{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST001898","ANALYSIS_ID":"AN003084","VERSION":"1","CREATED_ON":"August 10, 2021, 11:59 am"},

"PROJECT":{"PROJECT_TITLE":"Untargeted lipidomics study of African killifish embryos","PROJECT_SUMMARY":"Untargeted lipidomics study of African killifish embryos in the context of diapause","INSTITUTE":"Stanford University","LAST_NAME":"Contrepois","FIRST_NAME":"Kevin","ADDRESS":"300 Pasteur Dr","EMAIL":"kcontrep@stanford.edu","PHONE":"6506664538"},

"STUDY":{"STUDY_TITLE":"Evolution of diapause in the African killifish by remodeling ancient gene regulatory landscape","STUDY_SUMMARY":"Suspended animation (e.g. hibernation, diapause) allows organisms to survive extreme environments. But the mechanisms underlying the evolution of suspended animation states are unknown. The African turquoise killifish has evolved diapause as a form of suspended development to survive the complete drought that occurs every summer. Here, we show that gene duplicates – paralogs – exhibit specialized expression in diapause compared to normal development in the African turquoise killifish. Surprisingly, paralogs with specialized expression in diapause are evolutionarily very ancient and are present even in vertebrates that do not exhibit diapause. To determine if evolution of diapause is due to the regulatory landscape rewiring at ancient paralogs, we assessed chromatin accessibility genome-wide in fish species with or without diapause. This analysis revealed an evolutionary recent increase in chromatin accessibility at very ancient paralogs in African turquoise killifish. The increase in chromatin accessibility is linked to the presence of new binding sites for transcription factors, likely due to de novo mutations and transposable element (TE) insertion. Interestingly, accessible chromatin regions in diapause are enriched for lipid metabolism genes, and our lipidomics studies uncover a striking difference in lipid species in African turquoise killifish diapause, which could be critical for long-term survival. Together, our results show that diapause likely originated by repurposing pre-existing gene programs via recent changes in the regulatory landscape. This work raises the possibility that suspended animation programs could be reactivated in other species for long-term preservation via transcription factor remodeling and suggests a mechanism for how complex adaptations evolve in nature.","INSTITUTE":"Stanford University","LAST_NAME":"Contrepois","FIRST_NAME":"Kevin","ADDRESS":"300 Pasteur Dr","EMAIL":"kcontrep@stanford.edu","PHONE":"6506664538"},

"SUBJECT":{"SUBJECT_TYPE":"Fish","SUBJECT_SPECIES":"Nothobranchius furzeri;Aphyosemion striatum"},
"SUBJECT_SAMPLE_FACTORS":[
{
"Subject ID":"nfur.D1m.1",
"Sample ID":"M27_KV_E",
"Factors":{"Stage":"1 month diapause","Species":"N. furzeri"},
"Additional sample data":{"Number of Embryos":"30","Total Protein Abundance (ug)":"475","RAW_FILE_NAME":"pRPLC_M27_KV_E","RAW_FILE_NAME":"nRPLC_M27_KV_E"}
},
{
"Subject ID":"nfur.Dexit.1",
"Sample ID":"M12_EX_E",
"Factors":{"Stage":"diapause exit","Species":"N. furzeri"},
"Additional sample data":{"Number of Embryos":"10","Total Protein Abundance (ug)":"439","RAW_FILE_NAME":"pRPLC_M12_EX_E","RAW_FILE_NAME":"nRPLC_M12_EX_E"}
},
{
"Subject ID":"nfur.Dexit.2",
"Sample ID":"M9_EX_E",
"Factors":{"Stage":"diapause exit","Species":"N. furzeri"},
"Additional sample data":{"Number of Embryos":"10","Total Protein Abundance (ug)":"1529","RAW_FILE_NAME":"pRPLC_M9_EX_E","RAW_FILE_NAME":"nRPLC_M9_EX_E"}
},
{
"Subject ID":"nfur.PreD.Y.1",
"Sample ID":"Y8_KV_E",
"Factors":{"Stage":"pre diapause young","Species":"N. furzeri"},
"Additional sample data":{"Number of Embryos":"25","Total Protein Abundance (ug)":"595","RAW_FILE_NAME":"pRPLC_Y8_KV_E","RAW_FILE_NAME":"nRPLC_Y8_KV_E"}
},
{
"Subject ID":"nfur.NonD.1",
"Sample ID":"M21_DEV_E",
"Factors":{"Stage":"development","Species":"N. furzeri"},
"Additional sample data":{"Number of Embryos":"21","Total Protein Abundance (ug)":"453","RAW_FILE_NAME":"pRPLC_M21_DEV_E","RAW_FILE_NAME":"nRPLC_M21_DEV_E"}
},
{
"Subject ID":"Ast.PreD.Y.1",
"Sample ID":"AY10_KV_E",
"Factors":{"Stage":"pre diapause young","Species":"A. striatum"},
"Additional sample data":{"Number of Embryos":"11","Total Protein Abundance (ug)":"845","RAW_FILE_NAME":"pRPLC_AY10_KV_E","RAW_FILE_NAME":"nRPLC_AY10_KV_E"}
},
{
"Subject ID":"Ast.PreD.O.1",
"Sample ID":"AO13_KV_E",
"Factors":{"Stage":"pre diapause old","Species":"A. striatum"},
"Additional sample data":{"Number of Embryos":"17","Total Protein Abundance (ug)":"1193","RAW_FILE_NAME":"pRPLC_AO13_KV_E","RAW_FILE_NAME":"nRPLC_AO13_KV_E"}
},
{
"Subject ID":"Ast.PreD.O.2",
"Sample ID":"AO10_KV_E",
"Factors":{"Stage":"pre diapause old","Species":"A. striatum"},
"Additional sample data":{"Number of Embryos":"26","Total Protein Abundance (ug)":"1431","RAW_FILE_NAME":"pRPLC_AO10_KV_E","RAW_FILE_NAME":"nRPLC_AO10_KV_E"}
},
{
"Subject ID":"nfur.Dexit.3",
"Sample ID":"M7_EX_E",
"Factors":{"Stage":"diapause exit","Species":"N. furzeri"},
"Additional sample data":{"Number of Embryos":"10","Total Protein Abundance (ug)":"2203","RAW_FILE_NAME":"pRPLC_M7_EX_E","RAW_FILE_NAME":"nRPLC_M7_EX_E"}
},
{
"Subject ID":"Ast.PreD.Y.2",
"Sample ID":"AY3_KV_E",
"Factors":{"Stage":"pre diapause young","Species":"A. striatum"},
"Additional sample data":{"Number of Embryos":"9","Total Protein Abundance (ug)":"534","RAW_FILE_NAME":"pRPLC_AY3_KV_E","RAW_FILE_NAME":"nRPLC_AY3_KV_E"}
},
{
"Subject ID":"Ast.PreD.Y.3",
"Sample ID":"AY1_KV_E",
"Factors":{"Stage":"pre diapause young","Species":"A. striatum"},
"Additional sample data":{"Number of Embryos":"9","Total Protein Abundance (ug)":"644","RAW_FILE_NAME":"pRPLC_AY1_KV_E","RAW_FILE_NAME":"nRPLC_AY1_KV_E"}
},
{
"Subject ID":"nfur.D1m.2",
"Sample ID":"M29_KV_E",
"Factors":{"Stage":"1 month diapause","Species":"N. furzeri"},
"Additional sample data":{"Number of Embryos":"27","Total Protein Abundance (ug)":"845","RAW_FILE_NAME":"pRPLC_M29_KV_E","RAW_FILE_NAME":"nRPLC_M29_KV_E"}
},
{
"Subject ID":"Ast.PreD.Y.4",
"Sample ID":"AY2_KV_E",
"Factors":{"Stage":"pre diapause young","Species":"A. striatum"},
"Additional sample data":{"Number of Embryos":"13","Total Protein Abundance (ug)":"701","RAW_FILE_NAME":"pRPLC_AY2_KV_E","RAW_FILE_NAME":"nRPLC_AY2_KV_E"}
},
{
"Subject ID":"nfur.Dexit.4",
"Sample ID":"M8_EX_E",
"Factors":{"Stage":"diapause exit","Species":"N. furzeri"},
"Additional sample data":{"Number of Embryos":"10","Total Protein Abundance (ug)":"1219","RAW_FILE_NAME":"pRPLC_M8_EX_E","RAW_FILE_NAME":"nRPLC_M8_EX_E"}
},
{
"Subject ID":"nfur.D6d.1",
"Sample ID":"M18_D6_E",
"Factors":{"Stage":"6 day diapause","Species":"N. furzeri"},
"Additional sample data":{"Number of Embryos":"27","Total Protein Abundance (ug)":"967","RAW_FILE_NAME":"pRPLC_M18_D6_E","RAW_FILE_NAME":"nRPLC_M18_D6_E"}
},
{
"Subject ID":"nfur.PreD.O.1",
"Sample ID":"O9_KV_E",
"Factors":{"Stage":"pre diapause old","Species":"N. furzeri"},
"Additional sample data":{"Number of Embryos":"25","Total Protein Abundance (ug)":"850","RAW_FILE_NAME":"pRPLC_O9_KV_E","RAW_FILE_NAME":"nRPLC_O9_KV_E"}
},
{
"Subject ID":"nfur.D1m.3",
"Sample ID":"MA_M2_E",
"Factors":{"Stage":"1 month diapause","Species":"N. furzeri"},
"Additional sample data":{"Number of Embryos":"25","Total Protein Abundance (ug)":"805","RAW_FILE_NAME":"pRPLC_MA_M2_E","RAW_FILE_NAME":"nRPLC_MA_M2_E"}
},
{
"Subject ID":"Ast.PreD.O.3",
"Sample ID":"AO8_KV_E",
"Factors":{"Stage":"pre diapause old","Species":"A. striatum"},
"Additional sample data":{"Number of Embryos":"18","Total Protein Abundance (ug)":"1498","RAW_FILE_NAME":"pRPLC_AO8_KV_E","RAW_FILE_NAME":"nRPLC_AO8_KV_E"}
},
{
"Subject ID":"nfur.NonD.2",
"Sample ID":"M24_DEV_E",
"Factors":{"Stage":"development","Species":"N. furzeri"},
"Additional sample data":{"Number of Embryos":"12","Total Protein Abundance (ug)":"2052","RAW_FILE_NAME":"pRPLC_M24_DEV_E","RAW_FILE_NAME":"nRPLC_M24_DEV_E"}
},
{
"Subject ID":"nfur.D6d.2",
"Sample ID":"M4_D6_E",
"Factors":{"Stage":"6 day diapause","Species":"N. furzeri"},
"Additional sample data":{"Number of Embryos":"25","Total Protein Abundance (ug)":"839","RAW_FILE_NAME":"pRPLC_M4_D6_E","RAW_FILE_NAME":"nRPLC_M4_D6_E"}
},
{
"Subject ID":"nfur.D6d.3",
"Sample ID":"M5_D6_E",
"Factors":{"Stage":"6 day diapause","Species":"N. furzeri"},
"Additional sample data":{"Number of Embryos":"25","Total Protein Abundance (ug)":"1351","RAW_FILE_NAME":"pRPLC_M5_D6_E","RAW_FILE_NAME":"nRPLC_M5_D6_E"}
},
{
"Subject ID":"nfur.PreD.Y.2",
"Sample ID":"Y9_KV_E",
"Factors":{"Stage":"pre diapause young","Species":"N. furzeri"},
"Additional sample data":{"Number of Embryos":"25","Total Protein Abundance (ug)":"820","RAW_FILE_NAME":"pRPLC_Y9_KV_E","RAW_FILE_NAME":"nRPLC_Y9_KV_E"}
},
{
"Subject ID":"nfur.PreD.O.2",
"Sample ID":"O13_KV_E",
"Factors":{"Stage":"pre diapause old","Species":"N. furzeri"},
"Additional sample data":{"Number of Embryos":"25","Total Protein Abundance (ug)":"503","RAW_FILE_NAME":"pRPLC_O13_KV_E","RAW_FILE_NAME":"nRPLC_O13_KV_E"}
},
{
"Subject ID":"nfur.PreD.O.3",
"Sample ID":"O11_KV_E",
"Factors":{"Stage":"pre diapause old","Species":"N. furzeri"},
"Additional sample data":{"Number of Embryos":"25","Total Protein Abundance (ug)":"749","RAW_FILE_NAME":"pRPLC_O11_KV_E","RAW_FILE_NAME":"nRPLC_O11_KV_E"}
},
{
"Subject ID":"nfur.D1m.4",
"Sample ID":"M28_KV_E",
"Factors":{"Stage":"1 month diapause","Species":"N. furzeri"},
"Additional sample data":{"Number of Embryos":"28","Total Protein Abundance (ug)":"1063","RAW_FILE_NAME":"pRPLC_M28_KV_E","RAW_FILE_NAME":"nRPLC_M28_KV_E"}
},
{
"Subject ID":"nfur.PreD.O.4",
"Sample ID":"O8_KV_E",
"Factors":{"Stage":"pre diapause old","Species":"N. furzeri"},
"Additional sample data":{"Number of Embryos":"25","Total Protein Abundance (ug)":"798","RAW_FILE_NAME":"pPRLC_O8_KV_E","RAW_FILE_NAME":"nRPLC_O8_KV_E"}
},
{
"Subject ID":"nfur.NonD.3",
"Sample ID":"M10_DEV_E",
"Factors":{"Stage":"development","Species":"N. furzeri"},
"Additional sample data":{"Number of Embryos":"12","Total Protein Abundance (ug)":"1609","RAW_FILE_NAME":"pRPLC_M10_DEV_E","RAW_FILE_NAME":"nRPLC_M10_DEV_E"}
},
{
"Subject ID":"nfur.PreD.Y.3",
"Sample ID":"Y6_KV_E",
"Factors":{"Stage":"pre diapause young","Species":"N. furzeri"},
"Additional sample data":{"Number of Embryos":"26","Total Protein Abundance (ug)":"1031","RAW_FILE_NAME":"pRPLC_Y6_KV_E","RAW_FILE_NAME":"nRPLC_Y6_KV_E"}
},
{
"Subject ID":"nfur.D6d.4",
"Sample ID":"M19_D6_E",
"Factors":{"Stage":"6 day diapause","Species":"N. furzeri"},
"Additional sample data":{"Number of Embryos":"25","Total Protein Abundance (ug)":"909","RAW_FILE_NAME":"pRPLC_M19_D6_E","RAW_FILE_NAME":"nRPLC_M19_D6_E"}
},
{
"Subject ID":"nfur.NonD.4",
"Sample ID":"M13_DEV_E",
"Factors":{"Stage":"development","Species":"N. furzeri"},
"Additional sample data":{"Number of Embryos":"10","Total Protein Abundance (ug)":"1597","RAW_FILE_NAME":"pRPLC_M13_DEV_E","RAW_FILE_NAME":"nRPLC_M13_DEV_E"}
},
{
"Subject ID":"nfur.PreD.Y.4",
"Sample ID":"Y7_KV_E",
"Factors":{"Stage":"pre diapause young","Species":"N. furzeri"},
"Additional sample data":{"Number of Embryos":"25","Total Protein Abundance (ug)":"713","RAW_FILE_NAME":"pRPLC_Y7_KV_E","RAW_FILE_NAME":"nRPLC_Y7_KV_E"}
},
{
"Subject ID":"Ast.PreD.O.4",
"Sample ID":"AO3_KV_E",
"Factors":{"Stage":"pre diapause old","Species":"A. striatum"},
"Additional sample data":{"Number of Embryos":"25","Total Protein Abundance (ug)":"1161","RAW_FILE_NAME":"pRPLC_AO3_KV_E","RAW_FILE_NAME":"nRPLC_AO3_KV_E"}
}
],
"COLLECTION":{"COLLECTION_SUMMARY":"For each stage in each species, roughly 25-30 embryos were carefully dissected in ice-cold PBS using biological-grade tweezers (Electron Microscopy Sciences, 72700-D) to carefully remove the chorion, the enveloping layer, and the yolk without damaging the embryo body. Freshly dissected embryos were then quickly rinsed with ice-cold PBS, and all the PBS was carefully removed. Embryo bodies were then snap-frozen in liquid nitrogen and stored at -80°C. A total of 25-30 embryos for lipidomics.","SAMPLE_TYPE":"Embryo"},

"TREATMENT":{"TREATMENT_SUMMARY":"N/A"},

"SAMPLEPREP":{"SAMPLEPREP_SUMMARY":"Embryos for each stage of diapause and development were isolated from African turquoise and red-striped killifish (3-4 replicates for each stage) and lipid profiling was performed as previously described (PMID: 30532037, PMID: 32612231). Lipids were extracted in a randomized order via biphasic separation with cold methyl tert-butyl ether (MTBE), methanol and water. Briefly, 260 μl of methanol and 40 μl of water were added to the embryos and vortexed for 20 s. A lipid internal standard mixture was spiked in each sample (EquiSPLASH LIPIDOMIX, Avanti Polar Lipids (cat #: 330731), and d17-Oleic acid, Cayman chemicals (cat #: 9000432) to control for extraction efficiency, evaluate LC-MS performance and normalize LC-MS data. Samples were diluted with 1,000 μl of MTBE, vortexed for 10 s, sonicated for 30 s three times in a water bath, and incubated under agitation for 30 min at 4°C. After addition of 250 μl of water, the samples were vortexed for 1 min and centrifuged at 14,000g for 5 min at 20°C. The upper phase containing the lipids was collected and dried down under nitrogen. The dry extracts were reconstituted with 150 μl of 9:1 methanol:toluene."},

"CHROMATOGRAPHY":{"CHROMATOGRAPHY_SUMMARY":"Lipid extracts were analyzed in a randomized order using an Ultimate 3000 RSLC system coupled with a Q Exactive mass spectrometer (Thermo Fisher Scientific) as previously described (PMID: 32612231). Each sample was run twice in positive and negative ionization modes and lipids were separated using an Accucore C30 column 2.1 x 150 mm, 2.6 μm (Thermo Fisher Scientific) and mobile phase solvents consisted in 10 mM ammonium acetate and 0.1% formic acid in 60/40 acetonitrile/water (A) and 10 mM ammonium acetate and 0.1% formic acid in 90/10 isopropanol/acetonitrile (B). The gradient profile used was 30% B for 3 min, 30–43% B over 5 min, 43–50% B over 1 min, 55–90% B over 9 min, 90-99% B over 9 min and 99% B for 5 min. Lipids were eluted from the column at 0.2 mL/min, the oven temperature was set at 30°C, and the injection volume was 5 μL. Autosampler temperature was set at 15°C to prevent lipid aggregation.","CHROMATOGRAPHY_TYPE":"Reversed phase","INSTRUMENT_NAME":"Thermo Dionex Ultimate 3000 RS","COLUMN_NAME":"Thermo Accucore 2.1 x 150 mm, 2.6 μm"},

"ANALYSIS":{"ANALYSIS_TYPE":"MS"},

"MS":{"INSTRUMENT_NAME":"Thermo Q Exactive Orbitrap","INSTRUMENT_TYPE":"Orbitrap","MS_TYPE":"ESI","ION_MODE":"NEGATIVE","MS_COMMENTS":"LC-MS peak extraction, alignment, quantification and annotation was performed using LipidSearch software version 4.2.21 (Thermo Fisher Scientific). Lipids were identified by matching the precursor ion mass to a database and the experimental MS/MS spectra to a spectral library containing theoretical fragmentation spectra. To reduce the risk of misidentification, MS/MS spectra from lipids of interest were validated as follows: 1) both positive and negative mode MS/MS spectra match the expected fragments, 2) the main lipid adduct forms detected in positive and negative modes agree with the lipid class identified, 3) the retention time is compatible with the lipid class identified and 4) the peak shape is acceptable. The fragmentation pattern of each lipid class was experimentally validated using lipid internal standards. Single-point internal standard calibrations were used to estimate absolute concentrations for 431 unique lipids belonging to 14 classes using one internal standard for each lipid class. Importantly, we ensured linearity within the range of detected endogenous lipids using serial dilutions of internal standards spanning 4 orders of magnitude. Subsequently, median normalization (excluding TG and DG) was employed on lipid molar concentrations to correct for differential quantity of starting material. The normalized lipid intensities were well correlated with protein abundances measured using BCA Protein Assay Kit (Pierce, cat# 23225) suggesting good sample quality. One development (diapause escape) sample had an unexpectedly low protein concentration and thus was discarded. Lipid molar concentrations for a given class were calculated by summing individual lipid species molar concentrations belonging to that class. Fatty acid composition analysis was performed in each lipid class. Fatty acid composition was calculated by taking the ratio of the sum molar concentration of a given fatty acid over the sum molar concentration across fatty acids found in the lipids of the class. Subsequently, saturated fatty acids (SFA), mono-unsaturated fatty acids (MUFA) and poly-unsaturated fatty acids (PUFA) were grouped together for comparative analysis.","MS_RESULTS_FILE":"ST001898_AN003084_Results.txt UNITS:MS count Has m/z:Yes Has RT:Yes RT units:Minutes"}

}