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Overexpression of HmgD causes the failure of pupariation in Drosophila by affecting ecdysone receptor pathway.

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Archives of Insect Biochemistry and Physiology 68:123–133 (2008)
Overexpression of HmgD Causes the Failure of
Pupariation in Drosophila by Affecting Ecdysone
Receptor Pathway
Jing Chen, Hui Wang, and Yu-Feng Wang*
HmgD encodes Drosophila homologue of high mobility group proteins (HMGD), which are thought to have an architectural
function in chromatin organization. However, current opinions about the function of HMGD in Drosophila development are
controversial. Our previous studies have shown that ubiquitous overexpression of HmgD caused the formation of melanotic
tumors in the Drosophila larvae by prematurely activating the Ras-MAPK pathway. Here we report that under maternal control,
the viability of flies links with overexpression of HmgD, while under ubiquitous control, ActGal4, overexpressing HmgD animals, which display prolonged larval stages around day 13, developmentally stagnate in the larva-white pupa transition.
Ecdysone feeding did not rescue overexpressing HmgD animals. RT-PCR analyses show that overexpression of HmgD does not
affect the temporal expression pattern of ecdysone receptor gene EcR, whereas transcriptional patterns of some key regulatory
genes, such as E74A, E74B, E75A, E75B, βFTZ-F1, are changed greatly. These results suggest that ubiquitous overexpression
of HmgD results in the failure of pupariation neither by affecting the process of ecdysone synthesis and release nor by
abnormal EcR transcription, but by causing expression of EcR regulatory nuclear receptors out of schedule. The results led us to
postulate that overexpression of HMGD likely changes the signaling cascade of Drosophila metamorphosis by an interaction
between HMGD and DNA strands, and subsequently by an error of DNA binding abilities and transcriptional activities of some
nuclear receptor genes. Arch. Insect Biochem. Physiol. 68:123–133, 2008. © 2008 Wiley-Liss, Inc.
KEYWORDS: HmgD; overexpression; Drosophila; pupariation; ecdysone receptor pathway
INTRODUCTION
The gene HmgD encodes the Drosophila homologue of the high mobility group proteins, HMGD,
which is closely related to the vertebrate HMG-Box
(HMGB) proteins (formerly termed HMG-1/2).
They are relatively abundant proteins that bind to
DNA and bend DNA substantially, acting primarily as architectural facilitators in the assembly of
nuclear protein complexes (Thomas and Travers,
2001). Circumstantial evidence suggests that HMGD
reduces the compactness of chromatin packing during very early development of Drosophila when his-
tone H1 is absent. This loose structure could facilitate the rapid condensation and de-condensation
of chromatin required during the very short early
nuclear division cycles. As the maternal pool of
HMGD is titrated by rapid replication cycles and
concomitant chromatin assembly, and as histone
H1 undergoes synthesis, HMGD would functionally be suppressed by histone H1 around the midblastula transition (MBT) (Ner and Travers, 1994).
However, comparison of the DNA-binding capacity and specificity in recognition of DNA clearly
shows that HMGD does not share the same binding properties with histone H1. Moreover, HMGD
Hubei Key Laboratory of Genetic Regulation and Integrative Biology, College of Life Sciences, Central China Normal University, Wuhan, P. R. China
Contract grant sponsor: National Natural Science Foundation of China; Contract grant number: 30300035; Contract grant sponsor: Scientific Research Foundation
for the Returned Overseas Chinese Scholars, State Education Ministry; Contract grant number: (2004) 527.
*Correspondence to: Yu-Feng Wang, College of Life Sciences, Central China Normal University, 152 Luoyu Avenue, Wuhan 430079 P. R. China.
E-mail: yfengw@mail.ccnu.edu.cn
Received 5 September 2007; Accepted 4 January 2008
© 2008 Wiley-Liss, Inc.
DOI: 10.1002/arch.20237
Published online in Wiley InterScience (www.interscience.wiley.com)
124
Chen et al.
is expressed not only in adult females and early
embryos but also in later embryonic developmental stages, suggesting that HMGD may exert other
functions than that of a specific early embryonic
substitute of H1 (Renner et al., 2000).
In Xenopus, both linker histone B4 and HMGB1
protein show the same properties as HMGD during early embryogenesis (Dimitrov et al., 1993) and
in the assembly system in vitro (Nightingale et al.,
1996). However, HMGB1 protein does not associate with chromatin in cells during mitosis in early
mouse embryos, even when the level of histone
H1 is very low (Spada et al., 1998). In mice, a
single mutant in Hmgb1 or Hmgb2 did not result
in embryonic defects, except that a Hmgb1 mutant
pup died within 24 h due to hypoglycemia and
that Hmgb2 knockout mice decreased male fertility (Calogero et al., 1999; Ronfani et al., 2001).
Hmgb3-deficient mice are viable but erythrocythemic (Nemeth et al., 2003, 2005). Recently,
Ragab et al. (2006) showed that homozygous mutant HmgD/Z is viable and exhibits only minor morphological defects. Taken together, the function of
HMGB in development still remains uncertain.
In order to further analyse the function of HMGD
on the development of Drosophila, we generated
pUASpHmgD transgenic flies and overexpressed
HmgD under both maternal control and ubiquitous
control (ActGal4), respectively. We found that under
maternal control some effects on the viability of embryos appeared to link with the overexpression of
HmgD. While overexpression of HmgD under ActGal4
driver prematurely activated Ras-MAPK pathway and
consequently led to the formation of melanotic tumors in larvae (Chen et al., 2006). Here, we report
that ubiquitous overexpression of HmgD led to disruption of the temporal expression pattern of some
ecdysone receptor signaling pathway members, thus
resulting in the failure of Drosophila pupariation.
MATERIALS AND METHODS
Fly Stocks and Their Propagation
Drosophila melanogaster used in our lab was wildtype strain Canton S, obtained from Central China
Normal University. All transgenic flies were constructed in the Curie Institute (Paris, France). Larvae and flies were cultured on standard yeast/
glucose medium.
Plasmid Construction
The coding region of HmgD gene (GeneBank
Accession No. NM166480) was obtained by PCR
amplification from a cDNA library of Drosophila
0–4-h embryos (Clontech). The primers containing Asp 718 and BamH I restriction sites were
HmgD5 (5′-ggggtacccatATGTCTGATAAGCCAAAACGCC-3′) and HmgD3 (5′-cgggatcccgCTACTCGCTCTCATCATCGTC-3′). After digestion by enzymes
Asp 718 and BamH I, PCR-amplified fragments were
inserted into pUASp plasmids (Rorth, 1998; Rorth
et al., 1998), and sequenced from both sides to
confirm the transgenes (data not shown).
Generation of Transgenic Fly Strains and Genetics
Progeny embryos of W– fly (white eyes) were
collected at 25°C. Embryonic microinjections of
constructs to generate transgenic lines were performed as described in Bellaiche et al. (1996).
Progeny with red eyes were selected for balancing
and mapping insertions. CyO and Tm2 are balancers for chromosome II and chromosome III, respectively. Sp and Sb are markers of chromosome
II and chromosome III, respectively. nanos-Gal4,
NGT40 (Bloomington Stock Center), and ActGal4/
CyO-PscGFP (generated from ActGal4/CyO flies by
a genetic method) females were used to cross with
pUASp transgenic males for overexpressing HmgD.
Transgenic flies were confirmed by PCR with primers harboring two ends of the cloning sites of the
pUASp vector (Chen et al., 2006).
Selection of Mutated Larvae
Virgin females of ActGal4/Cyo-PscGFP, which
overexpress the target gene under the control of actin ubiquitous promoter ActGal4 when UAS (Upstream Activation Sequence) is present, were collected
and crossed with transgenic males pUASpHmgD A;
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Chromatin Structure in Drosophila Development
pUASpHmgD E carrying four copies of pUASpHmgD.
The eggs laid within 3–4 h were incubated at 25°C
for 24 h until all larvae hatched out. The first instar
overexpressing HmgD (pUASpHmgD A/ActGal4;
pUASpHmgD E/+) larvae were selected under the fluorescence stereomicroscope (Leica MZ16 F Germany)
from their lack of GFP fluorescence. The wild-type
larvae (Canton S) were used as control.
Ecdysone Feeding Experiments
20-hydroxyecdysone (20E, Sigma, St. Louis,
MO) was prepared to 12 mg/ml stock solution with
ethanol first. This stock solution (4.2 ml) was diluted in 95.8 ml sterile water and added to 0.05 g
dry yeast to make the yeast paste containing 0.5
mg/ml 20E. As a negative control, 4.2 ml ethanol
was diluted in 95.8 ml sterile water and added to
0.05 g dry yeast (Gates et al., 2004). One hundred
milliliters of sterile water containing 0.05 g dry
yeast was used as normal yeast medium.
First instar larvae were allowed to develop for
50 h after larvae hatching (ALH), a time point corresponding to the early third instar in wild type
animals. HmgD overexpressing larvae were divided
into two groups. One group was transferred to the
20E containing yeast paste, the other was transferred to the ethanol containing paste as control.
The same aged wild type larvae were cultured in
normal yeast medium. As positive controls, embryos from the cross E75 A81 / TM6B Tb UbiGFP×E75 ∆51/TM6B Tb Ubi-GFP (gift from C.S.
Thummel) were collected for 3 to 4 h. The embryos were maintained at 25°C and allowed to
hatch. E75A81/E75∆51 first instar larvae were selected
by their lack of GFP expression. The mid-second
instar larvae were transferred to food with or without 20E for 6 h and then returned to regular yeast
paste.
These larvae were fed until all wild type larvae
developed to pupae. Developmental stages were
observed and freshly corresponding yeast pastes
were provided three or four times a day. Around
the time of pupariation, the larvae were observed
every 15 min.
Archives of Insect Biochemistry and Physiology July 2008
125
Staging of HmgD Overexpressing Larvae and
White Pupae
The wandering third instar larvae were collected
around 66 h ALH (Andres et al.,1993) and staged
at approximately 14–22 h relative to puparium formation (–16 h in Fig. 3, taking 2 h difference into
account during egg laying). Four hours later, the
larvae were sampled as –10 h in figures. Stationary
larvae were estimated to be –3 h. Newly formed
white pupae were marked as 0 h relative to puparium formation and for future collection at various time points (Karim et al., 1993). The HmgD
overexpressing larvae were staged by synchronizing wild-type animals at puparium formation. Ten
to fifteen samples were collected per time point.
RT-PCR Analysis for Ecdysone Signal Pathway
Total RNAs were isolated using the SV total RNA
Isolation System (Promega) and were reversely
transcribed with M-MLV (Promega, Madison, WI)
with oligo(dT) primers (Tiangen, Beijing, China ).
rp49 was used to adjust the concentration of reverse-transcribed first-strand cDNAs. Well-adjusted
cDNAs were used as templates to amplify specific
fragments with gene-specific primers (Table 1).
Amplification conditions for PCR reactions were:
94°C 30 s; 50–61 (based on various primers), 45 s;
72°C, 1 min for 30 cycles.
TABLE 1. Primers Used for RT-PCR
Genes
Primer names
EcR
E74A
E74B
E75A
E75B
BR-C
Edg84A
âFTZ-F1
E93
EcR-Fwd
EcR-Rev
E74A-Fwd
E74A-Rev
E74B-Fwd
E74B-Rev
E75A-Fwd
E75A-Rev
E75B-Fwd
E75B-Rev
BRC-Fwd
BRC-Rev
Edg84A-Fwd
Edg84A-Rev
FTZ-Fwd
FTZ-Rev
E93-Fwd
E93-Rev
Sequences
5′-TTTCGGCATATAACCGAGATAAC-3′
5′-CGTATATGATCTATTATTCGCG-3′
5′-GCCGGCGACTGTGCCACCAAGCTG-3′
5′-CGGAGTGAGCAGCGTATTTTGCTC-3′
5′-GCGGACGTGGAGGCAGACGCCGAG-3′
5′-CGGAGTGAGCAGCGTATTTTGCTC-3′
5′-GCCAACACGGGGAGGAACACGCCA-3′
5′-CAGAACGATGGCGCAGAACAGGCC-3′
5′-ACAGTGCTGTGCCGCGTTTGCGGG-3′
5′-CAGAACGATGGCGCAGAACAGGCC-3′
5′-GCTCGAGCGGGCGCCGATGATG-3′
5′-AGGGGACACGGCGAGGTCACCG-3′
5′-GATGATGTCCGTGGATTCAACGCCG-3′
5′-GGCGTGGTGCACGGTAGTCTTCAG-3′
5′-TACCAGGACACCACCTCCTCACAC-3′
5′-AAGTCGATCACCTCGCCGCCGTG-3′
5′-AACGGTAGCAATGGCAACGGGC-3′
5′-CTTGTTGTGCGCCAAATCGCTGT-3′
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Chen et al.
Western Blot Analysis
Virgin females of ActGal4/Cyo-PscGFP were
crossed with two fly lines carrying two and four copies of transgene, respectively. HmgD overexpressing
first instar larvae were selected by their lack of GFP
fluorescence. Wild-type larvae were collected as controls. The larvae were allowed to develop to midthird-instar for extraction of proteins. The proteins
were separated by SDS-PAGE. HMGD levels were
detected by an antibody raised against the HMGB
domain of HMGD (rat anti-HMGD-100, 1:400).
BCIP/NBT staining was performed subsequently.
Statistics Analysis
Assays were repeated three times. Data were expressed as mean ± SE and the statistical significance of the results was assessed by one-way
analysis of variance (ANOVA).
RESULTS
The Effect of Overexpressing HmgD Under
Maternal Control
Dominant phenotypic marker (CyO, curly wings
located on chromosome II), were used to clearly
characterize the effect of overexpressing HmgD in
heterozygous transgenic flies carrying the insertion
of HmgD on chromosome II. These heterozygous
transgenic flies were crossed with NGT40 females
and the percentages of the flies with straight wings
(non Cyo, with Gal4) in the first generation from
these crosses (F1) was investigated. Our results
(Table 2) showed that percentages of straight wing
flies in F1 were significantly lower than that of controls (P < 0.01) except D line flies, indicating that
overexpressing HmgD decreases viability of flies.
Overexpression of HmgD Under the Ubiquitous Control
(Act-Gal4) Caused Defects in Transition From Third
Instar Larvae to White Pupae
Virgin females of ActGal4/Cyo-PscGFP, expressing Gal4 under control of actin ubiquitous promoter, were crossed with transgenic males to
investigate the effects of overexpressing HmgD on
the development of Drosophila. Western blotting
showed that compared with wild type, HMGD protein was overexpressed under the control of ActGal4,
and with the increase of transgenic pUASpHmgD
copies, the HMGD level also increased (Fig. 1).
When males carrying four copies of pUASpHmgD
were crossed with females of ActGal4/Cyo-PscGFP,
all progeny had curly (CyO) wings. This indicates
that the presence of two copies of pUASpHmgD
transgenes and ActGal4 together in the same fly induced 100% lethality, whereas their siblings (curly
wings), which did not overexpress HmgD due to a
lack of ActGal4, developed normally.
To investigate at which stage these flies died,
virgin females of ActGal4/Cyo-PscGFP were crossed
with the transgenic males of pUASpHmgD A;
pUASpHmgD E. The eggs laid within 4 h were incubated at 25°C for 24 h, while wild type eggs were
TABLE 2. Decreased Survival Rate Induced in Adults by HmgD
Expression Under Maternal Control*
Maternal genotype: NGT40 (25°C)
Paternal genotype
[+; +]/Cyo-Tm9:Sb
pUASpHmgD A /Cyo; Sb/Tm2:ry
[+;pUASpHmgD B]/Cyo-Tm9:Sb
[+;pUASpHmgD C]/ Cyo-Tm9:Sb
[+;pUASpHmgD D]/Cyo-Tm9:Sb
[Sp; pUASpHmgD E]/Cyo-Tm9:Sb
[pUASpHmgD A; pUASpHmgD E]/Cyo-Tm9:Sb
Flies with straight
wings (%) ± SE
Number of
flies analysed
±
±
±
±
±
±
±
166
239
105
156
149
168
238
69.7
59.3
58.0
62.4
65.8
56.9
58.0
0.9
1.2a
0.5a
1.2a
1.5
0.6a
1.2a
*A, B, C, D, E (in roman, not italics): different transgenic lines. Data are expressed
as mean ± SE and the statistical significance of the results was assessed using a
one-way analysis of variance (ANOVA).
a
The difference is significant, P < 0.01.
Fig. 1. Overexpression of HMGD was confirmed by Western blotting. A very weak signal was detectable in the 3rd
instar larvae of wild type (Lane 1). More protein was induced in the 3rd instar larvae by ActGal4 with transgenic 1
copy of pUASpHmgD (Lane 2) and 2 copies of pUASpHmgD
(Lane 3).
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Chromatin Structure in Drosophila Development
127
TABLE 3. The Larval Development of Drosophila Overexpressing HmgD Under the Control of Act-Gal4 Driver*
Fly line
Wild type
pUASpHmgD A /ActGal4:: pUASpHmgD E/+
Number of
L1 (d1)
Survival rate of
L2 (d2) (%)
Survival rate of
L3 (d3) (%)
Percentage of
pupae (d4) (%)
120
85
84.2
81.2
67.5
61.2
26.7
0
Percentage of
Survival rate of
pupae (d5) (%) adults (d10) (%)
52.5
5.9a
52.5
0
*A, E (in roman): different transgenic lines.
a
The percentage of malformed pupae. L1, L2, and L3: 1st, 2nd, and the 3rd instar larvae. Perhaps because these larvae were washed out from the
medium every day for counting, the survival rates of the 2st and the 3rd instar larvae were a little lower than normal.
collected and incubated in the same way as the
control. The HmgD overexpressing first instar larvae were selected under the fluorescence stereomicroscope by the lack of GFP fluorescence. All larvae
were allowed to develop in standard medium. The
number of living larvae and their pupariation status was assessed every day. We found that HmgD
overexpressing larvae developed in the same way
as wild type at the first and the second larval stages.
However, when wild type third instar larvae began
to pupate, HmgD overexpressing larvae crawled to
the walls of vials, while none of them pupariated
normally. Most HmgD overexpressing animals did
not enter the normal pupal stage and died in a
few days (Table 3). Very few malformed “pupae”
Fig. 2. Phenotypes of HmgD overexpressing animals.
Most of the HmgD overexpressing animals died, showing
old larval morphology (A). Few of them died just at the
beginning of metamorphosis as white pupae (B,C). Few
Archives of Insect Biochemistry and Physiology July 2008
formed in 5 days after larval hatching. Some of
these “pupae” were tanned a little, but most body
parts resembled larvae (Fig. 2A). Abnormal bodies
did not shorten like wild type animals at the beginning of pupariation (Fig. 2B and C). Although
some HmgD overexpressing larvae displayed normally tanned pupae, the heads still connected with
larval spicules and did not evert at all, and the larval mouth hooks were not ejected (Fig. 2D–F).
Ecdysone Feeding Did Not Rescue HmgD
Overexpressing Animals
The pupariation defects in HmgD overexpressing
animals could result from either a decrease in
other HmgD overexpressiing animals could form tanned
puparia, but larvae metamorphosis failed (D,E). F: Wild
type pupa. [Color figure can be viewed in the online edition which is available at www.interscience.wiley.com]
128
Chen et al.
ecdysone titer or a decrease in the ability of the
ecdysone signal to be transduced. To distinguish
these two possibilities, we examined the effects of
feeding ecdysone to HmgD overexpressing larvae,
which has shown to effectively rescue phenotypes
associated with ecdysone-deficient mutations in
Drosophila (Venkatesh and Hasan, 1997; Freeman
et al., 1999; Bialecki et al., 2002). Considering that
most HmgD overexpressing larvae developed to the
third instar larval stage and their size and vitality
did not show a significant difference from those
of wild type, early third instar HmgD overexpressing
larvae were used to perform ecdysone feeding experiments. When all synchronizingly developed
wild type larvae metamorphosed to pupae, no normal HmgD overexpressing pupae were observed.
All HmgD overexpressing larvae died in the same
way as described previously (Fig. 2). Feeding
ecdysone to E75A mutant second instar larvae,
however, had a dramatic effect on their development, rescuing 50% (n = 20) of them to pupal
stage, while almost all of E75A mutant second
instar larvae that were maintained on food without 20E failed to molt and develop to later stages.
This result is consistent with previous work
(Bialecki et al., 2002). Our results demonstrate
that ecdysone is not a limiting factor in HmgD
overexpressing animals.
Ubiquitous Overexpression of HmgD Disrupts the
Temporal Expression Pattern of Ecdysone Receptor
Signaling Pathway
Since HmgD overexpressing animals displayed
serious failure in pupariation and ecdysone feeding could not rescue HmgD overexpressing animals,
it logically increased possibilities that there could
be something wrong with the ecdysone signaling
pathway (Koelle et al., 1991; Yao et al., 1992, 1993).
The EcR-USP complex directly induces the transcription of primary-response genes, including early
genes originally defined as ecdysone-inducible puffs
in the larval salivary gland polytene chromosomes
(Ashburner et al., 1974). Our RT-PCR analyses
showed that the EcR gene transcription pattern in
HmgD overexpressing animals was virtually indis-
tinguishable from that in wild-type animals, when
rp49 was used as a quantitative control (Fig. 3).
Three well-characterized early genes Broad-Complex (BR-C), E74, and E75 encode transcription factors that transduce and amplify hormonal signals
(Dubrovsky, 2005). E74 encodes two isoforms of
an ETS domain transcription factor, designated
E74A and E74B (Burtis et al., 1990). E75 encodes
three orphan members of the nuclear receptor superfamily, designated E75A, E75B, and E75C
(Segraves and Hogness, 1990). Their transcription
patterns around metamorphosis were tested in
both HmgD overexpressing animals and wild type
animals. Although the timing and levels of early
gene BR-C expression in HmgD overexpressing animals were similar to that of wild type animals (data
not shown), the temporal expression profiles of
early genes E74 and E75 were altered significantly
in HmgD overexpressing animals compared with
wild type (Fig. 3). In wild type animals, E74A and
E75A were induced at the beginning of the white
pupa stage, and then repressed but still detectable,
and subsequently accumulated at the white pupa–
pupa transition around 8–10 h after pupariation
(Fig. 3). In contrast, expression of these two genes
was variable in HmgD overexpressing animals.
Neither E74A nor E75A transcription was detectable at the time corresponding to wild-type larva
pupariation. The expression patterns of these two
genes were compressed to only three time points,
corresponding to 2, 6, and 10 h after wild-type
pupariation (Fig. 3). In HmgD overexpressing animals, E74B was prematurely transcribed to the
time of puparium formation of wild type, whereas
E75B transcription was severely postponed from
mid-third-instar larval stage to the time point corresponding to white pupa and pupa stages of
wild-type (Fig. 3).
The early gene products activate a large group
of late genes, which are directly or indirectly involved in the metamorphic process (Thummel,
2002). We also examined expression patterns of
two late downstream genes Edg84A and βFTZ-F1,
which encode key transcription factors in ecdysone pathway cascades. Edg84A is expressed primarily in late white pupae and encodes pupa cuticle
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Chromatin Structure in Drosophila Development
Fig. 3. RT-PCR analysis of genes encoding key regulatory factors in the ecdysone cascades during late larval
and white pupa development in wild type and HmgD over-
Archives of Insect Biochemistry and Physiology July 2008
129
expressing animals, respectively. rp49 was used as a reference gene.
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Chen et al.
proteins that contribute to the synthesis of pupa
epidermis (Fechtel et al., 1988, 1989). The overexpression of HmgD severely affected expression
of Edg84A. In wild type, Edg84A transcription was
first detected at the beginning of pupariation and
the level was gradually increased and then kept
stable at the white pupa–pupa transition. However,
in HmgD overexpressing animals, the mRNA level
of Edg84A was dramatically repressed until the time
point relative to 10 h after puparium formation in
wild type (Fig. 3). βFTZ-F1 also encodes an orphan
member of the nuclear hormone receptor superfamily, which is a critical regulator of white pupa–pupa
transition in ecdysone cascades (Woodard et al.,
1994; Broadus et al., 1999). In wild type, βFTZ-F1
was expressed in a brief interval in the middle of
the white pupa stage (6 h in Fig. 3) (Lam et al.,
1999), while in HmgD overexpressing animals, it
was prematurely transcribed in the mid-third instar larval stage; then the transcript could not be
detected at the time of the late third instar and the
early white pupa stage relative to wild type. Only
at the time corresponding to the wild-type middle
pupa stage (6 h) was a very weak expression detected (Fig. 3).
In addition, the stage-specific E93 early gene was
also tested in our study. E93 is induced directly by
ecdysone in late white pupa salivary glands but
shows no response to the signal several hours earlier, in a late-third-instar larva (Baehrecke and
Thummel, 1995). In HmgD overexpressing animals,
E93 was expressed throughout the time relative to
the wild-type white pupa stage (data not shown),
while the transcript was only detected at the late
white pupa stage in wild type (Lam et al., 1999).
DISCUSSION
Ner and Travers (1994) showed that HMGD is
associated with condensed chromatin structures
during the first six rapid nuclear cleavage cycles of
the developing embryos. At that time, histone H1
is absent from these structures. With the accumulation of H1 from the seventh nuclear division onwards, the nuclei become more compact. This
compaction is paralleled by a reduction in the size
of mitotic chromatin, which implies that the condensed state of chromatin induced by HMGD may
be less compact than the H1-containing standard
chromatin fiber and that this state of chromatin
could facilitate rapid nuclear cycles. An in vitro
chromatin assembly system derived from pre-blastoderm Drosophila embryos showed that incorporation of purified HMGD into chromatin in a
manner similar to histone H1 alters the nucleosome repeat length, indicative of interaction with
the nucleosome linker DNA. Furthermore, histone
H1 can displace HMGD protein from chromatin
in a concentration-dependent manner (Ner et al.,
2001). Our results indicate that the overexpression
of the HmgD under maternal control influences the
viability of the embryos. However, these effects are
weak, since it does not result in 100% lethality.
Overexpression of HmgD under the control of a
ubiquitously expressed Gal4 protein caused severe
lethality at the late larval stage, from which we infer that overexpression of HmgD influences the later
larval stage rather than embryogenesis. Although
in Xenopus B4 and HMGB1 could substitute for histone H1 during early embryogenesis, single mutation in Hmgb1, Hmgb2, or Hmgb3 in mice did not
result in lethality during embryogenesis (Calogero
et al. 1999; Ronfani et al. 2001; Nemeth et al. 2003,
2005). Recently, Ragab et al. (2006) showed that
the homozygous mutant HmgD/Z is viable and
does not exhibit severe defects in Drosophila. Therefore, HMGB protein may not be essential for the
overall organization of chromatin, but is critical
for proper transcriptional control by specific transcription factors.
Ubiquitous overexpression of HmgD caused
pupariation failure in Drosophila. Pupariation is
tightly controlled and initiated by 20E. The latethird-instar pulse of 20E is propagated through a
genetic regulatory hierarchy, which defines the onset of metamorphic transition. 20E first binds to a
heterodimer receptor comprising the ecdysone receptor, EcR, and the gene product of usp (Koelle et
al., 1991), and subsequently activates a small group
of early genes (Dubrovsky, 2005). The products of
early genes then activate transcription of more than
100 “late genes” that control various aspects of
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Chromatin Structure in Drosophila Development
metamorphosis such as degeneration of larval tissues, differentiation of imaginal discs, and pupal
cuticle production. Three well-characterized early
genes are BR-C, E74 and E75, each of which encodes a set of protein isoforms of DNA binding
transcriptional regulators (Thummel, 2001). Our
feeding experiments confirm that the defect of
pupariation in overexpression of HmgD mutants
is not due to the reduction of 20E levels. RT-PCR
analysis indicates that it does not result from defects of EcR transcription, either. Among early
genes, the expression of both E74 and E75 is severely affected, while the transcription of BR-C does
not change in comparison with wild type. BR-C is
a key regulator in the initiation of, and progression through, metamorphosis. BR-C null mutants
die after a prolonged third instar. Misexpression
of BR-C during the second larval instar redirects
epidermal cells from larval to pupal cuticle production (Mugat et al., 2000; Gonzy et al., 2002;
Zhou et al., 2004). A similar expression pattern of
BR-C in HmgD overexpressing animals and wild
type indicates that overexpression of HmgD does
not affect the ecdysone signaling pathway at the
BR-C transcription level. E74A plays a role in puparium formation (Fletcher and Thummel, 1995).
E74B may play a role in prolonging larval muscles
in the pupal stage until they are required for a successful head eversion and body shortening during
puparium formation and in ecdysone induction of
imaginal disc evagination (Fletcher and Thummel,
1995). In HmgD overexpressing animals, failure in
pupariation, head eversion, and larval body shortening were observed, which may be involved in disruption of the expression pattern of E74 gene. E75
gene encodes three protein isoforms designated
E75A, E75B, and E75C (Segraves and Hogness,
1990). E75A mutation resulted in developmental
delay and molting defects, suggesting that E75A
may function in amplifying or maintaining the
ecdysteroid titer during larval development so as
to ensure proper temporal progression through the
life cycle (Bialecki et al., 2002). E75B may function by targeting gene regulation (White et al.,
1997). Interestingly, the transcription of E74A and
E75A displayed similar temporal shifts in HmgD
Archives of Insect Biochemistry and Physiology July 2008
131
overexpressing animals, suggesting that this mutation influences expression of early genes in the
ecdysone signaling pathway.
Edg84A, which encodes components of the pupal cuticle, was severely repressed in HmgD overexpressing animals. Our results show that expression
of Edg84A was very weak during the white pupa
stage, and was increased only at the time point corresponding to 10 h after wild-type puparium formation. Actually, most overexpressing HmgD
animals died at very late third instar stage with no
or little tanning. We infer that expression of this
gene must be repressed intensively. However, βFTZF1 was prematurely expressed in the mid-third instar larval stage and reduced in the middle white
pupa stage in HmgD overexpressing larvae compared with wild type. Although temporal expression patterns of both E75B and β FTZ-F1 are
changed in HmgD overexpressing animals, their
shifted expression still agrees with the point that
E75B can act as a repressor of the βFTZ-F1 competence factor during metamorphosis (White et al.,
1997). The altered temporal expression patterns of
early and later genes during larval and white pupa
stages in HmgD overexpressing animals suggest that
optimum expression of HmgD is required for the
ecdysone receptor pathway.
ACKNOWLEDGMENTS
We thank Dr. Nathalie Dostatni of the Curie
Institute for helping us to establish the transgenic
flies and for some suggestions. We also thank Dr.
Andrew A. Travers of the MRC Laboratory of Molecular Biology, Cambridge, for kindly providing
the HMGD antibody, Dr. Carl S. Thummel of the
University of Utah School of Medicine, for kindly
providing the flies of E75A81/TM6B Tb Ubi-GFP
and E75∆51/TM6B Tb Ubi-GFP, and Dr. Inaki IturbeOrmaetxe of SIB, The University of Queensland,
for a critical reading of the manuscript.
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