{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST002506","ANALYSIS_ID":"AN004128","VERSION":"1","CREATED_ON":"March 20, 2023, 7:35 am"},

"PROJECT":{"PROJECT_TITLE":"Lactate-dependent Transcriptional Regulation Controls Mammalian Eye Morphogenesis","PROJECT_SUMMARY":"Mammalian retinal metabolism favors aerobic glycolysis. However, the role of glycolytic metabolism in retinal morphogenesis remains unknown. Here we report that aerobic glycolysis is necessary for the early stages of retinal development. Taking advantage of an unbiased approach that combines the use of eye organoids and single-cell RNA sequencing we identified specific glucose transporters and glycolytic genes in retinal progenitors. Next, we determined that the optic vesicle territory of mouse embryos displays elevated levels of glycolytic activity. At the functional level, we found that removal of Glut1 (also known as Slc2a1) and Lactate dehydrogenase A (Ldha) gene activity from developing retinal progenitors arrested eye morphogenesis. Surprisingly, we uncovered that lactate-mediated upregulation of key eye-field transcription factors was controlled by the epigenetic modification of histone H3 acetylation through histone deacetylase (Hdac) activity. Our results identify a novel bioenergetic independent role of lactate as a signaling molecule necessary for mammalian eye morphogenesis.","INSTITUTE":"Northwestern University","LAST_NAME":"TAKATA","FIRST_NAME":"NOZOMU","ADDRESS":"303 East Superior Street, 10-220, Chicago, Illinois, 60611, USA","EMAIL":"nozomu.takata@northwestern.edu","PHONE":"13125036066"},

"STUDY":{"STUDY_TITLE":"Natural abundance of isotopic metabolite detection in mouse eye orgnaoids.","STUDY_SUMMARY":"Because the natural abundance of isotopic metabolites in the early eye organoids has not yet been reported, we have performed LC-MS/MS without exogenous isotopic labeling to show the natural abundance of isotopic carbons. Cell Name AES0145 : Rx-GFP K/I EB5 (RIKEN Cell Bank): Organoid method (PMID: 21475194).","INSTITUTE":"Northwestern University","LAST_NAME":"TAKATA","FIRST_NAME":"NOZOMU","ADDRESS":"303 East Superior Street, 10-220","EMAIL":"nozomu.takata@northwestern.edu","PHONE":"3125036066"},

"SUBJECT":{"SUBJECT_TYPE":"Mammal","SUBJECT_SPECIES":"Mus musculus","TAXONOMY_ID":"10090"},
"SUBJECT_SAMPLE_FACTORS":[
{
"Subject ID":"-",
"Sample ID":"Natural_abundance1",
"Factors":{"Genotype":"Wild-type","Treatment":"Control"},
"Additional sample data":{"RAW_FILE_NAME":"Natural_abundance-biological_independent1.raw"}
},
{
"Subject ID":"-",
"Sample ID":"Natural_abundance2",
"Factors":{"Genotype":"Wild-type","Treatment":"Control"},
"Additional sample data":{"RAW_FILE_NAME":"Natural_abundance-biological_independent2.raw"}
},
{
"Subject ID":"-",
"Sample ID":"Natural_abundance3",
"Factors":{"Genotype":"Wild-type","Treatment":"Control"},
"Additional sample data":{"RAW_FILE_NAME":"Natural_abundance-biological_independent3.raw"}
}
],
"COLLECTION":{"COLLECTION_SUMMARY":"Collect embryonic stem cell derived eye organoids without isotopic labeling. Three biologically independent repeated samples were collected.","COLLECTION_PROTOCOL_FILENAME":"Glucose_and_lactate_isotope_labeling.pdf","SAMPLE_TYPE":"Retina"},

"TREATMENT":{"TREATMENT_SUMMARY":"No treatment was perfomed."},

"SAMPLEPREP":{"SAMPLEPREP_SUMMARY":"The eye organoids were collected and washed twice with PBS before pellets were flash-frozen and stored at −80°C until metabolite extraction. Metabolite analysis was performed as described previously (PMID: 33931446). Briefly, for metabolite extraction, 80% methanol was used followed by the rapid freeze-thaw method to break the tissues. The supernatant underwent speedvac drying. The samples were prepared in 80% acetonitrile and were analyzed by High-Resolution Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS)."},

"CHROMATOGRAPHY":{"CHROMATOGRAPHY_TYPE":"HILIC","INSTRUMENT_NAME":"Q-exactive","COLUMN_NAME":"Waters XBridge BEH Amide (100 x 3.0mm, 3.5um)","SOLVENT_A":"95% water/5% acetonitrile; 10 mM ammonium hydroxide; 10 mM ammonium acetate, pH 9.0","SOLVENT_B":"100% acetonitrile","FLOW_GRADIENT":"0 min, 15% A; 2.5 min, 30% A; 7 min, 43% A; 16 min, 62% A; 16.1-18 min, 75% A; 18-25 min, 15% A with a flow rate of 150 μL/min","FLOW_RATE":"150 μL/mi","COLUMN_TEMPERATURE":"40"},

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

"MS":{"INSTRUMENT_NAME":"Thermo Q Exactive Orbitrap","INSTRUMENT_TYPE":"Orbitrap","MS_TYPE":"ESI","ION_MODE":"UNSPECIFIED","MS_COMMENTS":"The capillary of the ESI source was set to 275 °C, with sheath gas at 35 arbitrary units, auxiliary gas at 5 arbitrary units and the spray voltage at 4.0 kV. In positive/negative polarity switching mode, an m/z scan range from 60 to 900 was chosen and MS1 data was collected at a resolution of 70,000. The automatic gain control (AGC) target was set at 1 × 106 and the maximum injection time was 200 ms. The top 5 precursor ions were subsequently fragmented, in a data-dependent manner, using the higher energy collisional dissociation (HCD) cell set to 30% normalized collision energy in MS2 at a resolution power of 17,500. Besides matching m/z, metabolites are identified by matching either retention time with analytical standards and/or MS2 fragmentation pattern. Data acquisition and analysis were carried out by Xcalibur 4.1 software and Tracefinder 4.1 software, respectively (both from Thermo Fisher Scientific)"},

"MS_METABOLITE_DATA":{
"Units":"Peak area",

"Data":[{"Metabolite":"Total Ion Count","Natural_abundance1":"400443050025","Natural_abundance2":"304853183842","Natural_abundance3":"512026697581"},{"Metabolite":"1,3-diphosphoglycerate","Natural_abundance1":"0","Natural_abundance2":"0","Natural_abundance3":"0"},{"Metabolite":"1,3-diphosphoglycerate_13C1","Natural_abundance1":"0","Natural_abundance2":"0","Natural_abundance3":"0"},{"Metabolite":"1,3-diphosphoglycerate_13C2","Natural_abundance1":"0","Natural_abundance2":"0","Natural_abundance3":"0"},{"Metabolite":"1,3-diphosphoglycerate_13C3","Natural_abundance1":"0","Natural_abundance2":"0","Natural_abundance3":"0"},{"Metabolite":"2,3-diphosphoglycerate","Natural_abundance1":"20639","Natural_abundance2":"0","Natural_abundance3":"0"},{"Metabolite":"2,3-diphosphoglycerate_13C1","Natural_abundance1":"0","Natural_abundance2":"0","Natural_abundance3":"0"},{"Metabolite":"2,3-diphosphoglycerate_13C2","Natural_abundance1":"0","Natural_abundance2":"0","Natural_abundance3":"0"},{"Metabolite":"2,3-diphosphoglycerate_13C3","Natural_abundance1":"0","Natural_abundance2":"0","Natural_abundance3":"0"},{"Metabolite":"2/3-phosphoglycerate","Natural_abundance1":"0","Natural_abundance2":"15488","Natural_abundance3":"0"},{"Metabolite":"2/3-phosphoglycerate_13C1","Natural_abundance1":"0","Natural_abundance2":"0","Natural_abundance3":"0"},{"Metabolite":"2/3-phosphoglycerate_13C2","Natural_abundance1":"0","Natural_abundance2":"0","Natural_abundance3":"0"},{"Metabolite":"2/3-phosphoglycerate_13C3","Natural_abundance1":"0","Natural_abundance2":"0","Natural_abundance3":"0"},{"Metabolite":"2-HG","Natural_abundance1":"1604679","Natural_abundance2":"69559931","Natural_abundance3":"642376"},{"Metabolite":"2-HG_13C1","Natural_abundance1":"0","Natural_abundance2":"3028143","Natural_abundance3":"0"},{"Metabolite":"2-HG_13C2","Natural_abundance1":"0","Natural_abundance2":"0","Natural_abundance3":"0"},{"Metabolite":"2-HG_13C3","Natural_abundance1":"163650","Natural_abundance2":"0","Natural_abundance3":"0"},{"Metabolite":"2-HG_13C4","Natural_abundance1":"0","Natural_abundance2":"0","Natural_abundance3":"0"},{"Metabolite":"2-HG_13C5","Natural_abundance1":"89070","Natural_abundance2":"100999","Natural_abundance3":"0"},{"Metabolite":"3-phospho-serine","Natural_abundance1":"0","Natural_abundance2":"135530","Natural_abundance3":"0"},{"Metabolite":"3-phospho-serine_13C1","Natural_abundance1":"0","Natural_abundance2":"0","Natural_abundance3":"0"},{"Metabolite":"3-phospho-serine_13C2","Natural_abundance1":"0","Natural_abundance2":"0","Natural_abundance3":"0"},{"Metabolite":"3-phospho-serine_13C3","Natural_abundance1":"0","Natural_abundance2":"0","Natural_abundance3":"0"},{"Metabolite":"6-phospho-D-gluconate","Natural_abundance1":"0","Natural_abundance2":"0","Natural_abundance3":"0"},{"Metabolite":"6-phospho-D-gluconate_13C1","Natural_abundance1":"0","Natural_abundance2":"0","Natural_abundance3":"0"},{"Metabolite":"6-phospho-D-gluconate_13C2","Natural_abundance1":"0","Natural_abundance2":"0","Natural_abundance3":"0"},{"Metabolite":"6-phospho-D-gluconate_13C3","Natural_abundance1":"0","Natural_abundance2":"0","Natural_abundance3":"0"},{"Metabolite":"6-phospho-D-gluconate_13C4","Natural_abundance1":"0","Natural_abundance2":"0","Natural_abundance3":"0"},{"Metabolite":"6-phospho-D-gluconate_13C5","Natural_abundance1":"0","Natural_abundance2":"0","Natural_abundance3":"0"},{"Metabolite":"6-phospho-D-gluconate_13C6","Natural_abundance1":"0","Natural_abundance2":"0","Natural_abundance3":"0"},{"Metabolite":"acetoacetate / succinic semialdehyde_13C1","Natural_abundance1":"0","Natural_abundance2":"0","Natural_abundance3":"0"},{"Metabolite":"acetoacetate / succinic semialdehyde_13C2","Natural_abundance1":"0","Natural_abundance2":"0","Natural_abundance3":"0"},{"Metabolite":"acetoacetate / succinic semialdehyde_13C3","Natural_abundance1":"0","Natural_abundance2":"0","Natural_abundance3":"0"},{"Metabolite":"acetoacetate / succinic semialdehyde_13C4","Natural_abundance1":"0","Natural_abundance2":"0","Natural_abundance3":"0"},{"Metabolite":"acetoacetic acid / succinic semialdehyde","Natural_abundance1":"0","Natural_abundance2":"0","Natural_abundance3":"0"},{"Metabolite":"acetyl-CoA +","Natural_abundance1":"0","Natural_abundance2":"70066","Natural_abundance3":"0"},{"Metabolite":"acetyl-CoA_13C1 +","Natural_abundance1":"0","Natural_abundance2":"0","Natural_abundance3":"0"},{"Metabolite":"acetyl-CoA_13C2 +","Natural_abundance1":"0","Natural_abundance2":"0","Natural_abundance3":"0"},{"Metabolite":"aconitic acid -","Natural_abundance1":"81103","Natural_abundance2":"21274331","Natural_abundance3":"0"},{"Metabolite":"aconitic acid_13C1 -","Natural_abundance1":"0","Natural_abundance2":"941451","Natural_abundance3":"0"},{"Metabolite":"aconitic acid_13C2 -","Natural_abundance1":"0","Natural_abundance2":"0","Natural_abundance3":"0"},{"Metabolite":"adenosine","Natural_abundance1":"562372","Natural_abundance2":"614479","Natural_abundance3":"897648"},{"Metabolite":"adenosine_13C10","Natural_abundance1":"0","Natural_abundance2":"0","Natural_abundance3":"0"},{"Metabolite":"adenosine_13C5","Natural_abundance1":"0","Natural_abundance2":"0","Natural_abundance3":"0"},{"Metabolite":"adenosine_13C6","Natural_abundance1":"0","Natural_abundance2":"0","Natural_abundance3":"0"},{"Metabolite":"adenosine_13C7","Natural_abundance1":"0","Natural_abundance2":"0","Natural_abundance3":"0"},{"Metabolite":"adenosine_13C8","Natural_abundance1":"0","Natural_abundance2":"0","Natural_abundance3":"0"},{"Metabolite":"adenosine_13C9","Natural_abundance1":"0","Natural_abundance2":"0","Natural_abundance3":"0"},{"Metabolite":"ADP / dGDP","Natural_abundance1":"1015665","Natural_abundance2":"3855359","Natural_abundance3":"225620"},{"Metabolite":"ADP / dGDP_13C1","Natural_abundance1":"0","Natural_abundance2":"0","Natural_abundance3":"0"},{"Metabolite":"ADP / dGDP_13C10","Natural_abundance1":"0","Natural_abundance2":"0","Natural_abundance3":"0"},{"Metabolite":"ADP / dGDP_13C2","Natural_abundance1":"0","Natural_abundance2":"0","Natural_abundance3":"0"},{"Metabolite":"ADP / dGDP_13C3","Natural_abundance1":"0","Natural_abundance2":"0","Natural_abundance3":"0"},{"Metabolite":"ADP / dGDP_13C4","Natural_abundance1":"0","Natural_abundance2":"0","Natural_abundance3":"0"},{"Metabolite":"ADP / dGDP_13C5","Natural_abundance1":"0","Natural_abundance2":"0","Natural_abundance3":"0"},{"Metabolite":"ADP / dGDP_13C6","Natural_abundance1":"4508325","Natural_abundance2":"996940","Natural_abundance3":"78327"},{"Metabolite":"ADP / dGDP_13C7","Natural_abundance1":"71506","Natural_abundance2":"66354","Natural_abundance3":"0"},{"Metabolite":"ADP / dGDP_13C8","Natural_abundance1":"0","Natural_abundance2":"0","Natural_abundance3":"0"},{"Metabolite":"ADP / dGDP_13C9","Natural_abundance1":"0","Natural_abundance2":"0","Natural_abundance3":"0"},{"Metabolite":"AICAR","Natural_abundance1":"0","Natural_abundance2":"0","Natural_abundance3":"0"},{"Metabolite":"AICAR_13C5","Natural_abundance1":"0","Natural_abundance2":"0","Natural_abundance3":"0"},{"Metabolite":"AICAR_13C6","Natural_abundance1":"0","Natural_abundance2":"0","Natural_abundance3":"0"},{"Metabolite":"AICAR_13C7","Natural_abundance1":"0","Natural_abundance2":"0","Natural_abundance3":"0"},{"Metabolite":"AICAR_13C8","Natural_abundance1":"0","Natural_abundance2":"0","Natural_abundance3":"0"},{"Metabolite":"AICAR_13C9","Natural_abundance1":"0","Natural_abundance2":"0","Natural_abundance3":"0"},{"Metabolite":"a-KG","Natural_abundance1":"12585829","Natural_abundance2":"109741621","Natural_abundance3":"1787218"},{"Metabolite":"a-KG_13C1","Natural_abundance1":"460663","Natural_abundance2":"5101652","Natural_abundance3":"0"},{"Metabolite":"a-KG_13C2","Natural_abundance1":"41759","Natural_abundance2":"42528","Natural_abundance3":"0"},{"Metabolite":"a-KG_13C3","Natural_abundance1":"0","Natural_abundance2":"0","Natural_abundance3":"0"},{"Metabolite":"a-KG_13C4","Natural_abundance1":"0","Natural_abundance2":"0","Natural_abundance3":"0"},{"Metabolite":"a-KG_13C5","Natural_abundance1":"0","Natural_abundance2":"0","Natural_abundance3":"0"},{"Metabolite":"alanine","Natural_abundance1":"287755183","Natural_abundance2":"355797865","Natural_abundance3":"107817154"},{"Metabolite":"alanine_13C1","Natural_abundance1":"8384848","Natural_abundance2":"8103127","Natural_abundance3":"2270232"},{"Metabolite":"alanine_13C2","Natural_abundance1":"0","Natural_abundance2":"0","Natural_abundance3":"0"},{"Metabolite":"alanine_13C3","Natural_abundance1":"0","Natural_abundance2":"0","Natural_abundance3":"0"},{"Metabolite":"AMP / dGMP","Natural_abundance1":"282938","Natural_abundance2":"1977943","Natural_abundance3":"76374"},{"Metabolite":"AMP / dGMP_13C1","Natural_abundance1":"0","Natural_abundance2":"0","Natural_abundance3":"0"},{"Metabolite":"AMP / dGMP_13C10","Natural_abundance1":"0","Natural_abundance2":"0","Natural_abundance3":"0"},{"Metabolite":"AMP / dGMP_13C2","Natural_abundance1":"57995","Natural_abundance2":"513533","Natural_abundance3":"0"},{"Metabolite":"AMP / dGMP_13C5","Natural_abundance1":"0","Natural_abundance2":"0","Natural_abundance3":"0"},{"Metabolite":"AMP / dGMP_13C6","Natural_abundance1":"0","Natural_abundance2":"0","Natural_abundance3":"0"},{"Metabolite":"AMP / dGMP_13C7","Natural_abundance1":"0","Natural_abundance2":"0","Natural_abundance3":"0"},{"Metabolite":"AMP / dGMP_13C8","Natural_abundance1":"0","Natural_abundance2":"0","Natural_abundance3":"0"},{"Metabolite":"AMP / dGMP_13C9","Natural_abundance1":"0","Natural_abundance2":"0","Natural_abundance3":"0"},{"Metabolite":"aspartate","Natural_abundance1":"94581191","Natural_abundance2":"137017738","Natural_abundance3":"97158509"},{"Metabolite":"aspartate_13C1","Natural_abundance1":"3923175","Natural_abundance2":"5145744","Natural_abundance3":"3584794"},{"Metabolite":"aspartate_13C2","Natural_abundance1":"336067","Natural_abundance2":"123296","Natural_abundance3":"98908"},{"Metabolite":"aspartate_13C3","Natural_abundance1":"3923175","Natural_abundance2":"5145744","Natural_abundance3":"3584794"},{"Metabolite":"aspartate_13C4","Natural_abundance1":"0","Natural_abundance2":"0","Natural_abundance3":"0"},{"Metabolite":"ATP / dGTP","Natural_abundance1":"255517","Natural_abundance2":"2828488","Natural_abundance3":"35651"},{"Metabolite":"ATP / dGTP_13C10","Natural_abundance1":"0","Natural_abundance2":"0","Natural_abundance3":"0"},{"Metabolite":"ATP / 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}

}