Summary of Study ST001898
This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR001194. The data can be accessed directly via it's Project DOI: 10.21228/M8CD8G This work is supported by NIH grant, U2C- DK119886.
See: https://www.metabolomicsworkbench.org/about/howtocite.php
This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.
| Study ID | ST001898 |
| 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 |
| kcontrep@stanford.edu | |
| Phone | 6506664538 |
| Submit Date | 2021-08-05 |
| Raw Data Available | Yes |
| Raw Data File Type(s) | raw(Thermo) |
| Analysis Type Detail | LC-MS |
| Release Date | 2022-12-15 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
Project:
| Project ID: | PR001194 |
| Project DOI: | doi: 10.21228/M8CD8G |
| 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 |
Subject:
| Subject ID: | SU001976 |
| Subject Type: | Fish |
| Subject Species: | Nothobranchius furzeri;Aphyosemion striatum |
Factors:
Subject type: Fish; Subject species: Nothobranchius furzeri;Aphyosemion striatum (Factor headings shown in green)
| mb_sample_id | local_sample_id | Stage | Species |
|---|---|---|---|
| SA176108 | M27_KV_E | 1 month diapause | N. furzeri |
| SA176109 | M28_KV_E | 1 month diapause | N. furzeri |
| SA176110 | M29_KV_E | 1 month diapause | N. furzeri |
| SA176111 | MA_M2_E | 1 month diapause | N. furzeri |
| SA176112 | M4_D6_E | 6 day diapause | N. furzeri |
| SA176113 | M5_D6_E | 6 day diapause | N. furzeri |
| SA176114 | M19_D6_E | 6 day diapause | N. furzeri |
| SA176115 | M18_D6_E | 6 day diapause | N. furzeri |
| SA176116 | M13_DEV_E | development | N. furzeri |
| SA176117 | M10_DEV_E | development | N. furzeri |
| SA176118 | M24_DEV_E | development | N. furzeri |
| SA176119 | M21_DEV_E | development | N. furzeri |
| SA176120 | M7_EX_E | diapause exit | N. furzeri |
| SA176121 | M9_EX_E | diapause exit | N. furzeri |
| SA176122 | M12_EX_E | diapause exit | N. furzeri |
| SA176123 | M8_EX_E | diapause exit | N. furzeri |
| SA176124 | AO10_KV_E | pre diapause old | A. striatum |
| SA176125 | AO8_KV_E | pre diapause old | A. striatum |
| SA176126 | AO13_KV_E | pre diapause old | A. striatum |
| SA176127 | AO3_KV_E | pre diapause old | A. striatum |
| SA176128 | O11_KV_E | pre diapause old | N. furzeri |
| SA176129 | O8_KV_E | pre diapause old | N. furzeri |
| SA176130 | O13_KV_E | pre diapause old | N. furzeri |
| SA176131 | O9_KV_E | pre diapause old | N. furzeri |
| SA176132 | AY10_KV_E | pre diapause young | A. striatum |
| SA176133 | AY2_KV_E | pre diapause young | A. striatum |
| SA176134 | AY1_KV_E | pre diapause young | A. striatum |
| SA176135 | AY3_KV_E | pre diapause young | A. striatum |
| SA176136 | Y9_KV_E | pre diapause young | N. furzeri |
| SA176137 | Y8_KV_E | pre diapause young | N. furzeri |
| SA176138 | Y7_KV_E | pre diapause young | N. furzeri |
| SA176139 | Y6_KV_E | pre diapause young | N. furzeri |
| Showing results 1 to 32 of 32 |
Collection:
| Collection ID: | CO001969 |
| 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 ID: | TR001988 |
| Treatment Summary: | N/A |
Sample Preparation:
| Sampleprep ID: | SP001982 |
| 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. |
Combined analysis:
| Analysis ID | AN003083 | AN003084 |
|---|---|---|
| Analysis type | MS | MS |
| Chromatography type | Reversed phase | Reversed phase |
| Chromatography system | Thermo Dionex Ultimate 3000 RS | Thermo Dionex Ultimate 3000 RS |
| Column | Thermo Accucore (150 x 2.1mm,2.6um) | Thermo Accucore (150 x 2.1mm,2.6um) |
| MS Type | ESI | ESI |
| MS instrument type | Orbitrap | Orbitrap |
| MS instrument name | Thermo Q Exactive Orbitrap | Thermo Q Exactive Orbitrap |
| Ion Mode | POSITIVE | NEGATIVE |
| Units | MS count | MS count |
Chromatography:
| Chromatography ID: | CH002277 |
| 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. |
| Instrument Name: | Thermo Dionex Ultimate 3000 RS |
| Column Name: | Thermo Accucore (150 x 2.1mm,2.6um) |
| Column Temperature: | 30 |
| Flow Gradient: | 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. |
| Flow Rate: | 0.2 mL/min |
| Injection Temperature: | 15 |
| Sample Injection: | 5ul |
| Solvent A: | 60% acetonitrile/40% water; 0.1% formic acid;10 mM ammonium acetate |
| Solvent B: | 90% isopropanol/10% acetonitrile; 0.1% formic acid;10 mM ammonium acetate |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS002865 |
| Analysis ID: | AN003083 |
| Instrument Name: | Thermo Q Exactive Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| 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. |
| Ion Mode: | POSITIVE |
| MS ID: | MS002866 |
| Analysis ID: | AN003084 |
| Instrument Name: | Thermo Q Exactive Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| 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. |
| Ion Mode: | NEGATIVE |
