#METABOLOMICS WORKBENCH kcontrep_20210805_144954 DATATRACK_ID:2788 STUDY_ID:ST001898 ANALYSIS_ID:AN003083 VERSION 1 CREATED_ON 12-15-2022 #PROJECT PR:PROJECT_TITLE Untargeted lipidomics study of African killifish embryos PR:PROJECT_SUMMARY Untargeted lipidomics study of African killifish embryos in the context of PR:PROJECT_SUMMARY diapause PR:INSTITUTE Stanford University PR:LAST_NAME Contrepois PR:FIRST_NAME Kevin PR:ADDRESS 300 Pasteur Dr PR:EMAIL kcontrep@stanford.edu PR:PHONE 6506664538 PR:DOI http://dx.doi.org/10.21228/M8CD8G #STUDY ST:STUDY_TITLE Evolution of diapause in the African killifish by remodeling ancient gene ST:STUDY_TITLE regulatory landscape ST:STUDY_SUMMARY Suspended animation (e.g. hibernation, diapause) allows organisms to survive ST:STUDY_SUMMARY extreme environments. But the mechanisms underlying the evolution of suspended ST:STUDY_SUMMARY animation states are unknown. The African turquoise killifish has evolved ST:STUDY_SUMMARY diapause as a form of suspended development to survive the complete drought that ST:STUDY_SUMMARY occurs every summer. Here, we show that gene duplicates – paralogs – exhibit ST:STUDY_SUMMARY specialized expression in diapause compared to normal development in the African ST:STUDY_SUMMARY turquoise killifish. Surprisingly, paralogs with specialized expression in ST:STUDY_SUMMARY diapause are evolutionarily very ancient and are present even in vertebrates ST:STUDY_SUMMARY that do not exhibit diapause. To determine if evolution of diapause is due to ST:STUDY_SUMMARY the regulatory landscape rewiring at ancient paralogs, we assessed chromatin ST:STUDY_SUMMARY accessibility genome-wide in fish species with or without diapause. This ST:STUDY_SUMMARY analysis revealed an evolutionary recent increase in chromatin accessibility at ST:STUDY_SUMMARY very ancient paralogs in African turquoise killifish. The increase in chromatin ST:STUDY_SUMMARY accessibility is linked to the presence of new binding sites for transcription ST:STUDY_SUMMARY factors, likely due to de novo mutations and transposable element (TE) ST:STUDY_SUMMARY insertion. Interestingly, accessible chromatin regions in diapause are enriched ST:STUDY_SUMMARY for lipid metabolism genes, and our lipidomics studies uncover a striking ST:STUDY_SUMMARY difference in lipid species in African turquoise killifish diapause, which could ST:STUDY_SUMMARY be critical for long-term survival. Together, our results show that diapause ST:STUDY_SUMMARY likely originated by repurposing pre-existing gene programs via recent changes ST:STUDY_SUMMARY in the regulatory landscape. This work raises the possibility that suspended ST:STUDY_SUMMARY animation programs could be reactivated in other species for long-term ST:STUDY_SUMMARY preservation via transcription factor remodeling and suggests a mechanism for ST:STUDY_SUMMARY how complex adaptations evolve in nature. ST:INSTITUTE Stanford University ST:LAST_NAME Contrepois ST:FIRST_NAME Kevin ST:ADDRESS 300 Pasteur Dr ST:EMAIL kcontrep@stanford.edu ST:PHONE 6506664538 ST:SUBMIT_DATE 2021-08-05 #SUBJECT SU:SUBJECT_TYPE Fish SU:SUBJECT_SPECIES Nothobranchius furzeri;Aphyosemion striatum #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS nfur.D1m.1 M27_KV_E Stage:1 month diapause | Species:N. furzeri Number of Embryos=30; Total Protein Abundance (ug)=475; RAW_FILE_NAME=pRPLC_M27_KV_E; RAW_FILE_NAME=nRPLC_M27_KV_E SUBJECT_SAMPLE_FACTORS nfur.D1m.4 M28_KV_E Stage:1 month diapause | Species:N. furzeri Number of Embryos=28; Total Protein Abundance (ug)=1063; RAW_FILE_NAME=pRPLC_M28_KV_E; RAW_FILE_NAME=nRPLC_M28_KV_E SUBJECT_SAMPLE_FACTORS nfur.D1m.2 M29_KV_E Stage:1 month diapause | Species:N. furzeri Number of Embryos=27; Total Protein Abundance (ug)=845; RAW_FILE_NAME=pRPLC_M29_KV_E; RAW_FILE_NAME=nRPLC_M29_KV_E SUBJECT_SAMPLE_FACTORS nfur.D1m.3 MA_M2_E Stage:1 month diapause | Species:N. furzeri Number of Embryos=25; Total Protein Abundance (ug)=805; RAW_FILE_NAME=pRPLC_MA_M2_E; RAW_FILE_NAME=nRPLC_MA_M2_E SUBJECT_SAMPLE_FACTORS nfur.D6d.1 M18_D6_E Stage:6 day diapause | Species:N. furzeri Number of Embryos=27; Total Protein Abundance (ug)=967; RAW_FILE_NAME=pRPLC_M18_D6_E; RAW_FILE_NAME=nRPLC_M18_D6_E SUBJECT_SAMPLE_FACTORS nfur.D6d.4 M19_D6_E Stage:6 day diapause | Species:N. furzeri Number of Embryos=25; Total Protein Abundance (ug)=909; RAW_FILE_NAME=pRPLC_M19_D6_E; RAW_FILE_NAME=nRPLC_M19_D6_E SUBJECT_SAMPLE_FACTORS nfur.D6d.2 M4_D6_E Stage:6 day diapause | Species:N. furzeri Number of Embryos=25; Total Protein Abundance (ug)=839; RAW_FILE_NAME=pRPLC_M4_D6_E; RAW_FILE_NAME=nRPLC_M4_D6_E SUBJECT_SAMPLE_FACTORS nfur.D6d.3 M5_D6_E Stage:6 day diapause | Species:N. furzeri Number of Embryos=25; Total Protein Abundance (ug)=1351; RAW_FILE_NAME=pRPLC_M5_D6_E; RAW_FILE_NAME=nRPLC_M5_D6_E SUBJECT_SAMPLE_FACTORS nfur.NonD.3 M10_DEV_E Stage:development | Species:N. furzeri Number of Embryos=12; Total Protein Abundance (ug)=1609; RAW_FILE_NAME=pRPLC_M10_DEV_E; RAW_FILE_NAME=nRPLC_M10_DEV_E SUBJECT_SAMPLE_FACTORS nfur.NonD.4 M13_DEV_E Stage:development | Species:N. furzeri Number of Embryos=10; Total Protein Abundance (ug)=1597; RAW_FILE_NAME=pRPLC_M13_DEV_E; RAW_FILE_NAME=nRPLC_M13_DEV_E SUBJECT_SAMPLE_FACTORS nfur.NonD.1 M21_DEV_E Stage:development | Species:N. furzeri Number of Embryos=21; Total Protein Abundance (ug)=453; RAW_FILE_NAME=pRPLC_M21_DEV_E; RAW_FILE_NAME=nRPLC_M21_DEV_E SUBJECT_SAMPLE_FACTORS nfur.NonD.2 M24_DEV_E Stage:development | Species:N. furzeri Number of Embryos=12; Total Protein Abundance (ug)=2052; RAW_FILE_NAME=pRPLC_M24_DEV_E; RAW_FILE_NAME=nRPLC_M24_DEV_E SUBJECT_SAMPLE_FACTORS nfur.Dexit.1 M12_EX_E Stage:diapause exit | Species:N. furzeri Number of Embryos=10; Total Protein Abundance (ug)=439; RAW_FILE_NAME=pRPLC_M12_EX_E; RAW_FILE_NAME=nRPLC_M12_EX_E SUBJECT_SAMPLE_FACTORS nfur.Dexit.3 M7_EX_E Stage:diapause exit | Species:N. furzeri Number of Embryos=10; Total Protein Abundance (ug)=2203; RAW_FILE_NAME=pRPLC_M7_EX_E; RAW_FILE_NAME=nRPLC_M7_EX_E SUBJECT_SAMPLE_FACTORS nfur.Dexit.4 M8_EX_E Stage:diapause exit | Species:N. furzeri Number of Embryos=10; Total Protein Abundance (ug)=1219; RAW_FILE_NAME=pRPLC_M8_EX_E; RAW_FILE_NAME=nRPLC_M8_EX_E SUBJECT_SAMPLE_FACTORS nfur.Dexit.2 M9_EX_E Stage:diapause exit | Species:N. furzeri Number of Embryos=10; Total Protein Abundance (ug)=1529; RAW_FILE_NAME=pRPLC_M9_EX_E; RAW_FILE_NAME=nRPLC_M9_EX_E SUBJECT_SAMPLE_FACTORS Ast.PreD.O.2 AO10_KV_E Stage:pre diapause old | Species:A. striatum Number of Embryos=26; Total Protein Abundance (ug)=1431; RAW_FILE_NAME=pRPLC_AO10_KV_E; RAW_FILE_NAME=nRPLC_AO10_KV_E SUBJECT_SAMPLE_FACTORS Ast.PreD.O.1 AO13_KV_E Stage:pre diapause old | Species:A. striatum Number of Embryos=17; Total Protein Abundance (ug)=1193; RAW_FILE_NAME=pRPLC_AO13_KV_E; RAW_FILE_NAME=nRPLC_AO13_KV_E SUBJECT_SAMPLE_FACTORS Ast.PreD.O.4 AO3_KV_E Stage:pre diapause old | Species:A. striatum Number of Embryos=25; Total Protein Abundance (ug)=1161; RAW_FILE_NAME=pRPLC_AO3_KV_E; RAW_FILE_NAME=nRPLC_AO3_KV_E SUBJECT_SAMPLE_FACTORS Ast.PreD.O.3 AO8_KV_E Stage:pre diapause old | Species:A. striatum Number of Embryos=18; Total Protein Abundance (ug)=1498; RAW_FILE_NAME=pRPLC_AO8_KV_E; RAW_FILE_NAME=nRPLC_AO8_KV_E SUBJECT_SAMPLE_FACTORS nfur.PreD.O.3 O11_KV_E Stage:pre diapause old | Species:N. furzeri Number of Embryos=25; Total Protein Abundance (ug)=749; RAW_FILE_NAME=pRPLC_O11_KV_E; RAW_FILE_NAME=nRPLC_O11_KV_E SUBJECT_SAMPLE_FACTORS nfur.PreD.O.2 O13_KV_E Stage:pre diapause old | Species:N. furzeri Number of Embryos=25; Total Protein Abundance (ug)=503; RAW_FILE_NAME=pRPLC_O13_KV_E; RAW_FILE_NAME=nRPLC_O13_KV_E SUBJECT_SAMPLE_FACTORS nfur.PreD.O.4 O8_KV_E Stage:pre diapause old | Species:N. furzeri Number of Embryos=25; Total Protein Abundance (ug)=798; RAW_FILE_NAME=pPRLC_O8_KV_E; RAW_FILE_NAME=nRPLC_O8_KV_E SUBJECT_SAMPLE_FACTORS nfur.PreD.O.1 O9_KV_E Stage:pre diapause old | Species:N. furzeri Number of Embryos=25; Total Protein Abundance (ug)=850; RAW_FILE_NAME=pRPLC_O9_KV_E; RAW_FILE_NAME=nRPLC_O9_KV_E SUBJECT_SAMPLE_FACTORS Ast.PreD.Y.1 AY10_KV_E Stage:pre diapause young | Species:A. striatum Number of Embryos=11; Total Protein Abundance (ug)=845; RAW_FILE_NAME=pRPLC_AY10_KV_E; RAW_FILE_NAME=nRPLC_AY10_KV_E SUBJECT_SAMPLE_FACTORS Ast.PreD.Y.3 AY1_KV_E Stage:pre diapause young | Species:A. striatum Number of Embryos=9; Total Protein Abundance (ug)=644; RAW_FILE_NAME=pRPLC_AY1_KV_E; RAW_FILE_NAME=nRPLC_AY1_KV_E SUBJECT_SAMPLE_FACTORS Ast.PreD.Y.4 AY2_KV_E Stage:pre diapause young | Species:A. striatum Number of Embryos=13; Total Protein Abundance (ug)=701; RAW_FILE_NAME=pRPLC_AY2_KV_E; RAW_FILE_NAME=nRPLC_AY2_KV_E SUBJECT_SAMPLE_FACTORS Ast.PreD.Y.2 AY3_KV_E Stage:pre diapause young | Species:A. striatum Number of Embryos=9; Total Protein Abundance (ug)=534; RAW_FILE_NAME=pRPLC_AY3_KV_E; RAW_FILE_NAME=nRPLC_AY3_KV_E SUBJECT_SAMPLE_FACTORS nfur.PreD.Y.3 Y6_KV_E Stage:pre diapause young | Species:N. furzeri Number of Embryos=26; Total Protein Abundance (ug)=1031; RAW_FILE_NAME=pRPLC_Y6_KV_E; RAW_FILE_NAME=nRPLC_Y6_KV_E SUBJECT_SAMPLE_FACTORS nfur.PreD.Y.4 Y7_KV_E Stage:pre diapause young | Species:N. furzeri Number of Embryos=25; Total Protein Abundance (ug)=713; RAW_FILE_NAME=pRPLC_Y7_KV_E; RAW_FILE_NAME=nRPLC_Y7_KV_E SUBJECT_SAMPLE_FACTORS nfur.PreD.Y.1 Y8_KV_E Stage:pre diapause young | Species:N. furzeri Number of Embryos=25; Total Protein Abundance (ug)=595; RAW_FILE_NAME=pRPLC_Y8_KV_E; RAW_FILE_NAME=nRPLC_Y8_KV_E SUBJECT_SAMPLE_FACTORS nfur.PreD.Y.2 Y9_KV_E Stage:pre diapause young | Species:N. furzeri Number of Embryos=25; Total Protein Abundance (ug)=820; RAW_FILE_NAME=pRPLC_Y9_KV_E; RAW_FILE_NAME=nRPLC_Y9_KV_E #COLLECTION CO:COLLECTION_SUMMARY For each stage in each species, roughly 25-30 embryos were carefully dissected CO:COLLECTION_SUMMARY in ice-cold PBS using biological-grade tweezers (Electron Microscopy Sciences, CO:COLLECTION_SUMMARY 72700-D) to carefully remove the chorion, the enveloping layer, and the yolk CO:COLLECTION_SUMMARY without damaging the embryo body. Freshly dissected embryos were then quickly CO:COLLECTION_SUMMARY rinsed with ice-cold PBS, and all the PBS was carefully removed. Embryo bodies CO:COLLECTION_SUMMARY were then snap-frozen in liquid nitrogen and stored at -80°C. A total of 25-30 CO:COLLECTION_SUMMARY embryos for lipidomics. CO:SAMPLE_TYPE Embryo #TREATMENT TR:TREATMENT_SUMMARY N/A #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Embryos for each stage of diapause and development were isolated from African SP:SAMPLEPREP_SUMMARY turquoise and red-striped killifish (3-4 replicates for each stage) and lipid SP:SAMPLEPREP_SUMMARY profiling was performed as previously described (PMID: 30532037, PMID: SP:SAMPLEPREP_SUMMARY 32612231). Lipids were extracted in a randomized order via biphasic separation SP:SAMPLEPREP_SUMMARY with cold methyl tert-butyl ether (MTBE), methanol and water. Briefly, 260 μl SP:SAMPLEPREP_SUMMARY of methanol and 40 μl of water were added to the embryos and vortexed for 20 s. SP:SAMPLEPREP_SUMMARY A lipid internal standard mixture was spiked in each sample (EquiSPLASH SP:SAMPLEPREP_SUMMARY LIPIDOMIX, Avanti Polar Lipids (cat #: 330731), and d17-Oleic acid, Cayman SP:SAMPLEPREP_SUMMARY chemicals (cat #: 9000432) to control for extraction efficiency, evaluate LC-MS SP:SAMPLEPREP_SUMMARY performance and normalize LC-MS data. Samples were diluted with 1,000 μl of SP:SAMPLEPREP_SUMMARY MTBE, vortexed for 10 s, sonicated for 30 s three times in a water bath, and SP:SAMPLEPREP_SUMMARY incubated under agitation for 30 min at 4°C. After addition of 250 μl of SP:SAMPLEPREP_SUMMARY water, the samples were vortexed for 1 min and centrifuged at 14,000g for 5 min SP:SAMPLEPREP_SUMMARY at 20°C. The upper phase containing the lipids was collected and dried down SP:SAMPLEPREP_SUMMARY under nitrogen. The dry extracts were reconstituted with 150 μl of 9:1 SP:SAMPLEPREP_SUMMARY methanol:toluene. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Lipid extracts were analyzed in a randomized order using an Ultimate 3000 RSLC CH:CHROMATOGRAPHY_SUMMARY system coupled with a Q Exactive mass spectrometer (Thermo Fisher Scientific) as CH:CHROMATOGRAPHY_SUMMARY previously described (PMID: 32612231). Each sample was run twice in positive and CH:CHROMATOGRAPHY_SUMMARY negative ionization modes and lipids were separated using an Accucore C30 column CH:CHROMATOGRAPHY_SUMMARY 2.1 x 150 mm, 2.6 μm (Thermo Fisher Scientific) and mobile phase solvents CH:CHROMATOGRAPHY_SUMMARY consisted in 10 mM ammonium acetate and 0.1% formic acid in 60/40 CH:CHROMATOGRAPHY_SUMMARY acetonitrile/water (A) and 10 mM ammonium acetate and 0.1% formic acid in 90/10 CH:CHROMATOGRAPHY_SUMMARY isopropanol/acetonitrile (B). The gradient profile used was 30% B for 3 min, CH:CHROMATOGRAPHY_SUMMARY 30–43% B over 5 min, 43–50% B over 1 min, 55–90% B over 9 min, 90-99% B CH:CHROMATOGRAPHY_SUMMARY over 9 min and 99% B for 5 min. Lipids were eluted from the column at 0.2 CH:CHROMATOGRAPHY_SUMMARY mL/min, the oven temperature was set at 30°C, and the injection volume was 5 CH:CHROMATOGRAPHY_SUMMARY μL. Autosampler temperature was set at 15°C to prevent lipid aggregation. CH:INSTRUMENT_NAME Thermo Dionex Ultimate 3000 RS CH:COLUMN_NAME Thermo Accucore (150 x 2.1mm,2.6um) CH:COLUMN_TEMPERATURE 30 CH:FLOW_GRADIENT The gradient profile used was 30% B for 3 min, 30-43% B over 5 min, 43-50% B CH:FLOW_GRADIENT over 1 min, 55-90% B over 9 min, 90-99% B over 9 min and 99% B for 5 min. CH:FLOW_RATE 0.2 mL/min CH:INJECTION_TEMPERATURE 15 CH:SAMPLE_INJECTION 5ul CH:SOLVENT_A 60% acetonitrile/40% water; 0.1% formic acid;10 mM ammonium acetate CH:SOLVENT_B 90% isopropanol/10% acetonitrile; 0.1% formic acid;10 mM ammonium acetate CH:CHROMATOGRAPHY_TYPE Reversed phase #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Q Exactive Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:MS_COMMENTS LC-MS peak extraction, alignment, quantification and annotation was performed MS:MS_COMMENTS using LipidSearch software version 4.2.21 (Thermo Fisher Scientific). Lipids MS:MS_COMMENTS were identified by matching the precursor ion mass to a database and the MS:MS_COMMENTS experimental MS/MS spectra to a spectral library containing theoretical MS:MS_COMMENTS fragmentation spectra. To reduce the risk of misidentification, MS/MS spectra MS:MS_COMMENTS from lipids of interest were validated as follows: 1) both positive and negative MS:MS_COMMENTS mode MS/MS spectra match the expected fragments, 2) the main lipid adduct forms MS:MS_COMMENTS detected in positive and negative modes agree with the lipid class identified, MS:MS_COMMENTS 3) the retention time is compatible with the lipid class identified and 4) the MS:MS_COMMENTS peak shape is acceptable. The fragmentation pattern of each lipid class was MS:MS_COMMENTS experimentally validated using lipid internal standards. Single-point internal MS:MS_COMMENTS standard calibrations were used to estimate absolute concentrations for 431 MS:MS_COMMENTS unique lipids belonging to 14 classes using one internal standard for each lipid MS:MS_COMMENTS class. Importantly, we ensured linearity within the range of detected endogenous MS:MS_COMMENTS lipids using serial dilutions of internal standards spanning 4 orders of MS:MS_COMMENTS magnitude. Subsequently, median normalization (excluding TG and DG) was employed MS:MS_COMMENTS on lipid molar concentrations to correct for differential quantity of starting MS:MS_COMMENTS material. The normalized lipid intensities were well correlated with protein MS:MS_COMMENTS abundances measured using BCA Protein Assay Kit (Pierce, cat# 23225) suggesting MS:MS_COMMENTS good sample quality. One development (diapause escape) sample had an MS:MS_COMMENTS unexpectedly low protein concentration and thus was discarded. Lipid molar MS:MS_COMMENTS concentrations for a given class were calculated by summing individual lipid MS:MS_COMMENTS species molar concentrations belonging to that class. Fatty acid composition MS:MS_COMMENTS analysis was performed in each lipid class. Fatty acid composition was MS:MS_COMMENTS calculated by taking the ratio of the sum molar concentration of a given fatty MS:MS_COMMENTS acid over the sum molar concentration across fatty acids found in the lipids of MS:MS_COMMENTS the class. Subsequently, saturated fatty acids (SFA), mono-unsaturated fatty MS:MS_COMMENTS acids (MUFA) and poly-unsaturated fatty acids (PUFA) were grouped together for MS:MS_COMMENTS comparative analysis. MS:ION_MODE POSITIVE MS:MS_RESULTS_FILE ST001898_AN003083_Results.txt UNITS:MS count Has m/z:Yes Has RT:Yes RT units:Minutes #END