Mitochondrial Gene Expression Is Regulated at the Level of
Transcription during Early Embryogenesis of Xenopus
laevis*
Chandramohan V.
Ammini
and
William W.
Hauswirth
§¶
From the Departments of
Molecular Genetics and
Microbiology and § Ophthalmology, University of Florida,
Gainesville, Florida 32610
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ABSTRACT |
Mitochondrial transcription in the early
Xenopus laevis embryo resumes several hours before active
mtDNA replication, effectively decoupling mtDNA transcription and
replication. This developmental feature makes Xenopus
embryogenesis an appealing model system to investigate the regulation
of mitochondrial transcription. Studies reported here refine our
understanding of the timing, magnitude, and mechanism of this
transcriptional induction event. Northern analyses of six mitochondrial
mRNAs (normalized to mtDNA) reveal that transcript levels remain
basal between fertilization and gastrulation and then undergo a
coordinate induction, culminating in a 20-28-fold increase over egg
levels by 48 h of development. Measurement of mitochondrial run-on
transcription rates demonstrates a good correlation between
transcription rates and transcript levels, showing that transcription
itself is the primary determinant of transcript abundance. Experimental
increases in mitochondrial ATP and energy charge also correlate with
patterns of transcript levels and transcription rates, suggesting that
developmental changes in the biochemical composition of the
mitochondrial matrix could be regulating transcriptional activity.
Consistent with this idea, transcriptional run-on rates in mitochondria
of early embryos can be stimulated by the addition of tricarboxylic
acid cycle intermediates to the run-on reaction. However, mitochondria of later stages do not show this response to the addition of
metabolite. In combination, these data suggest that mitochondrial
transcription is under metabolic regulation during early
Xenopus embryogenesis.
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INTRODUCTION |
The Xenopus laevis egg stockpiles mitochondria and
mitochondrial DNA along with other organelles and macromolecules during its year-long development (1). This active period of mitochondrial biogenesis ceases with the completion of egg development and does not
resume until relatively late in embryogenesis, as measured by mtDNA
synthesis, cytochrome oxidase activity, and mitochondrial protein
accumulation (2). However, active mitochondrial transcription resumes
much earlier, as indicated by total mitochondrial RNA synthesis (2-4)
and mRNA accumulation (5). This apparent decoupling of
mitochondrial transcription from mtDNA replication makes this model
very useful for studying the nuances of transcriptional regulation of
mitochondrial genes.
The precise timing of the resumption of mitochondrial transcription in
the developing embryo remains controversial. Initial studies reported a
low rate of mitochondrial rRNA synthesis up to the gastrula stage (10 h
postfertilization), with the rRNA content per embryo doubling by
96 h of development (2). Microinjection of [32P]GTP
into fertilized eggs detected active mitochondrial RNA synthesis only
after the midblastula transition (3). Measurement of mtRNA synthesis
rates during the terminal stages of oogenesis and fertilization showed
that synthesis rates remained essentially unchanged by the events
associated with fertilization (6). In contrast, direct measurements of
mitochondrial RNA steady-state levels during early embryogenesis showed
that transcript levels decreased 5-10-fold in a coordinate fashion
within a few hours after fertilization, remaining depressed up to the
late neurula stage (24 h of development) and increasing thereafter (5).
Based on these results, a fertilization-induced shutdown of
mitochondrial gene expression during early embryogenesis was proposed.
Despite this uncertainty regarding the precise status of mitochondrial
gene expression during early Xenopus embryogenesis, this
model offers the opportunity to investigate mitochondrial gene
regulation paradigms other than that of modulating gene dosage (mtDNA).
Using mtDNA levels at each stage to normalize transcript levels, we
determine stage- and gene-specific transcriptional rates. The results
reveal that mitochondrial gene expression during early embryogenesis is
regulated primarily by transcription. The data also suggest that the
developmental cue(s) directing these changes is likely to be related
directly or indirectly to the changing biochemical composition of the
mitochondrial matrix during embryogenesis.
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EXPERIMENTAL PROCEDURES |
Plasmid Clones and Riboprobes--
Gene-specific DNA templates
for making hybridization probes and targets were constructed by cloning
polymerase chain reaction-amplified fragments into the
HincII site of
pBS1 (+/
) phagemid
(Stratagene, La Jolla, CA). Sense or antisense riboprobes were
synthesized from linearized clones using T3/T7 RNA polymerase kits
(Stratagene). The clones and their corresponding positions in the frog
genome (7) are as follows: pBSND1, nucleotides 4849-5715; pBSCYTb,
nucleotides 16417-17245; pBSCOII, nucleotides 9168-9790; pBSND4,
nucleotides 12409-13571; pBSND6, nucleotides 16110-15680.
Frogs, Eggs, Embryos, and Mitochondria--
Sexually mature male
and female X. laevis frogs were purchased from
Xenopus I, Ann Arbor, MI. Fertilized embryos were obtained using in vitro fertilization procedures (8, 9). Briefly, female frogs were injected with 700-1,000 units of human chorionic gonadotropin (Sigma) into the peritoneal cavity 10 h before eggs were required. Fertilization was done in batches of 300-400 eggs in
1 × F-1 (41.25 mM NaCl, 1.25 mM KCl, 0.25 mM CaCl2, 0.0625 mM
MgCl2, 0.5 mM Na2HPO4, 2.5 mM HEPES, pH 7.8), using sperm obtained from macerated
testes. During the first 8 h of growth, the 1 × F-1 buffer
was replaced with 0.2 × F-1 buffer in the Petri dishes. Afterward, the whole Petri dish was immersed in a large dish filled with 2-3 liters of 0.2 × F-1 buffer until hatching.
Developmental stages were identified according to Nieuwkoop-Faber
tables (10). Eggs and embryos were dejellied by gently swirling in 2%
cysteine solution (in 2 × F-1, pH 7.8) for 3-4 min followed by
repeated washing in 2 × F-1 (eggs) or 0.2 × F-1 (embryos).
Mitochondria were purified from sorted eggs and embryos by
modifications of a protocol used for isolation of heart muscle
mitochondria (11), as described below. Dejellied eggs and embryos were
homogenized with seven or eight strokes in MSE buffer (220 mM mannitol, 70 mM sucrose, 5 mM
MOPS, pH 7.0, 2 mM EGTA; 40 ml for 200-300 eggs or
embryos) in a glass Dounce homogenizer with a type B Teflon pestle. The
homogenate was spun twice at 1,500 × g for 10 min in a
JA20 rotor, and mitochondria were pelleted at ~9,000 × g. Mitochondria were washed and repelleted twice using MSE
buffer in Eppendorf tubes at 9,000 × g for 2 min.
Isolation of Mitochondrial Nucleic Acids--
For total RNA
isolation, 100 eggs or embryos were thawed in 10 ml of lysis buffer (4 M guanidine thiocyanate, 100 mM sodium acetate,
pH 5.0, 5 mM EDTA), incubated for 20 min at room
temperature to facilitate dissociation of nucleoprotein complexes,
extracted three or four times with phenol:chloroform:isoamyl alcohol
(25:24:1, pH 7.0), loaded on 5.7 M CsCl cushions, and spun
at 38,000 rpm for 24 h at 4 °C in an SW40 rotor to recover RNA
pellets. For simultaneous isolation of RNA and DNA, mitochondrial
pellets were resuspended in 400 µl of lysis buffer (50 mM
Tris, pH 7.0, 200 mM NaCl, 10 mM EDTA) and SDS
added to 1%. The lysates were then extracted twice each with phenol,
pH 7.0, phenol:chloroform:isoamyl alcohol (25:24:1), pH 7.0, chloroform, and ethanol precipitated.
RNA Analysis--
RNA samples were run in 1.2% MOPS (20 mM MOPS, pH 7.0, 5 mM sodium acetate, 0.1 mM EDTA) formaldehyde-agarose gels; blotted on nylon
N+ membranes (Amersham Pharmacia Biotech) with 10 × SSC; prehybridized in 6 × SSPE, 50% formamide, 5 × Denhardt's solution, 0.5% SDS, 200 µg/ml tRNA at 60 °C; and
hybridized overnight at 60 °C in a fresh aliquot of the same buffer
with radiolabeled riboprobes (50-100 ng/ml). Blots were washed in 40 mM Na+, 1% SDS, 1 mM EDTA at
60 °C for 1.5 h with three or four changes, and the blots were
exposed to film and PhosphorImager screens (Molecular Dynamics,
Sunnyvale, CA). In experiments with total RNA, 2 µg of total RNA was
electrophoresed per lane. For measuring RNA levels/unit of
mitochondrial genome, mtDNA levels in total nucleic acid samples were
quantitated by dot blotting. Samples were incubated with 3 µl of
RNase mixture (Ambion, Inc., Austin, TX) at 37 °C for 15 min to
degrade RNA; denatured in 0.4 M NaOH at 96 °C for 10 min; adjusted to 10 × SSC; blotted on nylon N+
membrane; and hybridized overnight in 7% SDS, 1% bovine serum albumin, 500 mM sodium phosphate, pH 7.1, 1 mM
EDTA (12) with mtDNA-specific riboprobes at 60 °C. Blots were washed
as above and quantitated. Mitochondrial DNA-normalized samples were
then run in formaldehyde-agarose gels for Northern analysis as
described above, and RNA levels were reported per unit of mitochondrial genome.
Mitochondrial Run-on Transcription--
The incubation
conditions for the run-on transcription assays were modifications of
reported protocols (13, 14). Mitochondria from eggs and staged embryos
were isolated in MSE buffer as above, washed once with run-on wash
buffer (40 mM Tris, 7.2, 50 mM NaCl, 10%
glycerol, 5 mM MgCl2, 1 mg/ml bovine serum
albumin), and incubated in run-on buffer (40 mM Tris, 7.2, 50 mM NaCl, 10% glycerol, 5 mM
MgCl2,, 2 mg/ml bovine serum albumin) with 50 µCi of
[
-32P]UTP at 30 °C for 30 min. Mitochondria were
then pelleted; lysed in 50 mM Tris, pH 7.5, 200 mM NaCl, 25 mM EDTA, 1% SDS; and the clarified
crude lysate was used to probe unlabeled riboprobe target panels at
60 °C overnight with the hybridization format described for Northern
analyses. To correct for differences in the yield of mitochondria
obtained from the different developmental stages, 10% of the
mitochondria in the run-on reactions were lysed, extracted twice with
phenol:chloroform:isoamyl alcohol (25:24:1) and chloroform, and then
processed for dot blotting (without ethanol precipitation) as described
in under "RNA Analysis." The unlabeled riboprobe target panels were
prepared with 1 µg of each transcript as described above. Because
in vitro transcription reactions always result in some level
of less than full-length transcripts of the same specific sequence,
membranes were trimmed below the specific RNA bands before
hybridization to simplify quantitation (see Fig. 3B).
Measurement of Adenine Nucleotides--
The total egg and
embryonic mitochondrial adenine nucleotide levels were measured by
reversed phase, ion-paired HPLC. Adenine nucleotides were extracted
from eggs, embryos, and isolated mitochondria by adapting a previous
protocol (15). Five dejellied eggs/embryos or double sucrose
gradient-purified mitochondria from 300 eggs or embryos were
homogenized in 12% trichloroacetic acid, incubated at room temperature
for 5 min, and centrifuged at 14, 000 × g for 5 min.
The clarified supernatant was extracted twice with 2-6 volumes of 0.5 M trioctylamine in cyclohexane, centrifuged, the aqueous
layer filtered through a 0.22-µm nylon centrifuge filter (Micron
Separations, Inc., Westborough, MA), and stored at
70 °C. HPLC was
done on a C18 silica HPLC column using an injection volume of 100 µl,
the mobile phase being 0.2 M sodium phosphate titrated to
pH 5.75 with tetrabutylammonium hydroxide, by the isocratic elution
mode. Signal amplification, peak detection, and area quantitations were
done using Nelson PE hardware and software (Perkin-Elmer). Standard
calibration curves for ATP, ADP, and AMP were generated using ultrapure
ATP, ADP, and AMP (Sigma). Protein pellets obtained after
trichloroacetic acid precipitation were resuspended in 10 mM Tris, pH 8.5, 0.5% SDS, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, and the clarified
supernatant was used for protein estimation (16).
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RESULTS |
To clarify the status of mitochondrial gene expression during
early Xenopus development, mitochondrial transcript levels
were measured initially exactly as reported previously (5). Identical amounts of total RNA (2 µg) from unfertilized eggs and staged embryos
were separated by electrophoresis on 1.2% formaldehyde-agarose gels,
blotted to nylon membranes, and probed with six gene- and strand-specific riboprobes (Fig. 1). All
transcripts showed a 1.5-2-fold decrease in steady-state levels by
14 h of development relative to that of the unfertilized egg,
returned to levels found in egg by 20 h in the case of NADH 1, cytochrome b, cytochrome oxidase II, and ATP synthetase 6 (Fig. 1 and Table I) and increased marginally thereafter. The levels of NADH4 and 6 continued to be low
even in the tadpole stage (50 h of development). The 12 S and 16 S rRNA
levels decreased only marginally during this window of development
(data not shown). Overall, these results are only in partial agreement
with the pattern of mitochondrial transcript levels reported in an
earlier study (5).

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Fig. 1.
Steady-state mitochondrial RNA levels during
early development of X. laevis. 2 µg of total RNA from
unfertilized eggs and embryonic stages at 5, 13, 20, 30, 38, and
50 h postfertilization was used for Northern analysis with the
indicated gene-specific riboprobes.
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Table I
Steady-state levels of mitochondrial mRNAs during early development
of X. laevis
Total egg or embryonic RNA was subjected to Northern analysis as shown
in Fig. 1, and the results of three experiments were used to calculate
the steady-state levels of the six mitochondrial transcripts. Each
transcript level is expressed relative to the level measured in the
unfertilized egg.
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One reason for this discrepancy could be the decision to normalize
steady-state mitochondrial RNA to total egg or embryonic RNA. In
addition to variation among animals, there are clear and dramatic
differences in nuclear transcription during early Xenopus embryogenesis after the midblastula transition (17). Therefore, using
total RNA to normalize mitochondrial transcripts will lead to a
progressive reduction in apparent mtRNA levels during embryogenesis. We
therefore sought an alternative normalization approach for mtRNA analysis.
A better and more logical reference for normalizing mitochondrial RNA
levels is the mitochondrial DNA itself because mtDNA is the
transcriptional template, and its concentration per embryo remains
essentially unchanged during the first 48 h of development (2). We
therefore isolated DNA and RNA simultaneously from purified
mitochondria of distinct developmental stages. Equal proportions of the
DNA/RNA mixture from each developmental stage were treated with RNase
to remove RNA, alkali denatured, and blotted to quantitate the level of
mtDNA in each sample (Fig.
2B). In control experiments,
DNase I digestion of these samples before alkali denaturation and
blotting completely eliminated the signal (not shown), confirming that
the values obtained represented mtDNA exclusively, without RNA
carryover.

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Fig. 2.
Steady-state levels of mitochondrial RNA/unit
of mitochondrial genome during early development. Panel
A, Northern analysis of mtDNA-normalized RNA samples from eggs and
embryos at 6 h, 9 h, 20 h, 30 h, 48 h, and 7 days postfertilization with the indicated gene-specific riboprobes.
Panel B, equal volumes of the samples used above were RNase
treated, alkali denatured, and probed on dot blots to quantitate the
amount of mtDNA in that sample. Digestion with DNase I completely
eliminated this signal (not shown).
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RNA samples normalized for mtDNA were then run on formaldehyde-agarose
gels, blotted, and hybridized with six gene-specific riboprobes (Fig.
2A), and the steady-state level of each transcript was
expressed relative to the level found in the unfertilized egg. The
results from two unrelated frogs are presented in Table II. Steady-state levels of all
transcripts remained very near levels found in the egg until 9 h
of development, increased slightly (1.5-4-fold) by 20 h,
increased moderately (6-8-fold) by 30 h, and increased
dramatically (13-60-fold in frog 1 and 17-23-fold in frog 2) by
48 h of development. The 96-h embryos displayed even higher
steady-state transcript levels, 21-100-fold over egg. By 7 days
postfertilization (5-day-old tadpole), however, steady-state levels
dropped to levels found in the 30-h embryo, 2-8-fold over egg. It is
apparent from Table II that there is substantial animal-to-animal variation in the magnitude of transcript levels. However, the pattern
of induction of mitochondrial gene expression during embryogenesis is
similar.
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Table II
Steady-state levels of mitochondrial RNA/unit of mtDNA in unfertilized
eggs and early embryos of X. laevis
Eggs and embryos derived from two unrelated frogs were subjected to
Northern analysis, and mitochondrial RNA levels normalized to the
corresponding mtDNA levels are presented. The steady-state level of
each transcript is expressed relative to the level found in
unfertilized eggs.
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Transcription Rates Correlate with Transcript Levels--
To
investigate whether the developmental control of mitochondrial gene
expression is regulated at the level of transcription, mitochondrial
run-on transcription assays were performed using isolated organelles
from unfertilized eggs and embryonic stages. To detect transcription
separately from the L- and H-strand promoters (LSP and HSP,
respectively), gene- and strand-specific RNAs corresponding to four
genes (Fig. 3A) were
synthesized in vitro and blotted to prepare identical
unlabeled target RNA panels (Fig. 3B). Run-on reactions were
performed in isotonic buffer without the addition of exogenous
nucleotides other than trace amounts of [32P]UTP.
Mitochondria from ~50 eggs or embryos at 5, 14, 20, 28, and 48 h
postfertilization were used in each mitochondrial run-on assay. After
the 30-min incubation, mitochondria were lysed, and the newly
synthesized, labeled RNA was used to probe the unlabeled target panels
(Fig. 3C). To account for differences in the yield of
mitochondria from each stage, 10% of each run-on reaction (before the
addition of radiolabel) was used to determine its mtDNA level (Fig.
3D), and these mtDNA quantitations were used to normalize measurements of promoter-specific (Fig.
4A) and overall (Fig. 4B) transcription rates at each stage.

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Fig. 3.
Mitochondrial run-on transcription in eggs
and embryos. Panel A, approximate genomic location of
the four genes (not drawn to scale) used as unlabeled targets are shown
along with the direction of transcription from the two promoters in the
D-loop region. Panel B, unlabeled L- and
H-strand sense transcripts, synthesized in vitro, were
electrophoresed on 1.2% formaldehyde-agarose gels (1 µg each except
2 µg for 16 S (H-strand) in the third lane from the
left) and blotted on nylon N+ filters. Note that
the filters were trimmed precisely below each ethidium-stained RNA band
before hybridization to aid in the quantitation of the bands. In this
hybridization format, LSP transcription is detected in the
first, second, and sixth lanes
(L-strand sense), whereas HSP transcription is detected in the
third, fourth, and fifth lanes
(H-strand sense). Panel C, radiolabeled mitochondrial run-on
transcription reactions were used to probe the unlabeled target panels.
Lane assignments are as in panel B. Numbers
correspond to the stages of egg (1), 5 h
(2), 14 h (3), 20 h (4),
28 h (5), and 48 h (6) embryos.
Panel D, mitochondrial DNA in 10% of mitochondria from each
run-on reaction shown in panel C, quantitated as described
in the Fig. 2B legend. Numbers correspond to
stages as described for panel C.
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Fig. 4.
Mitochondrial transcription rates in
unfertilized eggs and embryos. Promoter-specific (panel
A) and overall (panel B) transcription rates/unit of
mitochondrial genome are shown during early development, calculated
from quantitations of Fig. 3, C and D, and
expressed relative to the egg level.
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Relative to transcription in the unfertilized egg, there was only a
small stimulation in transcription of each gene at each embryonic stage
up to 20 h of development. Transcription from either LSP or HSP
was 2-fold higher than levels found in the unfertilized egg by 5 h, 4-5-fold higher by 14 h, and 4-6-fold higher by 20 h of
development (Fig. 4A). The 28-h embryo showed the largest increase in transcriptional activity, with transcription rates from the
LSP and HSP being 25-fold and 91-fold higher than levels in the egg
(Fig. 4A), respectively. Overall transcription was ~70-fold higher than the level in eggs (Fig. 4B).
Transcription in the 48-h embryo, although 2-fold less than the 28-h
stage, was still elevated significantly, being 15-fold and 44-fold
higher than the level in eggs for LSP and HSP, respectively (Fig.
4A).
A comparison of the overall transcription rates with steady-state
transcript levels is presented schematically in Fig.
5. The resumption of transcription in the
developing embryo (hatched bars in Fig. 5) correlates well
with the steady-state accumulation of transcripts (line in
Fig. 5) because increased rates of transcription precede the appearance
of increased RNA levels by a few hours. Transcription rates of the 14-h
and 20-h embryos were 5-6-fold higher than in eggs. Transcript levels
were 2-4-fold higher by 20 h and 6-8-fold higher by 30 h
than the level in eggs. The high transcriptional rate measured in the
28-h embryo (~70-fold over egg) is also consistent with the
substantial transcript accumulation observed by 48 h (13-60-fold
over egg) and 96 h (18-100-fold over egg) of development,
assuming unaltered RNA turnover during this period.

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Fig. 5.
Schematic comparison of mitochondrial
transcription rates and transcript levels during early development of
X. laevis. The line graph represents the pattern
of steady-state levels of mitochondrial transcripts (expressed relative
to egg levels). The bar graph represents overall
mitochondrial run-on transcription rates (also expressed relative to
egg levels) measured in mitochondria from each developmental
stage.
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Transcription Rates Correlate with the Energy Status of
Mitochondria--
To estimate the stage-specific metabolic status of
mitochondria, the adenine nucleotide content of double sucrose
gradient-purified mitochondria from unfertilized eggs and embryos was
measured by reversed phase, ion-paired, HPLC. These studies revealed a
2-3-fold increase in mitochondrial ATP levels in the 30- and 44-h
embryo over those of earlier stages (from 650-850 pmol/mg
mitochondrial protein to 1,400-2,500 pmol/mg mitochondrial protein).
The energy charge (ATP/ADP + AMP) also increased proportionally (from
0.12-0.17 to 0.33-0.4). In contrast, total cellular (egg or
embryonic) ATP levels remained constant at ~2 mM during
this time. This selective increase in mitochondrial ATP levels in later
developmental stages suggests an induction or shift of mitochondrial
metabolic activity during embryogenesis.
To test whether the induction of mitochondrial transcription in the
30-h embryo is related to the stimulation of mitochondrial metabolic
activity, metabolic intermediates were added to mitochondrial run-on
transcription reactions derived from 20-h embryos (a relatively un-induced stage for mitochondrial transcription) (Table
III). Equal aliquots (equivalent to 100 embryos) of mitochondria prepared from 20-h embryos were incubated in
basic buffer (condition 1); basic buffer + ADP (condition 2); basic
buffer +
-ketoglutaric acid (condition 3); basic buffer + sodium
phosphate, ADP, and
-ketoglutaric acid (condition 4); or basic
buffer + ATP (condition 5). The overall mitochondrial run-on
transcription rate showed a dramatic induction (8-22-fold over basic
buffer) with the addition of all of the substrates needed for
endogenous production of ATP in the mitochondria (condition 4 in Table
III). The addition of exogenous ATP (condition 5) or low level
production of endogenous ATP by the addition of the citric acid cycle
intermediate
-ketoglutaric acid (condition 3) caused a reproducible
5-7-fold stimulation over basic buffer. The addition of ADP alone
(condition 2) or ADP + phosphate (not shown) resulted in only a
2-3-fold enhancement in transcription rate. The addition of other
trichloroacetic acid cycle intermediates pyruvate, malate, or glutamate
also produced 6-10-fold stimulation of transcriptional activity (data
not shown). Similar experiments performed with mitochondria from 48-h
embryos (a transcriptionally active stage for mitochondria) in basic
buffer with sodium phosphate, ADP, and
-ketoglutaric acid showed
almost no stimulation of transcription rates (less than 1.5-fold; data not shown) compared with those seen with early embryos. This further suggests that changes in mitochondrial metabolism during development have profound effects on the mitochondrial transcriptional machinery. These results also suggest that transcriptionally quiescent
mitochondria of the 20-h embryo are fully competent for high level
transcription and are possibly awaiting a developmentally induced
change in the mitochondrial microenvironment for induction of
transcription to occur.
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Table III
Effect of the addition of mitochondrial metabolic intermediates and
inhibitors on the run-on transcription rate of mitochondria derived
from 20-h embryo
Labeled run-on products were used to probe unlabeled RNA target panels
as in Fig. 3C, and overall transcription rates are expressed
as the average of the gene-specific rates relative to the rate obtained
in basic buffer. Rates reported are the range obtained in independent
experiments with the embryos derived from two to three unrelated frogs.
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To explore further the specificity of the processes stimulating
mitochondrial transcription, the mitochondrial drugs antimycin A and
atractyloside, which block oxidative phosphorylation and ATP synthesis,
were tested for their effect on mitochondrial transcription (Table
III). Each inhibitor was added along with ADP, phosphate, and
-ketoglutaric acid (antimycin A, condition 6; atractyloside, condition 7) in the run-on reactions derived from 20-h embryonic mitochondria. Both drugs inhibited the transcription-inducing effects
of substrate addition substantially (compare condition 4 with condition
6 or 7). The addition of cyanide, a specific inhibitor of cytochrome
oxidase, also produced a similar inhibition (data not shown).
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DISCUSSION |
Aside from a simple response to mitochondrial genome content,
mechanisms regulating vertebrate mitochondrial gene expression remain
largely unstudied. Early embryogenesis of X. laevis provides an experimental window on this problem because mitochondrial
transcription precedes resumption of mtDNA synthesis by at least
24 h. The timing, magnitude, and nature of this early
transcriptional regulation were studied by measuring steady-state
levels of six mRNAs and the two mitochondrial rRNAs between
fertilization and 72 h of development. All mtRNA levels remained
relatively unchanged between fertilization and gastrulation (~10 h
postfertilization) but increased thereafter in a coordinate fashion and
culminated in a dramatic 13-60-fold (by 48 h) or 21-100-fold (by
96 h) increase over levels found in unfertilized eggs (Fig. 2 and
Table II). That this coordinate increase of steady-state mtRNA levels
is caused by an induction of mitochondrial transcription was
substantiated by measurements of mitochondrial run-on transcription
rates. Transcription rates in eggs and embryos remained at basal levels
between fertilization and gastrulation and then showed moderate
2-3-fold increases in the 14-h and 20-h embryos and a dramatic
~70-fold induction in the 28-h embryo (Figs. 3 and 4). The parallel
behavior of mitochondrial transcription rates and transcript levels
leads to the pivotal mechanistic question: what developmental stimuli
regulate this resumption of active mitochondrial transcription in the embryo?
In one scenario, the developmental cue could be a nuclear encoded
factor that is imported into mitochondria, stimulating transcription directly or indirectly, perhaps by inactivating inhibitors of transcription. Alternatively, stimulation of mitochondrial metabolic activities such as oxidative phosphorylation or substrate biosynthesis could indirectly activate transcription. The fact that transcription rates in mitochondria isolated from the 20-h embryo in which
mitochondrial transcription is uninduced could be stimulated by the
addition of trichloroacetic acid cycle intermediates such as
-ketoglutarate (Table III), malate, pyruvate, or glutamate, unlike
mitochondria isolated from the 48-h tadpole in which mitochondrial
transcription is already maximally induced, lends credence to the
metabolite induction scenario. The lower ATP content and energy charge
ratios of stages before 30 h are also consistent with this view.
Finally, the marked inhibitory effects of antimycin A and atractyloside on transcription rates (Table III) demonstrate the exquisite
sensitivity of mitochondrial transcription, either directly or
indirectly, to perturbations of energy metabolism.
Metabolic studies of carbon flow during amphibian development have
revealed developmental shifts in substrate utilization which may be
relevant to the transcriptional induction we report. Between the first
embryonic cleavage and gastrulation, embryos use amino acids from yolk
in preference to carbohydrates from stored glycogen as the primary
carbon source (18), with mitochondrial glutamate oxidation playing a
major role (19, 20). Glycolysis is not active during early embryonic
cleavage even though the glycolytic machinery is intact (21-23).
Fertilization also triggers transient increases in cytoplasmic calcium
(24-26) which are likely to have profound effects on mitochondrial
physiology because calcium regulates oxidative metabolism through
effects on several matrix dehydrogenases (27-30). It is also possible
that changes in mitochondrial ultrastructure could act as either the
cause or effect of these metabolic changes during development. Evidence
in support of this comes from electron micrographs of mature mouse
eggs, showing mitochondria to be condensed, electron-dense structures.
In contrast, mitochondria of four- to eight-cell embryos are swollen
and structurally distinct, with numerous transversely arranged cristae
(31, 32). These embryos exhibit a coincident rise in oxygen consumption and cyanide-sensitive ATP synthesis (33, 34). In the developing mouse
embryo, mitochondrial transcription is active from the two- to
four-cell embryonic stage onward, as indicated by a 25-50-fold increase in rRNA, cytochrome oxidase I, and cytochrome oxidase II
mRNA levels (35). Such de novo transcription appears to
play an essential role in mitochondrial differentiation during cleavage because inhibitors of mitochondrial RNA and protein syntheses also
block the normal growth and differentiation of mitochondrial cristae
(32). A similar mechanism of mitochondrial differentiation from an
"embryonic" to "adult" state is likely to exist during X. laevis embryogenesis in concert with the changes in the metabolic steady states of the cell. Results reported here provide a mechanistic link between mitochondrial metabolism and mitochondrial transcriptional regulation during early embryogenesis. Unraveling the precise details
of this link should help further understanding of the intricacies of
cellular-mitochondrial communication.
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ACKNOWLEDGEMENTS |
We thank Drs. Alfred Lewin, Maurice Swanson,
Thomas Rowe, Steven Ghivizzani, and Cort Madsen for helpful suggestions
during the course of this investigation. Our special thanks to Dr.
Thomas Hollinger for help with in vitro fertilization.
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FOOTNOTES |
*
This work was supported by grants from the Foundation
Fighting Blindness and Research to Prevent Blindness, Inc.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom correspondence should be addressed: Dept. of Molecular
Genetics and Microbiology, Box 100266, JHMHC, University of Florida,
Gainesville, FL 32610. Tel.: 352-392-0679; Fax: 352-392-3062; E-mail:
hauswrth{at}eye1.eye.ufl.edu.
 |
ABBREVIATIONS |
The abbreviations used are:
pBS, pBluescript;
ND1, ND4, and ND6, NADH dehydrogenase 1, 4, and 6, respectively;
CYTb, cytochrome b;
COII, cytochrome oxidase II;
MOPS, 4-morpholinepropanesulfonic acid;
HPLC, high performance liquid
chromatography;
ATPase 6, ATP synthetase 6;
LSP, light strand promoter;
HSP, heavy strand promoter.
 |
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