Multi-Omics analysis of the developing Lung
Geremy Clair
1 Fertilization
2 … initiates the developmental process…
3 …of an incredibly complex system
4 The Lung is incredibly complex
DNA RNA ProteinsLipids Metabolites
5 The LungMAP
RNA ProteinsLipids Metabolites Multiple spatial and temporal resolutions
E9.5 Wk4 Pseudoglandular E16.5 Wk17 Canalicular E17.5 Wk26 Saccular PND5 Wk36 Alveolar PND28 8 Years
Fluorescence and Mass spectrometry imaging 6 Transcription and translation from the textbooks
Genes
If there was only transcription mRNA and translation controlling protein abundance : the transcripts/proteins correlation would be very high. Proteins
7 Transcription and translation the real world
Epigenetic Control Nucleus Chromatin (DNA‐Met/Acetyl) remodeling
Protein turnover Genes
Proteic transcriptional regulations miRNA Transcription PTMs Splicing miRNA miRNA transport mRNA
Transport Cytoplasm Translation mRNA siRNA maturation Protein maturation RE Proteins Protein transport Translational siRNA Ouside Cell regulation 8 Anti-correlated mRNA and proteins during lung development
mRNA Protein
E16.5
E18.5
PND5
PND28
Protein and mRNA
abundance 9 Low High Post-transcriptional regulated functions
mRNA Protein
Functional enrichment : Post‐transcriptionally regulated processes
E16.5 Immune system process Lipid metabolic process Oxidation‐reduction process E18.5 Metabolic process Translation PND5 mRNA processing Covalent chromatin modification PND28 …
Protein and mRNA
abundance 10 Low High The lung proteome is extensively remodeled during normal development
8,932 proteins → 8,171 quantifiable → 5,515 significantly changing in abundance (68%)
E16.5
E18.5
PND5
PND28
11 Transcription regulators and signaling molecules orchestrate the development
1,569 transcription/translation regulators E16.5 and proteins involved in signaling
E18.5
PND5
PND28
12 Some TR and SM are potentially highly involved in the proteome remodeling
CLR unsupervised clustering method allowed us to identify 4 modules of transcriptions factors and signaling molecule that appear to be implicated in the global proteome remodeling
13 Lung Heterogeneity
Mouse E16.5 NKX2‐1 Acta2 Cspg4 DAPI
14 > 40 cell types
Mouse E16.5 NKX2‐1 Acta2 Cspg4 DAPI
15 rocklab.ucsf.edu Blending and averaging effects in omics
16 LCM
Isolated alveolar parenchyma
E16.5
PND7
PND28
17 Clair et al. Scientific report 2016. LCM and nano-proteomics
Isolated alveolar parenchyma
E16.5
PND7
PND28
18 Clair et al. Scientific report 2016. LCM and nano-proteomics (Optimal from 2,000 to 8,000 cells)
Isolated alveolar parenchyma
E16.5
PND7
PND28
Optimal after 2,000 cells
19 Clair et al. Scientific report 2016. Alveolar tissue proteomics
PCA
E16.5
PND7 Segregation of the biological replicates PND28 by time‐points
20 Clair et al. Scientific report 2016. Alveolar tissue proteomics during development
PCA >3,400 identified proteins
E16.5
PND7 Segregation of the biological replicates PND28 by time‐points
21 Clair et al. Scientific report 2016. Alveolar tissue proteomics during development
PCA >3,400 identified proteins
E16.5
PND7 Segregation of the biological replicates PND28 by time‐points
22 Clair et al. Scientific report 2016. > 300 low abundance regulatory molecules modulated during the alveolarization process
E16.5
PND7
PND28
23 Clair et al. Scientific report 2016. Sorting lung cell population
3 donors 20 month‐old Homogenization females
Polar metabolites Proteins Lipids
FACS cell sorting 24 Sorted-cells proteomics
PECAM GO BP: CD34 • Angiogenesis In line with : ICAM1 • ECM organization physiological role in ICAM2 • Cell‐cell adhesion angiogenesis and CADH5 • VEGF receptor signaling vascular permeability VWF pathway
EPCAM GO BP: 3 donors SP‐A In line with : • Lipid biosynthetic process ABCA3 physiological role 20 month‐old • Fatty acid metabolic process Muc1 • Oxidation‐reduction in surfactant females CDH1 • Translation production
PDGRFB
GO BP: CD45 • Innate immune response TLR8 • Inflammatory response FCERG In line with : • Apoptotic process MCR1 physiological role in • Fc‐gamma receptor signaling MRC2 pathway involved in immune responses MSRE phagocytosis MCEM1 IL‐16
25 Surface antigens in our proteomics data
Our data contains a lot of surface antigens that could be use to further sort sub‐cell types
26 Surface antigens enriched in epithelial cells
Our data contains a lot of surface antigens that could be use to further sort sub‐cell types
27 Surface antigens enriched in epithelial cells could help to further sort sub-populations
Our data contains a lot of surface antigens that could be use to further sort sub‐cell types
LAMP3/CD208 DAF/CD55
28 Human tissue imaging from : www.proteinatlas.org Lipidomics of sorted cells
3 donors 20 month‐old females
299 lipids identified across 21 subclasses
29 Lipidomics of sorted cells
3 donors 20 month‐old females
299 lipids identified across 21 subclasses
30 Surfactant lipids are enriched in EPI population
PC(16:0/16:0) PC(16:0/14:0)
3 donors 20 month‐old females
PG(16:0/16:0) PG(16:0/14:0)
299 lipids identified across 21 subclasses
31 Sphingolipids origin in lungs
32 Sphingolipids origin in lungs
33 Mass spectrometry imaging (Nano-DESI)
PC(16:0/16:0) Surfactant lipid m/z 756.5520
• Images hundreds of lipids and metabolites simultaneously PC(16:0/22:6) • No sample pre‐treatment Structural? • High spatial resolution (~10m) m/z 828.5520 • Accurate quantification
34 PNNL in LungMAP consortium
>10,000 proteins >800 lipids >150 polar metabolites Developmental Alveolar tissue expression expression
Organ specificity Cell specificity Sub‐cellular localization
35 ABCA3 responsible of pediatric lung disease
36 LungMAP ABCA3 protein ID Card
ABCA3 organ specificity (m.) ABCA3 Sub‐cellular localization (m.)
Cyto Cyto Mito Mito Nuc Nuc insol. sol. & LB & LB insol. Sol. insol. sol. ABCA3 Cell‐type specificity (h.) ABCA3 whole lung expression (m. & h.) IF imaging
mRNA expression
Etc.
37 MS-LungMAP website
38 Acknowledgments
Single Cell Proteomics, PNNL LungMAP Transcriptome Confocal Metabolomics, Lipidomics, Awesome team! Imaging HT‐ISH, MS Imaging Coordinating Center Duke & RTI
Sponsor CCHMC: PNNL & TACC Cincinnati Richland & Austin Charles Ansong
Human Lung Tissue 3‐D Systems Jennifer Kyle Procurement Imaging Biology Young‐Mo Kim Paul Piehowski Ahmed Mohieb Son Nguyen Sydney Dautel Erika Zink Ryan Sontag Julia Laskin URMC: Saban Inst. UAB & Yale Jason McDermott Rochester CHLA & USC & Pitt & UCSD Richard Corley
June 20, 2017 39