Data and code from: Early developmental decline in HSP expression affects subsequent response to transient heat exposure
Data files
Dec 16, 2025 version files 134.05 KB
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R_Script_-_All_Studies.R
55.05 KB
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README.md
6.38 KB
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StudyIData.xlsx
23.05 KB
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StudyIIData.xlsx
31.67 KB
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StudyIIIData.xlsx
17.90 KB
Abstract
Understanding physiological responses to short-term changes in temperature is of growing interest, considering the rising frequency and severity of transient temperatures such as heat waves. During the embryonic development of egg-laying vertebrates, inducible physiological responses to transient heat are likely critical to short-term survival but may also be energetically costly or disruptive to development. Inducible heat-shock proteins (HSPs) are conserved molecular chaperones that act to safeguard cellular protein homeostasis during transient stress. However, experiments in ectotherms have shown that overexpression of HSPs can increase embryonic mortality and reduce later thermotolerance. Yet, few studies have explored natural developmental patterns of HSP expression and heat inducibility in embryos of egg-laying ectothermic vertebrates. Using the red-eared slider turtle (Trachemys scripta), we characterized the response of five HSP genes in embryonic trunks following repeated 3-d transient heat wave exposures. Interestingly, we found that the expression of most HSPs naturally declined during early development and that warm temperatures amplified this decline, while also accelerating developmental rate. Only in a few instances did we observe induction of certain HSP genes during heat wave exposures, though this depended on the thermal history of the embryo. Specifically, induction of these genes during a particular heat wave was reduced in embryos that had already experienced two recent prior exposures relative to those experiencing it for the first or second time, suggesting repeated heat exposures can attenuate subsequent responses. The observed changes in HSP expression and inducibility may relate to an individual’s need to balance thermotolerance alongside extensive cellular differentiation and proliferation during early development. The effects of incubation temperature on these changes could also have important implications for how turtle embryos deal with subsequent heat stress and may be similarly present in other ectothermic vertebrates. Our study demonstrates the importance of considering ontogenetic changes in physiological responses to temperature even across embryonic development.
Descriptions of the Data and file structure
There are three separate Excel data files corresponding to each of the three studies/experiments described in the manuscript. These are referred to as 'StudyIData', 'StudyIIData', and 'StudyIIIData', corresponding respectively with study I, study II, and study III of the manuscript. Each Excel file contains gene expression values of 5 HSP genes (normalized to the housekeeping gene) as obtained from biological (embryonic trunks) samples in each study.
Excel Document "StudyIData.xlsx" Headers:
Sheet 1 'StudyI_MainSheet'
- EggID: identifies individual T. scripta eggs (i.e., embryos) used in the study. Note: these are distinct from eggs used in the other studies.
- Treatment: the treatment applied to that specific egg. 2 levels: RecurringHeat or CoolControl; individuals underwent either three repeated 3-day heat wave exposures ("RecurringHeat") or remained at consistent cool baseline conditions ("CoolControl").
- Day: Indicates which incubation day that embryo was sampled. 6 levels: individuals were sampled on either incubation day 14, 17, 20, 23, 26, or 29 (corresponding with just before (day of) and three days into the heat waves of the experimental group).
- GapdhCt_Mean: The mean cycle threshold (CT) value of the house-keeping gene, GAPDH, assessed from the RNA sample of each individual embryo using real-time quantitative polymerase chain reaction (qPCR).
- HSP110_NE: The normalized expression of HSPH1 (i.e., HSP110) assessed from each individual using qPCR.
- HSP90A_NE: The normalized expression of HSP90AA1 assessed from each individual using qPCR.
- HSP90B_NE: The normalized expression of HSP90B1 assessed from each individual using qPCR.
- HSP705_NE: The normalized expression of HSP70A5 assessed from each individual using qPCR.
- HSP70A8_NE: The normalized expression of HSP70A8 (i.e., HSC70) assessed from each individual using qPCR. Some samples had to be dropped due to errors in running the plate and are therefore marked as NA in this column. This should not cause major issues in the R script since generalized linear models are used. However, the second sheet in this excel file has these NA's removed if this version causes issues.
Sheet 2 'StudyI_HSP70A8_ONLY': The same data as the previous sheet except only samples with HSP70A8 (i.e., HSC70) are included. Those which were NA's for HSP70A8_NE on the previous sheet are excluded from this one. This is included just in case the NA's on the previous sheet cause issues when running the R script.
Excel Document "StudyIIData.xlsx" Headers:
Excel Sheet 'Study II':
- EggID: identifies individual T. scripta eggs (i.e., embryos) used in the study. Note: these are distinct from eggs used in the other studies.
- Treatment: the treatment applied to that specific egg. 4 levels: 3HW, 2HW, 1HW, or 0HW; individuals underwent either three repeated 3-day heat wave exposures on incubation days 15 - 17, 21 - 23, and 27 - 29 ("3HW"), two repeated 3-day heat waves on incubation days 21 - 23 and 27 - 29 ("2HW"), one 3-day heat wave on incubation day 27 - 29 ("1HW"), or remained at consistent cool baseline conditions ("0HW").
- SamplingDay: Indicates which incubation day that embryo was sampled. 6 levels: individuals were sampled on either incubation day 23, 26, 29, 32, or 35 (focusing around the final heat wave exposure of the experimental groups and 5 days afterwards).
- Stage: Represents the approximate embryonic stage estimated for each individual. Individuals that could not be accurately staged or for which staging was missed contain an 'NA' in this column.
- GapdhCTmean: The mean cycle threshold (CT) value of the house-keeping gene, GAPDH, assessed from the RNA sample of each individual embryo using qPCR.
- HSP70A5_NE: The normalized expression of HSP70A5 assessed from each individual using qPCR.
- HSPH1_NE: The normalized expression of HSPH1 (i.e., HSP110) assessed from each individual using qPCR.
- HSP90B1_NE: The normalized expression of HSP90B1 assessed from each individual using qPCR.
- HSP90AA1_NE: The normalized expression of HSP90AA1 assessed from each individual using qPCR.
- HSP70A8_NE: The normalized expression of HSP70A8 (i.e., HSC70) assessed from each individual using qPCR.
Excel Document "StudyIIIData.xlsx" Headers:
Excel Sheet 'Study III':
- EggID: identifies individual T. scripta eggs (i.e., embryos) used in the study. Note: these are distinct from eggs used in the other studies.
- Treatment: the treatment applied to that specific egg. 2 levels: FPT or MPT; individuals underwent either consistent warm incubation conditions corresponding with 'female producing temperatures' in this species (FPT; 31 ± 3°C) or consistent cool incubation conditions corresponding with 'male producing temperatures' in this species (MPT; 26 ± 3°C).
- Day: Indicates which incubation day that embryo was sampled. 3 levels: individuals were sampled on either incubation day 6, 12, or 18.
- AvgStage: Represents the average estimated embryonic stage for each treatment*day combination.
- EF1aCtMean: The mean cycle threshold (CT) value of the house-keeping gene, EF1a, assessed from the RNA sample of each individual embryo using qPCR.
- HSP90B1_NE: The normalized expression of HSP90B1 assessed from each individual using qPCR.
- HSPH1_NE: The normalized expression of HSPH1 (i.e., HSP110) assessed from each individual using qPCR.
- HSP70A5_NE: The normalized expression of HSP70A5 assessed from each individual using qPCR.
- HSP90AA1_NE: The normalized expression of HSP90AA1 assessed from each individual using qPCR.
- HSP70A8_NE: The normalized expression of HSP70A8 (i.e., HSC70) assessed from each individual using qPCR.
The included R Script file ('R_Script_-_All_Studies.R') code shows how we- for each given study- used linear models to test the effects of treatment and day on our normalized expression of each HSP gene, checked for assumption violations, conducted post-hoc analyses with FDR adjustments, and graphed for major results figures. The R Script reads in the order of Study I, II, and III and will remind you which Excel file to use. Ensure that the specific Excel file is in your set directory so that the R script can read it.
