This study highlights the advantages of a multi-omics approach to the
characterization of networks and pathways existing in a cell or tissue. The human placenta is a difficult organ to study at the early stages because few samples are available before
delivery. Understanding placental development is, however, crucial, as the placenta exerts maternal effects on the fetus that may be carried for life, and defects in its function can have devastating effects on maternal health. There remains a need for biomarkers and drug targets for the discovery and treatment of placental disorders.
Our analysis is a step forward in that direction. Using mouse placenta, we generated the largest reported placenta proteome and phosphoproteome, which allowed us to discover potentially novel genes and phosphosites that may play a role in critical placental processes. We also identified processes parallel to those found in previous literature that furthered our understanding of murine placental development, which is an established model for human placentation. This information could not have been obtained from any of the individual datasets alone with the same confidence and not at all in some cases, emphasizing the benefits of incorporating different levels of gene expression regulation.
We acknowledge that our study has limitations. For example, bias in the proteome due to technical limitations of proteomic technology; noise in the datasets due the cellular heterogeneity of the placenta; lack of strong evidence due to absence of extensive bioinformatic, statistical and experimental analyses; and possibly distorted findings due to the use of published transcriptome data rather than generating our own.
To overcome the limitations, future directions include incorporating single-cell multi- omics analyses to reduce noise. Analyzing invasive TGCs (Prl3d1+ cells) in both timepoints has the potential to reveal an unprecedented level of information regarding trophoblast invasion, for example. In addition, a stronger experimental design with more biological replicates will allow for more extensive statistical and bioinformatic analysis, such as coexpression analysis and the construction of gene regulatory networks. Finally, experimental validation is an essential aspect of future directions. Mouse knockouts of the genes identified in this study should be investigated for placental abnormalities associated with their putative function. In addition, the cell culture system and Matrigel invasion assays can be utilized to test for the impact of knocking down or overexpressing genes on invasive potential. The phosphosites can be mutated to phosphomimetic sites to validate and investigate the role of the sites in placental development. Finally, these findings should also be validated in human cell lines and placentas.
In summary, we utilized a systems-level integrative approach at two crucial timepoints of murine placentation to uncover regulatory differences between the timepoints. We identified genes that may be novel participants in invasion or nutrient transport, and novel phosphosites that may regulate the function of critical placental transcription factors.
ABBREVIATIONS
DEP Differentially Expressed Protein DET Differentially Expressed Transcript DEpP Differentially Expressed Phosphoprotein
p.c. post coitum
e embryonic day
IUGR Intrauterine Growth Restriction EVT Extravillous Trophoblast TGC Trophoblast Giant Cell
TGFβ Transforming Growth Factor Beta FGF4 Fibroblast Growth Factor 4 MMP Matrix Metalloproteinase
iEVT interstitial Extravillous Trophoblast enEVT endovascular Extravillous Trophoblast
TF Transcription Factor
hCG human Chorionic Gonadotropin
RNA Ribonucleic Acid
DNA Deoxyribonucleic Acid
ECM Extracellular Matrix
p-TGC parietal Trophoblast Giant Cell
TSC Trophoblast Stem Cell
ch-TGC channel Trophoblast Giant Cell spA-TGC spiral Artery Trophoblast Giant Cell
c-TGC canal Trophoblast Giant Cell s-TGC sinusoidal Trophoblast Giant Cell mRNA messenger Ribonucleic Acid RNA-seq Ribonucleic Acid sequencing
LC-MS/MS Liquid Chromatography Tandem Mass Spectrometry
cDNA complimentary DNA
iTRAQ isobaric Tag for Relative and Absolute Quantitation
TMT Tandem Mass Tag
MS Mass spectrometry
SILAC Stable Isotope Labelling by Amino acids in Cell culture QTL Quantitative Trait Loci
ART Assisted Reproductive Technology IVF In-Vitro Fertilization
FDR False Discovery Rate
FPKM Fragments Per Kilobase Mapped DEG Differentially Expressed Gene GOBP Gene Ontology Biological Process
EPC Ectoplacental Cone
IAA Iodoacetamide
ABC Ammonium Bicarbonate
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APPENDIX. GENE ONTOLOGY TERMS
Table A1. List of Gene Ontology Biological Process non-redundant terms for e7.5 common DEPs and DETs
geneset description PValue FDR
GO:0061458 reproductive system development 1.67E-07 0.000119
GO:0001667 ameboidal-type cell migration 4.27E-07 0.000152
GO:0051271 negative regulation of cellular component movement
1.24E-06 0.000296
GO:0040013 negative regulation of locomotion 2.27E-06 0.000405
GO:0001701 in utero embryonic development 3.28E-06 0.000468
GO:0048514 blood vessel morphogenesis 1.10E-05 0.001177
GO:0090130 tissue migration 1.15E-05 0.001177
GO:0048771 tissue remodeling 1.32E-05 0.00118
GO:0031589 cell-substrate adhesion 2.09E-05 0.001661
GO:0007015 actin filament organization 2.57E-05 0.001833
GO:0002064 epithelial cell development 3.70E-05 0.002399
GO:0009611 response to wounding 6.14E-05 0.003599
GO:0035239 tube morphogenesis 7.03E-05 0.003599
GO:0044706 multi-multicellular organism process 7.06E-05 0.003599
GO:0050673 epithelial cell proliferation 7.83E-05 0.003729
GO:0051235 maintenance of location 0.000132 0.005903
GO:0032102 negative regulation of response to external stimulus 0.000147 0.006173
GO:0002009 morphogenesis of an epithelium 0.000159 0.006313
GO:1901342 regulation of vasculature development 0.000184 0.006912
GO:0034330 cell junction organization 0.000276 0.009499
GO:0010638 positive regulation of organelle organization 0.000279 0.009499
GO:0048638 regulation of developmental growth 0.000305 0.009892
GO:0032970 regulation of actin filament-based process 0.000324 0.010036
GO:0016049 cell growth 0.000337 0.010036
GO:0042330 taxis 0.0004 0.011435
GO:0001503 ossification 0.000452 0.01234
GO:0007163 establishment or maintenance of cell polarity 0.000467 0.01234
GO:0040017 positive regulation of locomotion 0.000484 0.01234
GO:0048568 embryonic organ development 0.000577 0.013935
GO:0008637 apoptotic mitochondrial changes 0.000594 0.013935
GO:0090066 regulation of anatomical structure size 0.000605 0.013935
GO:0042476 odontogenesis 0.000872 0.019458
GO:0051272 positive regulation of cellular component movement
0.000963 0.020829
GO:0043112 receptor metabolic process 0.001166 0.024279
GO:0045927 positive regulation of growth 0.00119 0.024279
GO:0051493 regulation of cytoskeleton organization 0.001226 0.024314
GO:0071526 semaphorin-plexin signaling pathway 0.00128 0.0247
GO:0002697 regulation of immune effector process 0.001359 0.024877 GO:0044089 positive regulation of cellular component
biogenesis
0.001583 0.028252
GO:0071496 cellular response to external stimulus 0.001855 0.032305
GO:0031214 biomineral tissue development 0.002071 0.035204
GO:0002521 leukocyte differentiation 0.002413 0.040073
GO:0021675 nerve development 0.002527 0.040608
GO:0044723 single-organism carbohydrate metabolic process 0.002559 0.040608
GO:0010035 response to inorganic substance 0.00302 0.046869
GO:0001763 morphogenesis of a branching structure 0.003222 0.048941
GO:0002526 acute inflammatory response 0.003349 0.049702
GO:0007229 integrin-mediated signaling pathway 0.003429 0.049702
GO:0048863 stem cell differentiation 0.003481 0.049702
Table A2. List of Gene Ontology Biological Process non-redundant terms for e9.5 common DEPs and DETs
geneset description PValue FDR
GO:0016042 lipid catabolic process 3.19E-07 0.000228
GO:0008202 steroid metabolic process 1.04E-06 0.000372
GO:0097164 ammonium ion metabolic process 1.14E-05 0.00272
GO:0051180 vitamin transport 8.01E-05 0.014293
GO:0006898 receptor-mediated endocytosis 0.000183 0.024826
GO:0030258 lipid modification 0.000209 0.024826
GO:0097006 regulation of plasma lipoprotein particle levels 0.000327 0.030772
GO:0044282 small molecule catabolic process 0.000345 0.030772
GO:0035272 exocrine system development 0.000536 0.042495
Table A3. List of Gene Ontology Biological Process non-redundant terms for e7.5 exclusive DEPs
geneset description PValue FDR
GO:0050817 coagulation 2.08E-14 1.48E-11
GO:0050878 regulation of body fluid levels 1.94E-10 5.57E-08
GO:0009611 response to wounding 2.34E-10 5.57E-08
GO:0006790 sulfur compound metabolic process 8.34E-08 1.49E-05
GO:0007229 integrin-mediated signaling pathway 3.90E-07 5.57E-05
GO:0034109 homotypic cell-cell adhesion 2.54E-06 0.000302
GO:0010035 response to inorganic substance 4.10E-06 0.000418
GO:0031589 cell-substrate adhesion 7.44E-06 0.000664
GO:0072593 reactive oxygen species metabolic process 1.16E-05 0.000923
GO:0006091 generation of precursor metabolites and energy
1.42E-05 0.000959 GO:0006575 cellular modified amino acid metabolic
process
1.48E-05 0.000959
GO:0006631 fatty acid metabolic process 1.80E-05 0.001072
GO:0046486 glycerolipid metabolic process 2.32E-05 0.001274
GO:0072350 tricarboxylic acid metabolic process 3.21E-05 0.001639
GO:0002237 response to molecule of bacterial origin 7.38E-05 0.003292
GO:0048871 multicellular organismal homeostasis 8.07E-05 0.003391
GO:0006979 response to oxidative stress 9.41E-05 0.003598
GO:0048872 homeostasis of number of cells 9.68E-05 0.003598
GO:0051186 cofactor metabolic process 0.000101 0.003598
GO:1901565 organonitrogen compound catabolic process 0.000143 0.004866
GO:0009636 response to toxic substance 0.000237 0.007689