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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

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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

Mikko Nikinmaa^1,*, Lotta Leveelahti¹, Emma Dahl², Eeva Rissanen¹, Kalle T. Rytkönen¹ and Anssi Laurila²

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

View larger version (25K): [in this window] [in a new window]   Fig. 1. A map of locations where frog eggs were collected. SK, Skåne; UP, Uppland; NO, Norrbotten.

View larger version (9K): [in this window] [in a new window]   Fig. 2. A schematic representation of HIF-1 protein in Rana temporaria and other selected vertebrates. The crucial interaction domains and hydroxylation targets of the molecule are shown. The basic helix-loop-helix (bHLH) and the Per-ARNT-Sim (PAS) domains are involved in DNA binding and dimerization of the protein. The oxygen-dependent degradation (ODD) and the C-terminal transactivation domains (C-TAD) confer oxygen-dependent regulation. The figure also gives the length of the deduced HIF-1 protein and a comparison of its insertion/deletion patterns. In the block diagram, grey represents teleost-specific insertions that are absent from tetrapods. Black represents protein regions that are present in mammalian molecules, but to a variable degree absent from those of other vertebrates. In the schematic representation of the molecules, lines indicate gaps in the alignment. Accession numbers – Homo sapiens: GenBank, NM_001530; Gallus gallus: GenBank, AB013746; Rana temporaria: GenBank, EU262663; Xenopus laevis: GenBank: BC043769; Danio rerio: GenBank, AY326951; and Takifugu rubripes: Ensembl, SINFRUG00000154390.

View larger version (8K): [in this window] [in a new window]   Fig. 3. The predicted Homo sapiens, Xenopus laevis and Rana temporaria amino acid sequence of the area (amino acid residues 420–530, approximately) to which the antibody used for probing the HIF-1 was made. Arrows (at amino acids 432 and 528, human nomenclature) indicate the exact start and end of the peptide used for preparing the antibody.

View larger version (6K): [in this window] [in a new window]   Fig. 4. The levels of HSP70 and HSP90, and putative HIF in the last two developmental stages studied (3, Gosner stage 39; and 4, Gosner 42). For all proteins the level increases significantly (P<0.05) from stage 3 to stage 4. Values are means and s.e.m. (indicated as lines above the bars). The number of tadpoles was seven for developmental stage 3 and ten for stage 4 in the case of HSP70; four and nine, respectively, for HSP90, and five and 37, respectively, in the case of putative HIF.

View larger version (12K): [in this window] [in a new window]   Fig. 5. The levels of the three measured proteins at developmental stage 4 (Gosner 42) in the different populations at 13°C (A) and at 19°C (B). SK denotes the southernmost and NO northernmost population (SK, Skåne; UP, Uppland; NO, Norrbotten). At 13°C for HIF N=7 for SK, 7 for UP, and 8 for NO; for Hsp70, N=9, 8 and 8; and for Hsp90 N=7, 9 and 6. At 19°C N=8, 8 and 7 for HIF; N=7, 10 and 9 for HSP70; and N=8, 7 and 8 for HSP90. There was a significant (P<0.01, ANOVA) effect 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 (P<0.01) from the other two populations. At 19°C, there was an effect only in HSP90, where the Skåne population again differed (P<0.01) from the other two.

View larger version (15K): [in this window] [in a new window]   Fig. 6. The effect of rearing temperature on the measured protein levels (as indicated by antibody binding) at stage 4 (Gosner stage 42) for the populations studied. On the basis of two-way ANOVA, the effect of temperature was statistically significant for the putative HIF but not for HSP70 or HSP90 after the effect of sampling location was taken into account. Filled bars indicate 13°C, open bars indicate 19°C. SK, Skåne; UP, Uppland; NO, Norrbotten. (A) HIF. For SK N=7 at 13°C and N=8 at 19°C; for UP N=7 and 8, respectively; for NO N=8 and 7, respectively. (B) HSP70. For SK N=9 at 13°C and N=8 at 19°C; for UP N=8 and 10, respectively; and for NO N=8 and 9, respectively. (C) HSP90. For SK N=7 at 13°C and N=8 at 19°C; for UP N=9 and 7, respectively; and for NO N=6 and 8; respectively. Values are means and s.e.m. (indicated as lines above the bars).

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