DOI: 10.5061/dryad.zs7h44jnx
title: Data and code from: Early developmental decline in HSP expression affects subsequent response to transient heat exposure

description: 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.
description: # Data and code from: Early developmental decline in HSP expression
 affects subsequent response to transient heat exposure ## 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.