Temperature During Embryonic Development Has Persistent Effects on Thermal Acclimation Capacity in Zebraﬁsh

Temperature during embryonic development has persistent effects on thermal acclimation capacity in zebraﬁsh

Graham R. Scotta,b,1 and Ian A. Johnstonb

aDepartment of Biology, McMaster University, Hamilton, ON L8S 4K1, Canada; and bScottish Oceans Institute, School of Biology, University of St Andrews, St Andrews, Fife KY16 8LB, United Kingdom

Edited by George N. Somero, Stanford University, Paciﬁc Grove, CA, and approved July 19, 2012 (received for review March 27, 2012)

Global warming is intensifying interest in the mechanisms enabling swimming performance in subsequent life stages. In the wild, ectothermic animals to adjust physiological performance and cope zebrafish experience daily temperature fluctuations of ∼5 °C and with temperature change. Here we show that embryonic tempera- wide seasonal variation in temperature, from as low as 6 °C in ture can have dramatic and persistent effects on thermal acclima- winter to ∼38 °C in summer, across their native range (Ganges tion capacity at multiple levels of biological organization. Zebrafish and Brahmaputra river basins in southern Asia) (11). Natural populations typically reproduce during the monsoon season, when embryos were incubated until hatching at control temperature ∼ (T = 27 °C) or near the extremes for normal development (T = food is readily available and temperatures range from 23 °C to E E 31 °C (12). We used fish derived from a wild-caught strain and 22 °C or 32 °C) and were then raised to adulthood under common a large number of families to approximate the natural genetic conditions at 27 °C. Short-term temperature challenge affected aer- variation found in wild populations. We reared zebrafish em- obic exercise performance (Ucrit), but each TE group had reduced T bryos at three temperatures (22 °C, 27 °C, and 32 °C) spanning thermal sensitivity at its respective E. In contrast, unexpected dif- the range for normal reproduction and development (Fig. 1). ferences arose after long-term acclimation to 16 °C, when perfor- Embryonic temperature had striking and often unpredictable ∼ T mance in the cold was 20% higher in both 32 °C and 22 °C E effects on thermal acclimation that persisted to adulthood, even T groups compared with 27 °C E controls. Differences in performance after fish were raised from hatching at a common temperature PHYSIOLOGY after acclimation to cold or warm (34 °C) temperatures were par- (27 °C). These effects could be explained by differences in how tially explained by variation in fiber type composition in the swim- thermal acclimation affected the abundance and proportion of ming muscle. Cold acclimation changed the abundance of 3,452 of fiber types in the swimming muscle and in the expression of a 19,712 unique and unambiguously identified transcripts detected in subset of transcripts across the transcriptome. the fast muscle using RNA-Seq. Principal components analysis dif- ferentiated the general transcriptional responses to cold of the Results and Discussion

27 °C and 32 °C TE groups. Differences in expression were observed Embryonic Temperature Affects Thermal Sensitivity and Acclimation for individual genes involved in energy metabolism, angiogenesis, of Swimming Performance in Adult Zebrafish. We first assessed how cell stress, muscle contraction and remodeling, and apoptosis. swimming performance (“critical swimming speed”) in adult fish Therefore, thermal acclimation capacity is not fixed and can be from each embryonic temperature (TE) group was affected by modified by temperature during early development. Developmen- temperature after (i) short-term (1 d) transfer to 22 °C, 27 °C “ ” tal plasticity may thus help some ectothermic organisms cope with (control), or 32 °C ( thermal sensitivity ) and (ii) long-term ac- – the more variable temperatures that are expected under future climation (28 30 d) to 16 °C or 34 °C (Fig. 1). We used a strong climate-change scenarios. inference approach to distinguish between multiple competing hypotheses for how TE influences the temperature dependence of swimming performance (13). Two-factor ANOVA with orthog- environmental physiology | fish | muscle transcriptome | onal polynomial contrasts of T was used to determine the level functional genomics | high-throughput sequencing E of statistical support for various hypotheses: “beneficial developmental plasticity” (significant interactions of TE and swim emperature has profound effects on the physical and chem- temperature, TS), “hotter is better” or “colder is better” (signif- Tical processes that dictate how biological systems function. icant linear effects of TE with a positive or a negative contrast Ectothermic organisms—those that cannot regulate body tem- coefficient, respectively), “optimal intermediate temperature” perature using endogenous heat production—are particularly (significant quadratic effect of TE with downward concavity), and susceptible to changes in environmental temperature. Species “developmental buffering” (no effect of TE), which would reflect and populations can typically survive and perform across a finite canalization of embryonic development. range of temperatures, the breadth of which is a critical de- TE affected the short-term thermal sensitivity of swimming terminant of their distribution and abundance (1, 2). The phys- performance (Fig. 2A and Table S1). Performance was generally iological and molecular mechanisms underlying how ectotherms improved at the temperature zebrafish were raised as embryos, perform when faced with daily and seasonal temperature varia- consistent with the hypothesis that developmental plasticity is tion have attracted significant attention for decades, and this beneficial. This result was supported by statistically significant interest has intensified in an attempt to understand the potential ecological impacts of global climate change (3, 4). It is clear that temperature acclimatization can shift thermal optima and per- Author contributions: G.R.S. and I.A.J. designed research; G.R.S. performed research; G.R.S. formance breadth (5), which may increase fitness and improve analyzed data; and G.R.S. and I.A.J. wrote the paper. the viability of populations during warming (6–8). However, The authors declare no conflict of interest. much of what we know about the integrative mechanisms for this This article is a PNAS Direct Submission. ability comes from studies of juvenile and adult animals. There Data deposition: The sequence data reported in this paper has been deposited in the has been relatively little emphasis on how interactions between Array Express Archive (European Bioinformatics Institute; EBI) database, www.ebi.ac.uk/ar- temperature and the developmental program might affect phe- rayexpress (accession no. E-MTAB-1155). notypic plasticity in later life (9, 10). 1To whom correspondence should be addressed. E-mail: [email protected]. fi fl Here we use zebra sh as a model to investigate the in uence This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. of temperature change during embryonic development on 1073/pnas.1205012109/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1205012109 PNAS | August 28, 2012 | vol. 109 | no. 35 | 14247–14252 Downloaded by guest on September 27, 2021 interactions for total muscle area and the total transverse area of fast fibers (P = 0.006 and P = 0.040) and by the cold-induced increases in total muscle, slow fiber, and fast fiber transverse areas in only the 22 °C and 32 °C TE groups. It is unclear whether these differences are related to variation in the number of axial myotomes, because TE affects the rate of somite

Fig. 1. Fish were raised at one of three embryonic temperature (TE) treatments and then at a common postembryonic temperature until adulthood, after which they underwent one of ﬁve adult temperature treatments.

Swimming performance was measured in each TE group after each adult temperature treatment. Muscle phenotype was determined in ﬁsh acclimated to 27 °C, 16 °C, and 34 °C (*). Transcriptome-wide analysis of gene

expression was conducted for the 27 °C and 32 °C TE groups acclimated to † 27 °C and 16 °C ( ). Each individual was subject to one distinct path from left

to right. TE groups are offset for clarity.

TS × TE interactions in two-factor ANOVA with orthogonal polynomial contrasts (P = 0.049 overall and P = 0.036 for linear contrast) (Table S2). Compared with swim trials at 27 °C, only the 22 °C TE group sustained performance at 22 °C and only the 32 °C TE group increased performance at 32 °C. Differences between TE groups were not caused by differences in body mass or length (Table S3). TE also affected thermal acclimation capacity, but the effects were not always of predictable benefit (Fig. 2B and Table S1). Some results suggested that development at a specific TE might optimize performance at that temperature, such as the significant TS × TE interactions (P < 0.001 overall and for both contrasts) (Table S2) and the lower performance of the 22 °C TE group at 34 °C. However, there also appeared to be an overall benefitof developing at 32 °C on thermal acclimation across all temperatures. The 32 °C TE group performed better than the 27 °C TE group in the cold and the main effect for the linear contrast of TE was significant (P < 0.001) (Table S2). The former difference was specifically due to TE-induced variation in thermal acclimation capacity, rather than to persistent effects on short-term thermal sensitivity, because critical swimming speed after 1 d at 16 °C was similar in 32 °C and 27 °C TE groups (9.89 ± 0.15 and 9.05 ± 0.32 standard body lengths/s, n = 4) (Table S1). We next sought to determine whether the differences in acclimation capacity between TE groups were due to differences in muscle plasticity. We therefore examined swimming muscle phenotype using standard histological methods (Fig. 3 A–D) in the caudal region of the trunk (0.7 of standard body length) for the same fish as were swum at acclimation temperatures (TA) of 16 °C, 27 °C, and 34 °C (Fig. 1).

Embryonic Temperature Affects Thermal Acclimation of Muscle Phenotype. The mechanisms underlying the improvement in swimming capacity after thermal acclimation can include shifts in the proportion of muscle fiber types (14, 15), changes in mitochondrial content and capillarity of muscle fibers (16, 17), and adjustments in contractile properties mediated by differential expression of contractile protein isoforms (18, 19). We observed an overall increase in the transverse area of the entire swimming musculature after cold acclimation (two-factor fl ANOVA, P < 0.001) (Fig. 3E and Table S4). The number and Fig. 2. (A and B) Embryonic temperature (TE)in uenced aerobic swimming performance in adult zebrafish after (A) short-term temperature transfer totalareaofslowmusclefibers and the total area of fast fibers and (B) long-term thermal acclimation. Critical swimming speed (Ucrit) was also increased after cold acclimation (P = 0.016, P < 0.001, and fi fi < fi compared between sh swum at control temperature (27 °C) and sh P 0.001 in two-factor ANOVA) (Table S5). Because sh were transferred for 1 d and swum at 22 °C or 32 °C (A) or between control fish of similar body length and mass (Table S3), there was most and fish acclimated for 28–30 d and swum at 16 °C or 34 °C (B). Data are likely a change in body shape. However, the magnitude of this means ± SEM (n = 8). Bonferroni posttests detected significant differences

change differed between TE groups, in accordance with pre- (P < 0.05) from Ucrit at 27 °C within each TE group [using both one- and two- vious observations that TE affects body shape (20). The dif- factor (**) or just one (*)-factor ANOVA] and between TE groups within each † ferent responses to cold were reﬂected by signiﬁcant TA × TE swim temperature ( ).

14248 | www.pnas.org/cgi/doi/10.1073/pnas.1205012109 Scott and Johnston Downloaded by guest on September 27, 2021 and total area of intermediate fibers (P = 0.016, P < 0.001, P < 0.001, P = 0.007, and P < 0.001 in two-factor ANOVA) (Table S5). However, these increases in the proportion of aerobic fibers occurred only in 27 °C and 32 °C TE groups, and there were significant TA × TE interactions for the total area and average fiber size of intermediate fibers (P = 0.042 and P = 0.036). There was also an overall increase in the number of slow fibers in the 27 °C TE group (P = 0.023 for TE effect in two-factor ANOVA), as previously observed in the fast muscle (22). Overall differences between TE groups in total muscle area and the proportion of aerobic fiber types can partly explain the observed differences in swimming performance after thermal acclimation (Fig. 2B). The expansion of slow muscle that is afforded by the accentuated increase in total muscle area in the 22 °C and 32 °C TE groups should enhance sustained swimming performance in the cold. Although fast muscle is most commonly associated with burst swimming, it is also recruited at high sustainable swimming speeds (23), particularly in the cold (24), and could also contribute to differences in swimming performance between TE groups at 16 °C. The relative increase after warm acclimation in the abundance of slow and intermediate muscle, the main fiber types supporting aerobic exercise (25), is expected to help the 27 °C and 32 °C TE groups maintain high cruising speeds at warm temperatures.

Transcripts Involved in Energy Metabolism Are Strongly Induced in the Cold. Whole-transcriptome shotgun sequencing (RNA-Seq) of fast muscle mRNA was used to compare gene expression

between 27 °C and 32 °C TE groups at acclimation temperatures PHYSIOLOGY of both 27 °C and 16 °C. This two-factor design (2 TA × 2 TE; n = 4) allowed us to assess the main effects of TA and TE and their interaction. We uncovered 3,452 transcripts at Q < 0.05 (2,275 transcripts at Q < 0.01 and 1,378 transcripts at Q < 0.001; Q is calculated by adjusting the P value for multiple comparisons) whose expression changed significantly in response to cold acclimation (Dataset S1). Thirty-two transcripts were turned on in the cold (i.e., no transcript was observed in any 27 °C samples), 33 increased in abundance by more than 20-fold, 227 increased between 5- and 20-fold, 870 increased by 2- to 5-fold, and 688 increased by 1.5- to 2-fold (Q < 0.05). Many of the transcripts that exhibited large relative changes in expression were expressed at low levels (on the basis of mean normalized transcript counts) (Fig. S1). Surprisingly, cold decreased the expression of numerous transcripts: 6 decreased by more than 20-fold, 100 by 5- to 20-fold, 719 by 2- to 5-fold, and 659 by 1.5- to 2-fold (Q < 0.05). The cold-induced repression of so many transcripts is in stark contrast to previous findings from microarray studies of other species, where >90% of differentially expressed genes during cold acclimation were up-regulated (26, 27). We next performed Gene Ontology (GO) and Kyoto Ency- clopedia of Genes and Genomes (KEGG) analyses of the transcripts that increased in the cold by 1.5-fold at Q < 0.01. There < Fig. 3. Embryonic temperature influenced thermal acclimation of muscle was an overrepresentation (at Q 0.05) of several GO categories phenotype. (A–D) The axial swimming musculature was stained for (A) slow relating to carbohydrate and lipid metabolism and to mito- fibers (S58 antibody), (B) intermediate fibers (alkaline-resistant myosin- chondria (Fig. 4A and Table S6). A similar overrepresentation of ATPase activity), and (C) aerobic capacity (succinate dehydrogenase activity) KEGG pathways relating to energy metabolism was also observed to identify the three predominant fiber types (D; the blue box indicates the (1,100 metabolic, 10 glycolysis/gluconeogenesis, 30 pentose phos- area of the muscle shown in A–C). (Scale bar in A,50μm.) Serial sections of phate, 51fructose and mannose metabolism, 52 galactose me- the same muscle fiber are represented with letters (s, i, f). (E) The total tabolism, and 71 fatty acid metabolism pathways) (Table S7). transverse area of axial muscle, expressed relative to body mass2/3 (mm2·mg−2/3) Consistent with this observation were cold-induced increases in to normalize for isometric variation in body size. (F) The relative proportion transcripts for 8 of 10 enzymes in glycolysis; every enzyme in of muscle area composed of slow (s, hatched) and intermediate (i, un- fatty-acid oxidation; and several enzymes in the citric acid cycle, hatched) fiber types. See Fig. 2 for statistical details. oxidative phosphorylation, and other pathways of energy production (Fig. 4B). The transcript of the enzyme essential for lipid desaturation in the cold (scd, Δ9-desaturase) (28) increased, but formation but does not affect somite length in zebrafish during those involved in fatty acid synthesis were not generally altered. embryogenesis (21). There were clear examples of isoform switching (e.g., increased Acclimation to 34 °C increased the relative proportion of both cox4i1 and decreased cox4i2 for cytochrome oxidase subunit 4), slow and intermediate fiber types in the myotome (P = 0.013 and differential expression of splice variants (e.g., increased eno3-005 P < 0.001 in two-factor ANOVA) (Fig. 2F and Table S4). These and eno3-007 and decreased eno3-002 for enolase 3), and subunits changes were primarily caused by increases in the number and responding in opposite directions (e.g., subunits of NADH de- total area of slow muscle fibers and the number, average area, hydrogenase) (blue and orange enzymes in Fig. 4B). We could

Scott and Johnston PNAS | August 28, 2012 | vol. 109 | no. 35 | 14249 Downloaded by guest on September 27, 2021 Cold acclimation in fish is known to induce mitochondrial biogenesis and increase the capacity for energy production in the muscle (14). Although the molecular pathways regulating these changes are still poorly understood (30–32), the orthologs of several genes that regulate energy metabolism in mammals were induced in the cold (e.g., ppargc1al, ppargc1b, pparaa, rxrgb, and esrra). The expression of genes regulating angiogenesis in mammals also increased (e.g., vegfab, fgf1, and fgfrl1b), which could increase the supply of O2 to the more abundant mito- chondria in the muscle (16). There were also complex changes in the expression of numerous factors regulating myogenesis and muscle fiber phenotype (increases in myf5, myog, mef2cb, mef2d, akt2, and foxo3b; decreases in myod1, myf6, mef2a, akt2l, and capn1a and several insulin-like growth factors, binding proteins, and receptors). Many other increases in gene expression with cold acclimation were typical of those observed in multiple tissues in other fish species (26, 29), including an overrepresentation of transcripts involved in proteolysis (KEGG 3050 Proteasome) and RNA processing (KEGG 3040 Spliceosome). The high-mobility group genes have been proposed to be a global regulator of transcription in response to temperature change (26), and the expression of many of these genes increased in the cold (hmgb1a, hmgb1b, hmgb2a, hmgb3a, hmgb3b, hmg20b, and hbp1). Also induced in the cold were a significant number of transcripts involved in proton and ion transport, including 35 transcripts from 21 solute carrier families, possibly related to the regulation of solute and pH homeostasis. Genes involved in circadian rhythm pathways were strongly induced in the cold (KEGG 4710 Circadian rhythm: mammal), including the core clock genes per1a, per1b, per3, cry1a, cry1b, and cry2. The induction of the clock genes probably relates to the strong influence of temperature on the amplitude of circadian rhythms in transcription, a property that is thought to confer temperature compensation of the clock mechanism (33). A similar GO and KEGG analysis of the transcripts that decreased in the cold uncovered an overrepresentation of transcripts involved in protein translation (KEGG 3010 Ribosome) (Fig. 4A and Tables S8 and S9), another response that occurs in many tissues of other species (26, 29). Consistent with previous muscle-specific responses to cold (26, 29) was also an overrepresentation of transcripts involved in muscle contraction and the connective tissue matrix (KEGG 4512 ECM-receptor interaction) among repressed transcripts.

Transcriptional Response to Cold Is Altered by Embryonic Temper- ature. Principal components analysis (PCA) was next used to describe the general patterns in expression and confirm that TE altered the overall pattern of gene expression. When all transcripts were included in PCA (Fig. 5A), the predominant pattern Fig. 4. Genes involved in energy metabolism in the swimming muscle were of variation [principal component 1 (PC1) described 22.3% of strongly induced by cold acclimation. (A) The expression of several transcripts variation in the data] was strongly associated with TA (P < 0.001 changed in each Gene Ontology (GO) category that was significantly over- in two-factor ANOVA), but this variation was also influenced by represented among those transcripts that were responsive to cold. IM, in- TE (P = 0.006 for TA × TE interaction in PC1). The main sec- termembrane; Mito, mitochondrial. (B) Transcripts of enzymes involved in ondary pattern of variation [principal component 2 (PC2) de- carbohydrate and lipid catabolism were generally induced by cold acclimation scribed 15.0% of the variation] starkly contrasted the T groups, (blue). Transcripts for many enzymes involved in fatty acid, glucose, and glycogen E which responded to TA in opposing directions (P = 0.027 for synthesis did not change (white) or were repressed (orange) by cold acclimation. T × T interaction in PC2). PC3 described 10.1% of the vari- For some enzymes, alternate isoforms, splice variants, and/or subunits responded A E ation in expression but was not significantly associated with TA or in opposite directions (blue and orange). Embryonic temperature (TE)also influenced the expression in the cold of several genes involved in energy me- TE. Similar results were obtained when only transcripts that were fl affected by TA were included in PCA (Fig. S2): PC1 described tabolism (black). Enzymes are connected to represent the ux through each < pathway (connections based on NADH, NADPH, ADP, or ATP are not shown). 61.2% of the variation in the data and was affected by TA (P 0.001) and a TA × TE interaction (P = 0.003), and PC2 described 6.2% of the variation and was affected by a TA × TE interaction (P = 0.018). These PCA findings show that the differences in detect these events because RNA-Seq clearly distinguishes be- swimming performance (Fig. 2) and muscle fiber abundance (Fig. tween sets of transcripts that are similar in sequence and because 3) between TE groups are associated with overall differences in the our relatively substantial sequencing effort allowed us to detect transcriptomic response to temperature change. changes in those transcripts that were expressed at low levels. The expression of individual transcripts was shown in our two- Our results therefore provide an extremely comprehensive de- factor (2 TA × 2 TE) interactive design to be influenced by em- scription of the transcriptomic responses of energy supply bryonic temperature. One transcript was influenced by TE as pathways in the muscle to cold (26, 27, 29). a main effect, the ubiquitin-conjugating enzyme E2D 4 (ube2d4-

14250 | www.pnas.org/cgi/doi/10.1073/pnas.1205012109 Scott and Johnston Downloaded by guest on September 27, 2021 groups were still signiﬁcant for 50 transcripts at Q < 0.10 (including 26 at Q < 0.05) after correcting for multiple comparisons (Fig. S4). The majority (38) of transcripts were expressed at higher levels in the 32 °C TE group, consistent with the overall patterns described by PCA (Fig. 5A). The transcripts that were higher in the 32 °C TE group were involved in energy production and its regulation (pprc1-201 and apooa-203, shown in Fig. 5B,as well as tkt-001, odc1-201, tomm40l-001, slc3a2 transcript zgc:55813-201, and aldh1l2 transcript CT573393.2-201, all shown in Fig. 4B), in angiogenesis (wdr43-001 and possibly sgk2b-001), in the cell stress response (chac1-001, hspa5-201,andhyou1-001), or in stimulating muscle differentiation (raver1-202, sik1 transcript si:ch211-235e18.3-001, and cdc6 transcript zgc:174506-201). Among the transcripts that are higher in the 27 °C TE group were transcripts involved in muscle contraction (myha-001), in carbohydrate or fatty acid biosynthesis (tdh-002 and TECR transcript mecr-001, shown in Fig. 4B), in apoptotic signaling (zc3h12a transcript si:dkey-206d17.12-201), or in repressing muscle differentiation (bhlhe40-001). An additional transcript that was higher in the 27 °C TE group (arrdc3 transcript si:dkey-172m14.3-001) is thought to reduce energy expenditure and contribute to obesity in mammals (34). Expression of other transcripts involved in puri- nurgic signaling, transcriptional regulation, RNA processing, and translation also differed between groups. The overall pattern of the differences in gene expression suggests that the 32 °C TE group is better able to acclimate and maintain swimming performance in the cold at least in part because of an accentuated induction of energy production path-

ways. The ultimate cause of this heightened response could PHYSIOLOGY involve the dramatic difference in peroxisome proliferator-activated receptor (PPAR)γ coactivator-related 1 (pprc1) expression in the cold (Fig. 5B). Mammalian orthologs of this protein (PRC) and the related proteins PGC1α and PGC1β are vital regulators of mitochondrial biogenesis and substrate oxidation (35). The setd8b-201 transcript, which encodes a histone meth- Fig. 5. Embryonic temperature (TE) affected the transcriptional response to γ cold in the swimming muscle (detected using RNA-Seq). (A) Principal com- yltransferase whose transcription is activated by PPAR in ponents analysis identiﬁed two primary transcriptional responses to the mammals (36), is also expressed at higher levels in the 32 °C TE

cold, one in which each TE group responded in the same direction but by group in the cold. different magnitudes (principal component 1, PC1: significant effect of ac- fi climation temperature, TA, and TA × TE interaction) and one in which TE Developmental Plasticity Is Not Always Bene cial. Criticism of the groups responded in opposite directions (PC2: significant TA × TE inter- hypothesis that development (or acclimation) in a particular action). (B) Representative transcripts generally differed in abundance be- environment always improves performance in that environment, tween TE groups in a manner similar to PC1 (e.g., apooa) or PC2 (e.g., slc2a10 originally termed the beneficial acclimation hypothesis (37), led and fgf4). The units for these transcript abundance data are read counts to the development of explicit statistical tests of the multiple- normalized for differences in library size between samples. Means ± SEM competing hypothesis for how developmental temperature could (n = 4) are shown (some error bars for PC1 are too small to be seen, and the influence thermal sensitivity in adults (13). Using this method additional point for pprc1 is an outlier). (ANOVA with orthogonal polynomial contrasts) for short-term thermal challenges, we obtained statistical support for the hypothesis that development at a particular TE improves swimming 001, Q < 0.0001), which was expressed 4.8-fold higher on average performance at that temperature (Fig. 2A, significant TS × TE in in the 32 °C TE group compared with the 27 °C TE group (Fig. Table S2). The same was not necessarily true after long-term fi × S1). There was a signi cant TA TE interaction for an additional thermal acclimation, for which we also had strong support for the 10 transcripts (at Q < 0.10, 3 at Q < 0.05) (Fig. S3). Seven of hypothesis that development at hotter temperatures is better these transcripts responded to the cold in opposite directions (significant main effect for the linear contrast of TE in Table S2). between TE groups (slc2a10-201 and fgf4-201, shown in Fig. 5B, Therefore, our results are consistent with previous findings in as well as znfl2a-201, baz1a transcript CU928207.1-201, abhd15 other fish and invertebrates that the beneficial acclimation hy- transcript CR942805.2-201, and uncharacterized transcripts pothesis is often but not always supported (37, 38). BX324215.3-201 and CABZ01030119.1-201), whereas 3 responded in the same direction but by different magnitudes (mylz3- Implications for a Warming World. Developmental plasticity is well 006, pcsk2-201, and myh7b transcript si:ch211-24n20.3-201). known to alter adult phenotypes and to create phenotypic nov- Many of these transcripts are related to energy metabolism elties that facilitate evolution (39), but we still have much to learn (glucose transporter slc2a10 and abhd15, shown in Fig. 4B,as about its mechanistic basis (9, 40). We have taken an integrative well as pcsk2), muscle contraction (mylz3 and my7b), or cell approach working across levels of biological organization to un- proliferation/differentiation (fgf4), some of the main pathways derstand the physiological and transcriptomic bases of develop- that are altered by cold acclimation in general. mental plasticity for swimming performance, an important fitness- On the basis of the swimming performance results, our a priori related trait. Cryptic thermal sensitivity and acclimation capacity expectation was that gene expression should differ between TE were unmasked when zebrafish embryos grew at temperatures groups only at 16 °C. We therefore performed an additional near the natural developmental extremes of this species. This analysis that included only samples from fish acclimated to 16 °C developmental plasticity was based in large part on variation in (Fig. S1). In doing so, we uncovered a large number of transcripts the magnitude of acclimation responses that are shared across TE (1,441) that were differentially expressed between TE groups at groups. Our findings suggest that warming during brief embryonic P < 0.05 (Dataset S2). Differences in expression between TE windows may actually improve thermal hardiness later in life

Scott and Johnston PNAS | August 28, 2012 | vol. 109 | no. 35 | 14251 Downloaded by guest on September 27, 2021 across a range of temperatures, both hot and cold. The effects of results. mRNA-Seq library preparation (5 μg RNA), sequencing (paired-end embryonic temperature may become extremely important in the reads on an Illumina HiSEq 2000 sequencer), base calling, and read mapping future, as temperature variability is expected to increase along to the zebrafish genome (DanRe7, using GSNAP version 2011-03-28) were with average temperature (41). performed by The GenePool at the University of Edinburgh. Four biological replicates were sequenced for each of the four groups, yielding ≥5 million Materials and Methods analyzed reads per replicate. Transcripts were filtered to exclude those in Experimental Treatments. The experimental protocol is illustrated in Fig. 1. the bottom 40% of total read counts across all samples (these transcripts Embryos were reared until hatching at 22 °C, 27 °C, or 32 °C, after which they were generally observed in only approximately one to two replicates and at were raised to adulthood at 27 °C. Adult fish were then subjected to one of most 22 total counts across all 16 samples) as well as those that were not five temperature treatments before swimming performance, muscle histol- expressed in at least three of four replicates in at least one treatment group. ogy, and gene expression were assessed. Information on breeding, rearing, DESeq was used to assess differential expression across the transcriptome and acclimation treatments can be found in SI Materials and Methods. (42). PCA using read counts normalized to library size was performed for all Experimental treatments were approved by the Animal Welfare Committee of the expressed transcripts or for just those that were affected by TA at Q < of the University of St Andrews. 0.05 (data were centered and scaled). We report only the principal components that describe more than the average variance across all components. Swimming Performance and Muscle Histology. Critical swimming speed (Ucrit), GOSeq was used for GO and KEGG pathway analysis of transcripts that in- a standard metric of sustainable exercise performance in fish, was assessed creased or decreased by 1.5-fold (at Q < 0.01) in response to cold acclimation using standard protocols (32). U was determined by increasing water crit (43). Q values were calculated by adjusting P values for multiple com- speed by 0.3 body lengths/s (1 cm/s) every 5 min until the fish could no parisons, using Benjamini–Hochberg correction in both DESeq and GOSeq longer swim against the current, after which it was sampled for histology. analyses. More details on transcriptomic analyses are in SI Materials Muscle phenotype was then assessed in these fish, using standard histological methods (22). Fig. 1 describes the treatment groups subjected to the and Methods. swimming and histology measurements. Other details on these methods and analyses are in SI Materials and Methods. ACKNOWLEDGMENTS. The authors thank the editor and two anonymous referees for excellent comments on an earlier version of this paper, as well as I. P. Amaral, I. Johnston, and D. J. Macqueen for technical help and advice. Transcriptomics. Whole-transcriptome sequencing (RNA-Seq) was performed This work was funded by European Union-Seventh Framework Programme on total RNA extracted from the hypaxial fast muscle (see Fig. 1 for the Project Lifecycle, the Marine Alliance for Science and Technology for Scot- treatment details). We pooled the RNA from one male and one female for land (a pooling initiative from the Scottish Funding Council), and a Natural each biological replicate to eliminate sex as a factor in the analysis of our Sciences and Engineering Research Council Postdoctoral Fellowship (to G.R.S.).

1. Schulte PM, Healy TM, Fangue NA (2011) Thermal performance curves, phenotypic 23. Johnston IA, Moon TW (1980) Exercise training in skeletal muscle of brook trout plasticity, and the time scales of temperature exposure. Integr Comp Biol 51:691–702. (Salvelinus fontinalis). J Exp Biol 87:177–194. 2. Anguilleta MJ (2009) Thermal Adaptation: A Theoretical and Empirical Synthesis 24. Rome LC, Loughna PT, Goldspink G (1984) Muscle fiber activity in carp as a function of (Oxford Univ Press, Oxford, UK). swimming speed and muscle temperature. Am J Physiol 247:R272–R279. 3. Somero GN (2011) Comparative physiology: A “crystal ball” for predicting con- 25. Coughlin DJ, Rome LC (1996) The roles of pink and red muscle in powering steady sequences of global change. Am J Physiol Regul Integr Comp Physiol 301:R1–R14. swimming in scup, Stenotomus chrysops. Am Zool 36:666–677. 4. Pörtner HO, Farrell AP (2008) Ecology. Physiology and climate change. Science 322:690–692. 26. Gracey AY, et al. (2004) Coping with cold: An integrative, multitissue analysis of the 5. Fry FEJ, Hart JS (1948) Cruising speed of goldfish in relation to water temperature. J transcriptome of a poikilothermic vertebrate. Proc Natl Acad Sci USA 101:16970–16975. Fish Res Board Can 7:169–175. 27. Chen Z, et al. (2008) Transcriptomic and genomic evolution under constant cold in 6. Plaut I (2001) Critical swimming speed: Its ecological relevance. Comp Biochem Physiol Antarctic notothenioid fish. Proc Natl Acad Sci USA 105:12944–12949. A Mol Integr Physiol 131:41–50. 28. Murray P, Hayward SA, Govan GG, Gracey AY, Cossins AR (2007) An explicit test of the 7. Huey RB, Kingsolver JG (1993) Evolution of resistance to high temperature in ecto- phospholipid saturation hypothesis of acquired cold tolerance in Caenorhabditis el- – therms. Am Nat 161:357 366. egans. Proc Natl Acad Sci USA 104:5489–5494. 8. Burger R, Lynch M (1995) Evolution and extinction in a changing environment: A 29. Vornanen M, Hassinen M, Koskinen H, Krasnov A (2005) Steady-state effects of – quantitative-genetic analysis. Evolution 49:151 163. temperature acclimation on the transcriptome of the rainbow trout heart. Am J 9. Beldade P, Mateus ARA, Keller RA (2011) Evolution and molecular mechanisms of Physiol Regul Integr Comp Physiol 289:R1177–R1184. – adaptive developmental plasticity. Mol Ecol 20:1347 1363. 30. O’Brien KM (2011) Mitochondrial biogenesis in cold-bodied fishes. J Exp Biol 214:275–285. 10. Overgaard J, Kristensen TN, Mitchell KA, Hoffmann AA (2011) Thermal tolerance in 31. LeMoine CM, Genge CE, Moyes CD (2008) Role of the PGC-1 family in the metabolic widespread and tropical Drosophila species: Does phenotypic plasticity increase with adaptation of goldfish to diet and temperature. J Exp Biol 211:1448–1455. latitude? Am Nat 178(Suppl 1):S80–S96. 32. McClelland GB, Craig PM, Dhekney K, Dipardo S (2006) Temperature- and exercise- 11. López-Olmeda JF, Sánchez-Vázquez FJ (2011) Thermal biology of zebrafish (Danio induced gene expression and metabolic enzyme changes in skeletal muscle of adult rerio). J Therm Biol 36:91–104. zebrafish (Danio rerio). J Physiol 577:739–751. 12. Spence R, Gerlach G, Lawrence C, Smith C (2008) The behaviour and ecology of the 33. Lahiri K, et al. (2005) Temperature regulates transcription in the zebrafish circadian zebrafish, Danio rerio. Biol Rev Camb Philos Soc 83:13–34. clock. PLoS Biol 3:e351. 13. Huey RB, Berrigan B, Gilchrist GW, Herron JC (1999) Testing the adaptive significance 34. Patwari P, et al. (2011) The arrestin domain-containing 3 protein regulates body mass of acclimation: A strong inference approach. Am Zool 39:323–336. and energy expenditure. Cell Metab 14:671–683. 14. Johnston IA, Lucking M (1978) Temperature induced variation in the distribution of dif- 35. Scarpulla RC (2011) Metabolic control of mitochondrial biogenesis through the PGC-1 ferent types of muscle fibre in the goldfish (Carassius auratus). J Comp Physiol 124:111–116. family regulatory network. Biochim Biophys Acta 1813:1269–1278. 15. Jones PL, Sidell BD (1982) Metabolic responses of striped bass (Morone saxatilis)to 36. Wakabayashi K, et al. (2009) The peroxisome proliferator-activated receptor γ/reti- temperature acclimation. II. Alterations in metabolic carbon sources and distributions noid X receptor alpha heterodimer targets the histone modification enzyme PR-Set7/ of fiber types in locomotory muscle. J Exp Zool 219:163–171. 16. Egginton S, Sidell BD (1989) Thermal acclimation induces adaptive changes in sub- Setd8 gene and regulates adipogenesis through a positive feedback loop. Mol Cell – cellular structure of fish skeletal muscle. Am J Physiol 256:R1–R9. Biol 29:3544 3555. 17. Johnston IA, Maitland B (1980) Temperature acclimation in crucian carp (Carassius car- 37. Leroi AM, Bennett AF, Lenski RE (1994) Temperature acclimation and competitive fi fi assius L.): Morphometric analysis of muscle fibre ultra-structure. JFishBiol17:113–125. tness: An experimental test of the bene cial acclimation assumption. Proc Natl Acad – 18. Imai JI, Hirayama Y, Kikuchi K, Kakinuma M, Watabe S (1997) cDNA cloning of myosin Sci USA 91:1917 1921. fi heavy chain isoforms from carp fast skeletal muscle and their gene expression asso- 38. Deere JA, Chown SL (2006) Testing the bene cial acclimation hypothesis and its al- ciated with temperature acclimation. J Exp Biol 200:27–34. ternatives for locomotor performance. Am Nat 168:630–644. 19. Johnston IA, Leeuwen JL, Davies MLF, Beddow T (1995) How fish power predation 39. West-Eberhard MJ (2003) Developmental Plasticity and Evolution (Oxford Univ Press, fast-starts. J Exp Biol 198:1851–1861. Oxford, UK). 20. Sfakianakis D, Leris I, Kentouri M (2011) Effect of developmental temperature on 40. Moczek AP, et al. (2011) The role of developmental plasticity in evolutionary in- swimming performance of zebrafish (Danio rerio) juveniles. Environ Biol Fishes 90: novation. Proc Biol Sci 278:2705–2713. 421–427. 41. Rahmstorf S, Coumou D (2011) Increase of extreme events in a warming world. Proc 21. Schröter C, et al. (2008) Dynamics of zebrafish somitogenesis. Dev Dyn 237:545–553. Natl Acad Sci USA 108:17905–17909. 22. Johnston IA, et al. (2009) Embryonic temperature affects muscle fibre recruitment in 42. Anders S, Huber W (2010) Differential expression analysis for sequence count data. adult zebrafish: Genome-wide changes in gene and microRNA expression associated Genome Biol 11:R106. with the transition from hyperplastic to hypertrophic growth phenotypes. J Exp Biol 43. Young MD, Wakefield MJ, Smyth GK, Oshlack A (2010) Gene ontology analysis for 212:1781–1793. RNA-seq: Accounting for selection bias. Genome Biol 11:R14.

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