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First published online May 30, 2008
Journal of Experimental Biology 211, 1999-2004 (2008)
Published by The Company of Biologists 2008
doi: 10.1242/jeb.016816
Population origin, development and temperature of development affect the amounts of HSP70, HSP90 and the putative hypoxia-inducible factor in the tadpoles of the common frog Rana temporaria
1 Centre of Excellence in Evolutionary Genetics and Physiology, Department of
Biology, University of Turku, FI-20014 Turku, Finland
2 Population and Conservation Biology/Department of Ecology and Evolution,
Evolutionary Biology Centre, Uppsala University, Uppsala, Sweden
* Author for correspondence (e-mail: miknik{at}utu.fi)
Accepted 2 April 2008
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levels
(Rana temporaria HIF-1 was sequenced in the present study)
with immunoblotting. The levels of the studied proteins increased with
developmental stage. Also, the levels increased with latitude at the lower but
not at the higher developmental temperature. This shows that there is a clear
difference between the populations at the molecular level but that this
difference can be modified by the environmental conditions experienced during
development. The proteins analyzed may be involved in the regulation of
developmental processes. If this is the case, the tadpoles from the
northernmost population have the most advanced complement of regulatory
proteins at developmental stages approaching metamorphosis.
Key words: amphibia, development, ecophysiology, population biology
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INTRODUCTION |
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;
Angilletta et al., 2002;
Angilletta et al., 2003;
Blanckenhorn and Demont, 2004).
More knowledge is needed on the cellular and molecular responses associated
with these intraspecific variations, and on the intraspecific variation in
general, especially in vertebrates (see
Nikinmaa and Waser, 2007). One
natural system where the effects of large-scale climatic variation on
intraspecific variation can be studied conveniently is the development of the
frog Rana temporaria. The species ranges from southern Europe to the
northernmost parts of Scandinavia. Thus, populations along this latitudinal
gradient encounter very different conditions in terms of both mean temperature
and season length (Laugen et al.,
2003).
This environmental variation has been well utilized in genetic and
ecological studies, which have shown genetic differences in the rates of
development and growth, and in associated traits such as energetics and
behaviour (Merila et al.,
2000; Laugen et al.,
2003; Lindgren and Laurila,
2005). For example, when developing in the same conditions, the
tadpoles from higher latitudes develop and grow faster than those from lower
latitudes across a range of temperatures
(Merila et al., 2000;
Laugen et al., 2003;
Lindgren and Laurila, 2005).
This suggests that the more stringent time constraints in the north select for
increased growth and development (Merila
et al., 2000; Laugen et al.,
2003; Lindgren and Laurila,
2005). R. temporaria tadpoles often develop in small
ponds (e.g. Laurila, 1998),
where both the temperature and the oxygen levels can vary markedly. In
addition, Rana temporaria changes from an aquatic and gill-breathing
tadpole to a lung-breathing frog at metamorphosis. This transformation is
associated with a marked increase in ambient oxygen concentration. Both
temperature and oxygen levels may affect regulatory proteins during
development.
Although it has long been known that increases in temperature elicit a heat
shock response (Feder and Hoffmann,
1999; Sørensen et al., 2003), it has recently become
obvious that in poikilothermic vertebrates also a decrease in acclimation
temperature can cause an accumulation of heat shock proteins (HSPs)
(Rissanen et al., 2006).
Furthermore, heat shock proteins and the regulatory protein of
oxygen-dependent responses, hypoxia-inducible factor 1 (HIF-1),
interact [as originally described by Katschinski et al.
(Katschinski et al., 2002)].
This interaction may be functionally significant in temperature acclimation of
poikilothermic animals (Rissanen et al.,
2006), since the DNA binding of HIF-1, which is required
for the transcriptional effects of the protein, is increased during
acclimation of the crucian carp, Carassius carassius, to reduced
temperature. Furthermore, the results show coprecipitation of Hsps with
HIF-1 in an immunoprecipitation experiment, indicating that the
proteins form a complex (Rissanen et al.,
2006). Notably, vertebrate studies on Hsps or HIF have not
concentrated on the development of specimens from natural populations.
In this regard, the stringent temperature conditions during development of
northern R. temporaria populations may select for a tighter
regulatory system than in more southern populations. If both HSPs and
HIF-1 are required in the regulatory pathways of normal development,
and their levels increase with the tightness of regulation, one would expect
differences between populations, so that the northernmost populations are
characterized by the highest levels of the proteins.
Whereas commercial (mammalian) antibodies function reasonably against some
frog proteins, such as Hsps, our earlier experience with HIF-1
indicates poor cross-reactivity between antibodies designed for species from
other vertebrate groups. To date, Rana temporaria HIF-1 has
not been sequenced, but another frog, Xenopus laevis, has five
allelic variants of the upstream sequence before the HIF-1
(Sipe et al., 2004) gene in
the tadpole, suggesting complicated regulation of the transcription factor
during development.
In the present study we first cloned and sequenced Rana temporaria
HIF-1 so that the data could be used to choose the most suitable
antibody for later experiments. Second, we raised tadpoles from three
different populations from southern, mid and northern Sweden (the total
north-to-south distance between populations is approximately 1500 km) at two
temperatures, and measured the differences in HSP70, HSP90 and putative
HIF-1 levels (at constant total protein level) with immunoblotting
followed by image analysis. In the results, we have evaluated the effects of
developmental stage (before metamorphosis), temperature and population on the
HSP and HIF-1 levels to see if there are population-level or
temperature-induced differences in the levels of these regulatory proteins
during tadpole development.
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MATERIALS AND METHODS |
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)] and kept at 19°C. At Gosner stage 25 [external gills
fully absorbed (Gosner, 1960)]
80 individuals were taken at random from each population and placed into the
experiment. The tadpoles were placed individually in opaque 1.0 l jars filled
with 0.8 l RSW. The tadpoles were reared at two temperatures, 13 and 19°C,
with 40 individuals from each population in both treatments. The group
maintained at 19°C was placed in a temperature controlled room, and the
group maintained at 13°C was placed on a water bath connected to a cooler.
The photoperiod used in the experiment was 16 h:8 h light:dark. The water was
changed completely every 4 days and the animals were fed ad libitum
with chopped and lightly boiled spinach.
For molecular analysis, the tadpoles were snap frozen by placing them in
test tubes and dipping them in liquid nitrogen, whereafter they were kept at
–70°C. For analysis, the animals were sampled at the same
developmental stage at both temperatures. Since development at the lower
temperature is slower than at the higher temperature, the animals reaching a
given developmental stage at the lower temperature were older than at the
higher temperature. The whole development from Gosner stage 25 to 42 took
approximately 30 days at 19°C (SK 32–36 days, UP 27–31 days
and NO 20–24 days) and 60 days at 13°C (SK 55–65 days, UP
60–70 days and NO 48–58 days). Ten tadpoles in every population
and treatment were sampled for molecular analysis at Gosner stages 26 (stage
1), 34 (2), 39 (3) and 42 (4) (Gosner,
1960). The analyses given in the Results consider stage 3 and
especially 4, since there were fewer samples with adequate levels of
antibody-binding protein for the analyses in the earlier developmental
stages.
Cloning and sequencing Rana temporaria HIF-1
RNA extraction, cDNA synthesis and PCR reactions were conducted as
described by Rytkonen et al. (Rytkonen et
al., 2007) with the TRI reagent (Sigma, St Louis, MI, USA) and the
SMARTTM RACE cDNA Amplification Kit (Clontech, Palo Alto, CA, USA) from a
whole R. temporaria tadpole preserved in –70°C. Alignments
were conducted with ClustalW Multiple Alignment, version 1.4.
(Thompson et al., 1994) and
the primers were designed employing the internet-based Primer3 program
(Rozen and Skaletsky, 2000).
Forward 5'-CAARTCHGCYACVTGGAAGGT-3' and reverse
5'-CCTCCWSRATGCCACTGAG-3' universal primers designed from the
alignment of HIF-1 sequences from Homo sapiens [GenBank:
NM_001530], Xenopus laevis [GenBank: BC043769], Danio rerio
[GenBank: AY326951] and Oncorhynchus mykiss [GenBank: AF304864] were
used to obtain a primary fragment of the gene. Then, after a BLAST
verification, the sequence of this primary fragment was used to design
Rana temporaria HIF-1-specific primers for 5'-RACE
(rapid amplification of cDNA ends)
(5'-TGGCGGCTCAGGAAGGTTTTACTGTCCA-3') and 3'-RACE
(5'-CCGCCACAGCCTTGACATGAAGTTTTCC-3') reactions. The PCR fragments
were run in agarose gels and HIF bands were excised and cloned with pGEM-T
Easy Vector System I (Promega, Madison, WI, USA) and sequenced using the ABI
PRISMTM BigDye Terminator v 3.1 Cycle Sequencing Kit (Applied Biosystems,
Foster City, CA, USA).
Immunoblot analysis
Nuclear extracts were prepared as described in Soitamo et al.
(Soitamo et al., 2001) with
some modifications. Protein concentration was determined
spectrophotometrically using the Bio-Rad Protein Assay (Bio-Rad Laboratories,
Hercules, CA, USA). Protein (25 µg) was separated on 8% SDS-polyacrylamide
gel and transferred to a nitrocellulose membrane (Schleicher & Schuell,
Keene, NH, USA). Membranes were blocked for 1 h in 5% nonfat dry milk in PBS
with 0.3% Tween 20 at room temperature and incubated with primary antibodies
overnight at 4°C. Primary antibodies and the dilutions used were the
following: polyclonal anti-HIF-1 recognizing human HIF-1 amino
acids 432-528 (Novus Biologicals, Littleton, CO, USA) 1:1000, anti-HSP70,
clone 3A3, raised against human recombinant HSP70 (Affinity BioReagents,
Golden, CO, USA) 1:5000 and monoclonal anti-HSP90 against human HSP90
(Stressgen Biotechnologies, Ann Arbor, MI, USA) 1:500. Horseradish
peroxidase-conjugated anti-rabbit (HIF-1), anti-mouse (HSP70) and
anti-rat (HSP90) antibodies (Amersham Biosciences, Piscataway, NJ, USA) were
used as secondary antibodies. The proteins were detected using enhanced
chemiluminescence according to the manufacturer's instructions (enhanced
chemiluminescence; Amersham Biosciences). The signals were captured on X-ray
film, and the relative optical density of protein bands was quantified with
MCID 5+ image analyzer software (InterFocus Imaging, Cambridge, UK). Since
gel-to-gel variation may bias western blot quantifications; every effort was
made to avoid such bias in the present experiments. First, equal (protein)
loading was confirmed by staining gels with Coomassie Brilliant Blue. Second,
to account for differences in exposure, the bands obtained on every film were
related to the background of the film. Third, the loading order of samples in
the gels was randomized. Thus, samples from any group were divided among
several gels (and consequently several films), and every gel (and film)
contained samples from virtually all groups. Consequently, any differences
between groups cannot be caused by gel-to-gel variation.
Statistics
For statistics, SPSS14 software (Chicago, IL, USA) was used. The equality
of variances was tested with Levene's test. When two datasets were compared,
t-test for independent samples was used. When more than two datasets
were compared, the comparison consisted of an initial ANOVA (one-way or
two-way as appropriate) followed by a post hoc LSD test.
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RESULTS |
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has been deposited in a public data bank
(GenBank: EU262663), and its major features are given in
Fig. 2. The predicted R.
temporaria HIF-1 protein sequence has 80% identity to Xenopus
laevis, 70% identity to Gallus gallus, 65% identity to Homo
sapiens and 53% identity to Danio rerio sequences. The molecule
has 806 deduced amino acids. Thus, it is longer than the molecules of
teleosts, supporting the notion that the molecules in teleosts have
accumulated deletions which do not occur in tetrapods
(Rytkonen et al., 2007). Since
the molecule does not have major deletions or insertions as compared to the
human molecule in the region of amino acids 432–528 (the equivalent
sequences for human, Xenopus and Rana temporaria are given
in Fig. 3), we used a
polyclonal antibody, made to this amino acid region, to evaluate the changes
in the level of the protein during development at the two temperatures. The
antibody recognized a protein of the correct size, and thus we call the
recognized molecule `putative HIF'.
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The putative HIF, HSP70 and HSP90 levels increase with increasing latitude at 13° but not at 19°C
Fig. 5 gives the levels of
the three measured proteins at developmental stage 4 (Gosner 42) in the
different populations at 13°C and 19°C. There was a very significant
effect (ANOVA) of latitude on all the proteins at 13°C. Pairwise
comparisons with a post-hoc test (LSD test) indicated that the
Skåne population differed significantly from the other two populations.
At 19°C, there was an effect only in HSP90 levels, and the Skåne
population again differed from the other two.
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DISCUSSION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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), total
HSP70, which represents proteins involved in the control of native protein
folding (Li and Mivechi,
1999), and total HSP90, which represents proteins involved in the
control of cellular signalling (Neckers et
al., 1999) the levels (at a constant amount of total protein) were
higher in the tadpoles originating from the northernmost population than in
the southern tadpoles. Data on growth and the rate of development have
indicated that tadpole development and growth proceed faster in the northern
than in the southern populations when tadpoles are raised under the same
conditions (Merila et al.,
2000; Laugen et al.,
2003; Lindgren and Laurila,
2005). The available time for development is shorter at higher
than at lower latitudes (Laugen et al.,
2003). In the face of this, increased levels of regulatory
proteins enable larger flexibility in development, and increase the
effectiveness of controlling, for example any temperature-dependent response.
The findings of the present study are also compatible with the observation
that any genetic differences between populations can be masked by such
environmental variables as temperature
(Laugen et al., 2003), since
we only found a clear population-dependent difference in the protein response
in one of the two temperature treatments.
In this study we did not try to separate the constitutive and inducible
forms of HSPs. The constitutive functions, e.g. ensuring native protein
folding, taking part in transmembrane movements of protein and binding to
proteins involved in cellular signalling before activation, of both HSP70 and
HSP90 are important. Thus, if these regulatory functions increase with
development of tadpoles before metamorphosis, an increase in the total level
of HSPs (per unit amount of protein) can be expected. Earlier reports on frogs
(Xenopus) have indicated that the heat shock response can only be
induced after a period of development
(Krone and Heikkila, 1988),
but we are not aware of published reports indicating an increase in the total
HSP70 and HSP90 levels (per unit amount of protein) at temperatures which
should not elicit heat shock response. In a way, it is not surprising that
there is an increase in HSPs in tadpoles approaching metamorphosis, since
metamorphosis is associated with massive restructuring of tissues, during
which pronounced apoptosis must also occur
(Nakajima et al., 2005). HSPs
are intimately involved in the control of apoptosis
(Garrido et al., 2001;
Beere 2004;
Zhang et al., 2006). In this
regard it is interesting that there was no significant effect of temperature
on the level of either heat shock protein in the last developmental stage
studied, showing that, indeed, the temperatures employed did not induce the
classical heat shock response, and suggesting that any response observed may
be associated with the approaching metamorphosis. Further studies are clearly
warranted to evaluate if the increased HSP levels at the developmental stage
approaching metamorphosis are due to apoptosis (associated with tissue
restructuring) or if they persist even in air-breathing juvenile and adult
frogs. Provided that an increase in the level of HSPs indicates advanced
development, our results fit with earlier observations of more rapid
development of tadpoles from higher latitudes
(Merila et al., 2000). Also
the increased level of the putative HIF-1 protein in the northernmost
population suggests that there is a need for a more effective regulation of
transcription, which an increase in the level of, e.g. HIF enables, in an
environment that approaches the limit of tolerance of the species.
Although temperature, as such, did not significantly affect the HSP levels,
there was a significant increase in the putative HIF-1 level with
temperature. As temperature increases, the water oxygen level decreases
(Dejours, 1975), and since an
increased HIF-1 level is associated with any responses to aquatic
hypoxia (Nikinmaa and Rees,
2005), the result is as expected, provided that the putative
HIF-1 is, indeed, the regulatory transcription factor involved.
The HIF-1 sequence shows, as expected, a reasonably high similarity
to the Xenopus sequence. There are, in addition, a couple of
important points with regard to the evolution of the transcription factor in
vertebrates. First, it appears, as stated by Rytkonen et al.
(Rytkonen et al., 2007), that
the molecule is longer in tetrapods than in teleost fish. Second, all
vertebrates, apart from mammals, appear to have a short deletion after
proline564 (human nomenclature). The amino acid is involved in the
oxygen-dependent regulation of the stability of the molecule. Earlier studies
on Xenopus have indicated that the molecule is transcriptionally
regulated during the development of tadpoles
(Sipe et al., 2004). This
transcriptional regulation may be under the influence of environmental and
genetic factors, since the apparent regulatory region preceding the
transcribed gene shows marked allelic variation in Xenopus
(Sipe et al., 2004). At
present, it is not known if similar variation occurs in the control regions of
the Rana HIF-1 gene, if different populations show differences
in the control sequences, and if the different control sequences cause the
transcription of the gene to be different under different environmental
conditions. However, the studies of Sipe et al.
(Sipe et al., 2004) on
Xenopus laevis, those of Vuori et al.
(Vuori et al., 2004) on
Salmo salar, and the present one on Rana temporaria all
indicate that there is clear normoxic appearance of HIF-1 during
development, indicating the importance of this transcription factor in normal
vertebrate development.
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