seem to have a
dual role in both repression and activation of transcription, depending on the
locus and genetic context (Brock and van
Lohuizen, 2001). Second, distinct PcG proteins function in
different complex manners. Some PcG proteins are differentially distributed
from others on polytene chromosomes
(DeCamillis et al., 1992;
Franke et al., 1992;
Ng et al., 2000); and the
phenotypes of different PcG mutants are distinct (e.g.
Campbell et al., 1995;
Narbonne et al., 2004).
Finally, derepression of Hox genes is variably involved in different mutants
and in distinct tissues (e.g. Beuchle et
al., 2001; Dura and Ingham,
1988; Simon et al.,
1992; Choi et al.,
2000).
Although global misrouting of CNS axons has been well demonstrated in
ph mutant embryos (Smouse and
Perrimon, 1990), little is known about roles of PcG complexes in
specific neuronal developmental processes. Here, we report recovery of a new
ph recessive lethal mutation from genetic mosaic screens in adult fly
brains. Loss of subtype identity was evident in ph mutant clones
within otherwise phenotypically wild-type brains. Through metamorphosis, all
ph mutant neurons were transformed into cells with unidentifiable
projection patterns and indistinguishable gene expression profiles. But
postembryonic-born ph mutant neurons were never transformed without
experiencing the prepupal ecdysone peak. In addition, we detected limited
derepression of Hox genes in ph mutant neurons and requirement of
distinct PcG proteins for different aspects of neuronal development. Taken
together, we demonstrate that Drosophila Ph plays an essential role
in maintaining neuronal diversity through metamorphosis, showing possible
two-way interactions between steroid hormone signaling and the epigenetic
functions of PcG.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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); and (4)
elav-GAL4 (Luo et al.,
1994). (5) FRT19A, UAS-mCD8GFP was used to create
wild-type MARCM clones. (6) PcXT109,FRT2A/TM6C and (7)
E(z)731,FRT2A/Tm6C were used together with (8)
hs-FLP,UAS-mCD8GFP;FRT2A,tubP-GAL80;GAL-OK107, (9)
GAL4-C155,UAS-mCD8GFP,hs-FLP and (10) FRT2A,tubP-GAL80 to
create Pc and E(z) MARCM clones. Fly stocks used for mapping
the mutations include (11) ec,cv,ct,t/C(1)DX (BL-1163), (12)
y,ct,ras,f (BL-4362), (13)
Df(1)64c18,g,sd/Dp(1;2;Y)w[+]/C(1)DX (BL-936), (14)
Dp(1;f)R/y,dor (BL-761), (15) Df(1)w258-45/C(1)DX;
Dp(1;3)w[vco] (BL-1527), (16) Df(1)Pgd-kz (BL-1902), (17)
Df(1)Pgd35 (BL-1986), wapl2 (BL-5741), (18)
ph602 (BL-5444), (19) PgdG0385
(BL-11998) and (20) l(1)G0458 (BL-10112). Phenotypic rescue was
carried out with (21) P{ph-d+} transgene that
comprises the ph-d transcription unit
(Randsholt et al., 2000).
MARCM-based genetic screens
Our ongoing genetic mosaic screens have been reported before
(Lee et al., 2000;
Wang et al., 2002;
Zheng et al., 2003). Briefly,
MARCM clones of MB neurons, which are homozygous for EMS-mutagenized various
chromosome arms, were created and screened for abnormal neurogenesis or
neuronal morphogenesis. Mutant chromosomes with interesting phenotypes were
then recovered for future analysis.
Mapping by recombination and complementation
After learning that the l(X)MB342 line was homozygous lethal, we
first mapped the lethal mutation(s) using linkage analysis. Homologous
recombination was induced between the FRT19A, UAS-mCD8-GFP mutant
chromosome and an X chromosome carrying multiple visible mutations. Hemizygous
male progeny were collected and analyzed for viable recombination patterns.
Based on the recombination patterns, l(X)MB342 mutation(s) should be
between y (1A5) and w (3C1). Three duplications, Dp(1;f)R,
Dp(1;2;Y)w[+], and Dp(1;3)w[vco], were found to rescue the
lethality caused by homozygous l(X)MB342. Dp(1;2;Y)w[+] rescued
l(X)MB342 males were used to conduct complementation tests with
related deficiency or mutant lines.
Induction and phenotypic analysis of MARCM clones
Following induction of mitotic recombination at selected stages, MARCM
clones of GAL80-minus cells were created from heterozygous precursors and
examined at various later stages. Organisms were dissected in cold
phosphate-buffered saline and their brains were fixed and immunostained, as
previously described (Lee and Luo,
1999). MARCM clones were detected by the rat anti-mCD8 mAb (1:100,
Caltag). Immunofluorescent signals were collected by confocal microscopy and
then processed using Adobe Photoshop.
Immunohistochemistry
Fly brains were fixed and subjected to immunostaining following the
procedures as described previously (Lee et
al., 1999). Rabbit polyclonal antibodies against Lab (1:100), Pb
(1:100) and Dfd (1:30) were kindly supplied by T. Kaufman. The mouse
monoclonal antibodies anti-Abd-A (1:400) and anti-Ubx (1:30) were gifts from
I. Duncan and J. Müller, respectively. Other mouse monoclonal antibodies
used in this study, including anti-Abd-B (1A2E9) (1:100), anti-Antp (8C11)
(1:100) and anti-Scr (6H4.1) (1:100), were obtained from the Developmental
Studies Hybridoma Bank developed under Department of Biological Sciences, Iowa
City, IA 52242.
CNS organ culture
The culture protocol was adopted from Gibbs and Truman
(Gibbs and Truman, 1998).
Briefly, the CNSs were dissected from mid-third instar larvae and, following
removal of the ring glands, cultured in 200 µl of the Shield & Sang M3
Drosophila medium (Sigma) containing 7.5% heat-inactivated fetal calf
serum (Gibco) and 1% of penicillin (10,000 units/ml)-streptomycin (10 mg/ml)
solution (Sigma). Cultures were kept in a 25°C humidified culture
incubator and aerated with a mixture of 95% air and 5% CO2. Culture
medium was changed every 48 hours. 20-hydroxyecdysone (20E) (Sigma) was
dissolved in isopropanol with a concentration of 10 mg/ml to serve as a
storage solution. A final concentration of 1 µg/ml 20E was achieved by
adding this storage solution directly into culture medium. The same amount of
isopropanol was added into the culture medium in the control experiments.
After culture, the CNS was fixed in 4% paraformaldehyde in PBS.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). For example, using GAL4-OK107
(Connolly et al., 1996) in
MARCM, one can selectively label clones of GAL80-minus neurons in the
Drosophila olfactory learning/memory center, the mushroom bodies
(MBs) (e.g. Lee et al., 1999).
The MBs are paired neuropils (Fig.
1A). One MB is derived from four neuroblasts (Nbs), each of which
continuously undergoes asymmetric divisions to produce a similar set of MB
neurons through different developmental stages until fly eclosion
(Ito et al., 1997;
Lee et al., 1999). In the
presence of GAL4-OK107, when mitotic recombination results in loss of
GAL80 from one MB Nb, one would observe labeling of all the MB neurons
subsequently derived from the GAL80-minus MB Nb
(Fig. 1B). One MB Nb clone,
generated in newly hatched larvae, consists of about 400 neurons at the adult
stage and exhibits all basic MB morphological features. MB cell bodies are
clustered on the posterior dorsal surface of the protocerebrum. Their
dendrites elaborate immediately below cell bodies and constitute the calyx.
Axons then project through the peduncle into various subsets of MB lobes
(Fig. 1B).
To identify genes governing various aspects of neuronal development, we
have been generating MARCM clones of MB neurons that are homozygous for
various mutations in otherwise largely heterozygous organisms
(Lee et al., 2000;
Wang et al., 2002;
Zheng et al., 2003). Following
chemical mutagenesis, we screened about 800 X chromosomes
(Fig. 1C) and recovered one
mutated X chromosome that drastically altered the general patterns of MARCM
clones (Fig. 1D). We no longer
observed typical MB clones. Instead, there were multiple unrecognizable clones
of cells in the mosaic brains. It appears that clones of homozygous mutant
neurons undergo very rudimentary morphogenesis and fail to acquire any
specific projection patterns (Fig.
1D). In addition, homozygous mutant cells might constitutively
express GAL4 (see below), resulting in ubiquitous labeling of GAL80-minus
clones despite use of a MB-selective GAL4 driver.
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).
Interestingly, l(X)MB342 fails to complement with three of the five
lethal groups. It appears that ph, PgD and wapl, which are
located within a 40 kb genomic segment, are all mutated in the
l(X)MB342 mutant X chromosome
(Fig. 2A). However, no deletion
could be detected in any of these open reading frames (data not shown).
Furthermore, we found that a lethal P-element line, l(1)G0458
(BL-10112), that carries a P-element insertion within the promoter region of
ph-D (Peter et al.,
2002), also fails to complement with ph, PgD and
wapl mutations, suggesting possible presence of a local cis element
essential for normal expression of several contiguous genes.
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).
ph602 is a null ph allele that carries a 2 kb
deletion in ph-d and a complex genomic DNA rearrangement involving
both deletion and inversion in ph-p
(Boivin et al., 1999).
Interestingly, a transgene that specifically encodes Ph-d,
P{ph-d+} can largely rescue the mutant phenotypes
of l(X)MB342 clones (Fig.
2C). This is not only consistent with the notion that Ph-P and
Ph-D are functionally redundant (Dura et
al., 1987; Deatrick et al.,
1991; Saget and Randsholt,
1994), but also suggest further that loss of Ph functions accounts
for most, if not all, of the observed l(X)MB342 phenotypes. To
examine specifically roles of Ph proteins in development of distinct neurons,
we selectively focused phenotypic analysis on clones of
ph602 mutant neurons hereafter.
Ph is required for proper neuronal morphogenesis as well as establishment of cell type-specific gene expression patterns
In insect brains, postmitotic neurons of the same lineages often remain
associated; and distinct clones of neurons normally acquire different
characteristic projection patterns (e.g.
Fig. 1A,B;
Fig. 3A,D,I,L). One can, thus,
describe different clones of neurons based on the cell body positions and
neurite trajectories of individual clones. Although most
GAL4-OK107-labeled ph mutant neurons exist in discrete
clones, we are no longer able to determine clonal identity because of a lack
of recognizable neuropil structural features in the ph mutant clones.
In addition, without Ph, the MB GAL4 driver, GAL4-OK107, appears
labeling more GAL80-minus clones than it usually does
(Fig. 3N, compared with 3J). In
wild-type mosaic brains, GAL4-OK107 labels about 0.75±0.23
neuroblast clones per brain; and the majority of these clones consist of MB
neurons. By contrast, there are 8.56±2.44 clones of ph mutant
neurons per brain that could be labeled by GAL4-OK107 after
comparable induction of mitotic recombination.
To gain additional insights into the neuronal functions of Ph, we repeated
MARCM analysis of ph602 using various GAL4 drivers.
Different GAL4 drivers normally label different types of MARCM clones,
depending on GAL4 expression patterns. Thus, while the pan-neuronal
elav-GAL4 (Luo et al.,
1994) allows for visualization of all neuronal clones,
GAL4-OK107 (Connolly et al.,
1996), GAL4-NP225 (gift from K. Ito), ato-GAL4
(Hassan et al., 2000) and
GAL4-EB1 (Wang et al.,
2002) selectively label clones of MB neurons, antennal lobe
projection neurons (PNs), Atonal-positive dorsal cluster (DC) neurons and
specific ellipsoid body (EB) neurons, respectively, in wild-type mosaic brains
(Fig. 3I-L; data not shown for
GAL4-EB1). Interestingly, when such distinct GAL4 drivers were
individually used to label ph mutant clones, we detected either all
or none of ph mutant clones (Fig.
3M-P). First of all, similar numbers of elav-GAL4-labeled
clones exist between wild-type (8.85±3.15) and ph
(9.12±2.79) mosaic brains, suggesting that ph mutation does
not affect patterns of mitotic recombination or initial neurogenesis (compare
Fig. 3I with 3M). Second,
GAL4-OK107 and GAL4-NP225 label similar numbers of
ph mutant clones (8.56±2.44 and 7.87±2.62,
respectively) as the pan-neuronal elav-GAL4 does, probably owing to
ectopic activation of these GAL4 drivers in all ph mutant neurons
(compare Fig. 3N and 3O with
3M). Third, ato-GAL4 and GAL4-EB1, by contrast,
become suppressed and fail to label any ph mutant cells in adult
mosaic brains (Fig. 3P). In
summary, we no longer detect differential GAL4 expression in ph
mutant neurons of various origins. Moreover, instead of acquiring different
characteristic projections, distinct clones of ph mutant neurons are
typically found with similar simple bundles of neurites
(Fig. 3M-O). Endogenous genes
may also display either ubiquitous or no expression in ph mutant
clones, as evidenced by lack of fasciculin II
(Lin and Goodman, 1994)
expression (based on immunostaining with the 1D4 monoclonal Ab) in all the
examined ph mutant clones (data not shown). These phenomena
collectively suggest that ph mutant neurons lose their individual
identities and are uniformly transformed into indistinguishable abnormal
cells.
Regulation of gene expression by PcG complexes is thought to occur via
changes in local chromatin structures (reviewed by
Levine et al., 2004). We
wondered whether derepression versus inactivation of a given GAL4 driver
depends on its specific genomic locus. Jumping an existing ato-GAL4
transgene out of its original chromosome, we isolated 10 additional
independent ato-GAL4 drivers that maintain strong GAL4 expression in
Ato-positive CNS neurons but probably have the ato-GAL4 transgene
inserted in 10 different genomic domains. We observe that none of these
independent ato-GAL4 drivers is capable of labeling ph
mutant neurons in adult mosaic brains, yielding no evidence for involvement of
local chromatin structures in constitutive silencing of atonal.
Instead, silencing of ato-GAL4 in ph mutant neurons possibly
occurs following derepression of some ato-GAL4 repressor(s).
Remarkably, the aforementioned ph mutant phenotypes are not observed prior to pupal formation. First, in mosaic larval brains, most subtype-specific GAL4 drivers label different characteristic ph mutant clones. For example, although GAL4-NP225 failed to label anything (Fig. 3G), GAL4-OK107 and ato-GAL4 selectively labeled MB and DC clones, respectively (Fig. 3F,H). These observations suggest that derepression of GAL4-OK107 and inactivation of ato-GAL4 in ph mutant neurons must occur later. Second, despite various degrees of morphological defects, distinct mutant clones roughly retained their normal patterns of projections at the wandering larval stage (Fig. 3E-H compare with 3A-D). Thus, ph mutant neurons remain largely recognizable until sometime after pupal formation. What a coincident for the loss of neuronal subtype identity to take place during metamorphosis when the prepupal ecdysone peak activates various transcriptional hierarchs in distinct types of cells.
Ecdysone-dependent transformation of ph mutant neurons during early metamorphosis
If ph mutant neurons gradually transform regardless of
ecdysone-mediated metamorphosis, one would observe similar phenotypes in all
aged ph mutant clones no matter whether they were induced before or
after the prepupal ecdysone peak. To examine possible involvement of ecdysone
signaling in Ph-dependent maintenance of neuronal diversity, we generated
clones of ph mutant neurons at late larval versus early pupal stages
and checked their phenotypes 2 days and 2 weeks after eclosion. As expected,
we obtained many clones within the optic lobes upon induction of mitotic
recombination at the mid-3rd instar stage when optic lobe (OL) precursors were
actively dividing (Fig. 4A).
Distinct GAL4 drivers were again used to probe neuronal cell fates. For
example, all GAL4-OK107-labeled OL cells are negative for
GAL4-NP225 expression in wild-type mosaic brains (compare
Fig. 4A with 4B). By contrast,
OL clones of ph mutant neurons are positive for both
GAL4-OK107 and GAL4-NP225 expression
(Fig. 4C,D). Additional
phenotypes are observed in the morphologies of ph mutant OL clones.
For example, most mutant OL neurons are aberrantly aggregated and their
bundled neurites fail to defasciculate into any recognizable optic lobe
neuropils (Fig. 4C,D). We thus
conclude that all the ph mutant neuronal clones generated prior to
pupal formation have lost their original identities and evolved into
undistinguishable clones at the adult stage. We then generated MARCM clones 1
day after pupal formation and, by great contrast, detected no indication for
similar cell fate transformation even in much aged ph mutant neurons.
MB Nbs, different from other Nbs, continue producing post-mitotic neurons
through the entire pupal life and can be subject to mitotic recombination
until eclosion (Lee et al.,
1999). Unlike ph mutant OL clones, pupal-born MARCM
clones of ph mutant MB neurons are strongly labeled by
GAL4-OK107 while remaining negative for GAL4-NP225
expression, even in 2-week-old adults (compare
Fig. 4G with 4H). In addition,
we see no problem in identifying pupal-born ph mutant MB neurons.
They acquired basic MB neuronal trajectories despite subtle morphogenetic
defects (Fig. 4G), reminiscent
of larval-born ph mutant MB neurons examined before pupal formation
(Fig. 3F). Taken together,
these observations suggest that ph mutant neurons lose their
subtype-characteristic gene expression and neurite projection patterns
specifically during metamorphosis.
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To determine directly the role of ecdysone in the transformation of ph mutant neurons during early metamorphosis, we subsequently examined whether and how synthetic 20-hydroxyecdysone affects MARCM labeling of ph mutant clones in cultured fly brains. We first found that loss of neuronal diversity, as evidenced by appearance of multiple unrecognizable MARCM clones, inevitably occurred in ph mosaic brains that were cultured from the late wandering larval stage (empty gut stage) (data not shown). By contrast, transformation of ph mutant neurons depended on the availability of 20-hydroxyecdysone when culture of mosaic brains started before larval wandering (Fig. 6). We cultured ph mosaic brains for 4 days and detected no sign for loss of neuronal diversity in the absence of 20-hydroxyecdysone (Fig. 6A). But adding 20-hydroxyecdysone into the media from the beginning or even 4 days later efficiently led to transformation of ph mutant clones and appearance of multiple bizarre GFP-positive clones in cultured ph mosaic brains (Fig. 6B,C). Such ecdysone-dependent changes in the patterns of MARCM clones were never observed in wild-type mosaic brains that were cultured in parallel (data not shown). These results provide direct evidence for involvement of ecdysone signaling in transforming ph mutant neurons of various origins into a homogeneous population of abnormal cells.
Differential involvement of distinct PcG proteins in neuronal development
Ph is best known for its involvement with other PcG proteins in maintaining
the silent state of homeotic (Hox) genes (e.g.
Beuchle et al., 2001;
Choi et al., 2000). To
determine roles of general PcG functions in neuronal development, we conducted
loss-of-function mosaic analysis for two other PcG proteins, Polycomb (Pc) and
Enhancer of zeste [E(z)]. Two distinct multimeric PcG complexes, which each
contains different PcG proteins, have been identified
(Levine et al., 2004).
Multiple lines of evidence reveal that Ph forms complexes with Pc but not with
E(z) (e.g. Franke et al.,
1992; Shao et al.,
1999). However, neither Pc nor E(z) mutant
clones phenocopy Ph loss-of-function phenotypes
(Fig. 7). First, we no longer
observe ectopic expression of GAL4-OK107 in the neuronal clones, that
are normally negative for GAL4-OK107 expression, within Pc
or E(z) mosaic brains. This did help us identify variably deformed MB
clones. Second, MB clones homozygous for these different PcG mutations exhibit
distinct morphological anomalies. At the wandering larval stage, we
specifically observe exuberant disorganized `dendrites' in Pc Nb
clones (arrow in Fig. 7A).
Preferential involvement of Pc in confining dendritic growth is further
evidenced by acquisition of over-elaborated dendrites, but not axons, in the
single-cell clones of Pc mutant adult MB neurons
(Fig. 7F compared with 7E). By
contrast, no specific gross defect could be detected in either larval
E(z) mutant Nb clones (Fig.
7B) or adult E(z) mutant single-cell clones
(Fig. 7G). In addition,
although both Pc (Fig.
7C) and E(z) (Fig.
7D) mutant MB Nb clones exhibit many ectopic neurites at the adult
stage, the presence of calyx-like structures in the E(z) (inset in
Fig. 7D), but not Pc,
clones, together with significant reductions in the numbers of
normal-projecting axons strongly suggest that the wide-spreading neurites are
mostly misguided axons, but not overshooting exuberant dendrites, in the adult
E(z) mutant Nb clones (Fig.
7D). Interestingly, such defasciculation of neurites is in great
contrast with the aberrantly aggregated neuronal bundles of Ph mutant
clones (compare Fig. 7C,D with
Fig. 1D). Finally, we observe
ectopic labeling of glia-like cells only in E(z) mutant mosaic adult
brains (Fig. 7H). This
phenomenon suggests selective involvement of E(z) in governing glial identity.
Taken together, distinct PcG proteins are differentially involved in
regulating various aspects of brain development, suggesting specific roles of
Ph in maintaining neuronal diversity especially through metamorphosis.
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;
Beuchle et al., 2001). We
wondered whether similar mechanisms underlie abnormal development of PcG
mutant neurons. Using immunohistochemistry, we characterized expression of
eight Hox genes, including abd-A, abd-B, ubx, antp, pb, dfd, lab and
scr, in various PcG mutant clones at the wandering larval stage.
Distinct Hox genes are normally expressed in different characteristic
segmental domains. For example, expression of abd-B and ubx
is restricted to the terminus and one middle segment of the ventral ganglion,
respectively (Fig. 8A,H).
Examining whether any Hox gene became ectopically expressed in mosaic larval
CNSs, we found that only abd-B was misexpressed in ph mutant
clones, while misexpression of several Hox genes, including abd-A,
abd-B and antp, occurred in Pc mutant clones
(Fig. 8;
Table 1). Furthermore, when we
ectopically expressed abd-B in wild-type MARCM clones using
GAL4-C155/UAS-abd-B, no obvious defects were found in the axonal
projection patterns of various neurons (J.W., C.-H.J.L. and T.L.,
unpublished). These results suggest little involvement of Hox genes in the
unanimously abnormal responses of ph mutant neurons to ecdysone
signaling during early metamorphosis. In addition, only antp became
derepressed when E(z) was knocked out; and derepression of
antp was restricted to about 20% of E(z) mutant clones
(Table 1). Taken together,
derepression of distinct Hox genes occurs in different PcG mutant clones,
further suggesting differential involvement of distinct PcG proteins in
governing neuronal identities through development.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
Deregulation of multiple genes aberrantly occurs in ph mutant tissues
(e.g. Dura and Ingham, 1988;
Beuchle et al., 2001). A
similar mechanism probably underlies most of the abnormalities in ph
mutant neurons. In particular, there are multiple lines of evidence suggesting
mal-expression of various subtype-specific GAL4 drivers in ph mutant
clones. First, with respect to GAL4-OK107, GAL4-NP225 and elav-GAL4, the use
of various GAL4 drivers results in labeling of similar numbers of clones
(Fig. 3M-O). Second, we observe
clones in the central brain versus the optic lobe, depending on when mitotic
recombination is induced (compare Fig.
3N-O with Fig.
4C-D), which is the same as in wild-type mosaic brains (compare
Fig. 3B with
Fig. 4A). Third, ato-GAL4 and
GAL4-EB1 fail to label any clone (Fig.
3P, data not shown for GAL4-EB1), arguing against constitutive
expression of UAS-transgenes in mutant clones. Finally, examining clones
through development reveals no evidence for derivation of some clones from
other clones; and, instead, we constantly observe sudden labeling of
full-sized clones shortly after a big ecdysone pulse
(Fig. 5). Apparently, loss of
Ph function alone is short of causing the full spectrum of abnormalities. Mass
ecdysone is required for the pathological transformation of ph mutant
neurons in the Drosophila brain, raising several interesting
possibilities about mutual involvement between the epigenetic function of PcG
and the global nuclear signaling of steroid hormones.
|
|
;
Truman et al., 1994;
Bender et al., 1997;
Lee et al., 2000), but
ph mutant neurons of distinct origins become no longer
distinguishable after ecdysone signaling. Ecdysone mediates diverse biological
activities partially via binding to different heterodimeric receptors (e.g.
Schubiger et al., 1998;
Lee et al., 2000;
Cherbas et al., 2003). Its
conventional receptors consist of the nuclear receptor superfamily members
ecdysone receptor (EcR) and Ultraspiracle (USP; the Drosophila RXR)
(Yao et al., 1992;
Yao et al., 1993). There are
three documented EcR isoforms (Talbot et
al., 1993); and cells that express different EcR isoforms have
been shown to undergo different changes in response to the prepupal ecdysone
peak (Truman et al., 1994).
For example, abundant EcR-B1 exists selectively in the neurons that remodel
projections during early metamorphosis
(Schubiger et al., 1998;
Lee et al., 2000). As we
detected no change in EcR expression patterns in ph mutant neurons
(J.W., C.-H.J.L. and T.L., unpublished), it is unlikely that the aberrant
responses of ph mutant neurons to the prepupal ecdysone peak occur as
a result of derepression of specific EcR isoforms. In addition, derepression
of multiple Hox genes appears not involved either. Nevertheless, given the
involvement of Ph in silencing transcription, it remains possible that
derepression of other unidentified genes directly re-programs ecdysone-induced
transcriptional hierarchies, leading to transformation of ph mutant
neurons. Alternatively, it is possible that loss of the epigenetic function of
Ph may permit diffuse activation of prohibited genes by normal transcriptional
hierarchies. Moreover, massive steroid hormone signaling might directly modify
genomic imprinting when PcG functions are compromised.
It is generally thought that nuclear signaling of steroid hormones occurs
routinely through the life of an organism without affecting the memories of
most cells. However, high levels of sex hormones during pregnancy possibly
alter gene expression patterns permanently in the mammary gland even after
involution (Ginger et al.,
2001). Exposure to some hormonally active reagents during early
development also has the potential for imprinting long-lasting changes on the
action of related hormones (Mena et al.,
1992). In mammalian neurons, the estrogen receptor-
was
further found to silence gene expression in an epigenetic fashion and via
hypermethylation of the involved promoters
(Zschocke et al., 2002). All
these phenomena argue for the abilities of steroid hormones to modulate
genomic imprint, at least, under certain circumstances. Ecdysone-dependent
transformation of ph mutant neurons, thus, provides a possible model
system for characterizing the epigenetic functions of steroid hormones in
genetically malleable organisms. In addition, our demonstration of the unusual
potent epigenetic effects of ecdysone in ph mutant neurons suggests
complex mechanisms may underlie pathogenesis of other documented PcG
loss-of-function phenotypes.
Both derepression and inactivation of genes occur in transformed
ph mutant neurons, characterization of which offers some molecular
insights into this status of transformation. First, we no longer detected the
fine-tuning of gene expression in transformed cells; and all the examined
drivers appeared either fully on or completely off. Second, on or off could
not be simply attributed to the genomic locations of drivers, as evidenced by
constitutive silencing of the multiple independently inserted
atonal-GAL4 transgenes. Third, transformed cells retained neuron-type
morphologies and remained positive for the neuron-specific gene elav;
and ph mutant neurons had been earlier reported to acquire
normal-looking neurites in culture (Smouse
and Perrimon, 1990). Taken together, the transformation leads to
loss of subtype identity without affecting basic neuronal fates, abolishes the
genomic imprints governing fine controls over gene expression, and locks gene
expression in `on' or `off' possibly in a promoter-autonomous manner (largely
independent of its chromatin environment).
Finally, loss of Ph, Pc, versus E(z) results in distinct phenotypes in the
developing fly brain. Differences in their underlying pathological mechanisms
are well exemplified by differential derepression of distinct Hox genes in
different PcG clones. In addition, for a given PcG mutation, patterns of Hox
gene derepression vary from neural clones to wing disc clones
(Beuchle et al., 2001) and
visceral mesoderm (Choi et al.,
2000). It remains to be elucidated how distinct PcG functions are
governed in diverse cell type-characteristic manners.
|
ACKNOWLEDGMENTS |
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|
Footnotes |
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REFERENCES |
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