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Updated: Jul 23, 2025
Using Ex Vivo Upright Droplet Cultures of Whole Fetal Organs to Study Developmental Processes during Mouse Organogenesis
Published on: October 21, 2015
Spatiotemporal protein dynamics during early organogenesis in mouse conceptuses treated with valproic acid
Samantha Lapehn1, Justin A Colacino1, Craig Harris1
1Department of Environmental Health Sciences, University of Michigan, Ann Arbor, MI, United States.
Insights
Valproic acid (VPA) alters protein levels during early development, impacting pathways crucial for preventing neural tube defects (NTDs). This study reveals temporal protein changes in embryonic tissues, offering new insights into VPA
Area of Science:
- Developmental Biology
- Proteomics
- Toxicology
Background:
- Valproic acid (VPA) is an anti-epileptic drug linked to increased neural tube defect (NTD) risk.
- Prior research on VPA's teratogenic mechanisms overlooked temporal protein abundance changes.
- The visceral yolk sac (VYS) and embryo proper (EMB) are critical for early development and potential targets of VPA toxicity.
Purpose of the Study:
- To investigate the impact of VPA exposure on protein abundance in the mouse VYS and EMB during early organogenesis.
- To identify specific protein pathways affected by VPA that may contribute to NTDs.
- To analyze the temporal dynamics of protein alterations following VPA exposure.
Main Methods:
- Mouse whole embryo culture was used to expose embryos to VPA.
- Tandem mass tag (TMT) labeling followed by liquid chromatography and mass spectrometry quantified protein abundance.
- Proteins with altered abundance were analyzed for pathway enrichment and temporal patterns.
Main Results:
- Over 1500 proteins exhibited altered abundance in the EMB or VYS after VPA exposure.
- Significant proteins showed limited overlap between VYS and EMB, suggesting distinct tissue-specific responses.
- Pathway analysis revealed VPA affects convergent extension, Wnt signaling, cell migration, proliferation, death, and cytoskeletal organization.
Conclusions:
- VPA exposure causes significant temporal alterations in protein abundance within the VYS and EMB during critical developmental periods.
- These protein changes affect key pathways involved in embryonic development and NTD formation.
- Understanding these proteomic shifts provides novel insights into VPA's teratogenic mechanisms.
Abstract:
Valproic acid (VPA) is an anti-epileptic medication that increases the risk of neural tube defect (NTD) outcomes in infants exposed during gestation. Previous studies into VPA's mechanism of action have focused on alterations in gene expression and metabolism but have failed to consider how exposure changes the abundance of critical developmental proteins over time. This study evaluates the effects of VPA on protein abundance in the developmentally distinct tissues of the mouse visceral yolk sac (VYS) and embryo proper (EMB) using mouse whole embryo culture. Embryos were exposed to 600 μM VPA at 2 h intervals over 10 h during early organogenesis with the aim of identifying protein pathways relevant to VPA's mechanism of action in failed NTC. Protein abundance was measured through tandem mass tag (TMT) labeling followed by liquid chromatography and mass spectrometry. Overall, there were over 1500 proteins with altered abundance after VPA exposure in the EMB or VYS with 428 of these proteins showing previous gene expression associations with VPA exposure. Limited overlap of significant proteins between tissues supported the conclusion of independent roles for the VYS and EMB in response to VPA. Pathway analysis of proteins with increased or decreased abundance identified multiple pathways with mechanistic relevance to NTC and embryonic development including convergent extension, Wnt Signaling/planar cell polarity, cellular migration, cellular proliferation, cell death, and cytoskeletal organization processes as targets of VPA. Clustering of co-regulated proteins to identify shared patterns of protein abundance over time highlighted 4 h and 6/10 h as periods of divergent protein abundance between control and VPA-treated samples in the VYS and EMB, respectively. Overall, this study demonstrated that VPA temporally alters protein content in critical developmental pathways in the VYS and the EMB during early organogenesis in mice.
